BACKGROUND OF THE INVENTION
Combating the growth of neoplastic cells and tumors has been a major focus of biological and medical research. Such research has led to the discovery of novel cytotoxic agents potentially useful in the treatment of neoplastic disease. Examples of cytotoxic agents commonly employed in chemotherapy include anti-metabolic agents interfering with microtubule formation, alkylating agents, platinum-based agents, anthracyclines, antibiotic agents, topoisomerase inhibitors, and other agents.
Aside from merely identifying potential chemotherapeutic agents, cancer research has led to an increased understanding of the mechanisms by which these agents act upon neoplastic cells, as well as on other cells. For example, cholecalciferol (vitamin D) can effect differentiation and reduce proliferation of several cell types cells both in vitro and in vivo. The active metabolite of vitamin D (1,25-dihydroxycholecalciferol (hereinafter “1,25D3”)) and analogs (e.g., 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (Ro23-7553), 1,25-dihydroxy-16-ene-23-yne-26, 27-hexafluoro-19-nor-cholecalciferol (Ro25-6760), etc.) mediate significant in vitro and in vivo anti-tumor activity by retarding the growth of established tumors and preventing tumor induction (Colston et al., Lancet, 1, 188 (1989); Belleli et al., Carcinogenesis, 13, 2293 (1992); McElwain et al., Mol. Cell. Diff., 3, 31-50 (1995); Clark et al., J. Cancer Res. Clin. Oncol., 118, 190 (1992); Zhou et al., Blood, 74, 82-93 (1989)). In addition to retarding neoplastic growth, 1,25D3 induces a G0/G1-S phase block in the cell cycle (Godyn et al, Cell Proliferation, 27, 37-46 (1994); Rigby et al., J. Immunol., 135, 2279-86 (1985); Elstner et al., Cancer Res., 55, 2822-30 (1995); Wang et al., Cancer Res., 56, 264-67 (1996)). These properties have led to the successful use of 1,25D3 to treat neoplastic tumors (see Cunningham et al., Br. J. Cancer, 63, 4673 (1991); Mackie et al., Lancet, 342, 172 (1993), Bower et al., Proc. Am. Assoc. Cancer. Res., 32, 1257 (1991)).
In addition to its antineoplastic and cell-cycle blocking effects, 1,25D3 treatment can lead to hypercalcemia. As a result, 1,25D3 is typically administered for therapeutic applications (e.g., metabolic bone disease) at relatively low doses (e.g., about 1 μg/day to about 2 μg/day per patient) long term. To mitigate the effects of hypercalcemia, analogs have been developed which retain antiproliferative activity without inducing hypercalcemia. (See, e.g., Zhou et al., Blood, 73, 75 (1991); Binderup et al., Biochem. Pharmacol., 42, 1569 (1991); Binderup et al., page 192 in Proceedings of the 8th Workshop on Vitamin D, Paris France (Norman, A. et al., Eds., Walter de Gruyter, Berlin, (1991))). Many of these synthetic analogs are more potent than 1,25D3 in inhibiting neoplastic growth (for a review of many such analogs, see Calverley et al., “Vitamin D” in Antitumor Steroids (Blickenstaff, R. T., Ed., Academic Press, Orlando (1992))).
The platinum-based agents are widely utilized in chemotherapeutic applications. For example, cisplatin kills tumor cells via formation of covalent, cross- or intrastrand DNA adducts (Sherman et al. Chem. Rev., 87, 1153-81 (1987); Chu, J. Biol. Chem., 269, 787-90 (1994)). Treatment with such platinum-based agents thereby leads to the inhibition of DNA synthesis (Howle et al., Biochem. Pharmacol., 19, 2757-62 (1970); Salles et al., Biochem. Biophys. Res. Commun., 112, 555-63 (1983)). Thus, cells actively synthesizing DNA are highly sensitive to cisplatin (Roberts et al., Prog. Nucl. Acid Res. Mol. Biol., 22, 71-133 (1979); Pinto et al., Proc. Nat. Acad. Sci. (Wash) 82, 4616-19 (1985)). Such cells generally experience a growth arrest in G2 and eventually undergo apoptosis. This apoptotic effect is observed at drug concentrations insufficient to inhibit DNA synthesis (Sorenson et al., J. Natl. Cancer Inst., 82, 749-55 (1990)), suggesting that platinum agents act on neoplastic cells via multiple mechanisms. Some cells also demonstrate increased platinum sensitivity when in the G1 phase of the cell cycle (Krishnaswamy et al., Mutation Res., 293, 161-72 (1993); Donaldson et al., Int. J. Cancer, 57, 847-55 (1994)). Upon release from G0/G1-S block, such cells remain maximally sensitized through the remainder of the cell cycle.
Other chemotherapeutic agents act by different mechanisms. For example, agents interfering with microtubule formation (e.g., vincristine, vinblastine, paclitaxel, docetaxel, etc.) act against neoplastic cells by interfering with proper formation of the mitotic spindle apparatus (see, e.g., Manfredi et al., Pharmacol. Ther., 25, 83-125 (1984)). Thus, agents interfering with microtubule formation mainly act during the mitotic phase of the cell cycle (Schiff et al., Proc. Nat. Acad. Sci. U.S.A., 77, 1561-65 (1980); Fuchs et al., Cancer Treat. Rep., 62, 1219-22 (1978); Lopes et al., Cancer Chemother. Pharmacol., 32, 235-42 (1993)). Antimetabolites act on various enzymatic pathways in growing cells. For example, methotrexate (MTX) is a folic acid analog which inhibits dihydrofolate reductase. As a result, it blocks the synthesis of thymidylate and purines required for DNA synthesis. Thus, the primary impact of MTX is in the S phase of the cell cycle, but it can also impact RNA synthesis in G1 and G2 (Olsen, J. Am. Acad. Dermatol., 25, 306-18 (1991)). Of course, other cytotoxic agents can also be employed (e.g., taxanes such as docetaxel (e.g., TAXATERE®)).
Because of the differences in the biological mechanisms of various cytotoxic agents, protocols involving combinations of different cytotoxic agents have been attempted (e.g., Jekunen et al., Br. J. Cancer, 69, 299-306 (1994); Yeh et al., Life Sciences, 54, 431-35 (1994)). Combination treatment protocols aim to increase the efficacy of cytopathic protocols by using compatible cytotoxic agents. In turn, the possibility that sufficient antineoplastic activity can be achieved from a given combination of cytotoxic agents presents the possibility of reducing the dosage of individual cytotoxic agents to minimize harmful side effects. In part because the various cytotoxic agents act during different phases of the cell cycle, the success of combination protocols frequently depends upon the order of drug application (e.g., Jekunen et al., supra; Studzinski et al, Cancer Res., 51, 3451 (1991)).
There have been attempts to develop combination drug protocols based, in part, on vitamin D and derivatives thereof. For example, the inhibitory effect of the concurrent administration of 1,25D3 and platinum drugs on the growth of neoplastic cells has been studied (Saunders et al., Gynecol. Oncol., 51, 155-59 (1993); Cho et al., Cancer Res., 51, 2848-53 (1991)), and similar studies have focused on concurrent combinations of 1,25D3 and other cytotoxic agents (Tanaka et al., Clin. Orthopaed. Rel. Res., 247, 290-96 (1989)). The results of these studies, however, have been less than satisfactory. In particular, the optimal sequence of drug administration has not been achieved. Moreover, the application of these approaches in therapy would require the long-term application of high doses of 1,25D3 in some protocols, which, as mentioned, can precipitate significant side effects. Thus, there remains a need for an improved method of enhancing the efficacy of chemotherapeutic agents, particularly a need for an improved combination therapy, especially involving vitamin D and derivatives thereof.
BRIEF SUMMARY OF THE INVENTION
This invention relates to combination chemotherapy, particularly involving vitamin D or a derivative thereof. In one aspect, the invention provides a method of killing a cell by first administering to the cell vitamin D (or a derivative) and subsequently administering to the cell a cytotoxic agent. Where this strategy is applied to an intact tumor, the present invention provides a method of retarding the growth of the tumor by first administering vitamin D (or a derivative) to the tumor and subsequently administering the cytotoxic agent. A further aspect of the invention concerns a method of treating prostate cancer within a patient by co-administration of vitamin D (or a derivative) and a glucocorticoid to the patient. In yet a further aspect, the invention provides an improved method of treating a patient with vitamin-D involving the adjunctive administration of zoledronate.
In some applications, the inventive method is a useful therapy, particularly in the treatment of neoplastic or cancerous diseases. In other applications, the present invention provides a tool for further research pertaining to subjects including neoplastic cell growth, the control and regulation of the cell cycle, and the mechanism and efficacy of cytotoxicity and chemotherapy. In this respect, the inventive method is useful for the development of more refined therapies. The invention can best be understood with reference to the following detailed description.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In one embodiment, the invention provides a method of killing a cell (e.g., a targeted cell) by first administering vitamin D (or a derivative) to the cell and subsequently administering a cytotoxic agent to the cell. Any period of pretreatment can be employed in the inventive method; the exact period of pretreatment will vary depending upon the application for the inventive method. For example, in therapeutic applications, such pretreatment can be for as little as about a day to as long as about 5 days or more; more preferably, the pretreatment period is between about 2 and about 4 days (e.g., about 3 days). Following pretreatment, the inventive method involves administering a cytotoxic agent. However, in a preferred embodiment, a glucocorticoid (e.g., cortisol, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, etc.), diphenhydramine, rantidine, antiemetic-ondasteron, or ganistron can be adjunctively administered, and such agents can be administered with vitamin D (or a derivative). The cytotoxic agent can be administered either alone or in combination with continued administration of vitamin D (or a derivative) following pretreatment. While, typically, treatment ceases upon administration of the cytotoxic agent, it can be administered continuously for a period of time (e.g., periodically over several days) as desired.
The cell can be solitary and isolated from other like cells (such as a single cell in culture or a metastatic or disseminated neoplastic cell in vivo), or the cell can be a member of a collection of cells (e.g., within a tumor). Preferably, the cell is a neoplastic cell (e.g., a type of cell exhibiting uncontrolled proliferation, such as cancerous or transformed cells). Neoplastic cells can be isolated (e.g., a single cell in culture or a metastatic or disseminated neoplastic cell in vivo) or present in an agglomeration, either homogeneously or in heterogeneous combination with other cell types (neoplastic or otherwise) in a tumor or other collection of cells. Where the cell is within a tumor, the present invention provides a method of retarding the growth of the tumor by first administering vitamin D (or a derivative) to the tumor and subsequently administering the cytotoxic agent to the tumor. By virtue of the cytopathic effect on individual cells, the inventive method can reduce or substantially eliminate the number of cells added to the tumor mass over time. Preferably, the inventive method effects a reduction in the number of cells within a tumor, and, most preferably, the method leads to the partial or complete destruction of the tumor (e.g., via killing a portion or substantially all of the cells within the tumor).
Where the cell is associated with a neoplastic disorder within a patient (e.g., a human), the invention provides a method of treating the patient by first administering vitamin D (or a derivative) to the patient and subsequently administering the cytotoxic agent to the patient. This approach is effective in treating mammals bearing intact or disseminated cancer. For example, where the cells are disseminated cells (e.g., metastatic neoplasia), the cytopathic effects of the inventive method can reduce or substantially eliminate the potential for further spread of neoplastic cells throughout the patient, thereby also reducing or minimizing the probability that such cells will proliferate to form novel tumors within the patient. Furthermore, by retarding the growth of tumors including neoplastic cells, the inventive method reduces the likelihood that cells from such tumors will eventually metastasize or disseminate. Of course, when the inventive method achieves actual reduction in tumor size (and especially elimination of the tumor), the method attenuates the pathogenic effects of such tumors within the patient. Another application is in high-dose chemotherapy requiring bone marrow transplant or reconstruction (e.g., to treat leukemic disorders) to reduce the likelihood that neoplastic cells will persist or successfully regrow.
In many instances, the pretreatment of cells or tumors with vitamin D (or a derivative) before treatment with the cytotoxic agent effects an additive and often synergistic degree of cell death. In this context, if the effect of two compounds administered together in vitro (at a given concentration) is greater than the sum of the effects of each compound administered individually (at the same concentration), then the two compounds are considered to act synergistically. Such synergy is often achieved with cytotoxic agents able to act against cells in the G0-G1 phase of the cell cycle, and such cytotoxic agents are preferred for use in the inventive methods. While any such cytotoxic agent can be employed (as discussed herein), preferred cytotoxic agents are platinum-based agents (e.g., cisplatin, carboplatin, etc.). Without being bound by any particular theory, it is believed that the inventive method effects cytotoxicity of neoplastic cells by inducing a G0/G1-S phase block in the cell cycle, as mentioned herein. The cells are sensitized to cytotoxic agents able to act on cells in such a blocked stage. Alternatively, synchronization of the release of the cells from the block can render them collectively sensitive to the effects of agents acting later in the cell cycle.
As an alternative to vitamin D, any derivative thereof suitable for potentiating the cytotoxic effect of chemotherapeutic agents can by used within the context of the inventive method, many of which are known in the art (see, e.g., Calverley et al., supra). One preferred derivative is its natural metabolite (1,25D3). However, many vitamin D analogs have greater antitumor activity than the native metabolite; thus the vitamin D derivative can be such an analog of 1,25D3. Furthermore, where the inventive method is used for therapeutic applications, the derivative can be a nonhypercalcemic analog of 1,25D3, as such analogs reduce or substantially eliminate the hypercalcemic side effects of vitamin D-based therapy. For example, the analog can be Ro23-7553, Ro24-5531, or another analog. In some embodiments, other agents that attenuate (e.g., deactivate) MAP kinase, specifically by inducing MAPK phosphatase, can be used as equivalents of vitamin D (or a derivative).
Pursuant the inventive method, the vitamin D (or a derivative) can be provided to the cells or tumors in any suitable manner, which will, of course, depend upon the desired application for the inventive method. Thus, for example, for in vitro applications, vitamin D (or a derivative) can be added to the culture medium (e.g., mixed initially with the medium or added over time). For in vivo applications, vitamin D (or a derivative) can be mixed into an appropriate vehicle for delivery to the cell or tumor. Thus, for systemic delivery, vitamin D (or a derivative) can be supplied by subcutaneous injection, intravenously, orally, or by other suitable means. Of course, vitamin D (or a derivative) can be provided more directly to the tumor (e.g., by application of a salve or cream comprising vitamin D (or a derivative) to a tumor, by injection of a solution comprising vitamin D (or a derivative) into a tumor, etc.).
The dose of vitamin D (or a derivative) provided to the cells can vary depending upon the desired application. In research, for example, the dose can vary considerably, as dose-response analysis might be a parameter in a given study. For therapeutic applications, because the pretreatment period can be quite brief in comparison with standard vitamin D-based therapies, higher than typical doses (as discussed above) of vitamin D (or a derivative) can be employed in the inventive method without a substantial risk of hypercalcemia. Thus, for example, in a human patient, as little as 1 μg/day of vitamin D (or a derivative) (which as mentioned above, is within the normal dosage for 1,25D3) can be supplied to a patient undergoing treatment, while the maximal amount can be as high as about 20 μg/day (or even higher in some larger patients). Preferably, between about 4 μg/day and about 15 μg/day (e.g., between about 7 μg/day and about 12 μg/day) of vitamin D (or a derivative) is delivered to the patient. Typically, the amount of vitamin D (or a derivative) supplied will not be so great as to pose a significant risk of inducing hypercalcemia or provoking other toxic side effects. Hence, where non-hypercalcemic vitamin D derivatives are used, higher amounts still can be employed. Thus, 30 μg/day or more (e.g., about 40 μg/day or even 50 μg/day or more) non-hypercalcemic vitamin D derivative can be delivered to a human patient during pretreatment in accordance with the inventive method. Of course, the desired dose of vitamin D (or a derivative) will depend upon the size of the patient and the mode and timing of delivery. Vitamin D (or a derivative) can be delivered once a day, or several times a day, as desired, or it can be delivered discontinuously (e.g., every other day, or every third day). The determination of such doses and schedules is well within the ordinary skill in the art.
Any cytotoxic agent can be employed in the context of the invention; as mentioned, many cytotoxic agents suitable for chemotherapy are known in the art. Such an agent can be, for example, any compound mediating cell death by any mechanism including, but not limited to, inhibition of metabolism or DNA synthesis, interference with cytoskeletal organization, destabilization or chemical modification of DNA, apoptosis, etc. For example, the cytotoxic agent can be an antimetabolite (e.g., 5-flourouricil (5-FU), methotrexate (MTX), fludarabine, etc.), an anti-microtubule agent (e.g., vincristine, vinblastine, taxanes (such as paclitaxel and docetaxel), etc.), an alkylating agent (e.g., cyclophasphamide, melphalan, bischloroethylnitrosurea (BCNU), etc.), platinum agents (e.g., cisplatin (also termed cDDP), carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C), topoisomerase inhibitors (e.g., etoposide, camptothecins, etc.), or other cytotoxic agents (e.g., dexamethasone). The choice of cytotoxic agent depends upon the application of the inventive method. For research, any potential cytotoxic agent (even a novel cytotoxic agent) can be employed to study the effect of the toxin on cells or tumors pretreated with vitamin D (or a derivative). For therapeutic applications, the selection of a suitable cytotoxic agent will often depend upon parameters unique to a patient; however, selecting a regimen of cytotoxins for a given chemotherapeutic protocol is within the skill of the art.
For in vivo application, the appropriate dose of a given cytotoxic agent depends on the agent and its formulation, and it is well within the ordinary skill of the art to optimize dosage and formulation for a given patient. Thus, for example, such agents can be formulated for administration via oral, subcutaneous, parenteral, submucosal, intraveneous, or other suitable routes using standard methods of formulation. For example, carboplatin can be administered at daily dosages calculated to achieve an AUC (“area under the curve”) of from about 4 to about 15 (such as from about 5 to about 12), or even from about 6 to about 10. Typically, AUC is calculated using the Calvert formula, based on the glomerular filtration rate of creatinine (e.g., assessed by analyzing a plasma sample) (see, e.g., Martino et al., Anticancer Res., 19(6C), 5587-91 (1999)). Paclitaxel can be employed at concentrations ranging from about 50 mg/m2 to about 100 mg/m2 (e.g., about 80 mg/m2). Where dexamethasone is employed, it can be used within patients at doses ranging between about 1 mg to about 10 mg (e.g., from about 2 mg to about 8 mg), and more particularly from about 4 mg to about 6 mg, particularly where the patient is human.
Another embodiment of the invention provides a method of treating prostate cancer within a patient by adjunctively administrating vitamin D (or a derivative) and a glucocorticoid to the patient. Any vitamin D derivative and glucocorticoid can be employed in accordance with this aspect of the invention, many of which are discussed elsewhere herein and others are generally known in the art. Moreover, vitamin D (or a derivative) and the glucocorticoid are delivered to the patient by any appropriate method, some of which are set forth herein. Thus, they can be formulated into suitable preparations and delivered subcutaneously, intravenously, orally, etc., as appropriate. Also, for example, the glucocorticoid is administered to the patient concurrently, prior to, or following the administration of vitamin D (or a derivative). One effective dosing schedule is to delver between about 8 μg and about 12 μg vitamin D (or a derivative) daily on alternative days (e.g., between 2 and 4 days a week, such as Mon-Wed-Fri or Tues-Thus-Sat, etc.), and also between about 1 mg and 10 mg dexamethasone (e.g., about 5 mg) to a human patient also on alternative days. In such a regimen, the alternative days on which vitamin D (or a derivative) and on which the glucocorticoid are administered can be different, although preferably they are administered on the same days. Even more preferably, the glucocorticoid is administered once, by itself, prior to concurrent treatment. Of course, the treatment can continue for any desirable length of time, and it can be repeated, as appropriate to achieve the desired end results. Such results can include the attenuation of the progression of the prostate cancer, shrinkage of such tumors, or, desirably, remission of all symptoms. However, any degree of effect is considered a successful application of this method. A convenient method of assessing the efficacy of the method is to note the change in the concentration of prostate-specific antigen (PSA) within a patient. Typically, such a response is gauged by measuring the PSA levels over a period of time of about 6 weeks. Desirably, the method results in at least about a 50% decrease in PSA levels after 6 weeks of application, and more desirably at least about 80% reduction in PSA. Of course, the most desirable outcome is for the PSA levels to decrease to about normal levels (e.g., less than about 4 ng/ml for at least three successive measurements in a non-prostatectomized individual or less than about 0.2 ng/ml in a prostatectomized individual).
In all aspects of the invention that involve in vivo application, preferably the method is employed to minimize the hypercalcemic properties of vitamin D. One manner of accomplishing this is to employ a nonhypercalcemic analog, such as those discussed above. Alternatively, or in conjunction with the use of such analogs, an agent that mitigates hypercalcemia can be adjunctively delivered to the patient. While any such agent can be employed, bisphosphonates (e.g., alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, tiludronate, zoledronate, etc.) are preferred agents for adjunctive administration. Such agents can be administered in any suitable manner to mitigate hypercalcemia. Thus, they can be formulated into suitable preparations and delivered subcutaneously, intravenously, orally, etc., as appropriate. Also, such agents can be administered concurrently, prior to, or subsequent to vitamin D (or a derivative). The dosage of such agents will, of course, vary with the potency of the compounds and also to mitigate any unwanted side effects. Thus, for example, for administration to human patients, the dosage of bisphosphonates can vary between about 1 mg/day and 500 mg/day (e.g., between about 5 mg/day and 100 mg/day), such as between about 10 mg/day and about 50 mg/day, or even between about 30 mg/day and about 40 mg day, depending on the potency of the bisphosphonates. Generally, it is preferred to employ a more potent bisphosphonate, as less of the agent need be employed to achieve the antihypercalcemic effects. Thus, a most preferred bisphosphonate is zoledronate, as it is effective even at very low doses (e.g., between about 0.5 mg day and about 2 mg/day in human patients, or between about 5 μg/kg to about 25 μg/kg body weight).
Indeed, in another aspect, the invention provides an improved method of employing vitamin D (or a derivative) therapeutically by adjunctively administering zoledronate. The zoledronate can be delivered as an adjunct in conjunction with any protocol in which vitamin D (or a derivative) is employed, such as those discussed herein or otherwise employed. As an adjunct, the zoledronate can be delivered in any desired regimen (several times a day, daily, weekly, etc.), as desired. Preferably, the zoledronate is delivered as a pretreatment, e.g., several hours to several days before treatment with vitamin D (or a derivative) commences. More preferably, the zolendronate is adjunctively administered in an amount sufficient to mitigate the antihypercalcemic effects of vitamin D (or a derivative).
While one of skill in the art is fully able to practice the instant invention upon reading the foregoing detailed description, the following examples will help elucidate some of its features. Of course, as these examples are presented for purely illustrative purposes, they should not be used to construe the scope of the invention in a limited manner, but rather should be seen as expanding upon the foregoing description of the invention as a whole.