U.S. patent application number 10/429840 was filed with the patent office on 2003-11-20 for combination chemotherapy.
This patent application is currently assigned to University of Pittsburgh of the Commonwealth System of Higher Education. Invention is credited to Johnson, Candace S., Trump, Donald L..
Application Number | 20030216359 10/429840 |
Document ID | / |
Family ID | 25445023 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030216359 |
Kind Code |
A1 |
Johnson, Candace S. ; et
al. |
November 20, 2003 |
Combination chemotherapy
Abstract
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.
Inventors: |
Johnson, Candace S.;
(Pittsburgh, PA) ; Trump, Donald L.; (Pittsburgh,
PA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
University of Pittsburgh of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
25445023 |
Appl. No.: |
10/429840 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10429840 |
May 5, 2003 |
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09544724 |
Apr 6, 2000 |
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6559139 |
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09544724 |
Apr 6, 2000 |
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08921170 |
Aug 29, 1997 |
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6087350 |
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Current U.S.
Class: |
514/168 ;
424/649; 514/102; 514/170; 514/34; 514/492; 514/89 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/675 20130101; A61K 31/337 20130101; A61K 31/59 20130101;
A61P 43/00 20180101; A61K 31/573 20130101; A61K 33/243 20190101;
A61K 45/06 20130101; A61K 33/24 20130101; A61K 31/59 20130101; A61K
31/675 20130101; A61K 31/59 20130101; A61K 31/59 20130101; A61K
31/55 20130101; A61K 31/59 20130101; A61K 31/335 20130101; A61K
31/59 20130101; A61K 2300/00 20130101; A61K 31/675 20130101; A61K
2300/00 20130101; A61K 33/24 20130101; A61K 2300/00 20130101; A61K
31/337 20130101; A61K 2300/00 20130101; A61K 31/573 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/168 ; 514/34;
514/89; 514/102; 514/170; 514/492; 424/649 |
International
Class: |
A61K 033/24; A61K
031/675; A61K 031/704; A61K 031/66; A61K 031/56; A61K 031/28 |
Goverment Interests
[0002] This invention was made in part with Government support
under Grant Number RO1-CA67267 awarded by the National Cancer
Institute of the National Institutes of Health. The United States
Government may have certain rights in this invention.
Claims
1. A method of killing a cell within a patient comprising the steps
of (a) first administering to a cell within the patient vitamin D
or a derivative thereof and (b) subsequently administering at least
one cytotoxic agent to the cell, wherein the cell is susceptible to
said steps (a) and (b).
2. The method of claim 1, wherein step (a) further comprises the
administration of a glucocorticoid concurrently with the vitamin D
or a derivative thereof.
3. The method of claim 2, wherein the glucocorticoid is
dexamethasone.
4. The method of claim 2, wherein the patient is human and the
glucocorticoid is dexamethasone and is administered at a dosing
schedule of between about 1 mg and 10 mg on alternative days.
5. The method of claim 1, wherein the vitamin D or a derivative
thereof is administered from 1 to 3 days before the cytotoxic
agent.
6. The method of claim 1, wherein the vitamin D or a derivative
thereof is administered at least once daily for at least two
successive days.
7. The method of claim 1, wherein the vitamin D or a derivative
thereof is administered at least once daily on alternate days.
8. The method of claim 1, wherein the vitamin D or a derivative
thereof is a nonhypercalcemic analog of 1,25D.sub.3.
9. The method of claim 8, wherein the analog is Ro23-7553 or
Ro24-5531.
10. The method of claim 1, wherein the vitamin D or a derivative
thereof is 1,25D.sub.3.
11. The method of claim 1, wherein the patient is human and the
daily dose of the vitamin D or a derivative thereof is between
about 4 .mu.g and about 15 .mu.g.
12. The method of claim 11, wherein the daily dose of the vitamin D
or a derivative thereof is between about 8 .mu.g and about 12
.mu.g.
13. The method of claim 1, wherein the cytotoxic agent selectively
acts on cells in the G.sub.0-G.sub.1 phase of the cell cycle.
14. The method of claim 1, wherein the cytotoxic agent is a
platinum-based cytotoxic agent.
15. The method of claim 1, wherein the cytotoxic agent is a
glucocorticoid.
16. The method of claim 1, wherein the cytotoxic agent is
carboplatin, cisplatin, dexamethasone, paclitaxel, or
docetaxel.
17. The method of claim 1, wherein the cytotoxic agent is
carboplatin and is administered at a dose calculated to achieve AUC
of about 5.
18. The method of claim 1, wherein the cytotoxic agent is
paclitaxel and is administered at a dose of about 80
mg/m.sup.2.
19. The method of claim 1, wherein said cytotoxic agent is
docetaxel.
20. The method of claim 1, wherein the vitamin D or a derivative
thereof is 1,25D.sub.3 and wherein said cytotoxic agent is
docetaxel.
21. The method of claim 1, further comprising adjunctively
administering at least one bisphosphonate selected from the group
of bisphosphates consisting of alendronate, clodronate, etidronate,
ibandronate, pamidronate, risedronate, tiludronate, and
zoledronate.
22. A method of retarding the growth of a tumor within a patient
comprising the steps of (a) first administering to the tumor within
the patient a vitamin D or a derivative thereof and (b)
subsequently administering to the tumor at least one cytotoxic
agent, wherein the cell is susceptible to said steps (a) and
(b).
23. The method of claim 22, wherein step (a) further comprises the
administration of a glucocorticoid concurrently with the vitamin D
or a derivative thereof.
24. The method of claim 23, wherein the glucocorticoid is
dexamethasone.
25. The method of claim 23, wherein the patient is human and the
glucocorticoid is dexamethasone and is administered at a dosing
schedule of between about 1 mg and 10 mg on alternative days.
26. The method of claim 22, wherein the vitamin D or a derivative
thereof is administered from 1 to 3 days before the cytotoxic
agent.
27. The method of claim 22, wherein the vitamin D or a derivative
thereof is administered at least once daily for at least two
successive days.
28. The method of claim 22, wherein the vitamin D or a derivative
thereof is administered at least once daily on alternate days.
29. The method of claim 22, wherein the vitamin D derivative is a
nonhypercalcemic analog of 1,25D.sub.3.
30. The method of claim 29, wherein the analog is Ro23-7553 or
Ro24-5531.
31. The method of claim 22, wherein the vitamin D derivative is
1,25D.sub.3.
32. The method of claim 22, wherein the patient is human and the
daily dose of the vitamin D or a derivative thereof is between
about 4 .mu.g and about 15 .mu.g.
33. The method of claim 32, wherein the daily dose of the vitamin D
or a derivative thereof is between about 8 .mu.g and about 12
.mu.g.
34. The method of claim 22, wherein the cytotoxic agent selectively
acts on cells in the G.sub.0-G.sub.1 phase of the cell cycle.
35. The method of claim 22, wherein the cytotoxic agent is a
platinum-based cytotoxic agent.
36. The method of claim 22, wherein the cytotoxic agent is a
glucocorticoid.
37. The method of claim 22, wherein the cytotoxic agent is
carboplatin, cisplatin, dexamethasone, paclitaxel, or
docetaxel.
38. The method of claim 22, wherein the cytotoxic agent is
carboplatin and is administered at a dose calculated to achieve AUC
of about 5.
39. The method of claim 22, wherein the cytotoxic agent is
paclitaxel and is administered at a dose of about 80
mg/m.sup.2.
40. The method of claim 22, wherein said cytotoxic agent is
docetaxel.
41. The method of claim 22, wherein said vitamin D or a derivative
thereof is 1,25D.sub.3 and wherein said cytotoxic agent is
docetaxel.
42. The method of claim 22, further comprising adjunctively
administering at least one bisphosphonate selected from the group
of bisphosphates consisting of alendronate, clodronate, etidronate,
ibandronate, pamidronate, risedronate, tiludronate, and
zoledronate.
43. A method of treating a human patient with vitamin D or a
derivative thereof, wherein the patient has a condition responsive
to vitamin D and a cytotoxic agent, comprising the steps of (a)
first administering to the patient vitamin D or a derivative
thereof and (b) subsequently administering to the patient at least
one cytotoxic agent, wherein the dose of the vitamin D or a
derivative thereof exceeds 1 .mu.g/day.
44. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 4 .mu.g/day.
45. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 8 .mu.g/day.
46. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 12 .mu.g/day.
47. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 18 .mu.g/day.
48. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 30 .mu.g/day.
49. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 40 .mu.g/day.
50. The method of claim 43, wherein the dose of vitamin D or a
derivative thereof is at last about 50 .mu.g/day.
51. The method of claim 43, wherein the vitamin D or a derivative
thereof is administered orally.
52. The method of claim 43, wherein the vitamin D or a derivative
thereof is administered intravenously.
53. The method of claim 43, wherein step (a) further comprises the
administration of a glucocorticoid concurrently with the vitamin D
or a derivative thereof.
54. The method of claim 53, wherein the glucocorticoid is
dexamethasone.
55. The method of claim 53, wherein the glucocorticoid is
dexamethasone and is administered at a dosing schedule of between
about 1 mg and 10 mg on alternative days.
56. The method of claim 43, wherein the vitamin D or a derivative
thereof is administered from 1 to 3 days before the cytotoxic
agent.
57. The method of claim 43, wherein the vitamin D or a derivative
thereof is administered at least once daily for at least two
successive days.
58. The method of claim 43, wherein the vitamin D or a derivative
thereof is administered at least once daily on alternate days.
59. The method of claim 43, wherein the vitamin D or a derivative
thereof is a nonhypercalcemic analog of 1,25D.sub.3.
60. The method of claim 59, wherein the analog is Ro23-7553 or
Ro24-5531.
61. The method of claim 43, wherein the vitamin D or a derivative
thereof is 1,25D.sub.3.
62. The method of claim 43, wherein the cytotoxic agent selectively
acts on cells in the G.sub.0-G.sub.1 phase of the cell cycle.
63. The method of claim 43, wherein the cytotoxic agent is a
platinum-based cytotoxic agent.
64. The method of claim 43, wherein the cytotoxic agent is a
glucocorticoid.
65. The method of claim 43, wherein the cytotoxic agent is
carboplatin, cisplatin, dexamethasone, paclitaxel, or
docetaxel.
66. The method of claim 43, wherein the cytotoxic agent is
carboplatin and is administered at a dose calculated to achieve AUC
of about 5.
67. The method of claim 43, wherein the cytotoxic agent is
paclitaxel and is administered at a dose of about 80
mg/m.sup.2.
68. The method of claim 43, wherein said cytotoxic agent is
docetaxel.
69. The method of claim 43, wherein said vitamin D or a derivative
thereof is 1,25D.sub.3 and wherein said cytotoxic agent is
docetaxel.
70. The method of claim 43, further comprising adjunctively
administering at least one bisphosphonate selected from the group
of bisphosphates consisting of alendronate, clodronate, etidronate,
ibandronate, pamidronate, risedronate, tiludronate, and
zoledronate.
71. A method of treating prostate cancer within a patient in need
of such treatment comprising adjunctively administering vitamin D
or a derivative thereof and a glucocorticoid to the patient.
72. The method of claim 71, wherein the treatment is repeated.
73. The method of claim 71, wherein the vitamin D or a derivative
thereof and glucocorticoid are administered to the patient on
alternative days between 2 and 4 times a week.
74. The method of claim 71, wherein the glucocorticoid is
administered to the patient prior to the administration of the
vitamin D or a derivative thereof.
75. The method of claim 71, wherein the glucocorticoid is
administered to the patient following the administration of the
vitamin D or a derivative thereof.
76. The method of claim 71, wherein the vitamin D derivative is a
nonhypercalcemic analog.
77. The method of claim 71, wherein the vitamin D derivative is
1,25D.sub.3.
78. The method of claim 71, wherein the glucocorticoid is selected
from the group of glucocorticoids consisting of cortisol,
dexamethasone, hydrocortisone, methylprednisolone, prednisolone,
and prednisone.
79. The method of claim 71, wherein the glucocorticoid is
dexamethasone.
80. The method of claim 71, further comprising administering at
least one bisphosphonate to the cell selected from the group of
bisphosphonates consisting of alendronate, clodronate, etidronate,
ibandronate, pamidronate, risedronate, tiludronate, and
zoledronate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of co-pending U.S.
patent application Ser. No. 09/544,724, filed Apr. 6, 2000, which
is a continuation-in-part of U.S. patent application Ser. No.
08/921,170, filed Aug. 29, 1997, now U.S. Pat. No. 6,087,350.
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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
"1,25D.sub.3")) 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, 1,25D.sub.3 induces a
G.sub.0/G.sub.1-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 1,25D.sub.3 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)).
[0005] In addition to its antineoplastic and cell-cycle blocking
effects, 1,25D.sub.3 treatment can lead to hypercalcemia. As a
result, 1,25D.sub.3 is typically administered for therapeutic
applications (e.g., metabolic bone disease) at relatively low doses
(e.g., about 1 .mu.g/day to about 2 .mu.g/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
1,25D.sub.3 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))).
[0006] 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 G.sub.2
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 G.sub.1 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
G.sub.0/G.sub.1-S block, such cells remain maximally sensitized
through the remainder of the cell cycle.
[0007] 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 G.sub.1 and
G.sub.2 (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.RTM.)).
[0008] 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)).
[0009] 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
1,25D.sub.3 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 1,25D.sub.3 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 1,25D.sub.3 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
[0010] 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.
[0011] 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
[0012] 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.
[0013] 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).
[0014] 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.
[0015] 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 G.sub.0-G.sub.1 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 G.sub.0/G.sub.1-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.
[0016] 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 (1,25D.sub.3).
However, many vitamin D analogs have greater antitumor activity
than the native metabolite; thus the vitamin D derivative can be
such an analog of 1,25D.sub.3. Furthermore, where the inventive
method is used for therapeutic applications, the derivative can be
a nonhypercalcemic analog of 1,25D.sub.3, 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).
[0017] 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.).
[0018] 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 .mu.g/day of vitamin D (or a derivative) (which as mentioned
above, is within the normal dosage for 1,25D.sub.3) can be supplied
to a patient undergoing treatment, while the maximal amount can be
as high as about 20 .mu.g/day (or even higher in some larger
patients). Preferably, between about 4 .mu.g/day and about 15
.mu.g/day (e.g., between about 7 .mu.g/day and about 12 .mu.g/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 .mu.g/day or more (e.g., about 40
.mu.g/day or even 50 .mu.g/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.
[0019] 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.
[0020] 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/m.sup.2 to about 100
mg/m.sup.2 (e.g., about 80 mg/m.sup.2). 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.
[0021] 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 .mu.g
and about 12 .mu.g 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).
[0022] 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 .mu.g/kg to about 25 .mu.g/kg body
weight).
[0023] 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).
[0024] 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.
EXAMPLE 1
[0025] This example explains the materials and general methods
employed in the following examples.
[0026] Inbred female C3H/HeJ mice age 6-10 weeks were obtained from
Jackson Laboratories. The mice were virus antibody-free, age and
weight-matched for experimental use, and fed a balanced rodent
diet.
[0027] SCCVII/SF cells--a murine, rapidly growing,
non-metastasizing squamous tumor line--were maintained in vivo in
C3H/HeJ mice as described previously (McElwain et al., Mol. Cell.
Diff., 3, 31-50 (1995)) by s.c. inoculation of 5.times.10.sup.5
log-phase tissue culture cells in the right flank of the animal.
The SCCVII/SF cell line was maintained in vitro in RPMI-1640
supplemented with 12.5% inactivated fetal calf serum (FCS) and 1%
penicillin-streptomycin sulfate.
[0028] 1,25D.sub.3 and its non-hypercalcemic analog, Ro23-7553,
were initially stored in pure powder form in a sealed
light-protective vessel at 4.degree. C. For use, each drug was
reconstituted in 100% ethyl alcohol and maintained as described
(McElwain et al., Mol. Cell. Diff., 3, 31-50 (1995)). The cytotoxic
agents (carboplatin, cisplatin, and paclitaxel) were diluted in
0.9% saline and were injected i.p. at various doses in a total
volume of 0.2 ml, during the experimental protocols.
[0029] The in vitro cytotoxicity of drug on tumor cells was
determined via the in vitro clonogenic assay (McElwain et al., Mol.
Cell. Diff., 3, 31-50 (1995)) with minor modifications as described
herein. Briefly, murine SCCVII/SF cells were pre-treated with
either 2 nM or 4 nM 1,25D.sub.3 or Ro23-7553. While 1,25D.sub.3 or
Ro23-7553 are not stable for long periods in tissue culture media,
anti-proliferative effects are observed at 24 hr, 48 hr and 7 day
incubation times (McElwain et al., supra). After 48 hours
incubation with 1,25D.sub.3 or Ro23-7553, cells were treated for 2
hours with varying concentrations of cytotoxic agent, washed with
RPMI 1640 plus FCS, and plated in various dilutions in 6-well
tissue culture plates. Following a 7 day incubation at 37.degree.
C. in 5% CO.sub.2, monolayers were washed with saline, fixed with
100% methanol, and stained with 10% Giemsa; colonies were counted
under light microscopy. The surviving fraction was calculated by
dividing the cloning efficiency of treated cells by the cloning
efficiency of untreated controls.
[0030] The effect of 1,25D.sub.3 or Ro23-7553 alone and/or in
combination with various cytotoxic agents on tumor cells in vivo
was determined by a modification of the in vivo excision clonogenic
tumor cell survival assay (Johnson et al., Cancer Chemother.
Pharmacol., 32, 339-46 (1993)). Briefly, SCCVII/SF tumor bearing
animals at 14 days post implantation were treated i.p. for 3 days
with 1,25D.sub.3 or Ro23-7553 at either 0.5 mg/kg body weight/day
or at varying doses of 0.03125-0.5 mg/kg body weight/day. On day 3,
animals also received an i.p. dose of either 6 mg/kg body weight or
varying doses of 1-6 mg/kg body weight of cytotoxic agent. After 24
hours, aliquots of minced tumor were enzymatically dissociated for
60 min at room temperature with a mixture of type I collagenase
(37.5 mg/ml), DNAse (55 mg/ml) and EDTA (1%). Viable tumor cells
(determined by trypan blue staining) were then plated at various
dilutions. After 7 days incubation, colonies were counted, and
numbers of clonogenic cells per gram of tumor were counted. The
mean.+-.standard deviation (SD) cell yield, cloning efficiency, and
number of clonogenic cells for control (no treatment) tumors (n=40)
averaged 139.4.+-.38.2.times.10.sup.6 viable tumor cells/g tumor,
27.0.+-.0.56%, and 37.5.+-.13.3.times.10.sup.6 clonogenic tumor
cells/g tumor, respectively. The surviving fraction per gram of
tumor is defined as the number of clonogenic tumor cells per gram
of treated tumor divided by the number of clonogenic tumor cells
per gram of control (untreated) tumor. This assay is an accurate
measure of in vivo anti-tumor activity; a surviving fraction less
than 0.1 correlates with an actual decrease in tumor volume and an
increase in tumor regrowth delay (Braunschweiger et al., Cancer
Res., 48, 6011-16 (1988); Braunschweiger et al., Cancer Res., 51,
5454-60 (1991)).
[0031] The effect of 1,25D.sub.3 or Ro23-7553 alone and/or in
combination with various cytotoxic agents on tumor cells in vivo
was further assayed by measuring the delay of tumor growth (tumor
regrowth assay). SCCVII/SF tumor cells (5.times.10.sup.5) were
inoculated s.c. into the flank of the leg of C3H/HeJ mice. On day 9
post implantation, as the tumors were palpable (approximately
5.times.5 mm), animals were randomized for treatment with low dose
i.p. Ro23-7553 (0.214 .mu.g/kg body weight/day) or 1,25D.sub.3 (0.2
.mu.g/mouse) using a micro-osmotic pump for continuous delivery
over seven days. After 7 days, 6 mg/kg body weight cytotoxic agent
was injected i.p. Control animals received either treatment alone
or no treatment. Control (no treatment) animals were given
injection of vehicle (PBS) alone or sham pumps were implanted.
Tumor growth was assessed by measuring the tumor diameter with
calipers three times weekly. Tumor volumes were calculated by the
formula: volume=length.times.(width.sup.2)/2. Post-treatment
volumes were expressed as a fraction of pretreatment volume at the
time of initial treatment. Tumor regrowth delay was calculated as
the mean.+-.standard deviation of the difference in time for
treated and control tumor volumes to reach 4 times the pretreatment
volume.
EXAMPLE 2
[0032] This example demonstrates the potential for sensitizing
tumor cells to the effects of conventional cytotoxic cisplatin
therapy by pretreatment with a vitamin D derivative.
[0033] Between 0.2 .mu.g/ml and 0.8 .mu.g/ml cisplatin and
Ro23-7553 were tested alone and in combination using the in vitro
clonogenic assay for the SCCVII/SF tumor cell line as described
above. It was observed that pretreatment of cells with both 2 nM
and 4 nM Ro23-7553 significantly enhanced clonogenic cell kill when
compared to cisplatin alone or in concurrent administration (i.e.,
no pretreatment) of cisplatin in combination with Ro23-7553
(p<0.001 ANOVA). Significant enhancement of cisplatin-mediated
cytotoxicity was observed even at low doses of cisplatin.
EXAMPLE 3
[0034] This example demonstrates the enhancement of in vivo
cisplatin-mediated anti-tumor activity by pretreatment with a
vitamin D derivative.
[0035] The excision clonogenic kill assay was employed wherein
SCCVII/SF tumor bearing animals at 14 days post implantation were
treated i.p. for 3 days with 0.5 mg/kg body weight/day of
Ro23-7553. On the third day animals received varying doses of
cisplatin. After 24 hours, tumors were harvested, dissociated, and
plated for a 7 day incubation. It was observed that pretreatment
for 3 days with the Ro23-7553 before cisplatin resulted in a
significant enhancement of clonogenic cell kill when compared to
animals treated with cisplatin or Ro23-7553 alone (p<0.001
ANOVA). A significant increase in clonogenic tumor cell kill was
observed at each cisplatin dose tested as compared to cisplatin
alone.
[0036] To determine the effect of varying the Ro23-7553 dose in
this assay, SCC tumor-bearing mice were treated daily for 3 days
with from 0.03125 mg/kg body weight/day to 0.5 mg/kg body
weight/day Ro23-7553. On day 3, cisplatin was administered at 6
mg/kg body weight. It was observed that Ro23-7553 was capable of
significantly enhancing cisplatin-mediated tumor cell kill even at
the lowest doses tested as compared to cisplatin or Ro23-7553 alone
(p<0.01 ANOVA). No animals in either experimental approach
became hypercalcemic at any of the Ro23-7553 doses.
EXAMPLE 4
[0037] This example demonstrates the enhancement of in vivo
cisplatin-mediated anti-tumor activity by pretreatment with a
vitamin D derivative.
[0038] The tumor regrowth assay was employed wherein SCCVII/SF
tumor-bearing mice (day 9 post implantation) were treated with
Ro23-7553 administered continuously. At the end of Ro23-7553
administration, cisplatin was injected i.p. at 6 mg/kg body weight.
Control (no treatment) or single treatment animals were injected
with vehicle (PBS) or implanted with sham pumps depending on the
treatment group. All animals experienced a significant decrease in
fractional tumor volume when pre-treated with Ro23-7553 before
cisplatin as compared to treatment with either agent alone
(p<0.001 ANOVA). When tumor regrowth delay (mean.+-.SD of the
difference in time for treated and control tumors to reach 4.times.
pretreatment size) was examined, a significant increase was
observed in animals treated with Ro23-7553 plus cisplatin as
compared either to cisplatin or Ro23-7553 alone (see Table 1)
1TABLE 1 Effect of Ro23-7553 and Cisplatin on Tumor Regrowth Delay
Treatment Tumor Regrowth Delay Ro23-7553 1.8 .+-. 0.8 Cisplatin 4.4
.+-. 0.3 Ro23-7553/cisplatin 7.7 .+-. 0.4
EXAMPLE 5
[0039] This example demonstrates the potential for sensitizing
tumor cells to the effects of cisplatin therapy by pretreatment
with a vitamin D derivative and dexamethasone.
[0040] SCC cells were incubated with dexamaethasone, 1,25D.sub.3,
and /or cisplatin, and cell viability was determined via trypan
blue exclusion. It was observed that pretreatment with
dexamaethasone and 1,25D.sub.3 followed by cisplatin resulted in
greater growth inhibition than treatment with any agent alone or
pretreatment with 1,25D.sub.3 followed by cisplatin. These results
demonstrate that pretreatment with a vitamin D derivative and
dexamethasone enhances the antitumor effect of cisplatin.
EXAMPLE 6
[0041] This example demonstrates the potential for sensitizing
tumor cells to the effects of cisplatin therapy by pretreatment
with a vitamin D derivative and dexamethasone in vivo.
[0042] SC tumor-bearing mice were treated with dexamethasone on
days 0-3, 1,25D.sub.3 on days 1-3, and/or cisplatin on day 3.
Greater antiproliferative activity was observed for the
triply-treated animals than for animals treated with dexamethasone
followed by cisplatin (p<0.003 using the Mann-Whitley test) or
with 1,25D.sub.3 followed by cisplatin (p<0.05 using the
Mann-Whitley test). These results demonstrate that pretreatment
with a vitamin D derivative and dexamethasone enhances the
antitumor effect of cisplatin.
EXAMPLE 7
[0043] This example demonstrates that vitamin D derivatives can
up-regulate a MAPK phosphatase.
[0044] SCC cells were treated in vitro with 10 nM 1,25D.sub.3, and
untreated cells served as a control. The level of phosphorylated
MAPK was assessed at 24 and 48 hours post treatment. Using Western
Blot analysis, the amounts of MAPK, MEK (the kinase responsible for
phosphorylating MAPK), MKP-1 (a MAPK-specific phosphatase), and
growth factor receptors (EGF, PDGF, and IGF1 growth factor
receptors) were assessed as well. Additionally, the activity level
of MEK was assessed by quantitative in vitro kinase assays.
[0045] Cells treated with 1,25D.sub.3 had less phosphorylated MAPK
than untreated cells, but the amount of MAPK protein was not
affected. The treated cells had slightly less MEK protein present,
but the MEK activity profile was not significantly different from
the untreated cells. Additionally, the treated cells had
significantly higher amounts of EGF, PDGF, and IGF1 growth factor
receptors than untreated cells, as well as higher amounts of
MKP-1.
[0046] The results indicate that 1,25D.sub.3 does not inhibit MAPK
by inhibiting the upstream mitogenic signal from growth factor
receptors, but that it may inhibit this protein by up-regulating
MKP-1.
EXAMPLE 8
[0047] This example demonstrates the potential for sensitizing
tumor cells to the effects of conventional cytotoxic carboplatin
therapy by pretreatment with a vitamin D derivative.
[0048] Carboplatin and 1,25D.sub.3 were tested alone and in
combination using the in vitro clonogenic assay. It was observed
that pretreatment of cells with 2 nM 1,25D.sub.3 for 48 hours
significantly enhanced clonogenic cell kill when compared to
carboplatin alone or in concurrent administration (i.e., no
pretreatment) of carboplatin in combination with 1,25D.sub.3
(p<0.001 ANOVA).
EXAMPLE 9
[0049] This example demonstrates the enhancement of in vivo
carboplatin-mediated anti-tumor activity by pretreatment with a
vitamin D derivative.
[0050] The excision clonogenic kill assay was employed wherein
SCCVII/SF tumor bearing animals at 14 days post implantation were
treated i.p. for 3 days with 0.5 mg/kg body weight/day of
1,25D.sub.3. On the third day animals received varying doses of
carboplatin (between 25 mg/kg body weight and 100 mg/kg body
weight). After 24 hours, tumors were harvested, dissociated, and
plated for a 7 day incubation. It was observed that pretreatment
for 3 days with 1,25D.sub.3 before carboplatin resulted in a
significant enhancement of clonogenic cell kill when compared to
animals treated with carboplatin or 1,25D.sub.3 alone (p<0.001
ANOVA). A significant increase in clonogenic tumor cell kill was
observed at each carboplatin dose tested.
[0051] In a second experiment, the excision clonogenic kill assay
was employed wherein the SCCVII/SF tumor bearing animals at 14 days
post implantation were treated i.p. for 3 days with 1,25D.sub.3 at
varying doses. On the third day, animals received 50 mg/kg body
weight/day carboplatin. After 24 hours, tumors were harvested,
dissociated, and plated for a 7 day incubation. It was observed
that pretreatment with 1,25D.sub.3 before carboplatin resulted in a
significant enhancement of clonogenic cell even at the lowest doses
of 1,25D.sub.3. A significant increase in clonogenic tumor cell
kill was observed at each carboplatin dose tested as compared to
carboplatin alone (p<0.001 ANOVA). No animals became
hypercalcemic at any of the 1,25D.sub.3 doses tested.
EXAMPLE 10
[0052] This example demonstrates the potential for sensitizing
tumor cells to the effects of conventional cytotoxic paclitaxel by
pretreatment with a vitamin D analog.
[0053] Paclitaxel and 1,25D.sub.3 were tested alone and in
combination using the in vitro clonogenic assay as described above.
It was observed that pretreatment of cells with 1,25D.sub.3
significantly enhanced clonogenic cell kill when compared to
1,25D.sub.3 (p<0.001 ANOVA). It was also observed that
concurrent administration of 1,25D.sub.3 and paclitaxel did not
result in an enhancement of clonogenic cell kill over paclitaxel
alone.
EXAMPLE 11
[0054] This example demonstrates the enhancement of
paclitaxel-mediated in vivo anti-tumor activity by pretreatment
with 1,25D.sub.3.
[0055] The excision clonogenic kill assay was employed wherein
SCCVII/SF tumor bearing animals at 11 days post implantation were
treated i.p. for 3 days with 0.2 .mu.g/day of 1,25D.sub.3. On the
third day, animals received varying doses of paclitaxel. After 24
hours, tumors were harvested, dissociated, and plated for a 7 day
incubation. It was observed that pretreatment for 3 days with
1,25D.sub.3 before paclitaxel, at all doses, resulted in a
significant enhancement of clonogenic cell kill when compared to
animals treated with paclitaxel alone (p<0.001 ANOVA). A
significant increase in clonogenic tumor cell kill also was
observed at each paclitaxel dose tested as compared to paclitaxel
alone. No animals became hypercalcemic during these treatments.
EXAMPLE 12
[0056] This example demonstrates the enhancement of in vivo
paclitaxel-mediated anti-tumor activity by pretreatment with
1,25D.sub.3.
[0057] The tumor regrowth assay was employed wherein SCCVII/SF
tumor-bearing mice (day 7 post implantation) were treated with 0.2
.mu.g/mouse 1,25D.sub.3 administered continuously for between two
and eight days. At the end of 1,25D.sub.3 administration,
paclitaxel was injected i.p. at 40 mg/kg body weight. Control (no
treatment) or single treatment animals were injected with vehicle
(PBS) or implanted with sham pumps depending on the treatment
group. All animals experienced a significant decrease in fractional
tumor volume when pre-treated with 1,25D.sub.3 before paclitaxel as
compared to treatment with either agent alone (p<0.001
ANOVA).
EXAMPLE 13
[0058] This is an example of a clinical dosing schedule for
treatment with carboplatin and 1,25D.sub.3 in accordance with the
inventive method.
[0059] Patients with malignant tumors were subject to a treatment
regimen involving carboplatin and 1,25D.sub.3, but 48 hours prior
to treatment, each patient was placed on a low calcium diet
(250-300 mg/48 hr) and maintained on that diet for at least 7 days.
The treatment schedule for the patients is as indicated in Table
2.
2TABLE 2 Cycle Dosage 1,25D3 Dosage carboplatin 1 4 .mu.g, SQ, QD
1-3 AUC 5 .times. 1 2 6 .mu.g, SQ, QD 1-3 AUC 5 .times. 1 3 8
.mu.g, SQ, QD 1-3 AUC 5 .times. 1 4 11 .mu.g, SQ, QD 1-3 AUC 5
.times. 1 5 14 .mu.g, SQ, QD 1-3 AUC 5 .times. 1 6 18 .mu.g, SQ, QD
1-3 AUC 5 .times. 1 7 23 .mu.g, SQ, QD 1-3 AUC 5 .times. 1 8 30
.mu.g, SQ, QD 1-3 AUC 5 .times. 1 9 39 .mu.g, SQ, QD 1-3 AUC 5
.times. 1
[0060] According to this regimen, each cycle lasts four weeks. For
successive cycles, the dosage of 1,25D.sub.3 is increased by 30%.
For the first two cycles, the patients were divided into two groups
as follows:
[0061] Group 1. One day 1, this group of patients was given
carboplatin (intravenously as a 30minute infusion in 100 ml of
carrier) at a dose calculated to achieve AUC=5. 24 hours later, a
plasma sample was taken to determine carboplatin AUC, and the
patients were placed on a three-day regimen of subcutaneous
1,25D.sub.3 per the schedule indicated in Table 2.
[0062] Group 2. On day 1, this group of patients was placed on a
three-day regimen of subcutaneous 1,25D.sub.3. On day 3, the
patients were given carboplatin at a dose calculated to achieve
AUC=5. 24 hours later, a plasma sample was taken to determine
carboplatin AUC.
[0063] Following the first cycle, the groups switched between
pretreatment and post treatment. For the third and subsequent
cycles in this treatment, the patients were placed on a three-day
regimen of subcutaneous 1,25D.sub.3. On day 3, the patients were
given carboplatin at a dose calculated to achieve AUC=5. 24 hours
later, a plasma sample was taken to determine carboplatin AUC.
[0064] Following the first two cycles, patients were assessed to
determine the effect on carboplatin AUC by pretreatment vs. post
treatment with 1,25D.sub.3. The AUC of carboplatin was higher in
each patient when pretreated with 1,25D.sub.3 than when carboplatin
was given first (mean AUC=7.8 .mu.g/ml.multidot.hr.+-.1.3,
carboplatin D1; 6.7 .mu.g/ml.multidot.hr.+-.1.3, carboplatin D3).
Consistent with the change in AUC, myleosuppression was
consistently less in each patient when carboplatin was followed by
adjunctive 1,25D.sub.3.
EXAMPLE 14
[0065] This example demonstrates a method of treating prostate
cancer within a patient by adjunctively administrating a vitamin D
derivative and dexamethasone to the patient.
[0066] Thirty-two patients with androgen-independent prostate
cancer were selected on the basis of cancer progression despite
anti-androgen withdrawal therapy. The serum prostate-specific
antigen (PSA) concentration of each was measured, and they were
treated with 1,25D.sub.3 and dexamethasone on a regimen indicated
in Table 3.
3TABLE 3 Cycle Dosage 1,25D3 Dosage dexamethasone 1 (28 days) 8
.mu.g M-W-F each week 4 mg Sun (first week) 4 mg M-W-F each week 2
(28 days) 10 .mu.g M-W-F each week 4 mg M-W-F each week 3 (28 days)
12 .mu.g M-W-F each week 4 mg M-W-F each week
[0067] Patients were evaluated if they completed this regimen, and
of the initial 36 patients, 24 were evaluated based on the change
in serum PSA levels. Five of the patients exhibited at least a 50%
reduction in PSA levels after this treatment, while in the
remaining 19 the rate of disease progression was markedly
attenuated. No toxicity was observed in any of these patients.
These results indicate that co-administration of a vitamin D (or a
derivative) and dexamethasone can successfully treat prostate
cancer.
EXAMPLE 15
[0068] This example demonstrates that adjunctive administration of
zoledronate significantly decreases hypercalcemia mediated by
vitamin D-derivatives.
[0069] Normal C3H/HeJ mice were pretreated with 10 .mu.g/kg body
weight zoledronate and then treated with 0.25 .mu.g 1,25D.sub.3
once a day for three days. Control animals received 1,25D.sub.3
alone, and one group of animals received only zoledronate.
Following the last 1,25D.sub.3 treatment, blood was collected at 0,
24 and 48 hours from each mouse, and the serum calcium levels were
measures.
[0070] Initial calcium levels were significantly reduced in
experimental animals as compared to control animals (p=0.00002). 24
and 48 hours later, the serum calcium levels in the control animals
remained high (17.2.+-.1.1 mg/dl and 16.5.+-.1.1 mg/dl,
respectively), while the serum calcium levels in the experimental
animals remained reduced (14.7.+-.0.9 mg/dl and 13.4.+-.0.9 mg/dl
respectively). Additionally, experimental animals, as well as those
treated with zoledronate alone, exhibited less dehydration,
piloerection, and cachexia attributable to hypercalcemia than did
control animals. These results demonstrate that zoledronate
significantly decreases hypercalcemia mediated by vitamin D (or a
derivative).
EXAMPLE 16
[0071] This is an example of a clinical dosing schedule for
treatment with paclitaxel and 1,25D.sub.3 in accordance with the
inventive method.
[0072] Patients with malignant tumors were subject to a treatment
regimen involving paclitaxel and 1,25D.sub.3. Paclitaxel was
supplied as a sterile solution concentrate (6 mg/ml) in
polyethoxylated castor oil 50% and dehydrated ethanol USP 50%.
Immediately prior to use, this concentrate was diluted to achieve
the appropriate dose in volumes of either 0.9% NaCl injection, USP
or 5% dextrose injection, USP (DW5). Preparation was performed in
glass to avoid leaching of diethylhexylphthalate plasticizer.
1,25D.sub.3 was supplied as 0.5 .mu.g tablets from Hoffman-LaRoche
Pharmaceutical Corporatoin. The treatment schedule for the patients
was as indicated in Table 4.
4TABLE 4 Cycle Dosage 1,25D3 Dosage paclitaxel 1 4 .mu.g orally 80
mg/m2 IV 2 6 .mu.g orally 80 mg/m2 IV 3 8 .mu.g orally 80 mg/m2 IV
4 10 .mu.g orally 80 mg/m2 IV 5 13 .mu.g orally 80 mg/m2 IV
[0073] According to this regimen, each cycle lasted eight weeks.
For successive cycles, the dosage of 1,25D.sub.3 was increased by
30%. Within each cycle, 1,25D.sub.3 was administered once a day
(between 8 am and 12 am) for the first three days. On the third
day, 20 mg dexamethasone, 50 mg diphenhydramine, 50 mg rantidine,
and antiemetic-ondasteron (10 mg) or ganisteron (1 mg) was
administered intravenously 90 minutes following the 1,25D.sub.3
administration. Two hours following 1,25D.sub.3 administration, the
paclitaxel was administered intravenously in 250 ml neutral saline
over one hour. Following four days of rest (days 4-7), the
three-day treatment, four-day rest routine was repeated until the
last day of paclitaxel administration (day 45). The patients then
rested for the remainder of the cycle (days 46-63), following which
the next cycle was begun.
[0074] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0075] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0076] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *