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METHOD OF TREATING PSORIASIS

TECHNICAL FIELD

The present invention relates to a novel method of treating psoriasis, a disease of the skin. The method comprises using vitamin D-related compounds.

BACKGROUND ART

Psoriasis is a disease of the epidermis and a major cause of disability and disfigurement for between 2,000,000 and 8,000,000 persons in the United States. Of these, about 100,000 are severely affected.

The disease is diagnosed by the presence of scal¬ ing erythematous on the scalp and extender aspects of the arms and legs; psoriatic lesions often are accen¬ tuated on the sites of repeated trauma, such as the elbows and knees. The papules or plaques of psoriasis often contain a silvery-white micaceous scale that is relatively easily removed in layers. There is a several fold increase in the normal number of the basal cells of the epidermis. This increase in the basal cell population reduces the turnover time of the epidermis from the normal 27 days to 3-4 days. This shortened interval leads to the consequence that normal events of cell maturation or keratinization do

not occur, and this failure of maturation is reflected by an array of abnormal morphologic and biochemical changes. Numerous cytologic, histologic, histochemi- cal and biochemical alterations are now known to be the result, rather than the cause of the disease pro¬ cess. The only main fact known at this time about the fundamental cause of psoriasis is that the predisposi¬ tion to its development is genetically transmitted. (This introduction is basically taken from Harrison's Principles of Internal Medicine, 10th Edition, Volume 1, pages 256 and 257).

The treatment of psoriasis still remains the province of dermatologists. The most effective treat¬ ment in the control of localized psoriasis for most patients is the topical use of corticosteroids with a plastic wrap and ultraviolet light or sunlight expo¬ sures. On certain patients who have generalized psoriasis, it has been necessary to use a variety of systemic chemotherapeutic agents, especially metho- trexate; the latter has the capacity to inhibit cell replication without a proportionate inhibition of cell function; that is, keratinization. Photochemotherapy was introduced in 1974, in the so-called PUVA treat¬ ment. In this treatment, psoralen is administered two hours before total body irradiation with a special light system that emits predominantly long wave ultra¬ violet light. The light alone is ineffective in producing erythema or remission of psoriatic lesions; however, in the presence of one of the psoralens, the UV-A light becomes a potent photoactive agent and pro¬ duces a remission of psoriatic lesions after several

exposures. Photochemotherapy requires specialized knowledge and lighting systems delivering precisely measured amounts .of ultraviolet light.

Along quite different areas of research, Holick et al. (New England Journal of Medicine, 303; 349-354 (1980)) have studied the feasibility of using the skin as the organ for the synthesis and absorption of vita¬ min D metabolites. These investigators demonstrated that topical application of various vitamin D metabo¬ lites or pro-vitamin forms- followed by phototherapy results in elevated serum levels of dihydroxy-vitamin D ₃ . It was therefore suggested that topical applica¬ tion of vitamin D analogues may be an effective method of therapy for diseases involving calcium, phosphorus and bone metabolism problems. It is only recently, however, that it has become clear that the skin itself may be a target tissue for l,25-(OH) ₂ -D ₃ (Stumpf, W.E. et al. , Science, 206:1188-1190 (1979)). Cells iso¬ lated from the skin of rats, mice, and humans, and from cultured human skin fibroblasts and keratinocytes contain a high affinity (1.0 x 10~ M) low capacity receptor-like protein for 1,25-dihydroxy-vitamin D ₃ (Franceschi, et al. , Arch. Biochem. Biophys., 210

1-13 (1979); Simpson, R. U. et al. , P.N.A.S. USA, 77 5822 (1980); Colston, K. et al. , Endocrinology, 107

1916 (1980); Feldman, D. et al. , Journal of Clinical Endocrinology & Metabolism, 51; 1463 (1980); Eil, C. et al^, P.N.A.S. USA, 78; 2562 (1981); and Clemens, T.L. et al. , J. Clin. Endocr. Metab. 56; April 1983). A specific biological function for l,25-(OH) ₂ -vitamin D ₃ in the skin, however, has yet to be discovered.

Nevertheless, evidence has come forth supporting the concept that the dihydroxy metabolite of the vitamin does have _. biologic actions in the skin. This was accomplished by evaluating the biological activity of l,25-dihydroxy-D ₃ simultaneously in cultured human skin fibroblasts that either possessed or lacked a cytosolic receptor-like protein for the hormone (Clemens, T. L. et al. , J. Clin. Endocrinol. Metab., 56; April 1983). The receptor-negative skin fibro¬ blasts were obtained from a patient with a rare bone disorder called vitamin D dependent rickets, type II, a heritable disorder caused by a defective or complete absence of a cytoplas ic or nuclear receptor for 1,25-dihydroxy vitamin D. The dihydroxy metabolite of vitamin O-. caused a dose-dependent inhibition of cell growth in receptor . positive skin fibroblasts (about

40-50% reduction in cell growth was observed in cul- tures containing 10 —6 and 10—8 M of hormone and 12% in cultures containing 10 M of l,25-(OH) ₂ -D ₃ ) , and, by contrast, had absolutely no effect on the growth of receptor negative skin fibroblasts.

The aforementioned seemingly two divergent lines of research, the treatment of psoriasis on the one hand, and the effects of vitamin D ₃ on skin components on the other, remained heretofore unrelated until, by the present invention, they have been brought togeth¬ er.

DISCLOSURE OF THE INVENTION

This invention arose out of the initial observa¬ tion that when psoriatic cells were incubated in vitro

with 1,25-(OH) ~ ^D 3 ^at physiologic concentrations, they were resistant to growth inhibition effects, whereas at phar acologic .concentrations (10 -6 and 10-4 M) , the dihydroxy metabolite of vitamin D ₃ was capable of in¬ hibiting the cell growth of these psoriatic fibro¬ blasts. Thus vitamin D, as well as its homologues, analogues and hydroxylated metabolites, can be util- itized effectively in the treatment of psoriasis.

An accurate correlation between an .in. vitro test or tests and anti-psoriatic treatment In vivo has fur¬ ther been established. According to this correlation, the vitamin D compounds usable in the treatment of psoriasis are those capable of stimulating or inducing the differentiation of tumor or normal cell lines which possess receptors for 1,25-dihydroxyvitamin D ₃ . Normal cell lines include cultured rodent and human keratinocytes. Active compounds are also those capable of increasing the enzymatic activity of trans- glutaminase in the same cell system, or are those capable of inhibiting the cell growth ij ^* ι vitro of human skin fibroblasts. Details of these tests can be found below.

The vitamin D compounds, homologues, analogues or metabolites thereof which are useful in treating psoriasis are those which demonstrate activity in any of the _in vitro tests.

BEST MODE OF CARRYING OUT THE INVENTION

Generally, an active compound is one which induces differentiation at physiologic concentration of a

tumor or a normal cell line which possess receptors for 1,25-dihydroxyvitamin D ₃ . Among normal useable lines are for example human or rodent keratinocytes or fibroblasts. ^* Among tumor lines are HL-60 cell line, M-l cell line, breast tumor cells. A few tests will be described in further detail herein.

A first test is one which measures the differen¬ tiation of cultured keratinocytes. The assay is essentially the one described by Hoso i, et al. , "Regulation of Terminal Differentiation of Cultured Mouse Epidermal Cells by 1 ,25-dihydroxy Vitamin D-,," Endocrinology, 113; 1950 (1983) for mice, or that described by Clemens et ^' al. supra for the human sys¬ tem, both herein incorporated by reference. Briefly, epidermal cells are prepared from newborn C57BL mice by overnight treatment with trypsin at 4°C followed by separation of the epidermis from the der is with for¬ ceps. Cells are plated at a density of 10 cells per 4.5 cm well and grown in Eagle minimum essential medium (MEM) (supplemented with 10% fetal calf serum (FCS)). Cells can also be grown in low calcium medium. Eagle MEM, without calcium supplemented with 10% dial zed FCS. Calcium concentration of the low calcium medium can be from about 0.01-0.5 mM, whereas a conventional MEM plus 10% FCS usually may contain 1.0-2.0 mM calcium. Cells are incubated in a humi¬ dified C0 ₂ incubator at 37°C. All experiments are performed on the primary cultures. Twenty-four hours after plating, the medium is changed and the vitamin D compound is added at concentration of 0.12, 1.2 and 12 nm (0.05, 0.5 and 5.0 mg/ml, respectively). Control

cultures are supplemented with ethanol at a final concentration of 0.5%. The media with and without the vitamin D is. renewed every 3-4 days. (FCS con¬ tains 1 ,25-(OH) ₂ -D ₃ at 0.12 nm (Tanaka, H. et al. , Biochem. J. , 204; 713 (1982)). Therefore, the endoge¬ nous concentration of the vitamin in the control culture medium which contains 10% FCS is negligible.)

Differentiation of epidermal cells in culture is examined morphologically by

(1) counting the number of squamous and enucle¬ ated cells sloughed off into the medium,

(2) counting the number of squamous and basal cells attached ^' to the dishes,

(3) formation of a cornified envelope,

(4) the cell size and cell density, or

(5) morphological changes seen under a light microscope,or some or all of the above in combination.

Floating cells are collected from the medium. Then the cultures are washed with phosphate buffered saline (PBS) and attached cells are dissociated by treatment with 0.05% trypsin and 0.1% EDTA solution at 37°C for 20-30 min. Cell suspensions are then divided into two portions: one for counting the numbers of squamous and basal cells and the other for counting cornified envelopes. Since basal cells are small and round, whereas squamous and enucleated cells are large and flat, they are readily distinguishable in a hemocy- tometer. The method of Sun and Green (Cell, 9_: 511 (1976)) can be used to determine the presence of a ^' cornified envelope. The cells are resuspended in 10

mM Tris-HCl (pH 7.4) containing 1% beta-mercapto- ethanol and 1% sodium dodecylsulfate at a density of 5:30 x 10 cells/ml. The mixture stands for 10 minutes at room temperature and then insoluble cells are counted in a hemocytometer under a phase contrast microscope.

The size of cells can be measured in photographs with a stage micrometer as a standard. The density distribution of cells is measured by density gradient p centrifugation in Percoll . Epidermal cells 8-11 x 10 /ml are suspended in PBS containing 40% Percoll, placed in a 10 ml polycarbonate tube, and centrifuged at 15,000 x g at 3°C ^' for 30 minutes in an angled rotor. Fractions are collected by use of density marker beads. For light microscopic observation, cells grown in a glass cover slip are fixed with either 10% formalin or methanol/acetiσ acid (3:1) and stained with hemotoxiline and eothine or rhodanile blue.

In the presence of an active vitamin D compound useful for psoriatic treatment, differentiation of epidermal cells is markedly stimulated. Focal strati¬ fication is formed in places on top of the epidermal cell sheets. Stratified foci increase in number and size and contiguous foci coalesce. In the uppermost layer of stratified foci, cells produce an amorphous material staining red with hemotoxaline and eothine and rhodanile blue. Some cells are enucleated and some have a thick pycnoctic nucleus. Differentiated cells slough off into the medium so that the total

-9-

nu ber of cells attached to the dish decrease continu¬ ously with the time of cultivation. The fraction of attached basal cells decrease sharply in the presence of an active 'vitamin D compound. For example, close to 100% of the cells are basal cells on day 0, but only about 25% on day 3 and less than 10% after day 10. In a control culture on the other hand, more than 60% of the cells are basal cells during the first six days and usually 30-40% or so remain basal on day 10. In parallel with decrease of basal cells, the number of squamous cells increases in the vitamin D active treated cultures, first among the attached cell popu¬ lation and then among the sloughed off floating cells.

Epidermal di ferentiation can be quantified by counting cornified envelopes remaining after cell lysis with a solution containing 1% sodium dodecyl- sulfate and 1% beta-mercaptoethanol. When the cells are grown in the presence of 12 nM active vitamin D compound, the percentage of cells with a cornified envelope increases with time of cultivation. The percentage is greatest after 10 days in culture when about 60-70% of the cells have an envelope. In con¬ trast, the percentage of control cultures remain at 20% or less during a two week observation period.

The cells obtained in the presence of an active vitamin D compound for 3 days are larger and lighter than those in its absence. The diameter of cells in the treated cultures is usually about 25 + 10 mm, compared with about 17 + 5 mm in a control.

Cell density by Percoll gradient centrifugation indicates that, when grown in the presence of an

active vitamin D compound for 3 days, about 65% of the cells are collected 'in the lightest fraction with a density of about .1.017-1.027, whereas about 40% of the control cells ^' are recovered in this fraction. Concom- itantly, the number of cells in a heavier fraction (density) between about 1.06 and 1.08 decrease in the treated cultures. Similar results are obtained at day 7. Human keratinocytes can be grown by the method of Clemens et al. , supra, and analyzed in an identical manner.

A second test is that of inhibition of human skin fibroblasts. This test is found in Clemens et al. , J. Clin. Endocr. Metab., 56; 824 (1983), herein incorpo¬ rated by reference. Briefly, skin cells are isolated from surgically obtained normal human skin from mammary, face, thigh, etc. of a normal patient.

Normal skin biopsies are placed immediately in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin G (75 U/ml) , and streptomycin (50 ng/ml) . After removal of subcutane¬ ous fat and the deep reticular layer of the der is, the tissues are minced and placed in 10 ml 0.25% trypsin at 4°C overnight.

4 Fibroblasts are plated at 7-10 x 10 cells in

35-mm Costar dishes in DMEM containing 5% NBS. After attachment of cells (6 hours), the media are aspirated and replaced with fresh medium containing ethanol alone (0.01%) or ethanol (0.01%) containing compound at 10~ ¹⁰ , 10 ^"8 , 10 ^"6 , or 10 ^"4 M. At intervals there¬ after, cells are harvested from duplicate plates by trypsinization and counted in a Coulter counter.

Control and compound supplemented media are replaced at 4-day intervals. Normal foreskin fibroblasts, plated at 5 x 10 ⁴ cells/well (DMEM; 5% NBS) , can also be treated with ethanol (0.01%) alone or ethanol con¬ taining compound (10 -10~ ^" M) . After 4 days, fresh medium containing the appropriate sterol is replaced, and cells are counted 2 days later, 6 days after plating.

An alternative and perhaps faster and more accurate test of correlation for active vitamin D compounds is the iri vitro activity of transgluta- minase, in the keratinocyte culture. The enzymatic test is carried out according to standard transgluta- minase assays, Scott, K.F.F. et al. , J. Cell. Physiol.

111:111-116 (1982) . Any compound which when present at a concentration of 10 -12M to 10-3M increases the enzymatic activity by 25% or more, preferably 50% or more, most preferably 100% or more is considered an active compound.

Use of the HL-60 cells in an in vitro test is described in Shiina, et a_l. , Arch. Biochem. Biophys.

220;90 (1983). Use of the Ml cells in an in vitro test is described in Honma et al. , PNAS, USA 8_0:201-

204 (1983). Both of these references are herein incorporated by reference.

Any vitamin D compound which at n vitro concentrations of 10 -12 M to 10-3 M is capable of cellular differentiation or inhibiting of fibroblast growth by at least 25%, preferably 50% is considered active.

Among the preferred compounds usable in the present invention are those of the formula (I):

wherein the bond between carbons C-22 and C-23 is sing

Q ^a is

Q is

R is a ) group; wherein X is selected from the group consisting of hydrogen and -OH.

When the compounds of formula (I) have a double bond at position C-22, they are derivatives of vitamin D , whereas if the bond at that position is single. and there is a lack of C ₂₄ alkyl, they are derivatives of vitamin D ₃ . The latter are preferred. Preferred are those compounds derived from vita¬ mins D ₃ or D ₂ ; 1-hydroxy-vitamins D ₃ or D ₂ ; 1,25-dihy- droxy vitamins D ₃ and D ₂ , 24,25-dihydroxy vitamins D,

or D ₂ , 25,26-dihydroxy vitamins D ₃ or D ₂ ; 1,24,25-tri- hydroxy vitamins D ₃ or D u ₂ - .- Most preferred among these are vitamins, D ₃ ^• or D ₂ ; 1-hydroxy-vitamins D ₃ or D_ _^ , ₂ , and 1,25-dihydroxy-vitamins D ₃ or D 2 i especially

5,6-epoxy derivatives of vitamin D and its metabo- lites, as well as the side chain fluoro derivatives of

1, 25-(OH) ₂ vitamin D and 1 (OH) vitamin D.

Among other preferred compounds are water soluble derivatives of the aforementioned compounds of formula (I) obtained by solubilizing such compounds by attach¬ ing thereto glycosidic residues such as those dis¬ closed in Holick, U.S. Patent 4,410,515. Alternative methods of solubilization are by conjugating compounds of formula (I) to glycosyl orthoester residues, as disclosed in copending U.S. S.N. 607,117 by Holick et al. , filed 3 May 1984. The disclosures of the afore¬ mentioned patent and application are herein incorpor¬ ated by reference and made a part hereof.

Of interest are compounds of the formula (II):

(II)

2 wherein Y is hydrogen, fluorine, methyl or ethyl;

Z ² is F, H or X ²

Q ^a and Q have the same meanings as in formula (I)*;

R is a double bond or an epoxy group;

2

X is selected from the group consisting of hydrogen, and OR , where R is hydrogen or a straight or branched chain glycosidic residue containing 1-20 glycosidic uunniittss ppeerr rreessiidduuee,, oorr RR is an ortoester glycoside moiety of the formula (III).

where A represents a glucofuranosyl or glucopy- ranosyl ring;

2

R is hydrogen, lower alkyl, aralKyl, or aryl; and

R is hydrogen or a straight or branched chain glycosidic residue containing 1-20 glycosidic units per residue, with the proviso that at least one of the R is either a glycosidic residue or an ortho- ester glycoside moiety.

The vitamin D compounds are prepared or obtained according to the disclosures of the aforementioned references. In particular, the 5,6-epoxy derivatives of vitamin D ₃ are obtained as described in Jpn. Kokai Tokkyo Koho JP 58,216,178 [83,216,178], 15 December 1983.

The fluoro derivatives are made or obtained as described in Shiina, et al_. , Arch. Biochem. Biophys 220:90 (1983).

The compounds of the invention can be administered in any appropriate pharmacological carrier for oral, parenteral, or topical administration. They can be administered by any means that effects palliating conditions of psoriasis in humans. The dosage admin¬ istered will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. Generally, systemic daily dosage of active ingredient compounds will be from about 0.001 micrograms/kg to 100 micrograms/kg preferably 0.1 to 1.0 micrograms per kg of body weight. Normally, from 0.1 to 100 micrograms/kg per day, in one or more applications per day is effective to obtain the desired results. Topical dosage would be 0.001 micro-

2 grams to 100 microgra s/cm area of skin.

The compounds can be employed in dosage forms such as tablets, capsules, powder packets, or liquid solu¬ tions, suspensions or elixirs for oral administration, sterile liquid for formulations such as solutions or suspensions for parenteral use. Alternatively, the compounds can be present in a pharmacologically inert topical carrier such as one comprising a gel, an oint¬ ment or a cream, including such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters or mineral oils. Other possible carriers are liquid petrolatum, iso- propylpalmitate, polyethylene glycol ethanol 95%,

polyoxyethylene monolaurate 5% in water, sodium lauryl sulfate 5% in water, and the like. Materials such as anti-oxidants, hμmectants, viscosity stabilizers and the like may be added, if necessary.

The compounds can also be administered by means of pumps or tapes.

Having now generally described this invention, the same will be understood by reference to an example which is provided herein for purposes of illustration only and is not intending to be limited unless other¬ wise specified.

EXAMPLE

Skin biopsies from involved and uninvolved sites were obtained from psoriatic patients and therefrom were obtained cultured fibroblasts. An analysis of cultured fibroblasts from the psoriatic patients revealed that these possess high affinity low capacity receptors for l,25-(OH ₂ )-D ₃ and that the Kd and den¬ sity for these receptors in fibroblasts from the uninvolved areas were essentially no different from that found in cultured skin fibroblasts from normal subjects. In addition, the fibroblasts from involved sites possessed receptors for 1,25-dihydroxyvitamin D ₃ that have a normal affinity constant but possibly as much as 100% decrease in number of receptor sites when compared to the uninvolved fibroblasts.

It was next determined if cultured fibroblasts from psoriatic patients would respond to 1,25-dihy- droxyvitamin D ₃ by causing an inhibition of cell growth. Cultured human fibroblasts from normal and

psoriatic subjects were incubated with either no 1,25-dihydroxyvitamin D ₃ or 1,25-dihydroxy vitamin D ₃ at either lθ ^"10 , lθ ^"8 , lθ ^"6 , or lθ ^"4 M. Fibroblasts from the normal subjects responded as expected in a dose dependent manner. However, none of the fibro¬ blasts obtained from six different subjects with psoriasis responded to 1,25-dihydroxyvitamin D^ at 10 -8 M in a similar fashion as the controls. When psoriatic cells were incubated with l,25-(OH) ₂ -D ₃ at

10~ M, there was a small but significant effect on inhibiting cell growth in some of the subjects studied

(who were resistant to up to 10 —6 M of 1,25-dihydroxy vitamin D ₃ ). In one subject, a detailed time course and dose response revealed a very small response at

10 -6 M while 1,25-dihydroxy vitamin D ₃ at 10-4 M was very effective in inhibiting cell growth (Figure 1).