British Journal of Dermatology (1975) 92, 569.

Pharmacology and Treatment

Evaluation of coal tar fractions for use inpsoriasiform diseases using the mouse tail test

(I) HIGH AND LOW TEMPERATURE TARS AND THEIR

CONSTITUENTS

ROSANNE WRENCH AND ADAM Z.BRITTEN

Department of Pharmacy, The University of Aston in Birmingham B4 7ET

Accepted for publication 20 August 1974

SUMMARY

'High' and 'low' temperature tars were evaluated on parakeratotic mouse tail skin, which was usedas a model for the psoriatic keratinization process. The skin was examined histologically for signs ofinduced granular layers in previously scaly areas; epidermal thicknesses were also measured. It appearsthat the acidic (phenolic) fractions of coal tars induce granular layers and cause epidermal thickening,whereas neutral constituents alone only cause thickening. It is suggested that tar acids should befurther investigated for anti-psoriatic activity.

Whilst investigations into the aetiology of psoriasiform diseases continue, there is a great need forsafe, effective and cosmetically acceptable long-term treatments for the chronic stages of the disorders.Corticosteroids, which initially cause dramatic improvements, can produce telangiectasia and collagenatrophy when applied topically (Shuster, 1969); and prolonged therapy leading to systemic absorption,as well as parenteral administration itself, causes systemic side-effects by interference with endoge-nous steroids (Scroggins & Kligman, 1965; Carr & Tarnowski, 1966). Long-term treatments knownto be safe and effective are coal tar and dithranol (Pegum, 1972). However, dithranol causes stainingof the treated area and its use necessitates careful supervision. Coal tar preparations have been usedfor centuries in the treatment of many skin diseases (Kerr & Plein, 1953). Preparations containingcrude coal tar are messy and smelly, but these ointments remain therapeutically superior (Champion,1966) despite many attempts to isolate a 'therapeutically active fraction', and to improve cosmeticacceptability (Jaffrey, 1928; Obermayer & Becker, 1935; Downing & Bauer, 1948). However, many ofthese trials involved the screening of fractions in often poorly defined groups of patients. Hospitalizedpatients are well known to imdergo spontaneous remissions (Samitz, 1958). Also the reports of theseinvestigations are sadly lacking in histological evaluations of the effects of the tar fractions. Much of thevariability in response of the skin to the tar preparations can be attributed to the use of a wide rangeof tar fractions derived from tars of similarly variable sources. The content of the tar, and henceits therapeutic nature, depends on the mode of production; the two most important parameters beingthe temperature of production and the type of coal used.

'Low' temperature tar is obtained at about 500°C and 'high' temperature tar is formed at tempera-tures in excess of iooo°C. The former has a greater number of components in lower proportions than

569

570 Rosanne Wrench and Adam Z.Britten

the latter. It is unlikely that all of the hundreds of components present in coal tar are beneficial ornecessary in the treatment of psoriasis. The isolation of a single component or group of compoundspossessing the therapeutic activity is clearly desirable, ensuring the preparation of formulations ofreproducible activity. It could lead to cosmetically clean products and to materials of enhanced activityas a result of synthetic chemical analogues based on the parent active structures.

It was decided to reinvestigate the therapeutic nature of coal tar using well standardized fractions,and employing histological evaluations of their effects. This required a model resembling humanpsoriatic skin. An animal model which would allow standardization by species, strain, age, sex andweight would be preferable to the use of patients for the initial large-scale screen. One such modelwas suggested by the work of Jarrett & Spearman (1964) who used mouse tail epidermis as a modelfor the psoriatic keratinization process.

Jarrett and Spearman pointed out that mouse tall-scale epidermis, unlike the rest of mouse bodyskin, is histologically and biochemically similar to psoriatic skin. The skin looks scaly and histologicalexamination reveals that this horny layer is produced without the formation of a granular layer. Groupsof hairs occur on the interscale regions (Spearman, 1964). It is only in areas of hair production that agranular layer appears in the mouse tail; and only here is the scaly keratin absent, and fiaky 'basket-weave keratin' to be seen. The presence of a granular layer seems a prerequisite for ortho-keratiniza-tion (Jarrett & Spearman, 1964). This layer is absent in psoriatic and mouse tail skin, and both producea more solid and parakeratotic horny layer. It has been shown that compounds, such as vitamin A,which induce a granular layer in psoriasis, can also induce this layer in mouse tail scales (Lawrence &Bern, 1958; Jarrett & Spearman, 1964). The mouse tail epidermis thus presents a very useful experi-mental model for screening potential anti-psoriatic tar constituents for a specific effect of granularlayer induction.

MATERIALS AND METHODS

Male (Tuck's Original strain) mice of a specified weight range were treated daily with each tar oint-ment. Ointments were applied with cotton wool all around the tail, about i cm from the proximalend of the tail to about 2^ cm along its length. At the end of each run, animals were killed by stunningand dislocation of the cervical vertebrae. The tail skin was then cut along the ventral surface, strippedoff and flattened for 10 minutes in 70% alcohol. The histological processing described by Jarrett &Spearman (1964) was then followed. Wax sections of 7 ̂ were cut and stained with haematoxylin andeosin. Each scale section was examined for the presence of a granular layer and epidermal thicknesswas measured with an eyepiece micrometer to include the basal layer to granular layer region. Ineach often scales per mouse, five measurements were made so that fifty measurements constituted thenjean epidermal thickness for each mouse. These figures produced a group mean. Groups were com-pared by the students' r-test.

Tars and tar fractions usedThe National Coal Board supplied a high-temperature tar ('Avenue' tar) and also the constituent oilsand pitch separated by heating the tar to 36o°C. 'Coalite' tar, a low temperature tar, was similarlyprovided along with its oil and pitch fractions. Both pitches were discarded, because they are brittledense masses and are difficult to formulate. Pitch is also well known to contain a high proportion ofcarcinogens (Kennaway, 1924).

Tar acids. Acids were obtained by partitioning the tar and oil between 10% NaOH and a benzene/ether mixture. The aqueous alkaline phase was separated, acidified with concentrated HCl and thenextracted with an ether/benzene mixture. The ethereal layer was dried (anhydrous MgS04) and thesolvent was removed on a steam bath under reduced pressure.

Evaluation of tars for psoriasis $ji

Tar neutrals, neutrals + bases. The organic fraction left from the first separation above was shakenwith 10% HCl, the protonated basic compounds being run off in the aqueous layer, leaving the neutralorganic layer (i). The aqueous bases were made alkaline with concentrated NaOH. Bases were extrac-ted with ether, dried and the solvent removed under reduced pressure. The yield of bases was verylow (< i%) . For formulation these were combined with the neutral compounds obtained by themethod outlined below.

Tar Neutrals. The remaining neutral compounds (i) were dissolved in ether and benzene, washed,dried and recovered from the solvent under reduced pressure.

FormulationTars and fractions were incorporated into an ointment containing 5% wool fat: 95% yellow softparaffin. In between treatments ointments were kept at 4°C to minimize deterioration.

ExperimentsAvenue and Coalite tars and oils were applied at 10% for 14 and 21 days. These were then appliedalong with their constituent acid, basic and neutral constituents at 40% for a period of 28 days. Controlswere normal untreated mice and mice treated with the vehicle only.

RESULTS

Avenue/Coalite tars and oilsAvenue and Coalite tars and oils at 10% did not induce a granular layer in the tail scale regions. In allcases there was highly significant epidermal thickening which was similar in groups treated for 14and 21 days (Table i). Increased basophilia and cell hypertrophy were observed throughout the epi-dermis.

A ddjbase+neutral fractionsAfter 4 days of treatment, the mice treated with 40% Avenue oil acids became hyperactive and tendedto scatter when approached for a treatment, and this group was killed after 9 days as the tar was ob-viously irritant. The tails were darkly stained in this group after only one treatment; all mice treatedwith Avenue oils had very slightly stained tails.

Similar to the effect of Avenue oil acids, after two applications Coalite oil acid treated mice becamehyperactive; subsequent treatment led to necrosis of the tail below the treated area and so these ani-mals were killed after only eleven treatments. In the case of the mice treated with Coalite oils, towardsthe end of the treatment period of 28 days, their tails seemed to be showing signs of early necroticchanges. Skin staining by Coalite oil acids was seen after one treatment. After two treatments. Coaliteoil treated mice showed signs of staining, which was not so advanced as in the acid treated group. Attermination, these mouse tails were fairly patchily stained in all degrees, whereas staining by Coaliteoil acids was uniform all over the treated area.

HistologyAvenue oil acids induced granular layers of varying thicknesses in only two out of nine mice. In theremainder, the skin was too eroded and in some cases, was too badly peeling to determine any suchactivity. Sebaceous glands were damaged. There was increased basophilia of all the epidermal cells.

Similarly Coalite oil acids produced a great deal of epidermal damage, which was so great thatepidermal measurements were not possible and which prevented adequate determination of anygranular layer induction.

572 Rosanne Wrench and Adam Z.Britten

TABLE I. Effect of coal tar and oil on mouse epidermis

After 14 days

Control10% Avenue tar10% Avenue oils10% Coalite tar10% Coalite oilsVehicle II

After 21 daysControl10% Avenue tar10% Avenue oils10% Coalite tar10% Coalite oilsVehicle II

Initial no. mice/group, 10 j weight range

Epidermalthickness

(̂ im)

20-638330-838-731-2

mm

aef34-831-836-728-6

mm

s.e.mean

1-081-910-582-292-980-61

I-IO0-622-101-541-401-14

n

88

7668

1 0

999

1 0

1 0

28-32g

t

8-067937-803751-12

0-0411-704-518-384330-24

P

o-ooio-ooiO-OOI

0-005n.s.

n.s.O-OOI

O-OOI

0-001

O-OOI

n.s.

There was no significant difference between the same ointments at 14 and 21days; Avenue tar (14) and Avenue tar (21) t = 1-86 (not significant),s.e., Standard Error.

TABLE 2. Effect of coal tar and oil acid and neutral fractions on mouse epidermis

Control40% Avenue tar40% Avenue oils (A.O.)40% Avenue oil acids40% A.O. Bases + Neutrals40% A.O. Neutrals40% Coalite tar (C.T.)40% C.T. Acids40% C.T. Bases +Neutrals40% C.T. Neutrals40% Coalite oils (CO.)40% C O . Acids40% C O . Bases + Neutrals40% C O . Neutrals

Initial no. mice/group, 10;

Daystreated

282828

928282828282828I I

282 1

Epidermalthickness

(//m)

23-653-233-840-331-837-554464-042-641-640-1

393351

weight range 23-27g

s.e.mean

1-203-092-511-242-661-653-723923-612-391-79

not0-961-40

n

777978

96

71 0

1 0

measurable1 0

6

t

8-953-679542-816-687-08

10-58 •5-025-926-97

10-346-27

P

0001

0-005

O-OOI

0-05O-OOI

O-OOI

O-OOI

O-OOI

O-OOI

O-OOI

O-OOI

O-OOI

Evaluation of tars for psoriasis 573

In mice treated with Coalite tar acids, granular layers of one to three layers thickness were in-duced. In parts of the tail, the skin was eroded and peeling. Granular layers were also induced byCoalite tar. Coalite oils and in parts in a few Avenue oil treated mice. However, in the latter case,most mice suffered erosion of the skin.

Mice treated with neutral+bases and neutrals from Avenue oils. Coalite tar and Coalite oils failedto develop a granular layer. At 40%, all tars, oils and fractions caused highly significant epidermalthickening (Table 2).

DISCUSSION

There was no sign of granular layer induction until the tars and oils were applied at 40%. Althoughat these high concentrations there was associated extensive skin damage, granular layer induction wasdefinitely caused by the acid (phenolic) fractions of Avenue oil and of Coalite tar and oil. This tendencywas perhaps greater in the oil-acid fractions. Neutral and basic constituents and neutral compoundson their own seemed only to cause an increase in epidermal thickness with cell hypertrophy, and pos-sibly an associated increased basophilia.

Keratinization can be altered by rubbing, which stimulates mitosis (Spearman & Garretts, 1966).However, experiments involving rubbing the tail skin daily with the cotton wool applicator showedthat the rubbing when applying the preparation for about 30 s each day was not sufficient to causethe epidermal thickening (Wrench, 1973), which thus was attributed to the effects of the tars.

The epidermal thickening produced by the tars was not surprising, since tars are well known tocause epidermal thickening in animal and in human skin. In fact Gross et al. (1954) suggested that theseproperties could help protect against contact dermatitis by thickening the horny layer. However sincethe epidermis is already thickened in psoriatic conditions, this undesirable side-effect of the tars mustbe eliminated. The results of these trials with tars on the mouse tail skin indicated that the epidermalthickening effects of the tars could be reduced by omitting the neutral substances; the question of thebases cannot be settled without independent tests of the bases alone. Previous experiments with otheranti-psoriatic preparations, namely a commercial 'tar' preparation 'Psorox' (Fisons PharmaceuticalsLimited), and with dithranol also produced highly significant epidermal thickening and no scalegranular layers (Wrench & Britten, 1974). About 85% of the 'synthetic tar' ointment is composed ofneutral substances, the remainder being acidic and basic compounds. This again shows the tendencyof a predominantly neutral mixture to thicken mouse tail scale epidermis along with the failure toinduce scale granular layers. Dithranol (i, 8,9-trihydroxyanthracene) also failed to induce scale granularlayers but caused very significant epidermal thickening. In their experiments with vitamin A, Jarrett& Spearman (1964) found that the epidermal thickening, produced along with granular layer induction,can be reduced with concomitant treatment with triamcinolone, probably through its anti-mitoticeflFect. Their combination of vitamin A and a steroid has formed the basis of a rational approach tochronic psoriatic therapy.

Skin staining by tar preparations has long been quoted as a drawback in prescribing coal tar pre-parations for psoriasis. Our experiments show that skin staining seems to be produced predominantlyby the phenolic preparations, i.e. the only tar fraction to show therapeutic promise. This is of interestsince attempts to 'clean-up' coal tar preparations in the past have led to a decrease in efficacy. Hellier& Whitefield (1967) have already suggested that the reduced potency may be due to the loss of phenolsduring purifications of tar involving distillation. The phenolic anti-psoriatic drug, dithranol (i, 8, 9-trihydroxyanthracene) stains the skin. Loss of the phenolic character in the triacetoxyanthraceneanalogue is reported to reduce staining, but there is a great loss in efficacy (Hellier & Whitefield,1967).

574 Rosanne Wrench and Adam Z.Britten

Phenols have previously been reported as having a beneficial effect in psoriasiform diseases (Sulz-herger & Obadia, 1956). Thorne (1963) found that a preparation containing tar acids and bases inequal proportions produced greater improvement in psoriatics than did crude coal tar.

It is suggested that the tar phenols should he further investigated to locate the specifically anti-psoriatic components.

ACKNOWLEDGMENTS

We would like to thank Mr W.S.Wise* and Dr D.F.Williams of the National Coal Board for theiradvice and provision of samples. We would also like to thank the Coalite and Chemical Products Ltdfor the provision of samples of low temperature tar.

This work forms part of a thesis for Ph.D., Aston University (R.Wrench), under an S.R.C. Post-graduate grant and with additional financial aid from the National Coal Board.

* Formerly with the National Coal Board but now with the Agricultural Research Council.

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