动物实验,QTc间期 z-bo 2020-08-26 576

　　Introduction: The occurrence of pro-arrhythmic effects after the use of non-antiarrhythmic drugs is an important reason for drug development and development. At present, mitigation measures are in place, relying on the extension of the QTC interval as a surrogate indicator to assess the risk of drug-induced torsades de pointes. Compounds with affinity and inhibitory power have a weakening effect on hERG-mediated potassium currents. In preclinical studies, human therapeutic concentrations or super-therapeutic concentrations are used. If the effect on cardiac repolarization is not clearly understood, they will eventually be discarded. Numerous experiments in vitro and in vivo are used for drug screening and safety pharmacological evaluation, but very little information has guiding significance for the species, experimental conditions and clinical relevance. Sensitivity, specificity and predictive value have not been verified. In addition, limited attention has been paid to the basic concentration exposure relationship. Different from traditional empirical research, preclinical in vitro and in vivo models of drug development are necessary to predict the prolonged QT interval in humans when using therapeutic concentrations. This requires not only insight into the inherent properties of the compound, but also insight into the impact of interspecies differences on the QTc interval itself. Using the concentration-effect relationship of the compound and the relative drug concentration (up to the therapeutic dose) can accurately explain this difference. It is important to realize that the drug concentration-effect relationship varies significantly between species. Recently, Holzgrefe et al. used non-rodents extensively to study the impact of interspecies differences on QTc. Their analysis highlights the interspecies differences of QTc. The results showed that: using a correction factor based on the QT / RR relationship, the observation between the peak concentration time of the drug and the species after administration found similar changes in the QTc interval. Considering the significance of the biosafety assessment of non-human primates, the purpose of the investigation was to quantitatively evaluate the interspecies difference of moxifloxacin in cynomolgus monkeys, dogs and humans in causing QTc interval prolongation. This comparison is important because cynomolgus monkeys have similar QRc to humans and are the preferred species for humans in preclinical studies.

　　Method: "Retrospective" evaluation-reference compound (moxifloxacin) cynomolgus monkey experimental protocol Through crossover test (n=8), blood samples and ECG data were collected from awake telemetry cynomolgus monkeys. The oral dose of moxifloxacin was tested in four groups of 0 mg to 90 mg/kg, and 0 mg and 90 mg/kg were selected for the current study. Cynomolgus monkey venous blood samples were collected at 1, 2, 4, 8 and 24 hours after the drug to study the pharmacokinetics of moxifloxacin. The weight range of cynomolgus monkeys is 2.8-6.8 kg. The research was approved by the institutional ethics committee and was conducted in accordance with ethical standards and GLP procedures.

　　Experimental operation in dogs and humans: It has been published earlier that moxifloxacin has pro-arrhythmic effects on dogs and healthy subjects. The clinical data of moxifloxacin was obtained from a phase I trial of 137 healthy volunteers in a two-way crossover positive control group, single-blind, randomized, and placebo-controlled, all at a dose of 400 mg.

　　Dog, cynomolgus monkey and healthy volunteer sample biological analysis: The method for determining the serum concentration of monkey moxifloxacin was carried out according to the method described by Watson et al. Details of the bioanalysis of moxifloxacin are described in Chain and Dubois.

　　Cynomolgus monkey experiment "prospective" evaluation candidate molecule (nce05): Collect blood samples and ECG data from awake telemetry cynomolgus monkeys. Six monkeys (3 females, 3 males, weighing 2.8-6.8 kg) were taken orally in four doses: 0, 25, 40 and 80 mg/kg. The nce05 was investigated from the first 3 dose groups. After administration, blood samples were collected at 1.5, 4, 8 and 24 hours for pharmacokinetic analysis.

　　Human clinical trials: The first clinical phase, a randomized, double-blind, placebo-controlled, parallel group single-center study to evaluate the safety, tolerability, pharmacodynamics and pharmacokinetics of nce05. Doses of 1, 4, 14 or 30 mg were tested in four treatment groups.

　　Cynomolgus monkey biological sample analysis: Collect approximately 400 μL of blood at each sampling point, collect it in a tube containing heparin, and place it on ice. Serum was prepared by centrifugation for 30 minutes (3200g, 5min, 4°C). The serum was transferred to a 1.5ml EP tube and stored in a refrigerator at ?70°C for analysis.

　　Sample analysis of healthy volunteers: Collect blood samples from the forearm using vacuum blood collection tubes containing K2 EDTA. Blood was collected by centrifugation for 10 minutes at 4°C within 30 minutes. The serum is collected and frozen at -20°C for analysis. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to determine the total concentration of compounds in plasma.

　　Preclinical ECG recording: Preclinical pharmacodynamic data is collected by awake telemetry animals. Obtain ECG records by implanting radio telemetry equipment. The implantation process uses aseptic surgical techniques.

　　Clinical operation: 12-lead ECG is used for ECG monitoring, and Marquette ECG equipment is used to measure QT, RR, and HR. The subject was kept in a supine position when the ECG record was collected.

　　Data analysis: pharmacokinetic (PK) data: pharmacokinetic modeling and deconvolution techniques are used to interpolate and interpolate the concentration at the time of ECG assessment. The in vivo research and clinical pharmacokinetic data analysis of moxifloxacin in dogs adopts a nonlinear mixed effects model method. In contrast, the deconvolution method was used to assess the concentration of nce05 and moxifloxacin in individual cynomolgus monkeys. It should be noted that for the purpose of the current analysis, the sample of the lower limit of quantification is set to zero. In addition, to prevent numerical difficulties in the parameter evaluation process, the drug concentration of all nce05 research groups of 1 mg was also set to zero. The characteristics of the pharmacokinetic parameter model of moxifloxacin are shown in the table.

　　pharmacokinetic-pharmacodynamics (PKPD) model: PKPD model includes three components: individual correction factor for RR interval, diurnal variation of baseline QTc value and concentration-effect relationship oscillation function captured by linear function. Equation: QT=QTC0RRα+Acos(2π(t)/24)+slopeC. QTC 0 is the baseline QTc correction alone, and RR is the time interval between consecutive R waves. α is the correction factor of the individual heart rate, A is the amplitude of the circadian rhythm, t is the time, and φ is the phase, which is a linear concentration-effect relationship. slope [ms/nM] is the linear concentration-effect relationship, and C is the drug concentration at the QT measurement time. This type of parameter distinguishes between system and drug specific properties. Therefore, these parameters can be used to compare drug properties across species without the need for further correction factors. The electrocardiograms of dogs and monkeys are recorded in a continuous manner, the data is filtered to ensure the feasibility of the data set size, and the clinical data is jointly analyzed. Make the absorption, peak and elimination phases appear accurately in the balance data set.

　　Probability of QT interval prolongation: the threshold of QTc interval prolongation at a preset concentration of 10 ms, as a measure to compare drug effects across species. The size of this threshold is based on the assumption that if there are differences in sensitivity between species, it can explain the potential impact of pro-arrhythmic activity on the human body. The analysis uses the slope and the correction factor between the individual gender and the step function to analyze the formula. P≥10ms(atC)=step(0.00001F(Gender)slope10ms/C) 0.00001 is set to an arbitrarily small number to avoid calculation errors. 10 milliseconds is the threshold for QT interval prolongation, C is the drug concentration, and slope is per unit The drug concentration increases QT. Draw a probability curve for each compound. The probability that the QT interval of this concentration prolongs ≥ 10ms is 0.5 (Cp50), which is determined by linear regression.

　　Result: Pharmacokinetics: As described in the method described above, the drug concentration during ECG measurement is estimated or interpolated by population pharmacokinetic model or deconvolution. This technology provides the best data description and reduces the impact of uncertainty in personal predictions of poor PKPD concentration accuracy.

　　PKPD model: ECG measurement is for modeling, combined with predicting drug concentration. The diagnostic criteria and the goodness-of-fit graph show the performance of the comparative model for the three species. The specific parameters of the system (baseline QTc (qtc0), QT-RR index correction factor (α), amplitude (A) and phase (Ф) show the values of the two drugs in the same range in dogs and monkeys. Species and compounds The main difference is found in drug-specific parameters (ie slope slope). The QT-RR correction factors (α) obtained from moxifloxacin and nce05 differ significantly from person to person. On the other hand, the same Compared with sifloxacin, the slope of nce05 is not significantly different from zero, and no increase in QT was observed in the concentration range.

　　Interspecies comparison: The use of animal models to predict the effects of drugs on humans requires systematic identification of specific attributes. Therefore, the choice of model parameters is critical. In this system, the difference between specific parameters, drug-specific parameters and their respective variability is allowed. Although the evidence for the two compounds is limited, analysis of the system-specific physiological parameters of each species showed similar estimates. In contrast, these parameters vary significantly between different species.

　　describes a general model of drug action, which provides important advantages in terms of parameterization from the perspective of drug development. A goodness-of-fit graph is summarized to describe the relationship between the drug concentration and the probability that the QT interval prolongs ≥ 10 milliseconds. From the three probability curves of moxifloxacin, it can be seen that there is a significant change in the relationship between PKPD between preclinical animals and humans. Dogs and monkeys have lower Cp50 values. In addition, the slope of the relationship between drug concentration and QT interval is also different between different species of the same drug.

　　Discussion and conclusion: Our research found that the animal model method can be used to quantitatively evaluate the inter-species difference of moxifloxacin on QT interval prolongation. After comparing the effects of the drug on dogs and humans, the method was confirmed in a second non-clinical species The feasibility. From the perspective of drug development, the availability of a common PKPD model, taking into account the clinically relevant exposure range, can meet the pharmacological evaluation of the drug's human impact (QT interval extension). The use of reference compounds and new candidate molecules and a clear distinction between drug-specific and system-specific parameters. Our research results show that there are essential differences in the PKPD relationship between dogs and cynomolgus monkeys, which makes preclinical animal tests directly transformed into drug effects on humans, which is challenging.

　　PKPD model: The potential predictive tools of PKPD model for drug safety evaluation are limited. Our results show that the effect of moxifloxacin on QT interval prolongation occurs in different exposure ranges of dogs, cynomolgus monkeys and humans. These data can be integrated, as shown in the figure below, to estimate the probability that the QT interval will extend ≥ 10 milliseconds. Contrary to popular beliefs about the sensitivity of drug safety in pre-clinical animal testing, the human probability curve clearly shows a steep increase. Each compound within the therapeutic concentration range indicates a QT interval prolongation of ≥10 at this level. The risk of MS. In contrast, prolonged QT interval is more likely to occur in dogs and cynomolgus monkeys than in humans, indicating the difference in sensitivity of QT interval prolongation across species.

　　Drug screening experiment operation: The peak concentration of moxifloxacin is only three times different between cynomolgus monkeys and humans, and an 86-fold difference is observed for the new molecule nce05. This difference in exposure affects the comparison of PKPD relationships and may lead to other adverse events, thereby masking the main pharmacological (QT interval prolongation) effect. In addition, if the investigated compound shows an affinity that exceeds a specific ion channel, multiple interactions and potential antagonism may occur. The external source of variation, food intake in preclinical operations (usually 4 hours after administration) can greatly affect the QT interval, derived from the data of moxifloxacin. The RR difference was corrected and reduced most food-induced QT reduction. However, the remaining differences may mask the intrinsic effects of the candidate molecule, resulting in a small lengthening. Another important design aspect is the sampling plan, sampling interval and frequency to express the correct pharmacokinetic characteristics. For example, the nce05 standard experimental design has great limitations, such as the lack of samples 1.5 hours after the first dose, the highest peak concentration may occur during a period of time during the absorption process. The lack of this information impairs the estimation of peak levels and possible maximum drug effects.

　　Translational pharmacology: It can be seen that the difference in the concentration-effect relationship of the slope parameter, the probability of a difference in the corresponding approved therapeutic dose at the exposure level reaches ≥ 10MS to increase the QTc interval. More specifically, the slope of the linear concentration effect relationship between moxifloxacin species difference in cynomolgus monkeys (0.0016 ms/nm) and humans (0.0039 ms/nm) is smaller than that of dogs (0.00056). On the other hand, it is well known that there are differences in the concentration-effect relationship between different species in the steady-state process, such as different diurnal changes, differences in target expression and/or transmission mechanisms. Interspecies QTc interval prolongation has been widely investigated. From a drug development perspective, it provides opportunities for modeling and simulation of prospective evaluations using new compounds. A limiting factor in these analyses is that the model cannot be reused for any other compound or class of compounds.