# Patent

## Title
Method for alleviating pain using sphingosine-1-phosphate and related compounds, and assays for identifying such compounds

## Abstract

Methods for alleviating pain comprising administering to a subject sphingosine-1-phosphate, functional fragments and derivatives thereof, and other compounds, and assays for identifying such compounds.

## Background
BACKGROUND OF THE INVENTION
The invention concerns the use of S1P (Sphingosine-1-Phosphate). Other aspects of the invention concern a method for screening pharmaceuticals and methods for the treatment of pain.
Pain is a complex subjective sensation reflecting real or potential tissue damage and the affective response to it. Acute pain is a physiological signal indicating a potential or actual injury. Chronic pain can either be somatogenetic (organic) or psychogenic. Chronic pain is frequently accompanied or followed by vegetative signs, which often result in depression.
Somatogenetic pain may be of nociceptive origin, inflammatory or neuropathic. Nociceptive pain is judged to be commensurate with ongoing activation of somatic or visceral pain-sensitive nerve fibers. Neuropathic pain results from dysfunction in the nervous system; it is believed to be sustained by aberrant somatosensory processes in the peripheral nervous system, the CNS, or both. (For an overview of pain mechanisms, see for example Scholz and Woolf, 2002; Julius and Basbaum, 2001, Woolf and Mannion, 1999; Wood, J. D., 2000; Woolf and Salter, 2000.)
Chronic pain results in individual suffering and social economic costs of tremendous extent. Existing pharmacological pain therapies are widely unsatisfying both in terms of efficacy and of safety.
Up to now, two classes of analgesics are mainly employed for the treatment of pain: Non-opioid analgesics, mostly acetaminophen and NSAIDS (non-steroidal anti-inflammatory drugs) and opioid (narcotic) agonists (wherein “opioid” is a generic term for natural or synthetic substances that bind to specific opioid receptors in the CNS, producing an agonist action). Unfortunately both analgesic classes, opioids and non-opioids, have several unwanted side effects. The most serious side effects of opioids are the possibility of inhibition of the respiratory system and after long-term treatment the possibility of addiction (Schaible H. G., Vanegas H., 2000). NSAIDs, a major class of non-opioids, on the other hand, can induce a variety of gastrointestinal complications such as ulcers and bleeding, but also kidney damage (Schaible H. G., Vanegas H., 2000). It has been estimated that in the U.S.A. about 16.000 patients die every year because of severe gastro-intestinal complications caused by conventional NSAIDs.
In light of the severe drawbacks connected with state of the art pain treatments, there is a great need for novel classes of pain modulating drugs. Especially in light of the vast gap between the fast advancing understanding of the neurobiology of pain and the unmet clinical need to provide effective treatments without the drawbacks of state of the art treatments, efforts need to be directed to the discovery of new targets for novel classes of analgesics. Thus, it is the object of the present invention to provide a new means for the development and provision of a new class of pain modulating drugs.
This object is solved by the use of S1P or functional fragments or derivatives thereof for the preparation of pharmaceutical compounds that alleviate pain.



DESCRIPTION OF THE FIGURES
FIG. 1: PAM is highly expressed in spinal cord neurons. In situ hybridization using horizontal sections of spinal cords hybridized with sense or antisense probes against rat PAM.
FIG. 2: PAM is expressed in DRG neurons as well as in neuronal cells in rat spinal cord.
FIG. 2A: Immunohistochemical analysis of rat spinal cord sections. The sections were stained with anti-PAM antibody (green) and anti-NeuN or anti-GFAP (red) to visualize neurons or glia cells, respectively. The overlay of both signals is presented in the right panels. The objects were magnified 20×.
FIG. 2B: Immunohistochemical analysis of rat DRGs sections. The sections were stained with anti-PAM antibody (green) and anti-Neu68, anti-Histon or anti-GFAP (red) to visualize neurons, nuclei or glia cells, respectively. The overlay of both signals is presented in the right panels. The objects were magnified 40× except for the Histon staining, which was magnified 63×.
FIG. 3: PAM is differentially expressed in DRGs and spinal cord during different developmental stages. Quantitative RT-PCR (TaqmanTM) was used to detect PAM in RNA (40 ng) of spinal cord, and DRGs of embryonic rats day 16 (E16), postnatal day 0.5 (P0.5), 3 (P3), 5 (P5), 9 (P9) and adult rats. The mean±SEM of at least 3 determinations is shown.
FIG. 4: PAM is upregulated in the rat spinal cord after zymosan and formalin treatment.
Panel A: RT-PCR analysis with RNA (40 ng) of spinal cords from control animals or animals treated with zymosan after 24 h, 48 h and 96 h. The lower panel shows the mean±SEM of 7 experiments. Student T test: *p<0.001.
Panel B: Western blot analysis using a 7% SDS-PAGE gel with rat spinal cord lysates of control animals or animals treated with zymosan after 24 h, 48 h and 96 h (40 μg) with anti-PAM antibody and anti-ERK1/2.
Panel C: Quantitative RT-PCR analysis with RNA (40 ng) of spinal cords from control animals or animals treated with formalin for 1 hour.
FIG. 5: PAM inhibits Gαs-stimulated AC activity in spinal cord lysates.
Panel A: RT-PCR was used to determine AC isoform expression in spinal cord and DRG RNA (40 ng).
Panel B: Lysates of spinal cords or DRG (10 μg) were assayed for AC activity in the presence of 80 nM Gαs as described in Material and Methods. The mean±SEM of at least 3 determinations done in triplicates is shown.
FIG. 6: Intrathecal application of antisense ODNs against PAM increases nociceptive behavior.
Panel A: Adult rats were given intrathecal sense and antisense ODN as described. After formalin treatment, the spinal cord was removed and subjected to immunohistological analysis using anti-PAM antibodies (green) or anti NeuN (red).
Panel B: Formalin assay of animals treated with sense or antisense ODNs as described in the material section. The total amount of flinches over 1 hour is shown. The mean±SE of at least 4 determinations is shown.
Panel C: Formalin assay of animals treated with sense or antisense ODNs as described in the material section. The number of flinches during 1 hour is shown. The mean±SE of at least 4 determinations is shown.
FIG. 7: Protein (FIG. 7B), genomic (FIG. 7C) and coding nucleotide sequence of human PAM (FIG. 7A) according to NCBI accession numbers AAC39928 (protein sequence; SEQ ID No. 1), AF075587 (coding sequence; SEQ ID No. 2). Human PAM is located on Chromosome 13q22; its genomic sequence is publicly available under NT—024524.11 (Start: position 24679861; Stop: position 24962245; SEQ ID No 3); FIG. 7C shows the contiguous sequence from position 24679861 to position 24962245.
FIG. 8: EST-clone coding sequences for rat PAM:
FIG. 8A: AW921303 (corresponds to by 960-1394 of hs cDNA; SEQ ID No. 4)
FIG. 8B: AW918711 (corresponds to by 8188-8632 of hs cDNA; SEQ ID No. 5)
FIG. 8C: BQ201485 (corresponds to by 8966-9633 of hs cDNA; SEQ ID No. 6)
FIG. 8D: BE112881 (corresponds to by 10311-10830 of hs cDNA; SEQ Id No. 7)
FIG. 8E: AW441131 (corresponds to by 13569-14152 of hs cDNA; SEQ ID No. 8)
FIG. 8F: BF409872 (corresponds to by 13569-14807 of hs cDNA). (SEQ ID No. 9)
FIG. 9: Overview of domain structure of human PAM according to Guo et al., 1988/Grossberger et al., JBC 1999 and Scholich et al., JBC 2001)
FIG. 10: PCR Primers for rat PAM RT PCR.
FIG. 11: Antisense Oligodeoxynucleotides for inhibiting rat PAM expression and control oligonucleotide.
FIG. 12: Different PAM hs polypeptides for the use in the context of present invention. The polypeptides are fragments derived from the polypeptide according to SEQ ID No. 2.
FIG. 13: Different PAM hs polynucleotides for the use in the context of present invention.
FIG. 13A: cDNA sequence coding for the protein fragment according to SEQ ID No. 17 comprising nucleotide positions 1317 to 4366 of hs Pam cDNA (SEQ ID No. 24);
FIG. 13B: cDNA sequence coding for the protein fragment according to SEQ ID No. 18 comprising nucleotide positions 1482 to 3332 of hs Pam cDNA (SEQ ID No. 25);
FIG. 13C: cDNA sequence coding for the protein fragment according to SEQ ID No. 19 comprising nucleotide positions 1641 to 3341 of hs Pam cDNA (SEQ ID No. 26);
FIG. 13D: cDNA sequence coding for the protein fragment according to SEQ ID No. 20 comprising nucleotide positions 3142 to 4046 of hs Pam cDNA (SEQ ID No. 27);
FIG. 13E: cDNA sequence coding for the protein fragment according to SEQ ID No. 21 comprising nucleotide positions 3142 to 3446 of hs Pam cDNA (SEQ ID No. 28);
FIG. 13F: cDNA sequence coding for the protein fragment according to SEQ ID No. 22 comprising nucleotide positions 3228 to 3839 of hs Pam cDNA (SEQ ID No. 29);
FIG. 13G: cDNA sequence coding for the protein fragment according to SEQ ID No. 23 comprising nucleotide positions 3228 to 3341 of hs Pam cDNA (SEQ ID No. 30);
FIG. 14: PAM signaling according to the above findings.
FIG. 15: Structure of Sphingosine-1-Phosphate.
FIG. 16: mDNA and amino acid sequences of S1P receptors:
FIG. 16A: amino acid sequence of S1P1 (SEQ ID No. 31);
FIG. 16B: mRNA sequence of S1P1 (SEQ ID No. 32); the coding sequence starts at position 244 and ends at position 1392;
FIG. 16C: amino acid sequence of S1P2 (SEQ ID No. 33);
FIG. 16D: mRNA sequence of S1P2 (SEQ ID No. 34); the coding sequence starts at position 1 and ends at position 1062;
FIG. 16E: amino acid sequence of S1P3 (SEQ ID No. 35)
FIG. 16F: mRNA sequence of S1P3 (SEQ ID No. 36), the coding sequence starts at position 1 and ends at position 1137;
FIG. 16G: amino acid sequence of S1P4 (SEQ ID No. 37)
16h) mRNA sequence of S1P4 (SEQ ID No. 38), the coding sequence starts at position 23 and ends at position 1177;
FIG. 16I: amino acid sequence of S1P5 (SEQ ID No. 39)
16j) mRNA sequence of S1P5 (SEQ ID No. 40), the coding sequence starts at position 10 and ends at position 1206;
FIG. 17: Structure of FT720
FIG. 18: PAM translocates from the ER to the plasma membrane in HeLa cells after serum stimulation.
Panel A: Serum starved HeLa cells (24 hours) were fixed and stained with anti-PAM antibodies (green) and anti-Calnexin antibody (red) as described above.
Panel B: HeLa cells were treated with 10% fetal bovine serum for different times and stained with anti-PAM antibodies to monitor the subcellular localization.
FIG. 19: Serum-treatment of HeLa cells decreases AC activity through a PAM-dependent process.
Panel A: cAMP accumulation in HeLa cells was measured in the absence and presence of serum as described above. The mean±SEM (Standard error of the mean) of three determinations is shown.
Panel B: Adenylyl cyclase activity of HeLa cell lysates was measured in the absence and presence of serum as described in Materials and Methods section. The Gsα* (80 nM; grey bars)- and forskolin (100 μM; black bars)-stimulated specific activities of ACV were 124±10 pmol/mg/min and 464±89 pmol/min/mg, respectively. The basal activity was 11.75±2 pmol/mg/min. The mean±SE of at least 2 experiments each done in triplicates is shown. Student T test: *p<0.001.
Panel C: RT-PCR analysis of the AC isoform expression in serum starved HeLa cells and after 1 hour incubation with 10% FBS.
Panel D: Serum-starved HeLa cells were transfected with 3 M each of antisense, sense, and antisense ODN harboring three point mutations (3M-as) as described in above. Cells were harvested and subjected to Western analyses using 7% SDS-PAGE (30 μg protein) with anti-PAM antibody and anti-Hsp70 antibody.
Panel E: Serum-starved HeLa cells were treated with sense, antisense, and 3M-as ODNs as described above. After 24 hours the cells were incubated for 30 minutes with 10% FBS. Cells were harvested and AC activity in presence of Gαs* (80 nM; grey bars) and forskolin (100 μM; black bars) was determined as described above. Data from at least 3 experiments measured in triplicates are presented as the mean±SEM. Student T test: *p<0.01, **p<0.05.
FIG. 20: S1P induces translocation of PAM the plasma membrane and inhibits PAM-dependent AC enzyme activity.
Panel A: RT-PCR analysis of the S1P receptor isoform expression in serum starved HeLa cells.
Panel B: HeLa cells were treated with 0.5 μM S1P for 30 minutes and stained with anti-PAM antibodies to monitor the subcellular localization.
Panel C: Time dependence of PAM translocation in HeLa cells. Serum-starved HeLa cells were treated with 0.5 μM S1P for different times and stained with anti-PAM antibodies to monitor the subcellular localization. The percentage of cells exhibiting PAM-translocation is presented.
FIG. 21: S1P-treatment of HeLa cells decreases AC activity through a PAM-dependent process.
Panel A: cAMP accumulation in HeLa cells was measured in the absence (black bars) and presence (grey bars) of 0.5 μM S1P at varying times as described in Materials and Methods section. The mean±SEM of three determinations is shown.
Panel B: Serum-starved HeLa cells were treated for varying times with 0.5 μM S1P. Cells were harvested and AC activity in presence of 80 nM Gαs* was determined as described in Material and Methods. Data from at least 4 experiments measured in triplicates are presented as the mean±SE.
Panel C: Serum-starved HeLa cells were treated with sense, antisense, and 3M-as ODNs as described in the Materials and Methods section. After 24 hours the cells were incubated for the indicated times with 0.5 μM S1P. Cells were harvested and AC activity in presence of Gαs* (80 nM) was determined as described above. Data are presented as the mean±SEM of three determinations.
FIG. 22: PAM-translocation and PAM-dependent inhibition of AC enzyme activity is mediated by the PLC/PKC signaling pathway
Panel A: Serum-starved HeLa cells were treated with 0.5 μM S1P for 3 (grey bars) or 60 (black bars) minutes. Prior incubation with S1P the cells were incubated for 24 hours with 1 μg/μl pertussis toxin (PTX). AC activity in was determined presence of 80 nM Gαs*. Data from at least 2 experiments measured in triplicates are presented as the mean±SE.
Panel B: Serum-starved HeLa cells were treated with 0.5 μM S1P. Prior incubation with S1P the cells were incubated for 20 minutes with 10 μM U73122, RO31-8220, BAPTA-AM or U0126. AC activity in was determined presence of 80 nM Gαs*. Data from at least 2 experiments measured in triplicates are presented as the mean±SE.
Panel C: Western Blot analysis of HeLa cells treated with 0.5 μM S1P for 10 minutes. Prior incubation with S1P the cells were incubated for 20 minutes with 10 μM U73122, BAPTA-AM or U0126. Cells were harvested in boiling Laemmli buffer and subjected to Western analyses using anti-active ERK1/2 antibody. Anti-Hsp70 antibody was used as loading control.
Panel D: HeLa cells were treated with 0.5 μM S1P for 20 minutes and stained with anti-PAM antibodies to monitor the subcellular localization. Prior S1P-treatment were the cells incubated for 24 hours with 1 μg/μl pertussis toxin (PTX), for 20 minutes with, 1 μM GF109203X, 10 μM U0126, BAPTA-AM, U73122 or Ro31-223.
FIG. 23: Schematic of the proposed signaling pathway leading to PAM translocation and PAM-dependent inhibition of AC enzyme activity.
FIG. 24: Pain behaviour of adult rats in the formalin assay with and without application of S1P over a period of 60 minutes. 20 μl of 10 μM S1P or 20 μl PBS/DMSO were given to adult rats by intrathecal application 15 minutes prior to formalin injection. After formalin injection, flinches were counted in 5 minute intervals over a time-period of 60 minutes. The mean of six animal experiments+SEM is shown.



DETAILED DESCRIPTION OF THE INVENTION
The invention is based on findings of the inventors that demonstrate for the first time the implication of S1P in nociceptive processing and its ability to decrease pain (a U.S. Patent Application filed May 20, 2004 listing the same inventors, entitled “Method for alleviating pain using protein associated with Myc and related compounds, and assays for identifying such compounds,” the contents of which are incorporated herein by reference, describes a similar role for protein associated with Myc). The term functional with respect to S1P or the term S1P function refers to the ability of S1P to interact with at least one of its receptors and preferably to activate the receptor, or to lower intracellular cAMP levels or to mediate the translocation of PAM (Protein Associated with Myc) from the endoplasmic reticulum to the cellular membrane; more preferably it refers to its ability to enhance PAM activity (i.e. the ability of PAM to interact with AC and/or to lower AC activity and/or to decrease pain) and/or to inhibit AC activity and even more preferably to its ability to decrease pain. The term functional with respect to S1P receptors or the term S1P receptor function refers to the ability of S1P receptors to interact with S1P, more specifically to mediate the receptor-typical signal triggered by the S1P interaction and more specifically to influence pain processing triggered by the S1P interaction.
A fragment of S1P can be any fragment that is smaller than the wild type molecule according to FIG. 15. A fragment of PAM can be any fragment polypeptide or polynucleotide that is shorter than the corresponding wild type. A fragment of an S1P receptor can be any fragment polypeptide or polynucleotide that is shorter than the corresponding wild type.
A derivative of S1P or of a S1P fragment can be any modification of the molecule having S1P function or any other kind of modification, such as a chemical or biological modification e.g. leading to the stabilization of the molecule, or modulating its specific targeting to e.g. certain cells or facilitating its entry into or uptake by cells; one known modification being the hydroxylation or methylation of S1P. Useful are suitable modifications or additives for ensuring or facilitating its targeting to the site of need and its entering the cell. On the other hand, a local application, such as an intraspinal application using suitable catheters, etc. or the like is possible for ensuring its targeting to the spinal cord. Other useful additives include salts (for physiologically tolerable organic or anorganic salts, see, e.g. Remington's Pharmaceutical Sciences, p. 1418, 1985), buffers or the like for its stabilization, etc.
Since S1P is internalized by cells via specific receptors, it can be applied externally and will then be internalized specifically. A modulation of S1P targeting can e.g. be gained by cloning and expression of the S1P receptors in the cell of want. A cell type specific expression can be ensured using appropriate promoters/enhancers of genes which are known in the art.
The present invention is based on studies of the inventors, that demonstrate for the first time the surprising implication of S1P in sensitisation mechanisms within the spinal cord and dorsal root ganglia (DRGs).
S1P (Sphingosine-1-Phosphate) is a phosphorylated derivative of sphingosine, the structural backbone of all sphingolipids. This extracellular (serum-borne) sphingolipide known to regulate a variety of cellular processes by binding to one of five specific G-protein coupled receptors (GPCRs), named S1P1 to S1P5, that are differentially expressed in different tissues, each regulating specific cellular actions (for a review, see. e.g. Payne et al., 2002; and Spiegel and Milstien, 2000). Known functions of extracellular S1P include, e.g. the regulation of cellular migration, cell survival or angiogenesis. Apart from its extracellular actions it is also known to act as an intracellular messenger (for a review, see. e.g. Payne et al., 2002; and Spiegel and Milstien, 2000). Its implication in nociceptive processes, however has not been known so far.
PAM (Protein Associated with Myc) is a giant protein of 510 kDa. The protein, genomic and coding polynucleotide sequences of PAM are known in the state of the art and are, e.g. publicly available from the NCBI (National Centre for Biotechnology Information; National Library of Medicine, Building 38A, Bethesda, Md. 20894, USA) data base under the accession numbers AAC39928 (coding sequence; SEQ ID No. 1), AF075587 (protein sequence; SEQ ID No. 2). Human PAM is located on Chromosome 13q22; its genomic sequence is publicly available under NT—024524.11 (Start: position 24679861; Stop: position 24962245; SEQ ID No. 3). Alternatively, the protein and coding sequence are publicly available under KIAA0916 protein Accession NP—055872 (protein sequence) and NM—015057 (coding sequence).
For rat PAM, the following EST-clone coding sequences are publicly available:
AW921303 (corresponds to by 960-1394 of hs cDNA; SEQ ID No. 4)
AW918711 (corresponds to by 8188-8632 of hs cDNA; SEQ ID No. 5)
BQ201485 (corresponds to by 8966-9633 of hs cDNA; SEQ ID No. 6)
BE112881 (corresponds to by 10311-10830 of hs cDNA; SEQ ID No. 7)
AW441131 (corresponds to by 13569-14152 of hs cDNA; SEQ ID No. 8)
BF409872 (corresponds to by 13569-14807 of hs cDNA; SEQ ID No. 9).
PAM was originally identified by its ability to interact specifically with the transcriptional activating domain in the N-Terminus of Myc (Guo Q., et al., 1998). PAM has recently been described as a powerful inhibitor of AC activity (Scholich K., Pierre S., Patel T. B.: Protein associated with Myc (PAM) is a potent inhibitor of adenylyl cyclase. J. Biol. Chem. 2001, Dec. 14; 276 (50):47583-9), but there has been no evidence of its function in nociceptive processing and sensitisation, so far.
Rather, PAM is believed to be playing a role in presynaptic growth regulation: PAM mRNA has been known to be highly expressed in specific anatomical regions, including hippocampus, dentate gyrus and cerebellum. Both PAM and Myc expression in the brain of adult rats and mice is confined to the maturing Purkinje cells in the cerebellum and granule and pyramidal cells in the hippocampus (Ruppert C., et al., 1986; Yang H. et al., 2002). None of these cell types, however is known to be involved in pain processing and sensitisation.
PAM homologues in Drosophila (highwire) and C. elegans (rpm-1) have been shown to play a crucial role in presynaptic terminal organization (Zhen et al., 2000), the regulation of synaptic growth (Wan et al., 2000), synaptogenesis, and neurite growth and targeting (Schaefer et al., 2000). These findings led to the assumption that highwire, rpm-1 and their mammalian homolog PAM might act as negative regulators of synaptic growth (Chang et al., 2000; Jin Y. 2002). Accordingly, a dramatic increase in PAM expression in the cerebellum, hippocampus and dentate gyrus was found during the major synaptogenic period in these structures (Yang et al., 2002).
During brain development in rodents, PAM expression is turned on shortly after birth, up-regulated during the first two weeks, and, thereafter, PAM expression remains elevated during adulthood (Yang et al., 2002). So far, nothing has been known about the expression and regulation of PAM in the spinal cord and DRGs and its function in sensitisation mechanisms and regulation of pain.
Previously, it has been demonstrated that human PAM is a potent regulator of cyclic AMP (cAMP)-signaling and inhibits the enzyme activity of several adenylyl cyclase (AC; E.C.4.6.1.1) isoforms at nanomolar concentrations (Scholich et al. 2001).
The ubiquitous cyclic AMP (cAMP) second messenger system is one of different signal transduction mechanisms translating extracellular stimuli to intracellular signals and responses. Upon extracellular stimulation, G-protein coupled receptors (GPCRs) modulate plasma-membrane bound enzymes or ion channels via trimeric GTP-binding regulatory proteins (G-proteins). One of the enzymes modulated in its activity by GPCRs is the adenylyl cyclase (AC), a cAMP generating enzyme. Thus, the incoming extracellular stimuli influence the intracellular concentration of the intracellular mediator cyclic AMP. A rise in cAMP levels affects the cell by stimulating protein kinase A (PKA), which phosphorylates specific intracellular target proteins and thereby alters their activity.
Each type of cell has characteristic sets of GPCRs, enzymes modulated by those GPCRs, specific subsets of adenylyl cyclase (AC) and target proteins, that, acting together with more unspecific or generally occurring players (such as the ubiquitous cAMP), enable each cell to make its own distinctive response to incoming extracellular signals. It is for example known that the cyclic AMP (cAMP)-second messenger plays a major role in the regulation of synaptic plasticity (Bailey et al., 1996; Xia et al., 1997; Brandon et al., 1997); on the other hand it is involved in metabolic processes and cellular proliferation. Thus, the role of the ubiquitous cAMP messenger system and its different components varies according to different specializations of different tissue and cell types.
So far, 5 different GPCRs acting as SIP receptors are known in the art termed S1P1 to S1P5, (for a review, see, e.g. Spiegel, S., and Milstien, S., 2000). The protein and coding polynucleotide sequences of the different SIP receptors are known in the state of the art and are, e.g. publicly available from the NCBI (National Centre for Biotechnology Information; National Library of Medicine, Building 38A, Bethesda, Md. 20894, USA) data base under the accession numbers: NM—001400 (SEQ ID No. 32; nucleotide sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 mRNA (EDG1/S1P1); NP—001391 (SEQ ID No. 31, protein sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor (EDG1/S1P.sub.1); NM—004230 (SEQ ID No. 34, nucleotide sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor, 5 (EDG5/S1P2) mRNA; NP—004221 (SEQ ID No. 33, protein sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor, 5 (EDG5/S1P2); NM—005226 (SEQ ID No. 36, nucleotide sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor 3 (EDG3/S1P3) mRNA; NP:005217 (SEQ ID No. 35, protein sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor 3 (EDG3/S1P3); NM:003775 (SEQ ID No. 38, nucleotide sequence of Homo sapiens endothelial differentiation, G-protein-coupled receptor 6 (EDGE/S1P4) mRNA; CAA04118 (SEQ ID No. 37, protein sequence of Homo sapiens endothelial differentiation, G-protein-coupled receptor 6 (EDGE/S1P4); NM—030760 (SEQ ID No. 40, nucleotide sequence of homo sapiens endothelial differentiation sphingolipid G-protein-coupled receptor 8 (EDG8/S1P5) mRNA; NP—110387 (SEQ ID No. 39, protein sequence of homo sapiens endothelial differentiation sphingolipid G-protein-coupled receptor 8 (EDG8/S1P5).
Another aspect of the invention concerns S1P or a functional fragment or derivative thereof for the use as a medicament, preferably a medicament for the prevention or treatment of pain.
A further aspect of the invention concerns the use of S1P or functional fragments or derivatives thereof for the alleviation of pain. The term “alleviate,” as applied to pain, means the ability to prevent, lessen, or abolish pain, or to otherwise make pain more bearable.
Moreover, the use of S1P or functional fragments of derivatives thereof for identifying compounds that alleviate pain is encompassed by present invention. The compounds preferably have, mimic or enhance S1P activity. Most preferably they have the ability to alleviate pain.
The compounds can for example be identified by their ability to
a) mimic, restore, activate or enhance S1P function (i.e. its ability to interact with at least one of its receptors or fragments thereof and preferably to activate the receptor, or to lower intracellular cAMP levels or to mediate the translocation of PAM from the endoplasmic reticulum to the cellular membrane; more preferably it refers to its ability to enhance PAM activity and/or to inhibit AC activity and even more preferably to its ability to decrease pain) or PAM function (i.e. its ability to lower intracellular cAMP levels, to interact with other factors like AC, especially with AC, to inhibit AC or its ability to lower the pain perception) or

b) increase the serum level of S1P (e.g. by activating or enhancing the production of S1P or diminishing its extracellular degradation) or

c) enhance the expression of at least one S1P receptor (i.e. by activation of its transcription, transcript stabilisation, translation or its posttranslational processing; by modulation of its posttranslational modification or by activation of its stabilisation or inhibition of its degradation, etc.) or

d) interacti with enzymes responsible for production or degradation of S1P.

Another aspect of present invention regards a method of alleviating pain comprising administering to an individual a sufficient amount of S1P or a functional fragment or derivative thereof.
The compounds of the invention—including S1P and functional fragments and derivates thereof—may further be used to alleviate inflammation as well as pain, meaning that the compounds may be used to prevent, lessen, or abolish the inflammation or otherwise make it more bearable.
Administration should suitably be performed in a way that allows for targeting of S1P to the site of action (DRG or spinal cord), e.g. by systemical administration of S1P derivatives or formulations to the bloodstream (e.g. intravenous or oral application) or by local (e.g. intraspinal) application of S1P or its fragments or derivatives thereof.
Another aspect of the invention concerns a method of screening for pharmaceuticals useful for modulating and/or preventing pain, comprising the steps

    
    
        a. Providing a sample containing PAM or a functional fragment or derivative thereof and not containing S1P,
        b. Providing a second sample containing PAM or a functional fragment or derivative thereof containing as well S1P,
        c. Contacting at least the first sample with a compound,
        d. Measuring the PAM activity in the samples,
        e. Determining the ability of the compound to mimic S1P function.
    
    


The method can further comprise a step, wherein the cell is contacted with S1P instead of the compound and wherein the PAM activities according to c) and d) above are compared to the PAM activity in presence of S1P.
Another example refers to a method comprising the steps,

    
    
        a) providing two samples comprising a cell expressing an S1P receptor or a functional fragment or derivative thereof, and
        b) contacting one sample with the compound, and
        c) measuring the receptor activity in both samples.
    
    


A method according to another aspect of the invention comprises the steps,

    
    
        a) providing two samples comprising a cell expressing an S1P receptor or a functional fragment or derivative thereof, and
        b) contacting the samples with S1P, and
        c) contacting one sample with the compound, and
        d) measuring the receptor activity or the interaction of S1P and receptor in both samples.
        PAM or the S1P receptors can be derived from any sequence available that allows for their specific purpose according to the different aspects of the present invention. Preferably, PAM or the S1P receptors are of human.
    
    


For the different aspects of present invention it is also preferred, if PAM or the S1P receptors are isolated polypeptides or oligo- or polynucleotides. Isolated in the context of the different aspects of present invention means at least partially purified from a natural source or recombinant molecules (which can, of course, also be purified or partially purified).
An assay is any type of analytical method to monitor a biological process. For the use in drug screening, the assay needs to be reproducible and is preferably also scalable and robust. The assay is preferably suitable for high throughput screening of chemical substances for their ability of modulating (preferably diminishing) and/or preventing pain. High throughput screening mostly comprises the screening of approximately 500.000 different compounds for a certain ability. The type of assay depends e.g. on the type of molecule used (either polypeptide or polynucleotide) and the “read out”, i.e. the way in which S1P, PAM or S1P receptor activity is determined (see below).
Different types of such assays are commonly known in the state of the art and commercially available from commercial suppliers. Suitable assays encompass radioisotopic or fluorescent assays, for example fluorescence polarization assays (such as those offered commercially by Panvera, Perkin-Elmer life sciences (e.g. LANCE) or Packard BioScience (e.g. HTRF or ALPHAscreenTM)) for measuring the interaction of a labeled member with a non-labeled member (e.g. PAM or fragments thereof could be labeled and their interaction with AC could be monitored).
Simple biochemical assays are suitable, e.g. for determining the interaction between a potential pharmaceutical compound and a receptor or a functional receptor fragment or derivative. More elaborate assays are also capable of determining whether the compound is able to activate a given receptor and is thus mimicking S1P activity.
More examples include cell based assays, wherein a cell line stably (inducibly or not; chromosomal or episomal) or transiently expresses a recombinant protein of interest. These assays comprise e.g. reporter gene assays, wherein the regulation of a certain promoter or a signal transduction pathway of a member of a signal transduction cascade is measured according to the activity of a reporter enzyme, the expression of which is under the control of said certain promoter. For this type of assay, a recombinant cell line has to be constructed containing the reporter gene under the control of a defined promoter that is to be investigated itself or that is regulated by the signaling cascade under investigation. Suitable reporter enzymes are commonly known within the state of the art and comprise firefly luciferase, renilla luciferase (e.g. commercially available by Packard reagents), β-Galactosidase. Suitable cell lines depend on the aim of the assay but comprise mostly cell lines that are easy to transfect and easy to cultivate, such as, e.g. HeLA, COS, CHO, NIH-3T3, etc.
Assays for measuring the intracellular ion level comprise e.g. FLIPR (fluorometric imaging plate reader, commercially available from Molecular Devices) assays, wherein an argon laser light source combined with a cooled CCD camera allows for parallel measurements in 384 well plates transient ion signals (such as Ca2+, etc) within cells (e.g. neuronal cells or other cells (e.g. cells recombinantly or naturally expressing certain ion channels). FLIPR assays allow e.g. for monitoring of intracellular calcium using certain fluorochromes, such as Fluo-3, Fluo-4, or monitoring intracellular pH using BCECF or BCPCF pr specific FLIPR assay kits, or detecting membrane potential changes using e.g. DiBAC or specific FLIPR assay kits, or monitoring of membrane polarization. For the monitoring of other intracellular ions, e.g. zinc or sodium, other dyes known in the state of the art can be used. Other types of assays and other types of read outs are commonly known to persons with skills in the art.
For the measurement of cAMP levels, e.g. AlphaScreen, fluorescence polarization or HTRF technology is suitable.
For the determination of ion channel activity (which control e.g. intracellular ion concentrations and can thus be employed for measurement of intracellular ion concentrations) e.g. membrane potential sensitive assays and dyes can be used such as DiBAC or Molecular Devices' membrane potential assay kit on FLIPR technology; mitochondrial membrane polarization measuring JC-1 dye with FLIPR technology; ion sensitive dyes such as Fluo-3, Fluo-4 or Molecular Devices calcium assay kit for intracellular calcium concentration measurement; sodium sensitive dye e.g. from Molecular Probes for measurement of intracellular sodium; assays based on patch-clamping or atomic adsorption spectroscopy-based Rubidium ion efflux measurement for determining of intracellular potassium concentrations, and so on. Further automatical devices and analytical methods for detecting certain changes and states within cells are known to the person of skill in the art and comprise, e.g. the Acumen detector (fluorescence-based laser scanning reader that allows for 3 dimensional reconstitution of distribution of suitably labeled objects) by ACUMEN bioscience.
For measurement of GPCR activity, e.g. cAMP measurement, for example by means of the AlphaScreenTM cAMP detection system by Packard Bioscience, Ca2+ mobilisation-assays or reporter gene assays are suitable.
The PAM polypeptide is preferably a polypeptide that comprises or consists of the sequence according to SEQ ID No 2 or is encoded by a polynucleotide comprising or consisting of the sequence according to SEQ ID No 1 or 3. The S1P receptor polypeptides are preferably polypeptides comprising or consisting of one of the amino acid sequences according to SEQ ID No. 31, 33, 35, 37 or 39 or are encoded by polynucleotides comprising or consisting of one of the nucleotide sequences according to SEQ ID No. 32, 34, 36, 38 or 40 or by the coding sequences comprised within these mRNA sequences.
The PAM polynucleotide is preferably a polynucleotide comprising or consisting of the sequence according to SEQ ID No 1 or 3 or a polynucleotide comprising or consisting of a sequence that is able to hybridize with the above polynucleotides under stringent conditions. The S1P receptor polynucleotides are preferably polynucleotides comprising or consisting of the sequences according to SEQ ID NO. 32, 34, 36, 38 or 40, polynucleotides comprising or corresponding to the positions 244 to 1392 of SEQ ID No. 32, 1 to 1137 of SEQ ID No. 34, 1 to 1062 of SEQ ID No. 36, 23 to 1177 of SEQ ID No. 38 or 10 to 1206 of SEQ ID No. 40, or comprise or consist of a sequence that is able to hybridize with one of these polynucleotides under stringent conditions.
Stringency describes reaction conditions that influence the specificity of hybridisation or annealing of two single stranded nucleic acid molecules. Stringency, and thus specificity of a reaction depends, inter alia, of the temperature and buffer-conditions used for a reaction: Stringency, and thus specificity, can e.g. be increased by increasing the reaction temperature and/or lowering the ion strength of the reaction-buffer. Conditions of low stringence (and thus low reaction and hybridisation specificity) exist for example, if a hybridisation is performed at room temperature in 2×SSC-solution. Conditions of high stringency comprise e.g. a hybridisation reaction at 68° C. in 0.1×SSC and 0.1% SDS solution.
Hybridisation under conditions of stringency within the different aspects of present invention is preferably understood to be:

    
    
        1) Hybridising a labelled probe with a nucleic acid sample to be analysed at 65° C., or in the case of oligonucleotide probes, at 5° C. below the annealing or melting temperature of the duplex consisting of oligonucleotide and sample (annealing and melting temperature are in the following understood to be synonyms) over night in 50 mM Tris pH 7.5, 1M Nacl, 1% SDS, 10% Dextran Sulfate, 0.5 mg/ml denatured salmon or herring sperm DNA.
        2) Washing for 10 minutes in 2×SSC at room temperature.
        3) Washing for 30 minutes in 1×SSC/0.1% SDS at 65° C. (or in the case of oligonucleotides: 5° C. below the annealing temperature).
        4) Washing for 30 minutes in 0.1×SSC/0.1% SDS at 65° C. (or in the case of oligonucleotides: 5° C. below the annealing temperature).
    
    


Oligonucleotides for the use as hybridisation probes are polynucleotide and preferably DNA-fragments having a length of 15 to 30, preferably 20 nucleotides. The annealing temperature is determined according to the formula Tm=2×(number of A+T)+4×(number of G+C)° C.
For preparing a 2×SSC or a 0.1×SSC (or any other kind of SSC dilution), e.g. a 20×SSC solution is diluted accordingly. 20×SSC consists of 3M NaCl/0.3 M Na-Citrate×2H2O.
Before performing a hybridisation reaction, the polynucleotides are, if wanted after performing electrophoretic separation (then: Southern Blot (DNA) or Northern Blot (RNA)) or without electrophoretic separation (then: slot or dot Blot), transferred to a suitable membrane, e.g. a nylon or nitrocellulose membrane. Hybridisation is performed using a suitably labelled probe. Suitable labelling techniques are e.g. radioactive labelling or labelling using fluorescence dyes. The probe is a single stranded polyribo- or polydesoxyribonucleotide being single stranded naturally or being usually double stranded and having been made single stranded by denaturation. This probe binds to the DNA or RNA sample (which is also in single stranded state) by means of base pairing.
The PAM fragments are preferably fragments comprised within the above sequences ID No. 1, 2 or 3 and the derivatives are preferably derived from the above sequences ID No. 1, 2 or 3 or from fragments thereof. The S1P receptor fragments are preferably fragments comprised within the above sequences ID No. 31 to 40, more preferably fragments comprising or consisting of positions 244 to 1392 of SEQ ID No. 32, 1 to 1137 of SEQ ID No. 34, 1 to 1062 of SEQ ID No. 36, 23 to 1177 of SEQ ID No. 38 or 10 to 1206 of SEQ ID No. 40 and the derivatives are preferably derived from these sequences.
The functional fragments or derivatives thereof are preferably capable of inhibiting adenylyl cyclase (AC) activity, more preferably that of AC Type I, V or VI (with respect to the S1P receptors, most preferably capable of inhibiting AC activity when activated by S1P binding or binding of a molecule mimicking S1P).
According to a preferred embodiment of the different aspects of present invention, the functional fragments or derivatives of PAM comprise or consist of amino acids 400 to 1400, preferably 446 to 1062, 499 to 1065 or 1028 to 1231, and more preferably 1000 to 1300 and even more preferably 1000 to 1100 and even more preferably 1028 to 1065 of the human PAM sequence, preferably of the human PAM sequence according to SEQ ID No. 2, or if they are encoded by the respective polynucleotide fragments, especially if comprised within the sequences according to SEQ ID No. 2 or 3.
If the functional fragments or derivatives thereof are polynucleotides, it is preferred, if they comprise or consist of polynucleotides encoding the above polypeptide fragments. More specifically, it is preferred if they comprise or consist of positions 1482 to 3332 (encoding amino acids 446 to 1062) or 1641 to 3341 (encoding amino acids 498 to 1066) or 3228 to 3839 (encoding amino acids 1038 to 1231) of the human PAM cds. It is even more preferred, if the human PAM cds from which the fragments are derived has the sequence according to SEQ ID No. 2.
According to one preferred embodiment of present method for identifying pain modulating compounds, a cell expressing an S1P receptor and/or PAM, preferably a recombinant S1P receptor and/or PAM is used.
The cell can be any type of cell, e.g. a eucaryotic or prokaryotic single cell organism (such as bacteria, e.g. e. coli, or yeast, e.g. s. pombe or s. cerevisiae) or cell lines derived from multicellular organisms (such as HeLa, COS, NIH-3T3, CHO, etc), wherein mammalian cell lines are preferred.
According to another preferred embodiment, a modified cell, having a lower S1P receptor activity as compared to its unmodified state, is used. This way, it can be tested, if the chemical compounds to be tested for their ability of modulating (preferably diminishing) and/or preventing pain, are able to enhance or restore the lowered or totally abolished S1P receptor activity.
The modification can be any type of modification (stable or transient, preferably stable), that leads to a decrease of S1P receptor activity and/or PAM activity (i.e. their ability to lower intracellular cAMP levels, the translocation of PAM, to inhibit AC or their ability to lower the pain perception), S1P receptor or PAM transcript steady state level (i.e. by inhibition of S1P receptor or PAM transcription or transcript stabilisation) or S1P receptor or PAM protein steady state level (i.e. by inactivation of S1P receptor or PAM translation or its posttranslational processing; by modulation of its posttranslational modification or by inactivation of its stabilisation or by increase of its degradation). This can for example be achieved by using dominant negative mutants of S1P receptors or PAM, antisense oligonucleotides, RNAi constructs, by generating functional or genomic S1P receptor or PAM knock outs (which can e.g. be inducible) or other suitable techniques known within the state of the art. For an overview of the above techniques, see for example: Current protocols in Molecular biology (2000) J. G. Seidman, Chapter 23, Supplement 52, John Wiley and Sons, Inc.; Gene Targeting: a practical approach (1995), Editor: A. L. Joyner, IRL Press; Genetic Manipulation of Receptor Expression and Function, 2000; Antisense Therapeutics, 1996; Scherr et al, 2003.
According to a preferred embodiment, a PAM knock-out cell is used. Suitable cell lines for the generation of knock-outs are well known in the state of the art and comprise e.g Current protocols in Molecular Biology (2000) J. G. Seidman, Chapter 23, Supplement 52, John Wiley and Sons, Inc; or Gene Targeting a practical approach. (1995) Ed. A. L. Joyner, IRL Press.
The S1P activity can either be determined directly, e.g. by its ability (or the ability of its fragments and derivatives) to interact with at least one of its receptors or their functional fragments or to trigger the translocation of PAM to the cell membrane, or it can be determined indirectly, e.g. by its ability (or the ability of its functional fragments and derivatives) to lower intracellular cAMP levels, to modulate ion concentrations within the neurons, to inhibit AC function, or its ability to modulate, especially decrease pain perception. Suitable techniques for measuring the above parameters are well known in the state of the art (see also above): The cAMP levels can e.g. be measured by HTRF or ALPHAscreenTM, the ion concentrations can e.g. be estimated by patch clamping or suitable dyes, the pain perception can e.g. be measured by means of the formalin test or tests of mechanical or thermal hyperalgesia, or the hot plate test etc. The interaction with its receptors can e.g. be determined by cAMP measurement, Ca2+ mobilisation or reporter gene assays.
Another aspect of present invention concerns a method of identifying a compound that alleviates pain comprising

    
    
        a) Selecting a compound that modulates or mimics the activity of S1P as a test compound, and
        b) Administering said test compound to a subject to determine whether the pain is alleviated.
    
    


The subject can be any subject with the ability of perceiving pain, preferably it is a mammal, either a non-human mammal or a human (i.e. within a patient study).
The modulation preferably alleviates the pain, meaning that it prevents, lessens, or abolishes it; or that it otherwise makes the pain more bearable for the subject. According to one preferred embodiment of the invention, the compound is an S1P receptor agonist (for a review, see for example Mandala et al., Science 2002) and more preferably FTY 720 (2-Amino-2-(4-ocylphanyl)ethyl)propane-1,3-diol, see FIG. 17) or a functional derivative or analog (analogs are known in the art, see e.g. Brinkmann et al., JBC, 2002) thereof (i.e. a derivative or an analog having the above-indicated capability of modulating pain), preferably a phosphorylated derivative (see Mandala et al., Science 2002). Suitable are also physiologically acceptable salts of the compound or its derivatives or analogs.
According to yet another aspect of the invention a method of alleviating pain comprising administering a sufficient amount of a pharmaceutical compound with the ability to bind and activate at least one of the S1P receptors and/or the activity to activate PAM function to an individual is concerned within the scope of the application. One suitable example of such a compound is an S1P receptor agonist (for a review, see for example Mandala et al., Science 2002) and more preferably FTY 720 or a functional derivative or analog as defined above, preferably a phosphorylated derivative thereof, or a physiologically acceptable salt of the compound or its functional derivative or analog.
In the following, the invention is illustrated in more detail by means of examples and figures. However, the examples are not meant to limit the scope of the invention.
EXAMPLES
Investigation of PAM Expression Pattern and Function of PAM and S1P
1. Materials
S1P was purchased from Tocris (Ellisville, Mo.), the anti-Hsp70 antibody and the anti-Calnexin antibody from BD TransductionLabs (Bedford, Mass.). The anti-active ERK1/2 antibody was obtained from Promega (Madison, Wis.). Pertussis toxin, U0126, U73122, and Wortmannin from Tocris (Ellisville, Mo.), RO31-223, BAPTA-AM and GF109203X by Sigma (St. Louis, Mo.).
2. Preparation of Animal Sections:
Wild type Sprague Dawley rats were purchased from Charles River Wiga GmbH (Sulzfeld, Germany). The animals had free access to food and water prior to the experiments. They were maintained in climate- and light-controlled rooms (24+0.5° C.). Each animal was used at one occasion only. In all experiments the ethics guidelines for investigations in conscious animals were obeyed, and the procedures were approved by the local Ethics Committee. After killing, adult rats were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.2) for one hour. Tissues were cryostat-sectioned in the horizontal plane at a thickness of 14-16 μm. Sections were mounted on Superfrost Plus Slides (Fisher Scientific Co., Pittsburgh, Pa.) and stored at −80° C. until use.
3. Preparation of Riboprobes:
The riboprobes were generated as described previously (Yang et al., 2002). Antisense and sense riboprobes of rat PAM were obtained with T7 and T3 polymerases, after linearizing the plasmid with Hind III (antisense) and BamHI (sense), respectively (see Yang et al., 2002). In vitro transcription was performed in the presence of [35S] UTP-αS (ICN, Irvine Calif.), linearized PAM cDNA, NTP at 37° C. for 1 hour according to the manufacturer's recommendation (Promega, Madison, Wis.). The RNA transcripts were purified using RNA Probe Purification Kit (Pequlab, Erlangen, Germany).
4. In Situ Hybridization:
In situ hybridization was performed as described earlier (Yang et al. 2002): Sections were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.2), pretreated with 0.25% acetic anhydride and 0.1 M triethanolamine, rinsed with 0.2×SSC and dehydrated with serially increasing concentrations of alcohol. Sections were prehybridized with prehybridization solution (50% deionized formamide, 0.6 M sodium chloride, 10 mM Tris-HCl (pH 7.6), 50 mM EDTA, 0.025% sodium pyrophosphate, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinyl pyrrolidone, 10 mM DTT, and heat-denatured, heterologous nucleic acids (0.005% yeast tRNA, type X, 0.05% yeast total RNA, type I, 0.05% salmon testes DNA, type III)) for 2 hours at room temperature; hybridized with riboprobes in hybridization solution (2.5×106 cpm/section), 50% deionized formamide and 50% hybridization buffer containing 0.6 M sodium chloride, 10 mM Tris-HCl (pH 7.6), 50 mM EDTA, 0.025% sodium pyrophosphate, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinyl pyrrolidone, heat-denatured, heterologous nucleic acids (0.005% yeast tRNA, type X, 0.005% yeast total RNA, type I, 0.05% salmon testes DNA, type III), 100 mM DTT, 0.0005% polyadenylic acid, 10% dextran sulfate) at 50° C. overnight. Sections were rinsed with 2×, 1×, 0.5×SSC at RT (room temperature). After digestion in 20 μg/ml RNase A (Sigma, St. Louis, Mo.), sections were washed in 1× RNase buffer, 2×, 1×, 0.5×SSC at room temperature, and in 0.1×SSC overnight at 45° C. Sections were dehydrated with serially increasing concentrations of alcohol, exposed to Kodak Biomax MR film (Kodak, Rochester, N.Y.) for 3-7 days at −80° C.
5. Antibody Generation and Immunofluorescence Staining:
Antisera were raised commercially in rabbits using peptides consisting of amino acid residues 135-153 and 4601-4614 of human PAM corresponding to the SEQ ID No. 1, respectively (BioTrend, Cologne, Germany). The antiserum was commercially produced by BioTrend, Cologne, Germany, according to standard procedures. To monitor the distribution of PAM in spinal cord and DRG slices, the slices were permeablized in 0.1% Triton X-100 for 5 minutes. The slices were blocked for 1 hour in 3% BSA in PBS and then incubated for 1 hour with anti-PAM antiserum (1:50 dilution). This was followed by incubation with FITC-labeled goat anti-rabbit antibody in PBS containing 3% BSA. The slices were then washed with PBS and mounted using FluoromountTM.
6. RT-PCR:
Total RNA from rat spinal cords and DRGs was isolated by guanidinium isothiocyanate/phenol/chloroform extraction (Chomczynski and Sacchi 1987). 2 μg of total RNA were annealed with 0.6 μM of each of oligo (dT) primer and reverse-transcribed using reverse transcriptase (Promega, Madison, Wis.) for 30 minutes at 37° C. The cDNA was then immediately used for amplification. Oligonucleotide primers used for the amplification of rat GADPH were 5′′-GAAGGGTGGGGCCAAAAG-3′′ (sense; SEQ ID No. 10) and 5′′-GGATGCAGGGATGATGTTCT-3′′ (antisense; SEQ ID No. 11; Trajkovic et al. 2000). Oligonucleotide primers for the amplification of AC isoforms were chosen as published by Xu et al. (Xu et al. 2001). Primers for rat PAM were 5′′-GGTGGTGAAGCTCGCTGTGATGCT-3′′ (sense; SEQ ID No. 12) and 5′′-CGTGTGAGCATTTCTGCACAC TCC-3′ (antisense; SEQ ID No. 13). The PCR product corresponds to the human PAM cDNA nucleotides 13692-14064. The corresponding rat sequence was derived from the EST clone AW441131 (SEQ ID No. 8). For semiquantitative PCR, SAWDAY DNA Polymerase (Peqlab, Erlangen, Germany) was used. After an initial denaturation step at 95° C. for 5 minutes, 30 cycles were performed with 1 minute at 95° C., 30 seconds at 55° C., and 10 seconds at 72° C., followed by a final 10-minute extension step at 72° C. Quantitative PCR was performed using the TaqManTM system and reagents (Applied Biosystems, Weiterstadt, Germany) according to the instructions of the manufacturer.
67 Purification of Full Length PAM:
PAM purification was performed with some modifications as published previously (Scholich et al. 2001). Shortly, HeLa cells were grown in DMEM medium with 10% fetal bovine serum and 1% penicillin/streptoMycin. Confluent cells of forty 150 mm dishes were harvested with 1×PBS, 1 mM EDTA and pelleted for 5 minutes at 400×g. The cells were resuspended in TED buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT) containing 125 mM NaCl, 20 μg/ml of aprotinin, 20 μg/ml leupeptin, 1 mM benzamidine, 5 μg/ml soybean trypsin inhibitor and lysed by 2×5 seconds of sonication. The homogenate was centrifuged at 27000×g for 30 min at 4° C., and the supernatant was loaded on a Q-Sepharose XK16 column (Amersham, Pharmacia, Piscataway, N.J.) and eluted with a gradient of 150-350 mM NaCl in TED according to instructions of the manufacturer. The fractions were analyzed by Western blotting according to standard procedures; positive fractions were pooled and the NaCl concentration adjusted to 1 M. The protein was then loaded on a Phenyl-Sepharose XK16 column (Amersham, Pharmacia, Piscataway, N.J.) and washed with 300 mM NaCl in TED according to instructions of the manufacturer. The flow through and wash fractions contained PAM. They were pooled and the buffer exchanged for the aforementioned TED buffer containing 100 mM NaCl using Centricon 50 (Amicon, Beverly, Mass.) according to the manufacturer's instructions. The protein was then loaded on a MonoS 5/5 FPLC column (Amersham, Pharmacia, Piscataway, N.J.) and washed with the loading buffer (100 mM NaCl in TED) according to instructions of the manufacturer. The flow through was collected and applied to a Mono Q 5/5 FPLC column (Amersham, Pharmacia, Piscataway, N.J.). The protein was eluted with a gradient from 150-400 mM NaCl in TED. Positive fractions were pooled and the buffer exchanged for 50 mM Tris-HCl, pH 8.0, 1 mM DTT using Centricon 50 (Amicon, Beverly, Mass.) and stored at −80° C. The stored PAM was used within 3 weeks.
8. Expression and Purification of Recombinant Gsα:
The hexahistidyl tagged constitutively active Q213L mutant of Gsα (Gsα*) was expressed and purified as described in Graziano et al., 1991. To ensure maximal activation of the Gsα*, the G protein was incubated with 1 μM GTPγS in the presence of MgCl2 (25 mM) for 30 minutes prior to use in AC activity assays.
9. Adenylyl Cyclase Activity Assays (AC Activity Assays):
Spinal cords were lysed in 25 mM Hepes, pH 7.4, 1 mM EGTA and cell membranes were prepared as described by Kassis and Fishman. Aliquots were stored at −80° C. until use. AC activity assays were performed in a volume of 100 μl for 15 min at room temperature in the presence of 100 μM MgCl2 as previously described (Patel et al., 2002). Gsα* (80 nM) or forskolin (100 μM) were used to stimulate AC enzyme activity in membranes (10 μg protein).
10. Spinal Delivery of PAM Antisense and Sense Oligonucleotides:
Rats were anesthetized with ketamine (60 mg/kg i.p.) and midazolam (0.5-1 mg/kg i.p.). The skin was incised above the vertebral column from vertebrae Th13 up to L3. Muscle tissue around L2-3 was cleared away. The processus spinosus of L3 was removed and a laminectomy was done at L2. Polyethylene catheters (ID 0.28 mm, OD 0.61 mm) were then inserted into the peridural space so that the tip of the catheter reached Th9-10. The catheter was fixed with cyanacrylate glue, externalized in the neck region, and the skin was sutured.
11. Infusion of PAM Oligonucleotides:
The sequences of the oligodeoxynucleotides (ODNs) were chosen from the rat PAM sequence as follows. Sense: 5′-GACTGGTTTAGCAATGGC-3′(SEQ ID No. 14), antisense: 5′-GCCATTGCTAAACCAGTC-3′ (SEQ ID No. 15), and antisense ODN harboring three mutations (3M-as; mutations are underlined): 5′-GCAATTGCTAAATCAGTA-3′ (SEQ ID No. 16). Three days after surgery, rats were placed into a “freely moving system” (CMA, Stockholm, Sweden) and antisense (n=5) or sense (n=5) oligonucleotides (2.5 mg/ml in artificial cerebrospinal fluid) were infused through the catheter at a flow rate of 0.05-0.1 μl/min for 100 hours using a microinfusion pump (CMA, Stockholm, Sweden).
12. Formalin Test:
Within 15 min after stopping the infusion, the formalin test was performed. 50 μl of a 5% formaldehyde solution was injected subcutaneously (s.c.) into the dorsal surface of one hind paw. Flinches were counted in one-minute intervals up to 60 min starting right after formalin injection. Flinches of 5 min intervals were summarized as mean flinches per minute. To compare the nociceptive behavior between groups, the sum of flinches during the two phases of the one-hour observation period were submitted to the Students t-test. α was set at 0.05.
At the end of the formalin test, the rats were killed, the lumbar spinal cord and dorsal root ganglia (DRGs) were excised, snap frozen in liquid nitrogen and stored at −80° C. until further analysis. To determine PAM expression, spinal cord slices were analyzed immunhistochemically using the above anti PAM antibodies.
13. Zymosan-Evoked Inflammation:
For induction of an inflammation, 2.5 mg zymosan A (Sigma, St. Louis, Mo.) suspended in 30 μl 0.03 M phosphate buffered saline (PBS, pH 7.5) was injected subcutaneously into the midplantar region of the right hindpaw. Such intraplantar zymosan injection is known to induce a reliable model of thermal and mechanical hyperalgesia rats (Meller and Gebhart 1997). Rats were killed by cardiac puncture under deep isoflurane anesthesia 24-96 hours after the Zymosan injection. The lumbar spinal cord and dorsal root ganglia (DRGs) were excised, snap frozen in liquid nitrogen and stored at −80° C. until further analysis.
14. Immunofluorescence Staining:
To monitor distribution of PAM in HeLa cells, cells were grown on glass coverslips in DMEM with 10% FBS and 1% penicillin/streptoMycin (Gibco, Karlsruhe, Germany). To visualize translocation of PAM HeLa, cells were serum starved overnight and subsequently treated with 10% serum for 2 hours. Wherever indicated, cells were preincubated for 30 minutes in the presence of different concentrations of inhibitors. The cells were fixed in 4% paraformaldehyde (Sigma, Taufkirchen, Germany) in PBS for 10 minutes and then permeablized in 0.1% Triton X-100 for another 5 minutes. The coverslips were blocked for 1 hour in 3% BSA in PBS and then incubated for 1 hour with anti-PAM antibody (1:50 dilution). This was followed by incubation with FITC-labeled goat anti-rabbit antibody in PBS containing 3% BSA. The cells were then washed with PBS and mounted. For the analysis confocal (BioRad, Hercules, Calif.) and regular fluorescence microscopes (Nikon, Duesseldorf, Germany) were used.
15. cAMP Accumulation
Spinal cord samples were sonicated and centrifuged at 4° C. for 20 minutes with 18.000×g. The supernatant was used for cAMP measurements. The cAMP accumulation in the cells was determined by the cAMP Detection Kit (Assay Design Inc, Ann Arbor, Mich.) according to the manufacturers instructions.
16. Antisense Oligodeoxynucleotides
The sequences of the oligodeoxynucleotides (ODNs) were chosen as published previously (Scholich et al., 2001). Sense: 5′-CTGTTCATGCCGGTT-3′, antisense: 5′-AACCGGCATGAACAG-3′, and antisense ODN harboring three mutations (3M-as; mutations are underlined): 5′-AATCCGTATGAACAC-3′. HeLa-cells were plated on 35 mm dishes (300,000 cells) and grown in DMEM medium (Gibco, Karlsruhe, Germany) containing 10% FBS and 1% penicillin/streptoMycin for 24 hours. The ODNs (3 μM each) were introduced into the cells by transfections using Tfx20 (Promega, Madison, Wis.) in 1 ml serum-free medium according to the instructions of the manufacturer. Two hours later, 1 ml DMEM (Gibco, Karlsruhe, Germany) containing 10% FBS was added. The cells were then incubated for 6 hours. Then the medium was exchanged against serum-free DMEM (Gibco, Karlsruhe, Germany), followed by an incubation for 16 hours in serum-free DMEM (Gibco, Karlsruhe, Germany) before being treated with 10% serum or 500 M S1P. The cells were then used for Western blots by adding boiling 1× Laemmli buffer or they were harvested for AC activity assays as described above.
17. Purification or the PAM Activating Serum Factor:
188 ml fetal bovine serum (Gibco, Karlsruhe, Germany) were adjusted to a final concentration of 0.3 M NaCl. The serum was then loaded on a Phenyl-Sepharose 15/10 column (Amersham, Pharmacia, Piscataway, N.J.). After elution from this column, as well as after all following columns, the flowthrough and all eluted fractions were collected and analyzed for its ability to induce translocation of PAM in HeLa cells. The flowthrough was then loaded on a Q-Sepharose 15/10 column (Amersham, Pharmacia, Piscataway, N.J.). The column was washed with 400 mM NaCl in TE (50 mMTris/Cl pH 7.4, 0.5 mM EDTA) and eluted with 1 M NaCl in TE. The eluate was loaded on a Superdex 200 pg Gel filtration column (Amersham, Pharmacia, Piscataway, N.J.). Protein was eluted with TE according to instructions of the manufacturer and the fractions were analyzed for their ability to induce translocation of PAM in HeLa cells. Positive fractions were pooled, loaded on a MonoQ 5/5 FPLC column (Amersham, Pharmacia, Piscataway, N.J.) and washed with 400 mM NaCl in TE. The protein was eluted with a gradient from 400-1000 mM NaCl in TED. Positive fractions were pooled and loaded on a Superdex 50 pg Gel filtration column (Amersham, Pharmacia, Piscataway, N.J.) according to instructions of the manufacturer. Protein was eluted with TE according to instructions of the manufacturer and the fractions were analyzed for their ability to induce translocation of PAM in HeLa cells. Positive fractions were pooled, stored at −80° C. and used within 2 weeks for mass spectrometry or biochemical assays. S1P was detected employing phthaldialdehyd-labeling followed by HPLC separation as described by Caligan et al.
18. Results Demonstrating the Implication of PAM in Nociceptive Processing:
The above experiments of the inventors using RT-PCR, immunohistochemistry and in situ hybridisation, demonstrate for the first time that PAM is expressed in sensory neurons of the spinal cord as well as in dorsal root ganglia (DRGs) of adult rats. PAM mRNA was detected by RT-PCR at similar levels in the spinal cord and dorsal root ganglia throughout development (E14-adult). PAM-expression is up-regulated 24-48 hours after zymosan treatment of rats as shown by western blot and RT-PCR.
The major adenylyl cyclase isoforms, which are expressed in the spinal cord and DRGs, are AC type 5 and 6 and AC type 4 and 6, respectively. No major changes in AC isoform expression were observed after zymosan treatment in spinal cord. Hence, Gαs stimulated AC activity in membrane preparations from spinal cord and DRG was inhibited by PAM. Consequently, it was found that treatment with antisense but not sense oligonucleotides against PAM increased formalin induced paw flinching in adult rats. Accordingly cAMP accumulation in the spinal cord of rats treated with antisense oligonucleotides to PAM was elevated as compared to control rats.
Addition of purified PAM to spinal cord lysates resulted in an inhibition of Gαs-stimulated AC activity of spinal cord lysates from control and zymosan treated animals (FIG. 5b). At 30 nM Gαs-stimulated AC activity was decreased by 50% in spinal cord lysates of control animals and 70% in lysates derived from rats treated with zymosan for 96 hours.
To determine if PAM is expressed in the spinal cord, first in situ hybridization was performed. This led to the detection of a clear signal for PAM mRNA throughout the gray matter but not in the white matter of the spinal cord of adult rats (FIG. 1). To define more precisely the cell populations that express PAM in the spinal cord as well as in DRGs, antibodies against PAM using peptides corresponding to the amino acid residues 135-153 and 4601-4614 of human PAM were generated. The immunohistochemical analysis revealed that PAM was co-localized with anti-NeuN but not with anti-GFAP immunoreactivity (FIG. 2a). More specifically, PAM expression was detected predominantly in dorsal horn neurons (FIG. 2a) while non-neuronal cell populations exhibited very little PAM expression. Especially high PAM expression could be detected in DRG neurons (FIG. 2b). Here, PAM immunoreactivity was located in the axons as well as in the cell body of both large- and small-diameter neurons (FIG. 2b). Interestingly, no PAM was detected in the nuclei of the cells as demonstrated by co-staining with anti-Histone antibody (FIG. 2b). As could also be seen in the spinal cord, PAM expression was not detected in GFAP expressing cells.
Since PAM expression in the brain is differentially regulated during development in rats and mice (Yang et al. 2002), the inventors investigated if PAM mRNA expression also changes during development in rat spinal cord and DRG. To this end, PAM mRNA expression was determined using quantitative RT-PCR. PAM mRNA was found to be highly expressed in the spinal cord and DRG in late embryonic stages (E16) until shortly after birth (P0.5; FIG. 3).
Interestingly, shortly after birth the expression declined to 30-40% of the embryonic expression and then remained constant throughout adulthood (FIG. 3).
Next, it was examined if PAM expression in the spinal cord is regulated by nociceptive stimuli. Therefore, spinal PAM expression was monitored after zymosan and formalin injection in the hind paws of adult rats. PAM mRNA was up-regulated about two-fold at 24 and 48 hours after zymosan treatment (FIG. 4a). Accordingly, PAM expression was up-regulated at the protein level 24 hours after zymosan injection and stayed elevated for 96 hours (FIG. 4b). 96 hours after zymosan injection, PAM mRNA expression declined. This reduction in PAM mRNA expression was not reflected at the protein level (comp. FIGS. 4a and b). Notably, PAM mRNA was also upregulated in the spinal cord of rats 1 hour after formalin injection (FIG. 4c).
Since PAM is known to be a powerful inhibitor of adenylyl cyclases type 1, 5 and 6 (Scholich et al. 2001) it was investigated if PAM is able to inhibit AC activity in spinal cord and DRG lysates. Two major AC isoforms are detected by semi-quantitative RT-PCR in the spinal cord. These AC isoforms are type 5 and 6 (FIG. 5a). Notably, both isoforms are inhibited at nanomolar concentrations of PAM (Scholich et al. 2001). The AC isoform expression pattern in the spinal cord was not significantly altered 24-96 hours after zymosan injection. Interestingly, 1 hour after formalin injection a shift in AC isoform expression was detected (FIG. 5a). The mRNA of AC type 5 is down-regulated and the mRNA of AC type 3 and 9 are up-regulated.
As could be shown by the above experiments, addition of purified PAM to spinal cord lysates resulted in inhibition of Gαs-stimulated AC activity in spinal cord preparations. Addition of 30 nM PAM decreased Gαs-stimulated AC activity by 49% in spinal cord lysates of control animals (FIG. 5b). Gαs-stimulated AC activity in spinal cord lysates derived from rats 96 hours after zymosan injection exhibited higher sensitivity to PAM inhibition as compared to untreated animals (70% inhibition at 30 nM; FIG. 5b). In contrast, inhibition of AC activity by PAM in spinal cord lysates from animals treated for 1 hour with formalin was inhibited to a lesser extent (25% inhibition at 30 nM; FIG. 5b).
In DRGs the predominantly expressed AC isoforms are AC type 4 and 6 (FIG. 5a). Gαs-stimulated AC activity in DRG lysates was decreased by 32% in presence of 30 nM PAM in contrast to spinal cord lysates (FIG. 5b).
To examine a possible role of PAM in spinal nociceptive transmission, animals were infused with PAM sense and antisense oligonucleotides by lumbar intrathecal catheters before performing a formalin assay. PAM expression was decreased in spinal cord neurons as observed by immunohistochemistry (FIG. 6a). Infusion of PAM antisense oligonucleotides caused a significant increase of the nociceptive response following formalin injection as compared to PAM sense treatment (p=0.007; FIGS. 6b and c). The hyperalgesia in PAM antisense-treated rats was accompanied by increased licking and biting behavior. Since PAM is an inhibitor of AC activity (Scholich et al. 2001), basal, Gαs- and forskolin-stimulated AC activities were determined in spinal cord lysates of sense and antisense-ODN treated rats. The experiments showed a significant increase (20.7%) of the basal AC activity in antisense treated rats (Table 1). In contrast, no significant changes were detected in Gαs- and forskolin-stimulated AC activities indicating that the total amount of AC was not altered by the ODN treatment (Table 1).
The above experiments showed that PAM is localized in the cell body and axons of both spinal cord and DRG neurons (FIG. 2a,b). Only very little immunoreactivity was detectable in the nucleus suggesting different functions for PAM in neurons and cancer cell lines.
Central sensitization after prolonged nociceptive stimuli is based on neuronal and synaptic changes in the spinal cord (Woolf and Costigan 1999; Woolf and Salter 2000; Ji and Woolf 2001). The finding of the inventors that Pam is expressed in sensory neurons of the spinal cord and DRGs led to the newly formed hypothesis, that PAM could be implicated in synaptic changes during spinal nociceptive processing. The above findings that PAM was up-regulated after nociceptive stimuli (FIG. 4a) supported this hypothesis that PAM may play a role in synaptic changes during spinal nociceptive processing.
Furthermore, the surprising finding of the inventors, that PAM is expressed in sensory nervous of the spinal cord and DRGs led to the question whether PAM is capable of inhibiting AC activity in spinal cord and DRG, as well.
The above experiments showed for the first time that PAM is a potent inhibitor of Gαs-stimulated AC activity in spinal cord preparations (FIG. 5b). AC activity was decreased by 50% after addition of 30 nM PAM. To achieve comparable inhibition using the α-subunit of the inhibitory G-protein, Gαi, 200-800 nM Gαi has to be used (Wittpoth et al. 1999). The inhibitory action of PAM was even stronger in spinal cord preparations of animals treated for 96 hours with zymosan (FIG. 5b) and could be explained by the elevated amounts of endogenous PAM in the spinal cord after zymosan injection (FIG. 4a,b). Inhibition of Gαs-stimulated AC activity in spinal cord preparations from formalin-treated animals (25% inhibition) was less pronounced as compared to control or zymosan-treated animals (50% and 75%, respectively).
Notably, in animals treated with formalin for 1 hour a shift in AC isoform expression was observed (FIG. 5a). AC of type 3 and 9 are up-regulated while AC type 5 is down-regulated. To date it is not known if PAM is an inhibitor of AC type 3 and 9. Therefore, these isoforms may not be inhibited by PAM or the tested PAM concentrations were too low to achieve an inhibitory effect. Since PAM is a giant protein of 510 kDa, it is technically not possible to test PAM concentrations greater than 30 nM. Nonetheless, according to the dose response curves shown in FIG. 5b, higher PAM concentrations might result in a stronger inhibition of Gαs-stimulated AC activity in the tested spinal cord preparations.
Interestingly, PAM was a less effective inhibitor of AC enzyme activity in DRG than in spinal cord preparations. The different inhibitory efficiencies of PAM in spinal cord and DRG preparations are most likely due to the observed differences in AC isoform expression. The major AC isoforms that are expressed in the spinal cord are type 5 and 6 that are both strongly inhibited by PAM (FIG. 5a; (Scholich et al. 2001)). In DRGs AC type 4 and 6 are the dominant AC isoforms (FIG. 5a). Since it is unknown if PAM inhibits AC type 4 either this isoform is not inhibited by PAM or, again, the tested PAM concentrations were too low to achieve the inhibitory effect. However, according to the dose response curve shown in FIG. 5b it is seems likely that higher concentrations of PAM would result in a stronger inhibition of Gαs-stimulated AC activity in the DRG preparations.
Most surprising, however, were the findings, that PAM activity had an influence on the nociceptive behavior of the test animals: This could be demonstrated for the first time by experiments of the inventors showing a significant increase in basal AC activity (Table 1) and—more important—a significant increase of the nociceptive response following formalin injection as compared to PAM sense treatment (FIGS. 6b and c) when endogenous PAM expression in the spinal cord was decreased by infusing animals with PAM antisense oligonucleotides (FIG. 6a).
19. Determination of the Analgesic Effect of PAM
The above-listed evidence for the analgesic effect of PAM could for example be supported by the following hypothetic experiment: The analgesic effect of PAM, e.g. in the formalin model of acute pain, could be determined directly by intrathecal application of e.g. a peptide corresponding to amino acid residues 1028 to 1065. This peptide represents the minimal region found to be capable of mediating PAM-adenylyl cyclase interactions as determined by the yeast-two-hybrid system and AC activity assays. The peptide could be applied in a complex with the bioporter lipofection reagent (commercially available at Peqlab, Germany). This approach would allow the peptide to enter the tissue and mimic the actions of physiological PAM towards ACs.
20. Results Demonstrating the Influence of S1P on PAM Signaling
To investigate PAM expression and localization in HeLa cells, two antibodies against PAM where employed, which are directed against peptides corresponding to the amino acid residues 135-153 and 4601-4614 of human PAM. Comparison of immunohistological staining of rat brain showed that both antibodies recognized the same brain regions which also exhibit PAM mRNA expression (Yang et al., 2002). In serum starved HeLa cells both antibodies showed colocalization of PAM with calnexin, an endoplasmatic reticulum marker (FIG. 18a). After addition of serum to the cells, a partial translocation of PAM to the plasma membrane was observed (FIG. 18b). PAM appeared at the membrane 20-30 minutes after serum treatment and started to disappear from the membrane after 1 hour serum incubation. The cellular distribution of PAM in HeLa observed with the antibodies used differs from the cellular distribution described by Guo et al. Since Guo et al. used a portion of PAM to generate antibodies that includes common motifs for nuclear proteins cross-reactions with nuclear proteins by this antibody are possible.
Since PAM is a potent inhibitor of AC enzyme activity, it was next investigated if the translocation of PAM from the ER to the plasma membrane results in an inhibition of AC activity. Serum-treatment of HeLa cells reduced the intracellular cAMP accumulation (FIG. 19a). Additionally, serum-treatment decreased Gαs- and forskolin-stimulated AC activity to 56.7% and 64.7%, respectively, as compared to untreated cells (FIG. 19b). The observed decrease in AC activity was not due to a change in the AC isoform expression or due to an increased AC expression since no changes in the mRNA expression of AC isoforms was detected (FIG. 19c). To determine if the decrease in stimulated AC activity was mediated by PAM, the amount of endogenous PAM was decreased, employing antisense oligonucleotides against PAM as previously described in Scholich et al., 2001. As shown in FIG. 19d, in HeLa cells treated with antisense ODN the amount of PAM, as determined by Western Blot analysis, was decreased as compared to cells treated with sense or mutant antisense ODNs. Reprobing the same blot with anti-Hsp70 antibody showed that the loading of proteins was the same (FIG. 19d). However, the treatment of HeLa cells with antisense ODN reduced the serum-induced inhibition of Gαs- and forskolin-stimulated AC activity significantly (FIG. 19e). Importantly, transfection of HeLa cells with sense or mutated ODNs had no influence on the serum-induced inhibition of Gαs- and forskolin-stimulated AC activity (FIG. 19e). These data suggest that endogenous PAM exerts an inhibitory influence on AC activity after stimulation of HeLa cells with serum.
To identify the serum factor that induces PAM translocation to the plasma membrane the factor was purified using reverse phase-, anionic exchange- and gelfiltration-columns. After each purification step, the fractions were tested for their ability to induce PAM translocation and AC inhibition. According to the purification properties, the serum factor could be identified to be slightly hydrophobic (Elution from the Phenyl-Sepharose column with 0.3 M NaCl), possesses a strong negative charge (elution from MonoQ and Q-Sepharose columns at 0.7 M NaCl) and has an estimated molecular weight under 500 according to the retention time on the superdex 30 gelfiltration column. According to the physical properties several candidate substances were tested, from which only sphingosine-1-phosphate induced PAM translocation.
S1P can bind to a family of five G-protein coupled receptors. Therefore it was investigated by semi-quantitative RT-PCR if HeLa cells express S1P-receptors. The mRNA of four of the five S1P-receptor isoforms (S1P1-4) was detected in the HeLa cells (FIG. 20a). Next, it was tested if purified S1P exhibits the same properties toward PAM activation/translocation as serum. First, HeLa cells were treated with increasing concentrations of purified S1P. PAM translocation to the plasma membrane occurred in 70-90% of cells treated with 0.1-5 μM S1P treated cells. PAM appeared at the plasma membrane after 10 minutes incubation with 500 nM S1P and started to disappear after 1 hour of incubation (FIG. 3b,c). Most importantly, treatment of HeLa cells with 0.5 μM S1P reduced the intracellular cAMP content (FIG. 21a) as well as the Gαs-stimulated AC activity (FIG. 21b). Gαs-stimulated AC activity decreased within 3 minutes after incubation with S1P and before partly recovering. 5-10 minutes after begin of the S1P treatment, Gαs-stimulated AC activity decreased once more FIG. 4b). Antisense ODN against PAM eliminated the inhibition of Gαs-stimulated AC activity at 60 minutes after incubation with S1P (FIG. 21c). Taken together, these data demonstrate for the first time that inhibition of AC activity in HeLa cells by S1P is achieved through two different mechanisms: A fast PAM-independent AC inhibition (3-10 minutes S1P treatment) and a delayed, PAM-dependent AC inhibition (10-60 minutes S1P treatment).
It has been demonstrated that by binding to its respective receptors, S1P can potentially activate four different G proteins, Gi, Gq, G12, and G13 (Hla et al., Science, 2001; Kluk et all, BBA, 2002; Siehler and Manning, BBA, 2002; Spiegel and Milstein, JBC, 2002). From these, only Gi is pertussis toxin sensitive. Pertusis toxin-treatment eliminated the inhibitory effect of S1P on Gαs-stimulated AC activity (FIG. 22a) and PAM translocation to the plasma membrane (FIG. 22b). Thus, it seems likely that the inhibitory G-protein, Gi, is responsible for the fast, PAM-independent inhibition by S1P.
Next, further elements of the signal transduction pathway that causes translocation and activation of PAM were elucidated. Since S1P1-4 receptors have been described to couple to Gi, Gq and G12/13 (Hla et al., 2001; Kluk et al., 2002; Siehler et al., 2002; Spiegel et al., 2002) and translocation of PAM as well as AC inhibition is pertussis toxin-dependent (FIG. 22a) the above data suggest that PAM activation in HeLa cells depends on Gi activation. Previously it has been described that S1P can activate phospholipase C (PLC) as well as ERK1/2 signaling through activation of Gi (Kluk et al., 2002; Siehler et al., 2002; Spiegel et al., 2002). Thus, it was tested if PLC activation is involved in PAM translocation, and it could be found that PAM translocation and late phase AC inhibition was abolished in presence of the PLC inhibitor U73122 (FIG. 22a). PLC converts phosphatidylinositol 4,5-biphosphate to inositol 1,4,5-triphosphate (IP3), a calcium-mobilizing second messenger, and 1,2-diacylglycerol (DAG), an activator of protein kinase C (PKC) (Rebecchi et al., 2000; Wilde et al., 2001). Calcium imaging showed that S1P induced a PLC-dependent calcium increase in HeLa cells. However, this calcium decrease was not necessary for PAM translocation since pre-treatment with BAPTA-AM did not interfere with PAM translocation (FIG. 22a). Yet PKC inhibitors GF109203X and RO 31-8220 eliminated PAM translocation and AC inhibition, respectively (FIG. 22a). These data suggest that S1P activates PLC through the inhibitory G-protein, Gi. Subsequently, PLC actions result in a calcium-independent PKC activation which is necessary to mediate PAM translocation and the delayed S1P-induced AC inhibition.
Since it has also been shown that S1P can activate the ERK1/2 signaling pathway (Kluk et al., 2002; Siehler et al., 2002; Spiegel et al., 2002), it was tested if EK1/2 is activated by S1P in HeLa. ERK1/2 phosphorylation was detectable after incubation of HeLa with S1P using anti-active ERK and anti-phosphoTyr183 ERK antibodies (FIG. 22b). Surprisingly, ERK1/2 phosphorylation depended on PLC activation (FIG. 22b), Gi, and PKC activity (data not shown) but was independent of an increase in intracellular calcium. However, ERK1/2 activation was not necessary for PAM translocation or inhibition of AC activity (FIG. 22a). Altogether these findings show for the first time that S1P induces PAM translocation and subsequent inhibition of AC enzyme activity through a signaling cascade that includes Gi, PLC and PKC. Interestingly, S1P induced additionally ERK1/2 activation and an increase of intracellular calcium concentrations, both of which were not necessary for PAM translocation or inhibition of Gαs-stimulated AC activity.
The signal transduction pathways regulated by S1P are the focus of intensive research (Kluk et al., 2002; Siehler et al., 2002; Spiegel et al., 2002). It is well known that S1P receptors can inhibit AC activity through Gi dependent mechanisms (Kluk et al., 2002; Siehler et al., 2002; Spiegel et al., 2002). However, here, it was shown for the first time that inhibition of AC activity over prolonged times after S1P stimulation is not due to a direct inhibition of AC by the inhibitory G-protein since the delayed AC inhibition depended on PLC- and PKC-activation. Moreover, the data of the inventors suggest that the prolonged inhibition of AC activity in HeLa cells after S1P-treatment depends on the translocation/activation of PAM to the plasma membrane. This translocation is regulated by Gi activation and PLC/PKC signaling. Since PAM is a potent inhibitor of AC enzyme activity S1P-induced PAM-dependent AC inhibition may be a result of direct interactions between PAM and AC although this still has to be determined.
The above experiments of the inventors led for the first time to the finding that PAM is localized at the endoplasmatic reticulum in HeLa cells and translocates to the plasma membrane after serum treatment. PAM translocation was accompanied by a decrease in Gαs- and forskolin-stimulated AC activity as compared to untreated HeLa cells. AC inhibition was mediated by PAM since pretreatment of the cells with antisense oligonucleotides against PAM prevented AC inhibition. In the following we identified Sphingosine-1-phosphate (S1P) as the serum factor responsible for PAM translocation. Treatment of HeLa cells with 0.1-5 μM S1P induced PAM translocation to the plasma membrane in 80% of the cells within 10-30 minutes. S1P reduced AC activity by two separated mechanisms. Initial AC inhibition was not mediated by PAM but was pertussis toxin sensitive. After prolonged S1P-treatment AC inhibition depended on PAM translocation. S1P actions towards PAM translocation and PAM-mediated AC inhibition were pertussis toxin-sensitive and required PLC- and PKC-activation. Taken together, these data identified for the first time a regulator of PAM activity. Moreover, it could be shown that long-term inhibition of AC activity by S1P is mediated by the translocation of the AC-inhibitory protein PAM from the ER to the plasma membrane.
21. Determination of the Analgesic Effect of S1P
To determine the analgesic effect of S1P, S1P was delivered to the spinal chord by intrathecal application.
21 a) Implantation of Lumbar Intrathecal Catheters:
Rats were anesthetized with ketamine (60 mg/kg i.p.) and midazolam (0.5-1 mg/kg i.p.). The skin was incised above the vertebral column from vertebrae Th13 up to L3. Muscle tissue around L2-3 was cleared away. The processus spinosus of L3 was removed and a laminectomy was done at L2. Polyethylene catheters (ID 0.28 mm, OD 0.61 mm) were then inserted into the peridural space so that the tip of the catheter reached Th9-10. The catheter was fixed with cyanacrylate glue and was externalized in the neck region and the skin was sutured.
21 b) Infusion of PAM Oligonucleotides.
Three days after surgery rats were placed into a “freely moving system” (CMA, Stockholm, Sweden) 20 μl of 10 μM S1P were infused through the catheter.
21 c) Formalin Test:
Within 15 min after stopping the infusion the formalin test was performed. 50 μl of a 5% formaldehyde solution were injected subcutaneously (s.c.) into the dorsal surface of one hind paw. Flinches were counted in one minute intervals up to 60 min starting right after formalin injection. Flinches of 5 min intervals were summarized as mean flinches per minute. To compare the nociceptive behavior between groups the sum of flinches during the one-hour observation period were submitted to the Students t-test. At the end of the formalin test, the lumbar spinal cord and dorsal root ganglions (DRGs) were excised, snap frozen in liquid nitrogen and stored at −80° C. until further analysis.
21d) Results:
20 μl of 10 μM S1P or 20 μl PBS/DMSO were given to adult rats by intrathecal application 15 minutes prior to the formalin injection. Then, flinches were counted in 5 minute intervals over a period of 60 minutes. A significant decrease in the number of nociceptive responses for phase 2A (20 to 35 minutes after formalin injection) could be detected as compared to PBS/DMSO-treated animals (see FIG. 24). These experiments clearly demonstrated that exogenous S1P acts as an analgesic.
22. Determination of the Analgesic/Antinociceptive Effect of S1P Receptor Agonists
The analgesic/antinociceptive effect of S1P receptor agonists, e.g. FTY 720 could for example be supported by the following hypothetic experiment: The analgesic/antinociceptive effect of e.g. FTY 720, e.g. in the formalin model of acute pain, could be determined directly by intrathecal or intravenous application of e.g. FTY 720 and consecutive testing of its analgesic/antinociceptive effect by means of e.g. the flinch test. This approach would allow the molecule to enter the tissue and mimic the actions of physiological S1P towards ACs.
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STANDARD LITERATURE FOR LABORATORY METHODS
If not indicated otherwise, laboratory methods were or can be performed according to standard methods listed in the below standard literature:

    Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 545 pp or Current Protocols in Molecular Biology;
    Current Protocols in Molecular Biology; regularly updated, e.g. Volume 2000; John Wiley & Sons, Inc; Editors: Fred M. Ausubel, Roger Brent, Robert Eg. Kingston, David D. Moore, J. G. Seidman, John A. Smith, Kevin Struhl.
    Current Protocols in Human Genetics; regularly updated, e.g. Volume 2003; John Wiley & Sons, Inc; Editors: Nicholas C. Dracopoli, Honathan L. Haines, Bruce R. Korf, Cynthia C. Morton, Christine E. Seidman, J. G. Seigman, Douglas R. Smith.
    Current Protocols in Protein Science; regularly updated, e.g. Volume 2003; John Wiley & Sons, Inc; Editors: John E. Coligan, Ben M. Dunn, Hidde L. Ploegh, David W. Speicher, Paul T. Wingfield.
    Molecular Biology of the Cell; third edition; Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J. D.; Garland Publishing, Inc. New York & London, 1994;
    Gene Targeting: a practical approach (1995), Editor: A. L. Joyner, IRL Press
    Genetic Manipulation of Receptor Expression and Function; D. Accili, Wiley-Liss., USA, 2000; ISBN: 0-471-35057-5.
    Antisense Therapeutics, S. Agrawal, Humana Press, USA, 1996, ISBN: 0-89603-305-8.
    Remington's Pharmaceutical Sciences, Edition 17, 1985.

Abbreviations Used:



AC, adenylyl cyclase; Gαs, α subunit of the stimulatory G protein of adenylyl cyclase, Gαs*, constitutively active (Q213L) mutant of Gαs; Gαi, α subunit of the inhibitory G protein Gi; Gβγ, βγ subunits of heterotrimeric G proteins; ODN, oligodeoxynucleotide; PAM, protein associated with Myc; RCC1, regulator of chromosome condensation; S1P, sphingosin-1-phosphate; TED, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT.; RT, room temperature;
Table 1
Basal AC activity is elevated in spinal cord lysates of antisense treated rats. Spinal cords lysates (20 μg) were assayed for AC activity in the absence or presence of 80 nM Gαs or 100 μM forskolin as described above. The mean AC activity±SEM of spinal cord lysates from at least three rats per group, each measured twice in triplicates, is shown (ns=not significant).











TABLE 1








AC activity




Condition
(pmol/min/mg





















basal






sense

102.7 ± 6.6 



antisense
1
24.0 ± 6.2
(p ≦ 0.01)



Gαs



sense

398.8 ± 16.9



antisense

432.6 ± 17.2
(ns)



Forskolin



sense

283.9 ± 25.7



antisense

316.2 ± 24.3
(ns)

## Claims

1. A method of alleviating pain associated with nociceptive processing or sensitization in the spinal cord and dorsal root ganglia, the method comprising administering to a subject experiencing pain a compound that activates a sphingosine-1-phosphate receptor in the spinal cord or dorsal root ganglia or enhances PAM (protein associated with Myc) activity in the spinal cord or dorsal root ganglia, wherein the compound is sphingosine-1-phosphate or a salt thereof or FTY 720 or a salt thereof, thereby alleviating pain associated with nociceptive processing or sensitization in the spinal cord and dorsal root ganglia in the subject.

2. The method of claim 1, wherein the compound is administered intrathecally.

3. The method of claim 1, wherein the compound is administered intravenously.

4. The method of claim 1, wherein the compound is administered orally.

5. The method of claim 1, wherein the subject is a mammal.

6. The method of claim 5, wherein the mammal is a human.

7. The method of claim 1, wherein the compound is sphingosine-1-phosphate or a salt thereof.

8. The method of claim 7, wherein the compound is administered intrathecally.

9. The method of claim 7, wherein the compound is administered intravenously.

10. The method of claim 7, wherein the compound is administered orally.

11. The method of claim 7, wherein the subject is a mammal.

12. The method of claim 11, wherein the mammal is a human.

13. The method of claim 1, wherein the compound is FTY 720 or a salt thereof.

14. The method of claim 13, wherein the compound is administered intrathecally.

15. The method of claim 13, wherein the compound is administered intravenously.

16. The method of claim 13, wherein the compound is administered orally.

17. The method of claim 13, wherein the subject is a mammal.

18. The method of claim 17, wherein the mammal is a human.

19. The method of claim 1, wherein the pain is nociceptive pain.

20. The method of claim 19, wherein the nociceptive pain is chronic nociceptive pain.

21. The method of claim 19, wherein the nociceptive pain is acute nociceptive pain.