CA1149739A - Pharmaceutical and dietary composition - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Compositions and use of .gamma.-linolenic acid and related materials with ascorbic acid, or ethyl alcohol, or an opiate antagonist such as naloxone, nalorphine or levallorphan, for treating alcohlism or moderating the effects of taking alcohol.

Description

~97~g FIELD OF THE INVE~rION
__ ___ This invention relates to compositions for the treatment of certain diseases and disorders primarily, but not exclusively, in the field of human medicine.
GENERAL BACKGROUND
Considerable interest has been shown in recent years in the use of prostaglandin (PG) precursors in medicine.
For various reasons it is not practical to administer naturally-occurring prostaglandins such as PGE 1 and PGE 2 to patients. Consequently, considerable attention has focussed on the use of prostaglandin precursors including linoleic acid, ~-linolenic acid (GLA) and dihomo-y-linolenic acid (DGLA).
Conversion of these materials in the body is believed to be as shown in the following diagram:

cis- Linoteic Acid (9, 12 - oetadecadi~noic acid ) GLA
(6, q, 12 - octadecatrienoic acid ) ~GLA DGLA lseries ~ster ~ ~ (5~8~ eicosatri~n~icacid) --end~perQxtdes ~eserves (small~
1 series ~G.'s Large AA
AAester ~- (Arachidonic acid,i,e.
~eserv 5,B,11, 14 -eicosatetraenoic acid ) series ~ endoperoxides T~A2 PGf2a PGI2 PGE~etc, (Thromboxane A2) 2 Series PG s The broad outline of this pathway is well known, and lt brings out clearly that a ma~or function of essential fatty acids (EFAs) is to act as precursors for prostaglandins, 1 series PGs being formed from dihomo-y-linolenic acid (DGLA) and 2 series PGs S from arachidonic acid (AA)~ DGLA and AA are present in food in only small quantities, and the major EFA in food is linoleic acid which is first converted to y-linolenic acid (GLA) and then to DGLA and AA. The conversion of linoleic acid to GLA is blocked by a high fat and high carbohydrate diet, by ageing and for example by diabetes. Stores of AA in the body in the form of lipid esters are very large indeed. In contrast only small amounts of DGLA
ester are present.
INVENTION AND BACKGROUND
DGLA is the key substance. GLA is almost completely and very lS rapidly converted in the body to DGLA and so for practical purposes the oral administration of DGLA and GLA amounts to the same thing.
DGLA can be converted to a storage form, changed to arachidonic acid and thence to PGs of the 2 series, or converted to PGs of the 1 series.
There is increasing evidence that PGs of the 1 series play a vital role in a number of key areas. First, PGE 1 activates T lymphocytes. Defective T lymphocytes are believed to be involved in causing a wide range of allergic and inflammatory disorders and in making individuals susceptible to cancer and infections of all types. Second, PGE 1 is important in preventing over-production 973'~

of collagen and fibrous tissue, a factor which plays a raajor role in arthritis and the so-called collagen diseases. Third, PGE 1 levels are extremely low in patients with schizophrenia and are moderately low in patients with depression. Fourth, PGE 1 appears to be important in controlling cholesterol levels and necessary for the normal actions of insulin. Fifth, PGE 1 dilates blood vessels and may ~e expected to be helpful in any situation in which vessel spasm occurs. Sixth, PGE 1 appears to inhibit the production of

2-series PG's, levels of which are raised in a wide variety of inflammatory disorders. Seventh, PGE 1 increases production of cyclic AMP which has anti-inflammatory effects.
There are therefore very strong reasons, and this broadly is an aim of the present invention, for influencing the 1-series/2-series PG balance in the body in favour of l-series PG ' s and specifically for selectively enhancing formation of PGs of the l-series and particularly P OE 1. The diseases and disorders below are among those in which such action is indicated:
1. Situations in which defective T lymphocyte function has been described such as allergic and inflammatory disorders, multiple sclerosis, schi~ophrenia and cancer.
2. Situations in which regulation of collagen formation and breakdown is defective including rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and the various "collagen"
diseases.
25 3. Mental illnesses in which low PGE 1 levels have been reported, 73~3 ~ 6 -including depression and schi~ophrenia. In depression, platelet PGE 1 production is m~derately reduced whereas ln schizophrenia it is severely reduced.
4. Disorders of lipid and carbohydrate metabolism in particular diabetes mellitus and situations in which blood cholesterol levels are elevated.
5. ~isorders in which there is a tendency of blood vessels to go into spasm such as angina pectoris, myocardial infarction and Reynaud's syndrome.
lO 6. Disorders of inflammation in which there may be excessive production of 2-series PGs from arachidonic acid, often coupled with low levels of cyclic AMP.
Selective enhancement of l-series PG production has been explored in human platelets. The method is given in detail later herein but briefly human platelets are incubated with radioactive DGLA or arachidonic acid. The PGs produced during incubation are extracted by conventional means and separated by thin layer chromatography, and the amount of radioactlvity appearing in each PG or related substance is counted. POE 1, PGF 1~ and thromboxane Bl from DGLA, and PGE 2, PGF 2a and thromboxane B2 from AA are estimated. The results, as given herein, demonstrate the inventor's belief that the effects of various agents on AA and DGLA conversion can be quite different and that it is possible to selectively enhance formation of PGE l and other l-series PG compounds.
The effect is believed to be by influencing the conversion of DGLA

9~3~

to the l-series PGs.
The balance between l-series and 2-series PGs is, the inventor believes, significant in terms of overall control of the conversion pathways given earlier. Such control is not understood in detail but without restriction to the theory it appears first that PGE 2 is able to enhance the formation of l-series PG's, and second that PGE 1 is able to block arachidonic acid mobilisation from tissue stores. Thus the conditions for a negative feedback control loop exist; overproduction of PGE 2 from AA will activate PGE 1 synthesis, the PGE 1 will inhibit AA mobilisation, and production of 2-series PG's will drop. Further, TXA 2, an unstable product of the 2-series endoperoxides arising in 2-series PG
production, also appears to enhance l-series PG and in particular PGE 1 production. Thus again the activity of the 2-series PG
synthesis pathway gives rise indirectly to a material that controls that pathway.
The inventor has found that ethyl alcohol, at concentrations from 30 to 300 mg~, causes a dose dependent rise in formation of PGE 1.
The idea that ethyl alcohol may affect PG formation has several tLmes been considered but not with specific reference to 1 or 2 series PGs. The main experimental evidence to date has related to conversion of AA and it has been demonstrated that extremely high levels of ethanol can block the formation of thromboxane B2.
The relative effects of alcohol on AA and DGLA conversion have not been considered. The lnventor has found that starting at the threshold 20-30 mg%, alcohol has an effect on conversion of DGLA to 1 series PGs, greatly enhancing their production. There is no significant effect on conversion of AA to 2 series compounds until concentrations of aoove 300 mg~ are reached when the effect is an inhibition of thromboxane B2 formation i.e. the opposite action to the effect on the 1 series.
This effect of alcohol can account for a number of its effects, particularly in relation to those on mood. It means that in those who consume large amounts of alcohol there is a considerable danger of depletion of DGLA stores and therefore of a failure of adequate PGE 1 production following a period of excess PGE 1 formation.
This is a particular risk in alcoholics whose dietary intake of many foods is likely to be defective. There is therefore a strong case for ensuring that in those whose alcohol consumption is high, EFA intake should be such that body levels of DGLA are maintained.
In summary, alcohol over the range 30 to 300 mg~ causes a marked enhancement of up to 60% in the amount of 14C-DGLA converted to PGE 1. Up to 100 mg~ alcohol has little effect on arachidonate metabolism but at 300 mg~ it tends to inhibit conversion of arachidonate to PGs and particularly thromboxanes. The effects on the 1 and 2 series PGs are therefore opposite. The threshold of the effect is at about 20-30 mg% which is the concentration of alcohol in human plasma at which signs of mild intoxication first appear. 300 mg% is a concentration which produces "blind drunkenness".

g Alcoholic intoxication wlll therefore enhance formatlon of PGE 1 and deplete the limited body stores of DGLA. Post-intoxicatlon depression which is such a major factor in the development of chronic alcoholism may well be related to a fall of P OE 1 formation due to depletion of DGLA stores. The withdrawal syndrome in chronic alcoholics is often schizophrenia-like and may be caused by extremely severe DGL~ depletion. Further, there is evidence that depletion of PGE 1 formation is associated with increased production of fibrous tissue, so that the development of liver cirrhosis in some chronic alcoholics may be related to chronic depletion of DGLA. Incidentally the stimulation of PG~; 1 formatlon by ethanol explains the reported desirable effects of modest consumption of alcohol (insufficient to deplete DGLA) such as prevention of heart attacks and resistance to viral infections.
Thus DGLA and materials giving it in the body (the "oil", see later) are of value in at least three ways.
1. In mild to moderate consumers of alcohol, to help prevent depletion of body stores of DGLA, post-intoxication depression and other short and long term features such as elevated cholesterol levels related to essential fatty acid deficiency.
2. In chronic alcoholics undergoing withdrawal, to replenlsh DGLA stores and maintain PGE 1 formation, thus preventing the worst features of withdrawal.

3. In chronic continuing consumers of alcohol, to partially or completely avoid long term adverse effects such as cirrhosis of the liver.

3'73~3 MATE~IALS AND MET~IODS
The detailed technique with platelets ;s given below by way of example.
(1- C) arachidonic acid and (1- C) dihomo-y-linolenic acid were used, diluted with hexane to specific activities of about 5~Ci/~mol. One day expired (2 days old) human platelets were obtained and used within 48 hou~s of expiration. One unit was centrifuged at lOoo g for 15 minutes and the supernatant dxawn off.
The platelet pellet was resuspended in Tris-NaCl-EDTA buffer, made up of 0.15 M NaC1, 0.15 M Tris HCl at pH 7.4 and 0.077 M NaEDTA
~90:8:2 v/v/v). The platelets were recentrifuged, the supernatent removed and the pellet resuspended in Krebs-Henseleit buffer (without calcium) at pH 7.4. The washed platelet suspension contained about 1-2~ red blood cells. All glassware used in the preparation of the platelets was siliconized.
Four equal sized 1 ml aliquots of the platelet suspension, containing 109 platelets/ml were incubated with e.g. 0.5 ~Ci C-DGLA for five minutes. At the beginning of the incubation the material under test was added to the suspenslons. The reaction was stopped after five minutes by addition of 1/10 volume of 10~ formic acid. The suspension was then extracted three times with ethyl acetate and the fractions pooled and dried under vacuum. The extract was then taken up with 5 ml chlorofcrm/methanol (2/1, v/v).
Recovery of radioactive material in the extract was checked by taking 50 ~1 of the chloroform/methanol and counting by liquid scintillation. Recovery was in the range ~0-95~ in most experlments.
The chloro~orm/methanol extract was then reduced in volume to 1 ml under dry prepurified nitrogen. Thin layer chromatography was carriea out on 500 ~g precoated, prescored silica gel G uniplates (Analtech). Plates were activated by heating to loo& for 1 hour immediately prior to use. The solvent system was chloroform:methanol acetic acid:water (90:8:1:0.8). Reference compounds (PGs El and Fl and thromboxane Bl) were run at the same time and visualised by phosphomolybdic acid spray followed by brief heating. The bands on the plates corresponding to the reference PGE 1, PFGl and TXB 1 were scraped off and eluted with 20 ml acetone. Each elution was then evaporated to dryness and counted by liquid scintillation (Beckman 100 LS counter).
Using the same batch of platelets at the same time exactly similar experiments were carried out with C-AA and PGE 2, PGE2 and TXB2 as reference compounds. Three experiments were performed with DGLA and three with AA.
RELATIONS~IP TO PREVIOUS PROPOSALS
-The above approach may be used in combination with other materials as disclosed in the earlier patent applications of the inventor namely European Nos. 79300079.5 and 79300546.3 (Publication Nos. 0 003 407 and 0 004 770: U.S. Serial Nos. 004 924 and 029 058).
Among these materials are a number believed to act by enhancing mobilisation of DGLA reserves, including zinc, penicillin and B-lactam antibiotics generally, and also penicillamine, g~39 phenformin and levamisole when the other effects of these materials are acceptable.
Further, since there is evidence that thromboxane A2 may indirectly enhance formation of P OE 1, substances such as colchicine, amantadine and melatonin, and also griseofulvin, vinblastine, vincristine and interferon as discussed in the pending patent applications and believed to act through increasing the proauction or effect of thromboxane A2, can also be used in conjunction with the materials of the present invention.
As appears from the earlier patent applications, in searching for ways to regulate PGE 1 formation the inventor has previously concentrated on the conversion of DGLA stores to free DGLA since this is believed to be a key rate-limiting step and since it has also been believed that factors which regulate conversion of free arachldonate to PGs will also regulate conversion of free DGLA
to PGs. The present work has been more on the conversion of DGLA
and of AA to the respective PGs, and as noted above it has been found that the factors regulating the two PG pathways are in some respects quite different. The discoveries on which the present application i9 based however build on and add to the earlier inventions rather than superseding them.
THE PRESENT INVENTION
According to the present invention there is provided a pharmaceutical composition comprising y-linolenic acid and/or dihomo-y-linolenic acid, optionally in association with linoleic or other 73~

fat acids, said acids being present as such or as physiologlcally functional ester or other derivatives, thereof, ln combination with ethyl alcohol.
DOS~ RANGES
The dose range for alcohol in humans is:
outer limits S to 500 ml/day desirable dose 50 to 200 ml/day Dose ranges for materials auxiliary to those of the invention are discussed elsewhere herein. All the materials may be given in doses of for example one half, one third or one quarter of the above amounts.
The amounts are related to those quoted earlier for platelet and other experiments, though of course a precise relation cannot be given in view of variation in inactivation and excretion rates and volume of distribution.
PACKS
If it is not desired to have compositions comprising the active materials together, as listed above, packs may be prepared comprising the materials presented for separate or part joint and part separate administration in the appropriate relative amounts, and such packs are within the purview of the invention.
DIETARY COMPOSITIONS
The invention is chiefly described in terms of pharmaceutical compositions, but it will be understood that the y-linolenic and other acids, being in the nature of dietary supplements, could be incorporated in a dietary margarine or other foodstuffs; such food~tuffs, possibly containing other active materlals and generally referred to in this description as dietary or pharmaceutical compositio~s, are within the purview of the invention and thus of the term pharmaceutical compositions, packs or the like used in the claims.
_TERINARY APPLICATIONS
It will be understood that where a disorder of a kind calling for treatment in animals arises, the invention while described primarily in terms of human medicine and treatment is equally applicable in the veterinary field.
AMOUNTS OF ACTIVE MATERIALS tADJUNCTS TO PRESENT INVENTION) Amounts of ma~erials are:
Zinc 2.5 to 800 mg/day, preferably 10-80 mg calculated as zinc B-lactam antibiotics 0.5 to 10 g/day Penicillamine 50 mg to 10 g/day Phenformin 10 mg to 5 g/day Levamisole 10 mg to 2 g/day Colchicine 0.3 to 15 m~/day, preferably 0.6 to 2.4 mg Melatonin 10 mg to 5 g/day Amantadine 100 mg to 1000 mg/day Griseofulvin 0.5 to 5 g/day Vinblastine 0.5 to 5 mg/kg/week (average weight 70 kg) Vincristine 0.1 to 1.0 mg/kg/week taverage weight 70 kg) Interferon tby 1 x lO to 1 x 10 units /day in;ection) ~9739 Detailed discussion of suitable amount~ and forms of use ls contained ln the published patent applications referred to earller, to which reference may be made. In particular tha B-la~tam antibiotics are convenlently any of the known penicillin and cephalosporin antibiotics (including semi-synthetic antibiotics) such as, for example, penicillin G, penicillin N, penicillin V, cephalexin, cephalothin, ampicillin, amoxycillin, cloxacillin and cephaloglycin. Any of these may be used in the form of their physiologically functional non-toxic derivatives, for example alkali metal salts e.g. sodium and potassium salts, and salts with organic bases, and reference to an antibiotic herein includesreference to such derivatives.
AMOUNTS OF y-LINOLENIC AND OTHER ACIDS SPECIFICALLY
A preferred daily dosage for all purposes for an adult (weight ca 75 kg) is from 0.05 to 0.1 up to 1, 2, 5 or even 10 g as required of y-linolenic acid or equivalent weight calculated as y-linolenic acid or a physiologically functional derivative thereof. Amounts in particular may be 0.1 to 1.0 g daily. Corresponding doses of the Oenothera oil containing 8 to 10~ of y-linolenic acid, are easily calculated. In place of, or in addition to, y-linolenic acid, one may use dihomo-y-linolenic acid or a physlologically functional derivative thereof, in amounts equivalent in molar terms to y-linolenic acid and calculated as such. This dosage can for example be taken as a single dose or divided into 2, 3 or 4 sub-divisions thereof as convenient.

... , _, .. . . . . . ..

73~

F~RMS AND SOURCE OF y-LINOLENIC AND OTHER ACIDS
Convenient physioloyically functional derivatives of y-linolenic acid and dihomo-~-linolenic acid for use according to the invention for all the purposes described include the Cl-C4 alkyl te.g~ methyl) esters and the glycerides of the acids.
If desired, pharmaceutical compositions may be produced for use in the invention by associating natural or synthetic y-linolenic acid (or a physiologically functional derivative thereof) and/or dihomo-y-linolenic acid tor a physiologically functional derivative thereof), as such, with an acceptable pharmaceutical vehicle.
It is at present convenient to incorporate the y-linolenic acid into compositions in the form of an available oil having a high y-linolenic acid content, hence references to "oil" herein.
At the present time known natural sources of oils having a high y-linolenic acid content are few tthere are no known natural sources of significant amounts of dihomo-y-linolenic acid).
One source of oils currently available is the seed of Evening Primrose species such as Oenothera biennis L. and Oenothera lamarckiana, the oil extract therefrom containing ~-linolenic acid (about 8~) and linoleic acid (about 72%) in the form of their glycerides together with other glycerides (percentages based on total fatty acids).
Other sources of y-linolenic acid are Borage species such as Bora~o officinalis which, though current yield per acre is low, provide a richer source of y-linolenic acid than Oenothera oil. Recent studies on fungi which can be cultivated by fermentation promise a fungal oil source .
The seed oil extracts referred to above can be used as such or can for exa~ple if desired be iractionated to yield an oily composition containing the triglycerides of ~-linolenic and linoleic as the main fatty acid co~ponents, the y-linolenic acid content being if desired a major proportion. Seed oil extracts appear to have a stabilising effect upon any dihomo-~-linolenic acid or physiologically functional derivative thereof.
PHARMACEUTICAL PRESENTATION
The compositions according to the invention are conveniently in a form suitable for oral, rectal, parenteral or topical administration in a suitable pharmaceutical vehicle, as discussed in detail for example in Willia~s U.K. Patent Specification No. 1 082 624, to which reference may be made, and in any case very well-known generally for any particular kind of preparation.
Thus for example tablets, capsules, ingestible liquid or powder preparations, creams and lotions for topical application, or suppositories, can be prepared as re~uired. Injectable solutions of hydrolysed Oenothera oil may be prepared using albumin to solubilise the free acid.
Advantageously a preservative is incorporated into the preparations. a-Tocopherol in a concentration of about 0.1~ by weight has been found suitable for the purpose.
It will be understood that the absolute quantity of active ingredients present in any dosage unit should not exceed that appropriate to the rate and manner of administration to be employed but on the other hand should also desirably be adequate to allow the desired rate of administration to be achieved by a small number of doses. The rate of administration will moreover depend on the precise pharmacological action desired.
The following Examples serve to illustrate pharmaceutical compositions useful in treatment according to the invention:
EXAMPLES
Pharmaceutical compositions contain a unit dose of an oil extract from the seeds of Oenothera biennis L., and of one of the active materials of the present invention, optionally with methyl dihomo-y-linolenate and/or zinc oleate and/or penicillin V and /or any of the other active materials referred to herein directly or by cross reference to other patent applications of the inventor.
They may be presented by encapsulation of the natural oil in soft gelatin capsules by known methods.
The oil is extracted from the seeds by one of the conventional methods of extractlon such as cold pressure, screw pressure after partially cooking the seed, or solvent extraction~
~ractionation of a typical sample of this oil shows a yield of 97.0% in the form of methyl esters, with the relative proportions:

~9739 Palmltate 6.15 Stearate 1.6 Oleate 10.15 Linoleate 72.6 y-Linolenate 8.9 As preservative, -tocopherol is added to the oil in a concentration of 0.1%.
Gelatin capsules containing oil extracts prepared as described above, each having the following contents of active ingredients (0.5 g oil extract = ca 0.045 g y-linolenic acid), are prepared in conventional fashion.
The following are specific examples of capsules that may be given, two capsules three times a day, in treatment of the condition~ listed earlier.

Oil extract 0.5 g Zinc sulphate 10 mg Two capsules may be administered thrice daily with alcohol in any convenient form to give 50 to 200 ml/day in the treatment of alcoholism, giving a daily dose of y-linolenic acid of ca 0.27 g.
Capsules without zinc are an alternative. Zinc oleate 20 mg is an alternative to zinc sulphate.

. , ~ . . . . .

EXAMPLES 2(a) - 2(e) Similarly to Example 1 the following may be adminlstered:
(a) Oil extract 0.5 g Methyl aihomo-y-linolenate 10 mg Zinc sulphate 20 mg (b) Oil extract 0.5 g Penicillin V 0.25 g ~Levamisole 25 mg, penicillamine 100 mg or phenformin 25 mg are alternatives to penicillin here and in (c) and (d) below).
(c) Oil extract 0.5 g Penicillin V 0.25 g Zinc sulphate 10 mg (d) Oil extract 0.5 g Methyl dihomo-y-linolenate 10 mg Penicillin V 0.25 g Zinc sulphate 10 mg (e) 011 extract 0.5 g Nethyl dihomo-y-linolenate 10 mg It will be understood throughout that while a full theoretical discussion of what is believed to be the reason for the effectiveness of the compositions proposed is given to aid understanding, the invention is in no way to be limited by this discussion.
VALUE OF OIL + ETHANOL
The following results were obtained by the platelet techniques given in detail earlier:

~973~

Alcohol (ethanol) concentration mg/100 ml 0 33 100 300 DGLA conversion to P OE 1, PGFl and TXBl ~total100 160 200 230 as ~ of control) AA conversion to PGE2, PGF2 and TXB2 (total as % 100 110 90 100 of control) The variation in the figures for A~ conversion is not significant, but that for DGLA conversion shows a dose-related and substantial rise at these physiological ethanol concentrations, with the significance for PG metabolism and treatment of the effects of taking alcohol already discussed at length herein.

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition comprising .gamma.-linolenic acid and/or dihomo-.gamma.-linolenic acid, optionally in association with linoleic or other fat acids, said acids being present as such or as physiologically functional ester or other derivatives, thereof, in combination with ethyl alcohol.

2. A composition as in claim 1 in dosage unit or other form comprising 50 mg to 10 g preferably 0.1 to 1 g of said .gamma.-linolenic or dihomo-.gamma.-linolenic acid or derivative (calculated as .gamma.-linolenic acid) together with 5 to 500 ml, preferably 50 to 200 ml of ethyl alcohol or one half one third or one quarter of said amounts.

3. A composition according to claim 1 or 2, comprising further a material influencing the 1-series/2-series PG balance in the body in favour of 1-series PGs by enhancing conversion of DGLA
ester stores to free DGLA.

4. A composition according to claim 1 or 2,comprising further zinc, penicillamine, phenformin, levamisole, or a penicillin, cephalosporin or other .beta.-lactam antibiotic.

5. A composition according to claim 4 in dosage unit form comprising:

Zinc 2.5 to 800 mg/day, preferably 10-80 mg calculated as zinc, or .beta.-lactam antibiotics 0.5 to 10 g/day or Penicillamine 50 mg to 10 g/day, or Phenformin 10 mg to 5 g/day, or Levamisole 10 mg to 2 g/day

6. A composition according to claim 1 or 2, comprising further a material influencing the 1-series/2-series PG balance in the body in favour of 1-series PGs by enhancing the physiological production or effect of TXA2,

7. A composition according to claim 1 or 2, comprising further colchicine, amantadine, melatonin, griseofulvin, vinblastine, vincristine or interferon.

8. A composition according to claim 7 in dosage unit form comprising:
Colchicine 0.3 to 15 mg/day, preferably 0.6 to 2.4 mg, or Melatonin 10 mg to 5 g/day, or Amantadine 100 mg to 1000 mg/day, or Griseofulvin 0.5 to 5 g/day, or Vinblastine 0.5 to 5 mg/kg/week, or Vincristine 0.1 to 1.0 mg/kg/week, or Interferon 1 x 10 to 1 x 108 units /day