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(12) United States Patent (10) Patent No.: US 9,439,850 B2 Kelliher et al. (45) Date of Patent: *Sep. 13, 2016

(54) USE OF PUFAS FOR TREATING SKIN (2013.01); A61 K3I/573 (2013.01); A61 K NFLAMMATION 45/06 (2013.01); C07C 59/42 (2013.01) (58) Field of Classification Search (71) Applicant: Dignity Sciences Limited, Dublin (IE) CPC ...... A61 K9/0014; A61K 31/002: A61 K (72) Inventors: Adam Kelliher, London (GB); Angus 31/573; A61K 45/06; C07C 51/42 Morrison, Isle of Lewis (GB); Phil USPC ...... 514/560 Knowles, Cumbria (GB) See application file for complete search history. (73) Assignee: Dignity Sciences Limited, Dublin (IE) (56) References Cited (*) Notice: Subject to any disclaimer, the term of this U.S. PATENT DOCUMENTS patent is extended or adjusted under 35 4,273,763. A 6, 1981 Horrobin U.S.C. 154(b) by 0 days. 4,309.415 A 1/1982 Horrobin This patent is Subject to a terminal dis (Continued) claimer. FOREIGN PATENT DOCUMENTS

(21) Appl. No.: 14/203,837 EP O1394.80 5, 1952 (22) Filed: Mar. 11, 2014 EP OO35856 9, 1981 (Continued) (65) Prior Publication Data OTHER PUBLICATIONS US 2014/O194399 A1 Jul. 10, 2014 Conrow et al., Org. Proc. Res. & Dev. 15:301-304 (2011). Related U.S. Application Data (Continued) (63) Continuation of application No. 13/461,472, filed on May 1, 2012, now Pat. No. 8,729.126, which is a Primary Examiner — Deborah D Carr continuation of application No. 13/318,290, filed as (74) Attorney, Agent, or Firm — Perkins Coie LLP application No. PCT/GB2010/000844 on Apr. 29, 2010, now Pat. No. 8,536,223. (57) ABSTRACT (60) Provisional application No. 61/177,811, filed on May The present invention provides a compound which is a 13, 2009. polyunsaturated fatty acid (PUFA) derivative of formula (I), (30) Foreign Application Priority Data Apr. 29, 2009 (GB) ...... O9074 13.9 O

(51) Int. Cl. RO --~~ A6 IK 9/00 (2006.01) A6 IK 3L/202 (2006.01) or a pharmaceutically acceptable salt, or Solvate thereof, for (Continued) use in treating various skin disorders. (52) U.S. Cl. CPC ...... A61K 9/0014 (2013.01); A61K 3 1/202 16 Claims, 5 Drawing Sheets

US 9,439,850 B2 Page 2

(51) Int. Cl. EP 0309086 3, 1989 A 6LX 3/573 (2006.01) EP O409559 1991 A6 IK 45/06 (2006.01) E. 'S 28. C07C 59/42 (2006.01) EP 250818O 10, 2012 JP 2006219.454 8, 2006 (56) References Cited JP 2006.306812 11 2006 WO WO9816215 4f1998 U.S. PATENT DOCUMENTS WO WO9852556 11, 1998 WO WOO 103696 1, 2001 4,386,072 A 5, 1983 Horrobin et al. WO WOO2O1969 1, 2002 4,388,324 A 6, 1983 Horrobin WO WOO2O89791 11 2002 4,444,755 A 4, 1984 Horrobin WO WOO2092O71 11 2002 4,826,877 A 5, 1989 Stewart et al. WO WOO2O964.08 12/2002 4,888,326 A 12/1989 Horrobin WO WO2007079224 7/2007 4,898,885 A 2f1990 Horrobin WO WO2O08070129 6, 2008 4,965,075 A 10, 1990 Horrobin et al. WO WO 20080701.29 A2 * 6, 2008 ...... A61K 31,202 4,970,076 A 11/1990 Horrobin WO WO2008114141 9, 2008 4,996,233 A 2f1991 Horrobin WO WO2O10020603 2, 2010 4,997,657 A 3, 1991 Horrobin et al. WO WO2010.125330 11 2010 5,145,686 A 9, 1992 Horrobin et al. WO WO2010.125340 11 2010 5, 198,468 A 3, 1993 Horrobin WO WO2O13057284 4/2013 5,318,991 A 6, 1994 Horrobin et al. WO WO2O13057287 4/2013 5,324,748 A 6, 1994 Horrobin WO WO2013O82265 6, 2013 5,328,691 A 7, 1994 Horrobin et al. WO WO2O13112876 8, 2013 5,380,757 A 1/1995 Horrobin WO WO2O13124479 8, 2013 5,552,150 A 9, 1996 Horrobin et al. WO WO2O14022816 2, 2014 5,562.913 A 10, 1996 Horrobin 5,580,556 A 12/1996 Horrobin OTHER PUBLICATIONS 5,583,159 A 12/1996 Horrobin et al. 5,589,509 A 12/1996 Horrobin Desbois et al., “Antibacterial free fatty adds: activities, mechanisms 5,614,208 A 3/1997 Horrobin et al. of action and biotechnological potential.' Appl. Microbiol. 5,618,558 A 4, 1997 Horrobin et al. Biotechnol. (2010) 85:1629-1642. SE A 22 E. al Eishof et al. “Biocatalytic large-scale production of 12(S)- 5,709,885 A 1/1998 Hellen et al. hydroperoxy-9(Z), 116 octadecadienoic acid from hydrolysed saf 5,888,541 A 3, 1999 Horrobin et al. flower oil by a crude soybean-flour extract as lipoxygenase source.” 6,069,168 A 5, 2000 Horrobin et al. Rec. Trav. Chim Pays-Bas, 115, 11-12, p. 499-504 (1996). 6,177,470 B1 1/2001 Horrobin et al. Elshof et al., “Biocatalytic hydroxylation of linoleic acid in a 6.479,544 B1 1 1/2002 Horrobin double-fed batch system with lipoxygenase and cysteine,” 100:6. 6,630,157 B1 10/2003 Horrobin et al. pp. 246-251 (1999). 7:50 R: $39, Rani, et al. Erythema, Retrieved online Feb. 11, 2014). Retrieved from 8.293,790 B2 10/2012 Manku et al. Rhttp:/ummelu health medical aimed conditionery. 8,536,223 B2 9, 2013 Kelliher et al. 68 A ------2003/OO73930 A1 4/2003 Morrissey et al. Feldlaufer et al., Antimicrobial activity of fatty acids against 2003/0194446 A1 10, 2003 Akes et al. Bacillus larvae, the causative agent of American Foulbrood dis 2007/00 15438 A1 1/2007 Lange et al. ease.” Apidologie (1993). 24. pp. 95-99. 2008.01254.87 A1 5, 2008 Das et al. Huang et al., “Antimicrobial activity of n-6, n-7 and n-9 fatty acids 2008.0161275 A1 7/2008 Gorstrup and their esters for oral microorganisms,” Archives of Oral Biology, 2008/0213357 A1 9, 2008 Hebard et al. 55 (2010), pp. 555-560. 2009,0176284 A1 7/2009 Furihata et al. Kawashima et al., “Subchronic (13-week) oral toxicity study of 2010/O184727 A1 T/2010 Roach et al. dihomo-y-linolenic acid (DGLA) oil in rats.” Food and Chemical 2010/0278948 A1 11, 2010 Courtin Toxicology (2009). 2010/0317735 Al 12/2010 Hong et al. Kiistala et al., “In vivo Separation of Epidermis by Production of 2011 OO15415 A1 1/2011 Singh et al. Sucti ------? 2011/00 16585 A1 1/2011 Pereira et al. uction Blister," Lancet 1964: 1444-1445 (1964). 2011 OO39010 A1 2/2011 Rein et al. Kiistala, “Suction buster device for separation fo viable epidermis 2011/0059885 A1 3/2011 Lea et al. from dermis,” J. Invest. Dermatol. 50: 129-137 (1968). 2012/O142773 A1 6, 2012 Kelliher et al. Miller et al., “Dietary Supplementation with Ethyl Ester Concen 2012fO213824 A1 8, 2012 Kelliher et al. trates of Fish Oil (N-3) and Borage Oil (N-6) Polyunsaturated Fatty 2012fO232147 A1 9, 2012 Manku et al. Acids Induces Epidermal Generation of Local Putative Anti-Inflam 2012fO264705 A1 10, 2012 Manku et al. matory Metabolites.” The Journ. of Invest. Dermatol., vol. 96, No. 2013/0101533 A1 4/2013 Manku et al. 1, pp. 98-103 (1991). 2013/01O2575 A1 4/2013 Manku et al. Miller et al. "Guinea Pig Epidermins Generates Putative Anti 2013/0267,598 A1 10, 2013 Manku et al. Inflammatory Metabolites from Fish Oil Polyunsaturated Acids.” 2013/0274338 A1 10, 2013 Manku et al. Lipids, vol. 24, No. 12 (1989). 2013/0331448 A1 12/2013 Manku et al. Mosmann, J. Immunol.1. v. W Meth. vol. 65, pp. 55-63 (1983). Mundlein et al., "Comparison of Transepiderminal Water Losee FOREIGN PATENT DOCUMENTS (TEWL) Measurements with Two Novel Sensors Based on Different EP OO37175 10, 1981 Sensing Principles,” Sensors and Actuators A: Physical, 142(1):67 EP OO78434 5, 1983 72 (2008). EP O087863 9, 1983 Nijs et al., “Adult-onset asthma, is it really different?” Eur, Respir. EP O087864 9, 1983 Rev., vol. 22(127), pp. 44-52, Mar. 1, 2013. EP O087865 9, 1983 PCT/EP12/070809 International Search Report dated Jan. 22, 2013. EP O115419 8, 1984 PCT/EP12/070813 International Search Report dated Jan. 14, 2013. EP O 132089 1, 1985 PCT/EP2013/053677 International Search Report dated Jun. 19, EP O173478 3, 1986 2013. US 9,439,850 B2 Page 3

(56) References Cited Schreiner at al., “Transfer of Penicillin G and Ampicillin into Human Skin Blisters Induced by Suction.” Scand. Journ. Infect. Dis. OTHER PUBLICATIONS 14(Suppl.);233-237 (1978). Tope, “Multi-electrode radio frequency resurfacing of ex vivo PCT/GB2010/000844 International Search Report dated Jul. 19, hontan skin.” Dermatol. Surg., 25:348-52 (1999). Xue et al., “Optimization of Synthethic Conditions for the Prepa Fusoid too International Search Report dated Feb. 5, ration of Dihomo-gamma-Linolenic Acid from gamma-Linolenic Acid,” Journ Amer. Oil Chemists’ Soc., Jan. 2009, vol. 86, Issue 1, Fusososol International Search Report dated Mar. 29. pp. 77-82. Ziboh et al., “Dihomo-gama-linolenic acid 15 Fusos ossos International Search Report dated Feb. 4. hydroxyeicosatrienoic acid in skin inflammatory and proliferative processes,” pp. 198-206 (2001). s et al., “Microdialysis documents changes in the micromilieu Ziboh et al., Am. J. Clin. Nutr. 2000:71(suppl):361S-6S. of psoriatic plaques under continuous systemic therapy.” Experi mental Dermatology 20:130-133 (2011). * cited by examiner U.S. Patent Sep. 13, 2016 Sheet 1 of 5 US 9,439,850 B2

Figure 1 U.S. Patent Sep. 13, 2016 Sheet 2 of 5 US 9,439,850 B2

Figure 2 U.S. Patent Sep. 13, 2016 Sheet 3 of 5 US 9,439,850 B2

Figure 3 U.S. Patent Sep. 13, 2016 Sheet 4 of 5 US 9,439,850 B2

Figure 4 U.S. Patent Sep. 13, 2016 Sheet S of 5 US 9,439,850 B2

Figure 5 US 9,439,850 B2 1. USE OF PUFAS FOR TREATING SKN NFLAMMATION

FIELD OF THE INVENTION The present invention relates to novel methods of treating skin inflammation, in particular skin inflammation caused by atopic eczema, contact dermatitis, psoriasis or uremic pru ritis. EP-A-0085579 describes the use of DGLA in combina 10 tion with antipruritic lithium salts. EP-A-0173478 describes BACKGROUND OF THE INVENTION the use of DGLA in combination with anti-inflammatory . In these applications, treatment with lithium salts and glucocorticoids is Supplemented with Most common skin disorders and complaints comprise a DGLA, as both lithium salts and glucocorticoids are number of different components. Thus, many skin disorders 15 believed to block release of DGLA from endogenous stores involve (a) hyperproliferation, (b) inflammation and/or (c) in the body. dehydration. Hyperproliferation involves a state of abnor Advantageously, it has now been discovered that DGLA mally high cell division, which can lead to excess flaking can be used effectively as a monotherapy. skin. Inflammation involves Swelling and redness of the 5-Hydroxy-eicosa-6E.8Z.11Z-trienoic acid (5-HETrE)) is skin, as well as sensations of increased heat, and pain in the a commercially available PUFA derivative derived from skin. Dehydration involves loss of water from the skin and mead acid. 5-HETrE has the structure shown below. may be due, for example, to damage to the normally waterprooftop layer of the skin (epidermis). Inflammation of the skin (dermatitis) in mammals can result from a number of different etiologies. Dermatitis can 25 be caused by eczema, in particular atopic eczema (atopic HO dermatitis), disseminated neurodermatitis, flexural eczema, infantile eczema, prurigo diathsidue, contact dermatitis, (eg irritant contact dermatitis, allergic contact dermatitis and photocontact dermatitis), Xerotic eczema, Seborrheic 30 eczema, dyshidrosis, discoid eczema, Venous eczema, der matitis herpetiformis, neurodermatitis and autoeczematisa 8-Hydroxy-eicosa-9E,11Z.14Z-trienoic acid (8-HETrE) tion. Dermatitis can also be caused by skin inflammation resulting from exposure to radiation, in particular exposure is a commercially available PUFA derivative derived from 35 eicosa-8Z.11Z.14Z-trienoic acid (Dilhomo-y-linolenic acid to ultraviolet radiation. Other causes of dermatitis includes or DGLA). 8-HETrE has the structure shown below. uremic pruritis and autoimmune diseases, in particular lupus and psoriasis. Inflammation of the skin causes rashes, redness, skin edema (Swelling), itching, blistering, sensations of pain 40 and/or heat and can be unsightly. The itchiness caused by HO inflammation can lead to scratching. Scratching of skin that OH is already damaged in Some way can easily lead to the barrier of the epidermis being broken, resulting in bleeding, and secondary infection with pathogens. Such secondary 45 infection can require treatment with antibiotics. It is well known that when treating a skin condition that 15-Hydroxy-eicosa-8Z., 11Z,13E-trienoic acid (15-HE has a number of different components, i.e. hyperprolifera TrE) is a commercially available PUFA derivative derived tive, inflammatory and/or dehydrative components, a num from eicosa-8Z.11Z., 14Z-trienoic acid (Dilhomo-y-linolenic ber of different treatments may be used. Thus, in the 50 acid or DGLA). 15-HETrE has the structure shown below. treatment of psoriasis, for example, an antihyperprolifera tive may be used to treat the hyperproliferative component of the disease, an anti-inflammatory may be used to treat the inflammatory component and an emollient may be used to treat the dehydrative component. 55 The most common form of treatment for inflammation of the skin is oral and/or topical . There are, however, drawbacks associated with treatments. Common side effects associated with steroids include stunting of growth, thinning of the skin, muscle loss and osteoporosis. 60 15-HETrE is known to have antiproliferative properties The present invention relates to new methods for treating when applied directly to the skin (Xi, et al; Prostaglandins, skin inflammation, in particular skin inflammation caused by Leukotrienes and Essential Fatty Acids (2000) 62(1), 13 to atopic eczema, contact dermatitis, psoriasis or uremic pru 19). ritis, in mammals. 13-Hydroxy-octadeca-6Z.97,11E-y-trienoic acid (13-HO Eicosa-8Z., 11Z.14Z-trienoic acid (Dilhomo-y-linolenic 65 TrE(Y)) is a commercially available PUFA derivative derived acid or DGLA) is a commercially available polyunsaturated from gamma-linolenic acid (GLA). 13-HOTrE(Y) has the fatty acid (PUPA). DGLA has the structure shown below. structure shown below. US 9,439,850 B2 4

hydroxyl, - SR", and - NR'R" groups where R' and R" are the same or different and represent hydrogen or unsubstituted C-C, alkyl: said aryl, heteroaryl, carbocyclyl and heterocyclyl groups are the same or different and are each unsubstituted or substituted by 1, 2, 3 or 4 unsubstituted substituents which are the same or different and are selected from halogen atoms, and cyano, nitro, C-C alkyl, C-C, It has now been surprisingly found that DGLA, 5-HETrE, alkoxy, C-C alkenyl, C-C alkenyloxy, C-C 8-HETrE, 15-HETrE, 13-HOTrE(Y) and their derivatives are 10 haloalkyl, C-Chaloalkenyl, C-Chaloalkoxy, C-C, clinically useful in treating skin inflammation, in particular haloalkenyloxy, hydroxyl, C-C hydroxyalkyl, - SR" skin inflammation caused by atopic eczema, contact derma and —NR'R" groups wherein each R" and R" is the titis, psoriasis or uremic pruritis, by topical administration in same or different and represents hydrogen or unsubsti mammals. A particular finding of the present invention is 15 tuted C-C alkyl; that these compounds reduce the level of COX-2 enzymes in and wherein the PUFA derivative is in the form of a the skin when applied topically. The COX-2 family of racemate, a stereoisomer or a mixture of stereoisomers, enzymes have been strongly linked to inflammation and which compound is for use in treating skin inflammation in have been found to be present in increased amounts in a mammal, by topical administration. inflamed tissue. 2O DETAILED DESCRIPTION OF THE SUMMARY OF THE INVENTION INVENTION The present invention therefore provides a compound FIG. 1 shows the results of an immunohistochemistry which is a polyunsaturated fatty acid (PUFA) derivative of assay to determine the amount of COX-2 enzymes present in formula (I), 25 ex vivo porcine ear skin 0 and 6 hours after staining with diaminobenzidine. FIG. 2 shows the results of an immunohistochemistry O assay to determine the amount of COX-2 enzymes present in 30 ex vivo porcine ear skin, which has been treated with --- "S-1a1n ketoprofen in fish oil, O and 6 hours after staining with RO diaminobenzidine. FIG. 3 shows the results of an immunohistochemistry or a pharmaceutically acceptable salt, or Solvate thereof, assay to determine the amount of COX-2 enzymes present in wherein 35 ex vivo porcine ear skin, which has been treated with a -Alk- is —CH(OR)-transCH=CH-cis CH=CH representative compound of the invention, DGLA.. O and 6 CH-cis ICH=CH-CH , —(CH) CH(OR)- hours after staining with diaminobenzidine. transCH=CH-cis ICH=CH-CH-cis FIG. 4 shows the results of an immunohistochemistry CH-CH , —(CH)-cis ICH=CH-CH-cis assay to determine the amount of COX-2 enzymes present in CH=CH-transCH=CH-CH(OR)– or -CH 40 ex vivo porcine ear skin, which has been treated with a cis ICH=CH-CH-cis ICH=CH-transCH=CH representative compound of the invention, 15-HETrE, 0 and CH(OR)–: 6 hours after staining with diaminobenzidine. R is a hydrogen atom; or FIG. 5 shows the results of a Western Blotting analysis to R is a C-Calkyl, C-C alkenyl, C-C alkynyl, Co-Co determine the effect of DGLA (second bar) and 15-HETrE aryl, 5- to 10-membered heteroaryl, C-C, carbocyclyl 45 (third bar) on COX-2 expression in porcine skin relative to or 5- to 10-membered heterocyclyl group; or water (first) bar. Levels in control were assigned a value of R is a group of formula —CH2—CH(OR)—CH2— 100%. (OR), wherein R and R are each independently Preferably the alkyl, alkenyl, alkynyl and aliphatic groups hydrogen atoms or —(C=O)—R, wherein R is an are unsubstituted or substituted with 1, 2 or 3, preferably 1 aliphatic group having from 3 to 29 carbon atoms; or 50 or 2, more preferably 1, unsubstituted substituents which are R is a group of formula—(CHOCH), OH, wherein m the same or different and are selected from halogen atoms is an integer of from 1 to 200; and C-C alkoxy, hydroxyl, C-C haloalkyl, C-C, R is a hydrogen atom; or haloalkenyl, C-C haloalkyloxy and —NR'R'" wherein R' R is a group —(C=O)—Rs, wherein Rs is a C-C alkyl, and R" are the same or different and represent hydrogen or C-C alkenyl, C-C alkynyl, Co-Co aryl, 5- to 55 C-C alkyl. More preferred Substituents are halogen, C-C, 10-membered heteroaryl, C-C, carbocyclyl or 5- to alkoxy, hydroxyl and —NR'R" groups where R' and R" are 10-membered heterocyclyl group, or Rs is an aliphatic the same or different and represent hydrogen or unsubsti group having from 3 to 29 carbon atoms; or tuted C-C alkyl. Particularly preferred substituents include R is a group of formula—(CHOCH), OH, wherein n is hydroxyl and —NR'R" groups where R' and R" are the same an integer of from 1 to 200; 60 and represent hydrogen. and wherein When the alkyl, alkenyl, alkynyl and aliphatic groups said alkyl, alkenyl, alkynyl and aliphatic groups are the above are substituted by two or three substituents, it is same or different and are each unsubstituted or substi preferred that not more than two substituents are selected tuted with 1, 2 or 3 unsubstituted substituents which are from hydroxyl. More preferably, not more than one sub the same or different and are selected from halogen 65 stituent is selected from hydroxyl. atoms and C-C alkoxy, alkenyloxy, haloalkyl, C-C Most preferably, the alkyl, alkenyl and alkynyl groups haloalkenyl, C-C haloalkoxy, C-C haloalkenyloxy, above are unsubstituted. US 9,439,850 B2 5 6 As used herein, a C-C alkyl group is a linear or branched or 4 heteroatoms, selected from O, S and N. When the ring alkyl group containing from 1 to 6 carbon atoms, for contains 4 heteroatoms these are preferably all nitrogen example a C-C alkyl group containing from 1 to 4 carbon atoms. Examples include thienyl, furyl, pyrrolyl, imidazolyl, atoms, preferably a C-C alkyl group containing from 1 to thiazolyl, isothiazolyl pyrazolyl, oxazolyl, isoxazolyl, tri 2 carbon atoms. Examples of C-C alkyl groups include azolyl, thiadiazolyl, oxadiazolyl pyridinyl, pyridazinyl, methyl, ethyl, n-propyl, i-propyl. n-butyl, i-butyl and t-bu pyrimidinyl, pyrazinyl, triazinyl and tetrazolyl groups. Thie tyl. For the avoidance of doubt, where two alkyl groups are nyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, present in a compound of the present invention, the alkyl oxazolyl, isoxazolyl, triazolyl pyridinyl, pyridazinyl, groups may be the same or different. pyrimidinyl and pyrazinyl groups are preferred, e.g. pyrro As used herein, a C-C alkenyl group is a linear or 10 lyl, imidazolyl, thiazolyl, isothiazolyl pyrazolyl, oxazolyl, branched alkenyl group having at least one double bond of isoxazolyl, triazolyl pyridinyl, pyridazinyl, pyrimidinyl and either cis or trans configuration where applicable and con pyrazinyl groups. More preferred groups are thienyl, pyridi taining from 2 to 6 carbon atoms, for example a C-C, nyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl and triazi alkenyl group containing from 2 to 4 carbon atoms, such as nyl, e.g. pyridinyl, pyridaZinyl, pyrimidinyl, pyrazinyl, pyr —CH=CH or —CH2—CH=CH, CH, CH 15 rolyl and triazinyl, most preferably pyridinyl. CH=CH, -CH-CH=CH-CH, -CH=C(CH)– As used herein, a 5- to 10-membered heterocyclyl group CH and —CH2—C(CH)—CH2 preferably a C alkenyl is a non-aromatic, Saturated or unsaturated monocyclic or group having 2 carbon atoms. For the avoidance of doubt, polycyclic, preferably monocyclic, Cso carbocyclic ring in where two alkenyl groups are present in a compound of the which one or more, for example 1, 2, 3 or 4, of the carbon present invention, they may be the same or different. atoms are replaced with a moiety selected from N, O, S, As used herein, a C-C alkynyl group is a linear or S(O) and S(O), and wherein one or more of the remaining branched alkynyl group containing from 2 to 6 carbon carbon atoms is optionally replaced by a group —C(O)— or atoms, for example a C-C alkynyl group containing from —C(S)-. When one or more of the remaining carbon atoms 2 to 4 carbon atoms, preferably a C alkynyl group contain is replaced by a group —C(O)— or —C(S)—, preferably ing 2 carbon atoms. Exemplary alkynyl groups include 25 only one or two (more preferably two) Such carbon atoms —C=CH or —CH2—C=CH, as well as 1- and 2-butynyl, are replaced. Typically, the 5- to 10-membered heterocyclyl 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, ring is a 5- to 6-membered ring. 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl. For the Suitable heterocyclyl groups include azetidinyl, oxetanyl, avoidance of doubt, where two alkynyl groups are present in thietanyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, isox a compound of the present invention, they may be the same 30 azolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, or different. tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl. Preferably, said C-C alkyl group is a C-C alkyl group, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazi said C-C alkenyl group is a C alkenyl group and said nyl, hexahydropyrimidinyl, methylenedioxyphenyl, ethyl C-C alkynyl group is a C alkynyl group. enedioxyphenyl, thiomorpholinyl. S-oxo-thiomorpholinyl, As used herein, a halogen atom is chlorine, fluorine, 35 S.S.-dioxo-thiomorpholinyl, morpholinyl, 1,3-dioxolanyl, bromine or iodine. 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl, pyra As used herein, a C-C alkoxy group or C-C alkeny nyl, pyrazolinyl, thioxolanyl, thioXothiazolidinyl, 1 H-pyra loxy group is typically a said C-Calkyl (e.g. a C-C alkyl) Zol-5-(4H)-onyl, 1,3,4-thiadiazol-2(3H)-thionyl, oxopyrro group or a said C-C alkenyl (e.g. a C-C alkenyl) group lidinyl, OXothiazolidinyl, oxopyrazolidinyl. Succinimido and respectively which is attached to an oxygen atom. 40 maleimido groups and moieties. Preferred heterocyclyl A haloalkyl, haloalkenyl, haloalkoxy or haloalkenyloxy groups are pyrrolidinyl, imidazolidinyl, oxazolidinyl, isox group is typically a said alkyl, alkenyl, alkoxy or alkenyloxy azolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, group respectively which is Substituted by one or more said tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl. halogen atoms. Typically, it is substituted by 1, 2 or 3 said dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazi halogen atoms. Preferred haloalkyl and haloalkoxy groups 45 nyl, hexahydropyrimidinyl, thiomorpholinyl and morpholi include perhaloalkyl and perhaloalkoxy groups, such as nyl groups and moieties. —CX and —OCX, wherein X is a said halogen atom, for For the avoidance of doubt, although the above definitions example chlorine and fluorine. of heteroaryland heterocyclyl groups refer to an “N' moiety As used herein, a C-C alkylthio or C-C alkenylthio which can be present in the ring, as will be evident to a group is typically a said C-C alkyl group or a C-C, 50 skilled chemist the Natom will be protonated (or will carry alkenyl group respectively which is attached to a Sulphur a substituent as defined below) if it is attached to each of the atom, for example —S—CH. adjacent ring atoms via a single bond. As used herein, a C-C hydroxyalkyl group is a C-C As used herein, a C-C, carbocyclic group is a non alkyl group Substituted by one or more hydroxy groups. aromatic Saturated or unsaturated hydrocarbon ring having Typically, it is substituted by one, two or three hydroxy 55 from 3 to 7 carbon atoms. Preferably it is a saturated or groups. Preferably, it is substituted by a single hydroxy mono-unsaturated hydrocarbon ring (i.e. a cycloalkyl moi group. ety or a cycloalkenyl moiety) having from 3 to 7 carbon As used herein, a Co-Co aryl group is a monocyclic or atoms, more preferably having from 3 to 6 carbon atoms. polycyclic, preferably monocyclic, aromatic ring containing Examples include cyclopropyl, cyclobutyl, cyclopentyl and from 6 to 10 carbon atoms, for example a Caryl group 60 cyclohexyl and their mono-unsaturated variants, more par containing 6 carbon atoms. Examples of Such aryl groups ticularly cyclopentyl and cyclohexyl. A C-C, carbocyclyl include phenyl, naphthalene and aZulene. Phenyl is pre group also includes C-C, carbocyclyl groups described ferred. above but wherein one or more ring carbon atoms are As used herein, a 5- to 10-membered heteroaryl group is replaced by a group —C(O)—. More preferably, 0, 1 or 2 a monocyclic or polycyclic, preferably monocyclic, 5- to 65 ring carbon atoms (most preferably 0) are replaced by 10-membered aromatic ring, such as a 5- or 6-membered —C(O)—. Most preferably, said C-C, carbocyclyl group is ring, containing at least one heteroatom, for example 1, 2, 3 cyclohexyl. US 9,439,850 B2 7 8 Typically the aryl, heteroaryl, heterocyclyl and carbocy group is typically unsubstituted or Substituted with one clyl groups in R and Rs are unsubstituted or Substituted by hydroxyl group. The aliphatic group is preferably unsubsti 1, 2, 3 or 4 unsubstituted substituents, for example by 1, 2 tuted. or 3 unsubstituted substituents. Preferred substituents Aliphatic groups may be saturated, monounsaturated or include halogen atoms and C-C alkyl, C-C alkenyl, 5 polyunsaturated. Saturated aliphatic groups are preferred. C-C alkoxy, C-C alkenyloxy, C-C haloalkyl, C-C, Typically, saturated aliphatic groups have from 7 to 25 haloalkenyl, C-C haloalkoxy, C-C haloalkenyloxy, carbon atoms, preferably 11 to 17 carbon atoms. hydroxyl, mercapto, cyano, nitro, C-C hydroxyalkyl, Monounsaturated aliphatic groups typically contain a C-C hydroxyalkenyl, C-C alkylthio. C-C alkenylthio 10 single C=C double bond. The double bond has cis or trans and —NR'R" groups wherein each R" and R" is the same or configuration. The single double bond may be present at any different and represents hydrogen or C-C alkyl. More point in the aliphatic group, but is typically 7 or 9 carbon preferred Substituents include halogen atoms and unsubsti atoms from the end of the aliphatic group distal to the tuted C-C alkyl, C-C alkoxy, hydroxyl, C-C haloalkyl, 15 (C=O) group to which the aliphatic group is attached. C-C haloalkoxy, C-C hydroxyalkyl, cyano, nitro. —SR Typically, monounsaturated aliphatic groups have from 7 to and —NR'R" groups where R" and R" are the same or 25 carbon atoms, preferably 15 to 23 carbon atoms. different and represent hydrogen or unsubstituted C-C, Polyunsaturated aliphatic groups typically contain two or alkyl. More preferred substituents include halogen atoms, more C=C double bonds, for example 2, 3, 4, 5 or 6 C=C double bonds. Each double bond may have cis or trans hydroxyl groups and C-C alkyl and C-C alkoxy groups. configuration. The double bonds may be present at any point Most preferably, the aryl, heteroaryl, heterocyclyl and in the aliphatic chain, but typically, the C=C double bond carbocyclyl groups above are unsubstituted. furthest from the (C=O) group to which the aliphatic group When the aryl, heteroaryl, heterocyclyl and carbocyclyl is attached is 3, 6 or 9 carbon atoms from the end of the groups in R and Rs are substituted by two, three or four 25 aliphatic group distal to the (C=O) group to which the substituents, it is preferred that not more than two substitu aliphatic group is attached. Typically, polyunsaturated ali ents are selected from hydroxyl, cyano and nitro. More phatic groups have from 7 to 25 carbon atoms, preferably 15 preferably, not more than one substituent is selected from hydroxyl, cyano and nitro. to 23 carbon atoms. As used herein, a pharmaceutically acceptable salt is a salt 30 Typically, said aliphatic group is the group R, wherein with a pharmaceutically acceptable acid or base. Pharma R—COH is a fatty acid. Preferably, said fatty acid is lauric ceutically acceptable acids include both inorganic acids such acid, myristic acid, palmitic acid, Stearic acid palmitoleic as hydrochloric, Sulphuric, phosphoric, diphosphoric, hyd acid, cis-vaccenic acid, oleic acid, eicosenoic acid, erucic robromic or nitric acid and organic acids Such as citric, acid, nervonic acid, alpha-linolenic acid, Stearidonic acid, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, 35 eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acetic, methanesulphonic, ethanesulphonic, benzenesul acid, docosapentaenoic acid, docosahexaenoic acid, tetra phonic or p-toluenesulphonic acid. Pharmaceutically accept cosapentaenoic acid, tetracosahexaenoic acid, linoleic acid, able bases include alkali metal (e.g. sodium or potassium) gamma-linolenic acid, eicosadienoic acid, dihommo and alkali earth metal (e.g. calcium or magnesium) hydrox gamma-linolenic acid, arachidonic acid, docosadienoic acid, ides and organic bases such as alkyl amines, aralkyl amines 40 adrenic acid, docosapentaenoic acid, or mead acid. More and heterocyclic amines. preferably, said fatty acid is lauric acid, myristic acid, The term "solvate” refers to a complex or aggregate palmitic acid, or Stearic acid. formed by one or more molecules of a solute, i.e. com In one embodiment, the aliphatic group having 3 to 29 pounds of the invention or pharmaceutically-acceptable salts carbon atoms is the aliphatic group of a PUFA derivative of thereof, and one or more molecules of a solvent. Such 45 formula (I) as defined herein, i.e. the aliphatic group is of Solvates are typically crystalline Solids having a Substan formula —(CH)-Alk-(CH2)CH, wherein -Alk- is as tially fixed molar ratio of solute and solvent. Representative defined herein. Solvents include by way of example, water, methanol, etha nol, isopropanol, acetic acid, and the like. When the solvent In a preferred embodiment, the aliphatic group having 3 is water, the solvate formed is a hydrate. 50 to 29 carbon atoms is the aliphatic group of dihommo The compounds of the invention may contain a chiral gamma-linolenic acid or 15-hydroxyeicosatrienoic acid, i.e. center. Accordingly, they can be used in the form of a the aliphatic group is —(CH2). CH=CH-CH2— racemic mixture, an enantiomer, or a mixture enriched in CH=CH-CH-CH=CH-(CH2)CH, wherein all of one or more Stereoisomer. The scope of the invention as the C=C double bonds have cis configuration, or described and claimed encompasses the racemic forms of 55 —(CH)-cis ICH=CH-CH-cis CH=CH-trans the compounds of the invention as well as the individual CH=CH-CH(OH)–(CH)CH. Preferably, the aliphatic enantiomers, and stereoisomer-enriched mixtures. group having 3 to 29 carbon atoms is the aliphatic group of It will be appreciated that the term "or a pharmaceutically 15-hydroxyeicosatrienoic acid, i.e. the aliphatic group is acceptable salt or solvate thereof is intended to include all —(CH)-cis ICH=CH-CH-cis CH=CH-trans permutations of salts and Solvates, such as Solvates of 60 CH=CH-CH(OH)–(CH)CH. pharmaceutically-acceptable salts of compounds of the In a more preferred embodiment, the PUFA derivative of invention. formula (I) is of formula R'(C=O)C CH-CH(O(C=O) Rs and R may be an aliphatic group having 3 to 29 carbon R")—CH O(C=O)R', wherein each R' is the aliphatic atoms. Typically, the aliphatic group is not cyclic. The group of 15-hydroxyeicosatrienoic acid, i.e. R' is aliphatic group is typically linear or branched, preferably 65 —(CH)-cis ICH=CH-CH-cis CH=CH-trans linear. Typically the aliphatic group has 7 to 25 carbon CH=CH-CH(OH)–(CH)CH. Thus, the PUFA deriva atoms, more preferably 11 to 25 carbon atoms. The aliphatic tive of formula (I) is preferably US 9,439,850 B2

It is to be understood that the left hand side of the -Alk 15 R is an aliphatic group having from 3 to 29 carbonatoms, moiety is bonded to the unsaturated carbon chain bearing the as defined herein. Typically, said aliphatic group is Saturated. —COOR moiety and the right hand side of the -Alk-group Typically, R is an aliphatic group having 7 to 25 carbon is bonded to the saturated carbon chain. atoms, preferably 11 to 17 carbon atoms. Preferably, R is a Typically, -Alk- is —CH(OR)-transCH=CH-cis group R, wherein R-COH is auric acid, myristic acid, CH=CH-CH-cis ICH=CH-CH , —(CH) CH palmitic acid, or Stearic acid. (OR)-transCH=CH-cis ICH=CH-CH-cis Typically, R is a hydrogen atom; or R is a group CH=CH-, or —(CH)-cis ICH=CH-CH-cis —(C=O)—Rs, wherein Rs is a C-C alkyl, C-C alkenyl, CH=CH-transCH=CH-CH(OR)-. Preferably, -Alk C-C alkynyl, Caryl, 5- to 6-membered heteroaryl, C-C, is —(CH)-cis ICH=CH-CH-cis CH=CH-CH 25 carbocyclyl or 5- to 6-membered heterocyclyl group, or Rs cisCH=CH-. is an aliphatic group having from 3 to 29 carbon atoms; or Typically, R is a hydrogen atom; or R is a C-C alkyl, R is a group of formula—(CHOCH), OH, wherein n is as C-C alkenyl, C-C alkynyl, Caryl, 5- to 6-membered defined herein, wherein said alkyl, alkenyl and alkynyl heteroaryl, C-C carbocyclyl or 5- to 6-membered hetero groups are the same or different and are each unsubstituted cyclyl group; or R is a group of formula —CH2—CH 30 or substituted with 1, or 2 unsubstituted substituents which (OR)—CH2—(OR), wherein R and Ra are as defined are the same or different and are selected from halogen herein; or R is a group of formula —(CHOCH)OH, atoms, C-C alkoxy, hydroxyl, and —NR'R" groups where wherein m is as defined herein, wherein said alkyl, alkenyl R" and R" are the same or different and represent hydrogen and alkynyl groups are the same or different and are each or unsubstituted C-C alkyl; and said aryl, heteroaryl, unsubstituted or substituted with 1, or 2 unsubstituted Sub 35 carbocyclyl and heterocyclyl groups are the same or differ stituents which are the same or different and are selected ent and are each unsubstituted or substituted by 1, 2 or 3 from halogen atoms, C-C alkoxy, hydroxyl, and —NR'R'" unsubstituted substituents which are the same or different groups where R' and R" are the same or different and and are selected from halogen atoms, and cyano, nitro, represent hydrogen or unsubstituted C-C alkyl, and said C-C alkyl, C-C alkoxy, and —NR'R" groups wherein aryl, heteroaryl, carbocyclyl and heterocyclyl groups are the 40 each R' and R" is the same or different and represents same or different and are each unsubstituted or substituted hydrogen or unsubstituted C-C alkyl group. by 1, 2 or 3 unsubstituted substituents which are the same or Preferably, R is a hydrogen atom; or R is a group different and are selected from halogen atoms, and cyano, —(C=O)—Rs, wherein Rs is unsubstituted C-C alkyl, or nitro, C-C alkyl, C-C alkoxy, and —NR'R" groups R is a group —(C=O)—Rs, wherein Rs is an aliphatic wherein each R' and R" is the same or different and 45 group having from 3 to 29 carbon atoms; or R is a group of represents hydrogen or unsubstituted C-C alkyl group. formula—(CHOCH), OH, wherein n is as defined herein. Preferably, R is a hydrogen atom; or R is an unsubsti More preferably, R is a hydrogen atom; or R is a group tuted C-C alkyl group; or R is a group of formula —(C=O)—Rs, wherein Rs is an aliphatic group having —CH-CH(OR)—CH2—(OR), wherein R and R are from 3 to 29 carbon atoms; or R is a group of formula as defined herein; or R is a group of formula 50 —(CHOCH), OH, wherein n is as defined herein. —(CHOCH), OH, wherein m is as defined herein. Most preferably, R is a hydrogen atom. More preferably, R is a hydrogen atom; or R is a group n is typically an integer of from 5 to 150, preferably from of formula —CH2—CH(OR)—CH2—(OR), wherein R 10 to 50. and Ra are as defined herein, and wherein at least one of R When Rs is an aliphatic group having 3 to 29 carbon or R is —(C=O)—R, wherein R is as defined herein. 55 atoms, said aliphatic group is as defined herein. Typically, Most preferably, R is a hydrogen atom. said aliphatic group is saturated. Typically, Rs is an aliphatic m is typically an integer of from 5 to 150, preferably from group having 7 to 25 carbon atoms, preferably 11 to 17 10 to 50. carbon atoms. Preferably, Rs is a group R, wherein R is typically —(C=O)—R, wherein R is as defined R—COH is auric acid, myristic acid, palmitic acid, or herein. 60 Stearic acid. R is typically —(C=O)—R, wherein R is as defined In one embodiment, the PUFA derivative of formula (I) is herein. present as a racemic mixture of the R and Senantiomers. Preferably, both R and R are —(C=O) R, wherein In another embodiment, the PUFA derivative of formula each R may be the same or different and is as defined (I) is present as the Renantiomer. herein. 65 In another embodiment, the PUFA derivative of formula Typically, when R and R are both —(C=O)—R, then (I) is present as the Senantiomer. Rs is not an aliphatic group having 3 to 29 carbon atoms. Typically, the mammal is a human. US 9,439,850 B2 11 12 In a preferred embodiment, -Alk- is —CH(OR)-trans having 3 to 29 carbon atoms, wherein at least one of R or CH=CH-cis ICH=CH-CH-cis CH=CH-CH , R is —(C=O)—Re; and/or (c) R is a group —(C=O)— —(CH) CH(OR)-transCH=CH-cis CH=CH Rs, wherein Rs is a saturated aliphatic group having 3 to 29 CH-cis CH=CH , or —(CH)-cis CH=CH-CH carbon atoms. cisCH=CH-transCH=CH-CH(OR)—; R is a hydro 5 In a more preferred embodiment, both R and R are gen atom, an unsubstituted C-C alkyl group, or a group of hydrogen atoms. formula —CH, CH(OR)—CH2—(OR), wherein R and In a particularly preferred embodiment, -Alk is Ra are each independently hydrogen atoms or —(C=O)— —(CH)-cis ICH=CH-CH-cis CH=CH-trans R, wherein R is a linear aliphatic group having from 11 to CH=CH-CH(OR)—, and R and R are both hydrogen 25 carbon atoms, which aliphatic group is unsubstituted or 10 Substituted with one hydroxyl group, or R is a group of atoms. In this embodiment, the PUFA derivative of formula formula (I) is 15-HETrE and is represented by the formula —(CHOCH), OH, wherein m is an integer of from 5 to 150; and R is a hydrogen atom, a group —(C=O)—Rs. wherein Rs is unsubstituted C-C alkyl, or a group 15 —(C=O)—Rs, wherein Rs is a linear aliphatic group hav ing from 11 to 25 carbon atoms, which aliphatic group is unsubstituted or substituted with one hydroxyl group; or R is a group of formula —(CHOCH), OH, wherein n is an integer of from 5 to 150. In a more preferred embodiment. -Alk- is —(CH)-cis Typically, compounds of the invention are administered CH=CH-CH-cis ICH=CH-transCH=CH-CH as one or more treatments per day, preferably from 1 to 4 (OR)—: R is a hydrogen atom, an unsubstituted C-C, treatments per day, more preferably from 1 to 2 treatments alkyl group, or a group of formula —CH2—CH(OR)— per day. CH2—(OR), wherein R and R are each independently 25 hydrogen atoms or —(C=O)—R, wherein R is a linear Typically, compounds of the invention are administered at aliphatic group having from 11 to 25 carbon atoms, which a daily dosage of from 0.1 mg/m/day to 1 kg/m/day, aliphatic group is unsubstituted or Substituted with one preferably from 1 mg/m/day to 100 g/m/day, more pref hydroxyl group, or R is a group of formula erably from 10 mg/m/day to 10 g/m/day, most preferably —(CHOCH), OH, wherein m is an integer of from 5 to 30 from 100 mg/m/day to 1 g/m/day. 150; and R is a hydrogen atom, a group —(C=O)—Rs. Skin inflammation includes rashes, hives, blisters and/or wherein Rs is unsubstituted C-C alkyl, or a group wheals and may be caused by eczema, exposure to radiation, —(C=O)—Rs, wherein Rs is a linear aliphatic group hav automimmune diseases, and/or uremic pruritis. ing from 11 to 25 carbon atoms, which aliphatic group is In a preferred embodiment, the skin inflammation is unsubstituted or substituted with one hydroxyl group; or R 35 caused by atopic eczema, contact dermatitis, psoriasis or is a group of formula —(CHOCH), OH, wherein n is an uremic pruritis. integer of from 5 to 150. In a further preferred embodiment, the skin inflammation In a most preferred embodiment, -Alk- is —(CH2)-cis is caused by exposure of the skin to electromagnetic radia CH=CH-CH-cis ICH=CH-transCH=CH-CH tion. This includes, for example, exposure to Sunlight, heat, (OR)—: R is a hydrogen atom, a group of formula 40 X-rays or radioactive materials. Thus, in this embodiment, —CH2—CH(OR)—CH2—(OR), wherein R and Ra are the compound of the present invention is typically used to each independently hydrogen atoms or —(C=O)—R. treat Sunburn. wherein R is an unsubstituted linear, Saturated aliphatic The term eczema is applied to a wide range of skin group having from 11 to 17 carbon atoms, and wherein at conditions with a variety of aetiologies. In general, eczema least one of R or R is —(C=O)—Re; and R is a hydrogen 45 atom, or a group —(C=O)—Rs, wherein Rs is an unsub is categorised by inflammation of the epidermis. Common stituted linear, Saturated aliphatic group having from 11 to symptoms associated with eczema include dryness, recur 17 carbon atoms, or R2 is a group of formula ring skin rashes, redness, skin edema (Swelling), itching, —(CHOCH), OH, whereinn is an integer of from 10 to 50. dryness, crusting, flaking, blistering, cracking, oozing, and Typically, in the compounds of the present invention, 50 bleeding. Eczema includes atopic eczema (atopic dermati including the preferred embodiments set out above, (a) R is tis), contact dermatitis, Xerotic eczema, seborrhoeic derma a group of formula —CH2—CH(OR)—CH2—(OR), titis, dyshydrosis, discoid eczema, Venous eczema, derma wherein R and Reach independently represent a hydrogen titis herpetiformus, neurodermatitis, and atom or —(C=O)—R, wherein R is a saturated aliphatic autoeczematisation. Eczema is typically atopic eczema or group having from 3 to 29 carbon atoms, wherein at least 55 contact dermatitis. one of R or R is —(C=O)—R; or Atopic eczema is primarily aggravated by contact with or (b) R is a group of formula—(CHOCH), OH, wherein intake of allergens, which include animal hair and dander, m is as defined herein; and/or food allergens, for example nuts or shellfish, or drugs, for (c) R is a group —(C=O)—Rs, wherein Rs is a saturated example penicillin. aliphatic group having from 3 to 29 carbon atoms; or 60 Contact dermatitis includes allergic contact dermatitis, (d) R is a group of formula —(CHOCH), OH, wherein irritant contact dermatitis and photocontact dermatitis. n is as defined herein. Such compounds will be particularly Electromagnetic radiation includes radio waves, micro lipophilic, which is advantageous in some instances. waves, terahertz radiation, infrared radiation, visible light, In this preferred embodiment, preferably (a) R is a group ultraviolet radiation, X-rays and gamma rays. Electromag of formula —CH2—CH(OR)—CH2—(OR), wherein R 65 netic radiation is preferably infrared radiation, visible light, and R are each independently hydrogen atoms or ultraviolet radiation, X-rays and gamma rays, more prefer —(C=O)—R, wherein R is a saturated aliphatic group ably ultraviolet radiation, X-rays and gamma rays. US 9,439,850 B2 13 14 Autoimmune diseases can involve an autoimmune 0.0875 g/m/day or less, more preferably 0.035 g/m/day, response against the skin. Examples of Such autoimmune most preferably 0.0175 g/m/day or less. Compounds of the diseases are lupus and psoriasis. present invention are typically coadministered with clobeta Uremic pruritis is a disorder of the skin associated with Sol proprionate, wherein the daily dosage of chronic renal failure. It also frequently affects patients proprionate is 0.009 g/m/day or less, preferably 0.0045 undergoing dialysis treatment. g/m/day or less, more preferably 0.0018 g/m/day or less, Typically, the compound of the present invention is co most preferably 0.0009 g/m/day or less. administered with a . The compounds of the present invention are typically Suitable to be used for co-administration coadministered with a further therapeutic agent, which is not with compounds of the invention are clobetasol diproprion 10 a corticosteroid, which is effective in treating skin condi ate, diproprionate, halbetasol proprionate, tions/diseases. Such therapeutic agents are well known to diacetate, , , amcinon the skilled person and include, but are not limited to, ide, , acetonide, immunomodulators, antiobiotics, immunosuppressants and furoate, proprionate, acetonide, anti-itch drugs. Valerate, , triamci 15 Compounds of the invention are typically commercially malone acetonide, , , , available, or may be prepared by analogy with known , , , , methods. Thus, for example, both DGLA and 15-HETrE are acetate, , beclomethasone dipropi commercially available. These available fatty acids can onate, hydrocortisone, pivalate, methylpredni easily be derivatised to obtain PUFA derivatives of formula Solone aceponate, dexamethasone palmitoate, , (I) by known methods. , dipropionate, For example, PUFA derivatives of formula (I) as defined , methylprednisolone Suleptanate, , herein, wherein R is a C-C alkyl, C-C alkenyl, C-C, prednisolone farnesylate, , deprodone propi alkynyl, Co-Co aryl, 5- to 10-membered heteroaryl, C-C, onateloteprednol etabonate, betamethasone butyrate propi carbocyclyl or 5- to 10-membered heterocyclyl group; or R onate, , , dexamethasone sodium phos 25 phate, triamcinolone, betamethasone 17-valerate, is a group of formula —CH2—CH(OR)—CH2—(OR), betamethasone, betamethasone dipropionate, hydrocorti wherein R and Ra are as defined herein; or R is a group of Sone acetate, hydrocortisone sodium Succinate, prednisolone formula—(CHOCH), OH, wherein m is as defined herein, Sodium phosphate and hydrocortisone probutate. can be prepared by esterifying a compound of formula Preferred corticosteroids corticosteroids to be used for 30 co-administration with compounds of the invention are O clobetasol diproprionate, betamethasone diproprionate, hal betasol proprionate, , fluocinonide, hal cinonide, , desoximetaSone, triamcinolone --~~~ acetonide, mometasone furoate, fluticasone proprionate, 35 , , hydrocor wherein-Alk- is as defined herein and X is a leaving group, tisone butyrate, triamcinalone acetonide, desonide, and for example a halogen atom, a tosylate or mesylate group prednicarbate. with an alcohol of formula R' OH, wherein R is a C-C, Any reference to corticosteroids within the scope of the alkyl, C-C alkenyl, C-C alkynyl, Co-Co aryl, 5- to present invention includes a reference to salts or derivatives 40 10-membered heteroaryl, C-C, carbocyclyl or 5- to thereof which may be formed from the corticosteroids. 10-membered heterocyclyl group; or R' is a group of Examples of possible salts or derivatives include: sodium formula —CH-CH(OR)-CH (OR), wherein R and salts, Sulphobenzoates, phosphates, isonicotinates, acetates, Ra are as defined herein; or R." is a group of formula propionates, dihydrogen phosphates, palmitates, pivaiates, —(CHOCH), OH, wherein m is as defined herein, to fameSylates, aceponates, Suleptanates, prednicarbates, 45 obtain a PUFA derivative of formula (I) as defined herein. furoates or acetonides. In some cases the corticosteroids may Alternatively, X may be a hydroxyl group. In that case, the also occur in the form of their hydrates. reaction is preferably carried out under acidic conditions, or It is a particular finding of the present invention that in the presence of a suitable catalyst, for example pyridine. co-administration of a compound of the present invention Compounds of formula R' OH are typically commercially with a corticosteroid enables the amount of corticosteroid 50 available or may be prepared by analogy with known administered to be reduced, without reducing the efficacy of methods. the treatment. Thus, in a preferred embodiment, the com When - Alk- is —CH(OR)-transCH=CH-cis pound of the present invention is co-administered with a CH=CH-CH-cis ICH=CH-CR —(CH) CH corticosteroid, wherein the corticosteroid is administered at (OR)-transCH=CH-cisCH=CH-CH-cis a daily dosage of 50% or less, preferably 25% or less, more 55 CH=CH-, or —(CH), cisCH=CH-CH-cis preferably 10% or less, most preferably 5% or less, of the CH=CH-transCH=CH-CH(OR)—, PUFA derivatives recommended daily dosage of said corticosteroid in said of formula (I) as defined herein, wherein R is a group mammal. —(C=O)—Rs, wherein Rs is a C-C alkyl, C-C alkenyl, The person skilled in the art is well aware of the recom C-C alkynyl, a Co-Co aryl, a 5- to 10-membered het mended daily dosages of corticosteroids in mammals. For 60 eroaryl, a C-C, carbocyclyl or a 5- to 10-membered het example, the recommended daily dosage of hydrocortisone erocyclyl group, or Rs is an aliphatic group having from 3 to in humans is approximately 0.35 g/m/day. The recom 29 carbonatoms, can be prepared by treating a compound of mended daily dosage of clobetasol proprionate in humans is formula (I) as defined herein, wherein-Alk- is —CH(OR)- 0.009 to 0.018 g/m/day. transCH=CH-cis ICH=CH-CH-cis ICH=CH Thus, compounds of the present invention are typically 65 CH , —(CH) CH(OR)-transCH=CH-cis coadministered with hydrocortisone, wherein the daily dos CH=CH-CH-cis ICH=CH , or —(CH)-cis age of hydroxortisone is 0.175 g/m/day or less, preferably CH=CH-CH-cis ICH=CH-transCH=CH-CH US 9,439,850 B2 15 16 (OR)—, and R is a hydrogen atom, with a carboxylic acid cetyl alcohol, Stearyl alcohol and cetearyl alcohol) can be derivative Y (C=O) R's, wherein R's is a C-C alkyl, used. The total amount of thickener present is preferably C-C alkenyl, C-C alkynyl, a Co-Co aryl, a 5- to 10-mem about 3 wt % to about 12 wt % based on the total weight of bered heteroaryl, a C-C, carbocyclyl or a 5- to 10-mem the pharmaceutical composition. bered heterocyclyl group, or R's is an aliphatic group having Preservatives such as methylparaben, propylparaben and from 3 to 29 carbon atoms, and Y is a leaving group, for benzyl alcohol can be present in the pharmaceutical com example a halogen atom, a tosylate or mesylate group. position. The appropriate amount of Such preservative(s) is Compounds of formula Y—(C=O)—R's are typically com known to those skilled in the art. The pharmaceutical mercially available or may be prepared by analogy with composition may also comprise a fat-soluble antioxidant known methods. 10 Such as ascorbyl palmitate, tocopherol and/or ascorbic acid When - Alk- is —CH(OR)-transCH=CH-cis in the presence of lecithin. CH=CH-CH-cis ICH=CH-CH , —(CH) CH Optionally, an additional Solubilizing agent Such as ben (OR)-transCH=CH-cis ICH=CH-CH-cis Zyl alcohol, lactic acid, acetic acid, Stearic acid or hydro CH=CH-, or —(CH)-cis ICH=CH-CH-cis chloric acid can be included in the pharmaceutical compo CH=CH-transCH=CH-CH(OR)—, PUFA derivatives 15 sition. If an additional Solubilizing agent is used, the amount of formula (I) as defined herein, wherein R is a group of present is preferably about 1 wt % to about 12 wt % based formula—(CHOCH), OH, wherein n is as defined herein, on the total weight of the pharmaceutical composition. can be prepared by treating a compound of formula (I) as Optionally, the pharmaceutical composition can contain a defined herein, wherein - Alk- is —CH(OR)-trans humectant such as glycerin and a skin penetration enhancer CH=CH-cis ICH=CH-CH-cis ICH=CH-CH , such as butyl stearate, urea and DMSO. It is known to those —(CH) CH(OR)-transCH=CH-cis CH=CH skilled in the art that a single ingredient can perform more CH-cis CH=CH , or —(CH)-cis CH=CH-CH than one function in a cream, i.e., cetyl alcohol can serve cisCH=CH-transCH=CH-CH(OR) , and R is a both as an emollient and as a thickener. hydrogen atom, with a compound of formula In one embodiment, the pharmaceutical composition is in Z—(CHOCH), OH, wherein n is as defined herein and Z 25 the form of a cream. The cream typically consists of an oil is a good leaving group, for example a halogen atom, a phase and a water phase mixed together to form an emul tosylate or mesylate group. Compounds of formula Sion. Preferably, the cream comprises an oil-in-water emul Z—(CHOCH), OH are typically commercially available sion. Preferably, the amount of water present in a cream of or may be prepared by analogy with known methods. the invention is about 45 wt % to about 85 wt % based on The present invention also provides a pharmaceutical 30 the total weight of the cream. composition Suitable for topical administration comprising a Where the pharmaceutical composition is in the form of PUFA derivative, as defined herein, or a pharmaceutically an ointment, it typically comprises a pharmaceutically acceptable salt, or Solvate thereof, and a pharmaceutically acceptable ointment base Such as petrolatum, or polyethyl acceptable diluent or carrier, for use in treating skin inflam ene glycol 400 (available from Union Carbide) in combi mation, as defined herein, in a mammal, as defined herein. 35 nation with polyethylene glycol 3350 (available from Union Preferred pharmaceutical compositions are sterile and Carbide). The amount of ointment base present in an oint pyrogen free. ment of the invention is preferably about 60 wt % to about The pharmaceutical composition is typically in the form 95 wt % based on the total weight of the ointment. of a gel, ointment, cream or lotion. In the pharmaceutical composition of the present inven When said pharmaceutical composition is a gel it typi 40 tion, the amount of the PUFA derivative of formula (I) is cally comprises a hydrophilic polymer Such as cross-linked typically from 0.01 wt % to 50 wt %, preferably from 0.5 wt polyethylene glycol, cross-linked starch or polyvinyl pyr % to 25 wt %, more preferably from 1 wt % to 10 wt %, for rolidone. example about 5 wt %, based on the total weight of the An ointment, cream or lotion typically contains an aque pharmaceutical composition. ous phase and an oleaginous phase in admixture. 45 The compound/composition of the present invention is The pharmaceutical composition may additionally con formulated for topical administration and it may be admin tain one or more emollients, emulsifiers, thickeners and/or istered to a patient in an amount such that from 0.00001 to preservatives, particularly when it is a cream or ointment. 10 g, preferably from 0.0001 to 1 g active ingredient is Emollients suitable for inclusion in creams or ointments delivered per m of the area being treated. of the present invention are typically long chain alcohols, for 50 Pharmaceutical compositions of the present invention example a C8-C22 alcohol such as cetyl alcohol, stearyl may additionally comprise one or more corticosteroids as alcohol and cetearyl alcohol, hydrocarbons such as petrola defined herein. The amount of the corticosteroid present in tum and light mineral oil, or acetylated lanolin. The total the pharmaceutical composition of the invention is typically amount of emollient in the pharmaceutical composition is 50% or less, preferably 25% or less, more preferably 10% or preferably about 5 wt % to about 30 wt %, and more 55 less, most preferably 5% or less, of the recommended preferably about 5 wt % to about 10 wt % based on the total amount of said corticosteroid in a commercially available weight of the pharmaceutical composition. formulation. The skilled person is well aware of the amount The emulsifier is typically a nonionic Surface active agent, of corticosteroids present in various topical formulations. e.g., polysorbate 60 (available from ICI Americas), sorbitan For example, the amount of hydrocortisone present in most monostearate, polyglyceryl-4 oleate and polyoxyethylene(4) 60 commercially available formulations is typically 1% w/w. lauryl ether. Generally the total amount of emulsifier is about The amount of clobetasol proprionate present in most com 2 wt % to about 14 wt %, and more preferably about 2 wt mercially available formulations is typically 0.0525% w/w. % to about 6 wt % by weight based on the total weight of Thus, pharmaceutical compositions of the present inven the pharmaceutical composition. tion typically comprise hydrocortisone in an amount of 0.5% Pharmaceutically acceptable thickeners, such as 65 w/w or less, preferably 0.025% w/w or less, more preferably VeegumTM K (available from R. T. Vanderbilt Company, 0.01% w/w or less, most preferably 0.005% w/w or less. Inc.), and long chain alcohols (i.e. C8-C22 alcohols such as Pharmaceutical compositions of the present invention typi US 9,439,850 B2 17 18 cally comprise clobetasol proprionate in an amount of Typically, the pharmaceutical composition comprising a 0.026% w/w or less, preferably 0.013% w/w or less, more polyunsaturated fatty acid (PUFA) derivative of formula (II) contains the PUFA derivative of formula (II) as sole active preferably 0.0053% w/w or less, most preferably 0.0026% ingredient. w/w or less. Preferably, the pharmaceutical composition comprising a The pharmaceutical compositions of the present invention polyunsaturated fatty acid (PUFA) derivative of formula (II) typically comprise one or more further therapeutic agents, consists of the PUFA derivative of formula (II) and a which are not corticosteroids, as defined herein. The amount pharmaceutically acceptable diluent or carrier, as defined of the one or more further therapeutic agents, which are not herein. corticosteroids, present in the composition will be evident to Preferably, the PUFA derivative of formula (II) is of 10 formula R"(C=O)O CH, CH(O(C–O)R") CH, O the person skilled in the art. (C=O)R", wherein each R" is the same and is the aliphatic Pharmaceutical compositions of the present invention group of dihommo-gamma-linolenic acid, i.e. R" is may be prepared simply by admixing the ingredients in a —(CH) CH=CH-CH CH=CH-CH Suitable manner. CH=CH-(CH2)CH, wherein all of the C–C double The present invention also provides the use of a com bonds have cis configuration. Thus, the PUFA derivative of pound which is a PUFA derivative of formula (I) as defined formula (II) is preferably

herein or a pharmaceutically acceptable salt, or Solvate In a particularly preferred embodiment, R is a hydrogen thereof, in the manufacture of a medicament for use in 35 atom. In this embodiment, the PUFA derivative of formula treating skin inflammation, as defined herein, in a mammal, (II) is DGLA and is represented by the formula as defined herein, by topical administration.

The present invention also provides a method of treating skin inflammation, as defined herein, in a mammal, as defined herein, which method comprises administering to 40 the skin of said mammal atherapeutically effective amount of a compound which is a PUFA derivative of formula (I) as defined herein or a pharmaceutically acceptable salt, or solvate thereof. The present invention also provides a pharmaceutical 45 composition comprising a polyunsaturated fatty acid The present invention also provides the use of a pharma (PUFA) derivative of formula (II), ceutical composition comprising a polyunsaturated fatty acid (PUFA) derivative of formula (II) as defined herein in the manufacture of a medicament for use in treating skin II 50 inflammation, as defined herein, in a mammal, as defined O herein, by topical administration, wherein the composition is Substantially free of lithium salts and glucocorticoids. RO --~~ The present invention also provides a method of treating skin inflammation, as defined herein, in a mammal, as 55 defined herein, which method comprises administering to or a pharmaceutically acceptable salt, or Solvate thereof, the skin of said mammal a therapeutically effective amount wherein of a pharmaceutical composition comprising a polyunsatu -Alk- is —(CH)-cis ICH=CH-CH-cis CH=CH rated fatty acid (PUFA) derivative of formula (II) as defined CH-cis ICH=CH-; and herein, wherein the composition is substantially free of R is as defined herein; 60 lithium salts and glucocorticoids. which composition is for use in treating skin inflammation EXAMPLES in a mammal, by topical administration, and is Substantially free of lithium salts and glucocorticoids. Immunohistochemistry analyses were carried out to mea As the skilled person will appreciate, in this embodiment, 65 sure the degree of expression of COX-2 enzymes in ex vivo the pharmaceutically acceptable salt of the PUFA derivative porcine ear skin. The COX-2 family of enzymes have been of formula (II) is not a lithium salt. strongly linked to inflammation and have been found to be US 9,439,850 B2 19 20 present in increased amounts in inflamed tissue. Thus, a The results of this experiment are shown as FIG. 4. After decreased level of COX-2 in the skin corresponds with six hours, the amount of dark stain has reduced, indicating reduced inflammation of the skin. penetration of 15-HETrE into the viable epidermis and Example 1 activity against COX-2 expression in the skin. Example 5 Freshly excised porcine ears were immersed in iced Hank’s buffer for transport from the abattoir to the labora tory. Upon arrival, the porcine ears were first washed with A Western Blotting experiment was carried out to deter running tap water, and the full thickness skin was liberated mine the effect of topically applied representative com from underlying cartilage by blunt dissection using a scalpel 10 pounds of the invention DGLA and 15-HETrE on the and hairs were removed with an electrical razor. The skin expression of COX-2 in porcine skin. Water was used as a was used within 2 h of slaughter. The full thickness skin was control. cut into approximately 2 cmx2 cm and placed in Hanks The porcine skin membranes (as described in Example 1) balance salt to maintain skin viability. were gently cleaned with de-ionised water before being A2 mm strip of skin was obtained using a Surgical scalpel 15 and the skin strip then fixed using a 40% formaldehyde homogenized (Silverson, Chesham, UK) in a RIPA lysis Solution, dehydrated and set in molten wax. The sections buffer 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM were cut using a Shandon Finesse microtome (Thermo PMSF, 1 mM EDTA, 5 lug mL aprotinin, 5 ug mL' Scientific, Waltham, Mass., USA) to a thickness of 5um and leupeptin, 1% Triton X-100, 1% sodium deoxycholate, 0.1% were placed onto 2.5 cmx7.5 cmx1 mm pre-cleaned SDS). After 15 min incubation on ice, the lysates were microSlides. The section of skin was stained with diamin clarified by centrifugation at 14000xg for 2x15 mM and the obenzidine, which binds to COX-2 enzymes and the slides Supernatant stored at -80° C. for Subsequent protein analy visualised on a light microscope with image capture facility. S1S. Skin membranes were mounted in Franz diffusion cells Aliquots of 30 ug total protein were separated by SDS using Hanks buffer as receptor phase. Water was used as the 25 PAGE, transferred to nitrocellulose membranes using the donor phase. After 6 hours the skin was removed from the Franz cell apparatus, excess donor phase was removed and Trans-Blot electrophoretic Transfer Cell and briefly stained the skin wiped clean with a clean paper tissue. The diffu with Ponceau S to verify effective transfer. Immunoblots sional area was then cut into approximately 2 mm Strips were incubated for 1 h in a blocking solution tris-buffered using a Surgical scalpel and the skin Strips then using a 40% saline (TBS)-Tween 20 containing 5% (w/v) commercial formaldehyde solution, dehydrated and set in molten wax. 30 skimmed milk powder (Marvel) at room temperature. After The sections were cut using a Shandon Finesse microtome washing, the membrane was probed overnight at 4°C. with (Thermo Scientific, Waltham, Mass., USA) to a thickness of COX-2 antibody at 1:1000, 5-LOX at 1:1000 and iNOS at 5 um and were placed onto 2.5 cmx7.5 cmx1 mm pre 1:500 in (1:20 and 1:100 western blocking reagent and cleaned microslides. The section of skin was stained with Sodium azide solution respectively made up to Volume with diaminobenzidine, which binds to COX-2 enzymes, and the 35 TBS tween). Membranes were then incubated for 1 h with slides visualised on a light microscope with image capture HRP-conjugated anti-rabbit at 1:10000. For B-actin, mem facility. branes were probed with anti-actin and anti-mouse for 1 h The results of this experiment are shown as FIG.1. A dark each at room temperature at 1:10000. After 3x10 mM stain is present in the sample both after 0 hours and 6 hours, washes in TBS Tween, they were finally exposed to freshly indicating the continuing presence of COX-2 in the skin. 40 prepared Dura Substrate for chemiluminescence for appro This indicates that water has no anti-inflammatory activity. priate time before performing autoradiography. The results of this experiment are shown as FIG. 5. It can Example 2 be seen that a significant reduction in COX-2 expression is seen following dosing with DGLA and 15-HETrE, relative An experiment was carried out as described in Example 1, 45 to the control. except that the donor phase comprised ketoprofen, a known COX-2 inhibitor, in fish oil. What is claimed is: The results of this experiment are shown as FIG. 2. After 1. A topical composition comprising: six hours, the amount of dark stain has reduced, indicating at least one excipient; and penetration of ketoprofen into the viable epidermis and 50 a polyunsaturated fatty acid (PUFA) derivative of formula activity against COX-2 expression in the skin. (I): Example 3 (I) An experiment was carried out as described in Example 1, 55 O except that the donor phase comprised a representative compound of the invention, DGLA. ------> The results of this experiment are shown as FIG. 3. After RO six hours, the amount of dark stain has reduced, indicating penetration of DGLA into the viable epidermis and activity 60 or a salt thereof, wherein: against COX-2 expression in the skin. -Alk- is: —CH(OR)-transCH=CH-cis ICH=CH-CH Example 4 cisCH=CH-CH , —(CH), CH(OR)- transCH=CH-cis ICH=CH-CH-cis An experiment was carried out as described in Example 1, 65 CH=CH —(CH)-cis CH=CH-CH-cis except that the donor phase comprised a representative CH=CH-transCH=CH-CH(OR)-, or compound of the invention, 15-HETrE. R is a hydrogen atom; US 9,439,850 B2 21 22 R is: 3. The topical composition of claim 1, wherein - Alk- is a hydrogen atom, —(CH)-cis CH=CH-CH-cis ICH=CH-trans —(C=O)—Rs, wherein Rs is a C-C alkyl, C-C, CH=CH-CH(OR) . alkenyl, C-C alkynyl, Co-Co aryl, 5- to 10-mem 4. The topical composition of claim 1, wherein R is a 5 hydrogen atom. bered heteroaryl, C-C, carbocyclyl or 5- to 5. The topical composition of claim 1, wherein the PUFA 10-membered heterocyclyl group, or Rs is an ali derivative is present as the Renantiomer. phatic group having from 3 to 29 carbon atoms, or 6. The topical composition of claim 1, wherein the PUFA —(CHOCH), OH, wherein n is an integer from 1 to derivative is present as the Senantiomer. 200; 7. The topical composition of claim 1, wherein: wherein said alkyl, alkenyl, alkynyl and aliphatic groups 10 (a) R is —(C=O)—Rs, wherein Rs is a saturated ali are the same or different and are each unsubstituted or phatic group having from 3 to 29 carbon atoms; or Substituted with 1, 2 or 3 unsubstituted substituents (b) R is —(CHOCH), OH. which are the same or different and are selected from 8. The topical composition of claim 1 wherein the excipi ent comprises a preservative selected from methylparaben, halogen atoms and C-C alkoxy, C-C alkenyloxy, 15 propylparaben, benzyl alcohol, ascorbyl palmitate, and C-C haloalkyl, C-C haloalkenyl, C-Chaloalkoxy, ascorbic acid. C-C haloalkenyloxy, hydroxyl. —SR', and —NR'R'" 9. The topical composition of claim 1, wherein the excipi groups where R' and R" are the same or different and ent comprises an emollient, an emulsifier, a thickener, and/or represent hydrogen or unsubstituted C-C alkyl; a preservative. wherein said aryl, heteroaryl, carbocyclyl and heterocy 10. The topical composition of claim 1, wherein the clyl groups are the same or different and are each excipient comprises a solubilizing agent and/or a humectant. unsubstituted or substituted by 1, 2, 3 or 4 unsubstituted 11. The topical composition of claim 1, wherein the Substituents which are the same or different and are topical composition is in the form of a cream. selected from halogen atoms, and cyano, nitro, C-C, 12. A method of treating skin inflammation in a mammal alkyl, C-C alkoxy, C-C alkenyl, C-C alkenyloxy, 25 comprising administering to skin of said mammal the topical C-Chaloalkyl, C-Chaloalkenyl, C-Chaloalkoxy, composition of claim 1. C-C haloalkenyloxy, hydroxyl, C-C hydroxyalkyl, 13. A topical composition comprising 15-HETrE or an - SR" and - NR'R" groups wherein each R" and R" is ester thereof and an excipient. the same or different and represents hydrogen or unsub 14. The topical composition of claim 13, wherein the stituted C-C alkyl, 30 excipient comprises an emollient, an emulsifier, a thickener, wherein the PUFA derivative is in the form of a racemate, a preservative, a solubilizing agent, and/or a humectant. a stereoisomer or a mixture of stereoisomers; and 15. The topical composition of claim 13, wherein the wherein the topical composition is substantially free of topical composition is Substantially free of glucocorticoids. glucocorticoids. 16. The topical composition of claim 13, wherein the 2. The topical composition of claim 1, wherein R is a 35 topical composition is in the form of a cream. hydrogen atom. k k k k k