- INTRODUCTION: THE FIRST MEDICAMENT THAT KILLS CANCER CELLS BUT NOT HEALTHY CELLS
- I. EFFICACY
- II. SAFETY
- III. QUALITY
- BIBLIOGRAPHY
At therapeutic dose NSC 631570 has no appreciable adverse effects and does not damage healthy cells but only attacks cancer cells. Due to its very high therapeutic index of 1250 – in contrast to common cytostatics with a low TI of 1.4-1.8 – there is no danger of an overdose with Ukrain therapy (therapeutic index is the ratio between the toxic dose and the therapeutic dose of a drug, used as a measure of the relative safety of the drug for a particular treatment. From ‘The American Heritage® Dictionary of the English Language, 4th Edition’). Ukrain also does not cause necroses when administered intramuscular, which is a proof for its safety (37).
The Austrian Research Center Seibersdorf is the leading Austrian research institution in life sciences. Now it is a part of the Austrian Institute of Technology (AIT), the largest non-university research institution in Austria. At the Seibersdorf branch of AIT the state-of-art toxicological studies are performed according to the GLP guidelines (Good Laboratory Practice). Following GLP conformed studies with NSC 631570 were performed at the ARCS.
Acute Intravenous Toxicity with Rats
The study was performed in conformance with the EC-Guideline 92/69 and the OECD-Guideline 401. The test substance was administered undiluted as a slow intravenous injection to male and female Him:OFA rats at doses of 33 mg/kg, 57 mg/kg, or 100 mg/kg (only females) body weight (b.w.). The administration induced immediate effects, which, if not lethal, soon lead to an almost complete recovery. Most probable cause of death was a shift in the blood pH or in the blood ion homeostasis. Males were more susceptible than females. Based on the results obtained, the LD50 (intravenous) of the active substance was calculated as 43 mg/kg b.w. for males and 76 mg/kg b.w. for females (151, OEFZS-A-4483, October 1998).
Acute Intravenous Toxicity with Mice
The study was performed according to the Directive 92/69/EEC and the OECD Guideline 401, 1987. NSC 631570 was administered as a slow intravenous injection to three groups of five male and of five female Him:OF1, SPF mice at doses of 33 mg/kg b.w., 74 mg/kg b.w., and 165 mg/kg b.w. All animals in the high dose group and two males and three females in the mid dose group died. All other animals survived until 14 days after administration. All survived animals were normal at the end of the study. All animals were normal at necropsy. There was no marked sex difference in the response to the test substance. Based on the results obtained, the LD50 (intravenous) was calculated as 80 mg/kg b.w. for males and 68 mg/kg b.w. for females (OEFZS-L-0400, May 2000).
Acute Intramuscular Toxicity with Rats
The aim of the study was to reveal acute toxic effects of NSC 631570 after a single intramuscular administration to rats. The study was performed according to the EC-Directive 87/176/EWG and the OECD-Guideline 401, as far as these were useful for intramuscular administration. The test substance was administered undiluted as an intramuscular injection to five male and five female Him:OFA rats at the highest technically feasible dose for the intended route, i.e. 150 mg active ingredient per kg b.w. All animals survived until the scheduled termination of the study. Body weight gain was not impaired. Sedation and less pronounced disturbed locomotion was noted on the day of the administration. Local changes at the injection site (crusts) were present in about half of the animals. There was no sex difference in the response to the test substance. The authors concluded intramuscular injection of NSC 631570 in the highest feasible dose induced some transient signs of minor clinical importance but was otherwise well tolerated and did not become life threatening. Based on the results obtained in this study the LD50 (intramuscular) of NSC 631570 was calculated to be higher than 150 mg/kg b.w. (151, OEFZS-L-0194, October 1999).
Acute Oral Toxicity Study with Rats
This study aimed to investigate acute toxic effects of NSC 631570 after a single oral administration to rats and was performed according to the EC-Directive 92/69 and the OECD Guideline 401. NSC 631570 concentrated was administered undiluted once by stomach intubation to three groups of five female Him:OFA Sprague Dawley rats and to one group of five male Sprague Dawley rats at the doses of 450 mg/kg b.w., 810 mg/kg b.w., and 1500 mg/kg b.w. (females) or 810 mg/kg b.w. (males). All females in the highest dose group died spontaneously on the day of administration. All females in both other dosage groups as well as all males survived until the scheduled termination. All animals of the mid and high dosed groups were affected with most of the signs observed on the day of administration of the test substance. The effects lasted until maximal three days after administration. All surviving animals were normal at the end of the study. There was no relevant sex difference in the response to the test substance.
Hence the test substance induced immediately effects which, if not lethal, led to an almost complete recovery soon. The LD50 (oral, females) was calculated as 1110 mg/kg b.w. (151, OEFZS-L-0195, October 1999).
NSC 631570 was administered as a slow intravenous, intraarterial, paravenous or intramuscular injection to two male and two female rabbits per route to study its local tolerability. Normal saline was administered correspondingly to the other ear or muscle of each animal as a control. NSC 631570 was well tolerated at intravenous and intraarterial administration. The paravenous injection caused mild local irritation, and the intramuscular administration caused a mild to moderate local inflammation. There was no sex difference in the response to the test substance (OEFZS-A-4204, October 1997).
The study was performed to detect the possible production of micronuclei induced by NSC 631570 as a result of chromosomal damage or of damage to the mitotic apparatus in an in vivo test system. NSC 631570 was diluted with normal saline and administered once at doses of 1.25, 2.50, or 5.00 ml/kg b.w. intravenously to three groups of five male and five female Crl:NMRI BR mice each. Additionally, one high dose group of one male and one female spare animal was included to replace possible unscheduled deaths in the high dose group. Two negative control groups (normal saline) and one positive control group (thiotepa) were also included into the study. Preparation of bone marrow cells and investigations were performed in conformance with the OECD Guideline 474.
All animals survived until scheduled sacrifices. Thiotepa (positive control group) caused cytotoxicity and produced micronuclei in polychromatic erythrocytes. NSC 631570 did not cause cytotoxicity. All data were in the range of historical negative control data. There were no significant differences in micronucleated normochromatic erythrocytes (MNE) between the test substance group animals of both sexes and the corresponding negative controls, neither 24 nor 38 hours after administration. Also no statistically significant differences in the amounts of micronucleated polychromatic erythrocytes (MPE) were noted in any sex at any dose used. The rates of MPE were at both sampling times within the limits of historical negative control groups. No sex difference in the response to the test substance was noted. The authors concluded NSC 631570 not to cause cytotoxicity to the bone marrow at doses up to 5 mg/kg b.w. and not to produce micronuclei in the polychromatic erythrocytes in mice of both sexes at the doses used (OEFZS-L-0225, November 1999).
Similar results were obtained in the second micronucleus study with NSC 631570 concentrated (OEFZS-L-0224, November 1999).
Salmonella typhimurium Reverse Mutation Test
NSC 631570 was tested for mutagenic activity in the S. typhimurium reverse mutation test (Ames test) according to the OECD Guideline 471 and the EEC Guideline 92/69, part B14. The test substance was tested at the range of concentrations according to the direct plate incorporation method without and with external metabolisation system S9-mix. The bacterial strains S. typhimurium TA97a, TA98, TA100, and TA1535 were used as test system. Negative and positive controls were included and an independent repetition of the experiment was performed. The test substance was not toxic to the S. typhimurium strains used. According to the results obtained in this study, NSC 631570 is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102, and TA1535 (OEFZS-L-0003, January 1999).
As the source materials for the production of NSC 631570 - celandine alkaloids and thiotepa are toxic for liver cells, a study was initiated to evaluate the possible toxic potential of NSC 631570 to the liver. The study was performed in rats and proved NSC 631570 is not toxic to the rat liver (Müller 2004, original report).
Many other toxicity studies were performed in Eastern Europe (15, 16, 105, 123, 127).
The first toxicity studies were performed on Albino Swiss mice and Wistar rats. NSC 631570 was administered intraperitoneally at the single daily dose of 0.025, 0.05, and 0.1 of LD50 (e.g. 4.75, 9.5, 19 mg/kg in mice and 7, 14 and 28 mg/kg in rats) during three months. The treatment had no effect on the organ weight with except of spleen in rats where the organ weight was up to 3fold higher than in the control group (29). The estimation of catecholamines in brain revealed the three month administration of NSC 631570 at middle and higher dose diminished the dopamine concentration in mice and rats. The concentrations of noradrenalin, 5-hydroxytryptamine and 5-hydroxyindolacetat were not changed (29). The single administration of NSC 631570 had no effect on the serum prolactin concentration in rats. On the contrary, after the three month administration of NSC 631570 the serum prolactin concentration increased in all dose groups compared to the control (32).
Regarding blood count, the administration of NSC 631570 had similar effects in the middle and higher dose groups: increase of white blood cells (WBC) and decrease of platelets. The changes in the differential count were also observed, e.g. lymphocytes increase and neutrophil bands decrease. No significant changes in red blood cells (RBC) were observed compared to the control (33).
Also there were no changes in the transaminase activity in rats. In mice, increase of ALT and AST activity occurred in the middle and higher dose groups. A slight decrease of the total protein was observed in male mice in the groups 9.5 and 19.5 mg/kg, in female mice only in the group 19 mg/kg. The single administration of NSC 631570 did not affect the evaluated parameters (31).
In the experiments with hamster cells it was revealed NSC 631570 is not mutagen as well as not genotoxic and does not cause morphologic cell transformations (19).
Tests in mice and guinea pigs have revealed NSC 631570 not to cause anaphylactic sensitization (23).
In the tests in rats, NSC 631570 was confirmed not to exert embryotoxic or teratogenic effect. In hamsters, the embryotoxic effect was observed at the very high dosage of 28 mg/kg body weight (30).
In a series of experiments the effect of the six week administration of NSC 631570 in rabbits was studied (subacute toxicity).
The daily treatment at three various dosage caused no effect on the organ morphology and body mass of the animals. In blood count, the numbers of red blood cells and white blood cells did not change. In the high dosage group, the percentages of lymphocytes, monocytes, and eosinophils increased. The blood chemistry parameters were not affected apart from increase of urea and uric acid (124).
Among sex hormones, estradiol increased in some groups as did testosterone in the lowest dose male group. In females, progesterone increased only in the highest dose group (125).
NSC 631570 caused the increase of thyroid hormones in males. In females, triiodothyronine increased and thyroxin concentration did not change (126).
In a study in male Wistar rats the researchers revealed that the six day administration of NSC 631570 in both doses of 1 mg/kg and 2 mg/kg caused no significant changes in the following parameters: albumin, mucopolysaccharides, aminotransferase activity, and thymol test. Similarly unaffected compared to the control group was the liver tissue morphology. Significantly lower was the blood concentration of the middle molecules (216).
In the Wistar rats with implanted Guerin carcinoma, NSC 631570 was revealed to reduce the number of thiol groups in the tumor homogenates (121).
Pilot studies of pharmacokinetics gave first suggestions on the methods which should be used in the later studies (105, 127).
To estimate the plasma concentration and pharmacokinetic parameters of NSC 631570 at intravenous administration, first a method for its determination in plasma was developed (152). This enabled consequently the study of pharmacokinetics of NSC 631570 in rats. It was revealed the tumor tissue and the liver had the strongest affinity to NSC 631570. In brain and muscles the NSC 631570 accumulation was the least (153).
The examinations with physical methods (spectrometry, polarized light microscopy) revealed NSC 631570 to be resistant against the UV light of middle intensity. The absorption peaks 210 and 230 nm can be used for the detection of NSC 631570 in plasma (50, 81).
In the tests with the rat liver homogenates the oxygen consumption was detected by means of polarography. The addition of NSC 631570 caused complex redox reactions in the substrate. This was dependant on the NADP induced oxidation (82).
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