|Title of Invention||
AN ASSAY OF SCREENING PHARMACEUTICAL
|Abstract||ABSTRACT Use of SIP or functional fragments or derivatives thereof for the preparation of pharmaceutical compounds.|
Use of SIP
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 ah 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 antiinflammatory 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 HM 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.SA 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 modulate pain.
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. 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 figure 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; www.ncbi.nhm.nih.gov) 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:
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 beeri 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 alM 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.220.127.116.11) 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 dr 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 ubiquftous 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 alM 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 S1P receptors are known in the art termed S1Pi to S1P5, (for a review, see, e.g. Spiegel, SM and Milstien, S,, 2000). The protein and coding polynucleotide sequences of the different S1P 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; www.ncbi.nhm.nih.gov) 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 /S1Pi); NP_001391 (SEQ ID No.31, protein sequence of homo sapiens endothelial differentiation, sphingolipid G-protein-coupled receptor (EDG1 / S1P1),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 / S1 P3);NM:003775 (SEQ ID No.38, nucleotide sequence of Homo sapiens '. endothelial differentiation, G-protein-coupled receptor 6 (EDG6/ S1 P4)mRNA; CAA04118 (SEQ ID No.37, protein sequence of Homo sapiens endothelial differentiation, G-protein-coupled receptor 6 (EDG6/S1P4); NM_030760 (SEQ ID No.40, nucleotide sequence of homo sapiens endothelial differentiation sphingolipid G-protein-coupled receptor 8 (EDG8/ S1P5) mRNA; NPJ10387 (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 modulation of pain. This modulation is preferably a lessening or prevention or total inhibition.
Moreover, the use of S1P or functional fragments of derivatives thereof for identifying compounds that modulate pain is encompassed by present invention. The modulating compounds are preferably compounds that have, mimic or enhance S1P activity. Most preferably they have the ability to prevent, lessen or abolish 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 S1 P.
Another aspect of present invention regards a method of preventing or lessening pain comprising administering a sufficient amount of S1P or a functional fragment or derivative thereof to an individual-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 SIP.
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 ALPHAscreen™)) 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
promotor 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 promotor. For this type of assay, a
recombinant cell line has to be constructed containing the reporter gene under the
control of a defined promotor 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), li-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 i 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 FIuo-3, FIuo-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 (flureescence-based laser scanning reader that allows for 3dimensional 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 AlphaScreen™ 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 2xSSC-solution. Conditions of high stringency comprise e.g. a hybridisation reaction at 68°C in 0,1xSSC 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 50mM Tris pH 7,5,1M Nad, 1% SDS, 10% Dextran Sulfate, 0,5 mg/ml denatured salmon or hering sperm DNA.
2) Washing for 10 minutes in 2xSSC at room temperature.
3) Washing for 30 minutes in 1xSSC/0,1%SDS at 65°C (or in the case of oligonucleotides: 5CC below the annealing temperature).
4) Washing for 30 minutes in 0f1xSSC/0,1%SDS at 65°C (or in the case of oligonucleotides: 5°C below the annealing temperature).
)ligonucleotides for the use as hybridisation probes are polynucleotide and preferably )NA-fragments having a length of 15 to 30, preferably 20 nucleotides. The annealing emperature is determined according to the formula Tm=2x (number of A+T) + 4x number of G+C)°C.
ror preparing a 2xSSC or a 0,1xSSC (or any other kind of SSC dilution), e.g. a 20x 3SC solution is diluted accordingly. 20xSSC consists of 3M NaCI/6,3 M Na-Citrate x2H20.
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 mimiking 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 identifiying pain modulating cqmpounds, 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 S1 P. 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, Supplemtent 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 ALPHAscreen™, 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 modulates pain comprising
a) Selecting a compound that modulates or mimics the activity of SiP as a test compound, and
b) Administering said test compound to a subject to determine whether the pain is modulated.
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 is preferably a prevention, lessening or an abolishment of pain. According to one prefered embodiment of the invention, the compound is an S1P
receptor agonist (for a review, see for example Mandala et al.f Science 2002) and more preferably FTY 720 (2-Amino-2-(4-ocylphanyi)ethyl)propane-1,3-dioI, see figure 17) or a functional derivative or analog (analogs are known in the art, see e.g. Brinkmann et alM 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.f 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 lessening or preventing pain comprising administering a sufficient amount of apharmaceutical 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 alM 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
S1P was purchased from Tocris (Ellisville, MO), the anti-Hsp70 antibody and the anti-Calnexin antibody from BD TransductionLabs (Bedford, MA). The anti-active ERK1/2 antibody was obtained from Promega (Madison, Wl). Pertussis toxin, U0126, U73122, and Wortmannin from Tocris (Ellisville, MO),R031-223, BAPTA-AM and GF109203X by Sigma (St.Louis, MO).
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 Gcrs-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 a-subunit of the inhibitory G-protein, Gai, 200-800 nM Gai 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 Gas-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 Figure 5b, higher PAM concentrations might result in a stronger inhibition of Gas-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 Figure 5b it is seems likely that higher concentrations of PAM would result in a stronger inhibition of Gas-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 (Fig. 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 wouid 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 immunhistological 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 Gas- 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 Gas- 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 Gas- 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 NaCI), possesses a strong negative charge (elution from MonoQ and Q-Sepharose columns at 0.7 M NaCI) 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 (SIP1-4) 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 occured in 70-90% of cells treated with 0.1-5 pM 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 pM S1P reduced the intracellular cAMP content (Fig. 21a) as well as the Gas-stimulated AC activity (Fig. 21b). Gas-stimulated AC activity decreased within 3 minutes after incubation with S1P and before partly recovering. 5-10 minutes after begin of the S1P treatment, Gas-stimulated AC activity 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 Gas-stimulated AC activity (Fig. 22a) and PAM
translocation to the plasma membrane (Fig. 22b). Thus, it seems likely that the inhibitory G-protein, G1, 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 S1 P-M 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 Gj activation. Previously it has been described that S1P can activate phospholipase C (PLC) as well as ERK1/2 signaling through activation of Gj (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-triphophate (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,
i 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), Gj, 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
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 Gas-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 S IP-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 Gas- and forskolin-stimulated AC activity as compared to untreated HeLa cells. AC inhibition was mediated by PAM since pretreatment of the celis 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 \iU 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 AG 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 oligonuleotides.
Three days after surgery rats were placed into a "freely moving system" (CMA,
Stockholm, Sweden) 20 μl of 10 μ M 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.
21 d) Results:
20μlof 1OμM SI P or 20μl 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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■ ( Abbreviations used:
AC, adenylyl cyclase; Gas, a subunit of the stimulatory G protein of adenylyl cyclase, Gas*, constitutively active (Q213L) mutant of Gas; Gai, a subunit of the inhibitory G protein Gi; GBy, By 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-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT.; RT, room temperature;
Figure 1: PAM is highly expressed in spinal cord neurons. In it’s hybridization using horizontal sections of spinal cords hybridized with sense or antisense probes against rat PAM as described above.
Figure 2: PAM is expressed in DRG neurons as well as in neuronal cells in rat spinal
Panel A: Immunhistochemical 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 20x.
Panel B: Immunhistochemical 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 40x except for the Histon
staining, which was magnified 63x.
Figure 3: PAM is differentially expressed in DRGs and spinal cord during different » developmental stages. Quantitative RT-PCR (Taqman™) was used to detect PAM in RNA (40 μ g) 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.
Figure 4: PAM is upregulated in the rat spinal cord after zymosan and formalin treatment.
Panel A: RT-PCR analysis with RNA (40 μ g) of spinal cords from control animals or animals treated with zymosan after 24h, 48h and 96h. The lower panel shows the mean ± SEM of 7 experiments. Student T test: * p i 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 24h, 48h and 96h (40pg) with anti-PAM antibody and anti-ERK1/2.
Panel C: Quantitative RT-PCR analysis with RNA (40 μ g) of spinal cords from control animals or animals treated with formalin for 1 hour.
Figure 5: PAM inhibits Gas-stimulated AC activity in spinal cord lysates.
Panel A: RT-PCR was used to determine AC isoform expression in spinalcord and
DRG RNA (40 ng).
Panel B: Lysates of spinal cords or DRG (10μg) were assayed for AC activity in the
presence of 80 nM Gas as described in Material and Methods. The mean ± SEM of at
least 3 determinations done in triplicates is shown. (
Figure 6: Intrathecal application of antisense ODNs against PAM increases nociceptive
Panel A: Adult rats were given intrathecal sense and antisense ODN as described.
After formalin treatment, the spinal cord was removed and subjected to
immunhistological 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.
Figure 7: Protein, genomic and coding nucleotide sequence of human PAM 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); Figure 7C shows the contiguous sequence from position 24679861 to position 24962245.
Figure 8: EST-clone coding sequences for rat PAM:
Figure 8A: AW921303 (corresponds to bp 960-1394 of hs cDNA; SEQ ID No. 4)
Figure 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);
Figure 14: PAM signaling according to the above findings.
Figure 15: Structure of Sphingosine-1-Phosphate;
Figure 16: mDNA and amino acid sequences of S1P receptors
16a) amino acid sequence of S1Pi (SEQ ID No. 31);
16b) mRNA sequence of S1Pi (SEQ ID No. 32); the coding sequence starts at position
244 and ends at position 1392;
16c) amino acid sequence of S1P2 (SEQ ID No. 33);
16d) mRNA sequence of S1P2(SEQ ID No. 34); the coding sequence starts at position
1 and ends at position 1062;
16e) amino acid sequence of S1P3(SEQ ID No. 35)
16f) mRNA sequence of SIP3 (SEQ ID No. 36), the coding sequence starts at position 1
and ends at position 1137;
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;
16i) amino acid sequence of S1P5(SEQ ID No. 39)
16j) mRNA sequence of SI P5 (SEQ ID No. 40), the coding sequence starts at position
10 and ends at position 1206;
Figure 17: Structure of FT720
Figure 18: PAM translocates from the ER to the plasma membrane in HeLa cells after
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.
1. The use of S1P or functional fragments or derivatives thereof for the preparation
of pharmaceutical compounds.
2. The use of S1P or functional fragments or derivatives thereof for the modulation of pain:
3. The use of S1P or functional fragments of derivatives thereof for identifying compounds that modulate pain.
4. A method of lessening pain comprising administering a sufficient amount of S1P or a functional fragment or derivative thereof to an individual.
5. S1P or a functional fragment or derivative thereof for the use as a medicament.
6. SI P or a functional fragment or derivative thereof for the use as a medicament for the prevention or treatment of pain and/or inflammation.
7. The use according to claim 1, wherein the pharmaceutical compounds are compounds for the prevention or treatment of pain and/or inflammation.
8. The use according to claim 3, wherein the compounds lessen or abolish pain.
9. A method of screening 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. Measuring the PAM activity in-both samples
d. Contacting the first sample with a compound
e. Determining the ability of the compound to mimic S1P function.
10. A method of screening pharmaceuticals useful for modulating and/or preventing
pain, comprising the steps
a) providing two samples comprising a cell expressing an S1P receptor or a functional fragment or derivative thereof, anb) contacting one sample with the compound, and c) measuring the receptor activity in both samples.
11. A method of screening pharmaceuticals useful for modulating and/or preventing
pain, comprising 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. ,
12. A method of screening pharmaceuticals useful for modulating and/or preventing
pain, comprising the steps
' b. Providing a cell expressing a S1P receptor or a functional fragment or derivative thereof and expressing PAM or a functional fragment or derivative thereof, c. Contacting the cell with a compound,
f. Determining the S1P activity in the presence of compound,
g. Determining the S1P activity in the absence of compound, and
h. Comparing the S1P activity according to c) with that according to
13.The method according to claim 10, further comprising a step wherein the cell is contacted with S1P instead of the compound and wherein the PAM activites according to c) and d) above are compared to the PAM activity in presence of S1P
14. A method of screening pharmaceuticals useful for modulating and/or preventing pain, comprising the steps
a. Providing two samples containing S1P or a functional fragment or
derivative thereof and containing a S1P receptor or a functional fragment
or derivative thereof,
b. Contacting one of the samples with a compound,
c. Determining and comparing S1P activity in both samples.
15. The use, method or S1P according to one of the above claims, wherein the
functional fragments or derivatives thereof are capable of inhibiting adenylyl cyclase
16. The use, method or S1P according to claim 10, wherein the adenylyl cyclase is
adenylyl cyclase type I, V or VI.
17. The method according to one of the preceding claims, wherein the determination of S1P activity concerns its ability to interact with and / or activate a S1P receptor, to mediate PAM translocation, to activate PAM function or to inactivate AC function.
18. A method of identifying a compound that modulates 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, and
c) Determining whether the pain is modulated in the subject.
19. Use of a compound having the ability to activate at least one of the S1P receptors and / or having the ability to activate PAM function for the preparation of a medicament that modulates or prevents pain.
20. Use according to claim 19, wherein the modulation is a lessening or an abolishment of pain.
21. Use according to one of the claims 19 or 20, wherein the compound is
FTY 720 or a functional derivative, preferably a phosphbrylated derivative
thereof, or a salt of the compound of the derivative.
22. A Method of lessening or preventing pain comprising administering a sufficient amount of a pharmaceutical compound with the ability to bind and activate at least one of the S1 P. receptors and/or the activity to activate PAM function to an individual.
23. Method according to claim 22, wherein the compound is FTY 720 or a functional derivative, preferably a phosphorylated derivative thereof, or a physiologically acceptable salt of the compound or the derivative.
|Indian Patent Application Number||3193/CHENP/2005|
|PG Journal Number||09/2011|
|Date of Filing||30-Nov-2005|
|Name of Patentee||SANOFI-AVENTIS DEUTSCHLAND GMBH|
|Applicant Address||BRUNINGSTRASSE 50, D-65929 FRANKFURT AM MAIN, GERMANY|
|PCT International Classification Number||A61K 31/661|
|PCT International Application Number||PCT/EP04/05236|
|PCT International Filing date||2004-05-15|