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Title:  Combination therapy for lymphoproliferative diseases

United States Patent:  6,316,435

Inventors:  Byrd; John C. (Bethesda, MD); Grever; Michael R. (Highland, MD); Flinn; Ian W. (Lutherville, MD); Waselenko; Jamie K. (San Antonio, TX)

Assignee:  SuperGen, Inc. (Dublin, CA)

Appl. No.:  256666

Filed:  February 24, 1999

Abstract

Disclosed are methods and kits for treating lymphoproliferative diseases in a host including (co)administering to the host pentostatin, at least one alkylating agent and at least one methylated xanthine.

DETAILED DESCRIPTION OF THE INVENTION

While precise knowledge of the mechanism of the synergism due to the combination is not necessary to the practice of the invention, a brief discussion of the putative mechanism may be helpful to understanding of the invention. Of course, because precise knowledge of the mechanism is not necessary to the practice of the invention, the inventor expressly does not wish to be bound to any discussion of mechanism present herein.

Generally speaking, the combination of pentostatin, alyklating agents, and methylated xanthines appears to be synergistically more effective in treating lymphoproliferative diseases than monotherapy of any of the three pharmaceuticals alone. Several reasons for this might exist. First of all, the three pharmaceuticals have different putative mechanisms of action, as is discussed further below. This implies that the dosages of each of the three pharmaceuticals may be increased to a point where the total pharmaceutical delivery exceeds the amount that safely can be given of any one of the pharmaceuticals. Additionally, use of the inventive combination therapy may tend to reduce the chances of developing cross-resistance to the various mechanisms of action of the three pharmaceuticals. Such approaches of combination therapies (different than these described herein) in the treatment of other hematologic malignancies has lead to cure in a minority of patients.

At this point, a discussion of the individual components of the inventive combination therapy may be helpful.

While not wishing to be bound by a particular mechanism or explanation, it appears that pentostatin may act through the lymphocyte's adenosine deaminase (ADA) pathways. While ADA is a ubiquitous enzyme, it is found in higher concentrations in lymphoid tissue, particularly T-lymphocytes.

2'-deoxycoformycin (also referred to as DCF, pentostatin, or NIPENT) is a nucleoside analog produced by Streptomyces antibiotics, and has been shown to be a quasi-irreversible inhibitor of ADA. By favoring the predominance of deoxycytidine kinase (DCK) over the dephosphorylating enzyme 5-nucleotidase in lymphocytes it is presumed to induce a preferential accumulation of deoxyadenosine-5'-triphosphate (dATP). Dighiero, G., "Adverse and beneficial immunological effects of purine nucleoside analogues," Hematol Cell Ther, 38:575-581 (1996).

In humans, a genetic deficiency of adenosine deaminase may cause severe combined immunodeficiency. This enzyme is responsible for deamination of adenosine to inosine and deoxyadenosine to deoxyinosine in the purine salvage pathway. ADA deficiency is characterized by a selective lymphopenia of both T and B cells resulting in reduced cellular and humoral immune capacity, which may be attributed to the toxic effect of deoxyadenosine accumulation.

While the exact nature of the ADA pathway intervention seems unclear, it may be that pentostatin's inhibition of adenosine deaminase might mimic an ADA-deficient state. Lack of ADA is believed to lead to a build up of deoxyadenosine and adenosine triphosphate in the cell, thus fatally accelerating DNA strand breaks in the cell.

Under normal conditions, lymphocytes are continuously breaking and rejoining DNA. When this physiological process is accelerated by the effect of excess adenosine triphosphate, it leads to consumption of NAD for poly-ADP-ribose synthesis. This polymer is produced from nicotinamide adenosine dinucleotides (NAD) in a reaction catalyzed by the chromatin-associated poly(ADP-ribose) synthetase, leading to a depletion of the NAD content of the cell. This depletion induces a profound alteration of cellular reducing power, because of lethal ADP and ATP depletion. The result is programmed cell death through activation of a Ca++, Mg++, dependent endonuclease.

At doses of 5 mg/m2 for 3 consecutive day every 3 weeks, ECOG investigators have been able to show a 32% response rate in refractory lymphomas using pentostatin as a monotherapy. Johnson and colleagues found a 30% response rate in patients with refractory/relapsed B-CLL. Seventeen of 29 of the study patients had prior treatment with either 2-CDA or fludarabine, thus suggesting differential efficacy compared to other purine analogs. Responses seen after treatment with alkylators and other purine analogs demonstrate possible non-cross-resistance. S. Johnson et al., Phase In Evaluation of 2'deoxycoformycin (Pentostatin) in a Five Day Schedule for the Treatment of Relapsed/Refractory Chronic Lymphocytic Leukemia (Cll), Blood:590a (1996).

Most conventional alkylating agents are useful in the practice of this invention. Such alkylating agents include, but are not limited to nitrogen mustards such as chlorambucil, cyclophosphamide, ifosamide, estramustine, mechlorethamine, and melphalan; ethyleneimine derivatives such as triethylenethiophosphoramide (THIOTEPA7, Immunex Corp.); alkyl sulfonates such as busulfan; nitrosureas such as carmustine, lomustine, and streptozocin; triazines such as dacarbazine; metal salts such as platinum compounds (including cisplatin, tertplatin, carboplatin, etc.). In a preferable embodiment, the alkylating agent is a nitrogen mustard. In a more preferable embodiment, the alkylating agent is chlorambucil.

As discussed above, alkylating agents possess a different mechanism of action from pentostatin. Briefly, alkylating agents tend to induce DNA damage via DNA alkylation and the forming of DNA cross links.

In more detail, alkylating agents generally are a diverse group of compounds capable of forming molecular bonds with nucleic acids, proteins, and many molecules of low molecular weight. Alkylating agents are usually either electrophiles or generate electrophiles in vivo to produce polarized molecules with positively charged regions. These polarized molecules can then interact with electron-rich regions of most cellular molecules. The cytotoxic effect of the alkylating agents appears to relate primarily to the interaction between the electrophiles and DNA. This interaction may result in substitution reactions, cross-linking reactions, or strand-breaking reactions. The net effect of the alkylating agent's interaction with DNA is to alter the information coded in the DNA molecule. This alteration results in inhibition or inaccurate replication of DNA with resultant mutation or cell death.

Within the context of the invention, methylated xanthines refers to methylated xanthines, their derivatives and prodrugs thereof. In a preferable embodiment, the methylated xanthines include theophylline, caffeine, theobromine, and paraxanthine. In a more preferable embodiment, the methylated xanthine is theophylline. Methylated xanthines have been reported to enhance the lethal potential of many DNA-damaging agents. J. P. Murnane et al., Effects of Methylated Xanthines on Mammalian Cells Treated with Bifunctional Alkylating Agents, Nature 285:326-329 (1980). As a single agent, methylated xanthines have reduced cytotoxic effects (Binet et al). Methylated xanthines may be used in the practice of this invention to enhance the cytotoxic effects of the alkylating agents and the pentostatin.

Based upon the observation of absent CLL progression in a patient receiving the phosphodiesterase inhibitor theophylline, it was subsequently demonstrated (Br J Haematol 90:957, 1995) that theophylline markedly increased spontaneous apoptosis in CLL samples studied in vitro. Further investigation of this observation demonstrated that theophylline given together with chlorambucil (Blood 88: 2172, 1996) yielded in vitro synergistic apoptosis toward human CLL cells. The induction of theophylline-induced apoptosis correlated with an increased intracellular level of cAMP, a known second signal required for programmed cell death and down-regulation of bcl-2 which is a known inhibitor or apoptosis in CLL. (50) A preliminary report (Leukemia 9:2159, 1995) noted no activity utilizing theophylline as a single agent, but its combination with chlorambucil or cyclophosphamide in alkylator-refractory CLL yielded responses in 11 of 12 patients.

The mechanism by which methylated xanthines function remains unclear. Some investigators believe that they exert their potentiating effect either by directly inhibiting repair of damage in DNA or by causing override of the radiation-induced inhibition of DNA synthesis. S. R. et al., Override of the Radiation-induced Mitotic Block in Human Tumour Cells by Methylxanthines and its Relationship to the Potentiation of Cytotoxicity, Int. J. Radiat. Biol. 57:1105-1112 (1990). There is also evidence that methyl xanthine compounds can potentiate cytoxocity of chemotherapy in p53 deficient cells through abrogation of the G2 cell cycle checkpoint. Additionally, methylated xanthine administration may result in accumulation of intracellular cAMP that activates the apoptosis cascade. F. Mentz et al., Theophylline Synergies with Chlorambucil in Inducing Apoptosis of Bchronic Lymphocytic Leukemia Ceffs, Blood 88:2172-2182 (1996). Of course, precise understanding of the mechanism of action is not necessary to the practice of this invention.

Pentostatin may be obtained from commercial suppliers, including SuperGen, Inc. (San Ramon, Calif.) which supplies pentostatin under the trademark NIPENT. Alkylating agents and methylated xanthines according to the invention are available from commercial suppliers, based upon information present in, for example, the Physician's Desk Reference. A more preferable alkylating agent, chlorambucil, is available as LEUKERAN from Burroughs Wellcome. A preferable methylated xanthine, theophylline, is available in tablets from Rhone-Poulenc Rorer.

The alkylating agents according to the invention may be preferably administered in an amount effective to treat a lymphoproliferative condition in a host, alone or in combination. In a more preferable embodiment, the alkylating agents according to the invention may be administered in an amount ranging from about 1 mg/m2 to about the maximum tolerated dosage for the alkylating agent. In a still more preferred embodiment, the alkylating agents according to the invention may be administered in an amount ranging from about 10 mg/m2 to about 1000 mg/m2. In a yet more preferred embodiment, the alkylating agents according to the invention may be administered in an amount ranging from about 20 mg/m2 to about 40 mg/m2.

The methylated xanthines according to the invention may be preferably administered in an amount effective to treat a lymphoproliferative condition in a host, alone or in combination. In a more preferable embodiment, the methylated xanthines according to the invention may be administered in an amount ranging from about 1 mg/kg to about the maximum tolerated dosage for the methylated xanthine. In a still more preferred embodiment, the methylated xanthines according to the invention may be administered in an amount ranging from about 1 mg/kg to about 10 mg/kg. In a yet more preferred embodiment, the methylated xanthines according to the invention may be administered in an amount ranging from about 2 mg/kg to about 5 mg/kg. In an especially preferred embodiment, the methylated xanthines according to the invention are administered in a first dose at 5 mg/kg, followed by subsequent doses of 2-3 mg/kg every 6-8 hours. The serum level of theopylline will be approximately 10-20 ug/ml during treatment.

Pentostatin, according to the invention, may be preferably administered in an amount effective to treat a lymphoproliferative condition in a host, alone or in combination. In a more preferable embodiment, pentostatin according to the invention may be administered in an amount ranging from about 0.1 mg/m2 to about the maximum tolerated dosage for pentostatin. In a still more preferred embodiment, pentostatin according to the invention may be administered in an amount ranging from about 1 mg/m2 to about 4 mg/m2. In a yet more preferred embodiment, pentostatin according to the invention may be administered in an amount ranging from about 2 mg/m2 to about 3 mg/m2.

The alkylating agents, methylated xanthines and pentostatin according to the invention may be administered by a variety of routes, and may be administered or coadministered in any conventional dosage form. Coadministration in the context of this invention is defined to mean the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such coadministration may also be coextensive, that is, occurring during overlapping periods of time. (Co)administration may be taken to mean either coadministration or administration or both.

For example, the alkylating agents, methylated xanthines and pentostatin according to the invention may be administered via a coordinated cycle of medication. In a preferable embodiment, the methylated xanthines are administered for nine days, and the alkylating agents and pentostatin are administered on the eighth day of the nine day methylated xanthine regimen. In another preferable embodiment, this nine day cycle is repeated once every twenty-one days. In a still more preferable embodiment, the nine day cycle is repeated once every twenty-one days for a maximum of six cycles.

The alkylating agents, methylated xanthines and pentostatin according to the invention may be administered or coadministered in any conventional dosage form. For example, they may be administered or coadministered parenterally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, or intrathecally. The alkylating agents, methylated xanthines and pentostatin according to the invention may also be administered or coadministered in slow release dosage forms. Furthermore, alkylating agents, methylated xanthines and pentostatin may be administered or coadministered with conventional pharmaceutical excipients and additives. As alkyating agents and pentostatin may induce nausea and vomiting, addition of a serotonin blocking agent (Kytril or Odansetron) may be considered on the 8th day. Additionally, as infection may develop while receiving this therapy in previously treated patients, consideration of treating with prophylactic antibiotics should be given.

A wide variety of lymphoproliferative diseases may be treated in the practice of this invention, although greatest efficacy has been observed thus far in B-cell chronic lymphocytic leukemia (3 of 4 responses for 75% response rate). Such diseases include, but are not limited to, low-grade lymphoproliferative disorders, chronic lymphocytic leukemias, cutaneous T cell leukemias including Sezary, hairy cell leukemias, lymphomas, Non-Hodgkin's lymphomas, and large granular lymphocytic leukemias, hairy cell leukemia, splenic lymphoma with vilous lymphocytes, Waldenstrom's macroglobulinemia and p53 deficient hematologic malignancy tumors.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claim 1 of 49 Claims

What is claimed is:

1. A method of treating lymphoproliferative diseases in a host comprising (co)administering to the host a pharmaceutically effective and synergistic amount of pentostatin, chlorambucil and a methylated xanthine.

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