AU628113B2 - 2',3'-dideoxypurine nucleoside/purine nucleoside phosphorylase inhibitor combination therapy and composition - Google Patents

Description

Our Ref: 299181

AUSTRALIA

Patents Act FORM COMPLETE SPECIFIC

(ORIGINAL)

kTION 62- 1A Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: Applicant(s): Ciba-Geigy AG Klybeckstrasse 141 4002 BASLE

SWITZERLAND

ARTHUR S. CAVE CO.

Patent Trade Mark Attornerys Level 10, 10 Barrack Street SYDNEY NSW 2000 Address for Service: Complete specification for the invention entitled "2',3'-dideoxypurine nucleoside/purine nucleoside phosphorylase inhibitor combination therapy and composition".

The following statement is a full description of this invention, including the best method of performing it known to me:- 1 5020 1 la 2',3'-DIDEOXYPURINE NUCLEOSIDE/PURINE NUCLEOSIDE PHOSPHORYLASE INHIBITOR COMBINATION THERAPY AND COMPOSITION The invention relates to 2',3'-dideoxypurine nucleoside treatment of retrovirus infections, including human immunodeficiency virus (HIV) infections. It also relates to purine nucleoside metabolic pathway inhibitors, in particular purine nucleoside phosphorylase (PNP) inhibitors.

This invention involves the coadministration of a PNP inhibitor with an anti-HIV active dideoxypurine nucleoside for the treatment of HIV infections.

Acquired immunodeficiency syndrome (AIDS) is a fatal disease characterized by a spectrum of immunological disorders that develop after infection by the human immunodeficiency virus (HIV) (De Clercq, J. Med. Chem. 29,1561 (1986)). Several strategies for the treatment of AIDS are currently under investigation (Mitsuya Broder, Nature 325, 773 (1987)). To date, the most successful chemotherapeutic approach has involved 2',3'-dideoxynucleosides (ddN). These agents generate the 2',3'-dideoxy nucleotide (ddNT) analog, which in turn prevents HIV DNA synthesis and thereby HIV replication by functioning either as an inhibitor or as a substrate analog for reverse transcriptase. In the latter case, reverse transcriptase catalyzes the addition of the ddNT to the 3' end of a growing DNA chain thereby terminating further elongation due to the absence of a 3'-hydroxyl.

Both 3'-azido-3'.deoxythymidine (AZT) and dideoxycytidine (ddC) have proven efficacious in man for the treatment of HIV infection and both 2',3'-dideoxypyrimidine nucleoside analogs are reported to increase life span. Both, however, exhibit some serious toxic side effects. AZT can cause myelosuppresion and frequently severe anemia while ddC can result in peripheral neuropathy [Yarchoan et al., Lancet, 76 (1988)].

In comparison to AZT and ddC, 2',3'-dideoxypurine nucleosides, e.g. dideoxyinosine (ddl) and dideoxyadenosine (ddA) exhibit a more promising therapeutic index in cell culture (Mitsuya and Broder, Proc. Natl. Acad. Sci. 83, 1911 (1986)). ddI and ddA, however, are -2also less effective in the protection of H9 cells from HIV infectivity and ATH8 cells from the cytopathic effects of HIV. One reason for this decrease in potency may be metabolic instability. Incubation of whole cells with ddl or ddA radiolabelled in the purine ring was shown to yield the suspected anti-HIV agent 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) along with substantial amounts of labeled ATP and ADP. ddATP presumably was generated from conversion of ddl (ddA is rapidly converted to ddl in vivo) to ddIMP (2',3'-dideoxyinosine monophosphate) followed by reentry into the adenylate pathway via adenylsuccinate synthetase (Ahluwalia et al., Biochem Pharmac. 36, 3787 (1987)).

Labeled ATP and ADP, on the other hand, were speculated to arise from initial degradation of ddl to 2',3'-dideoxyribose-l'-phosphate (ddRP) and hypoxanthine (Hx) by Purine Nucleoside Phosphorylase (PNP). The labeled hypoxanthine is subsequently converted to adenylate nucleotides through the purine salvage pathway (Haertle et al., J.

Bio. Chem. 263, 5870 (1988)). ddl is known to be a PNP substrate, although in comparison to the natural PNP substrates, inosine and guanosine, ddl is substantially worse (1000x lower kcat/Km; Stoeckler et al., Biochem. 19, 102 (1980)). Hence, the fact that this relatively poor PNP substrate is degraded to a significant extent by PNP in cell culture suggests that either PNP exists in large amounts in these lymphocyte cell lines or/and that ddl phosphorylation to ddIMP is a relatively slow reaction.

In contrast to cell culture, the effect of PNP on ddl pharmacokinetics in the whole animal is unknown. Clearly, ddl elimination in vivo could occur by many different mechanisms including renal filtration, oxidation, conjugation, chemical hydrolysis etc. For a single enzyme to catalyze the degradation of a drug and thereby dictate its in vivo half-life, the drug would have to have ready access to the enzyme and the turnover rate would have to be significantly faster than the rate of all other clearance mechanisms. The fact that ddl is a very poor PNP substrate would therefore tend to suggest that the rate of ddl elimination in vivo is unlikely to be dependent on PNP activity and thus unlikely to be affected by a PNP inhibitor.

A wide variety of Purine Nucleoside Phosphorylase (PNP) inhibitors are known in the art.

A number of these are specifically disclosed in J. Stoeckler's Chapter in Developments in Cancer Chemotherapy, CRC Press, 1984, entitled "Purine Nucleoside Phosphorylase: A Target for Chemotherapy", pp. 35-60 and in Drugs of the Future 13, No. 7, 1988, "Purine Nucleoside Phosphorylase (PNP) Inhibitors: Potentially Selective Immunosuppressive Agents", Sircar and Gilbertsen, pp. 653-668; EP-A-193,454, EP-A-178,178, EP-A-156,559, EP-A-145,207 and EP-A-260,491.

-3- PNP deficiency or inhibition has been found to suppress T-cell mediated immunity as reported in the above literature and further in Cancer Research 46, February 1986, "Potentiation of 2'-Deoxyguanosine Cytotoxicity by a Novel Inhibition of Purine Nucleoside Phosphorylase, 8-amino-benzylguanine", pp. 519-523; Cancer Research 46, 1774-1778, April 1986; Agents and Actions 21, 3/4 (1987), pp. 253-256; Agents and Actions 22, 3/4 (1987), pp. 379-384; Agents and Actions 21, 3/4 (1987), pp. 272-274; Immunology 1986, 59, pp. 63-67; and Clinical Experimental Immunology (1986), 66, pp.

166-172. These references make it clear that PNP deficiency results in T cell death and compromises the host's immunological integrity. Cells deficient in PNP accumulate toxic levels of purine nucleotide triphosphates causing cell death. In fact, T cell- seem to be the most grossly affected cells by PNP deficient toxicity. As such a reduction of PNP activity in immune compromised patients, such as AIDS patients, would appear to be contraindicated.

Therefore, it would seem to be cortraindicated to administer PNP inhibitors as a means of overcoming a retrovirus, particularly a human immunodeficiency virus infection. PNP inhibitors might be expected to further compromise immune function in such infections.

S Notwithstanding the above, the instant invention is directed to the administration of a 2',3'-dideoxypurine nucleoside (or a derivative thereof) in combination with a purine nucleoside phosphorylase inhibitor so as to inhibit the metabolic degradation and to enhance the antiretroviral effect of said 2',3'-dideoxypurine nucleoside (or a derivative thereof) for the treatment of human immunodeficiency virus (HIV) infections.

An object of the invention is to provide a method for treating retrovirus infections, especially human immunodeficiency virus infection, and a composition therefor.

Another object of the invention is to provide an improved method of 2',3'-dideoxypurine nucleoside treatment of human immunodeficiency virus infections and other 2',3'-dideoxypurine nucleoside responsive conditions in mammals, including man.

t Yet another object of the invention is to provide a method of slowing the in-vivo metabolism or degradation of 2',3'-dideoxypurine nucleosides in mammals, including man, a method of increasing their duration of action, a method of potentiating their therapeutic effect, a method of reducing the effective dose, and a method to minimize the side effects

L

i -4observed during treatment with said 2',3'-dideoxypurine nucleosides.

A still further object of the invention is to provide a composition for treating a 2',3'-dideoxypurine nucleoside responsive condition in mammals, including man, with a heretofore subtherapeutic amount thereof.

Surprisingly, these and other objects are achieved by administering both a 2',3'-dideoxypurine nucleoside and a purine nucleoside phosphorylase inhibitor to a mammal having a 2',3'-dideoxypurine nucleoside responsive condition.

The instant invention is a combination therapy and a composition for the treatment of retrovirus infections, especially in humans, particularly human immunodeficiency virus infections. It requires the administration of a 2',3'-dideoxypurine nucleoside (ddPN) and a purine nucleoside phosphorylase inhibitor (PNP inhibitor) sufficiently close in time such that the PNP inhibitor prevents, or at least lessens, the rate at which the 2',3'-dideoxypurine nucleoside is metabolized by native purine nucleoside phosphorylase.

2',3'-dideoxypurine nucleosides for use in the invention are e.g. compounds of the formula

HOH

2 C 0 B

(I)

A

wherein A is hydrogen, azido, or halogen, preferably hydrogen, B represents the radical of a purine base bound to the dideoxy sugar residue by its 9-position and metabolizable esters of compounds of formula I or pharmaceutically acceptable salts thereof. Esters of the compound of formula I are compounds of formula I wherein hydroxy of the hydroxymethyl group is esterified with an acid, and include a) carboxylic acid esters of acids of the formula R 1 -COOH in which R 1 is selected from straight and branched alkyl, alkoxyalkyl, arylalkyl, aryloxyalkyl, and aryl, wherein aryl is phenyl or naphthyl which are unsubstituted or substituted by halogen, alkyl or alkoxy; b) sulfonate esters of sulfonic acids of the formula R 2

-SO

3 H wherein R 2 is alkyl or arylalkyl with aryl being defined as above; and c) phosphate esters of phosphoric acids of the formula i I1 00, 0 Coo 00 0 000 0'0 tOO* 0 D 90000

R

3 0

R

4 0 P -O

R

5 0 wherein at least one of R 3

-R

5 is other than hydrogen, R 3

-R

5 being selected from hydrogen and the groups set forth for R 1 above. In the above, the groups alkyl and alkoxy each have 1-18, preferably 1-7, more preferably 1-4, carbon atoms. Halogen represents preferably fluoro or chloro. The purine base radical B represents preferably adenin-9-yl, guanin-9-yl, hypoxanthin-9-yl, thiohypoxanthin-9-yl, thioguanin-9-yl or 2-aminoadenin-9-yl.

Preferred are the corresponding compounds of formula I wherein A represents hydrogen, B is adenin-9-yl, guanin-9-yl, hypoxanthin-9-yl, thiohypoxanthin-9-yl, thioguanin-9-yl or 2-aminoadenin-9-yl; and the hydroxy group is free, namely 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine, 2',3'-dideoxythioinosine, 2',3'-dideoxythioguanosine and 2',3'-dideoxy-2-aminoadenosine. Particularly preferred are the corresponding compounds wherein A is hydrogen; R is adenin-9-yl, guanin-9-yl, hypoxanthin-9-yl or thiohypoxanthin-9-yl and the hydroxy group is free, namely 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine and 2',3'-dideoxythioinosine. Most preferred are the corresponding compounds wherein A is hydrogen, B is adenin-9-yl or hypoxanthin-9-yl, and the hydroxy group is free, namely 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine.

The above compounds are known or can be made in accordance with techniques generally known in the art, as for example in EP-A-206,497, published December 30, 1986.

Specific examples of pharmaceutically acceptable derivatives of the compounds of formula that may be used in accordance with the present invention include the following esters and pharmaceutically acceptable alkali metal salts, preferably the monosodium salts: monophosphate; disodium monophosphate; diphosphate, triphosphate; acetate; 3-methylbutyrate; octanoate; palmitate; 3-chlorobenzoate; 4-methylbenzoate; hydrogen succinate; pivalate; and mesylate.

The potentiation of the anti-retroviral effect of a dideoxypurine nucleoside, e.g. of formula I, by a PNP inhibitor can be determined, e.g. in cell cultures H9 cells, ATH8 cells) exposed to a retrovirus HIV) according to methodology well-known in the art, such o( 0 0 0( 0o 0 00 000 Oo0 -6as described in Proc. Nat. Acad. Sci, U.S.A. 83, 1911 (1986). The potentiation can also be determined in vivo in rats) by measuring the increase in the plasma level of the dideoxypurine nucleoside which is achieved by prior or simultaneous administration of the particular PNP inhibitor, according to methodology well-known in the art.

The compounds according to the invention may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and transdermal. It will be appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the infection and the chosen active ingredient.

In general a suitable dose of ddPN will be in the range of about 0.01 to 20 mg per kilogram body weight of the recipient per day, preferably in the range of about 0.05 to mg per kilogram body weight per day and most preferably in the range of about 0.1 to mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing about 0.1 to 200 mg, preferably about 0.5 to 100 mg, and most preferably about to 50 mg of active ingredient per unit dosage form.

Ideally, the ddPN ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 1 to about 75 gM, preferably about 2 to gM, most preferably about 3 to about 30 gM.

While it is possible for the active ingredient to be administered alone it is preferable to administer such as a pharmaceutical formulation. The formulations of the present invention comprise at least one active ingredient, as herein defined, together with one or more acceptable carriers thereof and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) or transdermal administration.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by unifrrmly and -7intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary o:r paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the Sactive ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. This is particularly advantageous with the compounds of formula as such compournds are susceptible to acid hydrolysis.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the ddPN ingredient such carriers as are known in the art to be appropriate.

-8- Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterilie liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of a ddPN ingredient and/or a PNP inhibitor.

Purine nucleoside phosphorylase (PNP) inhibitors for use within the instant invention are of a wide variety of compounds, many of which are typically known in the art. A number of these are specifically disclosed in J. Stoeckler's Chapter in Developments in Cancer S Chemotherapy, CRC Press, 1984, entitled "Purine Nucleoside Phosphorylase: A Target for Chemotherapy", pp. 35-60 and in Drugs of the Future 13, No. 7, 1988, "Purine Nucleoside Phosphorylase (PNP) Inhibitors: Potentially Selective Immunosuppressive Agents", Sircar and Gilbertsen, pp. 653-668. See also US patent application 704,991, filed February 1985, corresponding to EP-A-193,454; US patent application 660,152, filed October 12, 1984 and US patent application 767,202, filed August 22, 1985, corresponding to EP-A-178,178; US patent application 593,063, filed March 26, 1984 and US patent application 698,905, filed February 11, 1985, corresponding to EP-A-156,559; US patent application 547,297, filed October 31, 1983 and US patent application 657,211, filed June 19, 1985, corresponding to EP-A-145,207; and US patent application 900,486, filed June 26, 1986 and US patent application 59,419, filed June 18, 1987, corresponding to EP-A-260,491; all of which are incorporated herein by reference.

While such disclosures constitute a large number of the PNP inhibitors, the instant invention is not so limited and can utilize any PNP inhibiting compound.

PNP inhibitors having an IC 5 0 of less than 5 tim for inhibition of PNP are preferred.

-9- A specific embodiment of the invention relates to use of a 2',3'-dideoxypurine nucleoside, e.g. 2',3'-dideoxyinosine or 2',3'-dideoxyadenosine, in combination with a 9-substituted guanine derivative, e.g. as disclosed in EP-A-178,178 (US patent 4,772,606), EP-A-156,559, Agents and Actions 21, 3/4 (1987) pp. 253-256 and Drugs of the Future 13, 653 (1988), or in combination with a 9-substituted-9-deazaguanine derivative of EP-A-260,491, e.g. of the formula R6

H

NJ

N

I/8 R7 N

(CH

2 -Ar n wherein R 6 is OH or SH; R 7 is hydrogen or NH 2

R

8 is hydrogen or NH 2 n is an integer of zero through four, and Ar is phenyl unsubstituted or substituted by halogen, alkyl of from one to four carbon atoms, hydroxy, alkoy of one to four carbon atoms, or trifluoromethyl, (ii) 2- or 3-thienyl, (iii) 2- or 3-furanyl; or a pharmaceutically acceptable salt thereof; and in particular a said compound wherein R 6 is OH and n is 1.

Illustrative examples of PNP inhibitors for use in combination are: formycin B, (b) 8-aminoguanine, 8-aminoguanosine, 1-p-D-ribofuranosyl- 1H-1,2,4-triazole-3carboxamidine, 8-amino-9-(1,3-dihydroxy-2-propoxymethyl)guanine, 9-deazaguanosine, 9-deazainosine, 5'-deoxy-5'-chloro-9-deazainosine, 5'-deoxy-5'-iodo-9-deazainosine, and 8-amino-9-deaza-9-(3-thienylmethyl)guanine, and 8-amino-9-benzylguanine, 8-amino-9-(2-thienylmethyl)guanine, and 8-amino-9-(3-thienylmethyl)guanine. Of these examples the PNP inhibitors of groups and are preferred. The most preferred PNP inhibitors are those of group Especially suitable PNP inhibitors include, without limitation, 8-aminoguanosine, 8-aminoguanine, and its 9-benzyl, 9-(2-thienylmethyl) and 9-(3-thienylmethyl) analogs, as well as 8-amino-9-deazaguanine and its 9-benzyl, 9 ienylmethyl) and 9-(3-thienylmethyl) analogs.

Purposely inhibiting the available PNP in a retrov' fected host, particularly a human immuno-deficiency virus infected host, can be tailored to allow 2',3'-dideoxypurine nucleosides to have a sufficient anti-retroviral effect in infected cells without any significant harm to uninfected cells.

Typically the PNP inhibitor of the invention is administered in an amount of about 0.5 to 150 mg/kg/day, which may be in as many divided doses as is convenient. In the most advantageous form, the PNP inhibitor dose administered inhibits no more than about 98 of the available PNP activity. The PNP inhibitor is administered at a dose which inhibits preferably about 50 to 98 more preferably about 65 to 95 and advantageously 75 to of the available PNP activity. Compositions of the PNP inhibitor may be formulated in the same way as compositions for the foregoing 2',3'-dideoxypurine nucleoside or the PNP inhibitor may simply be incorporated as a second active ingredient in the same formulation along with the 2',3'-dideoxypurine nucleoside.

The two active ingredients (ddPN and PNP inhibitor) may be administered simultaneously by the same or different routes, in the same or different formulations or may be administered at discrete points in time provided that there is effective PNP inhibition when the 2',3'-dideoxypurine nucleoside is present. The extent to which this time separation of administered active agents can be accomplished depends upon the amount of available PNP and the rate at which the PNP inhibitor is itself degraded. For these reasons, S the preferred dosage is in divided doses two to four times a day, most preferably with both agents being administered simultaneously.

The invention will be more fully understood with reference to the following examples which illustrate but do not limit the invention.

Example 1: 10000 Capsule Preparation: 2',3'-dideoxyadenosine 60 g 8-amino-9-(3-thienylmethyl)guanine 1400 g The two components are blended together and filled into capsules which are suitable for administration, e.g. 2 capsules every 8 hours, 3 times a day to an adult human.

Similarly prepared are formulations of other 2',3'-dideoxypurine nucleosides, e.g.

dideoxyinosine, in combination with a suitable PNP inhibitor.

CI_ -11 for 10000 capsules: 2',3'-dideoxyinosine 50 g 8-amino-9-deaza-9-(3-thienylmethyl)guanine (compound 9 of EP-A-260,491) 2000 g for 10000 capsules: 2',3'-dideoxyinosine 50 g 8-amino-9-(2-thienylmethyl)guanine 1500 g Example 2: The potentiation of the antiretroviral activity of dideoxy nucleosides by a PNP inhibitor can be determined in cell cultures, e.g. as follows: H9 cells [Popovic et al., Science 224, 497 (1984)] or ATH8 cells [Mitsuye and Broder, Proc. Natl. Acad. Sci. U.S.A. 83, 1911 (1986)] are treated with a particular dideoxypurine nucleoside (ddPN) either alone or in combination with an effective purine nucleoside phosphorylase (PNP) inhibiting amount of a PNP inhibitor. The cells are then exposed to the HIV virus, and after incubation for several days the number of viable cells are determined according to the experimental protocol outlined by Mitsuye and Broder, Proc.

Natl. Acad. Sci. U.S.A. 83, 1911 (1986). A greater number of viable cells in the cell culture treated with the combination of the ddPN and PNP inhibitor as compared to ddPN alone is indicative of potentiation of ddPN by the PNP inhibitor in protecting such cells from the cytopathic effect of the HIV virus.

Example 3: The effect of a purine nucleoside phosphorylase (PNP) inhibitor on the plasma concentration of a dideoxypurine nucleoside (ddPN) can be determined as follows: One group of Lewis rats are administered either p.o. or i.p. a non-toxic dose of the particular antiretroviral ddPN. Another group are administered the same dose of the particular ddPN after being administered either p.o. or i.p. a purine nucleoside phosphorylase inhibiting amount of a PNP inhibitor. Blood samples taken at various intervals are analyzed by HPLC for plasma concentration of the ddPN in both groups.

Elevation and/or prolongation of the plasma level of the ddPN in the group of rats treated with both the ddPN and PNP inhibitor is indicative of inhibition of the metabolic degradation of the ddPN and thus of the potentiation of the therapeutic effect of the particular ddPN by the administered PNP inhibitor.

For example, in rats administered 20 mg/kg i.v. of 2',3~-dideoxyinosine and about 5 to mg/kg i.v. of 8-amino-9-(2-thienylmethyl)guanine, plasma levels of 2,3-dideoxyinosine are increased and prolonged in comparison to rats administered the same dose of 2,3-dideoxyinosine alone.

Ot 0011 *0 $4 055 1 00 004k 0*1~ 0*0 0400 0 00 00 0 0 00 00 0 0 0* 00 4 *0 0 01151 0 11 00 0 00 13 In a concrete Example, it is demonstrated that didesoxyinosine (DDI) beta-phase half-life after i.v. application is potentiated when 8-amino-9-(2-thienylmethyl)guanine (PNP inhibitor) is coadministered a) as 0.5 mg/kg i.v. bolus or b) as a 10 mg/kg p.o. dose.

Without PNP inhibitor, the plasma half-life of DDI is 21.1 min. In case the plasma half-life increases to 74.1 min. In case plasma half-life increases to 46.8 min.

Detailed description of the assay: Purpose: To evaluate the plasma pharmacokinetics of DDI in the rat following i.v.

administration and the effects of concomitant administration of 8-amino-9-(2-thienylmethyl)guanine, a potent in vitro PNP inhibitor. Primary criterion is the beta-phase half-life of DDI in the presence and absence of p.o. or i.v. 8-amino-9-(2-thienylmethyl)guanine.

Materials: 1. Sprague-Dawley rats, male, mean wt. 303 58 g; 2. Dosing solutions: A) DDI in DMSO/saline at 25 mg/ml; B) DDI in DMSO/saline at 25 mg/ml plus 8-amino-9-(2-thienylmethyl)guanine at 1 mg/ml in 3% cornstarch solution; C) DDI (25 mg/ml) plus 8-amino-9-(2-thienylmethyl)guanine (0.5 mg/ml) in DMSO/saline; 3. HPLC grade methanol, water, Na 2 HP0 4 and NaH 2 P0 4 Bond-elut C18 solid phase cartridges (cartridges filled with reverse phase silica gel, Varian, USA).

Methods: TT 1) Animal surgery All rats used in the studies are surgically prepared 24 72 h prior to 2 -14 dosing by implantation of an indwelling carotid catheter which is exteriorized via a spring tether at the back of the neck. Animals are housed in metabolism cages for collection of urine (data not presented here).

2) Plasma sampling At 0.1, 0.16, 0.33, 0.65, 1, 2, 3, 6, 8 and 24 h post dose, blood is drawn by attaching a heparinized sampling syringe to the catheter. Plasma is immediately prepared, weighed and frozen pending analysis for DDI. From 1 6 h, blood cells from the previous timepoint are reinfused to preserve the hematocrit level throughout the study.

3) Dosing For i.v. dosing, rats are anesthetized with ether. The jugular is then exposed via a 1 cm incision, and the compound(s) are administered as an i.v. bolus at a volume of 1 ml/kg. Oral doses are administered by gavage (cornstarch solution) at a volume of ml/kg. In combination dosing, oral doses precedes the i.v. dosing by 6 10 min.

S 4) Dosing regimens For comparison of the kinetics of DDI, the following key is employed: Group number of animals Dosing protocol B 4 DDI, 25 mg/kg i.v. in DMSO/Saline E 4 as in B plus 8-amino-9-(2-thienylmethyl)guanine 10 mg/kg p.o.

F 1 DDI, 25 mg/kg i.v. in DMSO/saline G 3 as in F plus 8-amino-9-(2-thienylmethyl)guanine 0.5 mg/kg i.v.

Analytical analysis Sample preparation is achieved by the addition of an internal standard to the weighed plasma sample followed by application to Bond-elut solid phase C18 cartridges. Endogenous interferences are removed by washing with 2 ml 0.02 M ph 7 buffer, followed by 2 ml 5% methanol/water. The analytes of interest are eluted with 3 ml methanol/water, concentrated and reconstituted to a volume of 140 pl, of which 30 tl is injected onto the analytical column. Chromatographic separation is performed on a Supelcosil LC-18-DB column (reversed phase C18-silica gel, Supelco, USA) employing a P1EF AX 10% to 65% gradient (0.02 M pH 7 buffer to methanol) over 7.2 min followed by a 5 min

-L

I. 15 isocratic period. Retention time for DDI is 6.6 min under these conditions. Absolute errors and relative standard deviations are generally below indicating excellent accuracy and precision.

Protocol: Subgroups of a single shipment of rats are administered 8-amino-9-(2-thienylmethyl)guanine and/or DDI according to the Dosing Regimen.

Results: Evaluation of the DDI plasma concentration vs. time showes a biphasic decline in plasma levels, with a distributional phase dominant through 20 to 30 min followed by a slower (beta) elimination phase. In most cases, three or more data points are available for calculation of the beta-phase parameters.

Table: Pharmacokinetics of DDI in the rat Dosing regimen beta half-life (min) S.D.

B 21.1 min+/- 11.2 E 46.8 min 13.2 F 21.7 min G 74.1 min 14.0 Discussion: Group B vs group E: These two groups employ the same i.v. vehicles for the administration of DDI, thus any pK changes are isolated to the action of 8-amino-9-(2-thienylmethyl)guanine. The mean beta-phase half-life increases form 21.1 min in the DDI alone group to 46.8 min in the DDI plus inhibitor group, indicating clearly a substantial increase in the plasma half-life of DDI, presumably due to the inhibition of PNP by 8-amino-9-(2-thienylmethyl)guanine.

Group G vs. group F: Group F (1 rat) is run simply to eliminate the possibility that the change from 10% to 50% DMSO i.v. vehicle (necessary for dissolution of S^--7

L-

16 0*90 *991 900

OI,

019 0 p 8-amino-9-(2-thienylmethyl)guanine for i.v. dosing) would alter DDI kinetics. The vehicle change has no effect, as evidenced by the 21.7 min half-life for rat F and the 21.1 min mean half-life for group B. The administration of 8-amino-9-(2-thienylmethyl)guanine produces a definitive potentiation of DDI plasma pharmacokinetics. Mean DDI half-life increases from 21.1 min to 74.1 min, a nearly 4-fold increase.

Remark: In a multiple-dose co-administration study (DDI 20 mg/kg i.p. for 8 days in the presence and absence of daily doses of 10 mg/kg of the PNP inhibitor), mean DDI plasma levels are elevated 10 fold in the inhibitor-dosed group.

These data show that the systemic exposure of rats to DDI is increased when the PNP inhibitor is given simultaneously.

Example 4: This Example shows the effects of CGS 22695 (8-amino-9-(2-thienylmethyl)guanine administration on blood lymphocytes, splenic lymphocytes and thymus weights in rats in the presence and absence of coadministered dideoxyinosine (ddl).

Materials: 1. Sprague-Dawley rats, male 200-250 g for PNP inhibitor groups; 300 350 g for the cyclosporin groups.

2. PNP inhibitor suspended in 3% cornstarch.

3. ddl dissolved in 50% saline-DMSO.

4. Cyclosporin A (Sigma Chemicals, USA) suspended in 3% cornstarch.

LPS (lipopolysaccharide from S. typhosa) and ConA (concanavalin A) (Sigma Chemicals, USA); PHA (phytohemagglutinin) (Wellcome, GB).

6. Antibodies for FACScan analyses: mAbs to CD4 (clone W3/25, CD8 (clone 0x8) and B cells (clone MRC O=X-33) from Bioproducts for Science; goat anti-mouse Ig-FITC from Becton Dickinson.

17

METHODS:

1. WBC DIFFERENTIAL COUNTS DETERMINED ON A Technicon H-1 Hematology Analyzer. EDTA used as anticoagulant.

2. Spleen cells stimulated with 1.5 g/ml PHA, g/ml ConA, and 12.5 g/ml LPS and proliferative responses determined by incorporation of tritiated thymidine.

3. CD4/CD8 ratios determined with HD FACScan.

PROTOCOL:

Six groups of rats (4-5 animals/group) were administered PNP inhibitor, ddl, and/or cyclosporin A according to the following protocol.

Group I Vehicle control group. Administered cornstarch p.o. (1.0 ml/100 grams) and saline-DMSO i.p. (0.1 ml/100 grams) daily for 8 days.

SGroup II PNP inhibitor group. Adminstered mg/kg/day p.o. for 8 days.

Group III ddl group. Administered 20 mg/kg ddI i.p.

for 8 days.

Group IV ddl ;lus PNP inhibitor group. Administered mg/kg p.o. followed by 20 mg/kg ddl i.p.

1 hour later, for 8 days.

Group V Vehicle control group for cyclosporin. Same as group I.

Group VI Cyclosporin A administered 50 mg/kg p.o. for 8 days.

18 Prior to initial dosing blood samples were taken via cardiac punture under methoxyfluraneanesthesia and collected in EDTA. Blood samples were set aside for H-1 WBC differentials, FACScan lymphocyte analysis (DC4/CD8 ratios), and PNP analysis, and plasma harvested and frozen immediately for ddl and PNP analysis. Animals were weighed and dosing begun on day 0. On day 8 blood samples were obtained as above and animals sacrificed (C0 2 and spleens removed fro proliferation assays and thymuses removed and weighed.

In order to manage the blastogenic assays on a given S day it was necessary to stagger the six groups when initiating their dosing regimens. Consequently, groups I and II were begun together as were groups III and IV, and V and VI. Due to the day to day variation in the proliferation assays group I can only be compared with group II, group III with group IV, and V with VI. In addition, 'groups V and VI represent a different batch of animals than the remaining groups.

RESULTS:

Spleen Cell Proliferation.

SFigure 1 shows the results of spleen cell proliferations with Groups I and II. PNP inhibitor administration resulted in a 29% decrease (p<0.05) of PHA stimulated proliferation and a 15% decrease in ConA stimulated proliferation (p<0.01) when compared to the vehicle control group. LPS stimulated proliferation was not different between the two groups.

S- 19 Figure 2 indicates that there were no differences between Groups III and IV in proliferation responses to PHA and ConA. A slight but significant (p<0.05) decrease in LPS stimulated proliferation was noted between these two groups. Therefore, PNP inhibitor administration appeared to diminish the LPS response only when coadministered with ddl.

Figure 3 demonstrates the affect of cyclosporin A.

Administration of cyclosporin appeared to suppress the proliferative response to all 3 stimuli. Responses to PHA, ConA, and LPS were inhibited 81 51 and 57% respectively.

4 4 4I,4 Splenic And Blood Lymphocyte CD4/CD8 Ratios.

Peripheral Blood Group Spleen (day 7) Prebleed Post Treatment *1-2 I 1.4 0.3 2.3 0.4 1.9 0.2 II 1.4 0.2 2.1 0.1 2.0 0.1 III 1.4 1 0.3 2.0 0.2 1.9 0.2 IV 1.3 0.2 2.0 0.2 V N.D. N.D.

VI 0.8 0.1 1.8 0.3 2.6 0.6 N.D. not done.

Ratio represents the results of 3 of 5 in this group.

The CD8 cells were undetechable in the remaining 2 out of the 5 rats thus not allowing ratio determination.

There were no differences in CD4/CD8 ratios between groups I and II for both splenic lymphocytes and peripheral blood lymphocytes indicating that PNP inhibitor 20 administration did not alter the balance between T helper cells and T suppresser cells. In the case of peripheral blood lymphocytes drug administration did not alter the total number of lymphocytes or percentage of WBC (see table below). It is therefore apparent that PNP inhibitor administration was without affect on the circulating lymphocyte population.

There is a suggestion that CGS 22695AE administration, when given with ddl, alters the splenic CD4/CD8 ratios as can be seen when comparing groups III and IV.

S, The spleen cells from the cyclosporin treated animals 0* tend to have a lower CD4/CD8 ratio 1) than all of the treatment groups.

S*

Blood Lymphocyte Analyses.

S. Day 0 Day 7 SGroup* Total(10 6 ml) of WBC Total of WBC I 8.7 1.3 81.4 1.0 8.6 1.0 82.1 2.6 II 8.9 1.2 82.5 1.5 8.0 1.0 84.9 III 7.4 0.9 83.2 0.9 5.7 1.0 82.4 IV 7.8 1.1 83.4 2.0 6.1 0.5 80.8 3.2 V 9.6 1.2 84.3 0.7 6.5 1.0 74.7 7.1 VI 11.4 0.5 83.7 0.6 5.6 1.1 72.8 When comparing total or of WBC at day 0 and day 7 there are no marked changes with the single exception of group VI. It appears that cyclosporing administration reduced the total lymphocyte counts without altering the of WBC.

21 Thymus and Whole Body Weights.

Whole Body Group* Thymus (grams) Weight Gain/Loss I 0.62 0.10 46 II 0.59 0.05 54 III 0.55 0.06 26 IV 0.59 0.04 V 0.64 0.03 18 VI 0.60 0.05 -18 *Groups I-IV represent a single "batch" of animals.

Groups V-VI represent a separate (from I-IV) batch of animals.

There were no marked differences in thymus weights among the various groups except the cyclosporin group, which i r t experienced a weight loss. There appeared to be a lesser weight gain in ddl administered animals.

In summary, Spleen Cell Proliferation is a measure of immnunosuppression by a drug. Figure 1 shows that PNP Inhibitor causes immunosuppression (less spleen cell proliferation) than control. Figure 2 shows that when PNP inhibitor is added to DDI, no further immunosuppression results over the DDI alone. Figure 3 shows the positive control of a known immunosuppressant, cyclosporin A, on splean cell proliferation.

Claims (21)

1. A method of treating a mammal, including a human, having a retrovirus infection comprising administering to said mammal an anti-retroviral effective amount of a dideoxypurine nucleoside and a purine nucleoside phosphorylase inhibiting amount of a purine nucleoside phosphorylase inhibitor, with the proviso that said 2',3' dideoxypurine nucleoside (ddPN) and said purine nucleoside phosphorylase inhibitor (PNP inhibitor) are administered sufficiently close in time such that the PNP inhibitor prevents the metabolism of the 2',3'-dideoxypurine nucleotide by native purine nucleoside phosphorylase or lessens the rate at which the 2',3'-dideoxypurine nucleoside is metabolized by this enzyme. S 2. The method of claim 1 wherein said retrovirus is a human immunodeficiency virus. o3. The method of claim 1 wherein said 2',3'-dideoxypurine nucleoside is selected from compounds of the formula HOH2C 0 B A wherein A is hydrogen, azido, or halogen; B is the radical of a purine base bound through the 9-position thereof; and the hydroxy is free or derivatized as a metabolizable ester thereof; or a pharmaceutically acceptable salt thereof.

4. The method of claim 3 wherein the compound of formula I is selected from 2',3'-di- deoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine, 2',3'-dideoxythioinosine, 2',3'-dideoxythioguanosine and 2',3'-dideoxy-2-aminoadenosine. The method of claim 3 wherein the compound of formula I is selected from 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine and 2',3'-dideoxythioinosine. -23-

6. The method of claim 3 wherein the compound of formula I is 2',3'-dideoxyadenosine.

7. The method of claim 3 wherein the compound of formula I is 2',3'-dideoxyinosine.

8. The method of claim 1 wherein the purine nucleoside phosphorylase inhibitor is selected from formycin B, 8-amninoguanine, 8-aminoguanosine, 1-P-D-ribo- furanosyl- 1H- 1,2,4-triazole-3-carboxamidine, 8-amino-9-( 1,3-dihydroxy-2-propoxy- methyl)guanine, 9-deazaguanosine, 9-deazainosine, 5'-deoxy-5 '-chloro-9-deazainosine, 5'-deoxy-5'-iodo-9-deazainosine, and 8-amino-9-deaza-9-(3-thienylmethyl)guanine, and 8-am ino-9-benzylguanine, 8-amino-9-(2-thienylmethyl)guanine and 8-arnino-9-(3- thienylmethyl)guanine.

9. The method of claim 8 wherein said purine nucleoside phosphorylase inhibitor is selected from groups and The method of claim 8 wherein said purine nucleoside phosphorylase inhibitor is selected from group

11. The method of claim 1 wherein said 2',3'-dideoxypurine nucleoside is selectd from 3-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine, 2',3'-dideoxy- thioinosine, 2',3-dideoxythioguanosine and 2',3'-dideoxy-2-amninoadenosine and said purine nucleoside phosphorylase inhibitor is selected from formycin B, (b) 8-aminoguanine, 8-aminoguanosine, 1 -0-D-ribofuranosyl- 1H-I ,2,4-triazole-3-carbox- amidine, 8-amino-9-( 1,3-dihydroxy-2- propoxy-iethyl) guanine, 9-deazaguanosine, 9-deazainosine, 5'-deoxy-5'-chloro-9-deazainosine, 5'-deoxy-5 '-iodo--9-deazainosine, and 8-amino-9-deaza-9-(3-thienylmethyl)guanine, and 8-amino-9-benzylguanine, 8-amino-9-(2-thienylmethyl)guanine and 8-amino-9-(3-thienylmethyl)guanine.

12. The method of claim 11 wherein said 2',3'-dideoxypurine nucleoside is 2',3'-dideoxy- inosine.

13. The method of claim 11I wherein said 2',3'-dideoxypurine nucleoside is 2',3'-dideoxy- inosine and said purine nucleoside phosphorylase inhibitor is selected from 8-amino- 9-benzylguanine, 8-amino-9-(2-thienylmethyl)guanine and 8-aniino-9-(3-thienyl- C2~ methyl)guanine. I ~Fl r 24

14. The method of claim 1 wherein said anti-retroviral effective amount is 0.01 to mg/kg/day and said purine nucleoside phosphorylase inhibiting amount is 0.5 to 150 mg/kg/day. The method of claim 1 wherein said 2',3'-dideoxypurine nucleoside and said purine nucleoside phosphorylase inhibitor are administered simultaneously.

16. The method of claim 1 wherein said 2',3'-dideoxypurine nucleoside and said purine nucleoside phosphorylase inhibitor are administered at different points in time.

17. A method of enhancing the effectiveness of a 2',3'-dideoxypurine nucleoside in the treatment f a 2',3'-dideoxynucleoside responsive condition in a mammal except of a S human ,avin: said condition comprising administering to said mammal a purine nucleo- side pt-p :.tir i- inhibiting amount of a purine nucleoside phosphorylase inhibitor, with the pro'; a2',3'-dideoxypurine nucleoside (ddPN) and a purine nucleoside phosphorylase inhibitor (PNP inhibitor) are administered sufficiently close in time such that the PNP inhibitor prevents the metabolism of the 2',3'-dideoxypurine nucleotide by native purine nucleoside phosphorylase or lessens the rate at which the 2',3'-dideoxypurine nucleoside is metabolized by this enzyme.

18. A method of enhancing the effectiveness of 2',3'-dideoxyinosine in the treatment of a 2',3'-dideoxynucleoside responsive condition in a mammal except of a human having said condition comprising administering to said mammal a. purine nucleoside phosphorylase inhibiting amount of a purine nucleoside phosphorylase inhibitor, with the proviso that a 2',3'-dideoxypurine nucleoside (ddPN) and a purine nucleoside phosphorylase inhibitor (PNP inhibitor) are administered sufficiently close in time such that the PNP inhibitor prevents the metabolism of the 2',3'-dideoxypurine nucleotide by native purine nucleoside phosphorylase or lessens the rate at which the 2',3'-dideoxypurine nucleoside is metabolized by this enzyme.

19. A method of slowing the in-vivo decompositon of a 2',3'-dideoxypurine nucleoside in a mammal in need of a 2',3'-dideoxypurine nucleoside treatment except of a human comprising administering to said mammal a purine nucleoside phosphorylase inhibitor, with the proviso that a 2',3'-dideoxypurine nucleoside (ddPN) and a purine nucleoside phosphorylase inhibitor (PNP inhibitor) are administered sufficiently close in time such PLfj that the PNP inhibitor prevents the metabolism of the 2',3'-dideoxypurine nucleotide by 25 i native purine nucleoside phosphorylase or lessens the rate at which the 2',3'-dideoxypurine nucleoside is metabolized by this enzyme. A method of slowing the in-vivo decompositon of 2',3'-dideoxyinosine in a mammal in need of a 2',3'-dideoypurine nucleoside treatment except of a human comprising ad- ministering to said mammal a purine nucleoside phosphorylase inhibitor, with the proviso that a 2',3'-dideoxypurine nucleoside (ddPN) and a purine nucleoside phosphorylase inhibitor (PNP inhibitor) are administered sufficiently close in time such that the PNP inhibitor prevents the metabolism of the 2',3'-dideoxypurine nucleotide by native purine nucleoside phosphorylase or lessens the rate at which the 2',3'-dideoxypurine nucleoside is metabolized by this enzyme.

21. The method of claim 16 wherein said purine nucleoside phosphorylase inhibitor is administered sufficiently close in time to the administration of said 2',3'-dideoxypurine nucleoside such that a substantial portion of the native pi .n,e ucleoside phosphorylase is inhibited during a substantial portion of the time said 2',3'-dideoxypurine nucleoside is available for degradation by said native purine nucleoside phosphorylase.

22. The method of claim 1 wherein no more than 98 of the native purine nucleoside phosphorylase of a retroviral infected cell is inhibited by said administration of said purine nucleoside phosphorylase inhibitor.

23. The method of claim 1 wherein 50-98 of the native purine nucleoside phosphorylase of a retroviral infected cell is inhibited by said administration of said purine nucleoside phosphorylase inhibitor.

24. A composition for the treatment of a 2',3'-dideoxypurine nucleoside responsive condition comprising an effective amount of a 2',3'-dideoxypurine nucleoside and a purine nucleoside phosphorylase inhibiting effective amount of a purine nucleoside phosphorylase inhibitor. A composition according to claim 24 wherein said 2',3'-dideoxypurine nucleoside is selected from 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine, 2',3'-dideoxythioinosine, 2',3'-dideoxythioguanosine and 2',3'-dideoxy-2-aminoadenosine. A composition according to claim 24 wherein said purine nucleoside phosphorylase 11 Ii 26 inhibitor is selected from formycin B, 8-aminoguanine, 8-aminoguanosine, I -j-D-ribofuranosyl- 1H-1I,2,4-triazole-3-carboxamidine, 8-amino-9-( 1,3-dihydroxy-2- propoxymethyl)guanine, 9-deazaguanosine, 9-deazainosine, 5'-deoxy-5 '-chloro-9- deazainosine, 5'-deoxy-5'-iodo-9-deazainosine, and 8-amino-9-deaza-9-(3-thienyl- methyl)guanine, and 8-amino-9-benzylguanine, 8-amino-9-(2-thienylrnethyl)guanine and 8-arnino-9-(3-thienylmethyl)gua-nine.

27. A composition according to claim 24 wherein said 2',3'-dideoxypurine nucleoside is selected from 2',3'-dideoxyadenosine, 2',3'-dideoxygua-nosine, 2',3'-dideoxyinosine, 2',3'-dideoxythioinosine, 2',3'-dideoxythioguanosine and 2',3'-dideoxy-2-a-minoadenosine and said purine nucleoside phosphorylase inhibitor is selected from formycin B, (b) 8-aminoguanine, 8-aminoguanosine, 1 -J-D-ribofuranosyl- 1H-i ,2,4-triazole-3-carbox- amidine, 8-amino-9-(1 ,3-dihydroxy-2-propoxymethyl)guanine, 9-deazaguanosine, 9-deazainosine, 5'-deoxy-5'-chloro-9-deazainosine, 5'-deoxy-5'-iodo-9-deazainosine, and 8-amino-9-deaza-9-(3-thienylmethyl)guanine, and 8-amino-9-benzylguanine, 8-axnino-9-(2-thienylmethyl)guarnine and 8-amino-9-(3-thienylmethyl)guanine.

28. A composition according to claim 24 wherein said 2',3'-dideoxypurine nucleoside is 2',3'-dideoxyinosine.

29. A method of treating a mammal having a retrovirus infection by coadministration of dideoxyinosine and 8-amino-9-(2-thienylmethyl)guanine substantially as herein described with reference to Example 3. A composition for the treatment of a 2',3'-dideoxypurine nucleoside responsive condition substantially as herein described with reference to Example 1. DATED this 29th day of June, 1992. CIBA-GEIGY AG By Their Patent Attorneys DAVIES COLLISON CAVE