Patent Application
20130040882
The invention relates generally to the in vivo testing of the efficacy of a compound or composition, and particularly to the testing and biologically functionalizing of classical small molecules, natural products, genes, peptides and proteins by activity in vivo.
The invention further relates to medical uses of fibroblast growth factor 23 (FGF-23), FGF-23 fragments, FGF-23 C-terminal polypeptides, FGF-23 homologs and/or FGF-23 variants, in particular for the manufacture of a medicament for the treatment of diseases associated with deregulated angiogenesis or cell proliferative disorders.
Pharmaceutical companies are interested in evaluating and understanding the function and regulation of newly discovered genes and gene products (proteins), especially newly discovered genes and proteins, which could help in the understanding of the mechanisms linked to diseases or compounds action. In addition, the genes and gene products can become potential drugs or biomarkers. Grenet O, Pharmacogenomics J. 1(1):11-2 (2001).
However, a gene sequence alone does not provide information about the actual function of the protein in the cell or organism physiology. In addition, while the genome has a relatively well defined number of genes, there is no known limit to the possible number of protein variants. The potential number of proteins encoded by these genes is estimated to be from two to at least one-hundred times higher than the number of genes, since it has recently been found that proteins can also be produced by splicing at the protein level not just at the RNA level.
The current process of drug discovery proceeds from single target to single drug product. The current process is a long process, frequently with late attrition for lack of efficacy, wrong design or false indication.
Thus, there is a need in the art for a more efficient method for discovering and identifying drug candidates, gene targets and biomarkers.
The invention provides a discovery process for biologically functionalizing peptides, proteins, genes, small molecules and natural products using organism-wide gene expression profiling. The discovery process of the invention proceeds from single lead or drug to multiple targets and indications (as indicated by an impact on any target in the cascade chain of a pathway), and multiple drug products, thus providing rapid guidance to a correct human proof-of-concept.
The discovery process of the invention begins with administration of test substances to animals, followed by screening of the resulting gene expression in many organs obtained from the test animal. The invention can be used to biologically functionalize the entire genome of any organism where micoarray chips are available. The invention is not restricted to the type of compound to be functionalized. Small molecules, proteins, natural products, cDNA (for functionalizing any gene of interest), etc. are all susceptible to the strategy of the invention.
Since the discovery process of the invention is based on a non-preconceived hypothesis and whole organism multi-organ analysis, polypeptides can be selected for testing in the absence of any biological selection criteria other than peptide sequence. The resulting organism-wide pattern of the gene expression changes in the transcriptome provides an overview of the activities at the molecular and organism-wide levels. Accordingly, the unbiased approach of the invention regarding the administration of a compound can provide information about the physiological relationships throughout the entire body that are caused by the compound's administration.
The discovery process of the invention then integrates in vivo profiling with internal and external genomic databases to elucidate the function of unknown proteins, typically within few months. The unbiased approach of the invention in regards to the administration of a compound advantageously provides genomic signatures from multiple organs. The resulting data can be analyzed either by using tools that are known to those of skill in the art or by using tools that compare the compound signatures produced by the administration of the compound among the different organs. This multi-organ analysis is in contrast to standard approaches, which, since they do not use an unbiased approach of compound administration, do not result in a multi-organ identification of the function of the compound. Instead the standard approaches provide analysis on a case-by-case basis, which can make cross-experimental comparisons difficult. In contrast to the standard approaches, the identification of the function of the compound using the method of the invention allows for the identification of the function of the compound in many metabolic and regulatory pathways. In addition, the identification of the function of the compound using the method of the invention advantageously results in an understanding of the stability of the active compound in the body, a property of the administered compound which would otherwise not be predictable a priori using standard approaches.
The identification of the function of the compound using the method of the invention can be multi-step, as one step or part of the identification leads to another step or part of the identification, to provide a more complete understanding of the administered compound's activity in vivo. For example, the identification of compound function in one organ (such as the spleen) can lead to an understanding of the compound function in other organs. By contrast, the standard approaches, which rely on immediate access to tests on a limited number of organs, depend on anecdotal evidence from other experiments to further steps in the identification of compound function.
The invention is suitable for several stages of drug discovery, identifying both drug targets and biomarkers. The discovery process of the invention advantageously delivers an increased number of validated drug candidates and identified drug targets and biomarkers along with a savings in time, resources and animals. The discovery process of the invention advantageously integrates into one process standard exploratory tools with new genomic approaches. The discovery process of the invention can also be used for the reprofiling of safe compounds stopped after the initial stages of drug approval (e.g., Phase I) for re-indication. The invention can be used for adjusting the best fit for combination therapies, by optimally matching of gene expression signatures between two compounds, cancelling of side effects and the potentiation of efficacy. The discovery process of the invention can be used for the profiling of the more advanced development portfolio to guide the later stages of drug approval process (e.g., Phase II and Phase III).
In several embodiments, the process can be used to analyze tissues or body fluids (such as coronary heart disease, breast cancer and another indication; each compared to healthy controls). Plasma proteins that are differentially expressed between normal subjects and coronary artery diseased patients, with known or unknown function, are analyzed for potential target identification/validation, and biomarker identification.
In one embodiment, the discovery process of the invention begins with an in vivo screening of proteins, peptides and reference compounds in mice. Based on the results of the mice screening, an in vivo verification of selected proteins, peptides or reference compounds is then conducted in non-human primates or animal models of human pathology or disease. The comparison of the resultant information to a profile of reference drugs, with well characterized pharmacological activity, facilitates biological interpretation of the profiles of unknown compounds. In a particular embodiment, the selection rate for the proteins, peptides or reference compounds is ˜20%.
In one embodiment the discovery process of the invention combines in one process: (a) pre-screening in mice; (b) verification of selected proteins/peptides/reference compounds in monkeys; (c) large number of the analyzed tissues (up to 25 in mice, up to 120 in monkeys); (d) homogeneity of the tissue sample (e) high quality mRNA; (f) a genome-wide approach with hybridization chips; (g) powerful bioinformation tools for clustering and statistics; (h) the possibility of cross-assay meta-analysis; and (i) localization at the cellular level of the affected genes or pathways by in situ hybridization.
By use of the discovery process of the invention, it has now surprisingly been found that polypeptides relating to FGF-23 affect key genes controlling cellular differentiation and proliferation, as well as angiogenesis.
Fibroblast growth factors (FGFs) make up a large family of polypeptide growth factors that are found in organisms ranging from nematodes to humans. During embryonic development, FGFs have diverse roles in regulating cell proliferation, migration and differentiation. In the adult organism, FGFs are homeostatic factors and function in tissue repair and response to injury. Inappropriate expression of some FGFs can contribute to the pathogenesis of cancer.
Mouse FGF-23 has been identified by homology search in the GenBank Nucleotide Sequence Database with amino acid sequence of mouse FGF-15. Mouse FGF-23 and human FGF-23 are highly identical (−72% amino acid identity). Both, mouse and human FGF-23, cDNAs encode a protein of 251 amino acids, having a hydrophobic amino terminus (−24 amino acids) typical for secreted proteins, and a unique C-terminus having no homology to other FGF family members. In the mouse, FGF-23 mRNA is expressed in the brain, preferentially in the ventrolateral thalamic nucleus, and in the thymus at low levels.
Overexpression of FGF-23 or expression of mutated FGF-23 has been demonstrated to be associated with several pathological findings:
Recombinant FGF-23 induces hypophosphatemia in vivo as a result of urinary phosphate wasting (Shimada T., et al., Proc. Natl. Acad. Sci. U.S.A. 98: 6500-6505 (2001)).
FGF-23 overexpression has been observed in tumors that are responsible for oncogenic osteomalacia (OOM) (White K. E., et al., J. Clin. Endocrinol. Metab. 86: 497-500 (2001)).
Autosomal dominant hypophosphatemic rickets (ADHR) has been shown to be associated with mutations of FGF-23 within the 176-RXXXR-179 cleavage site, preventing degradation of FGF-23 (The ADHR Consortium, Nat. Genet. 26: 345-348 (2000)).
While OOM and ADHR have been demonstrated to be associated with FGF-23, a further disorder, X-linked hypophosphatemia (XLH), which is phenotypically similar to OOM and ADHR, has been shown to result from mutations in the PHEX gene. PHEX encodes a membrane-bound endopeptidase (The HYP Consortium, Nat. Genet. 11: 130-136 (1995)) and it is hypothesized that FGF-23 is a PHEX substrate, while FGF-23 ADHR mutant (FGF-23(R179Q) being undegradeable by PHEX.
Each of the above described syndromes is characterized by hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum calcitriol concentrations, normal serum concentrations of calcium and parathyroid hormone, and defective skeletal mineralization (Quarles L. D. and Drezner M. K., J. Clin. Endocrinol. Metab. 86: 494-496 (2001)).
Because both the overproduction and missense mutations of FGF-23 cause hypophosphatemia with renal phosphate wasting, it is concluded that FGF-23 is at least one of the causative factors of OOM and is an important regulator of phosphate and bone metabolism (Shimada T., Proc. Natl. Acad. Sci. USA 98: 6500-6505 (2001)). However, the molecular targets of FGF-23 or of FGF-23 proteolytic cleavage products are so far unknown, as is the mechanism of how FGF-23 or FGF-23-derived proteins or peptides cause renal and skeletal abnormalities (Quarles L. D., Am. J. Physiol. Endocrinol. Metab. 285: E1-9 (2003)).
The present invention thus relates the use of a polypeptide for the manufacture of a medicament for use in the treatment of a disease associated with deregulated angiogenesis, wherein the polypeptide is selected from the groups consisting of (a) fibroblast growth factor 23 (FGF-23) (SEQ. ID No: 1) or a fragment of FGF-23; (b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); or (c) a bioactive variant of any one of the polypeptides of (a) or (b).
In a further aspect, the present invention relates to the use of a polypeptide as defined above for the manufacture of a medicament for use in the treatment of a cell proliferative disorder.
In a further aspect, the present invention relates to a method for the treatment of a disease associated with deregulated angiogenesis or of a cell proliferative disorder comprising administering an effective amount of a polypeptide as defined above to a mammal including a human suffering from the disease or disorder.
In another aspect, the present invention relates to a pharmaceutical composition for use in a disease associated with deregulated angiogenesis or a cell proliferative disorder comprising a polypeptide as defined above and a pharmaceutically-acceptable carrier.
Introduction and overview. The classical discovery process in the pharmaceutical industry is based on targets (enzymes, receptors, cellular assays, animal and disease models, etc.). Chemicals or biological products are tested, in a high-throughput mode, on a battery of pre-selected different targets. The weakness of the classical approach are the “artificially disconnected” in vitro target models compared to the tightly interconnected and interdependent relationship of the different targets in a whole organism and the fact that biological activity on all non selected targets is missed.
By contrast, the invention is a “non pre-conceived hypothesis” discovery process to rapidly identify and analyze the biological activity of new products in the whole organism, multi-organs and whole transcriptome. All physiological interactions between the different organs or tissues are present and any cellular pathway or any potential targets could potentially be analyzed in a non artificial system.
The drug discovery process of the invention advantageously increases the capabilities in the field of proteomics and functionalization. Proteomics involves the systematic separation, identification and characterization of the proteins present in a sample of tissue, or in a biological fluid, at a given time. All biological processes, including diseases and responses to drugs, induce changes in proteins, and the global protein profile (the “proteome”) varies during the development of an organism, maturation of cell types or tissues, and progression or treatment of disease. Each cell type may express different patterns of proteins at different times. Each protein in turn may be modified chemically in an equally diverse number of ways to serve different cellular functions. As proteins derived from the same gene can be largely identical, and might differ only in small but functionally relevant details, protein identification tools not only identify a large number of proteins but also differentiate between close relatives.
The classical proteomics approach combines high resolution two-dimensional gel electrophoresis (2-DGE) with imaging software to quantitatively and qualitatively screen for proteins that differ in abundance, molecular weight (Mr) or charge between the gels. These protein differences can then be identified with high speed and sensitivity by using a combination of “state-of-the-art” mass spectrometry (MS) approaches and robotics, alongside sensitive bioinformatics search tools.
RNA transcripts represent the intermediate form between the DNA and the proteins that are among the most active molecules involved in the cellular functions. The total content of RNA is called the “transcriptome”. The high-density DNA chip technology gives potential access to the analysis of all the transcripts produced by a cell population or tissue at any determined time point. Genome-scale RNA expression analysis can thus provide new insights into the cellular events induced upon administration of an animal with peptides or other chemicals. This provides a broad view of the metabolic, signalling, regulatory or other biochemical pathways in the animal being tested. The analysis of the induced perturbations in cellular transcription gives a detailed molecular description of the activity of the administered compound.
An analysis of a transcriptome has become an approachable reality with the implementation of high throughput RNA quantification system. The high-density microarrays allow collecting thousands of information points of a transcriptome at once, reaching the order of magnitude of the probable number of genes expressed and producing a broad and detailed view of the cellular events.
As the changes of the different functions inside a cell are tightly interconnected, the changes in different organs inside an organism are linked. Applying gene profiling to different organs submitted to the same treatment gives a complete overview of the effects and modifications of the physiological status. The identification of common changes in organs with originally very different transcriptomes facilitates the elimination of the experimental noise. The presence or absence of identical signals can indicate if the treatment has a pleiotropic effect or is affecting a target organ. If a compound is targeting a primary organ, the other organs will reflect the functional modifications of the first organ impacted. This type of information can be collected in correlation to the pharmacological effect or to the potential toxic effects. The organism-wide pattern of expression changes can also provide useful information on the pharmacodynamic of the compound, precisely delineating the range of organs affected.
The accumulation of information in different organs not only helps to elucidate the precise mode of action but also provides a complete reconstruction of the compound-induced modifications at the organism scale.
Administration of compounds. Administration of the protein or other drug compound triggers multiple cascades of intracellular signalling events, involving complex networks (pathways) and relying on protein modifications such as phosphorylation, glycosylation, etc. These events eventually lead to modifications of gene expression levels. Administration of an active compound therefore leads to multiple and interdependent changes in the composition of the transcriptome.
In one embodiment, the test animal is a vertebrate. In a particular embodiment, the vertebrate is a mammal. In a more particular embodiment, the mammal is a primate, such as a cynomolgus monkey or a human. As used herein, the administration of an agent or drug to a subject or patient includes self-administration and the administration by another.
In more particular embodiments, natural or synthetic substances of biological or non-biological sources, e.g. amino acids, peptides, proteins, nucleotides, cDNAs, chemicals, can be administered to animals, e.g. mice (Mus musculus), rat (Rattus norvegicus), monkey (Macaca fascicularis), by methods known in the art, e.g. by injection, inhalation, or oral administration. Administration of those substances can be adjusted in terms of time of exposure and dosage, and combinations thereof. The “treatment group” of animals should receive a substance or a combination of substances in a vehicle compound suitable for administration of the substance or the combination of substances, while the “control” (or “baseline”) group should receive the vehicle compound only. During the treatment period biological specimen such as tissue pieces (e.g. obtained by biopsy), or body fluids, such as blood, urine, or saliva, can be sampled. At the end of the treatment time all animals of all groups can be sacrificed and biological specimen such as whole organs or pieces thereof can be sampled. All sampled specimen can be stored as known in the art for further analysis that include, but are not limited to, RT-PCR, Northern blotting, in-situ hybridization, gene expression profiling with microarrays.
As used herein, “direct administration” is the injecting, oral gavage, feeding or other administration of a compound, such as a protein, into animals. After some time, i.e. hours, days or weeks, organs and tissues are collected from the animals and the gene-expression profiles determined. This procedure is commonly used in pharmacotoxigenomics, pharmacogenomics and the like.
In one embodiment, the invention begins with differentially expressed proteins in plasma between normal subjects and coronary artery diseased patients with regard to the identification and validation of potential targets and the identification of biomarkers.
The drug discovery process of the invention is particularly amenable to the analysis of the smaller proteins of a proteome (ranging from 0.5 to 20 kDa) escaping the classical detection methods. Small molecular weight proteins can be readily synthesized by commercial methods (e.g., Microprot™ method, GeneProt, Geneva, Switzerland). Chemically-synthesized proteins can be rapidly produced and do not contain biological contaminants.
For mice, a minimal amount of the compound to be functionalized (only ca. 5 mg) is used.
As used herein, “indirect administration” is the injecting of a gene that codes for that protein (as a cDNA plasmid) and then doing the gene expression profiling. In one embodiment, the technology is the use of ‘naked’ DNA (a cDNA expression plasmid) injected into mice (or other animals). This technique is widely published for either DNA immunization (Kim J-M et al., Gene Ther. 10(15): 1216-24 (August 2003)) and delivery of genes for therapeutic purposes (Aliño S F et al., Gene Ther. 10(19):1672-9 (September 2003)). Among a number of techniques for gene transfer in vivo, intravenous injection or the direct injection of plasmid DNA into muscle are simple, inexpensive, and safe. Kim J-M et al., Gene Ther. 10(15): 1216-24 (August 2003). The important efficacy of nonviral genomic DNA opens a new avenue in the safety applications of human gene therapy. Aliño S F et al., Gene Ther. 10(19):1672-9 (September 2003).
Administration of naked DNA can be by methods known to those of skill in the art, see, U.S. Pat. Nos. 6,165,754; 6,309,370; 6,566,342; 6,620,617 and 6,651,655, and references cited therein.
Gene expression profiles. After a period of time (e.g., two weeks) of protein administration, the treated animals are necropsied. Selected tissues (e.g., 25 tissues for mice/120 tissues for monkeys) are dissected and rapidly snap-frozen for genomics analysis. Organ samples (e.g., fifty organs samples for monkeys) can be isolated for histopathological examinations and for gene expression localizations, such as by in situ hybridization. Initial studies have shown that for mice, 3-10 tissues out of twenty-five sampled tissues are generally sufficient to characterize a compound by gene expression and hybridization; for monkeys, twenty tissues out of 120 sampled tissues are generally sufficient.
In more particular embodiments, the methods of detecting the level of expression of mRNA are well-known in the art and include, but are not limited to, reverse transcription PCR, real time quantitative PCR, Northern blotting and other hybridization methods. A particularly useful method for detecting the level of mRNA transcripts obtained from a plurality of genes involves hybridization of labelled mRNA to an ordered array of oligonucleotides. Such a method allows the level of transcription of a plurality of these genes to be determined simultaneously to generate gene expression profiles or patterns.
As used herein, a gene expression profile is diagnostic when the increased or decreased gene expression is an increase or decrease (e.g., at least a 1.2-fold difference) over the baseline gene expression following administration of a compound. As used herein, a gene expression pattern is “higher than normal” when the gene expression (e.g., in a sample from a treated subject) shows a 1.2-fold difference (i.e., higher) in the level of expression compared to the baseline samples. A gene expression pattern is “lower than normal” when the gene expression (e.g., in a sample from a treated subject) shows a 1.2-fold difference (i.e., lower) in the level of expression compared to the baseline samples. In other embodiments, a 1.5-fold change may be used as the criteria.
Techniques for the detection of gene expression of the genes described by this invention include, but are not limited to northern blots, RT-PCT, real time PCR, primer extension, RNase protection, RNA expression profiling and related techniques. Techniques for the detection of gene expression by detection of the protein products encoded by the genes described by this invention include, but are not limited to, antibodies recognizing the protein products, western blots, immunofluorescence, immunoprecipitation, ELISAs and related techniques. These techniques are well known to those of skill in the art. Sambrook J et al., Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Press, Cold Spring Harbor, 2000). In one embodiment, the technique for detecting gene expression includes the use of a gene chip. The construction and use of gene chips are well known in the art. See, U.S. Pat. Nos. 5,202,231; 5,445,934; 5,525,464; 5,695,940; 5,744,305; 5,795,716 and 5,800,992. See also, Johnston, M. Curr Biol 8:R171-174 (1998); Iyer V R et al., Science 283:83-87 (1999) and Elias P, “New human genome ‘chip’ is a revolution in the offing” Los Angeles Daily News (Oct. 3, 2003).
Gene expression profiles can be generated using e.g. the Affymetrix microarray technology. Briefly, total or, preferably, polyA+-RNA from a biological sample is extracted using standard procedures known in the art, e.g. the RNeasy® kit (Qiagen, Md., USA). In a following step, double stranded cDNA is prepared in a process termed “reverse transcription (RT)” which is known in the art, using e.g. the “SuperScript Double-Stranded cDNA Synthesis Kit” (Invitrogen, CA, USA). In a subsequent step, termed “in-vitro transcription”, double stranded cDNA obtained in a previous step is labelled with a fluorochrome by methods known in the art, using e.g. the ENZO Labeling Kit (ENZO, NY, USA). Labelled RNA is hybridized to oligonucleotide microarrays. These are known in the art and consist of a surface to which probes that correspond in sequence to gene products (e.g. mRNAs, polypeptides, fragments thereof etc.) can be specifically hybridized or bound to a known position. Processing of the microarrays, including e.g. washing, staining, scanning, is performed according to the manufacturer's instructions. Hybridization intensity data detected by the scanner are automatically acquired and processed by analytical software components, e.g. the GENECHIP® software (Affymetrix, Calif., USA). Raw data is normalized to expression levels using a target intensity of 200.
Two elements of value in expression profiling are the quality and homogeneity of the tissue samples and the mRNA quality. For this purpose, the location of tissues to be sampled and each sample can be carefully dissected from the other surrounding tissues using a binocular microscope.
The samples are then transferred to a molecular biology laboratory for RNA extraction. The protocol for RNA extraction can be partially automated thus increasing the reproducibility and speed of this step. The extracted RNA can be stored for long periods of time in a frozen state and kept as an archive.
An aliquot of the extracted RNA is reverse transcribed to obtain a cDNA. In a second step, cDNA is transcribed in the presence of a fluorescent label to obtain cRNA. The composition of the cRNA obtained is identical to the original composition of the RNA in the samples, but each molecule now carries a fluorescent marker. The labelled mixture of cRNA is used for the hybridization process, e.g. using GeneChip® assays (Affymetrix, Santa Clara, Calif. USA). The raw data (obtained after laser-scanning of the chip) are processed by a specific algorithm condensing for each gene all available information in a unique value. This value called average difference represents the level of expression of the gene.
The information can be further refined by the use of complementary techniques. In situ hybridization, for example, can indicate precisely which cell type inside an organ is specifically expressing a given gene. This technique based on the detection of RNA is independent of the availability of an antibody. Quantitative PCR may also be used to confirm expression levels of particular genes of interest.
Analysis.
Mathematical and statistical processing of the data (clustering) help to reduce the complexity and the size of the data sets. Different types of clustering can be used to separate the different genes according to their behavioural similarity across the different conditions and to establish links between genes that may be related to the same biological phenomenon. Data processing also includes statistical tests to separate significant variations from experimental noise. However, the stringency of the various filtering steps must be modulated to integrate the biological nature of the data.
The list of different affected genes is then compared to the information collected in the scientific literature. The synthesis of the available knowledge related to the different genes, points to one or several signalling, metabolic or other biochemical pathways or to known modifications. Once a coherent picture has been reconstructed, the profiles may be associated with potential indications. The discrimination between the different hypotheses follows a process closely related to differential diagnosis.
During the analysis, a constant comparison between the expression data and the current knowledge on cell signalling and regulation is established. Such a permanent bridge provides an efficient way to refine the existing models, particularly in the field of intra- and inter-cellular signalling. The interdependence of gene expression changes is assessed in different organs and under different stimuli. New players in the pathways can be identified and the link between the already described players can be refined. Even if only a part of the cellular regulation depends on the RNA expression changes, the accumulation of expression data can help to build new and more accurate model of the cell functions. The information collected could help to identify the critical elements of the pathways to be used as target or biomarker.
Some of the expression profiles can be easily matched with existing information harvested from the general scientific knowledge. Linking this information with a potential indication or a potential side effect is then straightforward. Some combinations of expression changes are more difficult to translate into pharmacological information. In such cases, matching of the RNA expression profile of an unknown compound to the profile of a reference drug or disease may facilitate the interpretation. It may then not be necessary to reconstruct the entire cellular modifications to find a potential indication. The reference drugs and disease profiling will also help to build the critical mass of information into the database.
In more particular statistical analysis embodiments, microarray datasets can be analyzed by the use of analytical software components, such as GeneSpringe (Silicon Genetics, Calif., USA). Microarray datasets consist in part of probe set identifiers that refer to an oligonucleotide sequence that is bound to the glass slide and to which a labelled cDNA (see above) with complementary sequence binds if it is present in the tissue or body fluid sample. The scanned intensity of the signal that is detected and converted into numeric values by a software, for example MAS5 (Affymetrix, Calif., USA), is an indirect measure for the amount, or expression level of the cDNA present in the biological samples under investigation. The entity of gene expression levels as indicated by signal intensity values for all probe sets in a microarray dataset of a biological sample can be referred to as expression profile of that sample. In each microarray dataset, signal intensity values, cDNA or gene annotations, as well as quality parameters that can be created by software, for example MAS5 (Affymetrix, Calif., USA), are informational results associated with the probe set ID.
In the cDNA microarray system, expressions of genes from the experimental cells of interest are measured relative to the expressions of the same genes in a fixed reference or control cell type. To identify statistically relevant effects of a substance on the expression profile of samples or tissue or body fluid under investigation, probe sets can be filtered based on the associated values given by the software used to create the values, for example MAS5 (Affymetrix, Calif., USA). Filters can be based on quality parameters, expression level, changes of expression levels in the samples from treated versus control specimen, as well as significance. The resulting list of probe sets refers to such genes that experience a significant change in their expression level as a direct or indirect result of the treatment of the biological samples they are derived from.
The interpretation of such gene lists with regards to effects of a substance on biological systems and pathways is subject to the investigators knowledge and experience. Application of analytical software components such as GeneSifter® (VizXLabs, Seattle, Wash., USA) assists in the interpretation of such gene lists.
Moreover, we have developed software for a multiorgan analysis of the data generated by the method of the invention, which compares the compound signatures produced by the administration of the compound among the different organs.
New Uses Identified by the Drug Discovery Process of the Invention.
The present invention also provides for the use of a polypeptide for the manufacture of a medicament for use in the treatment of a disease associated with deregulated angiogenesis, wherein the polypeptide is selected from the groups consisting of (a) fibroblast growth factor 23 (FGF-23) (SEQ. ID No: 1) or a fragment of FGF-23; (b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); or (c) a bioactive variant of any one of the polypeptides of (a) or (b).
According to another aspect, the present invention provides for the use of a polypeptide as defined above for the manufacture of a medicament for use in the treatment of a cell proliferative disorder.
The term “polypeptide” as used herein, refers to a protein, peptide, oligopeptide or synthetic oligopeptide. These terms are intended to be used interchangeably. Any one of said terms refers to a chain of two or more amino acids which are linked together with peptide or amide bonds, regardless of post-translational modification such as glycosylation or phosphorylation. The polypeptides may also comprise more than one subunit, where each subunit is encoded by a separate DNA sequence.
The term “bioactive”, as used herein, refers to a molecule that elicits or affects a biological event. Such biological event may for example be related to a disease associated with deregulated angiogenesis or to a cell proliferative disorder.
A “bioactive polypeptide” of the invention includes FGF-23, fragments of FGF-23 such as fragments derived from the C-terminus of FGF-23. Also included are homologs which have an amino acid sequence having a percentage of identity of at least 50% to FGF-23 or fragments thereof and variants of FGF-23 or of FGF-23 fragments. The polypeptide according to the invention may comprise FGF-23 having the amino acid sequence of SEQ ID NO: 1. A fragment of FGF-23 may comprise at least 10 amino acids, preferably at least 15, 20, 25 or 30 amino acids. More preferably a fragment of FGF-23 may comprise at least 50, 60, or 70 amino acids. Most preferably a fragment of FGF-23 comprises 75 amino acids. Alternatively, a fragment of FGF-23 may comprise at least 80 or 100 amino acids, and most preferred at least 120 or 150 amino acids. In particular, the fragment may comprise at least 180 amino acids, such as e.g. 200 amino acids.
Such polypeptide may also be a proteolytic cleavage product of FGF-23 generated by proteases such as a membrane-bound endopeptidase including PHEX. A polypeptide according to the invention may comprise a C-terminal fragment of FGF-23. Such C-terminal fragment may comprise at least 15 amino acids of the C-terminus of FGF-23, preferably at least 25, at least 35 or 45, more preferably at least 55 or at least 65, most preferred at least 70, such as e.g. 75 amino acids. The at least 15 amino acids may comprise the most C-terminal at least 15 amino acids, it may also comprise the at least 15 amino acids within the C-terminal part of FGF-23. The polypeptide may comprise the at least 75 most C-terminal amino acids of FGF-23 and it may have the amino acid sequence of SEQ ID NO: 2, designated FGF-23 C-terminal polypeptide (FGF23CTP).
The polypeptide may also have an amino acid sequence having a percentage of identity of at least 50%, preferably at least 60%, more preferred at least 70% or 80%, and most preferably at least 90% such as 95%, 97%, or 99% identity with the amino acid sequence of any one of the aforementioned polypeptides.
The bioactive polypeptides of the present invention as described above may also be referred to as FGF-23, FGF-23 protein or polypeptide, FGF-23 derived or related polypeptides, FGF-23 C-terminal derived or related polypeptides, or FGF-23 C-terminal polypeptides.
Amino acid residues are referred to herein by their standard single-letter or three-letter notations: A (Ala) alanine; C (Cys) cysteine; D (Asp) aspartic acid; E (Glu) glutamic acid; F (Phe) phenylalanine; G (Gly) glycine; H(H is) histidine; I (Ile) isoleucine; K (Lys) lysine; L (Leu) leucine; M (Met) methionine; N (Asn) asparagine; P (Pro) proline; Q (Gin) glutamine; R (Arg) arginine; S (Ser) serine; T (Thr) threonine; V (Val) valine; W (Trp) tryptophan; Y (Tyr) tyrosine.
The term “percentage (%) of identity”, or like term, used in respect of the comparison of a reference sequence and another sequence (i.e. a “candidate” sequence), means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a number of subunit positions equivalent to the indicated percentage, the subunits being nucleotides for polynucleotide comparisons or amino acids for polypeptide comparisons. As used herein, an “optimal alignment” of sequences being compared is one that maximizes matches between subunits and minimizes the number of gaps employed in constructing an alignment. Percent identities may be determined with commercially available implementations of algorithms described by Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)(“GAP” program of Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, Wis.). Other software packages in the art for constructing alignments and calculating percentage identity or other measures of similarity include the “BestFit” program, based on the algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981) (Wisconsin Sequence Analysis Packge, Genetics Computer Group, Madison, Wis.). The percentage of identity may also be generated by WU-BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). WU-BLAST-2 used several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues in the aligned region. For example, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to five percent of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to five percent of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence of in one or more contiguous groups with in the references sequence. It is understood that in making comparisons with reference sequences of the invention that candidate sequence may be a component or segment of a larger polypeptide or polynucleotide and that such comparisons for the purpose computing percentage identity is to be carried out with respect to the relevant component or segment.
A polypeptide of the invention also includes a polypeptide fragment of a polypeptide of the invention. Such polypeptide fragment is meant to be a polypeptide having an amino acid sequence that entirely is the same in part, but not in all, of the amino acid sequence of a polypeptide of the invention. Such polypeptide fragment may be “free-standing,” or may be part of a larger polypeptide of which such polypeptide fragment forms a part or region, most preferably as a single continuous region. Preferably such polypeptide or polypeptide fragment retains the biological activity of the corresponding polypeptide of the invention.
The invention also includes functionally preserved variants of the polypeptides or polypeptide fragments described herein. Such variants may be made using methods standard in the art, for example, by conservative amino acid substitutions. Typically such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5 to 10, 1 to 5, or 2 amino acids are substituted, deleted or added, in any combination.
In various other embodiments, the polypeptide (fragment) or polypeptide variant may be linear or branched, it may comprise modified amino acids, it may be interrupted by non-amino acids, and/or it may be assembled into a complex of more than one polypeptide chain. As is well understood in the art, a polypeptide may be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. In some embodiments, polypeptides or polypeptide fragments contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
A polypeptide or a polypeptide fragment of the invention includes isolated naturally occurring polypeptides. Preferably, such a naturally occurring polypeptide has a frequency in a selected population of at least five percent, and most preferably, of at least ten percent. The selected population may be any recognized population of study in the field of population genetics. Preferably, the selected population is Caucasian, Negroid, or Asian. More preferably, the selected population is French, German, English, Spanish, Swiss, Japanese, Chinese, Korean, Singaporean of Chinese ancestry, Icelandic, North American, Israeli, Arab, Turkish, Greek, Italian, Polish, Pacific Islander, or Indian.
A polypeptide (fragment) of the invention may also include recombinantly produced polypeptides, synthetically produced polypeptides and a combination of such polypeptides of the invention, and fragments thereof. Means for preparing such polypeptides are well understood in the art. For instance, a polynucleotide fragment or a polypeptide of the invention can be isolated from body fluids including, but not limited to, serum, urine, and ascites, or synthesized by chemical or biological methods (for example, cell culture, recombinant gene expression). “Isolated”, if not otherwise specified herein includes the meaning “separated from coexisting material”.
Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to the production of polypeptides by recombinant techniques, to expression system which comprises a nucleic acid or nucleic acids encoding the polypeptides of the present invention, to host cells which are genetically engineered with such expression systems, and to methods to isolate the polypeptides.
Another embodiment provides that a polypeptide of the invention is encoded by a nucleic acid which hybridizes under stringent conditions to SEQ. ID No: 3 or to SEQ. ID No: 4. In some embodiments, the nucleic acid comprises at least 50, at least 75, at least 100, at least 125, or at least 150 nucleotides. Preferably the nucleic acid comprises at least 175 or at least 200 nucleotides. In particular it comprises 225 or 228 nucleotides. The nucleic acid may also comprise at least 300, or at least 400 or 500 nucleotides. Preferably it may comprise at least 600 or at least 700 nucleotides. Most preferably it comprises at least 750 nucleotides. Such nucleic acids may comprise contiguous nucleotides of SEQ ID NO: 3 or 4 or contiguous nucleotides able to hybridize to SEQ ID NO: 3 or 4 under stringent conditions.
The term “nucleic acid” means natural or semi-synthetic or synthetic or modified nucleic acid molecules. It refers to nucleotide sequences, oligonucleotides or polynucleotides including deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) and/or modified nucleotides. These terms are intended to be used interchangeably. RNA may be in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, and ribozymes. DNA may be in form of plasmid DNA, viral DNA, linear DNA, chromosomal or genomic DNA, cDNA, or derivatives of these groups. In addition these DNAs and RNAs may be single, double, triple, or quadruple stranded. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
“Stringent conditions” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends upon the ability of a denatured nucleic acid to reanneal when complementary strands are present in an environment near but below their melting temperature. The higher the degree of homology between the probe and the hybridizable sequence such as SEQ. ID No: 3 or 4, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. Moreover, stringency is also inversely proportional to salt concentrations. “Stringent conditions” are exemplified by reaction conditions characterized by: (1) low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) the use of a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C. Alternatively, stringent conditions can be: 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Protocols in Molecular Biology (1995).
Recombinant Manufacture of FGF-23 C-Terminal Polypeptides.
The nucleic acids described herein such as SEQ. ID No: 3 or 4 may be used in recombinant DNA molecules to direct the expression of the corresponding polypeptides in appropriate host cells. Because of the degeneracy in the genetic code, other DNA sequences may encode the equivalent amino acid sequence, and may be used to clone and express FGF-23 or fragments thereof. Codons preferred by a particular host cell may be selected and substituted into the naturally occurring nucleotide sequences, to increase the rate and/or efficiency of expression. The nucleic acid (e.g., cDNA or genomic DNA) encoding the desired FGF-23 or FGF-23 fragments such as FGF23CTP may be inserted into a replicable vector for cloning (amplification of the DNA), and/or for expression.
Expression Systems.
The polypeptide can be expressed recombinantly in any of a number of expression systems according to methods known in the art (Ausubel, et al., editors, Current Protocols in Molecular Biology, John Wiley Sons, New York, 1990). Such expression systems include chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a nucleic acid to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). In general, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art.
Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, a signal or secretion sequence, and a transcription termination sequence:
The expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Such sequences are well known for a variety of bacteria, yeast strains, and viruses.
Preferably, the expression vector contains a marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients e.g., the D-alanine racemase gene.
Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. Further, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably, two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by insertion of the appropriate homologous sequence in the vector. Constructs for integrating vectors are well known in the art.
An appropriate secretion signal may be incorporated into the desired polypeptide to allow secretion of the polypeptide into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, the alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders). In mammalian cell expression systems, mammalian signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders may be used to direct secretion of FGF-23 or fragments thereof such as FGF23CTP.
Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells, for example primary cells, including but not limited to stem cells. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptococci, Staphylococci, Streptomyces, and Bacillus subtilis; fungal cells, such as Saccharomyces cerevisiae, other yeast cells or Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
A host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a “prepro” form of the polypeptide, may also be important for correct insertion, folding and/or function.
FGF-23 or fragments thereof such as FGF23CTP may be produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding an FGF-23 or fragments thereof under the appropriate conditions to induce or cause expression of the protein or polypeptide. In a preferred embodiment of the invention a host cell is provided which is stably or transiently transfected with a nucleic acid of SEQ. ID No: 3 or 4 or transfected with a nucleic acid which hybridizes under stringent conditions to SEQ. ID No: 3 or 4. According to another embodiment of the invention said host cell is cultured to allow expression of FGF-23 or of an FGF-23 fragment, and the polypeptide is isolated from the cell culture.
Transformed host cells include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmic DNA expression vectors, yeast transformed with yeast expression vectors, and insect cells infected with a recombinant insect virus (such as baculovirus), and mammalian expression systems.
The appropriate conditions for expression of FGF-23 or fragments thereof such as FGF23CTP will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used together with insect cells are lytic viruses, and thus harvest time selection can be crucial for product yield.
The desired FGF-23 or FGF-23 fragment may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide. Such heterologous polypeptide is generally placed at the amino- or carboxyl-terminus of FGF-23 or of an FGF-23 fragment and may provide for an epitope tag to which an anti-tag antibody can selectively bind. Accordingly, such epitope tag enables FGF-23 or a fragment thereof to be readily purified by using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Examples of epitope tags are 6×His or c-myc tag. Alternatively FGF-23 or a fragment thereof may be expressed in the form of e.g. an GST-fusion protein. Appropriate constructs are generally known in the art and are available from commercial suppliers such as Invitrogen (San Diego, Calif.), Stratagene (La Jolla, Calif.), Gibco BRL (Rockville, Md.) or Clontech (Palo Alto, Calif.).
Evaluation of Gene Expression.
Gene expression may be evaluated in a sample directly, for example, by standard techniques known to those of skill in the art, e.g., Southern blotting for DNA detection, Northern blotting to determine the transcription of mRNA, dot blotting (DNA or RNA), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be used in assays for detection of nucleic acids, such as specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Such antibodies may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. Gene expression, alternatively, may be measured by immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to directly evaluate the expression of FGF-23 or of an FGF-23 fragment. Antibodies useful for such immunological assays may be either monoclonal or polyclonal, and may be prepared against a native sequence FGF-23 or FGF-23 fragments based on the DNA sequences provided herein.
Purification of Expressed Protein.
Expressed FGF-23 or an FGF-23 fragment such as FGF23CTP may be purified or isolated after expression, using any of a variety of methods known to those skilled in the art. The appropriate technique will vary depending upon the way of expression of FGF-23 or an FGF-23 fragment. The polypeptide may for example be recovered from culture medium in the form of a secreted potein or from host cell lysates. Cells can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or by use of cell lysing agents, whereas membrane-bound polypeptides may be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. The appropriate technique for polypeptide purification or isolation will also vary depending upon what other components are present in the sample. The degree of purification necessary will also vary depending on the use of FGF-23 or a fragment thereof. Contaminant components that are removed by isolation or purification are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other solutes. The purification step(s) selected will depend, for example, on the nature of the production process used and the particular FGF-23 or FGF-23 fragment produced.
Ordinarily, isolated FGF-23 or a fragment thereof will be prepared by at least one purification step. Well-known methods for purification include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, high performance liquid chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, affinity chromatography is employed for purification. For example, the FGF-23 or a fragment thereof such as FGF23CTP may be purified using a standard anti-FGF-23 C-terminal polypeptide antibody column. Ultrafiltration and dialysis techniques, in conjunction with protein concentration, are also useful (see, for example, Scopes, R., Protein Purification, Springer-Verlag, New York, N.Y., 1982). Well-known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification
Labeling of Expressed Polypeptide.
The nucleic acids, proteins and antibodies of the invention may be labeled. By labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound which is being detected.
Chemical Manufacture of FGF-23 and FGF-23 Fragments.
Polypeptides or fragments thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds alpha-amino- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are Nalpha-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivatized amino acids, and washing with dichloromethane and/or N,N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesis may also be carried out on machines such as the ABI 431A peptide synthesizer (Applied Biosystems). A polypeptide or a fragment thereof may be purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton T. E. (1984) Proteins, Structures and Molecular Properties, W H Freeman, New York N.Y.).
Variants.
Variants of the natural polypeptide may be desirable in a variety of circumstances. For example, undesirable side effects might be reduced by certain variants, particularly if the side effect activity is associated with a different part of the polypeptide from that of the desired activity. In some expression systems, the native polypeptide may be susceptible to degradation by proteases. In such cases, selected substitutions and/or deletions of amino acids which change the susceptible sequences can significantly enhance yields. Variants may also increase yields in purification procedures and/or increase shelf lives of proteins by eliminating amino acids susceptible to oxidation, acylation, alkylation, or other chemical modifications. Preferably, such variants include alterations that are conformationally neutral, i.e. they are designed to produce minimal changes in the tertiary structure of the variant polypeptides as compared to the native polypeptide, and (ii) antigenically neutral, i.e. they are designed to produce minimal changes in the antigenic determinants of the variant polypeptides as compared to the native polypeptide.
Manufacture of a Medicamen for Use in the Treatment of a Disease.
The aforementioned polypeptides may according to the invention be used for the manufacture of a medicament for use in the treatment of a disease associated with deregulated angiogenesis or a cell proliferative disorder.
The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
A “disorder” or a “disease” is any condition that would benefit from treatment with FGF-23 or a fragment of FGF-23 as defined above and further below. This includes both chronic and acute disorders, as well as those pathological conditions which predispose to the disorder or disease in question. Non-limiting examples of disorders or diseases to be treated herein include any condition which results from deregulated angiogenesis or from deregulated cell proliferation. Examples of diseases associated with deregulated angiogenesis include: ocular neovascularisation, such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, haemangioblastoma, haemangioma, arteriosclerosis, inflammatory diseases, such as rheumatoid or rheumatic inflammatory diseases, especially arthritis, such as rheumatoid arthritis, or other chronic inflammatory disorders, such as chronic asthma, arterial or post-transplantational atherosclerosis, endometriosis, and especially neoplastic diseases, for example so-called solid tumors and liquid tumors (such as leukemias).
A preferred example of a diseases associated with deregulated angiogenesis is selected from the group of retinopathies, age-related macular degeneration, haemangioblastoma, haemangioma, and tumors. A particularly preferred example of a diseases associated with deregulated angiogenesis is retinopathy.
Examples of cell proliferative disorders include: chronic or acute renal diseases, e.g. diabetic nephropathy, malignant nephrosclerosis, thrombic microangiopathy syndromes or transplant rejection, or especially inflammatory renal disease, such as glomeruloneephritis, especially mesangioproliferative glomerulonephritis, haemolytic-uraemic syndrome, diabetic nephropathy, hypertensive nephrosclerosis, atheroma, arterial restinosis, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and autoimmune diseases, acute inflammation, fibric disorders (e.g. hepatic cirrhosis), diabetes, endometriosis, chronic asthma, neurodegenerative disorders and especially neoplastic diseases such as adenocarcinoma, gliomas, leukemia, lymphoma, melanoma, myeloma, sarcoma, Kaposi's sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung (especially small-cell lung cancer), muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
A preferred example of a cell proliferative disorder is selected from the group of chronic or acute renal diseases, arteriosclerosis, atherosclerosis, psoriasis, endometriosis, diabetes, chronic asthma and cancer. A particularly preferred example of a cell proliferative disorder is cancer.
Another aspect of the invention relates to a method for the treatment of a disease associated with deregulated angiogenesis which comprises administering an effective amount of a polypeptide to a mammal including a human suffering from the disease, wherein the polypeptide is selected from the groups consisting of (a) FGF-23 (SEQ. ID No: 1) or a fragment of FGF-23; (b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); or (c) a bioactive variant of any one of the polypeptides of (a) or (b). Accordingly, a polypeptide as described above may be administered.
Another aspect of the invention relates to a method for the treatment of a cell proliferative disorder comprising administering an effective amount of FGF-23 or an FGF-23 fragment such as FGF23CTP as described above to a mammal including a human suffering from the disorder.
“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and from animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cattle, etc. Preferably, the mammal is human.
Pharmaceutical Composition.
Another aspect of the invention provides a pharmaceutical composition for use in a disease associated with deregulated angiogenesis or a proliferative disorder comprising FGF-23 or a fragment thereof according to the invention as described above and a pharmaceutically-acceptable carrier. The composition of the invention is administered in effective amounts.
The pharmaceutical composition may be used in the foregoing methods of treatment. Such compositions are preferably sterile and contain an effective amount of FGF-23 or an FGF-23 fragment such as FGF23CTP or a nucleic acid encoding the polypeptide for inducing the desired response in a unit of weight or volume suitable for administration to a patient.
An “effective amount” of FGF-23 or fragment thereof, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results including clinical results such as inhibiting premature or diabetic retinopathy, inhibiting angiogenesis, shrinking the size of the tumor, retardation of cancerous cell growth, decreasing one or more symptoms resulting from the disease or disorder, increasing the quality of life of those suffering from the disease or disorder, decreasing the dose of other medications required to treat the disease or disorder, enhancing effect of another medication, delaying the progression of the disease or disorder, and/or prolonging survival of patients, either directly or indirectly.
Such amounts will also depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
An effective amount can be administered in one or more administrations and may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
An effective amount of FGF-23 or an FGF-23 fragment or the pharmaceutical composition comprising the polypeptide of the invention, alone or in conjunction with another drug, compound, or pharmaceutical composition can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, topical or transdermal.
When administered, the pharmaceutical composition of the present invention is administered in pharmaceutically acceptable preparations. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into mammals including humans. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents.
When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The doses of polypeptide or nucleic acid encoding said polypeptide administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of a polypeptide or nucleic acid encoding the polypeptide, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.
Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
The following EXAMPLES are presented in order to more fully illustrate the preferred embodiments of the invention. These EXAMPLES should in no way be construed as limiting the scope of the invention, as defined by the appended claims.
In this EXAMPLE, 100 unknown chemically synthesized peptides are functionalized using the discovery method of the invention. Most of these peptides are present in human plasma.
As a control, twenty reference drugs are concurrently investigated with the discovery method of the invention. For this screening, one control and four treated groups of six males are treated for two weeks by daily administration by the subcutaneous route of the proteins. The reference drugs can be active for treating conditions in the areas of glaucoma, neuroprotection, neovascularisation, antiangiogenesis, acne, asthma and allergy, cardiovascular diseases, neurological disorder, pain, diabetes, hypercholesterolemia, osteoporosis and oncology.
The expectations from those selected active peptides are that (a) several potential therapeutic drugs could be identified; (b) target peptides for therapeutic antibodies could be identified; (c) new targets for research, deduced from the reconstructed biochemical pathways, could be identified; and (d) biomarkers, to be used to develop diagnostic tests could be identified. Bioinformatic investigations of gene expression in mice suggest therapeutic indications and insight through the analysis of molecular pathways and functions, which allow prioritizing for the verification of selected proteins/peptides in monkeys. The protein/peptide selection rate is approximately 20%. The selection/prioritization criteria are the type of activity, the therapeutic interest and the suspected toxicity.
In an initial analysis, following administration of a peptide (FGF23CTP, see below, EXAMPLE III) to mice, transcript level changes were observed in several organs which indicate that the compound is active and impacts on pathways involved in cell differentiation.
Introduction and Summary.
Five peptides with unidentified function were tested in mice to obtain biochemical and pharmacogenomic data that would allow a specification of their activity. Outbred CD-1 mice were treated with peptides GPA018, GPA019, GPA020, GPA022, and GPA023 for seven days, observed for clinical signs of treatment effects (mortality, clinical signs, body weight, food consumption, haematology, clinical biochemistry) and, after sacrifice, a selected set of tissues were used for gene expression profiling. A snap freezing sampling of the tissues was performed at necropsy at the end of the treatment period. These tissues were used for mRNA expression profiling and for histopathological analysis (formalin fixation). In addition, parameters investigated in a standard exploratory study were recorded. None of the peptides had any influence on clinical or pharmacogenomic parameters. Gene expression profiling revealed no significant changes between control and treated animals. It was concluded that the peptides were inactive and decided that no further investigations on these peptides would follow.
Treatment.
Peptides GPA018, GPA019, GPA020, GPA022, and GPA023 (GeneProt, Geneva, Switzerland; see,
Dosage forms were prepared once before the beginning of the treatment period. Each test item was dissolved in the vehicle (PBS) in order to achieve the required concentration. The dosage forms obtained were divided into aliquots and stored at −20° C. pending use. Two aliquots were prepared for each group and day. The aliquots for treatment were delivered twice on each day of treatment to the animal room.
In Vivo Examinations.
Animals were examined at least twice daily for mortality, food consumption and clinical observations. Body weight was recorded once per week.
Blood samples were collected from each animal. The serum samples were deep frozen (approximately −80° C.) until analyses for hormone determination.
For tissue sampling, animals were asphyxiated by carbon dioxide approximately 12 to 16 hours after the last injection. A gross macroscopic post-mortem examination was performed in order to specify the possible reduced size of main organs (with particular attention to lymphoid tissues). No treatment-related morphological changes were noticed.
Snap freezing of many organs was performed at necropsy at the end of the treatment period. Within 15 to 20 after the sacrifice, all sampling for snap freezing was performed.
Twenty-eight tissues were sampled, including brain, duodenum (caecum), liver, kidney, muscle and spleen (blood). For this EXAMPLE, male and female animals were used in the kidney and in the liver assays. All other tissue samples were derived from males only.
Samples for histopathology were fixed in phosphate-buffered 10% formalin. Bone demineralization was performed with 10% formic acid.
Samples for gene expression profiling were quick-frozen in liquid nitrogen immediately after excision, stored on dry ice and subsequently in a deep-freezer at approximately −80° C. until further use.
RNA Extraction and Purification.
Briefly, total RNA was obtained by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol®, Invitrogen Life Technologies, Carlsbad, Calif. USA) from each frozen tissue section and the total RNA was then purified on an affinity resin (Rneasy®, Qiagen) according to the manufacturer's instructions, and quantified. Total RNA was quantified by the absorbance at λ=260 nm (A260nm) and the purity was estimated by the ratio A260nm/A280nm. Integrity of the RNA molecules was confirmed by non-denaturing agarose gel electrophoresis. RNA was stored at approximately −80° C. until analysis. One part of each individual RNA sample was kept for the analysis of critical genes by means of Real-Time PCR.
GeneChip® Assays.
All GeneChip® assays were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Expression Analysis Technical Manual, (Affymetrix, Santa Clara, Calif. USA, 2003). Genome MG-U74Av2 expression probe array set (Affymetrix, Inc., San Diego, Calif. USA) were used.
Double stranded cDNA was synthesized with a starting amount of approximately 5 μg full-length total RNA using the Superscript Choice System (Invitrogen Life Technologies) in the presence of a T7-(dT)24 DNA oligonucleotide primer. Following synthesis, the cDNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. The purified cDNA was then transcribed in vitro using the BioArray® High Yield RNA Transcript Labeling Kit (ENZO, Farmingdale, N.Y., USA) in the presence of biotinylated ribonucleotides form biotin labelled cRNA. The labelled cRNA was then purified on an affinity resin (Rneasy®, Qiagen), quantified and fragmented. An amount of approximately 10 μg labelled cRNA was hybridized for approximately 16 hours at 45° C. to an expression probe array. The array was then washed and stained twice with streptavidin-phycoerythrin (Molecular Probes, Eugene, Oreg., USA) using the GeneChip® Fluidics Workstation 400 (Affymetrix).
The array was then scanned twice using a confocal laser scanner (GeneArray® Scanner, Agilent, Palo Alto, Calif. USA) resulting in one scanned image. This resulting “.dat-file” was processed using the MAS4 program (Affymetrix) into a “.cel-file”. The “.cel file” was captured and loaded into the Affymetrix GeneChip Laboratory Information Management System (LIMS). The LIMS database is connected to a UNIX Sun Solaris server through a network filing system that allows for the average intensities for all probes cells (CEL file) to be downloaded into an Oracle database. Raw data was converted to expression levels using a “target intensity” of 150. The data were checked for quality and loaded into the GeneSpring® software 5.0.3 (Silicon Genetics, Redwood City, Calif. USA) for analysis.
As a quality control, hybridizations were performed using GAPDH or β-actin probes.
Data Analysis.
RNA samples were studied by using the human Affymetrix MG-U74Av2 GeneChip®. On such chip platform, probe-sets for individual genes contain 20 oligonucleotide pairs, each composed of a “perfect match” 25-mer and a “mismatch” 25-mer differing from the “perfect” match oligonucleotide at a single base. After probe labelling, hybridization, and laser scanning, the expression level is estimated by averaging the differences in signal intensity measured by oligonucleotide pairs of a given probe (AvgDiff value). The image acquisition and numerical translation software used for this study was the Affymetrix Microarray Suite version 4 (MAS5). The numerical values were stored and transferred for the analysis into the Silicon Genetics GeneSpring® 5.0.3 software toolkit.
Various filtering and clustering tools in these programs were used to explore the datasets and identify transcript level changes that inform on altered cellular and tissue functions and that can be used to establish working hypotheses on the modes of action of the compound. All datasets were normalized to the median. To identify pleiotropic or unique effects, scripts provided by Silicon Genetics, were applied. Pleiotropic effects were also investigated manually by analyzing genes with an expression level of more than 60 in at least three of eight conditions (i.e. organs) and hereafter applying statistical analysis (parametric, assume variances not equal, p<0.1) to identify genes with significant changes in their expression between control and treated animals.
To identify significant changes in gene expression in individual organs, an expression restriction filter was applied (ADV >60 in 50% of the samples), and a statistical filter as described above was added.
In some instances, a fold change filter script (provided by Silicon Genetics) was used after the expression restriction to find genes with a change in gene expression above a certain level, and then a statistical filter applied.
The decision to consider a specific gene relevant is based on a conjunction of numerical changes identified by exploratory filtering and statistical algorithms as described above and the relationship to other modulated genes that point to a common biological theme. The weight of that relationship is assessed by the analyst through a review of the relevant scientific literature.
Gene Expression Profiling.
Despite a multitude of statistical approaches, no significant changes in the gene expression patterns of the treatment groups were observed. In every analysis the number of changed genes was always smaller than the number of genes that would have been found by chance. The range of fold change was usually very small, seldom exceeding 2-fold.
Interestingly, the strongest changes were gender-specific. Comparison of control males and control females, as well as treated males versus treated females, showed about 3 times as many genes changed than did control males or females versus treated males or females. In addition, the genelists of treatment specific effects in females and males showed little overlap.
In the GPA018 treated kidneys of males and females combined (eight samples per group), some possible influence on genes affected by or affecting TGFβ signalling were observed (TABLE 2A and TABLE2B). GPA018 shows some sequence similarity to the N-terminal region of mLTBP-2 (murine latent transforming growth factor binding protein-2). LTBP proteins aid the LAP (latency associated protein)-TGFβ complex to become secreted and binds it to the ECM (extracellular matrix)-structural protein fibrillin (Annes J P et al., J Cell Sci. 116(Pt2):217-24 (2003); Chen S et al., Nucl. Acids Res. 31(4):1302-10 (2003); Vehivilainen P et al., J. Biol. Chem. 278(27):24705-13 (2003)). However, the extent of the changes after treatment was very small, usually below 1.5-fold, and similar patterns were not observed when female or male groups were investigated separately.
Chem. 274(33): 23256-23262 (1999))
Biochem. J. 369(Pt2): 311-8 (2003))
Genomics 80(5): 465-72 (2002))
Chem.278(20): 18408-18 (2003))
Introduction and Summary.
The aim of this EXAMPLE was to identify for the peptide FGF23CTP modes of action with possible therapeutic applications by multi-organ microarray profiling in monkey. The peptide FGF23CTP (GPA006, GeneProt, Geneva, Switzerland) is derived from a unique COOH-terminal domain of the FGF-23. It is a unique 75-mer COOH-terminal peptide of FGF-23 with no homology to regions of other FGF family members. See, PCT patent application WO 02/088,358, the contents of which are incorporated by reference. In the brain FGF-23 transcripts are preferentially expressed in the thalamus (Yamashita T et al., Biochem. Biophys. Res. Commu., 277: 494-8 (2000)). Mutations in this region of the FGF-23 molecule were proposed as causative events in a renal phosphate wasting syndrome responsible for a form of autosomal dominant rickets (Saito H et al., Am. J. Pathol. 156: 697-707 (2002), White K E et al., Kidney Int. 60: 2079-86 (2001)). A similar paraneoplastic form of this syndrome was accompanied by ectopic expression of FGF-23 in the tumour tissue (Shimada T et al., Proc. Natl. Acad. Sci. USA 98: 6500-5 (2001)). FGF-23 is expressed in the ventrolateral thalamic nucleus of the brain. FGF23CTP has been derived in silico as a possible processing product of the fibroblast growth factor FGF-23 known to be involved in renal phosphate wasting syndromes. It was hypothesized that the 75-mer peptide could affect phosphate homeostasis.
FGF23CTP is given two weeks subcutaneously to cynomolgus monkeys and is found to affect critical pathways of cell differentiation in a multi-organ gene expression profiling analysis with high density human microarray assays. Comparison of the expression changes in sixteen different organs indicated common transcript level changes for genes involved in cell to cell signalling of growth and lineage determination, in cell cycling and in amino acid and ion transport. Although the FGF23CTP domain is unique and not found in other FGF family members, transcript levels for components of the FGF signalling pathway are affected in several organs. Serum protein levels of circulating IGF2-binding protein are decreased in the treated animals.
Genes involved in angiogenesis/vasculogenesis are found in to be impacted in several organs. The effect of FGF23CTP on angiogenesis is confirmed in a hypoxic vascular retinopathy mouse model (see EXAMPLE VIII).
Methods.
FGF23CTP is administered subcutaneously to cynomolgus monkeys for two weeks at a dose of 100 μg/day. At the end of the treatment period samples from all organs are subjected to snap freezing at necropsy and are analyzed with GeneChip® expression profiling.
Total RNA is extracted from these frozen tissues using TRIzol® reagent (Life Technologies) according to the manufacturer's instructions. Total RNA is quantified by the absorbance at λ=260 nm (A260nm) and the purity is estimated by the ratio A260nm/A280nm. Integrity is checked by denaturing gel electrophoresis. RNA is stored at −80° C. until analysis.
Good quality total RNA is used to synthesize double-stranded cDNA using the Superscript® Choice System (Technologies, Gaithersburg, Md. USA). The cDNA is then in vitro transcribed (MEGAscript™ T7 Kit, Ambion) to form biotin labelled cRNA. Next, 12 to 15 μg of labelled cRNA is hybridized to the Affymetrix Human U95A Version 2 expression probe arrays for 16 hours at 45° C. Arrays are then washed according to the EukGE-WS2 protocol (Affymetrix), and stained with 10 μg/ml of streptavidin-phycoerythrin conjugate (Molecular Probes). The signal is antibody-amplified with 2 mg/ml acetylated BSA (Life Technologies, Gaithersburg, Md. USA), 100 mM MES, 1 M [Na+], 0.05 Tween 20, 0.005% Antiofoam (Sigma), 0.1 mg/ml goat IgG and 0.5 mg/ml biotinylated antibody and re-stained with the streptavidin solution. After washing, the arrays are scanned twice with the Gene Array® scanner (Affymetrix).
The expression level is estimated by averaging the differences in signal intensity measured by oligonucleotide pairs of a given probe (AvgDiff value). The image acquisition and numerical translation software used was the Affymetrix Microarray Suite version 4 (MAS4).
To identify genes that are impacted by treatment, the dataset is initially filtered to exclude in a first wave of analysis genes whose values are systematically in the lower expression ranges where the experimental noise is high (at least an AvgDiff value of 80 in a number of assays corresponding to the smallest number of replicas of any assay point). In a second round of selection a threshold t-test p-value (0.05) identifies genes with different values between treated and non-treated based on a two component error model (Global Error Model) and, where possible, with a stepdown correction for multi-hypothesis testing (Benjamini and Hochberg false discovery rate).
The selected genelists are then compared with established genelists for pathways and cellular components using Fisher's exact test. Venn diagrams are used to identify the gene changes that are in common between the different organs. Expression profiles of highly relevant genes are used to find genes with correlated changes at individual assay points, using several distance metrics (standard, Pearson).
The decision to consider a specific gene relevant is based on a conjunction of numerical changes identified by exploratory filtering and statistical algorithms as described above and the relationship to other modulated genes that point to a common biological theme.
A filter is applied on the FGF23CTP rostral hypothalamus expression raw data from a raw value of at least 80.0 in at least four out of eight conditions.
A selection is made on treatment conditions for FGF23CTP rostral hypothalamus based on statistically significant differences with a p-value cutoff 0.05 and a multiple testing correction based on the Benjamini and Hochberg False Discovery Rate. This restriction tested 5134 genes. About 5.0% of the identified genes would be expected to pass the restriction by chance.
Results.
At the RNA level, it is clear that the compound affects key genes controlling cellular differentiation and proliferation, especially growth factors and growth factor receptors. Genes critically involved in angiogenesis are found in several organs. There is also a multi-organ effect on transcripts for components of the retinoblastoma cycling control checkpoint. The rostral hypothalamus shows the most pronounced changes especially for transport proteins and cytoarchitecture.
A cross comparison of organs analyzed reveals that FGF23CTP affects the same or closely related pathways and provokes similar cellular effects. No routine clinical or biochemical changes are observed in the treated animals. Surprisingly, no effect on phosphate metabolism is observed.
TABLES 3 to 6 show that at the RNA level FGF23CTP affects key genes controlling cellular differentiation and proliferation, especially growth factors and growth factor receptors (TABLE 3). Genes critically involved in angiogenesis are found to be altered in several organs upon treatment with FGF23CTP (TABLE 4). There is also a multi-organ effect on transcripts for components of the retinoblastoma cycling control checkpoint (TABLE 5). The rostral hypothalamus shows the most pronounced changes especially on transcripts corresponding to proteins involved in transport and cytoarchitecture (TABLE 6).
In particular, FGF23CTP affects several molecules that have been described to play a role in the pathogenesis of malignant proliferation of glial cells and precursors: Epidermal growth factor (EGF) (Hoi Sang U et al., J. Neurosurg. 82: 841-846 (1995); Wu C J et al., Oncogene 19: 3999-4010 (2000)), Bax (Streffer J R et al., J. Neurooncol. 56: 43-49 (2002); Martin S et al., J. Neurooncol. 52: 129-139 (2001)), connexin 43 (Huang R et al., Cancer Res. 62: 2806-2812 (2002); Soroceanu L et al., Glia 33: 107-117 (2001)), PKR (Shir A & Levitzki A, Nature Biotechnology 20: 895-900 (2002)), neurofibromin (NF1) (Cichowski K & Jacks T, Cell 104: 593-604 (2001); Gutmann, D H et al., Hum. Mol. Genet. 10: 3009-3016 (2001)).
Growth Factors and Growth Factor Receptors.
Several families of growth factors and related growth factor receptors including fibroblast growth factor (FGF)-receptors and other important extracellular signalling molecules for cell differentiation and maintenance, like members of the bone morphogenetic proteins (BMP), transforming growth factor (TGF), insulin-like growth factor (IGF), tumour necrosis factor (TNF) families are found to be impacted in more than one organ by treatment with FGF23CTP (TABLE 3).
Angiogenesis/Vasculogenesis.
Transcript level changes for genes specifically involved in angiogenesis/vasculogenesis are found in several organs of animals treated with FGF23CTP (TABLE 4).
Cycling: Retinoblastoma Checkpoint.
Transcript levels of genes involved in cell cycle control, especially those genes involved in the transition from the G1 to S-phase, are affected in several organs by treatment with FGF23CTP (TABLE 5). In particular, expression of those genes upstream or downstream of the retinoblastoma gene product (Rb) phosphorylation step, a major downstream control step for growth factor-induced proliferation, is altered. FGF23CTP also affects transcript levels for cyclin-dependent kinase 4 and cyclins D2, D3 and E2 involved in Rb phosphorylation. A second control level is also mobilized with cyclin kinase inhibitors like p191NK4D, p21CIP1 and p27Kip1. Also affected is the inhibitor of p53 and Rb, Mdm2. The targets of the retinoblastoma protein are also involved: E2F1, E2F2, E2F5 and their binding partner Dp-2.
Rostral Hypothalamus.
The rostral hypothalamus is the organ with the most pronounced changes in transcript levels in animals treated with FGF23CTP. TABLE 6 reflects genes of defined pathways and cellular actions with the most significant changes in this organ. In particular, effects on cytoarchitecture genes are especially pronounced in brain tissues.
As can be seen in the TABLE 7 (genelists per organ), there are genes representing these pathways with less pronounced changes in other organs. Effects on genes coding for transport molecules and intracellular signalling molecules are found in most organs. The effects on cytoarchitecture genes are especially pronounced in brain tissues. Shown in TABLE 7 are the Affymetrix chip probeset ID, p-value, and gene description of the 602 selected genes.
musculus protein encoded by GenBank Accession Number U67327; contains WD40 repeat motif, also
Drosophila bendless gene product, complete cds.
Drosophila melanogaster
Serum levels were determined for IGFB2 and found to be decreased, in agreement with the decreased transcript levels in several organs, including the liver.
Analysis.
Angiogenesis is further shown to be inhibited by FGF23CTP in a hyperoxia-induced angioproliferative retinopathy model (Aiello F P et al., Proc. Natl. Acad. Sci. USA, 92: 10457-61 (1995), Ozaki H et al., Am. J. Pathol. 156: 697-707 (2000)) in C57/B6 mice (see EXAMPLE VIII). The proliferative component of the retinal angiopathy induced through a relative ischemia by transition of postnatal mice from hyperoxic to normoxic conditions is significantly inhibited (p=0.018) by the intravitreal injection of the FGF23CTP (GPA006) peptide.
In the rostral hypothalamus, besides its effects on angiogenesis and cycling genes, FGF23CTP affects several molecules that have been described to play a role in the pathogenesis of malignant proliferation of glial cells and precursors (malignant brain tumours): epithelial growth factor (EGF; Hoi Sang U et al., J. Neurosurg. 82: 841-6 (1995), Wu C J et al., Oncogene 19: 3999-4010 (2000)), Bax (Streffer J R et al., J. Neurooncol. 56: 43-9 (2002); Martin et al., (2001)), connexin 43 (Huang R et al., Cancer Res. 62: 2806-12 (2002), Soroceanu Let al., Glia 33: 107-17 (2001)), PKR (Shir A & Levitzki A, Nature Biotechnology 20: 895-900 (2002)), NF1 (Cichowski K & Jacks T, Cell 104: 593-604 (2001), Gutmann D H et al., Hum. Mol. Genet. 10: 3009-16 (2001)).
The discovery method of the invention has been validated through a “blind” monkey trial using three peptides of well known pharmacological activity: (1) the somatostatin analogue SOM230, (2) gonadotrophin releasing hormone (GnRH), (3) and leukemia inhibitory factor (LIF). In each case, a “blind” test with 3 “unknown” polypeptides was performed. The results demonstrated the capacity of the gene expression analyst teams to identify, within four months, the pharmacological activities, most of the therapeutic indications and side effects, and even the identity of the proteins. These first results demonstrate that the discovery method of the invention usefully provide the art with advantage in the understanding of drug pharmacological mechanisms and the potential side effects, the selection of biomarkers and potential new indications.
For the verification of the selected human proteins or peptides in cynomolgus monkeys, one control and four treated groups (i.e., each of three peptides and placebo) of two males and two females are treated for two weeks by daily administration of the proteins dissolved in autologous serum (e.g., in each animal's own serum) through the subcutaneous route. The administration of the peptides was blinded. The amount of peptide administered was 100 μg/animal/day (5-6 mg of peptide total).
Drug profiling in these non-human primates was analyzed using gene expression profiling of more than 100 organs on Affymetrix U95 chips (each containing ⅓ of the human genome). In addition, extensive biochemistry and clinical chemistry screening (>60 parameters) and histopathology (ca. 60 organs) were performed.
Data mining procedures were then used. The data were probed to answer four questions: (1) Is the polypeptide worth further investigations? (2) What are the physiological pathways impacted? (3) What are the potential indications? (4) What is the likelihood of the identity of the polypeptide? Peptide 1. Peptide 1 was SOM230. SOM230 (pasireotide) has a chemical structure cyclo[4-(NH2—C2H4—NH—CO—O)Pro-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe] as follows:
Here, Phg means —HN—CH(C6H5)—CO— and Bzl means benzyl. See, PCT patent application WO 02/10192. SOM230 is a somatostatin analogue with binding affinities for the five somatostatin receptors except somatostatin receptor 4 (SSTR4). SOM230 has been developed for several indications, including those disclosed above for other somatostatin analogues. See, Lewis I et al., J. Med. Chem. 46(12):2334-44 (Jun. 5, 2003); Weckbecker G et al., Endocrinology 143(10): 4123-4130 (2002); Kneissel M et al., Bone 28:237-250 (2001); and Thomsen J S et al., Bone 25:561-569 (1999), the contents of which are incorporated herein by reference.
SOM230 was developed for the approved Sandostatin® indications, but as a more potent somatostatin analogue with a longer plasma half-life in vivo. Lewis I et al., J Med Chem 46(12): 2334-44 (Jun. 5, 2003); Weckbecker G et al., Endocrinology 143(10): 4123-30 (October 2002). In contrast with other analogues, SOM230 binds to all somatostatin receptors except SSTR4. The binding affinity for the different somatostatin receptors was a basis for defining the scope of possible new clinical indications for SOM230. Bruns C et al., Eur J Endocrinol 143(Suppl 1): S3-7 (2000); Bruns C et al., Eur J. Endocrinol. 146(5):707-16 (May 2002). In addition, other possible new indications were suggested due to the improved activity of SOM230 for growth hormone and IGF-1 regulation and its different inhibitory effects on insulin and glucagon secretions.
Following the blinded administration of peptide 1 (SOM230), the following results were obtained:
The underlined results in the TABLE above were identified for further investigation.
Based upon these results, the Data Mining Team made predictions as to the identity of the administered Peptide 1.
Based upon these results, SOM230 was selected for further development as a somatostatin analogue to treat the somatostatin-approved neuroendocrine indications (e.g., acromegaly, gastroenteropancreatic tumours) and also has potential for further indications with a pancreatic endocrine eetiology such as diabetic angiopathy and morbid obesity associated with hyperinsulinemia. IGF-1 serum level was proposed as surrogate marker. Other potential indications for SOM230 are therefore inflammation (e.g., psoriasis), pain and immunosuppression (e.g., chronic rejection).
Peptide 2.
Following the blinded administration of peptide 2 (gonadotrophin releasing hormone), the following results were obtained:
The underlined results in the TABLE above were identified for further investigation.
Based upon these results, the Data Mining Team made predictions as to the identity of the administered Peptide 2.
Based upon these results, the predicted indications for the administered Peptide 2 (gonadotropin releasing hormone) were be for gonadotrophin releasing hormone-related indications or for lutenizing hormone releasing hormone-related indications, such as antiproliferative disorders (cancer), ovarian and testicular functions (hypothalamic gonadotropc hypogonadism, fertility control and delayed or arrested puberty/precocious puberty), and growth hormone deficiencies.
Peptide 3.
Following the blinded administration of peptide 3 (leukemia inhibitory factor), the following results were obtained:
The underlined results were identified for further investigation.
Based upon these results, the Data Mining Team made predictions as to the identity of the administered Peptide 2.
Based upon these results, the predicted actions for the administered Peptide 3 would be to increase platelets, myeloid cells and megakaryocytes; to increase acute phase proteins (such as C-Reactive Protein (CRP) and haptoglobin); to decrease lipogenicity (with therapeutic indications for treating obesity and cardiovascular risk); to decrease transaminase, albumin and lactate dehydrogenase (LDH); and to decrease alkaline phosphatase, bone sialoprotein (BSP) and osteocalcin (with therapeutic indications for treating osteoarthritis and osteoporosis).
In summary, the three questions that were posed to the data miners could be answered almost fully by the data miners. All three polypeptides were identified as being worth further investigation. All of the physiological pathways known to be impacted by these three administered polypeptides were discovered (e.g. bone fractures for LIF, gastrointestinal tract for SOM230). Eighty % of their known potential indications were discovered and some new ones were identified.
Introduction and Summary.
Microarray gene expression assays were performed using tissues of monkeys treated with SOM230 at sub-therapeutic dose for 14 days. The assays were analyzed to identify the modes of actions of SOM230 with possible relationships to therapeutic applications. For a description of SOM230, see the EXAMPLE above.
All monkey tissues examined (thyroid, brown fat, pituitary, pancreas, liver, kidney, spleen) demonstrated changes in the genes regulated by the binding of the natural somatostatin 14 (SST-14) and somatostatin 28 (SST-28) to somatostatin receptors (SSTRs). The transcript profiles reflected the known somatostatin actions on the growth hormone/insulin-like growth factor 1 (GH/IGF-1), glucagon/insulin axes and on cell proliferation. However, the compound affected significantly the transcript levels of other related genes like insulin-like growth factor 2 (IGF-2) in the pituitary and kidneys. This could be a candidate biological marker (biomarker) of drug efficacy provided that the change in protein biosynthesis would be reflected in an easily accessible tissue like the blood. Other known effects of somatostatin and agonists on growth factors, cells of the immune system and the cardio-vascular and renal functions were also reflected by the changes in the profiles of these classes of genes after SOM230.
Origin of Tissue and Processing.
Male and female cynomolgus monkeys received subcutaneously SOM230 (100 μg/animal/day) or the vehicle for 14 days. On day 15, all animals were sacrificed and tissues for RNA extraction were immediately snap frozen and kept at −80° C. until processing.
RNA expression profiling was conducted by means of the HG-U95A gene expression probe array (Affymetrix; Santa Clara, Calif., USA), containing more than 12,600 probe sets interrogating primarily full-length human genes and also some control probe sets. The assays were conducted according to the recommendations of the manufacturer. Briefly, total RNA was obtained by acid guanidinium thiocyanate-phenol-chloroform extraction (TRIzol®, Invitrogen Life Technologies, San Diego, Calif., USA) from each frozen tissue section. The total RNA was then purified on an affinity resin (Rneasy®, Qiagen) and quantified. Double stranded cDNA was synthesized with a starting amount of approximately 5 μg full-length total RNA using the Superscript® Choice System (Invitrogen Life Technologies, Carlsbad, Calif. USA) in the presence of a T7-(dT)24 DNA oligonucleotide primer. Following synthesis, the cDNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. The purified cDNA was then transcribed in vitro using the BioArray® High Yield RNA Transcript Labelling Kit (ENZO, Farmingdale, N.Y. USA) in the presence of biotinylated ribonucleotides form biotin labelled cRNA. The labelled cRNA was then purified on an affinity resin (Rneasy®, Qiagen), quantified and fragmented. An amount of approximately 10 μg labelled cRNA was hybridized for 16 hours at 45° C. to an expression probe array. The array was then washed and stained twice with streptavidin-phycoerythrin (Molecular Probes,) using the GeneChip® Fluidics Workstation 400 (Affymetrix, Santa Clara, Calif. USA). The array was then scanned twice using a confocal laser scanner (GeneArray® Scanner, Agilent, Palo Alto, Calif. USA) resulting in one scanned image. This resulting “.dat-file” was processed using the MAS4 program (Affymetrix) into a “.cel-file”. The “.cel file” was captured and loaded into the Affymetrix GeneChip® Laboratory Information Management System (LEVIS). The LIMS database is connected to a UNIX Sun Solaris server through a network filing system that allows for the average intensities for all probes cells (CEL file) to be downloaded into an Oracle database (NPGN). Raw data was converted to expression levels using a “target intensity” of 150. The data were evaluated for quality control and loaded in the GeneSpring® software 4.2.4 (Silicon Genetics, Calif. USA) for analysis.
On the human Affymetrix HGU95Av2 chip, probe sets for individual genes contain 20 oligonucleotide pairs, each composed of a “perfect match” 25-mer and a “mismatch” 25-mer differing from the “perfect” match oligonucleotide at a single base. After probe labelling, hybridization, and laser scanning, the expression level was estimated by averaging the differences in signal intensity measured by oligonucleotide pairs of a given probe (AvgDiff value). The fold changes and directions were calculated for selected genes, from the differences of the AvgDiff values between control and treated.
To identify genes that were impacted by SOM230, the dataset was initially filtered to exclude in a first wave of analysis, genes whose values were systematically in the lower expression ranges where the experimental noise is high (at least 80 in a number of assays corresponding to the smallest number of replicas of any assay point). In a second round of selection a threshold p-value of 0.05 (based on a t-test) identified differences between treated and control based on a two component error model (Global Error Model) and, whenever possible, with a stepdown correction for multi-hypothesis testing (Benjamini and Hochberg false discovery rate). The decision to keep or reject a specific gene was based on the conjunction of numerical changes identified by comparative and statistical algorithms and the relationship to other modulated genes that point to a common biological theme. The weight of this relationship was assessed by the analyst through a review of the relevant scientific literature.
For the assay analysis described herein: (1) The increase and decrease in expression referred to the RNA expression level unless specifically stated. (2) If there were multiple probe sets representing the same gene, the probe set designed for sense target was favored. (3) The changes in gene expression indicated that a pathway, a cellular activity or component represented by an individual gene might be impacted. Understanding the functional implication is dependent on the information available on the biological context of the transcript level change (gene function, physiological variation, other gene changes, tissue, compound). RT-PCR is used to identify the extent of absolute change in mRNA levels, but this method in general does not add more information on the relevance of the transcript level changes.
Among the 12,600 genes per chip, about 100 genes were found to reflect the compound signature in a particular tissue. For clarity, they were divided in different classes and subdivided, with many overlaps, into functional categories in the following TABLE.
These results show that several signal transduction pathways were affected. They included the phosphatidyl inositol/PKC/phospholipases/calcium-calcineurin-calmodulin pathway, the Ras/MAPK kinase/ERK kinase dependent pathway, the JAK/STAT pathway, and adenylate/guanylate cyclases with their dependent pathways. The changes for the cell surfaces receptors included numerous G-protein coupled receptors, receptors for growth factors and glutamate receptors. The changes in ATP-dependent transport proteins involved ion channels and associated proteins. The compound also affected neuromediators/neuromodulators, pancreatic and gastrointestinal secretions, hormones, cytoskeletal proteins and enzymes/catalysts.
Examples of genes reflecting several SSTR signalling pathways in the pituitary are shown in TABLE 21. Selected genes from the primary gene lists were produced by a succession of filtering and statistical algorithms (t-test: p value: 0.05). The numerical values correspond to the AvgDiff (see above) of the relevant probe set for each assay with the range of observed values between brackets. Of particular interest in this analysis were the transcript level changes for molecules known to be closely associated with the binding of the natural peptides, SST-14 and SST-28, to the SSTRs.
The effects on the growth hormone/insulin-like growth factor-1 (GH/IGF-1) and glucagon/insulin axes (Macaulay V M, Br J Cancer 65: 311-20 (1992); Pollak M N & Schally A V, Proc Soc Exp Biol Med 217: 143-52 (1998)) were reflected in transcript level changes in several organs. The results are shown in TABLE 22. Beside the expected change in IGF-1 transcript level, there was an effect on insulin-like growth factor-2 (IGF-2) as well (in the pituitary and kidneys) that might be useful as a biological marker of SOM230 activity if reflected in the blood. The genes were selected as above in TABLE 21.
Other genes of interest affected by SOM230 were the transcript levels of growth factors (PDGF, FGF, EGF, TGFfβ), their receptors and factors of angiogenesis (PDGF, VEGF, thrombospondin) involved in tumour growth and spreading (Woltering E A et al., New Drugs 15: 77-86 (1997)). Also reported for somatostatin and analogues, genes involved in immunity were changed, i.e. cytokines (IL-1, TNF, IFN), regulators of T and B cell genesis and function (CD2 antigen, IL-2 receptor, B-lymphoid tyrosine kinase, IL-2 inducible T cell kinase, p56lck, RAG1, TCRζ chain precursor, RAG2, FLT 3 ligand) (van Hagen P M et al. Eur Clin Invest 24: 91-9 (1994)), as well as genes involved in blood pressure control and diuresis, i.e. atrial natriuretic peptide and its receptor guanylyl cyclase A, arginine vasopressin and its receptor (Aguilera G et al., Nature 292: 262-3 (1981); Aguilera G et al., Endocrinology 111: 1376-84 (1982); Ray C et al., Clin Sci (Loud) 84: 455-60 (1993); Cheng H et al., Biochem J364: 33-9 (2002)). A specific gene involved in the control of fat storage is the adrenergic 133 receptor in brown fat (Bachman E et al., Science 297: 843-45 (2002)).
Protein products of the above genes are useful as surrogate markers of the biological activity of SOM230, especially the findings for IGF-2 in the pituitary and kidneys.
To conclude, the gene profiling of monkey tissues treated with SOM230 at sub-therapeutic is a sensitive approach to identify signalling and effector pathways known for somatostatin. The finding of clear transcriptional signatures for this agonist argues for a comparison with gene expression changes induced by Sandostatin®.
Calcitonins are endogenous regulator of calcium homeostasis and can be used as anti resorptive agents for the treatment of hypercalcaemia-associated disorders. Various calcitonins, including e.g. salmon and eel calcitonin, are commercially available and are commonly employed in the treatment of e.g. Paget's disease and osteoporosis. See, U.S. Pat. Nos. 5,733,569 and 5,759,565, the contents of which are incorporated by reference. See also, U.S. Pat. Nos. 5,719,122, 5,175,146, and 5,698,6721, and U.S. Pat. Appln. 003015815. A version of calcitonin (Miacalcin®) is available as a nasal spray. Information regarding the administration of Miacalcin® (calcitonin-salmon) nasal spray is available in the Miacalcin® Prescribing Information (Novartis, November 2002).
Parathyroid hormone (PTH) is a polypeptide of 84 amino acids. Parathyroid hormone regulates bone remodeling and Ca2+ homeostasis. Parathyroid hormone is also a known paracrine activator of osteoclast differentiation and activity. PTS893 is an analogue of the endogenous parathyroid hormone, in which certain sites of chemical instability are eliminated within N-terminal parathyroid hormone fragments by making appropriate amino acid substitutions at particular residues which results in stable and biologically active human parathyroid hormone fragments. PTS893 [SDZ PTS 893; Leu8, Asp10, Lysll, A1a16, G1n18, Thr33, A1a34 human PTH 1-34 [hPTH(1-34)]] is a 34 amino acid parathyroid analogue that enhances bone mass and biomechanical properties. Kneissel M et al., Bone 28: 237-50 (March 2001); Stewart A F et al., J. Bone. Miner. Res. 15(8): 1517-25 (August 2000); Thomsen J S et al., Bone 25(5):561-9 (November 1999). N-terminal fragments of human parathyroid hormones include hPTH(1-34)OH muteins and hPTH(1-38)OH muteins. PTS893 comprises at least the first 27 N-terminal amino acid units of parathyroid hormone. Preferred parathyroid hormone derivatives are those comprising at least one amino acid unit replaced in one or more of the following positions of the parathyroid hormone sequence: 8-11, 13, 16-19, 21, 22, 29 to 34, particularly 8-11, 16-19, 33 and/or 34. These compounds exhibit desirable bone-forming properties both in vivo and in vitro which are equal to or above the level of natural PTH and its N-terminal fragments. See, European patent EP 0 672 057; published PCT patent application WO 94/02510; Kneissel M et al., Bone 28: 237-50 (March 2001); Stewart A F et al., J Bone Miner Res 15(8): 1517-25 (August 2000); Thomsen J S et al., Bone 25(5):561-9 (November 1999).
Introduction and summary. The purpose of this EXAMPLE was to evaluate the gene expression changes in cynomolgus monkeys following a two-week subcutaneous treatment with salmon calcitonin (sCT) at 50 μg/animal/day and PTS893 at 5 μg/animal/day to elucidate the mechanisms of action mediating their effects as well as the identification of biomarkers of therapeutic indications. This EXAMPLE is believed to be the first analysis that globally describes the molecular mechanisms of action of salmon calcitonin and a parathyroid hormone analogue by multiorgan-gene-profiling analysis in primates. This is also believed to be the first gene profiling analysis which describes the molecular mechanisms of action of hormonal-mediated bone remodeling by salmon calcitonin and PTS893.
In this EXAMPLE, salmon calcitonin and PTS893 were both found to have modulating effects on genes affecting the direct, autocrine, paracrine and endocrine regulation of the mesenchymal cell functions such as transforming growth factor betas (TGF-βs), insulin-like growth factors (IGFs), bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF). Both compounds also regulate the synthesis and degradation of extracellular matrix components. Salmon calcitonin also regulates estrogen receptor and steroidogenic factor, whereas PTS893 produced a strong up-regulation on nuclear receptors of the steroid/thyroid receptor family. These data therefore support the role of calcitonin as an anabolic agent.
In addition, salmon calcitonin and PTS893 also influenced some aspects of the mineralization of the extracellular matrix, since changes in amelogenin, dentin and ectonucleotide pyrophosphatases were observed.
In addition, PTS893 showed an effect on mediating the paracrine activation of osteoclast differentiation and activity, through cytokine and RANK ligand.
No significant differences in gene expression profiling were attributable to the fact of administering salmon calcitonin and PTS893 in combination, with respect to the single therapy.
Thus, gene profiling analysis in this EXAMPLE allowed the reconstruction of the pathways involved in calcitonin and parathyroid hormone signal transduction, triggered by protein-G-linked-receptor stimulation and their influence on cell cycle, as indicated by the changes observed in cyclins.
Animals.
A two-week subcutaneous treatment was carried out with salmon calcitonin (sCT), PTS893 or a combination of the two, each of which were dissolved in phosphate buffered saline (PBS) containing 9% autologous serum. Solvent was used as vehicle for the control group.
The animals used in this analysis were cynomolgus monkeys (Macaca fascicularis), supplied by Centre de Recherches Primatologiques, Port Louis, Mauritius. Two animals were used per group and sex. At the beginning of the treatment period, the animals were at least 24 months old, with a body weight of approximately 3 kg. Animals were kept under standard conditions for animal welfare. Animals were examined daily for mortality, food consumption and clinical observations. Body weight was recorded once per week. The dosages were 0 μg/animal/day (as the control), 50 μg/animal/day of salmon calcitonin and 5 μg/animal/day of PTS893.
In vivo examinations. No significant histopathological changes were observed. No relevant changes were observed other than a body weight decrease ranging from 8 to 12% in the salmon calcitonin group. A decrease in food consumption was also observed, although not always consistent with the decrease in body weight.
The animals to whom salmon calcitonin was administered presented with a decrease in body weight ranging between 8 to 12%, which can be attributed to a decrease in food consumption. An anorectic effect had previously been described for salmon calcitonin acting through amylin receptors Eiden S et al., J. Physiol. 541(pt3): 1041-1048 (2002); Lutz T A et al., Peptides 21 (2): 233-8 (2000). However, no signs of toxicity were observed here. Hormonal and lipid changes observed in this EXAMPLE are most probably related to a consequent metabolic adaptation.
No relevant changes in electrocardiograms (ECG) or blood pressure were observed.
Blood Sampling.
Animals were fasted overnight before blood collection but had free access to water. Blood samples were taken from a peripheral vein. Standard haematology and clinical chemistry analysis were performed once during pretest and at the end of the treatment period. Blood samples were collected from each animal at the same intervals as described for the clinical chemistry investigations. The serum samples were deep-frozen (approximately −80° C.) until analyses for hormone determination.
Clinical Chemistry and Hormone Determinations.
A slight anaemia was observed in all animals of the study, including the controls. This was attributed to the repeated blood sampling and not considered to be relevant.
Among the standard clinical chemistry tests performed, slight to moderate decreases in phosphorus and/or magnesium and a moderate to marked decrease in triglycerides were seen in the groups administered salmon calcitonin and PTS893.
No relevant changes were observed in the standard urinalysis tests performed.
The salmon calcitonin group presented with moderate decreases in serum somatomedin (S.MED, see TABLES 32 and 42).
Tissue Sampling.
Animals were killed by deep anaesthesia induced by intravenous injection of Pentothal®, followed by exsanguinations. All relevant tissues were sampled for histopathology and gene expression profiling. The following tissue samples were processed for analysis: liver, kidney, pituitary, muscle, bone, duodenum, spleen and trachea. Samples for histopathology were fixed in phosphate-buffered 10% formalin. Bone demineralization was performed with 10% formic acid. Tissue samples were embedded in Paraplast® and sectioned at 4 microns, for staining with haematoxylin and eosin. Samples for gene expression profiling were quickly frozen in liquid nitrogen immediately after excision, stored on dry ice and subsequently in a deep-freezer at approximately −80° C. until further use. All selected tissues for gene expression profiling were examined histopathologically.
Histopathology.
Histopathological examination of the tissues selected for gene profiling analysis exhibited a normal spectrum of incidental lesions which were in terms of severity and distribution of lesions not different to the controls in all groups of treatment.
A slightly higher incidence of inflammatory and regenerative changes in the kidneys of females administered salmon calcitonin was observed. These changes were not considered to be relevant, since no records of kidney toxicity exist after 40 years of calcitonin therapeutic use.
Bone sections were stained for osteonectin, osteopontin and osteocalcin and were evaluated histopathologically. Histomorphometry of the bone tissue was performed regarding parameters for bone resorption and synthesis (osteoid formation).
The osteonectin, osteopontin, and osteocalcin staining of the tibia showed no difference between the groups one (control) and two (salmon calcitonin). Osteonectin exhibited a major enlargement and deterioration of the epiphysial growth plate of animal no 2553 due to a severe non-treatment related pathological status (severe, subacute epiphysiolysis).
Histomorphometry of bone tissue was performed to determine parameters related to bone resorption and bone synthesis (osteoid formation).
The results (see, TABLES 34 and 35) showed that salmon calcitonin increased trabecular volume and thickness in about a 17% in tibia, but not in vertebra. PTS893 reduced the cortical thickness (18%) and increased the cortical porosity (54%) in tibia (T), but not in vertebra (V). In contrast, PTS893 induced an increase in osteoid volume (37% T, 213% V) and surface (49% T, 37% V), as well as an increase in the osteoblast surface (40% T, 24% V), in both tibia and vertebra, respectively.
Histomorphometry showed inconsistent results between tibial and vertebral bone, except for an increase in osteoid synthesis induced by PTS893. This effect is well documented for parathyroid hormone, when administered in a discontinuous way.
RNA Extraction and Purification.
A set of tissues was selected for gene expression profiling. These set included samples from kidney, bone, muscle, duodenum, pituitary and liver. Briefly, total RNA was obtained by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol®, Invitrogen Life Technologies, Carlsbad, Calif. USA) from each frozen tissue section and the total RNA was then purified on an affinity resin (RNeasy®, Qiagen) according to the manufacturer's instructions. Total RNA was quantified by the absorbance at λ=260 nm (A260 nm), and the purity was estimated by the ratio A260nm/A280nm. Integrity of the RNA molecules was confirmed by non-denaturing agarose gel electrophoresis. RNA was stored at approximately −80° C. until analysis. One part of each individual RNA sample was kept for the analysis of critical genes by means of Real-time PCR.
Hybridization Assay.
Transcript profiling by means of GeneChip® expression probe arrays was done as recommended by the manufacturer of the GeneChip® system (GeneChip Expression Analysis Technical Manual, Affymetrix Inc., Santa Clara, Calif. USA). HG-U95Av2 GeneChip® expression probe arrays (Affymetrix, Santa Clara Calif. USA) were used. Double stranded cDNA was synthesized with a starting amount of approximately 5 μg full-length total RNA using the Superscript Choice System (Invitrogen Life Technologies) in the presence of a T7-(dT) 24 DNA oligonucleotide primer. Following synthesis, the cDNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. The purified cDNA was then transcribed in vitro using the BioArray® High Yield RNA Transcript Labeling Kit (ENZO) in the presence of biotinylated ribonucleotides form biotin labeled cRNA. The labeled cRNA was then purified on an affinity resin (Rneasy®, Qiagen), quantified and fragmented. An amount of approximately 10 μg labeled cRNA was hybridized for approximately 16 hours at 45° C. to an expression probe array. The array was then washed and stained twice with streptavidin-phycoerythrin (Molecular Probes) using the GeneChip Fluidics. Workstation 400 (Affymetrix). The array was then scanned twice using a confocal laser scanner (GeneArray® Scanner, Agilent) resulting in one scanned image.
This resulting “.data-file” was processed using the Micro Array Analysis Suite version 4 (MAS4) program (Affymetrix) into a “.cel-file”. The “.cel file” was captured and loaded into the Affymetrix GeneChip Laboratory Information Management System (LIMS). The LIMS database is connected to a UNIX Sun Solaris server through a network filing system that allows for the average intensities for all probes cells (CEL file) to be downloaded into an Oracle database. Raw data was converted to expression levels using a “target intensity” of 150. The numerical values displayed are weighted averages of the signal intensities of the probe-pairs comprised in a probe-set for a given transcript sequence (AvgDiff value). The data were checked for quality and loaded into the GeneSpring® software versions 4.2.4 and 5 (Silicon Genetics, Calif. USA) for analysis.
Data Analysis.
Data analysis was performed with the Silicon Genetics software package GeneSpring version 4.2.1 and 5. Average difference values below 20 were set to 20. Various filtering and clustering tools in these programs were used to explore the data sets and identify transcript level changes that inform on altered cellular and tissue functions and that can be used to establish working hypotheses on the modes of action of the compound.
The threshold range for considering as up or down regulation was determined within the context of the biological interpretation of the study.
The information content of these data sets is a conjunction of numerical changes and biological information. The decision to consider a specific gene relevant was based on a conjunction of numerical changes identified by comparative and statistical algorithms and the relationship to other modulated genes that point to a common biological theme. The weight of that relationship was assessed by the analyst through a review of the relevant scientific literature.
Increase and decrease reported here refer to transcript abundance, unless specifically stated.
Gene Expression Profiling.
Multiorgan comparative gene profiling analysis was performed in the group administered salmon calcitonin at 50 μg/animal/day. The organs chosen for analysis were liver, kidney, pituitary, skeletal muscle, bone, duodenum, spleen and trachea.
In addition, the effect of PTS893 was assessed in bone.
Real-Time PCR.
Based on the DNA microarray data a set of transcripts was chosen for quantitative analysis by real time-PCR(RT-PCR).
Briefly, the method exploits the SyBr Green dye which intercalates into double stranded DNA. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the SyBr Green dye. Reactions are characterised by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of nucleic acid target, the sooner a significant increase in fluorescence is observed.
From each RNA sample, cDNA was made using an Applied Biosystem kit (Applied Biosystems # N808-0234) following the recommendation of the manufacturer. The PCR mixture was prepared using the SyBr Green Universal PCR Master Mix (Applied Biosystems #4309155) as follows: 5 μl cDNA template, 400 nM of each primer, 0.2 mM deoxynucleotide triphosphates, 1 mM MgC12 and 0.5 U Taq DNA polymerase, 5 μl SyBr Green PCR buffer and RNase free water up to a final volume of 50 μl. The PCR was performed using the ABI Prism 7700 Sequence Detection System, after a step at 95° C. for 10 min, the step-cycle program was performed for a total of 40 cycles as follows: 95° C. for 30 s, 60° C. for 1 min. A negative control was included: PCR reaction mixture with water in place of the cDNA sample.
The initial template concentration was determined based on the threshold cycle. The threshold cycle is the PCR cycle at which fluorescence is first detected above background and has been shown to be inversely proportional to the number of target copies present in the sample. Quantification was performed by calculating the unknown target concentration relative to an absolute standard and by normalizing to a validated endogenous control such as a housekeeping gene (β-actin). Results are presented as percentage of control, once the ratio between the numbers of molecule for the gene of interest divided by the number of molecule for beta-actin has been calculated.
Based on the DNA microarray data the following set of transcripts was chosen for quantitative analysis by RT-PCR: adhesion receptor CD44, angiopoietin, bone morphogenetic protein 5, carbonic anhydrase II, cartilage oligomeric matrix protein, cathepsin K, osteopontin, pre-pro-alpha-2 type I collagen, Spi-B and Y-box binding protein.
RT-PCR confirmed in most of the cases the changes observed in the gene profiling analysis, as it was the case for bone morphogenetic protein 5, carbonic anhydrase II, cathepsin K, cartilage oligomeric matrix protein, pre-pro-alpha-2 type I collagen, Spi-B and Y-Box binding protein. No changes were however detected in the level of expression of adhesion receptor CD44, angiopoietin-1 and osteopontin.
Analysis.
Calcitonin is known to exert an effect on the differentiation, survival and resorptive activity of osteoclasts, resulting in a decreased osteoclastic activity. Pondel M, Intl. J. Exp. Pathol. 81(6): 405-22 (2000). These effects could be reconstructed by multiorgan gene profiling (TABLE 39).
PU.1 is involved in the initial stages of osteoclastogenesis. Tondravi M M et al., Nature 386(6620): 81-4 (1997). CSF-1 is imperative for macrophage maturation; it binds to its receptor c-fms on early osteoclast precursors, providing signals required for their survival and proliferation. Teitelbaum SL, Science 289(5484):1504-1508 (2000).
Interestingly, PTS893 also regulates the genes implicated in osteoclast differentiation and survival, SPI1, CSF-1 and MMD. This osteoclast regulation has not been previously described.
Salmon calcitonin was shown to regulate the expression of the gene coding for osteoclast stimulating factor (OSF), which is an intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption. Reddy S et al., J. Cell Physiol. 177 (4): 636-45 (1998). This would imply an autocrine effect of salmon calcitonin in the regulation of the osteoclast function, which is described here for the first time.
In addition, salmon calcitonin seems to exert a paracrine regulation of the osteoclast resorptive activity, through the regulation of cystatin expression in the osteoblast. Carbonic anhydrase I, II, H+-ATPases and cathepsin K are the main effectors for dissolving bone mineral and matrix degradation. Blair H C et al., Biochem. (2002). Regulation of tubulins and PAK-4 genes can be related to the effect of calcitonin on osteoclast motility PAK 4. Zaidi M et al., Bone 30(5): 655-63 (2002); Jaffer Z M & Chemoff J, Intl. J. Biochem. Cell Biol. 34(7): 713-7 (2002).
These results show modulating effects of calcitonin on genes affecting the direct, autocrine, paracrine and endocrine regulation of the osteoblast function (TABLE 40). These data support the hypothesis that attributes a bone anabolic effect to calcitonin.
Three families of growth factors, the transforming growth factor betas (TGF-Ps), insulin-like growth factors (IGFs), and bone morphogenetic proteins (BMPs), are considered to be principal local regulators of osteogenesis. Bone morphogenetic proteins are thought to have their major effects on early precursor bone cell replication and osteoblast commitment. In contrast, TGB-Ps are thought to be the most potent inducers of committed bone cell replication and osteoblast matrix production, while IGFs appear to integrate and extend the effect of both factors. McCarthy T L et al., Crit. Rev. Oral Biol. Med. 11(4): 409-22 (2000). These results support the fact that both salmon calcitonin and PTS893 are able to regulate these local and systemic factors implicated in bone metabolism. The fact that salmon calcitonin regulates α2-HS glycoprotein (AHSG), which blocks TGF-β-dependent signalling in osteoblastic cells, also supports this role. Mice lacking AHSG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis. Szweras M et al., J. Biol. Chem., 277(22): 19991-19997 (2002).
Salmon calcitonin and PTS893 were also shown to modulate the expression of the genes coding for vascular endothelial growth factor (VEGF). VEGF is known for playing a key role in normal and pathological angiogenesis. The critical role of angiogenesis for successful osteogenesis during the endochondral ossification is well documented. VEGF indirectly induces proliferation and differentiation of osteoblasts by stimulating endothelial cells to produce osteoanabolic growth factors. Wang D S et al., Endocrinology 138(7): 2953-62 (1997). In addition, VEGF stimulates chemotactic migration of primary human osteoblasts, suggesting a functional role in bone formation and remodeling. Mayr-Wohlfahrt U et al., Bone 30 (3): 472-7 (2002).
The effects of parathyroid hormone on osteoblast for mediating both bone resorption and formation have been widely described. Swarthout J T et al., Gene 282(1-2):1-17 (2002). It was here possible to confirm the effect of PTS893 on cytokines like interleukin 6 (IL-6), which mediates the paracrine activation of osteoclast differentiation and activity. Greenfield E M et al., Life Sci. 65:1087-102 (1999). PTS893 also produced a strong up-regulation on nuclear receptors (steroid/thyroid family).
Both calcitonin and parathyroid hormone receptors belong to the G-protein receptor superfamily. After receptor stimulation, signal transduction is mediated by adenylate cyclase/cAMP/protein kinase, phospholipase C, phospholipase D, and MAPK (as a late effecter) pathways in the case of calcitonin, and by adenylate cyclase and phospholipase C in the case of parathyroid hormone. Gene profiling analysis allowed the reconstruction of these pathways, showing genes that were modulated by the treatment and that are localised at different levels of the signal transduction pathway.
Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signalling in osteoblasts. Smad pathway as well as Tob as one of their regulators were also identified as genes modulated by the sCT and PTS893 treatment, in agreement with the hypothesised effect of both compounds on BMP regulation of bone remodelling. Within this context, both compounds seem to exert a direct influence on cell cycle, since changes in cyclins and cyclin-related proteins could be also observed.
Both compounds regulate also synthesis and degradation of extracellular matrix components (TABLE 42).
Of particular interest is the regulation of the Y-Box binding protein (YB-1), which appears to be modulated by both treatments and in four out of six organs analysed in the salmon calcitonin group. YB-1 is a protein that interacts with a TGF-β response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-β addition to fibroblasts, suggesting a role for this protein in TGF-β signalling. Sun W et al., Matrix Biol. 20(8): 527-41 (2001).
In addition, salmon calcitonin and PTS893 regulated some aspects of the mineralization of the bone extracellular matrix, since changes in amelogenin, dentin and ectonucleotide pyrophosphatases were observed.
The compound of formula (II), 7-hydroxy-4-[4-(2-pyrrolidin-1-yl-ethoxy)-benzyl]-3-(2,4-dichloro-phenyl)-chromen-2-one, as shown:
The compound of formula (II) was developed for the treatment of bone-resorbing diseases generally, including osteoporosis, metastatic bone cancer, osteolytic lesions with orthopedic implants, Paget's disease, and bone loss associated with hyperthyroidism. The compound of formula (II) can also be used to treat other conditions associated with IL-6 including various cancers (e.g. breast cancer, prostate cancer, colon cancer, endometrial cancer, multiple myeloma, renal cell carcinoma, and cervical carcinoma) and arthritis (e.g. adjuvant-, collagen- and antigen-induced arthritis, particularly rheumatoid arthritis). See, published PCT patent applications WO 96/31206, WO 00/39120, WO 01/49673. The compound of formula (II) has estrogenic, antiestrogenic, antifertility and uterotropic activity.
The purpose of this EXAMPLE is to identify the “compound signature” of the compound of formula (II), to define a strategy for further development. The main concerns are the potential stimulation of the uterus and the induction of deep venous thrombosis (DVT). In addition, a goal of the EXAMPLE is to identify biomarkers for efficacy or risk assessment of the compound of formula (II).
Selective Estrogen Receptor Modulators (SERMs) are selective estrogen receptor modulators that act as agonists or antagonists depending on the target tissue. To increase our understanding of the direct tissue-specific effects of different SERMs and to elucidate the underlying molecular mechanisms, a study was started to perform a comprehensive in vivo gene expression profiling using DNA microarrays.
Gene expression microarray technology allows for the simultaneous detection and measurement of the expression of thousands of genes in a given cell or tissue sample in a single experiment. van de Rijn M & Gilks C B. Histopathology 44:97-108 (2004). This technique has significant advantages over previous methods for measuring gene expression, such as the reverse transcriptase-polymerase chain reaction and Northern blot analysis, which are limited to evaluation of small numbers of genes per experiment.
Because microarray technology enables rapid global gene expression profiling (GEP), it provides a powerful tool for analysis of a wide range of diseases, pharmacogenomic research, high throughput screening in drug discovery, and diagnostic screening for various diseases. Heller M J. Annual Review of Biomedical Engineering. 4:129-153 (2002). Gene expression profiling can be used to identify the genes whose altered expression may be directly related to causing a particular disease. By systematically evaluating the genes identified by such screens, researchers increase the likelihood of uncovering suitable targets for therapeutic intervention. In addition, the use of gene expression profiling in pharmacogenomics can be useful to identify potential undesirable effects of drug treatment by identifying changes in gene expression. Similarly, gene expression profiling may be useful in identifying patients that have a greater likelihood of achieving a meaningful outcome from a given therapeutic treatment. While methods for detecting changes in gene expression in response to therapy have been used, a method for scoring (or ranking) treatment agents based on their respective gene expression profilings has not been described.
Tamoxifen, a selective estrogen receptor modulator (SERM), is a nonsteroidal hormonally active agent that has agonistic and antagonistic effects in different target tissues; it is an antagonist in breast tissue and an agonist in the uterus. Kiang D T & Kennedy B J, Ann Intern Med. 87:687-690 (1997); Jordan V C & Allen K E, Eur. J. Cancer 16:239-251 (1980); Jordan V C et al., Breast Cancer Res Treat. 10:31-35 (1987); Gottardis M M et al., Cancer Res. 48:812-815 (1998); Fornander T et al., Lancet 1:117-120 (1989). Tamoxifen is an effective antitumour agent that is used in the treatment of estrogen-mediated breast cancer and as a chemopreventative agent; it decreases the risk of both invasive and noninvasive breast cancer. However, tamoxifen increases the risk for uterine hyperplasia and cancer through its proestrogenic effects on the endometrium. These undesired effects led to a search for SERMs with better safety profiles. Raloxifene, a second generation SERM that has a more favorable therapeutic and safety profile than tamoxifen, was subsequently developed. Cohen F J et al., Obstet. Gynecol. 95:104-110 (2000); Ring J et al. (The Desloratadine Study Group) Int J Dermatol. 40:1-5 (2001); Fugere P et al., Am J Obstet. Gynecol. 182:568-574 (2000). Raloxifene has a reduced antagonistic effect on the endometrium compared with tamoxifen is not associated with endometrial cancer risk.
Because various SERMs exhibit differential estrogenic effects on the uterus, we used this treatment effect as a model system to test and validate a new, unique method of scoring the relative pharmacological and toxicological activity of different therapeutic agents according to their gene expression profilings. Gene expression profiling has been used for dissecting the molecular mechanisms of physiology and disease in the uterus using tissue cultures, transgenic mice, and normal rats. However, few studies have been performed in primates and little is known of the gene expression profiling in monkey uterine tissue. Ace C I & Okulicz W C, Reprod Biol Endocrinol. 2:54 (2004); Marvanova M et al., FASEB J. 17:929-931 (2003); Zou J et al., Genome Biol. 3:research0020.1-research0020.13 (2002). Furthermore, no studies have incorporated a method for scoring (or ranking) therapeutic agents based on their respective gene expression profilings. If such a ranking system where available, it could have broad utility for a wide spectrum of therapeutic areas, and represent a significant advance for the discovery and development of new pharmacological entities. In the current EXAMPLE, changes in uterine gene expression profilings were explored in normal and OVX cynomolgus monkeys following tamoxifen and raloxifene treatment; results are compared with those obtained after treatment with estradiol. The estrogenic potency of each agent was then attributed a score based in its effect on uterine gene expression.
Animals.
Female sexually mature cynomolgus monkeys (Macaca fascicularis obtained from R. C. Hartelust B V, Tilburg, the Netherlands) approximately 48 months old were subjected to surgery (ovarectomy [OVX] or sham) 10 weeks prior to treatment and were treated for parasitic arthropods and helminthes, subjected to a tuberculin testing, and acclimated to the treatment facility for at least 14 days before beginning treatment.
All animals received tap water, filtered with a 0.22 μm filter, ad libitum. Approximately 180 g of OWM pelleted diet (Dietex France, SDS, Saint Gratien, France) was administered daily to each animal at least one hour after dosing except on the last day of treatment where the animals were fasted. Each animal received two fruits or vegetables daily.
Surgical Procedures. The ovariectomized cynomolgus monkey (Macaca fascicularis), which has been validated as a useful model predictive of outcomes in human clinical trials of estrogen and SERMs (Cline J M et al., Toxicol Pathol. 29:84-90 (2001)), was used as a model of estrogen deficiency. Animals were ovariectomized approximately 10 weeks before the first day of treatment. The ovaries were removed following anesthesia with a combined intramuscular injection of xylazine (Rompun®: 0.4 mL/animal, Bayer Pharma Division Sante Animale, Puteaux, France) and ketamine hydrochloride (Imalgéne®: 0.6 mL/kg, Mérial, Lyon, France). Sham-operated animals were subjected to the same surgical procedure, except for the removal of ovaries.
Estradiol Assay.
Estradiol serum levels were determined in each animal approximately 2 weeks after surgery in order to verify the effect of ovarectomy. Venous blood samples (approximately 1.5 mL) of unfasted animals were collected in tubes without anticoagulant and analyzed using radioimmunoassay (Sorin, Ecole Nationale Vétérinaire de Lyon, France).
Treatment Protocol.
Ovariectomized and sham-operated animals were divided into groups of 4 and treated as outlined below.
1An aqueous solution of 0.5% carboxymethylcellulose was used as a vehicle.
2Ethinyl estradiol was prepared in corn oil.
3Tamoxifen and raloxifene were prepared in an aqueous solution of 0.5% carboxymethylcellulose.
Tissue Preparation.
Gene expression was determined in the pituitary and uterus at the end of drug therapy. Tissues to be analyzed (pituitary glands and uterus) were excised, snap frozen in liquid nitrogen, and stored at −80° C. until RNA extraction was performed.
DNA Microarray Analysis.
Total RNA was obtained by acid guanidinium isothiocyanate-phenol-chloroform extraction (Trizol; Invitrogen Life Technologies, San Diego, Calif., USA) and purified on an affinity resin column (RNeasy; Qiagen, Hilden, Germany) according to manufacturer instructions. Chomczynski P & Sacchi N. Anal Biochem. 1987; 162:156-159. DNA microarray experiments were conducted as recommended by the manufacturer of the GeneChip system (Affymetrix, Inc. 2002) and as previously described. Lockhart D J, Dong H, Byrne M C, et al. Nat. Biotechnol. 1996; 14:1675-1680.
Previous studies have proven the validity of cross-species DNA chip analysis (Hacia J G et al. Nat Genet. 18:155-158 (1998)), therefore, the human gene expression probe arrays HGU133A (Affymetrix, Santa Clara, Calif., USA) containing 22,283 probe sets interrogating primarily annotated human genes were used. One GeneChip was used per tissue, per animal. The resulting image files (.dat files) were processed using the Microarray Analysis Suite 5 (MAS5) software (Affymetrix, Inc.). Tab-delimited files were obtained containing data regarding signal intensity (Signal) and categorical expression level measurement (Absolute Call).
Quantification of Drug Potency Relative to Specific Drug Action; GEP Scoring Axis.
To define a reference axis representing the desired pharmacological/toxicological action, we used a priori knowledge of 2 groups representing or approximating the extremes of the condition under study. These consisted of the OVX group (OVX untreated animals representing the one extreme) and OVX+estradiol (OVX animals treated with estradiol, an agent know to exercise a well-defined action, representing the other extreme). These two groups were used to quantitatively scale the axis. Other compounds were then positioned on this axis whereby the drugs could be ranked with regard to specific aspects of a pharmacological/toxicological action.
Selection of Features.
Generally, the data derived from an individual microarray, proteomic, or metabolite profiling experiment can be considered as a fingerprint. However, in practical terms, many of the features (e.g. genes) do not represent relevant information and only confound the analysis of the outcomes of such experiments. Thus, selection and elimination of the appropriate features is an important aspect of this methodology and the quantitative nature of this approach depends on the features used to define the axis of drug action.
In the case of a well-defined condition, it is possible to invoke a priori knowledge on the selection of features. If the molecular biology of the specific drug action is known, the features (e.g. genes, metabolites) to be used for the definition of the axis can be defined a priori. In the case of a less-well-defined condition, it is possible to use univariate filters (in this case, gene intensity >50, ≧1.5-fold change in expression vs. controls [OVX], and statistical significance and statistical methods to identify and exclude non-informative features.
Data Pretreatment.
Issues of transformation, centering (variable-wise), and scaling of the data were considered. In the case of microarrays, the data were log10-transformed and probe set-wise centered. Generally, no scaling was applied. When using only a small number of biologically identified features, these were scaled to unit variance.
Principal Component Analysis.
To calculate the axis of drug action, a principal component axis (PCA) was performed using only the 2 treatment groups that were at the extremes (the untreated OVX animals vs. the estrodiol-treated OVX animals). Massart D L et al., Handbook for Chemometrics and Qualimetrics, Part a. (Elsevier; 1997); Jolliffe I T. Principal Component Analysis. 2nd Edition (Springer; New York, N.Y., 2002). Only 1 principal component (PC) was calculated, which was a weighted linear combination of all features in the input data pointing in the direction of maximum variance. The weights of the probe sets in this linear combination were called “loadings” and their absolute value is a measure of their importance in explaining the difference between the 2 groups. Every measurement in the experiment (e.g. microarray) could then be represented by its coordinate (score) on the PC. Alternatively, to obtain an even better separation between the groups, the PLS-DA (Partial-Least-Squares Discriminate Analysis) method with a 0/1 dummy variable as response vector was used. Barker M & Rayens W. Journal of Chemometrics 17:166-173 (2004). Massart D L et al., Handbook of Chemometrics and Qualimetrics, Part b. (Elsevier; 1997). The next step was identification of the features that were important for the definition of the axis. This was based on the magnitude of their loadings and their statistical significance using error estimates based on cross-validation and/or normal probability plots of the loadings. Hunter W G & Hunter J S. Statistics for Experimenters: an Introduction to Design, Data Analysis, and Model Building. 1st Edition (Wiley; 1978). This enabled calculation of a new PCA model using only the important features.
If a pharmacological or toxicological action affected multiple biochemical pathways that were represented by grossly different numbers of features in the measurement, block scaling was used. Eriksson L et al., Multi-and Megavariate Data Analysis-Principles and Applications. (Umetrics Academy; 2001). This attributed the same influence to all pathways (groups of features) in the calculation of the PCA model. Hierarchical PCA could be applied to identify the most important pathways of drug action. Eriksson L et al., J Comput Aided Mol. Des. 16:711-726 (2002).
Applying the PCA Model.
Using the loadings of the final PCA model the scores of all measurements in the other treatment groups (tamoxifen and raloxifene) were calculated. The medians (or means) of the scores of the two extreme groups were used to scale the axis of drug action (0-100%). The score (estrogenic potency) of a treatment (drug/dose) was expressed on this scale for individuals or group averages (median or mean).
Statistical Analyses.
Score variances were used to estimate confidence intervals (Sachs L. Angewandte Statistik, 10. (Springer; Berlin, Germany, 2002)), and significant differences between groups were assessed with various statistical tests (e.g. t-test, u-test). Zar J H. Biostatistical Analysis. 4th Edition (Prentice Hall; 1998).
Comparative GEPs Following Estradiol, Tamoxifen, and Raloxifene Treatment. Pituitary Gland.
Ovariectomy induced the expected significant up-regulation of the genes encoding luteinising hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary gland (TABLE 45).
Compared with sham surgery, the transcript levels were 7.6- and 6.2-fold greater (LH and FSH, respectively) in untreated OVX animals. Administration of estradiol reversed the effects of ovariectomy, and decreased the expression of LH and FSH transcripts 16.4- and 73.4-fold, respectively, well below normal physiological values. Tamoxifen had a weak estrogenic effect, downregulating pituitary LH and FSH gene expression by 2.4- and 2.7-fold in OVX monkeys. Raloxifene had a minimal effect on gonadotropin expression, reducing LH and FSH transcripts by 1.2- to 1.3-fold.
Uterus.
The effect on uterine gene expression was subdivided by grouping genes in different logical categories; signal transduction, growth factors, extracellular matrix, and as a group, genes involved in cell-cycle, transport, and redox.
Signal Transduction Inducers.
Overall, an increased expression of genes encoding signal transduction proteins was observed following estradiol treatment in OVX monkeys (TABLE 46).
Progesterone receptor, secreted frizzled-related protein 1, secreted frizzled-related protein 4, Dishevelled associated activator of morphogenesis 1, wingless-type MMTV integration site (family, member 2B; wnt2B), and integrin (beta 5) were increased 9.8-, 7.1-, 4.2-, 3.1-, and 2.6, and 1.7 fold, respectively, compared with untreated OVX controls. These same genes were up regulated by 1.2- to 4.5-fold following tamoxifen therapy. In contrast raloxifene therapy was associated with more modest changes in gene expression for these same signal transduction proteins (0.9- to 1.9-fold). The remaining genes examined were all downregulated by estradiol, tamoxifen (except for LDL receptor adaptor protein), and raloxifine.
Growth Factors.
Estradiol increased the expression of several genes encoding growth factors (GFs) as outlined in TABLE 47.
The most prominent effects were observed for insulin-like GF binding protein 2 (36 kDa) (7.1-fold increase), and insulin-like GF 1 (somatomedin C) (5.3- to 6.9-fold increase), compared with OVX controls. Genes encoding these growth factors were also upregulated by 2.7- and 3.7- to 4.8-fold following tamoxifen therapy. Raloxifene caused minor increases of approximately 2-fold, similar to the sham group.
Estradiol also upregulated insulin-like growth factor binding protein 5, latent transforming growth factor beta binding protein 1, and cysteine-rich angiogenic inducer 61 by 5.2-, 3.6-, and 3.5-fold, respectively, compared with OVX controls. In contrast tamoxifen and raloxifene were associated with only modest changes in genes encoding these growth factors, similar to the levels observed in the sham group (TABLE 46).
Extracellular Matrix.
A number of diverse extracellular matrix proteins were upregulated by estradiol, as outlined in TABLE 47. Collagen type 1 alpha 2, collagen type III alpha 1, collagen type 1 alpha1, and collagen type IV alpha 1 were upregulated by estradiol treatment 13.2-, 6.8-, 6.6-, and 4.3-fold, respectively compared with OVX controls. Tamoxifen therapy also upregulated genes encoding these matrix proteins by 6.1-, 3.4-, 3.5-, and 3.2-fold, respectively. For the majority of the rest of the genes in this category, after treatment with tamoxifen or raloxifene, overall expression was brought back up to levels not that dissimilar to sham controls.
Transport, Redox, and Cell Cycle.
Estradiol increased expression of a variety of transport genes (solute carrier family 2 [facilitated glucose/fructose transporter]; member 5 ATPase; Na+/K+ transporting, beta 3 polypeptide; solute carrier family 2 [facilitated glucose/fructose transporter], member 5; solute carrier family 25 [mitochondrial carrier; adenine nucleotide translocator], member 5; ATPase, H+ transporting, lysosomal 38 kDa, VO subunit d isoform 1; ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c [subunit 9] isoform 3; solute carrier family 22 [organic cation transporter], member 1-like ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c [subunit 9] isoform 3; chloride intracellular channel 4) by 2- to 7-fold; genes encoding redox proteins (SET domain bifurcated 1, peroxiredoxin 1, and cytochrome c oxidase subunits VIa and Wk.) by approximately 2- to 3-fold; and genes encoding cell cycle proteins (CDC28 protein kinase regulatory subunit 2) by up to 4-fold in estradiol treated animals compared with OVX controls. Estrodial treatment downregulated expression of the gene for the redox protein, thioredoxin interacting protein, 3-fold and the gene for the cell cycle protein, cyclin-dependent kinase inhibitor 1C (p57, Kip2), by 2- to 4-fold compared with OVX control. Although slightly less potent, tamoxifen exerted similar effects whereas raloxifene had little or no effect on any of the transporter, redox, and cell cycle genes studied.
Estrogen-Induced Genes.
Several estrogen-induced genes (brain creatine kinase; cytoplasmic dynein; hippocalcin-like 1; light polypeptide 1; brain prostaglandin D2 synthase 21 kDa; hexokinase 1; and osteoblast cadherin 11, type 2) were upregulated by estradiol by 3- to 10-fold compared to OVX control. However, the significance of these changes to uterine physiology and pathology has yet to be defined.
Ranking of Drug Activity According to Uterine GEP.
To determine the relative estrogenic potency and score for a given estrogenic treatment, a potency axis was constructed by assigning a median GEP score of “0” for OVX controls (no estrogen) and a median GEP score of “100” for estradiol treated animals (maximum estradiol effect). The respective GEP scores for tamoxifen and raloxifene were plotted to determine their relative estrogenic potencies.
Application of different filtering parameters—gene intensity >50, statistically different change from OVX controls, and genes showing >1.5-fold changes from OVX control values—permitted ranking of the relative treatment potencies according to defined gene expression profiling (GEP) criteria. These approaches yielded more useful information than unsupervised GEP data analysis.
Of the 22,283 probes assayed per chip 225 individual genes showed statistically significant greater than 1.5-fold changes in expression level compared with OVX controls. Considering only this group of genes, tamoxifen showed a strong estrogenic response with a score of 59 and raloxifene had a weak estrogenic score of 23. In contrast, sham animals exhibit a baseline estrogenic score of 48.
When considering fold changes alone and disregarding statistical significance, 673 genes showed greater than 1.5-fold change in expression level compared with OVX controls. Tamoxifen exhibited a strong estradiol effect, with a score of 71, raloxifene still had a weak estrogenic score of 34, and baseline for sham animals was 66.
A further subanalysis was done using 123 genes that showed statistically significant greater than 1.5-fold changes in expression and are thought to have a role in uterine function. According to this analysis, tamoxifen retained a high estrogenic score of 69, raloxifene still had a weak estrogenic score of 20, and the sham baseline was 44. Using this approach, further subanalysis of specific gene groups showed that the estrogenic effects of tamoxifen (scores of 61 to 87) were consistently greater than those of raloxifene (scores of −11 to 32) in each category.
Discussion.
Previous studies have established that the OVX cynomolgus monkey model of estrogen deficiency is relevant to humans with respect to hormonal effects on the uterus. Cline J M et al., Toxicol Pathol. 29:84-90 (2001). Data from these studies showed increased endometrial hyperplasia following treatment with estradiol or tamoxifen, and were confirmed in the present EXAMPLE. Moreover, we showed that in this model, estradiol reversed the effects of OVX on gonadotropin gene upregulation in the pituitary (TABLE 45), providing further evidence that the OVX monkey represents a valid model for evaluating the gene expression profiling (GEP) of estradiol and SERMs.
Overall, the gene expression data reported in the present EXAMPLE correlated with the histopathological effects of estradiol and SERMs on the uterus. Experimental and clinical studies have demonstrated the unequivocal proliferative effects of estrogen and tamoxifen on the uterus and uterine tumour cells. Gottardis M M et al., Cancer Res.48:812-815 (1988). Tamoxifen induced an estrogen-like gene expression profiling in the uterus of OVX monkeys that was consistent with uterine cystic hyperplasia, stromal fibrosis, and increased progesterone receptor expression observed in ovariectomized macaques. Cline J M et al., Toxicol Pathol. 29:84-90 (2001). The minor changes in uterine GEPs of OVX monkeys following raloxifene therapy were in accord with studies showing its lack of pathological effects on the human uterus. Cohen F J et al., Obstet. Gynecol. 95:104-110 (2000).
In the uterus, the proliferative effects of tamoxifen are mediated in part by GFs. Increased expression of IGF-1 and related binding proteins following estrogen therapy is well documented and may be important for local paracrine stimulation of uterine tissue growth. Norstedt G et al., Acta Endocrinologica (Copenh).120:466-472 (1989); Stygar D et al., Reproductive Biology and Endocrinology. 1:40 (2003). Induction of cysteine-rich angiogenic inducer “61” and transforming growth factor beta genes has also been described in response to estrogen, and both growth factors appear to mediate endometrial growth and hyperplasia. Sampath D et al., Endocrinology. 142:2540-2548 (2001); Takahashi T et al., Cell Growth Differ. 5:919-935 (1994); Sartor B M et al., Reprod Toxicol. 9:225-231 (1995). These observations support data from the present EXAMPLE in which estradiol increased the expression of these growth factors in the uterus (TABLE 46).
Recent studies using gene expression profiling in human endometrium have identified an array of genes and gene families associated with the implantation process, which involves a complex interaction between the embryo and the maternal endometrium. Locally secreted wnt proteins initiate a cell signaling pathway that involves activation of membrane-associated frizzle receptors (FzRs), which in turn activate Dishevelled (Dvl) and regulation of nuclear gene transcription required for implantation. Frizzle-related proteins (FRPs) modulate wnt signaling at the wnt-FzR-ligand binding site, and secreted FRP4 (also know as FrpHE) inhibits wnt signaling. Although wnt genes are controlled by sex steroids, including estrogen, the relevance of these findings to estrogen-induced uterine growth is not known. Tulac S et al., J Clin Endocrinol Metab. 88:3860-3866 (2003). According to studies in cancer cell lines, wnt2B signaling appears to play a role in tumourigenesis. Katoh M, Int Mol. Med. 8:657-660 (2001); Ricken A et al., Endocrinology. 143:2741-2749 (2002). Moreover, increased expression of secreted FRP1 and secreted FRP4 was observed in human uterine leiomyomas, the uterus of OVX rats administered estradiol, and human proliferative endometrium. Fujita M et al., J Mol Endocrinol. 28:213-223 (2002); Fukuhara K et al., J Clin Endocrinol Metab. 87:1729-1736 (2002). Whereas stromal FRP4, which is upregulated by estrogen, was involved in endometrial proliferation, secreted FRP1 was associated with antiapoptotic and antitumour effects. These studies are relevant to data reported in the present EXAMPLE, which showed that increased uterine expression of genes encoding wnt2B, Dvl, and secreted FRP1 and FRP4 following estradiol therapy (TABLE 46). The data suggest estrogenic effects on the uterus may involve the wnt signaling pathway and its regulation by secreted FRP4. Tulac S et al., J Clin Endocrinol Metab. 88:3860-3866 (2003).
Long-term treatment with conjugated equine estrogens upregulates progesterone receptors in the endometrial stromal tissue of OVX cynomolgus macaques. Wang H et al., Reprod Biol Endocrinol. 1:7 (2003). Estradiol increased progesterone receptor gene expression in the present EXAMPLE (TABLE 46), further validating the OVX monkey model for gene expression profiling.
Estradiol also increased uterine expression of other functional classes of genes, including extracellular matrix proteins (TABLE 47) and genes generally associated with cell proliferation (e.g., cell cycle, redox, and transport; TABLE 48). These effects are likely related to estrogen-induced uterine hyperplasia, which is characterized by increased cell proliferation and tissue growth. Cline J M et al., Toxicol Pathol. 29:84-90 (2001).
Analysis of the gene expression profiling (GEP) profiles associated with estradiol and SERMs revealed important differences in the molecular mechanisms through which these agents affect the uterus. Thus, tamoxifen's gene expression profiling suggests that it acts primarily via estrogenic stimulation of growth factors, signal transduction mechanisms, extracellular matrix protein synthesis, and genes related to cell proliferation in the uterus. In contrast, raloxifene exhibited weak estrogenic activity with little or no effect on these molecular pathways as indicated by its gene expression profiling.
Further analysis of the gene expression profiling (GEP) data involved the development of a novel scoring system for ranking the relative estrogenic effects of tamoxifen and raloxifene. Across each of the seven different classes of genes expressed, tamoxifen ranked as the most estrogenic agent, with scores ranging from 69 to 87, compared with 100 for estradiol. Raloxifene was much less estrogenic by these criteria as shown by its low scores ranging from −11 to 32, compared with 100 for estradiol.
In conclusion, gene expression profiling (GEP) using DNA microarray combined with the novel method of ranking therapeutic agents described here is a powerful technique for defining and predicting the relative pharmacological and toxicological actions of new chemical entities, and represents a new paradigm for drug development in the pharmaceutical industry. Moreover, this procedure has enormous potential for identifying efficacy and safety biomarkers that can be used to refine the drug screening process, reducing the time and costs required for taking a drug from discovery to clinic.
Introduction and Summary.
The aim of this EXAMPLE is to further evaluate the effect of FGF23CTP on angiogenesis as shown in EXAMPLE III by using a hypoxic vascular retinopathy mouse model.
Methods.
Seven days old C57/B16J mice are put together with their nursing parent in a sealed container ventilated by a mixture of oxygen and compressed air to a final oxygen concentration of 75%±2% until day 12. Returned at room air, the animals develop a relative ischemia of the retina with neovascularization starting as early as day 14.
On day 12, when animals returned from the oxygen incubation, a volume of 2.0 μl is injected in the eye using a glass pipette with a diameter of about 150 μm at its tip. The right eye is injected with 6 microgram (μg) FGF23CTP (3 μg/tip and the left eye of the animal with PBS as individual control.
For the intravitreal injection of a substance at day 12, mice are anesthetized by inhalation of isoflurane. Five days later the animals are sacrificed after perfusion with dextran-fluorescein and retinal flatmounts are prepared. The coded whole mounts allow to evaluate the vascular changes of the retinal blindly. The proliferation score includes quantification of the proliferation including the papilla, vascular development, vasoconstriction, retinal bleeding and tortuosity of the vessels, features also seen in human retinal disease.
Animals.
In total, 44 animals are used for injection with FGF23CTP and PBS. Of those, 8 animals died during hyperoxia or after injection, 2 animals are not perfused, 5 animals are paraffin embedded for evaluation of neurons, of which only three could be examined, and from 29 of these animals retinal flatmounts are prepared.
Evaluation of the Angioproliferative Changes.
Evaluation of the angioproliferative changes in the retinal preparations is performed in a masked way on coded preparations. Retinopathy scoring system is adapted from Higgins, R. D., et al. (J. AAPOS 3: 114-116 (1999)) that was developed after modification of a scoring system used clinically in the neonatal intensive care unit. The following features are taken into consideration: neovascularisation of the optic disc, blood vessel tufts formation, large clusters of blood vessel tufts, central vasoconstriction and tortuosity of the vessels. Retinal haemorrhages are not taken into account as they may also result from the intraocular injection.
For each criteria, a defined number of points (P) is assigned, which are summed up to a total proliferation score. The higher the score, the worse the hypoxia induced retinopathy. In detail:
Neovascularisation of the optic disc: either 1 or 0 points are assigned in case optic disc proliferation is measured (1 P) or absence (0 P).
Blood vessel tufts formation: the score for blood vessel tufts is determined by dividing every clock hour of the preparation surface in 4 zones: Zone 1 (optic disc zone) is evaluated in addition; Zone 2: one point for every wing (maximal 4 P); Zone 3: one point for every clock hour (maximal 12 P); and Zone 4: one point for every clock hour (maximal 12 P). Thus, a maximum of 28 points is measured provided that no avascular areas exist, and a maximum of 12 points is calculated in case of complete avascularization.
Large clusters of blood vessel tufts: every large cluster of blood vessel tufts covering more than 3 sectors is counted as one point. Thus, maximal 8 points can be measured provided that no avascular areas exist, and maximal 4 points are assignable for complete avascularization.
Central vasoconstriction: 1 point is assigned to a vasoconstriction of zone 1 of greater than 50%; 1 point is allocated if the vasoconstriction is greater than 50% of every wing of zone 2 and 3. Thus, maximal 1 point can be assigned to zone 1, and maximal 4 points each for zones 2, 3 and 4. Since so far no vasoconstriction is ever observed for zone 4, a maximum of total 9 points (without zone 4) can be allotted.
Tortuosity of the vessels: 2 points are assigned if less than one-third of the vessels are tortuous; if the amount of tortuous blood vessels is between one-third and two-thirds, 4 points are allocated, and in case more than two-thirds of the vessels are tortuous, than 6 points are given.
The theoretical maximum of points is never reached, e.g. Zone 4 never shows avascular zones. Proliferation in avascular zones is never observed. This is in principle a mutual exclusion of criteria; the existence of vascular proliferations has a higher impact on the retinopathy score than large avascular areas. Therefore, the maximum score ever counted with this system is 38 points using a substance which is considered to enhance vascular proliferation.
The proliferation score relating to vascular changes on retinal flatmounts for individual animals treated with FGF23CTP (right eye) and PBS control (left eye) are measured as described above. The severity of retinopathy varies from animal to animal. TABLE 52 summarizes the determined proliferation score.
The proliferation scores of all animals are further displayed as box plot in
The proliferation scores for the separate factors are presented in TABLE 54, and the respective paired t-tests in TABLE 55 to 57.
Evaluation of the Toxicity.
From the five mice originally planed for evaluation of neuronal damage after injection of FGF23CTP in the right eye and PBS in the left eye, only three are studied due to damage to the lens in one eye and fixation problems in another animal.
The total thickness of the outer nuclear layer, the inner nuclear layer and the ganglion cell layer is measured using a standardized magnification after paraffin embedding and hematoxylin and eosin (HE)-staining. Three sections of each eye are measured at three locations each. TABLE 58 shows the result of the measurements in mm using a x200 magnification. Sections are encoded for evaluation.
Based on the measurements performed on the three animals, FGF23CTP is found not to induce any neuronal damage in the eye. In detail, there is no marked difference in the thickness of the neuronal layers of the inner nuclear layer, the outer nuclear layer or the ganglion cell layer five days after the injection of either FGF23CTP or PBS.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. In addition, all GenBank accession numbers, Unigene Cluster numbers and protein accession numbers cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each such number was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.
| Number | Date | Country | |
|---|---|---|---|
| 60518073 | Nov 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13152750 | Jun 2011 | US |
| Child | 13656084 | US | |
| Parent | 10578470 | Apr 2008 | US |
| Child | 13152750 | US |
