Efficient statistical inference for a parallel study with missing data by using an exact method

In a parallel group study comparing a new treatment with a standard of care, missing data often occur for various reasons. When the outcome is binary, the data from such studies can be summarized into a 2 × 3 contingency table, with the missing observations in the last column. When the missingness is neither related to the outcome of interest nor related to other outcomes from the study but it is covariate dependent with the sole covariate being treatment, this type of missing data mechanism is considered as missing at random. In 2016, Tian et al. proposed three statistics to test the hypothesis that the response rate is equivalent for a parallel group study with missing data. The asymptotic limiting distributions of these test statistics were used for statistical inference. However, asymptotic approaches for testing proportions generally do not have satisfactory performance with regard to type I error rate control for a clinical trial with the sample size from small to medium. For this reason, we consider an exact approach based on maximization to provide valid and efficient statistical inference for a parallel group study with missing data. Exact approaches can guarantee the type I error rate and they are computationally feasible in this setting. We conduct extensive numerical studies to compare the performance of the exact approach based on the three statistics for a one-sided hypothesis testing problem. We conclude that the exact approach based on the likelihood ratio statistic is more powerful than the exact approach based on the other two statistics. Two real clinical trial data sets are used to illustrate the application of the proposed exact approach.     

A strategy for preclinical formulation development using GastroPlus as pharmacokinetic simulation tool and a statistical screening design applied to a dog study.

The aim of this paper is to propose a pharmaceutical risk assessment strategy that goes beyond the usual characterisation of a clinical candidate molecule according to the biopharmaceutical classification system (BCS). This strategy was evaluated for a new CNS drug with poor solubility and good permeability. In a first step, GastroPlus was used to simulate the absorption process based on preformulation data. These input data involved a physicochemical drug characterisation including drug solubility measurements in simulated physiological media, as well as permeability determination. Further computer simulations were conducted to determine the sensitivity to changes of selected input values. Thus, oral bioavailability prediction was studied as a function of the particle size and drug solubility. The second part of the presented strategy for preclinical formulation development was to test specially designed formulations in a 2(3) screening factorial plan using the dog as the animal model. The factors were the dosage form, food effect and dose strength. One of the two experimental formulations was a capsule filled with the micronised drug, whereas the other formulation was a surfactant solution of the drug. Accordingly, a worst case formulation was compared with a best case drug solution over the clinically relevant dose range in fasted and fed dogs. The results of the computer simulation indicated that a fraction of the dose is dissolved in the stomach and precipitates partially in the small intestine. The simulation predicted almost full drug absorption during the GI transit time. Interestingly, the simulation implies that stomach drug solubility had little impact on overall fraction absorbed. The results also showed that changes of particle size and reference solubility within two orders of magnitude hardly affected the oral bioavailability. This in silico deduction was subsequently compared with the results of the dog studies. Indeed a surfactant drug solution showed no clear biopharmaceutical superiority over a solid capsule formulation on the average of both dose strengths in fasted and fed dogs. Despite the substantial variability of the in vivo data, the factorial screening design indicated marginal significant interaction between the dose level and feeding status. This can be viewed as a flag for the planning of further studies, since a potential effect of one factor may depend on the level of the other. In summary, the GastroPlus simulation together with the statistically designed dog study provided a thorough biopharmaceutical assessment of the new CNS drug. Based on these findings, it was decided to develop a standard granulate in capsules for phase I studies. More sophisticated formulation options were abandoned and so the clinical formulation development was conducted in a cost-efficient way.     

Drug-excipient compatibility testing using a high-throughput approach and statistical design

The aim of our research was to develop a miniaturized high throughput drug-excipient compatibility test. Experiments were planned and evaluated using statistical experimental design. Binary mixtures of a drug, acetylsalicylic acid, or fluoxetine hydrochloride, and of excipients commonly used in solid dosage forms were prepared at a ratio of approximately 1:100 in 96-well microtiter plates. Samples were exposed to different temperature (40 degrees C/ 50 degrees C) and humidity (10%/75%) for different time (1 week/4 weeks), and chemical drug degradation was analyzed using a fast gradient high pressure liquid chromatography (HPLC). Categorical statistical design was applied to identify the effects and interactions of time, temperature, humidity, and excipient on drug degradation. Acetylsalicylic acid was least stable in the presence of magnesium stearate, dibasic calcium phosphate, or sodium starch glycolate. Fluoxetine hydrochloride exhibited a marked degradation only with lactose. Factor-interaction plots revealed that the relative humidity had the strongest effect on the drug excipient blends tested. In conclusion, the developed technique enables fast drug-excipient compatibility testing and identification of interactions. Since only 0.1 mg of drug is needed per data point, fast rational preselection of the pharmaceutical additives can be performed early in solid dosage form development.     

Validation of an auditory startle response system using chemicals or parametric modulation as positive controls

Neurotoxicity regulatory guidelines mandate that automated test systems be validated using chemicals. However, in some cases, chemicals may not necessarily be needed to prove test system validity. To examine this issue, two independent experiments were conducted to validate an automated auditory startle response (ASR) system. In Experiment 1, we used adult (PND 63) and weanling (PND 22) Sprague-Dawley rats (10/sex/dose) to determine the effect of either d-amphetamine (4.0 or 8.0 mg/kg) or clonidine (0.4 or 0.8 mg/kg) on the ASR peak amplitude (ASR PA). The startle response of each rat to a short burst of white noise (120 dB SPL) was recorded over 50 consecutive trials. The ASR PA was significantly decreased (by clonidine) and increased (by d-amphetamine) compared to controls in PND 63 rats. In PND 22 rats, the response to clonidine was similar to adults, but d-amphetamine effects were not significant. Neither drug affected the rate of the decrease in ASR PA over time (habituation). In Experiment 2, PND 31 Sprague-Dawley rats (8/sex) were presented with 150 trials consisting of either white noise bursts of variable intensity (70-120 dB SPL in 10 dB increments, presented in random order) or null (0 dB SPL) trials. Statistically significant sex- and intensity-dependent differences were detected in the ASR PA. These results suggest that in some cases, parametric modulation may be an alternative to using chemicals for test system validation.     

Random and systematic medication errors in routine clinical practice: a multicentre study of infusions, using acetylcysteine as an example

AIMS: The nature and incidence of errors in prescribing and giving medicines have previously been estimated by trained observers, or by retrospective analysis of incidents in which patients have come to harm. We have examined prospectively in routine clinical practice the concentrations of intravenous infusions of a drug (acetylcysteine) which is given according to a complicated dosing schedule. METHODS: We prospectively collected samples before and, where possible, after the infusion of acetylcysteine in 66 anonymous patients requiring treatment for acetaminophen (paracetamol) overdose in four centres in the United Kingdom. We measured the concentration in each infusion bag, and deduced from the weight of the patient the percentage of the anticipated dose that had actually been given. RESULTS: The experimentally determined dose was within 10% of the anticipated dose in 68 of 184 individual bags (37%), and within 20% of the anticipated dose in 112 bags (61%). Doses in 17 bags were more than 50% from the anticipated doses. In three patients, values in all three bags appeared to be systematically wrong by 50% or more; in a further seven cases, individual bags differed by 50% or more from the anticipated value. The median difference between pre- and post-infusion samples was 0% [interquartile range -5.2% to +14.6%], but 9% showed a disparity of greater than +/- 50%. CONCLUSIONS: Our data suggest that there is large random variation in administered dosage of intravenous infusions. Systematic calculation errors occur in about 5% [95% confidence interval 2, 8%] of cases, and major errors in drawing up in a further 3% [1, 7%], with inadequate mixing in 9% [4, 14%]. While we have no evidence that patients were adversely affected, and while the regime of administration of the drug studied (acetylcysteine) is complicated, these data suggest that the delivered dose often deviates from the intended dose, and that methods of quality control are needed.     

Blood pressure as an example of a biomarker that functions as a surrogate

There are many important uses of biomarkers in drug development. An area of particular interest is the use of biomarkers as surrogate end points. Only a small minority of biomarkers are established surrogate end points. Blood pressure is an example of a surrogate end point accepted by both clinicians and regulators. It was a plausible surrogate because of the large epidemiologic databases demonstrating a correlation between elevated blood pressures and adverse cardiovascular outcomes. That plausibility has been supported, however, by the numerous placebo-controlled outcome studies evaluating several pharmacologically distinct agents that showed an effect on stroke and coronary heart disease outcomes from lowering blood pressure.     

正交试验设计实例分析

正交试验设计是使用正交表来安排多因素、多水平试验，并采用统计学方法分析实验结果的一种实验设计方法。对于多因素、多水平的问题，人们一般希望通过若干次的实验找出各因素的主次关系和最优搭配条件，用正交表合理地安排实验，可以省时、省力、省钱，同时又能得到基本满意的实验效果。因此，这种方法在改进产品质量、优化工艺条件及研发新产品等诸多方面广泛应用。但是，很多研究人员在使用该方法时，有些细节往往容易被忽视。作者以姜黄素的提取为例，具体阐述这一方法的使用和注意事项。     

Developing and testing adaptive treatment strategies using substance-induced psychosis as an example

Decisions concerning treatment changes pervade the management of chronic psychiatric disorders that resist definitive cure, yet empirical evidence for the comparative clinical effectiveness of treatment strategies remains underdeveloped. In this paper we exploit the example of psychosis following substance use to illustrate some new developments in clinical trials design that can provide the most solid evidence base for defining successful strategies. The intent is to explore the strengths and limitations of the methodological approach through a meaningful clinical example, with an emphasis on concepts and issues. Both methodology and clinical science are overviewed.     

The acoustic startle response as a measure of behavioral dependence in rats

A series of experiments was conducted to assess the sensitivity of the acoustic startle response to chronic morphine administration and naloxone-precipitated withdrawal. Rats were implanted with two subcutaneous pellets containing either 75 mg each of morphine or containing only placebo. In experiment 1, withdrawal induced by 0.05–0.2 mg/kg naloxone dose-dependently decreased the magnitude of the startle response. Physical dependence was confirmed by a naloxone-induced acute weight loss seen in morphine-implanted rats, but naloxone had no effect on startle or body weight in nondependent animals. In experiment 2, a modified procedure with fewer trials per session and fewer test days was employed. Naloxone (0.2 mg/kg) given 4–5 days after implantation induced large startle-response decreases in morphine-dependent rats while having no effect in placebo-implanted rats. Post-naloxone saline tests revealed no significant differences in startle between morphine and placebo groups. Startle scores were significantly higher in morphine-implanted rats than in placebo rats during a saline test given 3 days following pellet implantation. In a separate group of animals, however, acute IP injections of morphine from 0.3–10 mg/kg had no significant effect on startle amplitude. The effect of repeated pairings of withdrawal with the startle environment was assessed in experiment 3. Morphine-dependent rats startled significantly less if naloxone injections were given before the startle session than if they were administered 4 h later. Conditioned withdrawal effects, expressed during a final test session when all rats received saline, were observed for the body-weight measure but not for the startle response. These results suggest that the acoustic startle response may be a useful objective measure in evaluating physical dependence produced by substances of abuse.     

RNA as a target for drug design,the example of Tat-TAR interaction

One of the new targets in the battle against HIV-1 infection is the interaction between the viral transactivator and the transactivation response (TAR) element, which is necessary for HIV-1 replication. After an overview of the most recent structural studies of the Tat-TAR system, new TAR-targeted inhibitors are presented in several classes: antisense oligonucleotides, cationic peptides, intercalators and a large class of small RNA binding molecules. The method of library screening of RNA binding ligands in the search for new inhibitors is explained in detail. Inhibition of Tat-TAR interaction is considered as a realistic approach to develop new anti-HIV compounds. The RNA binding molecules in this review also demonstrate that the development of drugs that target RNA will become a feasible goal and that such compounds will be added in the future to the therapeutic arsenal to combat several diseases.     

试验设计的三要素

试验研究中不可缺少的三项内容分别是试验因素、受试对象和试验效应,通常将他们称为试验设计的三要素。如何正确把握三要素是试验设计的关键问题之一。     

Neurotoxicological and statistical analyses of a mixture of five organophosphorus pesticides using a ray design.

Environmental exposures generally involve chemical mixtures instead of single chemicals. Statistical models such as the fixed-ratio ray design, wherein the mixing ratio (proportions) of the chemicals is fixed across increasing mixture doses, allows for the detection and characterization of interactions among the chemicals. In this study, we tested for interaction(s) in a mixture of five organophosphorus (OP) pesticides (chlorpyrifos, diazinon, dimethoate, acephate, and malathion). The ratio of the five pesticides (full ray) reflected the relative dietary exposure estimates of the general population as projected by the US EPA Dietary Exposure Evaluation Model (DEEM). A second mixture was tested using the same dose levels of all pesticides, but excluding malathion (reduced ray). The experimental approach first required characterization of dose-response curves for the individual OPs to build a dose-additivity model. A series of behavioral measures were evaluated in adult male Long-Evans rats at the time of peak effect following a single oral dose, and then tissues were collected for measurement of cholinesterase (ChE) activity. Neurochemical (blood and brain cholinesterase [ChE] activity) and behavioral (motor activity, gait score, tail-pinch response score) endpoints were evaluated statistically for evidence of additivity. The additivity model constructed from the single chemical data was used to predict the effects of the pesticide mixture along the full ray (10-450 mg/kg) and the reduced ray (1.75-78.8 mg/kg). The experimental mixture data were also modeled and statistically compared to the additivity models. Analysis of the 5-OP mixture (the full ray) revealed significant deviation from additivity for all endpoints except tail-pinch response. Greater-than-additive responses (synergism) were observed at the lower doses of the 5-OP mixture, which contained non-effective dose levels of each of the components. The predicted effective doses (ED20, ED50) were about half that predicted by additivity, and for brain ChE and motor activity, there was a threshold shift in the dose-response curves. For the brain ChE and motor activity, there was no difference between the full (5-OP mixture) and reduced (4-OP mixture) rays, indicating that malathion did not influence the non-additivity. While the reduced ray for blood ChE showed greater deviation from additivity without malathion in the mixture, the non-additivity observed for the gait score was reversed when malathion was removed. Thus, greater-than-additive interactions were detected for both the full and reduced ray mixtures, and the role of malathion in the interactions varied depending on the endpoint. In all cases, the deviations from additivity occurred at the lower end of the dose-response curves.     

A review of regulatory risk assessment with formaldehyde as an example

Quantitative risk assessment may vary by over a millionfold depending upon the model used. The EPA currently uses models which project the highest potential risk. Furthermore, there is no consideration of the probability that a chemical is a human carcinogen. Such an approach may result in unrealistically high risk at exposures far below current ambient levels. In the case of formaldehyde, three alternative approaches to risk assessment are examined. One uses the maximum likelihood estimate of the multistage model, another uses the no observable adverse effect level divided by a safety factor of 100, and the third uses a probability estimate that the substance is carcinogenic at typical ambient exposures multiplied by EPA's upper bound estimate. The probability estimate is made from considerations of metabolism and pharmacokinetics, toxicology, short-term tests, animal tests, and epidemiology.     

Preformulation development of recombinant pegylated staphylokinase SY161 using statistical design

The goal of this study was to perform preformulation development of SY161 by using statistical design methods to understand the effects of buffer strength, NaCl concentration, and pH on conformation and stability of the protein. It was also important to elucidate interactions between these factors. A central composite design using a 2-level full-factorial study was performed. Secondary structure was evaluated using circular dichroism. Stability toward unfolding was investigated using high-sensitivity differential scanning calorimetry. Depegylation, aggregation, and protein loss were evaluated using SEC-HPLC with on-line light scattering, at time zero and after a 2-week stability study. Response surface plots clearly show optimal pH, NaCl, and buffer conditions. Interactions between pH and NaCl as well as pH and buffer concentration are observed. T_m is seen to be predictive of SY161 stability. Secondary structure changes were minimal and did not influence stability. Statistical design was very effective in providing an understanding of the effects of the formulation components on SY161 stability.     

Preparation of herbal tea as infusion or by maceration at room temperature using mistletoe tea as an example

Herbal tea can be prepared by infusion or maceration at room temperature resulting in different compositions of extractable constituents, which possibly influences the mode of action or safety profile. Knowledge on this topic is limited. The aim of this study was to investigate the substantial differences between infusion and maceration as recommended preparation methods for the preparation of herbal mistletoe tea, a traditional remedy against cardiovascular diseases. No active substances are known but analytical marker substances such as proteins, triterpenoids, phenylpropane derivatives and flavonoids can be quantified within the herb and the different herbal tea preparations. Whereas phenylpropane derivatives were completely extracted by infusion and maceration, neither method dissolved viscotoxins. 43% of mistletoe lectins were extracted by maceration, whereas by infusion they are inactivated by thermal degradation. By contrast, oleanolic acid and betulinic acid are present in higher concentrations in infusates compared with macerates, but even infusion extracted less than 2%. Infusion extracted 43% of flavonoid-like substances and maceration only 31%. In conclusion this study determines some differences between both extraction methods on the profile of solved substances. The relevance of it should be determined in studies dealing with the efficacy of herbal mistletoe tea.     

A modified rabbit ear chamber and an example of its application for intravital-microscopic study on acute effects of topical thermal stimulation

A pre-existing plastic rabbit ear chamber was modified to be applicable for intravital-microscopic observations in pharmacological as well as pathophysiological studies on inflammation. The chamber consists of a base disk, a mica (or glass) cover-slip and a holder ring. The base disk has a round-table with a central protrusion in which a heat conductor of platinum wire is so implanted near the protrusion that thermal stimulation can be given to the regenerated tissue between the table and the coverslip.     

The use of mechanistic DM-PK-PD modelling to assess the power of pharmacogenetic studies -CYP2C9 and warfarin as an example

AIM: To assess the power of in vivo studies needed to discern the effect of genotype on pharmacokinetics (PK) and pharmacodynamics (PD) using CYP2C9 and (S)-warfarin as an example. METHODS: Information on the in vitro metabolism of (S)-warfarin and genetic variation in CYP2C9 was incorporated into a mechanistic population-based PK-PD model. The influence of study design on the ability to detect significant differences in PK (AUC(0-12 h)) and PD (AUEC(0-12 h) INR) between CYP2C9 genotypes was investigated. RESULTS: A study size of 90 (based on the natural abundance of genotypes and uniform dosage) was required to achieve 80% power to discriminate the PK of (S)-warfarin between wild type (*1/*1) and the combination of all other genotypes. About 250 subjects were needed to detect a difference in anticoagulant response. The power to detect differences between specific genotypes was much lower. Analysis of experimental comparisons of the PK or PD between wild-type and other individual genotypes indicated that only 21% of cases (20 of 95 comparisons within 11 PD and four PK-PD studies) reported statistically significant differences. This was similar to the percentage expected from our simulations (20%, chi(2) test, P = 0.80). Simulations of studies enriched with specific genotypes indicated that only three and five subjects were required to detect differences in PK and PD between wild type and the *3/*3 genotype, respectively. CONCLUSION: The utilization of prior information (including in vivo enzymology) in clinical trial simulations can guide the design of subsequent in vivo studies of the impact of genetic polymorphisms, and may help to avoid costly, inconclusive outcomes.