U.S. patent application number 12/701093 was filed with the patent office on 2010-12-23 for inhibition of melanogenesis and melanoma metastasis with p-amino benzoic acid (paba).
This patent application is currently assigned to NEW YORK UNIVERSITY. Invention is credited to Peter C. Brooks, Danielle Morais, Dorothy Rodriguez.
Application Number | 20100323002 12/701093 |
Document ID | / |
Family ID | 32682384 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100323002 |
Kind Code |
A1 |
Brooks; Peter C. ; et
al. |
December 23, 2010 |
INHIBITION OF MELANOGENESIS AND MELANOMA METASTASIS WITH P-AMINO
BENZOIC ACID (PABA)
Abstract
The present invention relates to the inhibition of melanogenesis
with para-aminobenzoic acid (PABA) and its use in treating
melanotic cancer.
Inventors: |
Brooks; Peter C.; (Carmel,
NY) ; Morais; Danielle; (Bedford Hills, NY) ;
Rodriguez; Dorothy; (Pequannock, NJ) |
Correspondence
Address: |
FISH & RICHARDSON P.C. (NY)
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
NEW YORK UNIVERSITY
New York
NY
|
Family ID: |
32682384 |
Appl. No.: |
12/701093 |
Filed: |
February 5, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10746206 |
Dec 23, 2003 |
7691905 |
|
|
12701093 |
|
|
|
|
60436394 |
Dec 24, 2002 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/133.1; 424/623; 424/85.1; 424/85.2; 424/85.7; 514/110; 514/114;
514/171; 514/19.3; 514/252.18; 514/283; 514/34; 514/383; 514/393;
514/44A; 514/449; 514/463; 514/49; 514/492; 514/564; 514/567;
514/64 |
Current CPC
Class: |
A61P 35/04 20180101;
A61K 31/195 20130101; A61P 35/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/450 ;
514/567; 514/449; 514/19.3; 514/110; 514/492; 514/463; 514/283;
514/564; 514/34; 514/64; 424/85.7; 514/44.A; 514/49; 424/133.1;
424/85.2; 424/85.1; 514/252.18; 514/383; 514/114; 514/171; 514/393;
424/623 |
International
Class: |
A61K 31/196 20060101
A61K031/196; A61P 35/00 20060101 A61P035/00; A61K 31/337 20060101
A61K031/337; A61K 38/14 20060101 A61K038/14; A61K 31/675 20060101
A61K031/675; A61K 31/282 20060101 A61K031/282; A61K 31/366 20060101
A61K031/366; A61K 31/4745 20060101 A61K031/4745; A61K 31/198
20060101 A61K031/198; A61K 31/704 20060101 A61K031/704; A61K 9/127
20060101 A61K009/127; A61K 31/69 20060101 A61K031/69; A61K 38/21
20060101 A61K038/21; A61K 31/7088 20060101 A61K031/7088; A61K
31/7068 20060101 A61K031/7068; A61K 39/395 20060101 A61K039/395;
A61K 38/20 20060101 A61K038/20; A61K 38/19 20060101 A61K038/19;
A61K 31/496 20060101 A61K031/496; A61K 31/4196 20060101
A61K031/4196; A61K 31/661 20060101 A61K031/661; A61K 31/5685
20060101 A61K031/5685; A61K 31/4188 20060101 A61K031/4188; A61K
33/36 20060101 A61K033/36; A61P 35/04 20060101 A61P035/04 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This work was supported in part by NIH/NCI grant ROI
CA91645. Pursuant to the terms of that grant, the federal
government has certain rights to this invention.
Claims
1. A method for treating a mammal afflicted with melanoma
comprising administering to said mammal an effective amount of
p-aminobenzoic acid (PABA) in combination with radiation therapy or
one or more chemotherapeutic agent(s).
2. The method of claim 1 which comprises administering between 10
mg/day and 20 g/day of PABA to said mammal.
3. The method of claim 1 which comprises administering between 20
mg/day and 12 g/day of PABA to said mammal.
4. The method of claim 1, wherein PABA is administered by the oral,
subcutaneous, intramuscular, or intratumoral route.
5. The method of claim 1 wherein said mammal is a human.
6. The method of claim 1 comprising administering radiation
therapy.
7. The method of claim 6 which comprises administering said
radiation therapy in doses of between 1 cGy and 100 Gy of
radiation.
8. The method of claim 6 which comprises administering said
radiation therapy in doses of between 2 cGy and 20 Gy of
radiation.
9. The method of claim 1 comprising administering one or more
chemotherapeutic agent(s).
10. The method of claim 9, wherein the one or more chemotherapeutic
agents are selected from the group consisting of bleomycin,
cyclophosphamide, paclitaxel, docetaxel, carboplatin, and a
platinum coordination compound.
11. The method of claim 9, wherein the one or more chemotherapeutic
agents are selected from the group consisting of, podophyllotoxin,
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan,
nitrosurea, adriamycin, dactinomycin, daunorubicin HCl,
doxorubicin, doxorubicin HCL liposome injection, epirubicin
hydrochloride, bleomycin, plicomycin, mitomycin, etoposide,
tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, vincristin,
vinblastin, bortezomib (formerly known as PS-341), dicarbizide,
a-interferon (Intron A), Genasense G3139 (Bc12 antisense
oligonucleotide), gemcitabine HCL, capecitabine:
5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine, epithalones A
and B, oxaliplatin, inhibitors of the EGFR tyrosine kinase
(OSI-774), C225, trastuzamab, rituximab, aldesleukin, profimer
sodium, denileukin difitox, mitoxantrone hydrochloride, tamoxifen
citrate, filgrastim, Neupogen gemtuzumab ozogamicin, topotecan HCL,
imatinib mesylate, letrozole, toremifene citrate, etoposide
phosphate, amifostine, irinotecan HCL, alemtuzumab, busulfan,
bleomycin sulfate, exemestane, anastrozole, docetaxel,
temozolomide, and arsenic trioxide.
12. The method of claim 1, wherein the mammal with malignant
melanoma has metastatic malignant melanoma.
13. The method of claim 1, wherein the mammal with malignant
melanoma has recurrent malignant melanoma.
14. The method of claim 1, wherein the mammal with malignant
melanoma has non-responsive malignant melanoma.
15. A pharmaceutical composition comprising p-aminobenzoic acid
(PABA) and one or more chemotherapeutic agent(s) selected from the
group consisting of paclitaxel, docetaxel, carboplatin, and a
platinum coordination compound.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/746,206, filed on Dec. 23, 2003 which
claims priority under 35 U.S.C. .sctn.119(e) to U.S. provisional
patent application Ser. No. 60/436,394 filed on Dec. 24, 2002; both
of which are incorporated by reference herein in their
entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to the inhibition of
melanogenesis with para-aminobenzoic acid (PABA) and its use in
treating melanotic cancer.
BACKGROUND OF THE INVENTION
Melanoma
[0004] The incidence of melanoma in most developed countries has
risen faster, over the past 50 years; than any other cancer type.
(Houghton A N, (2002) Cancer Cell; 2:275-278.) Approximately 45,000
new cases of melanoma are diagnosed each year in the United States,
of which about 20% will eventually die secondary to metastatic
disease. (Buzaid, A C. (2002) Crit Rev Ocol Hematol 44:103-108.)
The prognosis for treatment of advanced melanoma is poor, with
patient survival dictated primarily by the pace of progress of the
disease. (Buzaid, supra.) Surgical intervention remains the most
effective treatment option, but only if the disease is diagnosed
and treated in its earliest stages. (Molife R et al. (2002) Crit
Rev Oncol Hematol 44:81-102.) Hence, survival chances are excellent
if melanoma is diagnosed and surgically removed in its earliest
stages. Each successive stage of disease progression, however,
witnesses a significant drop in the chance for survival as the
risks of relapse and recurrence increase. (Molife et al, supra.)
Thus, there is a need for new, more effective methods of treatment
that are distinct from surgical intervention or enhance the
efficacy of surgical intervention.
[0005] Clinical response rates to treatment are typically lower in
patients with melanoma than in patients with other cancers.
Clinical trials have shown malignant melanoma to be highly
resistant to both chemotherapy as well as radiation treatment. A
durable response rate of only about 10% was observed following
current treatment modalities. (Flaherty L E et al. (2002) Semin
Oncol 29:446-455.) An evaluation of biochemotherapy in previously
treated patients documented a 6% response rate. (Chapman, et al.
(2002) Melanoma Res. 12:381-387.)
[0006] The lack of therapeutic response to the existing treatment
protocols for melanoma, is due largely to its cellular,
biochemical, and molecular origins. (Ichihasyhi N et al. (2001) Br
J Dermatol 144:745-750; Heere-Ress et al. (2002) Int J Cancer
99:29-34; Sinha P et al. (2000) Electrophoresis 21:3048-3057.)
Melanomas arise from a very specific cell lineage: they are the
product of the malignant conversion of melanocytes, which are
themselves derived originally from mesenchymal neural crest cells.
In contrast, carcinomas arise from the malignant conversion of
epithelial cells. Furthermore, melanomas are not sex hormone
dependent, while many carcinomas are (e.g., androgen-dependent
prostate cancer and estrogen-dependent breast). Additionally,
melanomas carry out the process of melanogenesis, while carcinomas
exhibit this process rarely, if ever. One or more of the
characteristic properties of melanomas must account for the
resistance of melanomas to existing treatment protocols.
Melanogenesis
[0007] Melanogenesis is the process of synthesizing of melanin,
which is responsible for cell pigmentation. Melanocytes, located in
the skin, hair follicles, stria vascularis of the inner ear and
uveal tract of the eye, are the cells of origin for melanomas and
exhibit melanogenesis. Melanogenesis is a complex biochemical
process initiated by the hydroxylation of the amino acid
L-tyrosine, which results in the formation of
L-dihydroxyphenylalanine (L-DOPA). L-DOPA is converted, in turn, to
Dopachrome by the action of a specific melanocyte-associated
enzyme: tyrosinase. Further oxidation and reduction reactions
ultimately convert Dopachrome to melanin.
[0008] Studies have indicated that melanogenesis is associated with
the enhanced resistance of pigmented melanoma cells to radiation
therapy and to chemotherapy. (Kinnaert E et al. (2000) Radiation
Res 154:497-502; Slominski A et al., (1998) Anti-Cancer Res
18:3709-3716.) These treatments are thus rendered ineffective
against melanotic melanoma. A method to block melanogenesis would
provide a clinically useful approach to render melanoma cells more
sensitive to both chemotherapy and radiotherapy.
[0009] Studies have also shown that many of the intermediate
products produced during melanogenesis have toxic effects.
(Slominski A et al. (1998), supra; Riley P A (1991) Eur J Cancer
27:1172-1177; Prota G el at (1994) Melanoma Res 4:351-358.)
Intermediates of melanogenesis can contribute to, for example,
immunosuppression, fibrosis, and mutagenesis. Inhibition of
melanogenesis will therefore enhance the efficacy of cancer
treatments that require participation of the host's immune system,
e.g., the killing of melanoma cells damaged by radiation or
chemotherapy.
para-Aminobenzoic Acid
[0010] para-Aminobenzoic acid (PABA) has been commonly used in
sunscreens for its capacity to absorb ultraviolet radiation. PABA
has also been used in clinical trials for the treatment of
connective tissue diseases (e.g. scleroderma; dermatomyositis) and
in combination with salicylates for the treatment of rheumatic
fever. U.S. Pat. No. 6,368,598 (the '598 patent) suggested the use
of PABA as a non-essential part of a linking group in a drug
complex for the treatment of prostate cancers. As set forth in the
'598 patent, the function of PABA is to act as a leaving group that
is separated from the cytotoxic therapeutic portion of the drug
complex by the action of enzymes present at the site of the
intended therapeutic action. There is no suggestion, however, that
PABA has any anti-tumor activity or other therapeutic function on
prostate or other types of cancer. According to Holt, PABA can
increase methotrexate levels, activity, and side effects. (Holt G A
(1998) Food & Drug Interactions. Chicago: Precept Press,
170.)
[0011] para-Aminomethylbenzoic acid (PAMBA), a methylated
derivative of PABA, has been found to be useful as a proteinase
inhibitor for reducing the invasiveness of transplantable melanoma
metastases in hamsters (Zbytniewski Z, et al. (1977) Arch
Geschwulstforsch 47:400-404). The action of PAMBA is to inhibit
proteolysis by extracellular proteases, thus preserving the
extracellular matrix as a physical barrier that reduces the
invasiveness of cancer cells. Reducing invasiveness, however, does
not inhibit the growth of an established metastatic tumor. There is
no suggestion therefore that PAMBA inhibits the growth of primary
or metastatic melanoma. Nor is there any suggestion that PAMBA
inhibits melanogenesis, or that it can enhance the effect of
radiation or the activity of chemotherapeutic agents known to be
useful in treating melanoma.
[0012] Accordingly, primary and metastatic melanoma continue to be
difficult to treat with existing therapies. There is therefore a
continued need for new effective treatments for these conditions.
It has now been surprisingly discovered that PABA acts as a potent
inhibitor of melanogenesis and can be used to treat melanoma
effectively when administered alone or in combination with other
anti-cancer modalities such chemotherapy and radiation. This
finding is surprising because melanoma has different cellular
origins from other cancers, including other skin cancers, melanoma
is known to be highly resistant to treatments such as chemotherapy
and radiation, and because the concentrations of PABA that
inhibited melanoma cell growth in vitro and in vivo were found to
have the opposite effect of enhancing growth of a lung
carcinoma.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method of inhibiting
melanogenesis by administering an effective amount of PABA. In
certain embodiments, the invention provides methods of treatment
for primary and metastatic melanotic cancer by administering an
effective amount of PABA. In treating the aforementioned cancers,
PABA may be administered alone as the sole therapeutic agent or, in
combination with one or more additional therapies, such as, for
example radiation therapy or chemotherapy with one or more
chemotherapeutic agents. In one embodiment the invention provides a
method for treating metastatic malignant melanoma by administering
a combination of PABA, carboplatin, and paclitaxel.
[0014] Treatment of metastatic melanotic cancer with PABA may be
achieved by any mechanism, e.g., by preventing the growth of
melanotic cancer cells or by reducing the invasiveness of melanoma
cells, e.g., by proteinase inhibition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the effect of PABA on melanin levels secreted
in DMEM medium in which B16 melanoma cells were cultured alone or
in the presence of PABA. Melanin levels were determined by
absorbance at 405 nm wavelength (A) and absorbance at 660 nm
wavelength (B).
[0016] FIG. 2 shows the inhibition of tyrosinase activity by PABA.
Tyrosinase activity was assessed by measuring the formation of
Dopachrome from L-DOPA using absorbance at 475 nm wavelength.
[0017] FIG. 3 shows the effect of PABA on 1316 melanoma metastasis
in chick lungs. The number of melanoma metastases were measured by
counting the number of metastatic lung tumor lesions present in
lungs from sacrificed chick embryos.
[0018] FIG. 4 shows the effect of PABA on B16 melanoma tumor
growth.
[0019] FIG. 5 shows the effect of PABA and 10 Gy of ionizing
radiation on B16 melanoma cell proliferation. Cell proliferation
was determined by direct cell counts.
[0020] FIG. 6 shows the effect of PABA and 20 Gy of ionizing
radiation on human 1424 melanoma cell proliferation. Cell
proliferation was determined by direct cell counts.
[0021] FIG. 7 shows the effect of PABA and Taxol on B16 melanoma
cell proliferation. Cell proliferation was determined by direct
cell counts.
[0022] FIG. 8 shows the effect of PABA on Lewis Lung Carcinoma cell
proliferation.
[0023] FIG. 9 shows the effect of PABA on Lewis Lung Carcinoma
tumor growth.
[0024] FIG. 10 shows the effect of PABA on the treatment of
melanoma with ionizing radiation in an in vivo study of chick
embryos.
[0025] FIG. 11 shows the effect of PABA and paclitaxel on the
treatment of melanoma in an in vivo xenograft study of nude
(immunodeficient) mice.
[0026] FIG. 12 shows the effect of combination therapy with PABA
and radiation on the treatment of melanoma in Balb/c mice.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention relates to methods for the use of PABA
to inhibit melanogenesis and to treat melanotic cancer in mammals.
In various aspects of the invention, PABA can be administered
alone, in combination with one or more chemotherapeutic agents, or
in combination with radiation therapy. In one embodiment, PABA is
administered in combination with carboplatin and paclitaxel for the
treatment of melanoma in a mammal.
[0028] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used.
[0029] Certain terms are discussed below, or elsewhere in the
specification, to provide additional guidance to the practitioner
in describing the compositions and methods of the invention and how
to make and use them.
[0030] As used herein, "melanotic cancer" encompasses cancers in
which melanin and/or a melanocyte is present. The most common
melanotic cancer is melanoma. Other melanotic cancers include, for
example, melanotic neuroectodermal tumor of infancy, melanotic
malignant peripheral nerve sheath tumor, melanotic medulloblastoma,
melanotic neurilemoma, melanotic schwannoma, meningeal
melanocytoma, and melanotic ependymoma.
[0031] As used herein, an "effective amount" of an agent is an
amount sufficient to ameliorate at least one symptom associated
with a pathological, abnormal or otherwise undesirable condition,
an amount sufficient to prevent or lessen the probability that such
a condition will occur or re-occur, or an amount sufficient to
delay worsening of such a condition.
[0032] As used herein, "melanogenesis" means the process of
synthesis of melanin, including, for example, all enzymatic and
non-enzymatic reactions related to a chemical precursor of melanin,
an intermediate, or a byproduct of the process.
[0033] As used herein, the term "inhibit" means to decrease, limit,
or block the action or function of a process.
[0034] As used herein, the terms "treatment" or "treat" mean the
lessening or ameliorating of at least one abnormal or undesirable
condition associated with melanotic cancer. Treatment may, for
example, cause a reduction in the rate or amount of growth of a
melanotic tumor. Treatment also includes reducing or ameliorating
the undesirable symptoms of melanotic cancer. The foregoing are
merely non-limiting examples of the treatment of melanotic cancer.
Other means and outcomes for treating melanotic cancer are also
encompassed by the invention.
[0035] As used herein, the phrase "a mammal in need of such
treatment" refers to a mammal suffering from at least one abnormal
or undesirable condition or disorder associated with melanin
synthesis or with melanotic cancer.
[0036] The phrase "in combination with" refers to a method of
treatment in which two or more treatments are administered
collectively or according to a specific sequence, such that they
produce a desirable effect.
[0037] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce an allergic or similar toxicity (for example,
gastric upset, dizziness and the like) when administered to an
individual. Preferably, and particularly where a vaccine is used in
humans, the term "pharmaceutically acceptable" may mean approved by
a regulatory agency (for example, the U.S. Food and Drug Agency) or
listed in a generally recognized pharmacopeia for use in animals
(for example, the U.S. Pharmacopeia).
[0038] The phrase "recurrent malignant melanoma" means malignant
melanoma in which the patient's cancer enlarged in size and/or
underwent metastatic spread following completed cancer
treatment.
[0039] The phrase "non-responsive malignant melanoma" means
malignant melanoma in which the patient's cancer enlarged in size
and/or underwent metastatic spread during the period of time when
cancer treatment was in progress.
[0040] PABA is commercially available from, e.g., Sigma-Aldrich
Chemical Co., St. Louis, Mo.
[0041] The biosynthetic pathway of melanogenesis is a complex
process involving the ability of the tyrosinase enzyme to
hydroxylate a number of substrates including L-tyrosine and L-DOPA.
This process ultimately leads to the formation of melanin. In vitro
assays have been developed to measure tyrosinase activity,
including measuring the formation of Dopachrome from L-DOPA in the
presence of tyrosine. (Heidcamp W (1995) Cell Biology Lab Manual,
National Science Foundation.) Both L-tyrosine and L-DOPA have
chemical structures similar to PABA. Et was hypothesized that PABA
may be acting as a competitive substrate for tyrosinase, thus
inhibiting melanogenesis.
[0042] In one aspect of the invention, PABA is used to inhibit
melanogenesis in a mammal, preferably a human. Inhibition may be
obtained, without limitation, by administration of 10 mg/day to 20
g/day of PABA. Preferably, PABA is administered in amounts of 20
mg/day to 12 g/day.
[0043] In another aspect of the invention, PABA is used to treat
melanotic cancer in a mammal, preferably a human. Treatment may
comprise, without limitation, administration of 1.0 mg/day to 20
g/day of PABA. Preferably, PABA is administered in amounts of 20
mg/day to 12 g/day.
[0044] In another aspect of the invention, an effective amount of
PABA is administered in combination with radiation therapy to treat
melanoma. Treatment may comprise, without limitation,
administration of 10 mg/day to 20 g/day of PABA. Preferably, PABA
is administered in amounts of 20 mg/day to 12 g/day. Preferably,
radiation is administered in doses of 1 cGy to 100 Gy. More
preferably, radiation is administered in doses of 2 cGy to 20
Gy.
[0045] In a further aspect of the invention, an effective amount of
PABA is administered in combination with one or more
chemotherapeutic agents known for use in treating melanoma.
Treatment may comprise, without limitation, administration of 10
mg/day to 20 g/day of PABA. Preferably, PABA is administered in
amounts of 20 mg/day to 12 g/day. Also preferably, the
chemotherapeutic agent is selected from the group including
platinum complex, podophyllotoxin, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, catnptothecin, ifosfamide,
melphalan, chlorambucil, bisulfan, nitrosurea, adriamycin,
dactinomycin, daunorubicin HCl, doxorubicin, Doxil (doxorubicin HCl
liposome injection), Ellence (epirubicin hydrochloride), bleomycin,
plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel
(Taxol.RTM.), transplatinum, 5-fluorouracil, vincristin,
vinblastin, bortezomib (VELCADE.TM., formerly known as PS-341),
dicarbizide, a-interferon (Intron A), Genasense G3139 (Bc12
antisense oligonucleotide), Gemzar (gencitabine HCl), Xeloda
(capecitabine: 5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine),
epithalones A and B, oxaliplatin, inhibitors of the EGFR tyrosine
kinase (e.g. OSI-774), C225, Herceptin (trastuzamab), Rituxan
(rituximab), Proleukin (aldesleukin), Photofrin (profimer sodium),
Ontak (denileukin difitox), Novantrone (mitoxantrone
hydrochloride), Nolvadex (tamoxifen citrate), Neupogen
(filgrastim), Mylotart (gemtuzumab ozogamicin), Hycamtin (topotecan
HCl), Glecvec (imatinib mesylate), Femara (letrozole), Fareston
(toremifene citrate), Etopophos (etoposide phosphate), Ethyol
(amifostine), Camptosar (irinotecan HCl), Campath (alemtuzumab),
Busulfex (busulfan), Blenoxane (bleomycin sulfate), Aromasin
(exemestane), Arimidex (anastrozole), Taxotere (docetaxel), Temodar
(temozolomide), and Trisenox (arsenic trioxide).
[0046] More preferably, the one or more chemotherapeutic agents
selected are paclitaxel (Taxol.RTM., available from Bristol-Meyers
Squibb Co., Princeton, N.J.) and/or docetaxel (Taxotere.RTM.,
available from Aventis Pharmaceuticals, Inc., Bridgewater, N.J.).
Paclitaxel stabilizes mictotubules through the binding of tubulin,
which results in arrest of mitosis. In accordance with the
invention, paclitaxel is administered in standard doses well known
to those skilled in the art. Docetaxel binds free microtubules and
results in arrest of mitosis. In accordance with the invention,
docetaxel is administered in standard doses well known to those
skilled in the art.
[0047] Carboplatin (Paraplatin.RTM., available from Bristol-Meyers
Squibb Co., Princeton, N.J.) is a platinum coordination compound
that produces cell cycle non-specific interstrand DNA cross-links.
In accordance with the invention, carboplatin is administered in
standard doses well known to those skilled in the art. (See also
Physician's Desk Reference, 57.sup.th ed. 2003.) One method of
dosing carboplatin according to the invention includes a dose
calculation to meet a target area under the curve (AUC) of
concentration multiplied by time according to the Calvert formula
using an estimated glomerular filtration rate (GFR) derived from
the Jelliffe formula. The Calvert formula is: total dose
(mg)=(target AUC).times.(GFR+25). For the purposes of this dosing
method, GFR is considered the equivalent to creatinine clearance.
Creatinine clearance (Ccr) is estimated by the Jelliffe formula:
Ccr (ml/min)={98-[0.8 (age-20)]})Scr; where age=patient's age in
years from 20-80 and Scr=serum creatinine in mg/dl. For patients
younger than 20, 20 is substituted for the patient's actual age.
For patients older than 80, 80 is substituted for the patient's
actual age.
[0048] In a preferred embodiment, PABA is administered in
combination with carboplatin and paclitaxel. In one embodiment,
PABA is administered at a dose of 2 grams orally for 5 days prior
to the administration of carboplatin and is continued daily for a
total of 10 days. Carboplatin is administered on the sixth day of
PABA administration at a dose according to the Calvert formula with
a target AUC of 5 milligram/milliliter*minute Paclitaxel is
administered at a dose of 100 milligrams/meter.sup.2 intravenously
on the sixth day of PABA treatment. A treatment cycle begins on the
first day of PABA treatment and lasts 21 days. The interval between
treatment cycles is II days unless dose limiting toxicity ("DLT")
occurs. Dose limiting toxicity includes, for example, hematologic
toxicity, nausea/vomiting, mucositis, arthralgias and myalgias
peripheral neuropathy, and liver function test abnormalities.
[0049] In another embodiment according to the invention, the
combination of PABA, carboplatin, and paclitaxel is administered in
a regimen that includes escalating doses of paclitaxel. In one such
embodiment, treatment is initiated with PABA at a dose of 2 grams
orally for 5 days prior to the administration of carboplatin and is
continued daily for a total of 10 days. Carboplatin is administered
on the sixth day of PABA administration at a dose calculated
according to the Calvert formula with a target AUC of 5
milligram/milliliter*minute. A treatment cycle lasts 21 days and
begins on the first day of PABA treatment. Following a treatment
cycle, and in the absence of DLT, a second cycle is initiated with
the administration of PABA at a dose of 2 grams orally for 5 days
prior to the administration of carboplatin and is continued daily
for a total of 10 days. Carboplatin is administered on the sixth
day of PABA administration at a dose calculated according to the
Calvert formula with a target AUC of 5 milligram/milliliter*minute.
Paclitaxel is administered at a dose of 100 milligrams/meter.sup.2
intravenously on the sixth day of PABA administration. If the
patient tolerates this treatment cycle without DLT, a third
treatment cycle is initiated 11 days later with the administration
of PABA at a dose of 2 grams orally for 5 days prior to the
administration of carboplatin and is continued daily for a total of
10 days. Carboplatin is administered on the sixth day of PABA at a
dose calculated according to the Calvert formula with a target AUC
of 5 milligram/milliliter*minute. Paclitaxel is administered at a
dose of 125 milligrams/meter.sup.2 intravenously on the sixth day
of PABA treatment. If the patient tolerates this treatment cycle
without DLT, a fourth treatment cycle is initiated 11 days later
with the administration of PABA at a dose of 2 grams orally for 5
days prior to the administration of carboplatin and is continued
daily for a total of 10 days. Carboplatin is administered on the
sixth day of PABA treatment at a dose calculated according to the
Calvert formula with a target AUC of 5 milligram/milliliter*minute.
Paclitaxel is administered at a dose of 150 milligrams/meter.sup.2
intravenously on the sixth day of PABA treatment. If the patient
tolerates this treatment cycle without DLT, a fifth treatment cycle
is initiated 11 days later with the administration of PABA at a
dose of 2 grams orally for 5 days prior to the administration of
carboplatin and is continued daily for a total of 10 days.
Carboplatin is administered on the sixth day of PABA administration
at a dose calculated according to the Calvert formula with a target
AUC of 5 milligram/milliliter*minute. Paclitaxel is started at a
dose of 175 milligrams/meter.sup.2 intravenously on the sixth day
of PABA administration.
Pharmaceutical Compositions
[0050] For administration to patients according to the method of
the present invention, PADA may be formulated into a pharmaceutical
composition. The pharmaceutical composition may include additives,
such as a pharmaceutically acceptable carrier or diluent, a
flavorant, a sweetener, a preservative, a dye, a binder, a
suspending agent, a dispersing agent, a colorant, a disintegrant,
an excipient, a film forming agent, a lubricant, a plasticizer, an
edible oil or any combination of two or more of the foregoing.
[0051] Suitable pharmaceutically acceptable carriers or diluents
include, but are not limited to, ethanol; water; glycerol;
propylene glycol, aloe vera gel; allantoin; glycerin; vitamin A and
E oils; mineral oil; PPG2 myristyl propionate; magnesium carbonate;
potassium phosphate; vegetable oil; animal oil; and solketal.
[0052] Suitable binders include, but are not limited to, starch;
gelatin; natural sugars, such as glucose, sucrose and lactose; corn
sweeteners; natural and synthetic gums, such as acacia, tragacanth,
vegetable gum, and sodium alginate; carboxymethylcellulose;
hydroxypropylmethylcellulose; polyethylene glycol; povidone; waxes;
and the like.
[0053] Suitable disintegrants include, but are not limited to,
starch, e.g., corn starch, methyl cellulose, agar, bentonite,
xanthan gum, sodium starch glycolate, crosspovidone and the
like.
[0054] Suitable lubricants include, but are not limited to, sodium
oleate, sodium stearate, sodium stearyl fumarate, magnesium
stearate, sodium benzoate, sodium acetate, sodium chloride and the
like.
[0055] A suitable suspending agent is, but is not limited to,
bentonite, ethoxylated isostearyl alcohols, polyoxyethylene
sorbitol and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, agar-agar and tragacanth, or mixtures of two or more
of these substances, and the like.
[0056] Suitable dispersing and suspending agents include, but are
not limited to, synthetic and natural gums, such as vegetable gum,
tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone and
gelatin.
[0057] Suitable film forming agents include, but are not limited
to, hydroxypropylmethylcellulose, ethylcellulose and
polymethacrylates.
[0058] Suitable plasticizers include, but are not limited to,
polyethylene glycols of different molecular weights (e.g., 200-8000
Da) and propylene glycol.
[0059] Suitable colorants include, but are not limited to, ferric
oxide(s), titanium dioxide and natural and synthetic lakes.
[0060] Suitable edible oils include, but are not limited to,
cottonseed oil, sesame oil, coconut oil and peanut oil.
[0061] Examples of additional additives include, but are not
limited to, sorbitol, talc, stearic acid, dicalcium phosphate and
polydextrose.
Unit Dosage Forms
[0062] The pharmaceutical composition may be formulated as unit
dosage forms, such as tablets, pills, hard or soft shell capsules,
caplets, boluses, powders, granules, sterile parenteral solutions,
sterile parenteral suspensions, sterile parenteral emulsions,
elixirs, tinctures, metered aerosol or liquid sprays, drops,
ampoules, autoinjector devices or suppositories. Unit dosage forms
may be used for oral, parenteral, intranasal, sublingual or rectal
administration, or for administration by inhalation or
insufflation, transdermal patches, and a lyophilized composition.
In general, any delivery of active ingredients that results in
systemic availability of such ingredients can be used in practicing
the present invention. Preferably the unit dosage form is an oral
dosage form, most preferably a solid oral dosage form, therefore
the preferred dosage forms are tablets, pills, caplets and
capsules. Parenteral preparations (e.g., injectable preparations in
saline and preparations for powder jet systems) comprise another
embodiment of the invention.
[0063] Solid unit dosage forms may be prepared by mixing an active
agent of the present invention with a pharmaceutically acceptable
carrier and any other desired additives as described above. The
mixture is typically mixed until a homogeneous mixture of the
active agents of the present invention, the carrier and any other
desired additives is formed, i.e., until the active agent is
dispersed evenly throughout the composition. In this case, the
compositions can be formed as dry or moist granules.
[0064] Dosage forms with predetermined amounts of PABA may be
formulated starting with compositions with known quantities of PABA
using methods well known in the art. In a preferred embodiment a
dosage form is obtained by mixing compositions comprising known
quantities of PABA.
[0065] Dosage forms can be formulated as, for example, "immediate
release" dosage forms. "Immediate release" dosage forms are
typically formulated as tablets that release at least 70%-90% of
the active ingredient within 30-60 min when tested in a drug
dissolution test, e.g., U.S. Pharmacopeia standard <711>. In
a preferred embodiment, immediate dosage forms release 75% of the
active ingredients in 45 min.
[0066] Dosage forms can also be formulated as, for example,
"controlled release" dosage forms. "Controlled," "sustained,"
"extended" or "time release" dosage forms are equivalent terms that
describe the type of active agent delivery that occurs when the
active agent is released from a delivery vehicle at an
ascertainable and modifiable rate over a period of time, which is
generally on the order of minutes, hours or days, typically ranging
from about sixty minutes to about 3 days, rather than being
dispersed immediately upon entry into the digestive tract or upon
contact with gastric fluid. A controlled release rate can vary as a
function of a multiplicity of factors. Factors influencing the rate
of delivery in controlled release include the particle size,
composition, porosity, charge structure, and degree of hydration of
the delivery vehicle and the active ingredient(s), the acidity of
the environment (either internal or external to the delivery
vehicle), and the solubility of the active agent in the
physiological environment, i.e., the particular location along the
digestive tract. Typical parameters for dissolution test of
controlled release forms are found in U.S. Pharmacopeia standard
<724>.
[0067] Dosage forms can also be formulated to deliver active agent
in multiphasic stages whereby a first fraction of an active
ingredient is released at a first rate and at least a second
fraction of active ingredient is released at a second rate. In a
preferred embodiment, a dosage form can be formulated to deliver
active agent in a biphasic manner, comprising a first "immediate
release phase", wherein a fraction of active ingredient is
delivered at a rate set forth above for immediate release dosage
forms, and a second "controlled release phase," wherein the
remainder of the active ingredient is released in a controlled
release manner, as set forth above for controlled release dosage
forms.
[0068] Tablets or pills can be coated or otherwise compounded to
form a unit dosage form which has delayed and/or prolonged action,
such as time release and controlled release unit dosage forms. For
example, the tablet or pill can comprise an inner dosage and an
outer dosage component, the latter being in the form of a layer or
envelope over the former. The two components can be separated by an
enteric layer which serves to resist disintegration in the stomach
and permits the inner component to pass intact into the duodenum or
to be delayed in release.
[0069] Biodegradable polymers for controlling the release of the
active agents, include, but are not limited to, polylactic acid,
polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydro-pyrans,
polycyanoacrylates and cross-linked or amphipathic block copolymers
of hydrogels.
[0070] For liquid dosage forms, the active substances or their
physiologically acceptable salts are brought into solution,
suspension or emulsion, optionally with the usually employed
substances such as solubilizers, emulsifiers or other auxiliaries.
Solvents for the active combinations and the corresponding
physiologically acceptable salts, can include water, physiological
salt solutions or alcohols, e.g. ethanol, propane-diol or glycerol.
Additionally, sugar solutions such as glucose or mannitol solutions
may be used. A mixture of the various solvents mentioned may
further be used in the present invention.
[0071] A transdermal dosage form also is contemplated by the
present invention. Transdermal forms may be a diffusion-driven
transdermal system (transdermal patch) using either a fluid
reservoir or a drug-in-adhesive matrix system. Other transdermal
dosage forms include, but are not limited to, topical gels,
lotions, ointments, transmucosal systems and devices, and
iontohoretic (electrical diffusion) delivery system. Transdermal
dosage forms may be used for timed release and controlled release
of the active agents of the present invention.
[0072] Pharmaceutical compositions and unit dosage forms of the
present invention for administration parenterally, and in
particular by injection, typically include a pharmaceutically
acceptable carrier, as described above. A preferred liquid carrier
is vegetable oil. Examples of solid carriers are lactose, terra
alba, sucrose, cyclodextrin, talc, agar, pectin, acacia, stearic
acid and lower alkyl ethers of cellulose corn starch, potato
starch, talcum, magnesium stearate, gelatine, lactose, gums, and
the like. Injection may be, for example, intra-tumoral,
intravenous, intrathecal, intramuscular, intratracheal, or
subcutaneous. Intravenous injection is preferred.
[0073] The active agent also can be administered in the form of
liposome delivery systems, such as small unilamellar vesicles,
large unilamellar vesicles and multilamellar vesicles. Liposomes
can be formed from a variety of phospholipids, such as cholesterol,
stearylamine or phosphatidylcholines.
[0074] The pharmaceutical compositions of the present invention
also may be coupled with soluble polymers as targetable drug
carriers. Such polymers include, but are not limited to,
polyvinyl-pyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamidephenol,
polyhydroxy-ethylaspartamidephenol, and
polyethyl-eneoxideopolylysine substituted with palmitoyl
residues.
Administration
[0075] The pharmaceutical composition or unit dosage forms of the
present invention may be administered by a variety of routes such
as intravenous, intratracheal, subcutaneous, oral, intratumoral,
mucosal parenteral, buccal, sublingual, ophthalmic, pulmonary,
transmucosal, transdermal, and intramuscular. Unit dosage forms
also can be administered in intranasal form via topical use of
suitable intranasal vehicles, or via transdermal routes, using
those forms of transdermal skin patches known to those of ordinary
skill in the art. Oral administration is preferred. Also preferred
is administration by local intratumoral injection.
[0076] The pharmaceutical composition or unit dosage forms of the
present invention may be administered to a mammal, preferably a
human being, in need of cancer treatment. The pharmaceutical
composition or unit dosage form of the present invention may be
administered according to a dosage and administration regimen
defined by routine testing in light of the guidelines given above
in order to obtain optimal activity while minimizing toxicity or
side-effects for a particular patient. However, such fine turning
of the therapeutic regimen is routine in light of the guidelines
disclosed in this specification.
[0077] The dosage of the composition of the present invention may
vary according to a variety of factors such as the underlying
disease state, the individual's condition, weight, sex and age and
the mode of administration. For oral administration, the
pharmaceutical compositions can be provided in the form of scored
or unscored solid unit dosage forms.
[0078] The pharmaceutical composition or unit dosage form may be
administered in a single daily dose, or the total daily dosage may
be administered in a plurality of divided doses. In addition,
co-administration or sequential administration of other active
agents may be desirable. The pharmaceutical composition of the
invention may be combined with any known drug therapy, preferably
for the treatment of cancer.
[0079] For combination therapy, the pharmaceutical PABA composition
of the present invention and the other active agent(s) (e.g.,
chemotherapeutic agent(s)) may initially be provided as separate
dosage forms until an optimum dosage combination and administration
regimen is achieved. Therefore, the patient may be titrated to the
appropriate dosages for his/her particular condition. After the
appropriate dosage of each of the compounds is determined to
achieve the desired effect without untoward side effects, the
patient then may be switched to a single dosage form containing the
appropriate dosages of the pharmaceutical PABA composition and the
other active agent(s), or may continue with a dual (or multi)
dosage form.
[0080] The exact dosage and administration regimen utilizing the
combination therapy of the present invention is selected in
accordance with a variety of factors including type, species, age,
weight, sex and medical condition of the patient; the route of
administration; the renal and hepatic function of the patient; the
treatment history of the patient; and the responsiveness of the
patient. Optimal precision in achieving concentrations of compounds
within the range that yields efficacy without toxicity requires a
regimen based on the kinetics of the drug's availability to target
sites. This involves a consideration of the absorption,
distribution, metabolism, excretion of a drug, and responsiveness
of the patient to the dosage regimen. However, such fine tuning of
the therapeutic regimen is routine in light of the guidelines
disclosed in this specification.
EXAMPLES
Example 1
PABA Suppresses Secretion of Melanin in B16 Melanoma Cells Cultured
in Vitro
[0081] Growth of melanotic B16 melanoma cells (Engbring J A et al.
(2002) Cancer Res 62:3549-3554) in DMEM medium (available from,
e.g. Life Technologies, Inc., Rockville Md.) resulted in a change
of the color of the medium from red to brown due to the
accumulation of secreted melanin. Growth of melanotic B16 melanoma
cells in RPMI medium (available from, e.g. Life Technologies, Inc.,
Rockville Md.) did not result in a color change. The RPMI medium
remained red due to the inhibition of melanin secretion. One of the
major components in RPMI that is absent in DMEM is PABA. To assess
the effect of PABA on melanin secretion from B16 cells, PABA was
added to DMEM culture medium to achieve the same concentration as
that found in the RPMI medium: 0.1 mg of PABA per milliliter of
medium. Cells were grown in PABA-supplemented DMEM medium for seven
days. The PABA-supplemented medium remained red, while the
unsupplemented medium turned brown. These results suggest that PABA
inhibits the formation and/or secretion of melanin.
Example 2
Time and Concentration Dependent Inhibition of Melanin Synthesis
and Secretion
[0082] To further analyze the effects of PABA on B16 melanoma cell
melanin secretion, PABA was added at concentrations of 0.5 mg/day
and 1.0 mg/day to B16 melanoma cells cultured in DMEM. Samples of
the culture medium were then analyzed for melanin content by
measuring absorbance at wavelengths of 405 nm and 660 nm.
(Kowalczuk C et al. (2001) Inter J Rad Biol 77:883-890.) The
addition of PABA caused a time dependent and concentration
dependent inhibition of melanin secretion (FIGS. 1A and 1B).
Example 3
PABA Suppresses Intracellular Synthesis of Melanin within B16
Melanoma Cells
[0083] To determine whether PABA inhibits the intracellular
synthesis of melanin in B16 cells, cells were grown in DMEM in the
presence or absence of 0.1 mg/ml PABA. Following three weeks of
culture, cells were collected by centrifugation. Cell pellets from
cells cultured in the absence of PABA appeared black, as normal,
indicating the presence of melanin. B16 melanoma cells cultured in
the presence of PABA appeared much lighter in color, indicating a
reduction in melanin accumulation. These results indicate that PABA
inhibits melanin synthesis and/or accumulation within melanoma
cells.
Example 4
PARA Inhibits Tyrosinase Activity Dose Dependently
[0084] The reduction in cell-associated melanin observed in cells
treated with PABA suggested that PABA inhibits a step in the
biosynthetic pathway of melanin synthesis. The effects of PABA on
tyrosinase activity were evaluated by measuring the formation of
Dopachrome using a previously published method. (Heidcamp W (1995),
supra.) L-DOPA (8.0 mM) was resuspended in sodium citrate buffer
and 800 U of purified tyrosinase was added. Tyrosinase activity was
measured by monitoring the formation of Dopachrome by measuring the
optical density of the mixture at a wavelength of 475 nm. To assess
the effects of PABA, the reactions were performed in the presence
or absence of PABA. As a control, reactions were performed in the
presence of adenosine instead of PABA. Results are shown in FIG. 2.
Addition of PABA led to a dose dependent inhibition of Dopachrome
formation. These results indicate that PABA is a potent inhibitor
of tyrosinase and suggests that inhibition of tyrosinase is the
mechanism by which PABA inhibits melanin accumulation in cells.
Example 5
PABA Inhibits 1316 Melanoma Metastasis in the Chick Embryo
Model
[0085] The effect of PABA on the aggressiveness and invasiveness of
B16 melanoma was measured in a chick embryo model. (Brooks P C et
al. (1994) Cell 79: 1157-1164.) The effects of PABA on B16
experimental metastasis in vivo were evaluated to determine the
role melanin synthesis plays in the aggressiveness and invasiveness
of melanoma metastasis. B16 melanoma cells grown in the presence or
absence of 0.1 mg/ml PABA for one to three weeks were injected into
12-day old chick embryos. At the end of a 7-day incubation period,
the embryos were sacrificed and the number of metastatic lung tumor
lesions were quantified. A time dependent inhibition of B16
melanoma metastasis was observed for B16 melanoma cells grown in
the presence of PABA (FIG. 3). These results indicate that PABA
inhibits the growth of non-primary melanomas in vivo.
Example 6
Effect of PABA on B16 Melanoma Tumor Growth in the Chick Embryo
[0086] The effect of PABA on solid tumor growth in vivo were
evaluated using the chick embryo tumor growth assay. (Petitclerc E
et al. (2000). J Biol Chem 275:8051-8061.) B16 melanoma cells were
grown in the presence or absence of 0.1 mg/ml PABA for 6 weeks.
Cells were harvested and 2.5.times.10.sup.5 cells from each group
were inoculated in the chorioallantoic membranes (CAM) of 10-day
old chick embryos. The embryos were allowed to develop for a
further seven days. At the end of the 7-day incubation period, the
embryos were sacrificed and the tumors removed and wet weights
determined. Tumors formed from B16 melanoma cells treated with PABA
were, on average, 60% smaller than tumors derived from untreated
B16 melanoma cells (FIG. 4). These results indicate that treatment
of B16 melanoma cells with PABA inhibits tumor growth in vivo.
Example 7
PABA Enhances the Anti-Proliferative Effects of Radiation on B16
Melanoma Cells
[0087] Previous studies have suggested that the amount of
intracellular melanin present in a melanoma cell is inversely
related to the radiosensitivity of that cell. (Kinnaert E et al.
(2000) Radiation Res 154:497-502.) Thus, blocking melanin synthesis
or melanogenesis may cause melanoma cells to become much more
sensitive to radiation therapy. To test the effect of PABA on the
anti-proliferative effects of ionizing radiation, B16 melanoma
cells grown in the presence or absence of 0.1 mg/ml PABA were
treated with a single fraction dose of 10 Gy of ionizing radiation.
Cell proliferation was monitored by direct cell counts over a 3-day
incubation period. Results showed that PABA treatment increased the
anti-proliferative and cytotoxic effects of a single fraction dose
of radiation, compared with cells not receiving PABA (FIG. 5).
Example 8
PABA Enhances the Anti-Proliferative Effects of Radiation on Human
1424 Melanoma Cells
[0088] The radiation-enhancing effect of PABA on melanoma observed
in B16 melanoma cells was confirmed in the pigmented human melanoma
cell line G-361 (ATCC Number CRL-1424, (1978) Pediatr Res 12:485.)
The human G-361 melanoma cells are more resistant to radiation than
B16 melanoma cells and thus require a higher dose of ionizing
radiation to inhibit growth, compared to B16 cells. Human G-361
melanoma cells were treated with 20 Gy of radiation, either alone
or in combination with 0.1 mg/ml PABA, as described in example 7. A
single dose of ionizing radiation significantly inhibited the
proliferation of human G-361 melanoma cells (FIG. 6). In
comparison, the combination of PABA and radiation essentially
completely inhibited the proliferation of human G-361 melanoma
cells (FIG. 6). These results confirm that the combination of PABA
and radiation has enhanced anti-proliferative effects, compared to
radiation alone.
Example 9
PABA Enhances the Anti-Proliferative Effects of Taxol on 1316
Melanoma Cells
[0089] The ability of PABA to enhance the anti-proliferative effect
of the chemotherapeutic agent, Taxol, was tested in B16 melanoma
cells. Cells were grown in DMEM medium in the presence or absence
of 0.1 mg/ml PABA and/or 10.0 .mu.M paclitaxel (Taxol). Cell
proliferation was monitored by direct cell counts over a 48-hour
incubation period. Paclitaxel significantly inhibited B16 melanoma
cell proliferation, as compared to no treatment (FIG. 7). Combined
treatments of PABA and Taxol showed an enhanced anti-proliferative
effect on cells, compared to Taxol alone (FIG. 7). These results
indicate that PABA enhances the anti-tumor activity of Taxol.
Example 10
Effect of PABA on the Proliferation of Lewis Lung Carcinoma
Cells
[0090] The anti-proliferative effect of PABA on carcinoma cells was
tested on Lewis Lung Carcinoma (LLC) cells. (Young M R et al.
(2003) Int J Cancer 103:38-44.) Cells were grown in DMEM medium in
the presence or absence of 0.1 mg/ml PABA. Cell proliferation was
monitored by direct cell counts over a 48-hour incubation period.
The addition of PABA increased the proliferation of LLC cells (FIG.
8). In contrast to the effect of PABA on melanoma cells, these
results indicate that PABA enhances, rather than inhibits, the
proliferation of carcinoma cells.
Example 11
Effect of PABA on Lewis Lung Carcinoma Tumor Growth
[0091] The effect of PABA on the growth of carcinoma tumors was
tested using the Lewis Lung Carcinoma (LLC) tumor growth assay.
(Mauceri H J et al. (2002) Cancer Chemother Pharmacol 50:412-418.)
LLC cells were grown in DMEM medium in the presence or absence of
0.1 mg/ml PABA for 3 weeks. Cells were harvested and
2.times.10.sup.6 cells from each group were inoculated in the CAMs
of 10-day old chick embryos. The embryos were allowed to develop
for a further seven days. At the end of the 7-day incubation
period, the embryos were sacrificed and the tumors removed and wet
weights determined. Tumors formed from LLC cells treated with PABA
were, on average, significantly larger than tumors derived from
untreated LLC cells (FIG. 9). In contrast to the effect of PABA on
melanoma tumors, these results indicate that treatment of LLC cells
with PABA enhances, rather than inhibits, carcinoma tumor
growth.
Example 12
In Vivo Effects of PABA on the Treatment of Melanoma with Ionizing
Radiation
[0092] The chick embryo tumor growth assay (Petitclerc E et al.
(2000) J Biol Chem 275:8051-8061) was used to determine whether
PABA enhances the effect of ionizing radiation to inhibit tumor
growth in vivo. Four groups of chick embryos were studied. The
control group did not receive any treatment, a second group
received PABA alone, a third group received ionizing radiation
alone, and a fourth group received PABA and ionizing radiation.
There were 5 to 10 chick embryos in each group.
[0093] The groups were created in the following manner. B16F10
melanoma cells were cultured for 14 days in growth medium in the
absence of PABA or in the presence of PABA at a concentration of
100 .mu.g/ml. The cells were harvested, washed, and resuspended in
sterile PBS. The B16F10 melanoma cells were implanted on the CAMs
of 10-day-old chick embryos, which were then incubated for at least
24 hours. The incubated chick embryos (some that were cultured in
the absence of PABA and some that cultured in the presence of PABA)
were treated with a single fraction dose of ionizing radiation (5.0
Gy). The embryos were incubated for 7 days and then the chick
embryos of each group were sacrificed. The tumors were resected and
tumor growth was assessed by measuring the wet weights of the
resected tumors.
[0094] As shown in FIG. 10, the group that received PABA alone and
the group that received radiation alone showed about 75% inhibition
of tumor growth as compared to the control group. The group that
received both PABA and radiation showed about 90% inhibition of
tumor growth as compared to the control group.
[0095] This example shows that PABA alone and PABA in combination
with radiation therapy result in significant inhibition of melanoma
tumor growth in an in vivo model.
Example 13
In Vivo Xenograft Study of PABA and Paclitaxel for the Treatment of
Melanoma
[0096] 1.times.10.sup.6 B16F10 melanoma cells (cultured in the
absence of PABA) were implanted subcutaneously into Balb/c nude
mice. Intraperitoneal injections of PABA at a concentration of 50
mg/kg were started 3 days after implantation of the tumor cells and
continued daily. Starting on day 4 post-implantation, some
PABA-injected mice and some non-treated mice received
intraperitoneal injections of paclitaxel at a concentration of 20
mg/kg, which was continued every other day of the 16 day study. The
control group did not receive PABA or paclitaxel. Every 4 days,
groups of 10 animals were used for tumor growth assessments
starting at day 8.
[0097] At day 12, reduced tumor growth was observed for both the
paclitaxel and the paclitaxel plus PABA groups as compared to the
control group (FIG. 11). The combination of paclitaxel and PABA
showed significant tumor growth retardation with a mean tumor
volume of 613.+-.282 mm.sup.3. Paclitaxel alone resulted a mean
tumor volume of 1097.+-.612 mm.sup.3. The mean tumor volume for the
control group exceeded 1500 mm.sup.3. At day 16, the PABA plus
paclitaxel combination group was significantly different form the
control group (p=0.016) and the paclitaxel alone group (p=0.045)
(Student's t-test for unpaired data).
Example 14
In Vivo Xenograft Study of PABA and Radiation for the Treatment of
Melanoma
[0098] 1.times.10.sup.6 B16F10 melanoma cells (cultured in the
absence of PABA) were implanted subcutaneously into Balb/c nude
mice. Intraperitoneal injections of PABA at a concentration of 50
mg/kg were started 3 days after implantation of the tumor cells and
continued daily. Ten days following tumor cell implantation, mice
were either not irradiated or were irradiated with 3 fractions of 3
Gy every other day for a total dose of 9 Gy. The control group did
not receive PABA or radiation.
[0099] After 15 days, the animals were sacrificed and the tumors
were resected. The tumors were measured and their volumes were
calculated. As shown in FIG. 12, PABA alone had a minimal effect on
tumor growth as compared to control (P>0.100), fractionated
doses of ionizing radiation alone inhibited tumor growth by about
60% as compared to control (P<0.04), and a combination of PABA
and fractionated ionizing radiation inhibited tumor growth by about
95% as compared to control (P<0.002).
[0100] This example demonstrates that PABA enhances the tumor
growth-inhibiting effect of ionizing radiation on melanoma.
Example 15
Treatment of a Patient with Metastatic Malignant Melanoma with a
Combination of Carboplatin, Paclitaxel, and PABA
[0101] A 56 year old woman was diagnosed with malignant melanoma in
1998. A wide surgical excision of her left foot malignant melanoma
was performed along with a regional lymph node dissection. The
sentinel lymph node (the first lymph node to receive lymphatic
drainage from the area of the malignant melanoma) was positive for
malignant cells on microscopic examination, but the remaining lymph
nodes were negative for malignant cells. The patient declined
adjuvant treatment at that time in 1999, the patient developed a
malignant left thigh nodule, which was excised. A work-up for
metastatic disease was negative. The patient agreed to adjuvant
treatment with interferon. During the sixth month of interferon
treatment, the patient developed two malignant subcutaneous left
thigh nodules. A six-month treatment with Temodar and thalidomide
was undertaken. Following one disease-free year, multiple liver
lesions were visualized on routine follow-up CT scans. The patient
was restarted on Temodar but her cancer continued to progress.
[0102] The patient's treatment was changed to carboplatin. During
the second cycle of carboplatin, the patient's colon perforated and
an emergency colostomy was performed. Post-operative CT scans in
December 2002 showed extensive liver metastases, peritoneal
implants (indicative of metastatic lesions on the peritoneum), and
bulky retroperitoneal and pelvic lymph nodes (indicative of
metastatic spread to these lymph nodes).
[0103] Combination chemotherapy with carboplatin, paclitaxel, and
PABA was started. PABA was administered at a dose of 2 grams orally
for a total of ten days. On the sixth day of PABA administration,
carboplatin was administered at a dose calculated according to the
Calvert formula with a target AUC of 5 mg/ml*min. Paclitaxel was
administered on the sixth day of PABA administration at a dose of
100 mg/m.sup.2 intravenously. A treatment cycle was completed 21
days after the first administration of PABA. Following an 11 day
interval between treatment cycles, another identical treatment
cycle was started. A CT scan was performed following three
treatment cycles. The patient showed significant clinical
improvement and she returned to work in February 2003. CT scans
performed in March 2003 showed complete resolution of the liver
lesions and a greater than 50% reduction in the intra-abdominal
disease.
[0104] In August 2003, the patient decided to have her colostomy
reversed (an operation in which the continuity of the colon is
restored and the colostomy is closed). The carboplatin, paclitaxel,
and PABA combination therapy was stopped for 8 weeks. CT scans
prior to surgery showed no change compared to the March 2003 CT
scans.
[0105] This example demonstrates that combination treatment with
carboplatin, paclitaxel, and PABA is effective against metastatic
malignant melanoma in a patient whose melanoma both re-occurred
following completed chemotherapy and progressed while she on
chemotherapy.
[0106] The present invention is not to be limited in scope by the
specific embodiments described herein. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
[0107] The complete disclosure of all patents, patent applications,
publications, procedures, and the like cited throughout this
application, are incorporated herein by reference in their
entireties. In the case of inconsistencies in definitions, the
present application is controlling.
* * * * *