Patent Application: US-201515114854-A

Abstract:
the present invention relates to a chemotherapeutic drug combination containing heterodinuclear cu - mn 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 .

Description:
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 . 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 eiwiromnent . 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 plumnic 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 4pc ovemight and was used after being filtered through a 0 . 2 itriu filter . final concentration of the pluronic was determined as 0 . 05 %. this concern ation 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 . toxic effect of the prepared chemicals was determined by analyzing cell viability using the mts method efalvac 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 . 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 . 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 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 . in order to observe the effects of the developed drug . formulation on tumor formation , 20 × 10 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 . 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 &# 39 ; s test and p ≦ 0 . 05 was considered statistically significant . 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 ( fig1 ). fibroblast cells were used in order to determine the toxicity of the drug conibination 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 ( fig2 ). 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 ). 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 ( fig3 ). 0 . 5 mg / kg was selected as the concentration to be applied in animal experiments after tumor formation . 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 . following the experiment , tumors were formed by subcutaneously injecting twenty million cells to the dorsal region within a period of 30 - 40 days ( fig4 ). 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 temiinated . the tumors were weighed and pathological analyses were conducted ( table 4 - 5 ). 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 munber of lymphocytic cells were detected around the liver central vein ( fig5 ). 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 . 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 unlmomi 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 .) 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 &# 39 ; 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 . aboul - 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