AP312A

AP312A - Administering particular compounds against various parasites, mycoplasmas, other indications and other infections.

Info

Publication number: AP312A
Application number: APAP/P/1991/000316A
Authority: AP
Inventors: Patrick T Prendergast
Original assignee: Patrick T Prendergast
Priority date: 1990-06-21
Filing date: 1991-06-21
Publication date: 1994-02-19
Also published as: PH30703A; CA2045279A1; AP9100316A0; AU7775894A; EP0462621A3; AU7920491A; EP0462621A2; OA09370A

Abstract

One or more compouds are administered against

Description

ADMINISTERING PARTICULAR COMPOUNDS AGAINST VARIOUS PARASITES, KYCOPLASMAS, OTHER INDICATIONS AND OTHER INJECTIONS

This invention relates to administering compounds to beings afflicted with one or more parasites (each believed to be a haemoflagellate parasite of nan), and to administering compounds to beings afflicted with disease caused by such parasites. More particularly, but not limited thereto, this aspect of the invention relates to administering compounds to beings afflicted with disease caused by one or more of trypanosomia, lsishmanla, toxoplasma and/or enterocytozoon bieneusi. This invention also relates to specific forms in which said compounds can be provided to facilitate administering them.

Thia invention further relates to administering compounds to beings infected with Mycoplasma, to administering compounds to beings afflicted with disease caused by Mycoplasma, and to specific forms in which said compounds can be provided to facilitate administering them,

This invention further relates to administering compounds to beings suffering from any one or more of the following indications or infections (a) hairy leukoplakia, (b) oral candidosis, (c, mouth ulcerations-aphthous/herpetlc/bactsrial, (d) fungal Candida, (e) human papilloma virus, (f) molluscum eontagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (1) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes zoster, and (1) rotaviruses. More particularly, but not limited to, this aspect of the Invention relates to administering compounds to HIV Infected beings afflicted with oral disease. This invention also relates to specific forms in which said compounds can be provided to facilitate administering them,

This invention additionally relates to administering compounds to beings to combat viruses and prions (proteinaceous infectious particles) e.g. CMV, Herpes Simplex, Hepatitis 8, Scapie Creutzfeldt-Jakob Disease. Particularly, this aspect of the invention relates to administering compounds to beings suffering from certain retroviral infections, and to beings suffering from infection by retroviruses related to human imauno-deficisncy viruses (HXV) and to methods of prophylactic drug treatment of beings who may be suffering from such infec2 fcione. More particularly, this aspect of the invention relates to administering compounds to beings to reduce levels of HIV virus in blood cells such as the monocyte/macrophage and T lymphocytes of the person being treated. This invention also relates to specific forms in which said compounds can be provided to facilitate administering them.

BACKGROUND OF THE INVENTION

The haemoflagellate parasites of man, despite their close taxonomic relationship within the family Trypanosomatidae, a shared range of morphological types, and common elements of structure and biochemistry, cause a diverse variety of diseases including African and American trypanosomiasis, and leishmaniasis (a person was reportedly exposed to leishmania while in Ireland, an area previously thought to be free of leishmania). This diversity is partially a reflection of the different extracellular and intracellular sites of development adopted by these trypanosomatids . Currently available compounds for use against such parasites suffer from various drawbacks, most notably their unacceptable toxicity.

A considerable effort is currently underway to find more efficacious and less toxic compounds for the clinical treatment of leishmaniasis and trypanosomiasis. A proliferation of studies on the chemotherapy of Leishmania and rational biochemical approaches in studies on Trypanosoma brucei have recently produced promising new leads, but many of the problems associated with the treatment of South American trypanosomiasis {Chagas disease) remain intractable.

Hairy leukoplakia is apparently unique to patients infected with HIV. It consists of white warty like projections occurring particularly on the lateral aspects of the tongue and the cheeks. Its cause is unknown, but electron microscopic studies suggest that a virus (possibly Epstein-Barr or the human papilloma virus) may be

BAD original

AP 0 0 0 3 1 2 implicated. Although it is usually painless, its unsightliness worries many patients.

One of the hardest problems confronting the physician dealing with a patient with HIV antibodies is the difficulty of predicting which patients will progress to AIDS. But there are both clinical features and laboratory markers which may help. Clinical features of a poor prognosis are oral candidosis, herpes, herpes zoster, hairy leukoplakia, and the presence of fever, malaise, diarrhoea, or weight loss.

It is believed that human infection by the family of retroviruses known as HIV is deleterious to the health of infected persons. Examples of viruses which are currently believed to belong to the hiv family are the lymphadenopathy-associated virus (LAV) and the human Tlymphotrophic virus type III (HTLV-III). LAV and HTLVIII, which were discovered independently of each other, are now known to be the same virus and are referred to as Hiv-I. Although much is known about modes of transmission of such viruses from person to person, (THE NATURAL HISTORY OF HIV INFECTION IN A COHORT OF HOMOSEXUAL AND BISEXUAL MEN: A DECADE OF FOLLOW UP. Nancy A. Hessol,

G.W. Rutherford, A.R. Lifson, P.M. O'Malley, Dept. of Public Health, San Francisco, CA) there is currently controversy regarding particular interactions between the virus and the host cells in which they reside. Generally, a person who is infected by HIV develops antibodies to the virus and at some point, the immune system of the person becomes damaged and becomes ineffective in defending the body from diseases. This condition has come to be known as Acquired Immune Deficiency Syndrome, or AIDS. Eventually, because of the immune deficiency of his or her body, an AIDS patient is overcome by one or more of a group of opportunistic infections, for example, Kaposi’s Sarcoma and pneumocystis.

There is evidence that macrophage/monocyte infection is a factor in the progression of HIV infection, in initiating the brain damage that is known to occur in AIDS

BAD ORIGINAL

Nicholson et al. have effect in monocyte patients, and in triggering the collapse of the immune system as evidenced by eventual profound depletion of T4 lymphocytes. It has been demonstrated using anti-HIV p24 antibody that monocyte/macrophages can be infected with hiv. Up to 70% of cells from individual donors could be infected (Crowe et al., AIDS Research and Human Retroviruses 3, no. 2, (1987) 135).

proposed an HIV-III /LAV-induced function rather than (or in addition to) an intrinsic defect in surviving T cells to account for observed abnormalities in T cell assays that are monocyte-dependent such as pokeweed mitogen-induced antibody synthesis and proliferative responses to soluble antigens. These T cell assays have previously been reported as abnormal even when assayed as T cell subsets (The Journal of Immunology, 137, No. 1, (1986) 323).

Since it is well established that one of the first events that occurs when a foreign material (for example, a virus) enters the body is its uptake by mononuclear phagocytes, it is conceivable that these cells represent a primary target for HIV. Gartner et al. have shown that virus production by HTLV-III/LAV infected macrophages was high and long-lived, indicating that these cells may play a role in virus dissemination and persistence. They have demonstrated HTLV-III/LAV replication in macrophages was fully productive in the situations they evaluated (Science 233 (1986) 215).

Salahuddin et al. observed that in-vitro pulmonary macrophages can be infected with HTLV-III and appear to be less susceptible to the phytopathic effects of this retrovirus, which suggests that tissue macrophages should be considered as potential reservoirs of HTLV-III in-vitro (Blood 68, No. 1, (1986) 281).

Ho D.D. et al observed normal blood-derived monocytes/macrophages were found to be susceptible to infection in-vitro by HTLV-III. In addition, HTLV-III was recovered from monocytes/rnacrophages of patients infected with this virus. It was postulated therefore that HTLVbad original

AP 0 0 0 3 1 2

Ill-infected monocyte/macrophages may serve as a vehicle for the dissemination of virus to target organs and as a reservoir for viral persistence, as has been shown for other lentiviruses, including viena virus and caprine arthritis encephalitis virus (J. Clin Invest. 77, (198) 1712).

Anti-viral agents which inhibit replication of viruses have been known since the mid 1960's. (PROSPECTS FOR THE PREVENTION AND THERAPY OF INFECTIONS WITH THE HUMAN IMMUNODEFICIENCY VIRUS. Markus Vogt, Martin S. Hirsch, Infectious Disease Unit, Massachusetts General Hospital, Harvard Medical School, Boston). Several hundred or more of these agents are now known but azidothymidine (AZT, zidovudine) is the only drug which has received approval from the Federal Drug Administration in the United States for treatment against the virus of people with AIDS. The use of AZT in the treatment of AIDS patients suffers from many deficiencies. AZT is very expensive. Treatment with AZT often causes side effects in persons being treated with it and often the side effects are so severe that treatment with it must be halted altogether. (DEVELOPMENT OF HIV-VARIANTS WITH HIGHER RESISTANCE AGAINST AZT UNDER TREATMENT WITH AZT.

F. Zimmermann, L. Biesert, H von Briesen, Klinikum der Universitat, Frankfurt, FRG.) The long term effectiveness of treatment with AZT of AIDS patients is still unknown, although it is believed that AZT treatment will not result in the elimination of the virus from the body of an infected person. There is evidence that AZT-resistant strains of HIV are developing in AIDS patients being treated with AZT (F. Zimmermann and L. Biesert).

As a further background to particular aspects of the present invention, the compound N*- (a²-isopentenyl) adenosine, (IPA), which has formula la, illustrated below, has been used previously in clinical trials involving the treatment of cancer. (CYTOKININS AS CHEMOTHERAPEUTIC AGENTS, Annals of the New York Academy of Science, 25, 225-234 Mittleman, Arnold et al. (1975)). IPA is a

BAD ORIGINAL naturally occurring compound. For example, it has been shown to be an anticodon-adjacent nucleoei.de in certain tRNAs (Ν*- (Δ¹- ISOPENTENYL) ADENOSINE: THE REGULATORY EFFECTS OF A CYTOKININ ANO MODIFIED NUCLEOSIDE FROM t-RNA ON HUMAN LYMPHOCYTES. Biochimica et Biophyeica Acta, 281:468-500. Gallo, Robert C., et al. (1972)). IPA haa been shown to have cytqkinin properties, (Mittleman, et al.) to inhibit the growth of human leukemic myeloblasts, to inhibit the growth of cultured lymphocytes stimulated by phytohereagglutinin (PKA) at certain concentration! and to stimulate the growth of cultured lymphocytes stimulated by PKA at lower concentrations (Gallo, et al.). Further, IFA has been used in clinical experiments on humane as a chemotherapeutic agent (Mittleman, et al.}.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, one or more compounds according to Formula I (set forth below) is administered to a being (in particular, a human or an animal) afflicted with one or more parasites (e.g., lelshmanla, trypanosomia, toxoplasma and/or enterocytozoon bieneusi) and/or afflicted with disease caused by such parasites. Such administration of compound(s) Is effective against such parasites and diseases, and is of acceptable toxicity.

According to another aspect of the present invention, one or more compounds according to Formula I (set forth below) is administered to a being (in particular, a human or an animal) afflicted with one or more Mycoplasma (in particular, Mycoplasma arthritidis, Mycoplasma fermentans and Mycoplasma incognitus) and/or afflicted with disease caused by such Mycoplasmss.

According to another aspect of the present invention, one or more compound according to Formula I (set forth below) is administered to a being (in particular, a human or an animal) suffering from one or more of the following indication or infections (a) hairy leukoplakia, (b) oral candidosis, (c) mouth ulceratione-aphthous/herpetic/bacterial, (d) fungal Candida, (e) human papilloma virus, (f) molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes zoster, and (1) rotaviruses. Such administration

AP ο 0 0 3 1 2 of compound(g) ig affective agsinat »uch indication! or infection*.

According to another aspect of the preaent invention, one or more compound according to Formula I (set forth below) ia administered to a being (in particular, a human or an animal) auffaring from viral infection and/or affliction with prione (proteinaceous infectious particles) e.g. CHV, Herpes Simplex, Hepatitis B, Scapl* Creutxfeldt-Jakob Diteas*. Particularly, thia aspect of the invention relates to administering compounds to beings suffering iron certain retroviral infections, and to beings suffering from infection by retroviruses related to human immuno-deflciency viruses (HIV) and to methods of prophylactic drug treatment of beings who may be suffering from such Infection. Such administration of compound(s) is effective against such viral infection and/or prions,

The present invention furthermore provides method* of treatment, therapeutic or prophylactic, against any viral infection and materials which may be used in auch methods. Further, the invention provides treatment for an organism infected with (and/or exhibiting abnormal levels of): (a) hairy leukoplakia, (b) oral candidosls, (c) mouth ulcerations-aphthous/herpetic/bacterlal, (d) fungal Candida, (e) human papilloma virus, (f) molluscuro eontagiosum, (g) squamous oral carcinoma, (h) Kaposi’s sarcoma oral lesions, (i) periodontitis, (3) necrotizing gingivitis, (k) orofacial herpes zoster, (1) rotaviruses, parasites, diseases caused by such parasites, Mycoplasmas and diseases caused by Mycoplasmal, and HIV. The invention also provides the pharmaceutical formulations themselves. Certain embodiments of the invention provide methods of treating blood samples to reduce levels of HIV, parasites, Mycoplasmal or characteristics indicative of (a) - (1) or disease caused by parasites, relative to untreated samples.

Further, the invention provides a process for preventing alteration of morphology or function of a cell latently or actively infected by the HIV virus genome.

Such an example would be the ability of IFA to prevent expression of the tat gene product in epidermal Langerhan

BAD ORIGINAL s · · l. ί! I MA cell· and thus prevent or cause to regress resultant epidermal morphological abnormalities and tumors in patients infected with HIV.

The present invention employe one or more 5 compound according to Formula I:

Formula I wherein:

^Ri ^Ri

H, or

H# Rj CH₃, R₃ CKj and R4

and R₅ «7

CH₃ Cl, OH or a monophosphate group CH₃, CH₂OH or Cl

H or Br

AP Ο Ο Ο 3 1 2

and X₁ and X₂ are independently 5 selected fro» H, methyl, ethyl, hydroxyl, the halogens and carboxyl or R₄ = CHj

« or R₄ = CNH-R_eand

Re Cl or R, 5 (CH₂)_?CH₃;

and R₂ - OH and R₃ » OH, monophosphate, diphosphate or triphosphate group or R₂ and R₃ are linked to form a 3', 5’-cyclic monophosphate derivative, or a physiologically acceptable salt of any such compound.

Formula I is used herein to refer to all of such compounds and salts, as well as (when referring to administration against one or more parasite) polymer of IPA —

BAD ORIGINAL Q ι· t τ η τ* ιι(|Α 10 identified herein as Poly N6- Isopentenyl Adenosine--/ preferably comprising 2-3 monomers.

Listed below are chemical groups R₁-R₄ for especially preferred compounds la-ϊυ according to this invention.

la: R₁ = H, Rj - OH, ^R4 ⁼ CH, _ CH,

R₃ = OH and

CHj

H *3 CHj

S 2

Ν -(Δ -isopentenyl) adenosine lb:

R₄ H, Rj CH₂

H

OH, Rj = monophosphate, and CHj CHj

2

Ν -(Δ -isopentenyl) adenosine-5 ¹-monophosphate

Ic:

R₁ * Η, Rj and Rj are linked to form a

3', 5'-cyclic monophosphate derivative, and

R₄ =

CHj

H

CH,

2

N - (Δ-isopentenyl) adenosine-3', 5'cyclic monophosphate

Id: Rt - H, Rj « OH, Rj = OH, and R₄ - CHjC₆H₆N⁶-benzyladenosine le: R₁ = H, Rj = OH

Rj = monophosphate, and R„ « CH₂C₆H₆N⁶-benzyladenosine-5'-monophosphate

If: R₁ = H, Rj and R₃ are linked to form a

3', 5'-cyclic monophosphate derivative and r₄ = ch₂c₆h₆

N⁶-benzyladenosine-3', 5'-cyclic monophosphate

AP 0 0 0 3 1 2 lg: R₁ H, R₂ OHi R₃ ⁼ OH, ^s CHj lh;

Furfuryladenosine R, - H, R₂ = OH, R₃ ^R* ’ CH₂ monophosphate and

Ii:

N⁶-furfuryladenosine-5'-monophosphate

R₁ = H, Rg and Rj are linked to form a

3', 5'-cyclic monophosphate derivative, and R< = CH₂

N⁸-furfuryladenosine-3¹, 5'-cyclic monophosphate

Ij: Rt - H, R₂ = OH, R₃ = OH and

Cl

N- (purin-6-ylcarbamoyl)-o-chloroaniline ribonucleoside

Ik: R-, s h, R₂ s OH, R₃ - monophosphate and

Cl

N- (purin-6-ylcarbamoyl)-o-chloroaniline ribonucleoside-5'-monophosphate

II: R, « H, R₂ « OH, R₃ = OH and

N^e-adamantyladenosine ^AD ORIGINAL d

Im: R-, *4 s Μ η Π Β ΜΑ

monophosphate and

N⁶-adamantyladenoeine-5¹-monophosphate In; R, - H, R₂ OH, R₃ OH and

II

R₄ = CNH(CHj,)₇CH₃

N-(purin-6-ylcarbamoyl)-n-octylamine ribonucleoside

Io; R, H, R₂ = OH, R₃ = monophosphate and o

II

R₄ = CNH(CH₂)₇CH₃

N-(purin-6-ylcarbamoyl)-n-octylamine ribonucleoside-5'-monophosphate

Ip: R-! * H, R₂ and R₃ are linked to form a

3'-5'-cyclic monophosphate derivative, and ii

R₄ = CNH (CH₂) ₇CH₃

N-(purin-6-ylcarbamoyl)-n-octylamine ribonucleoside-3', 5'-cyclic monophosphate

N⁶-(a²-isopentenyl)-2-methy1thioadenosine

N¹-(4-hydroxy-3-methvl-trans-2-butenyl)j) adenosine

AP 0 0 0 3 1 2

Is:

R. · H, R₂ = OH, R₃ » ^R* ^e CH,

OH, and Cl

CHj it:

N⁶ -(3-chloro-fcEftnB-2-butenyl) adenosine R1 H, R2 OH, Rj = OH and ^R* - CH2 ,

H

N⁶ - (3-chloro-cis-2-butenvl) adenosine

Iu: R, - H, R_z * CH₃, Rj - CH₃ and R< H

The present invention also employs one or more metabolite of the family of compounds of Formula I. For example, preferred metabolites include:

S 2

Ν-(Δ -isopentenyl) adenine;

6-N-{3-methyl-3-hydroxybutylamino) purine; Adenine;

Hypoxanthine;

Uric Acid; and Methylated xanthines.

According to another aspect of this invention, there are provided specific forms in which the compounds of Formula I can be administered.

The present invention further provides use of any compound of Formula I in the manufacture or preparation of formulations, and especially pharmaceutical formulations, for use against (a) hairy leukoplakia, (b) oral candidosis, (c) mouth ulcerations-aphthous/herpetic/bacterial, (d) fungal Candida, (e) human papilloma virus, (f) molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes zoster, (1) rotaviruses, against parasites and diseases caused by such

DAD ORIGINAL parasites, against Mycoplasma* and diseases caused by Mycoplasma*, and againet HIV. The invention also provides the pharmaceutical formulations themselves,

This invention also relates to establishing improved lmmuno response in beings in whom immunodeficiency is considered a future risk.

MTAILES -DESCRIPTION ΡΓ ΤΗΣ INVENTION

Compounds uaed according to this invention are administered by any suitable route including enteral, parenteral, topical, oral, rectal, nasal or vaginal routes. Parenteral routes include subcutaneous, intramuscular, intravenous and sublingual administration. Topical routes Include buccal and sublingual administration.

Pharmaceutical formulations prepared according to the invention include at least one compound of Formula I contained in a macrophage specific liposome micell of suitable size to facilitate phagocytosis, a gelatine capsule, in tablet form, dragee, syrup, suspension, topical cream, suppository, injectable solution (such as a pharmaceutically acceptable solution which may include a carrier), or kits for the preparation of a syrup, suspension, topical cream, suppository or injectable solution just prior to use. Also, compounds of Formula I may be included in a composite which facilitate Its slow release Into the blood stream, e.g., a silicone disc, polymer beads or a transdermal patch.

Pharmaceutical preparations prepared according to the invention include the employment of compounds of Formula I in admixture with conventional excipients, that la, pharmaceutically acceptable organic or inorganic carrier substances which do not deleteriously react with the compounds. Suitable pharmaceutically acceptable carriers include (but are not limited to) water, salt solution», alcohols, gum arable, vegetable oils, gelatine, carbohydrates, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid mono- and di-glycerides, etc.

The preparative procedure may include the sterilization of the pharmaceutical preparations. The com-

pound* nay be mixed with auxiliary agent* such aa lubricant*, preservatives, stabilizers, salts for influencing osmotic pressure, etc., which do not react deleteriously with the compounds.

The present invention further provides use of any compound of Formula I combined with an adenosine deaminase inhibitor such as pentostatln to extend the half life of Formula I compounds in the blood stream.

Compounds of Formula Za (IPA) have especially low toxicity for pediatrics, and acceptable toxicity of adult humans, although it ls somewhat higher than for pediatrics. IPA can be stored dry almost indefinitely if protected from light and stored at -?5*C. IPA la photosensitive and deteriorates at room temperature, whether in a solid form or in aqueous or ethanolic solutions. It was found in experiments that the breakdown rate of IPA ia approximately 3t per month in a dark container at room temperature.

This invention includes the use of physiologically acceptable salts of Formula 1, for example, those derived from inorganic acids such as hydrochloric, sulphuric, phosphoric acid, etc., and organic sulphuric acids such aa p-toluenesulphonic acid, methanesulphonic acid, etc., and organic carboxylic acids such as acetic, oxalic, succinic, tartaric, citric, malic, maleic acid, etc.

The dosage of any one or more of compounds having Formula I which is effective in treatment against: (a) hairy leukoplakia, (b) oral candidosis, (c) mouth ulcerations-aphthous/herpetic/bacterial, (d) fungal Candida, (e) human papilloma virus, (f) molluscum contaglosum, (g> squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (i) periodontitla, (j) necrotizing gingivitis, (k) orofacial herpes zoster, (1} rotaviruses, parasites, diseases caused by such parasites, Mycoplasmaa and diseases caused by Mycoplasmas,and KIV will depend on many factors including the specific compound or combination of compounds being utilized, the mode of administration, and the organism being treated. Dosages of a particular compound or combinations of compounds,

BAD ORIGINAL each belonging to that class defined by Formula I, for a given host can be determined using conventional considerations, for example, by customary comparison of the differential activities of the subject compounds and of a known agent, that is, by means of an appropriate pharmacological protocol.

In one embodiment of the invention, a pharmaceutical formulation comprising a compound having Formula I is administered at the rate of 1 unit dose to 10 unit doses per day, and preferably 1 unit dose to 4 unit doses per day. The doses are given for periods of up to twelve weeks and in certain cases may be given for the life of the patient or, depending on the patient's medical requirements, at less frequent intervals.

In one aspect of the invention, a unit dose comprises 0.01 to 5000 mg of a formulation comprising a compound having Formula I. In one case study, daily dosages in the range of from 250 to 1800 mg of IPA were administered.

In one embodiment of the invention, the pharmaceutical formulation is administered orally in unit doses once per day when the compound is in a slow release form or up to eight unit doses per day when the compound is in its native form. Alternatively or additionally, the pharmaceutical formulation is administered intravenously in unit doses comprising a compound having Formula I in the range of 0.3 mg to 80 mg per Kg of body weight.

In one embodiment of the invention, a pharmaceutical formulation comprising a compound having Formula I is administered by the use of a trans-dermal patch.

In one embodiment of the invention, a pharmaceutical formulation comprising a compound having formula I is administered using an emulsifying or semi-emulsifying formulation to improve absorption from the small intestine. Such an emulsion may be formulated using a derivative of coconut oil, e.g. Miglyol 812.

In accordance with one aspect of this invention, it has been discovered (as confirmed in clinical trials,

OR»®**'¹’

AP 0 0 0 3 1 2 that formulations comprising one or more compound selected from the group consisting of compounds having Formula I, when administered to beings suffering from one or more of the following indication or infections: (a) hairy leukoplakia, (b) oral candidosie, (c) mouth ulceratior.s-aphthous/herpetic/bacterial, (d) fungal Candida, (e) human papilloma virus, (£) molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (1) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes soater, (1) rotaviruses, have beneficial effects.

For example, in one particular case study, a patient suffering from AIDS with indications of hairy leukoplakia showed complete relief of hairy leukoplakia upon administration of IPA {compound la) administrated in the form of a fluid oral dose or mouth wash with relief of the indication occurring within seven days. When the indication re-appeared, following a period of time (several days), the administration of the compound gave further relief.

In another case study, V.T., a 28 year old homosexual male, was diagnosed HIV positive in June, 1986. V.T. was also diagnosed as having hepatitis at that time. Beginning on May 7, 1990, IPA was administered orally to V.T. On May 7, 1990, V.T. was experiencing (1) weight loss, (2) night sweats, (3) thrush, (4) fungal infections, (5) pneumonia, (6) diarrhea, and (7) hairy leukoplakia.

Prior to treatment with IPA, V.T. had experienced diarrhea and night sweats for the previous six weeks. These symptoms cleared within 48 hours of the beginning of the treatment with IPA. Treatment with ipa resulted in a complete remission of hairy leukoplakia. V.T. experienced a weight gain of about 0.5 lbs per day over the first month of treatment with IPA (on May 7, 1990, V.T. weighed 119 lbs--on June 11 , 1990, he weighed 132 lbs). V.T.'s weight stabilized at 133 lbs. V.T.’s energy level and general health greatly improved. V.T.’s T4 count steadily increased from 207 (on May 7, 1990) to 330 (on June 21 , 1990). V.T.’s helper/suppressor ratio

BAD ORIGINAL ft improved from 0.53 (on May 7, 1990) to 0.65 (on June 21, 1990). Ae of July 26, 1990, V.T.'s helper/suppreesor ration was 0.93.

There were observed no permanent toxic aide effect· from administration of IPA to v.t.

Rotaviruses cause an estimated 140 million case· of gastroenteritis in Infants and children and 1 million deaths world-wide each year. They are also the most common cause of hospital-acquired gastroenteritis, infecting 13-27 % of children admitted to pediatric hospitals during rotavirus epidemics. Nosocomial rotavirus infections can significantly extend the hospital stey and increase morbidity and financial cost. Rotaviruses belong to the group of virus known as rtovlruses, it has no envelope, its shape is icosahedral, its approximate sire is 60-80 nm, and it has an RNA nucleic acid structure.

Mycoplasma· are small bacterium-like organisms that normally lead a parasitic existence in close association with animal cells. This invention is directed to administering compound(s) against Mycoplasma·, particularly Mycoplasma KrthritidiS, Mycoplasma Fermentans and Mycoplasma incognitus.

Xn accordance with another aspect of this invention, one or more compound selected from the group of compounds having Formula I is/are administered to a being afflicted with one or more parasites (each believed to be a haemoflegellate parasite of man), e.g., leishmania, trypanosomia, toxoplasma and/or enterocytozoon bieneusi and/or afflicted with disease caused by such parasites.

Xn accordance with a preferred aspect of this invention, parasites can be targeted by selecting one or more compound(s) which tend to Collect in a particular region of the being. For instance, IPA tends to collect in the human liver. This property can be very useful for use against particular parasites which have preferential areas in which to reside. For instance, by using IPA against T. brucei (believed to be the cause of sleeping sickness), which preferentially resides in the liver and spleen of a human, lower dosages can be used against the τ brucei in the liver.

Xt is believed that the present Invention kills and/or prevents replication and/or causes intracellular digestion of parasites in macrophages. This is especially believed to be true in the case of toxoplasmosis, whose mode of survival ie believed to be through macrophages. Thue, this inven·»

AP Ο Ο Ο 3 1 2 tion further relate· to administration to being· afflicted with any parasite whose mode of survival is similar to that of toxoplasmosis.

In-vitro screens in the experimental chemotherapy of leishmaniasis and trypanosomiasis must provide active or dividing populations of the mammalian stages of the parasite. They must provide a measure of drug activity that is readily quantified, and accurately show the activity of standard drugs at concentrations close to those achievable in serum or tissues (over a long timecourse if necessary). Inevitably, however, in-vitro screens ignore Important host factors involved in absorption, metabolism and pharmacokinetics of drugs, so in assessing different screens one must also bear in mind their accuracy in predicting in-vitro activity of new compounds, and whether or not the system has the facility to examine additional features of interest, such as variation in drug susceptibility between strains and subspecies, drug resistance, and effects of immune or metabolic components.

Leishmaniasis

Following initial infection, the amastigote is the only form present in the mammalian host, surviving and dividing In various macrophage populations. Differences between the promastigote and amastigote in form and habitat are not matched by known biochemical dissimilarities. However, the drug susceptibility of the cultured promastigote, used in many early chemotherapy screens, contrasts markedly with that of the amastigote (see Table 1, below), most importantly to the clinically used pentavalent antimonials - sodium stibogluconate (Pentostam) and meglumine antimonlate (Glucantime) .

Amastigote· of Leiehmania can be cultivated in a wide range of mammalian cells, some of which may be used in in-vitro screens. These include (1) a Sticker dog sarcoma (fibroblast) cell line, (2) transformed rodent macrophage cell lines, (3) primary isolated mouse peritoneal macrophages (MP mo), and (4) human monocyte-derived

BAD ORIGINAL macrophages (HM mo). The methods are similar for all models. Amaatigote or promastigote infected cells are maintained in a medium containing the drug for 4-7 days, at 37°C for L. donovani and 33-35°C for L. major and L. mexicana. Drug activity is then assessed by counting (I) the number of infected host cells and/or (II) the number of amastigotes/100 host cells. For some compounds, the method of counting can influence the measure of activity; for example, pentamidine was found to be inactive against L. donovani in MP mos by method (I) but active by method (II) .

South American Trypanosomiasis

The chemotherapeutic targets in Chagas disease are the non-dividing, tissue invading, bloodstream trypomastigotes, and the dividing intracellular amastigotes, often sequestered within the muscle and difficult to eliminate. In Table 2 (below), the activities of selected drugs, including clinically used compounds nifurtimox (Lampit) and benznidazole (Rochagan), are shown to indicate differences observed between in-vitro systems and between strains of T. cruzi. In-vitro chemotherapy screens have for many years used models involving three stages of the T. cruzi life cycle;

(1) Epimastigote. Dividing populations are easily cultivated in a range of defined and semi-defined media, but are equivalent to a vector stage of the life cycle.

(2) Trypomastigote. These are isolated from a rodent host and can be maintained as a non-dividing population for 24-48 h in media.

(3) Ajnastigote. Dividing populations can be cultivated in muscle cells, fibroblasts and macrophages, following infection with trypomastigotes. After the initial infection, there is normally a period of 4-5 days during which the amastigotes divide before transforming, via the epimastigote stage, back to trypomastigotes which escape from the cell. Drug activity is measured during this 4-5 day period. A

AP 0 0 0 3 1 2 dividing host cell population, low infection rate, and the presence of trypomastigote· in the overlay can complicate the interpretation of reculta.

Intracellular amastigote models can use a range of muscle (skeletal and cardiac) and fibroblast (e.g. HeLa, HePa) cell lines, macrophages, and chick embryo cells. The choice of host cell may be relevant to the assessment of drug activity; some drugs, such aa puromycin amino-nucleoside and ketoconazole may be metabolized prior to fixation by the amaetigote. Compounds that act on T. bruzi through the generation of free radical metabolites also interact with host cells, particularly macrophages. To overcome the problems of overgrowth of the monolayer and a low percentage of infected cells, often associated with the use of cell lines, irradiated rat L6 myoblasts are used.

African Trypanosomiasis

A compound for treating African trypanosomiasis must be active against the trypomastigotes of T. brucei gambiense and T. brucei rhodesiense in the blood and cerebro-spinal fluid (CSF), and against extravascular forms present in the brain, particularly the choroid plexus region. Although considerable progress has been made with the in-vitro cultivation of blood stream trypomastigotes for use in drug screens, the ability of compounds to pass through the blood-CSF barriers, as already demonstrated for IPA, and their activity against extravascular brain forms continue to be examined in rodents and primates.

Primary screening for new trypanocides has made extensive use of rodent systems, in which host survival time is a simple indicator of drug activity. In-vitro screens have used a variety of trypomastigote types (see Table 3, below). The procyclic trypomastigote of T. brucei, a form equivalent to that found in the tsetse midgut, is easy to culture but possesses many different biochemical and structural characteristics to the bloodstream trypomastigote and is- therefore of limited use. Most early in-vitro screens employed a short test in which bad original Q

4' bloodstream trypomastigotes were Isolated from an infected rodent and maintained for up to 24 h in a simple drugcontaining media. Two significant modifications of this technique include maintaining isolated bloodstream trypomastigotes in drug-containing media for 3-4 hours at 37°C in micro-titre plates and their subsequent viability assessed by the uptake of radiolabelled precursors or motility and infectivity to mice.

Culture systems developed in the last decade have used mammalian cell feeder layers to support the growth of dividing populations of bloodstream trypomastigotes of T. brucei spp. Some of these systems have been used in chemotherapy studies, including; (1) bovine fibroblast culture - T. brucei models to test some standard and experimental compounds, (2) murine bone marrow culture T.b. gambiense model to screen inosine analogues and platinum and hypolipidemic compounds, and (3, Microtus embryonic fibroblast culture (MEF) - T.b. rhodesiense model to test standard and experimental drugs. These systems give a good indication of in-vivo activity (see Table 3). They measure activity against dividing cells rather than specific macromolecular synthesis pathways and can be readily established in micro-titre plates. The MEF and bone marrow cell feeder layers can be used to grow a range of strains of T. brucei spp,, offering the possibility of examining variations in drug susceptibility and resistance between strains. The presence of a feeder layer, however, limits the opportunities for the automation of these systems.

in-vitro studies on isolated bloodstream trypomastigotes were also used in the rational biochemical approaches towards the chemotherapy of T. brucei infections involving inhibitors of carbohydrate catabolism salicyl hydroxamic acid plus glycerol), of polyamine synthesis (methylgyoxal bis-guanylhydrazone and - DFMO), and of surface coat glycoprotein synthesis (Tunicamycin). In these studies, activity of'the compounds are shown invitro and in-vivo, whereas the sensitivity of isolated o^^G

AP 0 0 0 3 1 2 bloodstream trypomaatigotes to toxic oxygen radicals generated by naphthoquinones was not shown by the parasites in mice. The activity of the standard anti-trypanosomals melarsoprol, pentamidine and suramin and tested compounds according to the present invention in in-vitro systems is shown in Table 3.

TflSQPl&gmoelg

Toxoplasma gondii:-protoxoon is the etiologic agent of toxoplasmosis. This sporoxoa is an Intracellular parasite Toxoplasma gondii that lives inside macrophages and has evolved a mechanism to avoid being killed by intracellular oxygen, metabolites and lysosomal enzymes.

The live parasite Toxoplasma gondii when ingested by the macrophage forms a membrane bound vesicle called a phagosome. This phagosome containing Toxoplasma parasite can inhibit the fusion to the phagosome of lysosomes which would cause its digestion and elimination. However, if dead Toxoplasma parasites are ingested by macrophages, the phagosome thus resulting cannot prevent lysosome fusion, and parasite digestion, if the macrophage is treated with IPA, it reduces RNA and protein synthesis both in the macrophage and in the parasite, but this imbalance in biosynthetic ability favours the macrophage as opposed to the Toxoplasma parasite since the intracellular macrophage lysosomes are already in position with specific receptors for their attachment to certain markers on the phagosome containing the Toxoplasma parasite. It is the function of the biosynthetic machinery of the parasite to synthesize molecules which block the ability of the lysosome to fuse with the Toxoplasma phagosome, thus IPA's ability to reduce RNA and de novo protein synthesis alters this dynamic synthetic balance and allows lysosomal fusion and digestion of the Toxoplasma Phagosome.

An experiment was conducted to evaluate the invitro effects of IPA on Toxoplasma Gondii by infecting human foreskin fibroblast monolayers (four replicates per condition) with RH strain T.· gondii in the presence of various concentrations of IPA dissolved in Eagle's minimum

BAD ORIGINAL ft ν ϊ r, c c o AA essential medium without fetal bovine serum. After an initial four hour period of infection, the media and any T, .gondii which had not entered the host cells were removed and replaced by media containing 1 μΜ tritiated uracil per milliter, and incubated overnight. The monolayers were lysed, nucleic acids precipitated by trichloroacetic acid, and the precipitates recovered on glass fiber filters. Radioactivity on the filters was measured in a Beckman scintillation spectrometer. Controls included fibroblast monolayers without T, gondii (cell control) and fibroblast monolayers infected by the same inoculum of T, condil as the IPA treated monolayers (Tg control). The results of our experiment are shown in Figure 24. The units of uracil incorporation are somewhat lower here because of the lower inoculum of T, gondii. Host cells have been tested and were free of mycoplasma --these experiments provide strong evidence of an inhibitory effect of IPA on the growth of T, gondii.

Enterocvtozoon Bieneusl Microsporidiosis

Chronic diarrhoea accompanied by wasting is one of the most frequent problems associated with HIV infection. Clinical evaluation of patients, treated with IPA including stool examinations and endoscopic biopsies, fails to identify any pathogen. A new genera and species of microspora, Enterocytozoon bieneusi, was identified in uitrastructurai studies of intestinal biopsies from AIDS patients with unexplained chronic diarrhoea prior to treatment with IPA.

E. bieneusi microsporidiosis (intestinal microsporidiosis) is diagnosed with increasing frequency in AIDS patients with chronic diarrhoea of unknown origin. Forty-seven cases have been reported in the United States, Africa and Europe. Reporting an incidence of 30% in a homosexual group, these findings suggested that E. bieneusi is an important cause of diarrhoea in AIDS patients. Until now, diagnosis depended upon light and electron microscopy studies of small bowel biopsies, particularly of the jejunum. Diagnosis is suspected on histological

AP 0 00 3 t 2 findings either on Giemaa-stained smears and/or hematoxylin-eosin or Giemsa-stained paraffin sections: identification of oval parasites measuring 2-9 pm and clusters of refractile spore· (1-1.5 pm) in villous enterocytes from juet above the mouth of the crypts to the tips of the villi. These organisms are located in the supranuclear position of the cytoplasm. Identification of E. bieneusi is confirmed by electron microscopy which reveals the following characteristic features: multinucleated proliferative and eporogonial plasmodia, measuring 4-6 μτη without parasitophorus vacuole and mitochondria; presence of numerous electron-dense precursors spores measuring 1.5 x 0.5 yim with a poorly differentiated endospore, coiled polar filament (5-7 coils) and electron lucent vacuole. Recently reported recovery of spores from faeces, would simplify diagnosis. Unlike other species of microspora, E. bieneusi has not been identified from different organs such as brain and kidney. A proportion of transient diarrhoeas of unknown etiology in AIDS patients may be related to E. bieneusi. Contamination of water with faeces and/or close human contact are involved in transmission. No treatment was known to be efficacious prior to this study using Isopentenyl Adenosine.

Compounds la and lb are particularly preferred for use against Enterocytozoon bieneusi.

it ★ w	* *	★	★ *	*
In-vitro	and in-vivo	models	were used	to test
activities of a	selection of	many	compounds.	Those

activities are summarized in Tables 1-3, showing variation between systems and species. Activities measured in the MP mo and HM mo models have shown the best correlation with results obtained in hamster and mouse models.

BAD ORIGINAL

T*bl· I. Activity of compound» »9*1net Le1»hm<n1a *pp, in In-vitro and 1n-v1vo »y»tama.

$ H Q 0 0 RA

Compound and Laithmama »bb.		'A-vitro	1n-vlvo
AM-PM tr*n\|forw- ation	AM ,n call Tinea	AM 1n novae or human «0»	mow»·/ ham tar modal a
Sodium ltiboe'uoonat· Ld.				4 4	4 4
Lm. Pantamldlna	•	4 4	•	4 4	4
Ld.	♦ 4	4 4	.	4	4
Lm. N$-l»opantany1 Afi»not1na	♦ 4	4 4	4	4 4	•
Ld.	4 4	4 4	-	4 4	4
Lm. FurFuryladanotma	♦	4 4	♦	4 4	•
Ld.	4 4	4 4	-	4	4
Lm.	* 4	4 4	4	4 4	4
N6-9an*y1ad»no»‘na Ld.	4 4	4 4		4	.
Jn.	4 4	4 4	4	4	•

V6-Iiop«nt<nyl Mtnoaine-S'monophoioHat·

Ld.	4 4	4	•	4	4
Lm.	-	4 4	4	4 4	•
Poly h6-’iop»ntany1 A0«no»1na
Ld.	4 4	4 4	•	4 4	4
Lm.	4 4	-	•	4 4	4

Activity

- no/poor activity# ♦ fair/moderate activity, ++ good/excellent activity

Ld. - L donovani.

Lm. - L major/L mexicana

PRO - Promastigote

AM - Amastigote

bad

OWO'^¹AP 0 0 0 3 1 2

Table 2.	Activity of compounds and 1n-vivo systems	aeainat T. crust «trains	in ir-vitro
		m-v1tro		m-vivo
Cemooune	ΕΡΣ	TRY	Intracellular AM	mouse model
Mfurtlnox BenaMduole	+(Y,WBH. ~rWBH,Sonya) Sonya)+(KR)+(Tu) «+(Y,KR, +(Y, Sonya) Sonya)	++(Y,W8H, Sonya) «-»(Y, Sonya)	++(Y,HSK,CL) ♦(Y.Ct.Tu, Sonya) ++(Y)+(Sonya) Sonya)
Νβ-Iaooentenyl Adenoaine ++(Y,KR, Sonya)	♦ (Y,Sonya)	♦♦(Y,Sonya)	++(Y)+(Sonye)
Furfuryladenoslne	++(YiHR, Sonya)	♦ (Y,Sonya)	♦ (*,Sonya)	Sonva) ++(Y)+(Sonya)
NS-Be nay1adenoa5ne	+(Y,MR, Sonya)	♦ (Y,Sonya)	♦ (Y,Sonya)	*(Y)+(Sonya)
M-Isopentenyl Adenosine-S'-Monophoephate +(Y,MR, ++(Y,Sonya) Sonya)	++{YiSonya)	++(Y)+(Sonya)
Poly NS-Iaopente.nyl Adenosine ++(Y, MR,	*(Y,Sonya)	++(Y,Sonya)	+(Y)+(Sonya)

Sonys) activity:

- no/poor activity, + fair/moderate activity, ++ good/excellent activity

T. cruzi strains in parenthesis EPI - Epimastigote TRY - Trypomastigote AM - Amastigote

Table 3. Activity of	compound*	agatnet T.b. brucel.	T.b. giro’ense	anc T.1
rbodestense	1η tn-vttro	and 1n-v1vO tyttems
		m-v1tno		ίft-vlvO
	procyC	!'C isolated	cultivated	nouM
Compound	TRY	bloodstream	bloodstream	model
		TRY	TRY
M»lar»oprol		4 4	4 4	4 4
Pentamidine		♦ 4	4 4	4 4
Sur+rnm	-	~\|4	4	4 4
N6-Jsopenteny1 Adenosine	4	4	4 4	4 *
Furfury1adeno*,ne	♦	4 4	4	4
N6-Benty1adenos1 ne	4 4		4 4	4 4
NS-l»opentenyl Ade-os 1ne-5'-Monophosphate
	♦ *	4	4	♦
Poly Νί-IsopentenyT Adenoaine	♦	4	4 4	4 4

activity:

- no/poor activity, ♦ fair/moderate activity, + + good/excellent activity

BAD ORIGINAL

Compound· of Formula I have been found to have valuable pharmaceutical properties as anti-viral agents.

A caee study of an AIDS patient, T.W., was conducted. Patient T.W. began treatment with IPA in February 1989, while in a nearly moribund condition, suffering from depression, exhaustion, bronchial infection, severe diarrhoea, severe weight loss (weight in February was 98 lbs.), complete loss of appetite and spiked fever. The case study reveals a major rapid recovery of T and B lymphocyte competence. By March 21, 1989, patient T.W. stated that he felt cured and abruptly (unilaterally) stopped taking IPA, resulting in dramatic deterioration of his condition. In May, 1989, patient T.W. resumed IPA therapy, and experienced dramatic recovery. At the end of May, 1989, patient T.W, again stopped taking IPA, and his condition again deteriorated. Again IPA treatment was restarted, and patient T.W.'s condition improved. In early June, 1989, patient T.W. again stopped IPA therapy and died several weeks later.

The study concluded that patient T.W. would have apparently enjoyed appreciable therapeutic benefits from IPA if he had conscientiously and continuously received IPA medication.

Also, compound la, IPA, has been found through in-vitro experiments to inhibit the replication of HIV-1 in monocyte/macrophage cells and in T4 lymphocytes as well as Langerhan cells at levels which are non-toxic to the cells themselves.

Studies have been carried out which show that

IPA reduces levels of HIV in HIV-infected macrophage cells treated with IPA relative to infected macrophage cells not treated with IPA. Toxicological studies have been carried out using uninfected cells (T and B lymphocytes and monocytes) which show that such cells can tolerate expo-

AP 0 0 0 3 1 2 eur· in-vitro to IPA at level· which reduce level· of HIV in HXV-infected macrophage cell·.

Studies have been carried out which indicate that IPA reduces in-vitro levels of HIV in HIV-infected K9, 61-66-45, and monocyte/macrophage cell· relative to similar cell· HXV-infected not treated with IPA.

Experiment· to show the in-vitro effect Of IPA on human leukocyte viability have been carried out.

Studies have been carried out that indicate that IPA, in ln-vltro experiments, reduce· level· of caprine arthritia encephalitis virus (CAEV) in Himalayan Tahr ovary cells.

Studies have been carried out which show that IPA reduces in-vitro levels of herpes simplex type 1 (HSV1) in an M413 cell line of human fibroblasts.

Studies have been carried out which show that IPA reduces levels of cytomegilovirus in cells in-vitro.

Studies have been carried out which show that IPA reduces levels of Epstern-Barr Virus (ESV) In P₃HR1 cells in-vitro.

The effectiveness of a particular regimen (of administration of a compound of Formula I) can be monitored by following over time the presences of parasites, Mycoplasma, HIV or characteristics indicative of: (a) hairy leukoplakia, (b) oral candidosis, (c) mouth ulcerationa-aphthous/herpetic/bacterial, (d) fungal Candida, (e, human papilloma virus, (£) molluscum contagiosum, (g) squamous oral carcinoma, (h) Kaposi's sarcoma oral lesions, (i) periodontitis, (j) necrotizing gingivitis, (k) orofacial herpes zoster, (1) rotaviruses, or diseases caused by parasites or Mycoplasma in blood samples of an organism being treated. There are available commercially kits for the detection of KIV antigens. The use of one such kit for detecting the antigen of HIV-1 was used as described in Example Ia of this invention. It will be possible to cause a reduction, over a period of about two months, in the level of detectable antigen in the blood serum of a patient by means of administration of compounds of Formula I. A better bad ORIGINAL ft ί Γ > Il 0 ί! HA

- 30 measure of the progression of th· level of infection would be the percentage Infected macrophage population. Monocytes /macrophage Cells obtained from either the blood or the lung during a course of treatment with compounds of Formula I will ahow a reduction in recoverable HIV antigen as the therapy progresses.

In another embodiment of the Invention, the method of the Invention comprises the step of treating a patient with known immune syetam booster or immune system modulator to enhance the production of T-cells by the bone marrow. The patient is treated with the immune system booster prior to administration of a pharmaceutical formulation comprising a compound having Formula I. in another case, the patient is treated with the immune system booster until the level of production of T-cells by the bone marrow (having been reduced by the infection) is stabilized or begins to increase. In particular, the immune system booster is administered until the level of T-4 cells is stabilized or begins to increase.

In another embodiment of the invention, the method includes the step of treating a patient with an immune system booster both prior to and simultaneously while a pharmaceutical formulation comprising a compound having Formula I is being administered.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 la a graph of the effect of various concentrations of IPA on the growth of Toxoplasma gondii in human fibroblasts.

Figures 2 and 3 are graphs of cytotoxicity and virus inhibition versus concentration of IPA in an assay.

Figure 4 is a graph of percentage increase or decrease in proliferation capability versus concentration of IPA in an assay.

Figure 5 is a graph of counts per minute versus concentration of IPA in an assay.

Tests were carried out by the present applicant to determine the effectiveness and toxicity of IPA against viral infections, as described in European Patent Application 355,825 (published February 28, 1990), the disclosure of which is incorporated herein by reference. Several of these tests are reproduced below.

AP Ο Ο Ο 3 1 2

- 31 EXPERIMENT 1a(d) Effects of IPA, AZT and ddC on Human Cell Viability

Human peripheral blood mononuclear cells were incubated for 18 hours in the presence of various concen5 trations of IPA and aridothymidine (A2T, at 37®C after which time cell viability was determined by visual counts of cells in the presence of trypan blue. The results are tabulated in Table ia(d) (i). Two controls were run, one with each series of experiments. No toxicity was observed for AZT concentrations between 0.1 μ and 6mM. There was a alight reduction in cell viability in the presence of 6mM IPA, but no toxic effect was observed using concentrations of from 0.1 μΜ to 3 μΜ.

BAD ORIGINAL &

- 32 TABLE laid) (1)

AZT 18 hours	IPA 18 hours
Concentration (μΜ)	Cells/ml (x10Ί	Viability (%)	Cells/ml (x10 )	Viability (1)
6000	3.8	95	2.4	75
3000	3.0	88	2.2	92
1000	2.2	100	2.8	82
300	2.4	92	2.2	79
100	2.0	91	3.8	90
30	3.0	100	3.8	95
10	1.8	90	3.0	94
3	2.0	100	2.6	87
1	1 .8	90	2.8	100
0.3	2.0	91	1.8	82
0.1	3.4	100	2.4	86
control	3.2	94	2.6	87

Experiments were also performed to determine the toxic effect of ipa, azt and ddC on other cell lines. The results are tabulated in Tables la(d) (ii) to la(d) (v).

Two lots of IPA (Sample 1 and Sample 2) were tested. Tests of Sample 1 over four months indicated there was no change in the compound.

AP Ο Ο Ο 3 1 2

- 33 TABLE laid) (iii

CELL LINE: H9, Continuous Exposure for 10 days

5	DRUG	CONCENTRATION <μΜ)	PERCENT VIABLE CELLS
	AZT	100	61
	AZT	10	77
	AZT	1	83
10	ddc	100	68
	ddC	10	79
	ddC Sample 1	1	93
	IPA	1000	12
15	I PA	300	18
	IPA	100	20
	I PA	30	56
	IPA	10	73
	IPA	3	84
20	IPA Sample 2	1	66
	IPA	1000	7
	IPA	300	21
	IPA	100	23
25	IPA	30	61
	IPA	10	78
	IPA	3	81
30	IPA CONTROL	1	84 87

bad original

TABLE ia(d? dill

CELL LINE: H9, HIV Chronically Infected, Continuous Exposure for 4 days

5	DRUG	CONCENTRATION (μΜ)	PERCENT VIABLE CELLS
	AZT	100	76
	AZT	10	Θ1
10	AZT	1	88
	ddC	100	66
	ddC	10	79
	ddC	1	91
	Sample 1
15	IPA	1000	51
	I PA	300	48
	IPA	100	60
	I PA	30	73
	IPA	10	85
20	IPA	3	89
	IPA	1	93
	Sample 2
	IPA	1000	57
	IPA	300	53
25	IPA	100	66
	IPA	30	78
	IPA	10	66
	IPA	3	89
	IPA	1	92
30	CONTROL		91

B^

AP 0 0 0 3 1 2

TABLE laid) (ivi

CELL LINE 81-66-45

DRUG	CONC (μΜ)	DAYS EXPOSED TO DRUG (PERCENT VIABLE CELLS)
TYPE OR EXPOSURE	1 DAY	4 DAYS/ CONT.	10 DAYS, CONT.	4 DAYS, 1 DAY EXP	10 DAYS, 1 DAY EXP
AZT	100	95	78	56	76	64
AZT	10	95	74	68	85	70
AZT	1	93	80	75	85	79
ddc	100	92	66	53	93	58
ddC	10	90	78	71	85	75
ddC	1	95	63	82	77	79
IPA	1000	58	52	18	73	38
IPA	300	74	65	26	72	51
IPA	100	66	60	34	84	59
IPA	30	62	56	44	92	68
IPA	10	81	85	70	84	79
IPA	3	75	85	78	90	88
IPA	1	80	84	81	89	81
IPA	1000	63	51	15	65	42
IPA	300	77	40	22	70	50
IPA	100	71	47	26	82	61
IPA	30	75	65	43	80	77
IPA	10	78	88	66	89	83
IPA	3	79	84	79	80	88
IPA	1	91	78	72	80	92
CONTROL	N/A	95	75	84	95	90

BAD ORIGINAL 0.

ς t t 0 ? t> RA

- 36 .

TABLE Ift(d) (v)

			MONOCYTE/MACROPHAGE CELLS
			4	4 DAYS,	10 DAYS,
TYPE	OR	1	DAYS,	10 DAYS, 1 DAY	1 DAY
EXPOSURE	DAY	CONT.	CONT. EXP	EXP
DRUG	CONC.	•
	(μΜ)	1 •

AZT	100	+ + + +	+ + ♦ +	4 + +	+ + + +	+ + +
AZT	10	+ + + +	+ + + +	+ + + +	+ + + ♦	+ + + +
AZT	1	+ + +	♦ + + +	+ + + +	+ + + +	+ + + +
ddc	100	+ ♦♦ +	♦ + + +	+ + +	+ + + +	+ + +
ddC	10	+ ♦♦ +	♦ + + +	+ + + +	+ + + +	+ + ♦♦
ddc	1	+ ♦ + +	+ + + +	+ + + ♦	+ + + +	+ + + +
IPA, OLD	1000	+ ♦	0	0	+	+ +
IPA, OLD	300	♦ +	+ +	+	+ ♦	+ +
IPA, OLD	100	+ +	+ +	♦ +	+ + +	+ + +
IPA, OLD	30	+ + ♦	♦ +	+ +	+ + +	+ + ♦
IPA, OLD	10	+ + +	+ + +	+ + ♦	4 + + +	+ + + +
IPA, OLD	3	+ + + +	+ + ♦	+ + +	+ + + +	+ + + +
IPA, OLD	1	+ + + +	+ ♦ + +	+ + + +	+ + + +	+ + + +
IPA, NEW	1000	+ ♦	0	0	+	+ +
IPA, NEW	300	+ +	+♦	+	+ +	+ +
IPA, NEW	100	♦ +	+ +	+ +	+ + +	+ + +
IPA, NEW	30	+ + +	+ +	+ +	+ ♦ +	+ ♦ +
IPA, NEW	10	+ + +	+ + ♦	+ + +	♦ + + +	+ + + +
IPA, NEW	3	+ + * +	♦ + +	4 + +	+ + + +	+ + ♦♦
IPA, NEW	1	+ + + +	+++ +	+ + + +	+ + + ♦	+ + + +
CONTROL	N/A	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +

Note: since monocyte/macrophage cells are tightly adherent to the wells, they cannot be trypsinized to stain and count. We were also unable to effectively observe dye uptake directly in the well, so the observations made above represent qualitative indications of the apparent health of the cells as well as the cell numbers.

Nevertheless, it is important to note that these monocyte/macrophage cells appear to be far more resistant to the toxic effect of IPA than the T cell lines, for example. Since the tahr cells used for CAEV testing were also relatively resistant to IPA, it may be that the most rapidly growing cells are most vulnerable.

AP 0 0 0 3 1 2

- 37 The Percent Dead Celle are taken from Tables Ia(d) (iii) and ia(d) (v) using the 1O-day value, and the therapeutic index have been calculated for each drug concentration. The result· of IPA are also graphed in Figure· 4 and 5. Cytotoxicity (the dashed line) is calculated a· the percent live cell·.

The availability of toxicity data collected in the same time frame a· required for determination of antiviral effectiveness allowed determination of values for two different cell lines. Figures 4 and 5 illustrate the curves obtained by plotting the cytotoxicity of IPA (as a percent of the control cell viability) versus the percent of inhibition of HIV-1 replication (as a percent of the control titer) when H9 and 81-66-45 cells were tested. The cytotoxicity data obtained with 10 days continuous exposure, using uninfected cells, were used since 10 days was the length of time required to carry out the infectivity test. As is apparent from Tables Ia(d) (ii) - Ia(d) (v), toxicity is much lower when the drug is added to cells for one day and then removed, or when cells are exposed for only 1 or 4 days. It is difficult to mimic in-vivo exposure times, particularly if metabolites are more toxic than the parent compound and are readily cleared in-vivo but not in-vitro.

Analysis of Figures 4 and 5 permit a list of the inhibition values for the two cell lines to be calculated as follows:

_BA0 original

Η9 Celle

81-66-45 Cells v * f Ου ο HA

	IC_M*	6 μΜ	6 μη»
		10 μΜ	10 μΜ
5		50 μΜ	40 μΜ
	ICjo/IDso	0.12	0.20

* ICjo - The concentration of IPA which inhibits the replication of HIV-1 by 50%.

** ^*** iC^o - The concentration of IPA which inhibits the replication of HIV-1 by 90%.

*** IDsq - The concentration of IPA which kills 50% of the cells.

Although the present invention has been de15 scribed in connection with preferred embodiments, it will be appreciated by those skilled in the art that additions, modifications, substitutions and deletions not specifically described may be made without departing from the spirit and scope of the invention defined in the appended claims.

Claims

I CLAIM:

AP 0 0 0 3 j 2

1- The use of at least one compound selected from the group having the formula:

wherein:

*’ «nd R, «1 H er CHjS and r₄ .

H_z or

I and Rj · CHj Cl, OH or a monophosphate group

R_e = CHj, CH₂OH or Cl

R_? « H or Br

APOOOJi2 and X, and X₂ are Independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl or R, = CH, or R, o «

or R« « CNH-Rg and ’CO)

Cl or R_fi » (CH₂)₇CH₃;

and R₂ » OH and Rj OH, monophosphate, diphosphate or triphosphate group ;BAD ORIGINAL _{s T} jΟ ί! ο ΑΑ or R₂ and R, are linked lo torm a 3’, 5'-cyclic mono-phosphate derivative, or a physiologically acceptable salt thereof, or a metabolite thereof, in the manufacture of a pharmaceutical formulation for use in the treatment of parasitic infection.
2. The use recited in claim 1, wherein said parasitic infection is by a haemoflagellate parasite.
3. The use recited in claim 1, wherein said at least one compound is Poly N6 Isopentenyl Adenosine.
4. The use recited in claim 1, wherein said parasitic infection is by a parasite having a mode of survival similar to that of Toxoplasma.
5. The use recited in claim 2, wherein said parasitic infection is by one or more kind of parasite being a member of the group of haemoflagellate parasites consisting of Leishmania, Trypanosomia and Toxoplasma.
6. The use recited in either of claims 2 or 5, wherein said compound is effective to kill and/or prevent replication and/or cause intracellular digestion of said one or more kind of haemoflagellate parasite in macrophages.
7. The use of at least one compound selected from the group having the formula:

o®'

AP Ο β Ο 3 1 2 wherein:

Η, R, . CH„ Rj . CHj and R, . H, _orRi H or CH₃S and r₅ - ch_J( cl, on or a monophosphate group R< · CH₃, ch₂oh or Cl R₇ = H or Br

BAD ORIGINAL £ or R. - H and X, and X₂ are independently selected from H, methyl, ethyl, hydroxyl, the halogen· and carboxyl ^{ΟΓ R}* ^CHZ or R,

I or R_t CNH-R_eand

Rt Cl or Re - (CH₂)₇CH₃;

and R₂ « OH and R₃ OH, monophosphate, diphosphate or triphosphate group or R₂ and R₃ are linked to form a 3', 5' -cyclic monophosphate derivative, or a physiologically acceptable salt thereof, or a metabolite thereof, in the manufacture of a pharmaceutical formulation for use in the treatment of an abnormal level of one or more kind of parasite and/or a disease resulting from said abnormal level.

OB

AP Ο Ο Ο 3 1 2
8. The use recited in claim 7, wherein said parasitic infection is by one or more kind of parasite being one or more kind of microsporidiosis.
9. The use recited in claim 8, wherein said one or more kind of parasite is Enterocytozoon bieneusi.
10. The use of at least one compound selected from the group having the formula:

wherein:

Ri H, R₂ CHj, R₃ CH₃ and R₄ H, or R, - H or CHjS

BAD ORIGINAL and R_s CH₃₍ Cl, OH or a monophosphate group

R< - CH₃, CH₂OH or Cl and X, and X₂ are independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl or R₄ - CH₂ or R, 0

K or R₄ = CNH-R_e and

Cl or Rg (CH₂)₇CH₃;

and R₂ * OH and R₃ « OH, monophosphate, diphosphate or triphosphate group

AP Ο Ο Ο 3 1 2 or R₂ and R₃ are linked to form a 3', 5'-cyclic mono-phosphate derivative, or a physiologically acceptable salt thereof, or a metabolite thereof, in the manufacture of a pharmaceutical formulation for use in the treatment of an infection by one or more kind of Mycoplasma.

5
11. A method comprising administering to a blood sample containing one or more kind of parasite an amount of a pharmaceutical formulation comprising at least one compound, or a physiologically acceptable salt thereof, or a metabolite thereof, said at least one compound being

10 selected from the group having the formula:

wherein:

Ri « H, R₂ - CH₃, R₃= K or CH₃S and R₄ - cHg

CH₃ and R₄

H, or

BAD ORIGINAL ft and R_s « CH₃ Cl, OH or a monophosphate group

R$ · CHj, CH₂OH or Cl

R, » H or Br or R, s H and X₁ and X₂ are independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl ^{or R<} ^CH* or R« cl· 7 o

K or R₄ - CNH-R₈ and •cO>

Cl or Rj » (CH₂)₇CH₃;

and R_z « OH and R₃ = OH, monophosphate, diphosphate or triphosphate group or R₂ and R₃ are linked to form a 3', 5’-cyclic monophosphate derivative,

AP000312 eaid amount of said pharmaceutical formulation being effective against said one or more kind of parasite.
12. A method comprising administering to a blood sample containing one or more kind of mycoplasma an amount

5 of a pharmaceutical formulation comprising at least one compound, or a physiologically acceptable salt thereof, or a metabolite thereof, said at least one compound being selected from the group having the formula:

wherein:

*1 ^Ri

H, or

H, R₂ ^e CHj, Rj ^b CHj and R₄H or CHjS

BAD ORIG'NAL and R_s CH₃₎ Cl, OH or a monophosphate group

R* - CH₃, CH₂OH or Cl

R, = H or Br or R, = H and X' and X₂ are independently selected from K, methyl, ethyl, hydroxyl, the halogens and carboxyl or R₄ - CH₂

Or R/ c. \ i

I or R₄ » CNH-R_eand or R_e « (ΟΗ₂)₇ΟΗ₃;

and R₂ « OH and R₃ « OH, monophosphate, diphosphate or triphosphate group θ'

II

AP ο Ο Ο 3 1 2 or R; and R₃ are linked to form a 3', 5' -cyclic monophosphate derivative, said amount of said pharmaceutical formulation being effective against said one or more kind of mycoplasma.
13. The use as recited in claim 10 or the method recited in claim 12, wherein said Mycoplasma is selected from the group consisting of Mycoplasma arthritidis, Mycoplasma fermentans and Mycoplasma incognitus.
14. The use of at least one compound selected from the group having the formula:

bad original S i> c o MA wherein:

»1

Ri » H, Rj CH₃, R₃ * CHj and R₄ = H, * H or CH₃S or and R_s » CHj₍ Cl, OH or a monophosphate group r« · CH₃, CHjOH or Cl r₇ β H or Br or R, = H and X, and X₂ are independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl or R₄ or Π₄

AFOOO 3 1 2 ο

« or R₄ - CNH-R_eand *« Cl or R_e (CH₂)₇CH₃;

and Rj - OH and R₃ » OH, monophosphate, diphosphate or triphosphate group or Rj and R₃ are linked to form a 3', 5' -cyclic monophosphate derivative, or a pharmacologically acceptable salt thereof, or a metabolite thereof, in the manufacture of a pharmaceutical formulation for use in the treatment of one or more of the following indications/ infections^hairy leukoplakia, mouth ulcerations - aphthous/herpetic/ bacterial, squamous oral carcinoam, Kaposi's sarcoma oral lesions, periodontitis, necrotizing gingivitis and orofacial herpes zoster.

/y

BAD ORIGINAL ft
15. The use of at least one compound selected from the group having the formula:

wherein:

^R1 “ H, R₂ « CH₃, R₃ = CH₃ and R₄R, H or CH₃S

H, or and R, ^C«* R_s

X and Rj CH₃ Cl, OH or a R_e = CH₃, CH₂OH or Cl R, H or Br /5monophosphate group

APOOO 3 1 2 and X, and X₂ are independently selected front H, methyl, ethyl, hydroxyl, the halogens and carboxyl

Or R₄ * CK₂ or R₄ «

I or R₄ CNH-Rg and

Cl or Re « (CH₂)₇CH₃;

and R₂ * OH and R₃ « OK, monophosphate, diphosphate or triphosphate group

Ii bad origin*¹- ϊ r ί ιι ο ό ΜΑ or R₂ and R₃ are linked to form a 3', 5'-cyclic mono-phosphate derivative or a physiologically acceptable salt thereof, or a metabolite thereof, in the manufacture of a pharmaceutical formulation for use in the treatment of one or more of the following infections! human papilloma virus and rotavi ruses.
16. A method comprising administering to a blood sample having one or more of the following indications/infections: hairy leukoplakia, mouth ulcerations-aphthous/herpetic/bacterial, squamous oral carcinoma, Kaposi's sarcoma oral lesions, periodontitis, necrotizing gingivitis, orofacial herpes 2oater, an amount of a pharmaceutical formulation comprising at least one compound, or a physiologically acceptable salt thereof, or a metabolite thereof, said at least one compound being selected from the group having the formula:

oh wherein:

R, => H, R₂ » CH₃,

R₃ CH₃ and R₄

H, or /7

AP Ο Ο Ο 3 1 2

R, - Η or CH₃S and R₄ - CH, l Rj >=<

and R_s = CH_3i Cl, OH or a monophosphate group R$ - CH₃, CHjOH or Cl Rj = H or Br and Xj and X₂ are Independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl or R₄ CH₂ or R₄ 0 »

or R₄ - CNH-R_e and

BAD ORIGINAL St

Cl ii i r l o u v ma or Rj · (CH₂)₇CH₃;

and R_z = OH and R₃ OH, monophosphate, diphosphate or triphosphate group or R₂ and R₃ are linked to form a 3', 5' -cyclic monophosphate derivative, said amount of said pharmaceutical formulation being effective against said one or more indications/infections.

5
17. A method comprising administering to a blood sample having one or more of the following infections: human papilloma virus and rotaviruses, an amount of a pharmaceutical formulation comprising at least one compound, or a physiologically acceptable salt thereof, or a

10 metabolite thereof, said at least one compound being selected from the group having the formula:

wherein:

R, * H, R₂ » CH₃, R₃ CH₃ and R^ = H, or R, - H or CH₃S

AP Ο Ο Ο 3 1 2 and R₄ and R_s * CH₃₍ Cl, OH or a monophosphate group Rj - CH₃, CHjOH Or Cl r₇ H or Br or R, H and X, and X₂ are independently selected from H, methyl, ethyl, hydroxyl, the halogen· and carboxyl or R₄ or R₄ »

or R₄ - CNK-Rg and

-cQ

Cl

BAD ORIGINAL

S’ t f, Ο Ο 0 RA or 8, . (CHj),CH,;

and R₂ OH and R_a » OH, monophosphate, diphosphate or triphosphate group or R₂ and R₃ are linked to form a 3', 5' -cyclic monophosphate derivative, said amount of said pharmaceutical formulation being effective against said one or more infections.
18. The use recited in any one of claims 1, 7, 10, 14 or 15 or the method recited in any one of claims 11, 12, 16 or 17, wherein said at least one compound has a combination of chemical groups R^ to R^ selected from the following combinations la to Iu:

la: R, - H, R_? - OH, R, - OH and lb:

Ic:

Id:

Ie:

R, Ra R, ® y, r« =

Rl R< = R, Ri H, R₂ » OH, Rj - monophosphate, and

H, Rj and R₃ are linked to form a 5'-cyclic monophosphate derivative

CHl

H ‘ CHj

H, Rj ® OH, Rj « OH, and CH₂C₆H₆

H, Rj - OH monophosphate, and R₄ « CHjCjHj

AP Ο Ο Ο 3 1 2

If: R, - Η, R₂ and R₃ are linked to form a

3', 5'-cyclic monophosphate derivative and R₄ * CHjC₆Hj lg : R, ³ H, R2 ⁼ OH, R, OH, R4 ⁼ CHj

Ih: R, - H, Rj = OH, Rj · monophosphate and R₄ - CH₂

Ii

Xj:

R, c h, R₂ and Rj are linked to form a 3', 5'-cyclic monophosphate derivative and R₄ ³ CH₂

R₃ ³ H, R₂ OH, Rj ³ OH and

0 (I

R4 » COT

Cl

Ik: R, ³ H, R₂ ³ OH, Rj ³ monophosphate and

II

COT

II: Rj ³ OH and

Cl

In: R, < H, R, « OH, R₃ » OH and

R₄ « CNH(CH₂,₇CHj

Σο: Ri ® Η, Rj * OH, R₃ β monophosphate and it

R₄ » CNK(CHj)₇CH₃

Ip: R, « H, Rj and R₃ are linked to form a

3', 5'-cyclic monophosphate derivative, and

R₄ iq: R,

R«

Ir: R,

R<

Is: Ri R<

It: R, ^R<

o tl

CNH(CHj)₇CH₃

CH₃S, Rj « OH, R₃ = OH and ch₂

H, Rj = OH, R₃ OH and

CH₃

CHjOH

H, Rj - OH, R₃ w OH and

CHj Cl

H CHj

H, Rj « OH, R₃ OH and iu: R, = H, R₂ » CH₃, Rj = CK₃ and R₄ » H

AP000J12
19. The use recited in any one of claims 1, 7, 10, 14 or 15 or the method recited in any one of claims 11, 12, 16 or 17, wherein said at least one compound has a combination of chemical groups R1 to R₄ as follows:

Za. R₁ * H, R₂ ^b OH, R3 ^— OH and R4 - CH; __CH₃

H CH₃
20. The use recited in any one of claims 1, 7, 10, 14 or 15 cr · the method recited in any one of claims 11, 12, 16 or 17, wherein said at least one compound has a combination of chemical groups R_] to R_d as follows:

Xb: R, - H, R₂ - OH, R₃ - monophosphate, and

R* - CH₂___* CH₃

H ***· CH₃
21. The use recited in any one of claims 1, 7, 10, 14 or 15 or the method recited in any one of claims 11, 12, 16 or 17, wherein said pharmaceutical formulationcomprises at least one metabolite of said at least one compound, said at least one metabolite being a member of the group consisting of:.

Ν®-(Δ²-isopentenyl) adenine;

6-N-(3-methyl-3-hydroxybutylamino) purine; Adenine;

Hypoxanthine;

Uric Acid; and

Methylated xanthines.

bad original

2?.. The use recited in any one of claims 1, 7, 10, 14 or 15 or the method recited $ 11, 12, 16 or 17, wherein said at least one compound is contained in a macrophage specific liposome micell.
23. The use recited in any one of claims 1, 7, 10, 14 or 15 or the method recited in an_? one of claims 11, 12, 16 or 17, wherein said pharmaceutical formulation ie in the form of a gelatine capsule, tablet, dragee, syrup, suspension, topical cream, suppository, injectable solution, or a kit for the preparation of a syrup, suspension, topical cream, suppository or injectable solution, or is contained a silicone disc, polymer beads or a transdermal patch.
24. The use recited in any one of claims 1, 7, 10, 14 or 15 or the method recited in any one of claims 11, 12, 16 or 17, wherein said pharmaceutical formulation further comprises an effective amount of an adenosine deaminase inhibitor.
25, An article comprising a macrophage specific liposome micell which contains at least one compound, or a physiologically acceptable salt thereof, or a metabolite thereof, said at least one compound being selected from the group having the formula:

wherein:

R₁ *1 = H, R₂ H or and R*

AP0 00 3 1 2 « CHj, Rj CHj and R* H, CHjS ’ Rj .X or and Rj CH₃₎ Cl, OH or a monophosphate group Rj - CHj, CHjOH or Cl R₇ - H or Br or R, s H and X, and X_? are independently selected from H, methyl, ethyl, hydroxyl, the halogens and carboxyl or R₄ or R₄ _bA0OWG'^NAL

Ο

H or R₄ = CNH-R₈ and *« -co)

Cl or Rg - (CH₂),CH₃;

and R₂ = OH and R₃ OH, monophosphate, diphosphate or triphosphate group or R₂ and R₃ are linked to form a 3', 5' -cyclic monophosphate derivative.