Patent Application
20180271960
The present invention relates to a method to treat cancer using an arginine depletor and an ornithine decarboxylase (ODC) inhibitor.
The hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.
Lung cancer is one of the most lethal cancers worldwide. Different drugs have been developed to treat lung cancer and some of the anti-cancer drugs may not be readily useful as a remedy to lung cancers.
In view of the demand for effectively treating lung cancers, improvements in method and compositions that treat lung cancers are desired.
One example embodiment is a method of treating lung carcinoma in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of an arginine reducing compound and a therapeutically effective amount of an ornithine decarboxylase (ODC) inhibitor to provide a combination therapy that has a synergistic therapeutic effect compared to an effect of the arginine reducing compound and an effect of the ODC inhibitor, in which each of the arginine reducing compound and the ODC inhibitor is administered alone.
Other example embodiments are discussed herein.
Example embodiments relate to methods and pharmaceutical composition that treat lung cancers.
Arginine, a semi-essential amino acid, is involved in many metabolic processes and is also important for growth of some cancer cells. Arginine depletion plays a useful role in the treatment of some cancers, but may not be proficient in treating other types of cancer. In fact, administration of arginase may even cause proliferation in some tumor cells.
The present inventors have determined that when arginase converts arginine into ornithine, certain types of cancer cells would upregulate ornithine decarboxylase (ODC). ODC then converts ornithine into polyamines, increasing the capability of these cancer cells to proliferate, invade and metastasize to new tissues.
The present inventors have further determined that even in ODC negative cells, the administration of arginase (and hence resulting in the increase of ornithine) may result in upregulation of ODC, inducing them to become ODC positive in certain cancer cells to result in increased polyamines.
In one example embodiment, the lung carcinoma is lung adenocarcinoma. In another example embodiment, the arginine reducing compound is a pegylated recombinant human arginase. By way of example, the recombinant human arginase has an amino acid sequence of SEQ ID NO:1.
In another example embodiment, the ODC inhibitor is difluoromethylornithine (DFMO). In yet another example embodiment, the arginine reducing compound and the ODC inhibitor are administered concurrently.
In an example embodiment, the cancer cells of the lung carcinoma are ODC positive. In one example embodiment, the cancer cells of the lung carcinoma are ODC negative. In another example embodiment, the cancer cells of the lung carcinoma are argininosuccinate synthase negative (ASS1−) or ornithine transcarbamylase negative (OTC−).
One example embodiment is therefore to treat lung cancer by blocking ODC in cancer cells in addition to depleting arginine by an administration of arginine depleting compound. A therapeutically effective amount of the arginine depleting compound and a therapeutically effective amount of an ODC blocking agent are administered to the subject, where the administration provides a synergistic therapeutic effect compared to an effect in treating lung cancer of the arginine depleting compound and an effect in treating lung cancer of the ODC blocking agent, in which each of the arginine depleting compound and the ODC blocking agent is administered alone.
In one example embodiment, the lung cancer is lung adenocarcinoma.
In another example embodiment, the arginine depleting compound is a pegylated recombinant human arginase. By way of example, the recombinant human arginase has an amino acid sequence of SEQ ID NO:1.
In another example embodiment, the ODC blocking agent is DFMO.
In an example embodiment, these cancer cells may be either ODC negative or ODC positive. In another example embodiment, these cancer cells are argininosuccinate synthase negative or ornithine transcarbamylase negative.
One example embodiment relates to a method for inhibiting proliferation of cancer cells of lung adenocarcinoma. The method includes contacting the cancer cells with an arginine depleting compound in combination with an ornithine decarboxylase (ODC) inhibitor. The combination provides a synergistic anti-cancerous effect compared to an effect of the arginine depleting compound and an effect of the ODC inhibitor, each administered alone
In an example embodiment, the arginine depleting compound is a pegylated recombinant human arginase. By way of example, the recombinant human arginase has an amino acid sequence of SEQ ID NO:1.
In another example embodiment, the ODC inhibitor is DFMO. In one example embodiment, the arginine reducing depleting compound and the ODC inhibitor are administered concurrently.
One example embodiment relates to a pharmaceutical composition for use in a synergistic treatment of lung cancer. The pharmaceutical composition includes an arginine depleting compound and an inhibitor of ODC.
In an example embodiment, the lung cancer is lung adenocarcinoma.
In another example embodiment, the arginine depleting compound is a pegylated recombinant human arginase. By way of example, the recombinant human arginase has an amino acid sequence of SEQ ID NO:1.
In one example embodiment, the inhibitor of ODC is DFMO.
In another example embodiment, an amount of the arginine depleting compound and an amount of the inhibitor of ODC are effective for therapy in a subject, and the subject is a human.
One example embodiment studies in vitro characterization of argininosuccinate synthase (ASS1) and ornithine transcarbamylase (OTC) expression in lung adenocarcinomas.
In one example embodiment, eight lung adenocarcinoma cell lines (i.e. H23, H358, HCC827, H1650, H1975, HCC2935, HCC4006, and A549), obtained from American Type Culture Collection (ATCC), are assessed for ornithine transcarbamylase (OTC) and argininosuccinate synthase (ASS1) protein expression by immunocytochemistry and Western blot. The level of expression is determined by normalizing against housekeeping protein (β-actin) in Western blot. Results of this study are presented in
As illustrated in
One example embodiment studies in vitro efficacy of PEG-BCT-100 and arginine deiminase (ADI) against lung adenocarcinoma.
In one example embodiment, eight lung adenocarcinoma cell lines (i.e. H23, H358, HCC827, H1650, H1975, HCC2935, HCC4006, and A549), obtained from ATCC, are used to assess the in vitro efficacies of PEG-BCT-100 and ADI. The inhibition ratio (IR) and the half maximal inhibitory concentration (IC50) values are determined by a MTT assay. Results of this study are presented in
As illustrated in
One example embodiment studies in vitro efficacy of PEG-BCT-100 as an intracellular arginine-depleting agent.
In one example embodiment, four lung adenocarcinoma cell lines (i.e. H358, H1650, H1975, and HCC4006), obtained from ATCC, are treated with PEG-BCT-100 at IC50 concentrations and 0.1 μg/μl. Internalization of PEG-BCT-100 by lung adenocarcinoma cells are assessed by immunocytochemistry using anti-PEG-antibodies. Detection is done using anti-rabbit Alexa 488 conjugated secondary antibody and visualization is performed using a fluorescent microscope. The PEG-BCT-100 treated lung adenocarcinoma cells are lysed in RIPA buffer for determination of arginine level by K7733 arginine ELISA kit from Immunodiagnostik. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in lung adenocarcinoma.
In one example embodiment, ten million lung adenocarcinoma cells from cell lines H1650, H1975, and HCC4006 are engrafted subcutaneously in BALB/cnude mice (4 weeks old with body weight of 10-14 g). Body weight, clinical signs and survival times are recorded.
Two groups of the mice, with eight mice in each group, are tested. Control group (negative control) receives physiological saline and treatment group receives 20 mg/kg of PEG-BCT-100. PEG-BCT-100 is administered via intraperitoneal (IP) injection, twice weekly, until euthanization. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 combined with α-Difluoromethylornithine (DFMO) in lung adenocarcinoma treatment.
In one example embodiment, ten million lung adenocarcinoma cells from cell lines H1650, H1975 and HCC4006 are engrafted subcutaneously in BALB/cnude mice (4 weeks old with body weight of 10-14 g). Body weight, clinical signs and survival times are recorded.
Four groups of the mice, with eight mice in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intraperitoneally, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
In H1650 xenograft, as shown in
In H1975 xenograft, as shown in
As illustrated in
In short, PEG-BCT-100, when combined with DFMO, produces a significant anti-tumor effect leading to prolonged survival in lung adenocarcinoma xenograft models.
One example embodiment studies in vivo efficacy of PEG-BCT-100 in SU-DHL-6 B cell lymphoma treatment.
In one example embodiment, the SU-DHL-6 human lymphoma cells are maintained in vitro as a suspension culture in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells growing in an exponential growth phase are harvested and counted for tumour inoculation.
One day before tumour inoculation, all mice are sub-lethally irradiated with 60Co (200 rad). Each mouse is inoculated subcutaneously at the right flank region with SU-DHL-6 tumour cells (5×106) in 0.1 ml of PBS/Matrigel (1:1) for tumour development. The treatments start when the mean tumour size reaches 125 mm3. The date of tumour cell inoculation is denoted as day 0.
Four groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and a combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in SU-DHL-10 B Cell lymphoma treatment.
In one example embodiment, SU-DHL-10 human lymphoma cells are maintained in vitro culture in RPMI1640 medium supplemented with 20% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells in an exponential growth phase are harvested and counted for tumour inoculation.
One day before tumour inoculation, all mice are sub-lethally irradiated with 60Co (200 rad). Each mouse is inoculated subcutaneously at the right flank region with SU-DHL-10 tumour cells (1×107) in 0.1 ml of PBS/Matrigel (1:1) for tumour development. The treatments start when the mean tumour volume reached 116 mm3. The date of tumour cell inoculation is denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v.
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in Kasumi-1 leukemia treatment.
In one example embodiment, the Kasumi-1 tumour cells are maintained in vitro as a suspension culture in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells growing in an exponential growth phase are harvested and counted for tumour inoculation.
All mice are γ-irradiated (200 rad) for 24 h before tumour cell injection. Kasumi-1 tumour cells (1×107) in 0.2 ml of PBS are mixed with cultrex in a 1:1 ratio. Each mouse is inoculated subcutaneously at the right flank region with the tumour cells suspension for tumour development. The treatments start when the mean tumour size reached 114 mm3. The date of tumour cell inoculation is denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
One example embodiment studies In vivo efficacy of PEG-BCT-100 in K562 leukemia treatment.
In one example embodiment, the K-562 tumour cells are maintained in vitro as a suspension culture in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells growing in an exponential growth phase are harvested and counted for tumour inoculation.
Each mouse is inoculated subcutaneously at the right flank region with K-562 tumour cells (5×106) in 0.1 ml of PBS/Matrigel (1:1) for tumour development. The treatments start when the mean tumour size reaches 125 mm3. The date of tumour cell inoculation is denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in Hep G2 hepatocellular carcinoma treatment.
In one example embodiment, the Hep G2 human liver cancer cells are maintained in vitro culture in RPMI1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells in an exponential growth phase are harvested and counted for tumour inoculation.
Each mouse is inoculated subcutaneously at the right flank region with Hep G2 tumour cells (1×107) in 0.2 ml of PBS (1:1 Matrigel) for tumour development. The treatments start when the mean tumour volume reaches 124 mm3. The date of tumour cell inoculation is denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in Hep 3B hepatocellular carcinoma treatment.
In one example embodiment, the Hep 3B tumour cells are maintained in vitro culture in EMEM medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells growing in an exponential growth phase are harvested and counted for tumour inoculation.
Each mouse is inoculated subcutaneously at the right flank region with Hep 3B tumour cells (5×106) in 0.1 ml of PBS (1:1 Matrigel) for tumour development. The treatments start when the mean tumour size reaches 119 mm3. The date of tumour cell inoculation is denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in MIA-PaCa-2 pancreatic cancer.
In one example embodiment, the MIA-PaCa-2 tumour cells are maintained in vitro culture in EMEM medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells growing in an exponential growth phase are harvested and counted for tumour inoculation.
Each mouse is inoculated subcutaneously at the right flank region with MIA-PaCa-2 tumour cells (5×106) in 0.1 ml of PBS (1:1 Matrigel) for tumour development. The treatments start when the mean tumour size reaches 124 mm3. The date of tumour cell inoculation was denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
One example embodiment studies in vivo efficacy of PEG-BCT-100 in CAPAN-1 pancreatic cancer.
In one example embodiment, the CAPAN-1 tumour cells are maintained in vitro culture in EMEM medium supplemented with 10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The tumour cells are routinely sub-cultured twice weekly. The cells growing in an exponential growth phase are harvested and counted for tumour inoculation.
Each mouse is inoculated subcutaneously at the right flank region with CAPAN-1 tumour cells (5×106) in 0.1 ml of PBS (1:1 Matrigel) for tumour development. The treatments start when the mean tumour size reaches 119 mm3. The date of tumour cell inoculation is denoted as day 0.
Four treatment groups of animals, with eight animals in each group, are tested. Control group (negative control) receives physiological saline, and the three treatment groups respectively receive 20 mg/kg of PEG-BCT-100 alone, 2% w/v DFMO in drinking water, and combination of 20 mg/kg of PEG-BCT-100 and 2% w/v DFMO. PEG-BCT-100 is administered intravenously, twice a week, until euthanization while DFMO is supplied in drinking water at 2% w/v. Results of this study are presented in
As illustrated in
As used herein, the term “arginine reducing compound” or “arginine depleting compound” means any compound that reduces arginine or depletes arginine. Examples include, but are not limited to, arginase or its analogs.
As used herein, the term “ornithine decarboxylase (ODC) inhibitor” or “ODC blocking agent” means any compound that inhibits or blocks ODC. Examples include, but are not limited to, DFMO or its analogs.
As used herein, the term “pegylated arginase”, “pegylated human arginase”, or “pegylated recombinant human arginase” refers to an arginase of the present invention modified by pegylation to increase the stability of the enzyme and minimize immunoreactivity.
In an example embodiment, the arginase is a recombinant human arginase I that has an amino acid sequence of SEQ ID NO:1 and a nucleic acid sequence of SEQ ID NO:2. In one example embodiment, the pegylated arginase has at least one polyethylene glycol (PEG) molecule that covalently links with an amino acid residue or with more than one amino acid residue of the arginase. By way of example, at least one PEG molecule covalently links with a lysine residue or with more than one lysine residues of the arginase. In another example embodiment, the PEG has a molecular weight of 5 KDa.
In one example embodiment, the pegylation of the arginase is achieved by covalently conjugating a PEG molecule with the arginase using a coupling agent. Examples of a coupling agent includes, but are not limited to, methoxy polyethylene glycol-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butyrate (mPEG-SBA), mPEG-succinimidyl succinate (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-succinimidyl glutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate, and mPEG-aldehyde. By way of example, the coupling agent is methoxy polyethylene glycol-succinimidyl propionate 5000 with an average molecular weight of 5K.
In an example embodiment, the pegylated recombinant human arginase, PEG-BCT-100, disclosed in this application includes a recombinant human arginase I that has an amino acid sequence of SEQ ID NO.1 and a nucleic acid sequence of SEQ ID NO.2, in which the recombinant human arginase I has at least one PEG molecule that covalently links with an amino acid residue or with more than one amino acid residue of the recombinant human arginase I. In one example embodiment, the recombinant human arginase I has about 6-12 PEG molecules per arginase. By way of example, the PEG molecule covalently links with a lysine residue or with more than one lysine residues of the recombinant human arginase I.
In another example embodiment, the pegylated recombinant human arginase, PEG-BCT-100, disclosed in this application includes a recombinant human arginase I that has an amino acid sequence of SEQ ID NO.3 and a nucleic acid sequence of SEQ ID NO.4, in which the recombinant human arginase I has six additional histidines at an amino-terminal end thereof, and at least one PEG molecule that covalently links with an amino acid residue or with more than one amino acid residue of the recombinant human arginase I. In an example embodiment, the six histidines are added for ease of purification. In one example embodiment, the recombinant human arginase I has about 6-12 PEG molecules per arginase. By way of example, the PEG molecule covalently links with a lysine residue or with more than one lysine residues of the recombinant human arginase I.
As used herein, the terms “combination therapy”, “combined treatment” or “in combination” means any form of concurrent or parallel treatment with at least two distinct therapeutic agents.
As used herein, the term “subject” means any mammal having cancer that requires treatment, includes but is not limited to human.
As used herein, the term “therapeutically effective amount” means the amount of the arginine reducing compound and/or the ornithine decarboxylase (ODC) inhibitor to be effective in treating cancer cells/disease of a particular type. A specific “therapeutically effective amount” will vary according to the particular condition being treated, the physical condition and clinical history of the subject, the duration of the treatment, and the nature of the combination of agents applied and its specific formulation.
As used herein, the term “synergistic” and its various grammatical variations means an interaction between the arginine reducing compound and the ODC inhibitor wherein an observed effect (e.g., cytotoxicity) in the presence of the drugs together is higher than the sum of the individual effects (e.g., cytotoxicities) of each drug administered separately. In one embodiment, the observed combined effect of the drugs is significantly higher than the sum of the individual effects.
The compounds or compositions of the present invention may be administered to a subject by a variety of routes, for example, orally, intrarectally or parenterally (i.e. subcutaneously, intravenously, intramuscularly, intraperitoneally, or intratracheally).
As used herein, the term “DFMO” means eflornithine or α-difluoromethylornithine.
As used herein, the term “ODC negative” means a cell is unable to express the enzyme, ornithine decarboxylase, either genotypically or phenotypically.
As used herein, the term “ODC positive” means a cell is able to express the enzyme, ornithine decarboxylase, either genotypically or phenotypically.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and disclose specific information for which the reference was cited in connection with.
The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/099642 | 9/21/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62221604 | Sep 2015 | US |
