Chemical stability of reactive skin decontamination lotion (RSDL®).

Reactive Skin Decontamination Lotion (RSDL®) is used for the decontamination of Chemical Warfare Agents and Toxic Industrial Compounds after dermal exposure. It has to be stockpiled over a long period and is handled in all climatic zones. Therefore stability is an essential matter of concern. In this work we describe a study to the chemical stability of RSDL® as basis for an estimation of shelf life. We analysed RSDL® for the active ingredient 2,3-butandione monoxime (diacetylmonooxime, DAM), the putative degradation product dimethylglyoxime (DMG) and unknown degradation products by means of a reversed phase high pressure liquid chromatography (HPLC). Calculations were done according to the Arrhenius equation. Based on the temperature dependent rate constants, the time span was calculated, until defined threshold values for DAM and DMG subject to specification and valid regulations were exceeded. The calculated data were compared to the ones gathered from stockpiled samples and samples exposed during foreign mission. The decline of DAM followed first order kinetics, while formation of DMG could be described by zero order kinetics. The rate constants were distinctively temperature dependent. Calculated data were in good accordance to the measured ones from stockpile and mission. Based on a specified acceptable DAM-content of 90% and a valid threshold value of 0.1% (w/w) for the degradation product DMG, RSDL® proved to be stable for at least four years if stored at the recommended conditions of 15°C-30°C. If continuously stored at higher temperatures shelf life will decrease markedly. Therefore RSDL® is an object for risk orientated quality monitoring during storage.

Generating Virtual Patients by Multivariate and Discrete Re-Sampling Techniques.

PURPOSE: Clinical Trial Simulations (CTS) are a valuable tool for decision-making during drug development. However, to obtain realistic simulation scenarios, the patients included in the CTS must be representative of the target population. This is particularly important when covariate effects exist that may affect the outcome of a trial. The objective of our investigation was to evaluate and compare CTS results using re-sampling from a population pool and multivariate distributions to simulate patient covariates. METHODS: COPD was selected as paradigm disease for the purposes of our analysis, FEV1 was used as response measure and the effects of a hypothetical intervention were evaluated in different populations in order to assess the predictive performance of the two methods. RESULTS: Our results show that the multivariate distribution method produces realistic covariate correlations, comparable to the real population. Moreover, it allows simulation of patient characteristics beyond the limits of inclusion and exclusion criteria in historical protocols. CONCLUSION: Both methods, discrete resampling and multivariate distribution generate realistic pools of virtual patients. However the use of a multivariate distribution enable more flexible simulation scenarios since it is not necessarily bound to the existing covariate combinations in the available clinical data sets.

Prediction of disease progression, treatment response and dropout in chronic obstructive pulmonary disease (COPD).

PURPOSE: Drug development in chronic obstructive pulmonary disease (COPD) has been characterised by unacceptably high failure rates. In addition to the poor sensitivity in forced expiratory volume in one second (FEV1), numerous causes are known to contribute to this phenomenon, which can be clustered into drug-, disease- and design-related factors. Here we present a model-based approach to describe disease progression, treatment response and dropout in clinical trials with COPD patients. METHODS: Data from six phase II trials lasting up to 6 months were used. Disease progression (trough FEV1 measurements) was modelled by a time-varying function, whilst the treatment effect was described by an indirect response model. A time-to-event model was used for dropout RESULTS: All relevant parameters were characterised with acceptable precision. Two parameters were necessary to model the dropout patterns, which was found to be partly linked to the treatment failure. Disease severity at baseline, previous use of corticosteroids, gender and height were significant covariates on disease baseline whereas disease severity and reversibility to salbutamol/salmeterol were significant covariates on Emax for salmeterol active arm. CONCLUSION: Incorporation of the various interacting factors into a single model will offer the basis for patient enrichment and improved dose rationale in COPD.

Analysis of the relationship between age and treatment response in migraine.

In migraine, headache severity varies with age. As a consequence, the effectiveness of medication may also depend on a patient's age. The purpose of this study was to assess the combined effect of age and drug treatment on headache characteristics. Using data from clinical trials of sumatriptan in adolescents and adults, we show how the interaction between age and drug exposure can be parameterised as a covariate on a Markov model that describes the decline of headache severity over three clinically defined stages (no relief, relief and pain-free status). The model explains important clinical observations: (i) the rates at which the pain relief and pain-free status were attained were found to be inversely related to age; (ii) in placebo-treated patients, the mean transit time from 'no relief' to 'relief' is 3 h for young adolescents and increases to 6 h for patients aged >or= 30 years; and (iii) sumatriptan reduces the transit time to 2 h, irrespective of age. These findings indicate that the therapeutic gain over placebo increases with age. Prospective studies of antimigraine drugs should take this relationship into account when extrapolating efficacy data from adults to adolescents.

Prediction of attack frequency in migraine treatment.

Usually limited information about the frequency of migraine episodes is derived from acute migraine trials. However, the design of some studies is such that they also provide relevant information about the attack frequency without the bias associated with patient expectations of treatment effect between attacks during prophylaxis trials. Using clinical data from repeated migraine attacks treated with placebo, naratriptan 2.5 mg or sumatriptan 100 mg, we show that attack and interictal periods can be described by a random probability distribution. Based on a gamma distribution, the mean interval between attacks was estimated to be 24 (17-34) days for placebo, 23 (18-29) days for naratriptan 2.5 mg and 22 (17-28) for sumatriptan 100 mg. These findings suggest that the interictal interval is not affected by abortive treatment with triptans. Interpretation of these results may be limited by the study type, yet the method represents a new tool for the evaluation of disease dynamics and treatment effect in the prophylaxis of migraine.

Prediction of headache response in migraine treatment.

Triptans are efficacious for the acute treatment of migraine attacks. Yet, defining a concentration-effect relation for these compounds is difficult as the dynamics of the migraine attack are not thoroughly understood. The objective of this investigation was to develop a disease model to predict measures of headache in randomized placebo-controlled clinical trials investigating oral sumatriptan as a paradigm compound. A hidden Markov model based on the states of response (no relief, relief, and pain free) and headache scores (observed variable) was used in conjunction with population pharmacokinetics. Model parameters were capable of predicting the course of headache relief, pain-free status and headache recurrence. It was shown that sumatriptan shortens mean transit times between states by up to 5 h. The potency of sumatriptan (EC(50)) was 9 ng/ml. These findings demonstrate the value of combining pharmacokinetic and efficacy information to model disease and characterize time-independent drug properties in a population of migraineurs.

Transdermal iontophoretic delivery of apomorphine in patients improved by surfactant formulation pretreatment.

The objective of the present study is to evaluate the efficacy and the safety of transdermal iontophoretic delivery of R-apomorphine, a potent dopamine agonist, in combination with surfactant pretreatment in patients with advanced Parkinson's disease. Iontophoresis patches were applied in 16 patients for 3.5 h, with 0.5 h of passive delivery followed by 3 h of current application at a current density of 250 microA/cm2. Eight of these patients were treated with a surfactant formulation prior to iontophoresis. The pharmacokinetics, pharmacodynamic effects, systemic and local side effects of R-apomorphine were assessed. The plasma concentration vs. time profiles upon iontophoresis of R-apomorphine were described successfully by a novel pharmacokinetic model. The model suggests that only 1.9% of the dose that has been released from the patch accumulated in the skin. The patients treated with the surfactant formulations showed a statistically significant increase of bioavailability (from 10.6+/-0.8% to 13.2+/-1.4%) and of the steady state input rate (from 75.3+/-6.6 to 98.3+/-12.1 nmol/cm2 h) compared to the control patients (iontophoresis without absorption enhancers). In five out of eight patients in the study group and in three out of eight patients in the control group, clinical improvement was observed.

Photonic nanopatterns of gold nanostructures indicate the excitation of surface plasmon modes of a wavelength of 50-100 nm by scanning near-field optical microscopy.

Scanning near-field optical microscopy images of metal nanostructures taken with the tetrahedral tip (T-tip) show a distribution of dark and bright spots at distances in the order of 25-50 nm. The images are interpreted as photonic nanopatterns defined as calculated scanning near-field optical microscopy images using a dipole serving as a light-emitting scanning near-field optical microscopy probe. Changing from a positive to a negative value of the dielectric function of a sample leads to the partition of one spot into several spots in the photonic nanopatterns, indicating the excitation of surface plasmons of a wavelength in the order of 50-100 nm in metal nanostructures.

Enhanced light confinement in a near-field optical probe with a triangular aperture.

We present a probe concept for scanning near-field optical microscopy combining the excellent background suppression of aperture probes with the superior light confinement of apertureless probes. A triangular aperture at the tip of a tetrahedral waveguide (full taper angle approximately 90 degrees ) shows a strong field enhancement at only one rim when illuminated with light of suitable polarization. Compared to a circular aperture of equivalent size, the resolution capability is doubled without loss of brightness. For a approximately 60 nm sized triangular aperture, we measured an optical resolution <40 nm and a transmission of approximately 10(-4).

A set-point model with oscillatory behavior predicts the time course of 8-OH-DPAT-induced hypothermia.

Agonists for the 5-hydroxytryptamine (HT)(1A) receptor induce a hypothermic response that is believed to occur by lowering of the body's set-point temperature. We have developed a physiological model that can be used to predict the complex time course of the hypothermic response after administration of 5-HT(1A) agonists to rats. In the model, 5-HT(1A) agonists exert their effect by changing heat loss through a control mechanism with a thermostat signal that is proportional to the difference between measured and set-point temperature. Agonists exert their effect in a direct concentration-dependent manner, with saturation occurring at higher concentrations. On the basis of simulations, it is shown that, depending on the concentration and the intrinsic efficacy of a 5-HT(1A) agonist, the model shows oscillatory behavior. The model was successfully applied to characterize the complex hypothermic response profiles after administration of the reference 5-HT(1A) agonists R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT) and S-8-OH-DPAT. This analysis revealed that the observed difference in effect vs. time profile for these two reference agonists could be explained by a difference in in vivo intrinsic efficacy.

Hyperplasia of proventricular duct epithelium in broilers.

Spontaneous non-leukotic tumours of the proventriculus are regarded as rare neoplasms of the digestive tract in domestic fowl. In a broiler flock, however, a high incidence of hyperplasia of glandular tissue in the proventriculus has been observed. The condition was correlated with a rather high mortality and condemnation rate. Macroscopically the proventriculi appeared swollen and had thickened, firm walls. Microscopically the epithelial cells lining the ducts of the proventricular glands showed a marked hyperplasia, and several ducts were conspicuously convoluted and formed prominent stellate lumina. Neither proliferation of glandular epithelial cells into the surrounding tissue nor metastases to other organs were detected, and the condition was classified as a hyperplasia of the duct epithelium. The aetiology of the hyperplasia and the striking mortality is unknown, although the presence of a toxic factor(s) in the litter of the chicken-house cannot be entirely ruled out.

Evaluation of resistance to Marek's disease in three white Plymouth Rock lines and in two reciprocal crosses.

Two White Plymouth Rock lines (WPR), both developed into lines resistant for Marek's disease (MD), and one commercial WPR line, supposedly susceptible to the disease, were mated in a number of combinations to evaluate resistance to MD in their pure- and crossbred progeny. For this purpose the pedigreed 1-day-old progeny chicks were exposed to 3-week-old spreader chicks which had been inoculated at 1-day-old with virulent MD virus (MDV) and reared with them for the whole experimental period of 15 weeks. Response to the challenge showed that resistance to MD in the cross-breds was intermediate to that of resistant and susceptible parental lines. It appeared that, besides a sex effect in the male progeny, the trait for MD resistance was most effectively transferred to the crossbred progeny by the resistant male parents. The rather high level of resistance to MD observed in the progeny of crossmatings between resistant males and susceptible females suggested that resistant males can be employed for upgrading MD resistance in (commercial) stock.

Age related resistance to avian leukosis virus. II. Influence of age at inoculation on mortality and congenital transmission.

Six age groups of specified pathogen free White Leghorn chickens housed in the same filtered air, positive pressure building, were inoculated at 1 day, 2, 4, 6, 8 and 10 weeks of age respectively, with a mixture of leukosis viruses of subgroups A and B. Birds which died during the experiment were examined for gross and microscopical lesions. The incidence of lymphoid leukosis (LL) in the various groups was inversely proportional to the age of first virus exposure, Le., mortality was 62.5% in the group inoculated at 1-day-old and decreased to zero in the group inoculated at 10 weeks of age. In the age group inoculated at 8 weeks, only one chicken succumbed to the disease. Congenital transmission of leukosis virus (LV) was detected only in chickens inoculated immediately after hatching or at 2 weeks of age. In the latter group, all leukosis virus positive embryos were derived from one hen only. A seventh age group, housed in a separate filtered air, positive pressure building, served as uninoculated controls. Challenge infection was performed on the same day with chickens of all groups at ages varying from 59 to 71 weeks. Testing of pooled embryo extracts collected between 1 and 15 weeks post challenge did not change the virus shedding pattern. Even the chickens of the control group which were challenged (for the first time) at 71 weeks of age remained negative for congenital transmission. The results of this experiment show that the chickens inoculated with LV at 4 weeks of age or older had a low incidence of LL (decreasing to zero when the chickens were inoculated at 10 weeks of age) but did not congenitally shed virus.

Age related resistance to avian leukosis virus. III. Infectious virus, neutralising antibody and tumours in chickens inoculated at various ages.

Viraemia and neutralising antibodies were determined in chickens of six age-groups following inoculation with leukosis virus of subgroups A and B at the age of 1 day, and 2, 4, 6, 8 and 10 weeks respectively. The birds were kept in a filtered air positive pressure (FAPP) house. A seventh age-group, accommodated in a separate FAPP-house, was used as an untreated control. Serum samples, received at biweekly intervals between 1-17 weeks post-inoculation, from birds of the groups inoculated at 4, 6, 8 and 10 weeks of age, showed at 1 week post-inoculation a transient viraemia followed by neutralising antibodies at the later sampling times. Neutralising antibody to subgroup A virus was detected in nearly all birds tested; this was not so for antibody to subgroup B. In all four groups the average titre of the former antibody was higher than that of the latter. Midway through the laying period birds of each group inoculated with leukosis virus, and some of the uninoculated controls, were challenged by infection with either subgroup A or B virus. At termination of the experiment survivors from each group were tested for the presence of leukosis virus. The virus recovery was performed with plasma samples, white blood cell preparations and explant cultures of various organs. The plasma samples were all negative; the great majority of blood cell specimens received from birds inoculated early with leukosis virus were positive, whereas the majority of the preparations from the birds inoculated later remained negative. The organ explants from the two youngest age groups were mostly leukosis virus-positive, from the birds inoculated at 4 weeks of age the spleen and kidney explants contained leukosis virus whereas in the groups inoculated at 6, 8 and 10 weeks of age only the spleen explants of birds challenged with subgroup A virus In a subsidiary experiment, started 4 months after the challenge infection, four birds from each group (two challenged with leukosis virus of subgroup A and two with subgroup B) were accommodated in isolators. The birds were challenged again, this time with Rous sarcoma virus (RSV) of the homologous subgroup used for the previous challenge. The tests for virus just prior to the challenge showed leukosis virus only in the white blood cell preparations from the birds in the three youngest age groups; the birds from the older groups were virus-negative. The serological tests after challenge showed neutralising antibodies to both subgroups in birds of nearly all groups. Tumour formation at the site of injection was mainly observed in the chickens challenged with RSV of subgroup B. The virological and serological results as well as the tumour response show that the immune system of birds between 0-4 weeks of age is insufficiently developed to cope with a controlled exposure with leukosis virus, whereas in birds of 4-10 weeks of age an adequate immunological response has developed. The significance of the presence of leukosis virus in sera, plasma, white blood cell preparations and organ explant cultures is mentioned. In programmes for the control of lymphoid leukosis in reproductive stock the use of information on virus and neutralising antibodies is recommended.

Horizontal transmission of lymphoid leukosis virus. Influence of age, maternal antibodies and degree of contact exposure.

Eight groups of 1-day-old or 8-week-old chickens were exposed by contact to lymphoid leukosis virus (LLV) infection. Five groups of about 60 spf chickens were used. Three groups of the same size were progeny from LLV vaccinated hens. Five groups were housed in one chicken house in close contact with a large number of immunologically tolerant chickens (virus "spreaders"). On two occasions infectious LLV was recovered from air/dust samples collected in this house. In the second house a small number of congenitally infected birds generated a mild degree of LLV exposure. It was demonstrated that infection by contact may lead to lymphoma formation and congenital virus transmission. The incidence of virus infection and LL mortality in the groups of birds exposed at 8 weeks of age were significantly lower than in chickens exposed at 1 day of age. In addition, about 100-fold differences in numbers of LLV-associated white blood cells were observed between both age groups. These results indicate that in addition to resistance to tumour formation, resistance to LLV infection develops in the chicken with increasing age. Maternal antibodies, present in three groups exposed at 1 day of age, reduced the rate of infection and the incidence of LL.

The development of two White Plymouth Rock lines resistant to Marek's disease by breeding from survivors.

A programme to develop resistance to Marek's disease (MD) in chickens in two non-inbred White Plymouth Rock (WPR) lines by breeding from survivors was initiated in 1968 and since then nine generations have been produced. In each generation only sires and dams which survived heavy exposure to virulent MD virus (MDV), Dutch strain K, either by inoculation (for the first two generations) or by contact-exposure (for the following seven generations) were used. In this long term trial a high level of resistance to MD was reached within five generations; thereafter no marked further increase in resistance could be obtained. Susceptibility to MD, reflected by MD mortality in each generation, was expressed as % incidence of MD. Statistical evaluation of MD susceptibility in the nine generations of the two lines revealed a significant interaction between MD mortality and two sources of variation, sex and generation. No overall line effect was found, but there was a significant line x generation interaction. The remaining interactions were not significant. Results indicate that breeding from survivors for development of resistance to MD is feasible. Exposure of birds to either artificial (by injection) or contact challenge with virulent strain K MDV changed lines of birds highly susceptible to MD into highly resistant lines in a few generations. Complete resistance to MD was not attained.

Age-related resistance to avian leukosis virus. I. Influence of age at exposure on mortality and congenital transmission.

Groups of White Leghorn chickens were inoculated at 1 day and at 2, 4, 6 and 8 weeks of age respectively with a mixture of leukosis viruses of subgroups A and B. The five infected groups were kept in a filtered air positive pressure house. A sixth group was accommodated separately in a similar house as a control. All birds which died or were removed were subjected to pathohistological examination; diagnosis of lymphoid leukosis was made upon either gross lesions plus microscopical lesions or microscopical lesions only. The incidence of lymphoid leukosis in the infected groups appeared inversely proportional to age of infection, i.e. the mortality due to lymphoid leukosis decreased from 54.3% in the group infected at 1-day-old to 7.4% in the group infected at 8 weeks of age. Prevalence of leukosis in the latter group may be attributed to a small number of chicks already infected vertically with the virus. Congenital transmission of leukosis virus was demonstrated in embryos in the groups infected at 1-day-old, 2, 4 and 6 weeks of age. In the latter group congenital transmission was extremely low; from 214 pooled embryo extracts (1007 embryos) only 2 (0.9%) contained leukosis virus. In the group infected at 8 weeks of age no virus was detected in the embryos. Congenital transmission of leukosis virus appeared to be related to age of infection, i.e. early infection went parallel with a high rate of transmission. The pattern of congenital transmission was erratic and the number of hens shedding leukosis virus continually was small. From the results in this trial it is concluded that both lymphoid leukosis and congenital transmission occur rarely if chickens (born free of leukosis virus) are kept free from infection during the first 6-8 weeks of life and subsequently are injected with a relatively high dose of leukosis virus.

The control of lymphoid leukosis in a flock White Plymouth Rock chickens.

Lymphoid leukosis (LL) was successfully controlled in a commercial basic breeding line of White Plymouth Rock chickens. The control method has been developed for breeder flocks and consists of three elements: --In the flock under study, homogenates of embryos from all eggs collected during a number of 14-day periods are tested for the presence of LL viruses. --Only eggs from hens that have been shown not to shed virus in their eggs are used for the production of progeny. The offspring are reared in isolation during the first two months of life, at which time the age-related resistance against tumour formation by LL viruses appears to be sufficiently developed. --The chickens are subsequently inoculated intramuscularly with LL viruses of subgroups A and B transferred to a conventional chicken house. The vaccination raises a solid immunity to horizontal LL virus exposure and, due to the age-related resistance, tumour formation does not follow. No excretion of LL viruses could be detected in three generations of White Plymouth Rock chickens to which the three elements of the control procedure were applied. Clinical disease was not observed in any of the chickens under notice.

The control of lymphoid leukosis in a flock White Plymouth Rock chickens.

Summary Lymphoid leukosis (LL) was successfully controlled in a commercial basic breeding line of White Plymouth Rock chickens. The control method has been developed for breeder flocks and consists of three elements: - In the flock under study, homogenates of embryos from all eggs collected during a number of I4-day periods are tested for the presence of LL viruses. - Only eggs from hens that have been shown not to shed virus in their eggs are used for the production of progeny. The offspring are reared in isolation during the first two months of life, at which time the age-related resistance against tumour formation by LL viruses appears to be sufficiently developed. - The chickens are subsequently inoculated intramuscularly with LL viruses of subgroups A and B transferred to a conventional chicken house. The vaccination raises a solid immunity to horizontal LL virus exposure and, due to the age-related resistance, tumour formation does not follow. No excretion of LL viruses could be detected in three generations of White Plymouth Rock chickens to which the three elements of the control procedure were applied. Clinical disease was not observed in any of the chickens under notice.

Quantitative pathogenicity of Marek's disease virus and vaccine virus strains in chickens: macroscopical aspects.

Two virulent strains (JM and K) and one vaccine strain (CVI 988) of Marek's disease virus (MDV), together with two vaccine strains of the herpesvirus of turkeys (HVT) (FC 126 and PB-THV 1), all in the cell-associated state, were administered intramuscularly at 3.7 log TCID50 per dose to day-old SPF White Leghorn chickens. A control group of chicks received uninfected cells. The pathological parameters studied were onset and duration of clinical symptoms, mortality, bird weight and macroscopical lesions of peripheral nerves and visceral organs. Data were obtained from females autopsied at the age of 3, 8 and 20 weeks, and from chickens which died. Virological and serological data were procured mainly from males taken at various ages. The results indicate a clear distinction between virulent and vaccine strains. MD vaccines had no significant influence on bird weight and caused no mortality or macroscopical lesions, whereas the virulent MDV strains produced all these effects. Macroscopical lesions caused by the virulent MDV strains were seen predominantly in nerves (in about 50% of birds succumbing to MD) and gonads (in 0% to 80% of such birds depending on sex and on strain of MDV). Differences between the two virulent strains could be demonstrated. Strain JM induced earlier incidence and shorter duration of clinical disease. With strain JM death occurred earlier in females than in males. Strain K caused significantly more macroscopical lesions in gonads, heart and liver. Under the conditions of the experiment, detection of macroscopical lesions after inoculation with a virulent MDV strain was possible 3 weeks after inoculation.