U.S. patent application number 10/628322 was filed with the patent office on 2004-07-29 for method for treating cancer with water-insoluble s-camptothecin of the closed lactone ring form and derivatives thereof.
Invention is credited to Giovanella, Beppino C., Hinz, Hellmuth R., Kozielski, Anthony J., Stehlin, John S..
Application Number | 20040147537 10/628322 |
Document ID | / |
Family ID | 27357265 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040147537 |
Kind Code |
A1 |
Giovanella, Beppino C. ; et
al. |
July 29, 2004 |
Method for treating cancer with water-insoluble S-camptothecin of
the closed lactone ring form and derivatives thereof
Abstract
A method of treating specific forms of cancer with
water-insoluble 20(S)-camptothecin compounds with the
closed-lactone ring intact and/or derivatives thereof is disclosed.
The method includes administering the compounds intramuscularly,
orally and transdermally.
Inventors: |
Giovanella, Beppino C.;
(Houston, TX) ; Hinz, Hellmuth R.; (Dripping
Springs, TX) ; Kozielski, Anthony J.; (Spring,
TX) ; Stehlin, John S.; (Houston, TX) |
Correspondence
Address: |
KILYK & BOWERSOX, P.L.L.C.
53 A EAST LEE STREET
WARRENTON
VA
20186
US
|
Family ID: |
27357265 |
Appl. No.: |
10/628322 |
Filed: |
July 28, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10628322 |
Jul 28, 2003 |
|
|
|
10052294 |
Jan 18, 2002 |
|
|
|
6624170 |
|
|
|
|
10052294 |
Jan 18, 2002 |
|
|
|
09638354 |
Aug 15, 2000 |
|
|
|
6342506 |
|
|
|
|
09638354 |
Aug 15, 2000 |
|
|
|
09080722 |
May 21, 1998 |
|
|
|
6166029 |
|
|
|
|
09080722 |
May 21, 1998 |
|
|
|
08767861 |
Dec 17, 1996 |
|
|
|
5889017 |
|
|
|
|
08767861 |
Dec 17, 1996 |
|
|
|
08474761 |
Jun 7, 1995 |
|
|
|
5652244 |
|
|
|
|
08474761 |
Jun 7, 1995 |
|
|
|
08301727 |
Sep 7, 1994 |
|
|
|
5552154 |
|
|
|
|
08301727 |
Sep 7, 1994 |
|
|
|
08002844 |
Jan 15, 1993 |
|
|
|
08002844 |
Jan 15, 1993 |
|
|
|
07820334 |
Jan 14, 1992 |
|
|
|
07820334 |
Jan 14, 1992 |
|
|
|
07432066 |
Nov 6, 1989 |
|
|
|
5225404 |
|
|
|
|
Current U.S.
Class: |
514/283 |
Current CPC
Class: |
A61K 9/7023 20130101;
A61K 31/47 20130101; A61K 31/4745 20130101 |
Class at
Publication: |
514/283 |
International
Class: |
A61K 031/4745 |
Claims
What is claimed is:
1. A method of treating or retarding a malignant tumor in a mammal
comprising administering subcutaneously to said mammal in need of
such treatment an effective amount of a water-insoluble compound
with a closed-lactone ring wherein said malignant tumor is selected
from the group consisting of breast cancer, colon cancer, lung
cancer, stomach cancer, ovarian cancer, pancreas cancer, prostate
cancer, osteosarcoma, melanoma, and bladder cancer; and wherein
said compound is selected from the group consisting of
20(S)-camptothecin, 9-nitro-20(S)-camptothecin, 9-amino-20
(S)-camptothecin, dimethylaminomethyl-10-hydroxy-20(S)-camptot-
hecin (topotecan), 7-ethyl-10-carbonyloxy-camptothecin
(camptothecin-11), 7-ethyl-10-hydroxy-20(S)-camptothecin,
10,11-methylenedioxy-20(S)-camptot- hecin,
9-chloro-20(S)-camptothecin, 9-bromo-20(S)-camptothecin,
9-hydroxy-20(S)-camptothecin, 11-hydroxy-20(S)-camptothecin,
10-hydroxy-20(S)-camptothecin, and mixtures thereof.
2. The method according to claim 1, wherein said compound is
selected from the group consisting of 20(S)-camptothecin,
9-Nitro-20(S)-camptothecin and 9-Amino-20(S)-camptothecin.
3. The method according to claim 1, wherein said effective amount
is from about 1 mg/per kg of body weight twice a week to about 4
mg/per kg of body weight twice a week in mice.
4. The method according to claim 1, wherein said effective amount
is administered daily from about 0.75 mg/per kg to about 1.5 mg/per
kg in mice.
5. The method according to claim 1, wherein said effective amount
is from about 12.5 mg/m.sup.2 of body surface per day to about 31.3
mg/m.sup.2 of body surface per day in humans.
6. The method of claim 1, wherein said compound is
20(S)-camptothecin.
7. The method, of claim 1, wherein said compound is
9-nitro-20(S)-camptothecin.
8. The method of claim 1, wherein said compound is
9-amino-20(S)-camptothe- cin.
9. The method of claim 1, wherein said compound is
9-nitro-20(S)-camptothe- cin and said malignant tumor is breast
cancer.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/052,294, filed Jan. 18, 2002, which is a
continuation of U.S. patent application Ser. No. 09/638,354, filed
Aug. 15, 2000 (incorporated in its entirety by reference herein),
now U.S. Pat. No. 6,342,506 B1, which is a divisional of U.S.
patent application Ser. No. 09/080,722, filed May 21, 1998, now
U.S. Pat. No. 6,166,029, which is a continuation of prior U.S.
patent application Ser. No. 08/767,861, filed Dec. 17, 1996, now
U.S. Pat. No. 5,889,017, which is a continuation of U.S. patent
application Ser. No. 08/474,761, filed Jun. 7, 1995, now U.S. Pat.
No. 5,652,244, which is a continuation of U.S. patent application
Ser. No. 08/301,727, filed Sep. 7, 1994, now U.S. Pat. No.
5,552,154, which is a continuation of U.S. patent application Ser.
No. 08/002,844, filed Jan. 15, 1993, now abandoned, which is a
continuation-in-part of U.S. patent application Ser. No.
07/820,334, filed Jan. 14, 1992, now abandoned, which is a
continuation-in-part of U.S. patent application Ser. No.
07/432,066, now U.S. Pat. No. 5,225,404.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for treating
cancer in mammals, more particularly, to a method for treating
malignant tumors such as lung, breast, stomach, pancreatic,
prostate, bladder, osteosarcoma and ovary carcinomas and malignant
melanomas.
BACKGROUND OF THE INVENTION
[0003] 20(S)-camptothecin (CPT), a plant alkaloid, was found to
have anticancer activity in 1966 (Wall, M., Wani, M. C., Cooke, C.
E., Palmer, K. H., McPhail, A. T. and Slim, G. A. "Plant antitumor
agents. I. The isolation and structure of camptothecin, a novel
alkaloidal leukemia and tumor inhibitor from Camptotheca
acuminata", J. Am. Chem. Soc. 88: 3888-3890, 1966).
[0004] During the sixties and seventies the sodium salt of CPT was
derived from CPT, and clinical trials of this chemically altered
CPT were carried out and then abandoned because of the high
toxicity and low potency of this compound (Gottlieb, J. A.,
Guarino, A. M., Call, J. B., Oliverio, V. T. and Block, J. B.
"Preliminary pharmacological and clinical evaluation of
camptothecin sodium salt (NSC 100880)", Cancer Chemother. Rep. 54:
461-470; 1979; Muggia, F. M., Creaven, P. J., Hansen, H. H., Cohen,
M. N. and Selawry, D. S. "Phase I clinical trials of weekly and
daily treatment with camptothecin (NSC 100880). Correlation with
clinical studies." Cancer Chemother. Rep. 56: 515-521; 1972;
Gottlieb, J. A. and Luce, J. K. "Treatment of malignant melanoma
with camptothecin (NSC 100880)." Cancer Chemother. Rep. 56:
103-105; 1972; and Moertel, C. G., Schutt, A. J., Reitemeier, R. J.
and Hahn, R. G. "Phase II study of camptothecin (NSC 100880) in the
treatment of advanced gastrointestinal cancer." Cancer Chemother.
Rep. 56: 95-101; 1972. All these trials were conducted using the
hydrosoluble, sodium salt derivative of CPT (CPT Na+), which was
administered via i.v. The research of the present inventors has
fully confirmed the ineffectiveness and the toxicity of CPT
Na+.
[0005] Experiments have demonstrated that the non-water soluble CPT
is nontoxic and highly effective as an anticancer agent.
Furthermore, the present inventors have demonstrated that the
intramuscular and oral administration provide unexpectedly better
results than the intravenous administration.
[0006] Drug therapies have been evaluated with respect to treating
human cancer, e.g., human cancer xenograft lines. Human tumors are
serially heterotransplanted into immunodeficient, so-called "nude"
mice, and the mice then tested for their responsiveness to a
specific drug. (Giovanella, B. C., et al., Cancer 52(7):1146
(1983)). The data obtained in these studies strongly support the
validity of heterotransplanted human tumors into immunodeficient
mammals, such as nude mice, as a predictive model for testing the
effectiveness of anticancer agents.
[0007] It was determined that 9-Amino-20(S)-Camptothecin (9AC) and
10,11-Methylendioxy-20(S)-Camptothecin (10,11MD) are capable of
having high anticancer activity against human colon cancer
xenografts (Giovanella, B. C., Wall, M. E., Wani, M. C., Nicholas,
A. W., Liu, L. F., Silber, R. and Potmesil, M. "Highly effective
topoisomerase-I targeted chemotherapy of human colon cancer in
xenografts." Science 246: 1046-1048; 1989). After this finding, the
present inventors extended these studies to other human cancers
growing as xenografts in nude mice as well as conducted studies on
the administration of CPT and its derivatives. It is important to
note that the fundamental difference between the chemical used by
the present invention (CPT) and the one used ineffectively and with
high attendant toxicity in the past (CPT Na+) is that CPT is
water-insoluble and CPT Na+ is water-soluble.
SUMMARY OF THE INVENTION
[0008] An object of the present invention is to provide a novel
method for treating cancers in mammals.
[0009] Additional objects and advantages of the present invention
will be set forth in part in the description which follows, and in
part will be apparent from the description, or may be learned by
practice of the present invention. The objects and advantages of
the present invention will be realized and attained by means of the
elements and combinations particularly pointed out in the appended
claims.
[0010] To achieve the objects and in accordance with the purpose of
the present invention, as embodied and broadly described herein,
the present invention relates to a method for treating malignant
tumors in a mammal which comprises administering intramuscularly,
orally or transdermally an effective amount of a water-insoluble
20(S)-Camptothecin (CPT) with the closed-lactone ring intact or a
derivative thereof or a mixture thereof.
[0011] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the present
invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1a shows the results obtained for CLO breast carcinomas
in nude mice treated once a week and twice a week with a suspension
of 20(S)-Camptothecin (CPT) in Intralipid 20 intramuscularly at the
dose of 4 mg/kg. The tumor size was measured in mm.sup.3 in
function of time.
[0013] FIG. 1b depicts the results of the same experiment as FIG.
1a in body weight in grams of the nude mice carrying CLO breast
carcinoma.
[0014] FIG. 2 shows the results obtained for SPA lung
adenocarcinomas in nude mice treated daily with CPT in Intralipid
20 administered orally at various doses.
[0015] FIG. 3 shows the results obtained for CAS colon carcinoma in
nude mice treated twice a week with CPT or its sodium salt (CPT
Na+) both at the dose of 4 mg/kg intravenously or intramuscularly.
All treatments in Intralipid 20 except for a group treated with CPA
Na+ in Phosphate Buffered Saline (PBS).
[0016] FIGS. 4 and 5 are graphs which show the results obtained for
LOV pancreatic carcinoma in nude mice treated twice a week with
20(S)-CPT in cottonseed oil administered intramuscularly at the
dose of 2.5 mg/Kg.
[0017] FIGS. 6 and 7 are graphs which show the results obtained for
BOL bladder carcinoma in nude mice treated twice a week with
20(S)-CPT in cottonseed oil administered intramuscularly at the
dose of 2.5 mg/Kg.
[0018] FIGS. 8 and 9 are graphs which show the results obtained for
PC-3 prostate carcinoma in nude mice treated twice a week with
20(S)-CPT in cottonseed oil administered intramuscularly at the
dose of 2.5 mg/Kg.
[0019] FIGS. 10 and 11 are graphs which show the results obtained
for BRO melanoma in nude mice treated with 20(S)-CPT or CPT
derivatives at various dosages by intrastomach means.
[0020] FIGS. 12 and 13 are graphs which show the results obtained
for TEP osteosarcoma in nude mice treated with 20(S)-CPT
intramuscularly at the dose of 2.5 mg/Kg.
[0021] FIGS. 14 and 15 are graphs which show the effectiveness of
20(S)-CPT on different tumor sizes.
[0022] FIGS. 16 and 17 are graphs which show the results obtained
for CLO breast carcinoma in nude mice treated with 20(S)-CPT or CPT
derivatives by transdermal application.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0023] CPT is a plant alkaloid isolated from Camptotheca acuminata.
Chemical derivatives of CPT can be prepared either in a
semisynthetic or totally synthetic way. (See, e.g., Wani, M. C. et
al., J. Med. Chem. 23:544, 1980; Wani, M. C. et al., J. Med. Chem.
30: 1774 (1987); and Wani, M. C. et al., J. Med. Chem. 30: 2317
(1987)).
[0024] In the examples discussed below, the CPT was obtained from
the Institute of Materia Medica, Academia Sinica, Shanghai, China,
and further purified using standard chromatographic techniques.
Camptothecin Sodium Salt (CPT Na+), 9-Nitro-20(S)-Camptothecin
(9NO.sub.2) and 9-Amino-20(S)-Camptothecin (9AC) were also
synthesized from CPT. CPT and derivatives thereof have to be
extensively purified prior to administering for use in the present
invention because: (1) the natural product contains several other
components which have a large degree of toxicity for example, in
mice, and (2) FDA regulations require such purifying for any drug
or compound to be used as a medicine. Methods of purification known
by those skilled in the art can be used, e.g.--dissolving the CPT
in a suitable solvent such as chloroform or methylene chloride and
then adsorbing onto a column containing silica gel and then
carrying out elution of the adsorbed materials by increasing the
polarity of the eluant by adding, e.g., methanol.
[0025] The purity of the compound can be tested by high performance
liquid chromatography (HPLC) and thin layer chromatography (TLC)
and other appropriate methods known in the art. The compound can
also be completely characterized using infrared (IR), ultraviolet
(UV) and nuclear magnetic resonance (NMR) spectroscopy and
elemental analysis.
[0026] Furthermore, the CPT and derivatives thereof used in the
present invention are water-insoluble and not administered in the
chemically and physically different sodium salt form. This
difference can easily be observed by looking at their elemental
analysis, NMR, UV and IR spectra and also by their different
physical behavior in HPLC and TLC experiments.
[0027] CPT and derivatives thereof of the closed lactone ring form
are used and are administered intramuscularly or orally, and in
such cases it was possible to obtain total remissions of a vast
spectrum of human cancers without the toxicity observed previously
with CPT Na+. The derivatives of CPT for use in the present
invention include, for example, 9NO.sub.2, and 9AC. A mixture of
CPT and derivatives thereof can also be used in the present
invention.
[0028] Other related derivatives can also be used in conjunction
with the method of the present invention. Examples include
dimethylaminomethyl-10-- hydroxy-20(S)-CPT (topotecan),
7-ethyl-10-[4-(1-piperdino)-1-piperdino]-ca- rbonyloxy-CPT
(CPT-11), 7-ethyl-10-hydroxy-20(S)-CPT, 9-amino-20(S)-CPT,
9-nitro-20(S)-CPT, 10,11-methylenedioxy-20(S)-CPT,
9-chloro-20(S)-CPT, 9-bromo-20(S)-CPT, 9-hydroxy-20(S)-CPT,
11-hydroxy-20(S)-CPT, and 10-hydroxy-20(S)-CPT.
[0029] Another method of administering the compounds of the present
invention is by a transdermal or transcutaneous route. One example
of such an embodiment is the use of a patch. In particular, a patch
can be prepared with a fine suspension of a compound disclosed in
the present application in, for example, dimethylsulfoxide (DMSO),
or a mixture of DMSO with cottonseed oil and brought into contact
with the skin of the tumor carrying mammals away from the tumor
location site inside a skin pouch. Other mediums or mixtures
thereof with other solvents and solid supports would work equally
as well. The patch can contain the CPT compound in the form of a
solution or a suspension. The patch can then be applied to the skin
of the patient, for example, by means of inserting it into a skin
pouch of the patient formed by folding and holding the skin
together by means of stitches, clips or other holding devices. This
pouch should be employed in such a manner so that continuous
contact with the skin is assured without the interference of the
mammal. Besides using a skin pouch, any device can be used which
ensures the firm placement of the patch in contact with the skin.
For instance, an adhesive bandage could be used to hold the patch
in place on the skin.
[0030] In the studies, the water-soluble derivatives of these
compounds proved to be ineffective as anticancer agents and very
toxic to mammals. Even the closed lactone ring forms of CPT and
derivatives thereof showed a great difference in activity,
depending on their route of administration. In the studies, the
intramuscular, transdermal and oral routes were unexpectedly
superior to other routes of delivery.
[0031] As used herein, the term "malignant tumor" is intended to
encompass all forms of human carcinomas, sarcomas and melanomas
which occur in the poorly differentiated, moderately
differentiated, and well differentiated forms.
[0032] In treating or retarding malignant tumors in mammals in
accordance with the present invention, the aforedescribed
camptothecin compounds are administered intramuscularly,
transdermally or orally, using commonly known methods, for example,
gelatin capsules for oral administration, as well as novel,
superior formulations, such as microsuspensions in lipid and in
lipid-like emulsions (e.g.--Intralipid 20, cottonseed oil and
peanut oil) for intramuscular administration and inclusion in
cholesterol pellets for subcutaneous long-term administration.
[0033] As used herein, an "effective amount" of CPT and derivatives
thereof described above is intended to mean that amount of the
compound which will inhibit the growth of, or retard cancer, or
kill malignant cells, and cause the regression and palliation of
malignant tumors, i.e., reduce the volume or size of such tumors or
eliminate the tumor entirely.
[0034] With mammals, including humans, the effective amounts can be
administered on the basis of body surface area. The
interrelationship of dosages for animals of various sizes, species
and humans (based on mg/M.sup.2 of body surface) is described by E.
J. Freireich et al., Cancer Chemother. Rep., 50(4):219 (1966). Body
surface area may be approximately determined from the height and
weight of an individual (see, e.g., Scientific Tables, Geigy
Pharmaceuticals, Ardsley, N.Y. pp. 537-538 (1970). An effective
amount of the camptothecin compounds in the present invention can
range from about 12.5 mg/m.sup.2 of body surface per day to about
31.3 mg/m.sup.2 of body surface per day.
[0035] The preferred effective amounts or dosages of CPT and/or
derivatives thereof in mice are about 1 to about 4 mg CPT/kg of
body weight twice a week for an intramuscular route and about 0.75
to about 1.5 mg CPT/kg/day for the oral route. Effective amounts or
dosages of CPT and/or derivatives thereof in mice are, for instance
about 1.5 mg/Kg/week to about 10 mg/Kg/week CPT for the transdermal
route. For all of the administering routes, the exact timing of
administration of the dosages can be varied to achieve optimal
results. Generally, when using Intralipid 20 as the carrier for the
CPT, the actual dosage of CPT reaching the patient will be less.
This is due to some loss of CPT on the walls of the syringes,
needles and preparation vessels, which is prevalent with the
Intralipid 20 suspension. When a carrier, such as cottonseed oil is
used, this above-described loss is not so prevalent because CPT
does not adhere as much to the surfaces of syringes, etc. For
instance and preferable, it has been found that generally about 2.5
mg CPT/kg of body weight twice per week using cottonseed oil,
administered by an intramuscular route, will deliver the same
amount to the patient as 4.0 mg CPT/kg of body weight twice per
week using Intralipid 20 as a carrier. Generally, about 1 mg to
about 4 mg of CPT and/or derivatives thereof is added to about 0.1
ml to about 1 ml of carrier. Levels of both CPT and 9NO.sub.2 were
well tolerated by mice in the examples set forth below without
weight loss or other signs of toxicity. These dosages have been
administered for up to six months continuously without any ill
effect.
[0036] Another important feature of the method provided by the
present invention relates to the relatively low or no apparent
overall toxicity of the camptothecin compounds administered in
accordance herein. Overall toxicity can be judged using various
criteria. For example, loss of body weight in a subject over 10% of
the initially recorded body weight (i.e., before treatment) can be
considered as one sign of toxicity. In addition, loss of overall
mobility and activity and signs of diarrhea or cystitis in a
subject can also be interpreted as evidence of toxicity. In one of
the examples which follow, the overall toxicity of the camptothecin
compounds of the present invention was evaluated. For example, CPT,
when administered twice per week in amounts of 4 mg/kg in
Intralipid 20 or 2.5 mg/kg in cottonseed oil, were found to be
therapeutically effective (resulting in complete reduction of
subcutaneous tumors) with no overall toxicity as described above.
This was also the case when administering for more than 35 weeks
the enormous total dose of 280 mg/kg.
[0037] Further, using 0.1 to 1.0 ml of cottonseed oil, in which CPT
or derivatives were suspended, up to a quantity of 1 to 4 mg/kg
were injected into the cavity of the stomach through a 27-gauge
needle once a day for as long as desired (mice have tolerated more
than 40 consecutive, daily injections) with no ill-effects.
[0038] The compounds of the present invention may be administered
in combination with pharmaceutically acceptable carriers or
diluents, such as Intralipid 10 or 20 or natural oils, or other
suitable emulsifiers for lipophilic compounds.
[0039] The present invention will be further clarified by the
following example, which is intended to be purely exemplary of the
present invention.
EXAMPLE 1
[0040] Swiss nude mice of the NIH high fertility strain were bred
and maintained pathogen-free in a laboratory. (Giovanella, B. C.
and Stehlin, J. S. "Heterotransplantation of human malignant tumors
in `nude` thymusless mice. I. Breeding and maintenance of `nude`
mice." J. Natl. Cancer Inst. 51: 615-619; 1973.)
[0041] Human malignant carcinomas of the colon, breast, lung,
ovary, stomach, pancreas, bladder, prostate, osteosarcoma and
malignant melanomas were heterotransplanted directly from a patient
into the nude mice and passaged serially. In particular, the tumors
CLO, MUR, CAS, SW 48, SQU and BRO as described in Giovanella, B.
C., Stehlin, J. S., Shepard, R. C. and Williams, L. J. "Correlation
between response to chemotherapy of human tumors in patients and in
nude mice." Cancer 52: 1146-1152; 1983), tumor SCH as described in
Heim, S., Mandahl, N., Arheden, K., Giovanella, B. C., Yim, S. O.,
Stehlin, J. S., Jr. and Mitelman, F. "Multiple karyotypic
abnormalities including structural rearrangements of 11P in cell
lines from malignant melanomas." Cancer Genet. Cytogent. 35: 5-20;
1988, and Verschraegen, C., Giovanella, B. C., Mendoza, J. T.,
Kozielski, A. J. and Stehlin, J. S., Jr. "Specific organ metastases
of human melanoma cells injected into the arterial circulation of
nude mice." Anticancer Res., In Press, and tumor PC-3 as described
in Invest. Urol. 17, 16-23, 1979, were used. Tumors DOY and HAR
(undifferentiated non-oat cell carcinomas of the lung), DIL (a
poorly differentiated squamous cell carcinoma of the lung), SPA (a
moderately differentiated adenocarcinoma of the lung), LAN (an
undifferentiated carcinoma of the ovary), and BRE (a moderately
differentiated adenocarcinoma of the stomach) were
heterotransplanted. For the experiments, the tumor tissue was
finely minced in complete MEM medium and 0.5 ml of a 10% v/v
suspension was inoculated subcutaneously on the upper back of
groups of 10-30 mice. When the tumors became palpable and
measurable in all the animals, they were divided into groups of 4-8
and treated with the desired dose of the drug in experiment or with
the vehicle only for the controls.
[0042] 9NO.sub.2 (also referred to as 9NC) and 9AC were synthesized
(Wani, M. C., Nicholas, A. W., Wall, M. E., "Plant Tumor Agents.
23. Synthesis and antileukemic Activity of Camptothecin analogues,
J. Med. Chem., 2358-2363, 1986) from CPT obtained from the
Institute of Materia Medica, Academia Sinica, Shanghai, China. The
derivatives were purified by chromatography and analyzed. A typical
sample preparation for IM-injection or oral administration using
Intralipid 20 or cottonseed oil for example includes the dispersion
of the test compound by sonication (3 pulses for 30 seconds each)
in Intralipid 20 or cottonseed oil at the standard concentration of
1 mg/ml using an Ultrasonic Processor, Model GE600 by Sonics and
Materials, Inc., CT. Intramuscular injections were performed
through a 27-gauge needle into the deep muscles of the posterior
legs of the mice twice a week. The animals received up to 70 such
injections consecutively without suffering ill effects except for
some local fibrosis. Intravenous injections, using Intralipid 20 as
the suspending agent, were performed through a 30-gauge needle in
one of the tail veins. No pulmonary embolism was observed after
over 200 injections.
[0043] Oral administration was achieved by mixing the required
amounts of drugs in Intralipid 20 or cottonseed oil with a
previously autoclaved dietary supplement composed of whole wheat
bread saturated with whole milk. The supplement containing the drug
was then thoroughly mixed to form a paste. Mice were fed 1 g of
dietary supplement containing the required dose of the drugs once a
day, and then 5 g of autoclaved mouse feed. (At this regime, the
animals initially lose about 4 g out of 30 g of body weight, regain
2 g within a few days, and stabilize at this level). This regime
can be carried on indefinitely. Actually, the mice fed in this
manner (a slightly lower maximum caloric intake) were healthier and
longer-lived than mice fed ad lib (Giovanella, B. C., Shepard, R.
C., Stehlin, J. S., Venditti, J. M. and Abbott, B. J. "Calorie
restriction: Effect on growth of human tumors heterotransplanted in
nude mice." J. Natl. Cancer Inst. 68: 249-257; 1982).
[0044] For the experiments and data reflected in FIGS. 10-11 and
one experiment in FIGS. 16-17, oral administration was accomplished
by injecting the mixture of CPT compound with cottonseed oil
directly into the stomach (1S).
[0045] Table 1 summarizes some of the results obtained by this
treatment with CPT and derivatives thereof.
1TABLE 1 NO. NO. TUMOR TREATMENT ANIMALS RESULTS TREATMENTS
OBSERVATIONS BREAST Ca. CLO IM CA 4 mg/kg 12 10/12 CR CR
58-29(.sup.1) Alive day 170; Control 6/6 dead from day 120. MUR IM
CA 4 mg/kg 6 5/6 CR 55 5/6 Alive day 180. Controls 1/6 alive. IM
9NO.sub.2CA 4 mg/kg 6 6/6 CR 54 4/6 Alive day 180. Controls 1/6
alive. COLON Ca. CAS IM 9-AC 4 mg/kg 6 5/6 CR 20 IV CA 4 mg/kg 6
6/6 NR 20 IM CA (Na.sup.+)4 mg/kg 6 6/6 NR 20 IV Ca(Na.sup.+) 4
mg/kg 12 12/12 NR 20 IM 9-AC 4, 8 mg/kg 6 3/3 CR 18 IV 9-A/C 4
mg/kg 3 3/3 CR 18 IM 9-NO.sub.2CA 4 mg/kg 6 2/3 CR 23 IM CA 4 mg/kg
6 5/6 GI 28 Growth inhibited for 34 days. Controls all dead day
105. IM CA 4 mg/kg 5 5/5 GI 24 Growth inhibited for 21 days.
Controls 4/6 dead day 20. IV CA 4 mg/kg 5 2/5 PR 24 IM CA 4, 6
mg/kg 10 9/10 CR 9 IV CA 4, 6, 8 mg/kg 15 NR 9 SW 48 IM CA 4 mg,kg
14 14/14.sup.(2) GI 1 .times./wk = 14T Growth inhibited for 30
days. 2 .times./wk = 29T SQU IM CA 4 mg/kg 7 7/7 GI 21 Growth
inhibited for 73 days. All alive day 112. Controls 3/7 alive. LUNG
Ca. SPA PO CA 4, 6, 8 mg/kg 12 10/12 CR 110 IM CA 4 mg/kg 6 47
Treated. All alive day 180. IM 9-NO.sub.2CA 4 mg/kg 6 9/12 CR 47
Treated. All alive day 180. Controls 6/6 dead day 153. DOY IM CA 4
mg/kg 6 6/6 CR 56 All treated. Alive day 190. Controls 6/6 dead day
146. HAR IM CA 4 mg/kg 5 4/5 CR 41 All treated. Alive day 167. IM
9-NO.sub.2CA 4 mg/kg 5 5/5 CR 41 All treated. Alive day 163.
Controls 3/4 alive. DIL IM CA 4 mg/kg 6 6/6 CR 48 All treated. IM
9-NO.sub.2CA 4 mg/kg 6 5/6 CR 48 Alive day 185. Controls 5/6 still
alive. OVARIAN Ca. LAN IM CA 4 mg/kg 8 8/8 CR 27 All treated. Alive
day 83; IM 9-NO.sub.2CA 4 mg/kg 8 8/8 CR 27 controls 1/8 alive. IM
9-NO.sub.2CA 4 mg/kg 12 12/12 CR 21 Alive day 66. Controls 2/6
alive; all treated. STOMACH Ca. BRE IM CA 6 mg/kg 5 5/5 CR 36 IV CA
6 mg/kg 5 5/5 PR 36 MAL. MELANOMA BRO IM CA 4 mg/kg 7 5/7 CR 51 All
treated. Alive day IM 9-AC 4 mg/kg 7 7/7 CR 15 170. Controls 7/7
dead day 60. IM 9-NO.sub.2CA 4 mg/kg 14 14/14 CR 21 Alive day 60.
Controls 2/7 alive. All treated. SCH IM CA 4 mg/kg 8 8/8 GI.sup.(3)
25 Growth inhibited for 60 days. IM 9-NO.sub.2CA 4 mg/kg 8 8/8 GI
25 Growth inhibited for 30 days. (.sup.1)5 treated once a week
.sup.(2)7 treated once a week .sup.(3)Tumor growth inhibited
CR--Complete remission defined as total disappearance of the
initial tumor, maintained for at least 30 days at the making of
this table NR--No response PR--Partial response GI--Growth
inhibited CA--Camptothecin 9-AC--9-Amino Camptothecin
9-NO.sub.2CA--9-Nitro Camptothecin W--Week D--Daily Unless
otherwise noted, all i.m. and i.v. doses are administered 2 .times.
week, all oral doses daily.
[0046] FIG. 1a shows the effectiveness of CPT against the human
breast cancer when administered intramuscularly. FIG. 1b indicates
that even prolonged intramuscular administration of the compound of
the present application do not have any effect on the body weight
of the mice at the dosage of 4 mg/kg using Intralipid 20 as the
carrier. FIG. 2 shows the results obtained by treating a human lung
cancer xenograft by oral administration of CPT. It is important to
note that it is possible to administer CPT in Intralipid 20 without
any toxicity at 8 mg/kg/day which is twice that of a 4 mg/kg/day
dose which was already sufficient to totally suppress the tumor
growth. FIG. 3 compares the effectiveness of the intramuscular
versus the intravenous route of administration of CPT and the
sodium salt derivative in side-by-side experiments. It can be seen
that the sodium salt of CPT is completely ineffective regardless of
its route of administration and regardless of the carrier used
(Intralipid 20 or PBS). Furthermore, FIG. 3 leads to the conclusion
that in comparing intramuscular administration with intravenous
administration, the intramuscular route is the effective route. In
other words, in viewing FIG. 3, the difference in anticancer
activity of CPT can be appreciated when comparing intravenous
administration versus intramuscular administration and the form of
CPT used. In fact, using CPT Na+, the inventors were never able to
eradicate human cancer xenografts, even when the most sensitive
human tumors were used. Raising the dose of CPT Na+ brought on
toxicity, causing first weight loss and then animal death without
achieving total tumor regression. These findings were very similar
to the unsuccessful clinical trials of the sixties and seventies
discussed above.
[0047] FIGS. 4 and 5 show the results obtained for LOV pancreatic
cancer. Seven nude mice were treated intramuscularly with 2.5 mg/Kg
body weight of 20(S)-CPT in cottonseed oil twice a week until day
100. Six additional nude mice were used as a control (cottonseed
oil only). After 125 days, all mice receiving 20(S)-CPT survived as
compared to only two of the six mice in the control group.
[0048] FIGS. 6 and 7 show the results obtained for BOL bladder
cancer. Six nude mice were treated intramuscularly with 2.5 mg/Kg
body weight 20(S)-CPT in cottonseed oil twice a week for 52 days.
Six additional nude mice were used as a control (cottonseed oil
only). All control mice were dead as of day 46, while all mice
receiving 20(S)-CPT survived when the experiment was concluded (day
52).
[0049] FIGS. 8 and 9 show the results obtained for PC-3 prostate
cancer. Six nude mice were treated intramuscularly, with 2.5 mg/Kg
body weight 20(S)-CPT in cottonseed oil twice a week for a total of
17 treatments. Six additional nude mice were used as a control
(cottonseed oil only). After day 83, all mice survived except for
one mouse receiving 20(S)-CPT.
[0050] FIGS. 10 and 11 show the results obtained for BRO-melanoma.
Five nude mice were treated inter-stomach with 1 mg/Kg body weight
20(S)-CPT in cottonseed oil per day for 5 consecutive days with 2
day intervals of no treatment (i.e., 5 days of treatment then 2
days no treatment, and then 5 days treatment and so on). Five nude
mice received 2 mg/Kg body weight 20(S)-CPT in cottonseed oil per
day for two consecutive days with 1 day intervals of no treatment.
Four additional groups of five mice each received in the exact same
manner either 9-NC or 9-AC and one additional group was a control
(cottonseed oil only) as reflected in FIGS. 10 and 11 and Table
2.
2TABLE 2 Control 20(S)-CPT (1) 20(S)-CPT (2) 9NC (1) 9NC (2) 9AC
(1) 9AC (2) # of mice 5 5 5 5 5 5 5 used amount -- 1 2 1 2 1 2
received (mg/Kg) timing of -- 5 days on 2 days on 5 days on 2 days
on 5 days on 2 days on admin. 2 days off 1 day off 2 days off 1 day
off 2 days off 1 day off # of mice 1 3 3 5 5 4 0 surviving after
day 72
[0051] FIGS. 12 and 13 show the results obtained for TEP
osteosarcoma. Four nude mice were treated intramuscularly with 2.5
mg/Kg body weight 20(S)-CPT in cottonseed oil twice a week for a
total of 14 treatments. Three additional nude mice were used as a
control (cottonseed oil only). After day 70, all mice survived
except for one mouse in the control group.
[0052] FIGS. 14 and 15 show the results of tumor response to
20(S)-CPT by treating two different sizes of CLO breast cancer
tumors. In one group of 10 nude mice, tumor growth was allowed to
get to one cm in size prior to treatment. In another group of ten
nude mice, a tumor growth was allowed to get to 2+ cm in size prior
to treatment. Each mouse in each group was then treated
intramuscularly with 2.5 mg/Kg body weight 20(S)-CPT in cottonseed
oil twice a week for 93 days. All mice survived when the experiment
was ended on day 104. The experiment shows that tumor size effects
the rapidity at which 20(S)-CPT regresses the tumor. In each group,
20(S)-CPT was not toxic to the mice and effectively regressed the
tumor size.
[0053] It is clear from these studies, that CPT and its derivatives
with the closed lactone ring have been demonstrated to possess an
astonishing level of anticancer activity. This applies both to the
spectrum of tumors covered and to the quality of the responses. The
method of the present invention has been able to block growth
completely and to totally regress human xenografts of carcinomas
(lungs, breast, colon, stomach, pancreas, bladder, prostate,
osteosarcoma and ovaries) and malignant melanomas. This has been
accomplished without any observable toxicity. Many of the mammals
which have been treated continuously for six months have shown no
ill effects nor regrowth of the tumor they once carried.
EXAMPLE 2
[0054] Swiss nude mice of the NIH high fertility strain were bred
and maintained pathogen-free in a laboratory. Human carcinoma of
breast were heterotransplanted as described before in Example 1.
For the experiment, CPT was purified to a purity of 99% as
determined by HPLC. Two samples were prepared in the following
manner:
[0055] 1) 7.5 mg of CPT was mixed with 1 mL of dimethyl sulfoxide
(DMSO) and after agitating for a minute, 4 mL of cottonseed oil was
added and the mixture was transformed into a fine suspension by
sonicating using an Ultrasonic Processor Model 2200 (Branson
Ultrasonics Corp., Danbury Conn. 06810-1961) at 60.degree. C. for
one hour to yield a standard concentration of 2.5 mg/kg/0.05
mL.
[0056] 2) 7.5 mg of CPT was dissolved in 5 mL DMSO with the help of
sonication as described above for a final concentration of 2.5
mg/kg/0.05 mL. The two control mixtures were prepared by mixing
cottonseed oil with DMSO in a ratio of 4:1, and the second control
mixture consisting of DMSO alone.
[0057] The patches for the transdermal application consisted of
sterile absorbent gauze from Johnson & Johnson (Skillman, N. J.
08558-9418) sheer bandages (3/4 in. by 3 in.), which were pulled
off from their adhesive backs and cut into thirds with the
appropriate dimensions of 16 mm (.+-.1 mm) by 8 mm (.+-.1 mm). The
appropriate amounts of suspended CPT were added. Tumor carrying
mice were anesthestized with Avertin composed of a mother solution
of 2.5 gm tribromoethanol and 50 mL tert. amyl alcohol, diluted
with distilled water in a ratio of 1:30. After having mixed this
solution, making sure that all had dissolved, the mixture was then
filtered through a disposable sterile syringe filter (25 mm, 0.45
{circumflex over ( )}mm) by Corning Glass Works, Corning, N.Y.
14831. To induce sleep for the duration of the surgery (10 to 20
minutes) 0.8 mL/30 gm animal of this solution was injected via I.P.
While asleep, the skin on the lower back of the animals was pulled
together forming a pouch followed by stapling it almost completely
shut, except for a small opening, through which the patch was
inserted. The patch, which was inserted away from the tumor, was
replaced twice a week using the same procedure above.
[0058] In further detail and as reflected in FIGS. 16 and 17, the
following experiment was conducted.
[0059] (a) four nude mice each were treated with 2.5 mg/Kg body
weight 20(S)-CPT in DMSO twice a week by means of the placement in
the pouch without the use of a patch.
[0060] (b) Three nude mice each were treated with DMSO in a pouch
as a control ("CONTROL DMSO pouch").
[0061] (c) Four nude mice each were treated with 2.5 mg/Kg body
weight 20(S)-CPT in DMSO once a week by means of a pouch.
[0062] (d) Four nude mice each were treated with DMSO in cottonseed
oil in a patch which was inserted in the pouch ("CONTROL
PATCH").
[0063] (e) Four nude mice each were treated interstomach with 2.0
mg/Kg body weight 20(S)-CPT in a medium of DMSO/cottonseed oil for
two day intervals with one day of no treatment in between (i.e., 2
days on then one day off and so on).
[0064] (f) Four nude mice each were treated with 5.0 mg/Kg body
weight 20(S)-CPT in a medium of DMSO/cottonseed oil twice a week by
means of a patch inserted into the pouch.
[0065] (g) Three nude mice each were treated with 5.0 mg/Kg body
weight 20(S)-CPT in a medium of DMSO/cottonseed oil once a week by
means of a patch inserted into the pouch.
[0066] The experiment was terminated at day 41. The survival rates
were as follows:
[0067] (a) four survived
[0068] (b) three survived
[0069] (c) three out of four survived
[0070] (d) four survived
[0071] (e) four survived
[0072] (f) four survived
[0073] (g) three survived.
[0074] As can be seen from FIGS. 16 and 17, the body weight of the
animals is more effected by the oral (I.S.) route than by the
trans-cutaneous or transdermal route, although none of the animals
in either group have shown any visual toxicity. Furthermore, when
observing the tumor size, it may be pointed out that both the oral
and the transdermal route of application showed good results in
tumor regression.
[0075] The difference in applying CPT in a medium of DMSO versus
DMSO/cottonseed oil shows that there is some dependence on the type
of patch used in the experiments, and modifications in the
application system may be made where the patch will show results
superior to the oral route.
[0076] Other embodiments of the present invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the present invention disclosed herein. It is
intended that the specification and examples be considered as
exemplary only, with a true scope and spirit of the invention being
indicated by the following claims.
* * * * *