| Objective: To observe the pharmacokinetic characteristics, toxicity reaction, toxictarget organs and probable delayed toxicity for NT injection in Beagle dogs after single orrepeated intravenous infusion administration. Provide a basis for the clinical application ofNT injection.Methods:18Beagle dogs in pharmacokinetic study were randomly arranged into3groups,3per sex in each group. The dosages were low dose group (5mg/kg), middle dosegroup (15mg/kg) and high dose group (45mg/kg). Animals in middle dose group wereadministrated for four continuous days and other groups were administrated as a single dose.The duration for intravenous infusion was set as30min by infusion pump. In the first andthe last administration for mid group, about1ml blood was collected respectively beforeand after dosing at5,15,30,35,45,60,90,120,150,180min from subcutaneous vein (theforelimb or hind legs). Plasma was separated from blood through the centrifugal and usedto detect drug concentration.40Beagle dogs were used in long-term toxicity study; animalswere randomly divided into5groups,4per sex in each group. The5groups were controlgroup (saline), excipients control group, low dose group (10mg/kg), middle dose group (30mg/kg) and high dose group (90mg/kg). Anmimals in the normal saline control group wereinfused by0.9%sodium chloride injection and animals in excipients control group wereinfused by the vehicle of the NT injection. The dosing volume was9ml/kg and the durationof intravenous infusion was set as30min by infusion pump. Animals were dosed once dailyfor28continuous days. The physiological signs were detected, including body weight, bodytemperature, electrocardiogram, ophthalmology, blood counts, coagulation function, bloodbiochemistry, urinalysis and so on. Animals in high dose group were euthanasia after dosingfor4days as the servere toxicity symptom were observed.3animals per sex in each groupwere euthanasia on day29. The other2animals (1per sex) in each group were stoppeddosing for4weeks recovery observation. The gross observation and histopathologyevaluation for dead or euthanasia animals were conducted after necropsy.Results: The pharmacokinetic characteristics were similar between male and female. After single dosing, the half-time for low, mid and high dose group were respectively2.74±0.95,4.44±2.27and11.00±2.24min. Cmax were respectively927.87±187.74,4458.84±1259.01,21260.54±10915.83ng/ml. AUC0-180minwere respectively385.03±89.93,1657.16±535.55,9227.954301.73h ng/kg. Cmax and AUC were increased with the doselevel increase in the dose range of5-45mg/kg. The dosage ratio among3groups was1:3:9.The ratio of Cmax and AUC0-180minwere respectively1:4.81:22.91and1:4.30:23.97.After4days continuous dosing, the half-time for midlle group was11.24±10.80min, Cmaxand AUC0-180minwere respectively2539.73±703.48ng/ml and1407.59319.57h ng/kg, themean accumulation index was0.90. In long term toxicity study, one animal in high dosegroup died on day5after dosing and the remaining7animals were euthanasia based onmoribund. Abnormal reaction related to dose administration was not observed in the lowdose group and mid dose group except one animal in mid dose group, the adverse reactionincluding overmany saliva, leftovers, little or no waste, languid, dental debris, flush gum,overmuch eyes secretion were noted for this animal. Adverse symptom from respiratorysystem, nervous system and gastrointestinal tract were noted on day2after dosing for allanimals in the high dose group: respiratory system, nervous system and gastrointestinaltract. Main abnormal reaction was shortness of breath, difficulty breathing, abdominalbreathing, overmuch nasal secretion, languid, prone position, Weakness, spasm, soft orloose stools, bloody stools, nausea, vomiting, filthy anus, little or no waste, leftovers,overmuch eyes secretion. These clinical symptoms showed an increasing trend withextended dosing time. Animals in this group were died or euthanasia on day5. Duringthe study, there was no toxicological change noted in low and middle dose group forbody weight, body temperature, electrocardiogram, eye exam and urinalysis. Bloodcounts, coagulation function and blood biochemical in low dose group did not showsignificance toxicological change. Comparing with normal saline control group animalsin the5thday after dosing, the body weight and temperature in high dose group werereduced. ECG waveform for some animals was abnormal, such as high P wave. This wasconsistent with the right atrium and ventricular dilatation. However, the clear pathologicalchanges in right atrial and ventricular were not found by histopathology examination. AST,CK results appeared normal. Comparing with normal saline control group, Mono increasewas noted on the5th,14thand28thday in middle dose group, and also noted in high dosegroup on the5thday. FIB increase was noted on the5th,28thday in middle dose group.FIB, APTT was increased and PT was decresed in high dose group on5thday. In low dose group, TP was increased on the14th,28thday, GLB was increased on the5th,14th,28thday, and A/G was decreased on the14th,28thday. In high dose group, A/G wasdecreased and ALP, TP, GLB, TCH was increased on the5thday. The other bloodcounts and blood biochemistry results did not show any significance toxicologicalchange in middle and high dose group. The histopathology evaluation showed thatswollen dark red or red and white lung with or without the foamy liquid in trachea,smaller thymus and spleen were noted for8animals in high dose group on5thday(necropsy day). The morphology characteristices under the microscopic were expansionand hyperemia in alveolar walls capillaries, the visible serous exudate in alveolarcavities. There were a large number of red blood cells, a certain amount of cellulose, asmall amount of neutrophils and alveolar macrophages. The lymphocyte in the thymus,spleen, lymph node showed exinanition/necrosis accompanied by the decrease in thenumber of lymphocytes. At the end of the dosing and recovery period, the test articlerelated pathological changes were not noted in low and middle dose group. The Cmaxand AUC（0-60min）increased with the increasing dose level in the dose range of10~90mg/kg. The ratio of Cmaxand AUC（0-60min）among3groups were respectively1:4.98:15.08and1:5.60:19.50after the first dosing. The ratio of Cmaxand AUC（0-60min）between low and middle dose group were respectively1:4.35and1:4.83af ter dosingfor28days. Both of the ratios of Cmaxand AUC（0-60min）between the first and28thdosingwere in the range of0.90~1.3. Accumulation was not observed obviously. During thedosing period,1animal in excipients control group,2animals in low dosing group and2animals in middle dosing group appeared swelling on dosing site(lasted only1day), theother animals were normal. The incidence and duration of the swelling was not relatedto dose level. The result of microscopic examination for dosing site showed that veininflammation happened in every group. In the normal saline control group and high dosegroup, subcutaneous bleeding was found. The incidence and degree of pathological changesfor vein inflammation and subcutaneous bleedingis were similar in each group and was notconsidered as dose level related. It should be a mechanical damage caused by the needle.Conclusion: NT was fast eliminated in Beagle dogs. Blood drug concentration wasincreased with the increase of dose level. The blood drug concentration increase rate isgreater than the dose level increase rate. But there was no accumulation and no genderdifferences. The safety dose for repeated dose (4weeks) NT injection was10mg/kg for Beagle dogs. The lethal dose was90mg/kg. The main toxicity symptom under high dosewas polypnea, pant, abdominal respiration, dyspnea, lie prostrate, weakness, spasm, soft orloose stool, hematochezia, nausea, vomit, dirty crissum, leftovers, shorter PT wave, extendAPTT wave, Mono, FIB, TP and GLB increasing, A/G reducing. Toxic reaction in themiddle dose group could disappear after4weeks recovery. The specific toxic targetorgan was lung and possible toxic target organs were thymus, spleen and lymph nodesafter repeated dose NT injection to Beagle dogs for4weeks. |
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