Tissue-engineered mucosa is a suitable model to quantify the acute biological effects of ionizing radiation.

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.

Analysis of a sleep-dependent neuronal feedback loop: the slow-wave microcontinuity of the EEG.

Increasing depth of sleep corresponds to an increasing gain in the neuronal feedback loops that generate the low-frequency (slow-wave) electroencephalogram (EEG). We derived the maximum-likelihood estimator of the feedback gain and applied it to quantify sleep depth. The estimator computes the fraction (0%-100%) of the current slow wave which continues in the near-future (0.02 s later) EEG. Therefore, this percentage was dubbed slow-wave microcontinuity (SW%). It is not affected by anatomical parameters such as skull thickness, which can considerably bias the commonly used slow-wave power (SWP). In our study, both of the estimators SW% and SWP were monitored throughout two nights in 22 subjects. Each subject took temazepam (a benzodiazepine) on one of the two nights. Both estimators detected the effects of age, temazepam, and time of night on sleep. Females were found to have twice the SWP of males, but no gender effect on SW% was found. This confirms earlier reports that gender affects SWP but not sleep depth. Subjectively assessed differences in sleep quality between the nights were correlated to differences in SW%, not in SWP. These results demonstrate that slow-wave microcontinuity, being based on a physiological model of sleep, reflects sleep depth more closely than SWP does.

A pharmacodynamic Markov mixed-effects model for the effect of temazepam on sleep.

BACKGROUND: A hypnogram shows how sleep travels through its various stages in the course of a night. The sleep stage changes can be quantified to study sedative drug effects. METHODS: Hypnograms from 21 patients with primary insomnia were collected during a randomized, placebo-controlled crossover study of 20 mg temazepam. A separate daytime session was performed to determine the pharmacokinetics of 20 mg temazepam and its effect on saccadic eye movement and electroencephalogram. A first-order Markov model was developed to describe the probability of sleep stage changes as a function of time after drug intake and time after last sleep stage change. The influence of temazepam concentration on the probability to change sleep stage was incorporated into the model. RESULTS: Transitions between sleep stages were profoundly influenced by the time of the night and by the time since the last change of sleep stage. Temazepam reduced the time spent awake. This effect could be attributed to four mechanisms: (1) transition to "deeper" sleep was facilitated, (2) transition to "lighter" sleep was inhibited, (3) regardless of sleep stage, the transition to wake state was inhibited, and (4) return to sleep was facilitated. Daytime sensitivities to temazepam, measured with the surrogate markers saccadic peak velocity and electroencephalogram beta activity, each correlated with one of the transition probabilities influenced by temazepam. CONCLUSIONS: By the development of a Markov model for these non-ordered six categorical data, the effect of temazepam on the sleep-wake status could be interpreted in terms of known mechanisms for sleep generation and benzodiazepine pharmacology.

Characterization of the pharmacodynamic interaction between parent drug and active metabolite in vivo: midazolam and alpha-OH-midazolam.

The pharmacodynamic interaction between midazolam and its active metabolite alpha-OH-midazolam was investigated to evaluate whether estimates of relevant pharmacodynamic parameters are possible after administration of a mixture of the two. Rats were administered 10 mg/kg of midazolam, 15 mg/kg of alpha-OH-midazolam, or a combination of 3.6 mg/kg of midazolam and 35 mg/kg of alpha-OH-midazolam. Increase in the 11.5- to 30-Hz frequency band of the electroencephalogram was used as the pharmacodynamic endpoint. The pharmacodynamics of midazolam and alpha-OH-midazolam after combined administration were first analyzed according to an empirical and a competitive interaction model to evaluate each model's capability in retrieving the pharmacodynamic estimates of both compounds. Both models failed to accurately estimate the true pharmacodynamic estimates of midazolam and alpha-OH-midazolam. The pharmacodynamic interaction was subsequently analyzed according to a new mechanism-based model. This approach is based on classical receptor theory and allows estimation of the in vivo estimated receptor affinity and intrinsic in vivo drug efficacy. The relationship between stimulus and effect is characterized by a monotonically increasing function f, which is assumed to be identical for midazolam and alpha-OH-midazolam. The pharmacodynamic interaction is characterized by the classical equation for the competition between two substrates for a common receptor site. This mechanism-based interaction model was able to estimate the pharmacodynamic parameters of both midazolam and alpha-OH-midazolam with high accuracy. It is concluded that pharmacodynamic parameters of single drugs can be estimated after a combined administration when a mechanistically valid interaction model is applied.

Relevance of arteriovenous concentration differences in pharmacokinetic-pharmacodynamic modeling of midazolam.

In the present investigation, the extent of arteriovenous concentration differences of midazolam in rats was quantified, and the consequences of these differences on the pharmacodynamic estimates were determined. The arterial concentration-effect relationships where analyzed by a traditional-effect compartment model that characterizes the delay between blood and the effect site with the rate constant k(eo). Venous concentration-effect relationships where analyzed according to the traditional model and an extended-effect compartment model that, by incorporating an additional rate constant k(vo), can characterize the delay between the arterial and venous sampling site. Significant hysteresis was observed in the arterial but not the venous concentration-effect relationships. Rate constants for k(eo), k(vo) and terminal half-life were (mean +/- S.E.M.) 0.32 +/- 0.062, 0.093 +/- 0.013 and 0.0217 +/- 0.0008 min-1, respectively, indicating the existence of significant arteriovenous concentration differences. Pharmacodynamic estimates as determined on basis of the arterial concentrations and the traditional-effect compartment model were EC50 = 104 +/- 1 ng/ml, Emax = 151 +/- 4 microV/sec and gamma = 0.83 +/- 0.06. Analysis of the venous concentration-effect relationships on basis of the traditional- or extended-effect compartment model led to similar pharmacodynamic estimates, indicating that the observed arteriovenous concentration differences did not result in biased pharmacodynamic estimates. This is due to the fact that the effect relevant elimination rate constant of midazolam is relatively small compared with its k(eo). The observed results are consistent with earlier reports based on computer simulations.

Pharmacodynamics of temazepam in primary insomnia: assessment of the value of quantitative electroencephalography and saccadic eye movements in predicting improvement of sleep.

BACKGROUND AND OBJECTIVE: Quantitative electroencephalographic parameters and saccadic eye movements are frequently used as pharmacodynamic measures of benzodiazepine effect. We investigated the relationship between these measures and the hypnotic effect. METHODS: The correlation between the pharmacodynamic measures and sleep quality was determined in 21 patients with primary insomnia. The pharmacokinetic-pharmacodynamic relationships were characterized after administration of 20 mg oral temazepam. The hypnotic effect was determined on the basis of polysomnographic sleep recordings and a subjective sleep evaluation questionnaire. Correlations between pharmacodynamic measures and the improvement of sleep were investigated. RESULTS: The pharmacokinetic-pharmacodynamic relationships for the parameters derived from electroencephalography and saccadic eye movements showed considerable interindividual variability. Administration of temazepam led to a significant improvement in the objective parameters sleep period efficiency, wake time after sleep onset, and sleep efficiency and in the subjective assessment of sleep quality. No significant correlations were observed between the pharmacokinetic-pharmacodynamic-derived parameters and the improvement in objective or subjective sleep parameters. CONCLUSION: In subjects with primary insomnia the administration of 20 mg oral temazepam results in changes in both the pharmacodynamic measures and in quality of sleep. No individual correlations between the pharmacodynamic measures and quality of sleep were observed. We concluded that the investigated pharmacodynamic measures are of value in the first assessment of clinical efficacy and for the selection of the dose(s) to be investigated in subsequent trials that aim at showing clinical efficacy. However, the conclusive quantification of clinical efficacy should be performed only on the basis of the clinical end point itself.

The impact of arteriovenous concentration differences on pharmacodynamic parameter estimates.

In many pharmacodynamic investigations venous drug concentrations are measured and linked to effect-site concentrations by means of a traditional first-order effect-compartment model to estimate pharmacodynamic (PD) parameters. This analysis ignores the underlying physiology that arterial blood supplies both the venous sampling site and effect site. Recently, an extended effect-compartment model has been proposed that reflects physiology by postulating a first-order rate constant of equilibrium between arterial and effect-site concentrations (ke0) as well as first-order rate constant between arterial and venous concentrations (kv0). In the current paper, we evaluate the bias in PD parameter estimates if venous drug concentrations are measured and linked to effect-site concentrations by a traditional effect compartment as a function ke0, kv0, and the drug's elimination half-life (T1/2); we present an analytical solution to the differential equations characterizing the extended effect-compartment model; and we evaluate the performance of the extended effect-compartment model to estimate pharmacodynamic parameters on the basis of venous drug concentrations. Time profiles of venous drug concentrations and drug effect were simulated for a wide range of different values of the half-life of ke0 (T1/2,e0), the half-life of kv0 (T1/2,v0), and T1/2. The simulations showed that a significant bias (up to 90%) in PD parameter estimates occurred for certain values of T1/2,e0, T1/2,v0, and T1/2 if venous drug concentrations are linked to effect-site concentrations by a traditional effect-compartment model. This model misspecification is not apparent from the results of the fitting procedure. The extended effect-compartment model provided unbiased but imprecise PD parameter estimates. The extended effect-compartment model was also able to analyze instances in which the venous concentrations equilibrate slower with the arterial concentrations than the effect-site concentrations, and proteresis is observed in the concentration--effect relationship. It is concluded that if the apparent T1/2 of the drug in the time period in which the decline in pharmacological effect is most pronounced is greater than 5 times T1/2,e0 and T1/2,e0 is greater than T1/2,v0 there is no need to model the underlying arteriovenous equilibrium delay. Under these conditions a traditional first-order link between venous and effect-site concentrations will yield accurate and reliable (less than 10% bias) estimates of the PD parameters such as Emax, EC50 and N. If T1/2 is less than 5 times T1/2,e0 or if T1/2,v0 is greater than T1/2,e0, the underlying arteriovenous equilibration delay needs to be taken into account in the model to obtain unbiased estimates of the PD parameters. This applies for almost all values of T1/2.v0. Arteriovenous equilibration delay can be best taken into account by measuring arterial blood concentrations. If this is not possible, the extended effect-compartment link model can be used. However, a large number of effect measurements needs to be obtained to estimate the model parameters accurately.

Solving inconsistencies in the analysis of receptor-ligand interactions.

According to the occupation model, the observed sigmoidicity in receptor-binding studies frequently yields non-integer values for the number of molecules that bind per receptor, which may suggest inconsistencies of the model. Here, Bertil Tuk and Michael van Oostenbruggen re-examine the derivation of the model and pinpoint the origin of the observed inconsistencies, thereby demonstrating that these inconsistencies may lead to substantial error in estimates of receptor affinity, number of molecules that bind per receptor and cooperativity. A reformulated model allows more information to be derived from receptor-binding experiments, yielding integer estimates of the number of molecules that bind per receptor and quantitatively estimating the degree of cooperativity.

Pharmacokinetic and pharmacodynamic interactions of bretazenil and diazepam with alcohol.

1. Interaction between alcohol and bretazenil (a benzodiazepine partial agonist in animals) was studied with diazepam as a comparator in a randomized, double-blind, placebo controlled six-way cross over experiment in 12 healthy volunteers, aged 19-26 years. 2. Bretazenil (0.5 mg), diazepam (10 mg) and matching placebos were given as single oral doses after intravenous infusion of alcohol to a steady target-blood concentration of 0.5 g l-1 or a control infusion of 5% w/v glucose at 1 week intervals. 3. CNS effects were evaluated between 0 and 3.5 h after drug administration by smooth pursuit and saccadic eye movements, adaptive tracking, body sway, digit symbol substitution test and visual analogue scales. 4. Compared with placebo all treatments caused significant decrements in performance. Overall, the following sequence was found for the magnitude of treatment effects: bretazenil+alcohol > diazepam+alcohol > or = bretazenil > diazepam > alcohol > placebo. 5. There were no consistent indications for synergistic, supra-additive pharmacodynamic interactions between alcohol and bretazenil or diazepam. 6. Bretazenil with or without alcohol, and diazepam+alcohol had marked effects. Because subjects were often too sedated to perform the adaptive tracking test and the eye movement tests adequately, ceiling effects may have affected the outcome of these tests. 7. No significant pharmacokinetic interactions were found. 8. Contrary to the results in animals, there were no indications for a dissociation of the sedative and anxiolytic effects of bretazenil in man.

In-vivo buccal delivery of fluorescein isothiocyanate-dextran 4400 with glycodeoxycholate as an absorption enhancer in pigs.

Buccal delivery of fluorescein isothiocyanate labeled dextran 4400 (FD4) was investigated in-vivo in pigs. The delivery device consisted of an application chamber with a solution of FD4 and was adhered to the buccal mucosa for 4 h using an adhesive patch. A randomized crossover study including intravenous administration and buccal delivery without and with 10 mM sodium glycodeoxycholate (GDC) as absorption enhancer was performed in five pigs. After buccal administration, steady state plasma levels were rapidly reached. Coadministration of 10 mM GDC increased the absolute bioavailability of FD4 from 1.8 +/- 0.5% to 12.7 +/- 2.0%. Since FD4 is a macromolecular and hydrophilic compound such as peptide and protein drugs, buccal delivery would provide an adequate alternative to the parenteral administration of these drugs.

A comparison of the concentration-effect relationships of midazolam for EEG-derived parameters and saccadic peak velocity.

1. Concentration-effect relationships of midazolam were assessed in an open study in six healthy volunteers. Saccadic eye movements and EEG parameters derived by fast Fourier transform (FFT) and aperiodic analysis (AP) were used to quantify drug effects. 2. Midazolam was infused at a rate of 0.6 mg kg-1 h-1 for a maximum of 15 min. Hypnotic effects were avoided by terminating infusions when subjects could no longer perform the eye movement test properly. 3. Wake-sleep transitions could be recognized through frequent observation of eye movements. The dose needed to reach maximum conscious sedation averaged 0.10 mg kg-1, ranging from 0.06 to 0.13 mg kg-1. 4. Sigmoidal concentration-effect relationships were found for EEG beta-amplitudes in five of six subjects, with average EC50 values (+/- s.d.) of 120 +/- 54 ng ml-1 for FFT and 104 +/- 40 ng ml-1 for AP. For the 'total number of waves' in the beta frequency range (AP) an average (n = 6) EC50 of 63 +/- 37 ng ml-1 was found. Changes in EEG alpha-amplitudes were found in three subjects, resulting in an average EC50 value of 55 +/- 32 ng ml-1. 5. For saccadic peak velocity (PV) concentration-effect relationships were linear in five subjects and sigmoidal in one. The maximal measured decrease in PV averaged -44 +/- 9%. 6. The differences in concentration-effect relationships for various effect parameters call for further studies with emphasis on the external validity and reproducibility of data. In such studies the dose needed to reach wake-sleep transition may be used as a relevant clinical end-point.

Pharmacokinetic-pharmacodynamic modeling of the central nervous system effects of midazolam and its main metabolite alpha-hydroxymidazolam in healthy volunteers.

The pharmacodynamics of midazolam and its main metabolite alpha-hydroxymidazolam were characterized in individual subjects by use of saccadic eye movement and electroencephalographic (EEG) effect measurements. Eight healthy volunteers received 0.1 mg/kg midazolam intravenously in 15 minutes, 0.15 mg/kg alpha-hydroxymidazolam intravenously in 15 minutes, 7.5 mg midazolam orally and placebo in a randomized, double-blind, four-way crossover experiment. Plasma concentrations of midazolam, alpha-hydroxymidazolam and 4-hydroxymidazolam were measured by gas chromatography. The amplitudes in the 11.5 to 30 Hz (beta) frequency band were used as EEG effect measure. The concentration-effect relationships were quantified by the sigmoid maximum effect model. The median effective concentrations of midazolam and alpha-hydroxymidazolam were (mean +/- SE) 77 +/- 15 and 98 +/- 17 ng/ml, respectively, for the EEG effect measure. For peak saccadic velocity the values were 40 +/- 7 ng/ml for midazolam and 49 +/- 10 ng/ml for alpha-hydroxymidazolam. The maximum effect values were similar for both compounds. The effects observed after oral administration of midazolam could not be predicted accurately by an additive and competitive interaction model. It seems that alpha-hydroxymidazolam is highly potent with respect to the measured effects and contributes significantly to those effects of midazolam after oral administration.

[Scanning electron microscopy in gastric adenocarcinoma and intestinal metaplasia]. / Microscopía electrónica de barrido en adenocarcinoma gástrico y metaplasia intestinal.

In recent years scanning electron microscopy has been used in gastric biopsy studies, contributing to better recognition of intestinal metaplasia and carcinoma, as a complement to light and transmission electron microscopy. During the second half of 1983, 53 cases of gastric carcinoma were diagnosed at the Department of Pathology of Hospital Mexico, of which six were studied ultrastructurally. A pattern similar to that of intestinal epithelium was found in cases of intestinal metaplasia. Well differentiated adenocarcinomas showed marked tumor cell proliferation with irregular "projections". In poorly differentiated carcinomas, changes were limited to areas where tumor cells invaded the epithelial surface. In summary, scanning electron microscopy is of great help in research and diagnosis of pathologic changes occurring in mucosal surfaces.