WO1992002530A1

WO1992002530A1 - Hexa-n-(o-hydroxybenzyl) neomycin b for inhibiting human retroviruses and for the treatment of aids

Info

Publication number: WO1992002530A1
Application number: PCT/US1991/004849
Authority: WO
Inventors: William Gary Tarpley; Roman P. Holysz
Original assignee: The Upjohn Company
Priority date: 1990-07-30
Filing date: 1991-07-16
Publication date: 1992-02-20
Also published as: JPH05509306A; AU8230091A; AU640511B2; EP0541597A1; CA2085367A1

Abstract

N-poly-(o-hydroxybenzyl) Neomycin B or salts and/or hydrates thereof, can be used to treat humans infected with one or more than one strain of a human immunodeficiency virus.

Description

Hexa-N-(o-hyαroxybenzyl )Neomyci B for inhibiting human retroviruses and for the treatment of AIDS

FIELD OF THE INVENTION

This invention relates to N-poly-(o-hydroxybenzyl) Neomycin B or salts and/or hydrates thereof, as a drug to treat a human cell system, including a human patient, possibly infected with a human retrovirus (HRV) to prevent or retard the further replication of the HRV in that human system or patient.

BACKGROUND OF THE INVENTION

An estimated one to one and one-half million people in the United States are infected with a human retrovirus, the human immunodeficiency virus type I, HIV-1, which is the etiological agent of acquired immunodeficiency syndrome, AIDS, Norman, C, Science, 661-662 (1986). Of those infected, an estimated two hundred and fifty thousands people will develop AIDS in the next five years, Curran, J.W., et al., Science, 1352-1357 (1985). On March 20, 1987, the FDA approved the use of the compound, zidovudine (AZT), to treat AIDS patients with a recent initial episode of pneumocystis carinii pneumonia, AIDS patients with conditions other than pneumocystis carinii pneumonia or patients infected with the virus with an absolute CD4 lymphocyte count of less than 200/mm³ in the peripheral blood. AZT is a known inhibitor of viral reverse transcript- ase, an enzyme necessary for human immunodeficiency virus replication.

U.S. Patent 4,724,232 claims a method of treating humans having acquired immunodeficiency syndrome utilizing 3'-azido-3'-deoxy-thymidine (azidothymidine, AZT). European Patent Application 88306456.0 (Publication No. 0 0300 687; 25.01.89 Bulletin

89/04) describes a method of inhibiting human immunodeficiency virus utilizing a therapeutically effective amount of an antiviral agent to attack the first splice acceptor site of tat m gene of HIV.

Oligodeoxyyribonucleosidemethylphosphonates (OMPs) are disclosed as useful antiviral agents for the treatment of HIV. Neomycin B free base is well known and commercially available from various sources, including for example Sigma (Sigma #N 1876); February 1986, page 869.

SUMMARY OF THE INVENTION

This invention is a method for treating a human infected with one or more than one strain of a human immunodeficiency virus which comprises administering an effective amount of a N- poly-(o-hydroxybenzyl) Neomycin B, or salts and/or hydrates thereof, to the infected human. Examples of alkali metal salt forms of N-poly-(o-hydroxybenzyl) Neomycin B include the sodium, potassium and lithium salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The term human retrovirus (HRV) includes human immunodeficiency virus type I, human immunodeficiency virus type II, or strains thereof, as well as human T cell leukemia virus 1 and

2 (HTLV-1 and HTLV-2) strains apparent to one skilled in the art, which belong to the same viral families and which create similar physiological effects in humans as various human retroviruses

Patients to be treated would be those individuals: 1) infected with one or more than on strain of a human retrovirus as determined by the presence of either measurable viral antibody antigen in the serum and 2) having either a symptomatic AIDS defining infection such as disseminated histoplasmosis, ii) isopsoriasis, iii) bronchial and pulmonary candidiasis includin pneumocystic pneumonia iv) non-Hodgkin's lymphoma or v) Kaposi's sarcoma and being less tha

* sixty years old; or having an absolute CD4 lymphocyte count of less than 200/mm³ in th peripheral blood. Treatment would consist of maintaining an inhibitory level of the compound use according to this invention in the patient at all times and would continue until the occurrence o a second symptomatic AIDS defining infection indicates alternate therapy is needed.

The structural formula of N-poly-(o-hydroxybenzyI) Neomycin B is illustrated in Chart I where R is -(2-hydroxy)phenyl.

This compound as depicted in the structure charts as the free base, however phar macologically acceptable salts and/or hydrates thereof, can be used and administered in practicin the method claimed in this invention. Pharmacologically acceptable salts refers to those salts o the compounds claimed in this invention which would be readily apparent to a manufacturin pharmaceutical chemist to be equivalent to the parent compound in properties such as formulation stability, patient acceptance and bio availability.

Those skilled in the art would know how to formulate the compounds used to practice th method claimed in this invention into appropriate pharmaceutical dosage forms. Examples of th dosage forms include oral formulations, such as tablets or capsules, or parenteral formulations, such as sterile solutions.

When the compound used to practice the method claimed in this invention are administered orally, an effective amount is from about 1 to 100 mg per kg per day. A typical unit dose for a 70 kg human would be from about 50 mg to 1000 mg, preferably 200 mg to 1000 mg taken one to four times per day. Either solid or fluid dosage forms can be prepared for oral administration. Solid compositions are prepared by mixing the compounds used to practice the method claimed in this invention with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, or functionally similar pharmaceutics diluents and carriers. Capsules are prepared by mixing the compounds used to practice the method claimed in this invention with an inert pharmaceutical diluent and placing the mixture into an appropriately sized hard gelatin capsule. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compounds used to practice the method claimed in this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum. Syrups are prepared by dissolving the compounds used to practice the method claimed in this invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preserva- tives. Elixirs are prepared using a hydroalcoholic vehicle such as ethanol, suitable sweeteners such as sugar or saccharin and an aromatic flavoring agent. Suspensions are prepared with an aqueous vehicle and a suspending agent such as acacia, tragacanth, or methyl cellulose.

When the compound used to practice the method claimed in this invention are administered parenterally, it can be given by injection or by intravenous infusion. An effective amount is from about 1 to 100 mg per kg per day. Parenteral solutions are prepared by dissolving the compounds used to practice the method claimed in this invention in water and filter sterilizing the solution before placing in a suitable sealable vial or ampule. Parenteral suspensions are prepared in substantially the same way except a sterile suspension vehicle is used and the compounds used to practice the method claimed in this invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle.

The exact route of administration, dose, or frequency of administration would be readily determined by those skill in the art and is dependant on the age, weight, general physical condition, or other clinical symptoms specific to the patient to be treated. Patients to be treated would be those individuals: 1) infected with one or more than one strain of a human immunodeficiency virus as determined by the presence of either measurable viral antibody or antigen in the serum and 2) having either a symptomatic AIDS defining infection such as i) disseminated histoplasmosis, ii) isoporiasis, iii) bronchial and pulmonary candidiasis including pneumocystis pneumonia, iv) non-Hodgkin's lymphoma, or v) Kaposi's sarcoma and being less than sixty years old; or having an absolute CD4 lymphocyte count of less than 200/mm³ in the peripheral blood. Treatment would consist of maintaining an inhibitory level of the compounds of this invention in the patient at all times and would continue until the occurrence of a second symptomatic AIDS defining infection indicates alternate therapy is needed. Preparation 1: Preparation of N-poly-(o-hydroxybenzyl) Neomycin B Part A:

12.33 g. (20mM) of Neomycin B free base is dissolved in 25 ml. of water. The solution is diluted with 50 ml of methanol. The resulting solution is mixed with an aqueous methanol solution of salicylaldehyde (SAL; salicylic aldehyde; 2-hydroxybenzaldehyde) consisting of 17.1 g. (140 mM) of the SAL, 100 ml methanol and 200 ml water). The resulting solution is heated to boiling, then allowed to stand at room temperature for

48 hours. The mixture is heated to boiling and 250 ml of hot water added and the mixture refrigerated at 0-5° C. for 24 hours. The supernatant is decanted and the resinous product dissolved in the methanol :dioxane (80:20), slurried with 2 g. of DARCO G60 charcoal and filtered though diatomaceous earth (Celite). The filtrate is added with stirring to 300 ml of ether to precipitate the desired Neomycin B Schiff base. The mixture is refrigerated overnight, and the supernatant decanted and the solid dried jn vacuo. Part B:

The product of Part A is dissolved in 200 ml methanol and hydrogenated at roo temperature at 50 psi initial pressure for 24 hours with 1.0 g of PtO₂ (Adams' catalyst). T pressure drop is 7.0 psi per 100 mM. The product is filtered through diatomaceous earth (Celit and the filtrate concentrated jn vacuo to about 50 ml and resulting concentrate added slowly to 30 ml of ether. The resulting mixture is refrigerated overnight, filtered/decanted and the solid produ - dried initially at room temperature in vacuo for several days and finally overnight at 50° C. in vacuum oven to yield 9.5 g of N-poly-(o-hydroxybenzyl) Neomycin B. Analytical results: N, 6.5 eq. wt. 216.1; Molecular Weight (MW) 1296.6; IR 3260, 1605, 1586, 1485, 1045, 1030, 750. Without further elaboration, those skilled in the art can practice the present invention t its fullest extent. The following detailed examples further describe how to use the compound claimed in this invention to treat humans infected with one or more than one strain of a huma immunodeficiency virus. These examples are merely illustrative and are not limitations of th preceding disclosure. Those skilled in the art will promptly recognize appropriate variations fro the examples. In each example, any compound claimed in this invention could replace th compound used in the particular example. Example 1 Hard Gelatin Capsules

One thousand two-piece hard gelatin capsules for oral use, each capsule containing 50 m of N-poly-(o-hydroxybenzyl) Neomycin B, are prepared from the following: N-poly-(o-hydroxybenzyl) Neomycin B 50 gm

Lactose 100 gm

Cornstarch 20 gm

Talc 20 gm

Magnesium Stearate 2 gm The N-poly-(o-hydroxybenzyl) Neomycin B is added to the other ingredients, mixed an encapsulated in the usual manner. Example 2 Tablets

One thousand tablets, each containing 50 mg of N-poly-(o-hydroxybenzyl) Neomycin B are prepared from the following: N-poly-(o-hydroxybenzyl) Neomycin B 50 gm

Lactose 75 gm

Cornstarch 50 gm

Magnesium Stearate 4 gm

Light liquid petrolatum 5 gm The N-poly-(o-hydroxybenzyI) Neomycin B is added to the other ingredients, mixed and slugged. The slugs are broken down by forcing through a number sixteen screen. The resulting granules are then compressed into tablets. Example 3 Parenteral solution A sterile aqueous solution for parenteral intravenous injection containing 150 mg of l-(4- chlorobenzoyl)-N-poly-(o-hydroxybenzyl) Neomycin B in one liter of solution is prepared from the following:

N-poly-(o-hydroxybenzyl) Neomycin B 150 mg

Water for injection, qs 1000 mg The N-poly-(o-hydroxybenzyl) Neomycin B is sterilized, added to the sterile water, filled into sterile containers and sealed.

The utility of this invention is demonstrated by the ability of N-poly-(o-hydroxybenzyl) Neomycin B to practice the method claimed in this invention to inhibit HIV-1 tat mediated transactivation, enzyme essential for human immunodeficiency virus replication. HIV contains various genes, including "tat" which encodes a trans-activator (TAT) that functions in the infected cell by increasing the levels of steady-state viral mRNA as well as the translational utilization of this mRNA. TAT is both essential for HIV replication and not structurally related to any normal cellular protein. "A Rapid, Quantitative Bioassay Based on the Human Immunodeficiency Virus Trans-Activator" is described in AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol. 5, Number 5, 1989, pp 507-516. The results (% Inhibition in sCD4 levels) in Table I.

The anti-HIV utility of this invention is further demonstrated by determining the levels of HIV p24 and HIV RNA in culture supematants of human peripheral blood lymphocytes (PBLs) 3 and 4 days after HIV infection. The HIV infectivity experiments were conducted in primary cultures of (PBLs) with HTLV-IIIB. Cultures were infected in triplicate with HIV, Compound 1 added, and 3 to 4 days after infection the levels of HIV p24 synthesized and released were quantified by an enzyme-linked immunosorbent assay (ELISA) [R. Maiolini and R. Masseyeff, J. Immunol. Methods 8, 223 (1975)] using a monoclonal capture antibody to HIV p24 (DuPont, Wilmington, DE) and recombinant HIV p24 calibration standard (MicroGenSys, West Haven, CT). The amounts of HIV RNA synthesized in these infected cultures were also determined by hybridization analysis with a HIV-specific -32P labeled probe. The absolute HIV RNA levels were determined by normalizing hybridization values to values obtained from a standard preparation of HIV RNA hybridization in parallel. The results are reported in Table II.

The proliferation of mitogen-stimulated PBLs 4 days after exposure to Compound 1 was quantified relative to nondrug-treated controls to ensure that the infectivity experiments were conducted at non-cytotoxic concentrations of drug. At 40 ug/ml of Compound 1 did not inhibit PBL proliferation. The results are reported in Table III. CHART I

Compound No. 1

H

TABLE I

INHIBITION OF HIV-1 tat MEDIATED TRANSACTIVAΗON

ug/ml sCD4 in Culture Supernatant % Inhibition

(ug/ml) in sCD4 Levels

20 6.48 83

10 22.15 43

43.8

38.7

TABLE II Inhibition of HIV-1 Replication of U-6466 % Inhibition

U-6466 (Mg/ml) p24 HIV RNA

Day Day

3 4 3 4

40 4

0.4 0.04

Claims

1. A method for inhibiting the replication of a human retrovirus (HRV) in a human infected with one or more than one strain of a human retrovirus comprising the systemic administration o an amount effective to inhibit the replication of the HRV of a N-poly-(o-hydroxybenzyl) Neomyci B of Formula I

where R is -(2-hydroxy)phenyl; or salts and/or hydrates thereof, to said human.

2. A method according to Claim 1 where the human is infected with human immunodeficiency virus type I.

3. A method according to Claim 1 where the human is infected with human immunodeficiency virus type ϋ.

4. A method according to Claim 1 where the administration is oral.

5. A method according to Claim 1 where the administration is parenteral.

6. A method according to Claim 1 where the compound has Molecular Weight (MW) of about 1296.6.

7. A method of treating a human having acquired immunodeficiency syndrome comprising the systemic administration of an effective inhibitory amount of a N-poly-(o-hydroxybenzyl)

Neomycin B of Formula I

where R is -(2-hydroxy)phenyl; or salts and/or hydrates thereof, to said human.

A N-poly-(o-hydroxybenzyl) Neomycin B of Formula I:

wherein R is -(2-hydroxy)phenyl; or salts and/or hydrates thereof.

9. A compound according to Claim 8 having a molecular weight (MW) about 1296.6. 10. Use of a N-poly-(o-hydroxybenzyl) Neomycin B of Formula I

where R is -(2-hydroxy)phenyl, or salts and/or hydrates thereof, to prepare a medicament fo inhibition of a human retrovirus (HRV) in a human infected with one or more strain of a huma retrovirus.