Chemotherapeutic drug combination

Abstract

The present invention relates to a chemotherapeutic drug combination containing heterodinuclear Cu (II)-Mn(II) complex. The objective of the present invention is to provide a drug combination, which will be actively used in chemotherapy, is anticarcinogenic and has no side effects, facilitates penetration into the cell, shows selectivity only for cancer cells and does not harm healthy cells, and is not toxic for the tissues and organs in the body, and which can prepared easily and provides a definitive and fast treatment.

Claims

1. A chemotherapeutic drug combination comprising a combination of a polynuclear Schiff base complex and a pluronic; wherein the polynuclear Schiff base complex is heterodinuclear Cu (II)-Mn(I) complex; wherein the pluronic is P85; wherein the combination of the polynuclear Schiff base complex and the pluronic enables penetration of the chemotherapeutic drug combination selectively for cancer cells while increasing viability in healthy cells.

2. The chemotherapeutic drug combination according to claim 1, a concentration of the polynuclear Schiff base complex is 0.5 mg/Kg or less.

3. The chemotherapeutic drug combination according to claim 1, a concentration of the polynuclear Schiff base complex is 0.5 mg/Kg.

4. A chemotherapeutic drug combination, consisting of a combination of a polynuclear Schiff base complex and a pluronic; wherein the polynuclear Schiff base complex is heterodinuclear Cu (II)-Mn(I) complex; wherein the pluronic is selected from at least one of F68, P106, P407, P85, and P123.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The “Chemotherapeutic drug combination” developed to fulfill the objectives of the present invention is shown in the accompanying figures wherein

(2) FIG. 1 is the representation of the toxicity of the drug combination on TRAMP cells (*P<0.05; n: negative control, p=positive control).

(3) FIG. 2 is the representation of the toxicity of the drug combination on L929 cells (*P<0.05; n: negative control, p=positive control).

(4) FIG. 3 is the toxicity observed after application of 1 mg/kg of the drug combination. (A) Liver tissue focal necrosis. (B) Kidney tissue lymphatic infiltration, (C) Hydrophobic degeneration in kidney tubular tissue, ling/kg drug combination.

(5) FIG. 4 is the representation of the tumor formation at the dorsum after 20×10.sup.6 TRAMP cell injection.

(6) FIG. 5 is the hepatic portal field focus of inflammation. It is the toxicity observed in the liver following the thug administration.

(7) In the scope of the present invention, a new chemotherapeutic drug is developed by combining a chemical molecule, which is Schiff-base derivative, with a block copolymer, which specifically recognizes cancer cells and speeds up penetration of the drug. In the drug combination, while the polymeric substance serves as a supporting molecule, Schiff-base is used as the active molecule group. In the drug combination that is prepared, pluronic P85 polymer, which facilitates penetration of the drug into the cell, is used. Thus, by enabling the penetration of the drug selectively only for cancer cells, it is enabled to obtain toxic effect without harming the healthy cells.

DETAILED DESCRIPTION OF THE INVENTION

(8) Preparation of the Chemotherapeutic Drug Combination

(9) Polynuclear Schiff (heterodinuclear Cu (II)-Mn(II) complex) base complex is used as the chemical molecule in preparation of the chemotherapeutic drug formulation. This complex was used in combination with pluronics. The combination was prepared at different concentrations in in vitro and in vivo conditions and applied in the experiments. The Schiff base solution and pluronic solution, which were prepared separately for the experimental studies, were used in combination.

(10) In Preparation of the Combination Schiff base was dissolved in DMSO (1 mg/ml) main stock solution and a homogenous solution was obtained. Since Schiff base is photosensitive, the processes including Schiff base were performed in a dark environment.

(11) The prepared Schiff base solution was used either in PBS or cell culture medium or by injecting to animals at concentrations in the range of 0.0001-10 μg ml for in vitro conditions and at concentrations in the range of 0.001 mg/kg-10 mg/kg for in vivo conditions. The pluronic which is the polymer solution is weighed at a ratio of 10% and the main stock solution was prepared and used. This polymer solution was incubated at 4° C. overnight and was used after being filtered through a 0.2 μm filter. Final concentration of the pluronic was determined as 0.05%. This concentration ma vary between 0.0001 and 2%. Finally, Schiff base derivative and pluronic were mixed and allowed to rest at room temperature for 15 minutes. Then medium was added thereon and it was made ready for use. The drug combination of the present invention will be denoted with the code CMSB1422113 in commercial applications. When injecting to animals, it was made up to a total volume with PBS and prepared in an injectable form. In the drug combination, Pluronic is selected from at least one of F68, F127, P106, P407, P85, P123.

(12) Characterization Studies

(13) Determining Cell Toxicity

(14) Toxic effect of the prepared chemicals was determined by analyzing cell viability using the MTS method (Yalvac et al., 2009). The drug combination was prepared at concentrations of 5 μM, 2 μM, 1 μM, 0.5 μM and was applied on the cancer cells which were counted and seeded on 96-well culture plates (5000 cells/Well). Toxicity response was determined by measuring cell viability for 3 days, formazan crystals are formed in MTS substance applied cells as a result of mitochondrial dehydrogenase enzyme activity as an indicator of cell viability. The color change was evaluated by measuring absorbance by ELISA device. The obtained values were analyzed using Microsoft Office Excel database.

(15) In Vivo Toxicology Analyses

(16) After the in vitro experiments were completed, in vivo toxicology analyses were performed in order to observe the effects of the drug formulation in the system of a living organism system. Acute toxicological analyses were performed as previously stated in the literature (Uckun et al, 2002). The prepared formulation was first tried on prostate cancer. Toxicology studies were conducted on C57 mice which are used in experiments. Four different doses of 0.1 mg/kg, 0.5 mg/kg and 1 mg/kg were administered intraperitoneally to male C57 mice. 7 days later the mice were sacrificed (acute toxicology). Blood parameters and histopathological analyses were completed.

(17) Forming In Vivo Tumor Model

(18) Visible prostate cancer tumor tissue was formed in the animals at the dorsal region by modifying the protocol which was described previously in the literature (Young et 2007). 20×10.sup.6 TRAMP cells were administered to male C57 mice subcutaneously. Tumor formation process was monitored. At the end of an approximately 44 day process, the tumors became visible.

(19) Inhibiting In Vivo Tumor Formation

(20) In order to observe the effects of the developed drug formulation on tumor formation, 20×10.sup.6 TRAMP cells were subcutaneously injected to 20 kg weighing C57 mice to the dorsal region near the tail. After one week following the injection, 0.5 mg/kg Schiff base derivative and 500 mg/kg plutonic were combined and injected to the animals which were separated as the experimental group. Saline was administered to the control group animals. At the end of 13 injections when death began in the control group animals, the experiment was terminated. Tumor photographs were taken and the pathological examination was completed.

(21) Statistical Analysis

(22) The results obtained were statistically evaluated by one-way analysis of variance (ANOVA) using GraphPad Prism 5 software. The difference between the groups was determined by Tukey's test and p≦0.05 was considered statistically significant.

Experimental Results

(23) In order to show the toxic effects of the drug combination of the present invention, toxicology tests were performed using the cancer and healthy cell lines. TRAMP (Mouse prostate cancer cells) prostate cancer cells, whose model will be formed in animals, were used as the cancer cell line. L929 (Mouse fibroblast cells) fibroblast cells were used as the healthy cell line. The drug combination was applied on the cells at concentrations of 5 μM. 2 μM, 1 ρM, 0.5 μM and the effects were examined at the end of three day toxicology analyses. Toxic effect was started to be observed at the end of the second day at four concentrations, and after three days of analysis, a significant amount of toxicity (at the same level with the positive control) was determined when compared with the negative control group (FIG. 1).

(24) Fibroblast cells were used in order to determine the toxicity of the drug combination at the same concentrations on healthy cells. Toxicity was observed at the end of three days at the two highest concentrations (5 μM and 2 μM), while no toxicity was observed at the concentrations of 1 μM and 0.5 μM (FIG. 2).

(25) The findings obtained showed that while the drug combination killed the cancer cells, it increased viability in the healthy cells. In vivo toxicology analyses were performed in order to support the findings and start in vivo studies. 0.5 mg/kg was determined to be the highest non-toxic concentration after blood parameters and enzyme analyses, (Table 1-2-3).

(26) After necroscopy, tissue and organ weights of the animals were measured and analyses were performed to find out Whether there were any anomalies. Tissue and organ samples were pathologically examined and while no toxicity was found at concentrations of 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, a low level of toxicity was observed at the concentration of 1 mg/kg. Focal necrosis was observed in the liver tissue and hydrophobic degeneration in hepatocytes. Lymphatic infiltration was observed in the kidneys and hydrophobic degeneration in the kidney tubules (FIG. 3). 0.5 mg/kg was selected as the concentration to be applied in animal experiments after tumor formation.

(27) TABLE-US-00001 TABLE 1 Blood parameter (enzyme, protein, etc.) results obtained after toxicity analysis of the drug combination at a concentration of 0.5 mg/kg. G3 Parameter (0.5 mg/kg) Control Description AST (U/L) 255 176 The enzyme is a significant indicator of liver function. ALT (U/L) 101 100 The enzyme is a significant indicator of liver function CK (U/L) 804 775 The enzyme is a significant indicator of renal function Amylase 415 383 The enzyme is a significant indicator of pancreatic (U/L) function. Bilirubin 0.06 0.05 The enzyme is a significant indicator of liver damage. (mg/dl) Albumin 2.70 2.43 The enzyme is a significant indicator of liver damage. (mg/dl) Creatinine 0.58 0.48 The enzyme is a significant indicator of renal damage. (mg/dl) Urea 45.75 40.50 The enzyme is a significant indicator of renal and cardiac (mg/dl) damage. Protein 6.48 6.33 The enzyme is a significant indicator of renal damage. (g/dl)

(28) TABLE-US-00002 TABLE 2 Blood cells results obtained after toxicity analysis of the drug combination at a concentration of 0.5 mg/kg. Parameter G3 (0.5 mg/kg) Control WBC 6.97 7.17 RBC 7.54 7.89 PLT 708.75 709.00 EOS# 0.06 0.07 EOS % 0.90 0.80 LYM# 4.46 4.93 LYM % 72.11 73.78 BAS# 0.06 0.05 BAS % 0.02 0.02 MON# 0.44 0.41 MON % 4.98 4.73 NEU# 1.34 1.13 NEU % 21.43 19.00

(29) TABLE-US-00003 TABLE 3 Blood parameter results obtained after toxicity analysis of the drug combination at a concentration of 0.5 mg/kg. G3 Parameter (0.5 mg/kg) Control Description Hgb (g/dL) 14.88 15.05 is an indicator of the hemoglobin values in blood. MCV (fL) 51.15 51.80 is an indicator of the average size of the oxygen carrying cells. MCH (pg) 19.48 19.58 is an indicator of the hemoglobin content in erythrocytes. MCHC (g/dL) 39.23 38.38 is an indicator of the average hemoglobin concentration in erythrocytes. RDW-SD 29.43 27.93 is an indicator of the erythrocyte distribution width. (fL) MPV (fL) 5.95 5.98 is an indicator of platelet size. PDW 14.93 14.75 is an indicator of the platelet distribution width. HCT % 44.03 51.75 is a measure of the hemoglobin and erythrocyte content. RDW-CV % 14.28 14.23 is an indicator of the erythrocyte distribution width. PCT % 0.45 0.44 is an indicator of the platelet ratio in blood.

(30) Prostate tumor was formed experimentally on C57 mice in order to reproduce the clinical picture. In the pilot study, tumor formation was induced by using different numbers of cells.

(31) Following the experiment, tumors were formed by subcutaneously injecting twenty million cells to the dorsal region within a period of 30-40 days (FIG. 4).

(32) After the tumor formation was successfully completed, experiments in the scope of the treatment were started. Following one week after the tumor cell was injected, the drug combination prepared by using Schiff base at a dose of 0.5 mg/kg, was intraperitoneally administered to the animals once every four days. At the end of 13 injections, when death began in the control (physiological saline application) group animals, the experiment, was terminated. The tumors were weighed and pathological analyses were conducted (Table 4-5).

(33) Prostatic adenocarcinoma formation was analyzed by using Gleason scoring. In the group to which the drug was administered, no toxicological effect was encountered except a mild congestion in the spleen. A small number of lymphocytic cells were detected around the liver central vein (FIG. 5).

(34) TABLE-US-00004 TABLE 4 Results of pathological examination of tumor tissue of control group animals Group (control) Tumor Gleason Score C1 Prostatic adenocarcinoma Gleason score 9 (4 + 5) C2 Prostatic adenocarcinoma Gleason score 9 (4 + 5) solid tumor-diffuse necrosis C3 Prostatic adenocarcinoma Gleason score 9 (5 + 5) solid tumor, necrotic and a plurality of apoptotic cells C4 Prostatic adenocarcinoma Gleason score 9 (4 + 5), small tumor. necrotic areas and apoptotic cells C5 Prostatic adenocarcinoma Gleason score 10 (5 + 5) C6 Tumor tissue was monitored Gleason score could not be in a limited area. assigned C7 Prostatic adenocarcinoma Gleason score 10 (5 + 5) tumor solid pattern C8 Prostatic adenocarcinoma 9 (5 + 4) solid pattern

(35) TABLE-US-00005 TABLE 5 Results of pathological examination of tumor tissue of experimental group animals Group (Drug Administration) Tumor Gleason Score D1 No tumor D2 No tumor D3 edematous tissue, chronic inflamation and fibrosis are present, no tumor D4 Prostatic adenocarcinoma Gleason score 9 (5 + 4) D5 Prostatic adenocarcinoma Gleason score 10 (5 + 5), solid pattern tumor is present D6 No tumor D7 No tumor D8 Prostatic adenocarcinoma Gleason score 10 (5 + 5)

(36) With the present invention, a new easily-prepared drug combination is obtained which can be actively used in chemotherapy and which is not toxic to the other tissues and organs of the body. The present invention can provide a definitive and rapid treatment by enabling the drug to penetrate the cancer cells rapidly and at a large quantity. Many drugs which are developed for chemotherapy have a great number of side effects. Problems such as gastrointestinal system disorders such as mouth ulcers, taste changes, irritation in the inner lining of the bowel, diarrhea, appetite problems and nausea, anemia, hemorrhage problems, fall in blood counts (damage on division of bone marrow cells), fatigue, hair loss, nervous system disorders, pain, reproductive system disorders, disorders occurring on the skin and nails are experienced following chemotherapy. The drug combination of the present invention is capable of eliminating these kinds of side effects.

APPLICATION OF THE INVENTION

(37) The present invention provides a treatment for the cancer types seen in human body by reducing the side effects. The formulation developed within the scope of the invention can be effective on all cancer types particularly prostate cancer. It can be used for treatment of AIDS-related cancer types, breast cancer and derivatives thereof, gastrointestinal tract-related cancer types, endocrine and neuroendocrine cancers, eye cancer, genitourinary system cancer types, gynecological cancer types, prostate cancer and derivatives thereof, germ cell cancer, head and neck cancer, hematologic cancer and leukemia, musculoskeletal system cancers, neurological cancers, respiratory system and thoracic cancers, skin cancer, cancers of unknown origin, cancer types observed in childhood (acute lymphoblastic leukemia, acute myeloid leukemia), cancer types observed in women (breast, cervical, endometrial, ovarian, uterus, vaginal, vulval etc.)

(38) The invention is effective in treatment and prevention of cancer types such as astrocytoma, glioma, lung cancer, hepatoma, colon cancer, osteoid cancer, pancreatic cancer skin cancer, cervical cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal cancer, colon carcinoma, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penial cancer, prostate cancer, urinary bladder cancer, kidney or ureter cancer, renal cell carcinoma, pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor and hypophysis adenoma.

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