Bismuth(III) complexes as adjuvants in the treatment of cancer using platinum-based chemotherapy

Abstract

The present disclosure relates to methods of using cisplatin active agents in which reduced organ toxicity is observed are provided. In the subject methods, an effective amount is administrated to the host before administration of an effective amount of cisplatin active agents. The cisplatin toxicity reducing agent comprising of stable bismuth(III) complexes or pharmaceutically acceptable salts reduces the levels of undesired toxicity of cisplatin active agents without compromising their anticancer activity. Also provided are methods for use in practicing the subject methods in the treatment of different disease conditions.

Claims

1. A method of administrating to a subject in need thereof an effective amount of a cisplatin active agent, comprising administering to a host an effective amount of a cisplatin toxicity reducing agent to reduce toxicity of the cisplatin active agent to a host organ, wherein the host organ is selected from a kidney, liver, heart, nerves, bladder, reproductive organs, blood, stomach, intestines, bone marrow, or lung, the cisplatin toxicity reducing agent comprises bismuth (Ill) complexes, or pharmaceutically acceptable salts thereof, with bismuth (Ill) coordinating to Ligand L2: ##STR00006##

2. The method according to claim 1, wherein the dosage of cisplatin toxicity reducing agent is 4 to 6 fold of the cisplatin active agent.

3. The method according to claim 2, wherein the cisplatin active agent and the cisplatin toxicity reducing agent are administrated as separate formulations.

4. The method according to claim 1, wherein the cisplatin active agent comprises cisplatin and its derivative/analogues or other metallo-anticancer drugs.

5. The method according to claim 1, wherein the cisplatin toxicity reducing agent is administrated prior to administration of the cisplatin active agent.

6. The method according to claim 1, wherein the cisplatin toxicity reducing agent comprises a dimeric bismuth(III) compound.

7. A method of treating a host suffering from tumors or a cellular proliferative disease condition, comprising administrating to the host effective amounts of a cisplatin active agent together with a cisplatin toxicity reducing agent to reduce organ toxicity of the cisplatin active agent to a host organ, wherein the host organ is selected from a kidney, liver, heart, nerves, bladder, reproductive organs, blood, stomach, intestines, bone marrow, or lung, the cisplatin toxicity reducing agent comprises bismuth (Ill) complexes, or pharmaceutically acceptable salts thereof, with bismuth (Ill) coordinating to Ligand L2: ##STR00007##

8. The method according to claim 7, wherein a dosage of cisplatin toxicity reducing agent is 4 to 6 fold of the cisplatin active agent.

9. The method according to claim 8, wherein the cisplatin active agent and the cisplatin toxicity reducing agent are administrated as separate formulations.

10. The method according to claim 7, wherein the cisplatin active agent comprises cisplatin and its derivative/analogues or other metallo-anticancer drugs.

11. The method according to claim 7, wherein the cisplatin toxicity reducing agent is administrated prior to administration of said cisplatin active agent.

12. The method according to claim 7, wherein the cisplatin toxicity reducing agent comprises a dimeric bismuth(III) compound.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 Provides the chemical structure of the representative bismuth(III) complex, BicitZ

(2) FIG. 2 Depicts (a) the anti-apoptotic properties of BicitZ in HK-2 cells treated with cisplatin and control group of HK-2 cells. (b) HK-2 cells treated with cisplatin (20 μM). (c) pretreated with BicitZ and then treated with the same concentration of cisplatin as (b).

(3) FIG. 3 Depicts the anti-apoptotic properties of bismuth(III) complexes in MIHA cells treated with bismuth(III) complexes with increasing concentrations (1-100 μM) in the absence and presence of cisplatin (100 μM). MIHA cells treated with culture medium served as a control. The respective survival rate was measured by cell proliferation kit XTT. All data are in triplicates.

(4) FIG. 4 Depicts the anti-apoptotic properties of bismuth(III) complexes in HK-2 cells treated with bismuth(III) complexes with increasing concentrations (1-100 μM) in the absence and presence of cisplatin (10 μM). HK-2 cells treated with culture medium served as a control. The respective survival rate was measured by cell proliferation kit XTT. All data are in triplicates.

(5) FIG. 5 Provides survival curves of cisplatin-treated mice with pretreatment of Bi-L1, Bi-L12 and BicitZ, respectively, showing the protective role of bimusth(III) complexes on cisplatin induced nephrotoxicity. All bismuth complexes (50 mg/kg) were administered orally on Day −2, Day −1, Day 0, Day 2 and Day 4. Cisplatin (30 mg/kg) was I.P. injected on Day 0. Kaplan-Meier survival curves show a higher survival rate of mice with pretreatment of bismuth(III) complexes, n=8. (Log-rank (Mantel-Cox) test, ***P=0.0004)

(6) FIG. 6 Provides graphs showing the effect of pretreatment of bismuth(III) complexes on renal function of mice with administration of cisplatin, which was measured by the level of BUN (blood urea nitrogen). All the experimental conditions were kept the same as described in survival assay. All the mice were sacrificed on day 5 and serums were collected for BUN assay. The BUN level was measured using Urea Nitrogen Kit (Stanbio Laboratory, USA, Stanbio, Ref. 2020-430). Each BUN represents as the mean±SD. *P<0.05 **P<0.01 and ***P<0.001.

(7) FIG. 7 Provides images showing that BicitZ has no interference on cisplatin's chemotherapeutic effect in tumor bearing mice. 0.2 Million SKNLP neuroblastoma cells transfected with luminescent plasmid were inoculated into the adrenal gland of SCID beige mice. BicitZ (100 mg/kg) was administered orally on Day −1, Day 0 and every two days since. Cisplatin (7.5 mg/kg) was i.p. injected at Day 0, Day 7 and Day 14. Tumor growth was monitored by luminescent of the transfected tumor cells.

(8) FIG. 8 Provides images showing that Bi-L1 and Bi-L12 have no interference on cisplatin's chemotherapeutic effect in tumor bearing mice. SKNLP neuroblastoma cells (˜2×10.sup.5 cells) were transfected with luminescent plasmid were inoculated into the adrenal gland of BALB/c nude mice. Bi-L1 and Bi-L12 (100 mg/kg) were administered orally at Day −1, Day 0 and every two days since. Cisplatin (7.5 mg/kg) was i.p. injected on Day 0, and Day 7. Tumor growth was monitored by luminescent of the transfected tumor cells.

(9) FIG. 9 Provides photos of the dissected tumors after tumor-bearing mice receiving treatment of PBS, cisplatin and cisplatin with BicitZ.

(10) FIG. 10 Provides size of the dissected tumors after tumor-bearing mice receiving treatment of PBS, cisplatin and cisplatin with Bi-L1 and Bi-L12.

(11) FIG. 11 Provides images showing that administration of Bi-L1, Bi-L12 and BicitZ delays SKNLP tumor cells metastases in lung and liver. The mouse livers and lungs were collected from the same experiment as in FIG. 7 and FIG. 8.

DETAILED DESCRIPTION

(12) Described herein are methods of using bismuth(III) pharmaceutical agents in which reduced host organ toxicity is observed for a cisplatin active agent. In the subject methods, an effective amount of bismuth complexes is pre-administrated to the host several days prior to the administration of a cisplatin active agent. Also provided are pharmaceutical compositions, dosages and pretreatment time of the same compositions for use in practicing the subject methods as well as application of the subject methods in different disease conditions.

(13) A host that suffers from disease or condition requiring the treatment of cisplatin typically has a form of cancer. As used herein, the term cancer refers to or describes the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancers.

(14) The subject matter herein relates to administration of cisplatin toxicity reducing agents to a host that suffers from disease or condition requiring the treatment of cisplatin. The toxicity reducing agents of interest are stable bismuth(III) complexes or pharmaceutically acceptable salts. By bismuth(III) complexes is meant that complexes with bismuth(III) coordinated to but not limited to N, O, S containing ligands including but not limited to current clinically used bismuth antiulcer drugs for the treatment of H. pylori infection e.g. citrate based bismuth drugs (CBS e.g. De-Nol and RBC e.g. Pylorid) as well as bismuth subsalicylate (BBS). The ligands involved in the preparation of tested bismuth(III) complexes are shown below but not limited to them.

(15) ##STR00001## ##STR00002## ##STR00003##

(16) In one specific embodiment, the subject method to reduce cisplatin induced organ toxicity in kidney proximal tubular cells and tumor-bearing mice is pre-administration of a composition comprising an effective amount of a bismuth(III) zinc(II) citrate complex. The restriction of dietary zinc leads to reduced bismuth uptake and bismuth-induced metallothionein in rat kidney. Moreover, cancer patients often suffer zinc deficiency. The bismuth(III) zinc(II) citrate complex is described herein as BicitZ prepared by solubilization of bismuth citrate with zinc hydroxide and can be represented by the following dimeric structural formula:

(17) ##STR00004##
or a pharmaceutically acceptable salt thereof, wherein:
O without clear originality is from water or other citrate, in addition, NH.sub.3 may be also included.

(18) In the embodiment of citrate-based molecules and the bismuth(III) center usually exists as a dimer in solution. This does not exclude a possibility of formations of other molecular structures such as monomers or polymers at different pH values. Moreover, the bismuth(III) center at the embodiment of citrate-based molecules carries net negative charges, thus at least one counter-cation is required to neutralize the charge of the bismuth(III) centers. Accordingly, the phrase “pharmaceutically acceptable salt,” as used herein, includes the salts formed from charged bismuth(III) complex and counter-cation

(19) Taken the dimer form of one embodiment as instance, as used herein, the term “citrate-based bismuth molecule” refers to a molecule of the following chemical structure:

(20) ##STR00005##
wherein, “A” represents the “counter-cation”, which refers to an ion associated with a negatively charged citrate-based bismuth(III) complex, is selected from a group consisting of Zn.sup.2+ or NH.sub.4.sup.+, m*n=2.

(21) As used herein, the term “citrate-based bismuth(III) zinc(II) complex (BicitZ)” refers to the complex of bismuth(III) bound to citrate molecule. The structure of the citrate based bismuth(III) complex can be a monomer, dimer or aggregated polymer.

(22) As described herein, the term “bismuth pharmaceutical agents” is meant by bismuth(III) based complexes with pharmaceutically acceptable carrier, which refers to a carrier combination of carrier ingredients approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, mammals, and more particularly in humans. Non-limiting examples of pharmaceutically acceptable carriers include liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water is a common vehicle when the compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions.

(23) As summarized above, methods of administration of cisplatin toxicity reducing bismuth(III) complexes to a host that suffers from disease or condition requiring the treatment of cisplatin active agents are provided. An important feature of the subject methods is that bismuth complexes as cisplatin toxicity reducing agents need to be pre-administrated prior to administration of cisplatin active agents. By “pre-administrated” is meant that bismuth complexes as cisplatin toxicity reducing agents are administrated several days e.g. at least 1 day, at least 2 days or at least 3 days depending on the amounts administrated prior to administration of cisplatin active agents. Thus, the toxicity reducing bismuth(III) complexes are typically administrated before cisplatin treatment begins.

(24) In the subjected methods, reduction of cisplatin toxicity by bismuth(III) complexes is pre-administrated to a host that requires treatment of cisplatin active agents with an effective amount of a cisplatin toxicity reducing agents. By “cisplatin toxicity” is meant that toxicity induced by cisplatin or its derivatives/analogues such as carboplatin, ormaplatin, oxalipaltin, or other platinum-based anticancer drug as well as other metal containing anticancer drugs e.g. arsenic trioxide (Trisenox®).

(25) The dosage cisplatin toxicity reducing agent is an amount effective to reduce organ toxicity of the cisplatin active agent, and/or eliminate organ toxicity of the cisplatin active agent. The dosage cisplatin toxicity reducing agent is typically greater than the dosage of the cisplatin active agent. In one embodiment, the dosage of cisplatin toxicity reducing agent is 2 or more fold of the cisplatin active agent. In another embodiment, the dosage of cisplatin toxicity reducing agent is 4 to 6 fold of the cisplatin active agent. In yet another embodiment, the dosage of cisplatin toxicity reducing agent is 4 to 14 fold of the cisplatin active agent.

(26) According to the subject matter herein, cisplatin and any analogues/derivatives or other metal-containing anticancer drugs whose toxicity is reduced when bismuth(III) complexes are administrated prior to administration of cisplatin active agents. Cisplatin toxicity reducing bismuth complexes are suitable to use in the subject methods as they exert no effects on the efficacy of cisplatin in mouse model described in the experimental section below. As described in FIG. 7 and FIG. 8, pretreatment of specific embodiments including BicitZ, Bi-L1 and Bi-L12, at 100 mg/kg exhibits no interference on cisplatin's chemotherapeutic effect (7.5 mg/Kg) in SCID beige tumor-bearing mice.

(27) As indicated above, an effective amount of cisplatin toxicity reducing bismuth complexes are employed in the subject method. The amounts of cisplatin toxicity reducing bismuth(III) complexes may vary according to administrated dosages of cisplatin. The bismuth(III) complexes can be pre-administrated at a single high dosage or at lower multiple dosages before injection of cisplatin. In one embodiment, bismuth(II) complexes was administered orally prior to i.p. injection of cisplatin. Mice exhibit significant improvement in survival rate compared with those without pretreatment of the embodiment as shown in FIG. 5.

(28) By cisplatin toxicity reducing bismuth(III) complexes is meant those bismuth complexes that reduce unwanted toxicity of cisplatin or its derivatives or other metal-containing anticancer drugs. Those bismuth complexes when pre-administrated to a host before administration of cisplatin active agents can reduce the toxicity of cisplatin active agents including but not limited to nephrotoxicity as described in one embodiment (FIG. 6), in which pretreatment of a bismuth complex reduced cisplatin-induced nephrotoxicity as manifested by the decreased levels of blood urea nitrogen (BUN).

(29) According to the subject method, pre-administration of cisplatin toxicity reducing bismuth(III) complexes to a host that requires treatment of cisplatin active agents reduced host organ toxicity. The term “organ” refers to but not limited to kidney, liver, heart, nerves, bladder, reproductive organs, blood, stomach, intestines, bone marrow, or lung.

(30) Regarding the underlying mechanism of bismuth complexes as a protector of cisplatin induced organ toxicity, it was found in previous studies that bismuth can selectively reduce cisplatin-induced nephrotoxicity without compromising its antitumor activity, and this effect is positively correlated to the renal expression level of a small protein-metallothionein (Cancer Res 1987, 15:983-987). Metallothionein (MT) is a family of cysteine-rich, low molecular weight proteins with the capability of binding to physiological Zn.sup.2+ and Cu.sup.2+, xenobiotical Bi.sup.3+, Pt.sup.2+, Cd.sup.2+ etc. and is universally expressed, but relatively rich in liver and kidney. Bi.sup.3+ has been shown to be able to displace Zn.sup.2+ and Cd.sup.2+ from the MT II even at extremely low pH, and binds to MT II with a stoichiometry of bismuth: MT=7:1 (J Biol Chem 1999, 274:29094-29101). The affinity of platinum to MT was demonstrated to be approximately 107 and 30 times more firm than zinc and cadmium respectively (J Inorg Biochem 1997, 65:295-298). However, reduced renal toxicity observed in animal model might not be attributable to increased binding of Pt(II) to renal MTs induced by bismuth, instead, the anti-oxidant activity of MT might be responsible for the reduction of cisplatin-induced renal toxicity (Biochem Pharmacol 1991, 41:369-375). Since renal superoxide dismutase (SOD) activity is increased in rats that received cisplatin, leading to peroxidative damage, but not in rats receiving Bi.sup.3+ prior to cisplatin. Taken together it can be the anti-oxidative stress property of MTs that reduces the cisplatin-induced nephrotoxicity.

(31) In our recent study, the intracellular amounts of bismuth under bismuth treatment are found to relate to glutathione (Proc Natl Acad Sci, 2015, 112: 3211-3216). The exact mechanism underlying bismuth capability of protecting cisplatin-induced organ toxicity is still not fully understood. It is not known whether thiolate-containing biomolecules other than MTs might also play roles in mitigation of cisplatin or its analogue induced organ toxicity.

EXAMPLES

(32) The following examples illustrate the subject invention. Unless otherwise indicated in the following examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Centigrade, and pressure is at or near atmospheric pressure.

Example 1: Preparation of Bismuth(III) Complexes

(33) In general, the preparation of bismuth(III) complexes are conducted by coordinating bismuth(III) ions to N, O and/or S-contained ligands via wet chemistry route. Herein, we take the synthesis of bismuth(III) zinc(II) citrate complex, BicitZ, as instance. Typically, zinc hydroxide solution was mixed with bismuth substrate, followed by adjusting pH to 7.2 using diluted ammonia and 18% HCl solution. The solubility of the complex is over 100 mM in water and is highly dependent on pH and ionic strength. Crystal structure of these complexes is not available yet, however, it is likely to assume similar structures as clinically used antiulcer drugs CBS or RBC (J Am Chem Soc 2003, 125:12408-12409). In these structures, bismuth(III) coordinates with tridentate citrate, forming a well-defined dinuclear unit [Bi(cit).sub.2].sup.2− with additional O.sup.2−, OH.sup.− and H.sub.2O ligands as shown in FIG. 1. Other cations such as NH.sup.4+ serve as counter ions to balance negative charge bismuth(III) center. Zinc(II) may play a similar role to stabilize the structure.

Example 2: Bismuth(III) Complexes Exert Potent Protective Role Against Cisplatin Caused Cytotoxicity in Human Cell

(34) To evaluate the in vitro protection effect of test bismuth(III) complexes against cisplatin stress, two nontumorigenic normal human cell lines, HK-2 (human kidney proximal tubular cell) and MIHA (human hepatocyte cell) are incorporated herein for cell proliferation kit II XTT assay.

(35) The XTT assay was carried out according to the manufacturer's instruction (Roche Diagnostics, USA). Typically, 1-2×10.sup.4 cells per well were grown in flat-bottom 96-well plates at 37° C. for an overnight under a humidified atmosphere of 5% CO.sub.2. Cells pellets were washed with PBS and treated with bismuth(III) complexes at various concentrations (1-100 μM) for 2 hours prior to the exposure to cisplatin (10 μM and 100 μM for HK-2 and MIHA cell line, respectively) for 2 days. Cells grown under culture medium alone were used as a negative control. After a fixed incubation time, 50 μl of the XTT labeling mixture was added to each well and the cells were incubated for 2 hours. The formation of formazan dyes, produced only by metabolic active cells, was detected spectrophotometrically at 490 nm.

(36) As shown in FIG. 3 and FIG. 4, bismuth(III) complexes show no or slight cytotoxicity towards two cell lines except Bi-L15 and Bi-L16 when used alone. Less than 40% cells survived after exposure to cisplatin only. However, upon the co-treatment, tested bismuth(III) complexes were able to counteract the cisplatin induced cytoxicity at different degrees. In particular, the cell viabilities were increased distinctly by 46.3%, 34.0% and 16.7% for HK-2 and 29.0% 14.7% and 28.5% for MIHA, respectively, when Bi-L1, Bi-L-12 and BicitZ were co-treated with cisplatin.

Example 3: Pre-Administration of Bismuth(III) Complexes Shows the Protective Property on Cisplatin Induced Nephrotoxicity in Mice

(37) Given the potent in vitro effect of Bi-L1, Bi-L12 and BicitZ, their protective roles against cisplatin caused nephrotoxicity was further examined in an in vivo murine model.

(38) In a typical experiment, 4 groups of 6 to 8-week-old female BALB/c mice (Laboratory Animal Unit, the University of Hong Kong) were housed and given free access to water and food. All animal experiments were done in accordance with the University of Hong Kong Guideline for Animal Care and Experimentation. Cisplatin (30 mg/kg) was injected i.p. on Day 0. 50 mg/kg of Bi-L1, Bi-L-12, BicitZ and PBS (serves as a control) were administered orally in a 200-μL aliquot on Day −2, −1, 0, 2 and 4. Survival rates of different groups of mice were monitored; Kaplan-Meier estimates of the survival curves were calculated and plotted. n=8 (FIG. 3). Significant increases in the survival rates of the mice are noted for the group with pre-treatment of bismuth(III) complexes prior to injection of cisplatin.

(39) It is also noted that the levels of blood urea nitrogen (BUN) in cisplatin-treated mice is increased significantly compared with those of PBS and Bi(III) complexes pre-administered groups. The pre-administration of Bi(III) complexes significantly reduced the BUN levels, suggesting a renal protective role of Bi(III) complexes on cisplatin-treated mice as shown in FIG. 6.

Example 4: Pre-Administration of Bismuth(III) Complexes Exhibits no Interference on Cisplatin's Anti-Cancer Effectiveness

(40) To evaluate whether Bi(III) complexes affect cisplatin's in vivo anti-tumor activity, SKNLP neuroblastoma cells transfected with luminescent plasmid were implanted into mice. Groups of tumor-engrafted mice were administered orally with Bi-L1, Bi-L12, BicitZ (50 mg/Kg) and PBS (as control) on Day −1, Day 0 (2 hours prior to cisplatin treatment) and every two days post cisplatin (7.5 mg/kg) treatment on Day 0, respectively. SCID Berge mice were used in the BicitZ-treated group and received cisplatin treatment on Day 0, 7 and 14. BALB/c nude mice were used in Bi-L1- or Bi-L12-treated group and received cisplatin treatment on Day 0 and 7.

(41) The results show that pre-administration of BicitZ (FIG. 7), Bi-L1 and Bi-L12 (FIG. 8) exerts negligible effects on cisplatin's anti-cancer activity in tumor engrafted mice. The sizes of the dissected tumor also confirmed this statement (FIG. 9 and FIG. 10). Further histology image of mouse liver and lung as present in FIG. 11 shows that pre-administration of Bi(III) complexes reduced or have no interference on tumor extensive metastasis in comparison with the control and cisplatin alone groups.

(42) With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.

(43) Other than in the operating examples, or where otherwise indicated, all numbers, values and/or expressions referring to quantities of ingredients, reaction conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term “about.”

(44) While the invention has been explained in relation to certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.