WO2003097084A1

WO2003097084A1 - Ddah modulators in the treatment of pain

Info

Publication number: WO2003097084A1
Application number: PCT/GB2003/002088
Authority: WO
Inventors: Keiko Mizuno; Karl Peter Giese
Original assignee: University College London
Priority date: 2002-05-16
Filing date: 2003-05-15
Publication date: 2003-11-27
Also published as: AU2003233998A1; GB0211297D0

Abstract

Use of a modulator of DDAHI methylarginase activity and/or expression for the manufacture of a medicament for treating pain in a human or animal wherein the modulator has activity which increases DDAHI methylarginase activity and/or expression.

Description

PAIN TREATMENT

Field of the invention This invention relates to agents for the treatment of pain in humans and animals. Background of the invention

Arginine residues in proteins are methylated by a family of protein arginine N-methyltransferases (PRMTs). These enzymes catalyze the methylation of guanidino nitrogens of arginine to produce N monomethyl-L-arginine (L-NMMA), N^'⁰ dimethyl-L-arginine (asymmetric dimethylarginine; ADMA) and N^⁰ dimethylarginine (symmetric dimethylarginine; SDMA). Proteolysis of proteins containing these residues releases free methylarginines. Although the biological role of methylarginine residues is unclear, free L-NMMA and ADMA, but not SDMA, are inhibitors of all three isoforms of nitric oxide synthase (NOS).

Free methylarginines are found in cell cytosol, plasma and tissues and their concentrations differ between tissues and between regions within a single tissue or organ. Elevated concentrations of ADMA have been detected in endothelial cells repopulating blood vessels damaged by balloon injury, in the plasma of patients or experimental animals with hyperlipidaemia, renal failure or athersclerosis, and in patients with schizophrenia or multiple sclerosis. Altered biosynthesis of nitric oxide (NO) has been implicated in the pathogenesis of all of these conditions and it is possible that the accumulation of endogenous ADMA underlies the inhibition of NO generation. The production of methylarginines is probably an obligatory step in protein turnover, and rates of production may show tissue specific and temporal variations. However, L-NMMA and ADMA, but not SDMA, are actively metabolised to citrulline and methylamines by the action of dimethylarginine dimethylaminohydrolase (DDAH). Certain tissues which express NOSs also appear to express DDAH. Pharmacological inhibition of DDAH increases the concentration of ADMA in endothelial cells and inhibits NO-mediated endothelium-dependent relaxation of blood vessels. These observations suggest that DDAH activity ensures that the local concentration of ADMA does not normally rise sufficiently to affect NO generation, and that changes in DDAH activity could actively alter NOS activity.

WO 00/44888 discloses the expression in humans of two functionally active methylarginases, designated DDAHI and DDAHII. The document describes the cloning of polynucleotides encoding the DDAHI and DDAHII isoforms and a study of the expression patterns of these two methylarginases via RNA blotting. The studies revealed that DDAHI has a tissue distribution in humans which is similar to that of the neuronal isoform of nitric oxide synthase (nNOS), whilst DDAHII is highly expressed in vascular tissues which also express endothelial NOS (eNOS). Summary of the Invention

It has now been found that inflammatory pain is associated with down- regulated DDAHI expression in the thalamus. Changes in DDAHI activity and/or expression therefore appear to contribute to altered sensory signalling in pain conditions. Accordingly, modulation, in particular upregulation of DDAHI methylarginase activity and/or expression has use in the therapeutic or prophylactic treatment of pain.

Accordingly, the present invention provides the use of a modulator of DDAHI methylarginase activity and/or expression for the manufacture of a medicament for treating pain in a human or animal where in the modulator has activity which increases DDAHI methylarginase activity and/or expression.

The invention further provides a method for treating pain in a human or animal comprising administering a therapeutically or prophylactically effective amount of such a modulator to the human or animal.

In pain treatment, the DDAHI modulator may be co-administered with one or more other agents, including NOS inhibitors such as methylarginines. Accordingly, the invention also provides products containing a modulator and a methylarginine, as a combined preparation for simultaneous, separate or sequential use in treating pain in a human or animal.

The invention further provides a method of identifying an agent for treating pain in a human or animal, comprising:

(a) assaying a test substance for activity which modulates DDAHI methylarginase activity and/or expression; and -_* -J-

(b) screening a modulator identified in (a) for efficacy in treating pain in a test animal. Brief Description of the Figures

Figure 1 shows the expression pattern of DDAHI mRNA in adult rat forebrain. Frontal sections are arranged rostrocaudally (A-D). In situ hybridisations were performed with antisense S-labelled DDAHI cDNA which was prepared by in vitro transcription using T3-RNA polymerase and ³⁵S-UTP. Note that DDAHI mRNA is predominantly expressed in thalamic nuclei.

Figure 2 shows levels of DDAHI mRNA expression in rat cerebellum, thalamus, midbrain and cortex at given time points after CFA-induced inflammation. Brief Description of the Sequences

SEQ ID NO: 1 is a nucleic acid sequence encoding human DDAHI.

SEQ ID NO:2 is an amino acid sequence of human DDAHI.

SEQ ID NO:3 is a nucleic acid sequence encoding human DDAHII SEQ ID NO:4 is an amino acid sequence of human DDAHII.

SEQ ID NO:5 is a nucleic acid sequence encoding S. coelicolor DDAH

SEQ ID NO:6 is an amino acid sequence of S. coelicolor DDAH

SEQ ID NO:7 is a nucleic acid sequence encoding P. aeruginosa DDAH

SEQ ID NO: 8 is an amino acid sequence of P. aeruginosa DDAH SEQ ID NO:9 is a nucleic acid sequence encoding P. aeruginosa arginine deiminase

SEQ ID NO: 10 is an amino acid sequence of P. aeruginosa arginine deiminase

SEQ ID NOT 1 is a nucleic acid sequence encoding M. tuberculosis DDAH SEQ ID NO: 12 is an amino acid sequence of M. tuberculosis DDAH

Detailed Description of the Invention

Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises" "comprising" "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements and integers not specifically recited, where the context allows. The present invention is concerned with agents for the treatment of pain in a human or animal. An agent of the invention comprises a modulator having activity which increases DDAHI activity and/or expression levels.

Typically a modulator of the invention has an activity which directly or indirectly causes enhancement of DDAHI activity and/or expression. In one embodiment, the modulator enhances the methylarginase activity of DDAHI. Such an activator may act at the level of, for example, substrate binding to or association with DDAHI, or product release or disassociation from DDAHI. Alternatively or additionally, the activator may affect the chemical reaction that converts substrate to product.

For example, a modulator may act by binding to DDAHI. Binding may induce a conformational change in DDAHI, producing a better fit or stronger binding between the enzyme and its substrate, thus increasing the rate of catalysis.

Alternatively, an activator may bind to a substrate of DDAHI, such as L- NMMA or ADMA, causing for example, a conformational change in the substrate to promote binding to DDAHI, and increase the rate of the enzyme substrate reaction.

A modulator may also act by facilitating or promoting disassociation of DDAHI and the products of the methylarginase reaction, for example by causing a conformational change that releases the products more readily from the enzyme. This may occur, for example as a result of agent binding to DDAHI, substrate, or a DDAHI/substrate or DDAHI/product complex. The effect is to promote recycling of active enzyme, thus increasing productivity and DDAHI activity.

An activator of the invention may act at the level of DDAHI expression. Such a modulator may cause its effect at any level of expression, for example, at initiation, progression, or termination of transcription, during transport of DDAHI mRNA from the nucleus to the cytoplasm, during processing and/or translation of DDAFII mRNA. Alternatively, or additionally, a modulator of the invention, may increase the stability of the DDAHI protein. For example, the modulator may reduce the rate at which the protein is degraded or inactivated. A modulator of expression may bind to a DDAHI gene either (i) 5' to the coding sequence, and/or (ii) in the coding sequence; and/or (iii) 3' to the coding sequence. Thus an activator may bind to the promoter, increasing the rate of transcription. The modulator may bind and activate a protein factor required for transcription of the gene, enhancing its activity. The modulator may itself comprise such a transcription factor or a polynucleotide encoding such a transcription factor. An activatory agent may bind to and inhibit a protein factor that causes down- regulation of DDAHI transcription. Preferably, the present modulator is specific in its modulation of transcription from a DDAHI gene and has substantially no effect on transcription from other genes. An activator according to the invention may bind to the untranslated or translated regions of DDAHI mRNA, to enhance initiation or progress of translation, to nuclear factors that bind to the mRNA and/or transport the mRNA to the cytoplasm, or to translation factors that contribute to translating the mRNA to protein.

The present modulator may comprise a polypeptide which has DDAHI methylarginase activity.

A modulator or activator of the invention may comprise a polynucleotide. The polynucleotide may encode a molecule with activator activity such as any of those described above, for example a protein or mRNA molecule. The activator molecule thus becomes available upon expression of the polynucleotide, for example, in a target cell. Alternatively the polynucleotide may itself have activator activity. A polynucleotide may thus, for example, encode an activator protein (including a transcription factor or a polypeptide having DDAHI methylarginase activity) or activator mRNA, or the polynucleotide may itself be an activator.

In general, a modulator for use in the invention may be identified by assaying a test substance for activity which increases DDAHI activity and/or expression. A typical assay comprises: (i) contacting a test substance with a source of DDAHI activity and/or expression, under conditions that would permit DDAHI activity and/or expression in the absence of the test substance; and (ii) determining whether the test substance causes an increase in the activity and/or expression of the DDAHI. A modulator which enhances DDAHI methylarginase activity can be identified using a microtitre plate assay comprising: (i) admixing a test substance, a labelled methylarginase substrate ([¹⁴C]L-

NMMA or [¹⁴C]L-ADMA) and a buffer solution in a well of the microtitre plate; (ii) adding a preparation of DDAHI enzyme to the well; (iii) incubating under conditions suitable for DDAHI activity in the absence of the test substance; (iv) terminating any enzyme reaction and assaying for any [' C] citrulline produced; (v) carrying out, in a second well of the microtitre plate, a control reaction in the absence of the test substance; and

(vi) assessing any modulatory effect of the test substance on the basis of

[¹⁴C]citrulline production in the presence and absence of the test substance.

The DDAHI enzyme used in the assay may be derived from any DDAH expressing species. Preferably mammalian DDAHI is used. Human DDAHI enzyme, having the amino acid sequence in SEQ ID NO:2 or an allelic variant thereof may be used. An allelic variant will be a variant which will occur naturally and which will function as a methylarginase in a substantially similar way to the specified protein. Human DDAHI for use in the assay may be prepared recombinantly according to the methods set out in WO 00/44888. Fungal or bacterial DDAH may be used. For example, a DDAH polypeptide having the amino acid sequence in any of SEQ ID NOs: 6, 8, 10 or 12, or an allelic variant thereof may be used. Suitable DDAH enzymes are described in WO 00/44888. The DDAH preparation used in the assay may be a purified protein preparation, or may be a cell lysate of a recombinantly expressing bacterial strain, for example an E. coli strain. In the assay, the buffer may be any biological buffer that can provide buffering capability at a pFI of about 7, preferably 6.5, such as HEPES buffer. Where an in vitro buffer is used, sodium phosphate buffer is preferred.

The contents of the well of the microtitre plate may be incubated for 30 minutes to 2 hours, typically for about 1 hour. The incubation temperature may be any temperature at which, in the absence of the test substance, the DDAHI enzyme used in the assay is active. Typically the temperature is in the range of from 25°C to 37°C. If human DDAHI is used in the assay, the assay is best carried out at 37°C. Any enzyme reaction may be terminated by adding a cation exchange resin. For example, 50% (w/v) Dowex (trade mark) 50X8-400 may be used.

The methylarginase activity to be assayed is the ability of the DDAHI enzyme to catalyse the metabolism of L-NMMA or ADMA methylarginines to citrulline. Measures of enzymatic activity are generally known to those skilled in the art, including equilibrium constants, reaction velocities of the appearance of reaction products or the consumption of reaction substrates, reaction kinetics, thermodynamics of reaction, spectrophotometric analysis of reaction products, detection of labelled reaction components, etc. See, generally, Segel, Biochemical Calculations, 2nd Edition, John Wiley and Sons, New York (1976); Suelter, A Practical Guide to Enzymology, John Eiley and Sons, New York (1985). In the above assay, enzyme activity is monitored by measuring [¹⁴C] citrulline production following incubation of the methylarginase with [¹⁴C]L-NMMA or [¹⁴C] ADMA (Leiper, J.M., Santa-Maria, J., Chubb, A., MacAllister, R.J., Charles, I.G., Whitley, G.S. and Vallance, P. Biochem J (1999) 343 Ptl . 209-214). [^l4C]citrulline may be assayed by scintillation counting.

As described above, a control assay is carried out to assess the activity of the DDAHI under the reaction conditions but in the absence of the test substance. Thus it is possible to assess the effect of the test substance on enzyme activity. The requirements for a reliable control assay will be known to those skilled in the art.

For example, the control is preferably carried out in parallel with the test assay. The volume of test substance should be replaced with buffer or other reaction-neutral substance so that the total reaction volume and the concentration of reaction components is the same in both the test and the control assay. Preferably, multiple repetitions are made of both the test and the control assay. Also, as a control, the samples may be assayed for any other enzyme to exclude the possibility that a test substance is a general modulator of enzyme activity.

An activator of DDAHI transcription can be identified using a reporter gene assay. Typically such an assay comprises, (i) providing a test construct comprising a DDAHI promoter operably linked to a second "reporter" polynucleotide to be expressed in the form of mRNA; (ii) contacting a test substance with the test construct under conditions, which in the absence of the test substance, would permit expression of the second polynucleotide in the form of mRNA. (iii) carrying out a control assay under the same reaction conditions but in the absence of the test substance.

(iv) determining whether the test substance enhances expression from the construct.

A DDAHI promoter for use in the assay may be isolated via methods known in the art. Any eukaryotic DDAHI promoter may be used but preferably a mammalian promoter, in particular a human DDAHI promoter is used. A human DDAHI gene promoter, isolated using the nucleic acid sequence in SEQ ID NOT may be used. The promoter sequence may be fused directly to a reporter gene coding sequence or via a linker. The linker sequence may comprise a sequence having enhancer characteristics, to boost expression levels. The reporter assay should be carried out under conditions that allow expression of the reporter polynucleotide as mRNA in the absence of the test substance. For example, the assay should include a suitable RNA polymerase and nucleotides to allow transcription of the reporter sequence. Levels of reporter, mRNA in the presence and absence of the test substance may be compared to assess any modulatory effect of the test substance on transcription from the DDAHI promoter. Preferably the assay is carried out under conditions which mimic those to which the DDAHI promoter is exposed in vivo. It is particularly preferred that the assay includes one or more protein factors, such as transcription factors which interact with the DDAHI promoter in vivo. For example, the assay may comprise extract from the cells from which the DDAHI promoter is derived. Preferably the assay comprises neuronal, in particular, thalamic cell extract.

Preferably one or more control assays is carried out using any other promoter in place of the DDAHI promoter, to exclude the possibility that a test substance is a general activator of transcription. Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, 1989, Molecular Cloning: a laboratory manual. A modulator which affects the stability of DDAHI mRNA or DDAHI protein may be identified in a stability assay. In a suitable assay, DDAHI mRNA or DDAHI protein is incubated in the presence and absence of a test substance, under conditions which mimic those to which the DDAHI mRNA or protein is exposed in vivo. Conditions may mimic those to which DDAHI mRNA or protein is exposed in a neuronal cell, in particular, a thalamic cell. Such an assay may thus comprise neuronal, in particular, thalamic cell extract. Typically, assay temperatures are those found in vivo, for example 25 to 37°C, in particular 37°C. mRNA or protein of any of the DDAH enzymes described above in relation to the other assays may be used. For example, human DDAHI having the amino acid sequence in SEQ ID NO:2, or the corresponding mRNA, produced according to the methods in WO 00/44888 may be used. Samples are removed at regular time intervals and assayed for the presence of DDAHI mRNA or protein. This can be done by any suitable means, for example, by gel analysis or spectrophotometrically. In this way, any alteration in the rate of degradation of DDAHI mRNA or protein, associated with the test substance, can be detected.

A modulator of the invention which acts at the level of translation of DDAHI may be identified using an in vitro translation system. Suitable translation systems are available in the art: for example, the rabbit reticulocyte system. DDAHI mRNA is used in the system in the presence and absence of a test substance, under conditions which permit translation of the mRNA in the absence of the test substance. Preferably conditions will mimic those to which DDAHI mRNA is exposed in vivo. In particular, neuronal cell, especially thalamic cell extract may be included in the assay. The effect of a test substance is assessed by monitoring the production of DDAFII protein using any suitable means, for example, by gel analysis, or by spectrophotometer. mRNA encoding any of the eukaryotic DDAHI molecules described above may be used for example, mammalian DDAHI mRNA. Preferably human DDAHI mRNA, for instance, human DDAHI mRNA corresponding to SEQ ID NOT or 2 is used. A modulator of DDAHI activity and/or expression is one which produces a measurable or detectable increase in methylarginase activity and/or expression in at least one of the assays described above. Generally such activators are those which enhance DDAHI methylarginase activity and/or expression by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the activator of lμg ml^"1, lOμg ml^"1, lOOμg ml^"1, 500μg ml^"1, lmg ml^"1' lOmg ml^"1 or lOOmg ml^"1. Preferred modulators are those which enhance the activity of DDAHI by a least 50% when used at a concentration of from 1 to lOμm in the microtitre plate activity assay. Thus in one embodiment, that assay may be used to identify such an activator.

The percentage activation represents the percentage increase in expression/activity in a comparison of assays carried out in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage enhancement and concentration of inhibitor may be used to define an activator of the invention, with greater activation at lower concentrations being preferred. In general, it is preferred that the present modulator is selective in its modulatory activity. For instance, the modulator is preferably a selective activator of DDAHI methylarginase activity and/or expression, having substantially no effect on the methylarginase activity and/or expression of DDAHII.

A modulator may be tested for an effect on DDAHII activity and/or expression using one or more of the assays as described above in relation to DDAHI. Preferably mammalian DDAHII is used, in particular human DDAHII. For instance, human DDAHII having the amino acid sequence in SEQ ID NO: 4 or an allelic variant thereof may be used in the activity assay or stability assay.

The human DDAHII nucleic acid sequence in SEQ ID NO: 3 or mRNA corresponding to the sequence, or a human DDAHII promoter sequence isolated using this sequence, may be used in an expression assay.

In general, such a selective modulator shows no detectable modulation of DDAHII activity or expression in the above assays. Typically, for example, an activator for use in the invention will cause less than 50% activation, more preferably less than 20% activation, generally less than 5% activation of DDAHII activity or expression in a particular assay. Preferably, such a selective modulator causes at least 2x, 4x, 6x, lOx, lOOx, lOOOx or at least 10⁶ x greater percentage activation of DDAHI methylarginase activity and/or expression at a given concentration of modulator than the percentage activation of DDAHII, at that same concentration. Additionally or alternatively, it is preferred that the modulatory substance of the invention has substantially no inhibitory effect on the expression or activity of NOS, in particular nNOS.

Generally, the modulator has substantially no effect on the transcription or translation of the NOS gene or on the activity of the NOS enzyme. A candidate DDAHI activator may be tested for an effect on NOS activity by an assay which generally involves:

(i) contacting the DDAHI modulatory substance with a NOS, such as nNOS, and a substrate and cofactor therefor under conditions which, in the absence of the substance, would be expected to allow NOS activity; and (ii) determining whether, or to what extent, NOS activity takes place.

A suitable assay for inhibition of NOS activity uses a microtitre plate and measures NOS activity by determining the change in absorbance as NADPH is converted to NADP⁺. This assay comprises:

(a) adding a candidate substance, a known NOS inhibitor (for example a nNOS inhibitor such as L-NMMA) and a buffer solution to separate microtitre wells;

(b) adding to each well NOS enzyme such as nNOS, cofactor(s) therefor, L- arginine and buffer; and

(c) determining the change in absorbance in each well.

Typically, the buffer is a HEPES buffer capable of maintaining a pFI of about 7, preferably about 7.4. The cofactors comprise oxyhemoglobin, NADPH and BH₄. They may also comprise CaCl₂, MgCL, FMN, FAD and/or CaM.

The NOS may be a naturally occurring form of NOS or may be a variant which retains NOS activity, for example variants produced by mutagenesis techniques. NOS used in the assay is preferably of mammalian origin, for example rodent (including rat and mouse) or primate (such as human). Preferably, the NOS is of human origin. Preferably nNOS is used. The NOS, in particular nNOS, may be obtained from mammal cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines. Preferably, the NOS used in the assay is recombinant. More preferably, it is obtained by expression in S 21 cells according to the methodology in Charles et al., Methods in Molecular Biology (edited by M.A. Titheradge, Humana Press, Totowa), vol 100, pgs 51-60.

The known NOS, preferably nNOS, inhibitor may be any substance which inhibits activity of NOS or nNOS enzyme. Typically, an inhibitor of nNOS enzyme is used. Competitive, non-competitive, _^reversible and irreversible inhibitors are suitable.

Suitable inhibitors include L-arginine analogues, thiocitrullines, indazole derivatives, imidazole derivatives, hydrazine derivatives, thioureas, thiazoles, biotin derivatives and phenyl-substituted thiopene amidines.

Examples of suitable L-arginine analogues include methyl-L-arginine, N - nitro-L-arginine methyl esther (L-NAME), N^G-monomethyl-L-arginine (L-NMMA), N^G-amino-L-arginine (L-NAA), N^w,N^w-dimethyl-L-arginine (ADMA), N^w,N^w2- dimethyl-L-arginine (SDMA), N^vv-ethyl-L-arginine (L-NEA), N^w-methyl-L- homoarginine (L-NMHA), N^w-nitro-L-arginine (L-NOARG), N^δ-iminoethyl-L- ornithine (L-NIO), N -iminoethyl-L-lysine (L-homo-NIO) and L-canavanine (L- CAN).

Examples of suitable thiocitrullines include S-methyl-L-thiocitrulline (SMTC), L-thiocitrulline (L-TC) and L-S-ethyl-thiocitrulline (Et-TC).

Examples of suitable indazole derivatives include indazole and 7-substituted indazoles such as 7-nitroindazole and 3-bromo-7-nitroindazole. Examples of suitable hydrazine derivatives include aminoguanidine.

Examples of suitable imidazole derivatives include phenyl substituted imidazoles such as 1-phenyl-imidazole.

Examples of suitable thioureas include S-methylisothiourea sulphate, δ-(S- methylisothioureido)-L-norvaline (L-MIN), S-ethylisothiourea (SETU) and S- isopropylisothiourea (SIPT).

Examples of suitable thiazoles include 2-amino-thiazole and 2-amino-4,5- dimethyl thiazole. Examples of suitable biotin derivatives include 2-iminobiotin. The above NOS inhibitors are commercially available, or may be made by analogy with known methods.

Step (c) of the assay may be carried out by reading the difference in absorbance between 420 and 405 nm. Typically, this is done by a spectrophotometer. Comparison of the well containing the candidate substance with the control wells containing a known NOS inhibitor (100% inhibition) and no inhibitor (0% inhibition) allows % inhibition achieved by the candidate compound to be calculated. A microtiter assay as set out above is described in detail in Dawson &

Knowles, Methods in Molecular Biology (edited by M.A. Titheradge, Humana Press, Totowa), vol 100, Chapt. 22, pgs 237-242.

A modulator may be tested for an effect on NOS expression using the transcription or translation assays described above in relation to DDAH expression. Such an assay may use a polynucleotide encoding any naturally occurring form of NOS or a variant which retains NOS activity. Preferably the polynucleotide encodes a mammalian NOS, for example a rodent (including rat or mouse) NOS or a primate (including human) NOS. Preferably a polynucleotide sequence encoding nNOS, especially human nNOS is used. Suitable polynucleotides are known in the art. For example, a sequence of human nNOS may be found at GenBank Ul 7327, coding sequence 686-4990.

Preferably a modulator for use in the present invention causes no detectable inhibition of NOS, and especially nNOS, expression or activity in the above assays. Typically a modulator will achieve less than 50% inhibition, more preferably less than 20 % inliibition, generally less than 5% inhibition of NOS, for example nNOS, in the assay. Generally, such results are achieved at a concentration of modulator of 0.01 to 10 μ , for example 0.1 to 5, or 1 to 2 μm.

In one embodiment it is preferred that an activator for use in the invention shows at least lOOx, lOOOx or at least 10 x greater percentage activation of DDAHI methylarginase activity and/or expression at a given concentration of activator than the percentage inhibition of NOS, especially nNOS at that same concentration. In one embodiment, a modulator of the invention has substantially no effect, activatory or inhibitory, upon NOS, especially nNOS activity and/or substantially no effect upon NOS, especially nNOS, expression.

A suitable assay for an effect on NOS activity again uses a microtitre plate, and measures NOS activity by determining the change in absorbance as NAPDH is converted to NADP⁺. Such an assay comprises:

(a) adding a test substance and a buffer solution to separate microtitre wells;

(b) adding to each well, NOS enzyme, such as nNOS, cofactor(s) therefore, L-arginine and buffer; and

(c) determining the change in absorbance in each well.

Suitable buffers, cofactors, and NOS are described above in relation to the inhibition assay. Again step (c) may be carried out by reading the difference in absorbance between 420 and 405 nm, typically by spectrophotometer. Comparison of the wells allows any modulation by the test substance to be determined.

A modulatory effect on expression may be assessed using the transcription or translation assays described above.

Preferably, a modulator of the invention causes no detectable change in NOS and especially nNOS, activity and/or no detectable change in NOS, especially nNOS expression, in the assays. Typically, a modulator will cause less than 50% change, more preferably less 20% change, such as a less than 5% change in NOS, especially nNOS, activity or expression in an assay.

In a particularly preferred embodiment, the present modulator is a selective activator of DDAHI but not DDAHII methylarginase activity and/or expression, but has substantially no inhibitory effect on the expression or activity of NOS, especially nNOS.

In the above assays, suitable test substances include antibodies, combinatorial libraries, defined chemical identities, chemical compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display libraries (e.g. phage display libraries). Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Test substances may include polynucleotides. Where a potential modulator comprises a polynucleotide encoding an activatory molcule, such as a protein or mRNA, the encoded molecule may be used a the "test substance" in as assay. A modulator may include a single substance or a combination of two, three or more substances. For example, a modulator may refer to a single peptide, a mixture of two or more peptides or a mixture of a peptide and a defined chemical entity.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show modulatory activity tested individually. Test substances may be used at a concentration of from 1mm to lOOOmM preferably from ImM to lOOmM, more preferably from ImM to lOmM.

In one embodiment, a modulator of the invention comprises a polypeptide having DDAH activity or a polynucleotide encoding such a DDAH polypeptide. In general, the DDAH polypeptide has DDAH methylarginase activity. Typically a DDAH polypeptide for use as a modulator comprises the amino acid sequence set out in SEQ ID NO: 2, 4, 6, 8, 10 or 12 or a substantially homologous sequence, or a fragment of either said sequence and has methylarginase activity. Methylarginase activity may be assayed according to the method described above.

In general, the naturally occurring amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12 is preferred. SEQ ID NOS: 2, 4, 6, 8, 10 and 12 set out the amino acid sequences of human DDAHI, human DDAHII, S. coelicolor DDAH, P. aeruginosa DDAH, P. aeruginosa arginine deiminase and M. tuberculosis DDAH respectively. In particular, the amino acid sequence of a human DDAHI, such as that in SEQ ID NO:2, is preferred. In one embodiment, a DDAH polypeptide for use as an activatory agent may comprise: (a) the polypeptide sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12; (b) an allelic variant or species homologue thereof; or

(c) a protein with at least 70, at least 80, at least 90, at least 95, at least 98 or at least 99% sequence identity to (a) or (b).

An allelic variant will be a variant which will occur naturally, for example, in a human, bacterium or yeast and which will function in a substantially similar manner to the protein of SEQ ID NO: 2, 4, 6, 8, 10 or 12, for example it acts as a methylarginase. Similarly, a species homologue of the protein will be the equivalent protein which occurs naturally in another species and which can function as a methylarginase. Allelic variants and species homologues can be obtained using the sequences of SEQ ID NOs 1 to 12 and procedures known in the art on a suitable cell source e.g. a human or bacterium cell. Suitable methods are described for example, in WO 00/4488. It will be possible to use a probe based on any of SEQ ID NOs 1 to 2 or 5 to 12 to probe libraries made from human or bacterial cells in order to obtain clones encoding the allelic or species variants. The clones can be manipulated by conventional techniques to generate a suitable DDAH polypeptide which can then be produced by recombinant or synthetic techniques known per se.

A DDAH polypeptide for use as a modulator preferably has at least 60% sequence identity to the protein of SEQ ID NO: 2, more preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, at least 100 contiguous amino acids or over over the full length of SEQ ID NO: 2, 4, 6, 8, 10 or 12.

Preferably a DDAH polypeptide for use as a modulator according to the invention is a DDAHI polypeptide, such as a mammalian, in particular a human DDAHI polypeptide. Thus, a particularly preferred DDAH polypeptide has an amino acid sequence as defined above, where (a) is SEQ ID NO:2.

As described above, the sequence of the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10 or 12 and of allelic variants and species homologues can be modified to provide a DDAH polypeptide for use according to the invention. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. The modified polypeptide generally retains activity as a methylarginase, as defined herein. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

A modulatory DDAH polypeptide of the invention may also include fragments of the above-mentioned full length polypeptides and variants thereof, including fragments of the sequence set out in SEQ ID NO: 2, 4, 6, 8, 10 or 12. Such fragments typically retain activity as a methylarginase.

Any of the above DDAH polypeptides may be chemically modified, e.g. post-translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell.

As described above, in one embodiment, a modulator of the invention comprises a polynucleotide. The polynucleotide may encode an activator molecule, such as a protein or mRNA with activator activity.

A modulator of the invention may comprise a polynucleotide encoding a DDAH polypeptide. In general the DDAH polypeptide hasDDAH methylarginase activity. Such a polynucleotide may encode any of the above DDAH polypeptides. In particular, such a polynucleotide typically: (a) encodes a polypeptide that has the properties of a methylarginase, which polynucleotide is selected from: (1) the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 1 1 ; (2) a sequence which hybridises selectively to the complement of a sequence defined in (1); and (3) a sequence that is degenerate as a result of the genetic code with respect to a nucleic sequence defined in (1) or (2); or (b) is a sequence complementary to a polynucleotide defined in (a).

SEQ ID NOS: 1, 3, 5, 7, 9 and 11 set out the sequences of human DDAHI, human DDAHII, S. coelicolor DDAH, P. aeruginosa DDAH, P. aeruginosa arginine deiminase and M. tuberculosis DDAH respectively.

A DDAH polynucleotide for use according to the invention also includes a variant of the coding sequence of SEQ ID NO: 1, 3, 5, 1 , 9 or 1 1, the expression product of which can function as a methylarginase. Such a variant thus has the ability to catalyze the production of citrulline from methylarginines. Typically a DDAH polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the complement of the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11.

A suitable DDAH polynucleotide and the complement of the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 can hybridize at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a DDAH polynucleotide of the invention and the complement of the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P. Selective hybridisation may typically be achieved using conditions of low stringency (0.03M sodium chloride and 0.03M sodium citrate at about 40°C), medium stringency (for example, 0.03M sodium chloride and 0.03M sodium citrate at about 50°C) or high stringency (for example, 0.03M sodium chloride and 0.03M sodium citrate at about 60°C).

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 11 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1, 3, 5, 7, 9 or 11. Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define a DDAH polynucleotide suitable for use in the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The coding sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9 or 1 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. The modified polynucleotide generally encodes a polypeptide which has methylarginase activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A DDAH polynucleotide for use as a modulator of the invention may comprise DNA or RNA. The polynucleotide may include synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. The DDAH polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotide.

A DDAH polynucleotide for use in the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. A polynucleotide may also be cloned by standard techniques. A polynucleotides is typically provided in isolated and/or purified form.

A DDAH polynucleotide will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques.

Typically, this will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the gene which it is desired to clone. Primers may, for example be based on any of SEQ ID NOs 1 to 12. The primers are brought into contact with mRNA or cDNA obtained from a cell and a polymerase chain reaction is performed under conditions which bring about amplification of the desired region.

The amplified fragment is isolated (e.g. by purifying the reaction mixture on an agarose gel) and the amplified DNA is recovered. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

Such techniques may be used to obtain all or part of the DDAH polynucleotides described herein. Genomic clones corresponding to the cDNA of SEQ ID NOS: 1, 3, 5, 7, 9 or 11 containing, for example, introns and promoter regions may also be produced using recombinant means, for example using PCR

(polymerase chain reaction) cloning techniques, starting with genomic DNA from for example a bacterial, an animal or a human cell.

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, 1989, Molecular Cloning: a laboratory manual.

DDAH polynucleotides which do not have 100% sequence identity to the sequence of SEQ ID NOS: 1, 3, 5, 7, 9 or 11 but fall within the scope of the invention can be obtained in a number of ways: 1. Other human allelic variants of the human DDAHI and DDAHII sequences given in SEQ ID NOS: 1 and 3 may be obtained for example by probing genomic DNA libraries made from a range of individuals, for example individuals from different populations, or individuals with different types of disorder related to aberrant NO metabolism, using probes as described above.

In addition, homologues of SEQ ID NO: 1, 3, 5, 7, 9 or 11 may be obtained from other animals particularly mammals (for example mice and rabbits) or fish (for example Fugu) or insects (for example D. melanogaster)oτ other invertebrates (for example C. elegans), plants (for example A. thaliana), bacteria and yeasts and such homologues and fragments thereof in general will be capable of selectively hybridising to the coding sequence of SEQ ID NOS: 1 and 3 or its complement. Such sequences may be obtained by probing cDNA or genomic libraries from dividing cells or tissues or other animal species with probes as described above. Degenerate probes can be prepared by means known in the art to take into account the possibility of degenerate variation between the DNA sequence of SEQ ID NOS: 1 , 3, 5, 7, 9 or 1 1 and the sequences being probed for under the selective hybridization conditions given above. 2. Allelic variants and species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding likely conserved amino acid sequences. Likely conserved sequences can be predicted from aligning the amino acid sequences of, for example, SEQ ID NOs 2, 4, 6, 8, 10 and/or 12. The primers will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. 3. Alternatively, polynucleotides may be obtained by site directed mutagenesis of SEQ ID NO: 1, 3, 5, 7, 9 or 11 or allelic variants thereof. This may be useful where, for example, silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

Preferably, a DDAH polynucleotide for use as a modulator in the invention encodes a DDAHI polypeptide, such as a mammalian DDAHI, in particular, a human DDAHI. Thus in a particularly preferred embodiment, a DDAH polynucleotide for use as a modulator is as defined above, where (a) (1) is the coding sequence of SEQ ID NOT .

A modulator of the invention may comprise a double stranded polynucleotide comprising a polynucleotide as described above and its complement. A polynucleotide modulator of the invention may encode an activatory molecule other than a DDAH polypeptide, for example, a transcription factor that positively regulates DDAHI transcription.

In general, a polynucleotide modulator which comprises sequence encoding an activator molecule (such as a DDAH polypeptide including those described above) also includes one or more sequence elements which are capable of providing for expression of the coding sequence in a host cell. For example, the polynucleotide coding sequence is generally capable of being expressed in a target cell of the human or animal to which the polynucleotide modulator is administered.

In one embodiment, the polynucleotide coding sequence is incorporated in a replicable recombinant vector. Typically, the coding sequence is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, a plasmid, virus or phage vector, adapted to be used in vivo. Typically a vector is provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide coding sequence and optionally a regulator of the promoter.

A promoter or other expression regulation signal may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium and the β-actin promoter. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter including the SV40 large T antigen promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR) . Viral promoters are readily available in the art. Preferably the polynucleotide encoding a modulatory molecule (for example a DDAH polypeptide having methylarginase activity) is operably linked to an inducible promoter. Expression of the activator may then be controlled as necessary. Thus, in use to combat pain in a human or animal, expression may be induced at a time when modulatory activity is needed, for example, when pain is felt or expected. In another preferred embodiment the polynucleotide encoding a modulatory molecule is operably linked to a tissue-specific promoter. Thus modulatory activity may be restricted to particular cells or tissues in which the activator may be expressed. A neuronal cell specific promoter, in particular a thalamic cell specific promoter is particularly preferred. For example, a DDAHI promoter may be used, especially a human DDAHI promoter.

A vector may include sequences flanking the polynucleotide coding sequence which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell.

Suitable viral vectors for use in the invention include herpes simplex viral vectors (for example as disclosed in WO 98/04726 and WO 98/30707) and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses (such as HPV- 16 or HPV- 18). Preferably an HSV based viral vector is used (Lachman RH and Efstathiou S (1997) Molecular Medicine Today 404-414). Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate a polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression. A modulator or activator, identified according to any of the above assays can be further tested for efficacy in a suitable test animal.

Accordingly in one aspect the present invention provides a method of identifying an agent for use in the treatment of pain, the method comprising or sometimes consisting essentially of: ^'

(a) assaying a test substance for activity which increases DDAHI methylarginase activity and/or expression;

(b) screening a modulator identified in (a) for efficacy in treating pain in a test animal. Step (a) may be carried out according to any of the DDAH modulation assays described above. In one embodiment, step (a) further comprises assaying a test substance for selective modulatory activity according to the DDAHI/DDAHII and/or DDAHI/NOS selectivity assays described above. A selective activator identified in step (a) may then be put forward for screening in step (b). Typically any initial batch screening of test substances is carried out in step

(a), such that typically only a particularly preferred modulator is put forward for screening in step (b). One or more modulators may be administered to a suitable test animal in step (b).

In general, step (b) comprises or may consist essentially of : (i) administering a modulator identified in step (a) to a test animal; and

(ii) determining whether the modulator reduces or prevents pain in the animal.

Protocols for assessment of pharmacological agents by animal testing are known in the art. In general, the present method will comprise administering a test modulatory agent to a test animal, and monitoring any effect of the modulator in the animal, in particular, any effects on pain sensitivity or pain processing, using appropriate tests. The animal test may also be used to monitor other potential effects of a modulator, in particular any side effects. The method may thus comprise monitoring the toxicity or metabolism of a modulator. Methods for monitoring side effects are known in the art, for example by monitoring appetite, weight, social behaviour, mortality. Thus, efficacy, toxicity, pharmacokinetic properties and/or potential therapeutic effects of metabolites of a modulator may be assessed in the test animal. _

A test modulator may be formulated with a standard carrier and/or excipient as is routine in the pharmacological art, and as fully described in Remington's Pharmaceutical 17^th Ed. 1985. A modulator may be administered to the test animal by enteral or parenteral routes, such as via oral, buccal, anal, pulmonary, nasal, vaginal, intravenous, intra-arterial, intrahepatic, intramuscular, intraperitoneal, subcutaneous or other appropriate administration routes. A test modulator may be present in the food or drinking water or may be administered using an osmotic minipump.

An activator may be administered to a test animal at any appropriate dosage. A typical dose may be from 0.1 to 50 mg/kg body weight, for example from 0.5 to 30 mg/kg body weight, 1 to 20 mg/kg body weight or 1 to 10 mg/kg body weight. Various doses may be used to determine a more/most effective dose. For polynucleotide modulators which encode an activatory molecule (such as a polypeptide or mRNA), either the encoded molecule or the polynucleotide may be tested. Preferably, the polynucleotide modulator is administered and assessed. Methods of polynucleotide administration are known in the art and are described below. Where a polynucleotide modulator is administered by viral vector, the dose is typically in the range of from 10 to 10¹⁰ pfu, preferably from 10⁷ to 10⁹ pfu, more preferably about 10⁸ pfu for adenoviral vectors. When injected, typically l-2ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. When the polynucleotide is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg. A test animal may be dosed with a modulator prophylactically or therapeutical ly on one or more occasions. Typically, a modulator may be administered biweekly, weekly, twice weekly, daily or two, three or more times a day, for example at hourly or at two, three or four hourly intervals.

The test animal is generally any laboratory bred animal suitable for testing of pharmacological or pharmaceutical agents. Such animals include non-human primates and rodents. Typically the animal is a rodent, such as a rat, mouse, guinea- pig, hamster, ferret or gerbil. In a preferred embodiment, the animal is an animal pain model. In such a model the animal has typically been treated in such a way as to mimic a particular pain state or pain type, and thus reflect some element of a clinical pain syndrome. The animal model is typically intended to replicate, as far as possible one or more of the pathophysiological changes present in an animal experiencing or subject to pain.

Preferably the animal is an animal model of neuropathic pain. In particular, a partial nerve injury model is preferred. Several such models are known, including the Chung spinal segmental nerve model, (Kim S H and Chung J M Pain (1992) 50: 355-363), the Bennett chronic construction injury model (Bennett, G J and Xie Y K Pain (1988) 33: 87-107), the Seltzer partial sciatic nerve injury model (Seltzer Z, Dubner R and Shir Y Pain (1990) 43: 205-218) and the Decosterd spared nerve injury model (Decosterd I and Woolf C J Pain (2000) 00: 1-10). Preferably the spared nerve injury model is used.

There are a number of established tests in the art for measuring pain or assessing pain sensitivity or processing in an animal subject. Any of these tests are suitable for use in the invention. Tests may be qualitative or quantitative. Preferably a quantitative test is used. Several of the tests are based on behavioural responsiveness of a test animal. Such tests may, assess for example, mechanical allodynia, mechanical hyperalgesia, cold allodynia or heat hyperalgesia. Suitable tests include but are not limited to the von Frey filament test (Tal M and Bennett, G J Pain (1994) 57: 375-382), the pin-prick test (Decosterd I, Buchser E, Gilliard N, Saydoff J, Zurn A D and Aebischer P Pain (1998) 76:159-166), the acetone drop test (Choi Y, Yoon Y W, Na H S, Kim S H and Chung, J M Pain (1994) 59: 369-376), the radiant heat test (Hargreaves K, Dubner R, Brown F, Flores C and Joris J Pain (1988) 32: 77-88) and the hot plate test (Lee DE, Kim S J and Zhuo M. Brain Res (1999) 845: 1 17-121). These named tests focus on peripheral and spiral mechanisms of pain processing. Pain processing at higher levels may be assessed using a behavioural test of conscious nociceptive processing as described by Mauderli (Mauderli A P, Acosta-Rua A and Vierck C J. J Neurosci Methods (2000) 97 : 19- 29). In such a test, an animal, typically a rat, makes a conscious choice under a mild thermal stimulus, thus involving thalamic and neocortical function. Any one or more of the above tests may be used to determine pain in the present method. Preferably two or more tests are used.

Pain in an animal may also be assessed in a qualitative way. Often this involves observation of animal behaviour. Suitable aspects of behaviour to monitor include animal stance, weight bearing, flexion/extension of limbs, weight loss, appetite, sleep cycles, social interaction, and grooming habits. For example, an animal subject to pain in a paw may refrain from weight bearing on the affected paw. In one embodiment the present method comprises monitoring pain sensitivity in a test animal before and after administration of a test modulatory agent, and determining any reduction in sensitivity associated with the agent. Typically more than one animal is tested. Administration of a modulator will preferably reduce or prevent sensitivity to the pain. Thus, pain sensitivity is typically less after administration of the agent, the reduction in sensitivity being statistically significant. Suitable statistical tests for analysing quantitative test results, are known in the art. Preferably the present method comprises the use of one or more controls. A control animal is typically subject to the same experimental conditions e.g. environment and testing regime as a test animal. There are several possible types of control animal. One type of control animal is not administered with the modulator, but may receive a placebo, preferably administered by the same route as the test agent. Another type of control is administered with a known anti-pain agent instead of the modulator. Where the test modulatory agent is being administered to an animal pain model, a control is typically carried out using an animal which is not so modelled. A naive and/or sham control animal may be used. For example, in the spared injury model, which involves surgical nerve injury, a control may be an animal which has not been surgically operated at all (a naive animal) and/or an animal which has undergone surgery to expose but not injure the nerves (a sham animal).

Preferably more than one control animal is used of any particular type. Preferably more than one type of control is used. Other requirements of controls are known to those skilled in the art.

Typically, an agent for treating pain according to the invention provides a statistically significant reduction in pain sensitivity in a test animal compared to a control which has not been contacted with the agent. In one embodiment, the effect of the present agent is comparable to or an improvement over that of a known agent.

Preferably, the present agent provides a statistically significant reduction in pain sensitivity compared to a known agent. Typically, an agent of the invention has an effect which is more immediate and/or longer lasting than a known agent.

Thus the method of the invention may be used to develop a product suitable for treating pain. Accordingly there is also provided an agent for treating pain, identified in accordance with the invention.

In general it is preferred that a modulatory agent of the invention is capable of passage across the blood-brain barrier. Thus in one embodiment a small modulatory molecule, in particular a lipid soluble molecule is preferred.

An agent of the invention is useful for combating pain in a human or animal. An agent may act to alleviate existing pain in an individual or may be administered in anticipation of a painful condition. Thus treatment may be therapeutic or prophylactic. Preferably the agent is suitable for use in a mammal. In particular, it is preferred that the subject for treatment is human. However, the invention will also be of veterinary use for treating livestock and domestic animals. For example, the invention may be of use for treating cattle, pigs, sheep, horses, dogs, cats or rabbits. Generally the pain to be treated is one in which pain signals are processed at least in part by the thalamus. For example, the pain may be "fast pain" for example, sharp pain, pricking pain, electric pain, or "slow pain", for example, burning pain, aching apin, throbbing pain, nauseous pain.

Attempts have been made to classify different types of pain. Among those classes which have been broadly identified, but which nevertheless overlap to an extent, are acute and chronic pain.

A definition of acute pain (Halpern (1984) Advances in Pain Research and Therapy Vol.7, Ed. C. Bendetti et al, pi 47) which is not intended to be limiting, is as a constellation of unpleasant sensory, perceptual and emotional experiences of certain associate autonomic (reflex) responses, and of psychological and behavioural reactions provoked by injury or disease. Tissue injury provokes a series of noxious stimuli which are transduced by nociceptors to impulses transmitted to the spinal cord and then to the upper part of the nervous system. Examples of acute pain are dental pain, post-operative pain, obstetric pain, headaches, neuralgia and myalgia.

A definition of chronic pain (Halpern (1984) ibid.) also not limiting, is pain that persists beyond the usual course of an acute disease or beyond a reasonable time for an injury to heal. Chronic pain is typically a result of persistent dysfunction of the nociceptive pain system. Examples of chronic pain include trigeminal neuralgia, post-herpetic neuralgia (a form of chronic pain accompanied by skin changes in a dermatomal distribution following damage by acute Herpes Zoster disease), diabetic neuropathy, causalgia, "phantom limb" pain and pain associated with osteoarthritis, rheumatoid arthritis and cancer. Some of these, for example, trigeminal neuralgia, diabetic neuropathic pain, causalgia, phantom limb pain and central post-stroke pain, have also been classified as neurogenic pain. One non-limiting definition of neurogenic pain is pain caused by dysfunction of the peripheral or central nervous system in the absence of nociceptor stimulation by trauma or disease. Physiological and pathological pain have been defined in terms of their origin in the plasticity of the nervous system. The latter is defined in turn as the alteration in structure or function of the nervous system caused by development, experience or injury and can be either adaptive or maladaptive (Cervero, F.(1991) Ewr. J. Neurosci Suppl 4, 162). Adaptive plasticity underlies the ability of the nervous system to compensate for damage or to produce changes in function which are appropriate to environmental change. Physiological pain, considered to be a sensation which reflects specific peripheral stimuli, is based on adaptive plasticity.

Maladaptive plasticity comprises those changes in the nervous system which lead to a disruption of function and therefore effectively constitute a disease state. Pathological pain is considered to be a sensation resulting from changes within the nervous system which bring about an alteration in the way in which information from the periphery, some of which is quite normal, is handled. Pathological pain is therefore based on maladaptive plasticity (Woolf, C.J. (1989) Br. J. Anaesth. 63, 139-146). Maladaptive plasticity of the nociceptive system has also been shown, in experimental models, to be present in states of chronic pain. For instance, multiple injections of hyperalgesic substances such as PGΕ into the paw of a rat have been shown to induce sustained hyperalgesia to mild pressure (e.g. Nakamura-Craig and Smith (1989) Pain 38, 91-98; Ferreira et al (1990) Pain 42, 365-371; Nakamura- Craig and Gill (1991) Neurosci. Lett. 124, 49-51).

A number of animal models have been developed of neuropathic pain in particular peripheral neuropathic pain, which suggest that this pain is often associated with partial denervation (Decosterd I and Woolf C J (2000) Pain 00 : 1- 10). Such models mimic in particular, pain associated with, for example diabetic neuropathy, postherpetic neuralgia, toxic neuropathies, compression neuropathies and trauma, characterised by spontaneous lancinating, burning pain and shock like pain, as well as pain hypersensitivity including tactile allodynia, pinprick hyperalgesia and hyperpathia.

The present modulatory agent can be used in a method of treating, including preventing, pain in a human or animal, the method comprising administering thereto a therapeutically or prophylactically effective amount of the agent. The modulator can be used in the therapeutic or prophylactic treatment of pathological conditions in which pain occurs. The condition of a human or animal to which the modulator is administered can thereby be improved.

The present modulatory agent is useful for treating or preventing different types of pain including chronic pain and acute pain. Examples of chronic pain which can be targeted using the present inhibitors include trigeminal neuralgia, postherpetic neuralgia, painful diabetic neuropathy, causalgia, central post-stroke pain, "phantom limb" pain, atypical facial pain, back pain, headaches, neuralgia and pain associated with osteoarthritis, rheumatoid arthritis and cancer.

Examples of acute pain which can be treated using the present methods include dental pain, post-operative pain, obstetric pain, headaches, neuralgia and myalgia. In particular, the agents can be administered pre-operatively to counteract the acute pain associated with surgical operations, including dental surgery and labour pain. In a preferred embodiment the method of the invention comprises administering a therapeutically effective amount of an agent to a patient prior to a dental operation, for example a tooth extraction. Virtually no analgesic agents currently in use are effective in controlling pain when administered pre-operatively in this way. The invention also provides a method for treating pain according to which the present modulatory agent is administered jointly together with one or more other agents. For example, a modulator may be administered with a known anti-pain agent. In one embodiment the one or more other agents comprises an inhibitor of NOS activity suach as a methylarginine. For example, a modulator of DDAHI may be used in conjunction with L-NMMA and/or ADMA. This approach may radically alter the activity profile of L-NMMA and/or ADMA and may result in L-NMMA and/or ADMA having an increased inhibitory effect for NOS.

A modulator may be administered in parallel with another agent (such as methylarginine), or the agents may be administered sequentially, one following on from the other. Alternatively, a modulator and another agent (for example methylarginine), while acting together for the same purpose, may be administered separately. Thus, the invention provides products containing a modulator of DDAHI methylarginase activity and/or expression and a further agent (such as methylarginine) as a combined preparation for simultaneous, separate or sequential use in the treatment of pain in a human or animal .

As described, a modulator may be used to improve the condition of a patient experiencing pain or to prevent or reduce future pain. The formulation of a modulator for administration in preventing or alleviating pain will depend largely upon the nature of the exact modulator. Formulation is also influenced, for example, by whether a pharmaceutical or veterinary use is intended, and by the requirement for passage across the blood-brain barrier. A modulator may also be formulated for simultaneous, separate or sequential use with another substance such as a methylarginine. In general, it is preferred that the modulator is directed or administered to neuronal cells, especially those of the CNS.

In one embodiment, activity of the modulator of the invention is restricted only to particular cell types. For example, activity may be limited to neuronal cells, particularly those of the CNS, in particular thalamic cells. A modulator may be formulated for uptake specifically by such cells. Alternatively, a modulatory agent may be administered directly to such cells. For example, an agent may injected into the thalamus. Administration of a modulator may be in a variety of dosage forms. Thus, a modulator may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. A modulator may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. A modulator may also be administered as a suppository. Preferably an agent is administered to the CNS, for example by direct intracranial, in particular intrathalamic, injection. Alternatively, it is preferred that the agent is injected intravenously. There may then be passage of the agent across the blood brain barrier, in particular to the thalamus. A physician will be able to determine the required route of administration for each particular patient.

A modulator is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

Generally, a modulator of the invention is provided in a pharmaceutical pack. Typically the product comprises a pharmaceutical composition including a modulator and a pharmaceutically acceptable carrier or diluent, together with instructions for use of the product in the treatment of pain. The product may also include one or more other agents for use in conjunction with the modulator, together with instructions for use in pain therapy. These may include, for example, a known anti- pain agent. In one embodiment the one or more other agents comprises an inhibitor of NOS activity, in particular, a methylarginine such as L-NMMA or ADMA. A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. The dose will be influenced by whether the modulator is administered to ease existing pain, or to protect against futμre pain, as well as by the severity of the pain itself A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the pain and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. A daily dose may be given in one or more than one, for example, 2, 3 or 4 administrations.

As described, a modulator of the invention may be administered as a polynucleotide. The polynucleotide may comprise coding sequence for a molecule with modulatory activity, for example a protein or mRNA. The polynucleotide is generally constructed so as to permit expression of the modulatory molecule in a target cell. For example, the coding sequence may be operably linked to a suitable promoter sequence, which will direct expression in a target cell. Alternatively the polynucleotide may itself have modulatory activity.

Generally it is preferred that a polynucleotide of the invention is directed towards or administered to neuronal cells, especially those of the CNS, in particular the thalamus. Thus it is preferred that the modulatory activity of the polynucleotide can be expressed in a neuronal cell of the CNS, expecially in a thalamic cell. A modulatory agent of the invention may comprise a polynucleotide encoding a modulatory product under the control of an inducible promoter. Expression of the modulator can then be restricted to a limited time only, for example until the cause of the pain is removed. Once a painful condition has been treated, the inducer is removed and expression of the modulator ceases. This will clearly have clinical advantages, for example, in the treatment of acute or short-lived pain.

The activity of a polynucleotide agent according to the invention may be restricted to particular cell or tissue types. In particular it is preferred that activity is limited to neuronal cells, particularly those of the CNS, especially cells of the thalamus. As described above, where an agent includes a polynucleotide encoding a modulatory product, the polynucleotide coding sequence may be operably linked to a tissue or cell specific promoter, thus limiting expression of the modulatory product to a particular cell or tissue. For example, a thalamus-specific promoter may be used. Alternatively, a polynucleotide agent may be administered to a specific cell or tissue type. For example, a polynucleotide agent may be applied by direct injection into a neuronal cell, especially a neuronal cell of the CNS, in particular of the thalamus. Tissue or cell specific activity may have particular benefit where the activatory activity is toxic in other cell types.

A polynucleotide may be administered as a naked nucleic acid construct. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Examples of these transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). A nucleic acid according to the invention may also be administered using a viral vector. Preferably such a virus-based vector will allow passage across the blood brain barrier and/or direct delivery to neurons, in particular a neuron of the CNS, for example by intracranial injection. Suitable viral vectors have been described above.

Typically, a polynucleotide modulator of the invention is mixed with a transfection agent to produce a composition. Preferably the naked polynucleotide construct, viral vector comprising the polynucleotide or polynucleotide composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. A polynucleotide composition according to the invention may be formulated for parenteral, intramuscular, intravenous, subcutaneous, or transdermal administration. Preferably administration is intravenous. The polynucleotide agent may then cross the blood brain barrier for passage into the CNS, in particular the thalamic cells. Alternatively it is preferred that administration is directly into neurons, in particular neurons of the CNS. Such administration is typically by needle injection. For example, a polynucleotide agent may be injected through the eye into the optic nerve. Alternatively, the agent may be injected intracranially, for example intra-thalamically. A nucleic acid of the invention may also be administered by needleless injection. A polynucleotide pharmaceutical composition is administered in such a way that the polynucleotide, for example viral vector can be incorporated into cells at an appropriate area. When the polynucleotide is delivered to cells by a viral vector, the amount of virus administered is in the range of from 10⁶ to 10¹⁰ pfu, preferably from

7 0 8

10 to 10 pfu, more preferably about 10 pfu for adenoviral vectors. When injected, typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. When the polynucleotide is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg.

Thus, a modulatory agent of the invention may be formulated and administered according to any of the above means. The routes of administration and dosages described above are intended only as a guide since a skilled physician will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.

The following Examples illustrate the invention. Examples

Materials and Methods

Unless indicated otherwise, the methods used are standard biochemistry and molecular biology techniques. Examples of suitable methodology textbooks include Sambrook et al; Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al; Current Protocols in Molecular Biology (1995), John Wiley & Sons Inc.

Example 1. Detection of DDAHI mRNA in adult rat brain by in situ hybridization

For tissue preparation, adult rat brain was dissected, rapidly frozen, sectioned on a cryostat (12-15 μm), thaw mounted onto subbed (gelatin-chrom alum) slides and fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS).

To prepare a probe, a 429 bp PCR fragment, derived by PCR differential display and containing 3 ' non-coding sequences of rat DDAHI cDNA (corresponding to position 2670-3008, Genbank accession number: D86041) was subcloned into the pCR-Script (Stratagene) described as CB15-1. ³⁵SUTP-labeled antisense (CB15-1 linearized with BamHI as template) and sense (CB15-1 linearized with Notl as template) transcripts were generated using T3 or T7 RNA polymerase. In all experiments, sense strand control probe was included as a measure of nonspecific background.

For the in situ hybridization, coronal sections of adult brain were first acetylated to reduce non-specific probe binding by immersion for 10 min in fresh 0.1 M triethanolamine and 25 mM acetic anhydride. After rinsing the sections in 2xSSC and dehydration, they were each covered with 40 μl of hybridization solution, consisting of 4xSSC, 50% Denhardt's, 1% SDS, 250 μg/ml yeast tRNA, 25 μg/ml polyadenylic acid, 25 μg/ml polycytidylic acid, 0.1 M DTT, 10% dextran sulfate and 0.2 ng/μl radiolabeled RNA. A glass coverslip was placed on top of each section and the slides were hybridized at 50°C in a moist chamber. After 3 h, slides were immersed in 4x SSC, 20mM DTT to soak off coverslips and rinsed in 4xSSC. Slides were digested with RNase solution (20 μg/ml RNase A in 0.5 M NaCl, 0.01 M Tris, 1 mM EDTA pH8.0) for 30 min at 37°C, washed in 2xSSC, 20 mM β- mercaptoethanol at room temperature for 2 h, and washed in 0.1 x SSC for 1 h at 60°C. Slides were dehydrated, dried and exposed to X-ray film. The in situ hybridisation analysis showed that DDAHI mRNA is prominently expressed in thalamus and is detectable in cortex (Figure 1). In the cortex, DDAHI mRNA signal was detected in the deep layer. In thalamic nuclei, strongest DDAHI mRNA signal was detected in the anterior dorsal thalamic nucleus and the central medial of thalamic nucleus. A strong signal was also observed in the ventroanterior thalamic nucleus and lateral subnuclei of the thalamus. DDAHI mRNA signal was also expressed strongly in the choroid plexus located in the walls of lateral or third ventricles.

Example 2. Regulation of DDAHI mRNA expression during CFA induced inflammation To induce inflammation male Sprague-Dawley rats (200g) were deeply anaesthetised under halothane (2%) and intraplantar CFA (Complete Freund's Adjuvant 100 μl) was injected into the rat left hindpaw. Rats were killed and dissected at 6 h and 12h after CFA-induced inflammation. Total RNA from brain areas such as cortex, thalamus, midbrain and cerebellum was extracted from individual animals including control rats.

Ribonuclease protection assays were carried out on total RNA extracts using the III kit (Ambion) to detect mRNA of DDAHI. The template for a DDAHI radiolabelled riboprobe was generated by linearized CB15-1 with Sphl using T3 RNA polymerase. ³²P-labeled DDAHI riboprobe, as used for in situ hybridisation (see above), was mixed to either tRNA (5 μg as negative control) or total RNA (5 μg) from cerebellum, thalamus, midbrain or cortex and incubated overnight at 42°C. Approximately the same amount of total RNA was used for each reaction according to protection assays with β-actin riboprobe. After hybridization, reaction solutions were treated with RNase mixture for 30 min at 37°C and then RNase was inactivated. Protected RNA pellets were separated on denaturing 4% acrylamide gel, gel was dried and exposed to X-ray film.

The expression of DDAHI mRNA in thalamus was found to be down- regulated 6 and 12 h after induction of inflammatory pain by CFA administration (Figure 2). Total RNA was extracted from rat tissues of a control animal (lane A), and animals 6h (lane B) and 12h (lane C) after CFA-induced inflammation. Ribonuclease protection analyses were performed with a DDAHI riboprobe that was hybridised to either tRNA (lane T) or total RNA from cerebellum, thalamus, midbrain or cortex. Approximately the same amount of RNA was used for each reaction acording to protection assays with a β-actin riboprobe (data not shown). Consistent with the in situ hybridisations, DDAHI mRNA expression was low in neocortex. DDAHI mRNA expression was also observed to be low in cerebellum. In midbrain DDAHI mRNA expression was observed to be moderate. As seen in in situ hybridisations, DDAHI was expressed highly in thalamus, and DDAHI decreased substantially after the induction of inflammatory pain. This down- regulation thus appears to be specific to the thalamus because it was not observed in cortex and cerebellum.

SEQUENCE LISTING

<110> UNIVERSITY COLLEGE LONDON

<120> PAIN TREATMENT

<130> N.84927A

<150> GB 02112977 <151> 2002-05-16

<160> 12

<170> Patentln Ver. 3.0

<210> 1 <211> 858 <212> DNA <213> H. Sapiens

<220>

<221> CDS

<222> (1).. (858)

<400> 1 atg gcc ggc etc ggc cac ccc tec gcc ttc ggc egg gcc ace cac gcc 48

Met Ala Gly Leu Gly His Pro Ser Ala Phe Gly Arg Ala Thr His Ala 1 5 10 15 gtg gtg egg gcg eta ccc gag teg etc tgc cag cac gcg ctg aga age 96 Val Val Arg Ala Leu Pro Glu Ser Leu Cys Gin His Ala Leu Arg Ser 20 25 30 gcc aag ggc gag gag gtg gac gtc gcc cgc gcg gaa egg cag cac cag 144 Ala Lys Gly Glu Glu Val Asp Val Ala Arg Ala Glu Arg Gin His Gin 35 40 45 etc tac gtg ggc gtg ctg ggc age aag ctg ggg ctg cag gtg gtg gag 192 Leu Tyr Val Gly Val Leu Gly Ser Lys Leu Gly Leu Gin Val Val Glu 50 55 60 ctg ccg gcc gac gag age ctt ccg gac tgc gtc ttc gtg gag gac gtg 240 Leu Pro Ala Asp Glu Ser Leu Pro Asp Cys Val Phe Val Glu Asp Val 65 70 75 80 gcc gtg gtg tgc gag gag acg gcc etc ate ace cga ccc ggg gcg ccg 288 Ala Val Val Cys Glu Glu Thr Ala Leu He Thr Arg Pro Gly Ala Pro 85 90 95 age egg agg aag gag gtt gac atg atg aaa gaa gca tta gaa aaa ctt 336 Ser Arg Arg Lys Glu Val Asp Met Met Lys Glu Ala Leu Glu Lys Leu 100 105 110 cag etc aat ata gta gag atg aaa gat gaa aat gca act tta gat ggc 384 Gin Leu Asn He Val Glu Met Lys Asp Glu Asn Ala Thr Leu Asp Gly 115 120 125 gga gat gtt tta ttc aea ggc aga gaa ttt ttt gtg ggc ctt tec aaa 432 Gly Asp Val Leu Phe Thr Gly Arg Glu Phe Phe Val Gly Leu Ser Lys 130 135 140 agg aea aat caa cga ggt get gaa ate ttg get gat act ttt aag gac 480 Arg Thr Asn Gin Arg Gly Ala Glu lie Leu Ala Asp Thr Phe Lys Asp 145 150 155 160 tat gca gtc tec aea gtg cca gtg gca gat ggg ttg cat ttg aag agt 528 Tyr Ala Val Ser Thr Val Pro Val Ala Asp Gly Leu His Leu Lys Ser 165 170 175 ttc tgc age atg get ggg ect aac ctg ate gca att ggg tct agt gaa 576 Phe Cys Ser Met Ala Gly Pro Asn Leu He Ala He Gly Ser Ser Glu 180 185 190 tct gca cag aag gcc ctt aag ate atg caa cag atg agt gac cac cgc 624 Ser Ala Gin Lys Ala Leu Lys He Met Gin Gin Met Ser Asp His Arg 195 200 205 tac gac aaa etc act gtg ect gat gac ata gca gca aac tgt ata tat 672 Tyr Asp Lys Leu Thr Val Pro Asp Asp He Ala Ala Asn Cys He Tyr 210 215 220 eta aat ate ccc aac aaa ggg cac gtc ttg ctg cac cga ace ccg gaa 720 Leu Asn He Pro Asn Lys Gly His Val Leu Leu His Arg Thr Pro Glu 225 230 235 240 gag tat cca gaa agt gca aag gtt tat gag aaa ctg aag gac cat atg 768 Glu Tyr Pro Glu Ser Ala Lys Val Tyr Glu Lys Leu Lys Asp His Met 245 250 255 ctg ate ccc gtg age atg tct gaa ctg gaa aag gtg gat ggg ctg etc 816 Leu He Pro Val Ser Met Ser Glu Leu Glu Lys Val Asp Gly Leu Leu 260 265 270 ace tgc tgc tea gtt tta att aac aag aag gta gac tec tga 858 Thr Cys Cys Ser Val Leu He Asn Lys Lys Val Asp Ser 275 280 285

<210> 2 <211> 285 <212> PRT <213> H. Sapiens

<400> 2

Met Al a Gly Leu Gly Hi s Pro Ser Al a Phe Gly Arg Al a Thr Hi s Al a 1 5 10 15

Val Val Arg Ala Leu Pro Gl u Ser Leu Cys Gi n Hi s Al a Leu Arg Ser 20 25 30 Ala Lys Gly Glu Glu Val Asp Val Ala Arg Ala Glu Arg Gin His Gin 35 40 45

Leu Tyr Val Gly Val Leu Gly Ser Lys Leu Gly Leu Gin Val Val Glu 50 55 60

Leu Pro Ala Asp Glu Ser Leu Pro Asp Cys Val Phe Val Glu Asp Val 65 70 75 80

Ala Val Val Cys Glu Glu Thr Ala Leu He Thr Arg Pro Gly Ala Pro 85 90 95

Ser Arg Arg Lys Glu Val Asp Met Met Lys Glu Ala Leu Glu Lys Leu 100 105 110

Gin Leu Asn He Val Glu Met Lys Asp Glu Asn Ala Thr Leu Asp Gly 115 120 125

Gly Asp Val Leu Phe Thr Gly Arg Glu Phe Phe Val Gly Leu Ser Lys 130 135 140

Arg Thr Asn Gin Arg Gly Ala Glu He Leu Ala Asp Thr Phe Lys Asp 145 150 155 160

Tyr Ala Val Ser Thr Val Pro Val Ala Asp Gly Leu His Leu Lys Ser 165 170 175 ^

Phe Cys Ser Met Ala Gly Pro Asn Leu He Ala He Gly Ser Ser Glu

180 185 190

Ser Ala Gin Lys Ala Leu Lys He Met Gin Gin Met Ser Asp His Arg

195 200 205

Tyr Asp Lys Leu Thr Val Pro Asp Asp He Ala Ala Asn Cys He Tyr

210 215 220

Leu Asn He Pro Asn Lys Gly His Val Leu Leu His Arg Thr Pro Glu

225 230 235 240

Glu Tyr Pro Glu Ser Ala Lys Val Tyr Glu Lys Leu Lys Asp His Met

245 250 255

Leu He Pro Val Ser Met Ser Glu Leu Glu Lys Val Asp Gly Leu Leu

260 265 . 270

Thr Cys Cys Ser Val Leu He Asn Lys Lys Val Asp Ser 275 280 285

<210> 3 <211> 858 <212> DNA <213> H sapiens

<220>

<221> CDS

<222> (1) (858)

<400> 3 atg ggg acg ccg ggg gag ggg ctg ggc cgc tgc tec cat gcc ctg ate 48

Met Gly Thr Pro Gly Gl u Gly Leu Gly Arg Cys Ser Hi s Al a Leu H e 1 5 10 15 egg gga gtc cca gag age ctg gcg teg ggg gaa ggt gcg ggg get ggc 96 Arg Gly Val Pro Glu Ser Leu Ala Ser Gly Glu Gly Ala Gly Ala Gly 20 25 30 ctt ccc get ctg gat ctg gcc aaa get caa agg gag cac ggg gtg ctg 144 Leu Pro Ala Leu Asp Leu Ala Lys Ala Gin Arg Glu His Gly Val Leu 35 40 45 gga ggt aaa ctg agg caa cga ctg ggg eta cag ctg eta gaa ctg cca 192 Gly Gly Lys Leu Arg Gin Arg Leu Gly Leu Gin Leu Leu Glu Leu Pro 50 55 60 ect gag gag tea ttg ccg ctg gga ccg ctg ctt ggc gac acg gcc gtg 240

Pro Glu Glu Ser Leu Pro Leu Gly Pro Leu Leu Gly Asp Thr Ala Val

65 70 75 80 ate caa ggg gac acg gcc eta ate acg egg ccc tgg age ccc get cgt

He Gin Gly Asp Thr Ala Leu He Thr Arg Pro Trp Ser Pro Ala Arg

85 90 95 agg cca gag gtc gat gga gtc cgc aaa gcc ctg caa gac ctg ggg etc 336

Arg Pro Glu Val Asp Gly Val Arg Lys Ala Leu Gin Asp Leu Gly Leu

100 105 110 cga att gtg gaa ata gga gac gag aac gcg acg ctg gat ggc act gac 384 Arg He Val Glu He Gly Asp Glu Asn Ala Thr Leu Asp Gly Thr Asp 115 120 125 gtt etc ttc ace ggc egg gag ttt ttc gta ggc etc tec aaa tgg ace 432 Val Leu Phe Thr Gly Arg Glu Phe Phe Val Gly Leu Ser Lys Trp Thr 130 135 140 aat cac cga gga get gag ate gtg gcg gac acg ttc egg gac ttc gcc 480 Asn His Arg Gly Ala Glu He Val Ala Asp Thr Phe Arg Asp Phe Ala 145 150 155 160 gtc tec act gtg cca gtc teg ggt ccc tec cac ctg cgc ggt etc tgc 528

Val Ser Thr Val Pro Val Ser Gly Pro Ser His Leu Arg Gly Leu Cys 165 170 175 ggc atg ggg gga ect cgc act gtt gtg gca ggc age age gac get gcc 576

Gly Met Gly Gly Pro Arg Thr Val Val Ala Gly Ser Ser Asp Ala Ala 180 185 190 caa aag get gtc egg gca atg gca gtg ctg aea gat cac cca tat gcc 624 Gin Lys Ala Val Arg Ala Met Ala Val Leu Thr Asp His Pro Tyr Ala 195 200 205 tec ctg ace etc cca gat gac gca get get gac tgt etc ttt ctt cgt 672 Ser Leu Thr Leu Pro Asp Asp Ala Ala Ala Asp Cys Leu Phe Leu Arg 210 215 220 ect ggg ttg ect ggt gtg ccc ect ttc etc ctg cac cgt gga ggt ggg 720 Pro Gly Leu Pro Gly Val Pro Pro Phe Leu Leu His Arg Gly Gly Gly 225 230 235 240 gat ctg ccc aac age cag gag gca ctg cag aag etc tct gat gtc ace 768

Asp Leu Pro Asn Ser Gin Glu Ala Leu Gin Lys Leu Ser Asp Val Thr

245 250 255 ctg gta ect gtg tec tgc tea gaa ctg gag aaa get ggc gcc ggg etc 816

Leu Val Pro Val Ser Cys Ser Glu Leu Glu Lys Ala Gly Ala Gly Leu

260 265 270 age tec etc tgc ttg gtg etc age aea cgc ccc cac age tga 858 Ser Ser Leu Cys Leu Val Leu Ser Thr Arg Pro His Ser 275 280 285

<210> 4 <211> 285 <212> PRT <213> H sapiens

<400> 4

Met Gly Thr Pro Gly Glu Gly Leu Gly Arg Cys Ser His Ala Leu He 1 5 10 15

Arg Gly Val Pro Glu Ser Leu Ala Ser Gly Glu Gly Ala Gly Ala Gly 20 25 30

Leu Pro Ala Leu Asp Leu Ala Lys Ala Gin Arg Glu His Gly Val Leu 35 40 45

Gly Gly Lys Leu Arg Gin Arg Leu Gly Leu Gin Leu Leu Glu Leu Pro 50 55 60

Pro Glu Glu Ser Leu Pro Leu Gly Pro Leu Leu Gly Asp Thr Ala Val 65 70 75 80

He Gin Gly Asp Thr Ala Leu He Thr Arg Pro Trp Ser Pro Ala Arg 85 90 95

Arg Pro Glu Val Asp Gly Val Arg Lys Ala Leu Gin Asp Leu Gly Leu 100 105 110

Arg He Val Glu He Gly Asp Glu Asn Ala Thr Leu Asp Gly Thr Asp 115 120 125 Val Leu Phe Thr Gly Arg Glu Phe Phe Val Gly Leu Ser Lys Trp Thr

130 135 140

Asn His Arg Gly Ala Glu He Val Ala Asp Thr Phe Arg Asp Phe Ala

145 150 155 160

Val Ser Thr Val Pro Val Ser Gly Pro Ser His Leu Arg Gly Leu Cys 165 170 175

Gly Met Gly Gly Pro Arg Thr Val Val Ala Gly Ser Ser Asp Ala Ala

180 185 190

Gin Lys Ala Val Arg Ala Met Ala Val Leu Thr Asp His Pro Tyr Ala

195 200 205

Ser Leu Thr Leu Pro Asp Asp Ala Ala Ala Asp Cys Leu Phe Leu Arg

210 215 220

Pro Gly Leu Pro Gly Val Pro Pro Phe Leu Leu His Arg Gly Gly Gly

225 230 235 240

Asp Leu Pro Asn Ser Gin Glu Ala Leu Gin Lys Leu Ser Asp Val Thr 245 250 255

Leu Val Pro Val Ser Cys Ser Glu Leu Glu Lys Ala Gly Ala Gly Leu 260 265 270

Ser Ser Leu Cys Leu Val Leu Ser Thr Arg Pro His Ser 275 280 285

<210> 5

<211> 777

<212> DNA

<213> S coelicolor

<220>

<221> CDS

<222> (1) (777)

<400> 5 gtg ccc age aag aag gcc ctg gtc cgc cgc ccc age ccc agg etc gcc 48

Val Pro Ser Lys Lys Ala Leu Val Arg Arg Pro Ser Pro Arg Leu Ala 1 5 10 15 gaa gga ctg gtg aea cac gtc gag egg gag cag gtc gat cac ggc ctg 96 Glu Gly Leu Val Thr His Val Glu Arg Glu Gin Val Asp His Gly Leu 20 25 30 gcc etc gaa cag tgg gac gcc tac gtc gag gcc etc gga gca cac ggc 144 Ala Leu Glu Gin Trp Asp Ala Tyr Val Glu Ala Leu Gly Ala His Gly 35 40 45 tgg gag act ctg gag gtg gac ccg gcc gag tac tgt ccg gac teg gtc 192 Trp Glu Thr Leu Glu Val Asp Pro Ala Glu Tyr Cys Pro Asp Ser Val 50 55 60 ttc gtc gag gac gcc gtc gtc gtg ttc cgc aac gtc gcg ctg ate acg 240 Phe Val Glu Asp Ala Val Val Val Phe Arg Asn Val Ala Leu He Thr 65 70 75 80 egg ccc ggc gcc gag teg egg cgc gcg gag acg gcc ggc gtc gag gag 288 Arg Pro Gly Ala Glu Ser Arg Arg Ala Glu Thr Ala Gly Val Glu Glu

85 90 95 gcc. gtg gcc gg etc ggc tgc teg gtg aac tgg gtg tgg gag ccg ggc 336 Ala Val Ala Arg Leu Gly Cys Ser Val Asn Trp Val Trp Glu Pro Gly 100 105 110 ace ctg gac ggc ggc gac gtc ctg aag ate ggc gac acg ate tac gtg 384

Thr Leu Asp Gly Gly Asp Val Leu Lys He Gly Asp Thr He T r Val

^■ 115 120 125 gga cgc ggc ggc egg ace aac gcg gcc ggt gtc cag cag ttg egg gcg 432

Gly Arg Gly Gly Arg Thr Asn Ala Ala Gly Val Gin Gin Leu Arg Ala

130 135 140 gcg ttc gag ccg ctg ggc gcc egg gtc gtc gcc gtg ccc gtg age aag 480 Ala Phe Glu Pro Leu Gly Ala Arg Val Val Ala Val Pro Val Ser Lys 145 150 155 • 160 gtg ctg cat ctg aag teg gcg gtc ace gcg ctg ccg gac ggg acg gtg 528 Val Leu His Leu Lys Ser Ala Val Thr Ala Leu Pro Asp Gly Thr Val 165 170 175 ate ggg cac ate ccg ctg acg gac gtg ccc teg ctg ttc ccc cgt ttc 576 He Gly His He Pro Leu Thr Asp Val Pro Ser Leu Phe Pro Arg Phe 180 185 190 ctg ccg gtg ccg gag gag teg ggg gcg cac gtg gtg ctg etc ggc ggg 624 Leu Pro Val Pro Glu Glu Ser Gly Ala His Val Val Leu Leu Gly Gly 195 200 205 age agg ctg ctg atg gcg gcg age gcg ccc aag acg gcg gag ctg etc 672

Ser Arg Leu Leu Met Ala Ala Ser Ala Pro Lys Thr Ala Glu Leu Leu

210 215 220 gcc gat etc ggt cac gag ccg gtg etc gtc gac ate ggg gag ttc gag 720 Ala Asp Leu Gly His Glu Pro Val Leu Val Asp He Gly Glu Phe Glu 225 230 235 240 aag ctg gag ggc tgt gtg acg tgc etc teg gtc agg ctg cgc gag ctg 768 Lys Leu Glu Gly Cys Val Thr Cys Leu Ser Val Arg Leu Arg Glu Leu 245 250 255 tac gac tga 777 Tyr Asp

<210> 6 <211> 258 <212> PRT

<213> S. coelicolor

<400> 6

Val Pro Ser Lys Lys Ala Leu Val Arg Arg Pro Ser Pro Arg Leu Ala 1 5 10 15

Glu Gly Leu Val Thr His Val Glu Arg Glu Gin Val Asp His Gly Leu 20 25 30

Ala Leu Glu Gin Trp Asp Ala Tyr Val Glu Ala Leu Gly Ala His Gly 35 40 45

Trp Glu Thr Leu Glu Val Asp Pro Ala Glu Tyr Cys Pro Asp Ser Val 50 55 60

Phe Val Glu Asp Ala Val Val Val Phe Arg Asn Val Ala Leu He Thr 65 70 75 80

Arg Pro Gly Ala Glu Ser Arg Arg Ala Glu Thr Ala Gly Val Glu Glu 85 90 95

Ala Val Ala Arg Leu Gly Cys Ser Val Asn Trp Val Trp Glu Pro Gly 100 105 110

Thr Leu Asp Gly Gly Asp Val Leu Lys He Gly Asp Thr He Tyr Val 115 120 125

Gly Arg Gly Gly Arg Thr Asn Ala Ala Gly Val Gin Gin Leu Arg Ala 130 135 140

Ala Phe Glu Pro Leu Gly Ala Arg Val Val Ala Val Pro Val Ser Lys 145 150 155 160

Val Leu His Leu Lys Ser Ala Val Thr Ala Leu Pro Asp Gly Thr Val 165 170 175

He Gly His He Pro Leu Thr Asp Val Pro Ser Leu Phe Pro Arg Phe 180 185 190

Leu Pro Val Pro Glu Glu Ser Gly Ala His Val Val Leu Leu Gly Gly 195 200 205

Ser Arg Leu Leu Met Ala Ala Ser Ala Pro Lys Thr Ala Glu Leu Leu 210 215 220

Ala Asp Leu Gly His Glu Pro Val Leu Val Asp He Gly Glu Phe Glu 225 230 235 240

Lys Leu Glu Gly Cys Val Thr Cys Leu Ser Val Arg Leu Arg Glu Leu 245 250 255

Tyr Asp <210> 7

<211> 765

<212> DNA

<213> P. aeruginosa

<220>

<221> CDS

<222> (1). (765)

<400> 7 atg ttc aag cac ate ate get cgc acg ccc gcc cgc age ctg gtc gac 48

Met Phe Lys His He He Ala Arg Thr Pro Ala Arg Ser Leu Val Asp

1 5 10 15 ggc ctg ace tec age cac etc ggc aag ccg gac tac gcc aag gcc ctg 96 Gly Leu Thr Ser Ser His Leu Gly Lys Pro Asp Tyr Ala Lys Ala Leu 20 25 30 gag cag cac aac gcc tac ate cgc gcc ttg cag ace tgc gac gtg gac 144 Glu Gin His Asn Ala Tyr He Arg Ala Leu Gin Thr Cys Asp Val Asp 35 40 45 ate ace ctg ctg ccg ccg gac gaa cgc ttc ccc gac teg gtg ttc gtc 192 He Thr Leu Leu Pro Pro Asp Glu Arg Phe Pro Asp Ser Val Phe Val 50 55 60 gag gac ccg gtg etc tgc ace teg cgc tgc gcc ate ate ace cgc ccc 240 Glu Asp Pro Val Leu Cys Thr Ser Arg Cys Ala He He Thr Arg Pro 65 70 75 80 ggc gcc gaa teg egg cgc ggc gag ace gag ate ate gag gaa ace gtg 288 Gly Ala Glu Ser Arg Arg Gly Glu Thr Glu He He Glu Glu Thr Val 85 90 95 cag cgc ttc tat ccg ggc aag gtc gag cgc ate gag gca ccc ggc acg 336 Gin Arg Phe Tyr Pro Gly Lys Val Glu Arg He Glu Ala Pro Gly Thr 100 105 110 gtg gaa gcc ggc gac ate atg atg gtc ggc gac cac ttc tac ate ggc 384 Val Glu Ala Gly Asp He Met Met Val Gly Asp His Phe Tyr He Gly 115 120 125 gaa teg gcc cgc ace aac gcc gag ggc gcc egg cag atg ate gcg ate 432 Glu Ser Ala Arg Thr Asn Ala Glu Gly Ala Arg Gin Met He Ala He 130 135 140 ctg gag aaa cat ggc etc age ggc teg gtg gtg cgc ctg gaa aag gtc 480 Leu Glu Lys His Gly Leu Ser Gly Ser Val Val Arg Leu Glu Lys Val 145 150 155 ^' 160 ctg cac ctg aag ace ggg etc gcc tac ctg gaa cac aac aac ctg ctg 528 Leu His Leu Lys Thr Gly Leu Ala Tyr Leu Glu His Asn Asn Leu Leu 165 170 175 gcc gcc ggc gag ttc gtc age aag ccg gag ttc cag gac ttc aac ate 576 Ala Ala Gly Glu Phe Val Ser Lys Pro Glu Phe Gin Asp Phe Asn He 180 185 190 ate gag ate ccc gaa gag gag tec tac gcc gcc aac tgc ate tgg gtc 624 He Glu He Pro Glu Glu Glu Ser Tyr Ala Ala Asn Cys He Trp Val 195 200 205 aac gaa agg gtg ate atg ccc gcc ggc tat ccc egg ace cgc gag aag 672 Asn Glu Arg Val He Met Pro Ala Gly Tyr Pro Arg Thr Arg Glu Lys 210 215 220 ate gcc cgc etc ggc tac egg gtg ate gag gtg gac ace tec gaa tat 720 He Ala Arg Leu Gly Tyr Arg Val He Glu Val Asp Thr Ser Glu Tyr 225 230 235 240 cgc aag ate gac ggc ggc gtc agt tgc atg teg ctg cgc ttc tga 765 Arg Lys He Asp Gly Gly Val Ser Cys Met Ser Leu Arg Phe

245 250 255

<210> 8

<211> 254

<212> PRT

<213> P aeruginosa

<400> 8

Met Phe Lys His He He Ala Arg Thr Pro Ala Arg Ser Leu Val Asp 1 5 10 15

Gly Leu Thr Ser Ser His Leu Gly Lys Pro Asp Tyr Ala Lys Ala Leu 20 25 30

Glu Gin His Asn Ala Tyr He Arg Ala Leu Gin Thr Cys Asp Val Asp 35 40 45

He Thr Leu Leu Pro Pro Asp Glu Arg Phe Pro Asp Ser Val Phe Val 50 55 60

Glu Asp Pro Val Leu Cys Thr Ser Arg Cys Ala He He Thr Arg Pro 65 70 75 80

Gly Ala Glu Ser Arg Arg Gly Glu Thr Glu He He Glu Glu Thr Val 85 90 95

Gin Arg Phe Tyr Pro Gly Lys Val Glu Arg He Glu Ala Pro Gly Thr 100 105 110

Val Glu Ala Gly Asp He Met Met Val Gly Asp His Phe Tyr He Gly 115 120 125

Glu Ser Ala Arg Thr Asn Ala Glu Gly Ala Arg Gin Met He Ala He 130 135 140

Leu Glu Lys His Gly Leu Ser Gly Ser Val Val Arg Leu Glu Lys Val 145 150 155 160

Leu His Leu Lys Thr Gly Leu Ala Tyr Leu Glu His Asn Asn Leu Leu 165 170 175

Ala Ala Gly Glu Phe Val Ser Lys Pro Glu Phe Gin Asp Phe Asn He 180 185 190

He Glu He Pro Glu Glu Glu Ser Tyr Ala Ala Asn Cys He Trp Val 195 200 205

Asn Glu Arg Val He Met Pro Ala Gly Tyr Pro Arg Thr Arg Glu Lys 210 215 220

He Ala Arg Leu Gly Tyr Arg Val He Glu Val Asp Thr Ser Glu Tyr 225 230 235 240

Arg Lys He Asp Gly Gly Val Ser Cys Met Ser Leu Arg Phe 245 250

<210> 9

<211> 1257

<212> DNA

<213> P aeruginosa

<220>

<221> CDS

<222> (1) (1257)

<400> 9 atg age acg gaa aaa ace aaa ctt ggc gtc cac tec gaa gcc ggc aaa 48

Met Ser Thr Glu Lys Thr Lys Leu Gly Val His Ser Glu Ala Gly Lys 1 5 10 15 ctg cgc aaa gtg atg gtc tgc teg ccc gga etc gcc cac cag cgc ctg 96 Leu Arg Lys Val Met Val Cys Ser Pro Gly Leu Ala His Gin Arg Leu 20 25 30 ace ccg age aac tgc gac gag ttg ctg ttc gac gac gtg ate tgg gtg 144 Thr Pro Ser Asn Cys Asp Glu Leu Leu Phe Asp Asp Val He Trp Val 35 40 45 aac cag gcc aag cgc gac cac ttc gac ttc gtc ace aag atg cgc gag 192 Asn Gin Ala Lys Arg Asp His Phe Asp Phe Val Thr Lys Met Arg Glu 50 55 60 cgc ggc ate gac gtc etc gag atg cac aat ctg ctg ace gag ace ate 240 Arg Gly He Asp Val Leu Glu Met His Asn Leu Leu Thr Glu Thr He 65 70 75 80 cag aac ccg gaa gcg ctg aag tgg ate etc gat cgc aag ate ace gcc 288 Gin Asn Pro Glu Ala Leu Lys Trp He Leu Asp Arg Lys He Thr Ala 85 90 95 gac age gtc ggc ctg ggc ctg ace age gag ctg cgc tec tgg ctg gag 336 Asp Ser Val Gly Leu Gly Leu Thr Ser Glu Leu Arg Ser Trp Leu Glu 100 105 110 age ctg gag ccg cgc aag ctg gcc gag tac ctg ate ggc ggc gtc gcc 384 Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu He Gly Gly Val Ala 115 120 125 get gac gac ctg ccc gcc age gaa ggc gcc aac ate etc aag atg tac 432 Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn He Leu Lys Met Tyr 130 135 140 cgc gag tac ctg ggc cat tec age ttc ctg ctg ccg ccg ttg ccg aac 480 Arg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu Pro Pro Leu Pro Asn 145 150 155 160 ace cag ttc ace cgc gac ace act tgc tgg ate tac ggc ggc gtg ace 528 Thr Gin Phe Thr Arg Asp Thr Thr Cys Trp He Tyr Gly Gly Val Thr 165 170 175 ctg aac ccg atg tac tgg ccg gcg cga cga cag gaa ace ctg ctg ace 576 Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gin Glu Thr Leu Leu Thr 180 185 190 ace gcc ate tac aag ttc cac ccc gag ttc gcc aac gcc gag ttc gag 624 Thr Ala He Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu 195 200 205 ate tgg tac ggc gac ccg gac aag gac cac ggc tec teg ace ctg gaa 672 He Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu 210 215 220 ggc ggc gac gtg atg ccg ate ggc aac ggc gtg gtc ctg ate ggc atg 720 Gly Gly Asp Val Met Pro He Gly Asn Gly Val Val Leu He Gly Met 225 230 235 240 ggc gag cgc tec teg cgc cag gcc ate ggt cag gtc gcc cag teg ctg 768 Gly Glu Arg Ser Ser Arg Gin Ala He Gly Gin Val Ala Gin Ser Leu 245 250 255 ttc gcc aag ggc gcc gcc gag egg gtg ate gtc gcc ggc ctg ccg aag 816 Phe Ala Lys Gly Ala Ala Glu Arg Val He Val Ala Gly Leu Pro Lys 260 265 270 tec cgc gcc gcg atg cac ctg gac ace gtg ttc age ttc tgc gac cgc 864

Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285 gac ctg gtc acg gtc ttc ccg gaa gtg gtc aag gaa ate gtg ccc ttc 912

Asp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu He Val Pro Phe

290 295 300 age ctg cgc ccc gat ccg age age ccc tac ggc atg aac ate cgc cgc 960 Ser Leu Arg Pro Asp Pro Ser Ser Pro Tyr Gly Met Asn He Arg Arg 305 310 315 320 gag gag aaa ace ttc etc gaa gtg gtc gcc gaa tec etc ggc ctg aag 1008

Glu Glu Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu Lys

325 330 335 aaa ctg cgc gtg gtc gag ace ggc ggc aac age ttc gcc gcc gag cgc 1056

Lys Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg

340 345 350 gag caa tgg gac gac ggt aac aac gtg gtc tgc ctg gag ccg ggc gtg 1104 Glu Gin Trp Asp Asp Gly Asn Asn Val Val Cys Leu Glu Pro Gly Val 355 360 365 gtg gtc ggc tac gac cgc aac ace tac ace aac ace ctg ctg cgc aag 1152 Val Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys 370 375 380 gcc ggc gtc gag gtc ate ace ate age gcc age gaa ctg ggt cgc ggt 1200

Ala Gly Val Glu Val He Thr He Ser Ala Ser Glu Leu Gly Arg Gly

385 390 395 400 cgc ggc ggc ggc cac tgc atg ace tgc ccg ate gtc cgc gac ccg ate 1248

Arg Gly Gly Gly His Cys Met Thr Cys Pro He Val Arg Asp Pro He

405 410 415 gac tac tga 1257 Asp Tyr

<210> 10

<211> 418

<212> PRT

<213> P aeruginosa

<400> 10

Met Ser Thr Gl u Lys Thr Lys Leu Gly Val Hi s Ser Gl u Al a Gly Lys 1 5 10 15

Leu Arg Lys Val Met Val Cys Ser Pro Gly Leu Al a Hi s Gi n Arg Leu 20 25 30

Thr Pro Ser Asn Cys Asp Gl u Leu Leu Phe Asp Asp Val H e Trp Val 35 40 45

Asn Gi n Al a Lys Arg Asp Hi s Phe Asp Phe Val Thr Lys Met Arg Gl u 50 55 60

Arg Gly H e Asp Val Leu Gl u Met Hi s Asn Leu Leu Thr Gl u Thr H e 65 70 75 80

Gi n Asn Pro Gl u Al a Leu Lys Trp H e Leu Asp Arg Lys H e Thr Al a 85 90 95

Asp Ser Val Gly Leu Gly Leu Thr Ser Gl u Leu Arg Ser Trp Leu Gl u 100 105 110

Ser Leu Glu Pro Arg Lys Leu Ala Glu Tyr Leu He Gly Gly Val Ala 115 120 125

Ala Asp Asp Leu Pro Ala Ser Glu Gly Ala Asn He Leu Lys Met Tyr 130 135 140

Arg Glu Tyr Leu Gly His Ser Ser Phe Leu Leu Pro Pro Leu Pro Asn 145 150 155 160

Thr Gin Phe Thr Arg Asp Thr Thr Cys Trp He Tyr Gly Gly Val Thr 165 170 175

Leu Asn Pro Met Tyr Trp Pro Ala Arg Arg Gin Glu Thr Leu Leu Thr 180 ^" 185 190

Thr Ala He Tyr Lys Phe His Pro Glu Phe Ala Asn Ala Glu Phe Glu 195 200 205

He Trp Tyr Gly Asp Pro Asp Lys Asp His Gly Ser Ser Thr Leu Glu 210 215 220

Gly Gly Asp Val Met Pro -He Gly Asn Gly Val Val Leu He Gly Met 225 230 235 240

Gly Glu Arg Ser Ser Arg Gin Ala He Gly Gin Val Ala Gin Ser Leu 245 250 255

Phe Ala Lys Gly Ala Ala Glu Arg Val He Val Ala Gly Leu Pro Lys 260 265 270

Ser Arg Ala Ala Met His Leu Asp Thr Val Phe Ser Phe Cys Asp Arg 275 280 285

Asp Leu Val Thr Val Phe Pro Glu Val Val Lys Glu He Val Pro Phe 290 295 300

Ser Leu Arg Pro Asp Pro Ser Ser Pro Tyr Gly Met Asn He Arg Arg 305 310 315 320

Glu Glu Lys Thr Phe Leu Glu Val Val Ala Glu Ser Leu Gly Leu Lys 325 330 335

Lys Leu Arg Val Val Glu Thr Gly Gly Asn Ser Phe Ala Ala Glu Arg 340 345 350

Glu Gin Trp Asp Asp Gly Asn Asn Val Val Cys Leu Glu Pro Gly Val 355 360 365

Val Val Gly Tyr Asp Arg Asn Thr Tyr Thr Asn Thr Leu Leu Arg Lys 370 375 380

Ala Gly Val Glu Val He Thr He Ser Ala Ser Glu Leu Gly Arg Gly 385 390 395 400 Arg Gly Gly Gly His Cys Met Thr Cys Pro He Val Arg Asp Pro He 405 410 415

Asp Tyr

<210> 11

<211> 1014

<212> DNA

<213> M tuberculosis

<220>

<221> CDS

<222> (69) (986)

<400> 11 atgtatcaat ggaaaatacg caacgaccat cgtttgattg tgaaatcaga gccaaatatc 60 gttggttt atg acg gat tec tac gtc get get gcc cgt eta ggg tea ect 110 Met Thr Asp Ser Tyr Val Ala Ala Ala Arg Leu Gly Ser Pro

1 5 10 gca cgc cgc ace ccc egg acg egg egg tat gca atg ace ccg ccg gcc 158

Ala Arg Arg Thr Pro Arg Thr Arg Arg Tyr Ala Met Thr Pro Pro Ala

15 20 25 30 ttc ttt gcc gtc gca tac gcg ate aac ccc tgg atg gac gtc ace gcg 206

Phe Phe Ala Val Ala Tyr Ala He Asn Pro Trp Met Asp Val Thr Ala

35 40 45 cca gtc gac gtc caa gtc gcg caa gca cag tgg gag cac etc cac cag 254

Pro Val Asp Val Gin Val Ala Gin Ala Gin Trp Glu His Leu His Gin

50 55 60 ace tat ctt egg eta ggc cac age gtg gat ctg ate gag ccc att tec 302

Thr Tyr Leu Arg Leu Gly His Ser Val Asp Leu He Glu Pro He Ser

65 70 75 ggg tta ccg gac atg gtg tac ace gcc aac ggt ggg ttc ate gcg cac 350

Gly Leu Pro Asp Met Val Tyr Thr Ala Asn Gly Gly Phe He Ala His

80 85 90 gac ate gcc gtg gtc gcc egg ttc egg ttc ccc gaa cga get ggt gag 398

Asp He Ala Val Val Ala Arg Phe Arg Phe Pro Glu Arg Ala Gly Glu

95 100 105 110 tct aga gcc tat gcc age tgg atg tec teg gtc gga tat cgc ccg gtg 446

Ser Arg Ala Tyr Ala Ser Trp Met Ser Ser Val Gly Tyr Arg Pro Val

115 120 125 ace ace cgc cac gtc aac gag gga cag ggc gac ctg ctg atg gtt ggc 494

Thr Thr Arg His Val Asn Glu Gly Gin Gly Asp Leu Leu Met Val Gly

130 135 140 gaa agg gtg ttg gcg ggc tac ggc ttt cgc aea gac cag cgc gca cac 542

Glu Arg Val Leu Ala Gly Tyr Gly Phe Arg Thr Asp Gin Arg Ala His 145 150 155 gcc gaa ate gcc gcg gtg ctt ggt ctg ccg gtg gtc tec etc gag ttg 590

Ala Glu He Ala Ala Val Leu Gly Leu Pro Val Val Ser Leu Glu Leu 160 165 170 gtc gac cca egg ttc tat cac ctg gac ace gcg ctg gcc gtg etc gac 638

Val Asp Pro Arg Phe Tyr His Leu Asp Thr Ala Leu Ala Val Leu Asp

175 180 185 190 gac cac acg ate gcc tac tac ccg ccg gcg ttc agt acg gca gcg cag 686

Asp His Thr He Ala Tyr Tyr Pro Pro Ala Phe Ser Thr Ala Ala Gin

195 200 205 gaa cag ttg teg gcg ctg ttc ccc gac gcg att gtg gtc ggc agt gcc 734

Glu Gin Leu Ser Ala Leu Phe Pro Asp Ala He Val Val Gly Ser Ala 210 215 220 gac gcg ttc gtg ttc gga etc aac gcc gtc tct gac ggt ctg aac gta 782

Asp Ala Phe Val Phe Gly Leu Asn Ala Val Ser Asp Gly Leu Asn Val 225 230 235 gtg ctt ccg gtc gcg gcc atg ggt ttt gcg gcg cag tta cgc gca gcc 830

Val Leu Pro Val Ala Ala Met Gly Phe Ala Ala Gin Leu Arg Ala Ala 240 245 250 ggc^{^}ttc gag ccg gtc ggt gtc gat ctg tec gag ctg etc aag ggc ggc 878

Gly Phe Glu Pro Val Gly Val Asp Leu Ser Glu Leu Leu Lys Gly Gly

255 260 265 270 ggt tec gtc aag tgc tgc acg ctg gag ata cac cca tga caa ate teg 926

Gly Ser Val Lys Cys Cys Thr Leu Glu He His Pro Gin He Ser

275 280 285 egg atg cca etc agg cca eta tgg cac tgg teg aaa ggc atg cag cgc 974

Arg Met Pro Leu Arg Pro Leu Trp His Trp Ser Lys Gly Met Gin Arg 290 295 300 aea att att cgc cgctgcctgt ggtggcggcc agcgctga 1014

Thr He He Arg 305

<210> 12

<211> 282

<212> PRT

<213> M. tuberculosis

<400> 12

Met Thr Asp Ser Tyr Val Al a Al a Ala Arg Leu Gly Ser Pro Al a Arg 1 5 10 15 Arg Thr Pro Arg Thr Arg Arg Tyr Ala Met Thr Pro Pro Ala Phe Phe 20 25 30

Ala Val Ala Tyr Ala He Asn Pro Trp Met Asp Val Thr Ala Pro Val 35 40 45

Asp Val Gin Val Ala Gin Ala Gin Trp Glu His Leu His Gin Thr Tyr 50 55 60

Leu Arg Leu Gly His Ser Val Asp Leu He Glu Pro He Ser Gly Leu

65 70 75 80

Pro Asp Met Val Tyr Thr Ala Asn Gly Gly Phe He Ala His Asp He

85 90 95

Ala Val Val Ala Arg Phe Arg Phe Pro Glu Arg Ala Gly Glu Ser Arg 100 105 110

Ala Tyr Ala Ser Trp Met Ser Ser Val Gly Tyr Arg Pro Val Thr Thr 115 120 125

Arg His Val Asn Glu Gly Gin Gly Asp Leu Leu Met Val Gly Glu Arg 130 135 140

Val Leu Ala Gly Tyr Gly Phe Arg Thr Asp Gin Arg Ala His Ala Glu 145 150 155 160

He Ala Ala Val Leu Gly Leu Pro Val Val Ser Leu Glu Leu Val Asp 165 170 175

Pro Arg Phe Tyr His Leu Asp Thr Ala Leu Ala Val Leu Asp Asp His 180 185 190

Thr He Ala Tyr Tyr Pro Pro Ala Phe Ser Thr Ala Ala Gin Glu Gin 195 200 205

Leu Ser Ala Leu Phe Pro Asp Ala He Val Val Gly Ser Ala Asp Ala 210 215 220

Phe Val Phe Gly Leu Asn Ala Val Ser Asp Gly Leu Asn Val Val Leu 225 230 235 240

Pro Val Ala Ala Met Gly Phe Ala Ala Gin Leu Arg Ala Ala Gly Phe 245 250 255

Glu Pro Val Gly Val Asp Leu Ser Glu Leu Leu Lys Gly Gly Gly Ser 260 265 270

Val Lys Cys Cys Thr Leu Glu He His Pro 275 280

Claims

-39-CLAIMS

1. Use of a modulator of DDAHI methylarginase activity and/or expression for the manufacture of a medicament for treating pain in a human or animal wherein the modulator has activity which increases DDAHI methylarginase activity and/or expression.

2. Use according to claim 1 wherein the modulator binds to DDAHI.

3. Use according to claim 1 wherein the modulator binds to a DDAHI promoter:

4. Use according to claim 1 wherein the modulator comprises a polypeptide having DDAHI methylarginase activity.

5. Use according to claim 1 wherein the modulator comprises a polynucleotide which encodes a polypeptide having DDAHI methylarginase activity.

6. Use according to any one of the preceding claims wherein DDAHI is human DDAHI.

7. Use according to any one of the preceding claims wherein the modulator does not modulate DDAHII methylarginase activity or expression.

8. Use according to any one of the preceding claims wherein the modulator does not inhibit the activity or expression of NOS.

9. Use according to claim 8 wherein NOS is nNOS.

10. A method for treating pain in a human or animal, comprising administering to the human or animal a therapeutically or prophylactically effective amount of a modulator as defined in any one of the preceding claims.

11. A method according to claim 10 wherein the modulator is administered to a human or animal that is experiencing pain.

12. A modulator of DDAHI methylarginase activity and/or expression, as defined in any one of claims 1 to 9, for use in treating pain in a human or animal.

13. Products containing a modulator as defined in any one of claims 1 to 9 and a methylarginine, as a combined preparation for simultaneous, separate or sequential use in treating pain in a human or animal.

14. A method of identifying an agent for treating pain in a human or animal, comprising -40-

(a) assaying a test substance for activity which increases DDAHI methylarginase activity and/or expression; and

(b) screening a modulator identified in (a) for efficacy in treating pain in a test animal.

15. A method according to claim 14 wherein step (b) comprises:

(i) administering a modulator identified in step (a) to a test animal; and

(ii) determining whether the modulator reduces or prevents pain in the animal.

16. A method according to claim 14 or 15, further comprising: (c) formulating the agent identified in (b) for administration to a human or animal in need of pain treatment.