CA2124852A1 - Use of a known chemical compound for the production of a pharmaceutical composition for topical application - Google Patents

Abstract

The compound diflunisal (5-(2,4-difluorophenyl)-salicylic acid) of formula (I) is used for the production of a pharmaceutical composition for treatment of inflammatory and/or autoimmune diseases, such as psoriasis, Crohn's disease and uveitis. Preferably compositions, such as cremes and ointments, for local use on skin surfaces, eye agents, compositions for local treatment of the intestinal canal, vaginal agents, compositions for local use in the oral cavity and compositions for inhalation are produced.

Description

WO93/10791 ~ 4 8 5 2 PCT/DK92/00~8 Use of a known chemical compound for the product~on of a pharmaceutical composition for topical application .

The present invention concerns the use of the compound di-flunisal (5-(2,4-difluorophenyl)-salicylic acid) of the formula:

COOH
0 F~ ~
~=~011 F

for the production of a pharmaceutical composition for toplcal treatment of inflammatory and/or autoimmune dis-eases, such as psoriasis, Crohn's disease and uveitis.

An example of these dise~ses is psorias~s which is a cro-nlc skin disease of unknown etiology. The indidence is about 2-3% in most western countries. The disease i8 here-ditary, presumably polymerically conditioned, with irregu-larly occurring manifestations that may be caused meohani-cally, but probably also by other ~nfluences, infections, psychic strain, etc. The onget of the disease is usually at the age of 20-30. It manifests itself as re~ular, slightly infiltrated, hyperamic patches which are covered by stearin-like scales consisting of many layers. The dis-ease is localized to the epidermis, and it is character-ized by increased cell turnover. The normal cell turnover,which means the time it takes for a recently formed cell to get from the basal cell layer to the horny layer at the surface of the skin, i~ usually about 4 weeks, but in case of psoriasis it ~ust takes 2-3 days.

WO93/10791 PCT/DK92/003~x '~12~85 ~

According to the clinical nature of the disease a distinc-tion is made between a number of different clinical forms, all of wh~ch are comprised by the desi~nation "psoriasis"
in the present context.
A plurality of modes of treatment have been employed in the course of time with a view to keepin~the disease under control (l): Thus, drugs are used for topical appli-cation, such as corticosteroids, calcipotricl, anthralin (dithranol) and coal tar. In severe cases systemic treat-ment with drugs is used, such as methotrexate or aromatic retinoids, and phototherapy alone or in combination with psoralens. Combinations of these systemic and topical modes of treatment have been in general use in recent years.

The more effective treatments usually lnvolve a high risk of serious side effects. Topically applied corticosteroids are moreover rendered more or less lneffective after a re-latlvely short time. Anthralin is difficult to administerfor the patient owing to discoloration and a considerable risk of inducing serious irritations, although these draw-backs have been mitigated to some extent after the intro-duction of the so-called minute therapy, comprising apply-ing the drug to the skin for just 10-30 minu~es. Topical treatment with tar is no~ used very much in Europe, in contrast to the widespread use in the USA where the treat-ment is considered rather effective when the tar applica-tion is followed by UVB iradiation (Goecksrmann's treat-ment). However, the treatment with tar is inexpedient,~ecause the tar composition is greasy and malodorous.

Systemic use of methotrexate is probably one of the most effective methods for the treatment of psoriasis in pa-tients who are not in the fertile age, but it involves apotential risk of liver side effects, and it is therefore W093/10791 ~ 12 ~ 8 5 2 PCT/DK92/0036X

to be supervised earefully.

Retinoids may be used, but serious side effeets in this eonneetion have been reported. These eomprise induetion of liver side effeets and in some eases artieular deforma-tions. Etretlnate (Tigason0), which is so far the only aromatie retinoid introdueed for the treatment of psoria-sis, is moreover teratogenic, and sinee its half life in the body is long, it is recommended that pregnaney during the treatment or within the first 12 months after the ees-sation of the treatment should be interrupted by abortion.

The D vitamin analog eompound ealeipotriol is another applieable agent for the treatment of psoriasis, beeause the compound lnhibits the proliferation of the epidermal eells (2). However, it ls marketed as an ointment, whieh may be inconvenlent to use for the patient, and the medi-e~ment ls not reeo _ ended for use on pregnant women owlng to the still limited experlenee.
The T eell inhibiting substanee cyelosporin A has a elear effeet on lnflammatery diseases, sueh as e.g. psoriasis (3). However, the substanee has a number of adverse sys-temie side effeets and i8 moreover diffieult to use lo-eally.

Thus, there is still a need for new modes of treatment andmedieaments against psoriasis, just as there is need for new eompositions for eonvenient topical treatment of other local inflammatory diseases, such as e.g. colitis ulce-rosa, Crohn's disease, uveitis, contact dermatitis and cutaneous herpes simplex.

In ease of these diseases as well as psoriasis it is be-lieved that lmmunologieal mechanisms are involved in the pathogene~is, where part~cularly T lymphoeytes play a part W093/l079l PCT/DK92/003~
21'~48$2 '' (4,5,6,7). Such diseases are moreover characterized by a generally increased cell proliferation of e.g. inflamma-tory cells, including T cells, and possibly of epidermal cells.
Therefore, a topical drug against psoriasis and other inflammatory and/or autoimmune diseases must satisfy the followlng requirements:

- It must exert an inhibitory effect on the cell proli-feration (also on T lymphocytes) without simultaneously being decidedly cell toxic; cf. the effect of e.g. con-ventional cytostatics and cyclosporin A;

- it must be absorbable to a considerably degree from external and internal body surfaces for local activity, and - lt must not involve side effects in the form of local lrrltatlon, discoloration, etc.

It has surprisingly been found that the compo~nd 5-(2,4-dlfluorophenyl)-salicylic acid (diflunisal) of the formu-la shown above has the above-mentioned desirable combina-tlon of properties. The compound has thus been found tohave an extremely potent effect with respect to inhibition of cell proliferation, also on ~ cells, whereas structu-rally related compounds, such as COOll acetyl~alicyllc acid ~OcOcH, WO93~10791 ~ 1 2 4 8 5 2 PCT/DK92/0036X

CO~2 salicylamide ~ O~
H

CONCH,CO,H
salicyl glycine ~OH and coo~
5-amidosalicylic acid NHCOCH ~

have been found to have no activity in therapeutically re-levant concentrations. On the other hand, a certain inhi bltory effect on T cell proliferation can be observed with sulfasalazine (SASP):

~ oo~

~ ~ 2 ~ ~=~ ~ ON

However r this property is observed only to a very limited degre~ for the metabolites of sulfasalazine:

coo~
5-aminosalicylic a~id (5-ASA) ~ ~ and ~2 sulfapyridine ~N~ ~2 WOs3fl079l PCT/DK92/0036~

2~2i~8$2 :

and to some extent in the azo compound HOOC COO~
5 olsalazine ~10 ~ ~ I`I=N ~ OII

In addition to the relatively modest activity of sulfa-salazine and olsalazine with respect to inhibition of cell proliferation, these compounds moreover give rise to in-convenience in the form of discoloration and discomfort when applied to the skin, and they will therefore not be expedient as drugs.
However, dlflunlsal haæ the necessary effect with respect to inhibition of cell proliferation of disease-relevant cells, and ~ince the compound has moreover been found to be absorbed surprisingly easily through the skin without glving rise to inconvenient side effects, it is extremely suitable for the production of topical drugs against pso-riasis and other inf lammatory diseases. In a~dition to ~eing useful for application on skin, dlflunisal can be formulated as eye agents for the treatment of uveitis and a~ agents intended for local treatment in the intestinal canal, as well as optionally as agents that can be used on other body surfaces.

Diflunisal is a well-known medicament of analgesic and antiinflammatory activity (8). In Denmark the substance is known from the analgesic compositions DiflonidO and Dono-bid0, both of which are in tablet form. The substance has not yet been marketed in the form of compositions for to-pical admin~stratlon, although other modes of administra-tion for diflunlsal than the oral one have been studied.The article (9) mentions a rectal dose form of diflunisal, W093/1079l PCT/DK92/OO~X

in which the base material is fat~y compounds and poly-ethylene glycols, but it is stated that the rectal ab-sorption is poor with respect to oral administration be-cause of the poor solubility of diflunisal. According to (10) composltions for e.g. vaginal and rectal administra-tion of diflunisal can be prepared with a view to treat-ment of lnflammatory diseases, but it is necessary here to use hydrogenated phospholipids and a fatty base material to increase the bioavailabllty of diflunisal which has poor absorbability through mucosal membranes. It has also been attempted to increase the penetration of diflunisal through the skin by using more readily soluble salts of diflunisal instead of free diflunisal, e.g. the diethyl-ammonium salt (11). Finally, the compound has been used for topical treatment of acne in combination preparations, which morQover contain an antimicrobial agent in the form of a llpophilic l-substltuted imidazole; see reference (12).

Reference (13) describes pharmaceutical compositions for the treatment of i.a. psoriasis, preferably for topical appllcation. These compositions comprise the substances 4-aminosalicylic acid (4-ASA), 5-aminosalicylic acid (5-ASA) or unctional derivatives thereof. Diflunisal is not com-prised by the patent claims of this reference. The clini-cal results with respect to the action of 5-ASA on psoria-sis are still uncerS~ain~ and 5-ASA moreover gives rise to dlscoloration of the skin after oxidation with the oxygen o the air. Although the compound has a well-documented useful activity against Crohn's disease and colitis ulce-rosa, the mode of action has still not been fully ex-plained.

According to reference (14) diflunisal can be used per-3S orally against psoriasis. The mode of action is stated to be ba8ed on inh~bition of lipoxygenase, interfering with wog3/lo791 2 8 - PCT/DK92/~36~

the biotransformation of arachidonie acid to LTB4 (leuko-triene B4 and 5-HETE (5-hydroxyeicosatS.8.10.14ltetraeno-ate). Thls i8 said to eounter the psoriasis attaek whieh gives high araeh~donic acid and 5-HETE levels. However, the referenee eontains no doeumentation of the actual aehievement of a favourable aetivity against psoriasis, and $f the oral administration of ~flunisal is to give the asserted effeet, it is necessary to adminis~er ex-tremely large doses (of the order of 200-1200 mg every 6th or 12th hour).

Aeeording to the present invention diflunisal can be for-mulated for a plurality of different modes of administra-tion in eonneetion with the present use of dlflunisal for the production of topleal drugs agalnst inflammatory ~nd/or autoimmune diseases.

As stated in the followlng table 1, whleh is eopied from refesenee (15), drug forms for topieal applieation may occur in ~arious states (solutions, emulsions, suspen-slons) based on various vehieles. As a drug form eontain-lng diflunisal lrrespective of state and vehiele, the use o$ the following ones is preferably eontemplated: cremes ~ineludlng foam), gels, liniments (including lotions~, pastes, powders, ointments, shampoo, stieks, supposi-tories, enemas, eontrolled release formulations, vaginal agents, aerosols for inhalation, oral agents for loeal use in the oral eavity, eye drops and eye ointments, drug forms for topieal application may occur in various states, as illustrated in the table.

. W093/10791 PCT/DK92/0036~ `, g Drugs for toplcal application. Taxonomy according to state and drug forms.
S
State Vehicle Drug form Solution Liniment, Shampoo (hydrophilic or hydrophobic) Liquid Suspension Liniment (hydrophilic or hydrophobic) Emulsion (o/w,w/o) Liniment Semi-solid to Hydrophobic gel Ointment semi-liquid water-emulslfying single phase gel system Hydrophilic gel Ointment, gel Semi-solid to w/o emulsion Creme ~emi-liquid o/w emulsion double phase system Suspension Ointment, Paste __.._.... . .
Solid Powdered mixturo Powder The activity of various NSAID (NSAID = Non Steroid Anti Inflammatory Drug) including diflunisal, on the cell pro-liferation of a number of cell types ~ncorporated in in-flammatory and immunological mechanisms is shown below.
Further, data for formulation and application of diflu-nisal to the skin are presented.
Methods The following methods are used for partly studying the 3~ activity of diflunisal in vitro and partly studying the~uitability for formulation as a drug:

WO 93/10791 PC~/DK92/0036~

2~2~S52 - 10-.

1. Antigen-specific T cell proliferation Inbred mice of either the Balb/c type or the ~lab/k type were used ln all the experiments-. The mice were immunized with a single in~ection in hind paws and tail stub with 0.1 ml of a water-in-oil emulsion containing equal parts of phosphate buffered salt water (PBS) and complete Freund's ad~uvant containing Mycobacterium butyricum (Dif-co) and in some experiments 1 mg/ml antigen in the form of a peptide (MP7 having the sequence PELFEALQKLFKHAY ) ( 16 ) .
Ten days later the mice were killed, and lymph glands in the pelvis and the groin were removed. Single cell suspen-sions of these were produced by passage through a steelnet. Antigen (P.7D (100 ~g/ml) or MP7 (30 ~g/ml)) as well as the various NSAID compositions were then admixed with microtiter plate cultures of the lymph gland cells con-taining 5 x 105 cells per well. The ce~ls were cultured in RPMI contain$ng penicillin, streptomycin, 2-mercapto etha-nol, glutamine, sodium pyruvate and 0.5% sy,ngenic fresh-drawn mouse serum. After 72 hours 1 ~Ci 3H-thymidine was added to each w~ll, and the cells were harvested af~er 90 hours. The 3H-thymidine incorporation, as an expression of the T cell poliferation, was then determ~ned by liquid scintillation count.

2. LPS and Con A stimulation of B and T lymphocyte roliferation, respectively Mouse spleens from either Balb/c or Balb/k mice were re-moved, and single cell suspensions were produced as above.
Erythocytes were ly~ed with hypotonic salt water and the spleen cells were washed twice in RPMI. The cells were then cultured in the same media as stated above in round bottom microtiter plates for 22 hours together with either wo 93Jl0791 2 1 2 ~ 8 5 2 PCT/DK92/0036X

lipopolysaccharide (LPS) (100 ~g/ml) or concanavalin A
(Con A) (1 ~g/ml) and dilutions of NSAID compositions as well as 1 ~Ci 3H-thymidine. The cells were finally har-vested and were then analyzed by liquid scintillation counts as described above.

3. Inhibition of roliferation of WeHi and X63 cells P

WeHi and X63 cells, respectively, were cultured in RPMI
with the same additions as above in microtiter plates with 5 x 104 cells per well. However, mouse serum was replaced by 10% foetal caif serùm in the medium~ The cells were cultured for 4 hours toyether with NSAID dilutions and 1 ~Ci H-thymidine per well, and they were then harvested and counted as described above.

All the NSAID compQs~tions were examined in concentrations of from 15.6 to 1000 ~M, and all the determinations were performed ln triplicate, ~ust as all the experiments were reproduced at least three times.

The percenta~e inhibition of LPS, Con A and antigen stimu-lated cells was calculated according to the following for-mula:

NSAID treat~d c-llr non-~tloul~ted cell~
cPn CP~ .
lnhlbltlon - 100 ~ 100 NSAID non-tr-~ted cell- no--tloul-ted cell~

The inhibition of WeHi and X63 cellc (which do not have to be stimulated) was calculated as follows: -NSAID tre-ted cell~

lnhlbltlo ~ 100 - ~ 100 W093/l0791 PCT/DK92/00~ ;
212485~ .

StudY of the absorption of diflunisal through the skin The abs~rption of diflunisal through the skin was measured indirectly by secret~on of the substance in the urine.
To design a suitable HPLC analysis for determination of free diflunisal and the glucuronidized derivatives there-of, respectively, an initial experiment was performed. 500 mg of diflunisal were administered orally, and a urine sample was collected subsequently. The urine sample was treated wlth ~-glucuronidase to obtain cleavage of both phenol and acyl glucuronides to free diflunisal. The total amount of diflunisal in the urine could then be determined quantitatively by means of standards of diflunisal chroma-tographed under similar circumstances.

Two serles of experiments were performed. One comprisedapplying to an individual who does not suffer from psoria-sis or other skln diseases, a solution of diflunisal in propylene glycol (25 mg/ml). 1 ml of solution was applied to each arm in the morning and in the evening for 3 days.
The total daily dose was 50 mg of diflunisal. Urine samples were collected over four subsequent days.

In the second series of experiments a non-aqueous foam fsrmulation was applied to the same test person as de-scribed in example 1, containing diflunisal dissolved in propylene glycol (24 mg/ml). 2.5 ml were applied to each arm in the morning and in the evening for six days. The total applied daily dose was 240 mg of diflunisal. Urine samples were collected during the entire period and for another two days. The content of diflunisal in the various day diureses appears from table 3.

~093/10791 21 2 4 8 ~ ~ PCT~DK92/0036X

Results and discussion -In vitro activity on a plurality of disease-relevant cell types.

Sulfasalazine (SASP) and 5-aminosalicylic aaid (5-ASA) have been used very successfully in the treatment of Crohn's disease and colitis ulcerosa. As appears from figs. la and lb, SASP has a direct inhlbitory activity on both antigen-specific as well as Concanavalin A (Con A) stimulated murine T cell proliferation. However, this pro-perty is only seen to a limited degree for the metabolites of SASP (5-ASA ana sulfapyridine (SP)). An activity of olsalazine (OS), consisting of an azo compound between two 5-ASA molecules, is likewise seen.

Similar (but indirect) results have previously been re-ported for SASP, SP and 5-ASA, it being found that SASP
was capable of inhibiting antibody production from murine spleen cells. It was believed that this was an indirect consequence of a simultaneously reduced production of interleukin-2, which is produced by T cells (17).

However, it has b~en found that this inhibiting activity 25 i8 not specific for T cells, but, on the contrary, the proliferation of other cells is inhibited to the same ex-tent. Fiss. 2a and 2b show partly the effect on Con A
stimulatéd T lymphocytes and partly the effect on LPS
stimulated B lymphocytes from mouse spleens. There is no difference in the inhibition of the two cell types, in contrast to the effect of the T cell specific substance cyclosporin A (CSP).

Further, pronounced inhibition of the proliferation of two rapidly growing tumor cells, X63 and WeHi, is demon-strated, said cells being a B cell and a monocyte/macro-WO 93/10791 PCI /DK92/00.~
'~124852 `,,.

phage cell line, respectively. The last-mentioned cell type plays an important part in inflammatory processes.
SASP inhibits 50% of the cell division over a period of 4 hours in concentrations from 700 to 1000 ~M. The effect of SP and 5-ASA on these cells is e~en less pronounced ( fig.
3). All experlments were performe~ with substance concentrations of the same order as the plasma concentration after a normal therapeutic peroral administration.
Using the same in vitro experiments a plurality of other derivatives were then examined with a view to finding more potent substances.

As will be seen from figs. 4a and 4b, salicylic acids, derivatised w~th e.g. an acetyl group on the OH group (acetylsalicylic acid), or in which the carboxylic acid group has been converted to an amide (salicylamide and salicyl glycine), have no noticeable effect on the cell proliferation, like 5-ASA.

Studies of salicylic acid derivatives with substitutions in the 5-position (figs. 5a-d) show that 5-phenylsalicylic acid (PS) (18), diflunisal and SASP have an extremely good effect in relation to the unsubstituted compounds salicy-lic acid and 5-amidosalicylic acid which have practically no effect. ~hiæ activlty, which is the result of a substi-tution with a very hydrophobic phenyl compound in salicy-lic acid, can possibly be ascribed to a better abillty of diflunisal and PS to penetrate cell membranes. Compared with the SASP dose response curve, the diflunisal and PS
curve is much steeper, and a 100% inhibition in concen-trations down to approx. 125 ~M is shown. The same degree of inhibition was seen on all examined cell types. How-ever, SASP and PS were somewhat less potent inhibitors ofthe proliferation of WeHi and X63 (figs. 5c and 5d).

~12 4 8 5 2 PCT/DK92/0036X

The general antiinflammatory properties of the substances are largely believed to be due to inhibition of cyclooxy-genase, but it has also been described that they are cap-able of interrupting the oxydative phosphorylation in the mitochondria (19), which leads to reduced ATP production and thereby inhibited cell function. However, for this effect to be achieved with salicylic acid, rather large doses are necessary. Various derivatives of salicylic acid have been studied in the experiments descr~bed below.
Owlng to an assumption that the mechanism causing the ob-served effect of diflunisal might be ascribed to inter-ruption of the oxidative phosphorylation in the mitochon-drla, two other NS~ID compositions were moreover studied, viz. diclofenac and indomethacin, which are known to effectively cause such interruption. Figs. 6a and 6b show a comparlson of the effect of dlclofenac, indomethacin, PS
and aiflunisal on the proliferation of WeHi and X63 cells, respectively. As will be seen, these compounds are sub-stantially equipotent with respect to the inhibitingeffect on the cell proliferation. However, PS is somewhat less potent, and diclofenac and indomethacin, which are not salicylic acid derivatives, but acetic acid deriva-fives, and which are therefore not chemically analogous to diflunisal, appear to exhibit less effective absorption through the skin. This makes these substances less inter-esting in the present context. Furthermore, indomethacin and diclofenac are known to be locally i~ritating, and diclofenac moreover exhibits photosensibility, which makes the substances unuseful for local application.

Diflunisal is absorbed almost completely after oral ad-mini~tration of doses of 50-500 mg. The oral bioavail-abillty is stated in the literature to be 100% based on recovery ln the urine after 96 hours. Diflunisal is greatly bound to plasma proteins (20).

WO93/l07gl PCT/DK92/003~
212~52 - 16 -The elimination is concentration dependent and depends upon con~ugation with glucuronic acid. About 80-95% of an oral dose is secreted in the urine 72-96 hours after ad-mln1stration, malnly as phenol (64%) and acyl glucuronides (20~)- ~ ;;

The use of diflunisal for the treatment of psoriasis will be conditional upon the substance penetrating into the skin.

Penetration experiments were performed with two formula-tions: A solution of diflunisal in propylene glycol (25 mg/ml) and a foam formulation, as stated in example 1.

Application of diflunisal in propylene glycol was followed by collection of urine r~und the clock, which was analyzed on HPLC for diflunlsal content. As appears from table 2, dlflunisal can be absorbed very well through the skin and then be ~ecreted in the urine.

~093/10791 ~12 ~ 8 S 2 PCT/DK92/0036X

. -Urine Total amount samples Urine Urine of diflunisal Day collected volume conc. secreted per at (ml) (~g/ml) 24 hours (mg) _ , .
1 16:30 430 1.33 . 1 . 1 _ 22:45 380 1.3~ _ 2 06:45 610 l.75 _ 11:00 410 5.79 _ 15:45 380 6.36 _ 19~00 650 2.17 _ 10 . O
_ 21:00 610 2.19 _ 23:00 62~ 2.21 -3 04.-00 740 3.55 _ 07:00 300 7.54 12.5 _ 21:00 550 13.8 . _ .... , 4 06:10 700 fi.7 4-7 . --Dlflunisal has thus been present in the epidermis~ where the substance may give r~se to inhibition of the prolife-ration of the epidermal cells as well as the pathogenic T
ce118 .

After application of diflunisal in a foam formulation si-milar analyses of the conten~ of diflunisal in the urine were performed. It appears from the results in table 3 that diflunisal is absorbed excellently through the skin here too. This experiment extended over a longer period WO93~10791 21 æ 4 ~ 5 2 PCT/DK92/00' than the preceeding one, and after day 3 where a steady-state condition occurred, it can be seen that about 10% of the applied dose of diflunisal is secreted. The observed decline on day 5 is due to the fact that only a single dose was applied on this day.

Total amount Urine Urine of diflusnial Day volume conc. secreted per (ml) (~g/ml) 24 hours (mg) 12400 1.63 3.9 22700 6.1116.5 3 9 W 7.2828.4 42400 10.7025.7 _ 3100 3.5210.9 6 4000 6.5626.2 7 3200 7.7924.9 8 3250 2.18 7.1 In future formulations of diflunisal a number of factors must be taken into consideration:

When formulating cremes (including foam) it is possible i.a. to vary the lipophilicity and the viscosity of the base, whereby the drug release from the creme can be con-trolled. Likewise, it is possible to vary the foam quality by varying the content of isobutane. The formulation can al~o be varied with respect to penetration enhancing sub-stances, so-called enhancers, e.g. ethanol and Azone0.

vo 93/10791 21 2 4 8 5 2 PCI~/DK92/0036~

-- 19 -- t :
When formulating gels it is possible to formulate a hydro-phobic as well as a hydrophilic gel. It applies to both of them that they will dry on the skin after application, thereby forming a firm brittle film. Hydrophilic gels have the advantage that they are not greasy. They can moreover be removed by ordinary washing, which is of importance in case of e.~. application on heary skln portions.

When formulating ointments a distinction i8 made between hydrophobic, hydrophilic and water emulsifying ointments.
Nydrophobic ointments are ointments based on lipophilic vehicles, typically hydrocarbons, vegetable oils, semi-synthetic fats, waxes and alkyl polysiloxanes. Hydrophobic ointments absorb moisture from the skin only to a limited extent, and they therefore have an occlusive effect. They also have a good sk$n co~tact and have a softening effect on the horny layer after application for an extended pe-riod of time, which is desirable when the horny layer is thlck. It applies to all types of ointments that stabi-lizing vehicles may be added, e.g. antioxidants, preserva-tives, and aromatic substances. Hydrophobic ointments also have a positive effect on scaly skin areas. Hydrophilic ointments are based on carriers which are miscible with water, most freguently polyethylene glycols. Water emulsi-fying ointments are based on lipophilic vehicles admixed with lanolin or other w/o emulsifying substances, e.g.
~orbitan esters.

Concerning eye agents the aqueous formulations are the most common dosage form. Since diflunisal is sparingly soluble in water at a neutral pH, eye drops containing -diflunisal will preferably be suspensions. The advantage of these is that the particles, after application, settle ln the con~unctlval 8ack of the eye in which they are dis-801ved 810wly. This provides a depot effect. When using a drug form contalning particulate diflunisal (suspension), m~cronized diflunisal is preferably to be used, which means that the greater part of the particles is smaller than 5 ~m and all the particles are smaller than 25 ~M.

Diflunisal can also be formulated,"in a controlled release form for the treatment of inflammatory intestinal di-seases, cf. reference (21). Formulation of enemas is also poss1ble.

The invention will be illustrated more fully by the fol-lowing formulation examples.

In the examples all percentages are percentages by weight.

Cremes Diflunlsal O.Ol - lO ~
Cetostearyl alcohol 0 - lS %
Cetomacrogol lO00 0 - 3 %
Cetanol 0 - 15 %
Isopropyl myristate 0 - 15 %
Propylene glycol lO - 50 %
White solid paraffin 0 - lS %
Paraffin oil 0 - 15 %
Preservatives q.s.
Ethanol 0 - lO %
Water 0 - 60 Non-aqueous foam:

vo 93Jl0791 2 1 2 ~ ~ 5 2 PCT/DK9~/0036X

Diflunisal 0.01 - 10 %
Propyle~e glycol 86 - 96 Polawax 0.8 ~
Bri~ 76 3.3 % ~:
Preservative q.s.
Isobutane 5.5 %
Nltrogen to 6 bars Aqueous foam: :
Diflunisal 0.01 - 10 %
Polawax 0.5 _ 5 % ~-~
Polysorbate 20 0.1 - 3 %
Propylene glycol 30 - 50 %
Softigen 767 10 - 30 %
Preservative q.s. ~.
Water 20 - 35 %
Isobutane 3 - 6 ~ ~
Nitrogen to 5 - 7 bars -The produced foam bases are packed in aluminium cans which `
are closed with a valve, following which a dosing device is applied, which makes it possible to dispense doses from 1 to 5 ml. Such a valve with a dosing device is available e.g. from Lablabo, 5 rue Roger Salengro, 92120 Montrou~e, France.

-wog3/1079~ 12 ~ ~ 5 2 PCT/DK92/00~

Gels (a) Hydrophilic gels:

Diflunisal 0.01 - 10 "Carbomern ` J
(acrylic acid polymer) 0 - 5 %
Sodium carboxymethylcellulose 0 - 5 %
Methylcellulose 0 - 5 ~
Natural starches 0 - 25 %
Silicates 0 - 5 %
Bentonite 0 - 5 %
Preservatives q.s.
Water 75 - 99 %
C~lycerol 75 _ 99 %
Propylene glycol 75 - 99 %
. . .
(b) Hydrophobic gels:

Diflunisal 0.01 - 10 %
Polyethylene 10 %
Paraffin oil 90 *

Liniments, including lotions (a) Diflunisal 0.01 - 10 %
Propylene glycol 50 - 99 %
Isopropyl myristate 0 - 10 %
Sodium hydroxide 1 N q.s.
Hydrochloric acid 1 N~.s.
Glycerol 0 - 10 %

~093/l07gl 21 2 ~ ,~ S 2 PCT/DK92/~36X

(b) Diflunisal 0.01 - 10 %
"Carbomer" 0.5 - 2 ~
Cellulose rubber 0.5 %
Polysorbate 80 0.1 %
Glycerol 85% 0 - 5 %
Propylene glycol 5 %
Preservative q.s.
Water 84 - 94 %

(c) Difluni8al 0.01 - 10 ~
Glycerol 85% 0 - 10 %
Propylene glycol0 - 10 %
Sorbitol 0 - 10 %
Sodium chloride 0 - 5 %
Ethanol o - 99 %
Water o - 99 %
Preservative g. 8.

(d) Diflunisal 0.01 - 10 %
Cetomacrogol 10000.8 %
Cetostearyl alcohol3.2 %
Clyceryl stearate 3 %
Mineral oil 15 ~
Polysorbate 80 0.6 %
Glycerol 85% 3 %
Propylene glycol 5 %
Preservative q.s Water 59 - 69 2~2~SS~ '.

Pastes Dlflunisal Q.01 - 25 %
Vaseline 0 - 99 %
Lanolin 0 - 99 %
Mineral oil 0 - 10 %

Powders Diflunisal 0.01 - 10 %
Sterilizable starch90 - 99 %
u, Ointments '-(a) Hydrophobic ointments:

Diflunisal 0.01 - 10 %
Paraffin 1 - 10 ~
White wax 1 - 10 %
Cetostearyl alcohol 1 - 10 ~
White vaseline 80 - 90 %

Diflunisal 0.01 - 10 %
Lanolin 1 - 10 %
Paraffin 1 - 10 ~
Cetostearyl alcohol 1 - 10 %
White vaseline 80 - 90 %
, `~093/l0791 212 4 8 5 2 PCT/DK92/~36X

(b) Water-emulsifying ointments: .

Diflunisal 0.01 - 10 % ::
Anhydrous lanolin 20 %
White vaseline 70 - 80 %

(c) Hydrophllic olntments: `

Dlflunisal 0.01 - 10 %
Macrogol 300 65 ~ :~
Macrogol 4000 25 - 35 %
.;
Diflunisal 0.01 - 10 ~ `~
Mbcrogol 400 60 %
Macrogol 3350 30 - 40 %
',.
EXAMPLE 7 .
~' Shampoo Diflunisal 0.01 - 10 % :, Texapon SBN0 50 % .:
Sodium chloride 3 %
Preservative q.s.
Water 37 - 47 %

The content may be increased to make the shampoo softer and thus more gentle to the skin.

, WOg3/107gl PCT/DK92/~? ; l~

4~52 26 -'.
EXAMPLE 8 ~
, (a) EYe drops Diflunisal ;- 0.01 - 10 %
Preservative, e.g.
benzalconium chloride0.00 - 0.5 % ;~

Methylcellulose 0 - 1 % :~.
Polyvinyl alcohol 0 - 2 %
Sodium hydroxlde to pH 7-9 Sodium chloride 0 - 0.9 %
Tween 0 - 1 % . ;
Polysorbate 0 - 1 % ~ f i Sterile water up to 100%

(b) Eye ointments Diflunisal 0.01 - 10 % ~.
Va~elin 80 %
Paraffin oll up to 100 % .

Diflunisal 0.01 - 10 %
Cetanol 0 - 15 % --~
Lanolin 0 - 10 %
Paraffin oil 0 - 15 %
White vaseline 0 - 50 %
Sterile water 0 - 60 %

:

`
''''.'' ~12~8~2 ~093/10791 PCT/DK92/0036 Description of figures -Fig. l: Study of the ability of various SASP analog sub-stances to inhibit the T cell activation. a. In-hibition of the antigen-specific T cell response to the synthetic peptide MP7 (PELFEALQKLFKHAY) with various concentrations of SASP, SP and 5-ASA. b. Inhibition of Con A stimulated T cells from spleens with various concentrations of SASP, SP, 5-ASA and Olsalazine.

Fig. 2: Study of whether various analog substances of SASP specifically inhibit T-cells. a. Inhibition of Con A stimulated T cells from spleens with various concentrations of SASP, SP, 5-ASA, Olsa-lazine and CSP. b. Inhibition of LPS stimulated B
cells from spleens with the same substances. The unit on the top x-axes is the concentration range o~ CSP.

Fig. 3: Study of the effect of various SASP analog sub-stancss on two immunologically relevant tumor lines. a. Inhibition of the growth of WeHi cells (monocyte/macrophage line) of SASP, SP, 5-ASA and Olsalazine. b. Inhibition of the growth of X-63 cells (myeloma line) with the same substances.

Fig. 4: Study of the proliferation inhibition of T and B
cells by addition of salicylic acid analog sub-3~ stances derivatised on the carboxylic acid group and the phenol group, respectively. a. Inhibition of Con A stimulated T cells from spleens with various concentrations of acetylsalicylic acid, W093/10791 PC~/DK92/003 2~-?,48s~

salicylamide, salicyl glycine and 5-ASA. b. Inhi-bition of LPS stimulated B cells with the same substances.

Fig. 5: Study of the proliferation inhibition of T and ~ , cells by addition of salicylic acids derivatised in the 5-position. a. Inhibition of Con A stimu-lated T cells from spleens with various concen-trations of 5-ASA, 5-amidosalicylic acid, 5-phenylsalicylic acid, diflunisal, salicylic acid and SASP. b. Inhibition of LPS stimulated B cells with the same substances. c. Inhibition of WeHi cells with the same substances. d. Inhibition of X-63 cells with the same substances.

Fig. 6: Study of the proliferation lnhibition of tumor cells by addition of inhibitors of the oxidative phosphorylation. a. Inhibition of WeHi cells with diclofenac, indomethacin, diflunisal and phenyl-salicylic acid. b. Inhibition of X-63 cells wlth the same substances.

`

VO93~l0791 21 2 4 8 5 2 PCT/DK92/~36~ ;

References:

1. E.M. Farber & L.S.O. Nall, Drugs 28 (4), 324-346 (1984) S ~.
2. A.M. Kissmeyer & L. Binderup, Blochem. Pharmacol. 4 (11), 1601-1606 (1991) 3. W. M~ller & U. Graf, Switzerland. Med. Wochenschr. 111 (12), 408-413 (1981) 4. O. Baadsgaard et al., J. Invest. Dermatol. 95 (5), 32-34 (1990) 5. A.B. Hawthorne & C.J. Hawkey, Drugs 38 (2), 267-288 (1989) 6. S. Silvennoinen-Kassinen, Scand. J. Immunol. 12 (1), ;, 61 65 (~980) A
~0 '.

7. A.M. McLeod & A.W. Thomson, Medicina (Florence) 10 (3), 329-332 (1990) 8. K.F. Tempero et al., Br. J. Clin. Pharmacol. 4, 31-36 (1977) 9. F. Moolenaar et al., Int. J. Pharm. 19 (2), 161-167 (1984) 10. EP Patent Application No. 0 275 526 11. DE Offenlegungsschrift No. 36 05 345 W093/10791 PCT/DK92/003 s c~
S ~

12. EP Patent Application No. 0 270 316 13. Danish Patent Application No. 1622/88 (DAK-Laboratoriet A/S) 14. US Patent Specification No. 4 483 854 (Julius Diamond) 15. H. G~elstrup Kristensen: "Almen Farmaci II", 2nd ed., Dansk Farmaceutforenings Forlag, Copenhagen (1991) 16. S. Mouritsen et al., Scand. J. Immunol. 28, 261 (1988) 17. M. Fujiwara, Japan. J. Pharmacol. 54 (2), 121-131 ( 1990 ) 18. ~ynthetized by Henning Lund, assistant professor at the University of Arhus, according to N.N. Vorozhtsov and A.T. Troshchenko, Zh. Obsuch. Khim. 8. 424-429 ~1938); melting point 215-216 C.

19. P. Mc~ougall et al., Biochem. Pharmacol. 32 (17), 2595-2598 (1983) 20. ~. N~rnberg et al., Clin. Pharmacokin. 20 ~1), 81-89 (1991).

21. USA Patent Specification No. 4 496 553 (S0ren Halskov~

Claims (10)

P a t e n t C l a i m s :

1. Use of diflunisal (5-(2,4-difluorophenyl)-salicylic acid) of the formula for the production of a pharmaceutical composition for topical treatment of inflammatory and/or autoimmune di-seases, such as psoriasis, Crohn's disease anf uveitis, said composition containing 0.01-10% by weight of diflu-nisal in combination with ordinary, pharmaceutically ac-captable vehicles and excipients.

2. Use according to claim 1, c h a r a c t e r i z e d in that compositions are produced for local use on skin surfaces in the form of cremes (including foam), gels, sticks, plasters, ointments, shampoo, liniments, powders or pastes.

3. Use according to claim 1, c h a r a c t e r i z e d in that compositions are produced for use as an eye agent in the form of eye drops or an eye ointment.

4. Use ac ording to claim 1, c h a r a c t e r i z e d in that compositions are produced for local treatment of the intestinal canal in the form of controlled release formulations, suppositories or enemas.

5. Use according to claim 1, c h a r a c t e r i z e d in that compositions are produced in the form of cremes, gels, liniments, powders, pastes or vagitories for local treatment of inflammation in the vagina.

6. Use according to claim 1, c h a r a c t e r i z e d in that compositions are produced for local use in the oral cavity in the form of lozenges, mouth rinsing water or mouth ointments.

7. Use according to claim 1, c h a r a c t e r i z e d in a composition is produced in the form of an inhalation spray for local treatment of the respiratory passages.

8. A process for local treatment of psoriasis, c h a -r a c t e r i z e d by applying to the patient a topical composition containing 0.01-10% by weight of diflunisal in combination with ordinary, pharmaceutically acceptable ve-hicles and excipients, on the affected part.

9. A process for local treatment of Crohn's disease, c h a r a c t e r i z e d by administering to the patient a composition for local treatment of the intestinal canal in the form of controlled release formulations, supposi-tories or enemas, said composition containing 0.01-10% by weight of diflunisal in combination with ordinary, pharma-ceutically acceptable vehicles and excipients.

10. A process for local treatment of uveitis, c h a -r a c t e r i z e d by administering to the patient a topical agent in the form of eye drops or an eye ointment containing 0.01-10% by weight of diflunisal in combination with ordinary, pharmaceutically acceptable vehicles and excipients.