S1P RECEPTOR MODULATORS

Abstract

The current invention is based on the determination that a S1P receptor modulator compound of formula (I):

##STR00001##

decreases the heart rate of a subject to which it is administered by about 5 beats/min or less daily, or about 4 beats/min or less daily, or about 3 beats/min or less daily, or about 2 beats/min or less daily, wherein the S1P receptor modulator is administered at an initial daily dosage which is substantially the same as the standard daily therapeutic dosage.

Claims

1. A method of treating or preventing a disease or disorder by administering to a human subject in need thereof a medicament comprising an S1P receptor modulator, whereby said medicament decreases the heart rate of the subject by about 5 beats/min or less daily, or about 4 beats/min or less daily, or about 3 beats/min or less daily, or about 2 beats/min or less daily; wherein the disease or disorder is selected from the group consisting of pruritis, pain, multiple sclerosis, ulcerative colitis, psoriasis, dermatitis and acne; wherein the S1P receptor modulator is administered at an initial daily dosage which is substantially the same as the standard daily therapeutic dosage; wherein the level of lymphopenia is ≤70%; and wherein the S1P receptor modulator is a compound of formula (I): ##STR00017## wherein R.sub.1 is selected from the group consisting of hydrogen, deuterium, halogen, CN, CF.sub.3, —COOH, amide, sulphonamide, alkoxy, aryloxy, nitro, and a C.sub.1-6 alkyl group, said alkyl group optionally comprising one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-nitrogen double bond, a carbon-nitrogen triple bond, heterocycle, aryl, alkyl and cycloalkyl (C.sub.3-7); wherein R.sub.2 is selected from the group consisting of hydrogen, deuterium, halogen, CN, CF.sub.3, alkoxy, aryloxy, and a C.sub.1-4 alkyl group, said alkyl group optionally comprising one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-nitrogen double bond, a carbon-nitrogen triple bond, heterocycle, aryl, and C.sub.3-7 cycloalkyl; wherein R.sub.3 is selected from the group consisting of hydrogen, deuterium, halogen, alkoxy, aryloxy, and a C.sub.1-6 alkyl group, said alkyl group optionally comprising one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-nitrogen double bond, a carbon-nitrogen triple bond, heterocycle, aryl, alkyl, and C.sub.3-7 cycloalkyl; preferably R.sub.3 is selected from the group consisting of Me, OMe, OEt, OPr, O-iPr, O-isobutyl, O-isopentyl, O-cyclopentyl, O-allyl, O-benzyl and ##STR00018## wherein R.sub.4 is selected from the group consisting of hydrogen, deuterium, halogen, CN, CF.sub.3, and a C.sub.1-4 alkyl group, said alkyl group optionally comprising one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a carbon-carbon double bond, a carbon-carbon triple bond, a carbon-nitrogen double bond, a carbon-nitrogen triple bond, heterocycle, aryl, alkyl, and C.sub.3-7 cycloalkyl; wherein A, independently in each occurrence, represents a carbon or nitrogen atom with the proviso that a ring has no more than two nitrogen atoms; wherein L is selected from the group consisting of hydrogen, deuterium, F, Cl, Br and a C.sub.1-3 alkyl; wherein R is selected from the group consisting of H, COOH, C.sub.1-4 alkyl and C.sub.1-4 hydroxy-alkyl; wherein R′ and R″ are independently selected from H and C.sub.1-4 alkyl; wherein R′″ is selected from OH, —OPO.sub.3H.sub.2 and physiologically acceptable salts; wherein represents an optional bridging group; or a pharmaceutically acceptable salt thereof.

2. A method according to claim 1, wherein the S1P receptor modulator is a compound of formula (II) ##STR00019## wherein R1 is selected from hydrogen, deuterium, halogen, CN, CF3, —COOH, amide, sulphonamide, alkoxy, aryloxy, nitro and an alkyl chain (C1-5), said alkyl chain optionally containing one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a multiple bond, heterocycle, aryl, and cycloalkyl (C3-7); wherein R2 is selected from hydrogen, deuterium, halogen, CN, CF3, an alkyl chain (C1-4) said alkyl chain optionally containing one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a multiple bond, heterocycle, aryl, and cycloalkyl (C3-7); wherein R3 is selected from hydrogen, deuterium, halogen, alkoxy, aryloxy, an alkyl chain (C1-7), said alkyl chain optionally containing one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a multiple bond, heterocycle, aryl, and cycloalkyl (C3-7); wherein R4 is selected from hydrogen, deuterium, halogen, CN, CF3, an alkyl chain (C1-4), said alkyl chain optionally containing one or more of deuterium, O, S, NR′ (R′═H, alkyl, cycloalkyl), halogen, a multiple bond, heterocycle, aryl, and cycloalkyl (C3-7); wherein L is selected from hydrogen, deuterium, F, Cl, Br and alkyl (C1-3).

3. A method according to claim 2, wherein the compound of formula (II) has R1 selected from F, Cl, Br, CN, CF3, NO2, Me, OMe, OEt, OPr, O-iPr, O-isobutyl, O-isopentyl, O-cyclopentyl, O-allyl, O-benzyl and; R2 selected from H, deuterium, F, Cl, Br, CN, CF3, NO2, Me, OMe, OEt, OPr, O-iPr, O-isobutyl, O-isopentyl, O-cyclopentyl, O-allyl, O-benzyl and; R3 selected from H, deuterium, Pr, butyl, OMe, OEt, OPr, OiPr, O-isobutyl, O-isopentyl, O-butyl, O-pentyl, O-cyclopentyl, O-allyl, O-benzyl and; R4 selected from H, deuterium, Me and Et; and L selected from H, deuterium, Me and Cl.

4. A method according to claim 2, wherein the compound of formula (II) has R1 selected from F, Cl, Br, CN, CF3, Me, NO2, OMe, OEt, OPr, O-iPr, O-isobutyl, O-isopentyl, O-cyclopentyl, O-allyl, O-benzyl and; R2 is H; R3 selected from H, deuterium, Pr, butyl, OMe, OEt, OPr, OiPr, O-isobutyl, O-isopentyl, O-butyl, O-pentyl, O-cyclopentyl, O-allyl, O-benzyl and; R4 selected from H, deuterium, Me and Et; and L is H.

5. A method according to claim 1, wherein the compound of formula (I) or formula (II) is selected from the group consisting of: ##STR00020## ##STR00021## ##STR00022## ##STR00023##

6. A method according to claim 1, wherein the difference between the initial daily dosage and the standard daily therapeutic dosage is less than 25%, or less than 15%, or less than 10%, or less than 5%.

7. A method according to claim 1, wherein the initial daily dosage is the same as the standard daily therapeutic dosage.

8. A method according to claim 1, wherein the standard daily therapeutic dosage of S1P receptor modulator is up to 70 mg.

9. A method according to claim 1, wherein the standard daily therapeutic dosage of S1P receptor modulator is up to 24 mg.

10. A method according to any claim 1, wherein the standard daily therapeutic dosage of S1P receptor modulator is between 0.5 mg and 12 mg.

11. A method according to claim 1, wherein the administration of the medicament does not cause a substantial decrease in heart rate.

12. A method according to claim 1, wherein the administration of the medicament does not cause bradycardia.

13. A method according to claim 1, wherein the level of lymphopenia is ≤25%.

14. A method according to claim 1, wherein the level of lymphopenia is ≤50%.

15. A method according to claim 1, wherein the medicament is administered to a subject who was previously under treatment with an alternate S1P1 modulator or agonist, and/or wherein said patient is currently undergoing discontinuation or cessation of treatment with an alternate S1P modulator or agonist.

16. A method according to claim 15, wherein said discontinuation or cessation of treatment is due to a bradycardia and/or lymphopenia event.

17. A method according to claim 1, wherein the medicament is an oral or injectable or systemic formulation, selected from a pill, a tablet, a capsule, a solution and a syrup.

18. A method according to claim 1, wherein the subject is susceptible to heart failure, arrhythmias, high grade atrio-ventricular blocks, sick sinus syndrome, has a history of Syncopal episodes or a combination thereof.

19. A method according to claim 18, wherein the subject is undergoing beta blocker or anti-arrhythmic treatment by receiving anti-arrthymic drugs.

20. A method according to claim 1, wherein the subject has undergone an interruption or treatment break from another S1P receptor modulator/agonist.

21. A method according to claim 20, wherein said treatment break is greater than 4, 6, 8, 10, 12, or 14 days.

22. A method according to, wherein the medicament is a slow release formulation, administered topically, by implantation or injection or via a medical device.

23. A method according to claim 1, wherein the medicament treats pain, selected from the group consisting of joint pain, arthritis pain, gout pain, back pain, muscle pain, neuropathy, neurologic pain, migraine, cancer pain, sports injury pain and wound pain.

24. A method according to claim 1, wherein the medicament comprises the S1P receptor modulator as a composition with another pharmaceutically active compound selected from immune suppressant/modulators agents, neuromodulators, anti-inflammatory agents, antipathogens, pain modulators, pruritus modulators, opioids, cannabinoids, antibacterial agents, antiviral agents and antifungal agents.

25. A method according to claim 1, wherein the medicament is in the form of a topical formulation selected from a solid, a patch, a powder, a liquid, a semisolid, an ointment, a gel, a spray, an aerosol, an inhaler and a lotion.

26. A method according to claim 1, wherein the medicament is administered topically, orally, transdermally, parenterally, intranasally, ocularly or rectally.

27. A method according to claim 1, wherein the medicament is applied topically.

28. A method according to claim 27, wherein the medicament comprises the S1P receptor modulator in an amount between 0.01% and 30% by weight.

29. A method according to claim 28, wherein the medicament comprises the S1P receptor modulator in an amount of about 3% by weight.

30. A method according to claim 27, wherein the medicament is applied to up to 1000 cm.sup.2 of body surface area per 1 g of medicament, and wherein the standard daily therapeutic dosage of S1P receptor modulator is ≤3 g.

31. A method according to claim 30, wherein the standard daily therapeutic dosage of S1P receptor modulator is ≤1.5 g.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0170] FIG. 1 illustrates various S1P receptor modulator pathways.

[0171] FIG. 2 illustrates the effect of a compound of Formula I on markers of inflammation and pruritis.

[0172] FIG. 3 illustrates the effect of a compound of Formula I on pruritus scores.

[0173] FIG. 4 illustrates the effect of a compound of Formula I on heart rate of human subjects over 72 hours, at a 12 mg dose.

[0174] FIG. 5 illustrates the anti-mould activity of the compound of formula I solution.4

[0175] FIG. 6 illustrates the effect of compound of formula I on the secretion of cytokines TNFα and IL6.

[0176] FIG. 7 confirms that the compound of formula (I) did not inhibit or impair wound healing.

DETAILED DESCRIPTION OF EMBODIMENTS

[0177] The following is a detailed description of the disclosure provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0178] Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and compositions are now described.

[0179] It must also be noted that, as used in the specification and the appended claims, the singular forms ‘a’, ‘an’ and ‘the’ include plural referents unless otherwise specified. Thus, for example, reference to ‘S1P receptor modulator’ may include more than one S1P receptor modulator, and the like.

[0180] Throughout this specification, use of the terms ‘comprises’ or ‘comprising’ or grammatical variations thereon shall be taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof not specifically mentioned.

[0181] Unless specifically stated or obvious from context, as used herein, the term ‘about’ is understood as within a range of normal tolerance in the art, for example within two standard deviations of the mean. ‘About’ can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein in the specification and the claim can be modified by the term ‘about’.

[0182] Any methods provided herein can be combined with one or more of any of the other methods provided herein.

[0183] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0184] Reference will now be made in detail to exemplary embodiments of the disclosure. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure.

EXAMPLES

[0185] The compound of formula (I) used in the below examples was:

##STR00016##

Example 1: Activity at Spingosine-1-Phosphate (S1P) Receptor

[0186] Compounds of formula (I) showed S1P receptor activity, especially type 1 receptor agonistic activity. The S1P′ assay system was GTPgama-S.sup.35 binding in membranes from CHO K1 cells, expressing S1P′ human receptor. The compounds were tested and generated a concentration-effect (dose response) curves at these receptors. The analysis provided efficacy (E.sub.max) and potency (EC.sub.50) of selected compounds of Formula (I) relative to S1P and demonstrated an EC.sub.50 of <2 nM at the S1P.sub.1 receptor. The compounds of Formula I has low tendency to degrade the S1P.sub.1 receptor. The compounds of formula (I) are selective against the S1P2, S1P3, S1P4 and S1P5 receptors.

[0187] Comparatively, the endogenous ligand S1P and drug FTY-720 has activity at S1P1 receptor in a GTPγS assay with EC50 nM of 1.2 nM and 2 nM respectively while the receptor degradation ability is 302 nM and 0.34 nM respectively (Lucas et al, Journal of Biomolecular Screening, 2013, 1-10 pp). The known S1P receptor modulators have low activity v/s degradation margin FTY720=0.17; BAF-312=2.95, Ponesimod <2 while the compound of formula (I) has a margin greater than 100 (Samuvel J et al, PLOS ONE, 2015, DOI:10.1371/journal.pone.0141781; Piali L et, JPET, 2011, 337, 547-556; Lucas S et al, Journal of Biomolecular Screening, 2014, Vol. 19(3) 407-416; Gatfield J et al, Immunomodulation, 28th ECTRIM, 10-13 Oct. 2012, Lyon, France).

Example 2: Correlation with Lymphopenia and Systemic Exposure of Compound of Formula (I) in Humans and Mice

[0188] Treatment of a compound of formula (I) (dose of ˜0.17 mg/kg) in humans resulted in a systemic exposure of 18.8 ng/mL with no induction of significant lymphopenia. In comparison, treatment of a compound of formula I (dose of 0.3 mg/kg) in mice resulted in a systemic exposure of 6.36 ng/mL with a measured lymphopenia of 40% at 6 hours post dose. This is surprising and suggests that the link between lymphopenia and the systemic exposure of S1P receptor modulators such as the compound of formula (I) differs between mice and humans.

[0189] It is notable that the delivery of a compound of formula (I) had a dose proportional systemic exposure. The 8 mg daily dosing of compound of formula (I) for one week resulted in a measured lymphopenia of only up to 40%, which is a dosage 16 times higher than the equivalent FTY720 therapeutic dose in humans.

Example 3: Effects of a Compound of Formula (I) In-Vivo on Heart Rate

[0190] Treatment of a compound of Formula I in humans resulted in a dose proportional increase in systemic exposure in humans with 0.5 mg=0.6 ng/mL; 2 mg=2.4 ng/mL; 6 mg=7.1 ng/mL; 12 mg=18.8 ng/mL; however, there was no dose proportional or dose relevant bradycardia event, a common side effect observed in prior art S1P1 modulators/agonists where the decrease of heart rate is sustained for several hours.

[0191] The observance of bradycardia with S1P1 selective agonists has been reported to be species specific. S1P1 receptor selective agonists do not induce bradycardia in rodents; however the S1P1 selective agonists tested in humans resulted in significant bradycardia in patients (Juif P-E. et al, Int. J. Mol. Sci. 2017, 18, 2636; doi:10.3390/ijms18122636; Pali L. et al, Pharmacol Res Perspect, 2017; e00370. wileyonlinelibrary.com/journal/prp2|1 of 12; Rey M et al, PLOS ONE, September 2013|Volume 8|Issue 9|e74285). Surprisingly the S1P1 receptor modulator of formula (I) did not induce bradycardia in humans when tested at different dose levels ranging from 0.5 mg dose to 12 mg dose (12 mg data shown in FIG. 4).

Example 4: Efficacy of a Compound of Formula (I) in an Animal Model of Excision Wound

[0192] Two groups with six Wistar rats in each group were anaesthetized with a dose of 80 mg/kg of ketamine (i.p.) and the back of the animals were shaved. One excision wound was inflicted by cutting away (with a sterile scalpel) a 600-700 mm.sup.2 section of the full thickness of the skin from a predetermined area. The wound was left undressed to the open environment. Group 1 rats were untreated and served as sham control. Group 2 animals were treated with a compound of Formula I in an ointment formulation (3% w/w). The ointment (0.20 g/animal wound) was applied topically twice daily. The compound of formula (I) did not inhibit or impair the wound healing (FIG. 7).

Example 5: Efficacy of a Compound of Formula (I) In-Vitro for Inflammation

[0193] Treatment of a compound of formula (I) (1-5 μg/mL) to the cultured microglial (BV2 cells) and macrophages (Raw cells) was made 4 hrs prior to treatment with lipopolysaccharide (LPS) at 500 ng/mL. After 16-18 hr of drug treatment the cytokines (TNF.sub.α, IL.sub.1β and IL6) were measured in culture media by ELISA and expression levels of cyclooxygenase2 (Cox-2), inducible nitroxide synthase (iNOS) or beta actin were analysed in cell homogenates by western analysis. There was a significant reduction of proinflammatory cytokines (p<0.05) when treated with a compound of formula (I).

Example 6: Efficacy of Compound of Formula (I) in In-Vivo for Pruritus and Inflammation

[0194] The efficacy of the compound of formula (I) was assessed in an animal model of experimental autoimmune encephalomyelitis (EAE), which is a widely-accepted model of demyelinating diseases such as MS. The cDNA was prepared from spleen obtained from Vehicle treated (Control), EAE induced (no treatment) and EAE induced (a compound of formula (I) treated) animals (n=5 each group) at the recovery (Day 27 from 1.sup.st day of MBP immunization). The treatment with a compound of Formula I (3 mg/kg) was initiated on the 11.sup.th day from MBP immunization. cDNA was synthesized using Bio-Rad cDNA synthesis kit. RT-PCR was performed for IL-31, IL1β and IFNγ. Primers were obtained from Qiagen and the samples were analysed on Bio-Rad CFX96Real-Time System. Administration of a compound of formula (I) significantly reduced markers of inflammation (Figure-2). Statistical Significance of the data is represented by p value. *denotes p≤0.05, **denotes p≤0.01.

[0195] In an atopic dermatitis and psoriasis disease models the compound of formula (I) downregulates the cytokines IL17, IL23, IL2.

Example 7: Efficacy of Compound of a Formula (I) in an Animal Model of Formalin Induced Nociception (Pain)

[0196] Forty male Sprague Dawley rats were assigned to different treatment and control groups containing 8 animals/group. The groups were treated with different doses of a compound of Formula I and received either 0.03, 0.3 and 3.0 mg/kg dose, administered orally 30 min before formalin injection. Tramadol hydrochloride (30 mg/kg) was administered orally 30 min before formalin injection as a positive control. Deionized water was used as a negative control (Vehicle) and administered orally 30 min before formalin injection. Individual rats were then gently restrained and formalin (5% in v/v in saline, 50 μl, s.c.) was injected into the plantar surface of the left hind paw using a 27G needle. Post-formalin nociceptive behaviour, paw flinching was recorded for 60 min, in bins of 5 min. The intra plantar injection of formalin (50 μl, 5% v/v) induced marked flinching of the ipsilateral rat paw with typical biphasic response in the vehicle/distilled water treated animals. The compound of formula (I) at 0.03 mg/kg, 0.3 mg/kg and 3 mg/kg when administered orally 30 min before formalin treatment inhibited nociceptive behaviour dose. Inhibition of nociceptive behaviour at 0.3 mg/kg (33%, p<0.05) and 3.0 mg/kg (45%, p<0.01) were statistically significant compared to vehicle control.

Example 8: Efficacy of Compound of a Formula (I) in an Animal Model of Paclitaxel Induced Neuropathic Pain

[0197] Forty-eight male Sprague Dawley Rats were assigned to different treatment and control groups containing 8 animals per group. All animals, except the Naive control were injected with Paclitaxel (2 mg/kg) intraperitoneally on four alternate days (D0, D2, D4 and D6). For prophylactic intervention, the compound of Formula I was dosed orally at dose of 0.3, 1 and 3 mg/kg/day when the treatment was initiated along with paclitaxel treatment and the dosage was continued until day 15 post first paclitaxel injection. For therapeutic intervention, the compound of Formula I was dosed orally at dose of 3 mg/kg/day when the treatment was initiated after establishment of neuropathic pain i.e. on day 16 post first paclitaxel injection and was continued for the next 7 days.

[0198] For behavioural testing each rat was allowed to acclimatize individually to a transparent enclosure on elevated wire mesh for at least 15 min. Behavioural testing was performed using an Electronic von Frey Aesthesiometer (IITC Life Science) to evaluate paw withdrawal threshold to mechanical stimulus. Paclitaxel injections in negative control (Paclitaxel+vehicle treated) resulted in significant reduction in hind paw withdrawal threshold to mechanical stimulus indicating development of paclitaxel-induced neuropathic pain. Initiation of treatment with the compound of Formula I in concurrence with paclitaxel injections (prophylactic treatment) inhibited the development of paclitaxel induced neuropathic pain dose dependently, of which the maximum effect was observed at the 3 mg/kg/day dose. Withdrawal of the compound of formula (I) treatment on day 15th post first paclitaxel injection did not cause any reversal of the drug's anti-allodynic effect until 7 days post last drug treatment (day 22 of study).

[0199] The day 16 score was reduced in the preventive model by >60% (p<0.001) at the 1 and 3 mg/kg dose levels and a similar reduction was observed in the therapeutic model. In the therapeutic treatment model, initiation of treatment with the compound of formula (I) at an oral dose of 3 mg/kg/day after establishment of paclitaxel induced neuropathic pain, resulted in reversal of neuropathic pain in time dependent manner, with the maximum effect recorded after the 5th daily dose with a sustained effect thereafter. Based on these findings it can be concluded that treatment with the compound of formula (I) is effective both as a prophylactic and therapeutic and has an anti-allodynic effect in paclitaxel induced neuropathic pain.

Example 9: Efficacy of Compound of a Formula (I) in an Animal Model of Multiple Sclerosis

[0200] The oral treatment with a compound of formula (I) was conducted in an animal model of multiple sclerosis. EAE was induced in female Lewis rats with guinea pig MBP (25 μg/rat). Rats that developed EAE were divided into 3 groups (n=6) and compound of Formula I was administered as oral (1.3 mg/kg) or FTY720 (1 mg/kg) orally every day until day 26. Both the compound of Formula I and FTY720 showed similar efficacy in reducing clinical signs of disease, while the lymphopenia was less in animals treated with the compound of formula (I). Further, the lymphopenia observed in compound of formula (I) treated rats was short-term and quickly reversed whereas the lymphopenia observed in FTY720 treated rats was induced at a high level over a long-term period.

Example 10: Efficacy of Compound of a Formula (I) in In-Vivo Inflammation

[0201] The efficacy of compound of formula (I) was determined in an animal model of dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH), an inflammatory model. The animals (n=9) were treated with the vehicle or a compound of formula (I), receiving a twice daily dose of 3 mg/kg. The efficacy end points were measured as Ear thickness before challenge and 24 h after challenge. Ear weight was measured 24 h after challenge. At sacrifice, right ear samples were collected and used for tissue MPO activity. Administration of a compound of formula (I) significantly reduced ear thickness (˜70%, p<0.0001) and ear weight (˜50%, p<0.01) as well as MPO activity.

Example 11: Efficacy of Compound of a Formula (I) in an Animal Model of Stroke

[0202] The compound of Formula I was assessed in an animal model of stroke, namely the middle cerebral artery occlusion (MCAO) model. The effect of a compound of Formula I on infarctions (TTC staining) and BBB leakage (Evan's blue extravasation) ischemia for 60 min and reperfusion for 72 h was assessed. The oral dose of 1, 3 and 5 mg/kg of a compound of formula (I) significantly reduced the infarct volume, infract area and sensory motor function and blood brain barrier leakage.

Example 12: Efficacy of Compound of a Formula (I) in an Animal Model of Sepsis

[0203] Sepsis was induced in female Sprague-Dawley rats by LPS (5 mg/kg) administration and treated orally with compound of Formula I (3 mg/Kg) 1 h after and every 24 h thereafter to determine the effect of a compound of formula (I) in this animal model of sepsis (LPS mediated systemic inflammation). Animals were sacrificed at 24 and 72h post LPS treatment. There was positive impact on the body temperature and organ histopathology.

Example 13: Efficacy of Compound of a Formula (I) in an Animal Model of Ulcerative Colitis

[0204] The effect of a compound of formula (I) was assessed in an animal model of Ulcerative colitis. 24 Balb/C Mice were divided into two groups; control and treatment (n=6). Acute colitis was induced in all groups by adding 2.5% w/v Dextran Sulphate Sodium (DSS) treatment in drinking water for 5-7 days. A compound of Formula I was administered at dose of 3 mg/kg body weight by oral route as a repeated dose for 3 or 6 days, consecutively. The daily administration of a compound of Formula I resulted in improvement of DSS-induced colitis, and significantly reduced the microscopic changes observed in colon. Gross pathological observations revealed that day 3 group animals were slightly emaciated and the anal areas were soiled with blood in both groups. During day 6 these observations were reduced to 16.66% in control and 0.00% in a compound of Formula I group.

[0205] The length of colon measured revealed that the mean colon length of control groups was short than the mean colon length of treatment groups. The mean colon lengths were 9.67 cm, 9.88 cm, 11.20 cm and 13.02 cm in day 3 control group, day 6 control group, day 3 a compound of Formula I treated group and day 6 a compound of Formula I treated group respectively. Histopathological evaluation of colon indicated the control animal displayed a severe increase in thickness of mucosa of 2/6 animals, minimal in ⅙ animals and a mild increase in 3/6 animals. In case of a compound of Formula I treated animals, the severity was reduced and the incidence of increase in thickness of mucosa was mild in 3/6 animals and minimal in 2/6 animals. The mucus secreting goblet cells in control animals were absent in 3/6 animals, whereas the number of mucus secreting goblet cells was increased moderately ( 4/6) in a compound of Formula I treated animals. The severity of presence of haemorrhages with desquamated cells seen in lumen of colon of control animals was reduced in a compound of Formula (I) treated animals. Histopathological evaluation on day 6 revealed reduction in infiltration of MNC and was minimal and focal in ⅚ animals and moderate in ⅙ control animals. In the compound of formula (I) treated group animals, it was minimal in 2/6 animals and mild in ⅙ animals. Thickness of mucosa was also markedly reduced in compound of Formula I treated animals compared to control animals. There was moderate ( 4/6 animals) to minimal (⅙ animals) increase in mucus secreting goblet cells in a compound of Formula I treated animals as compared with those of controls.

Example 14: Efficacy of Compound of a Formula (I) in an Animal Model of Epilepsy

[0206] In an animal model of epilepsy, Sprague Dawley rats (n=6) were treated with the compound of Formula I or vehicle, dosed 1 hour prior to kainic acid administration (10 mg/kg, IP). One-hour post kainic acid administration, the rats were observed for behavioural changes (grooming, rearing, hind limb scratching, urination, defecation, wet dog shakes, jaw movements, salivation, head nodding), incidence and latency of convulsions and mortality until 1 hour. There was significant reduction in seizures (70%, p<0.005) in the compound of formula (I) treated group. The changes observed in the hippocampal region of control animals included, minimal to mild neuronal cell death which included vaculations in neuronal cells particularly at C3 and C1 regions proving the compound of Formula I is neuroprotective.

Example 15: Efficacy of Compound of a Formula (I) in Arthritis Patients with Pain

[0207] Fifteen arthritis patients who were experiencing pain were treated with the compound of Formula I@60 mg daily dosing as a topical treatment for seven consecutive days. The drug appeared to be safe and well tolerated and there were no serious or significant adverse events (AEs) observed (all AEs were in the mild to moderate category). The subjects blood parameters, urine alysis, vital signs, physical examination, ECG were normal. In all participants treated with compound of formula (I) (n=12) there was an overall positive response to patient global assessment of response to therapy (PGART) scores observed at Day 3 (41.7% participants) and at Day 7 (58.3% participants). In participants with osteoarthritis a positive PGART response was observed by Day 3 (28.6% participants) and at Day 7 (57.1% participants). The placebo participants (n=3) did not received a positive response.

[0208] The numerical rating scale for pain scoring (NRS) in a compound of Formula I treated group (n=12) reduced from 7.1±1.38 (Day 1; baseline) to 5.8±2.12 (Day 3) and 4.8±2.72 (Day 7). The mean NRS score change from baseline was −1.3±1.54 (Day 3) and −2.3±2.06 (Day 7) points in arthritis participants treated with compound of Formula I. In osteoarthritis participants, the mean change in pain score from Day 1 to Day 7 was of −1.9±1.46 and −1.9±1.57 for pain felt and worst pain felt in 24 hours, respectively. This reduction in NRS score in OA participant was found to be significant (p=0.0153 and 0.0205, respectively). The topical application of a compound of Formula I in participants with arthritis for 7 days appeared to be safe, efficacious, and well tolerated and resulted in significant and fast reductions in the pain scores over time. There was no systemic change in the absolute lymphocyte count. The systemic exposure on day 7 ranged from 0.42 ng/mL to 2.44 ng/mL (average 0.79 ng/mL).

Example 16: Efficacy of Compound of a Formula (I) in Psoriasis Patients

[0209] Twelve psoriasis patients were treated with the compound of Formula I@30 mg daily dosing under occlusion as topical treatment for 28 consecutive days. A significant reduction compared to baseline in the overall local psoriasis severity index (LPSI) score were noted in a compound of formula (I) group at each visit from Day 7 through to Day 28. The LPSI score reduced from 5.8 (day 1) to −2.1 (day 28) (p<0.0016), and no significant change was observed in the placebo group. Some subjects in the compound of Formula I group showed a reduction in plaque area, as assessed using image analysis of clinical photographs. The systemic exposure after topical applications was recorded below the limit of quantification (BLQ) <0.400 ng/mL. There was no systemic change in the absolute lymphocyte count. The study drug appears to be well tolerated as evidenced by adverse events, laboratory blood parameters, urinalysis, vital signs, physical examination and ECG.

Example 17: Efficacy of Compound of a Formula (I) in Atopic Dermatitis Patients

[0210] Atopic dermatitis patients were treated with the compound of Formula I@60 mg daily dosing as topical treatment for 28 consecutive days. A reduction compared to baseline in the overall eczema area & severity index (EASI) score and pruritus score (FIG. 3) were noted with a compound of formula (I). There was no systemic change in the absolute lymphocyte count. The compound of formula I appears to be well tolerated as evidenced by adverse events, laboratory blood parameters, urinalysis, vital signs, physical examination and ECG. The systemic exposure by week 3 was ≤1 to 2.5 ng/mL with no lymphopenia event occurred.

Example 18: Efficacy of Compound of a Formula (I) in Healthy Human Subjects

[0211] Human subjects were treated in cohorts of different dose levels (each cohort=8 human subjects) with a compound of formula (I)@0.5 mg, 2 mg, 6 mg and 12 mg single oral dose. The drug was safe with no bradycardia events reported after 72 hours of clinical monitoring and ECGs (12 mg dose shown in FIG. 4 for one of the cohorts).

[0212] One cohort (8 human subjects) was treated daily with 8 mg oral dosing for 7 consecutive days. The systemic exposure reached to mean value of 53.3 ng/mL and only mild lymphopenia was observed (Table 1).

TABLE-US-00001 TABLE 1 Effects of compound of formula (1) on absolute lymphocyte count (ALC), CD3, CD4 and CD8 counts in healthy human subjects. Subject No ALC Change (%) CD3 Change (%) CD4 Change (%) CD8 Change (%) 101 +40 −12.58 −10.91 −13.79 102 −22.58 −5.77 −8.21 0 103 −15.56 −12.36 −25.3 −7.06 104 −20 −11.86 −7.87 −11.86 109 −33.33 −18.75 −22.41 −19.05 110 −27.66 −30.32 −33.62 −27.87 111 −35.71 −30.58 −32 −31.74 112 0 −0.6 −5.2 −1.67

Example 19: Systemic Exposure of Compound of a Formula (I) in Human Subjects after Topical Application

[0213] The body surface area of an adult is around 16,000 cm.sup.2 to 18,000 cm.sup.2 and the skin surface area varies from 10,000 cm.sup.2 to 20,000 cm.sup.2 (https://hypertextbook.com/facts/2001/IgorFridman.shtml, and references cited within). Human subjects received a topical application of 1 g of 3% by weight formulation per 150 cm.sup.2 of body surface area (over the skin) twice daily for 21 days. The resulting systemic exposure was approximately 2.2 ng/ml. In another cohort, the systemic exposure via local application of 2 g of 3% or 6% by weight formulation with the amount of compound of formula (I) as 60 mg or 120 mg over the joint area for one week did not exceed 2.5 ng/ml.

Example 20: Preparation of Tablet Formulation of a Compound Formula (I)

[0214] Lactose monohydrate was passed through U.S. Mesh size #40 and collected in a clean sterile poly lined container, maize starch was then added and passed through U.S. Mesh size #100. Following this, microcrystalline cellulose was passed through U.S. Mesh size #40 mesh and the composition was dry-mixed. Povidone solution was used as a binder solution (polyvinylpyrrolidone K 30) and subsequently filtered through the U.S. Mesh size #100. The granules were dried in a hot oven (55 to 60 deg. C) for approximately 25 to 30 minutes or until the granules had dried to the desired levels. Then granules were sifted through U.S. Mesh size #20 and collected in clean and sterile polylined containers. Colloidal silicon dioxide was added and the mixture subsequently sifted through U.S. Mesh size #40, and mixed for 3 minutes. Then, magnesium stearate was sifted through U.S. Mesh size #60, and mixed for 3 minutes. The mixture was then compressed into the desired tablet formulation. Table 2 lists the ingredients used in the above described process.

TABLE-US-00002 TABLE 2 Pharmacopoeia Standard Standard Ingredients grade quantity/mg quantity/kg Compound of Formula 1 IH 2.00 0.0200 Lactose monohydrate BP 28.250 0.2825 Maize starch BP 21.600 0.2160 Microcrystalline cellulose BP 28.250 0.2825 Sodium starch glycolate BP 2.700 0.0270 Povidone (PVP K 30) BP 3.600 0.0360 Purified water* IH q.s. 0.2800 Colloidal silicon dioxide USP 0.540 0.0054 Magnesium Stearate BP 0.360 0.0036 *Not present in the final product

Example 21: 3% w/w Ointment Composition of S1P1 Agonist of Formula (I), Free Base, for Topical Use

[0215] A mixture of Vaseline (30.8 g) and Gelucire 50/13 pellets (4 g) was melted and stirred at ˜70° C. until homogenous (˜15 min). A solution of compound of formula (I), free base, (1.2 g) in anhydrous DMSO (4 ml) was added to the mixture with vigorous stirring. The mixture was allowed to cool to room temperature and the resultant ointment (40 g), contained 3% (w/w) of free base of a compound of formula (I).

Example 22: 3% w/w Gel Composition HCl Salt of Formula (I), for Topical Use

[0216] A mixture of H.sub.2O (4.85 g) and propylene glycol (4.85 g) and cellosize PCG 10 (0.3 g) was prepared. The mixture was allowed to stir overnight at room temperature to give a transparent viscous gel (10 g). This gel (6 g) was mixed with EtOH (4 g) and the resulting mixture was stirred at ˜70° C. for 2 h. To it a hydrochloride salt of a compound of formula (I) (0.45 g), dissolved in anhydrous DMSO (3 g) was added at once and EtOH was added to give a final mass of 15 g. The resulting mixture was stirred for 1 hour at ˜70° C., to give a transparent colourless gel with excellent stability and spread ability.

Example 23: 3% w/w Gel Composition of Formula (I), Mesylate Salt, for Topical Use

[0217] When the hydrochloride salt of a compound of formula (I) of Example 7 was substituted for the mesylate salt of a compound of formula (I), an identical process gave the title composition.

Example 24: 3% Liquid Composition of Formula (I), Mesylate Salt, for Topical Use

[0218] A mesylate salt of the compound of formula (I) (0.3 g) was dissolved in 50% aqueous DMSO (4 g) and this was diluted to 10 g with EtOH, to give the title formulation as a colourless liquid (10 g).

Example 25: 1% Liquid Composition of Formula (I), HCl Salt, with Polyvinyl Pyrrolidone (PVP) for Topical Use

[0219] A HCl salt of a compound of formula (I) (0.05 g) was dissolved in 80% aqueous EtOH (4.45 ml). To it, polyvinyl PVP (0.5 g) was added and the mixture was stirred until completely homogenous (˜1 h) at room temperature to give a stable colourless solution, which formed a film after application to the skin.

Example 26: 0.5% Sterile Aqueous Solution of Formula (I), Mesylate Salt, for Injection/Liquid Oral Formulation/Drops for Eye and Ear Administration

[0220] To a sterile container with a mesylate salt of a compound of formula (I), (0.005 g), sterile isotonic solution was added (1 ml) via syringe and the resulting mixture was stirred at room temperature by shaking until homogenous, which may be used for injection, eye or ear drops or orally.

Example 27: Topical Patch Formulation of Formula (I)

[0221] A compound of formula (I) and other ingredients including solubility enhancers or permeation enhancers such as but not limited to DMSO, polyvinyl pyrrolidones (PVPs), glycyryl laurates, lauryl lactate, aerosol, eudragit may be dissolved in solvent (ethanol, propanol, isopropanol). An adhesive is added and mixed until homogenous. The homogenous slurry at optimal temperature may be casted onto a release layer (silicone or fluoropolymer coated polyester film and dried.

Example 28: 3% w/w Ointment Composition of S1P1 Agonist of Formula (I), Free Base, in Combination with 1% Nicotinamide and 2% Vitamin E for Topical Use

[0222] A compound of formula (I) as a free base, (0.6 g), nicotinamide (0.2 g),vitamin E (d isomer; 0.4 g), Gelucire 50/13 pellets (2 g), polysorb 20 (0.6 g) in anhydrous DMSO (2 ml) were stirred at ˜55° C. until homogenous (˜30 min). Melted Vaseline was added to make a final weight of 20 g. This was vigorously stirred for 15 min at ˜50° C., cooled to room temperature to give an off-white ointment.

Example 29: 3% w/w Ointment Composition of S1P1 Agonist of Formula (I), Free Base, and 0.05% w/w of Betamethasone for Topical Use

[0223] A mixture of Vaseline (30.78 g) and Gelucire 50/13 pellets (4 g) was melted and stirred at ˜70° C. until homogenous (˜15 min). To it a solution of compound of formula (I), free base, (1.2 g) and betamethasone (0.02 g) in anhydrous DMSO (4 g) was added with vigorous stirring. The mixture was allowed to cool to room temperature to give cloudy ointment (40 g), containing 3% (w/w) of free base of a compound of formula (I) and 0.05% of betamethasone.

Example 30: 2% w/w Gel Composition HCl Salt of Formula (I) and 1% Diclofenac for Topical Use

[0224] A solution of solution of H.sub.2O (4.85 g) and propylene glycol (4.85 g) and cellosize PCG 10 (0.3 g) was prepared. The mixture was allowed to stir overnight at room temperature to give a transparent viscous gel (10 g). This gel (6 g) was mixed with EtOH (3.9 g) and the resulting mixture was stirred at ˜70° C. for 2 h. To it a mixture of hydrochloride salts of a compound of formula (I) (0.3 g) and diclofenac (0.15 g), dissolved in anhydrous DMSO (4.5 g) was added at once and EtOH was added to give a final mass of 15 g. The resulting mixture was stirred for 1 hour at ˜70° C., to give the titled product as a transparent colourless gel with excellent stability and spreadability.

Example 31: Use of a Topical Formulation of a Compound of Formula (I) in Wound Patients

[0225] A 68-year old male patient presented with a second degree burn wound on the inner surface of his middle finger of his left hand, suffering from swelling, blistering and pain at the site of injury. A topical gel formulation of compound of formula (I) was applied topically to the site of injury. After 10 minutes, the swelling and blistering had visibly reduced dramatically and the patient reported significant pain relief.

[0226] A 49-year old male patient presented with a scratch wounds on hands while cleaning gutters. These become inflamed and painful in 3 hours. The ointment formulation of compound of formula (I) was applied topically to the site of injury, the swelling and pain reduced dramatically and the patient reported significant pain relief. No adverse side effects were reported in either case.

Example 32: Alternative Formulations of Compounds of Formula I

[0227] The compound of formula I is soluble in water as salt form such as HCl, mesylate salt giving a stable clear solution. The compound of formula I free base (free amine form) is insoluble in water. To solubilize the free base in water the novel technology AvignaSOL (N,N-Dimethyl hexanamide; patent number: U.S. Pat. No. 9,186,338B2) and its higher derivatives such as octinamide, decamide which improve the absorption of various poorly soluble drugs was used. AvignaSOL as 1-70% ww % assisted the solubilization of free base as 1 gm/20 mL of water. The solubility of free base in water assisted its direct use in various formulation such as creams, gels, solutions which together with the permeation enhancing effect will be harnessed to improve skin penetration and/or bioavailability and in altering the pharmacokinetics and pharmacodynamics profile of free base.

[0228] The AvignaSOL having a log P of ˜1.6 hinder the release by increasing the hydrophobicity of the delivery system. The solubilized compound of Formula I-AvignaSOL does not precipitate in phosphate buffer pH 6.8. Hence AvignaSOL can be used to increase the bioavalibility of compound of formula I but not limited via intestinal and/or via dermal. The combination of compound of formula I with other ingredients including sustained release enhancer with or without AvignaSOL were prepared as novel formulation techniques to improve and/or alter the pharmacokinetics and pharmacodynamics profile of compound of Formula I.

[0229] To improve the pharmacokinetics profile such as half-life (T½) and Cmax the compound of formula I was formulated in various ingredients and solvents listed in Table 6:

TABLE-US-00003 TABLE 6 Compound of formula I formulations Range S.No Ingredient Function % w/w  1 AvignaSOL and higher derivatives octa and deca form Solubulizer 0-40%  2 Lactose Diluent 0-30%  3 Dicalcium phosphate Diluent 0-30%  4 Isomalt Diluent 0-30%  5 Microcrystalline cellulose Diluent 0-30%  6 Partially Pregelatinized Maize starch Binder 0-20%  7 Hydroxy propyl cellulose Binder 0 to 40%  8 Polyvinyl pyrrolidone Binder 0-15%  9 Ethyl cellulose Binder/Release retardant 0-20 % 10 Hydroxy propyl methyl cellulose K100 M Release retardant 0-30% 11 Hydroxy propyl methyl cellulose K200 M Release retardant 0-30% 12 Hydroxy propyl methyl cellulose K15M Release retardant 0-30% 13 Hydroxy propyl methyl cellulose K4 M Release retardant 0-30% 14 Poly ox N-750 Release retardant 0-30% 15 Polyox WSR-301 Release retardant 0-30% 16 Poly ox WSR-303 Release retardant 0-30% 17 Poly ox WSR-205 Release retardant 0-30% 18 PolyoxN1105 Release retardant 0-30% 19 Polyox WSR-N-12K Release retardant 0-30% 20 Polyox WSRN60K Release retardant 0-30% 21 EudragitRL 100 Release retardant 0-30% 22 EudragitRS 100 Release retardant 0-30% 23 Glyceryl behenate Binder/ Release retardant 0-30% 24 Caranauba wax Release retardant 0-30% 25 Xanthan gum Release retardant 0-30% 26 Colloidal silicon dioxide Flow improver 0-1% 27 Magnesium stearate Lubricant 0-3 % 28 Sodium stearyl fumarate Lubricant 0-3% 29 Polyvinyl alcohol Tablet coating agent 0 to 20% 30 Hypromellose Tablet coating sustained 0 to 20% release agent 31 Polyethylene glycol Tablet coating agent 0 to 10% 32 Titanium dioxide Tablet coating agent 0 to 10% 33 Talc Tablet coating agent 0 to 10% 34 hydroxy ethylcellulose In gel and tablet coating 0-20% Skin permeation enhancers for topical formulations 1 AvignaSOL NA 0 to 40% 2 Isopropyl myristate NA 0 to 20% 3 Dimethyl isosorbide. NA 0 to 20 % 4 Propylene Glycol NA 0 to 40% 5 Glycerol NA 0 to 40% 6 Glycofural NA 0 to 40% Other ingredient 11 Tween 80 0-20% 12 Glyceryl monostearate 0-20% 13 Glyceryl distearate 0-20% 14 Dimethyl isosorbide 0-20% 15 Stearic acid 0-20% 16 Cetyl alcohol 0-15% 17 Stearyl alcohol 0-15% 18 Purified water 0-98% 19 Cetearyl alcohol 0-15% 23 Isopropyl myristate 0-20% 24 Petroleum jelly 0-95% 25 Paraffin light oil 0-95% 26 SPAN 80 0-20% 27 SPAN 60 0-20% 28 Decyl glycoside 0-20%

[0230] i) Preparation of ointment: White soft paraffin, cetyl alcohol, glyceryl monostearate and light liquid paraffin was heated to 70 to 75° C. with mechanical stirring for 15 minutes when a clear solution (solution A) was obtained. Separately a clear solution (solution B) of propylene glycol, compound of formula I and stearic acid was prepared by heating with stirring for 15 minutes at 90 to 95° C. with magnetic stirrer. This solution B was added to solution A (maintained at 90 to 95° C.) with stirring by a mechanical stirrer. This mixture was then stirred at 380 rpm at 75 to 85° C. for 15 minutes. Then the mixture was cooled with stirring by mechanical stirrer at 380 rpm at 40 to 45° C. for 30 minutes and then at 300 rpm at 25 to 30° C. (ambient conditions) for additional 60 minutes to give a homogenous off-white ointment.

TABLE-US-00004 Ingredients % w/w Range % w/w Compound of formula I 3.00 1 to 6 Propylene glycol 17.00 5 to 20 White soft paraffin 48.70 20 to 80 (petroleum jelly) Cetyl alcohol 5.00 2 to 7 Stearic acid 1.80 0.5 to 4 Glyceryl mono stearate 6.00 2 to 8 Light liquid Paraffin 18.50 7 to 35 Total 100.00 (petroleum jelly proportion was adjusted)

[0231] ii) Preparation of gel or spray: To the mixture of propylene glycol, glycerol, water with or without the dimethyl isosorbide was added the compound of formula I and stirred (60 to 70° C.) till the solution is clear. To this was added the hydroxy ethyl cellulose or HPMCK100 or hydroxy propyl cellulose or hydroxy methyl cellulose (Cellosize) and the content was stirred at 55° C. for 1 hr to give a clear gel or solution.

TABLE-US-00005 Ingredients % w/w Range %w/w Compound of formula I 3.00 1 to 6 Propylene glycol 15.00 5 to 20 Glycerol 5.00 2 to 20 Dimethyl isosorbide 1.50 0 to 5 Water 74.30 40 to 90 Hydroxy propyl cellulose 0.1 for spray and 1.20 0.1 to 4 Or Hydroxy ethyl cellulose for gel Total 100.00 (water proportion was adjusted)

[0232] iii) Preparation of extended-release granules: Compound of formula I was dissolved in AvignaSOL by bath sonication and then triturated with mixture of Isomalt and MCC PH101 in a mortar. Further other ingredients: ethyl cellulose, HPMC K100 M and Pregelatinized starch were added by trituration in a mortar and then co-sifting through #30 mesh-3 times. Then the solution of 5 ml of IPA: Water (80:20) containing ethyl cellulose was added to granulate the mixture. In another experiment the compound of formula I was blended with Isomalt, MCCPH101, ethyl cellulose 7 cps, HPMC K100 M and Pregelatinized starch and then co-sifting through #30 mesh-3 times. Then the solution of 5 ml of IPA: Water (80:20) containing ethyl cellulose was added to granulate the mixture. The wet granules were sieved through #20 mesh and dried in a hot air oven at 65° C. for 2.5 hrs. The granules passing through #20 mesh and retained on #60 mesh was used for further dissolution study at 37 to 40° C., which exhibited an in-vitro release as follows: 1 hr (27%) and 19 hrs (71.4%).

Example 33: The Anti-Mould (Antifungal) Activity of Compound of Formula I

[0233] The compound of formula I solution as 0% ww, 1% ww and 3% ww as I mL water solution was added to a glass jar (5 mL) containing the fresh wheat flour bread (1 gm). The content was mixed well in order to wet the bread with solution. The content was kept in open glass cylinder for 14 days at room temperature and light (normal conditions) and the photos of Day 1 and Day 14 were taken (FIGS. 5A and B respectively).

[0234] The anti-mould activity of the compound of formula I was demonstrated by the jars containing compound of formula I preventing mould growth at day 14, as seen by the black colouring in the 0% jar. The mould is common in many skin indications and other human, animal and plant diseases. For example, Rhizopus stolonifera.

Example 34: Topical Efficacy of Compound of Formula I for Pain in Freund's Adjuvant and Deep Incision Wound in Rat

[0235] The total 24 female Sprague Dawley Rats were assigned to two test and two control groups having 6 animals per group. Animals from G1 and G2 received CFA on Day 1 by intraplanter route followed application of test item (0 and 3% respectively), onto injection site from Day 1 to Day 7. Group G3 and G4 animals received deep incision through the skin and fascia of the plantar foot followed by application of test item (0 and 3% respectively), onto the incision site from Day 1 to Day 7. Clinical signs, body weight and pain assessment parameters like Von Frey estimation and FOB was evaluated and the pain reduction results at Day 7 are summarized in Table 7. Compound of formula I treatment inhibited the CFA induced and incision wound (neuropathic) pain in rats and has anti allodynic effect recorded at day 7 in CFA (complete pain relief) and deep incision induced neuropathic pain (67% relief).

TABLE-US-00006 TABLE 7 Pain reduction results at Day 7 CFA mediated pain Incision mediated pain Placebo (G1) 3% CFI (G2) Placebo (G3) 3% CFI (G4) 0.17 0.00 0.33 0.11

Example 35: In Vitro Efficacy of Compound of Formula I in Acne Bacteria Induced Inflammation

[0236] RAW 264.7 cell line (monocyte cells) challenged with the heat-killed Propionibacterium acne (P. acne) and incubated for 1 hour and were divided into four groups as G1=not challenged with P. acne, G2=challenged with P. acne but untreated, G3=challenged with P. acne and treated with 1 μM of compound of formula I, G3=challenged with P. acne and treated with 3 μM of compound of formula I. The ELISA method was used to analyze the effect of compound of formula I on the secretion of proinflammatory cytokines the TNFα and IL6. Compound of formula I inhibits the cytokines in P. acne challenged monocyte cells. The results are shown in below FIG. 6. The compound of formula I inhibit the secretion of TNFα@1 μM=˜42% and @3 μM=˜69% and IL6@1 μM=˜27% and @3 μM=˜42% compared to untreated in P. acne challenged cell line.

[0237] It is to be understood that while the present disclosure has been described in conjunction with the specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications will be apparent to those skilled in the art to which the disclosure pertains. Therefore, the following examples are put forth so as to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed compositions and are not intended to limit the scope of the disclosure.

[0238] All documents cited are herein fully incorporated by reference for all jurisdictions in which such incorporation is permitted and to the extent such disclosure is consistent with the description of the present disclosure.