THE USE OF 5-HT7 RECEPTOR ANTAGONISTS INCLUDING SOME ATYPICAL

ANTIPSYCHOTICS AS ANTIPRURITIC AGENTS

Field of Invention

The present invention refers to importance of targeting of 5-HT7 receptors in the treatment of pruritus and the use of 5-HT7 receptor antagonists including some atypical sntipsychotics at peripheral and central levels for the treatment of pruritus.

Background of Invention

The treatment of chronic pruritus in skin diseases such as atopic dermatitis, contact dermatitis, psoriasis or xerosis (dry skin) or in allergic conjunctivitis, cholestatic liver disease, kidney disease and hemorrhoids is difficult in clinic with the current available drugs (Stander et al., 201 1 ; Davidson and Giesler, 2010; Stander et al., 2008). Chronic pruritus is a worldwide clinical problem with the estimated prevalence of 20 to 27 % of all adults worldwide (Weisshaar E and Dalgard F., 2009; Dalgard et al., 2007). The scratching behaviour to relieve an itch in pruritus can lead to skin/mucosal damage or inflammation, impaired quality of life and sometimes to depression or even suicide of the sufferers (Stander et al., 2010).

The current antipruritic pharmacotherapeutic agents consist of mainly corticosteroids, immunosuppressive agents, antihistamines and antiallergic drugs (Stander et al. , 2008). The efficacy of corticosteroids and immunosuppressive agents have been proven, but their use are generally associated with serious side effects. The efficacy of antihistamines and antiallergic agents are limited (Stander et al., 2008). Thus, the development of new and targeted antipruritic agents against pruritus with high efficacy and low side effect profile is mandatory.

Recent studies have been suggested that itch spesific mediators exist not only in the- skin but also in the central nervous system (Potezieri and Undem, 201 1 ; Cevikbas et al., 2010). The identity of itch-spesific mediators in the central nervous system provide a novel therapeutic approach for antipruritic drug development and expand current strategies from targeting the skin (such as glucocorticoids, antihistamines and antiallergic drugs) to the central nervous system (Cevikbas et al., 2010; Tey and Yosipovitch, 201 1 ; Stander et al., 2008).

Pruritus is mediated by free nerve endings of sensory C-fibers that are located at the dermo- epidermal junction and transmitted from skin to the dorsal-horn of the spinal cord, up the spinothalamic tract to the thalamus and sensorial cortex, analogies to pain sensation (Stander et al., 201 1 ; Davidson and Giesler, 2010; Cevikbas et al. , 2010; Potezieri and Undem, 201 1 ). Recent studies showed that not only the main mediator histamine but also more diverse molecular mediators are involved in the pathogenesis of pruritus (Stander et al. , 201 1 ; Davidson and Giesler, 2010). A positron emission tomography study demonstrated that intradermal histamine induce activation in thalamus and somatosensorial cortex (Drizezga et al., 2001 ). It is known that intradermal administration of histamine induces pruritus in human and laboratory animals and this effect is reversed by antihistaminic drugs (Potezieri and Undem, 201 1 ). However, ineffectiveness of antihistaminic drugs in a variety of pruritic diseases, such as atopic dermatitis, support the notion that many other substances, apart from histamine, are involved in the pruritus-spesific pathophysiological mechanism (Davidson and Giesler, 2010)

Serotonin is another important mediator of itch in human. While serotonin evoke a weaker itch sensation than histamine when applied to healthy human skin, it produces strong and robust itching behaviour (scratching) in mice and rats (Akiyama et al., 2009a; Shimada et al. , 2006). However, it has been reported that serotonin exerts much stronger itch sensation when administered to lesioned skin in patients with atopic dermatitis. 5-HT stimulates action potential discharge in a subset of human cutaneous C-fibres (Potenzieri and Undem, 2012). Serotonin mediates its biological effects via 5-HT receptors (Millan, 2002). Currently, there are seven families of 5-HT receptors (5-HT1 -7) (Millan, 2002). Based on studies using selective 5-HT receptor subtype agonists and antagonists, it appears that HT1 , 5-HT2 and 5-HT3 receptors are involved in the processing of itch (Potenzieri and Undem, 201 1 ; Akiyama et al., 2010; Kim et al., 2008; Akiyama et al., 2009b; Yamaguchi et al, 1999;). It is noteworthy that the demonstration of pretreatment with ketanserin, a 5-HT2A receptor antagonist, blocks intradermal serotonin and 12(S)-HPETE, a 12-lipoxygenase metabolite of arachidonic acid, -induced scratching implicate that 5-HT2A receptor is involved in the pathophysiology of pruritus (Potezieri and Undem, 201 1 ; Yamaguchi et al, 1999; Kim et al, 2008).

US Patent No. 5,521 , 183 A discloses use of 5-HT ligands as antipruritic agents. In this patent application, 5-HT ligands consist of 5-HT1 , 5-HT2, 5-HT3 and 5-HT4 receptor agonists, partial agonists and antagonists as antipruritic agents.

One of the most recently identified subtypes of 5-HT receptors is 5-HT7 receptor (Leopoldo et al. , 2007). 5-HT7 receptors have been shown to couple G _s protein and stimulate cAMP formation (Leopoldo et al, 201 1 ; Millan, 2002). Selective agonist induced activation of 5-HT7 receptors increase cAMP and cause excitation in primary afferent neurons (Millan, 2002; Leopoldo et al., 201 1 ). Particulary high levels of 5-HT7 receptors have been detected in thalamus, hypothalamus, cortex and cerebellum. Important roles for 5-HT7 receptors have been suggested in depression, anxiety, epilepsy, schizophrenia and sleep disturbances (Matthys et al., 201 1 ; Leopoldo et al. , 201 1 ; Stahl, 2010). Beside these data, immunocytochemical studies have shown that 5-HT7 receptors are highly expressed in the superficial layers of spinal cord dorsal horn (lamina I ve II) which is consistent with a predominant role of the 5-HT7 receptors in the control of pain (Doly et al., 2005). A variety of studies provide evidence that 5-HT7 agonists have analgesic effect and a patent application has been filed as the use of 5-HT7 receptor agonists for the treatment of pain. However, some studies provide evidence that 5-HT7 receptor agonists may exert hyperalgesic rather than an analgesic effect.

Although itch and pain are separate sensory modalities, owing to sharing of similar neuronal afferent pathways by itch and pain, there is a close interaction between these two sensations (Davidson and Giestler, 2010). Reducing of itch sensation by the painful counter stimuli (e.g. scratching), itch inducing side effects of strong analgesic opioids, the activation of nociceptive primary afferent fibers by itch producing agents, relieving of both pain and itch sensation by surgical anterolateral funiculus lesion, and individuals with congenital insensitivity to pain are also insensitive to itch, all together suggest that itch and pain should be strongly interconnected (Davidson and Giestler, 2010).

The present invention is related to the role of 5-HT7 receptors in pruritus. While the previous studies provide evidence that 5-HT1 , 5-HT2 and 5-HT3 receptors participate in the processing of pruritus (Potezieri and Undem, 201 1 ; Akiyama et al., 2010; Kim et al., 2008; Akiyama et al., 2009b; Yamaguchi et al., 1999), due to our knowledge, there is no study evaluating the role of 5-HT7 receptors in pruritus. Additionally, some atypical antipsychotic agents possess 5-HT7 receptor antagonist properties, in addition to 5-HT2A and D2 receptor antagonism. Our invention provides a method for treating pruritus using any pharmaceutical formulation which contain 5-HT7 receptor antagonist alone including some atypical antipsychotic agents or its combination with other drugs.

In this invention, preferred embodiments of selective 5-HT7 receptor antagonist as an antipruritic agent encompasses the use of the compound with higher affinity by a factor of at least 30 to 100 over the other 5-HT receptors (5-HT1 , 5-HT2, 5-HT3, 5-HT4, 5-HT5 and 5-HT6) in peripheral tissues or central nervous system. The selectivity of any compounds to 5-HT7 receptors is calculated by binding studies and expressed as a pKi value. V.Pittala and D.Pittala (201 1 ) are described selective 5-HT7 receptor antagonists in Mini Rev Med Chem. ; 1 1 (3);1 108- 21 .

After cloning 5-HT7 receptors, it had been reported that many of old drugs have also 5-HT7 receptor antagonist properties. Especially, several drugs used in psychosis, atypical antipsychotics including risperidone, its metabolite 9-OH-risperidon, clozapine, olanzapine, amisulpride and lurasidone known to be antagonist for other 5-HT receptor subtypes (especially 5-HT2 _A ), were also found to have very high affinity and antagonist properties for 5-HT7 receptors. Thus, atypical antipsychotics may take the advantage of a combination of spesific activity at 5-HT2A and 5-HT7 receptors in related to serotonin mechanism in the pathophysiology of pruritus. It is interesting to note that an old antihistaminic and antipruritic agent, cyproheptadine, also displays 5-HT7 receptor antagonistic effect. In this invention, preferred embodiments of using some atypical antipsychotic as an antipruritic agent encompasses the use of the atypical antipsychotic with higher affinity to 5-HT7 and 5- HT2A receptor by a factor of at least 30 to 100 over the other 5-HT receptors (5-HT1 , 5-HT3, 5- HT4, 5-HT5 and 5-HT6) in peripheral tissues or central nervous system. The selectivity of any atypical antipsychotics to 5-HT7 and 5-HT2A receptors (dual inhibition) are calculated by binding studies and expressed as a pKi value .

In this invention, using a selective 5-HT7 receptor agonist, LP44 (Monti et al., 2008; Leopoldo et al., 2007), and a selective competitive 5-HT7 receptor antagonist, SB 269970, we firstly demonstrate the important role of 5-HT7 receptors in pathogenesis of pruritus. Secondly, we demonstrate that olanzapine and risperidone, two atypical antipsychotic which show, elicit highly potent antipruritic effect via their 5-HT7 receptor antagonist properties or dual antagonist properties of 5-HT7 and 5-HT2A receptors. Owing to the present patent application describing the importance of 5-HT7 receptor system in the processing of pruritus, the present patent application protects the use of all compounds with 5-HT7 receptor antagonistic efficacy as antipruritic agents including some atypical antipsychotics. These agents consist of selective 5- HT7 receptor antagonists, SB 269970, SB 258719, SB-691673, DR-4004, DR-4365 and LY 215840 and atypical antipsychotics, risperidone, 9-OH-risperidon, olanzapine, clozapine, amisulpride, lurasidone.

Brief Description of The Invention

The present invention is related to the role of 5-HT7 receptors in treatment of pruritus and mainly the use of selective 5-HT7 receptor antagonists including some atypical antipsychotics (dual inhibitor of 5-HT7 and 5-HT2A ^" receptor) or 5-HT7 receptor modulating drugs as an alternative antipruritic agent in treatment of pruritic diseases.

The present invention provides a method for treating pruritic patients by administering any pharmaceutical formulation containing therapeutically effective amount of 5-HT7 receptor antagonists or any drugs modulating 5-HT7 receptors .

This invention also provides a pharmaceutical composition which comprise admixing a pharmaceutical acceptable carrier and a pharmaceutical acceptable amount of chemical compound which act as 5-HT7 receptor antagonist or modulate 5-HT7 receptors.

The intradermal injection of the 5-HT7 receptor agonist produced dose-dependent itching behaviour (scratching) in mice. Systemic or spinal administration of the 5-HT7 receptor antagonist totally blocked intradermally injected serotonin, histamine and 5-HT7 receptor agonist induced pruritic behaviour. Additionally, systemic or spinal administration of olanzapine and risperidone also potently blocked intradermally injected serotonin, histamine and 5-HT7 receptor agonist induced pruritic behaviour. The present invention refers to 5-HT7 receptors as a new therapeutic target for pruritus. The present invention describes antipruritic mechanism of action of 5-HT7 antagonist at the level of peripheral tissues and central nervous sytem.

This invention protects the production of any antipruritic pharmaceuticals which contain 5-HT7 receptor antagonist or any other drugs which modulate 5-HT7 receptors as an active ingredient. According to this invention, any antipruritic formulation which comprise aferomentioned active ingredient as well as pharmaceutically acceptable auxiliary material and/or additives, known to those skilled in the art, to be suitable for the particular mode of administration may be prepared, oral, topical or parenteral dosage formulation.

Any pharmaceutical compound of the present invention can be used to treat itchy skin conditions, such as eczema (atopic dermatitis), contact dermatitis, psoriasis, urticeria, sun burn, insect bites and stings, contact with poisonous plants (e.g. poison ivy), biliary disease and jaundice, cholestasis, kidney disease, xeroderma, group of ichthyosis skin diseases, uremia, delusional parasitosis, itching during pregnancy, hodgkine lymphoma related itch, parasites and infections-induced itch, allergic conjuctivitis and thyroid disease related itch, diabetic itching and drug induced itch as a side effects of medication.

Objective of invention

The aim of this invention is to use the 5-HT7 receptor antagonist including some atypical antispsychotics as an active ingredient for the treatment of pruritus, a serious problem associated with a number of skin disease such as atopic dermatitis, contact dermatitis, urticeria or systemic diseases such as chronic liver and kidney failure. Another aim of this invention to use 5-HT7 receptor antagonists including some atypical antispsychotics as alternative antipruritic agents for the treatment of resistant pruritus with the current available drugs.

Description of the drawings :

Figure 1. SB 269970 (10 pg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.05% serotonin. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after serotonin injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.05% serotonin alone) group is statistically significant (P < 0.05).

Figure 2. SB 269970 (10 pg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.05% serotonin. The total number of scratches was counted over a 30 min time period after intradermal injection of 0.05% serotonin. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.05% serotonin alone) group is statistically significant (P< 0.05). Figure 3. SB 269970 (20 mg/kg) was administered intraperitoneal^ (i.p.) 20 min prior to the intradermal injection of 0.05% serotonin. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after serotonin injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.5% serotonin alone) group is statistically significant (P < 0.05).

Figure 4. SB 269970 (20 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.05% serotonin. The total number of scratches was counted for 30 min after intradermal injection of 0.05% serotonin. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.05% serotonin alone) group is statistically significant (P < 0.05). Figure 5. SB 269970 (10 pg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.25% histamine. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after histamine injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (2.5% histamine alone) group is statistically significant (P < 0.05).

Figure 6. SB 269970 (10 pg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.25% histamine. The total number of scratches was counted for 30 min after intradermal injection of 0.25% histamine. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the control (2.5% histamine alone) group is statistically significant (P < 0.05).

Figure 7. SB 269970 (20 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.25% histamine. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after histamine injection. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the control (0.25% histamine alone) group is statistically significant (P < 0.05).

Figure 8. SB 269970 (20 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.25% histamine. The total number of scratches was counted for 30 min after histamine injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.25% histamine alone) group is statistically significant (P < 0.05).

Figure 9. The scratch-inducing activity of intradermal injection of LP 44, a selective 5-HT7 receptor agonist. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after 0.025, 0.1 , 0.2 and 0.3% LP 44 injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the 0.9% saline control group is statistically significant (P < 0.05).

Figure 10. Dose response for the scratch inducing effect of LP 44, a selective 5-HT7 agonist. The total number of scratches was counted for 30 min after intradermal injection of 0.025, 0.1 , 0.2 and 0.3% LP44. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the 0.9% saline control group is statistically significant (P < 0.05).

Figure 11. SB 269970 (10 μg/^ μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.2% LP 44. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after LP 44 (0.25%, 0.1 %, 0.2% and 0.3%) injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent (s.e.m.). Symbol* indicates that difference from the control (2% LP 44 alone) group is statistically significant (P < 0.05).

Figure 12. SB 269970 (10 Mg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.2% LP 44. The total number of scratches was counted for 30 min after intradermal injection of 0.2% LP 44. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent (s.e.m.). Symbol * indicates that difference from the control (0.2% LP 44 alone) group is statistically significant (P < 0.05).

Figure 13. SB 269970 (20 mg/kg) was administered intraperitoneal^ (i. p.) 20 min prior to the intradermal injection of 0.2% LP 44. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after LP 44 injection. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the control (0.2% LP 44 alone) group is statistically significant (P < 0.05).

Figure 14. SB 269970 (20 mg/kg) was administered intraperitoneally (i. p.) 20 min prior to the intradermal injection of 0.2% LP 44. The total number of scratches was counted for 30 min after intradermal injection of 0.2% LP 44. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the control (0.2% LP 44 alone) group is statistically significant (P < 0.05).

Fig 15. Olanzapine or risperidone (10 pg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.05% serotonin. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after 0.05% serotonin injection. All data are presented as means of the scratches + standart error of mean (s.e. m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.05% serotonin alone) group is statistically significant (P < 0.05).

Figure 16. Olanzapine or risperidone (10 g/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.05% serotonin. The total number of scratches was counted over a 30 min time period after intradermal injection of 0.05% serotonin. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.05% serotonin alone) group is statistically significant (P< 0.05).

Figure 17. Olanzapine or risperidone (0.75 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.05% serotonin. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after serotonin injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the control (0.05% serotonin alone) group is statistically significant (P < 0.05)

Figure 18. Olanzapine or risperidone (0.75 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.05% serotonin. The total number of scratches was counted for 30 min after intradermal injection of 0.05% serotonin. All data are presented as means of the scratches t standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.5% serotonin alone) group is statistically significant (P < 0.05).

Figure 19. Olanzapine or risperidone (10 pg/10-μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.25% histamine. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after histamine injection. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.25% histamine alone) group is statistically significant (P < 0.05).

Figure 20. Olanzapine or risperidone (10 pg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.25% histamine. The total number of scratches was counted for 30 min after intradermal injection of 0.25% histamine.. All data are presented as means of the scratches t standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol * indicates that difference from the control (0.25% histamine alone) group is statistically significant (P < 0.05).

Figure 21. Olanzapine or risperidone (0.75 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.25% histamine. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after histamine injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.25% histamine alone) group is statistically significant (P < 0.05).

Figure 22. Olanzapine or risperidone (0.75 mg/kg) was administered intraperitoneal^ (i.p.) 20 min prior to the intradermal injection of 0.25% histamine. The total number of scratches was counted for 30 min after histamine injection. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.25% histamine alone) group is statistically significant (P < 0.05).

Figure 23. Olanzapine or risperidone (10 Mg/10 μΙ) was administered intrathecal^ (i.th.) 10 min prior to the intradermal injection of 0.2% LP 44. The total number of scratches was counted for 30 min after intradermal injection of 0.2% LP 44. All data are presented as means of the scratches ± standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.2% LP 44 alone) group is statistically significant (P < 0.05).

Figure 24. Olanzapine or risperidone (0.75 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.2% LP 44. The time course of scratching behaviour was observed during each 10 min interval of the 30 min session after LP 44 injection. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.2% LP 44 alone) group is statistically significant (P < 0.05).

Figure 25. Olanzapine or risperidone (0.75 mg/kg) was administered intraperitoneally (i.p.) 20 min prior to the intradermal injection of 0.2% LP 44. The total number of scratches was counted for 30 min after intradermal injection of 0.2% LP 44. All data are presented as means of the scratches + standart error of mean (s.e.m.). Error bars represent s.e.m. Symbol ^* indicates that difference from the control (0.2% LP 44 alone) group is statistically significant (P < 0.05).

Detailed description of the invention

Animal Experiments

Male Balb-C mice, weighing 25-30 gr, were used in this study to evaluate the efficacy of 5-HT7 agonist and antagonist in pruritus. Animals received an intradermal injection of 0.05% serotonin or 0.25% histamine (50 μΙ in isotonic saline) into the rostral back of interscapular area via Hamilton microinjector to induce itch behaviour (1 mg substance dissolved in 1 ml solution equal 0.1 %). Additionally, LP-44, which display selectivity for the 5-HT7 receptor over the 5- HT1A and the 5-HT2A receptors (200- and >1000-fold, respectively) (Monti et al. , 2008), injected intradermal^ at a dose of 0.025%, 0.1 %, 0.2% and 0.3% into the rostral back of interscapular area in a volume of 50 μΙ in isotonic saline via Hamilton microinjector to evaluate any itching behaviour. To induce pruritic behaviour (strach), pru itogenic chemicals, 0.05% serotonin, 0.25% histamine and 0.2% LP 44 were administered intradermal^ to mice. The selective 5-HT7 receptor antagonist, SB-269970, which displayed at least 100-fold selectivity for the 5-HT7 receptor subtype over the other 5-HT receptor subtypes (Thomas et al. , 2002), was given intrathecal^ (i.th.) at the dose of 10 or intraperitoneally at the dose of 20 mg/kg prior to the aferomentioned intradermal^ injected pruritogens. In the second part of experiments, olanzapine, risperidone were given i.th. at the dose of 10 or intraperitoneally at the dose of

0.75 mg/kg prior to intradermal injection of 50 μΙ of 0.05% serotonin, 0.25% histamine and 0.2% LP44.

The experiments are performed as follows:

1. Animals were injected intradermal^ with pruritogenic agents, serotonin (0.05%), histamine (0.25%) and LP 44 (0.025%, 0.1 %, 0.2% and 0.3 %). Immediately after injections, animals were returned to an acrylic cage for observation of itching responses (scratch). The time course and total number of scratches were observed during each 10-min interval of the entire 30 min observation (0-10, 10-20 and 20-30 min).

2. Mice were pretreated with i.th. injection of SB 269970 (10 pg/10 μΙ) 10 min prior to the intradermal injection of serotonin (0.05%), histamine (0.25%) and LP 44 (0.2%), and the time course and total number of scratches were observed during each 10-min interval of the entire 30 min observation (0-10, 10-20 and 20-30 min).

3. Mice were pretreated with intraperitoneal injection of SB 269970 (20 mg/kg) 20 min prior to the intradermal injection of serotonin (0.05%), histamine (0.25%) and LP 44 (0.2%), the time course and total number of scratches were observed during each 10-min interval of the entire 30 min observation (0-10, 10-20 and 20-30 min).

4. Mice were pretreated with i.th. injection of olanzapine or risperidone (10 pg/10 μΙ) 10 min prior to the intradermal injection of serotonin (0.05%), histamine (0.25%) and LP 44 (0.2%), and the time course and total number of scratches were observed during each 10-min interval of the entire 30 min observation (0-10, 10-20 and 20-30 min).

5. Mice were pretreated with intraperitoneal injection of olanzapine or risperidone (0.75 mg/kg) 20 min prior to the intradermal injection of serotonin (0.05%), histamine (0.25%) and LP 44 (0.2%), the time course and total number of scratches were observed during each 10-min interval of the entire 30 min observation (0-10, 10-20 and 20-30 min).

Intrathecal drug administration to the subjects

Intrathecal injections were performed with the method of Hylden and Wilcox (1980) in which a 30-gauge needle is inserted into the lumbar space between the L5 and L6 vertebrae of unanesthetized mice. 5-HT7 antagonist was dissolved in 0.9% sterile saline and injected as a volume of 10 μΙ. The spinal 10 pg dose of SB 269970 was selected as an effective dose from our previous laboratory studies (Dogrul et al., 2006, 2009).

Statistical analysis

Results are expressed as mean and standart error of mean (n=10-15). We assessed the significance of any differences in groups using two-way repeated-measures analysis of variance. The Dunnet post-hoc test was used to compare more than two groups at corresponding time-points. Comparison of two groups was evaluated using the unpaired Student's t-test. Statistical significance was taken as P < 0.05.

Experimental Drugs

LP 44, serotonin hydrochloride and histamine dihydrochloride were purchased from Sigma- Aldrich (USA). SB 269970 was purchased from Tocris. They were dissolved in 0.9% saline

The blocking effects of pretreatments with systemic and spinal administration of 5-HT7 antagonist, SB 269970, on intradermal serotonin and histamine-induced pruritic responses

When 0.9% saline was injected, the number of total scratches were found to be 3.85 ± 0.53 within 30 min after injection.

We firstly administered serotonin as a pruritogen. Intradermal injection of 0.05% serotonin elicited a significant increase in the number of scratches at 10, 20 and 30 mins after the injection (Figure 1 ). The spinal (intrathecal, i.th.) administration of SB 269970 (10 Mg/10 μΙ) significantly inhibited serotonin-induced scratches at 10, 20 and 30 mins after serotonin injection (Figure 1 ). While the total number of scratching behaviors in response to serotonin (0.05%) alone was 38.63 ± 6.79 within 30 min, intrathecal pretreatment with SB 269970 significanly inhibited serotonin-induced scratches to 3.86 ± 2.14 (Figure 2).

Systemically, intraperitoneal (i.p.) administration of SB 269970 (20 mg/kg) significantly inhibited serotonin-induced scratches at 10 and 20 mins after serotonin injection (Figure 3). While serotonin (0.05%) alone displayed a total scratching number of 38.28 ± 4.9 within 30 min, systemic i.p. SB 269970 (20 mg/kg) pretreatment significanly inhibited serotonin-induced scratches to 6 (Figure 4).

The second pruritogenic agent we administered was histamine. Intradermal injection of 2.5% histamine elicited a significant increase in the number of straches at 10, 20 and 30 mins after the injection (Figure 5). The i.th. administration of SB 269970 (10 pg/10 μΙ) significantly inhibited histamin-induced straches at 10 and 20 mins after histamine injection. While the number of scratching behaviors in response to histamine (0.25%) alone was 23.50 ± 2.9 within 30 min, intrathecal pretreatment with SB 269970 significanly inhibited histamine-induced scratches to 8.57 ± 2.6 (Figure 6).

Systemically, i.p. administration of SB 269970 (20 mg/kg) significantly inhibited histamine- induced straches at 10 and 20 mins after histamine injection (Figure 7). While histamine (0.25%) alone displayed a total scratching number of 22.6 ± 2.51 within 30 min, systemic 269970 (20 mg/kg) pretreatment significanly inhibited histamine-induced scratches to 8.1 ± 2.4 (Figure 8).

The pruritus inducing effects of intradermal injection of 5-HT7 receptor agonist.

When injected intradermal^, the selective 5-HT7 receptor agonist LP 44 (0.1 %, 0.2% and 0.3%) produced a dose dependent and statistically significant scratching behaviour at 10, 20 and 30 mins after the injection when compared to control group (Figure 9). The lowest dose of LP 44 (0.025%) elicited a significant scratching effect at 10 and 20 mins after the injection (Figure 9). When the total number of scratches within 30 min after LP 44 injection were evaluated, LP 44, at all doses used (0.025%, 0.1 %, 0.2% and 0.3%), dose dependenly and significanly increased the number of scratches when compared to the 0.9% saline groups (P<0.001 ) (Figure 10). 0.2% and 0.3% LP 44-induced total number of scratches were 91 .6 ± 8.3 and 106.5 ± 13.2, respectively.

The blockade of intradermal 5-HT7 agonist induced pruritic behaviour by systemic and spinal administration of SB 269970, a 5-HT7 antagonist

Intradermal injection of LP 44 (0.2%) elicited an increase in scratching behaviors at 10, 20 and 30 mins after the injection when compared to control group. The spinal injection of SB 269970 totally blocked_ LP-44 (0.2%) induced pruritic behaviour (Figure 1 1 ). When the total number of scratches for 30 min after LP 44 (0.2%) injection were evaluated, while the total number of scratches in response to LP 44 (0.2%) alone was 98.7 ± 6.9, intrathecal SB 269970 (10 pg/10 μΙ) pretreatments significantly inhibited LP 44 (0.2%)-induced scratches to 5.6 ± 1 .7 (Figure 12). Systemically (intraperitoneal) injection of SB 269970 (20 mg/kg) significantly inhibited LP 44 (0.2%)-induced pruritic behaviour at 10, 20 and 30 mins after LP 44 injection (Figure 13). When the total number of scratches within 30 min after LP 44 (0.2%) injection were evaluated, intraperitoneal SB 269970 (20 mg/kg) pretreatments decreased the number of scratching behaviors of LP 44 (0.2%) to 19.6 ± 3.6 (Figure 14).

The blocking effect of systemic and spinal administration of two atypical antipsychotic olanzapine and risperidone, on intradermal serotonin, histamine and 5-HT7 agonist- induced pruritic responses The i.th. administration of olanzapine and risperidone (10 pg/10 μΙ) significantly inhibited 0.05% serotonin -induced straches at 10, 20 and 30 mins after serotonin injection (Fig 15) and decreased the total number of straches to 1 .4 ± 2.58 and 0 (zero) within 30 min, respectively (Figure 16). Systemically, i.p. administration of olanzapine and risperidone (0.75 mg/kg) significantly inhibited 0.05% serotonin -induced straches at 10, 20 and 30 mins after serotonin injection (Fig 17). The i.p. pretreatment with olanzapine and risperidone significanly inhibited serotonin-induced scratches to 2.5 ± 1.8 and 0 (zero) within 30 min, respectively (Figure 18).

The i.th. administration of olanzapine and risperidone (10 pg/10 μΙ) significantly inhibited 0.25% histamine-induced straches at 10 and 20 mins after histamine injection (Figure 19) and decreased the total number of straches to 3.25 ± 2.4 and 1 .25 ± 0.73 within 30 min, respectively (Figure 20). Systemically, i.p. administration of olanzapine and risperidone (0.75 mg/kg) significantly inhibited 0.25% histamine-induced straches at 10 and 20 mins after histamine injection (Figure 21 ) and the total number of straches decreased to 0 (zero) and 0.25 ± 0.86 within 30 min, respectively (Figure 22).

Similar to serotonin, i.th. administration of olanzapine and risperidone (10 pg/10 μΙ) significantly inhibited 0.2% LP 44-induced straches at 10, 20 and 30 min mins after LP 44 injection (Figure 23). The i.th. pretreatment with olanzapine and risperidone significanly inhibited LP 44-induced scratches to 1 .5 ± 1 .8 and 2 ± 1 .8 within 30 min, respectively (Figure 24). Systemically, i.p. administration of olanzapine and risperidone (0.75 mg/kg) significantly inhibited 0.2% LP 44- induced ¹ straches at 10 and 20 mins after LP 44 injection (Figure 25) and the total number of straches decreased to 1 .2 ± 1.4 (zero) and 3.2 ± 2.1 within 30 min, respectively (Figure 26).

The present invention is based on the finding that 5-HT7 receptor antagonist is useful in the treatment of pruritus at peripheral and central nervous system levels in preclinical pruritus models. Any pharmaceutical dosage form containing 5-HT7 receptor antagonist including some atypical antipsychotics as an active ingredient is useful in treating pruritus, complicated with some diseases such as eczema (atopic dermatitis), contact dermatitis, psoriasis, urticeria, sun burn, insect bites and stings, poison oak, poison ivy, biliary disease and jaundice, cholestasis, kidney disease, xeroderma, group of ichthyosis skin diseases, uremia, psychogenic itch such as delusional parasitosis, itching during pregnancy, hodgkine lymphoma related itch, parasites (e.g. , pinworms, scabies, lice, etc.) and infections related itch, allergic conjuctivitis, thyroid disease related itch, diabetic itching and itching as a side effect due to using a variety of therapeutic drugs.

The selected compound in the present invention has 5-HT7 receptor antagonistic activity as an active ingredient and the pharmaceutical carrier or vehicle suitable for the admininistration of this compound which is known to those skilled in the art for the particular mode of administration including oral, parenteral, topical, intradermal, transdermal, nasal sprey, inhalation sprey or any other pharmaceutical forms.

Oral pharmaceutical dosage forms are either solid, semisolid or liquid. Capsules, tablets and granules comprising a 5-HT7 receptor antagonist as an essential active ingredient may be provided with the combination of pharmaceutically acceptable carriers used in solid form including lactose, cellulose, cellulose-derivates, sucrose, starch, talk, gelatine, sodium alginate, polyvinylpyrrolidone, magnesium oxide and other carriers in semisolid forms. Type of tablets or capsules can be sustained release forms. Liquid oral dosage forms include solution, emulsion, suspension, elixir or syrups. Parenteral administration of formulation of present invention include sterile intravenous, subcutaneous, intramuscular and intrathecal preparations, such as ampule and lyophilised ampules. For topical administration, 5-HT7 antagonist may be formulated in the form of cream, pomade, lotion, gel, microemulsion, solution, suspansion or transdermal patch.

In the present invention, antipruritic 5-HT7 antagonists may be combined with other antipruritic drugs. 5-HT7 antagonists may increase the antipruritic efficacy of each of steroids, antihistaminics or immunosuppressives, such as tacrolimus.

In the present invention, we used selective 5-HT7 receptor agonist LP 44 (Monti et al. , 2008; Leopoldo et al., 2007) and selective 5-HT7 receptor antagonist SB 269970 to identify the role of 5-HT7 receptor system in pruritus. In the second part of study, we tested risperidone and olanzapine (having 5-HT7 antagonist properties in addition to 5-HT2A antagonist) as an antipruritic agents. Although we used only one 5-HT7 receptor agonist and 5-HT7 antagonist, two atypical antipsychotics, our approach identified the role of 5-HT7 antagonism on pruritic process, our patent protect use of all the 5-HT7 antagonists as antipruritic agents. These 5-HT7 antagonists include SB 269970, SB 258719, SB-691673, DR-4004, DR-4365 and LY 215840, amisulpride, risperidone, 9-OH-risperidon, clozapine olanzapine and lurasidone.