Electrochemical Characterization of Redox Probes Confined in 3D Conducting Polymer Networks.

In this manuscript we present a versatile platform for introducing functional redox species into tailor-made 3D redox polymer networks. Electrochemical characterization based on cyclic voltammetry is applied to verify the immobilization of the redox species within the conducting networks. Ultimately this strategy shall be extended to (photo)electrocatalytic applications which will profit from the conducting polymer matrix. Soluble precursor copolymers are synthesized via radical copolymerization of vinyltriphenylamine (VTPA) with chloromethylstyrene (CMS) in different ratios, whereas CMS is subsequently converted into azidomethylstyrene (AMS) to yield poly(VTPA-co-AMS) copolymers. Spin-coating of poly(VTPA-co-AMS) on gold electrodes yields thin films which are converted into stable polymer network structures by electrochemical crosslinking of the polymer chains via their pendant triphenylamine groups to yield N,N,N',N'-tetraphenylbenzidine (TPB) crosslinking points. Finally, the resulting redox-active, TPB-crosslinked films are functionalized with ethynylferrocene (EFc) as a representative redox probe using a click reaction. Main experimental tools are polarization modulation infrared reflection absorption spectroscopy and scan rate dependent cyclic voltammetry. Especially the latter proves the successful conversion and the immobilization of redox probes in the polymer matrix. The results are compared with the reference system of azide-terminated self-assembled monolayers on gold substrates, allowing to distinguish between free and immobilized EFc species.

Correlation of genotype, phenotype, and mRNA expression of CYP2D6 and CYP2C19 in peripheral blood leukocytes (PBLs).

INTRODUCTION: The genetic polymorphism of drug metabolizing enzymes of the cytochrome P450 (CYP) families, especially CYP2D6 and CYP2C19, is the most important cause of variable responses of many drugs. Enzyme activity ranges from complete deficiency, so called poor metabolizers (PMs), to an ultrafast metabolism. While PMs and extensive metabolizers (EMs) can be well distinguished by genotyping, phenotyping is necessary to subdivide EMs from intermediate metabolizers (IMs). The aim of the study was to evaluate if messenger RNA (mRNA) concentration for CYP-enzymes in peripheral blood leukocytes (PBLs) will be predictive of systemic enzyme activity, allowing an easy and safe determination of metabolic activity. METHODS: The genotype, phenotype, and mRNA-expression in PBLs were evaluated in 124 healthy Caucasian volunteers (males and females, age range 23 - 59 years) on three occasions (every 4 weeks). Genotyping was performed by Taqman allelic discrimination on the most common null alleles for CYP2D6 (*3, *4, *6, *7, and *8) and CYP2C19 (*2 and *3). For phenotyping CYP2D6, dextromethorphan/dextrorphan metabolic ratios were determined in collected urine (8 hours) after administration of 30 mg dextromethorphan. For phenotyping CYP2C19, we used the plasma concentration ratio of omeprazole/hydroxyomeprazole 4 hours after ingestion of 40 mg omeprazole. mRNA-expression in PBLs for CYP2D6 and CYP2C19 was measured by Taqman real-time PCR before medication and 4 hours afterwards. RESULTS: Genotyping for CYP2D6 and CYP2C19 showed a regular distribution of EMs and PMs compared to studies of a comparable population. The median dextromethorphan/dextrorphan metabolic ratio was 0.47 in EMs/IMs and 2.29 in PMs. The median omeprazole/hydroxyomeprazole metabolic ratio was 3.06 in EMs/IMs and 35.29 in PMs. CYP2D6 and CYP2C19 mRNA expression was detected without evidence of correlation to the respective metabolic ratio. CONCLUSION: The results do not support the concept of using mRNA expression profiles for CYP2D6 and CYP2C19 enzymes in PBLs for prediction of systemic enzyme activity.

Pharmacodynamic Effects of an Angiotensin II Receptor-Antagonist in Phase I-Comparison between Healthy Subjects and Patients with Hypertension.

UNLABELLED: Biomarkers are increasingly used to provide decision making data early in phase I by showing Proof of Mechanism or Proof of Concept (PoM/PoC). For antihypertensive agents, the administration of multiple doses (md) to hypertensive patients is assumed to be necessary for an early go/no-go decision. We compared the effects of an Angiotensin II receptor antagonist (ARA) on Plasma Renin and blood pressure (BP) following an oral single dose (sd) and once daily md for seven days to healthy volunteers and patients with essential hypertension (diastolic BP 95 mmHg to 114 mmHg; systolic BP 130 mmHg to 200 mmHg). METHODS: 5-12 healthy male subjects/dose received 10 mg to 300 mg ARA sd and 50 to 300 mg md for 7 days; patients (9-10/dose) received 20 mg-400 mg ARA for 7 days. The studies were designed as randomized, single-blind, placebo-controlled, group comparison or crossover dose-escalation studies. Plasma Renin and BP were monitored up to 24 hours after dosing. RESULTS: Plasma Renin showed a high interindividual variability in both healthy volunteers and patients. Healthy subjects showed a dose- and time-related increase in plasma Renin after sd from 40 mg to 300 mg and md of 50 mg to 300 mg (p < 0.05 for doses of 200 mg and 300 mg). In patients, increases in plasma Renin occurred at 8 hours and beyond starting at sd of 100 mg and md of 50 mg (p < 0.05 for the dose of 400 mg). While healthy volunteers showed no relevant decrease in BP, in hypertensive patients a reduction in BP in doses of 100 mg to 400 mg occurred (p < 0.05); effects were more pronounced after md compared to sd. CONCLUSION: Early PoM for an antihypertensive agent can be shown by use of laboratory biomarkers following sd to healthy subjects. PoC can be achieved after sd in hypertensive patients. Administration of sd to healthy volunteers is sufficient for an early go/no-decision.

The applications of biomarkers in early clinical drug development to improve decision-making processes.

Selecting, evaluating and applying biomarkers in drug discovery and exploratory drug development do substantially shorten the time to reach a critical decision point. Biomarkers are most useful in the early phase of clinical development when measurement of clinical endpoints or true surrogates may be too time-consuming or cumbersome to provide timely proof of principle or dose-ranging information. The use of biomarkers in early drug development helps to streamline clinical development by determining whether the drug is reaching and affecting the molecular target in humans, delivering findings that are comparable to preclinical data, and by providing a measurable endpoint that predicts desired or undesired clinical effects. Critical decisions such as candidate selection, early proof of mechanism or proof of concept, dose ranging and patient stratification as well as the assessment of development risks regarding safety, toxicity and drug interactions can be based on measurement of appropriate biomarkers that are biologically and/or clinically validated. Preclinical and phase I development plans can be focused to support an early s.d. or m.d. biomarker study in healthy volunteers or mildly diseased patients, thus saving both resources and time. Dose estimates and patient stratification may reduce the size and duration of clinical studies in later phases of development, and safety and toxicity biomarkers may help to stop or continue a programme early on. Even if a biomarker fails in the validation process there may still be a benefit of having used it as more knowledge about pathophysiology of the disease and the drug may be obtained. Thus, appropriateness of biomarkers depends on the stage of development, development strategy and the medical indication. Examples of biomarkers in exploratory clinical development are given for the development of new drugs in various indications.

Mechanism-based pharmacokinetic-pharmacodynamic modeling-a new classification of biomarkers.

In recent years, pharmacokinetic/pharmacodynamic (PK/PD) modeling has developed from an empirical descriptive discipline into a mechanistic science that can be applied at all stages of drug development. Mechanism-based PK/PD models differ from empirical descriptive models in that they contain specific expressions to characterize processes on the causal path between drug administration and effect. Mechanism-based PK/PD models have much improved properties for extrapolation and prediction. As such, they constitute a scientific basis for rational drug discovery and development. In this report, a novel classification of biomarkers is proposed. Within the context of mechanism-based PK/PD modeling, a biomarker is defined as a measure that characterizes, in a strictly quantitative manner, a process, which is on the causal path between drug administration and effect. The new classification system distinguishes seven types of biomarkers: type 0, genotype/phenotype determining drug response; type 1, concentration of drug or drug metabolite; type 2, molecular target occupancy; type 3, molecular target activation; type 4, physiological measures; type 5, pathophysiological measures; and type 6, clinical ratings. In this paper, the use of the new biomarker classification is discussed in the context of the application of mechanism-based PK/PD analysis in drug discovery and development.

Concentration-effect relationships of alcohol in a computerised psychometric test system.

The purpose of this study was to validate a computerised psychometric test system by demonstrating the change of the test variables of the Bochum Diagnostic System after administration of alcohol. Twenty-four healthy young male or female volunteers participated in a doubleblind, randomised, placebo-controlled study in a 3-way cross-over design. The volunteers took single doses of placebo, 0.4 and 0.7 g alcohol/kg body weight (females: 10% less) in a random sequence. Psychometric tests were performed before as well as 1 and 3 h after the administration of alcohol. The effects of alcohol on the psychometric performance were most pronounced 1 h after administration. At this time a significant dose-dependent impairment of performance in the concentration test, the simple and complex reaction tests, the vigilance test and the coordination test was observed. 3 h after administration, significant effects were only observed in the complex reaction test and the concentration test. Maximal alcohol serum concentrations of 0.47 +/- 0.05 % per thousand and of 0.90 +/- 0.15 % per thousand were reached after administration of 0.4 g/kg and 0.7 g/kg, respectively. The correlations between individual serum alcohol concentrations and differences of psychometric variables from baseline were significant. It is concluded that the tests of the Bochum Diagnostic System can quantitatively measure the effects of alcohol. The most sensitive tests are the complex reaction test and the concentration test.

Influences of caffeine, acetazolamide and cognitive stimulation on cerebral blood flow velocities.

Assessment of cerebral blood flow velocities (CBFV) can be used as a non-invasive tool to evaluate specific drug effects, like caffeine (CAF), acetazolamide (AA) as well as cognition. Their influences on each others CBFV were evaluated in detail, using a randomized, double-blind, double-dummy, placebo-controlled three-fold cross-over study design in 18 right-handed healthy male volunteers. CBFV (maximal, mean, minimal) and pulsatility index of both middle cerebral arteries were recorded by transcranial Doppler ultrasound simultaneously, during a verbal memory test, oral CAF, intravenous AA or placebo. AA led to increase in CBFV of 25-32%. Caffeine resulted in decreased V(mean) and V(min) of 10-13%. Cognitive stimulation resulted in a slight increase of CBVF of about 4%, but was overruled by effects of AA and CAF. We conclude that pharmacological effects can easily be assessed by TCD during clinical pharmacological studies of vasoactive drugs. However intraindividual variability and effects of neuropsychological stimulation needs to be taken into account.

[Clinical-pharmacological aspects to accelerate the development process from the preclinical to the clinical phase/2nd communication: promising strategies]. / Klinisch-pharmakologische Aspekte zur Beschleunigung des Drug Development-Prozesses von der Präklinik zur Klinik. 2. Mitteilung: Erfolgversprechende Strategien.

To improve the transition from research to development a critical evaluation of the individual project by research and disease area teams is required to include input from pharmacology, toxicology, pharmacokinetics, galenics, clinical pharmacology, clinical as well as regulatory experts and marketing. Decisions on the individual development strategy should be made prior to the start of development and all projects should be reviewed at predefined stages throughout the product development life cycle. This ensures consistency of decision-making not only during the development of individual products but throughout the entire development pipeline. Studies in the exploratory stage of drug development should be designed for decision making in contrast to later clinical trials in the confirmatory stage that require power for proof-of-safety and proof-of-efficacy. The more thorough and profound studies have been carried out during this exploratory stage of drug development, the earlier a decision can be made on the continuation or discontinuation of further development, thus saving development time and money and assessing and considerably reducing the risk for the patients and increasing the success rate of the project in the later confirmatory effectiveness trial with an adequate number of subjects receiving the new therapy under typical conditions of use. Strategies which may be helpful to improve the quality of decisions in drug discovery and drug development are: discovery experiments should be done to critically evaluate the compound, the "killer" experiments should be done as early as possible, continuous effort on preclinical disease models is necessary to improve predictability of efficacy in patients ("humanized" research): genomic technology should be used to identify novel, disease-related targets and to characterise preclinical test systems, improvement of knowledge and experience concerning the relevance of new technologies for the clinical picture, genotyping of clinical trial patients to select patient groups which are likely to respond to treatment (pharmacogenomics), modelling and simulation of preclinical and clinical trials, integration of pharmacokinetic and pharmacodynamic principles into drug development, assessment of the interaction potential (CYP-450, trasporter proteins and others), increasing use of biomarker/surrogate marker for rapid clinical feedback, involvement of the target population as soon as possible, applying statistical data analysis techniques for proving effectiveness, co-operation with high quality centers. To reach this goal clinical pharmacology must be fully integrated in the whole process from the candidate selection to its positioning within the market.

[Clinical-pharmacological aspects to accelerate the development process from the preclinical to the clinical phase/1st communication: The contribution of clinical pharmacology]. / Klinisch-pharmakologische Aspekte zur Beschleunigung des Drug Development-Prozesses von der Präklinik zur Klinik.

To improve the transition from research to development a critical evaluation of the individual project by research and disease area teams is required to include input from pharmacology, toxicology, pharmacokinetics, galenics, clinical pharmacology, clinical as well as regulatory experts and marketing. Decisions on the individual development strategy should be made prior to the start of development and all projects should be reviewed at predefined stages throughout the product development life cycle. This ensures consistency of decision-making not only during the development of individual products but throughout the entire development pipeline. Studies in the exploratory stage of drug development should be designed for decision making in contrast to later clinical trials in the confirmatory stage that require power for proof-of-safety and proof-of-efficacy. The more thorough and profound studies have been carried out during this exploratory stage of drug development, the earlier a decision can be made on the continuation or discontinuation of further development, thus saving development time and money and assessing and considerably reducing the risk for the patients and increasing the success rate of the project in the later confirmatory effectiveness trial with an adequate number of subjects receiving the new therapy under typical conditions of use. Strategies which may be helpful to improve the quality of decisions in drug discovery and drug development are: discovery experiments should be done to critically evaluate the compound, the "killer" experiments should be done as early as possible, continuous effort on preclinical disease models is necessary to improve predictability of efficacy in patients ("humanized" research): genomic technology should be used to identify novel, disease-related targets and to characterise preclinical test systems, improvement of knowledge and experience concerning the relevance of new technologies for the clinical picture; genotyping of clinical trial patients to select patient groups which are likely to respond to treatment (pharmacogenomics), modelling and simulation of preclinical and clinical trials, integration of pharmacokinetic and pharmacodynamic principles into drug development, assessment of the interaction potential (CYP-450, trasporter proteins and others), increasing use of biomarker/surrogate marker for rapid clinical feedback, involvement of the target population as soon as possible, applying statistical data analysis techniques for proving effectiveness, co-operation with high quality centers. To reach this goal clinical pharmacology must be fully integrated in the whole process from the candidate selection to its positioning within the market.