| QO-58, one of pyrazolo[1,5-a]pyrimidin-7(4H)-ones(PPOs), is a novelopeners of KCNQ2 /3 potassium channels and have been found to haveinhibitory effect on the sodium channel. Preliminary pharmacodynamics studysuggests that it has the effect of anticonvulsants, antiepileptic and analgesia.Because of QO-58’ poorly water-soluble, research and development of thelysine salt QO-58 lysine salt was synthesized to improve its solubility. In orderto evaluate QO-58 lysine preclinical safety, this research will study itssubchronic toxicity and toxicokinetics, which will provide guidance for furtherpre-clinical toxicity research and the design of clinical trials.Part 1 The subchronic toxicity of QO-58lysineObjective: Observe the subchronic toxicity of QO-58 lysine after4-weeks repeated oral administration in SD rats to provide the reference for itsforward research and development.Methods: Sprague-Dawley rats(80), weighing 180-220 g. The animalswere observed for general condition for 7 d during the quarantine andacclimation period to confirm that there were no abnormalities. The animalswere divided into four groups(each group contained 10 animals per sex) atrandom by sex and weight, then they were kept in separate cages.QO-58 lysine,from small, medium and large dose group of 66, 200, 600 mg/kg wasrespectively given to the test groups, and the control group was given forsolvent of 0.5% CMC-Na. Above all animals were administered by oralgavages for the volume of 2 ml / 100 g based on individual daily body weightsonce daily for 4 weeks. During the administration and recovery period,appearance signs and activities of animals were observed daily. Body weightand food consumption for each rat was measured once a week. The 24 h afterlast dosing, 12 rats(16 animals per sex) from each group were dissected, andthe changes in the hematology and clinical chemistry indexes between the testgroups and the control group were observed. Then gross anatomy andhistopathological examination was carried out. Following necropsy, the heart,lungs, liver, spleen, kidney, adrenal, thymus, testis, epididymis, uterus, ovariesand brain were individually isolated and weighed to calculate the ratios oforgan weight to body weight. The rest of the animals were dissected after the2-week recovery period and were examined of the above indexes. Dataprocessing and statistical analysis were performed using by SPSS 13.0software and Excel 2003.Results1 General situation: Because of my unskilled intragastric operation orexcessive anesthesia, individual death occurred in each group. Compared tocontrols, the test group exhibited no obvious changes in external appearance,behavior and daily activities. With regard to food consumption or bodyweights, rats tested for all dosages showed no significant difference from thosein the control group during the administration and recovery period.2 Hematology: After 4-week repeated low-, medium- and high-doseQO-58 lysine, compared with the control group, monocyte percentage(MO%)and the prothrombin time(PT) following 4 weeks exposured to the low doseof QO-58 lysine were significantly increased compared with those of controlrats(P < 0.05), mean corpuscular hemoglobin(MCH) significantly decreased(P < 0.05). After 2 weeks recovery period, MO % is still on the high side(P <0.05) and the other parameters returned to normal. The indicators ofmedium-dose were no significant difference(P > 0.05). MO% and PT ofhigh-dose group were obviously increased(P < 0.05), neutrophil percentage(NE%) significantly decreased(P < 0.05) and platelet count(PLT)significantly increased(P < 0.05) after 2 weeks recovery period. Due to theincrease of MO% with a dose-response relationship, animals may beassociated with inflammatory reaction.3 Biochemistry: After 4-week repeated low-, medium- and high-doseQO-58 lysine, compared with the control group, animals showed a significantincrease in total bilirubin(T-BIL)(P < 0.01) a decrease in albumin(ALB)(P< 0.05) of low-dose group. Animals of the medium-dose group after 4-weekoral administration showed an obvious decrease in aspartate aminotransferase(AST), creatinekinase(CK), chloride(Cl-) and potassium(K+)(P < 0.05 or P< 0.01) and an obvious increase in T-BIL and glucose(GLU)(P < 0.01, P <0.05) compared to control animals. With respect to the high-dose group,animals only showed an indecrease in T-BIL(P < 0.01) and ALB(P < 0.05).After a 2-week recovery period, only there were obvious decrease in CK andAST(P < 0.05) and the changes were considered to have no biologicalsignificance.4 Organ weight and ratios of organ weight to body weight: After 4-weekrepeated low-, medium- and high-dose QO-58 lysine, with regard to organweight from both male rats and female rats, there were no significantdifferences between the test group and the control group. But ratios of heartweight to body weight from male rats of high-dose group were smaller thanthe control group(P<0.05). After a 2-week recovery period, kidney weightsfrom female rats of low-dose group showed a obvious decrease(P<0.05),adrenal weights from male rats of medium-dose group and heart weights frommale rats and spleen weights from female of high-dose group all showed asignificant increase compared with the control group(P<0.05). Ratios of heartweight to body weight from male rats of low-dose group were bigger(P<0.05)and ratios of spleen weight to body weight from female rats of high-dosegroup were smaller(P<0.05) than the control group.5 Histopathology: After 4-week repeated low-, medium- and high-doseQO-58 lysine, animals of control group appeared different levels of trachea andpneumonia inflammatory cells infiltration, spleen medullary hematopoiesisand individual liver, heart and kidney lesions. Animals of low-, medium- andhigh-dose appeared spleen medullary hematopoiesis(58.3%-83.3%), tracheainflammatory cells infiltration( 58.3%-91.7%), and bronchial lunginflammatory change(25%-58.3%)after a 4-week oral administration. Fromthe incidence and degree there were no significant difference compared withcontrol group. These lesions had been reduced or disappeared after a 2-weekrecovery period. Because the above changes were also found in control group,and there was no dose-response relationship, those changes might beassociated with drug delivery operation and were not caused by drugs.Conclusion: After a 4-week oral administration of QO-58 lysine, eachgroup appears inflammatory changes in the trachea, lung, which might beassociated with gastric operation, and no obvious drug-related toxicity isfound. But the phenomenon that T-BIL rises in dose dependent and heartweight increases is needed to pay attention in the future research.Part 2 The toxicokinetics of QO-58lysineObjective: Study the toxicokinetics of QO-58 lysine after single andmultiple(4 weeks) oral administration and analyze the change rule of plasmaconcentration and distribution of accumulation of QO-58 lysine in rats.Methods:1 Sprague-Dawley rats(80), weighing 180-220 g. The animals wereobserved for general condition for 7 d during the quarantine and acclimationperiod to confirm that there were no abnormalities. The animals were dividedinto four groups(each group contained 10 animals per sex) at random by sexand weight, then they were kept in separate cages. Drug dose and groupingwere consistent with the subchronic toxicity study. The first day ofadministration, 10 rats(5 animals per sex) taken from each group randomlywere used to do the single administration toxicokinetics. At 0 h beforeadministration and 1 h, 5 h, 10 h, 15 h, 24 h, 48 h after administration, a totalof seven points, the blood samples were obtained from inner canthus of ratscontinuously. After the blood samples of last point were taken, all rats werekilled and its main organs were take out, such as heart, liver, lung, spleen,kidney, stomach, muscle, brain, fat and bladder. The organs were detectedwhether there was still a drug distribution in vivo after homogenate. The restof rats(40) were administered by oral gavages for 4 weeks consecutively atthe same period of subchronic toxicity experiment. At 0 h beforeadministration and 1 h, 5 h, 10 h, 15 h, 24 h, 48 h after administration, a totalof seven points, the blood samples were obtained from inner canthus of ratscontinuously. After the blood samples of last point were taken, all rats werekilled and its main organs were take out, such as heart, liver, lung, spleen,kidney, stomach, muscle, brain, fat and bladder. The organs were detectedwhether there was still a drug distribution in vivo after homogenate.2 The plasma concentration of QO-58 lysine was detected by HP-LC/MS/MS method. LC conditions: Phenomenex C18 chromatographiccolumn(50 x 2.0 mm, 3μm), a 35 ℃ column temperature, acetonitrile- 10 m M ammonium acetate solution(53:47) for the mobile phase, a 0.3m L·min-1flow rate, a 5 μL injection volume;MS conditions: electrospray ion source,positive ion ionization mode, MS scan mode of more reactive ion monitoring.AUC0-48, Cmax and Tmax of toxicokinetics parameters were computed byDAS3.2.6 software.Results:1 The linear range of blood concentration of QO-58 lysine was 0.05~250mg/L, and standard curve equation was Y=135000X-211( r=0.9959), thelower limit of quantification(LLOQ) was 0.05 mg/L; Intra- and inter-dayprecisions of low concentration(0.1mg/L) were well(RSD<20%) andaccuracy was within ±15%. Accuracy and intra- and inter-day precisions ofmedium concentration(25 mg/L) and high concentration(250 mg/L) wereboth well(RSD<15%). The matrix effects and the recoveries of extractioncould satisfy the requirement of analytic method. The analyte was found to bestable after 15 days storage at-20℃, after three cycles of freeze(-20℃) andthaw(room temperature), and after stay 2 h at room temperature.2 Animal sexes had no obvious effect on drug maximum concentration(Cmax) and area under the curve(AUC), that is difference between male andfemale is not obvious. The results of Cmax and AUC from both the male andfemale rats of different groups were similar and AUC is linearly related to thedose. But the value of Cmax, Tmax and AUC from repeated dosage test wassmaller than that from a single dosage test. The results of organizationconcentration distribution after 48 h showed that drug left in the liver andkidney and other major organs were not found obvious residue withdrawalafter 48 h.Conclusion: This experiment determined the QO-58 lysine toxicokineticsparameters by HPLC/MS/MS method for the first time, and the method has astrong specificity, high sensitivity and easy to operate. The value of Cmax andAUC of QO-58 lysine was significantly bigger than that of QO-58 compound,which shows absorption by oral admonition of QO-58 lysine is better than thatof QO-58 compound. The toxicokinetics parameters of a single dosage andrepeated dosage test are different, suggesting that this drug may have influenceon the liver metabolic enzymes. The results of toxicokinetical tissuedistribution and a linear result of AUC related to the dose show that the drugdoes not cause accumulation phenomenon in the body. |
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