BIOMARKERS FOR ASSESSING TREATMENT OF SIALIC ACID DEFICIENCY DISEASES AND CONDITIONS

TECHNICAL FIELD

The present invention relates to biomarkers for determining responsiveness and monitoring the treatment of sialic acid deficiency diseases and conditions, e.g., Hereditary Inclusion Body Myopathy (HIBM).

BACKGROUND

Sialic acid is the only sugar that contains a net negative charge and is typically found on terminating branches of N-glycans, O-glycans, and glycosphingolipids (gangliosides) (and occasionally capping side chains of GPI anchors). The sialic acid modification of cell surface molecules is crucial for many biological phenomena including protein structure and stability, regulation of cell adhesion, and signal transduction. Sialic acid deficiency disorders such as Hereditary Inclusion Body Myopathy (HIBM or HIBM type 2), Nonaka myopathy, and Distal Myopathy with Rimmed Vacuoles (DMRV) are clinical diseases resulting from a reduction in sialic acid production.

HIBM is a rare autosomal recessive neuromuscular disorder caused by a specific biosynthetic defect in the sialic acid synthesis pathway. Eisenberg et al., Nat. Genet. 29:83-87 (2001). The disease manifests between the ages of 20 to 40 with foot drop and slowly progressive muscle weakness and atrophy. Patients may suffer difficulties walking with foot drop, gripping and using their hands, and normal body functions like swallowing. Histologically, it is associated with muscle fiber degeneration and formation of vacuoles containing 15-18 nm tubulofilaments that immunoreactive like β-amyloid, ubiquitin, prion protein and other amyloid-related proteins. Askanas et al., Curr Opin Rheumatol. 10:530-542 (1998). Both the progressive weakness and histological changes initially spare the quadriceps and certain other muscles of the face. However, the disease is relentlessly progressive with patients becoming incapacitated and wheelchair-confined within one to two decades. There are no treatments currently available. Other causative mutations were identified for HIBM in the gene GNE, which encodes the bifunctional enzyme UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK). Studies of an Iranian-Jewish genetic isolate mapped the mutation associated with HIBM to chromosome 9p12-13. Argov et al., Neurology 60:1519-1523 (2003). Eisenberg et al., Nat. Genet. 29:83-87 (2001). DMRV is a Japanese variant, allelic to HIBM. Nishino et al., Neurology 59:1689-1693 (2002).

The current assessment of HIBM patients requires the use of a muscle biopsy and the assessment of sialylation of muscle bound glycoproteins such as PSA-NCAM. Ricci et al., Neurology, 66(5), 755-8 (2006); Broccolini et al., Neurology 75 265-272 (2010); Tajima et al., The American Journal of Pathology, 166(4) 1121-1130 (2005); Nemunaitis et al., J Gene Med, 12(5) 403-12 (2010). Muscle biopsies cannot be assessed regularly, are difficult to quantify and cannot be used reliably for regular management or drug development studies. Assessment can also be done genotypically.

Given the problems associated with current methods for diagnosing HIBM and determining responsiveness to and/or monitoring treatment of HIBM patients, there is a need for methods which allow for diagnosing and monitoring treatment of HIBM patients.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention include methods for monitoring responsiveness or efficacy of a treatment with a sialic acid deficiency treatment in a subject suffering from a sialic acid deficiency. The methods comprise detecting the level of one or more sialic acid replacement therapy (SAT) biomarkers in a biological sample from the subject treated for a sialic acid deficiency, wherein an increase or decrease in the level of one or more SAT biomarkers indicates efficacy of the treatment with the sialic acid deficiency treatment. In some embodiments, the method is conducted within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, the presence or absence of a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level indicates efficacy of treatment with the sialic acid deficiency treatment a short period of time after the treatment starts.

Embodiments of the present invention also include methods for determining the treatment regimen for treating a sialic acid deficiency comprising detecting the level of one or more SAT biomarkers in a biological sample from a subject treated for a sialic acid deficiency and determining a treatment regimen based on an increase or decrease in the level of one or more SAT biomarkers in the biological sample. In some embodiments, the method is conducted before the treatment. In some embodiments, the treatment is adopted if an increase or decrease in the baseline level of one or more SAT biomarkers in the biological sample is present compared to a predetermined standard level. In some embodiments, the method is conducted within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, the determination is based on the presence or absence of a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level in the biological sample a short period of time after the treatment starts.

Embodiments of the present invention also include methods for predicting the treatment efficacy of a sialic acid deficiency treatment, comprising detecting the level of one or more SAT biomarkers in a biological sample from a subject, wherein an increase or decrease in the level of one or more SAT biomarkers compared to a predetermined standard level is predictive of the treatment efficacy of the sialic acid deficiency treatment. In some embodiments, the method is conducted before the treatment. In some embodiments, the treatment is predicted to be effective if an increase or decrease in the baseline level of one or more SAT biomarkers in the biological sample is present compared to a predetermined standard level. In some embodiments, the method is conducted within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, the presence or absence of a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level is predictive of the treatment efficacy of the sialic acid deficiency treatment.

Embodiments of the present invention also include methods for determining whether a subject with a sialic acid deficiency is suitable for a sialic acid deficiency treatment comprising detecting the level of one or more SAT biomarkers in a sample from the subject, wherein an increase or decrease in the level of one or more SAT biomarkers compared to a predetermined standard level indicates a subject is suitable for a sialic acid deficiency treatment. In some embodiments, the method is conducted before the treatment. In some embodiments, the subject is predicted to be suitable for the treatment if an increase or decrease in the baseline level of one or more SAT biomarkers in the biological sample is present compared to a predetermined standard level. In some embodiments, the method is conducted within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, the presence or absence of a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level indicates a subject is suitable for the sialic acid deficiency treatment.

Embodiments of the present invention provide methods for treating a subject with a sialic acid deficiency. These methods comprise detecting the level of one or more SAT biomarkers in a biological sample from the subject and administering a sialic acid deficiency treatment to the subject if the level of one or more SAT biomarkers is increased or decreased compared to a predetermined standard level. In some embodiments, the detecting step is conducted before the treatment. In some embodiments, a sialic acid deficiency treatment is administered to the subject if the baseline level of one or more SAT biomarkers is increased or decreased compared to a predetermined standard level. In some embodiments, the method is conducted within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, a sialic acid deficiency treatment is continued to be administered to the patient if there is a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level.

Embodiments of the present invention provide methods for treating a subject with a sialic acid deficiency. These methods comprise receiving information on the level of one or more SAT biomarkers in a biological sample from the subject, and administering a sialic acid deficiency treatment to the subject if the level of one or more SAT biomarkers is increased or decreased compared to a predetermined standard level. In some embodiments, the information is collected before the treatment. In some embodiments, the sialic acid deficiency treatment is administered to the subject if an increase or decrease in the baseline level of one or more SAT biomarkers in the biological sample is present compared to a predetermined standard level. In some embodiments, the information is collected within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, a sialic acid deficiency treatment is continuously administered to the patient if there is a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level.

Embodiments of the present invention provide methods for providing data comprising detecting the level of one or more SAT biomarkers in a sample from a subject and providing the information regarding the level of one or more biomarkers to a healthcare provider for diagnosis or treatment of the subject. In some embodiments, the data is collected before the treatment. In some embodiments, the data is collected within a short period of time after the treatment starts. For example, the data is collected right after at least one administration of the sialic acid deficiency treatment.

Embodiments of the present invention provide methods of providing useful information for predicting or determining the treatment efficacy of a sialic acid deficiency treatment comprising determining the level of one or more SAT biomarkers from a biological sample of a subject and providing the level of one or more SAT biomarkers to an entity that provides a prediction or determination of the treatment efficacy based on an increase or decrease in the level of one or more of the SAT biomarkers in a subject. In some embodiments, the information is collected before the treatment. In some embodiments, the determination is based on an increase or decrease in the baseline level of one or more SAT biomarkers in the biological sample compared to a predetermined standard level. In some embodiments, the information is collected within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. In some embodiments, the determination is based on the presence or absence of a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level in the biological sample.

Embodiments of the present invention provide a combination of tests useful for predicting or determining the treatment efficacy of sialic acid deficiency treatment comprising a first test for detecting the level of one SAT biomarker from a biological sample from a subject and a second test for detecting the level of a second SAT biomarker from a biological sample, wherein the first SAT biomarker is different from the second SAT biomarker.

Embodiments of the present invention provide for kits comprising reagents for detecting the level of one or more SAT biomarkers in a biological sample and an instruction for using the SAT biomarker according to any of the methods described herein.

Embodiments of the present invention provide for a collection of level of a panel of SAT biomarkers. In some embodiments, the SAT biomarkers comprise at least two or more SAT biomarkers of the present invention. In some embodiments, the SAT biomarkers are selected from those listed in Tables 2-14. In some embodiments, the SAT biomarkers are selected from AgRP, AR, BDNF, CD40-L, CgA, Cortisol, CK-MB, EGF, ENA-78, FGF-4, IGFBP-6, IL-3, IL-5, IL-7, IL-8, Kallikrein 5, LAP TGF-b1, M-CSF, MIP-3 alpha, MMP-1, MMP-3, MMP-9, MPO, Myoglobin, NT proBNP, NSE, Nr-CAM, PAI-1, PDGF-BB, S100-A4, S100-A6, ErbB3, SGOT, RANTES, Thrombospondin-1, TG and VEGF-C. In some embodiments, SAT biomarkers can include but are not limited to Chromogranin-A (CgA), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78) Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-9, total (MMP-9, total), Myoglobin, Neuronal Cell Adhesion Molecule (Nr-CAM), Plasminogen Activator Inhibitor 1 (PAI-1), Receptor tyrosine-protein kinase erbB-3 (ErbB3), and Vascular Endothelial Growth Factor C (VEGF-C).

Embodiments of the present invention provide for an array comprising probes for detection of at least two or more biomarkers. In some embodiments, the SAT biomarkers comprise at least two or more SAT biomarkers of the present invention. In some embodiments, the SAT biomarkers are selected from those listed in Tables 2-14. In some embodiments, the SAT biomarkers are selected from AgRP, AR, BDNF, CD40-L, CgA, Cortisol, CK-MB, EGF, ENA-78, FGF-4, IGFBP-6, IL-3, IL-5, IL-7, IL-8, Kallikrein 5, LAP TGF-b1, M-CSF, MIP-3 alpha, MMP-1, MMP-3, MMP-9, MPO, Myoglobin, NT proBNP, NSE, Nr-CAM, PAI-1, PDGF-BB, S100-A4, S100-A6, ErbB3, SGOT, RANTES, Thrombospondin-1, TG and VEGF-C. In some embodiments, the array is a microarray. In some embodiments, SAT biomarkers can include but are not limited to Chromogranin-A (CgA), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78) Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-9, total (MMP-9, total), Myoglobin, Neuronal Cell Adhesion Molecule (Nr-CAM), Plasminogen Activator Inhibitor 1 (PAI-1), Receptor tyrosine-protein kinase erbB-3 (ErbB3), and Vascular Endothelial Growth Factor C (VEGF-C).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a diagram of intracellular sialic acid metabolism.

FIG. 2. LAP TGF-b1 as an example of biomarker showing trend of normalization when the baseline level in HIBM patients was higher than normal controls. Serum level of the analyte at week 0 and week 24 from individual patient (black lines) within each dosage group was plotted. Levels of LAP TGF-b1 in 6 g/day dosage group declined at a faster rate (average delta, bolded line) towards normal level (green line) compared to placebo. FIG. 2A depicts change in serum biomarker levels in HIBM patients of placebo group between week 0 and week 24. FIG. 2B depicts change in serum biomarker levels in HIBM patients of 6 g/day group between week 0 and week 24.

FIG. 3. NCAM as an example of biomarker showing trend of normalization when the baseline level in HIBM patients was lower than normal controls. Serum level of the analyte at week 0 and week 24 from individual patient (black lines) within each dosage group was plotted. Levels of NCAM in 6 g/day dosage group increased at a faster rate (average delta, bolded line) towards normal level (green line) compared to placebo. FIG. 3A depicts change in serum biomarker levels in HIBM patients of placebo group between week 0 and week 24. FIG. 3B depicts change in serum biomarker levels in HIBM patients of 6 g/day group between week 0 and week 24.

FIGS. 4A-4D. Delta (A) of serum biomarkers between week 0 and week 24 in each dosage group. For analytes with baseline levels in HIBM patients higher than normal controls (marked as RED), the slope of delta always displayed a downward trend from placebo to 6 g/day group (x-axis), indicating a more favorable change in the 6 g/day group moving toward normal levels. In contrast, for analytes with baseline levels lower than normal controls (marked as BLUE), the slope of delta always displayed a upward trend.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms shall have the following meanings:

The verb “comprise” as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

The term “a” or “an” refers to one or more of that entity; for example, “a gene” refers to one or more genes or at least one gene. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements.

The invention provides isolated, chimeric, recombinant or synthetic polynucleotide sequences. As used herein, the terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein and encompass DNA, RNA, cDNA, whether single stranded or double stranded, as well as chemical modifications thereof. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5′-monophosphate form) are referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide. In some embodiments, the isolated, chimeric, recombinant or synthetic polynucleotide sequences are derived from gene markers of the present invention.

Single letter amino acid abbreviations used herein have their standard meaning in the art, and all peptide sequences described herein are written according to convention, with the N-terminal end to the left and the C-terminal end to the right.

The invention provides probes and primers that are derived from the nucleic acid sequences of the biomarker genes. The term “probe” as used herein refers to an oligonucleotide which is capable of specific annealing to the amplification target. The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A/T vs. G/C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

The terms “array” or “matrix” refer to an arrangement of addressable locations or “addresses” on a device. The locations can be arranged in two-dimensional arrays, three-dimensional arrays, or other matrix formats. The number of locations may range from several to at least hundreds of thousands. Most importantly, each location represents a totally independent reaction site. A “nucleic acid array” refers to an array containing nucleic acid probes, such as oligonucleotides or larger portions of genes. The nucleic acid on the array is preferably single-stranded. Arrays wherein the probes are oligonucleotides are referred to as “oligonucleotide arrays” or “oligonucleotide chips.” A “microarray,” also referred to herein as a “biochip” or “biological chip,” is an array of regions having a density of discrete regions of at least about 100/cm², and preferably at least about 1000/cm². The regions in a microarray have typical dimensions, for example, diameters, in the range of between about 10-250 μm, and are separated from other regions in the array by about the same distance. None limiting examples of compositions and methods for making and using arrays are described in U.S. Pat. Nos. 5,202,231, 5,695,940, 5525464, 5445934, 5744305, 5677195, 5800992, 5871928, 5795716, 5700637, 6054270, 5807522, and 6110426, each of which is incorporated by reference herein in its entirety for all purposes.

The present invention is based at least in part, on the surprising discovery that specific biomarkers can be employed to evaluate, predict, and determine efficacy of treatment for a sialic acid deficiency treatment. Compared to previous technologies, which relied on muscle tissue biopsies, this discovery is less invasive and thus much easier to use for regularly monitoring responsiveness or efficacy of a sialic acid therapy (SAT) in a subject, determining whether a subject with a sialic acid deficiency is suitable for sialic acid deficiency treatment and using that information to improve treatment of such subjects, among other methods described herein. In some embodiments, the prediction or determination is made before the treatment. In some embodiments, the prediction or determination is based on an increase or decrease in the baseline level of one or more SAT biomarkers in the biological sample compared to a predetermined standard level. In some embodiments, the prediction/determination is made within a short period of time after the treatment starts. Within the short period of time, when there is no obvious alteration of symptoms of the diseases due to the treatment yet, it is hard to predict or determine the efficacy of the treatment and if the treatment is suitable for the subject by other methods. However, using the methods of the present invention, the prediction or determination can be quickly made based on the presence or absence of a normalization or stabilization in the level of one or more SAT biomarkers toward a predetermined standard level in the biological sample.

As used herein, the term “marker” or “biomarker” encompasses a broad range of intra- and extra-cellular events as well as whole-organism physiological changes. A marker may be represent essentially any aspect of cell function, for example, but not limited to, levels or rate of production of signaling molecules, transcription factors, metabolites, gene transcripts as well as post-translational modifications of proteins. Marker may include partial and/or whole genome analysis of transcript levels, rates, and/or stability, and partial and/or whole proteome analysis of protein levels, activity and/or modifications. A signature may refer to a gene or gene product which is up- or down-regulated in a subject to be treated compared to clinically normal subjects. A signature may also refer to a gene or gene product which is up- or down-regulated in a treated subject having the disease compared to an untreated subjects. That is, the gene or gene product is sufficiently specific to the treated cell that it may be used, optionally with other genes or gene products, to identify, predict, or detect efficacy of a small molecule. Thus, in some embodiments, a signature is a gene or gene product that is characteristic of efficacy of a compound in a diseased cell or the response of that diseased cell to treatment by the compound.

As used herein, sialic acid deficiency treatment refers to any treatment that can either increase the endogenous sialic acid level, and/or activating the sialic acid signaling transduction pathway.

As used herein, the term “sialic acid” refers to sialic acid, any functional derivatives thereof, analogs thereof, any anomers thereof, such as those disclosed in U.S. Pat. Nos. 4,694,076, 8,293,888, 6,288,041, 5,783,564, 7,875,708, 5,712,254, 5,438,125, 4,935,506, 5,077,397, RE34,091, 5,243,035, 4,918,177, 6,444,649, 4,968,786, 5,834,423, 5,621,086, 5,034,516, 7,413,729, 4,990,603, 4,914,035, 5,350,841, 8,217,154, 7,807,824, 5,519,007, 5,792,858, 5,459,031, 8,097,591, 5,453,272, 4,457,865, 5,233,033, 7,867,541, 7,951,410, 5,792,842, 4,520,111, 5,849,717, 5,679,321, 5,851,395, 5,750,508, 5,192,661, 5,658,880, 5,405,753, 4,963,653, 5,679,645, 8,323,654, 5,330,897, 5,334,514, 5,908,766, 7,803,583, 5,660,992, 8,148,335, and WO/2013/063149, each of which is incorporated by reference in its entirety. As used herein, the word “derivative” as used herein includes derivatives, analogs, prodrugs, and unnatural precursors.

In some embodiments, the invention provides methods for monitoring responsiveness or efficacy of a sialic acid deficiency treatment in a subject suffering from a sialic acid deficiency comprising detecting the level of one or more sialic acid therapy (SAT) biomarkers in a biological sample from the subject treated for a sialic acid deficiency, wherein an increase or decrease in the level of one or more SAT biomarkers indicates efficacy of treatment with the sialic acid deficiency treatment. The term “sample” or “biological sample” as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. The sample may be a sample which is derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white blood cells), tissue or biopsy samples (e.g., tumor biopsy), urine, peritoneal fluid, and pleural fluid, patient derived xenografts (PDXs), or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

In some embodiments, a collection of activity profiles of a panel of biomarkers is provided. As used herein, the term “activity profile” refers to a set of data representing distinctive features or characteristics of one or more gene markers. Such features or characteristics include, but are not limited to, transcript abundance, transcript stability, transcription rate, translation rate, post-translation modification, protein abundance, protein stability, and/or protein enzymatic activity, etc. In some embodiments, the activity profile comprises data related to gene expression level of each biomarker. In some embodiments, the collection comprises activity profiles is obtained from a specific population of subjects. In some embodiments, the specific population of subjects consists of clinically normal subjects.

In some embodiments, the collection comprises activity profiles that are statistically homogeneous in one or more aspects, e.g., statistically homogeneous in one or more quantitative or semi-quantitative parameters describing the features and characteristics of the activity profiles. In some embodiments, the quantitative parameters include, but are not limited to, transcript abundance, transcript stability, transcription rate, translation rate, post-translation modification, protein abundance, protein stability, and/or protein enzymatic activity, etc. Whether a group of activity profiles are statistically homogeneous or not in one or more aspects can be determined by any suitable statistic test and/or algorithm known to one skilled in the art.

In some embodiments, one or more of the biomarkers increase its activity in response to the treatment. In some embodiments, one or more of the biomarkers decrease its activity in response to the treatment. In some embodiments, one or more of the biomarkers remains its activity in response to the treatment. As used herein, the term “gene activity” refers to gene expression level, RNA activity level, or protein activity level. As used herein, the term “RNA activity level refers to mRNA abundance, synthesis rate, and/or stability, etc. As used herein, the term “protein activity level” refers to protein abundance, synthesis rate, stability, enzymatic activity, phosphorylation rate, etc.

In some embodiments, one or more of the biomarkers in a subject increases its activity and goes toward the level of a predetermined standard level. In some embodiments, one or more of the biomarkers in a subject decreases its activity and goes toward the level of a predetermined standard level. As used herein, when the level of a biomarker goes toward the level of a predetermined standard level, it is called normalization. In some embodiments, the normalization biomarkers of the present invention include, but art not limited to Chromogranin-A (CgA), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78) Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Myoglobin, Neuronal Cell Adhesion Molecule (Nr-CAM), and Plasminogen Activator Inhibitor 1 (PAI-1). In some embodiments, one or more of the biomarkers in a subject been treated with a drug has a less change in its activity compared to the same biomarker in a subject treated with a placebo which goes further away from a predetermined standard level. In some embodiments, the same biomarker in a subject treated with a drug reduces its speed of going away from the predetermined standard level compared to that of a placebo treatment. As used herein, when the level of a biomarker reduces its speed of going away from the level of a predetermined standard level, it is called stabilization. In some embodiments, the stabilization biomarkers of the present invention include, but art not limited to Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-9, total (MMP-9, total), Receptor tyrosine-protein kinase erbB-3 (ErbB3), and Vascular Endothelial Growth Factor C (VEGF-C).

In some embodiments, the collection of activity profiles of one or more gene markers of the present invention is obtained from one or more tests. The test can be performed by the subject himself/herself, by a doctor, by a nurse, by a test lab, by a healthcare provider, or any other parties capable of doing the test. The test results containing the collection of activity profiles can be then analyzed by the same party or by a second party, such as the subject himself/herself, a doctor, a nurse, a test lab, a healthcare provider, a physician, a clinical trial personnel, a hospital, a lab, a research institute, or any other parties capable of analyzing the test to determine if the subject is responsive to the drug.

In some embodiments, the present invention provides methods for determining the treatment regimen for treating a sialic acid deficiency. The methods include detecting the level of one or more SAT biomarkers in a biological sample from a subject treated for a sialic acid deficiency and determining a treatment regimen based on an increase or decrease in the level of one or more SAT biomarkers in the biological sample. In some embodiments, the treatment regimen is continued when the level of one or more biomarkers of the present invention goes toward the level of a predetermined standard level, or reduces the speed of going away from the predetermined standard level compared to that of a placebo treatment.

In some other embodiments, the present invention provides methods for predicting the treatment efficacy of a sialic acid deficiency treatment. The method includes detecting the level of one or more SAT biomarkers in a biological sample from a subject, wherein an increase or decrease in the level compared to a predetermined standard level is predictive of the treatment efficacy of the sialic acid deficiency treatment. In some embodiments, the level of one or more SAT biomarkers in the biological sample from the subject is detected before the treatment, and the treatment is determined to be effective if the baseline level of one or more SAT biomarkers is increased or decreased compared to a predetermined standard level. In some embodiments, the level of one or more SAT biomarkers in the biological sample from the subject is detected a short period of time after the treatments. For example, the detecting step is conducted right after at least one, two, three, four, five, six, seven, eight, nine, ten, or more administrations of the sialic acid deficiency treatment. Each administration is spaced by a half day, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, or more. Within such short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet, but the efficacy of the treatment be can be predicted based on the level of one or more biomarkers compared to the predetermined standard level. In some embodiments, the level of one or more biomarkers in a subject group treated with placebo is also included as a control. In some embodiments, the treatment is determined to be effective if one or more biomarkers of the present invention goes toward the level of a predetermined standard level, or reduces the speed of going away from the predetermined standard level compared to that of a placebo treatment.

In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at last nineteen, at least twenty, at least twenty five, at least thirty, at least thirty five, at least forty, at least forty five, at least fifty, at least fifty five, at least sixty, at least sixty five, at least seventy, at least seventy five, at least eighty, at least eighty five, at least ninety, at least ninety five, at least one hundred or more biomarkers of the present invention provide a pattern that indicates the treatment is effective. The more biomarkers that give such a pattern, the more accurate the prediction is.

In some embodiments, the methods of the present invention comprise detecting the level of one or more biomarkers in a biological sample of a subject before the treatment. In some embodiments, the methods of the present invention comprise detecting the level of one or more biomarkers in a biological sample of a subject after the treatment. In some embodiments, the detecting step is conducted within a short period of time after the treatment starts. In some embodiments, within the short period of time, there is no obvious alteration of symptoms of the diseases due to the treatment yet. For example, the detecting step is conducted right after at least one administration of the sialic acid deficiency treatment. In some embodiments, the short period of time lasts for about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, one year, two years, three years, or more.

In yet some other embodiments, the present invention provides methods for determining whether a subject with a sialic acid deficiency is suitable for a sialic acid deficiency treatment. The methods include detecting the level of one or more SAT biomarkers in a sample from the subject, wherein an increase or decrease in the level of one or more SAT biomarkers compared to a predetermined standard level indicates a subject is suitable for a sialic acid deficiency treatment. In some embodiments, the patient is determined to be suitable for the treatment when the level of one or more biomarkers of the present invention in the patient goes toward the level of a predetermined standard level, or reduces the speed of going away from the predetermined standard level compared to that of a placebo treatment.

In yet further embodiments the present invention provides methods for treating a subject with a sialic acid deficiency. The methods include detecting the level of one or more SAT biomarkers in a biological sample from the subject and administering a sialic acid deficiency treatment to the subject if the level of one or more SAT biomarkers is increased or decreased compared to a predetermined standard level. In some embodiments, the dosage of the treatment is determined by monitoring the level of one or more SAT biomarkers. In some embodiments, the dosage of the treatment is modified according to the level of one or more SAT biomarkers in the sample. In some embodiments, the dosage of the treatment can be raised when the subject is less sensitive to the treatment compared to a clinically normal group of subjects. In some embodiments, the dosage can be reduced when the subject is more sensitive to the treatment compared to a clinically normal group of subjects.

In still other embodiments, the present invention provides methods for providing data. These methods include detecting the level of one or more SAT biomarkers in a sample from a subject and providing the information regarding the level of one or more SAT biomarkers to a healthcare provider for diagnosis or treatment of the subject. In some embodiments, the biological sample is received from a healthcare provider.

In yet other embodiments, the present invention provides methods of providing useful information for predicting or determining the treatment efficacy of a sialic acid deficiency treatment comprising determining the level of one or more SAT biomarkers from a biological sample of a subject and providing the level of one or more SAT biomarkers to an entity that provides a prediction or determination of the treatment efficacy based on an increase or decrease in the level of one or more of the SAT biomarkers in a subject. In some embodiments the subject is treated with the sialic acid deficiency treatment. In some embodiments the level of one or more SAT biomarkers is determined before treatment with the sialic acid deficiency treatment. In some embodiments the level of one or more SAT biomarkers is determined after treatment with the sialic acid deficiency treatment. In some embodiments the level of one or more SAT biomarkers is compared between before treatment with the sialic acid deficiency treatment and after treatment with the sialic acid deficiency treatment. In some embodiments the level of one or more SAT biomarkers is determined before and/or after treatment with the sialic acid deficiency treatment and is compared to a predetermined standard level.

In still other embodiments, the present invention provides a combination of tests useful for predicting or determining the treatment efficacy of a sialic acid deficiency treatment comprising a first test for detecting the level of one SAT biomarker from a biological sample from a subject and a second test for detecting the level of a second SAT biomarker of said biological sample, wherein the first SAT biomarker is different from the second SAT biomarker.

Sialic acid deficiencies of the present invention refers to symptoms associated with any abnormal activity of the sialic acid when compared to a normal subject, which include but are not limited to Hereditary Inclusion Body Myopathy (HIBM), Nonaka myopathy, or Distal Myopathy with Rimmed Vacuoles (DMRV). In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM). In some embodiments, the sialic acid deficiency is due to a mutation and/or deficiency in the GNE gene, which encodes the bi-functional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase. In some cases, the sialic acid deficiency is due to a homogeneous or homozygous mutation and/or deficiency in the GNE gene. In some embodiments, the sialic acid deficiency is due to a heterogeneous or heterozygous mutation and/or deficiency in the GNE. In some embodiments, the sialic acid deficiency is not due to a mutation and/or deficiency in the GNE gene.

SAT biomarkers contemplated for use with the methods of the present invention can include but are not limited to those listed in Tables 2-14.

In some embodiments, SAT biomarkers can include but are not limited to 6Ckine, Agouti-Related Protein (AgRP), Aldose Reductase, Alpha-1-Antichymotrypsin (AACT), Alpha-2-Macroglobulin (A2Macro), Amphiregulin (AR), Angiogenin, Angiotensin-Converting Enzyme (ACE), Angiotensinogen, Apolipoprotein A-I (Apo A-I), Apolipoprotein A-II (Apo A-II), Apolipoprotein B (Apo B), Apolipoprotein C-I (Apo C-I), Apolipoprotein C-III (Apo C-III), Apolipoprotein E (Apo E), AXL Receptor Tyrosine Kinase (AXL), B cell-activating factor (BAIT), Brain-Derived Neurotrophic Factor (BDNF), Calbindin, Carcinoembryonic Antigen (CEA), CD40 Ligand (CD40-L), CD5 Antigen-like (CD5L), Cellular Fibronectin (cFib), Chromogranin-A (CgA), Collagen IV, Complement C3 (C3), Cortisol (Cortisol), Creatine Kinase-MB (CK-MB), E-Selectin, EN-RAGE, Endoglin, Endostatin, Eotaxin-1, Epidermal Growth Factor (EGF), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Erythropoietin (EPO), Factor VII, Fatty Acid-Binding Protein, adipocyte (FABP, adipocyte), Fatty Acid-Binding Protein, heart (FABP, heart), Fatty Acid-Binding Protein, liver (FABP, liver), Fibrinogen, Fibroblast Growth Factor 4 (FGF-4), Galectin-3, Glucagon-like Peptide 1, total (GLP-1 total), Glutathione S-Transferase alpha (GST-alpha), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Haptoglobin, HE4, Heparin-Binding EGF-Like Growth Factor (HB-EGF), Hepatocyte Growth Factor (HGF), Hepatocyte Growth Factor receptor (HGF receptor), Human Epidermal Growth Factor Receptor 2 (HER-2), Immunoglobulin A (IgA), Insulin-like Growth Factor-Binding Protein 2 (IGFBP-2), Insulin-like Growth Factor-Binding Protein 3 (IGFBP-3), Insulin-like Growth Factor Binding Protein 4 (IGFBP-4), Insulin-like Growth Factor Binding Protein 6 (IGFBP-6), Intercellular Adhesion Molecule 1 (ICAM-1), Interferon-inducible T-cell alpha chemoattractant (ITAC), Interleukin-2 receptor alpha (IL-2 receptor alpha), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 receptor subunit beta (IL-6R beta), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-15 (IL-15), Kallikrein 5, Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Lectin-Like Oxidized LDL Receptor 1 (LOX-1), Leptin, Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha), Macrophage Inflammatory Protein-3 alpha (MIP-3 alpha), Macrophage-Stimulating Protein (MSP), Malondialdehyde-Modified Low-Density Lipoprotein (MDA-LDL), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3), Matrix Metalloproteinase-9, total (MMP-9, total), Mesothelin (MSLN), MHC class I chain-related protein A (MICA), Monocyte Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 2 (MCP-2), Monocyte Chemotactic Protein 4 (MCP-4), Monokine Induced by Gamma Interferon (MIG), Myeloid Progenitor Inhibitory Factor 1 (MPIF-1), Myeloperoxidase (MPO), Myoglobin, Neuron-Specific Enolase (NSE), Neuronal Cell Adhesion Molecule (Nr-CAM), Neutrophil Gelatinase-Associated Lipocalin (NGAL), Pepsinogen I (PGI), Peptide YY (PYY), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Pregnancy-Associated Plasma Protein A (PAPP-A), Progesterone, Prostatic Acid Phosphatase (PAP), Protein S100-A4 (S100-A4), Protein S 100-A6 (S100-A6), Receptor for advanced glycosylation end products (RAGE), Receptor tyrosine-protein kinase erbB-3 (ErbB3), Resistin, Serum Glutamic Oxaloacetic Transaminase (SGOT), Sortilin, Stem Cell Factor (SCF), Stromal cell-derived factor-1 (SDF-1) Superoxide Dismutase 1, soluble (SOD-1), T-Cell-Specific Protein RANTES (RANTES), T Lymphocyte-Secreted Protein 1-309 (1-309), Tenascin-C (TN-C), Thrombomodulin (TM), Thrombopoietin (TPO), Thrombospondin-1, Thyroglobulin (TG), Thyroxine-Binding Globulin (TBG), Tissue Factor (TF), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), Tumor Necrosis Factor alpha (TNF-alpha), Tumor Necrosis Factor Receptor I (TNFR1), Urokinase-type Plasminogen Activator (uPA), Urokinase-type Plasminogen Activator Receptor (uPAR), Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor B (VEGF-B), Vascular Endothelial Growth Factor C (VEGF-C), Vascular Endothelial Growth Factor D (VEGF-D), Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3), Vitamin K-Dependent Protein S (VKDPS), Vitronectin and von Willebrand Factor (vWF). See for example, Table 2 biomarkers.

In some embodiments, SAT biomarkers can include but are not limited to Agouti-Related Protein (AgRP), Amphiregulin (AR), Brain-Derived Neurotrophic Factor (BDNF), CD40 Ligand (CD40-L), Chromogranin-A (CgA), Cortisol (Cortisol), Creatine Kinase-MB (CK-MB), Epidermal Growth Factor (EGF), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Fibroblast Growth Factor 4 (FGF-4), Insulin-like Growth Factor Binding Protein 6 (IGFBP-6), Interleukin-3 (IL-3), Interleukin-5 (IL-5), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Kallikrein 5, Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage Inflammatory Protein-3 alpha (MIP-3 alpha), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3), Matrix Metalloproteinase-9, total (MMP-9, total), Myeloperoxidase (MPO), Myoglobin, N-terminal prohormone of brain natriuretic peptide (NT proBNP), Neuron-Specific Enolase (NSE), Neuronal Cell Adhesion Molecule (Nr-CAM), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Protein S100-A4 (S100-A4), Protein S100-A6 (S100-A6), Receptor tyrosine-protein kinase erbB-3 (ErbB3), Serum Glutamic Oxaloacetic Transaminase (SGOT), T-Cell-Specific Protein RANTES (RANTES), Thrombospondin-1, Thyroglobulin (TG) and Vascular Endothelial Growth Factor C (VEGF-C). See for example, Table 7.

In some embodiments, SAT biomarkers can include but are not limited to Brain-Derived Neurotrophic Factor (BDNF), CD40 Ligand (CD40-L), Chromogranin-A (CgA), Creatine Kinase-MB (CK-MB), Insulin-like Growth Factor Binding Protein 6 (IGFBP-6), Interleukin-3 (IL-3), Interleukin-5 (IL-5), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-9, total (MMP-9, total), Myoglobin, Neuron Specific Enolase (NSE), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Receptor tyrosine-protein kinase erbB-3 (ErbB3), T-Cell-Specific Protein RANTES (RANTES), Thrombospondin-1 and Vascular Endothelial Growth Factor C (VEGF-C). See for example, Table 8.

In some embodiments, SAT biomarkers can include but are not limited to Brain-Derived Neurotrophic Factor (BDNF), CD40 Ligand (CD40-L), Chromogranin-A (CgA), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Matrix Metalloproteinase-9, total (MMP-9, total), Myoglobin, Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Receptor tyrosine-protein kinase erbB-3 (ErbB3), T-Cell-Specific Protein RANTES (RANTES) and Thrombospondin-1. See for example, Table 9.

In some embodiments, SAT biomarkers can include but are not limited to 6Ckine, Adiponectin Agouti-Related Protein (AGRP), Aldose Reductase, Alpha-1-Antichymotrypsin (AACT), Alpha-1-Antitrypsin (AAT), Alpha-1-Microglobulin (A1Micro), Alpha-2-Macroglobulin (A2Macro), Alpha-Fetoprotein (AFP), Amphiregulin (AR), Angiogenin, Angiopoietin-2 (ANG-2), Angiotensin-Converting Enzyme (ACE), Angiotensinogen, Annexin A1 (ANXA1), Apolipoprotein A-I (Apo A-I), Apolipoprotein A-II (Apo A-II), Apolipoprotein A-IV (Apo A-IV), Apolipoprotein B (Apo B), Apolipoprotein C-I (Apo C-I), Apolipoprotein C-III (Apo C-III), Apolipoprotein D (Apo D), Apolipoprotein E (Apo E), Apolipoprotein H (Apo H), Apolipoprotein(a) (Lp(a)), AXL Receptor Tyrosine Kinase (AXL), B cell-activating factor (BAFF), B Lymphocyte Chemoattractant (BLC), Bcl-2-like protein 2 (Bcl2-L-2), Beta-2-Microglobulin (B2M), Betacellulin (BTC), Bone Morphogenetic Protein 6 (BMP-6), Brain-Derived Neurotrophic Factor (BDNF), Calbindin, Calcitonin, Cancer Antigen 125 (CA-125), Cancer Antigen 15-3 (CA-15-3), Cancer Antigen 19-9 (CA-19-9), Cancer Antigen 72-4(CA-72-4), Carcinoembryonic Antigen (CEA), Cathepsin D, CD 40 antigen (CD40), CD40 Ligand (CD40-L), CD5 (CD5L), Cellular Fibronectin (cFib), Chemokine CC-4 (HCC-4), Chromogranin-A (CgA), Ciliary Neurotrophic Factor (CNTF), Clusterin (CLU), Collagen IV, Complement C3 (C3), Complement Factor I-I, Connective Tissue Growth Factor (CTGF), Cortisol (Cortisol), C-peptide, C-Reactive Protein (CRP), Creatine Kinase-MB (CK-MB), Cystatin-C, Endoglin, Endostatin, Endothelin-1 (ET-1), EN-RAGE Eotaxin-1, Eotaxin-2, Eotaxin-3, Epidermal Growth Factor (EGF), Epidermal Growth Factor Receptor (EGFR), Epiregulin (EPR), Epithelial cell adhesion molecule (EpCam), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Erythropoietin (EPO), E-Selectin, Ezrin, Factor VII, Fas Ligand (FasL), FASLG Receptor (FAS), Fatty Acid-Binding Protein, adipocyte (FABP, adipocyte), Fatty Acid-Binding Protein, heart (FABP, heart), Fatty Acid-Binding Protein, liver (FABP, liver), Ferritin (FRTN), Fetuin-A, Fibrinogen, Fibroblast Growth Factor 4 (FGF-4), Fibroblast Growth Factor basic (FGF-basic), Fibulin-1C (Fib-1C), Follicle-Stimulating Hormone (FSH), Galectin-3, Gelsolin, Glucagon, Glucagon-like Peptide 1, total (GLP-1 total), Glucose-6-phosphate Isomerase (G6PI), Glutamate-Cysteine Ligase Regulatory subunit (GCLR), Glutathione S-Transferase alpha (GST-alpha), Glutathione S-Transferase Mu 1 (GST-M1), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Growth Hormone (GH), Haptoglobin, HE4, Heat Shock Protein 60 (HSP-60), Heparin-Binding EGF-Like Growth Factor (HB-EGF), Hepatocyte Growth Factor (HGF), Hepatocyte Growth Factor receptor (HGF receptor), Hepsin, Human Chorionic Gonadotropin beta (hCG), Human Epidermal Growth Factor Receptor 2 (HER-2), Immunoglobulin A (IgA), Immunoglobulin E (IgE), Immunoglobulin M (IGM), Insulin, Insulin-like Growth Factor Binding Protein 4 (IGFBP4), Insulin-like Growth Factor Binding Protein 5 (IGFBP5), Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Insulin-like Growth Factor-Binding Protein 1 (IGFBP-1), Insulin-like Growth Factor-Binding Protein 2 (IGFBP-2), Insulin-like Growth Factor-Binding Protein 3 (IGFBP-3), Intercellular Adhesion Molecule 1 (ICAM-1), Interferon gamma (IFN-gamma), Interferon gamma Induced Protein 10 (IP-10), Interferon-inducible T-cell alpha chemoattractant (ITAC) Interleukin-1 alpha (IL-1 alpha), Interleukin-1 beta (IL-1 beta), Interleukin-1 receptor antagonist (IL-1ra), Interleukin-10 (IL-10), Interleukin-12 Subunit p40 (IL-12p40), Interleukin-12 Subunit p70 (IL-12p70), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-16 (IL-16), Interleukin-18 (IL-18), Interleukin-2 (IL-2), Interleukin-2 receptor alpha (IL-2 receptor alpha), Interleukin-25 (IL-25), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-6 receptor (IL-6r), Interleukin-6 receptor subunit beta(IL-6R beta), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Kallikrein 5, Kallikrein-7 (KLK-7), Kidney Injury Molecule-1 (KIM-1), Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Lectin-Like Oxidized LDL Receptor 1 (LOX-1), Leptin, Luteinizing Hormone (LH), Lymphotactin, Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage inflammatory protein 3 beta (MIP-3 beta), Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha), Macrophage Inflammatory Protein-1 beta (MIP-1 beta), Macrophage Inflammatory Protein-3 alpha (MIP-3 alpha), Macrophage Migration Inhibitory Factor (MIF), Macrophage-Derived Chemokine (MDC), Macrophage-Stimulating Protein (MSP), Malondialdehyde-Modified Low-Density Lipoprotein (MDA-LDL), Maspin, Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-10 (MMP-10), Matrix Metalloproteinase-2 (MMP-2), Matrix Metalloproteinase-3 (MMP-3), Matrix Metalloproteinase-7 (MMP-7), Matrix Metalloproteinase-9 (MMP-9), Matrix Metalloproteinase-9, total (MMP-9, total), Mesothelin (MSLN), MHC class I chain-related protein 1 (MICA), Monocyte Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 2 (MCP-2), Monocyte Chemotactic Protein 3 (MCP-3), Monocyte Chemotactic Protein 4 (MCP-4), Monokine Induced by Gamma Interferon (MIG), Myeloid Progenitor Inhibitory Factor 1 (MPIF-1), Myeloperoxidase (MPO), Myoglobin, Nerve Growth Factor beta (NGF-beta), Neuron Specific Enolase (NSE), Neuronal Cell Adhesion Molecule (Nr-CAM), Neuropilin-1Neutrophil Gelatinase-Associated Lipocalin (NGAL), N-terminal prohormone of brain natriuretic peptide (NT proBNP), Nucleoside diphosphate kinase B (NDK B), Osteopontin, Osteoprotegerin (OPG), Pancreatic Polypeptide (PPP), Pepsinogen I (PGI), Peptide YY (PYY), Peroxiredoxin 4 (Prx-IV), Phosphoserine Aminotransferase (PSAT), Placenta Growth Factor (PLGF), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Pregnancy-Associated Plasma Protein A (PAPP-A), Progesterone, Proinsulin (Intact), Proinsulin (Total), Prolactin (PRL), Prostasin, Prostate-Specific Antigen Free (PSA-f), Prostatic Acid Phosphatase (PAP), Protein S100-A4 (S100-A4), Protein S100-A6 (S100-A6), Pulmonary and Activation-Regulated Chemokine (PARC), Receptor for advanced glycosylation end products (RAGE), Receptor tyrosine-protein kinase erbB-3 (ErbB3), Resistin, S100 calcium-binding protein B (S100-B), Secretin, Serotransferrin (Transferrin), Serum Amyloid P-Component (SAP), Serum Glutamic Oxaloacetic Transaminase (SGOT), Sex Hormone-Binding Globulin (SHBG), Sortilin, Squamous Cell Carcinoma Antigen-1 (SCCA-1), Stem Cell Factor (SCF), Stromal cell-derived factor-1 (SDF-1), Superoxide Dismutase 1, Soluble (SOD-1), T Lymphocyte-Secreted Protein 1-309 (1-309), Tamm-Horsfall Urinary Glycoprotein (THP), T-Cell-Specific Protein RANTES (RANTES), Tenascin-C (TN-C), Testosterone (Total), Tetranectin, Thrombomodulin (TM), Thrombopoietin, Thrombospondin-1, Thyroglobulin (TG), Thyroid-Stimulating Hormone (TSH), Thyroxine-Binding Globulin (TBG), Tissue Factor (TF), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), Tissue type Plasminogen activator (tPA), TNF-Related Apoptosis-Inducing Ligand Receptor 3 (TRAIL-R3), Transforming Growth Factor alpha (TGF-alpha), Transforming Growth Factor beta-3 (TGF-beta-3), Transthyretin (TTR), Trefoil Factor 3 (TFF3), Tumor Necrosis Factor alpha (TNF-alpha), Tumor Necrosis Factor beta (TNF-beta), Tumor Necrosis Factor Receptor 2 (TNFR2), Tumor Necrosis Factor Receptor I (TNF R1), Tyrosine kinase with Ig and EGF homology domains 2 (TIE-2), Urokinase-type Plasminogen Activator (uPA), Urokinase-type Plasminogen Activator Receptor(uPAR), Vascular Cell Adhesion Molecule-1 (VCAM-1), Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor B (VEGF-B), Vascular Endothelial Growth Factor C (VEGF-C), Vascular Endothelial Growth Factor D (VEGF-D), Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3), Vitamin K-Dependent Protein S (VKDPS), Vitronectin, von Willebrand Factor (vWF) and YKL-40.

In some embodiments, SAT biomarkers can include but are not limited to 6Ckine, Adiponectin, Agouti-Related Protein (AgRP), Aldose Reductase, Alpha-1-Antichymotrypsin (AACT), Alpha-1-Antitrypsin (AAT), Alpha-1-Microglobulin (A1Micro), Alpha-2-Macroglobulin (A2Macro), Alpha-Fetoprotein (AFP), Amphiregulin (AR), Angiogenin, Angiopoietin-2 (ANG-2), Angiotensin-Converting Enzyme (ACE), Angiotensinogen, Apolipoprotein(a) (Lp(a)), Apolipoprotein A-I (Apo A-I), Apolipoprotein A-II (Apo A-II), Apolipoprotein A-IV (Apo A-IV), Apolipoprotein B (Apo B), Apolipoprotein C-I (Apo C-I), Apolipoprotein C-III (Apo C-III), Apolipoprotein D (Apo D), Apolipoprotein E (Apo E), Apolipoprotein H (Apo H), AXL Receptor Tyrosine Kinase (AXL), B cell-activating factor (BAFF), B Lymphocyte Chemoattractant (BLC), Beta-2-Microglobulin (B2M), Betacellulin (BTC), Brain-Derived Neurotrophic Factor (BDNF), C-Peptide, C-Reactive Protein (CRP), Calbindin, Cancer Antigen 125 (CA-125), Cancer Antigen 15-3 (CA-15-3), Cancer Antigen 19-9 (CA-19-9), Cancer Antigen 72-4 (CA 72-4), Carcinoembryonic Antigen (CEA), Cathepsin D, CD5 Antigen-like (CD5L), CD 40 antigen (CD40), CD40 Ligand (CD40-L), Cellular Fibronectin (cFib), Chemokine CC-4 (HCC-4), Chromogranin-A (CgA), Ciliary Neurotrophic Factor (CNTF), Clusterin (CLU), Collagen IV, Complement C3 (C3), Complement Factor H—Related Protein 1 (CFHR1), Cortisol (Cortisol), Creatine Kinase-MB (CK-MB), Cystatin-C, E-Selectin, EN-RAGE, Endoglin, Endostatin, Eotaxin-1, Eotaxin-2, Eotaxin-3, Epidermal Growth Factor (EGF), Epidermal Growth Factor Receptor (EGFR), Epiregulin (EPR), Epithelial cell adhesion molecule (EpCam), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Ezrin, Factor VII, Fas Ligand (FasL), FASLG Receptor (FAS), Fatty Acid-Binding Protein—adipocyte (FABP, adipocyte), Fatty Acid-Binding Protein—heart (FABP, heart), Fatty Acid-Binding Protein—liver (FABP, liver), Ferritin (FRTN), Fetuin-A, Fibrinogen, Fibroblast Growth Factor 4 (FGF-4), Fibroblast Growth Factor basic (FGF-basic), Fibulin-1C (Fib-1C), Follicle-Stimulating Hormone (FSH), Galectin-3, Gelsolin, Glucagon, Glucagon-like Peptide 1-active (GLP-1 active), Glucagon-like Peptide 1—total (GLP-1 total), Glucose-6-phosphate Isomerase (G6PI), Glutathione S-Transferase alpha (GST-alpha), Glutathione S-Transferase Mu 1 (GST-M1), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Growth Hormone (GH), Growth-Regulated alpha protein (GRO-alpha), Haptoglobin HE4, Heat Shock Protein 60 (HSP-60), Heparin-Binding EGF-Like Growth Factor (HB-EGF), Hepatocyte Growth Factor (HGF), Hepatocyte Growth Factor receptor (HGF receptor), Hepsin, Human Chorionic Gonadotropin beta (hCG), Human Epidermal Growth Factor Receptor 2 (HER-2), Immunoglobulin A (IgA), Immunoglobulin E (IgE), Immunoglobulin M (IgM), Insulin, Insulin-like Growth Factor-Binding Protein 1 (IGFBP-1), Insulin-like Growth Factor-Binding Protein 2 (IGFBP-2), Insulin-like Growth Factor-Binding Protein 3 (IGFBP-3), Insulin-like Growth Factor Binding Protein 4 (IGFBP4), Insulin-like Growth Factor Binding Protein 5 (IGFBP5), Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Intercellular Adhesion Molecule 1 (ICAM-1), Interferon gamma (IFN-gamma), Interferon gamma Induced Protein 10 (IP-10), Interferon-inducible T-cell alpha chemoattractant (ITAC), Interleukin-1 alpha (IL-1 alpha, Interleukin-1 beta (IL-1 beta), Interleukin-1 receptor antagonist (IL-1ra), Interleukin-2 (IL-2), Interleukin-2 receptor alpha (IL-2 receptor alpha), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interleukin-6 (IL-6), Interleukin-6 receptor (IL-6r), Interleukin-6 receptor subunit beta (IL-6R beta), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-12 Subunit p40 (IL-12p40), Interleukin-12 Subunit p70 (IL-12p70), Interleukin-13 (IL-13), Interleukin-15 (IL-15), Interleukin-16 (IL-16), Interleukin-17 (IL-17), Interleukin-18 (IL-18), Interleukin-23 (IL-23), Kallikrein 5, Kallikrein-7 (KLK-7), Kidney Injury Molecule-1 (KIM-1), Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Lectin-Like Oxidized LDL Receptor 1 (LOX-1), Leptin, Luteinizing Hormone (LH), Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage-Derived Chemokine (MDC), Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha), Macrophage Inflammatory Protein-1 beta (MIP-1 beta), Macrophage Inflammatory Protein-3 alpha (MIP-3 alpha), Macrophage inflammatory protein 3 beta (MIP-3 beta), Macrophage Migration Inhibitory Factor (MIF), Macrophage-Stimulating Protein (MSP), Malondialdehyde-Modified Low-Density Lipoprotein (MDA-LDL), Maspin, Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3), Matrix Metalloproteinase-7 (MMP-7), Matrix Metalloproteinase-9 (MMP-9), Matrix Metalloproteinase-9—total (MMP-9, total), Matrix Metalloproteinase-10 (MMP-10), Mesothelin (MSLN), MHC class I chain-related protein A (MICA), Monocyte Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 2 (MCP-2), Monocyte Chemotactic Protein 3 (MCP-3), Monocyte Chemotactic Protein 4 (MCP-4), Monokine Induced by Gamma Interferon (MIG), Myeloid Progenitor Inhibitory Factor 1 (MPIF-1), Myeloperoxidase (MPO), Myoglobin, N-terminal prohormone of brain natriuretic peptide (NT proBNP), Nerve Growth Factor beta (NGF-beta), Neuron-Specific Enolase (NSE), Neuronal Cell Adhesion Molecule (Nr-CAM), Neuropilin-1, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Osteopontin, Osteoprotegerin (OPG), Pancreatic Polypeptide (PPP), Pepsinogen I (PGI), Peptide YY (PYY), Phosphoserine Aminotransferase (PSAT), Placenta Growth Factor (PLGF), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Progesterone, Proinsulin (Intact), Proinsulin (Total), Prolactin (PRL), Prostasin, Prostate-Specific Antigen, Free (PSA-f), Protein S100-A4 (S100-A4), Pulmonary and Activation-Regulated Chemokine (PARC), Receptor for advanced glycosylation end products (RAGE), Receptor tyrosine-protein kinase erbB-3 (ErbB3), Resistin, S 100 calcium-binding protein B (S100-B), Serotransferrin (Transferrin), Serum Amyloid P-Component (SAP), Sex Hormone-Binding Globulin (SHBG), Sortilin, Squamous Cell Carcinoma Antigen-1 (SCCA-1), Stem Cell Factor (SCF), Stromal cell-derived factor-1 (SDF-1), Superoxide Dismutase 1, soluble (SOD-1), T-Cell-Specific Protein RANTES (RANTES), T Lymphocyte-Secreted Protein 1-309 (1-309), Tamm-Horsfall Urinary Glycoprotein (THP), Tenascin-C (TN-C), Testosterone (Total), Tetranectin, Thrombomodulin (TM), Thrombospondin-1, Thyroglobulin (TG), Thyroid-Stimulating Hormone (TSH), Thyroxine-Binding Globulin (TBG), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), Tissue type Plasminogen activator (tPA), TNF-Related Apoptosis-Inducing Ligand Receptor 3 (TRAIL-R3), Transforming Growth Factor alpha (TGF-alpha), Transforming Growth Factor beta-3 (TGF-beta-3), Transthyretin (TTR), Trefoil Factor 3 (TFF3), Tumor Necrosis Factor alpha (TNF-alpha), Tumor Necrosis Factor beta (TNF-beta), Tumor Necrosis Factor Receptor I (TNF R1), Tumor necrosis factor receptor 2 (TNFR2), Tyrosine kinase with Ig and EGF homology domains 2 (TIE-2), Urokinase-type Plasminogen Activator (uPA), Urokinase-type plasminogen activator receptor (uPAR), Vascular Cell Adhesion Molecule-1 (VCAM-1), Vascular Endothelial Growth Factor (VEGF), Vascular endothelial growth factor B (VEGF-B), Vascular Endothelial Growth Factor C (VEGF-C), Vascular endothelial growth factor D (VEGF-D), Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), Vascular endothelial growth factor receptor 3 (VEGFR-3), Vitamin D-Binding Protein (VDBP), Vitamin K-Dependent Protein S (VKDPS), Vitronectin, von Willebrand Factor (vWF) and YKL-40.

In some embodiments, SAT biomarkers can include but are not limited to 6Ckine, Aldose Reductase, Alpha-1-Microglobulin (A1Micro), Alpha-2-Macroglobulin (A2Macro), Angiogenin, Angiopoietin-2 (ANG-2), Angiotensin-Converting Enzyme (ACE), Apolipoprotein(a) (Lp(a)), Apolipoprotein A-I (Apo A-I), Apolipoprotein A-II (Apo A-II), Apolipoprotein A-IV (Apo A-IV), Apolipoprotein B (Apo B), Apolipoprotein C-III (Apo C-III), Apolipoprotein D (Apo D), Apolipoprotein H (Apo H), AXL Receptor Tyrosine Kinase (AXL), B cell-activating factor (BAFF), B Lymphocyte Chemoattractant (BLC), Beta-2-Microglobulin (B2M), Brain-Derived Neurotrophic Factor (BDNF), C-Peptide, Carcinoembryonic Antigen (CEA), Cathepsin D, CD5 Antigen-like (CD5L), CD 40 antigen (CD40), CD40 Ligand (CD40-L), Cellular Fibronectin (cFib), Chromogranin-A (CgA), Clusterin (CLU), Complement C3 (C3), Complement Factor H—Related Protein 1 (CFHR1), Cortisol (Cortisol), Creatine Kinase-MB (CK-MB), Cystatin-C, E-Selectin, EN-RAGE, Endoglin, Endostatin, Eotaxin-1, Epidermal Growth Factor (EGF), Epidermal Growth Factor Receptor (EGFR), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Factor VII, FASLG Receptor (FAS), Fatty Acid-Binding Protein—adipocyte (FABP, adipocyte), Fetuin-A, Fibrinogen, Galectin-3, Gelsolin, Glucagon-like Peptide 1—total (GLP-1 total), Glutathione S-Transferase Mu 1 (GST-M1), Granulocyte Colony-Stimulating Factor (G-CSF), Hepatocyte Growth Factor (HGF), Hepsin, Human Epidermal Growth Factor Receptor 2 (HER-2), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Insulin-like Growth Factor-Binding Protein 2 (IGFBP-2), Insulin-like Growth Factor Binding Protein 4 (IGFBP4), Insulin-like Growth Factor Binding Protein 5 (IGFBP5), Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Intercellular Adhesion Molecule 1 (ICAM-1), Interferon gamma Induced Protein 10 (IP-10), Interferon-inducible T-cell alpha chemoattractant (ITAC), Interleukin-1 alpha (IL-1 alpha), Interleukin-1 receptor antagonist (IL-1ra), Interleukin-2 receptor alpha (IL-2 receptor alpha), Interleukin-6 receptor (IL-6r), Interleukin-6 receptor subunit beta (IL-6R beta), Interleukin-7 (IL-7), Interleukin-12 Subunit p40 (IL-12p40), Interleukin-15 (IL-15), Interleukin-16 (IL-16), Kallikrein 5, Kidney Injury Molecule-1 (KIM-1), Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Lectin-Like Oxidized LDL Receptor 1 (LOX-1), Leptin, Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage-Derived Chemokine (MDC), Macrophage inflammatory protein 3 beta (MIP-3 beta), Macrophage Migration Inhibitory Factor (MIF), Macrophage-Stimulating Protein (MSP), Maspin, Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3), Matrix Metalloproteinase-9—total (MMP-9, total), Mesothelin (MSLN), Monocyte Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 2 (MCP-2), Monocyte Chemotactic Protein 4 (MCP-4), Myeloid Progenitor Inhibitory Factor 1 (MPIF-1), Myeloperoxidase (MPO), Myoglobin, Neuron-Specific Enolase (NSE), Neuronal Cell Adhesion Molecule (Nr-CAM), Neuropilin-1, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Osteopontin, Osteoprotegerin (OPG), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), ProgesteroneProinsulin (Intact), Proinsulin (Total), Protein S100-A4 (S100-A4), Pulmonary and Activation-Regulated Chemokine (PARC), Receptor for advanced glycosylation end products (RAGE), Receptor tyrosine-protein kinase erbB-3 (ErbB3), Resistin, Serotransferrin (Transferrin), Serum Amyloid P-Component (SAP), Sortilin, Squamous Cell Carcinoma Antigen-1 (SCCA-1), Stem Cell Factor (SCF), Stromal cell-derived factor-1 (SDF-1), Superoxide Dismutase 1, soluble (SOD-1), T-Cell-Specific Protein RANTES (RANTES), Tenascin-C (TN-C), Tetranectin, Thrombomodulin (TM), Thrombospondin-1, Thyroxine-Binding Globulin (TBG), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), Tissue type Plasminogen activator (tPA), Transthyretin (TTR), Trefoil Factor 3 (TFF3), Tumor Necrosis Factor alpha (TNF-alpha), Tumor Necrosis Factor Receptor I (TNF R1), Tumor necrosis factor receptor 2 (TNFR2), Urokinase-type plasminogen activator receptor (uPAR), Vascular Cell Adhesion Molecule-1 (VCAM-1), Vascular Endothelial Growth Factor C (VEGF-C), Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2), Vascular endothelial growth factor receptor 3 (VEGFR-3), Vitamin K-Dependent Protein S (VKDPS), Vitronectin, von Willebrand Factor (vWF) and YKL-40. See for example, Table 10a and Table 10b.

In some embodiments, SAT biomarkers can include but are not limited to B Lymphocyte Chemoattractant (BLC), Brain-Derived Neurotrophic Factor (BDNF), CD40 Ligand (CD40-L), Chromogranin-A (CgA), Creatine Kinase-MB (CK-MB), Eotaxin-1, Epidermal Growth Factor (EGF), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Glucagon-like Peptide 1, total (GLP-1 total), Hepatocyte Growth Factor (HGF), Interleukin-12 Subunit p40 (IL-12p40), Kidney Injury Molecule-1 (KIM-1), Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-9—total (MMP-9, total), Monocyte Chemotactic Protein 4 (MCP-4), Myeloperoxidase (MPO), Myoglobin, Neuron-Specific Enolase (NSE), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Proinsulin (Total), Protein S100-A4 (S100-A4), Receptor tyrosine-protein kinase erbB-3 (ErbB3), Squamous Cell Carcinoma Antigen-1 (SCCA-1), T-Cell-Specific Protein RANTES (RANTES), Thrombospondin-1, Tumor Necrosis Factor alpha (TNF-alpha) and Vascular Endothelial Growth Factor C (VEGF-C). See for example, Table 11.

In some embodiments, SAT biomarkers can include but are not limited to Brain-Derived Neurotrophic Factor (BDNF), Creatine Kinase-MB (CK-MB), Epidermal Growth Factor (EGF), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Hepatocyte Growth Factor (HGF), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-9-—total (MMP-9, total), Myoglobin, Neuron-Specific Enolase (NSE), Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Receptor tyrosine-protein kinase erbB-3 (ErbB3), T-Cell-Specific Protein RANTES (RANTES), Thrombospondin-1, and Vascular Endothelial Growth Factor C (VEGF-C). See for example, Table 12.

In some embodiments, SAT biomarkers can include but are not limited to Brain-Derived Neurotrophic Factor (BDNF), Creatine Kinase-MB (CK-MB), Epidermal Growth Factor (EGF), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), Hepatocyte Growth Factor (HGF), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-9—total (MMP-9, total), Myoglobin, Plasminogen Activator Inhibitor 1 (PAI-1), Platelet-Derived Growth Factor BB (PDGF-BB), Receptor tyrosine-protein kinase erbB-3 (ErbB3), T-Cell-Specific Protein RANTES (RANTES), Thrombospondin-1 and Vascular Endothelial Growth Factor C (VEGF-C). See for example, Table 13.

In some embodiments, SAT biomarkers can include but are not limited to Chromogranin-A (CgA), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78) Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-9, total (MMP-9, total), Myoglobin, Neuronal Cell Adhesion Molecule (Nr-CAM), Plasminogen Activator Inhibitor 1 (PAI-1), Receptor tyrosine-protein kinase erbB-3 (ErbB3), and Vascular Endothelial Growth Factor C (VEGF-C). See for example, Table 14. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or more of the biomarkers provides a level pattern that indicates efficacy of the treatment. In some embodiments, Chromogranin-A (CgA), Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78) Lactoylglutathione lyase (LGL), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Myoglobin, Neuronal Cell Adhesion Molecule (Nr-CAM), and/or Plasminogen Activator Inhibitor 1 (PAI-1) provides a “normalization” level pattern after the treatment. In some embodiments, Macrophage Colony-Stimulating Factor 1 (M-CSF), Matrix Metalloproteinase-9, total (MMP-9, total), Receptor tyrosine-protein kinase erbB-3 (ErbB3), and/or Vascular Endothelial Growth Factor C (VEGF-C) provides a “stabilization” level pattern after the treatment.

In some embodiments, SAT biomarkers can include but are not limited to Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), CD40 Ligand (CD40-L), Interleukin-3 (IL-3), Interleukin-5 (IL-5), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage Inflammatory Protein-3 alpha (MIP-3 alpha), Myeloperoxidase, T-Cell-Specific Protein RANTES (RANTES), Vascular Endothelial Growth Factor C (VEGF-C), Agouti-Related Protein (AgRP), Thrombospondin-1, Platelet-Derived Growth Factor BB (PDGF-BB), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3) and Matrix Metalloproteinase-9 (MMP-9), Brain-Derived Neurotrophic Factor (BDNF), Chromogranin-A (CgA), Receptor tyrosine-protein kinase erbB-3 (ErbB-3), Neuron-Specific Enolase (NSE), Neural cell adhesion molecule (Nr-CAM), Epidermal Growth Factor (EGF), Fibroblast Growth Factor 4 (FGF-4), Kallikrein 5, Plasminogen Activator Inhibitor 1 (PAI-1), Serum Glutamic Oxaloacetic Transaminase (SGOT), Creatine Kinase-MB (CK-MB), Myoglobin, N-terminal prohormone of brain natriuretic peptide (NT proBNP), Protein S100-A4 (S100-A4), Protein S100-A6 (S100-A6), Insulin-like Growth Factor Binding Protein 6 (IGFBP-6), Thyroglobulin, Amphiregulin and Cortisol.

In some embodiments, SAT biomarkers are associated with muscle inflammation and fibrosis. Muscle inflammation and fibrosis associated biomarkers can include but are not limited to Epithelial-Derived Neutrophil-Activating Protein 78 (ENA-78), CD40 Ligand (CD40-L), Interleukin-3 (IL-3), Interleukin-5 (IL-5), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), Macrophage Colony-Stimulating Factor 1 (M-CSF), Macrophage Inflammatory Protein-3 alpha (MIP-1 alpha), Myeloperoxidase, T-Cell-Specific Protein RANTES (RANTES), Vascular Endothelial Growth Factor C (VEGF-C), Agouti-Related Protein (AgRP), Thrombospondin-1, Platelet-Derived Growth Factor BB (PDGF-BB), Matrix Metalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3) and Matrix Metalloproteinase-9 (MMP-9). See for example, Table 2. In some embodiments, the SAT biomarkers are associated with muscle inflammation and fibrosis. Muscle inflammation and fibrosis associated biomarkers can include but are not limited to CD40 Ligand (CD40-L), Interleukin-3 (IL-3), Interleukin-5 (IL-5), Interleukin-7 (IL-7), Interleukin-8 (IL-8), Latency-Associated Peptide of Transforming Growth Factor beta 1 (LAP TGF-b1), T-Cell-Specific Protein RANTES (RANTES), Vascular Endothelial Growth Factor C (VEGF-C), Agouti-Related Protein (AgRP), Thrombospondin-1, Platelet-Derived Growth Factor BB (PDGF-BB) and Matrix Metalloproteinase-9 (MMP-9). See for example, Table 2 (biomarkers highlighted in red and bolded).

In some embodiments, the SAT biomarkers are associated with muscle and nerve development. Muscle and nerve associated biomarkers can include but are not limited to Brain-Derived Neurotrophic Factor (BDNF), Chromogranin-A (CgA), Receptor tyrosine-protein kinase erbB-3 (ErbB-3), Neuron-Specific Enolase (NSE), Neural cell adhesion molecule (Nr-CAM), Epidermal Growth Factor (EG), Fibroblast Growth Factor 4 (FGF-4), Kallikrein 5 and Plasminogen Activator Inhibitor 1 (PAI-1). See for example, Table 3. In some embodiments, the muscle and nerve associated biomarkers and include but are not limited to Brain-Derived Neurotrophic Factor (BDNF), Chromogranin-A (CgA), Receptor tyrosine-protein kinase erbB-3 (ErbB-3), Neuron-Specific Enolase (NSE), Fibroblast Growth Factor 4 (FGF-4), and Plasminogen Activator Inhibitor 1 (PAI-1). See for example, Table 3 (biomarkers highlighted in red and bolded).

In some embodiments, the SAT biomarkers are associated with muscle damage. Muscle damage associated biomarkers can include but are not limited to Serum Glutamic Oxaloacetic Transaminase (SGOT), Creatine Kinase-MB (CK-MB) and Myoglobin. See for example, Table 4. In some embodiments, the muscle damage associated biomarkers include but are not limited to myoglobin. See for example, Table 4 biomarkers (biomarkers highlighted in red and bolded).

In some embodiments, the SAT biomarkers are other biomarkers, such as biomarkers associated with cardiovascular risk, Calcium binding protein, tissue morphology, EGF and TGF-a, the stress response and suppression of the immune system. Such other biomarkers can include but are not limited to N-terminal prohormone of brain natriuretic peptide, Protein S100-A4 (S100-A4), Protein S100-A6 (S100-A6), Insulin-like Growth Factor Binding Protein 6 (IGFBP-6), Thyroglobulin, Amphiregulin and Cortisol. See for example, Table 5. In some embodiments, other biomarkers include but are not limited to Insulin-like Growth Factor Binding Protein 6. See for example, Table 5 (biomarkers highlighted in red and bolded).

In some embodiments, the level of one or more than one SAT biomarkers is determined or detected. The levels of individual SAT biomarkers can be determined using a variety of methods know to those of skill in the art, including but not limited to those described in the present application. Additionally, the levels of more than one SAT biomarker can be determined in order to generate a composite of the level of more than one SAT biomarker. In some embodiments, the methods of the present invention comprise determining a composite level of a panel of selected SAT biomarkers. In some embodiments, the methods comprise determining a composite level of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more selected SAT biomarkers. The composite can contain any of the SAT biomarkers described by the present application.

A composite of the level of SAT biomarkers can include any collection of information regarding the level of more than one SAT biomarker. Information can include nucleic acid or protein information, or a combination of information regarding both nucleic acid and/or protein levels. Generally, the composite includes information regarding the increase or decrease in the level of selected SAT biomarkers.

In some embodiments, the information in the composite can include index values based on the increase or decrease in the biomarker level. For example, the level of each SAT biomarker in a group of SAT biomarkers can be assigned an index value based on the increase or the decrease in the SAT biomarker. In some embodiments, the larger the increase or decrease, the larger the index value. In some embodiments, the index values can be compiled as group to generate a composite. In some embodiments, the composite is compared to a predetermined standard. In some embodiments, comparison of the composite to a predetermined standard is indicative of treatment efficacy of treatment with the therapeutic entity. In some embodiments, comparison of the composite to a predetermined standard is predictive of treatment efficacy of treatment with the therapeutic entity. In some embodiments, comparison of the composite to a predetermined standard can be used to modify the treatment regimen of treatment with a therapeutic entity. The composite can also contain information regarding the increase of selected SAT biomarkers and the decrease of other selected SAT biomarkers. The composite can also contain information regarding the change in biomarker levels before and after treatment with a therapeutic entity and such information can be used to modify the treatment regimen of treatment with the therapeutic entity. In some embodiments treatment efficacy is determined by determining the increase of selected SAT biomarkers and the decrease of other selected biomarkers, wherein the biomarkers that increase are not the same as the biomarkers that decrease.

The SAT biomarkers for the methods of the present invention can include both the nucleic acid and protein forms of the biomarkers. Methods for detecting the levels of nucleic acids and proteins are well known in the art and any standard methods for detection of nucleic acid or protein levels can be employed with the methods of the present invention and used for detecting the levels of SAT biomarkers. The SAT biomarkers can also include small nucleotide polymorphisms (SNPs).

Methods for detecting the levels of nucleic acids, such as RNA or DNA have been well described and are well known to those of skill in the art. Methods for detecting RNA can include but are not limited to RT-PCR, northern blot analyses, gene expression analyses, microarray analyses, gene expression chip analyses, hybridization techniques (including FISH), expression beadchip arrays, and chromatography as well as any other techniques known in the art. Methods for detecting DNA can include but are not limited to PCR, real-time PCR, digital PCR, hybridization (including FISH), microarray analyses, SNP detection assays, SNP genotyping assays and chromatography as well as any other techniques known in the art.

Methods for detecting proteins and polypeptides can include but are not limited to spectrophotometric determination of protein concentration, quantitative amino acid analysis, protein concentration assays, chromatography assays, western blot analyses, gel electrophoresis, (followed by staining procedures including but not limited to Coomassie Blue, Silver stain, Syber Green, Syber Gold), hybridization, multiplex cytokine assays, immunoassays, ELISA, bicinchoninic acid (BCA) protein assays, Bradford protein assays, and Lowry protein assays as well as any other techniques known in the art. Protein detection can also include detecting the levels of stable or active proteins and methods such as kinetic assays, kinase assays, enzyme assays and post-translation modification assays (for example, assays for determining phosphorylation and glycosylation state) can also be employed.

Methods for quantitating nucleic acid and protein levels have been well described. Methods can include traditional methods, such as western blot quantization and immunoassays as well as computer based methods, such as microarray assay or genechip assay analyses, for analyzing SAT biomarker levels. Immunoassays can include for example, the Human Discovery 250+ Immunoassay from Myriad RBM (commercially available from Myriad RBM, Texas, USA). These analyses can include protein level analyses as well as gene expression level analyses. These standard methods known in the art can be employed to determine whether the level of a SAT biomarker has increased or decreased. An increase or decrease in the level of the SAT biomarker can be based on a comparison of the level of the biomarker with a predetermined standard level or by comparison of the biomarker level after treatment with the therapeutic entity to the SAT biomarker level before treatment with the therapeutic entity, e.g. a sialic acid deficiency treatment. By way of non-limiting examples, Tables 2-14 show relative changes for exemplary SAT biomarkers contemplated for use with methods of the present invention.

In some embodiments detection of the level of the SAT biomarker can include detection of the level of proteins, nucleic acids, nucleic acid presence or absence (gene presence or absence), gene expression or SNP presence or absence. In other embodiments the biomarker can include SNPs. In some embodiments detection of the level of the one or more SAT biomarker can include detection of the presence or absence of one or more SNPs in the SAT biomarker.

In some embodiments, the level of the one or more SAT biomarkers is determined prior to treatment with the therapeutic entity and the level is then determined again after treatment with the therapeutic entity. In other embodiments the level of the one or more SAT biomarkers is determined after treatment with the therapeutic entity and compared to a predetermined standard level. In yet other embodiments the level of the one or more SAT biomarkers is measured prior to treatment with the therapeutic entity and compared to a predetermined standard level.

As used herein, the term “predetermined standard level” or “predetermined activity profiles” refers to standardized data or data set representing the average, representative features or characteristics of one or more biomarkers in a specific population. Such features or characteristics include, but are not limited to, transcript abundance, transcript stability, transcription rate, translation rate, post-translation modification, protein abundance, protein stability, and/or protein enzymatic activity, etc. In some embodiments, the specific population of subjects are consisting of about 5, about 10, about 20, about 50, about 100, about 200, about 300, about 400, about 500, about 1000, about 5000, about 10K, or more individual subjects. The predetermined activity profile can be a standardized data or data set collected before, during, or after the specific population of subjects has been all exposed to a drug. In some embodiments, the specific population is consisting of clinically normal subjects. As used herein, the term “clinically normal subject” refers to a subject without, or substantially without the symptoms associated with sialic acid deficiencies. Predetermined standard levels of SAT biomarkers can be defined using a variety of methods known to those of skill in the art. Generally, standard levels for a biomarker are determined by determining the level of a SAT biomarker in a sufficiently large number of samples obtained from normal, healthy control subjects, for example Table 2 which describes clinically normal SAT biomarker levels. Further, standard level information can be obtained from publically available databases, as well as other sources. (See, e.g., Bunk, D. M., Clin. Biochem. Rev., 28(4):131-137 (2007); Suraj Peril, et al., Genome Res. 13: 2363-2371 (2003); Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005).) For example, in some embodiments, an increase or decrease of the level of one or more SAT biomarkers in a sample obtained from a subject treated with a sialic acid deficiency treatment is determined by comparing the level of one or more SAT biomarkers to a predetermined standard level, such as for example a level as described in Table 2.

As used herein, a subject is “responsive” to a drug for treating sialic acid deficiencies when the level of one or more of the biomarkers of the present invention increases or decreases toward a pre-determined standard level when the subject is exposed to a the drug, or when the drug modifies the speed of level changes of one or more biomarkers of the present invention compared to a placebo.

For methods related to detection, quantitation and comparison of biomarker levels, see, e.g., Current Protocols in Molecular Biology, Ed. Ausubel, Frederick M. (2010); Current Protocols in Protein Science Last, Ed. Coligan, John E., et al. (2010); Current Protocols in Nucleic Acid Chemistry, Ed. Egli, Martin (2010); Current Protocols in Bioinformatics, Ed. Baxevanis, Andreas D. (2010); and Molecular Cloning: A Laboratory Manual, Third Edition, Sambrook, Joseph (2001), all of which are incorporated herein by reference in their entirety.

In certain embodiments, when measuring biomarkers or other indicators of treatment, an “increased” or “decreased” amount or level may include a “statistically significant” amount. A result is typically referred to as statistically significant if it is unlikely to have occurred by chance. The significance level of a test or result relates traditionally to the amount of evidence required to accept that an event is unlikely to have arisen by chance. In certain cases, statistical significance may be defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true (a decision known as a Type I error, or “false positive determination”). This decision is often made using the p-value: if the p-value is less than the significance level, then the null hypothesis is rejected. The smaller the p-value, the more significant the result. Bayes factors may also be utilized to determine statistical significance (see, e.g., Goodman S., Ann Intern Med. 130:1005-13, 1999). In some embodiments, an “increased” or “decreased” amount or level is about 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, or 50× more or less the amount of a predetermined standard, or the amount of a determined time point relative to a previous or earlier timepoint.

Methods for obtaining biological samples are well known in the art and any standard methods for obtaining biological samples can be employed. Biological samples that find use with the methods of the present invention include but are not limited to blood (including but not limited to serum, blood, plasma, whole blood and derivatives thereof), skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy samples. (See, e.g., Clinical Proteomics: Methods and Protocols, Vol. 428 in Methods in Molecular Biology, Ed. Antonia Vlahou (2008).) In some embodiments, the biological sample is selected from blood (including but not limited to serum, blood, plasma, whole blood and derivatives thereof), skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy sample. In some embodiments, the biological sample is a blood sample.

According to the methods of the present invention, the term “subject,” and variants thereof as used herein, includes any subject that has, is suspected of having, or is at risk for having a sialic acid deficiency disease or condition. Suitable subjects (or patients) include mammals, such as laboratory animals (e.g., mouse, rat, rabbit, guinea pig), farm animals, and domestic animals or pets (e.g., cat, dog). Non-human primates and, preferably, human patients, are included. A subject “at risk” may or may not have detectable disease, and may or may not have displayed detectable disease prior to the diagnostic or treatment methods described herein. “At risk” denotes that a subject has one or more so-called risk factors, which are measurable parameters that correlate with development of a condition of sialic acid deficiency, which are described herein. A subject having one or more of these risk factors has a higher probability of developing a sialic acid deficiency than a subject without these risk factor(s). One example of such a risk factor is an increase or decrease in a SAT biomarker as compared to a “clinically normal” sample.

The term “effective amount” refers to the amount of one or more compounds that renders a desired treatment outcome. An effective amount may be comprised within one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint.

The term “therapeutically effective amount” as used herein, refers to the level or amount of one or more agents needed to treat a condition, or reduce or prevent injury or damage, optionally without causing significant negative or adverse side effects. For instance, a therapeutically effective amount includes an amount of a pharmaceutical formulation including for example one or more compounds in the sialic acid biosynthesis pathway sufficient to produce a desired therapeutic outcome (e.g., reduction of severity of a disease or condition).

A “prophylactically effective amount” refers to an amount of an agent (e.g., a pharmaceutical formulation including one or more compounds in the sialic acid biosynthesis pathway) sufficient to prevent or reduce severity of a future disease or condition when administered to a subject who is susceptible and/or who may develop a disease or condition.

By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

The term “disorder” or “disease” used interchangeably herein, refers to any alteration in the state of the body or one of its organs and/or tissues, interrupting or disturbing the performance of organ function and/or tissue function (e.g., causes organ dysfunction) and/or causing a symptom such as discomfort, dysfunction, distress, or even death to a subject afflicted with the disease.

The term “subject”, “individual” or “patient” refers to an animal, for example, a mammal and includes, but is not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

The term “derivative” as used herein includes derivatives, analogs, prodrugs, and unnatural precursors.

The terms “treating” and “treatment” as used herein refer to an approach for obtaining beneficial or desired results including clinical results, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. A treatment is usually effective to reduce at least one symptom of a condition, disease, disorder, injury or damage. Exemplary markers of clinical improvement will be apparent to persons skilled in the art. Examples include, but are not limited to, one or more of the following: decreasing the severity and/or frequency one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, increasing production of sialic acid, the sialylation precursor CMP-sialic acid (e.g., increasing intracellular production of sialic acid) and restoring the level of sialylation in muscle and other proteins, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life.

“Prophylaxis,” “prophylactic treatment,” or “preventive treatment” refers to preventing or reducing the occurrence or severity of one or more symptoms and/or their underlying cause, for example, prevention of a disease or condition in a subject susceptible to developing a disease or condition (e.g., at a higher risk, as a result of genetic predisposition, environmental factors, predisposing diseases or disorders, or the like). Prophylaxis includes prophylaxis of HIBM myopathy in which chronic disease changes in the muscles are irreversible, and for which animal model data suggests that prophylactic treatment prior to such irreversible damage confers a significant treatment benefit.

The phrase “determining the treatment efficacy” or “determining the efficacy of treatment” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject, including for example clinical results or minimal changes or improvements as discussed above. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a subject. For example, evidence of treatment efficacy can include but is not limited to general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression, decreasing the severity and/or frequency one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), delay or slowing the progression of the disease, ameliorating the disease state, increasing production of sialic acid, the sialylation precursor CMP-sialic acid (e.g., increasing intracellular production of sialic acid) and restoring the level of sialylation in muscle and other proteins, decreasing the dose of one or more other medications required to treat the disease, and/or increasing the quality of life (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)

Certain embodiments include treatment of a condition of sialic acid deficiency, and related therapeutic agents and pharmaceutical compositions/formulations. Such treatments include but are not limited to replacement therapies, which typically achieve increased sialic acid levels by administering an agent that directly or indirectly increases one or more components of the sialic acid biosynthesis pathway (see, e.g., FIG. 1).

Also included as part of such replacement therapies are gene therapies. Such gene therapies can incorporate one or more genes involved directly or indirectly in the sialic acid biosynthesis pathway. Exemplary components of the sialic acid biosynthesis pathway that can be used as part of gene therapies include mannosamine, N-acetyl mannosamine (ManNAc), ManNac-6-phosphate (ManNAc-6-P), UDP-GlcNAc, N-acetylneuraminic acid (NeuAc), NeuAc-9-phosphate (NeuAc-9-P), sialic acid (i.e., 5-N-acetylneuraminic acid), and CMP-sialic acid. Hence, certain treatments include the direct administration of one or more of these components as compounds, or as derivatives or pharmaceutically acceptable salts thereof, including extended release formulations of such compounds (see, e.g., U.S. Application No. 61/363,995; and PCT/US2011/043910, each of which is incorporated by reference in its entirety). The term “derivative” as used herein includes derivatives, analogs, prodrugs, and unnatural precursors of a given compound. In specific embodiments, the compound in the sialic acid biosynthesis pathway or a derivative thereof does not include glucose or a pharmaceutically acceptable salt thereof.

As one example, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof include ManNAc or a derivative thereof (see, e.g., U.S. Application No. 2010/0249047 and WO 200/8150477, which are incorporated by reference in their entireties). Structures of such ManNAc and derivatives thereof include, but are not limited to, those defined by the formula below:

embedded image

wherein: R₁, R₃, R₄, or R₅is hydrogen, lower alkanoyl, carboxylate or lower alkyl; and R₂is lower alkyl, lower alkanoylalkyl, lower alkyl alkanoyloxy.

The term lower alkyl refers to (C₁-C₆)alkyl. A lower alkyl includes methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, (C₃-C₆)cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), (C₃-C₆)cycloalkyl(C₁-C₆)alkyl (e.g., cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl), (C₁-C₆)alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy) (C₂-C₆)alkenyl (e.g., vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl), (C₂-C₆)alkynyl (e.g., ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl), (C₁-C₆)alkanoyl (e.g., acetyl, propanoyl or butanoyl), halo(C₁-C₆)alkyl (e.g., iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl), hydroxy(C₁-C₆)alkyl (e.g., hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxy butyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl), C₆)alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl), (C C₆)alkylthio (e.g., methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio), and/or (C₂-C₆)alkanoyloxy (e.g., acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy).

In some embodiments, R₂is methyl, and R₁, R₃, R₄, and R₅is hydrogen. In some embodiments, the ManNAc or derivative thereof is N-acetyl mannosamine (ManNAc). In some embodiments, the ManNAc or derivative thereof is N-levulinoylmannosamine (ManLev) or N-azidoacetylmannosamine (ManNAz).

In some embodiments, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof include N-acetylneuraminic acid (NeuAc) or a derivative thereof. Structures of such NeuAc or derivatives thereof include, but are not limited to, those defined by the formula below:

embedded image

wherein each R₁, R₂, R₃, R₅, R₆, or R₇is independently hydrogen, lower alkanoyl, carboxylate or lower alkyl; and R₄is lower alkyl, lower alkanoylalkyl or lower alkyl alkanoyloxy.

In some embodiments, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof include sialic acid or a derivative thereof, including for example both N- or O-substituted derivatives of neuraminic acid such as N-acetylneuraminic acid (Neu5Ac or NANA). In some embodiments, the sialic acid or derivative thereof is sialic acid. In some embodiments, the sialic acid or derivative thereof is a sialic acid analog such as N-levulinoyl sialic acid (SiaLev), N-azidoacetyl sialic acid (SiaNAz). In some embodiments, the sialic acid or derivative thereof is bound as a glycoconjugate. In some embodiments, the sialic acid or derivative thereof is an unnatural precursor such as sialylactose. In some embodiments the sialic acid or derivative thereof is conjugated to an immunoglobulin. Specific embodiments include a sialic acid extended release formulation (see, e.g., U.S. Application No. 61/363,995; and PCT/US2011/043910). In specific embodiments, the extended release formulation is a formulation of Table 1, below.

TABLE 1

Quantitative Formula for Sialic Acid 325 mg and 500 mg sustained

release Tablets Prototypes.

mg/Tablet

ProCR

Ingredient
Vendor
Hypromellose
% w/w

Sialic Acid (N-
Food and
325.0
43.3

Acetylneuraminic
BioResearch

acid)
Center, Inc

Hypromellose,
Colorcon
191.3
25.5

Type 2208

(Methocel ® K100

M Premium CR)

Polyethylene Oxide

—
25.5

WSR (Polyox)

Sodium Alginate
FMC
159.0
21.2

(Protanal ® LF
Biopolymer

120M)

Carrageenan
FMC
31.5
4.2

(Viscarin GP-209)
Biopolymer

Microcrystallline
JRS Pharma
39.8
5.3

Cellulose and

Colloidal Sillicon

Dioxide (ProSolv ®

SMCC HD 90)

Magnesium
Mallinckrodt
3.8
0.5

Stearate

(HyQual ®),

Vegatable Source

Product Code 2257

Total for 325 mg Strength

750.4
100

Total for 500 mg Strength

1154.5
100

In one variation, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof is an ester of a compound in the sialic acid biosynthesis pathway. In one aspect, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof is an ester of sialic acid or MaNAc. In a particular variation, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof is an ester of sialic acid. In one aspect, the one or more compounds in the sialic acid biosynthesis pathway or derivative thereof is a prodrug of sialic acid. See also WO 2010/131712, published Nov. 18, 2010, for derivatives of compounds in the sialic acid biosynthesis pathway, which is incorporated herein by reference in its entirety and specifically with respect to compounds (e.g., derivatives of compounds in the sialic acid biosynthesis pathway) detailed therein.

In one aspect, a derivative of one or more compounds in the sialic acid biosynthesis pathway (e.g., a derivative of sialic acid or MaNAc) is an effective substrate replacement for sialic acid, such as in an subject who has or is suspected of having a condition of sialic acid deficiency. A derivative of one or more compounds in the sialic acid biosynthesis pathway (e.g., a derivative of sialic acid or MaNAc), or an extended release formulation comprising a derivative of one or more compounds in the sialic acid biosynthesis pathway (e.g., a derivative of sialic acid or MaNAc) may exhibit any one or more of the following characteristics: (i) capable of delivering to an individual in need thereof a therapeutically effective amount of one or more compounds in the sialic acid pathway or derivatives thereof over a period of greater than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; (ii) capable of delivering to an individual in need thereof a substantially constant therapeutically effective amount of one or more compounds in the sialic acid pathway or derivatives thereof over a period of greater than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; (iii) capable of delivering to an individual in need thereof a therapeutically effective amount of one or more compounds in the sialic acid pathway or derivatives thereof with a T_maxof between about any of 2-4 hours, 3-4 hours, 6-8 hours, 6-12 hours, 6-15 hours, 12-18 hours, or 18-24 hours; (iv) capable of delivering to an individual in need thereof a therapeutically effective amount of one or more compounds in the sialic acid pathway or derivatives thereof with a C_maxof about 0.5-100 μg/mL; (v) capable of delivering to an individual in need thereof a therapeutically effective amount of one or more compounds in the sialic acid pathway or derivatives thereof with a trough level of about 0.1-20 ng/mL; (vi) capable of delivering to an individual in need thereof a therapeutically effective amount of one or more compounds in the sialic acid pathway or derivatives thereof with less than about any of 10%, 20%, 30%, 40%, 50%, 60%, or 70% excreted after one hour; (vii) capable of delivering to an individual in need thereof between about any of 0.01-750 mg/kg/day, 0.5-500 mg/kg/day, 1-250 mg/kg/day, 2.5-100 mg/kg/day, or 5-50 mg/kg/day of one or more compounds in the sialic acid pathway or derivatives thereof or a pharmaceutically acceptable salt of the foregoing; (viii) capable of delivering to an individual in need thereof between about any of 0.01-750 mg/kg/day, 0.5-500 mg/kg/day, 1-250 mg/kg/day, 2.5-100 mg/kg/day, or 5-50 mg/kg/day of one or more compounds in the sialic acid pathway or derivatives thereof or a pharmaceutically acceptable salt of the foregoing; (ix) has an absolute bioavailability of about 1 to about 50%; (x) has a bioavailability based on sialic acid levels in the urine of about 0.5 to about 100%; and (xi) has a mean residence time (MRT) of at least about 3.5 hours.

As noted above, gene replacement therapy is also contemplated. Any gene involved (e.g., directly, indirectly) in the sialic acid biosynthesis pathway can be utilized (see FIG. 1). As one example, certain embodiments include methods for increasing sialic acid production by providing a subject with a wild-type GNE-encoding nucleic acid sequence that is optionally operably linked to a regulatory element, such as a promoter and/or enhancer sequence (see U.S. Application No. 2011/027373; WO 2008/097623; and U.S. Application No. 2009/029811, which are incorporated by reference in their entireties). This gene replacement therapy targets GNE/MNK, which is defective in HIBM patients, typically due to an autosomal recessive mutation of the GNE gene (see, e.g., Nemunaitis et al., The Journal of Gene Medicine 12:403-12, 2010). The GNE gene encodes the bi-functional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase.

Various viral vectors that can be utilized for gene replacement therapy include adenovirus, herpes virus, vaccinia, adeno-associated virus (AAV), or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus, or is a lentiviral vector. The preferred retroviral vector is a lentiviral vector. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.

“Non-viral” delivery techniques for gene therapy can also be used including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄precipitation, gene gun techniques, electroporation, liposomes, lipofection, and the like. Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention can be accomplished using any of the available methods of transfection. Lipofection can be accomplished by encapsulating an isolated DNA molecule within a liposomal particle and contacting the liposomal particle with the cell membrane of the target cell. Liposomes are self-assembling, colloidal particles in which a lipid bilayer, composed of amphiphilic molecules such as phosphatidyl serine or phosphatidyl choline, encapsulates a portion of the surrounding media such that the lipid bilayer surrounds a hydrophilic interior. Unilammellar or multilammellar liposomes can be constructed such that the interior contains a desired DNA molecule.

A desirable clinical or non-clinical outcome of the treatment(s) described herein includes, but is not limited to, increased production of sialic acid, restored level of sialylation in muscle and other proteins, increased muscle function, increased muscle strength (e.g., muscle strength of the quadriceps), increased muscle tensile force, improved muscle movement, improved limb movement, muscle growth, increased muscle stamina, decrease in muscle fatigability, decrease in muscle atrophy, decrease in neuronal atrophy, increase in pulmonary function, reduction in proteinuria (e.g., lower amounts of protein in the urine), reduction in hematuria (e.g., lower amounts of red blood cells in the urine) increased activity, stable disease (e.g., preventing or delaying the worsening of the disease), and/or increase or elongation of overall survival. The clinical outcome(s) will then be considered, and a decision as to whether the patient is suitable for the therapy will be made accordingly, taking into account the patient's specific situation and the relevance of the clinical or non-clinical outcomes.

Various pharmaceutical formulations comprising one or more therapeutic agents may be used in any of the methods described herein. In particular, provided herein are pharmaceutical formulations comprising one or more therapeutic agents (e.g., those described herein) and a pharmaceutically acceptable carrier, diluent, and/or excipient. Examples of suitable carriers, excipients, and diluents include, but are not limited to, sugars, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum such as xanthan gum, guar gum, or gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyethylene glycols, polyvinylpyrrolidone, phospholipics, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, mineral oil, lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, disintegrating agents, antioxidants, surfactants, and/or flavoring agents.

Pharmaceutical formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

The pharmaceutical formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

In some embodiments, when the therapeutic agent is a nucleic acid, the therapeutic agent may be used and delivered to a system in connection with an appropriate delivery vehicle (such as a liposome or lipid nanoparticle). In specific aspects, the nucleic acid is administered in conjunction with a lipid nanoparticle. Particular embodiments include a human non-viral GNE-plasmid embedded in cationic liposomes (e.g., GNE Lipoplex). Merely for illustrative purposes, these and other embodiments can be administered via intramuscular injection (e.g., biceps and extensor carpi radialis longus), intravenously (IV), or via intrahepatic (the major organ of SA synthesis) injections.

For topical administration, the pharmaceutical formulation may be a cream, milk, gel, dispersion, or microemulsions, lotion thickened to a greater or lesser extent, impregnated pad, ointment or stick, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap.

The pharmaceutical formulation may be a food supplement or incorporated into food or drink item such as a nutritional bar, snack bar, cookie, candy, cereal, pudding, ice cream, frozen confectionary, chewing gum, drink mix, soda pop, liquid supplement, sauce, salad dressing, gravy, jelly, jam, spread, margarine, peanut butter, nut spread, frosting, and the like. In essence, can be used in any food, composition or supplement in which sugar is employed. Hence, the therapeutic agent and/or derivatives thereof can be used as a partial or full substitute for sugar.

Such food supplements, drinks and food items can include any other food ingredient including, for example, flour, oil, cream, butter, sugar, salt, spices and the like. In addition, the food supplements, drinks and food items can include vitamins and nutrients commonly found in other nutritional supplements.

Various routes of administration may be used in any of the methods described herein. In some embodiments of any of the methods described herein, the therapeutic agent can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular, intraperitoneal, intraarticular, intraarterial, intrasynovial, or infusion techniques), rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes.

Administration of the therapeutic agents in accordance may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the therapeutic agent may be essentially continuous over a pre-selected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

In certain of the methods described herein, the therapeutic agent is formulated for various forms of administration by any of the methods well known to the pharmaceutical arts. See, e.g., WO 2008/150477 and US 20090298112, incorporated herein in their entireties. The therapeutic agent may be administered, for example, at a dose of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 200 to 400 mg/kg, at least about 1 mg/kg to about 25 mg/kg, or at least about 5 mg/kg to about 40 mg/kg, or at least about 1 mg/kg to 200 mg/kg, at least about 1 mg/kg to about 1000 mg/kg, at least about 200 mg/kg to about 1000 mg/kg, at least about 400 mg/kg to about 1000 mg/kg, or at least about 600 mg/kg to about 1000 mg/kg of body weight, although other dosages may provide beneficial results. The amount administered will vary depending on various factors including, but not limited to the disease, the weight, the physical condition, the health, the age of the mammal, whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.

In some embodiments, the methods of the present invention include determining that the subject is suitable for sialic acid deficiency treatment based upon an increase or decrease in one or more SAT biomarkers. Optionally, in some embodiments, the increase or decrease in one or more biomarkers has been maintained for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 weeks or at least about 1 month, 2 months, 3 months or 6 months or more prior to said determination.

In some embodiments the methods of the present invention include maintaining, increasing or reducing the dosage amount and/or frequency of the treatment upon an increase or decrease in one or more SAT biomarkers. Optionally, in some embodiments the increase or decrease in one or more SAT biomarkers has been maintained for at least about 1, 2, 3, 4, 5, 6, 7 days or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 weeks or at least about 1 month, 2 months, 3 months or 6 months or more prior to maintaining, increasing or reducing the dosage amount and/or frequency of the treatment. In some embodiments, the dosage of a drug for treating sialic acid deficiencies can be tested, determined, or modified in view of the patient's response to the drug reflected by the level of one or more biomarkers of the present invention. In some embodiments, the dosage can be increased if the level of the biomarkers indicates that the subject is more responsive to a higher dosage of the drug. In some embodiments, the dosage can be decreased if the biomarkers indicates that the subject is more sensitive to the drug compared to the average subjects.

The dosage amount can be increased, merely by way of example, by about 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20× or more, relative to the previous dosage. The dosage frequency can be increased, merely by way of illustration, by about 1, 2, 3, 4, 5 or more dosages per day, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dosages per week, relative to the previous dosing schedule. As noted above, the dosage amount can be increased separately or in combination with the dosage frequency, and vice versa, optionally until a desired level or range of one or more biomarkers or other treatment indicators is achieved. In some embodiments, the drug is administered to a subject at about 1 g/day, 2 g/day, 3 g/day, 4 g/day, 5 g/day, 6 g/day, 7 g/day, 8 g/day, 9 g/day, 10 g/day, or more.

Provided herein are kits and/or articles of manufacture comprising packaging material and at least one vial comprising an agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein the agent is used to determine and/or detect the level of one or more SAT biomarkers in a biological sample. Also provided herein are kits and/or articles of manufacture comprising packaging material and at least one vial comprising a therapeutic agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent. Further provided herein are kits and/or articles of manufacture comprising packaging material and at least one vial comprising an agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein the agent is used to the level of one or more SAT biomarkers in a biological sample and at least one vial comprising a therapeutic agent with the prescribed buffers and/or preservatives, optionally in an aqueous diluent.

Further provided herein are kits and/or articles of manufacture for use in treating an individual suffering from a sialic acid deficiency, identifying an individual as suitable or not suitable for treatment, and/or selecting an individual for treatment based on the level of one or more SAT biomarkers in a biological sample from the individual. The kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable agent(s) to determine the level of one or more SAT biomarkers, one or more therapeutic agent(s) and instructions for use thereof. In some embodiments, the kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable antibodies or probes, one or more therapeutic agent(s) and instructions for use thereof.

Provided herein are also kits and/or articles of manufacture for use in monitoring responsiveness or lack of responsiveness to treatment in an individual and/or identifying an individual as suitable or not suitable to continue treatment with a therapeutic agent based on level of one or more SAT biomarkers in a biological sample from the individual. The kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable agent to determine level of one or more SAT biomarkers, one or more therapeutic agent and instructions for use thereof. In some embodiments, the kit and/or article of manufacture comprises, or alternatively consists essentially of, or yet further consists of, one or more suitable antibody or probe, one or more therapeutic agent and instructions for use thereof. In some embodiments, the level of one or more SAT biomarkers indicates that the individual is non-responsive to current treatment, and might need optimization of treatment. In some embodiments, the level of one or more SAT biomarkers is compared between biological samples obtained before and after treatment and provides an indication that the individual is responsive or non-responsive to treatment. In some embodiments, the level of one or more SAT biomarkers is compared between biological samples obtained before or after treatment and a predetermined standard level and provides an indication that the individual is responsive or non-responsive to treatment.

In some embodiments, the biological sample is a blood sample (e.g., serum sample). In some embodiments, the biomarker is a SAT biomarker. In some embodiments, the sialic acid deficiency is Hereditary Inclusion Body Myopathy (HIBM).

The kits and/or articles of manufacture can include all or some of the positive controls, negative controls, reagents, antibodies and probes described herein for determining the level of one or more SAT biomarkers in a biological sample.

As amenable, these suggested kit and/or article of manufacture components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit and/or article of manufacture components may be provided in solution or as a liquid dispersion or the like.

Included within the scope of the invention are DNA arrays or microarrays containing a plurality of sequences that hybridize under stringent hybridization conditions to one or more of the gene sequences of the biomarkers. An example of a substrate containing one or more probes of interest is a plurality of DNA probes that are affixed to a substrate. In certain embodiments, the substrate may comprise one or more materials such as gel, nitrocellulose, nylon, quartz, glass, metal, silica based materials, silica, resins, polymers, etc., or combinations thereof. Typically, the DNA probes comprise about 10-50 bp of contiguous DNA. In certain embodiments, the DNA probes are from about 20 to about 50 bp of contiguous DNA. In certain embodiments, the present invention relates to kits which comprising a microarray directions for its use. The kit may comprise a container which comprises one or more microarrays and directions for their use.

The biological sample may also be analyzed for gene expression of one or more gene markers using methods that can detect nucleic acids including, but not limited to, PCR (polymerase chain reaction); RT-PCT (reverse transcriptase-polymerase chain reaction); quantitative or semi-quantitative PCR, etc.

In certain embodiments, the levels of gene expression are measured by detecting the protein expression products of the genes or DNA sequences. The levels of protein products may be measured using methods known in the art including the use of antibodies which specifically bind to a particular protein. These antibodies, including polyclonal or monoclonal antibodies, may be produced using methods that are known in the art. These antibodies may also be coupled to a solid substrate to form an antibody chip or antibody microarray. Antibody or protein microarrays may be made using methods that are known in the art.

In some embodiments, the methods of the present invention can be applied on a dosage basis. For example, for each pre-determined dosage of the same drug, one or more biomarkers of the present invention can be used to determine if a specific subject is responding to a specific drug at the pre-determined dosage.

In some embodiments, the methods of the present invention can be applied on an administration method basis. For example, for each pre-determined drug administration method of the same drug, one or more biomarkers of the present invention can be used to determine if a specific subject is responding to the multi-kinase inhibitor by using the pre-determined drug administration method. None limiting examples of a route for administration include, mucosal, enteral, parental, transdermal/transmucosal, and inhalation. In one embodiment, the mucosal route is via the nasal, oropharyngeal, ocular, or genitourinary mucosa. In another embodiment, the enteral route is oral, rectal or sublingual. Still in another embodiment, the parenteral route is any one of intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and submucosal injection or infusion. Still in another embodiment, the transdermal/transmucosal route is topical. Still in another embodiment, the inhalation route is intranasal, oropharyngeal, intratracheal, intrapulmonary or transpulmonary.

In some embodiments, the methods of the present invention can be applied on a drug combination basis. For example, for each pre-determined drug combination of a drug for treating sialic acid deficiencies, and a drug for treating other diseases, or a combination of two or more drugs for treating sialic acid deficiencies, one or more biomarkers of the present invention can be used to determine if a specific subject is responding to the drug combination.

In some embodiments, the methods of the present invention can be applied on a formulation basis. For example, for each pre-determined drug formulation of a given drug, one or more biomarkers of the present invention can be used to determine if a specific subject is responding to the multi-kinase inhibitor by using the pre-determined drug formulation.

The biomarkers and associated methods of the present invention can be used for all suitable purposes. In some embodiments, they are used in prospective clinical trial. In some embodiments, they are used in clinical treatment/prevention practice.

All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference in its entirety.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES
Example 1
Phase 1 Clinical Trial
Use of Myriad RBM Human Discovery MAP250+ v1.0 to Identify Biomarkers that May be Involved in Heredity Inclusion Body Myopathy and Change During Treatment with Sialic Acid

HIBM is a genetic disorder characterized by progressive muscle weakness and wasting that develops in young adults. Muscle wasting usually starts around the age of 20-30 years, although young onset at 17 and old onset at 52 has been recorded. It can progress to marked disability within 10-15 years, confining many patients to the wheelchair. The weakness and severity can vary from person to person. In some, weakness in the legs is noticed first. In few others, the hands are weakened more rapidly than the legs. Weakness is progressive, which means the muscle become weaker over time. The quadriceps are relatively spared, and remain strong until the late stages of disease.

The current status of biomarker discovery for HIBM or other muscle diseases remains at early stage. One of the challenges is to identify useful surrogate markers in serum that can accurately reflect the changes in muscle and predict the effect of treatments on the disease.

Prior to this study, there had been limited biomarker data available for HIBM patients. No other studies have been done with the Human Discovery MAP250+ v1.0 to assess HIBM and to evaluate the efficacy of treatment in the disease. The present invention represents a major step toward identifying clinically relevant serum biomarkers that can be developed and used in clinical trials.

This study related to the search for serum biomarkers that are relevant to HIBM. The Human Discovery MAP250+ v1.0 quantitative immunoassay system from Myriad RBM was used (commercially available from Myriad). The system contains more than 250 serum markers (analytes) that cover dozens of biochemical pathways.

The immunoassay system is a product of Myriad RBM. The Human Discovery MAP250+ v1.0 platform consists of an extensive list of protein markers that are designed to be used in drug discovery and diagnostic development. The system is a multiplexed immunoassay and requires only a small sample volume.

Results of the 258 analytes (proteins markers) for the 18 HIBM patient serum samples were compiled. The mean, median and standard deviation were calculated. Data from 126 “clinically normal” individuals were used for comparison (see FIG. 1 of U.S. Provisional Application Ser. No. 61/779,929 filed on Mar. 13, 2013, and Table 2 below) in order to demonstrate statistical difference using Wilcoxon Rank Sum test. In addition, the ratios of median values between HIMB and normal samples were listed.

TABLE 2

Level of potential biomarkers in clinically normal individuals

RBM
RBM

Low Serum
High Serum

Reference
Reference
Normal
Normal
Normal

Positive

258 Analytes
Units
Range
Range
Mean
SD
Median
Min
Max
Count

6Ckine

pg/mL
250
895
536
182
520
201
1200
126

Adiponectin
ug/mL
1.1
11
4.4
2.6
3.8
1
14
126

Agouti-Related

pg/mL
54
317
196
86
183
54
719
126

Protein (AGRP)

Aldose Reductase

ng/mL
0.72
6.5
3.4
1.7
3.4
0
12
125

Alpha-1-

ug/ml
465
2120
866
595
752
435
6400
126

Antichymotrypsin

(AACT)

Alpha-1-Antitrypsin
mg/mL
0.55
2.7
1.6
0.55
1.6
0
3
126

(AAT)

Alpha-1-
ug/ml
6.7
18
11
2.9
11
6
23
126

Microglobulin

(A1Micro)

Alpha-2-

mg/mL
1.00
7.9
3.2
1.8
2.9
1
8
126

Macroglobulin

(A2Macro)

Alpha-Fetoprotein
ng/mL
<LOW>
15
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

(AFP)

Standard
Standard
Standard
Standard
Standard
Standard

Amphiregulin

pg/mL
<LOW>
733
505
225
484
200
1060
24

(AR)

Angiogenin

ng/mL
379
1200
623
191
588
260
1600
126

Angiopoietin-2
ng/mL
1.0
7.0
3.2
1.5
3.0
0
9
126

(ANG-2)

Angiotensin-

ng/ml
50
157
92
30
87
43
197
126

Converting

Enzyme (ACE)

Angiotensinogen

ng/mL
35
1340
555
366
493
30
1440
126

Annexin A1
ng/mL
<LOW>
296
163
64
161
44
323
86

(ANXA1)

Apolipoprotein A-I

mg/mL
0.75
2.1
1.3
0.36
1.3
1
3
126

(Apo A-I)

Apolipoprotein A-II

ng/ml
228
1150
534
274
448
189
1560
126

(Apo A-II)

Apolipoprotein A-IV
ug/ml
7.8
90
38
22
34
5
134
126

(Apo A-IV)

Apolipoprotein B

ug/ml
1000
3550
2013
714
1830
835
5130
126

(Apo B)

Apolipoprotein C-I

ng/ml
139
484
275
92
261
116
598
126

(Apo C-I)

Apolipoprotein C-III

ug/mL
56
529
163
106
135
52
758
126

(Apo C-III)

Apolipoprotein D
ug/ml
77
348
192
71
178
52
481
126

(Apo D)

Apolipoprotein E

ug/ml
6.5
109
45
29
38
3
165
126

(Apo E)

Apolipoprotein H
ug/mL
176
524
296
87
282
143
589
126

(Apo H)

Apolipoprotein(a)
ug/mL
23
3310
781
875
506
1
3760
122

(Lp(a))

AXL Receptor

ng/mL
4.3
16
9.7
3.0
9.5
2
20
126

Tyrosine Kinase

(AXL)

B cell-activating

pg/mL
407
1150
684
191
654
259
1380
126

factor (BAFF)

B Lymphocyte
pg/ml
<LOW>
60
19
42
8.6
6
245
67

Chemoattractant

(BLC)

Bcl-2-like protein 2
ng/mL
<LOW>
2.5
1.7
2.5
1.1
1
16
34

(Bcl2-L-2)

Beta-2-
ug/mL
1.0
2.8
1.7
0.47
1.6
1
3
126

Microglobulin

(B2M)

Betacellulin (BTC)
pg/mL
<LOW>
185
124
117
84
51
875
78

Bone
ng/mL
<LOW>
0.61
0.27
0.15
0.17
0
1
68

Morphogenetic

Protein 6 (BMP-6)

Brain-Derived

ng/mL
0.13
2.1
0.57
0.69
040
0
7
126

Neurotrophic

Factor(BDNF)

Calbindin

ng/ml
<LOW>
2.4
2.6
0.42
2.4
2
3
7

Calcitonin
pg/mL
<LOW>
17
8.3
5.0
6.6
3
20
67

Cancer Antigen 125
U/mL
<LOW>
35
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

(CA-125)

Standard
Standard
Standard
Standard
Standard
Standard

Cancer Antigen 15-
U/mL
1.2
9.6
4.5
2.2
4.3
1
12
126

3 (CA-15-3)

Cancer Antigen 19-
U/mL
<LOW>
52
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

9 (CA-19-9)

Standard
Standard
Standard
Standard
Standard
Standard

Cancer Antigen 72-
U/mL
<LOW>
21
19
52
3.7
2
279
30

4(CA-72-4)

Carcinoembryonic

ng/mL
0.40
5.6
2.2
2.6
1.6
0
28
124

Antigen (CEA)

Cathepsin D
ng/mL
230
590
357
89
346
208
814
126

CD 40 antigen
ng/mL
0.26
0.68
0.44
0.11
0.42
0
1
126

(CD40)

CD40 Ligand

ng/mL
<LOW>
0.055
0.031
0.064
0.019
0
0
54

(CD40-L)

CD5 (CD5L)

ng/ml
771
3450
1624
647
1450
581
4580
126

Cellular

ug/mL
<LOW>
1.1
0.66
0.39
0.63
0
2
27

Fibronectin (cFib)

Chemokine CC-4
ng/mL
0.76
14
3.8
3.6
2.7
0
27
126

(HCC-4)

Chromogranin-A

ng/mL
<LOW>
321
79
113
13
5
372
52

(CgA)

Ciliary
pg/mL
<LOW>
12
14
2.6
13
12
17
4

Neurotrophic Factor

(CNTF)

Clusterin (CLU)
ug/ml
190
526
302
84
286
112
577
126

Collagen IV

ng/mL
45
198
93
43
78
26
331
126

Complement C3

mg/mL
1.2
3.3
2.2
0.58
2.1
1
4
126

(C3)

Complement Factor
ug/ml
900
5670
3333
1375
3330
704
6810
126

H

Connective Tissue
ng/ml
<LOW>
1.9
1.1
0.57
0.71
0
3
29

Growth Factor

(CTGF)

Cortisol (Cortisol)

ng/ml
10
221
101
52
94
10
250
123

C-peptide
ng/ml
0.22
5.5
1.4
1.5
1.1
0
12
126

C-Reactive Protein
ug/mL
<LOW>
8.0
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

(CRP)

Standard
Standard
Standard
Standard
Standard
Standard

Creatine Kinase-

ng/mL
0.12
3.5
1.1
0.93
0.80
0
6
125

MB (CK-MB)

Cystatin-C
ng/ml
550
1720
1035
286
980
330
1780
126

Endoglin

ng/mL
2.2
6.2
3.9
1.0
3.8
1
7
126

Endostatin

ng/mL
52
122
79
19
77
33
144
126

Endothelin-1 (ET-1)
pg/mL
<LOW>
25
18
3.9
17
13
35
53

EN-RAGE

ng/mL
3.5
43
15
11
11
3
67
126

Eotaxin-1

pg/mL
<LOW>
100
42
30
28
11
202
86

Eotaxin-2
pg/mL
190
2670
839
626
653
86
3040
126

Eotaxin-3
pg/mL
<LOW>
137
266
262
150
137
796
6

Epidermal Growth

pg/mL
<LOW>
41
12
44
4.7
2
432
102

Factor (EGF)

Epidermal Growth
ng/mL
3.4
5.9
4.4
0.62
4.4
3
6
126

Factor Receptor

(EGFR)

Epiregulin (EPR)
pg/mL
<LOW>
44
<LOW>
<LOW>
<LOW>
44
44
1

Epithelial cell
pg/mL
<LOW>
155
109
34
105
67
197
31

adhesion molecule

(EpCam)

Epithelial-Derived

ng/mL
0.095
0.90
0.38
0.39
0.29
0
4
126

Neutrophil-

Activating Protein

78 (ENA-78)

Erythropoietin

pg/mL
<LOW>
33
14
8.4
15
2
41
95

(EPO)

E-Selectin

ng/mL
6.1
34
19
7.7
19
1
47
126

Ezrin
ng/mL
<LOW>
4.4
5.1
1.0
5.1
4
6
2

Factor VII

ng/mL
66
633
310
167
304
54
855
126

Fas Ligand (FasL)
pg/mL
<LOW>
121
54
29
47
9
146
122

FASLG Receptor
ng/mL
2.3
24
10
12
8.0
2
134
123

(FAS)

Fatty Acid-Binding

ng/mL
2.9
39
14
11
12
2
80
126

Protein, adipocyte

(FABP, adipocyte)

Fatty Acid-Binding

ng/mL
<LOW>
8.3
2.1
2.5
1.5
0
13
74

Protein, heart

(FABP, heart)

Fatty Acid-Binding

ng/mL
7.9
116
37
28
31
5
193
126

Protein, liver

(FABP, liver)

Ferritin (FRTN)
ng/ml
15
792
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

Standard
Standard
Standard
Standard
Standard
Standard

Fetuin-A
ug/ml
753
2170
1269
363
1200
697
2490
126

Fibrinogen

mg/mL
<LOW>
0.046
0.028
0.010
0.025
0
0
86

Fibroblast Growth

pg/mL
44
371
210
108
201
27
1030
125

Factor 4 (FGF-4)

Fibroblast Growth
pg/mL
<LOW>
92
114
63
92
73
277
11

Factor basic (FGF-

basic)

Fibulin-1C (Fib-1C)
ug/mL
16
47
27
7.5
26
13
50
126

Follicle-Stimulating
mIU/mL
1.7
63
11
16
4.7
1
68
126

Hormone (FSH)

Galectin-3

ng/mL
3.4
19
11
4.1
10
3
27
125

Gelsolin
ug/mL
44
93
66
14
65
38
114
126

Glucagon
pg/ml
<LOW>
<LOW>
<LOW>
<LOW>
<LOW>
0
0
0

Glucagon-like

pg/ml
<LOW>
6.1
2.8
2.0
2.4
1
9
52

Peptide 1, total

(GLP-1 total)

Glucose-6-
ng/mL
<LOW>
24
20
20
15
4
83
17

phosphate

Isomerase (G6PI)

Glutamate-Cysteine
ng/mL
<LOW>
12
6.0
2.6
5.1
3
17
68

Ligase Regulatory

subunit (GCLR)

Glutathione S
-
ng/ml
1.3
79
18
19
11
1
117
126

Transferase alpha

(GST-alpha)

Glutathione S-
ng/mL
<LOW>
13
4.7
3.2
4.2
1
14
82

Transferase Mu 1

(GST-M1)

Granulocyte

pg/mL
<LOW>
19
8.6
5.6
7.3
3
48
122

Colony-

Stimulating Factor

(G-CSF)

Granulocyte-

pg/mL
<LOW>
8.5
11
4.0
8.5
8
18
5

Macrophage

Colony-

Stimulating Factor

(GM-CSF)

Growth Hormone
ng/mL
<LOW>
4.6
1.0
2.0
0.35
0
17
103

(GH)

Haptoglobin

mg/mL
0.21
5.4
1.9
1.4
1.5
0
7
126

HE4

pM
<LOW>
78
38
18
37
16
98
106

Heat Shock Protein
ng/ml
<LOW>
29
15
33
6.7
3
213
41

60 (HSP-60)

Heparin-Binding

pg/mL
<LOW>
43
29
20
23
12
94
18

EGF-Like Growth

Factor (HB-EGF)

Hepatocyte Growth
ng/mL
0.31
8.0
3.1
2.1
2.6
0
11
123

Factor (HGF)

Hepatocyte

ng/mL
40
87
63
12
64
36
109
126

Growth Factor

receptor (HGF

receptor)

Hepsin
pg/mL
465
1250
815
187
820
227
1360
126

Human Chorionic
mIU/mL
<LOW>
5.5
2.3
5.9
1.4
0
56
92

Gonadotropin beta

(hCG)

Human Epidermal

ng/mL
0.33
0.92
0.59
0.17
0.57
0
1
126

Growth Factor

Receptor 2 (HER-2)

Immunoglobulin A

mg/mL
1.5
8.0
3.8
1.7
3.4
1
10
126

(IgA)

Immunoglobulin E
IU/mL
<LOW>
180
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

(IgE)

Standard
Standard
Standard
Standard
Standard
Standard

Immunoglobulin M
mg/mL
0.79
6.6
2.9
1.4
2.6
1
8
126

(IGM)

Insulin
uIU/mL
0.34
32
5.4
11
2.3
0
98
124

Insulin-like

ng/mL
258
816
520
153
511
138
1000
126

Growth Factor

Binding Protein 4

(IGFBP4)

Insulin-like Growth
ng/mL
100
216
148
30
145
78
250
126

Factor Binding

Protein 5 (IGFBP5)

Insulin-like

ng/mL
13
326
126
82
105
9
420
126

Growth Factor

Binding Protein 6

(IGFBP6)

Insulin-like Growth
ng/mL
<LOW>
61
11
24
3.7
1
215
109

Factor-Binding

Protein 1 (IGFBP-1)

Insulin-like

ng/mL
21
193
80
49
70
16
278
126

Growth Factor-

Binding Protein 2

(IGFBP-2)

Insulin-like

ng/mL
983
3910
2768
738
2805
139
4370
126

Growth Factor-

Binding Protein 3

(IGFBP-3)

Intercellular

ng/mL
<LOW>
195
98
48
97
28
242
121

Adhesion Molecule

1 (ICAM-1)

Interferon gamma
pg/mL
<LOW>
4.3
2.0
1.7
1.3
1
11
61

(IFN-gamma)

Interferon gamma
pg/ml
107
721
313
244
272
52
2360
126

Induced Protein 10

(IP-10)

Interferon-

pg/mL
<LOW>
43
15
10
11
5
57
108

inducible T-cell

alpha

chemoattractant

(ITAC)

Interleukin-1 alpha
ng/mL
<LOW>
0.0040
0.0021
0.0019
0.0018
0
0
85

(IL-1 alpha)

Interleukin-1 beta
pg/mL
<LOW>
0.52
0.63
0.12
0.65
1
1
5

(IL-1 beta)

Interleukin-1
pg/mL
<LOW>
135
61
35
51
14
242
96

receptor antagonist

(IL-1ra)

Interleukin-10 (1L-
pg/mL
<LOW>
7.9
4.0
2.0
3.7
2
14
72

10)

Interleukin-12
ng/mL
<LOW>
0.18
0.15
0.051
0.12
0
0
25

Subunit p40 (IL-

12p40)

Interleukin-12
pg/mL
<LOW>
<LOW>
<LOW>
<LOW>
<LOW>
0
0
0

Subunit p70 (IL-

12p70)

Interleukin-13 (IL-
pg/mL
13
58
41
12
41
3
132
124

13)

Interleukin-15 (IL-

ng/mL
<LOW>
0.61
0.35
0.20
0.29
0
2
68

15)

Interleukin-16 (IL-
pg/mL
99
330
208
62
202
52
393
126

16)

Interleukin-18 (IL-
pg/mL
86
538
216
102
194
68
713
126

18)

Interleukin-2 (IL-2)
pg/mL
<LOW>
15
15
7.5
13
10
33
13

Interleukin-2

pg/mL
637
2970
1444
556
1390
566
3880
126

receptor alpha (IL-

2 receptor alpha)

Interleukin-25 (IL-
pg/mL
<LOW>
25
11
6.6
8.7
4
45
78

25)

Interleukin-3 (IL-

ng/mL
<LOW>
0.025
0.011
0.0089
0.0092
0
0
76

3)

Interleukin-4 (IL-

pg/mL
<LOW>
50
26
12
22
9
54
74

4)

Interleukin-5 (IL-

pg/mL
<LOW>
5.5
2.9
2.6
2.2
1
23
76

5)

Interleukin-6 (IL-6)
pg/mL
<LOW>
5.0
3.5
6.1
2.0
1
41
43

Interleukin-6
ng/mL
9.5
39
23
7.4
21
5
41
126

receptor (IL-6r)

Interleukin-6

ng/mL
118
271
185
37
185
109
347
126

receptor subunit

beta(IL-6R beta)

Interleukin-7 (IL-

pg/mL
<LOW>
40
21
26
14
4
185
54

7)

Interleukin-8 (IL-

pg/mL
3.9
289
51
69
20
3
344
125

8)

Kallikrein 5

ng/mL
0.17
2.0
0.96
0.51
0.89
0
3
126

Kallikrein-7 (KLK-7)
pg/mL
<LOW>
1170
626
312
531
287
1890
86

Kidney Injury
ng/ml
0.019
0.69
0.16
0.34
0.089
0
3
126

Molecule-1 (KIM-1)

Lactoylglutathione

ng/mL
1.6
6.3
3.4
1.4
3.1
0
11
126

lyase (LGL)

Latency-

ng/mL
0.49
2.6
1.1
0.55
0.99
0
5
126

Associated Peptide

of Transforming

Growth Factor

beta 1 (LAP TGF-b1)

Lectin-Like

ng/mL
<LOW>
0.62
0.60
0.13
0.53
0
1
15

Oxidized LDL

Receptor 1 (LOX-1)

Leptin

ng/mL
0.34
39
12
14
8.6
0
96
126

Luteinizing
mIU/mL
1.2
22
6.1
5.1
4.2
1
24
126

Hormone (LH)

Lymphotactin
ng/mL
<LOW>
<LOW>
0.18
0.11
0.13
0
0
3

Macrophage

ng/mL
0.051
0.17
0.100
0.033
0.092
0
0
126

Colony-

Stimulating Factor

1 (M-CSF)

Macrophage
pg/mL
42
551
268
616
171
32
6760
126

inflammatory

protein 3 beta (MIP-

3 beta)

Macrophage

pg/mL
30
159
63
37
56
25
339
126

Inflammatory

Protein-1 alpha

(MIP-1 alpha)

Macrophage
pg/mL
41
751
210
995
93
21
11100
126

Inflammatory

Protein-1 beta

(MIP-1 beta)

Macrophage

pg/ml
<LOW>
176
87
88
73
14
680
106

Inflammatory

Protein-3 alpha

(MIP-3 alpha)

Macrophage
ng/mL
0.0092
0.17
0.056
0.046
0.044
0
0
123

Migration Inhibitory

Factor (MIF)

Macrophage-
pg/mL
216
674
414
124
414
122
826
126

Derived Chemokine

(MDC)

Macrophage-

ng/mL
111
1100
506
264
474
82
1350
126

Stimulating

Protein (MSP)

Malondialdehyde-

ng/mL
<LOW>
289
228
98
148
148
446
23

Modified Low-

Density

Lipoprotein

(MDA-LDL)

Maspin
pg/mL
<LOW>
2590
1327
582
1160
863
3820
76

Matrix

ng/ml
<LOW>
2.2
1.2
0.97
0.95
0
10
117

Metalloproteinase-

1 (MMP-1)

Matrix
ng/ml
0.15
2.6
0.71
0.66
0.55
0
4
126

Metalloproteinase-

10 (MMP-10)

Matrix
ng/mL
<LOW>
24
14
6.4
13
5
43
73

Metalloproteinase-2

(MMP-2)

Matrix

ng/mL
1.0
13
4.3
3.8
3.1
1
28
126

Metalloproteinase-

3 (MMP-3)

Matrix
ng/ml
2.0
7.5
3.8
2.3
3.4
1
24
126

Metalloproteinase-7

(MMP-7)

Matrix

ng/mL
<LOW>
13
7.9
4.8
4.5
5
19
13

Metalloproteinase-

9 (MMP-9)

Matrix
ng/ml
49
202
119
54
114
42
569
126

Metalloproteinase-9,

total (MMP-9, total)

Mesothelin

nM
26
127
63
31
56
12
252
126

(MSLN)

MHC class I chain-

pg/mL
<LOW>
52
<LOW>
<LOW>
<LOW>
52
52
1

related protein 1

(MICA)

Monocyte

pg/mL
55
238
131
49
130
46
312
126

Chemotactic

Protein 1 (MCP-1)

Monocyte

pg/ml
17
109
33
24
28
9
234
126

Chemotactic

Protein 2 (MCP-2)

Monocyte
pg/mL
<LOW>
9.8
3.9
5.0
2.3
1
27
33

Chemotactic Protein

3 (MCP-3)

Monocyte

pg/ml
294
1240
707
272
697
146
1660
126

Chemotactic

Protein 4 (MCP-4)

Monokine Induced

pg/ml
462
3020
1119
825
937
359
5730
126

by Gamma

Interferon (MIG)

Myeloid

ng/mL
0.33
1.8
0.80
0.50
0.67
0
4
126

Progenitor

Inhibitory Factor 1

(MPIF-1)

Myeloperoxidase

ng/mL
46
629
199
139
158
46
716
124

(MPO)

Myoglobin

ng/mL
5.0
43
15
12
11
4
89
126

Nerve Growth
ng/mL
<LOW>
0.048
0.051
0.032
0.034
0
0
9

Factor beta (NGF-

beta)

Neuron Specific

ng/mL
<LOW>
43
14
11
10
2
84
112

Enolase (NSE)

Neuronal Cell

ng/mL
<LOW>
3.4
1.6
0.89
1.5
0
6
119

Adhesion Molecule

(Nr-CAM)

Neuropilin-1
ng/mL
107
245
167
36
161
103
319
126

Neutrophil

ng/ml
<LOW>
209
106
48
88
40
263
102

Gelatinase-

Associated

Lipocalin (NGAL)

N-terminal
pg/ml
<LOW>
425
288
1245
56
29
10200
70

prohormone of brain

natriuretic peptide

(NT proBNP)

Nucleoside
ng/mL
<LOW>
3.3
1.6
0.68
1.4
0
4
121

diphosphate kinase

B(NDK B)

Osteopontin
ng/ml
1.6
18
5.1
6.8
3.6
1
66
126

Osteoprotegerin
pM
2.6
7.6
4.6
1.4
4.2
2
8
126

(OPG)

Pancreatic
pg/ml
12
349
104
102
70
8
493
126

Polypeptide (PPP)

Pepsinogen I (PGI)

ng/mL
29
281
110
62
106
5
390
126

Peptide YY (PYY)

pg/mL
17
236
133
57
128
17
299
123

Peroxiredoxin 4
ng/mL
0.14
1.1
0.51
0.26
0.47
0
2
126

(Prx-IV)

Phosphoserine
ng/mL
0.81
8.1
2.9
3.7
2.1
1
39
126

Aminotransferase

(PSAT)

Placenta Growth
pg/ml
<LOW>
43
24
11
22
12
67
72

Factor (PLGF)

Plasminogen

ng/mL
6.9
115
32
29
23
4
166
126

Activator Inhibitor

1 (PAI-1)

Platelet-Derived

pg/ml
<LOW>
960
271
327
167
42
2390
79

Growth Factor BB

(PDGF-BB)

Pregnancy-

mIU/mL
<LOW>
0.018
0.0086
0.0048
0.0074
0
0
64

Associated Plasma

Protein A (PAPP-A)

Progesterone

ng/ml
<LOW>
15
6.0
4.2
4.8
1
30
119

Proinsulin, Intact
pM
<LOW>
10
5.8
3.6
5.5
2
22
43

Proinsulin, Total
pM
<LOW>
41
15
15
11
4
93
68

Prolactin (PRL)
ng/ml
1.4
43
7.6
11
4.7
1
80
126

Prostasin
ng/mL
89
421
206
95
182
74
804
126

Prostate-Specific
ng/mL
<LOW>
0.27
0.068
0.089
0.041
0
0
97

Antigen, Free (PSA-f)

Prostatic Acid

ng/mL
<LOW>
0.89
0.46
0.22
0.44
0
1
114

Phosphatase (PAP)

Protein S100-A4

ng/mL
<LOW>
19
6.6
7.8
4.6
0
63
102

(S100-A4)

Protein S100-A6

ng/mL
1.9
20
6.9
5.1
6.0
2
40
124

(S100-A6)

Pulmonary and
ng/mL
35
182
91
39
84
27
227
126

Activation-

Regulated

Chemokine (PARC)

Receptor for

ng/mL
0.41
6.3
2.5
1.7
2.0
0
7
125

advanced

glycosylation end

products (RAGE)

Receptor tyrosine-

ng/mL
0.25
3.6
2.2
0.85
2.3
0
4
126

protein kinase

erbB-3 (ErbB3)

Resistin

ng/ml
1.4
5.6
2.7
1.1
2.3
1
7
126

S100 calcium-
ng/mL
<LOW>
0.85
0.38
0.37
0.18
0
1
33

binding protein B

(S100-B)

Secretin
ng/mL
<LOW>
0.71
4.3
3.0
4.6
1
7
4

Serotransferrin
mg/dl
195
534
327
90
305
188
657
126

(Transferrin)

Serum Amyloid P-
ug/mL
12
42
25
8.0
24
9
50
126

Component (SAP)

Serum Glutamic

ug/mL
1.4
5.3
2.5
0.97
2.2
1
8
126

Oxaloacetic

Transaminase

(SGOT)

Sex Hormone-
nmol/L
20
133
57
31
51
16
220
126

Binding Globulin

(SHBG)

Sortilin

ng/mL
2.8
8.6
5.1
1.5
4.9
1
9
126

Squamous Cell
ng/mL
0.079
1.8
0.50
0.65
0.36
0
6
125

Carcinoma Antigen-

1 (SCCA-1)

Stem Cell Factor

pg/mL
105
421
212
92
199
24
879
126

(SCF)

Stromal cell-

pg/mL
720
2620
1614
523
1670
516
3730
126

derived factor-1

(SDF-1)

Superoxide

ng/mL
3.6
17
7.5
4.7
6.2
3
43
126

Dismutase 1,

Soluble (SOD-1)

T Lymphocyte
-
pg/mL
33
2650
453
1713
116
22
17700
125

Secreted Protein I-

309 (I-309)

Tamm-Horsfall
ug/ml
0.016
0.079
0.049
0.018
0.049
0
0
126

Urinary

Glycoprotein (THP)

T-Cell-Specific

ng/mL
0.31
5.2
1.4
1.7
1.1
0
14
126

Protein RANTES

(RANTES)

Tenascin-C (TN-C)

ng/mL
189
927
466
241
423
184
2160
126

Testosterone, Total
ng/ml
0.20
16
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical

Standard
Standard
Standard
Standard
Standard
Standard

Tetranectin
ug/mL
9.4
20
14
2.8
14
8
21
126

Thrombomodulin

ng/ml
2.9
8.5
5.1
1.4
4.8
2
9
126

(TM)

Thrombopoietin

ng/mL
<LOW>
2.8
1.6
0.62
1.5
0
3
72

(TPO)

Thrombospondin-1

ng/mL
85
3380
876
1160
584
44
8460
125

Thyroglobulin

ng/mL
3.9
223
57
99
34
2
993
126

(TG)

Thyroid-Stimulating
uIU/mL
0.15
4.0
1.5
2.2
1.1
0
23
125

Hormone (TSH)

Thyroxine-Binding

ug/mL
35
113
65
18
63
27
131
126

Globulin (TBG)

Tissue Factor (TF)

ng/mL
<LOW>
2.1
1.8
0.81
1.9
0
3
8

Tissue Inhibitor of

ng/mL
42
114
71
19
70
35
136
126

Metalloproteinases

1 (TIMP-1)

Tissue type
ng/mL
0.28
1.8
0.93
0.47
0.81
0
2
126

Plasminogen

activator (tPA)

TNF-Related
ng/mL
6.0
35
16
7.2
14
5
40
126

Apoptosis-Inducing

Ligand Receptor 3

(TRAIL-R3)

Transforming
pg/mL
<LOW>
<LOW>
<LOW>
<LOW>
<LOW>
0
0
0

Growth Factor alpha

(TGF-alpha)

Transforming
pg/mL
<LOW>
13
68
93
13
13
243
8

Growth Factor beta-

3 (TGF-beta-3)

Transthyretin (TTR)
mg/dl
13
66
33
13
32
9
69
126

Trefoil Factor 3
ug/ml
0.018
0.14
0.064
0.030
0.058
0
0
125

(TFF3)

Tumor Necrosis

pg/mL
<LOW>
21
6.0
6.3
3.8
2
37
62

Factor alpha

(TNF-alpha)

Tumor Necrosis
pg/mL
<LOW>
22
12
8.9
10
6
61
52

Factor beta (TNF-

beta)

Tumor Necrosis
ng/mL
3.4
11
5.5
1.6
5.3
2
12
126

Factor Receptor 2

(TNFR2)

Tumor Necrosis

pg/mL
692
2020
1246
394
1215
518
3190
126

Factor Receptor I

(TNF RI)

Tyrosine kinase
ng/mL
8.4
23
15
3.6
15
5
28
126

with Ig and EGF

homology domains

2 (TIE-2)

Urokinase-type

pg/mL
381
1450
827
290
822
45
1810
126

Plasminogen

Activator (uPA)

Urokinase-type

ng/mL
0.51
3.5
1.5
0.84
1.3
0
6
125

Plasminogen

Activator

Receptor(uPAR)

Vascular Cell
ng/mL
366
1200
612
214
561
303
1510
126

Adhesion Molecule-

1 (VCAM-1)

Vascular

pg/mL
282
659
430
99
414
235
804
126

Endothelial

Growth Factor

(VEGF)

Vascular

ng/mL
<LOW>
11
7.0
2.9
5.6
3
14
41

Endothelial

Growth Factor

B(VEGF-B)

Vascular

ng/mL
2.2
5.8
3.9
1.0
3.8
2
9
126

Endothelial

Growth Factor C

(VEGF-C)

Vascular

pg/mL
<LOW>
1320
634
289
606
177
1620
116

Endothelial

Growth Factor

D(VEGF-D)

Vascular

pg/mL
<LOW>
175
68
40
70
27
285
102

Endothelial

Growth Factor

Receptor 1

(VEGFR-1)

Vascular

ng/mL
3.7
7.9
5.8
1.2
5.8
2
9
126

Endothelial

Growth Factor

Receptor 2

(VEGFR-2)

Vascular

ng/mL
12
89
44
19
42
8
92
126

Endothelial

Growth Factor

Receptor 3

(VEGFR-3)

Vitamin K-

ug/ml
16
35
23
5.8
22
14
54
126

Dependent Protein

S (VKDPS)

Vitronectin

ug/ml
844
2530
1791
420
1780
745
2870
126

von Willebrand

ug/mL
13
93
40
20
37
9
117
126

Factor (vWF)

YKL-40
ng/mL
17
88
35
22
28
15
173
126

In total, 18 confirmed HIBM (hereditary inclusion body myopathy) patient serum samples from the phase 1 clinical trial were used to test and identify biomarkers that were different in protein levels from normal serum samples.

Data from the patient samples were compared to 126 “clinically normal” serum samples using Wilcoxon Rank Sum test. Of the 258 analytes tested, as a first cut 134 analytes had a p value of <0.05 (see FIG. 3 of U.S. Provisional Application Ser. No. 61/779,929 filed on Mar. 13, 2013), which are also those biomarkers in Table 2 underlined and bolded. From the 134 analytes, as a second cut 31 of them had >4 fold difference in the medians and 6 others might be related to HIBM. As a third cut, additional analyses by pathway distinction were performed (20 analytes showing relevant biology to HIBM were underlined and bolded in Tables 3-6 below): muscle inflammation/fibrosis, muscle & nerve development, muscle damage and other markers related to muscle.

TABLE 3

Muscle Inflammation and Fibrosis

Fold

Analyte
Symbol
Changes
p-value*
Primary Pathway
Note

Epithelial-Derived
ENA-78
4.5
<0.001
Inflammation

Neutrophil-Activating

Protein 78

CD40 Ligand
CD40-L
24.1
<0.001
Inflammation

Interleukin-3
IL-3
10
<0.001
Inflammation
Response to chronic

inflammation

Interleukin-5
IL-5
9.6
<0.001
Inflammation
Response to chronic

inflammation

Interleukin-7
IL-7
8
<0.001
Inflammation

Interleukin-8
IL-8
−5.3
<0.001
Inflammation
2 fold reduction in median,

5.3 reduction in mean

Latency-Associated
LAP TGF-b1
10.1
<0.001
Inflammation
TGF beta important role in

Peptide of

fibrosis and modifier in

Transforming Growth

muscular dystrophy

Factor beta 1

Macrophage Colony-
M-CSF
5.2
<0.001
Inflammation
Response to chronic

Stimulating Factor 1

inflammation, similar

pathway as interleukins

Macrophage
MIP-3 Alpha
−4.2
<0.001
Inflammation
Similar pathway to

Inflammatory Protein-3

interleukins

alpha

Myeloperoxidase
MPO
3.9
<0.001
Inflammation
3.8 fold increase in median,

3.9 fold increase in mean.

Might be used to measure

muscle damage due to

neutrophil infiltration

T-Cell-Specific Protein
RANTES
23.8
<0.001
Inflammation
Elevated in mdx diaphragm

RANTES

Vascular Endothelial
VEGF-C
8.9
<0.001
Inflammation/nerve
Similar to PDGF; elevated

Growth Factor C

development
in DMD; improve muscle

repair and reduce fibrosis

Agouti-Related
AgRP
ND
ND
Neuroendocrine
Undetectable in all HIBM

Protein

<54 pg/mL

response to
but detectable in all normal

inflammation

Thrombospondin-1
THBS1
14.3
<0.001
Inflammation/fibrosis
Binds to ECM proteins;

chemotactic factors for

inflammation in some

muscle diseases

Platelet-Derived
PDGF-BB
54.4
<0.001
Inflammation/fibrosis
Elevated in DMD and

Growth Factor BB

PDGF-receptor increased

in regenerating muscles of

mdx mice

Matrix
MMP-1
5.2
<0.001
Fibrosis
3.5 fold increase in median,

Metalloproteinase-1

5.2 increase in mean

Matrix
MMP-3
3.8
<0.001
Fibrosis
Did not meet the 4-folds

Metalloproteinase-3

cutoff criteria

Matrix
MMP-9, total
4.4
<0.001
Fibrosis
Increased in DMD

Metalloproteinase-9,

total

*Wilcoxon rank sum test

TABLE 4

Muscle and Nerve Development

Fold

Primary

Analyte
Symbol
Changes
p-value*
Pathway
Note

Brain-Derived
BDNF
38.8
<0.001
Nerve & muscle
Striatum BDNF decreased in

Neurotrophic Factor

growth
mdx mice

Chromogranin-A
CgA
19.7
<0.001
Nerve

development

Receptor tyrosine-
ErbB3
−5
<0.001
Nerve
Neuregulin family essential for

protein kinase erbB-3

development
neuromuscular junction and

myogenesis

Neuron-Specific
NSE
−7.7
<0.001
Nerve
Might be an indicator of

Enolase

development/function
neuronal regeneration

Neural cell adhesion
NCAM
−2.1
<0.001
Nerve & muscle
The data might corroborate the

molecule

function
muscle NCAM Western blot

data

Epidermal Growth
EGF
4.8
<0.001
Cell growth
More useful to look at erbB-3

Factor

Fibroblast Growth
FGF-4
ND
ND
Cell growth
Undetectable in all HIBM but

Factor 4

<44 pg/mL

detectable in all normal. Might

have some biological meaning.

Other FGFs elevated in DMD

and potential role in fibrosis

Kallikrien5
KLK5
3.87
<0.001
Serine protease
Did not meet the 4-folds cutoff

criteria

Plasminogen Activator
PAI-1
7.3
<0.001
Serine protease
Inhibition improves muscle

Inhibitor 1

inhibitor
regeneration

*Wilcoxon rank sum test

TABLE 5

Muscle Damage Markers

Fold

Primary

Analyte
Symbol
Changes
p-value*
Pathway
Note

Serum Glutamic
SGOT
4.2
<0.001
Cell damage
Blood test already covers

Oxaloacetic

many markers for

Transaminase

muscle/organ damage

Creatine Kinase-MB
CK-MB
6.8
<0.001
Muscle damage
Already assayed in blood test

Myoglobin
MB
7.5
<0.001
Muscle damage
Another marker to assess

muscle damage in addition to

CK

*Wilcoxon rank sum test

TABLE 6

Other Related Markers

Fold

Primary

Analyte
Symbol
Changes
p-value*
Pathway
Note

N-terminal prohormone
NT proBMP
5.6
<0.001
Biomarker for
Too many “low” in the normal

of brain natriuretic

cardiovascular
and the range for many HIBM

peptide

risk
samples is below least

detectable dose (LDD). The

difference between median and

mean in normal is 5 times.

Protein S100-A4
S100-A4
4
<0.001
Ca binding
Activates MMPs and nerve

protein
development, mRNA

expression increased in DMD

Protein S100-A6
S100-A6
3.6
<0.001
Ca binding
Did not meet the 4-folds cutoff

protein
criteria

Insulin-like Growth
IGFBP-6
5.1
<0.001
Tissue
IGF important for muscle

Factor Binding

morphology
growth and mass

Protein 6

Thyroglobulin
TG
−4
<0.001
Tissue
3.7 fold increase in median, 4

morphology
fold increase in mean

Amphiregulin
AR
−4.4
<0.001
Related to EGF
Too many “low” in the normal

and TGF-a;
and the range for many HIBM

growth factor
samples is below least

for fibroblasts,
detectable dose (LDD).

Schwann cells

Cortisol
Cortisol
4.7
<0.001
Stress response
Might be elevated in many

Suppress
conditions unrelated to muscle

immune system
diseases

*Wilcoxon rank sum test

A total of 37 markers with p value <0.05 and >4 fold difference in medians were identified (Table 7). These markers were further grouped into 3-4 major biological pathways using bioinformative software in which 20 of them showed relevant biology to HIBM. The list can be further narrowed down to: a total of 18 markers in which >50% of normal samples are above “least detectable dose (LDD)” or more than 5 fold changes in median (Table 8). A total of 11 markers had p<0.05 and significantly greater than 5 fold changes in median; >80% of the patient samples are above 5 times the LDD, showing the robustness of the assay (Table 9). From the box graph analysis, the distribution of normal vs. HIBM was wide apart, partly due to the large difference in median ratio.

TABLE 7

37 markers w/p <0.05 and greater or close to 4 fold changes in median, therefore

having a median ratio of HIBM/normal at >4 or <0.25

(Rows in grey: HIBM patients)

median ratio:

(Rows in white: normal)
mean
median
SD
p value
HIBM/normal

Agouti-Related Protein (AgRP)
<LOW>
<LOW>
<LOW>

Agouti-Related Protein (AgRP)
196.44
183
85.8
N/A
N/A

Amphiregulin (AR)
161.81
113.5
102.9

Amphiregulin (AR)
505
484
219.88
1.83E−07
0.23

Brain-Derived Neurotrophic Factor
14.91
15.5
7.86

(BDNF)

Brain-Derived Neurotrophic Factor
0.57
0.4
0.69
8.30E−07
38.8

(BDNF)

CD40 Ligand (CD40-L)
0.86
0.47
0.82

CD40 Ligand (CD40-L)
0.03
0.02
0.06
0.00065
24.09

Chromogranin-A (CgA)
270.67
248
146.43

Chromogranin-A (CgA)
78.99
12.6
111.66
8.10E−05
19.68

Cortisol (Cortisol)
494.83
438.5
209.32

Cortisol (Cortisol)
101.08
94.1
51.43
5.30E−07
4.66

Creatine Kinase-MB (CK-MB)
6.57
5.45
4.77

Creatine Kinase-MB (CK-MB)
1.09
0.8
0.93
0.00019
6.83

Epidermal Growth Factor (EGF)
78.23
22.5
90.62

Epidermal Growth Factor (EGF)
11.81
4.66
43.9
0.00818
4.83

Epithelial-Derived Neutrophil-
1.77
1.3
1.21

Activating Protein 78 (ENA-78)

Epithelial-Derived Neutrophil-
0.38
0.29
0.39
0.00019
4.45

Activating Protein 78 (ENA-78)

Fibroblast Growth Factor 4 (FGF-4)
<LOW>
<LOW>
<LOW>

Fibroblast Growth Factor 4 (FGF-4)
209.62
201
107.22
N/A
N/A

Insulin-like Growth Factor Binding
523
535
227.78

Protein 6 (IGFBP-6)

Insulin-like Growth Factor Binding
125.97
104.5
81.83
1.40E−06
5.12

Protein 6 (IGFBP-6)

Interleukin-3 (IL-3)
0.11
0.09
0.08

Interleukin-3 (IL-3)
0.01
0.01
0.01
0.00012
10.03

Interleukin-5 (IL-5)
23.1
21
20.1

Interleukin-5 (IL-5)
2.91
2.18
2.61
0.001
9.63

Interleukin-7 (IL-7)
112.13
112.5
67.84

Interleukin-7 (IL-7)
21.15
14
25.51
0.0001
8.04

Interleukin-8 (IL-8)
9.96
10.5
3.21

Interleukin-8 (IL-8)
51.32
20.1
68.95
8.60E−10
0.52

Kallikrein 5
3.72
3.35
1.57

Kallikrein 5
0.96
0.89
0.51
2.40E−05
3.76

Latency-Associated Peptide of
9.31
10
2.61

Transforming Growth Factor beta 1

(LAP TGF-b1

Latency-Associated Peptide of
1.09
0.99
0.54
2.70E−10
10.13

Transforming Growth Factor beta 1

(LAP TGF-b1)

Macrophage Colony-Stimulating
0.52
0.48
0.18

Factor 1 (M-CSF)

Macrophage Colony-Stimulating
0.1
0.09
0.03
7.10E−08
5.22

Factor 1 (M-CSF)

Macrophage Inflammatory Protein-
22.5
17.5
8.45

3 alpha (MIP-3 alpha)

Macrophage Inflammatory Protein-
87.3
72.8
87.51
6.80E−11
0.24

3 alpha (MIP-3 alpha)

Matrix Metalloproteinase-1 (MMP-
5.94
3.3
5.22

1)

Matrix Metalloproteinase-1 (MMP-
1.15
0.95
0.96
0.00149
3.46

1)

Matrix Metalloproteinase-3 (MMP-
11.36
11.5
5.88

3)

Matrix Metalloproteinase-3 (MMP-
4.25
3.05
3.78
0.00011
3.77

3)

Matrix Metalloproteinase-9, total
493.44
501
191.67

(MMP-9, total)

Matrix Metalloproteinase-9, total
119.36
114
54.23
3.10E−07
4.39

(MMP-9, total)

Myeloperoxidase (MPO)
783.18
592
571.62

Myeloperoxidase (MPO)
199.48
158
138.16
0.00087
3.75

Myoglobin
120.67
85.5
74.72

Myoglobin
14.96
11.35
11.91
2.00E−05
7.53

N-terminal prohormone of brain
344.76
315
259.74

natriuretic peptide (NT proBNP)

N-terminal prohormone of brain
287.81
56.25
1236.2
0.72661
5.6

natriuretic peptide (NT proBNP)

Neuron-Specific Enolase (NSE)
1.73
1.35
1.29

Neuron-Specific Enolase (NSE)
13.96
10.46
11.33
6.70E−20
0.13

Neuronal Cell Adhesion Molecule
0.72
0.57
0.47

(Nr-CAM)

Neuronal Cell Adhesion Molecule
1.61
1.45
0.89
4.30E−07
0.39

(Nr-CAM)

Plasminogen Activator Inhibitor 1
169.89
171.5
75.22

(PAI-1)

Plasminogen Activator Inhibitor 1
32.1
23.4
28.96
7.00E−07
7.33

(PAI-1)

Platelet-Derived Growth Factor BB
9775
9085
5091.84

(PDGF-BB)

Platelet-Derived Growth Factor BB
271.3
167
324.68
6.10E−07
54.4

(PDGF-BB)

Protein S100-A4 (S100-A4)
24.48
18
24.42

Protein S100-A4 (S100-A4)
6.58
4.56
7.81
0.01004
3.95

Protein S100-A6 (S100-A6)
21.96
21.5
10.86

Protein S100-A6 (S100-A6)
6.88
6
5.06
2.40E−05
3.58

Receptor tyrosine-protein kinase
0.75
0.46
0.69

erbB-3 (ErbB3)

Receptor tyrosine-protein kinase
2.15
2.29
0.85
7.00E−08
0.2

erbB-3 (ErbB3)

Serum Glutamic Oxaloacetic
10.12
9.25
3.88

Transaminase (SGOT)

Serum Glutamic Oxaloacetic
2.46
2.2
0.97
2.70E−07
4.21

Transaminase (SGOT)

T-Cell-Specific Protein RANTES
23.26
25
11.73

(RANTES)

T-Cell-Specific Protein RANTES
1.44
1.05
1.73
6.30E−07
23.81

(RANTES)

Thrombospondin-1
8488.11
8355
4265.59

Thrombospondin-1
875.94
584
1155.01
1.10E−06
14.31

Thyroglobulin (TG)
14.33
9.4
15.77

Thyroglobulin (TG)
57.16
33.55
98.25
1.60E−05
0.28

Vascular Endothelial Growth Factor
29.78
33.5
8.09

C (VEGF-C)

Vascular Endothelial Growth Factor
3.89
3.75
1.04
2.30E−10
8.93

C (VEGF-C)

TABLE 8

reduced to 18 markers after removing markers that have >50% of normal samples at

least detectable dose or less than 5 fold changes in median

(Rows in grey: HIBM patients)

median ratio:

(Rows in white: normal)
mean
median
SD
p value
HIBM/normal

Brain-Derived Neurotrophic Factor
14.91
15.5
7.86

(BDNF)

Brain-Derived Neurotrophic Factor
0.57
0.4
0.69
8.30E−07
38.8

(BDNF)

CD40 Ligand (CD40-L
0.86
0.47
0.82

CD40 Ligand (CD40-L
0.03
0.02
0.06
0.00065
24.09

Chromogranin-A (CgA
270.67
248
146.43

Chromogranin-A (CgA
78.99
12.6
111.66
8.10E−05
19.68

Creatine Kinase-MB (CK-MB
6.57
5.45
4.77

Creatine Kinase-MB (CK-MB
1.09
0.8
0.93
0.00019
6.83

Insulin-like Growth Factor Binding
523
535
227.78

Protein 6 (IGFBP-6)

Insulin-like Growth Factor Binding
125.97
104.5
81.83
1.40E−06
5.12

Protein 6 (IGFBP-6)

Interleukin-3 (IL-3
0.11
0.09
0.08

Interleukin-3 (IL-3
0.01
0.01
0.01
0.00012
10.03

Interleukin-5 (IL-5
23.1
21
20.1

Interleukin-5 (IL-5
2.91
2.18
2.61
0.001
9.63

Latency-Associated Peptide of
9.31
10
2.61

Transforming Growth Factor beta 1

(LAP TGF-b1)

Latency-Associated Peptide of
1.09
0.99
0.54
2.70E−10
10.13

Transforming Growth Factor beta 1

(LAP TGF-b1)

Macrophage Colony-Stimulating
0.52
0.48
0.18

Factor 1 (M-CSF)

Macrophage Colony-Stimulating
0.1
0.09
0.03
7.10E−08
5.22

Factor 1 (M-CSF)

Matrix Metalloproteinase-9, total
493.44
501
191.67

(MMP-9, total)

Matrix Metalloproteinase-9, total
119.36
114
54.23
3.10E−07
4.39

(MMP-9, total)

Myoglobin
120.67
85.5
74.72

Myoglobin
14.96
11.35
11.91
2.00E−05
7.53

Neuron-Specific Enolase (NSE)
1.73
1.35
1.29

Neuron-Specific Enolase (NSE)
13.96
10.46
11.33
6.70E−20
0.13

Plasminogen Activator Inhibitor 1
169.89
171.5
75.22

(PAI-1)

Plasminogen Activator Inhibitor 1
32.1
23.4
28.96
7.00E−07
7.33

(PAI-1)

Platelet-Derived Growth Factor BB
9775
9085
5091.84

(PDGF-BB)

Platelet-Derived Growth Factor BB
271.3
167
324.68
6.10E−07
54.4

(PDGF-BB)

Receptor tyrosine-protein kinase
0.75
0.46
0.69

erbB-3 (ErbB3)

Receptor tyrosine-protein kinase
2.15
2.29
0.85
7.00E−08
0.2

erbB-3 (ErbB3)

T-Cell-Specific Protein RANTES
23.26
25
11.73

(RANTES)

T-Cell-Specific Protein RANTES
1.44
1.05
1.73
6.30E−07
23.81

(RANTES)

Thrombospondin-1
8488.11
8355
4265.59

1155.01

Thrombospondin-1
875.94
584
8.09
1.10E−06
14.31

Vascular Endothelial Growth Factor
29.78
33.5
1.04

C (VEGF-C)

Vascular Endothelial Growth Factor
3.89
3.75

2.30E−10
8.93

C (VEGF-C)

TABLE 9

11 markers w/p <0.05 and significantly greater than 5 fold changes in median. Also,

>80% of the samples above 5 times the least detectable dose (LDD)

(Rows in grey: HIBM patients)

median ratio:

(Rows in white: normal)
mean
median
SD
p value
HIBM/normal

Brain-Derived Neurotrophic Factor (BDNF)
14.91
15.5
7.86

Brain-Derived Neurotrophic Factor (BDNF)
0.57
0.4
0.69
8.30E−07
38.8

CD40 Ligand (CD40-L)
0.86
0.47
0.82

CD40 Ligand (CD40-L)
0.03
0.02
0.06
0.00065
24.09

Chromogranin-A (CgA)
270.67
248
146.43

Chromogranin-A (CgA)
78.99
12.6
111.66
8.10E−05
19.68

Latency-Associated Peptide of
9.31
10
2.61

Transforming Growth Factor beta 1 (LAP

TGF-b1)

Latency-Associated Peptide of
1.09
0.99
0.54
2.70E−10
10.13

Transforming Growth Factor beta 1 (LAP

TGF-b1)

Matrix Metalloproteinase-9, total (MMP-9,
493.44
501
191.67

total)

Matrix Metalloproteinase-9, total (MMP-9,
119.36
114
54.23
3.10E−07
4.39

total)

Myoglobin
120.67
85.5
74.72

Myoglobin
14.96
11.35
11.91
2.00E−05
7.53

Plasminogen Activator Inhibitor 1 (PAI-1)
169.89
171.5
75.22

Plasminogen Activator Inhibitor 1 (PAI-1)
32.1
23.4
28.96
7E−07
7.33

Platelet-Derived Growth Factor BB (PDGF-
9775
9085
5091.84

BB)

Platelet-Derived Growth Factor BB (PDGF-
271.3
167
324.68
6.10E−07
54.4

BB)

Receptor tyrosine-protein kinase erbB-3
0.75
0.46
0.69

(ErbB3)

Receptor tyrosine-protein kinase erbB-3
2.15
2.29
0.85
7E−08
0.2

(ErbB3)

T-Cell-Specific Protein RANTES
23.26
25
11.73

(RANTES)

T-Cell-Specific Protein RANTES
1.44
1.05
1.73
6.30E−07
23.81

(RANTES)

Thrombospondin-1
8488.11
8355
4265.59

Thrombospondin-1
875.94
584
1155.01
1.10E−06
14.31

The system, along with the results generated will facilitate the establishment of a panel of markers to monitor treatment response in HIBM. The invention is also useful for diagnostic purpose, such that, a panel of unique and specific serum markers that can be used to differentiate HIBM from other muscle diseases. In addition, the system, along with the results generated will enable identification of new drug targets in HIBM.

Example 2
Phase 2 Clinical Trial

The plan for further work is to test phase 2 patient samples at both baseline (before treatment), after 24 weeks of treatment and 48 weeks of treatment. As used herein, the term “baseline level” refers to a standard control for “normal” levels (i.e., patients without disease), but can also be comparative, e.g., where low baseline levels is compared to the levels of other subjects having the disease.

Forty-seven (47) HIBM patient serum samples collected at week 24 from phase 2 study were tested using the Human Discovery MAP250+ v2.0 quantitative immunoassay system developed by Myriad RBM. The results were compared to the serum samples of the same 47 HIBM patients collected at week 0 (baseline) which were conducted earlier with the same immunoassay system.

Patients were unblinded and the results were organized into the 3 assigned dosage groups, placebo (n=14), 3 g/day (n=17) and 6 g/day (n=14). The major focus of the analysis was 1) to assess changes (delta, A) in levels of analytes between baseline and week 24 in each dosage group, 2) whether the changes correlate to dosage levels.

Two patients did not provide serum samples at week 24 and therefore, the final number of patients being analyzed was 45.

Previous analysis of the baseline serum samples identified 31 potential analytes that showed significant changes (>4 folds change in mean or median with a p-value <0.05) in the patients compared to 126 normal controls. The current analysis also included an additional analyte, Neural Cell Adhesion Molecule (NCAM) that was thought to be an important biomarker for sialylation in HIBM. See FIG. 9 of U.S. Provisional Application Ser. No. 61/779,929 filed on Mar. 13, 2013 which shows 243 analytes (markers) in the Human Discovery MAP250 quantitative immunoassay system tested in 47 HIBM patients. Data from the patient samples were compared to 126 “clinically normal” serum samples using Wilcoxon Rank Sum test. Of the 243 analytes tested, as a first cut 140 analytes had a p value of <0.05 (see FIG. 10 of U.S. Provisional Application Ser. No. 61/779,929 filed on Mar. 13, 2013, which are also those biomarkers in Table 10a and Table 10b). From the 140 analytes, as a second cut 31 of them had >4 fold difference in the medians and 6 others might be related to HIBM (see FIG. 11 of U.S. Provisional Application Ser. No. 61/779,929 filed on Mar. 13, 2013, which are also those biomarkers in Table 11). As a third cut, additional analyses by pathway distinction were performed, and 16 analytes of the 31 had >50% of HIBM and normal samples are above LDD or greater than 5 fold changes in median (see FIG. 12 of U.S. Provisional Application Ser. No. 61/779,929 filed on Mar. 13, 2013, which are also those biomarkers in Table 12):

TABLE 10a

Phase II Clinical Trial - 140 analytes (biomarkers) which had a p value of <0.05 (126 Normal Controls)

change in
change in

mean
median

Normal
Normal
Normal

Positive
p-
(HIBM/
(HIBM/

140 markers with p < 0.05.
Units
Mean
SD
Median
Min
Max
Count
value
nomral)
normal)

6Ckine
pg/mL
536
182
520
201
1200
126
0.0001
0.85
0.89

Aldose Reductase
ng/mL
3.4
1.7
3.4
0
12
125
0.0000
3.45
2.94

Alpha-1-Microglobulin
ug/mL
11
2.9
11
6
23
126
0.0467
0.93
0.92

(A1Micro)

Alpha-2-Macroglobulin
mg/mL
3.2
1.8
2.9
1
8
126
0.0000
0.47
0.52

(A2Macro)

Angiogenin
ng/mL
623
191
588
260
1600
126
0.0000
0.66
0.64

Angiopoietin-2 (ANG-2)
ng/mL
3.2
1.5
3.0
0
9
126
0.0000
1.32
1.38

Angiotensin-Converting Enzyme
ng/mL
92
30
87
43
197
126
0.0000
0.71
0.70

(ACE)

Apolipoprotein(a) (Lp(a))
ug/mL
781
875
506
1
3760
122
0.0000
0.39
0.45

Apolipoprotein A-I (Apo A-I)
mg/mL
1.3
0.36
1.3
1
3
126
0.0000
1.25
1.25

Apolipoprotein A-II (Apo A-II)
ng/mL
534
274
448
189
1560
126
0.0000
0.62
0.72

Apolipoprotein A-IV (Apo A-IV)
ug/mL
38
22
34
5
134
126
0.0000
0.44
0.33

Apolipoprotein B (Apo B)
ug/mL
2013
714
1830
835
5130
126
0.0000
0.65
0.76

Apolipoprotein C-III (Apo C-III)
ug/mL
163
106
135
52
758
126
0.0000
1.43
1.52

Apolipoprotein D (Apo D)
ug/mL
192
71
178
52
481
126
0.0000
0.65
0.69

Apolipoprotein H (Apo H)
ug/mL
296
87
282
143
589
126
0.0000
0.84
0.88

AXL Receptor Tyrosine Kinase
ng/mL
9.7
3.0
9.5
2
20
126
0.0044
1.17
1.15

(AXL)

B cell-activating factor (BAFF)
pg/mL
684
191
654
259
1380
126
0.0000
1.22
1.27

B Lymphocyte Chemoattractant
pg/mL
19
42
8.6
6
245
67
0.0006
3.63
8.17

(BLC)

Beta-2-Microglobulin (B2M)
ug/mL
1.7
0.47
1.6
1
3
126
0.0000
0.77
0.80

Brain-Derived Neurotrophic
ng/mL
0.57
0.69
0.40
0
7
126
0.0000
34.58
50.06

Factor (BDNF)

C-Peptide
ng/mL
1.4
1.5
1.1
0
12
126
0.0000
1.77
2.14

Carcinoembryonic Antigen (CEA)
ng/mL
2.2
2.6
1.6
0
28
124
0.0000
0.46
0.52

Cathepsin D
ng/mL
357
89
346
208
814
126
0.0001
0.81
0.81

CD5 Antigen-like (CD5L)
ng/mL
1624
647
1450
581
4580
126
0.0000
1.56
1.70

CD 40 antigen (CD40)
ng/mL
0.44
0.11
0.42
0
1
126
0.0000
2.10
2.13

CD40 Ligand (CD40-L)
ng/mL
0.031
0.064
0.019
0
0
54
0.0000
57.13
82.90

Cellular Fibronectin (cFib)
ug/mL
0.66
0.39
0.63
0
2
27
0.0000
3.23
2.92

Chromogranin-A (CgA)
ng/mL
79
113
13
5
372
52
0.0000
4.36
25.95

Clusterin (CLU)
ug/mL
302
84
286
112
577
126
0.0000
0.85
0.91

Complement C3 (C3)
mg/mL
2.2
0.58
2.1
1
4
126
0.0000
0.57
0.57

Complement Factor H - Related
ug/mL
3333
1375
3330
704
6810
126
0.0000
0.70
0.69

Protein 1 (CFHR1)

Cortisol (Cortisol)
ng/mL
101
52
94
10
250
123
0.0000
2.21
2.20

Creatine Kinase-MB (CK-MB)
ng/mL
1.1
0.93
0.80
0
6
125
0.0000
11.37
10.90

Cystatin-C
ng/mL
1035
286
980
330
1780
126
0.0000
0.78
0.83

E-Selectin
ng/mL
19
7.7
19
1
47
126
0.0000
0.46
0.43

EN-RAGE
ng/mL
15
11
11
3
67
126
0.0003
2.90
2.28

Endoglin
ng/mL
3.9
1.0
3.8
1
7
126
0.0000
0.84
0.90

Endostatin
ng/mL
79
19
77
33
144
126
0.0003
1.23
1.26

Eotaxin-1
pg/mL
42
30
28
11
202
86
0.0000
4.90
7.39

Epidermal Growth Factor (EGF)
pg/mL
12
44
4.7
2
432
102
0.0000
23.55
53.22

Epidermal Growth Factor
ng/mL
4.4
0.62
4.4
3
6
126
0.0008
0.92
0.94

Receptor (EGFR)

Epithelial-Derived Neutrophil-
ng/mL
0.38
0.39
0.29
0
4
126
0.0000
6.42
6.85

Activating Protein 78 (ENA-78)

Factor VII
ng/mL
310
167
304
54
855
126
0.0016
1.26
1.21

FASLG Receptor (FAS)
ng/mL
10
12
8.0
2
134
123
0.0007
1.55
1.88

Fatty Acid-Binding Protein,
ng/mL
14
11
12
2
80
126
0.0001
1.50
1.60

adipocyte (FABP, adipocyte)

Fetuin-A
ug/mL
1269
363
1200
697
2490
126
0.0000
0.58
0.62

Fibrinogen
mg/mL
0.028
0.010
0.025
0
0
86
0.0000
2.48
2.51

Galectin-3
ng/mL
11
4.1
10
3
27
125
0.0000
1.26
1.25

Gelsolin
ug/mL
66
14
65
38
114
126
0.0000
0.77
0.77

Glucagon-like Peptide 1, total
pg/mL
2.8
2.0
2.4
1
9
52
0.0011
6.09
4.71

(GLP-1 total)

Glutathione S-Transferase Mu 1
ng/mL
4.7
3.2
4.2
1
14
82
0.0000
1.38
1.53

(GST-M1)

Granulocyte Colony-Stimulating
pg/mL
8.6
5.6
7.3
3
48
122
0.0433
1.16
1.20

Factor (G-CSF)

Hepatocyte Growth Factor (HGF)
ng/mL
3.1
2.1
2.6
0
11
123
0.0000
4.59
5.04

Hepsin
pg/mL
815
187
820
227
1360
126
0.0000
1.32
1.28

Human Epidermal Growth Factor
ng/mL
0.59
0.17
0.57
0
1
126
0.0002
0.81
0.80

Receptor 2 (HER-2)

Immunoglobulin A (IgA)
mg/mL
3.8
1.7
3.4
1
10
126
0.0000
0.57
0.58

Immunoglobulin M (IgM)
mg/mL
2.9
1.4
2.6
1
8
126
0.0000
0.65
0.57

Insulin-like Growth Factor-
ng/mL
80
49
70
16
278
126
0.0000
0.65
0.60

Binding Protein 2 (IGFBP-2)

Insulin-like Growth Factor
ng/mL
520
153
511
138
1000
126
0.0000
0.70
0.71

Binding Protein 4 (IGFBP4)

Insulin-like Growth Factor
ng/mL
148
30
145
78
250
126
0.0006
0.88
0.91

Binding Protein 5 (IGFBP5)

Insulin-like Growth Factor
ng/mL
126
82
105
9
420
126
0.0000
2.90
3.49

Binding Protein 6 (IGFBP6)

Intercellular Adhesion Molecule 1
ng/mL
98
48
97
28
242
121
0.0002
1.26
1.25

(ICAM-1)

Interferon gamma Induced Protein
pg/mL
313
244
272
52
2360
126
0.0101
1.45
1.28

10 (IP-10)

Interferon-inducible T-cell alpha
pg/mL
15
10
11
5
57
108
0.0001
3.88
3.82

chemoattractant (ITAC)

Interleukin-1 alpha (IL-1 alpha)
ng/mL
0.0021
0.0019
0.0018
0
0
85
0.0000
1.99
2.23

Interleukin-1 receptor antagonist
pg/mL
61
35
51
14
242
96
0.0000
2.87
3.11

(IL-1ra)

Interleukin-2 receptor alpha (IL-2
pg/mL
1444
556
1390
566
3880
126
0.0000
1.64
1.71

receptor alpha)

Interleukin-6 receptor (IL-6r)
ng/mL
23
7.4
21
5
41
126
0.0000
1.30
1.34

Interleukin-6 receptor subunit beta
ng/mL
185
37
185
109
347
126
0.0003
0.89
0.88

(IL-6R beta)

Interleukin-7 (IL-7)
pg/mL
21
26
14
4
185
54
0.0463
0.59
0.71

Interleukin-12 Subunit p40 (IL-
ng/mL
0.15
0.051
0.12
0
0
25
0.0000
3.41
4.14

12p40)

Interleukin-15 (IL-15)
ng/mL
0.35
0.20
0.29
0
2
68
0.0001
1.43
1.71

Interleukin-16 (IL-16)
pg/mL
208
62
202
52
393
126
0.0000
1.99
1.87

Kallikrein 5
ng/mL
0.96
0.51
0.89
0
3
126
0.0000
3.64
3.60

Kidney Injury Molecule-1 (KIM-
ng/mL
0.16
0.34
0.089
0
3
126
0.0000
0.16
0.25

1)

Lactoylglutathione lyase (LGL)
ng/mL
3.4
1.4
3.1
0
11
126
0.0000
5.04
4.21

Latency-Associated Peptide of
ng/mL
1.1
0.55
0.99
0
5
126
0.0000
9.75
11.14

Transforming Growth Factor beta

1 (LAP TGF-b1)

Lectin-Like Oxidized LDL
ng/mL
0.60
0.13
0.53
0
1
15
0.0000
3.56
3.18

Receptor 1 (LOX-1)

Leptin
ng/mL
12
14
8.6
0
96
126
0.0005
1.67
1.98

Macrophage Colony-Stimulating
ng/mL
0.100
0.033
0.092
0
0
126
0.0000
5.02
4.90

Factor 1 (M-CSF)

Macrophage-Derived Chemokine
pg/mL
414
124
414
122
826
126
0.0007
1.22
1.23

(MDC)

Macrophage inflammatory protein
pg/mL
268
616
171
32
6760
126
0.0038
1.72
2.23

3 beta (MIP-3 beta)

Macrophage Migration Inhibitory
ng/mL
0.056
0.046
0.044
0
0
123
0.0292
1.80
1.42

Factor (MIF)

Macrophage-Stimulating Protein
ng/mL
506
264
474
82
1350
126
0.0000
0.48
0.37

(MSP)

Maspin
pg/mL
1327
582
1160
863
3820
76
0.0000
1.99
2.11

Matrix Metalloproteinase-1
ng/mL
1.2
0.97
0.95
0
10
117
0.0000
6.89
7.24

(MMP-1)

Matrix Metalloproteinase-3
ng/mL
4.3
3.8
3.1
1
28
126
0.0000
2.47
2.95

(MMP-3)

Matrix Metalloproteinase-9, total
ng/mL
119
54
114
42
569
126
0.0000
5.51
5.18

(MMP-9, total)

Mesothelin (MSLN)
nM
63
31
56
12
252
126
0.0375
0.85
0.78

Monocyte Chemotactic Protein 1
pg/mL
131
49
130
46
312
126
0.0000
3.44
3.15

(MCP-1)

Monocyte Chemotactic Protein 2
pg/mL
33
24
28
9
234
126
0.0379
1.27
1.23

(MCP-2)

Monocyte Chemotactic Protein 4
pg/mL
707
272
697
146
1660
126
0.0000
4.75
4.84

(MCP-4)

Myeloid Progenitor Inhibitory
ng/mL
0.80
0.50
0.67
0
4
126
0.0000
2.14
2.23

Factor 1 (MPIF-1)

Myeloperoxidase (MPO)
ng/mL
199
139
158
46
716
124
0.0000
5.24
4.69

Myoglobin
ng/mL
15
12
11
4
89
126
0.0000
10.16
8.81

Neuron-Specific Enolase (NSE)
ng/mL
14
11
10
2
84
112
0.0000
0.18
0.11

Neuronal Cell Adhesion Molecule
ng/mL
1.6
0.89
1.5
0
6
119
0.0000
0.52
0.50

(Nr-CAM)

Neuropilin-1
ng/mL
167
36
161
103
319
126
0.0000
0.84
0.88

Neutrophil Gelatinase-Associated
ng/mL
106
48
88
40
263
102
0.0000
2.27
2.61

Lipocalin (NGAL)

Osteopontin
ng/mL
5.1
6.8
3.6
1
66
126
0.0131
1.41
1.72

Osteoprotegerin (OPG)
pM
4.6
1.4
4.2
2
8
126
0.0328
1.11
1.13

Plasminogen Activator Inhibitor 1
ng/mL
32
29
23
4
166
126
0.0000
6.12
8.16

(PAI-1)

Platelet-Derived Growth Factor
pg/mL
271
327
167
42
2390
79
0.0000
57.62
93.41

BB (PDGF-BB)

Progesterone
ng/mL
6.0
4.2
4.8
1
30
119
0.0000
2.00
2.10

Proinsulin, Intact
pM
5.8
3.6
5.5
2
22
43
0.0010
3.11
3.10

Proinsulin, Total
pM
15
15
11
4
93
68
0.0046
3.61
5.14

Protein S100-A4 (S100-A4)
ng/mL
6.6
7.8
4.6
0
63
102
0.0183
7.73
8.44

Pulmonary and Activation-
ng/mL
91
39
84
27
227
126
0.0249
0.83
0.76

Regulated Chemokine (PARC)

Receptor for advanced
ng/mL
2.5
1.7
2.0
0
7
125
0.0000
1.91
2.03

glycosylation end products

(RAGE)

Receptor tyrosine-protein kinase
ng/mL
2.2
0.85
2.3
0
4
126
0.0000
0.29
0.14

erbB-3 (ErbB3)

Resistin
ng/mL
2.7
1.1
2.3
1
7
126
0.0000
0.67
0.65

Serotransferrin (Transferrin)
mg/dl
327
90
305
188
657
126
0.0000
0.73
0.76

Serum Amyloid P-Component
ug/mL
25
8.0
24
9
50
126
0.0000
0.69
0.71

(SAP)

Sortilin
ng/mL
5.1
1.5
4.9
1
9
126
0.0000
1.47
1.48

Squamous Cell Carcinoma
ng/mL
0.50
0.65
0.36
0
6
125
0.0000
3.05
4.47

Antigen-1 (SCCA-1)

Stem Cell Factor (SCF)
pg/mL
212
92
199
24
879
126
0.0000
1.55
1.55

Stromal cell-derived factor-1
pg/mL
1614
523
1670
516
3730
126
0.0000
2.29
2.26

(SDF-1)

Superoxide Dismutase 1, soluble
ng/mL
7.5
4.7
6.2
3
43
126
0.0000
3.19
2.72

(SOD-1)

T-Cell-Specific Protein RANTES
ng/mL
1.4
1.7
1.1
0
14
126
0.0000
12.69
17.14

(RANTES)

Tenascin-C (TN-C)
ng/mL
466
241
423
184
2160
126
0.0130
0.82
0.80

Tetranectin
ug/mL
14
2.8
14
8
21
126
0.0171
1.10
1.05

Thrombomodulin (TM)
ng/mL
5.1
1.4
4.8
2
9
126
0.0000
0.81
0.84

Thrombospondin-1
ng/mL
876
1160
584
44
8460
125
0.0000
11.19
16.34

Thyroxine-Binding Globulin
ug/mL
65
18
63
27
131
126
0.0000
0.57
0.61

(TBG)

Tissue Inhibitor of
ng/mL
71
19
70
35
136
126
0.0000
1.94
2.01

Metalloproteinases 1 (TIMP-1)

Tissue type Plasminogen activator
ng/mL
0.93
0.47
0.81
0
2
126
0.0000
1.60
1.61

(tPA)

Transthyretin (TTR)
mg/dl
33
13
32
9
69
126
0.0000
0.67
0.66

Trefoil Factor 3 (TFF3)
ug/mL
0.064
0.030
0.058
0
0
125
0.0377
3.67
1.42

Tumor Necrosis Factor alpha
pg/mL
6.0
6.3
3.8
2
37
62
0.0304
5.47
9.66

(TNF-alpha)

Tumor Necrosis Factor Receptor I
pg/mL
1246
394
1215
518
3190
126
0.0096
1.16
1.23

(TNF RI)

Tumor necrosis factor receptor 2
ng/mL
5.5
1.6
5.3
2
12
126
0.0290
0.91
0.92

(TNFR2)

Urokinase-type plasminogen
ng/mL
1.5
0.84
1.3
0
6
125
0.0065
0.81
0.85

activator receptor (uPAR)

Vascular Cell Adhesion
ng/mL
612
214
561
303
1510
126
0.0094
0.89
0.92

Molecule-1 (VCAM-1)

Vascular Endothelial Growth
ng/mL
3.9
1.0
3.8
2
9
126
0.0000
9.07
9.60

Factor C (VEGF-C)

Vascular Endothelial Growth
ng/mL
5.8
1.2
5.8
2
9
126
0.0000
1.23
1.22

Factor Receptor 2 (VEGFR-2)

Vascular endothelial growth
ng/mL
44
19
42
8
92
126
0.0004
1.35
1.41

factor receptor 3 (VEGFR-3)

Vitamin K-Dependent Protein S
ug/mL
23
5.8
22
14
54
126
0.0000
0.65
0.69

(VKDPS)

Vitronectin
ug/mL
1791
420
1780
745
2870
126
0.0000
1.34
1.33

von Willebrand Factor (vWF)
ug/mL
40
20
37
9
117
126
0.0010
1.32
1.31

YKL-40
ng/mL
35
22
28
15
173
126
0.0105
0.79
0.89

TABLE 10b

Phase II Clinical Trial - 140 analytes (biomarkers) which had a p value of <0.05 (47 HIBM samples)

change in
change in

mean
median

HIBM
HIBM
HIBM

Positive
p-
(HIBM/
(HIBM/

140 markers with p < 0.05.
Units
Mean
SD
Median
Min
Max
Count
value
nomral)
normal)

6Ckine
pg/mL
456.49
82.91
460.00
260.00
664.00
47
0.0001
0.85
0.89

Aldose Reductase
ng/mL
11.67
5.01
9.90
5.60
27.00
47
0.0000
3.45
2.94

Alpha-1-Microglobulin
ug/mL
10.42
2.20
10.00
6.50
16.00
47
0.0467
0.93
0.92

(A1Micro)

Alpha-2-Macroglobulin
mg/mL
1.54
0.34
1.50
0.92
2.60
47
0.0000
0.47
0.52

(A2Macro)

Angiogenin
ng/mL
413.00
136.72
379.00
192.00
903.00
47
0.0000
0.66
0.64

Angiopoietin-2 (ANG-2)
ng/mL
4.20
1.23
4.10
1.90
7.70
47
0.0000
1.32
1.38

Angiotensin-Converting
ng/mL
64.83
16.95
61.00
37.00
105.00
47
0.0000
0.71
0.70

Enzyme (ACE)

Apolipoprotein(a) (Lp(a))
ug/mL
305.24
314.28
226.00
5.40
1740.00
47
0.0000
0.39
0.45

Apolipoprotein A-I (Apo A-I)
mg/mL
1.62
0.29
1.60
1.10
2.40
47
0.0000
1.25
1.25

Apolipoprotein A-II (Apo A-II)
ng/mL
331.30
57.00
322.00
253.00
536.00
47
0.0000
0.62
0.72

Apolipoprotein A-IV (Apo A-
ug/mL
16.51
20.08
11.00
6.30
127.00
47
0.0000
0.44
0.33

IV)

Apolipoprotein B (Apo B)
ug/mL
1304.43
405.44
1390.00
387.00
2090.00
47
0.0000
0.65
0.76

Apolipoprotein C-III (Apo C-
ug/mL
233.91
86.35
205.00
141.00
538.00
47
0.0000
1.43
1.52

III)

Apolipoprotein D (Apo D)
ug/mL
124.73
32.04
122.00
75.00
199.00
40
0.0000
0.65
0.69

Apolipoprotein H (Apo H)
ug/mL
249.57
50.16
248.00
137.00
388.00
47
0.0000
0.84
0.88

AXL Receptor Tyrosine Kinase
ng/mL
11.38
3.35
11.00
5.20
21.00
47
0.0044
1.17
1.15

(AXL)

B cell-activating factor (BAFF)
pg/mL
834.85
165.90
831.00
486.00
1190.00
47
0.0000
1.22
1.27

B Lymphocyte
pg/mL
70.50
4.50
70.50
66.00
75.00
2
0.0006
3.63
8.17

Chemoattractant (BLC)

Beta-2-Microglobulin (B2M)
ug/mL
1.31
0.27
1.30
0.88
1.90
47
0.0000
0.77
0.80

Brain-Derived Neurotrophic
ng/mL
19.72
4.80
20.00
8.80
31.00
47
0.0000
34.58
50.06

Factor (BDNF)

C-Peptide
ng/mL
2.49
1.32
2.30
0.73
6.10
47
0.0000
1.77
2.14

Carcinoembryonic Antigen
ng/mL
0.98
0.55
0.82
0.36
3.40
47
0.0000
0.46
0.52

(CEA)

Cathepsin D
ng/mL
288.72
100.80
281.00
136.00
610.00
47
0.0001
0.81
0.81

CD5 Antigen-like (CD5L)
ng/mL
2530.21
1024.10
2470.00
1150.00
6160.00
47
0.0000
1.56
1.70

CD 40 antigen (CD40)
ng/mL
0.92
0.32
0.89
0.48
2.60
47
0.0000
2.10
2.13

CD40 Ligand (CD40-L)
ng/mL
1.75
0.90
1.60
0.12
3.90
47
0.0000
57.13
82.90

Cellular Fibronectin (cFib)
ug/mL
2.14
0.90
1.85
0.89
4.30
40
0.0000
3.23
2.92

Chromogranin-A (CgA)
ng/mL
344.73
164.73
327.00
30.00
871.00
41
0.0000
4.36
25.95

Clusterin (CLU)
ug/mL
257.40
29.27
259.00
210.00
330.00
47
0.0000
0.85
0.91

Complement C3 (C3)
mg/mL
1.25
0.23
1.20
0.79
1.90
47
0.0000
0.57
0.57

Complement Factor H -
ug/mL
2328.70
793.31
2290.00
879.00
3900.00
47
0.0000
0.70
0.69

Related Protein 1 (CFHR1)

Cortisol (Cortisol)
ng/mL
223.43
87.78
207.00
120.00
643.00
47
0.0000
2.21
2.20

Creatine Kinase-MB (CK-MB)
ng/mL
12.36
10.04
8.70
3.30
47.00
47
0.0000
11.37
10.90

Cystatin-C
ng/mL
812.17
125.92
817.00
521.00
1190.00
47
0.0000
0.78
0.83

E-Selectin
ng/mL
8.87
4.33
8.20
0.23
23.00
47
0.0000
0.46
0.43

EN-RAGE
ng/mL
43.93
49.68
26.00
7.00
291.00
47
0.0003
2.90
2.28

Endoglin
ng/mL
3.31
0.67
3.40
2.00
4.60
47
0.0000
0.84
0.90

Endostatin
ng/mL
96.66
29.09
97.00
40.00
172.00
47
0.0003
1.23
1.26

Eotaxin-1
pg/mL
207.05
39.57
209.00
148.00
299.00
21
0.0000
4.90
7.39

Epidermal Growth Factor
pg/mL
278.28
157.61
248.00
44.00
656.00
46
0.0000
23.55
53.22

(EGF)

Epidermal Growth Factor
ng/mL
4.03
0.52
4.10
2.90
4.80
47
0.0008
0.92
0.94

Receptor (EGFR)

Epithelial-Derived Neutrophil-
ng/mL
2.46
1.34
2.00
0.67
6.40
47
0.0000
6.42
6.85

Activating Protein 78 (ENA-

78)

Factor VII
ng/mL
391.04
136.04
367.00
127.00
722.00
47
0.0016
1.26
1.21

FASLG Receptor (FAS)
ng/mL
16.23
8.02
15.00
6.30
56.00
45
0.0007
1.55
1.88

Fatty Acid-Binding Protein,
ng/mL
20.78
9.05
19.00
2.50
50.00
47
0.0001
1.50
1.60

adipocyte (FABP, adipocyte)

Fetuin-A
ug/mL
742.28
108.37
738.00
473.00
989.00
47
0.0000
0.58
0.62

Fibrinogen
mg/mL
0.07
0.01
0.06
0.06
0.10
8
0.0000
2.48
2.51

Galectin-3
ng/mL
13.41
2.68
13.00
8.30
19.00
47
0.0000
1.26
1.25

Gelsolin
ug/mL
50.83
16.58
50.00
22.00
96.00
47
0.0000
0.77
0.77

Glucagon-like Peptide 1, total
pg/mL
16.99
13.62
11.50
7.60
60.00
16
0.0011
6.09
4.71

(GLP-1 total)

Glutathione S-Transferase Mu
ng/mL
6.50
0.00
6.50
6.50
6.50
5
0.0000
1.38
1.53

1 (GST-M1)

Granulocyte Colony-
pg/mL
10.00
3.11
8.80
6.30
20.00
44
0.0433
1.16
1.20

Stimulating Factor (G-CSF)

Hepatocyte Growth Factor
ng/mL
14.18
8.41
13.00
2.80
35.00
47
0.0000
4.59
5.04

(HGF)

Hepsin
pg/mL
1074.19
169.08
1050.00
728.00
1390.00
47
0.0000
1.32
1.28

Human Epidermal Growth
ng/mL
0.47
0.16
0.45
0.17
0.88
47
0.0002
0.81
0.80

Factor Receptor 2 (HER-2)

Immunoglobulin A (IgA)
mg/mL
2.14
0.86
1.95
0.56
4.20
46
0.0000
0.57
0.58

Immunoglobulin M (IgM)
mg/mL
1.84
1.17
1.50
0.53
7.00
47
0.0000
0.65
0.57

Insulin-like Growth Factor-
ng/mL
51.98
28.71
42.00
18.00
169.00
47
0.0000
0.65
0.60

Binding Protein 2 (IGFBP-2)

Insulin-like Growth Factor
ng/mL
364.77
74.83
362.00
230.00
556.00
47
0.0000
0.70
0.71

Binding Protein 4 (IGFBP4)

Insulin-like Growth Factor
ng/mL
129.87
28.79
132.00
75.00
205.00
47
0.0006
0.88
0.91

Binding Protein 5 (IGFBP5)

Insulin-like Growth Factor
ng/mL
364.96
146.83
365.00
69.00
880.00
47
0.0000
2.90
3.49

Binding Protein 6 (IGFBP6)

Intercellular Adhesion
ng/mL
123.66
34.06
122.00
50.00
281.00
47
0.0002
1.26
1.25

Molecule 1 (ICAM-1)

Interferon gamma Induced
pg/mL
455.23
333.07
349.00
149.00
2150.00
47
0.0101
1.45
1.28

Protein 10 (IP-10)

Interferon-inducible T-cell
pg/mL
59.24
63.01
42.00
26.00
435.00
42
0.0001
3.88
3.82

alpha chemoattractant (ITAC)

Interleukin-1 alpha (IL-1 alpha)
ng/mL
0.00
0.00
0.00
0.00
0.01
40
0.0000
1.99
2.23

Interleukin-1 receptor
pg/mL
173.81
49.70
159.00
97.00
313.00
42
0.0000
2.87
3.11

antagonist (IL-1ra)

Interleukin-2 receptor alpha
pg/mL
2363.19
639.55
2380.00
1020.00
4180.00
47
0.0000
1.64
1.71

(IL-2 receptor alpha)

Interleukin-6 receptor (IL-6r)
ng/mL
29.60
8.49
28.00
13.00
53.00
47
0.0000
1.30
1.34

Interleukin-6 receptor subunit
ng/mL
164.81
30.28
163.00
94.00
241.00
47
0.0003
0.89
0.88

beta (IL-6R beta)

Interleukin-7 (IL-7)
pg/mL
12.50
5.16
10.00
10.00
24.00
6
0.0463
0.59
0.71

Interleukin-12 Subunit p40 (IL-
ng/mL
0.50
0.14
0.48
0.30
0.91
36
0.0000
3.41
4.14

12p40)

Interleukin-15 (IL-15)
ng/mL
0.50
0.08
0.49
0.42
0.70
12
0.0001
1.43
1.71

Interleukin-16 (IL-16)
pg/mL
415.51
165.09
377.00
157.00
1050.00
47
0.0000
1.99
1.87

Kallikrein 5
ng/mL
3.50
1.63
3.20
0.84
8.80
47
0.0000
3.64
3.60

Kidney Injury Molecule-1
ng/mL
0.02
0.01
0.02
0.02
0.05
14
0.0000
0.16
0.25

(KIM-1)

Lactoylglutathione lyase (LGL)
ng/mL
17.26
16.54
13.00
2.80
102.00
47
0.0000
5.04
4.21

Latency-Associated Peptide of
ng/mL
10.63
3.05
11.00
5.00
19.00
47
0.0000
9.75
11.14

Transforming Growth Factor

beta 1 (LAP TGF-b1)

Lectin-Like Oxidized LDL
ng/mL
2.12
1.04
1.70
0.93
4.70
32
0.0000
3.56
3.18

Receptor 1 (LOX-1)

Leptin
ng/mL
20.51
12.97
17.00
2.60
52.00
47
0.0005
1.67
1.98

Macrophage Colony-
ng/mL
0.50
0.16
0.45
0.26
0.96
45
0.0000
5.02
4.90

Stimulating Factor 1 (M-CSF)

Macrophage-Derived
pg/mL
505.45
157.29
507.00
111.00
1110.00
47
0.0007
1.22
1.23

Chemokine (MDC)

Macrophage inflammatory
pg/mL
460.60
242.36
382.00
209.00
1620.00
47
0.0038
1.72
2.23

protein 3 beta (MIP-3 beta)

Macrophage Migration
ng/mL
0.10
0.12
0.06
0.01
0.57
42
0.0292
1.80
1.42

Inhibitory Factor (MIF)

Macrophage-Stimulating
ng/mL
240.23
163.74
177.00
63.00
800.00
47
0.0000
0.48
0.37

Protein (MSP)

Maspin
pg/mL
2640.00
630.17
2450.00
1970.00
4520.00
11
0.0000
1.99
2.11

Matrix Metalloproteinase-1
ng/mL
7.93
4.92
6.90
1.10
21.00
47
0.0000
6.89
7.24

(MMP-1)

Matrix Metalloproteinase-3
ng/mL
10.50
5.50
9.00
2.60
25.00
47
0.0000
2.47
2.95

(MMP-3)

Matrix Metalloproteinase-9,
ng/mL
657.09
197.30
591.00
326.00
1310.00
47
0.0000
5.51
5.18

total (MMP-9, total)

Mesothelin (MSLN)
nM
53.38
23.82
44.00
21.00
119.00
47
0.0375
0.85
0.78

Monocyte Chemotactic Protein
pg/mL
450.26
176.19
409.00
215.00
1220.00
47
0.0000
3.44
3.15

1 (MCP-1)

Monocyte Chemotactic Protein
pg/mL
41.81
25.01
35.00
14.00
148.00
47
0.0379
1.27
1.23

2 (MCP-2)

Monocyte Chemotactic Protein
pg/mL
3361.28
1297.41
3370.00
1140.00
7310.00
47
0.0000
4.75
4.84

4 (MCP-4)

Myeloid Progenitor Inhibitory
ng/mL
1.72
1.09
1.50
0.69
7.40
47
0.0000
2.14
2.23

Factor 1 (MPIF-1)

Myeloperoxidase (MPO)
ng/mL
1045.60
901.05
741.00
193.00
4380.00
47
0.0000
5.24
4.69

Myoglobin
ng/mL
152.09
112.88
100.00
47.00
551.00
47
0.0000
10.16
8.81

Neuron-Specific Enolase
ng/mL
2.51
6.41
1.10
0.32
45.00
47
0.0000
0.18
0.11

(NSE)

Neuronal Cell Adhesion
ng/mL
0.84
0.37
0.72
0.22
1.80
47
0.0000
0.52
0.50

Molecule (Nr-CAM)

Neuropilin-1
ng/mL
140.19
30.15
142.00
90.00
229.00
47
0.0000
0.84
0.88

Neutrophil Gelatinase-
ng/mL
240.57
82.96
229.00
87.00
494.00
47
0.0000
2.27
2.61

Associated Lipocalin (NGAL)

Osteopontin
ng/mL
7.22
3.25
6.20
3.30
16.00
33
0.0131
1.41
1.72

Osteoprotegerin (OPG)
pM
5.08
1.37
4.70
3.30
11.00
47
0.0328
1.11
1.13

Plasminogen Activator
ng/mL
196.30
56.14
191.00
88.00
316.00
47
0.0000
6.12
8.16

Inhibitor 1 (PAI-1)

Platelet-Derived Growth Factor
pg/mL
15631.91
4560.00
15600.00
7260.00
24600.00
47
0.0000
57.62
93.41

BB (PDGF-BB)

Progesterone
ng/mL
12.13
5.63
10.00
6.50
28.00
38
0.0000
2.00
2.10

Proinsulin, Intact
pM
18.00
2.35
17.00
16.00
22.00
4
0.0010
3.11
3.10

Proinsulin, Total
pM
54.75
10.33
55.00
43.00
66.00
4
0.0046
3.61
5.14

Protein S100-A4 (S100-A4)
ng/mL
50.87
46.18
38.50
9.70
149.00
10
0.0183
7.73
8.44

Pulmonary and Activation-
ng/mL
75.30
39.78
64.00
24.00
201.00
47
0.0249
0.83
0.76

Regulated Chemokine (PARC)

Receptor for advanced
ng/mL
4.77
3.05
4.00
1.00
16.00
47
0.0000
1.91
2.03

glycosylation end products

(RAGE)

Receptor tyrosine-protein
ng/mL
0.62
0.69
0.31
0.18
3.30
47
0.0000
0.29
0.14

kinase erbB-3 (ErbB3)

Resistin
ng/mL
1.78
0.81
1.50
0.44
5.50
47
0.0000
0.67
0.65

Serotransferrin (Transferrin)
mg/dl
238.47
35.67
230.00
178.00
370.00
47
0.0000
0.73
0.76

Serum Amyloid P-Component
ug/mL
17.26
5.01
17.00
8.00
32.00
47
0.0000
0.69
0.71

(SAP)

Sortilin
ng/mL
7.57
1.83
7.20
4.40
12.00
47
0.0000
1.47
1.48

Squamous Cell Carcinoma
ng/mL
1.53
0.19
1.60
1.20
1.80
7
0.0000
3.05
4.47

Antigen-1 (SCCA-1)

Stem Cell Factor (SCF)
pg/mL
328.40
112.43
308.00
164.00
620.00
47
0.0000
1.55
1.55

Stromal cell-derived factor-1
pg/mL
3703.28
677.81
3780.00
454.00
4790.00
47
0.0000
2.29
2.26

(SDF-1)

Superoxide Dismutase 1,
ng/mL
23.93
18.88
17.00
8.60
91.00
47
0.0000
3.19
2.72

soluble (SOD-1)

T-Cell-Specific Protein
ng/mL
18.26
7.29
18.00
3.70
36.00
47
0.0000
12.69
17.14

RANTES (RANTES)

Tenascin-C (TN-C)
ng/mL
381.17
174.87
340.00
87.00
1100.00
47
0.0130
0.82
0.80

Tetranectin
ug/mL
15.92
3.68
15.00
9.10
23.00
47
0.0171
1.10
1.05

Thrombomodulin (TM)
ng/mL
4.10
0.93
4.00
2.30
7.10
47
0.0000
0.81
0.84

Thrombospondin-1
ng/mL
9800.64
2558.16
9540.00
3930.00
14700.00
47
0.0000
11.19
16.34

Thyroxine-Binding Globulin
ug/mL
37.36
7.45
38.00
22.00
55.00
47
0.0000
0.57
0.61

(TBG)

Tissue Inhibitor of
ng/mL
138.49
23.17
140.00
99.00
189.00
47
0.0000
1.94
2.01

Metalloproteinases 1 (TIMP-1)

Tissue type Plasminogen
ng/mL
1.49
0.73
1.30
0.46
3.30
47
0.0000
1.60
1.61

activator (tPA)

Transthyretin (TTR)
mg/dl
22.04
3.91
21.00
15.00
33.00
47
0.0000
0.67
0.66

Trefoil Factor 3 (TFF3)
ug/mL
0.23
0.54
0.08
0.04
3.50
47
0.0377
3.67
1.42

Tumor Necrosis Factor alpha
pg/mL
33.00
7.12
37.00
23.00
39.00
3
0.0304
5.47
9.66

(TNF-alpha)

Tumor Necrosis Factor
pg/mL
1448.77
459.65
1500.00
162.00
2480.00
47
0.0096
1.16
1.23

Receptor I (TNF RI)

Tumor necrosis factor receptor
ng/mL
5.04
1.18
4.90
2.90
8.30
47
0.0290
0.91
0.92

2 (TNFR2)

Urokinase-type plasminogen
ng/mL
1.21
0.50
1.10
0.35
2.90
47
0.0065
0.81
0.85

activator receptor (uPAR)

Vascular Cell Adhesion
ng/mL
545.23
113.03
518.00
338.00
862.00
47
0.0094
0.89
0.92

Molecule-1 (VCAM-1)

Vascular Endothelial Growth
ng/mL
35.32
6.73
36.00
22.00
46.00
47
0.0000
9.07
9.60

Factor C (VEGF-C)

Vascular Endothelial Growth
ng/mL
7.09
1.30
7.00
3.50
9.70
47
0.0000
1.23
1.22

Factor Receptor 2 (VEGFR-2)

Vascular endothelial growth
ng/mL
60.15
26.03
59.00
13.00
120.00
47
0.0004
1.35
1.41

factor receptor 3 (VEGFR-3)

Vitamin K-Dependent Protein
ug/mL
15.06
3.27
15.00
8.00
26.00
47
0.0000
0.65
0.69

S (VKDPS)

Vitronectin
ug/mL
2395.32
466.22
2360.00
1550.00
3470.00
47
0.0000
1.34
1.33

von Willebrand Factor (vWF)
ug/mL
52.87
21.82
48.50
21.00
100.00
46
0.0010
1.32
1.31

YKL-40
ng/mL
27.64
14.42
25.00
7.00
89.00
47
0.0105
0.79
0.89

TABLE 11

Phase II Clinical Trial - 31 analytes (biomarkers) which had >4 fold difference in the medians

(31 markers with 1) p < 0.05, 2) >4 folds change in mean or median between HIBM samples

and normal controls, therefore having a mean or median HIBM/normal >4 or <0.25, 47 HUBM Samples)

Positive
Normal

Units
HIBM Mean
HIBM SD
HIBM Median
Min
Max
Count
Mean

B Lymphocyte
pg/mL
70.50
4.50
70.50
66.00
75.00
2
19

Chemoattractant

(BLC)

Brain-Derived
ng/mL
19.72
4.80
20.00
8.80
31.00
47
0.57

Neurotrophic Factor

(BDNF)

CD40 Ligand
ng/mL
1.75
0.90
1.60
0.12
3.90
47
0.031

(CD40-L)

Chromogranin-A
ng/mL
344.73
164.73
327.00
30.00
871.00
41
79

(CgA)

Creatine Kinase-
ng/mL
12.36
10.04
8.70
3.30
47.00
47
1.1

MB (CK-MB)

Eotaxin-1
pg/mL
207.05
39.57
209.00
148.00
299.00
21
42

Epidermal Growth
pg/mL
278.28
157.61
248.00
44.00
656.00
46
12

Factor (EGF)

Epithelial-Derived
ng/mL
2.46
1.34
2.00
0.67
6.40
47
0.38

Neutrophil-

Activating Protein

78 (ENA-78)

Glucagon-like
pg/mL
16.99
13.62
11.50
7.60
60.00
16
2.8

Peptide 1, total

(GLP-1 total)

Hepatocyte Growth
ng/mL
14.18
8.41
13.00
2.80
35.00
47
3.1

Factor (HGF)

Interleukin-12
ng/mL
0.50
0.14
0.48
0.30
0.91
36
0.15

Subunit p40 (IL-

12p40)

Kidney Injury
ng/mL
0.02
0.01
0.02
0.02
0.05
14
0.16

Molecule-1 (KIM-

1)

Lactoylglutathione
ng/mL
17.26
16.54
13.00
2.80
102.00
47
3.4

lyase (LGL)

Latency-Associated
ng/mL
10.63
3.05
11.00
5.00
19.00
47
1.1

Peptide of

Transforming

Growth Factor beta

1 (LAP TGF-b1)

Macrophage
ng/mL
0.50
0.16
0.45
0.26
0.96
45
0.100

Colony-Stimulating

Factor 1 (M-CSF)

Matrix
ng/mL
7.93
4.92
6.90
1.10
21.00
47
1.2

Metalloproteinase-1

(MMP-1)

Matrix
ng/mL
657.09
197.30
591.00
326.00
1310.00
47
119

Metalloproteinase-

9, total (MMP-9,

total)

Monocyte
pg/mL
3361.28
1297.41
3370.00
1140.00
7310.00
47
707

Chemotactic Protein

4 (MCP-4)

Myeloperoxidase
ng/mL
1045.60
901.05
741.00
193.00
4380.00
47
199

(MPO)

Myoglobin
ng/mL
152.09
112.88
100.00
47.00
551.00
47
15

Neuron-Specific
ng/mL
2.51
6.41
1.10
0.32
45.00
47
14

Enolase (NSE)

Plasminogen
ng/mL
196.30
56.14
191.00
88.00
316.00
47
32

Activator Inhibitor

1 (PAI-1)

Platelet-Derived
pg/mL
15631.91
4560.00
15600.00
7260.00
24600.00
47
271

Growth Factor BB

(PDGF-BB)

Proinsulin, Total
pM
54.75
10.33
55.00
43.00
66.00
4
15

Protein S100-A4
ng/mL
50.87
46.18
38.50
9.70
149.00
10
6.6

(S100-A4)

Receptor tyrosine-
ng/mL
0.62
0.69
0.31
0.18
3.30
47
2.2

protein kinase erbB-

3 (ErbB3)

Squamous Cell
ng/mL
1.53
0.19
1.60
1.20
1.80
7
0.50

Carcinoma Antigen-

1 (SCCA-1)

T-Cell-Specific
ng/mL
18.26
7.29
18.00
3.70
36.00
47
1.4

Protein RANTES

(RANTES)

Thrombospondin-1
ng/mL
9800.64
2558.16
9540.00
3930.00
14700.00
47
876

Tumor Necrosis
pg/mL
33.00
7.12
37.00
23.00
39.00
3
6.0

Factor alpha (TNF-

alpha)

Vascular
ng/mL
35.32
6.73
36.00
22.00
46.00
47
3.9

Endothelial Growth

Factor C (VEGF-C)

change
change

Normal

in
in

Normal
Normal
Controls

mean
median

Normal
Normal
Controls
Controls
Positive
p-
(HIBM/
(HIBM/

SD
Median
Min
Max
Count
value
nomral)
normal)

B Lymphocyte
42
8.6
6
245
67
0.0006
3.63
8.17

Chemoattractant

(BLC)

Brain-Derived
0.69
0.40
0
7
126
0.0000
34.58
50.06

Neurotrophic Factor

(BDNF)

CD40 Ligand
0.064
0.019
0
0
54
0.0000
57.13
82.90

(CD40-L)

Chromogranin-A
113
13
5
372
52
0.0000
4.36
25.95

(CgA)

Creatine Kinase-
0.93
0.80
0
6
125
0.0000
11.37
10.90

MB (CK-MB)

Eotaxin-1
30
28
11
202
86
0.0000
4.90
7.39

Epidermal Growth
44
4.7
2
432
102
0.0000
23.55
53.22

Factor (EGF)

Epithelial-Derived
0.39
0.29
0
4
126
0.0000
6.42
6.85

Neutrophil-

Activating Protein

78 (ENA-78)

Glucagon-like
2.0
2.4
1
9
52
0.0011
6.09
4.71

Peptide 1, total

(GLP-1 total)

Hepatocyte Growth
2.1
2.6
0
11
123
0.0000
4.59
5.04

Factor (HGF)

Interleukin-12
0.051
0.12
0
0
25
0.0000
3.41
4.14

Subunit p40 (IL-

12p40)

Kidney Injury
0.34
0.089
0
3
126
0.0000
0.16
0.25

Molecule-1 (KIM-

1)

Lactoylglutathione
1.4
3.1
0
11
126
0.0000
5.04
4.21

lyase (LGL)

Latency-Associated
0.55
0.99
0
5
126
0.0000
9.75
11.14

Peptide of

Transforming

Growth Factor beta

1 (LAP TGF-b1)

Macrophage
0.033
0.092
0
0
126
0.0000
5.02
4.90

Colony-Stimulating

Factor 1 (M-CSF)

Matrix
0.97
0.95
0
10
117
0.0000
6.89
7.24

Metalloproteinase-1

(MMP-1)

Matrix
54
114
42
569
126
0.0000
5.51
5.18

Metalloproteinase-

9, total (MMP-9,

total)

Monocyte
272
697
146
1660
126
0.0000
4.75
4.84

Chemotactic Protein

4 (MCP-4)

Myeloperoxidase
139
158
46
716
124
0.0000
5.24
4.69

(MPO)

Myoglobin
12
11
4
89
126
0.0000
10.16
8.81

Neuron-Specific
11
10
2
84
112
0.0000
0.18
0.11

Enolase (NSE)

Plasminogen
29
23
4
166
126
0.0000
6.12
8.16

Activator Inhibitor

1 (PAI-1)

Platelet-Derived
327
167
42
2390
79
0.0000
57.62
93.41

Growth Factor BB

(PDGF-BB)

Proinsulin, Total
15
11
4
93
68
0.0046
3.61
5.14

Protein S100-A4
7.8
4.6
0
63
102
0.0183
7.73
8.44

(S100-A4)

Receptor tyrosine-
0.85
2.3
0
4
126
0.0000
0.29
0.14

protein kinase erbB-

3 (ErbB3)

Squamous Cell
0.65
0.36
0
6
125
0.0000
3.05
4.47

Carcinoma Antigen-

1 (SCCA-1)

T-Cell-Specific
1.7
1.1
0
14
126
0.0000
12.69
17.14

Protein RANTES

(RANTES)

Thrombospondin-1
1160
584
44
8460
125
0.0000
11.19
16.34

Tumor Necrosis
6.3
3.8
2
37
62
0.0304
5.47
9.66

Factor alpha (TNF-

alpha)

Vascular
1.0
3.8
2
9
126
0.0000
9.07
9.60

Endothelial Growth

Factor C (VEGF-C)

TABLE 12

Phase II Clinical Trial - 16 analytes (biomarkers) which had >50% of HIBM and normal

samples are above LDD or greater than 5 fold changes in median between HIBM samples and

normal controls, 3) >50% of HIBM and normal samples above least detectable dose (LDD).

Positive
Normal

Units
HIBM Mean
HIBM SD
HIBM Median
Min
Max
Count
Mean

Brain-Derived
ng/mL
19.72
4.80
20.00
8.80
31.00
47
0.57

Neurotrophic Factor

(BDNF)

Creatine Kinase-
ng/mL
12.36
10.04
8.70
3.30
47.00
47
1.1

MB (CK-MB)

Epidermal Growth
pg/mL
278.28
157.61
248.00
44.00
656.00
46
12

Factor (EGF)

Epithelial-Derived
ng/mL
2.46
1.34
2.00
0.67
6.40
47
0.38

Neutrophil-

Activating Protein

78 (ENA-78)

Hepatocyte Growth
ng/mL
14.18
8.41
13.00
2.80
35.00
47
3.1

Factor (HGF)

Latency-Associated
ng/mL
10.63
3.05
11.00
5.00
19.00
47
1.1

Peptide of

Transforming

Growth Factor beta

1 (LAP TGF-b1)

Matrix
ng/mL
7.93
4.92
6.90
1.10
21.00
47
1.2

Metalloproteinase-1

(MMP-1)

Matrix
ng/mL
657.09
197.30
591.00
326.00
1310.00
47
119

Metalloproteinase-

9, total (MMP-9,

total)

Myoglobin
ng/mL
152.09
112.88
100.00
47.00
551.00
47
15

Neuron-Specific
ng/mL
2.51
6.41
1.10
0.32
45.00
47
14

Enolase (NSE)

Plasminogen
ng/mL
196.30
56.14
191.00
88.00
316.00
47
32

Activator Inhibitor

1 (PAI-1)

Platelet-Derived
pg/mL
15631.91
4560.00
15600.00
7260.00
24600.00
47
271

Growth Factor BB

(PDGF-BB)

Receptor tyrosine-
ng/mL
0.62
0.69
0.31
0.18
3.30
47
2.2

protein kinase erbB-

3 (ErbB3)

T-Cell-Specific
ng/mL
18.26
7.29
18.00
3.70
36.00
47
1.4

Protein RANTES

(RANTES)

Thrombospondin-1
ng/mL
9800.64
2558.16
9540.00
3930.00
14700.00
47
876

Vascular
ng/mL
35.32
6.73
36.00
22.00
46.00
47
3.9

Endothelial Growth

Factor C (VEGF-C)

change
change

Normal

in
in

Normal
Normal
Controls

mean
median

Normal
Normal
Controls
Controls
Positive
p-
(HIBM/
(HIBM/

SD
Median
Min
Max
Count
value
nomral)
normal)

Brain-Derived
0.69
0.40
0
7
126
0.0000
34.58
50.06

Neurotrophic Factor

(BDNF)

Creatine Kinase-
0.93
0.80
0
6
125
0.0000
11.37
10.90

MB (CK-MB)

Epidermal Growth
44
4.7
2
432
102
0.0000
23.55
53.22

Factor (EGF)

Epithelial-Derived
0.39
0.29
0
4
126
0.0000
6.42
6.85

Neutrophil-

Activating Protein

78 (ENA-78)

Hepatocyte Growth
2.1
2.6
0
11
123
0.0000
4.59
5.04

Factor (HGF)

Latency-Associated
0.55
0.99
0
5
126
0.0000
9.75
11.14

Peptide of

Transforming

Growth Factor beta

1 (LAP TGF-b1)

Matrix
0.97
0.95
0
10
117
0.0000
6.89
7.24

Metalloproteinase-1

(MMP-1)

Matrix
54
114
42
569
126
0.0000
5.51
5.18

Metalloproteinase-

9, total (MMP-9,

total)

Myoglobin
12
11
4
89
126
0.0000
10.16
8.81

Neuron-Specific
11
10
2
84
112
0.0000
0.18
0.11

Enolase (NSE)

Plasminogen
29
23
4
166
126
0.0000
6.12
8.16

Activator Inhibitor

1 (PAI-1)

Platelet-Derived
327
167
42
2390
79
0.0000
57.62
93.41

Growth Factor BB

(PDGF-BB)

Receptor tyrosine-
0.85
2.3
0
4
126
0.0000
0.29
0.14

protein kinase erbB-

3 (ErbB3)

T-Cell-Specific
1.7
1.1
0
14
126
0.0000
12.69
17.14

Protein RANTES

(RANTES)

Thrombospondin-1
1160
584
44
8460
125
0.0000
11.19
16.34

Vascular
1.0
3.8
2
9
126
0.0000
9.07
9.60

Endothelial Growth

Factor C (VEGF-C)

TABLE 13

Phase II Clinical Trial - 16 markers with 1) p < 0.05, 2) >5 folds change

in median between HIBM samples and normal controls, 3) >50% of HIBM and normal

samples above least detectable dose (LDD), 4) >80% of HIBM samples above 5x LDD.

HIBM
HIBM
HIBM

Positive
Normal
Normal

Units
Mean
SD
Median
Min
Max
Count
Mean
SD

Brain-Derived
ng/mL
19.72
4.80
20.00
8.80
31.00
47
0.57
0.69

Neurotrophic

Factor

(BDNF)

Creatine
ng/mL
12.36
10.04
8.70
3.30
47.00
47
1.1
0.93

Kinase-MB

(CK-MB)

Epidermal
pg/mL
278.28
157.61
248.00
44.00
656.00
46
12
44

Growth Factor

(EGF)

Epithelial-
ng/mL
2.46
1.34
2.00
0.67
6.40
47
0.38
0.39

Derived

Neutrophil-

Activating

Protein 78

(ENA-78)

Hepatocyte
ng/mL
14.18
8.41
13.00
2.80
35.00
47
3.1
2.1

Growth Factor

(HGF)

Latency-
ng/mL
10.63
3.05
11.00
5.00
19.00
47
1.1
0.55

Associated

Peptide of

Transforming

Growth Factor

beta 1 (LAP

TGF-b1)

Matrix
ng/mL
7.93
4.92
6.90
1.10
21.00
47
1.2
0.97

Metalloproteinase-1

(MMP-1)

Matrix
ng/mL
657.09
197.30
591.00
326.00
1310.00
47
119
54

Metalloproteinase-9,

total (MMP-9,

total)

Myoglobin
ng/mL
152.09
112.88
100.00
47.00
551.00
47
15
12

Plasminogen
ng/mL
196.30
56.14
191.00
88.00
316.00
47
32
29

Activator

Inhibitor 1

(PAI-1)

Platelet-
pg/mL
15631.91
4560.00
15600.00
7260.00
24600.00
47
271
327

Derived

Growth Factor

BB (PDGF-

BB)

Receptor
ng/mL
0.62
0.69
0.31
0.18
3.30
47
2.2
0.85

tyrosine-

protein kinase

erbB-3

(ErbB3)

T-Cell-
ng/mL
18.26
7.29
18.00
3.70
36.00
47
1.4
1.7

Specific

Protein

RANTES

(RANTES)

Thrombospondin-1
ng/mL
9800.64
2558.16
9540.00
3930.00
14700.00
47
876
1160

Vascular
ng/mL
35.32
6.73
36.00
22.00
46.00
47
3.9
1.0

Endothelial

Growth Factor

C (VEGF-C)

change
change

in
in

mean
median
Myriad

Normal

Positive
p-
(HIBM/
(HIBM/
RBM

Median
Min
Max
Count
value
nomral)
normal)
LDD
5xLDD

Brain-Derived
0.40
0
7
126
0.0000
34.58
50.06
0.023
0.12

Neurotrophic

Factor

(BDNF)

Creatine
0.80
0
6
125
0.0000
11.37
10.90
0.91
4.55

Kinase-MB

(CK-MB)

Epidermal
4.7
2
432
102
0.0000
23.55
53.22
3.0
15.00

Growth Factor

(EGF)

Epithelial-
0.29
0
4
126
0.0000
6.42
6.85
0.033
0.17

Derived

Neutrophil-

Activating

Protein 78

(ENA-78)

Hepatocyte
2.6
0
11
123
0.0000
4.59
5.04
0.20
1.00

Growth Factor

(HGF)

Latency-
0.99
0
5
126
0.0000
9.75
11.14
0.056
0.28

Associated

Peptide of

Transforming

Growth Factor

beta 1 (LAP

TGF-b1)

Matrix
0.95
0
10
117
0.0000
6.89
7.24
0.45
2.25

Metalloproteinase-1

(MMP-1)

Matrix
114
42
569
126
0.0000
5.51
5.18
1.2
6.00

Metalloproteinase-9,

total (MMP-9,

total)

Myoglobin
11
4
89
126
0.0000
10.16
8.81
2.0
10.00

Plasminogen
23
4
166
126
0.0000
6.12
8.16
1.1
5.50

Activator

Inhibitor 1

(PAI-1)

Platelet-
167
42
2390
79
0.0000
57.62
93.41
127
635.00

Derived

Growth Factor

BB (PDGF-

BB)

Receptor
2.3
0
4
126
0.0000
0.29
0.14
0.030
0.15

tyrosine-

protein kinase

erbB-3

(ErbB3)

T-Cell-
1.1
0
14
126
0.0000
12.69
17.14
0.033
0.17

Specific

Protein

RANTES

(RANTES)

Thrombospondin-1
584
44
8460
125
0.0000
11.19
16.34
57
285.00

Vascular
3.8
2
9
126
0.0000
9.07
9.60
1.3
6.50

Endothelial

Growth Factor

C (VEGF-C)

Analytes were excluded from analysis if <50% of the HIBM patient samples were below least detectable dose (LDD). Within each dosage group, individual values with >5× difference to the mean of the dosage group were considered as outliers and thus removed from analysis.

Out of the 32 analytes, the levels of eleven (11) analytes showed signs of improvement at week 24 (see Table 14), mainly moving toward normal levels (normalization) as defined by the 126 normal controls used in the study. The 6 g/day group also showed a more favorable change in the levels of these analytes compared to the placebo group at week 24.

The analytes (biomarkers) with improvement in levels are summarized in Table 13. Upon treatment, levels of these analytes show an overall trend of either stabilization or normalization. As used herein, the term normalization indicates that the level of a given biomarker after drug treatment moves toward a normal level, and the change in is larger compared to that of a placebo treatment. As used herein, the term stabilization indicates that the level of a given biomarker after drug treatment may still move away from normal level, but at a slower rate compared to that of a placebo treatment. For example, LAP TGF-b1 is an example of biomarker showing trend of normalization when the baseline level in HIBM patients is higher than normal controls, see FIG. 2. NCAM is another example of biomarker showing trend of normalization when the baseline level in HIBM patients is lower than normal controls, see FIG. 3.

TABLE 14

Summary of biomarkers showing improvement in levels at week 24

fold change

between
average
average
average

normal
baseline and
Δweek 0-
Δweek 0-
Δweek 0-

Analytes
level
normals
24 (placebo)
24 (3 g/day)
24 (6 g/day)
overall trend

Chromogranin-A
79
4.4
31.67
64.00
5.83
stabilization

(CgA) (ng/mL)

Epithelial-Derived
0.38
6.4
−0.41
−0.26
−0.42
normalization

Neutrophil-Activating

Protein 78 (ENA-78)

(ng/mL)

Lactoylglutathione
3.4
5
−3.53
0.25
−8.27
normalization

lyase (LGL) (ng/mL)

Latency-Associated
1.1
9.8
−0.10
−0.44
−1.00
normalization

Peptide of

Transforming Growth

Factor beta 1 (LAP

TGF-b1) (ng/mL)

Macrophage Colony-
0.100
5
0.08
0.10
0.02
stabilization

Stimulating Factor 1

(M-CSF) (ng/mL)

Matrix
119
5.5
534.93
449.18
444.64
stabilization

Metalloproteinase-9,

total (MMP-9, total)

(ng/mL)

Myoglobin (ng/mL)
15
10.2
−21.64
−48.88
−38.00
normalization

Neuronal Cell
1.6
−1.9
−0.17
−0.07
0.02
normalization

Adhesion Molecule

(Nr-CAM) (ng/mL)

Plasminogen
32
6.1
4.14
0.82
−0.36
normalization

Activator Inhibitor 1

(PAI-1) (ng/mL)

Receptor tyrosine-
2.2
−3.5
−0.39
−0.28
−0.16
stabilization

protein kinase erbB-3

(ErbB3) (ng/mL)

Vascular Endothelial
3.9
9.1
6.57
1.35
6.43
stabilization

Growth Factor C

(VEGF-C) (ng/mL)

normalization: level moved toward normal level and the change in 6 g/day group was larger compared to placebo

stabilization: level might still move away from normal level but at a slower rate in 6 g/day group compared to placebo

FIG. 4 shows Delta (A) of serum biomarkers between week 0 and week 24 in each dosage group (placebo, 3 g/day, and 6 g/day). Analytes with baseline levels in HIBM patients higher than normal controls include PAI-1, LGL, myoglobin, ENA-78, TGfb1, M-CSF1, MMP-9, and CgA. For these biomarkers, the slope of delta displays a downward trend from placebo to higher dosage group (x-axis), indicating a more favorable change in the higher dosage group moving toward normal levels. In contrast, analytes with baseline levels lower than normal controls include NCAM and ErbB3, the slope of delta always displayed a upward trend.

Example 3
Phase 2 Biomarkers

Forty-seven (47) HIBM patient serum samples from phase 2 study were tested using the Human Discovery MAP250+ quantitative immunoassay system developed by Myriad RBM.

The results of analysis are shown in FIGS. 1-6 (phase 1) of 61/779,929 filed on Mar. 13, 2013, and FIGS. 8-13 (phase 2) of 61/779,929 filed on Mar. 13, 2013, which is herein incorporated by reference in its entirety for all purposes. The same criteria used to narrow down the list of potential biomarkers of interest in the phase 1 study were applied to the phase 2 patients.

A summary of the comparison between the results for phase 1 and phase 2 patients using the same criteria to narrow down the potential list of biomarkers of interest is shown in Table 14 below.

TABLE 15

Phase 1 and Phase 2 Comparison

same markers

identified

phase 2
phase 1
in both

patients
patients
phase 1 and 2

n = 47
n = 18
patients

# of markers tested
243
258
238*

1st cutoff: markers had a p-value
140
134
89

of <0.05

2nd cutoff: markers with >4 folds
31
34
20

change in mean or median

3rd cutoff: markers in which >50%
16
18
12

of HIBM and normal samples above

least detectable dose (LDD), or >5

folds change in median.

4th cutoff: markers with >5 folds
15
11
9

change in median. Also >80% of

HIBM samples were above 5x LDD.

*20 markers discontinued and 5 additional markers offered for the phase 2 patients study

	Number	Date	Country
	61704373	Sep 2012	US
	61779929	Mar 2013	US

BIOMARKERS FOR ASSESSING TREATMENT OF SIALIC ACID DEFICIENCY DISEASES AND CONDITIONS

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Provisional Applications (2)