Patent Application
20130209438
The presently disclosed subject matter pertains to the use of prostatic acid phosphatase (PAP) compositions for the treatment of pain.
Pain affects more Americans than heart disease, diabetes and cancer combined. In fact, about 50 million Americans suffer from chronic pain and spend about $100 billion for treatments per year. Unfortunately, many of the strongest available analgesics have serious side-effects including addiction, dependence and increased risk of heart attack and stroke. Moreover, many chronic pain conditions cannot be effectively treated with existing medications. Considering the revenue of drugs like CELEBREX® ($2.8 billion in 2004; G.D. Searle & Co., Skokie, Ill., United States of America) and VIOXX® ($1.4 billion in 2004, Merck & Co., Inc., Whitehouse Station, N.J., United States of America), an effective treatment for chronic pain would significantly benefit human health. Accordingly, there is an unmet need for effective pain treatments.
In some embodiments, a method is provided for treating pain in an animal by administering a composition or a pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active fragment, variant or derivative thereof, or a therapeutically effective amount of an activity enhancing PAP modulator. In some embodiments, all types of pain are treated including, but not limited to, pain characterized by one or more of: chronic pain, chronic inflammatory pain, neuropathic pain, chronic neuropathic pain, allodynia, hyperalgesia, nerve injury, trauma, tissue injury, inflammation, cancer, viral infection, Shingles, diabetic neuropathy, osteoarthritis, burns, joint pain or lower back pain, visceral pain, trigeminal neuralgia, migraine headache, cluster headache, headache, fibromyalgia and pain associated with childbirth.
In some embodiments, a method is provided for treating an animal for a disorder characterized at least in part by an excess of lysophosphatidic acid, comprising administering to the animal a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active fragment, variant or derivative thereof, or a therapeutically effective amount of an activity enhancing PAP modulator.
In some embodiments, the animal is a human.
In some embodiments, the PAP is selected from the group consisting of human PAP, bovine PAP, rat PAP and mouse PAP, and active fragments, variants and derivatives thereof.
In some embodiments, the PAP or the active fragment, variant or derivative thereof, comprises one or more modifications selected from the group consisting of one or more: conservative amino acid substitutions; non-natural amino acid substitutions, D- or D,L-racemic mixture isomer form amino acid substitutions, amino acid chemical substitutions, carboxy- or amino-terminus modifications, conjugation to biocompatible molecules including fatty acids and PEG and conjugation to biocompatible support structures including agarose, sepharose and nanoparticles.
In some embodiments, the PAP is obtained by recombinant methods.
In some embodiments, the PAP or the activity enhancing modulator of the PAP is administered via one or more of injection, oral administration, a surgically implanted pump, stem cells, viral gene therapy, naked DNA gene therapy. In some embodiments, the injection is intravenous injection, epideral injection, or intrathecal injection. In some embodiments, the administration is via intrathecal injection of PAP-expressing embryonic stem cells. In some embodiments, the administration is by intrathecal injection about once every 3 days. In some embodiments, the administration is in combination with one or more of adenosine, adenosine monophosphate (AMP) or an AMP analogue. In some embodiments, the administration is in combination with a known analgesic. In some embodiments, the known analgesic is an opiate. In some embodiments, the administration is via viral gene therapy using a retroviral, adenoviral, or adeno-associated viral vector transfer cassette comprising a nucleic acid sequence encoding the PAP or active variant or fragment thereof.
In some embodiments, a method is provided for treating cystic fibrosis in an animal, the method comprising administering to the animal a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active fragment, variant or derivative thereof, or a therapeutically effective amount of an activity enhancing PAP modulator. In some embodiments the administering is by aerosolizing in the lungs.
In some embodiments, a method is provided for increasing levels of adenosine in the lungs of an animal having a disorder characterized at least in part by a deficiency in adenosine or adenosine receptor function, the method comprising administering to the animal a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active fragment, variant or derivative thereof, or a therapeutically effective amount of an activity enhancing PAP modulator.
In some embodiments, an isolated PAP peptide is provided. The peptide can be selected from the group consisting of human PAP, cow PAP, rat PAP and mouse PAP, and fragments, variants, and derivatives thereof. In some embodiments, an isolated nucleotide sequence is provided that encodes the PAP peptide. In some embodiments, an expression vector is provided that comprises the nucleotide sequence. In some embodiments, a host cell is provided that comprises the expression vector. In some embodiments, a retroviral, adenoviral, or adeno-associated viral vector transfer cassette is provided that comprises a nucleotide sequence encoding the PAP or active variant or fragment thereof.
In some embodiments, a composition is provided comprising the PAP peptide, or an active fragment, variant or derivative thereof, wherein the composition is prepared for administration to animals, or as a pharmaceutical formulation for administration to humans.
In some embodiments, a method is provided for screening for a small molecule modulator of PAP activity by measuring the activity of a PAP in the presence and absence of a candidate small molecule and identifying as PAP modulators the candidate small molecules that cause either an increase or a decrease in the PAP activity.
In some embodiments, a kit is provided for the treatment of pain in animals, comprising a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active fragment, variant or derivative thereof, and a surgically implantable pump apparatus for delivery of PAP to local tissue.
In some embodiments, a method is provided for diagnosing an individual's response to a pain medicine, comprising identifying one or more single nucleotide polymorphisms (SNPs), insertions, deletions and/or other types of genetic mutations in and around a PAP genomic locus in the individual; and correlating the SNPs, insertions, deletions and/or other types of genetic mutations with a predetermined response to the pain medicine.
In some embodiments, a method is provided for diagnosing an individual's threshold for pain, comprising identifying one or more single nucleotide polymorphisms (SNPs) insertions, deletions and/or other types of genetic mutations in and around a PAP genomic locus in the individual; and correlating the SNPs, insertions, deletions and/or other types of genetic mutations with a predetermined threshold for pain.
In some embodiments, a method is provided for diagnosing an individual's propensity to transition from acute to chronic pain, comprising identifying one or more single nucleotide polymorphisms (SNPs) insertions, deletions and/or other types of genetic mutations in and around a PAP genomic locus in the individual; and correlating the SNPs, insertions, deletions and/or other types of genetic mutations with a predetermined threshold for pain.
In some embodiments, a method is provided for diagnosing an individual's response to a pain medication, threshold for pain or propensity to transition from acute to chronic pain, the method comprising correlating differences in PAP expression levels in the individual and a control population, and correlating the extent of differential expression with a predetermined response to a pain medication or a predetermined threshold for pain.
Accordingly, it is an object of the presently disclosed subject matter to provide methods and compositions for the treatment of pain and cystic fibrosis. These and other objects are achieved in whole or in part by the presently disclosed subject matter.
Objects of the presently disclosed subject matter having been stated above, other objects and advantages will become apparent upon a review of the following descriptions, figures and examples.
(A1R) are required for bovine prostatic acid phsophatase (bPAP) anti-nociception. Wild-type mice (open circles, n=7) and A1R−/− mice (dark squares, n=7) were tested for thermal (
In accordance with the presently disclosed subject matter, methods and compositions are provided for the treatment of pain and cystic fibrosis. In some embodiments, the protein called Prostatic Acid Phosphatase (PAP) is provided for the treatment of these disorders. PAP protein is highly effective at treating chronic inflammatory and neuropathic pain in animal models when injected intrathecally (into spinal cord). A single injection of PAP protein can produce analgesia for up to three days. Such a single administration that relieves pain for three days is a vast improvement over existing pain treatments.
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
Following long-standing patent law convention, the terms “a” and “an” mean “one or more” when used in this application, including the claims.
Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical region of parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term “animal” refers to any animal (e.g., an animal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
“Amino acid sequence” and terms such as “peptide”, “polypeptide” and “protein” are used interchangeably herein, and are not meant to limit the amino acid sequence to the complete, native amino acid sequence (i.e. a sequence containing only those amino acids found in the protein as it occurs in nature) associated with the recited protein molecule. The proteins and protein fragments of the presently disclosed subject matter can be produced by recombinant approaches or can be isolated from a naturally occurring source.
Similarly, all genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences by GENBANK® Accession No., are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
The term “LPA” stands for lysophosphatidic acid.
A “modulator” of PAP is referring to a small molecule that can modulate PAP catalytic activity. PAP modulators can be either activators or inhibitors of PAP activity.
The term “PAP” means a protein having prostatic acid phosphatase activity (E.C. 3.1.3.2.). The term “ACPP” (i.e., acid phosphatase, prostate) is herein used interchangeably with “PAP”. The GENBANK® database discloses amino acid and nucleic acid sequences of PAPs from various species, some of which are summarized in Table 1, below.
H. sapiens
H. sapiens
M. musculus
M. musculus
B. taurus
R. norvegicus
R. norvegicus
A “recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements which permit transcription of a particular nucleic acid in a cell. The recombinant expression cassette can be part of a plasmid, virus, or other vector. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed, a promoter, and/or other regulatory sequences. In some embodiments, the expression cassette also includes, e.g., an origin of replication, and/or chromosome integration elements (e.g., a retroviral LTR).
A “retrovirus” is a single stranded, diploid RNA virus that replicates via reverse transcriptase and a retroviral virion. A retrovirus can be replication-competent or replication incompetent. The term “retrovirus” refers to any known retrovirus (e.g., type c retroviruses, such as Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV) and Rous Sarcoma Virus (RSV). “Retroviruses” of the presently disclosed subject matter also include human T cell leukemia viruses, HTLV-1 and HTLV-2, and the lentiviral family of retroviruses, such as, but not limited to, human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and equine immunodeficiency virus (EIV).
Several terms herein can be used interchangeably. Thus, “virion”, “virus”, “viral particle”, “viral vector”, “viral construct”, “vector particle”, “viral vector transfer cassette” and “shuttle vector” can refer to virus and virus-like particles that are capable of introducing nucleic acid into a cell through a viral-like entry mechanism. Such vector particles can, under certain circumstances, mediate the transfer of genes into the cells they infect. Such cells are designated herein as “target cells”. When the vector particles are used to transfer genes into cells which they infect, such vector particles are also designated “gene delivery vehicles” or “delivery vehicles”. Retroviral vectors have been used to transfer genes efficiently by exploiting the viral infectious process. Foreign genes cloned into the retroviral genome can be delivered efficiently to cells susceptible to infection or transduction by the retrovirus. Through other genetic manipulations, the replicative capacity of the retroviral genome can be destroyed. The vectors introduce new genetic material into a cell but are unable to replicate.
PAP is a member of the histidine acid phosphatase superfamily. Histidine acid phosphatases contain a highly conserved RHGXRXP (SEQ ID NO: 1) motif located within the active site. PAP can be made catalytically inactive, for example, by methods including heat denaturation and by incubating the protein with diethylpyrocarbonate (DEPC), which chemically modifies all histidine residues, or by mutating the active site histidine residue (His12) to alanine (McTigue and Van Etten, 1978; Ostanin et al., 1994). As its name implies, PAP is predominantly expressed in prostate, although the presently disclosed subject matter shows PAP is also expressed at high levels in small diameter DRG neurons (Examples 3-5,
Fluoride-Resistant Acid Phosphatase (FRAP) is a classic histochemical marker of many small-diameter dorsal root ganglia (DRG) neurons and is implicated in pain mechanisms. The molecular identity of FRAP was unknown. Using genetic approaches, the presently disclosed subject matter demonstrates that a transmembrane isoform of Prostatic Acid Phosphatase (PAP, EC 3.1.3.2) is FRAP. Pain-sensing peptidergic and nonpeptidergic nociceptive neurons of mice and humans express PAP suggesting an unanticipated role for PAP in pain (Examples 3-5).
PAP and FRAP have many features in common. For example, FRAP is localized to plasma membrane, golgi and endoplasmic reticulum by electron microscopy, and is particularly enriched near the presynaptic membrane of DRG neurons (Csillik and Knyihar-Csillik, 1986; Knyihar-Csillik et al., 1986; Knyihar and Gerebtzoff, 1970). These ultrastructural data are consistent with the fact that transmembrane PAP is the predominant isoform in DRG (Examples 3-5). PAP and FRAP are also both reversibly inhibited by L-tartrate (
Several groups have also found that PAP dephosphorylates lysophosphatidic acid (LPA) to monoglyceride (MG) and inorganic phosphate (
Lysophosphatidic Acid (LPA) is a potent lysophospholipid mediator that regulates many biological processes, including proliferation, differentiation, survival, and pain (Brindley et al., 2002; Inoue et al., 2004; Moolenaar, 2003; Moolenaar et al., 2004; Tigyi et al., 1994). LPA is released from platelets upon wounding as well as from neurons and other cells (Eichholtz et al., 1993; Sugiura et al., 1999; Xie et al., 2002).
There are four well-characterized LPA receptors, called LPA1, LPA2, LPA3 and LPA4 (Anliker and Chun, 2004; Noguchi et al., 2003; Takuwa et al., 2002). These receptors couple to diverse downstream signaling molecules and are expressed in many cells throughout the body. LPA1 and LPA3 are also expressed in DRG neurons (see Example 5; Inoue et al., 2004; Renback et al., 2000). In addition, Lee et al. found a fifth LPA receptor called LPA5 and demonstrated that it is also expressed in DRG (Lee et al., 2006). LPA receptor activation is routinely measured using calcium imaging, Mitogen Activated Protein Kinase (MAPK) pathway activation, Elk1 transcriptional activation, and RhoA/ROCK pathway activation (Mills and Moolenaar, 2003). LPA receptor signaling is terminated by either receptor desensitization or by dephosphorylation (degradation) of LPA. There are currently several known phosphatases that dephosphorylate LPA extracellularly: 1) PAP; 2) Lysophosphatidic Acid Phosphatase (LPAP; also known as ACP6); and 3) Lipid Phosphate Phosphatases 1 through 3 (LPP1-3), also known as Phosphatadic Acid Phosphatase type 2A-C (PPAP2A-C) (Brindley et al., 2002; Hiroyama and Takenawa, 1999; Pyne et al., 2005; Tanaka et al., 2004). Using calcium imaging as readout, over-expression of LPP1 was shown to inhibit LPA-receptor signaling via dephosphorylation of LPA (Pilquil et al., 2001; Zhao et al., 2005). PAP has not been studied using such cell-based assays.
LPA has several well-documented direct effects on DRG neurons and pain-related behaviors (Park and Vasko, 2005). Elmes and colleagues found that intracellular calcium levels were increased in small-diameter DRG neurons following stimulation with LPA (Elmes et al., 2004). LPA was also shown to increase action potential duration and frequency in wide dynamic range neurons located in the dorsal spinal cord, and to increase nociceptive flexor responses when injected into the hindpaw (Elmes et al., 2004; Renback et al., 1999). When injected into skin, LPA has been shown to cause itching/scratching behaviors (Hashimoto et al., 2006; Hashimoto et al., 2004). Itch signals are transmitted from the periphery to the CNS by small diameter DRG neurons (Han et al., 2006; Schmelz et al., 1997).
Intrathecal injection of LPA has been shown to cause profound allodynia and thermal hyperalgesia that persisted for several days in mice (intrathecal=i.t.=into spinal cord cerebrospinal fluid (“CSF”)) (Inoue et al., 2004). Additionally, Inoue and colleagues demonstrated, using pharmacological and genetic approaches, that LPA receptor signaling was required for the initiation of neuropathic pain. Inoue and colleagues found that LPA1−/− mice failed to develop allodynia and thermal hyperalgesia after nerve injury. They also found that neuropathic pain could be blocked by intrathecal injection of LPA1 antisense oligonucleotides, intrathecal injection of Botulinum toxin C3 exoenzyme (BoTXC3 inhibits RhoA, which is activated downstream of LPA1), and by systemic pharmacological inhibition of ROCK (which is downstream of RhoA) (Inoue et al., 2004). Although not conclusive, their studies suggested LPA1 receptor activation in DRG was required for these effects.
Intrathecal LPA injections have also been shown to cause demyelination in sciatic nerve and up-regulation of the α2δ1 subunit of the voltage-gated calcium channel (Caα2δ1) (Inoue et al., 2004). Caα2δ1 is up-regulated in DRG in neuropathic pain models and is the target for the drug gabapentin (Field et al., 2006; Luo et al., 2001; Maneuf et al., 2006). Gabapentin is frequently prescribed to treat neuropathic pain in humans (Baillie and Power, 2006; Dworkin et al., 2003). Taken together, these studies indicate that LPA signaling plays a direct role in the physiology of DRG neurons, sensitization of nociceptive circuits, and promotion of pathological pain states.
While the presently disclosed subject matter is not limited to any particular mechanism, the following is one proposed model. In healthy, uninjured animals PAP functions to dephosphorylate (degrade) LPA and maintain LPA receptors (LPA-R) in an inactive, non-signaling state (
Glutamate receptor activation is also required to initiate neuropathic pain (Davar et al., 1991). LPA signaling could facilitate glutamate release by sensitizing or depolarizing neurons (Chung and Chung, 2002). After nerve injury, PAP expression and FRAP activity precipitously declines and remains low in DRG neurons (Example 3) (Costigan et al., 2002; Csillik and Knyihar-Csillik, 1986). Without PAP, LPA concentrations would be higher in injured animals compared to healthy animals. These abnormal LPA concentrations could chronically activate LPA receptors on DRG neurons. This chronic activation could sensitize DRG neurons and contribute to the allodynia and hyperalgesia that persists for days following nerve injury (during the maintenance phase) (
The presently disclosed subject matter demonstrates that bovine PAP inactivates LPA (Example 7;
Again, without being bound to any one mechanism of action, the presently disclosed subject matter further relates to the ability of PAP to act as a ectonucleotidase and suppress pain by generating adenosine. As described in Example 13, the in vivo effects of PAP on acute and chronic pain appear to mimic the effects of i.t. adenosine and A1-receptor (A1R) antagonists. See
Examples 10-11 demonstrate that PAP functions as an analgesic in mice for a period of 3 days after injection into cerebrospinal fluid.
Accordingly, PAP is provided as a treatment for chronic pain, including but not limited to neuropathic and inflammatory pain in animals and humans. PAP, an active variant, fragment or derivative thereof, or a small molecule modulator of PAP is provided in the presently disclosed subject matter. PAP, or an active variant, fragment or derivative thereof, can be administered by intrathecally injecting purified PAP protein or by administering (via all possible routes) small-molecule modulators to activate PAP that is normally present on pain-sensing neurons. These treatments could be used pre- or post-operatively to treat surgical pain; to treat pain associated with childbirth; to treat chronic inflammatory pain (osteoarthritis, burns, joint pain, lower back pain) to treat visceral pain, migraine headache, cluster headache, headache and fibromyalgia and to treat chronic neuropathic pain. Neuropathic pain is caused by nerve injury, including but not limited to injuries resulting from trauma, surgery, cancer, viral infections like Shingles and diabetic neuropathy.
The secreted isoform of human PAP protein is commercially available and PAP circulates in the blood of males (Vihko et al., 1978a). This suggests injection of PAP protein into patients suffering from pain will be well-tolerated. Moreover, PAP is a “druggable” protein, as selective PAP inhibitors have been previously identified by pharmaceutical companies (Beers et al., 1996). PAP activators or allosteric modulators are also provided in this disclosure as effective drugs for the treatment of pain. Methods for identifying small-molecule modulators of PAP are provided in this disclosure. Such methods include high-throughput screens (HTS) for PAP modulators using the biochemical and cell-based assays of the presently disclosed subject matter, including the assay described in Example 12. In some embodiments, large compound libraries are screened to identify drugs that activate PAP at very low doses. PAP is considered to be expressed in many fewer tissues than LPA receptors, and small molecules that increase PAP activity can be used to treat neuropathic pain and inflammatory pain and other human diseases, such as cystic fibrosis, with more specificity and fewer side effects.
While the presently disclosed subject matter is not limited to any particular mechanism, in one model PAP causes the analgesic effect disclosed herein by catalyzing the conversion of adenosine monophosphate (AMP) to adenosine. Experimental results show that PAP can dephosphorylate AMP in spinal cord tissue. In addition, adenosine is analgesic and reduces allodynia in humans suffering from neuropathic pain (Lynch et al., 2003; Sjolund et al., 2001). AMP is converted to adenosine when injected into rodent spinal cord and causes analgesia via adenosine receptor activation (Patterson et al., 2001). Thus, in some embodiments of the presently disclosed subject matter PAP is co-administered with AMP for the treatment of pain. In some embodiments, AMP analogs that can be dephosphorylated by PAP to adenosine are co-administered with PAP. In some embodiments, these analogs are more stable in biological tissues, are lipophilic, and have favorable drug metabolism and pharmacokinetics (DMPK). In some embodiments of the presently described subject matter, the administration of PAP for the treatment of pain is in combination with one or more of adenosine, adenosine monophosphate (AMP), an AMP analogue, an adenosine kinase inhibitor, adenosine kinase inhibitor 5′-amino-5′-deoxyadenosine, adenosine kinase inhibitor 5-iodotubercidin, an adenosine deaminase inhibitor, adenosine deaminase inhibitor 2′-deoxycoformycin, a nucleoside transporter inhibitor, nucleoside transporter inhibitor dipyridamole. In some embodiments of the presently described subject matter, the administration of PAP for the treatment of pain is in combination with one or more known analgesic, including, but not limited to, an opiate (e.g., morphine, codeine, etc.).
Adenosine and adenosine receptor agonists are being tested in the art as treatments for cystic fibrosis (CF). In some embodiments, PAP is aerosolized into the lungs of patients to convert endogenous AMP to adenosine and thus to serve as a treatment for CF.
There are several pain conditions that differentially affect males and females (Craft et al., 2004; Giles and Walker, 1999). PAP expression is androgen regulated in prostate (Porvari et al., 1995). In some embodiments of the presently disclosed subject matter, PAP is useful to treat and diagnose a variety of pain conditions that impact human health. In some embodiments, a method is provided for diagnosing an individual's response to a pain medicine comprising identifying one or more single nucleotide polymorphisms (SNPs), insertions or deletions in and around a PAP genomic locus in the individual; and correlating the SNPs with a predetermined response to the pain medicine. In some embodiments, a method is provided for diagnosing an individual's threshold for pain, comprising identifying one or more single nucleotide polymorphisms (SNPs), insertions or deletions in and around a PAP genomic locus in the individual; and correlating the SNPs with a predetermined threshold for pain. In some embodiments, a method is provided for correlating the differential expression of PAP in male and female DRG neurons with pain response, the method comprising: determining the extent to which a PAP is differentially expressed in male and female DRG neurons; and identifying a differential response to pain or to a pain medicine between the males and females; and correlating the extent of differential expression with the differential response to pain or to the pain medicine.
Preparations of PAP protein for use in embodiments of the presently disclosed subject matter can be prepared using a variety of methods. Human PAP is commercially available from Sigma-Aldrich and other vendors. Production of the PAP generally requires quality control to ensure the preparation is sterile, endotoxin free and acceptable for use in humans.
Recombinant methods of obtaining suitable preparations of PAP or active PAP variants, fragments or derivatives are also suitable. Using a PAP cDNA (such as the cDNAs described in Example 1), recombinant protein can be produced by one of the many known methods for recombinant protein expression (see, e.g. Vihko et al., 1993). Isolated nucleotide sequences encoding for the PAP peptide of the presently disclosed subject matter and expression vectors comprising these nucleotides are provided. Host cells comprising the expression vectors are also provided. The presently disclosed subject matter includes viral vector transfer cassettes, such as but not limited to, adenoviral, adeno-associated viral, and retroviral vector transfer cassettes comprising a nucleotide sequence encoding a PAP or active variant or fragment thereof.
Active PAP variants and fragments can be produced using mutagenesis techniques, including site-directed mutagenesis (Ostanin et al., 1994), somatic hypermutation (Wang and Tsien, 2006) and generation of deletion constructs, to evolve versions of hPAP that are more stable or have a higher kcat for substrates like LPA and AMP. Active PAP variants, fragments or derivatives of the presently disclosed subject matter can comprise one or more modifications including conservative amino acid substitutions; non-natural amino acid substitutions, D- or D,L-racemic mixture isomer form amino acid substitutions, amino acid chemical substitutions, carboxy- or amino-terminus modifications and conjugation to biocompatible molecules including fatty acids and PEG.
The term “conservatively substituted variant” refers to a peptide comprising an amino acid residue sequence substantially identical to a sequence of a reference peptide in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the activity as described herein for the reference peptide (e.g., of the PAP). The phrase “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting peptide displays the activity of the reference peptide as disclosed herein.
Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
Peptides of the presently disclosed subject matter also include peptides comprising one or more additions and/or deletions or residues relative to the sequence of a peptide whose sequence is disclosed herein, so long as the requisite activity of the peptide is maintained. The term “fragment” refers to a peptide comprising an amino acid residue sequence shorter than that of a peptide disclosed herein.
PAP, and particularly a smaller molecular weight active PAP variant, fragment or derivative, can be obtained by chemical synthesis using conventional methods. For example, solid-phase synthesis techniques can be used to obtain PAP or an active variant, fragment or derivative thereof.
In some embodiments, PAP preparations are provided where PAP protein or an active PAP variant, fragment or derivative is complexed to an immobile support including supports such as agarose, sepharose, and nanoparticles. Through such immobilization, PAP is protected from degradation and remains in situ for longer periods of time. In this manner, the three day window of PAP analgesia observed herein in some embodiments can be extended to weeks or months.
PAP can be administered by a variety of methods for the treatment of pain and cystic fibrosis in animals. The PAP, the active variant, fragment or derivative thereof, and/or the PAP modulator can be administered via one or more of injection, oral administration, suppository, a surgically implanted pump, aerosolizing into the lungs, stem cells, viral gene therapy, or naked DNA gene therapy. Injection can include any type of injection, such as, but not limited to, intravenous injection, epideral injection or intrathecal injection.
In some embodiments, a small molecule modulator of PAP activity is administered by oral administration.
In some embodiments, a therapeutically effective amount of a composition or pharmaceutical formulation comprising a PAP, or an active variant, fragment or derivative thereof, is administered to the animal or human by injection. Any suitable method of injection, such as intrathecal, intravenous, intraarterial, intramuscular, intraperitoneal, intraportal, intradermal, epideral, or subcutaneous can be used. In some embodiments, PAP is dispersed in any physiologically acceptable carrier that does not cause an undesirable physiological effect. Examples of suitable carriers include physiological saline and phosphate-buffered saline. The injectable solution can be prepared by dissolving or dispersing a suitable preparation of the active PAP in the carrier using conventional methods. In some embodiments, PAP is provided in a 0.9% physiological salt solution. In some embodiments, PAP is provided enclosed in liposomes such as immunoliposomes, or other delivery systems or formulations that are known in the art.
In some embodiments, a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active variant, fragment or derivative thereof, is provided through a surgically implantable pump apparatus for delivery of PAP to local tissue. In some embodiments, the surgically implantable pump apparatus is an intrathecal drug delivery system comprising an implantable infusion pump and an implantable intraspinal catheter. See, for example, the commercially available apparatus used to deliver opiates for chronic pain treatment (Medtronic, Minneapolis, Minn., United States of America). In some embodiments, a kit is provided for the treatment of pain in animals, comprising a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active variant, fragment or derivative thereof, and a surgically implantable pump apparatus for delivery of PAP to local tissue.
In some embodiments, an animal is treated with PAP for cystic fibrosis. In some embodiments, the animal is administered a composition or pharmaceutical formulation comprising a therapeutically effective amount of a PAP, or an active variant, fragment or derivative thereof, or a therapeutically effective amount of an activity enhancing modulator of a PAP wherein the PAP composition is aerosolized in the lungs.
In some embodiments, an animal is administered a PAP, or an active variant or fragment thereof, through intrathecal injection of embryonic stem (ES) cells expressing PAP (see, e.g., Wu et al., 2006). This method employs derivation of patient-specific ES cells by somatic cell nuclear transfer (SCNT). The feasibility of this approach has been demonstrated in animal models. Cells are produced that can be differentiated into hematopoietic stem cells (HSCs), neurons or other cell types in vitro and transplanted into a subject animal or human.
In some embodiments, the therapeutically effective amount of PAP, or an active variant, fragment or derivative thereof, can be administered once daily. In some embodiments, the dose is administered twice or three times weekly. In some embodiments, administration is performed once a week or biweekly.
In some embodiments, the therapeutically effective amount of a PAP or active variant or fragment thereof is administered by methods known to those of skill in the art as “gene therapy”. Gene therapy as used herein refers to a general method for treating a pathologic condition in a subject by inserting an exogenous nucleic acid into an appropriate cell(s) within the subject. The nucleic acid is inserted into the cell in such a way as to maintain its functionality, for example, so as to maintain the ability to express a particular polypeptide. In some embodiments, a therapeutically effective amount of a PAP is administered via viral gene therapy using a viral vector transfer cassette (e.g., a retroviral, adenoviral or adeno-associated viral cassette) comprising a nucleic acid sequence encoding the PAP or active variant or fragment thereof.
With respect to the methods of the presently disclosed subject matter, a preferred subject is a vertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. The subject treated by the presently disclosed methods is desirably a human, although it is to be understood that the principles of the presently disclosed subject matter indicate effectiveness with respect to all vertebrate species which are included in the term “subject.” In this context, a vertebrate is understood to be any vertebrate species in which treatment of a disorder is desirable. As used herein “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter.
As such, the presently disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos or as pets (e.g., parrots), as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
In some embodiments, a subject's genotype can be used to determine valuable information for predicting the subject's response to pain and/or to pain medication. As used herein, the term “genotype” means the genetic makeup of an organism. Expression of a genotype can give rise to an organism's phenotype, i.e. an organism's physical traits. The term “phenotype” refers to any observable property of an organism, produced by the interaction of the genotype of the organism and the environment. A phenotype can encompass variable expressivity and penetrance of the phenotype. Exemplary phenotypes include but are not limited to a visible phenotype, a physiological phenotype, a susceptibility phenotype, a cellular phenotype, a molecular phenotype, and combinations thereof. The phenotype can be related to pain response and/or a response to pain medication. A particular subject's genotype can be compared to a reference genotype or the genotype of one or more other subjects to provide valuable information related to current or predictive phenotypes.
“Determining the genotype” of a subject, as used herein, can refer to determining at least a portion of the genetic makeup of an organism and particularly can refer to determining a genetic variability in a subject that can be used as an indicator or predictor of phenotype. The genotype determined can be the entire genome of a subject, but far less sequence is usually required. In some embodiments, determining the genotype comprises identifying one or more polymorphisms, including single nucleotide polymorphisms (SNPs), insertions, deletions and/or other types of genetic mutations in and around a PAP genomic locus in the subject. As used herein, the term “polymorphism” refers to the occurrence of two or more genetically determined alternative variant sequences (i.e., alleles) in a population. A polymorphic marker is the locus at which divergence occurs. Exemplary markers have at least two alleles, each occurring at a frequency of greater than 1%. A polymorphic locus may be as small as one base pair (e.g., a single nucleotide polymorphism (SNP)).
In some embodiments, the presently disclosed subject matter provides a method for diagnosing an individual's response to a pain medicine, comprising identifying one or more SNPs, insertions, deletions and/or other types of genetic mutations in and around a PAP genomic locus in the individual; and correlating the SNPs, insertions, deletions and/or other types of genetic mutations with a predetermined response to the pain medicine. For example, an individual's (or a population subset's) response to a pain medicine can be compared to the response to the pain medicine in a control population. Then, it can be determined if the individual (or population subset) has one or more genetic variations related to the PAP gene. In some embodiments, certain genetic variations can be correlated to an ability to respond to pain or to a pain medication. For example, genetic variations can be statistically correlated to particular pain response behaviours. Thus, in some embodiments, the presently disclosed subject matter provides a method for diagnosing an individual's (or a population subset's) threshold for pain and/or propensity to transition from acute to chronic pain, comprising identifying one or more single nucleotide polymorphisms (SNPs) insertions, deletions and/or other types of genetic mutations in and around a PAP genomic locus in the individual; and correlating the SNPs, insertions, deletions and/or other types of genetic mutations with a predetermined threshold for pain or propensity to transition from acute to chronic pain. In some embodiments, the method involves correlating differences in PAP expression in male and female DRG neurons, identifying a differential response to pain or to pain medicine between males and females, and correlating the extent of differential expression with the differential response to pain or to pain medicine.
Various methods of determining genetic variations such as SNP's are known in the art. For example, U.S. Pat. No. 6,972,174, provides a method of determining SNP's based on polymerase chain extension reactions adjacent to potential SNP sites. U.S. Pat. No. 6,110,709 describes a method for detecting the presence or absence of an SNP in a nucleic acid molecule by first amplifying the nucleic acid of interest, followed by restriction analysis and immobilizing the amplified product to a binding element on a solid support. PCT International Patent Publication WO9302212 describes another method for amplification and sequencing of nucleic acid in which dideoxy nucleotides are used to create amplified products of varying lengths. The varying length products are then separated and visualized by gel electrophoresis. PCT International Patent Publication WO0020853 further describes a method of detecting single base changes using tightly controlled gel electrophoretic conditions to scan for conformational changes in the nucleic acid caused by sequence changes.
The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.
Molecular Biology.
The full-length expression construct of ACPP-transmembrane isoform (mouse PAP) (nt 64-1317 from GENBANK® accession # NM—207668; SEQ ID NO: 2) was generated by RT-PCR amplification, using C57BL/6 mouse trigeminal cDNA as template and Phusion polymerase (New England BioLabs, Beverly, Mass., United States of America). PCR products were cloned into pcDNA3.1 (Invitrogen, Carlsbad, Calif., United States of America) and completely sequenced. Isoform-specific in situ hybridization probes of ACPP, secreted variant (nt 1544-2625 from GENBANK® accession # NM—019807; SEQ ID NO: 3) and ACPP, transmembrane variant (nt 1497-2577 from GENBANK® accession # NM—207668; SEQ ID NO: 4) were generated by PCR amplification, using C57BL/6 mouse genomic DNA as template and Phusion polymerase. Probes were cloned into pBluescript-KS (Stratagene, La Jolla, Calif., United States of America) and completely sequenced.
A pFastBAC baculovirus expression vector was generated that contains the secreted isoform of mouse PAP (nt 64-1206 from GENBANK® accession # NM—019807; SEQ ID NO: 5) fused to a carboxyl-terminal thrombin cleavage site-hexahistidine tag. Similarly, a pFastBAC baculovirus expression vector was generated that contains the secreted isoform of human PAP (nt 43-1200 from GENBANK® accession # NM—001099; SEQ ID NO: 6) fused to a carboxyl-terminal thrombin cleavage site-hexahistidine tag.
In Situ Hybridization.
In situ hybridization was performed as described in Dong et al. using digoxygenin-labeled antisense and sense (control) riboprobes.
Cell Culture.
HEK 293 cells were grown at 37° C., 5% CO2, in Dulbecco's Modified Eagle's Medium (DMEM), high glucose, supplemented with 1% penicillin, 1% streptomycin and 10% fetal bovine serum. For transfections, 6×105 cells were seeded per well in 6-well dishes. Cells were cotransfected with 0.5 μg ACPP-transmembrane isoform and 0.5 μg farnesylated EGFP (EGFPf) using Lipofectamine Plus (Invitrogen, Carlsbad, Calif., United States of America). Twenty-four hours post transfection, samples were imaged for intrinsic EGFPf fluorescence to confirm that all cells were transfected. Cells were then fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) and stained using FRAP histochemistry.
Tissue Preparation.
All procedures involving vertebrate animals were approved by Institutional Animal Care and Use Committees at the University of North Carolina at Chapel Hill and at the University of Oulu.
For FRAP histochemistry, wild-type and PAP−/− adult male mice, ages 6-12 weeks, were anesthetized with pentobarbital and perfused transcardially with 20 mL 0.9% saline (4° C.) followed by 25 mL fixative (4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.3 at 4° C.). The spinal column was dissected then cryoprotected in 20% sucrose, 0.1 M phosphate buffer, pH 7.3 at 4° C. (for 2-3 days). Spinal cord encompassing the lumbar enlargement (L4-L6 region) and L4-L6 DRG were carefully dissected and frozen in OCT.
For immunofluorescence staining, wild-type adult male mice were sacrificed by cervical dislocation or decapitation. Lumbar spinal cord and DRG (L4-L6) were dissected then postfixed for 6 hr and 2 hr, respectively. Tissues were cryoprotected in 20% sucrose, 0.1 M phosphate buffer, pH 7.3 at 4° C. for 24 hours, frozen in OCT, sectioned with a cryostat at 15-20 μm, and mounted on Superfrost Plus slides. Slides were stored at −20° C. Free-floating sections were sectioned at 30 μm and immediately stained.
FRAP Histochemistry.
FRAP/Thiamine Monophosphatase (TMPase) histochemistry was performed essentially as described by Shields et al., 2003, with modifications suggested by Silverman and Kruger, 1988. Cells or tissue sections were washed twice with 40 mM Trizma-Maleate (TM) buffer, pH 5.6, then once with TM buffer containing 8% (w/v) sucrose. To precipitate lead on cells and axons bearing FRAP, samples were incubated at 37° C. for 2 hr in TM buffer containing 8% sucrose (w/v), 6 mM thiamine monophosphate chloride, 2.4 mM lead nitrate. Lead nitrate must be made fresh immediately prior to use. To reduce nonspecific background staining, samples were washed once with 2% acetic acid for one minute. Samples were then washed three times with TM buffer, developed for 10 seconds with 1% sodium sulfide, washed several times with PBS, pH 7.4, and mounted in Gel/Mount (Biomeda Corp., Foster City, Calif., United States of America). Images were acquired using a Zeiss Axioskop and Olympus DP-71 camera.
When assaying HEK 293 cells for FRAP activity, duplicate samples were stained with and without 0.1% Triton X-100 in the initial TM wash. FRAP histochemical staining was stronger in detergent permeabilized cells, presumably detecting intracellular stores of TM-PAP in the endoplasmic reticulum and golgi apparatus.
Immunofluorescence.
Free-floating and slide-mounted sections were washed 3 times with 50 mM Tris base, 460 mM NaCl, 0.3% Triton X-100, pH 7.6 (TBS+TX; the high-salt concentration was essential for optimal PAP antibody staining), blocked for 60 minutes in TBS+TX4 containing 10% goat serum, then incubated overnight at 4° C. with primary antibodies diluted in blocking solution The antibodies used included: 1:1000 rabbit anti-GFP (A-11122, Molecular Probes, Eugene, Oreg., United States of America), 1:1000 chicken anti-GFP (GFP-1020, Ayes Labs, Tigard, Oreg., United States of America), 1:250 mouse anti-NeuN (MAB377, Chemicon, Billerica, Mass., United States of America), 1:800 guinea pig anti-CGRP (T-5027, Peninsula Laboratories, Inc., San Carlos, Calif., United States of America), 1:750 rabbit anti-CGRP (T-4032, Peninsula Laboratories, Inc., San Carlos, Calif., United States of America), 1:1000 rabbit anti-P2X3 (AB5895, Chemicon, Billerica, Mass., United States of America), 1:300 guinea pig anti-P2X3 (GP10108, Neuromics, Edina, Minn., United States of America), 1:100 mouse anti-PKCγ (clone PKC66, Cat. #13-3800, Zymed Laboratories, Inc., South San Francisco, Calif., United States of America), 1:1000 rabbit anti-PKCγ (c-19, Cat. # sc-211, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., United States of America), 1:1000 rabbit anti-human PAP (Biomeda Corporation, Foster City, Calif., United States of America).
Biomeda Anti-PAP antibody specificity was confirmed by: a) absence of staining when primary antibody was excluded, and b) absence of staining in DRG and spinal cord sections from PAP−/− mice. Mrgprd-expressing cells and axons were visualized by staining tissue from MrgprdΔEGFPf mice with anti-GFP antibodies. Sections were then washed three times with TBS+TX and incubated for 2 hours at room temperature with secondary antibodies. All secondary antibodies were diluted 1:250 in blocking solution, and were conjugated to Alexa-488, Alexa-568, or Alexa-633 fluorochromes (Molecular Probes, Eugene, Oreg., United States of America), or to FITC, Cy3, or Cy5 fluorochromes (Jackson ImmunoResearch, West Grove, Pa., United States of America). To detect IB4-binding, 1:100 Griffonia simplicifolia isolectin GS-IB4-Alexa 488 (1-21411, Molecular Probes, Eugene, Oreg., United States of America) was included during secondary antibody incubations. It was necessary to amplify the anti-PAP antibody signal by using secondary antibodies conjugated to biotin, then using either 1:250 Streptavidin-Cy3 (Jackson ImmunoResearch, West Grove, Pa., United States of America); or the Tyramide Signal Amplification kit (New England Nuclear, Boston, Mass., United States of America, following manufacturers protocol).
Following staining, sections were washed three times with TBS+TX, followed by three PBS washes and wet-mounted in Gel/Mount (Biomeda Corporation, Foster City, Calif., United States of America). Images were obtained using a Leica TCS-NT confocal microscope (Leica Microsystems, Wetzlar, Germany). All cell counts are represented as percentages +/−Standard Error of the Mean (SEM).
Immunofluorescence Combined with FRAP Histochemistry.
To demonstrate overlap between immunofluorescence and FRAP histochemistry in DRG, adjacent 15 μm sections were stained with anti-PAP antibodies, and for FRAP histochemistry. Similar methods have been used previously to co-localize FRAP with antibody and lectin markers and, due to the use of adjacent sections, underestimates the number of coexpressing cells (Dodd et al., 1983; Nagy and Hunt, 1982; Silverman and Kruger, 1988; Silverman and Kruger, 1990). Technical limitations prevented sequential processing of the same DRG section for immunofluorescence and FRAP histochemistry.
To demonstrate overlap in spinal cord tissue, identical sections were first stained with anti-PAP antibodies, imaged using confocal microscopy, and then the same sections were stained histochemically for FRAP and imaged by transmitted light microscopy. This procedure is based on a published method (Wang et al., 1994). Fluorescence and transmitted light images were overlaid in Photoshop by scaling and rotating the images as necessary.
Behavior.
C57BL/6 male mice, 2-3 months old, were purchased from Jackson Laboratories (Bar Harbor, Me., United States of America) for all behavioral experiments involving PAP protein injections. All mice were acclimated to the testing room, equipment and experimenter for one day before behavioral testing. The experimenter was blind to genotype and drug treatment during behavioral testing.
Thermal sensitivity was measured by heating one hindpaw with a Plantar Test apparatus (IITC) following the Hargreaves method (Hargreaves et al., 1988). The radiant heat source intensity was calibrated so that a paw withdrawal reflex was evoked in ˜10 seconds, on average, in wild-type C57BL/6 mice. Cutoff time was 20 s. One measurement was taken from each paw per day to determine paw withdrawal latency. To perform the tail immersion assay, mice were gently restrained in a towel and the distal one-third of the tail was immersed in 46.5° C. water. Latency to withdrawal the tail was measured once per mouse. Mechanical sensitivity was measured using semi-rigid tips attached to an Electronic von Frey apparatus (IITC) as described elsewhere (Cunha et al., 2004; Inoue et al., 2004). Three measurements were taken from each paw (separated at 5 min. intervals) then averaged to determine paw withdrawal threshold in grams.
To induce persistent inflammatory pain, 20 μL Complete Freunds Adjuvant (CFA, Sigma) was injected into one hindpaw, centrally beneath glabrous skin, with a 27 G needle. The spared nerve injury (SNI) model of neuropathic pain was performed as described (Shields et al., 2003).
Intrathecal Injections.
hPAP, bPAP and vehicle controls were injected into the lumbar region of unanesthetized mice as described (Fairbanks, 2003).
A mouse PAP (secreted isoform; nt 64-1206 from GENBANK® accession # NM—019807; SEQ ID NO: 5) baculovirus expression construct was made containing a thrombin cleavage site and hexahistidine purification tag at the C-terminus using the clone described in Example 1 and standard procedures in the art. The recombinant mouse PAP was purified using a fee-for-service Protein Purification core facility. A hPAP (secreted isoform; nt 43-1200 from GENBANK® accession # NM—001099; SEQ ID NO: 6) expression construct was similarly constructed having a thrombin-hexahistidine C-terminal tag. Large quantities of recombinant hPAP protein could be produced with this construct using procedures that are known to the art. Recombinant hPAP protein is useful as a drug in human clinical trials and can be used to assess safety of intrathecal hPAP in humans.
For nearly fifty years, it has been known that many small-diameter DRG neurons contain an acid phosphatase, commonly referred to as FRAP or Thiamine Monophosphatase (Csillik and Knyihar-Csillik, 1986; Knyihar-Csillik, 1986; Colmant, 1959). FRAP was used to mark nonpeptidergic DRG neurons and their unmyelinated axon terminals in lamina II of spinal cord, as well as a subset of peptidergic (CGRP+, Substance P+) neurons (Hunt and Mantyh, 2001; Carr et al., 1990). Use of FRAP as a marker waned when it was found that certain lectins, like Griffonia simplicifolia Isolectin B4 (IB4), also marked nonpeptidergic neurons and co-localized with FRAP (Silverman and Kruger, 1988). Moreover, the gene encoding FRAP was never unequivocally identified.
In the early 1980s, Dodd and co-workers partially purified FRAP protein from rat DRG using chromatography (Dodd et al., 1983). The partially purified FRAP protein was similar in molecular weight to human prostatic acid phosphatase (PAP) and was inhibited by L(+)-tartrate, a non-selective inhibitor of several acid phosphatases. These biochemical experiments hinted that FRAP might be PAP. However, antibodies raised against the partially purified FRAP protein and antibodies against human PAP did not immunostain small-diameter DRG neurons and their axon terminals in lamina II of the spinal cord (Silverman and Kruger, 1988, Dodd et al., 1983). These inconclusive immunohistochemical findings cast doubt as to whether or not PAP was identical to FRAP.
To resolve this ambiguity, the relationship between FRAP and PAP was re-examined using molecular, genetic and immunohistochemical techniques. PAP is expressed as either a secreted protein or as a type 1 transmembrane (TM) protein, with the catalytic acid phosphatase domain localized extracellularly (Kaija et al., 2006; Roiko et al., 1990). See
To determine if either PAP isoform was expressed in small-diameter DRG neurons like FRAP, in situ hybridization was performed with isoform-specific probes. These studies revealed that TM-PAP was expressed in a subset of small-diameter DRG neurons (see
Next, the extent to which FRAP histochemical activity was dependent on PAP enzymatic activity was directly tested. To do this, mouse TM-PAP was over-expressed in HEK 293 cells, and the cells were stained using FRAP histochemistry. While control cells transfected with empty vector did not show signs of staining, cells transfected with TM-PAP were heavily stained when the plasma membrane was left intact or was permeabilized with detergent. This indicated that TM-PAP was sufficient for FRAP histochemical activity and that TM-PAP could dephosphorylate substrates extracellularly. Similar results were obtained when TM-PAP was transfected into Rat1 fibroblasts.
DRG and spinal cord tissues from PAPΔ3/Δ3 (henceforth referred to as PAP−/−) knock-out mice were also analyzed. In these mice, deletion of exon 3 causes PAP protein truncation and complete loss of PAP catalytic activity in prostate. Strikingly, FRAP histochemical staining of DRG neurons and axon terminals in spinal cord were abolished in PAP−/− mice.
Absence of FRAP staining was not due to developmental loss of neurons or axon terminals in PAP−/− mice. Wild-type and PAP−/− mice had equivalent numbers of P2X3+ neurons relative to all NeuN+ neurons in lumbar ganglia (43.4+/−1.9% verses 42.4+/−1.9% 5 (s.e.m.); not significantly different, paired t-test; n=1500 NeuN+ neurons counted per genotype). P2X3 marks nonpeptidergic DRG neurons and is extensively co-localized with PAP. Moreover, confocal image analysis revealed no gross anatomical differences between genotypes (n=2 mice from each genotype) when spinal cord was examined using antibodies to CGRP (to mark peptidergic nerve endings), isolectin B4 (IB4, to mark non-peptidergic nerve endings) and antibodies to protein kinase C-γ (PKCγ, to mark interneurons in laminas Ilinner and III).
These data indicate that PAP is the only acid phosphatase in DRG and spinal cord with FRAP-like activity. Moreover, these gain- and loss-of function experiments conclusively demonstrate that FRAP in small-diameter DRG neurons is encoded by PAP.
Experiments were performed to show that PAP is similarly expressed in human DRG tissue. FRAP histochemical activity is located in small diameter DRG neurons in humans (Silverman and Kruger, 1988a). RT-PCR was performed using total RNA from human DRG (Clontech, Palo Alto, Calif., United States of America) as a template, and intron-spanning primers to human PAP (intron-spanning primers ensure that the amplification product originates from cDNA, not genomic DNA). A band of the correct size was obtained after only 30 cycles. This finding, combined with published FRAP histochemical data, strongly suggest human small-diameter (presumably nociceptive) neurons express PAP.
PAP protein and FRAP histochemical activity were also found to co-localize at the cellular level in DRG neurons. To do this, several commercially available anti-human hPAP antisera were purchased and tested on mouse prostate (positive control), DRG and spinal cord tissues (no commercially available anti-mouse or anti-rat PAP antibodies exist). One rabbit polyclonal antiserum stained prostate epithelial cells, small-diameter DRG neurons and axon terminals within lamina II of the spinal cord; precisely where FRAP histochemistry was observed. Small diameter trigeminal ganglia neurons and axons in lamina II of nucleus caudalis were also labeled by the antibody. Trigeminal neuron staining suggests PAP could be effective at treating pain associated with the head, such as headache or dental pain. Antibody specificity was confirmed by: a) absence of staining when primary antibody was excluded, and b) absence of staining in DRG and spinal cord sections from PAP−/− mice.
Expression of TM-PAP suggested that PAP protein is localized extracellularly, on the plasma membrane of DRG neurons (Quintero et al., 2007). This was confirmed by surface labeling of live, dissociated mouse DRG neurons using the anti-PAP antibody.
DRG neurons and spinal cord were double-labeled with antibodies to determine if PAP was expressed in peptidergic or nonpeptidergic nociceptive circuits (Table 2). Mouse L4-L6 DRG neurons and lumbar spinal cord sections were double-labeled with antibodies against various sensory neuron markers and with antibodies against PAP. Tissue from adult MrgprdΔEGFPf mice was used to identify Mrgprd-expressing neurons (Zylka et al., 2005). IB4 and MrgprdΔEGFPf are markers of nonpeptidergic neurons and endings while CGRP is a marker of peptidergic neurons and endings. These studies revealed that PAP protein was primarily localized to nonpeptidergic neurons and their axon terminals in lamina II of the mouse spinal cord.
Table 2 shows the results of quantitative analysis of PAP and sensory neuron marker colocalization studies within mouse L4-L6 DRG neurons. Images were acquired by confocal microscopy. At least 350 cells were counted per combination. Cell counts from confocal images revealed that virtually all nonpeptidergic DRG neurons co-expressed PAP: 91.6% of all IB4+ (n=497 cells counted), 99.2% of all Mrgprd+ (n=357 cells counted), and 92.6% of all P2X3+ neurons (n=824 cells counted) expressed PAP (Zylka et al., 2005). A smaller percentage (17.1%) of peptidergic CGRP+ neurons (n=1364 cells counted) expressed PAP. This preferential expression of PAP in nonpeptidergic neurons is consistent with previous studies that used FRAP histochemistry in combination with sensory neuron markers (Hunt and Mantyh, 2001; Carr et al., 1990).
Predominant expression of the transmembrane isoform of PAP in DRG is consistent with ultrastructural studies (Csillik and Knyihar-Csillik, 1986) showing that FRAP is localized to the membrane of small-diameter DRG neurons. Thus, TM-PAP and FRAP share the same cellular and subcellular localization in DRG neurons (membrane associated) further suggesting PAP encodes FRAP. When taken together, these findings solve a fifty-year-old mystery, and demonstrate that FRAP in nociceptive neurons is equivalent to PAP.
Microarray analysis has demonstrated that numerous genes are up- or down-regulated in rat DRG three days after sciatic nerve transection (Costigan et al., 2002) and following nerve injury in a neuropathic pain model (Davis-Taber, 2006). The microarray dataset presented in Costigan et al. (presented in Costigan et al. as Supplemental
Expression of LPA receptors was analyzed in DRG neurons to confirm a role for PAP in regulation of LPA receptor signaling. At the time these studies were begun, RT-PCR experiments indicated that LPA1 was the only LPA receptor in DRG (Inoue et al., 2004; Renback et al., 2000). To examine expression of these receptors in more detail, in situ hybridization was performed with antisense LPA1 and LPA3 riboprobes. These experiments revealed that LPA1 was expressed in all mouse DRG neurons while LPA3 was expressed in a subset of small diameter DRG neurons. To determine if LPA3 was co-expressed with Mrgprd, fluorescent double in situ hybridization was performed with antisense Mrgprd and LPA3 riboprobes using previously published methods (Zylka et al., 2003). The experiment revealed that all Mrgprd+ neurons expressed LPA3. Conversely, almost all LPA3+ cells expressed Mrgprd (although there were a few LPA3+ only cells). In summary, all DRG neurons express LPA1 while Mrgprd+ neurons co-express LPA1 and LPA3. These data suggest that all DRG neurons have the potential to signal via LPA receptors. Since Mrgprd+ neurons also express PAP (see Table 2), LPA receptor signaling can be modulated by increasing and decreasing PAP protein levels.
A way to quantify PAP activity was needed so that reproducible amounts of active PAP protein could be added to cultured cells or injected into live mice for the experiments described below. To accomplish this, two well-established methods were tested for measuring PAP activity: 1) a colorimetric assay using para-nitrophenyl phosphate (p-NPP) hydrolysis; and 2) a fluorometric assay using difluoro-4-methylumbelliferyl phosphate (DiFMUP) hydrolysis (commercially available as the EnzChek Acid Phosphatase kit from Invitrogen, Carlsbad, Calif., United States of America). Based on direct comparisons, it was determined that the fluorometric assay was much more sensitive than p-NPP for quantification of PAP activity. Exemplary data with purified bovine PAP (bPAP, secreted isoform) and mouse PAP (mPAP) are presented in
Previous studies found that human PAP dephosphorylates LPA in test tubes (Hiroyama and Takenawa, 1999; Tanaka et al., 2004). Although it is assumed that dephosphorylated LPA can no longer activate LPA receptors, this was never formally demonstrated using more biologically-meaningful, cell-based assays. To prove that PAP inactivates LPA, 1 μM LPA was incubated with an excess (0.2 mU) of bovine PAP in a test tube for 1.5 hr at 37° C. (“a” in
Since exogenous bPAP could block LPA-evoked signaling, it was hypothesized that LPA-evoked signaling could be acutely reduced in Rat1 cells that over-expressed PAP. To test this hypothesis, a fluorescently tagged mPAP construct was generated by fusing the yellow fluorescent protein Venus to the C-terminus of TM-PAP (Nagai et al., 2002). This allowed direct visualization of live cells that were transfected with PAP-Venus. It was demonstrated that PAP-Venus had phosphatase activity by staining transfected cells using FRAP histochemistry. The catalytically active fusion construct was then transfected into Rat1 cells and LPA-evoked changes in intracellular calcium were measured with the calcium-sensitive dye Fura2-AM. As can be seen in
To support the hypothesis that PAP modulates LPA signaling by dephosphorylating LPA, a phosphatase-dead mouse PAP expression construct (PAP-mutant) was engineered by mutating the active site residue Histidine 12 to Alanine, and then fusing the fluorescent protein Venus to the C-terminus (to permit visualization of cells transfected with this PAP-mutant). First, it was confirmed that the PAP mutant construct was expressed and membrane localized as effectively as wild-type PAP-Venus. Second, it was confirmed that the PAP-mutant construct lacked phosphatase activity using Fluoride-Resistant Acid Phosphatase (FRAP) histochemistry. Then, Rat1 fibroblasts were transfected with PAP or PAP-mutant, and the cells loaded with the calcium-sensitive dye Fura2-AM. The cells were then stimulated with 100 nM LPA. Calcium responses were compared in PAP transfected cells to untransfected cells in the same field of view. As can be seen in
An abnormal amount of LPA stimulates the nociceptive system and initiates neuropathic pain including allodynia and hyperalgesia. See
In addition, PAP expression and FRAP activity are down-regulated after nerve injury. Accordingly, injection of PAP after nerve injury can restore PAP activity and reduce allodynia during the maintenance phase of neuropathic pain. See
Dose Selection.
An initial dose of 100 mU PAP intrathecally (i.t.) was chosen based on the finding that 1 pmol of fluorometric substrate is degraded by 1 U of bovine PAP per minute. If it is assumed that bPAP hydrolyzes the fluorometric substrate as efficiently as LPA, then this equals a of 1 μmol of LPA hydrolyzed/U bPAP/minute. LPA (1 nmol, i.t.) caused behavioral allodynia and hyperalgesia that was equal in magnitude to that seen after nerve injury (Inoue et al., 2004). If it is assumed that a similar amount of LPA is released by platelets after nerve injury, then to degrade 1 nmol LPA in 1 minute, 1 mU of bPAP would be required. Thus, a 100 mU dose of PAP represents 100-fold excess, and accounts for diffusion and dilution in CSF and spinal cord parenchyma.
The direct lumbar puncture method was used to intrathecally (i.t.) inject 5 μL of approximately 100 mU PAP (Sigma, St. Louis, Mo., United States of America) dissolved in 0.9% saline between the lumbar 5 and 6 regions of mouse spinal cord (Fairbanks, 2003). Intrathecal injection was chosen because PAP protein is unlikely to reach spinal cord tissue if injected intraperitoneally. Bovine serum albumin was purchased from Sigma (St. Louis, Mo., United States of America, Catalog Number P8361, expressed in Pichia pastoris, >4000 U/mg protein). Morphine sulfate (Sigma, St. Louis, Mo., United States of America, Catalog Number M8777) was diluted into 0.9% saline.
Intrathecal injection of bPAP or hPAP had no obvious side effects. For example, no paralysis, muscle weakness, lethargy, excitability, infection or death was observed for the duration of the behavioral testing period (up to 14 days in some cases). It was expected that bPAP and hPAP protein would be well tolerated in vivo, because PAP protein is located extracellularly in the spinal cord (on the axons of PAP+ neurons). In addition, because PAP was being injected into the CNS (i.e. behind the blood-brain-barrier), and the CNS is immune privileged, an immune response seemed unlikely. Signs of immune and microglial activation can be monitored using molecular markers.
PAP activity can also be increased using additional methods such as by plasmid or viral transduction, or by injecting cell lines that over-express the secreted isoform of PAP.
PAP can be inactivated by heat-denaturation, DEPC-treatment or by introducing a catalytically inactive point mutation (His12→Ala) into recombinant protein.
bPAP inhibits LPA-evoked sensitization in vivo. To prove that bovine PAP protein (bPAP) (purchased from Sigma, St. Louis, Mo., United States of America) is non-toxic when injected i.t., and to prove that bPAP can modulate LPA-evoked signaling in vivo, four groups of wild-type C57BL/6 male mice were injected (i.t.) with: 1) vehicle, 2) 20 μU bPAP, 3) 1 nmol LPA, or 4) 1 nmol LPA+20 μU bPAP. It was found that 20 μU bPAP could dephosphorylate 1 nmol LPA when incubated at 37° C. for 10 min.; therefore, all samples were incubated at 37° C. for 10 min. prior to injection.
First, mechanical sensitivity was measured with an electronic von Frey apparatus (IITC). Then, thermal sensitivity was measured using the Hargreaves method (radiant heating of hindpaw; IITC Plantar Test Apparatus). As can be seen in
This significant increase in thermal sensitivity was reproduced with additional vehicle- and bPAP-injected mice. Mechanical sensitivity in these same animals was not significantly different when compared to vehicle controls (with the exception of the 6 hour time point). These findings show that bPAP has analgesic properties in vivo.
No significant thermal analgesia was observed in LPA+bPAP-injected mice (except at the 1 day time point). This difference between LPA+bPAP-injected mice and bPAP-injected mice could be due to incomplete dephosphorylation of LPA prior to injection or could be due to the presence of monoglyceride and inorganic phosphate in the LPA+bPAP sample (dephosphorylation of LPA produces monoglyceride and inorganic phosphate). Body weight was stable for the entire experimental period indicating no loss of appetite or infection. Overall, these experiments indicate that i.t. injection of bPAP is non-toxic and well-tolerated in mice.
bPAP and hPAP are Analgesic In Vivo.
To determine pain-related functions for PAP, bovine bPAP was injected into spinal cord of wild-type mice. These mice were then tested before and up to 5 days post injection for thermal sensitivity using the Hargreave's method (radiant heating of hindpaw) and mechanical sensitivity using an electronic Von Frey apparatus. Mice injected with bPAP showed significantly increased latency to withdraw their hindpaws from the thermal stimulus for up to 3 days compared to vehicle-injected controls. See
Active and heat-inactivated hPAP were used to directly test if PAP catalytic activity is required for the analgesic effect.
Next, PAP antinociception was compared to the commonly used opioid analgesic morphine using the same behavioral assay for sensitivity to a noxious thermal stimulus. The dose dependency of morphine antinociception is shown in
Complete Freund's Adjuvant (CFA) Inflammatory Pain Model.
The Complete Freund's Adjuvant (CFA) inflammatory pain model was used to determine if PAP could reverse chronic mechanical and thermal inflammatory pain. The baseline mechanical sensitivity of adult (2-3 months old), age-matched, weight-matched male C57BL/6 mice was quantified by probing glabrous skin (right hindpaw) with an electronic von Frey apparatus (IITC). The Hargreave's method, which entails radiant heating of the hindpaw (IITC Plantar Test Apparatus), was used to test thermal sensitivity in the same group of mice (Hargreaves et al., 1988). Baseline thermal and mechanical sensitivity was determined prior to injection of test compounds. The mice were then injected with 20 μL CFA. One day later, all mice showed profound thermal and mechanical hypersensitivity in the CFA-injected hindpaw. Half of the mice were then intrathecally injected with 1.3 mg/mL BSA (control) and the other half with bPAP (see
Strikingly, bPAP significantly reversed inflammatory pain caused by thermal and mechanical stimuli. See
PAP Treatment of Neuropathic Pain.
The extent to which intrathecal injection of PAP protein can block maintenance of neuropathic pain was determined. The main difference between blocking initiation and maintenance of neuropathic pain has to do with when PAP is injected relative to the spared nerve injury (SNI) surgery. See
The spared nerve injury (SNI) model was used to produce a neuropathic-like pain state in mice. Surgeries were performed in the animal facility following published procedures (Shields et al., 2003). In brief, mice were anesthetized with halothane, the sural and peroneal branches of the right sciatic nerve were ligated, then ˜1 mm from each nerve cut. The tibial nerve was spared. This causes profound mechanical allodynia in the right hindpaw but little thermal hyperalgesia (Shields et al., 2003).
The right (control-untreated) and left (injured) hindpaws were tested for mechanical sensitivity (using the von Frey method; described above) and thermal sensitivity (Hargreave's method; described above) before surgery (baseline) and post SNI-surgery. Active bPAP or hPAP was injected i.t. using a dose that was empirically found to have maximal phosphatase activity but minimal side effects. An equivalent amount of inactive hPAP protein was injected to prove that the observed analgesic effects were due to PAP phosphatase activity. Injections (i.t.) were performed as described above 5-6 days after surgery (maintenance experiments). Statistical tests (t-tests) were used to determine the significance of differences in thermal and/or mechanical sensitivity between control and experimental animals. For the injured paw, i.t. injection of bPAP caused a decrease in thermal (see
These data suggest chronic pain can be treated in humans and other animal subjects by intrathecally injecting purified PAP protein or by administering small-molecule allosteric modulators to activate PAP normally present on pain-sensing neurons. These drug treatments can be used pre- or post-operatively to treat surgical pain; to treat chronic inflammatory pain (e.g., osteoarthritis, burns, joint pain, lower back pain); and to treat chronic neuropathic pain.
PAP was generally thought to function only in the prostate (Ostrowski and Kuciel, 1994). However, the presently disclosed data suggests that PAP can also function in nociceptive neurons. To further evaluate pain-related functions for PAP, age-matched wild-type C57BL/6 and PAP−/− male mice (backcrossed to C57BL/6 for 10 generations) were evaluated using acute and chronic pain behavioral assays. No significant differences between genotypes were found using a measure of mechanical sensitivity (electronic von Frey) or several different measures of acute noxious thermal sensitivity. See Table 4.
In contrast, PAP−/− mice showed significantly greater thermal hyperalgesia and mechanical allodynia relative to wild-type mice in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain. See
Since PAP−/− mice showed enhanced hyperalgesia and allodynia in the CFA inflammatory pain model, the ability of hPAP treatment to rescue these enhanced thermal and mechanical phenotypes in PAP−/− mice was examined. Intrathaceal injection of hPAP increases thermal withdrawal latency in the control (right) paw of PAP−/− (PAP KO) mice to the same extent as wild-type mice. See
The anti-nociceptive effects of PAP require catalytic activity. Without being bound to any one theory, this suggests that PAP generates, via dephosphorylation, a molecule that regulates nociceptive neurotransmission in the spinal cord. PAP and TMPase can dephosphorylate many different substrates (Dziembor-Gryszkiewicz et al., 1978; Sanyal and Rustioni, 1974; Silverman and Kruger, 1988b; Vihko, 1978b). One possible substrate is AMP. Dephosphorylation of AMP produces adenosine, a molecule that inhibits nociceptive neurotransmission in spinal cord slices and has well-studied analgesic properties in mammals (Li and Perl, 1994; Liu and Salter, 2005; Post, 1984; Sawynok, 2006).
Prior to the presently disclosed subject matter, there was no direct proof that PAP or TMPase could generate adenosine from AMP. Instead, production of adenosine was inferred by measuring production of inorganic phosphate (Vihko, 1978b). To directly test whether PAP could generate adenosine from AMP and other adenine nucleotides, PAP was incubated with 1 mM AMP, ADP or ATP at pH 7.0 for 4 h. Adenine nucleotides and adenosine were detected using high performance liquid chromatography (HPLC) and UV absorbance (Lazarowski et al., 2004). These studies revealed that PAP can rapidly dephosphorylate AMP and, to a much lesser extent ADP, to adenosine. See
Next, the extent to which PAP could dephosphorylate extracellular AMP in HEK 293 cells, DRG neurons and spinal cord was studied using AMP enzyme histochemistry. HEK 293 cells transfected with TM-PAP were heavily stained whereas control cells were not (see
Adenosine mediates anti-nociception through Gi-coupled A1-adenosine receptors (A1Rs) (Lee and Yaksh, 1996; Sawynok, 2006). To directly test whether A1Rs were required for PAP anti-nociception, wild-type C57BL/6 and A1-adenosine receptor knockout mice (A1R−/−, Adora1−/−; backcrossed to C57BL/6 mice for 12 generations), were i.t. injected with hPAP. Then noxious thermal and mechanical sensitivity was measured (Hua et al., 2007; Johansson et al., 2001). Strikingly, hPAP increased thermal paw withdrawal latency for three days in wild-type mice but was without effect in A1R−/− mice. See
The responses of wild-type and A1R−/− mice were also tested using the CFA chronic inflammatory pain model and the SNI neuropathic pain model. Reproducing previous findings (Wu et al., 2005), A1R−/− mice showed greater thermal hyperalgesia compared to wild-type mice after CFA injection and after nerve injury (but before PAP injection). See
A high-throughput biochemical assay was developed to identify drugs that modulate PAP activity. This assay relies on the use of pure hPAP protein as well as a fluorometric PAP substrate (difluoro-4-methylumbelliferyl phosphate (DiFMUP); commercially available from Invitrogen). Dephosphorylation of DiFMUP by hPAP was monitored using fluorometric microplate readers (such as FLIPR or Flexstation). First, appropriate concentrations of hPAP protein and DiFMUP substrate were identified for use in 96-well plates, then 2,000 compounds (NCl Diversity Set) were screened to identify small-molecules that enhanced (activators) or suppressed (inhibitors) hPAP reaction rate. Using data from this screen, a Z-factor was calculated of 0.86 (this figure can range from 0-1; with 0.5 being the cutoff for a useful HTS. 0.86 is a very high value and indicates the assay is highly reproducible and has a large signal-to-noise ratio) (Zhang et al., 1999). From the screen, 6 candidate hPAP inhibitors and 3 candidate hPAP activators were identified. Fresh compounds (ordered from NCl) were obtained and dose-response experiments performed. These experiments confirmed that all 9 candidates were in fact activators or inhibitors. The extent to which these compounds were specific for hPAP was assessed by testing the effects of these compounds on hPAP, bPAP, potato acid phosphatase and bovine alkaline phosphatase. Thus, activators and inhibitors of hPAP can be identified using a reproducible, miniaturized, and economical HTS. The assay is useful to identify additional small molecule modulators of PAP.
The references listed below as well as all references cited in the specification, including patents, patent applications, journal articles, and all database entries (e.g., GENBANK® Accession Nos., including any annotations presented in the GENBANK® database that are associated with the disclosed sequences), are incorporated herein by reference to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.
It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/003,205, filed Nov. 15, 2007; the disclosure of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61003205 | Nov 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12743110 | Jun 2010 | US |
| Child | 13868541 | US |
