AU640511B2 - Hexa-n-(o-hydroxybenzyl) neomycin b for inhibiting human retroviruses and for the treatment of aids - Google Patents

Description

OPI DATE 02/03/92 AOJP DATE 09/04/92 APPLN. ID 82300 91 PCT NUMBER PCT/US91/04849 TREATY (PCT) INTERNA (51) International Patent Classification 5 (11) International Publication Number: WO 92/02530 C07H 15/232, A61K 31/71 Al (43) International Publication Date: 20 February 1992 (20.02.92) (21) International Application Number: PCT/US91/04849 (72) Inventors; and Inventors/Applicants (for US only) TARPLEY, William, (22) International Filing Date: 16 July 1991 (16.07.91) Gary [US/US]; 5093 Chickadee, Kalamazoo, MI 49002 HOLYSZ, Roman, P. [US/US]; 4209 Old Colony Priority data: Road, Kalamazoo, MI 49008 (US).

559,851 30 July 1990 (30.07.90) US (74) Agent: JAMESON, William, The Upjohn Company, Parent Application or Grant Kalamazoo, MI 49001 (US).

(63) Related by Continuation US 559,851 (CON) (81) Designated States: AT (European patent), AU, BB, BE Filed on 30 July 1990 (30.07.90) (European patent), BF (OAPI patent), BG, BJ (OAPI patent), BR, CA, CF (OAPI patent), CG (OAPI patent), (71) Applicant (for all designated States except US): THE UP- CH (European patent), CI (OAPI patent), CM (OAPI JOHN COMPANY [US/US]; 301 Henrietta Street, Kal- patent), CS, DE (European patent), DK (European paamazoo, MI 49001 tent), ES (European patent), FI, FR (European patent), GA (OAPI patent), GB (European patent), GN (OAPI patent), GR (European patent), HU, IT (European patent), JP, KP, KR, LK, LU (European patent), MC, MG, A ML (OAPI patent), MN, MR (OAPI patent), MW, NL S(European patent), NO, PL, RO, SD, SE (European patent), SN (OAPI patent), SU, TD (OAPI patent), TG 6451 (OAPI patent), US.

Published With international search report.

(54)Title: HEXA-N-(O-HYDROXYBENZYL) NEOMYCIN B FOR INHIBITING HUMAN RETROVIRUSES AND FOR THE TREATMENT OF AIDS I (57) 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.

See back of page WO 92/02530 PCT/US91/04849 Hexa-N-(o-hyoroxybenzyl)Neomycin 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, Science, 661-662 (1986). Of those infected, an estimated two hundred and fifty thousands people will develop AIDS in the next five years, Curran, 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/mm 3 in the peripheral blood. AZT is a known inhibitor of viral reverse transcriptase, 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 (azidothymidi; i, 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 mI 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 Npoly-(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 WO 92/02530 PC1'/US91/04849 -2families 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 one strain of a human retrovirus 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) isopsoriasis, iii) bronchial and pulmonary candidiasis including pneumocystic 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/mm 3 in the peripheral blood. Treatment would consist of maintaining an inhibitory level of the compound used according to 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.

The structural formula of N-poly-(o-hydroxybenzyl) 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 pharmacologically acceptable salts and/or hydrates thereof, can be used and administered in practicing the method claimed in this invention. Pharmacologically acceptable salts refers to those salts of the compounds claimed in this invention which would be readily apparent to a manufacturing 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 the method claimed in this invention into appropriate pharmaceutical dosage forms. Examples of the 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 pharmaceutical 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- WO 92/02530 PCT/US91/04849 -3tives. 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. Parenter' juspeasions 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/mm 3 in the peripheral blood. Treatment would consist of maintaining an inhibitory level of the compounds of this invention in the patient at ;11 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 0-50 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 in vacuo.

WO 92/02530 PCT/US91/04849 -4- Part B: The product of Part A is dissolved in 200 ml methanol and hydrogenated at room temperature at 50 psi initial pressure for 24 hours with 1.0 g of PtO 2 (Adams' catalyst). The pressure drop is 7.0 psi per 100 mM. The product is filtered through diatomaceous earth (Celite) and the filtrate concentrated in vacuo to about 50 ml and resulting concentrate added slowly to 300 ml of ether. The resulting mixture is refrigerated overnight, filtered/decanted and the solid product dried initially at room temperature in vacuo for several days and finally overnight at 500 C. in a vacuum oven to yield 9.5 g of N-poly-(o-hydroxybenzyl) Neomycin B. Analytical results: N, 6.58; 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 to its fullest extent. The following detailed examples further describe how to use the compounds claimed in this invention to treat humans infected with one or more than one strain of a human immunodeficiency virus. These examples are merely illustrative and are not limitations of the preceding disclosure. Those skilled in the art will promptly recognize appropriate variations from the examples. In each example, any compound claimed in this invention could replace the 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 mg 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 and 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 WO 92/02530 PCT/US91/04849 The N-poly-(o-hydroxybenzyl) Neomycn 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 1-(4chlorobenzoyl)-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. 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 supernatants 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) 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.

WO 92/02530 WO 9202530PCI'/US91/64849 -6- CHART I Compound No. 1

H

E2N CH 2

R

R

CH2

NH

CH

2

NH

H O

H

R-CH

2 N CH 2

H

OHi

CH

2

R

WO 92/02530 PCi-IUS9 1/04849 -7- TABLE I INHIBITION OF HIV-1 tat MEDIATED TRANSACTIVATION uglml sCD4 in Culture Supernatant (ug/mi) 6.48 22.15 43.8 38.7 Inhibition in sCD4 Levels WO 92/02530 WO 9202530PC T/US9I /04849 TABLE II Inhibition of HIV-I Replication of U-6466 Inhibition p24 HIV RNA Day Day U-6466 (Mg/mi) 3 4 3 4 4 0.4 0.04 WO 92/02530 PCrl US9 1/04849 -9- TABLE III U-6466 (ug/ml) 4 0.4 0.04 Cell Viability 93 92 96

Claims (10)

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 of an amount effective to inhibit the replication of the HRV of a N-poly-(o-hydroxybenzyl) Neomycin B of Formula I H H CD 2 R 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 II.

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

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 WO 92/02530 PCT/US9 1/04849 -11- H zN CH 2 R R-CB 2 where R is -(2-hydroxy)phenyl; or salts and/or hydrates thereof, to said human.

8. A N-poly-(o-hydroxybenzyl) Neomycin B of Formula 1: H pz N CH 2 R H 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. WO 92/02530 WO 9202530PCr/US91 /048d9 -12- 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 medicamient for inhibition of a human retrovirus (HRV) in a human infected with one or more strain of a human retroviras. INTERNATIONAL SEARCH REPORT In~arnational Applica.,on No I)CT/US 91 /048A9 I. CLAkSSIrICAILO\OF SUBJECTNIMATTER i if several classification symsoos appi5. indicate allit' Accorat.s! to Internationsi Patent classification IIPC1 or to both sationai Classitication and IPC 0 07 H 15/232 A 61 K 31/71 11. FIELD)S SEARCIIEI) Minimum Documentation Searched' Classification bstem Classification Symbols C 07 H A 61 K Documentation Searched other than Minimum Documentation to the Extent that ouch Documents arc Included to the Fields Searchedg Ill. DOCUMEFNTS CONSIDERED TO BE RLELEVAINT 9 Categors Citation of Document. 11 %ith indication. %here appropriate, of the relevant patsaeesil Relevnt to Claim \o.13 Y Journa~l of Antibiotics, vol. 26, no.

10, 1973, 2-10 W.T. Shier et al. Chemistry and biochemistry of the neornycins. XVI synthesis and bioactivity of hexa-n-benzyineomycins t pages 547-550, see the whole article YThe Journal of Antibiotics, vol. XLII, no. 1, 8-10 1989, Y. Take et al. Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian ryeloblastosis virus reverse transcriptases and cellular DNA polymerases t t pages 107-115, see whole article, especially page 110, table 1 Special categorie of cited documents 0 later document published after the international filing date or priority date and not in conflict with the application but docitmentf defininR the general state of the art which it not cited to understand the prtnciple or theory underlying the considered to he of particular relevance Invention 'El earlier document but published on or after the International X' document of particular relevace: the claimed Invention filing date cannot be considered novel or cannot be considered to ILI document which may throw doubts on priority clainmis) or involve an inventive step n which is cited to esiablish the publication date of another document of particular relevance: the Claimed inventlon-' citation or other Special reason is specified) cannot be considered to involve an inventive step when the '0 document referring to an oaml disclosure. une. exhibition or document Is combined with one or more other such docu- other means ments. ouch combination being ouvious to a person skilled document published prior to the international Miing date but In the art. later than the prioriry date claimed W. document memnber of the same patent family- IV. CERTIFICATION Date of the ACtual1 Coinpiios of the International Search Date of Mailing of this International Search Report- 03-10-1991 2 4. 10. 91 lnternationai Scorching Autsoriry Signature of uhoie Offic' EUROPEAN PATENT OFFICE van cer Haas form i'CT11SA:210 ttma lneij Januarv 19iI51 International FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET llcatlon No. PCTI U.S91 /04849 I V. OBSERVATION WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE 1 This International search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons 1. Claim numbers 1-7 because they relate to sublect matter not required to be searched by this Authority, namely Please see rule 39.1(iv) PCT. Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods. 2 UI Claim numbers because they relate to parts of th internlional application that do not comply with the prescnbed requirements to such an extent that no meaningful International search can be c. med out, specifically 3. O Claim numbers the second and third sentences of PCT Rule E 4(a). because they are dependent claims and are not drafted in accordance with VIO. OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authonty found multiple Inventions in this International application as follows: 1. I] As all required additional search fees were timely paid by the applicant, this Internationa search report covers all searchable claims of the International application 2. O As only some of the required additional search fees were timely paid by the applicant,thir international search report covers only those claims of the International application for which fees were paid, specifically claims: 3. D No required additional search fees were timely paid by the applicant. Consequently, this International search report is restricted to the invention first -entloned in the claims; it is covered by claim numbers: 4. As all 5earchable clairs could be searched without effort justifying an add'tional fee, the International Searching Authonty did not invie payment of any additional fee. Remark on Protest I The additional search fees were accompanied by applicant's protest. i]No protest accompanied the payment of ad'itional search fees. Form PCT/ISA/210 (supplemental sheet P9412B 05/91