METHOD OF INHIBITING BIOSYNTHESIS OF EIf5A

Abstract: Inhibition of the intracellular biosynthesis of EIf-5A comprises administration of a polyamine of formula R1N1(R2)(CH2)mN2(R3)(CH2)nN3(R4)(CH2)mN4(R5)R6 (I), R1N1H(CH2)vN2H(CH2)wN3H(CH2)xN4H(CH2)yN5H(CH2)zN6HR6 (I'), (I'') or (I''') in an amount sufficient to deplete the intracellular supply of spermidine (required for EIf-5A biosynthesis) but insufficient to affect polyamine homeostasis: R, R6 = H (except for (I)) alkyl or 1-12C aralkyl; R2-R5 = H, R1 or R6; R7 = H, alkyl, aryl or 1-12C aralkyl; m, n = 3-6; v-z = 3-10; R8-R13 = H, alkyl, aryl, arylalkyl or cycloalkyl such that any alkyl chains are optionally interrupted by \- 1 etheric O; N1-N4 = nitrogen atoms capable of protonation at physiological pH; a, b = 0-4, but both may not be 0; A-C = bridging groups which effectively maintain the distance between the N atoms such that the polyamine: (i) is capable of uptake by a target cell or is capable of binding to \- 1 polyamine site of a receptor located within or on the surface of a cell upon administration; (ii) upon uptake by the target cell, competitively binds via an electrostatic interaction between the positively charged nitrogen to biological counter-ions such that the polyamine on binding to the biological counter ion, functions in a manner biologically different from the intracellular polyamines; and where \- 1 of the bridging groups may contain \- 1 CH(OH) group which is not ~a to either of the nitrogen atoms.

Description: BACKGROUND OF THE INVENTION The initiation factor, EIf5A, is unique in that it is the only known cellular protein that contains the amino acid hypusine (Hpu) [N ε -(4-amino-2-hydroxybutyl)lysine], an unusual naturally occurring amino acid, having the structure: Hypusine was first isolated from bovine brain extracts by Shiba et al in 1971 [ Biochim. Biophys. Acta. , Vol. 244, pages 523-531 (1971)]. The molecule has two chiral centers at positions 2 and 9, each of which can be classified R or S by the Cahn-Ingold-Prelog method. The post-translational formation of the (2S, 9R) diastereomer: has been shown to occur on a precursor protein of the eukaryotic initiation factor 5A, i.e., EIf5A (formerly called eIF-4D) [Cooper et al, Proc. Natl. Acad. Sci. U.S.A. , Vol. 80, pages 1854-1857 (1983); and Safer, Eur. J. Biochem., Vol. 186, pages 1-3 (1989)]. EIf5A is biosynthesized by the post-translational aminobutylation of lys-51 of the precursor polypeptide followed by hydroxylation which results in hypusine at residue 51. In the mid-1970's, EIf5A was shown to stimulate ribosomal subunit joining and to enhance 80 S-bound Met-t-RNA i reactivity with puromycin [Anderson et al, FEBS Lett., Vol. 76, pages 1-10 (1977); and Kemper et al, J. Biol. Chem., Vol. 251, pages 5551-5557 (1976)]. Later in 1983, Cooper et al, supra , suggested that a hypusine-modified protein serves as an important initiation factor in all growing eukaryotic cells. In 1986, Park et al [ J. Biol. Chem., Vol. 261, pages 14515-14519 (1986)] isolated the EIfSA protein from human red blood cells and elucidated the amino acid sequence surrounding the single hypusine residue, as Thr-Gly-Hpu-His-Gly-His-Ala-Lys. EIf5A has also been found to be essential to HIV replication [Bevec et al, J. Proc. Natl. Acad. Sci. U.S.A. , Vol. 91, pages 10829-10833 (1994); and Ruhl et al, J. Cell Biol. , Vol. 123, pages 1309-1320 (1994)]. The initial step in the biosynthesis of EIf5A in the cell requires spermidine as the aminobutyl donor. Document EPA 0349224 describes compounds and pharmaceutical compositions which have an anti-neoplastic, anti-viral, anti-retroviral or anti-psoriasis effect. Document WO 96/12480 discloses a method for treating and preventing vascular proliferative disorders comprising administering a particular polyamine in an effective amount. A method of preventing or retarding the growth of cancerous cells and other pathological cells is described in document US 5541230. Document US 5498522 describes the use of polyamine analogs to treat malignant solid tumours and methods for evaluating the responses of individuals undergoing such treatment. Compositions and methods for the in vitro and in vivo treatment of mammalian cells to initiate cell growth associated with malignant and non-malignant diseases are described in document WO 94/15596. An article "Diamine and triamine analogs and derivatives as inhibitors of deoxyhypusine syhthase: synthase and biological activity" published in the Journal of Medical Chemistry, volume 38, number 16, 1995, pages 3053-3061, XP002110818 discloses the use of particular di and triamines to reduce the synthesis of EIf5A. It is an object of the present invention to provide a method of inhibiting or preventing intracellular biosynthesis of EIf5A. SUMMARY OF THE INVENTION The above and other objects are realized by the present invention, one embodiment of which relates to the use of a polyamine having the formula wherein: R 1 and R 6 may be the same or different and are H, alkyl or aralkyl having from 1 to 12 carbon atoms, provided that R 1 and R 6 are not H; R 2 -R 5 may be the same or different and are H, R 1 or R 6 ; m is an integer from 3 to 6, inclusive; n is an integer from 3 to 6, inclusive; or a salt thereof with a pharmaceutically acceptable acid, in the preparation of a medicament for the treatment of HIV by the inhibition or prevention of the intracellular biosynthesis of EIf5A. The polyamine may be diethylhomospermine or diethylnorspermine. The medicament is preferably a pharmaceutical composition in unit dosage form and the amount of polyamine in said composition is sufficient to provide a dosage in the range from 5 to 200 mg/m 2 . BRIEF DESCRIPTION OF THE DRAWINGS Figures 1-6 are graphic depictions of the results obtained from employing the composition of the invention in the method of the invention. DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated on the discovery that polyamines of the above formula, when administered to human or non-human mammals, suppress the intracellular biosynthesis of spermidine by depleting the cell of the enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), thereby depleting the supply of intracellular spermidine available to initiate synthesis of EIf5A. As a result, depending on the dosage of polyamine administered, the amount of intracellular EIf5A produced may be severely limited or eliminated. Suitable polyamines for use in the compositions and methods of the present invention are those described in U.S. Patent Nos. 5,091,576; 5,393,757; and 5,510,390. Methods for the preparation of the polyamines are also disclosed therein. Hydroxy-substituted polyamines suitable for use in the methods and compositions of the invention and methods for their production are described in U.S. patent application serial no. 08/595,877 filed February 6, 1996. It will be understood that those skilled in the art, given the disclosure herein of the invention, will be able to determine, without the exercise of undue experimentation, the dosage of polyamine necessary to reduce the intracellular production of EIf5A to a desired level in any particular application while not severely disrupting intracellular polyamine homeostasis. Generally, dosages in the range of from 5 to 200 mg/m 2 will be sufficient. It will be appreciated that while the agents described above form acid addition salts and carboxy acid salts, the biological activity thereof will reside in the agent itself. These salts may be used in human medicine and presented as pharmaceutical formulations in the manner and in the amounts (calculated as the base) described herein, and it is then preferable that the acid moiety be pharmacologically and pharmaceutically acceptable to the recipient. Examples of such suitable acids include (a) mineral acids, i.e. , hydrochloric, hydrobromic, phosphoric, metaphosphoric and sulfuric acids; (b) organic acids, i.e., tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, gulonic, succinic and aryl-sulfonic acids, e.g., p-toluenesulfonic acid. The pharmaceutical compositions of the invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally. The active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient. It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present invention. Illustrative of such methods, vehicles and carriers are those described, for example, in Remington's Pharmaceutical Sciences , 4th ed. (1970), Those skilled in the art, having been exposed to the principles of the invention, will experience no difficulty in determining suitable and appropriate vehicles, excipients and carriers or in compounding the active ingredients therewith to form the pharmaceutical compositions of the invention. The therapeutically effective amount of active agent to be included in the pharmaceutical composition of the invention depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc. Generally, an amount of active agent is included in each dosage form to provide from 0.1 to 250 mg/kg, and preferably from 0.1 to 100 mg/kg. While it is possible for the agents to be administered as the raw substances, it is preferable, in view of their potency, to present them as a pharmaceutical formulation. The formulations of the present invention for human use comprise the agent, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Desirably, the formulations should not include oxidizing agents and other substances with which the agents are known to be incompatible. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the agent with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agent with the carrier(s) and then, if necessary, dividing the product into unit dosages thereof. Formulations suitable for parenteral administration conveniently comprise sterile aqueous preparations of the agents which are preferably isotonic with the blood of the recipient. Suitable such carrier solutions include phosphate buffered saline, saline, water, lactated ringers or dextrose (5% in water). Such formulations may be conveniently prepared by admixing the agent with water to produce a solution or suspension which is filled into a sterile container and sealed against bacterial contamination. Preferably, sterile materials are used under aseptic manufacturing conditions to avoid the need for terminal sterilization. Such formulations may optionally contain one or more additional ingredients among which may be mentioned preservatives, such as methyl hydroxybenzoate, chlorocresol, meta-cresol, phenol and benzalkonium chloride. Such materials are of special value when the formulations are presented in multi-dose containers. Buffers may also be included to provide a suitable pH value for the formulation. Suitable such materials include sodium phosphate and acetate. Sodium chloride or glycerin may be used to render a formulation isotonic with the blood. If desired, the formulation may be filled into the containers under an inert atmosphere such as nitrogen or may contain an anti-oxidant, and are conveniently presented in unit dose or multi-dose form, for example, in a sealed ampoule. EXAMPLE 1 The CEM-SS cell line derived from a human T4-lymphoblastoid line was chosen as a test model because it is CD4 positive and is susceptible to infection with HIV. The cells were treated with a 1 µm dose of diethylhomospermine that resulted in only a mild inhibition of cell growth, i.e., a concentration estimated to result in only 25% growth inhibition (IC 75 ). The results over 72-hour, 96-hour and 144-hour treatment programs are set forth graphically in Fig. 1 which shows the decrease in the levels of spermidine (SPD) and spermine (SPM) in the cell line as a result of treatment with diethylhomospermine (DEHSPM). The results of the 96-hour treatment on the levels of hypusine in the cell line are set forth in Fig. 2 (control) and Fig. 3. The reduced level of hypusine reflects the decreased production of EIf5A in the cells as a result of treatment with diethylhomospermine. Fig. 4 shows the effect on the levels of hypusine in the cell line after 72, 96 and 144 hours of treatment with diethylhomospermine. EXAMPLE 2 Several human patients were selected for a five (5) day protocol of treatment with diethylnorspermine. Polyamine levels [spermidine (SPD), diethylnorspermine (DENSPM) and hypusine (Hpu)] were measured on day one and used as pretreatment controls. Each patient then received the drug for five days and a second sample was taken. In several cases, samples were taken in the middle of the dosing schedule. The dose is contained in the first column of the following table under "Patient." For example, "1-094" means this patient received a dose of 94 mg/m 2 once per day for five days; "1-118" means this patient received a dose of 118 mg/m 2 once per day for five days, etc. The polyamine levels were again determined on day five. The results are set forth in the following table. As can be seen from the results set forth in the foregoing table, the levels of spermidine were lowered in each patient's cell line, resulting in a decreased production of EIf5A as demonstrated by the reduced levels of hypusine in each case. The results for hypusine levels in patient 1-094 are graphically depicted in Fig. 5 [hypusine level on day Monday (control)] and Fig. 6 [hypusine level on day Friday (after treatment)].

Claims: The use of a polyamine having the formula: wherein: R 1 and R 6 may be the same or different and are H, alkyl or aralkyl having from 1 to 12 carbon atoms, provided that R 1 and R 6 are not H; R 2 - R 5 may be the same or different and are H, R 1 or R 6 ; m is an integer from 3 to 6, inclusive; n is an integer from 3 to 6, inclusive; or a salt thereof with a pharmaceutically acceptable acid, in the preparation of a medicament for the treatment of HIV by the inhibition or prevention of the intracellular biosynthesis of EIf5A. The use of a polyamine as claimed in claim 1, wherein said polyamine is diethylhomospermine. The use of a polyamine as claimed in claim 1, wherein said polyamine is diethylnorspermine. The use of a polyamine as claimed in any preceding claim, wherein said medicament is a pharmaceutical composition in unit dosage form. The use of a polyamine as claimed in claim 4, wherein the amount of polyamine in said composition is sufficient to provide a dosage in the range from 5 to 200 mg/m 2 .

Inventors: BERGERON RAYMOND J JR, BERGERON, RAYMOND, J., JR.
Applicants: UNIV FLORIDA, UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INC.
Application year: 1997
Main classification: A61K31
IPC codes: A61K31, C07D239, A61P31, A61P43