CA1289883C - Topical therapeutic composition containing oxidation inhibitor system - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A substantially non-toxic, stable, topical therapeutic composition is provided which comprises:
(a) a therapeutically active component which is susceptible to oxidative degradation:
(b) an oxidative degradation-inhibitory amount of a redox system containing (1) a water soluble polymer containing a plurality of reducing moieties covalently bound thereto and (2) a water soluble polymer containing a plurality of oxidizing moieties-covalently bound thereto;
and (c) an aqueous vehicle base compatible with the therapeutically active component.

Description

~'~89883 TOPICAL T~lERAPEllTIC COMPOSITION CONTAINING
OXIDATIO~l INIll~ITO~. SYSTEM _ _ Backqround of the Invention This invention relates to compositions containing a therapeutic component which is susceptible to oxidative degradation. More particularly, this invention relates to such compositions especially formulated for topical application in which the oxidation-prone therapeutic 10 ingredient is a proteinaceous biological response modifier such as an interferon or other lymphokine.
Since their discovery in 1957, the interferons, a complex family of several species and multiple subspecies of hormone-like cellular proteins, have been widely 15 investigated for their potent antiviral, antiproliferative and immunomodulating properties.
The major interferon species are now designated alpha, beta and gamma according to their origin and mode of ~induction. Leukocytes are the primary producers of the 20 alpha species, fibroblasts mainly that of the beta species and T lymphocytes are the principal source of the gamma species. Collectively, the alpha and beta interferons are classified as Type I interferons, Type II being immune interferons. Each of these interferon species have been 25 produced on an industrial scale employing recombinant techniques in bacteria and yeast with purification being accomplished by means of classical techniques or by monoclonal immunosorbent antibody. General discussions of these interferons can be found in various texts and 30 monographs, including The Interferon System, by W.E. Stewart, - :

` ~ ~ 1289883 1 II, Sprinyer-Verlag, New York ~1979); Interferon lg81, Vol.

3, edited by Ion Gresser, Academic Press, New York (1981);
and Interferon TheraP~, World Health Organization TechniCal Reports Series 676, World Health Organization, Geneva, 1982.
For over a decade, interferons of all types have been employea in clinical trials. Originally, they were applied against viral pathogens, but subse~uently their use has been extended to include treatment of a variety of malignant diseases. An important factor in the clinical 10 application of interferons and other lymphokines is the method of administration. Systemic administration, by either intravenous of intramuscular injection, has been used most fre~uently with some success. Among the problems inherent in this method of administrations is that the 15 interferon can come into contact with uninfected or nonmalignant cells causing unwanted side effects.
Accordingly, the preferred approach would be to deliver interferon directly to the affected tissues or organs. In some cases, this can be accomplished by direct injection 20 into the diseased site. In other cases, e.g., eye disease and diseases like herpes genitalis, herpes labialis, herpes zoster and adenovirus induced keratitis and condyloma, all of which produce skin lesions, local topical application is the preferred method of administration.
The topical administration of interferon has proved to be a formidable problem for a number of reasons.
First, interferon is a protein with a higher molecular weight than the molecular weights of therapeutic agents previously administered in topical preparations, e.g., 3o procaine, nitroglycerin, etc. In general, large molecular ~' ~

- ` ~2839~a3 1 weight proteins have a much smaller solu~ion diffusion coefficient than low molecular weight substances, a difference which generally becomes exacerbated in semi-solid media. Accordingly, the vehicle used to administer 5 interferon locally must be able to hold the high molecular weight interferon in suspension during packaging, shipping and application, and yet also be able to release the interferon fro~ the vehicle in a reasonable length of time once it has been applied to the diseased site. Second, the 10 vehicle must not adversely affect the activity of the interferon by direct chemical action, precipitation or immobilization, any of which would preclude interaction of the interferon with the diseased site.
Third, and in many ways the most difficult 15 objective to achieve, the vehicle should allow the interferon preparation a sufficiently long shelf-life at room and body temperatures to allow for convenient shipping, handling and administration by the patient. In ge~eral terms, if a therapeutic agent is to be administered 20 topically, the asent and its vehicle should satisfy the following shelf-life conditions: ~1) the agent should retain a significant fraction of its therapeutic effect when held at room temperature (e.g., about 22C) for a period of approximately fourteen days and (2) the agent should also 25 retain a significant fraction of its activity when held at body temperature (37C) for a period of approximately one day. The fourteen day requirement at room temperature allows for shipping~ handling and retailing of the preparation. The one-day requirement at body temperature 30 allows the patient to carry the product on his person and apply it throughout the day when needed.

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Interferons produced by recombinant-DNA techniques 1 or from natural sources when in a crude or partially purified form are notoriously temperature-labile substances.
For example, ~Ioller, et al. reported a. the Third Annual International Congress for Interferon ResearCh that 5 even at ~C, a human leu~ocyte interferon gel lost 80% of its activity in just ~wo weel;s. (~ioller, B.R., Johannesen, P., Ostller, ~., Ulmsteen, U., Ilastrup, J. and ~erg, K., "Initial Evaluation o. Topical Treatment of Dysplasiâ of the Cervical Epithelium with a ~uman Leukocyte Interferon Gel,n, Third Annual International Congress for Interferon P~esearch, 1982.) Plainly, .his is far from the fourteen days at 22C
and one day at 37C stability re~uirements which an interferon preparation should achieve to satisfy co.7~mercial requirements. Presently available data suggest that highly purified interferon, in particular, highly purified gamma-interferon, may also be terDperature-labile. The problem of temperature-related instability of the lympho~ines generally and the interferons in particular is further aggravated by 20 the tendency of t~ese proteinaceous therapeutics to undergo oxidative degradation both during storage and during application. In the case of topical preparations, the tendency toward oxidative degradation is particularly troublesome since it is diCicult, if not impossible, to 25 exclude those conditions, e.g., the presence OL atmospheric ox~7gen, which make such degradation possible once the preparation has been applied to the diseased site.
~ 7hile it might appear to be a simple enough solution to this problem to add an antioxidant to the to~ical preparation to inhibit or forestall oxidative :
.

~3 ~2~ a3 _5_ 1 degradation of the active therapeutic component therein, in practice this is not an acceptable approach with many known antioxidant agents which tend to be somewhat toxic (even if only mildly so) and, being of relatively low molecular 5 weight, are readily absorbed through the skin. (For various types of antioxidants which are commercially available, reference may be made to Rirk-Othmer, The Encyclopedia of Chemical Technoloqy, 3rd Ed., Vol. 2, pp. 132-141.) Even aside from the problem of toxicity, it is generally 10 undesirable to treat with a drug composition containing any bio-active component which is not absolutely essential to achieve the desired therapeutic effect.
Accordingly, the monomeric interferon composition of U.S. Patent No. 4,432,895 containing a reducing and 15 oxidizing agent, i.e., a redox reagent, such as cysteine and cystine, cysteamine and cystamine, and the like, might not be a likely candidate for a preparation to be applied topically. Indeed, U.S. Patent No. 4,432,895 makes provision for the removal of the redox reagent from the 20 interferon composition prior to its therapeutic use by known chromatographic procedures or by dialysis.
In view of this state of the artl it is clear that a vehicle for use in topically administering interferon or other drug susceptible to oxidative degradation has a heavy 25 burden to carry in terms of providing a toxicologically acceptable and therapeutically stable preparation.
SVMMARY OE THE IMVENTION
It is an object of the present invention to overcome the problems in the prior art regarding providing 30 suitable vehicles for the topical administration of ~ ~289883 `-`~

1 therapeutic compositions containing an active ingredient which is susceptible to oxidative degradation.
It is a particular ob~ect of the present invention to provide topical administration vehicles for proteinaceous 5 drugs such as the lymphokines, and especially the interferons, which will hold the drug in suspension during packaging, shipping and application and yet will readily release the drug at the diseased site.
It is still another object of the invention to 10 provide topical administration vehicles which do not significantly diminish the activity of the therapeutically active component therein by direct chemical action, precipitation, immobilization or other mechanisms, both during manufacture of the preparation and thereafter.
It is a further particular object of the invention to provide interferon topical administration vehicles in which the interferon exhibits an extended shelf-life at both room temperature and body temperatures. More particularly, it is an object of the invention to provide interferon 20 topical administration vehicles in which the interferon maintains a substantial fraction of its biological activity for a period of approximately fourteen days or longer at room temperature and approximately one day or longer at body temperature.
In accordance with the present invention, the foregoing and other objects are achieved by providing a substantially non-toxic, stable topical therapeutic composition which comprises:
~a) a therapeutically active component which is 3o susceptible to oxidative degradation;

' .

- - ~
~ . '' ' ~ ' :

, ~3 128988 1 (b) an oxidative degradation-inhibitory amount of a redox syste~ containing (1) a water soluble polymer containing a plurality of reducing moieties~covalently bound thereto and (2) a water soluble polymer containing a plurality of oxidizing moieties covalently bound thereto;
and, ~ (c) an aqueous vehicle base compatible with the therapeutically active component.
Topical therapeutic compositions formulated with a redox system in accordance with the teachings of this invention retain high levels of therapeutic effectiveness despite relatively lengthy periods of storage and/or application to a diseased site. Due to the relatively large 15 size of the water soluble polymer ~lolecule which in effect serves as an "anchor" for the oxidizable and reducible moieties constituting the active principle of the redox system,there will be little tendency of these moieties to be absorbed through the skin where the topical therapeutic 20 composition is applied. Such would not be the case where the moieties were present in the composition in their chemically uncombined form; their relatively small molecular size would permit them to pass through the skin in appreciable quantities thus presenting the risk of toxic or 25 other undesirable side effects.
~ESC~IPT~Q~L~ T~ F~ ~QDIMENTS
The present invention can be practiced with any therapeutic agent which is intended for topical application and which is prone to a loss in potency due to oxidative 3o degradation. Tn general, the stability of topical ~ 1~89~83 -~

1 therapeutic compositions containing proteinaceous swbstances as the active ingredient can be significantly improved by incorporating a redox system as taught herein.
The lymphokines represent a particularly important 5 class of proteinaceous therapeutics which are advantageously ~ormulated into topical preparations as described herein.
Of the lyn~phokines which have been studied to date, the interferons have received the greatest amount of attention from the medical and scientific communities and therefore 10 the present invention will be hereinafter illustrated with specific reference to them. However, it shall be understood that the teachings herein as applied to the interferons apply as well and to the same extent to any other therapeutic substances which are susceptible to oxidative 15 degradati0n The invention is applicable to all types of interferons including natural interferons, interferons produced by recombinant DNA technology, and interferons produced by chemica1 synthesis. Also, the invention can be 20 used with crude, semi-purified and purified interferons.
Examples of the more common types of interferons with which the invention can be used include alpha, beta and gamma interferons of human and animal origin. Each of these three types of interferons can be produced by a variety of 25 techniques. For example, a method ~or producing alpha-interferon is described by Cantell, et al. in Methods in Enzymology, Vol. 78, pages 29-38 (1981). 5imilarly, beta-interferon production schemes are described by Leong and Horoszewicz in Methods in Enzymoloay, Vol. 78, pages 3o 87-101 (1981), and by Van Damme and Billiau in ~ethods in EnzymoloaY, Vol. 78, pages 101-119 (1981). A method - ' ' .

, ~9883 _9_ 1 for producing gamma-interferon is described by Johnson, et al. in Methods in En~vmolo~y, Vol 78, pages 158-162 (1981).
In general, interferon produced by these and other methods is supplied as a liquid.
S The amount of interferon which is topically administered in any particular case, as well as the frequency at which the interferon is administered, will depend upon such factors, well known to the art, including the : interferon used, the disease being treated and the patient's response to interferon treatment.
For alpha and beta interferons, unit strengths have been established by the National Institutes of Health (United States Department of Health and Human Services, Bethesda, Maryland). In terms of these unit strengths, dosage levels for ointment preparations using crude or partially purified natural interferons can range from about 10,000 NIH Units/gram of ointment to 1,000,000 NIH
Units/gram, while for purified natural and recombinant DNA
interferons, the dosage levels can be as high as 50,000,000 NIH Units/gram. Preferred dosage levels for alpha and beta interferons in ointments are generally between about 25,000 and about 500,000 NIH Units/gram of ointment.
The conveniently obtained dosage levels for liquid preparations exhibit a similar variability as a function of A

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l the source of the interferon used. Thus, a formulation incl~ding crude or partially purified natural alpha or beta interferons can have a strength of between approximately 25,000 and 2,000,000 NIH Units per milliliter of preparation, 5 with a particularly preferred dosage level being between approximately lO0,000 and l,000,000 NIH ~nits per milliliter. If desired, even higher levels, e.g., 50,000,000 NIH Units/milliliter, can readily be obtained with purified natural and recombinant DNA interferons.
For gamma-interferon, unit strengths have not yet been established. Gamma-interferon is commercially available from a number of sources, including Interferon Sciences, Inc., (New Brunswick, Mew Jersey), the assianee of the present application, and l~eloy Laboratories 15 (Springfield, Virginia). The strengths of these commercial preparations are given in units established'by in-house standards. In terms of these units, ointment and liquid gamma-interferon preparations generally contain similar concentration levels to those given above for alpha and beta 20 interferons.
In addition to applying one interferon at a time, the present invention is also applicable to the administration of mixtures of interferons, including interferons of different types, interferons from different 25 sources and interferons produced by different methods of manufacture. For example, it is known that alpha and beta interferons, as well as possibly other interferon combinations, e.g., mixing different recombinantly produced alpha-interferons, can have a synergistic effect. The 30 present invention specifically encompasses the topical administration of such synergistic combinations.

~ 121~9883 1 ~5 previously stated, the oxidative degradation-inhibitor component of the topical preparations herein comprises a redox system containing a reducing agent and an oxidizing agent. Both the reducin~ agent and the oxiaizing 5 agent are provided in the form of water soluble polymers which have been chemically modified in hnown and conventional ways to contain the reducing and oxidizinq moieties covalently bound thereto. As used herein, the term "polymer" shall be inclusive of homopol~7mers as ~7ell as lO random, block and graft copolyrners.
E~amples of ~?ater solu~le polymers which can be modified to contain reducing and oxidizing moieties in accordance with this invention include any ol the water soluble cellulose ethers, in particular, carbo~yethyl 15 cellulose and carboxymethyl cellulose; mixed ethers such as carboxyalkylhydroxyalkyl ethers, e.g., carboxymethyl hydroxyethyl cellulose, hydroxyalkyl celluloses such as hydroxyethyl cellulose and hydroxypropyl cellulose; al~yl ~hydroxyalkyl celluloses such as methyl hydroxypropyl 20 cellulose; al~yl celluloses such as methyl cellulose, e.hyl cellulose and propyl cellulose; alkyl carboxyallcyl celluloses such as ethyl carboxymethyl cellulose; cellulose sulfate esters, e.g., those disclosed in U.S. Patent Nos.
3,702,843 and 4,141,746; polyacrylamide homopolymers and 25 copolymers such as those described in U.S. Patent Nos.
3,702~843 and 4,141,746; polyacrylamide homopolymers and copolymers such as those described in U.S. Patent Nos.
2,625,529; 2,740,522; 2,729,557; 2,~31,041; 2,909,508;
3,818,998; and, 4,103,742; guar gum; heteropolysaccharides 3o such as those produced by fermentation of carbohydrates 1 by Xanthomonas cam~est lS~ Xanthomonas begonia, XanthomOnaS
~aseoli, Xanthomonas hederae, Xanthomonas incanae, Xanthomonas carotae and Xanthomonas translucens;
polyalkylene glycols, e.g., those of the formula 5 HO(CH2CH2O)nH wherein n can vary from about 20 to about 225,000 polymethacrylic acid, olefin-maleic acid copolymers; amino-containing polymers such as polyethyleneimine, polyvinyl amine, and the like; polyvinyl alcohol, and the like. The molecular weights of the 10 suitable water soluble polymers and copolymers can vary widely.
Hydroxyethyl cellulose (HEC) is especially advantageous in that it is approved for use in pharmaceutical formulations and also serves as a thickener 15 for aqueous-based media such as the topical therapeutic compositions herein.
The monomeric units (400 to 10,000 or more~
comprising the HEC molecule can be represented by the structure:
Ol!

C H~ I H 2 Cl /2 /CH2 c~ cl 1l2 /o tC112 H~/~ /~ H
~ 0~ ~
~,/H 13 \ O
H OH
CH2 ~ -CH2 He'C - hydroxyethyl cellulose ~ . -~ ~2898~3 1 While all of the hydroxyl groups are available for reaction, there is a greater likelihood of reaction at the hydroxyl groups which terminate the ethoxy ether chains. The terminal hydroxy groups of HEC can be reacted directly with 5 a reducing/oxidizing agent to covalently bind the agent to the polymer. For example, HEC can be reacted with a reducing/oxidizing agent possessing at least one carboxylic acid group under esterification conditions to bind the agent to the polymer through ester linkages. Alternatively, the 10 hydroxyl groups of the HEC can first be modified with a difunctional intermediate, e.g., a dicarboxylic acid such as maleic acid, to provide pendant carboxylic acid groups, the latter then being reacted with a reducing/oxidizing agent possessing a reactive group such as a hydroxy, thiol or 15 amine group. Other procedures for preparing functionalized celluloses which can be sued herein are described in U.S.
Patent No. 4,137,399. These direct and indirect chemical procedures whereby reducing/oxidizing moieties can be covalently bound to HEC apply with obvious modifications to 20 other water soluble polymers including those prevlously cited above.
Reducing agents which can be used herein include amines such as 6-ethoxy-1,2-dihydro-2,2,~-trimethylquino-line, N,N'-diphenyl-p-phenylenediamine, N,N'-di-beta-naphthyl 25 -p-phenylenedia~nine and alkylated diphenylamines such as monooctyl diphenylamine and dioctyl diphenylamine; phenols such as the alkylated hydroquinones and the bisphenols;
mercaptoethanoic mercaptans such as mercaptoethanoic acid, mercaptopropionic acid, dithioglycerine, glutathione, 3o pantetheine, mercaptoethanol, dithiothreitol, thioglycolic a ~28~883 1 acid and thiosorbitol, reduced alpha-amino acids such as cysteine, cysteamine, and the like.
The di ulfides constitute a particularly advantageous class of reducing agents which can be used to 5 modify the foregoing water soluble polymers. In addition to the preferred disulfide, dithiodipropionic acid, which possesses the structure ~HO-C-(C}12)2-S 3 other suitable disulfides for the practice of this invention include those of the structures:
[Ho cH2 I c ( c112 n 3 ~Ho_cH2_~_c_(CH2)n o (CH2 n 3 r 1l L HO-CH2-1-C-CH2-CH-CH2-S~

[ 1~ ~
rR

LHC-CH2-C1~2 C~2 J

wherein n is an integer of from 1 to about 10.

a 1289~83 -~

1 Direct or indirect methods of covalently bonding these additional disulfides to the backbone of the water soluble polymer can be utilized as previously explained. While monomeric functionalized thiol groups can be used, it is 5 necessary to protect the thiol group with a blocking agent employing known procedures (e.g., those described in Milkowski, et al. Or~anic ~vnthesis, 59, 190 (19 );
Pastuszak, et al. J. Am. Chem. ~oc., 46, 1~6~ (1981); Veber, et al. J. Am Chem. Soc., 94, 5456 (1972); Marbach, et al.
10 Helv. Chem. Acta, 57, ~03 (1974~; and Ruegg, et al. ~iochem.
J., 1979, 127 (1979) followed by deprotection to provide the free thiol oxidizing agent.
In the oxidized form, the foregoing can serve as '.he oxidizing moieties of the water soluble polymers herein.
15 For example, in the case of a preferred reducing agent, dithiodipropionic acid, reacted with a preferred water soluble polymer, ~EC, the resulting polymer can take any and all of the following forms (only monomeric units shown):
' 1l o ~ o c-tcH2)2 S-S-(CH2)2 c ~
ClH2 0 C~ CH2 Cl 1/ o ~ H 2 c~ IcH2 ~0 fH2 C1~2 H O

OH CH2\ OH

KEC - intramolecular dithiodipropionic acid es.er , . .

~ lZ89~

Cl~ C1~2 110 OCI~ o -->~ ~ O ~ r Il \ H 110 / \ A
\ ,~ H
~O H o / '12 Cl H 2 O~
C H~2 1 2 CH2 o C~H2 \HI 2 O-C-(CH2)2-S-S (CH2)2 ~CI
CH

CH~ \lH2 Cl 2 O~

~ O ~ 2 ~k~o ~k' o H OH 2 \ OH

Hl::C - intermolecular dithiodipropiorlic acid ester ' , : . '. ' - - - ~
- ~ . -~ ~z~

-17 ~

o-c-~C~ 2-s-s--~c~1l)2 1!
o\
c~ Cj"2 o ~ 2 C"2 ~'/ 'X~

2 _ 2 CH2 o\ 2 0 CH~ Cl~l2 2 o/ H 2 C~'2 C~2 ,0, ~o-c (ca212-s~S-~C~(2~2-c-o O\
/ /ll~22 ' C~"2 o\
C~ CH2 o C " 2 ~=,~
20 u oll CH20~ 0"
C"2--C"2 !II:C ~ ixed intramolecular/intermolecular dithiodipropionic acid ester 3o ' :

~ 1289883 ~

1 Cellulose polymers which have been modified to contain disulfide groups are known from U.S. Patent ~os. 2,418,940, 2,418,938 and 3,485,815 and can be prepared by the methods which are disclosed therein.
Upon undergoing reduction, the disulfide linkage in each of the above forms will be disrupted thereby providing the following reducing component of the redox system herein (only monomeric unit shown):
o , ~0-C--(CH2~5H

C~ CH2 ~0 C1~2 15 CH~ 1~2 2 ~1 o~CH2 o ,1 1~
~_ ~ O ~_ H >~

H OH CH O OH
2 \

HEC - mercaptopropionic acid ester The reducing moieties of the redox system, such as the one 25 shown above, upon undergoing oxidation, will form the disulfide linkage.
In much the same way, the other reducing moieties herein will undergo oxidation to provide the oxidizing moieties of the redox system.
3o In the case of the interferons, the active forms of ~7hich comprise a chain of 165 amino acids and one or two disulfide bridges, it is necessary to maintain a balance . ' , .:. , ~ . . . ` ' : , ~ . ~ . . . - , - ' ' - ---89~383 -~, 1 between the polymeric reducing agent and the polymeric oxidizing agent such that the reducing agent can intercept any oxygen which would otherwise get at the interferon causing it to degrade and the oxidizing agent will help to 5 preserve the disulfide bonds of the active forms of interferon. The ratio of reducing agent to oxidizing agent is not critical, the optimum ratio for a particular interferon composition being readily determined by those skilled in the art employing routine laboratory procedures.
10 In general, a molar ratio of reducing moieties to oxidi~ing moieties in the range of from about 30:1 to about 1:1 and preferably from about 10:1 to about 2:1 can be used with good results.
The amount of redox system necessary to impart 15 significantly improved stability against oxi~ative degradation can vary widely ~ith from about O.Q0001 moles to about Q.02 moles redox system per 10 to ~0 ml of interferon source liquid being suitable in most cases.
The aquepus vehicle base used with the present 20 invention must simultaneously satisfy the following criteria; (1) it must be capable of holding the high molecular weight interferon or interferons in suspension and yet be able to release the interferon or interferons at the diseased site, (2) it must be compatible with the interferon 25 or interferons being administered ~o that the activity of the interferon is not adversely a~fected, (3) it must be acceptable to the patient, i.e., it should be non-irritating, non-toxic and should have an appropriate odor, color and texture and t4) it must have suitable 3o rheological properties so that it can be easily manufactured and dispensed into containers and subsequently applied to the diseased site by the patient.

i X ~9 ~3 l The topical therapeutic compositions herein can advantageously be formulated with a water soluble polyalkylene qlycol, a particularly preferred class of which is the polyethylene glycols. By combining different 5 molecular weight polyethylene glycols, e.g., polyethylene ~lycols having molecular weights between 300 and 20,000, a wide range of viscosities and aqueous solution capacities (e.g., 15-25%) for this ~ype of base can be obtained. One such formulation comprises a 60:40 mixture of a 400 lO molecular weight polyethylene glycol and a 3350 molecular ~leight polyethylene glycol. This mixture produces a particularly suitable vehicle base for administering interferons.
To use these preferred polyethylene glycol-15 containing bases with interferons, however, requires special handling procedures for incorporating the interferon into the vehicle base. Pt room temperature and below, the various mixtures of polyethylene glycols preferred for use ~ith the present invention are semi-solid pastes rather than 20 liquids. In contrast, interferons, as stated above, are typically supplied as liquids. The two components can be combined by mechanical mixing but such mixinq often results in a non-homogeneous ointment and can denature the interferon. In accordance with the lnvention and as 25 described in more detail in connection with the example presented below, it has been found that a superior polyethylene glycol-based ointment can be prepared by combining the interferon with the polyethylene glycols when both components have been heated to an elevated temperature, 3o e.g., 45C. Surprisingly, it has been found that this procedure does not significantly deactivate the interferon, : ~. ' ' ~ . . -.

~, 1~898B3 ~

1 notwithstanding its proteinaceous character, which one would expect ~lould lead to heat sensitivity.
Jn order to maintain the consistency of the final preparation ~ithin a suitable range for application to the 5 diseased site, it has been found that the amount of interferon source liquid added to polyethylene glycol vehicles bases such as the ~0:40 base described above should be kept in the range of from about 0.01 to about 0.25 milliliters per gram of base, and most preferably in the lO range from about 0.05 to about 0.15 milliliters per gram of base.
Another particularly preferred water-soluble vehicle base for use with the present invention employs hydroxethyl cellulose as a thickening agent for the topical 15 prepara,ion. The HEC gel can be prepared as follows: .he polymer powder is ~etted and dispersed in glycerin as a suspended slurry, the liquid phase which can ir,clude the interferon source liquid is added to ,he slurry and after a short period with gentle agitation, a gel which thickens 20 into a homogenous mixture is obtained. Further addition of liquids, including the interferon source liquid, to the already formed gel will be eventually absorbed with slow mixing.
Since, as discussed above, interferons are normally supplied as liquids, this means that a homogeneous mixture can be easily obtained. Also, it has been found that hydroxyethyl cellulose-containing vehicle bases, after they fully gel, give the final preparation a pleasing feel and appearance. Moreover, interferon preparations using 3o this vehicle base have been found to have especially high initial interferon titers.

~ 128~88~ ~

l A preferred topical ointment base using hydroxethyl cellulose includes between about l and about 5 weight percent of hydroxethyl cellulose, for example, a viscosity of 2200 centipoise, between about lO and about 50 5 weight percent of glycerine and between about 49 and about 85 weight percent of water. To this vehicle base is preferably added between about lO and about 40 milliliters per hundred grams of base of interferon source liquid. A
particularly preferred hydroxyethyl cellulose vehicle base lO includes between about 2 and about 3 weight percent of hydroxyethyl cellulose, between about ~0 and about 38 weight percent of glycerine and between about 60 and about 78 weight percent of water. In combination with this base, preferably between about 15 and about 20 milliliters of 15 interferon source liquid are used per hundred grams of base.
For interferon source liquids having low titers, the quantity of water used in the vehicle base can be reduced and higher amounts of source liquid can be admixed with the base. In this way!, the titer of the final preparation can 20 be adjusted without adversely affecting the preparation's rheological properties. In place of glycerine and water, the hydroxethyl cellulose-containing vehicle base can include polysorbate and other similar wetting agents.
Instead of HEC, other celluloses and their derivatives, such 25 as sodium CMC, methylcellulose and hydroxpropyl cellulose can be used.
In addition to an interferon component and an aqueous vehicle base component, the topical preparations of the present invention can optionally include one or more 3o protease inh~bitors. These inhibitors can be included in the preparations for the purpose of reducing the rate of - . . .
-- , 12898~33 1 decay of the biological activity of the interferon component due to proteolytic agents. The major source of interferon decay in topical preparations, especially at elevated temperatures, i.e., room or body temperature, is digestion 5 of the interferon by proteolytic enzymes introduced into the preparation as part of the interferon source liquid. Once in the preparation, these contaminating enzymes over time destroy the therapeutic potency of the interferon.
The proteolytic enzymes found in interferon source 10 liquids come from the human serum, leukocytes or other biological materials used to manufacture the interferon, 2S
well as possibly from contaminating microorganisms.
Typically these enzymes are of the "serine" protease type in that they have a critical serine residue at their active 15 site. Examples of such proteolytic enzymes include trypsin, plasmin, thrombin, leukocyte elastease, kallikrien, and cathepsin. In some cases (e.g., plasmin and thrombin), the interferon source liquid may not actually include the active proteolytic enzyme, but rather, an inactive precursor which 20 is slowly converted to the active form, especially at elevated temperatures.
To control the effects of these proteolytic enzymes, the topical preparations of the present invention advantageously include one or more protease inhibitors which 25 interact ~ith the proteolytic enzymes and prevent them from digesting and thus inactivating the interferon in the interferon source liquid.
Various proteolytic inhibitors of human, animal or plant origin can be used in the practice of the present 3o invention. Because of the highly conserved nature of both protease inhibitors and the active sites of the proteolytic ~ a~ ~

1 enzymes typically present in interferon source liquids, the inhibitor need not be from the same species as the proteolytic enzyme, but can be from a different species or even of plant origin. For example, soybean trypsin 5 inhibitor (also referred to herein as "soybean inhibitor~
and abbreviated "STIn) is capable of inhibiting trypsins from such diverse sources as humans, cows, salmon, stingrays, barracudas and turkeys. Also, in addition to inhibiting trypsins, STI will inhibit bovine and chicken 10 chymotrypsin, human plasmin, human kallikrein and cocoonase, as well as blocking the conversion of prothrombin to thrombin.
Protease inhibitors particularly preferred for use with the present invention include STI, alphal-trypsin 15 inhibitor (hereinafter abbreviated alphal-AT), MalPha-tosyl-lysine chloromethyl ketone (hereinafter abbreviated "TLCRn), phenylmethylsulfonyl fluoride (hereinafter abbreviated "PMSFn), NalPha-tosylphenylalanine chloromethyl ketone (hereinafter abbreviated "TPCKn), alpha-2~
20 macroglobulin, and mixtures thereof. Of these inhibitors, STI is particularly preferred because of its low cost and alphal-AT is most preferred, especially when prepared from human serum, because of the reduced chance of allergic reactions. A suitable technique for purifying human alphal-25 AT is described by J. Travis, et al. in Methods inEnzy~oloqy, ~Q, pages 754-765. The other preferred protease inhibitors listed above are commercially available from various sources including Sigma Chemical Company, St. Louis, ~o. (STI, TLCK, and PMSF), Chemical Dynamics Corp., South 30 Plainfield, New Jersey (TPCK) and Boehringer Mannheim Biochemicals, Indianapolis, Indiana (alpha-2-macroglobulin).

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1 The amount of protease inhibitor which can be included in the topical preparation depends upon the amount and type of proteolytic enzyme present in the interferon source liquid, and the particular inhibitor used. For 5 example, crude alpha-interferon sold by Interferon Sciences, Inc. (~ew Brunswick, Mew Jersey, Catalog ~1100) following concentration in a known and conventional manner typically contains approximately 100 mg of protein per milliliter of liquid. Of this lQ0 milligrams, up to 1~ may be proteolytic lO enzymes generally of the type found in human plasma (e.g , thrombin, plasmin, etc.). These enzymes have molecular weights in the range of 25,000 to 100,000 daltons. In comparison, STI, for example, has a molecular weight of approximately 20,000 daltons. Accordingly, since this 15 inhibitor generally forms a 1:1 complex with its target proteolytic enzymes, the addition of 1 mg of inhibitor per milliliter of interferon source liquid can be expected to produce a multifold molar excess of STI on the order 0.25 milligrams per milliliter of crude concentrated 20 alpha-interferon source liquid is sufficient to guarantee an extended interferon half-life, especially at elevated temperatures, such as, 37C. Since this particular interferon source liquid has an activity of approximately 2 x 106 Units per milliliter and since it is preferable to 25 have between 25,000 and 500,000 Vnits per gram of ointment, this means an STI addition rate of between approximately 0.003 and 0.06 milligrams inhibitor per gram of ointment.
(E25,000 Units/gram]/~2 x 106 Units/ml] x [0.25 mg/ml~
= 0.003 mg/gm; E~oo,ooo Units/gram]/ 2 x 106 Units/ml]
30 x Eo.25 mg/ml] = 0.06 mg/gm). Similarly, for a liquid preparation using this particular interferon source liquid ~ ~289883 1 and havin~ a strength of between 200,000 and 2,000,000 ~nits per r,lilliliter o, preparation, between approximately 0.025 and 0.25 milligrams of this inhibitor is us~d per milliliter of solution. ([200,000 Units/ml]/[2 x 106 Units/ml] x [0.25 5 mg/ml~ = 0.025 mg/ml; ~2,000,000 Units/ml]/[2 x 106 Units/ml] x [0.025 mg/ml] = 0.25 mg/ml).
As will be evident to persons of ordinary skill in the art, a similar procedure to that described above can be used to determine the appropriate addition levels for otl-,er lO inhibitors and other interferon source liquids.
In addition to including one or r,lore interferons and a vehicle base, the interferon preparations of the present invention can include various optional components.
For example, it is generally desirable to include one or 1~ more preservative in the preparation to prevent microbial srowth. ~xamples of preservatives which have been found compatible with interferons include benzalkonium chloride and methyl and propylparabens. Also, the preparations can - include non-interferon type therapeutic agents in addition 20 to the one or more interferons. Other optional components which can be included in the preparation are various coloring agents and protein stabilizing agents such as glycerol, sucrose, sorbitol and mannitol.
Of the following examples, Examples 1 to 3 are 25 illustrative of the preparation of the water soluble polymeric reducing and oxidizing agents comprising the redox system herein and Examples ~ to 6 are illustrative of various topical interferon compositions containing polymeric redox systems in accordance with this invention.
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~ ~2~3~883 ~

This example illustrates the preparation of HEC-cysteine hydrochloride reducing agents of different cysteine content.
Hydroxyethyl Cellulose (109) was dissolved in 500 ml water. A water solution of cysteine hydrochloride monohydrate was added to the stirred HEC solution and the resulting solution was refluxed under nitrogen for three hours. Water was removed under vacuum and dry product was lO ground in a mill. Four water soluble polymer compounds differing in cysteine hydrochloride content were obtained:
.

Active Cysteine Polymer Compound m.p. rClContents ~/W~l 1 150-160 (d) 24.6 2 170-180 ~d) 8.1 3 190-220 (d) 4.3 4 , 240-270 (d) o.g Compounds 1-4 were titrated with iodine and thiosulfate solution in order to determine the reducing activity of the products as well as the amount of active SH
groups in a given amount of product. All compounds 25 demonstrated signiicant reducing activity. In addition, Compound 1 was dialyzed against water under nitrogen to determine its stability; the dialyzed product (m.p.
215-220C) remained active as a reducing agent after three days o~ dialysis.
3o :' l2ass~3 ~E 2 This example illustrates the preparation o~ an ~EC-cystine hydrochloride oxidizing agent. Both reactions were carried out in dimethyl formamide (D~IF~ solution. Tn 5 the case of the second reaction, a reducing agent was added;
the first compound was obtained without a reducing agent.
Reducing agents which are used to rupture the disulfide crosslinks and to form mercapto groups can be any of the following compounds: sodium tetraborate, dithiothreitol, lO dithioerythritol, glutathione, mercaptoethanol, 2,3-dimercaptoproponal, thioglycolate and thioglycolic acid.
The compounds were obtained in the following way:
Polymer Com~ound 5 HEC (lOg) was dissolved in 250 ml of DMF. Cystine 15 hydrochloride was added into a stirred solution followed by addition of thionyl chloride in DMF. The reaction mixture was stirred and heated on a water bath for 4 hours and left overnight. Half of the DMF was removed under vacuum and the resultant solution was dialyzed against water under 20 nitrogen. The product was dried under vacuum, m.p.
218-219C.
Polymer Compound 6 The reaction mixture obtained as described above was alkalized with lN NaOH and an aqueous solution of NaBH4 25 reduciny agent was introduced. When the evolution of hydrogen ceased ~0.50-1.0 hr.), lN HCl was added to decompose excess NaBH4. Half of the solvent was removed under vacuum and the resultant sclution was dialyzed against water under nitrogen. The product was dried under vacuum, 3o m.p. 216-218C.

.
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-, , , . ~ ':'~ -- : : -- , .

~ 1;~8~883 ~

1 Reducing activity of these compounds was observed with iodine solution. Compound 5 did not exhibit reducing activity whereas Compound 6 exhibited significant reducing activity.

3o .~ .. .. : ' , , ' - ' ~ 12B9883 1 EXAMPL~_~
This example illustrates the preparation of ~EC-dithiodipropionic acid ester reducing agent.
Dithiodipropionic acid (5g) was heated on a water 5 bath with thionylchloride (15 ml) for 5 hours. Two drops of DMF were added. Excess thionyl chloride was removed under vacuum. HEC (lOg) was dissolved in 250 ml of DMF and dithiodipropionyl chloride in DMF was dropped into the stirred solution. The reaction mixture was heated for 5 10 hours and left overnight.
It was then alkalyzed with lN NaOH followed by introduction of an aqueous solution of NaBH4 with stirring for one hour. Excess NaBH4 was decomposed with lN HCl and half of solvent was removed under vacuum. The resultant 15 solution was dialyzed against water under nitrogen. The product was dried under vacuum, m.p. 230-235C. The product exhibited significant reducing activity.

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q ~289~3 -~

1 EXAMP~E 4 Preparation of Crude Concentrated alpha-Interferon Source Li~luid Crude concentrated alpha-interferon source liquid 5 was prepared from Natural Crude alpha-Interferon sold by Interferon Sciences, Inc., (New Brunswick, New Jersey, Catalog #1100) as follows. The pH of the commercial product was checked and, if necessary, adjusted to 7.0-7.2 using sodium hydroxide. The p~-l adjusted material was then 10 concentrated using a hollow fiber filter having a lO,Q00 molecular weight cutoff and run at 20 p.s.i. until the volume of the product was 1/50 of the starting volume. The concentrated product was then clarified by centrifugation at 18-20,000 x g and finally sterile filtered.

3o 1 X~lPr,~ 5 Preparation of an Interferon Tol)ical Ointn,ent ~avinq a ~olyethylene ~lycol Vehicle Base An interferon ointment having a polyethylene 5 glycol vehicle base is prepared as follows.
Sixty grams of polyethylene glycol 400 liquid and 40 grams of polyethylene glycol 3350 powder, both obtained from Fisher Scientific, Fair Lawn, Mew Jersey, are mixed together in a sterile glass beaker and then autoclaved at 10 121C. for 40 minutes. While still molten, the beaker is submerged in a 50C. water bath set inside a laminer flow hood. The mixture is slowly stirred with a sterile propeller-type stirring blade and its temperature was adjusted to approximately 45C. ~ifteen milliliters of 15 frozen crude concentrated alpha-interferon source li~uid prepared in accordance with ~xample 4 above is thawed at 4C
and then heated in a water bath to a te~perature of approximately 45C. The interferon is then added to the - molten polyethylene glycol mixture and the two components 20 are stirred together. Thereafter, 4 gm of re~ox system containing 2.0 gm of PEG-thiopropionic acid (1~
thiopropionic acid bound3 oxidizing agent and 2.0 gm of PEG
- dithiodipropionic acid (0.1~ thiopropionic acid bound) reducing agent are combined with the polyethylene 25 glycol-interferon mixture. Stirring is resumed and continued until the mixture is homogeneous and has an even color.
Using a syringe, pipet or equivalent, aliquots of the interferon/polyethylene glycol mixture are placed in - 3o sterile aluminum ointment tubes which have been prechilled : .

1 to 4C. The open ends of the tubes are covered with alcohol-swabbed parafilm and the tubes are placed in a -20C
freezer. After approximately 20 minutes at -20C, the tubes are crimped under a laminer flow.
To fill a large number of tubes, it has been found preferable to perform the filling step in batches so as to minimize the amount of time during which the ointment stands at room temperature. Other larger filling equipment which is well known in the art can also be used.
To demonstrate that the heating of the interferon source liquid to 45C to facilitate it mixing with the polyethylene glycol mixture did not significantly decrease the activity of the interferon, a sample of crude concentrated alpha-interferon source liquid, prepared in 15 accordance ~ith Example 1, was heated to 45C, held at that temperature for 1 hour, and then stored at -20C. The changes in the titer of this sample over time were compared to those of an unheated sample. The titers of the heated and unheated interferon samples were essentially identical, - 20 thus establishing that the heating step used to prepare polyethylene glycol-based ointments does not destroy the ~iological activity of the interferon.
To compare the quality of an ointment produced by mixing the interferon and the polyethylene slycols at 25 elevated temperature with the quality of an ointment produced by mechanical mixing of these components at room temperature, a batch of ointment was prepared by adding 45 milliliters of crude concentrated alpha-interferon source -liguid to 300 grams of the 60:40 polyethylene glycol 3o ~ 89883 -~

1 mixture, described above, which had been cooled to room temperature The two components were placed in a sealable plastic bag and the contents were mixed by kneading the bag by hand and rolling the bag using a cylindrical bar. The 5 kneading and rolling was carried out for a period of approximately 30 minutes, after which the ointment was compared with the ointment prepared by heating, as described above. The mechanically mixed ointment was in general found to have a non-uniform color and consistency indicating that 10 a homogeneous dispersion of the interferon throughout the polyethylene glycol mixture had not been achieved. In comparison, the ointment prepared by heating the interferon and the polyethylene glycols exhibited uniform color and consistency throughout the ointment.

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~ 89883 ~

Preparation of an Interferon Topical Ointment Havina a ~TEC Vehicle ~ase An interferon ointment having a hydroxyethyl 5 cellulose vehicle base is prepared as follows (for 100 gms):
Prepar~tion of ~tock P
First, 2.5 grams of high ViSCOsity hydroxyethyl cellulose (2200 centipoise) (Polysciences, ~arrington, Pennsylvania) are measured into a beaker. AggregateS of the lO hydroxyethyl cellulose powder are broken up. ~ext, 10.0 grams of USP glycerine are added to the hydroxethyl cellulose powder and the two components are mixed to form a uniform slurry. Then 60.~ milliliters of purified water and 3.0 gm of redox system containing 1.5 gm ~EC-cysteine (1%
15 cysteine bound) reducing agent and 1.5 gm ~EC-cystine (0.1%
cystine bound) oxidizing agent are added to the hydroxyethyl cellulose/glycerine mixture. The water is then added.
Thereafter, the solution is mixed rapidly until the gel - thickens. I
PreParation of Stock B
0.06 grams of propyl paraben and 0.25 grams of methyl paraben are added to lQ grams of sterile glycerin which is at approximately 65-75C. Care is taken to insure that the mixture does not reach a temperature above about 25 75C so as not to inactivate the propyl and methyl parabens.
The parabens are mixed in the glycerin until no paraben powder is observed in the mixture. Stock B is then cooled to room temperature. At t~is time, stock A and stock B are mixed together to form a homogeneous gel. The mixture is 3O then placed in an ice bath and cooled with mixing until a temperature of 4C is reached.

2~9883 1 A sterile interferon stock solution is formulated as follows:
Thirteen milliliters of the foregoing sterile-filtered interferon stock solution are then combined with 5 .52 milliliters of a 50 mg/ml, sterile-filtered, soybean trypsin inhibitor solution (Sigma Chemical Company, St.
Louis, Missouri). This solution is then added to the cooled vehicle base/paraben mixture and the combination is mixed until a uniform dispersion is visually observed. The gel is 10 then loaded into a tube filling machine (various types of this equipment are known to the art). The gel is dispensed into sterile aluminum ointment tubes which are then crimped closed.

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Claims (17)

1. A substantially non-toxic, stable, topical therapeutic composition which comprises:
(a) a therapeutically active component which is susceptible to oxidative degradation;
(b) an oxidative degradation-inhibitory amount of a redox system containing (1) a water soluble polymer containing a plurality of reducing moieties covalently bound thereto and (2) a water soluble polymer containing a plurality of oxidizing moieties covalently bound thereto; and (c) an aqueous vehicle base compatible with the therapeutically active compound.

2. The therapeutic composition of Claim 1 wherein the therapeutically active component is a protein.

3. The therapeutic composition of Claim 1 wherein the therapeutically active component is a lymphokine.

4. The therapeutic composition of Claim 1 wherein the therapeutically active agent is one more natural or recombinant interferon.

5. The therapeutic composition of Claim 4 containing from about 10,000 to about 10,000,000 IU per gram.

6. The therapeutic composition of Claim 4 containing from about 50,000 to about 500,000 IU per gram.

7. The therapeutic composition of Claim 1 wherein the water soluble polymer is derived from a polyethylene glycol of the formula HO(CH2CH2O)nH wherein n is from about 20 to about 225,000.

8. The therapeutic composition of Claim 1 wherein the water soluble polymer is derived from hydroxyethyl cellulose.

9. The therapeutic composition of Claim 1 wherein the reducing moieties are derived from cysteine or mercaptopropionic acid.

10. The therapeutic composition of Claim 1 wherein the oxidizing moieties are derived from the oxidation of cysteine or mercaptopropionic acid ester moieties to provide cystine or dithiodipropionic acid ester, respectively.

11. The therapeutic composition of Claim 1 wherein the vehicle base is a water soluble polyethylene glycol.

12. The therapeutic composition of Claim 1 wherein the vehicle base is an aqueous hydroxyethyl cellulose.

13. A method for inhibiting the oxidative degradation of a therapeutic agent susceptible thereto which comprises combining said therapeutic agent with an oxidative degradation-inhibiting amount of a redox system containing (a) a water soluble polymer containing a plurality of reducing moieties covalently bound thereto and (b) a water soluble polymer containing a plurality of oxidizing moieties covalently bound thereto.

14. The method of Claim 13 wherein the therapeutic agent is a natural or recombinant interferon.

15. The method of Claim 14 wherein the water soluble polymer is derived from a polyethylene glycol or a hydroxyethyl cellulose.

16. The method of Claim 15 wherein the reducing moieties are derived from cysteine or mercaptopropionic acid.

17. The method of Claim 15 wherein the oxidizing moieties are derived from cystine or dithiodipropionic acid.