WO2011049433A1 - Diagnosis and treatment of pruritis - Google Patents

Abstract

The present invention relates to means and methods for diagnosis and treatment of pruritis, in particular diagnosis and treatment of histamine independentpruritis such as cholestatic pruritus.

Description

Diagnosis and treatment of pruritis

Field of the invention

The present invention relates to the field of medicin and pharmacy. More specifically, the invention relates to means and methods for diagnosis and treatment of pruritis, in particular diagnosis and treatment of histamine independent pruritis such as cholestatic pruritus. Background of the invention

Pruritus is an unpleasant sensation that elicits the desire to scratch. For many it is a short-lasting annoyance - occurring after insect bites, parasitic affections or contact with certain plants - that serves as a physiological defence mechanism to protect the body from potentially harmful external agents. For others, pruritus is a chronic and seriously disabling symptom accompanying a broad range of systemic disorders such as chronic liver diseases, chronic renal failure, malignancies, infections, endocrine and hematological diseases (Greaves, 2005, Dermatol Ther 18, 323-327; Paus et al, 2006, J Clin Invest. 116, 1174-1186; Ikoma et al, 2006, Nat Rev Neurosci. 7, 535-547). Despite the recent discovery of itch-specific neuronal pathways, novel itch mediators and their receptors (Yosipovitch et al., 2003, Lancet 361 , 690-694; Paus et al., 2006, supra; Steinhoff et al, 2006, J Invest Dermatol. 126, 1705-1718; Sun et al, 2007, Nature. 448, 700-703) the pathogenesis of pruritus remains enigmatic in many disorders. This lack of knowledge results in a lack of treatment options for countless distressed patients. Indeed, excessive and chronic itching can lead to sleep deprivation, depression, suicidal ideations and may completely disrupt a patient's quality of life (Sheehan-Dare et al, 1990, Br J Dermatol 123, 769-774; Hashiro et al, 1997, J Dermatol Sci 14, 63-67). In patients with chronic cholestatic liver diseases pruritus is often refractory to all medical treatments and can in severe cases be considered as an indication for liver transplantation even in the absence of liver failure (Heathcote et al, 2000, Hepatology 31, 1005-1013; Neuberger and Jones, 2001 , Eur J Gastroenterol Hepatol. 13, 1393-1394) [Beuers et al, EASL guidelines 2009]. After liver transplantation the underlying disease is cured and the accompanying symptoms disappear. Cholestatic conditions, independent of their primary cause, such as intrahepatic cholestasis of pregnancy (ICP), benign recurrent intrahepatic cholestasis (BRIC), progressive familiar intrahepatic cholestasis (PFIC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), Alagille syndrome, drug-induced cholestasis, alcoholic hepatopathy, chronic viral hepatitis B (HBV) and C (HCV) infections , hilar lymphadenop athy and obstructive malignancy such as cholagniocellular carcinoma (CCC), pancreatic cancer, and metastases often induce pruritus. These cholestatic liver disorders are characterized by an impairment of hepatocellular and/or cholangiocellular secretory function and bile flow (Beuers, 2006, Nat Clin Pract Gastroenterol Hepatol 3, 318-328). In the past, both enhanced serum levels of bile salts and opioids have been held responsible for the induction of cholestatic pruritus (Jones and Bergasa, 1990, Hepatology. 11, 884-887). However, neither correlations between itch intensity and bile salt or opioid levels nor a causative link could ever be established.

Autotaxin (ATX) was originally identified in the conditioned medium of human A2058 melanoma cells and described as an autocrine cell motility factor (Stracke et al., 1992, J Biol Chem 267, 2524-2529; Murata et al, 1994, J Biol Chem 269, 30479- 30484). Beside malignant melanomas, ATX is overexpressed in several other tumor entities and has been linked to tumor cell proliferation, motility and formation of metastasis (Mills and Moolenaar, 2003, Nat Rev Cancer 3, 582-591). Physiologically, ATX is required in angiogenesis and neuronal development, as indicated by ATX- deficient mice which are embryonic lethal due to vascular malformation and neuronal abnormalities (Tanaka et al, 2006, J Biol Chem 281, 25822-25830; van Meeteren et al, 2006, Mol Cell Biol 26, 5015-5022). Sequencing ATX disclosed a phosphodiesterase domain, which is essential for its motility- stimulating activity (Murata et al, 1994, supra; Lee et al. 1996, J Biol Chem 271, 24408-24412). Only recently, ATX was identified as lysophospholipase D, an extracellular enzyme which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC) as well as sphingosine-1 -phosphate (SIP) from sphingosine-phosphorylcholine (SPC) (Umezu- Goto et al, 2002, J Cell Biol 158, 227-233; Tokumura et al, 2002, J Biol Chem 277, 39436-39442). LPA is a small but potent bioactive phospholipid which has a wide variety of effects in many cell types ranging from cytoskeletal organization and cell migration to cytokine production and platelet activation (Mills and Moolenaar, 2003, supra; Hama et al, 2004, J Biol Chem 279, 17634-17639; van Meeteren and Moolenaar, 2007, Prog Lipid Res 46, 145-160). The enzymatic activity of ATX is believed to be responsible for the production of (local and systemic) LPA and LPA signals through at least seven different G protein-coupled receptors (GPCRs) on the plasma membrane of many cell types (Mills and Moolenaar, 2003, supra; Aoki et al., 2008, Biochim Biophys Acta 1781 , 513-518). Most interest in ATX has so far been directed towards its functions in cancer and early development.

It is an object of the present invention to provide for means and methods for diagnosis and treatment of pruritis, in particular diagnosis and treatment of histamine independent pruritis such as cholestatic pruritus.

Description of the invention

In a first aspect, the present invention relates to a method for diagnosing pruritus in a subject, more preferably to a method for diagnosing histamine independent pruritus in a subject. Preferably the method of the invention comprises determining the level of at least one of LPA and autotaxin in a sample from the subject.

Pruritus, also known as itching, is an unpleasant sensation that evokes the desire or reflex to scratch and can be induced by a variety of stimuli, including e.g. mechanical, chemical, thermal, and electrical stimulation. Wound healing after injury or surgery is typically accompanied by local itch perception. LPA promotes re- epithelialization and healing of cutaneous wounds by stimulating proliferation and migration of fibroblasts (Balazs, L., Okolicany, J., Ferrebee, M., Tolley, B. & Tigyi, G. Am J Physiol Regul Integr Comp Physiol 280, R466-472 (2001)). Pruritus may be a symptom accompanying a broad range of systemic disorders (also known as symptomatic pruritus), such as allergy, infection (e.g. parasitic infections), jaundice, chronic renal disease, skin irritation, chronic liver diseases, malignancies (such as cancer, e.g. tumors of the skin [e.g. melanoma], leukemia, carcinoid and lymphoma [Hodgkin's lymphoma and non-Hodgkin's lymphoma]), endocrine and hematological diseases (Greaves, 2005, Dermatol Ther 18, 323-327; Paus et al, 2006, J Clin Invest. 1 16, 1 174-1 186; Ikoma et al., 2006, Nat Rev Neurosci. 7, 535-547). In addition, pruritus may occur without a known cause (also known as other forms of pruritus: Stander S, et al. Clinical classification of itch: a position paper of the International Forum for the Study of Itch. Acta Derm Venereol. 2007;87(4):291-4 ). In a preferred embodiment, a method of the present invention relates to diagnosis of histamine-independent pruritus, such as pruritus as a symptom accompanying systemic disorders (e.g. infection (e.g. parasitic infections), jaundice, chronic renal disease, skin irritation, chronic liver diseases, malignancies (such as cancer, e.g. tumors of the skin (e.g. melanoma), leukemia, carcinoid and lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma)), endocrine and hematological diseases). Histamine- independent pruritus differs from histamine-dependent pruritus as histamine is a biogenic amine that is known to trigger inflammatory responses and to increase the permeability of capillaries to white blood cells and other proteins.

In a more preferred embodiment, a method of the present invention relates to diagnosis of pruritus induced by and/or associated with malignancies (such as cancer, e.g. tumors of the skin (e.g. melanoma), leukemia, carcinoid and lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma)) or cholestatic diseases such as intrahepatic cholestasis of pregnancy (ICP), benign recurrent intrahepatic cholestasis (BRIC), progressive familiar intrahepatic cholestasis (PFIC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), Alagille syndrome, drug-induced cholestasis, alcoholic hepatopathy, chronic viral hepatitis B (HBV) and/or C (HCV) infections, hilar lymphadenopathy and obstructive malignancy such as cholagniocellular carcinoma (CCC), pancreatic cancer, and metastases.

A subject to be diagnosed using the method of the present invention, preferably is a subject with at least one of cholestasis, uremia, wound healing, a keloid, a malignancy (such as cancer, e.g. tumors of the skin (e.g. melanoma), leukemia, carcinoid and lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma)) , an endocrinological disorder (e.g. hyperthyreosis, hypothyreosis, hyperparathyroidism) and atopic dermatitis (also known as "prurigo Besnier," "neurodermitis," "endogenous eczema," "flexural eczema," "infantile eczema," and "prurigo diathsique). More preferably the subject is a subject with cholestasis and even more preferably a subject with cholestasis wherein the primary cause of the cholestasis is selected from the group consisting of intrahepatic cholestasis of pregnancy (ICP), benign recurrent intrahepatic cholestasis (BRIC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hilar lymphadenopathy and obstructive malignancy.

In a preferred embodiment a method of the present invention may be used to diagnose systemic or local pruritis. Lysophosphatidic acid (LPA) is a small but potent bioactive phospholipid derivative which has a wide variety of effects in many cell types ranging from cytoskeletal organization and cell migration to cytokine production and platelet activation (Mills and Moolenaar, 2003, supra; Hama et al., 2004, J Biol Chem 279, 17634-17639; van Meeteren and Moolenaar, 2007, Prog Lipid Res 46, 145-160). ATX activity is believed to be mainly mediated by the enzymatic formation of LPA and LPA acts through at least seven different G protein-coupled receptors (GPCRs) (Mills and Moolenaar, 2003, supra; Aoki et al, 2008, Biochim Biophys Acta 1781, 513-518).

Autotaxin (also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2), ATX, ATX-X, PDNP2, LysoPLD, FLJ26803, AUTOTAXIN and PD-IALPHA) is a secreted enzyme that has lysophospholipase D activity that converts lysophosphatidylcholine into LPA as well as sphingosine-phosphorylcholine (SPC) into sphingosine-1 -phosphate (SIP) (Umezu-Goto et al, 2002, J Cell Biol 158, 227-233; Tokumura et al, 2002, J Biol Chem 277, 39436-39442) (E.C. 3.6.1.9 en 3.1.4.1).

In an embodiment, in a method of the invention at least one of: a) an LPA level in a sample that is at least about 110%, at least about 115%, at least about 118%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 145%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 200%, at least about 250% or more of the average LPA level in samples from corresponding healthy control subjects; and, b) a autotaxin level that is at least about 105%, at least about 110%, at least about 115%, at least about 118%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 150%), at least about 170%), at least about 190%), at least about 220%), at least about 250%), at least about 215%, at least about 300%) or more of the average autotaxin level in samples from corresponding healthy control subjects, is indicative of pruritus. Preferably, average LPA and average autotaxin levels from corresponding healthy control subjects are determined from at least 3, at least 5, at least 10 or more control subjects. A corresponding healthy control subject is herein understood to mean a subject of the same species as the subject, preferably with the same gender as the subject and more preferably matched. For example, if the subject is a pregnant female (woman), then the healthy control subjects are preferably pregnant females (women) who do not suffer from pruritus. In another example, if the subject is a male suffering from primary biliary cirrhosis, then the healthy control subjects are preferably men with primary biliary cirrhosis but without pruritus.

In a preferred embodiment, in a method according to the invention at least one of: a) a plasma LPA level that is at least about 110%, at least about 1 15%, at least about 118%, at least about 120%, at least about 125%, at least about 130%, at least about 140%, at least about 145%, at least about 150%, at least about 160%, at least about 170%), at least about 180%), at least about 200%, at least about 250% of the average plasma LPA in corresponding healthy control subjects; and, b) a plasma autotaxin level that is at least about 105%, at least about 1 10%, at least about 1 15%, at least about 118%, at least about 120%, at least about 125%, at least about 130%, at least about 140%), at least about 150%), at least about 170%), at least about 190%), at least about 220%), at least about 250%), at least about 275%), at least about 300%) of the average plasma autotaxin level in corresponding healthy control subjects, is indicative of pruritus. In an even more preferred embodiment, in a method according to the invention at least one of: a) a plasma LPA level that is at least 110% of the average plasma LPA in corresponding healthy control subjects; and, b) a autotaxin level that is at least 110% of the average plasma autotaxin level in corresponding healthy control subjects, is indicative of pruritus.

As used herein, a subject is meant to be an individual. The subject can include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) and birds. In a preferred embodiment, the subject is a mammal such as a primate or a human, even more preferably the subject is a human.

In a preferred embodiment the subject is a pregnant female. The present inventors found that enhanced LPA and/or autotaxin levels may be used as a diagnostic tool to distinguish between intrahepatic cholestasis of pregnancy (ICP) and other forms of pruritus, such as histamine-dependent pruritus, in pregnant subjects. This is of great importance as ICP women are the only group of pruritic pregnant subjects that have an enhanced risk for adverse fetal outcome, such as increased fetal morbidity and mortality (particularly from chronic placental insufficiency), preterm labor, fetal distress and intrauterine death. Thus, enhanced LPA and autotaxin levels could be regarded as a parameter for differential diagnosis. Women with ICP may be advantageously treated with ursodeoxycholate, which partially ameliorates pruritus, partially improves the underlying disease and partially reduces fetal morbidity and mortality.

A sample to be used in a method of the invention may originate from any tissue from the subject that is to be diagnosed, such as blood, serum, plasma or a biopsy (preferably of the itching site). Preferably, the sample is blood, serum or plasma, since this kind of samples can easily be obtained and since LPA and/or autotaxin are extracellular molecules and therefore present in the circulation. Therefore, it is a preferred embodiment of the invention that at least one of the plasma LPA and the plasma autotaxin level is determined.

LPA levels in a sample may be determined in several ways known by a skilled person in the art, such as by HPLC-MS (as described elsewhere in this application), thin-layer chromatography (TLC), and enzymatically (Kishimoto T, et al. A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method. Clin Chim Acta. 2003 Jul l ;333(l):59-67). Autotaxin levels in a sample may be determined in various ways known to a skilled person, for example by enzyme immuno assay (EIA), enzyme-linked immunosorbent assay (ELISA), radio immuno assay (RIA), SDS-PAGE and Western blotting (described elsewhere in this application), any other immunological assay (Nakamura K, et al. Validation of an autotaxin enzyme immunoassay in human serum samples and its application to hypoalbuminemia differentiation. Clin Chim Acta. 2009 Jul;405(l-2): 160-2), or by an autotaxin activity assay (Nakamura et al. Measurement of lysophospholipase D/autotaxin activity in human serum samples. Clin Biochem. 2007 Feb;40(3-4):274-7) (described elsewhere in the present application). In a preferred embodiment, the level of autotaxin in a sample is determined by the level of autotaxin activity.

Since both autotaxin and its substrate, lysophosphatidylcholine (LPC), are present in the circulation, it is preferred that a sample, in particular when the sample is blood, serum, plasma or the like, prior to determination of the LPA level, is maintained or stored under conditions that prevent or reduce hydrolysis of LPC to LPA. The skilled person is well aware how samples may be handled in order to reduce LPA formation after sample collection as much as possible. Examples of conditions that prevent or reduce hydrolysis of LPC to LPA are e.g. keeping freshly isolated samples on ice, (immediately) freezing in liquid nitrogen, processing the freshly isolated samples in a minimum required amount of time, freezing the sample in liquid nitrogen and/or storing the sample at about -20°C, more preferably at about -80°C, even more preferably at about -169°C. In a preferred embodiment, an autotaxin inhibitor is added to the sample before maintenance or storage in order to prevent or reduce hydrolysis of LPC to LPA. Examples of autotaxin inhibitors that may be added to the sample in order to prevent or reduce hydrolysis of LPC to LPA are: FTY720 (= fingolimod) (2-Amino-2-(2-(4- octylphenyl) ethyl)propan-l,3-diol; van Meeteren et al. (2008) Cancer Lett 266:203- 208), HA130 (see formula [1]), BrP-LPA (l(R)-Bromo-3(S)-hydroxy-4- (palmitoyloxy)butyl]phosphonate); Zhang et al. (2009) Cancer Res Epub), S32826 ([4- (tetradecanoylamino)benzyl]phosphonic acid; Ferry et al. (2008) J Pharmacol Exp Ther 327:809-819), VPC8a202 (phosphonate derivate; Cui et al. (2007) Bioorg Med Chem Lett 17: 1634-1640), Damnacanthal (Moulharat et al. (2008) Chem Biol Interact 172: 115-124), Hypericin (4,5,7,4',5',7'-Hexahydroxy-2,2'-dimethylnaphthodianthrone; Giganti et al. (2008) JBC 283:7776-7789) and derivatives of these compounds. As the skilled person will understand, it is not preferred to use an autotaxin inhibitor in order to prevent or reduce hydrolysis of LPC to LPA if autotaxin activity levels are to be determined in the same sample.

Formula [1]

HA130 (Formula [1]) can be prepared by the method as described in the examples.

In a second aspect, the present invention relates to an agent that reduces triggering of an LPA receptor for use in the treatment or prevention of pruritus, preferably histamine independent pruritus, in a subject. In this aspect, the present invention also relates to use of an agent that reduces triggering of an LPA receptor for the manufacture of a medicament for the treatment or prevention of pruritus, preferably histamine independent pruritus, in a subject. Further, the present invention relates to the treatment or prevention of pruritus, preferably histamine independent pruritus, comprising administration of a therapeutically effective amount of an agent that reduces triggering of an LP A receptor to a subject in need thereof. A therapeutically effective amount is herein understood to mean an amount that results in prevention of pruritus or in a reduction of itching of at least 5, preferably at least 10, more preferably at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90% or more. Most preferably the amount results in 100% reduction of itching as determined by visual analog scale (VAS) or scratching activity monitor system (SAMS). The SAMS device is based on piezoelectric crystals that are attached to a fingernail and permit registration of every scratch independently of other body movements (Stein H et al. Pruritometer 1 : Portable measuring system for quantifying scratching as an objective measure of cholestatic pruritus. Biomed Tech (Berl). 1996 Sep;41(9):248-52; Bijak M et al. Pruritometer 2: portable recording system for the quantification of scratching as objective criterion for the pruritus. Biomed Tech (Berl). 2001 May;46(5): 137-41).

In a preferred embodiment, an agent that reduces triggering of an LPA receptor is an autotaxin inhibitor or an LPA receptor inhibitor. In an embodiment, the agent being an autotaxin inhibitor is herein defined as a compound that at a concentration of 17000 nM or less, more preferably at a concentration of less than 5000 nM, even more preferably at a concentration of less than 3000 nM, even more preferable at a concentration of less than 2000, 1000, 700, 500, 400, 300, 250, 200, 150, 100, 50, 20 or 10 nM of autotaxin inhibitor results in at least 50% inhibition of autotaxin as compared to a control sample without the agent in the assay described by Nakamura, K., et al. Clin Biochem 40, 274-277 (2007). In short, 2 μΐ of serum is incubated with 78 μΐ buffer containing 1 mmol/L of LPC 14:0, 500 mmol/L NaCl, 5 mmol/L MgCl₂, lOO mmol/L Tris (pH = 9.0) and 0.05% Triton X-100 for 60 min at 37° C . Subsequently, the phosphodiesterase activity of ATX is determined by the amount of liberated choline which is detected by an enzymatic photometric method using choline oxidase (2 U/ml), horseradish peroxidase (1.6 U/ml), and homovanillinic acid as substrate for peroxidase. After addition of both enzymes in a buffer (consisting of 20 mmol/L CaCl₂, 2 mmol/L homovanillinic acid, 50 mmol/L MOPS (pH = 8.0) and 0.1%) Triton X-100), 190 μΐ of the choline oxidase/horseradish peroxidase comprising buffer is added to 20 μΐ of the serum comprising buffer. Then, the increase in fluorescence is monitored at 37°C on a Novostar analyzer (BMG Labtech GmbH, Offenburg, Germany; excitation 320 nm, emission 405 nm). In order to measure the inhibiting capacity of an agent, the agent is added to the serum.

Optionally, to study interference of bile salts and steroids on ATX activity, either serum or recombinant ATX can be incubated with above-mentioned buffer set to pH = 7.4. Stock solutions of bile salts and steroids are prepared in dimethylsulfoxide (DMSO), diluted 1 : 100 in buffer containing serum or rATX and incubated for 3 h at 37°C. 1% DMSO has been found to have no detectable effect on ATX activity.

A preferred autotaxin inhibitor is selected from the group consisting of FTY720 (= fingolimod) (2-Amino-2-(2-(4-octylphenyl) ethyl)propan-l,3-diol; van Meeteren et al. (2008) Cancer Lett 266:203-208; Novartis), HA130 (see formula [ 1 ] above), BrP-LPA (l(R)-Bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate); Zhang et al. (2009) Cancer Res Epub), S32826 ([4-(tetradecanoylamino)benzyl]phosphonic acid; Ferry et al. (2008) J Pharmacol Exp Ther 327:809-819), VPC8a202 (phosphonate derivate (s. paper); Cui et al. (2007) Bioorg Med Chem Lett 17: 1634-1640), Damnacanthal (Moulharat et al. (2008) Chem Biol Interact 172: 115-124), Hypericin (4,5,7,4',5',7'-Hexahydroxy-2,2'-dimethylnaphthodianthrone; Giganti et al. (2008) JBC 283:7776-7789) and derivatives of these compounds. In principle an autotaxin inhibitor with a lower IC50 is preferred with respect to an autotaxin inhibitor with a higher IC50. Therefore, S32826, BrP-LPA and HA 130-2 are more preferred autotaxin inhibitors (see table 1).

Table 1. Autotaxin inhibitors that may be used in the invention

* 73% inhibition of ATX at 1 μΜ

In another embodiment, the agent being an LPA receptor inhibitor is herein defined as a compound that at a concentration of 25000 nM or less, more preferably at a concentration of less than 10000 nM, even more preferably at a concentration of less than 7000 nM, even more preferable at a concentration of less than 6000, 5000, 4000, 3000, 2000, 1000, 750, 700, 500, 400, 300, 250, 200, 150, 100, 50, 30, 20 or 10 nM of LPA receptor inhibitor results in at least 50% inhibition of the LPA receptor as compared to a control sample without the agent in an assay as described in any of the following references Ohta et al. (2003) Mol Pharmacol 64: 994-1 005 ; Fischer et al . (2001) Mol Pharmacol 60:776-784; Beck et al. (2008) Bioorg Med Chem Lett 18: 1037- 1041; Virag et al. (2003) Mol Pharmacol 63: 1032-1042; Heise et al. (2001) Mol Pharmacol 60: 1173-1180; Zhang et al. (2009) Cancer Res Epub; Yamamoto et al. (2007) Bioorg Med Chem Lett 17:3736-3740; Fells et al. (2008) Bioorg Med Chem 16:6207-6217; Liliom et al. (2006) BBA 1761 : 1506-1514.

A preferred LPA receptor inhibitor is selected from the group consisting of: Κ Ϊ 1 6 4 2 5 ( 3-(4-[4-([l-(2-Chlorophenyl)ethoxy]carbonylamino)-3-methyl-5- isoxazolyl]benzylthio)propanoic acid; Ohta et al. (2003) Mol Pharmacol 64: 994-1005), DGPP 8:0 (di-octyl glycerol pyrophosphate; Fischer et al. (2001) Mol Pharmacol 60:776-784), Tetradecyl Phosphonate (Tetradecyl Phosphonate; catalogue number 10007565 Cayman), Compound 35 (Beck et al. (2008) Bioorg Med Chem Lett 18: 1037-1041), FAP-12 (fatty acid phosphate 12:0; Virag et al. (2003) Mol Pharmacol 63: 1032-1042), VPC 12249 (2-substituted N-oleoyl ethanolamide phsophoric acid compounds; Heise et al. (2001) Mol Pharmacol 60: 1173-1180), BrP-LPA (1(R)- Bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate; Zhang et al. (2009) Cancer Res Epub), Compound 34 (3-(4-{4-[l-(2-chloro-cyclopent-l-enyl)- ethoxycarbonylamino]-isoxazol-3-yl}-benzylsulfanyl)-propionic acid; Yamamoto et al. (2007) Bioorg Med Chem Lett 17:3736-3740), NSC161613 (Fells et al. (2008) Bioorg Med Chem 16:6207-6217), H2L5105099 (Fells et al. (2008) Bioorg Med Chem 16: 6207-6217), FMP (farnesyl phosphate; Liliom et al. (2006) BBA 1761 : 1506-1514) and derivatives of these compounds. In principle an LPA receptor inhibitor with a lower IC50 is preferred with respect to an LPA receptor inhibitor with a higher IC50. It is currently believed that there are 7 different LPA receptors (Aoki J. et al. (2008) BBA 1781 : 513-516), but it is not known through which of the receptors LPA may result in pruritus. In Table 2, the results are shown for LPAi, LPA₂ and LPA₃. Table 2. LP A receptor inhibitors that may be used in the invention

* K| values

** 2.9 and 130, respectively - depending on test

assay

Pruritus is a common symptom in lymphoma patients especially in those with Hodgkin's disease (Rubenstein, M. & Duvic, M. Int J Dermatol 45, 251-256 (2006)). Approximately every third patient suffers from this disabling symptom generally at those skin parts being closely located to affected lymph nodes (Rubenstein, M. & Duvic, M. Int J Dermatol 45, 251-256 (2006)). ATX inhibitors and LPA receptor blockers which are currently developed for the treatment of cancer patients to reduce disease progression and formation of metastasis (Peyruchaud, O. Anticancer Agents Med Chem 9, 381-391 (2009)) might also represent a novel class of anti-pruritic drugs.

A subject to be treated or to be prevented of (histamine independent) pruritus according to the invention, is a subject as defined above.

Delivery of an agent that reduces triggering of an LPA receptor or a medicament according to the invention may be via any administration route, preferably via oral administration, topical administration, transdermal or via a parental route e.g., injection or infusion by subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intra-arterial or intralesional routes. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the figures

Figure 1 : Cytosolic free calcium [ Ca²⁺]i is increased in human neuroblastoma cell line SH-SY5Y by serum of patients with cholestatic pruritus more than by serum of healthy controls, (a) Serum induced an increase in [Ca²⁺]i which was dose-dependent. Note that even high serum dilutions of up to 1 :512 induced an increase o f [Ca²⁺]i. (b) Sera of women with ICP (ICP) induced higher increases of [Ca²⁺]i compared to gestation- matched normal pregnancies (PC) and age-matched female controls (HC). ** p<0.01 (c) Sera of PBC patients with and without pruritus induced a higher increase in [Ca²⁺]i compared to age-matched healthy female controls (HC). ** p<0.01 (d,e,f) Effect of different pre-treatments of serum on [Ca²⁺]i in neuronal cells. U: untreated; E: 90% ethanol precipitation; Prot K: incubation for 24 hours with proteinase K; 100 kD, 10 kD: flow through of 100 kD and lO kD-filter, respectively, of untreated serum; 100 kD+E, 3 kD+E: flow through of 100 kD and 3 kD-filter, respectively, of ethanol- precipitated serum; lOO kD+E+CA, lO kD+E+CA, 3 kD+E+CA: flow through of 100 kD, 10 kD, and 3 kD-filter, respectively, of ethanol-precipitated serum which was resuspended in 2 mM cholate suspension; WP pH 7.4, LP pH 7.4, WP pH 1.0, LP pH 1.0: Water phase (WP) and lipid phase (LP) of serum treated by Bligh and Dyer lipid extraction at physiological and acidic pH values, (g) Pre-treatment of neuronal cells with pertussis toxin (PTX) and LPA receptor blocker ΚΪ16245; U: untreated.

Figure 2: Serum levels of LPA and ATX are elevated in pruritic patients with various cholestatic disorders, (a) LPA 18: 1 is enhanced in ICP women compared to gestation- matched regular pregnant women (PC). * p<0.05 (b) ATX activity measured as choline release is highly enhanced in ICP compared to pregnant controls (PC) and healthy female controls (HC). *** p<0.0001 (c) ATX protein content was enhanced similar to the ATX activity. Recombinant ATX (rATX) was used as positive control. (d,e) ATX activity was highly enhanced in cholestatic women and men suffering of pruritus compared to non-pruritic cholestatic patients and healthy controls. *** p<0.0001 (f) Increased ATX activity correlated with ATX content in serum of cholestatic patients. Recombinant ATX (rATX) was used as positive control, (g) ATX activity in patients with chronic hepatitis C was enhanced compared to controls but similar to those of other chronic cholestatic disorders.

Figure 3: ATX activity, but neither histamine, nor bile salts or μ-opioid activity in serum are correlated with itch intensity of patients with cholestatic itch, (a) ATX activity closely correlated with the itch intensity represented as visual analogue scale ranging from 0 (no pruritus) to 10 (most severe form of pruritus), (b) No correlation could be observed between histamine levels and itch intensity, (c) Total serum bile salts did not correlate with itch intensity, n.s. not significant, (d) Total μ-opioid activity was comparable in female and pregnant controls compared to women with either ICP, PBC without pruritus or PBC with pruritus. Only two PBC patients with pruritus presented with high μ-opioid levels, n.s. not significant, (e) In PBC patients undergoing nasobiliary drainage (on day 0), ATX activity similarly dropped with pruritus scores and rose upon reappearance of pruritus several weeks later (day 15-144). Data are shown as percent changes of baseline values. * p<0.05 (f) ATX activity in bile of patients undergoing nasobiliary drainage or control bile, (g) ATX protein could not be detected on western blot. Recombinant ATX (rATX) was used as positive control, (h) Bile salts were incubated with recombinant ATX in the absence or presence of different bile salts using the following concentrations: 10 μΜ, 25 μΜ, 100 μΜ, and 500 μΜ. Both chenodeoxycholate (CDC) and ursodeoxycholate (UDC) significantly inhibited ATX activity already at low concentrations, whereas cholate (C), deoxycholate (DC) and lithocholate (LC) hardly influenced activity. Similar results were obtained for the glycine and taurine conjugates, of all these bile salts (data not shown). LC precipitated at concentrations above 50 μΜ and higher concentrations could not be analyzed. * p<0.05, ** p<0.01, *** pO.001 Figure 4: Induction of scratch responses by LP A in vivo and inhibition of ATX activity in vitro by bile salts, (a) Intradermal injections of LPA (50 nmol in 50 μί) led to increased scratching behaviour compared to vehicle injections being significant during the first 15 minutes, (b) Dose-dependent scratching behaviour after intradermal injections of LPA. Injections were performed in 7 mice. * p<0.05, ** p<0.01, *** p<0.001

Examples

Example 1

1. Materials and methods

1.1 Human subjects

Peripheral venous blood was obtained from healthy donors and patients after they provided informed consent with approval from the Medical Ethical Committee of the University of Amsterdam, the Netherlands. Sera of healthy pregnant women and women with intrahepatic cholestasis of pregnancy were collected after providing written consent at the Institute of Reproduction and Developmental Biology, Imperial College, London, UK.

1.2 Animals

Female C57BL/6J mice (6-8 weeks of age) were used for intradermal and intravenous injections. A Teflon-coated magnet was implanted in each hind paw of C57BL/J6 mice one week prior to experiments. Mice were shaved at the back of the neck where intradermal injections were applied. All mouse experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Academic Medical Center, University of Amsterdam, the Netherlands.

1.3 Intradermal injections

Mice were given 120 min to acclimate to the chamber surrounded by a magnetic coil before they were briefly removed from the chamber and intradermally injected with saline (50 μί) or lysophosphatidic acid (8-200 nmol in 50 μί). Movements of the magnets being implanted in the hind paws induced an electric current in the magnet field, which was registered by an oscillograph. Mice scratch themselves with their hind paws with a typical frequency between 10-20 Hz enabling a computer-based software analysis of the scratch movements. The number of scratch bouts was analyzed as previously described (Inagaki, N., et al.. Eur J Pharmacol 448, 175-183 (2002)). In short, we used a computer-based software to count scratch movements using a low cutoff frequency of 10 Hz, a high cut-off frequency of 20 Hz, a threshold level of 300 mV and a minimum of 4 beats per bout. The analysis procedure was validated with compound 48/80 (Inagaki, N., et al. Eur J Pharmacol 448, 175-183 (2002)) showing a positive predictive value of 95% at a sensitivity of 50%.

1.4 Cell culture

HEK293 cells were obtained from ATCC. Cells were maintained in Dulbucco's modified Eagle medium (DMEM) with 10% (v/v) fetale bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (0.2 mmol/L) at 37°C in a humidified atmosphere of 5% C0₂/95%> air. ATX-overexpressing HEK293 cells were cultured in the same medium and selected by the addition of hygromycin B (100 μ^πΛ).

1.5 Materials

C ell culture media were from Lonza (Basel, Switzerland). Stearoyl- lysophosphatidic acid (LPA 18: 1) and myristoyl-lysophospahtidyl choline (LPC 14:0) were purchased at Avanti Lipids (Alabaster, USA). Choline oxidase, horseradish peroxidase, homovanillinic acid, pertussis toxin, ionomycine, and from Sigma- Aldrich (St. Louis, USA). Ki 16245, Tetradecoyl-phosphonate, and ATX-antibody for Western Blotting were obtained from Cayman (Ann Arbor, USA). Indo-1 AM was from Invitrogen (Carlsbad, USA), Microcon filters from Millipore (Billerica, USA).

1.6 Fluorimetric measurement of intracellular free calcium

SH-SY5Y cells (ATCC; CRL-2266) were detached, washed twice and suspended in HEPES -buffered Hank's balanced salt solution (HBSS) containing 10 mmol/L NaCl, 130 mmol/L KC1, 5.56 mmol/L glucose, 10 mmol/L HEPES, 0.441 mmol/L NaH₂P0₄, 0.336 mmol/L Na₂HP0₄, 4.17 mmol/L NaHC0₃ and 0.811 mmol/L MgS0₄, pH 7.4. Cell suspensions were incubated with a final concentration of 10 μιηοΙ/L indo-1 AM for 30 min at 37°C. Cells were washed, re-suspended in HEPES-buffered HBSS and incubated for another 30 min at 25°C to allow for dye de-esterification. After another wash step, cells were re-suspended again in HEPES-buffered HBSS and stored on ice until required. Analysis were performed in UV-sensitive 96-well-plates using the fluorimeter Novostar analyzer (BMG Labtech GmbH, Offenburg, Germany; excitation 320 nm, emission 405 nm and 520 nm). Cell suspensions were allowed to adapt to 37°C for 10 min before serum or serum extracts were added. For experiments using receptor inhibitors cells were pre-incubated with those substances for 10 min prior to addition of serum. Cytosolic free calcium [Ca²⁺]i were calculated after calibration with ionomycin (10 μιηοΙ/L) and EGTA (5 mmol/L) according to Grynkiewicz et al, 1985, J Biol Chem 260, 3440-3450.

1.7 Purification of recombinant autotaxin

Human ATX cDNA was cloned with a C/N-terminal His-tag in a pcDNA3 vector. HEK293T cells were stably transfected with pcDNA3-ATX-His and selected by addition of hygromycin B (100 μg/ml). Medium of ATX overexpressing HEK293T cells was harvested and incubated with Ni-NTA beads (Qiagen, Germany) at 4°C overnight. For fast protein liquid chromatography (FPLC) a low-imidazol (20 mmol/L) and high-imidazol (500 mmol/L) buffer containing 500 mmol/L NaCl, 5 mmol/L CaCl₂, 5 mmol/L MgCl₂, 100 mmol/L Tris (pH = 8.0) was used. Ni-NTA beads were put in a 5 -ml column and recombinant ATX was eluted using a continuously increasing imidazol gradient ranging between 20-500 mmol/L. Fractions of 0.5 ml were collected and ATX activity herein determined as outlined below. Those fractions containing high ATX activity were pooled and dialyzed five times to reduce imidazol content to concentrations below 5 μιηοΙ/L. Dialysis was performed using a 30 kD filter ultrafilration membranes (DIAFLO^®, Micropore, UK) under 2.5 bar N₂-pressure at 4°C. Purification of recombinant ATX was analyzed by Coomassie brilliant blue R-250 staining.

1.8 Autotaxin activity assay

ATX activity was analyzed as described recently (Nakamura, K., et al. Clin Biochem 40, 274-277 (2007)). In short, serum samples were incubated with a buffer containing 1 mmol/L of LPC 14:0, 500 mmol/L NaCl, 5 mmol/L MgCl₂, 100 mmol/L Tris (pH = 9.0) and 0.05% Triton X-100 for 60 min at 37°C. The phosphodiesterase activity of ATX was determined by the amount of liberated choline which was detected by an enzymatic photometric method using choline oxidase (2 U/ml), horseradish peroxidase (1.6 U/ml), and homovanillinic acid as substrate for peroxidase. After addition of both enzymes in a buffer (consisting of 20 mmol/L CaCl₂, 2 mmol/L homovanillinic acid, 50 mmol/L MOPS (pH = 8.0) and 0.1% Triton X-100) the increase in fluorescence was monitored at 37°C on a Novostar analyzer (BMG Labtech GmbH, Offenburg, Germany; excitation 320 nm, emission 405 nm). To study interference of bile salts and steroids on ATX activity either serum or recombinant ATX were incubated with above-mentioned buffer set to pH = 7.4. Stock solutions of bile salts and steroids were prepared in dimethylsulfoxide, diluted 1 : 100 in buffer containing serum or rATX and incubated for 3 h at 37°C. 1% DMSO had no effect on ATX activity.

1.9 LP A determination by HPLC-MS

As internal standards myristoyl-LPA (LP A 14:0) and myristoyl-PA (PA 14:0) were added at a final concentration o f 1 μΜ to serum samples. Lipids were subsequently extracted from 100 of serum by one-phase lipid extraction using 1 mL of methanol/chloroform (1 : 1, vol/vol). The extraction fluid was evaporated to dryness (45°C, vacuum) and reconstituted in methanol/chloroform. HPLC-MS analysis was performed.

1.10 Bile salt determination

Total serum bile salt levels were determined using Diazyme total bile acids kit

(Diazyme Laboratories, Poway, CA) according to the manufacturer's instructions. The change in absorption was monitored at 37°C for 30 min on a Novostar analyzer (BMG Labtech GmbH, Offenburg, Germany; absorption 410 nm).

1.11 Histamine determination

Serum histamine concentrations were measured by a competitive enzyme immunoassay (Immunotech, Marseille, France) based on the competition between free acylated histamine and alkaline phosphatase acylated histamine conjugate. The intensity of the color after addition of paranitrophenyl phosphate as substrate was measured at 37°C on a Novostar analyzer (BMG Labtech GmbH, Offenburg, Germany; absorption 410 nm) .

1.12 Determination of μ-opioid activity

Total serum μ-opioid activity was determined exactly as described by Swain et al (Swain, M.G., et al. Gastroenterology. 103, 630-635. (1992)).

1.13 SDS-PAGE and Western Blotting

To concentrate ATX, 50 μΐ of serum samples were first incubated for 4 hours at

4°C with immuno -precipitating ATX-antibody 5A5 bound to sepharose. After washing, sepharose beads were incubated for 10 min at 37°C with SDS-Loading buffer containing β-mercapto-ethanol and spin down. Equal amounts of supernatant were separated by SDS-PAGE, blotted on PVDF membranes, blocked with 5% skim milk in PBS/0.05% Tween-20, and incubated with anti-ATX (1 :250, Cayman) overnight. Proteins were visualized with horseradish peroxidase-conjugated immunoglobulins and detected by enhanced chemoluminescence (Amersham, Buckinghamshire, UK).

1.14 Statistical analysis

Statistical differences were evaluated for two groups by Student's t-test and for three or more groups by one-way analysis of variance (AN OVA) using SPSS (version 16.0). Pearson's correlation coefficient and corresponding p-values were calculated to assess the relationship between tested parameters. Statistical significance is displayed as p<0.05 (one asterisk), p<0.01 (two asterisks) or p<0.0001 (three asterisks). All data are expressed as means ± standard deviations.

2. Results

2.1 Neuron-activating serum factor identified as LP A

To identify potential pruritogens in cholestasis, we screened sera of pruritic patients for activation of different neuronal cell lines measured as rise in intracellular free calcium concentrations. In the human neuroblastoma cell line SH-SY5Y we observed a dose dependent rise in intracellular calcium concentrations due to the addition of serum (Fig la). Interestingly, sera of women with intrahepatic cholestasis of pregnancy (ICP) suffering from pruritus showed a significant stronger neuronal activation compared to pregnant controls (PC) and healthy female controls (HC) (Fig lb). Similarly, sera of patients with primary biliary cirrhosis (PBC) suffering from pruritus induced a higher rise in [Ca²⁺]i levels than sera of PBC patients without pruritus and healthy controls (HC) (Fig lc). We further analyzed the serum samples to identify the neuron-activating serum factor. Pre-treatment of serum with 90% ethanol (E) or proteinase K (prot K) hardly diminished the calcium release, indicating that the serum factor was not a peptide or protein (Fig Id). Serum samples were centrifuged through filters to estimate the molecular size. The factor could pass a 100 kD filter, but not a 10 kD filter. Interestingly, pre-treatment of serum with 90% ethanol (E) enabled the factor to partially pass through the 10 kD and even a 3 kD filter (Fig le). This observation could be explained by a strong binding of the factor to albumin (molecular weight approximately 60 kD). Thus, like unconjugated bilirubin, the substance appeared to be partially forced into solution upon ethanol-induced precipitation of albumin. Total recovery of the serum factor through a 10 kD filter was achieved by addition of cholate (CA) above its critical micelle concentration enabling a hydrophobic substance to be completely solved in an aqueous solution. As those micelles have a diameter of approximately 4.4 kD, they barely pass a 3 kD filter. Hence, we were dealing with a small, hydrophobic substance. Its chemical properties were further analyzed by a two-phase Bligh and Dyer lipid extraction. At neutral pH the compound presented an amphiphilic character but solved better in the lower aqueous phase (LP). Lowering the pH to 1.0 and thus protonating the serum factor led to a recovery of the substance mainly in the upper lipid phase (UP) (Fig If). The increased hydrophobicity upon protonation could be explained by uptake of protons to phosphate- or sulfate-groups of the molecule. As pertussis toxin diminished the rise in [Ca²⁺]i, signalling was assumed to occur via a G-protein coupled receptor. These observations rendered LPA a likely candidate because LPA has been reported to increase [Ca²⁺]i in neuronal cells (Simpson et al, 2002, Assay Drug Dev Technol 1, 31- 40). Pre-treatment of the neuronal cells with ΚΪ16425, a specific LPA-receptor inhibitor, significantly reduced the [Ca²⁺]i rise induced by serum of pruritic patients, indicating that LPA was the unknown neuronal stimulus in our blood samples (Fig lg). 2.2 LPA and ATX are enhanced in pruritus of cholestasis

Analyzing the LPA content in serum samples by mass spectrometry indeed showed significant higher concentrations of LPA 18: 1 in sera of women with intrahepatic cholestasis of pregnancy (ICP) compared to gestation matched pregnant controls (PC) (Fig 2a). Similar differences were shown for other LPA species including LPA 16:0, 18:0, 18:2, 20:3 and 20:4 (data not shown). LPA is formed in blood through cleavage of choline from lysophosphatidylcholine (LPC) by autotaxin (ATX), which could recently be identified as lysophospho lipase D (Umezu-Goto et al., 2002, J Cell Biol 158, 227-233; Tokumura et al, 2002, J Biol Chem 277, 39436-39442). As LPC is present in blood in high concentrations (above 100 μΜ), the amount of LPA in blood primarily depends on ATX activity. Therefore, we analyzed whether ATX activity in blood also correlates with the occurrence of itch. ATX activity was analyzed as described recently (Nakamura et al., 2007, Clin Biochem 40, 274-277). Indeed, we observed a markedly increased ATX activity in parallel with elevated serum LPA in women with pruritus due to ICP (Fig 2b).. The enhanced ATX activity correlated with the ATX protein content in serum of these patients (Fig 2c). We subsequently studied whether this observation could be extended to other forms of cholestasis. Therefore, sera of patients with different cholestatic disorders with and without pruritus were analyzed. Quite strikingly, we found that ATX activity and protein concentrations were highly enhanced in patients of both genders suffering from pruritus, compared to cholestatic patients without pruritus (Fig 2d-f). ATX serum levels were also increased in our group of HCV patients when compared to healthy controls, but were lower than in pruritic patients and not different from non-pruritic cholestatic patients (Fig 2g). 2.3 ATX activity correlates with intensity of pruritus

Pruritus is a subjective perception which differs from one person to another. Thus, quantification of this symptom is difficult but can be achieved by the use of visual analogue scales (VAS). Patients quantified their itch intensity at the time point of blood drawing on a scale ranging from 0 (no pruritus) to 10 (most severe form of pruritus). Next we analyzed the correlation between itch intensities and the ATX activity in serum of these patients by linear regression analysis. We found a strong correlation between ATX activity and itch intensity objectified by the patient's scoring on a VAS (Fig 3a). In contrast, other agents discussed as potential pruritogens in cholestasis in the past (Bergasa, N.V. J Hepatol 43, 1078-1088 (2005); Kremer, A.E., Beuers, U., Oude-Elferink, R.P. & Pusl, T. Drugs 68, 2163-2182 (2008)) did not show any correlation with itch intensity in our patient cohort: (i) serum histamine (Fig 3b), (ii) serum bile salts (Fig 3c), and (iii) serum μ-opioid activity (Fig 3d) did not show any correlation with VAS scores, and serum μ-opioid activity was not enhanced in ICP patients compared to regular pregnancies. A direct role of histamine and bile salts as pruritogens has already been questioned in the past (Bergasa, N.V. J Hepatol 43, 1078- 1088 (2005); Jones, E.A. & Bergasa, N.V. Hepatology. 11, 884-887. (1990); Beuers, U., Gerken, G. & Pusl, T. Hepatology 44, 280-281 (2006)). Surprisingly, only a few PBC patients suffering from pruritus showed increased serum μ-opioid levels in line with the antipruritic effect of μ-opioid antagonists in some patients (Bergasa, N.V., et al. Ann Intern Med. 123, 161-167. (1995)) but questioning a key causative role of opioids for the pathogenesis of cholestatic pruritus in our cohort (Spivey, J.R., Jorgensen, R.A., Gores, G.J. & Lindor, K.D. Am J Gastroenterol 89, 2028-2032. (1994)). In some patients with PBC, extensive long-lasting pruritus was intractable and did not adequately respond to any medication recommended (Kremer, A.E., Beuers, U., Oude-Elferink, R.P. & Pusl, T. Drugs 68, 2163-2182 (2008); EASL Clinical Practice Guidelines: Management of cholestatic liver diseases. J Hepatol 51, 237-267 (2009)). These patients underwent nasobiliary drainage for 2-7 days as an experimental treatment of pruritus (Beurers et al. (2006) Hepatology 44:280-281). All four patients experienced dramatic reduction or complete relief of pruritus within 24 hours which lasted for several days or weeks. Interestingly, concomitant with the relief of pruritus ATX activity dropped and rose back to pre-treatment values when pruritus returned (Fig 3e).

2.4 Inhibition of ATX activity by bile salts

The increased ATX levels in serum of patients with cholestatic itch could either be caused by increased ATX expression or by reduced clearance of the enzyme. Although ATX has a short half life of several minutes in circulation (Jansen, S., et al. Cancer Lett (2009)), it can be easily detected in serum, suggesting a continuous synthesis and release. Expression of ATX has been demonstrated in endothelial cells, adipocytes and liver tissue (Ferry, G., et al. J Biol Chem 278, 18162-18169 (2003); Kanda, H., et al. Nat Immunol 9, 415-423 (2008); Giganti, A., et al. J Biol Chem 283, 7776-7789 (2008)). However, the source of circulating ATX and its regulation remains to be elucidated. Recently, liver sinusoidal endothelial cells were shown to efficiently remove ¹²⁵I-labelled ATX from the circulation and to degrade this enzyme (Jansen, S., et al. Cancer Lett (2009)). This finding is supported by our observation that neither ATX protein nor ATX activity were detected in bile (Fig 3f,g).

In chronic cholestatic liver diseases such as PBC pruritus often develops at early stages months to years before the onset of jaundice (Kremer, A.E., Beuers, U., Oude- Elferink, R.P. & Pusl, Drugs 68, 2163-2182 (2008)), whereas this perception often subsides or even disappears in end-stage liver disease when cholestasis is most pronounced. In addition, itch is the first and defining symptom in women with ICP, who have only mildly elevated serum BS concentrations (in our group: 15 μΜ). Interestingly, ATX activity was suppressed by the 3,7-dihydroxy-bile salts chenodeoxycholate (CDC), ursodeoxycholate (UDC) and their glyco- and tauro- conjugates (glycochenodeoxycholate (GCDC), taurochenodeoxycholate (TCDC), glycoursodeoxycholate (GUDC), and tauroursodeoxycholate (TUDC)). Other bile salts such as cholate (trihydroxy-BS) (C), lithocholate (monohydroxy-BS) (LC), deoxycholate (7,12-dihydroxy-BS) (DC) and their conjugates hardly influenced the enzymatic activity of ATX (Fig 3h). The bile salts inhibiting ATX showed a IQ well within the pathophysiological range of serum BS levels during advanced cholestasis. This inhibitory effect could explain the enigmatic amelioration of pruritus in end-stage liver disease as well as the therapeutic effect of ursodeoxycholate that is regarded as first line treatment for patients with ICP because it was shown to reduce cholestasis and to ameliorate pruritus (EASL Clinical Practice Guidelines: Management of cholestatic liver diseases. J Hepatol 51, 237-267 (2009); Kondrackiene, J., Beuers, U. & Kupcinskas, L. Gastroenterology 129, 894-901 (2005)).

2.5 Induction o f pruritus by LP A

To investigate the potential pruritogenic effect of LPA in vivo we used female C57BL/J6 mice. A Teflon-coated magnet was implanted in the hind paw of C57BL/6J mice. Their cage was put in a magnetic coil, and movements were continuously recorded. Intradermal injection of LPA, but not the carrier, induced significant scratching behaviour shown as number of scratch bouts per 5-minute intervals (Fig. 4a), which was in line with previous reports (Hashimoto et al, 2004, Pharmacology 72, 51-56; Kim et al, 2008, Eur J Pharmacol 583, 92-96). The scratching behaviour due to the intradermal injection of LPA increased dose-dependently (Fig. 4b).

3. Discussion

Although being a serious problem for patients and clinicians alike, pruritus has for a long time being largely neglected by researchers. But only the discovery of itch- specific sensory neurons in the skin by Schmelz et al. revolutionized the research field of pruritus (Schmelz et al, 1997, J Neurosci. 17, 8003-8008). These primary sensory neurons only responded to histamine, but were insensitive to mechanically-induced pain stimuli. Recently, another class of itch-specific sensory neurons has been described that mediates pruritic stimuli independent of histamine (Davidson et al, 2007, J Neurosci. 27, 10007-10014). Thus, pruritoceptive nerve fibres seem to consist of different subsets of neurons as already known for nociceptive nerve fibres. This could explain the varying characters of itch sensations in different diseases ranging from tickling over "burning and painful" to agonizing pruritus (Stander and Schmelz, 2006, Eur J Pain 10, 473-478). In diverse systemic disorders including cholestatic liver diseases, renal insufficiency, endocrine, hematologic and metabolic diseases, various infections as well as certain malignancies pruritus is commonly reported.

In the case of cholestatic disorders, bile salts and endogenous opioid peptides have been hypothesized as pruritogens (Jones and Bergasa, 1990, supra). However, in line with previous reports (Murphy et al. (1972) Gut. 13 :201-206; Ghent et al. (1977) Gastroenterology 73: 1125-1 130; Bartholomew et al. (1982) Clin Sci (Lond) 63 :65-73; Spivey et al. (1994) Am J gastroenterol 89:2028-2032) we have found no correlation between the severity of itch and these two parameters. In contrast, we have found strong evidence that the occurrence of itch is associated with increased systemic levels of the signalling lipid mediator, lysophosphatidic acid. The serum level of this factor closely correlated with the occurrence of itch. Moreover, this correlation extends to the serum enzyme autotoxin that is responsible for the production of this factor. Autotaxin is a lysophospholipase D that converts lysophatidylcholine into lysophosphatidic acid. Not only did increased autotaxin levels correlate with the occurrence of itch, but the level of serum autotaxin also closely correlated with the extent of itch perception as objectified by the patient's scoring on a visual analogue scale, which was determined long before determination of serum autotaxin levels in these patients. Moreover, we found that relief of itch in PBC patients by interruption of the enterohepatic cycle also significantly decreased the serum autotaxin levels. We subsequently confirmed the finding reported by others that intradermal injection of LPA in mice caused causes short-lasting but significant scratching behaviour. Taken together these findings strongly suggest that autotaxin plays a causative role in the induction of itch during cholestasis.

The subsequent question that rises is that of the source of increased autotaxin levels. In principle enhanced levels could be caused by increased ATX expression or by reduced clearance of the enzyme. It has been suggested that the liver plays an important role in the latter process. Although we did not yet investigate this possibility, we observed that human hepatocytes have a 5-fold increased expression of autotaxin when incubated with chenodeoxycholate, a potent inductor of FXR signalling. This suggested that during cholestasis the liver becomes an important source of circulating ATX. If this is the primary mechanism, there should be a clear correlation between the level of circulating bile salts and the extent of itch, but we showed that this is clearly not the case. Interestingly, however, we also showed that certain bile salts (3,7-dihydroxy bile salts) inhibit ATX with a IQ that is well within the pathophysiological range of serum bile salt levels during advanced cholestasis. Hence, this effect may very well mask the inductive effect of bile salts on ATX expression, because at higher serum bile salt levels the induction of expression may be annihilated by the high serum bile salt levels. In this context it is important to mention that ursodeoxycholate also inhibited ATX activity; this bile salt is administered to many patients with ICP and PBC because it was shown to reduce cholestasis and relieve the itch.

In cholestatic disorders, bile salts have been among the hypothesized pruritogens (Jones et al. (1990) Hepatology 1 1 :884-887). During cholestasis, bile salts but also various other substances accumulate in circulation and tissue. Indeed, bile salts being injected intradermally induced pruritus in healthy individuals (Kirby et al. (1974) Br Med J 4:693-695) and anion exchange resins such as cholestyramine which bind bile salts in the gut ameliorated pruritus (Datta et al. (1966). Gastroenterology 50:323-332) However, pruritus is neither seen in every patient with cholestatic liver disease (Murphy et al. (1972) Gut 13:201-206) nor does a correlation exist between itch intensity and bile salt concentrations in serum or tissue (Ghent et al. supra. ; Bartholomew et al. supra). In line with these results, we confirmed that total bile salt levels did not correlate with itch intensity in our large patient group.

The putative role of endogenous opioids in the pathogenesis of cholestatic pruritus was upon the clinical observation that pruritus improved upon treatment with μ-opioid-receptor antagonists such as naltrexone (B ergasa et al . ( 1 992) Gastroenterology 102:544-549; Wolfhagen et al. (1997) Gastroenterology 113: 1264- 1269). Indeed, elevated serum concentrations of endogenous opioids were observed in plasma of cholestatic patients (Thornton et al. (1988) Bmj 297: 1501-1504) and in rats made cholestatic by bile duct resection (Swain et al. (1992) Gastroenterology 103: 630- 635). Nevertheless, a correlation between pruritus scores and opioid concentrations has never been shown (Spivey et al. (1994) Am J Gastroenterol 89:2028-2032). We measured total μ-opioid activity in sera of healthy female controls, pregnant women, intrahepatic cholestasis of pregnancy (ICP) and primary biliary cirrhosis (PBC) patients with and without pruritus. Although some patients with PBC had elevated levels there was no correlation with itch, indicating that μ-opioids are unlikely to play a key role in the pathogenesis of cholestatic pruritus. Thus, there must be other factors causing pruritus in cholestasis, which are only enhanced in those patients suffering of this bothersome perception and which correlate with itch intensity.

We hypothesized that among various substances being normally secreted into bile there exist one or more pruritogens that accumulate in circulation and tissues during cholestasis. These pruritogens may either directly or indirectly activate pruritoceptive neurons. It is very likely that these pruritogens undergo an enterohepatic circulation as its interruption by nasobiliary drainage or external biliary diversion dramatically improves pruritus: within hours the agonizing perception subsides (Emerick et al. (2002) Hepatology 35 : 1501-1506; Beuers et al. (2006) Hepatology 44:280-281 ; Stapelbroek et al. (2006) Hepatology 43 :51-53). By screening blood samples of pruritic cholestatic patients for the presence of substances being able to activate neuronal cell lines we identified lysophosphatidic acid (LPA), a small but highly bioactive phospholipids (Mills et al. (2003) Nat Rev Cancer 3:582-591; Aoki et al. (2008) Biochim Biophys Acta 1781 :513-518), as a possible pruritogen. Among its various actions, LPA is known to be increased during pregnancy and its concentrations correlate with the gestational week (Tokumura et al. (2002) Biol Reprod 67: 1386- 1392). Here we show that pregnant women suffering from intrahepatic cholestasis of pregnancy (ICP) have even significantly higher LPA levels compared to gestational matched pregnancies.

Circulating LPA is in large parts formed through cleavage of choline from lysophosphatidylcholme by autotoxin (ATX) (Tanaka et al. (2006) J biol chem 281 :25822-25830; Van Meeteren et al. (2006) Mol Cell Biol 26:5015-5022), an enzyme that was shown to be identical to lysophospholipase D (Umezo-Goto et al. (2002) J Cell Biol 158:227-233; Tokumura et al. (2002) J biol chem 277:39436-39442). Hence, inappropriate storage of blood samples may easily lead to artificially high LPA concentrations. To avoid this pitfall, we decided to determine the ATX activity in blood as a more reliable parameter of LPA production. Doing so, we observed that blood samples of ICP women showed highly significant increased ATX activities compared to pregnant controls and female controls. Regular pregnant women are however not cholestatic and thus represent not a proper control group. Analyzing the ATX activity in sera of cholestatic patients of both genders, we found that ATX activity and protein content was only enhanced in those cholestatic patients suffering of pruritus. Intriguingly, ATX activities in these patients highly correlated with itch intensities. Even more striking, in PBC patients treated with nasobiliary drainage for long-lasting, otherwise intractable pruritus, ATX activity dropped parallel to tremendous reduction or relief of the bothersome perception. As this is an invasive procedure, the drainage has to be removed after some days (Beuers et al. (2006) Hepatology 44:280-281 ; Stapelbroek et al. Hepatology 43:51-53). Pruritus re-appeared in all patients after several weeks. And indeed, ATX activity rose back to baseline values in all these patients upon reappearance of pruritus. Thus, ATX closely correlated with the itch perception and might represent a key mediator of pruritus in cholestasis.

Thus, in those cholestatic patients suffering of pruritus a compound accumulates that increases the content of ATX in circulation by a yet unknown mechanism. Interestingly, in primary biliary cirrhosis pruritus generally develops month to years before the onset of jaundice (Kremer et al. (2008) Drugs 68:2163-2182), whereas this perception often improves or even disappears in end-stage liver diseases when cholestasis is most pronounced. This clinical observation has also been used to discard bile salts as well as opioids as key pruritogens. Surprisingly we found that high levels of bile salts inhibited ATX activity which could explain this enigmatic amelioration of pruritus in end-stage liver diseases.

In vivo we could show that intradermal application of LPA led to scratch responses in mice which was in line with previous reports (Hashimoto et al. (2004) Pharmacology 72:51-56; Kim et al. (2008) Eur J Pharmacol 583 :92-96). Furthermore, in a mouse model of benign recurrent intrahepatic cholestasis (BRIC) (Paulusma et al. (2009) J Biol Chem 284:9947-9954) chronic cholestasis is accompanied by increased ATX levels in serum, although we could not observe increased scratching behaviour. Why the transition from enhanced ATX activity to pruritus in this mouse model does not take place remains to be elucidated. However, one has to consider that humans act differently from mice and that increased scratching behaviour has never been observed in any cholestatic mouse model.

Several other pathophysiological conditions are associated with pruritus in which ATX and LPA might play a potential role in the pathogenesis of itch. Wound healing after injury or surgery is typically characterized by itch perception. LPA promotes re-epithelialization and healing of cutaneous wounds by proliferation and migration of fibroblasts (Balazs et al. (2001) Am J Physiol Regul Integr Comp Physiol 280:R466-472). High local concentrations of LPA might thus be able to elicit itch- specific neurons leading to the well-known desire to scratch a wound during its healing process. Pruritus is also a common symptom in lymphoma patients especially in those with Hodgkin's disease. Approximately every third patients suffers from this disabling symptom generally at those skin parts being closely located to affected lymph nodes (Rubenstein et al. (2006) Int J Dermatol 45:251-256). Recently, EBV-infected Hodgkin lymphoma cells have been shown to highly express ATX (Baumforth et al. (2005) Blood 106:2138-2146). Thus, these cells might release high amounts of ATX forming high local concentrations of LPA which not only enhance tumor growth but may also activate the neuronal itch pathway. Intriguingly, patients with Hodgkin disease suffering of intense pruritus had a significant shorter survival time than that of mild and non-itching cases (Gobbi et al. (2009) Cancer 51 : 1934-2146). Furthermore, increased expression of ATX has been reported in several other tumor entities such as malignant melanoma, kidney, colon, and breast cancer (Mills et al. (2003) Nat Rev Cancer 3:582- 591). The enhanced formation of ATX mainly in tumors located in the skin, e.g. malignant melanomas, could explain the onset of pruritus in those diseases. It is attractive to speculate whether our results of pruritus in cholestasis may also hold for the pathogenesis in other systemic diseases.

The molecular elucidation of pruritus in systemic diseases will have major impact for novel treatment options of this agonizing symptom. ATX inhibitors and LPA receptor inhibitors which are currently developed for the treatment of cancer patients to reduce disease progression and forming of metastasis (Peyruchaud et al. (2009) Anticancer Agents Med Chem 9:381-391) might also represent a novel class of anti-pruritic drugs. Our model of ATX and LPA provides insight in the pathogenesis of cholestatic pruritus and opens possibilities for causative therapy of a yet insufficiently treatable perception.

Example 2

Preparation of HA130

Materials and Methods. All chemicals were obtained from Sigma- Aldrich and used without further purification. Dry N,N-dimethylformamide (DMF) was obtained by treatment with molsieves (4 A). Analytical TLC was performed on aluminium sheets precoated with silica gel 60 F254. Column chromatography was carried out on silica gel (0.035-0.070, 90A, Acros). For isolation by centrifugation, a Heraeus Multifuge 3 S-R centrifuge was used. Products were spun at 4400 x g, at 4 °C for 5 min. Nuclear magnetic resonance spectra ('H-NMR and COSY) were determined in deuterated dimethyl sulfoxide (d₆-DMSO) using a Bruker ARX 400 Spectrometer (1H: 400 MHz) at 298 K, unless indicated otherwise. Peak shapes in the NMR spectra are indicated with the symbols 'd' (doublet), 'dd' (double doublet), V (singlet), 'bs' (broad singlet) and 'm' (multiplet). Chemical shifts (δ) are given in ppm and coupling constants J in Hz. DMSO (δ=2.50 ppm) was used as internal reference. LC-MS measurements were performed on a system equipped with a Waters 2795 Seperation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190-750 nm), Waters Alltima C18 Column (2.1x100 mm) and an LCT™ Orthogonal Acceleration Time o f F light Mass Spectrometer. Samples were run at a flow rate of 0.40 mL min^"1 using gradient elution (water/acetonitrile/formic acid) from 950/50/10 (v/v/v) to 50/950/10 (v/v/v).

Synthesis of 3-[(4-formylphenoxy)methyl]benzene boronic acid (formula [2]) To a solution of 4-hydroxybenzaldehyde (0.185 g, 1.52 mmol) and KOH (0.1 16 g, 2.07 mmol) in DMSO (1.4 mL), 4-bromomethylphenylboronic acid (0.302, 1.41 mmol) was added. The reaction mixture was stirred at room temperature and after 4 h the reaction mixture was poured into 0.5 M HC1 solution. The title product was isolated as a precipitate by centrifugation, washed and dried under high vacuum. Yield: 57 %. 1H-NMR: δ =9.87 (s, 1H, c), 8.01 (s, 2H, OH), 7.88 (m, 3H, d+i₂), 7.77 (d, J 7.8, 1H, ii), 7.51-7.35 (m, 2H, j+k), 7.21 (d, J 8.7, 2H, g), 5.22 (s, 2H, h). MS: m/z [M+H]⁺ calc. 257.10, obs. 257.28. LC: R_t=7.90.

Formula [2]

Synthesis of 3-(4-fluorobenzyl)-l,3-thiazolane-2,4-dione (formula [3]). To 2 ,4- thiazolidinedione (1.01 g, 7.77 mmol) dissolved in dry DMF (40 mL), sodium hydride as solid (0.333 g, 8.33 mmol) and 4-fluorobenzylbromide (6.39 mmol) were added and the suspension was stirred for 22 h at room temperature. The reaction mixture was concentrated in vacuo at 328 K. Subsequently, the crude product was purified by column chromatography (dichloromethane) to afford the title product in homogeneous form.

Yield: 53 %. 1H-NMR: δ =7.34-7.14 (m, 4H, x+y), 4.65 (s, 2H, z), 4.26 (s, 2H, b). MS: m/z [M+H]⁺ calc. 226.03, obs. 226.04. LC: R_t=8.22. Formula [3]

Synthesis of (2,4-dioxo-l,3-thiazolan-5-yliden) benzoic acids and benzene boronic acid. Piperidine (12 μΐ_^, 0.207 mmol) and 3-[(4-formylphenoxy)methyl]benzene boronic acid (0.352 mmol) were added to a solution of 3-(4-fluorobenzyl)-l,3- thiazolane-2,4-dione (66.0 mg, 0.293 mmol) in ethanol (2.5 mL); the solution was refluxed for 22 h. Upon cooling to room temperature the product precipitated.

Centrifugation and washing with ethanol yielded the homogeneous title compound.

3-[(4-{[3-(4-fluorobenzyl)-2,4-dioxo-l,3-thiazolan-5- yliden]methyl}phenoxy)methyl]benzene boronic acid (HA130)

Yield: 64 %. 1H-NMR: δ =8.08 (bs, 2H, OH), 7.92 (s, 1H, c), 7.87 (s, 1H, i₂), 7.76 (d, J 7.4, 1H, i), 7.60 (d, J 7.1, 2H, d), 7.50-7.16 (m, 10H, d+g+k+j+x+y), 5.18 (s, 2H, h), 4.82 (s, 2H, z). MS: m/z [M +H]⁺ calc. 464.11, obs. 464.40. LC: R_t=10.72.

Formula [1]

Claims

Claims

1. A method for diagnosing (histamine independent) pruritus in subject, wherein the method comprises determining the level of at least one of LPA and autotaxin in a sample from the subject, wherein the sample preferably is blood, serum, plasma or a biopsy.

2. A method according to claim 1, wherein at least one of the plasma LPA and the plasma autotaxin level is determined.

3. A method according to claim 1 or claim 2, wherein at least one of:

a) a plasma LPA level that is at least 110% of the average plasma LPA in corresponding healthy control subjects; and,

b) a plasma autotaxin level that is at least 1 10% of the average plasma autotaxin level in corresponding healthy control subjects,

is indicative of pruritus.

4. A method according to any one of claims 1 - 3, wherein the level of autotaxin is determined by determination of the level of autotaxin activity.

5. A method according to any one of claims 1 - 3, wherein prior to determination of the LPA level the sample is maintained or stored under conditions that prevent or reduce hydrolysis of LPC to LPA, preferably by adding an autotaxin inhibitor to the sample before maintenance or storage.

6. A method according to any one of claims 1 - 5, for diagnosis of systemic or local pruritus.

7. A method according to any one of claims 1 - 6, wherein the subject is a subject with at least one of cholestasis, uremia, wound healing, a keloid, a malignancy, an endocrinological disorder and atopic dermatitis, preferably wherein the subject is a subject with cholestasis wherein the primary cause of the cholestasis is selected from the group consisting of intrahepatic cholestasis of pregnancy (ICP), benign recurrent intrahepatic cholestasis (BRIC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hilar lymphadenopathy and malignancy.

8. An agent that reduces triggering of an LPA receptor for use in the treatment or prevention of (histamine independent) pruritus in a subject.

9. Use of an agent that reduces triggering of an LPA receptor for the manufacture of a medicament for the treatment or prevention of (histamine independent) pruritus in a subject.

10. An agent according to claim 8 or a use according to claim 9, wherein the agent is an autotaxin inhibitor or an LPA receptor inhibitor.

11. An agent or use according to claim 10, wherein the agent is an autotaxin inhibitor, which is herein defined as a compound that at a concentration of 17000 nM or less results in at least 50% inhibition of autotaxin as compared to a control sample without the agent in the assay described by Nakamura, K., et al. Clin Biochem 40, 274- 277 (2007).

12. An agent or use according to claim 10, wherein the autotaxin inhibitor is selected from the group consisting of: FTY720 (= fmgolimod), HA130, BrP-LPA, S32826, VPC8a202, Damnacanthal, Hypericin and derivatives of these compounds.

13. An agent or use according to claim 10, wherein the agent is LPA receptor inhibitor, which is herein defined as a compound that at a concentration of 25000 nM or less results in at least 50% inhibition of the LPA receptor as compared to a control sample without the agent in an assay as described in any of the following references Ohta et al. (2003) Mol Pharmacol 64: 994-1005; Fischer et al. (2001) Mol Pharmacol 60:776-784; Beck et al. (2008) Bioorg Med Chem Lett 18: 1037-1041; Virag et al. (2003) Mol Pharmacol 63: 1032-1042; Heise et al. (2001) Mol Pharmacol 60: 1173-1180; Zhang et al. (2009) Cancer Res Epub; Yamamoto et al. (2007) Bioorg Med Chem Lett 17:3736-3740; Fells et al. (2008) Bioorg Med Chem 16:6207-6217; Liliom et al. (2006) BBA 1761 : 1506-1514.

14. An agent or use according to claim 10, wherein the LPA receptor inhibitor is selected from the group consisting of: ΚΪ16425, DGPP 8:0, Tetradecyl Phosphonate, Compound 35 , FAP-12, VPC12249, BrP-LPA, Compound 34, NSC161613, H2L5105099, FMP and derivatives of these compounds.

15. An agent or use according to any one of claims 8 - 13, wherein the subject is a subject with at least one of cholestasis, uremia, wound healing, a keloid, a malignancy, an endocrinological disorder and atopic dermatitis, preferably wherein the subject is a subject with cholestasis wherein the primary cause of the cholestasis is selected from the group consisting of intrahepatic cholestasis of pregnancy (ICP), benign recurrent intrahepatic cholestasis (BRIC), progressive familiar intrahepatic cholestasis (PFIC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), Alagille syndrome, drug-induced cholestasis, alcoholic hepatopathy, chronic viral hepatitis B (HBV) and/or C (HCV) infections, hilar lymphadenopathy and obstructive malignancy such as cholagniocellular carcinoma (CCC), pancreatic cancer, and metastases.