Reproductive toxicity and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Possible effects on the next generation after long-term exposure (subcutaneous administration) of male rats to very high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. Two dose regimes were applied: TCDD-25 (initial dose: 25 g/kg body wt; maintenance dose: 5 g/kg body wt, once weekly) and TCDD-75 (initial dose: 75 g/kg body wt; maintenance dose: 15 g/kg body wt). Male rats were treated for 10 weeks before mating and then throughout the entire 12 week mating period. They were mated to unexposed virgin females. One group of pregnant females was used for teratological evaluations, and another group was allowed to deliver. No significant differences were observed in the number of implantations or fetuses per litter, and resorption rate, and fetal weight between the controls and TCDD-treated groups. No gross-structural anomalies occurred in any of the fetuses sired by TCDD-treated males. In the TCDD-25 group an increased frequency of two types of variations was observed which also occur in controls: incompletely ossified fingers (TCDD-25=5.1%, controls=2.6%), and incompletely ossified ossa zygomatica (TCDD-25=1.8%, controls=0.5%). In the TCDD-25 group a slight but statistically significant increase was observed in the rate of stillbirths (TCDD-25=1.3%, controls=0.1%), apparently due to an unusually low frequency occurring in the controls (overall historical controls=0.6%). There was no difference in postnatal mortality (TCDD-25=1.3%, controls=1.3%). Taken together, despite the very high doses of TCDD used, the data do not provide evidence for biologically significant paternally-mediated developmental toxicity in the fetuses and newborn.     

Population PK modelling and simulation based on fluoxetine and norfluoxetine concentrations in milk: a milk concentration-based prediction model

Aims

Population pharmacokinetic (pop PK) modelling can be used for PK assessment of drugs in breast milk. However, complex mechanistic modelling of a parent and an active metabolite using both blood and milk samples is challenging. We aimed to develop a simple predictive pop PK model for milk concentration–time profiles of a parent and a metabolite, using data on fluoxetine (FX) and its active metabolite, norfluoxetine (NFX), in milk.

Methods

Using a previously published data set of drug concentrations in milk from 25 women treated with FX, a pop PK model predictive of milk concentration–time profiles of FX and NFX was developed. Simulation was performed with the model to generate FX and NFX concentration–time profiles in milk of 1000 mothers. This milk concentration-based pop PK model was compared with the previously validated plasma/milk concentration-based pop PK model of FX.

Results

Milk FX and NFX concentration–time profiles were described reasonably well by a one compartment model with a FX-to-NFX conversion coefficient. Median values of the simulated relative infant dose on a weight basis (sRID: weight-adjusted daily doses of FX and NFX through breastmilk to the infant, expressed as a fraction of therapeutic FX daily dose per body weight) were 0.028 for FX and 0.029 for NFX. The FX sRID estimates were consistent with those of the plasma/milk-based pop PK model.

Conclusions

A predictive pop PK model based on only milk concentrations can be developed for simultaneous estimation of milk concentration–time profiles of a parent (FX) and an active metabolite (NFX).     

A population model of early rheumatoid arthritis disease activity during treatment with methotrexate,sulfasalazine and hydroxychloroquine

Aims

To develop a population model describing the disease activity (DAS28) time course in patients with early rheumatoid arthritis (RA) treated with triple disease-modifying anti-rheumatic drug (DMARD) therapy (methotrexate, sulfasalazine and hydroxychloroquine).

Methods

DAS28 was obtained in 263 patients with early RA from initiation of therapy until 60 weeks. Using nonmem®, base models (DAS28 vs. time) and covariate influences were investigated for the population.

Results

The best model was an exponential model of DAS28 vs. time that was additive to baseline DAS28, with covariance between parameters, and a combined residual error model. Age and patient smoking history were covariates significantly affecting response to therapy. Population estimates were baseline DAS28 (5.7), extent of change in DAS28 (−2.8) and the half-life of disease activity (6.2 weeks; time to steady disease state achieved within approximately 30 weeks). Older individuals exhibited more severe baseline DAS28, described by a power function centred around 57 years (baseline DAS28 for 40- and 70-year-old patients were 5.4 vs. 5.8, respectively) and current smokers took longer to achieve a steady disease state (approximately 50 weeks). There was considerable within-patient random variability in DAS28 over time (empirical 90% CI for DAS28 in a population typical patient at 60 weeks: 1.8, 4.2 with median value of 2.8).

Conclusions

This is the first report of a disease activity model for early RA treated with triple DMARD therapy. Smoking and age were identified as covariates.     

Clinical endpoint sensitivity in rheumatoid arthritis: modeling and simulation

The commonly used efficacy endpoints in Rheumatoid Arthritis (RA) clinical trials are American College of Rheumatology 20 % improvement criteria (ACR20), ACR50, and ACR70 response rates, and the 28-joint disease activity score (DAS28). Longitudinal models to quantitate the exposure–response relationships for ACRs and DAS28 score were developed for four biologics used for the management of RA. The models were then used to simulate the clinical outcome at various time points following different treatment regimens. Discriminative sensitivity of these endpoints was assessed using a power analysis. The trial simulation and subsequent power analysis showed that both ACR20 and DAS28 exhibit much lower power in distinguishing between two doses investigated compared with distinguishing treatment effect over placebo/Methotrexate (MTX) control. ACR20 response rate is generally more powerful in detecting treatment effect over placebo/MTX control as compared to DAS28. The findings of current study provide useful information which will help future clinical trial design for the treatment of patients with RA.     

Thalidomide derivatives and the immune system. I. Changes in the pattern of integrin receptors and other surface markers on T lymphocyte subpopulations of marmoset blood

Treatment of marmosets (Callithrix jacchus) with thalidomide (Thd) or its derivative EM12 (which is also teratogenic, but more stable to hydrolysis) resulted in the lack of reaction of adhesion surface receptors (integrins) on T lymphocytes in venous blood. Lymphocyte subsets appeared, for example CD4+CD2–, which are not found under normal conditions, (a) There was no clear effect of the treatments on the total number of leukocytes or lymphocytes or on the total number of CD4+ or CD8+ T lymphocytes. (b) A decrease in the percentage of the cytotoxic T cells carrying the CDw29 marker (CD8+CD56+CDw29+) at a dose as low as 5 mg EM12/kg bw, and an increase in the percentage of suppressor cells carrying the CDw29 marker (CD8+CD56-CDw29+) at 10 mg EM12/kg bw were found. Similar effects were induced by Thd at somewhat higher doses, while supidimide (Sup) was less active even at the very high dose of 100 mg/kg bw. Especially at the lower doses these effects occurred with a lag phase and persisted after discontinuation of the dosing. Alterations induced in helper T cell subpopulations by Thd or EM12 were less impressive (no significant effect was observed with 5 mg EM12/kg bw). Some changes were observed at higher dose levels in the CD4+CD45RA+CDw29+ cells and the CD4+CD45RA-CDw29 cells. (c) The most significant effect, reduction in the reactivity of CD2+, was detectable subsequent to daily oral doses as low as 10 mg Thd/kg or 1 mg EM12/kg bw. Peak plasma concentrations to be expected under these experimental conditions are less than 1 /ml. (d) The surface receptors found to be affected include among others: CD2 (LFA-2) and CD11 a (LFA-1) and CD18 (LFA-1). Clearly, CD4+ cells were found to be more susceptible to the loss of the integrin receptors than CD8+ cells. (e) The effect persisted for several weeks subsequent to the discontinuation of the dosing. (f) A rough estimate of the relative potency to reduce the CD2 receptor in the marmoset suggests EM12 to be five to ten times more potent than Thd. Sup, a Thd derivative reported to exhibit no or a low teratogenic potency, was found to be at least five times less potent than Thd. (g) The alterations of surface adhesion receptors by the substances studied in this investigation were not confined to T lymphocytes. We also observed similar effects on B lymphocytes, monocytes, and neutrophils, and many other cell types carrying such receptors might be affected. Interferences with the many functions of integrins with fundamental biological importance may open a new dimension of pharmacological or toxic effects.     

Acute and 30-day oral toxicity studies of administered carnosic acid

Purpose

Increasing interest in carnosic acid (CA) is due to its pharmacological properties. The aim of this study was to evaluate the acute and 30-day oral toxicity of CA.

Methods

The acute oral toxicity study in Kuming mice design followed the OECD-guidelines 423, and a 30-day chronic oral toxicity study in Wistar rats based on the enhanced OECD test guideline 407 were performed.

Results

The oral lethal dose (LD50) for mice was 7100 mg/kg of body weight in the acute toxicity study. The histopathological changes were observed in the heart, liver and kidney for the survival mice treated with a single dose CA. For the sub chronic toxicity study, CA administered for 30 days produced slightly reductions in the weight gain pattern, which did not reach the significant level when compared with the control values. With respect to serum biochemistry test, decreased total serum protein levels, but conversely increased aspartate aminotransferase (AST) levels were detected in the high-dose and moderate-dose groups. Histopathologically, light pathological changes were observed in the heart, liver, and kidney of rats treated with the high-dose CA.

Conclusion

The present work suggests that a short-term oral administration of CA has a relatively low toxicity profile.     

Toxic properties of the mushroom Cortinarius orellanus

The toxins of the mushrooms Cortinarius orellanus (Fries) and Cortinarius speciosissimus (Kühn & Romagn) were isolated by extraction procedures and Sephadex chromatography. All intermediate and end products of the purification process were tested in mice for acute toxicity after oral and i.p. administration. In both species a fluorescent main toxin and a nonfluorescent compound of minor toxicity were found. The main toxin of both species was identified by mass spectrometric and nuclear magnetic resonance analyses as the 2,2-bipyridine- 3,3,4,4-tetrol-1,1-dioxide, which is identical to orellanine. The purified compound was toxic when administered either orally or i.p. When given orally the LD₅₀ was 33 mg/kg body weight in mice. The oral LD₅₀ of Cortinarius orellanus (2.20 g dried mushroom/kg) and of Cortinarius speciosissimus (3.12 g/kg) depended on the orellanine content (14 mg/g in Cortinarius orellanus and 9 mg/g in Cortinarius speciosissimus). The second toxic component was ineffective in mice when given orally. It caused acute toxicity when administered i.p., but toxicity was low when compared to the main toxin. Thus it appears to be of minor importance. Intraperitoneal testing of both isolated toxins and of the extract containing the whole toxic potential of the mushrooms revealed that toxicity, time dependency and expression of toxicosis is accounted for by the sum of these two toxins. No peptidic main toxin as described by other mycologists could be detected.     

Astragalus polysaccharides alleviates glucose toxicity and restores glucose homeostasis in diabetic states via activation of AM PK

Aim:

To establish the mechanism underlying the improvement of glucose toxicity by Astragalus polysaccharide (APS), which occurred via an AMP activated protein kinase (AMPK)-dependent pathway.

Methods:

In vivo and in vitro effects of APS on glucose homeostasis were examined in a type 2 diabetes mellitus (T2DM) rat model. The T2DM rat model was duplicated by a high-fat diet (58% fat, 25.6% carbohydrate, and 16.4% protein) and a small dose of streptozotocin (STZ, 25 mg/kg, ip). After APS therapy (700 mg·kg⁻¹·d⁻¹, ig) for 8 weeks, blood glucose, glycosylated hemoglobin, and serum insulin were measured. Insulin sensitivity was evaluated by the comprehensive analysis of oral glucose tolerance tests (OGTT) and HOMA IR index. Hepatic glycogen was observed by the PAS staining method. The expression and activity of skeletal muscle AMPKα and acetyl-CoA carboxylase (ACC), and the phosphorylation of hepatic glycogen synthase (GS), the glycogen synthase (GS),were measured by Western blotting. Glucose uptake was measured with the 2-deoxy-[³H]-D-glucose method in C2C12 cells.

Results:

The hyperglycemia status, insulin sensitivity, glucose uptake, and activation level of AMPK in diabetic rats were improved in response to APS administration. APS could also alleviate glucose toxicity in cultured mouse cells by the activation of AMPK.

Conclusion:

APS can alleviate glucose toxicity by increasing liver glycogen synthesis and skeletal muscle glucose translocation in the T2DM rat model, via activation of AMPK.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

Polyhalogenated dibenzo-p-dioxins and dibenzofurans and the immune system. 4. Effects of multiple-dose treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on peripheral lymphocyte subpopulations of a non-human primate (Callithrix jacchus)

Non-human primates (Callithrix jacchus) were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) over a period of 30 weeks, and lymphocyte subpopulations of venous blood were monitored using monoclonal antibodies and flow cytometry (FACScan). There was no clear-cut change in the total lymphocyte population during this study. In the first part of the study the new-world monkeys (marmosets) were treated for 24 weeks with a weekly dose of 0.3 ng TCDD/kg body wt. At the end of this treatment period a level corresponding to an actual cumulative dose of about 2.5–2.7 ng TCDD/kg body wt was expected. The percentage and the absolute number of the CD4⁺CDw29⁺ cells (helper inducer or memory cells) surmounted the physiologically occurring increase. Concomitantly the percentage of the CD4⁺CD45RA⁺ cells (suppressor-inducer or naive cells) decreased. There was, at the same period, no change in the total T cell population (CD2⁺ cells) or in the cells carrying the CD8 or the CD4 epitope. When increasing the weekly dose to 1.5 ng TCDD/kg body wt, a transient increase in the percentage and the absolute number of the CD8⁺CD56⁺ cell population (cytotoxic T cells) was observed 3 weeks after the increase in dosing. At this time the expected decrease in the percentage or the absolute number of CD4⁺CDw29⁺ cells was just detectable and this decline was at its maximum 6 weeks after switching to the higher weekly doses. The reduction in the percentage and the absolute number of CD4⁺CDw29⁺ cells persisted 5 weeks after discontinuation of the dosing, but this cell population was again within normal limits 7 weeks later. Because the two subpopulations are changed in opposite directions, the ratio CD4⁺CDw29⁺/CD4⁺CD45RA⁺ is a very sensitive measure of the effect induced by TCDD. There was a pronounced decrease in the percentage of the CD20⁺ cells (B₁ cells), but their percentage and number rapidly normalized, in contrast to the CD4⁺CDw29⁺ cells, when the dosing was discontinued. At the end of the treatment period the apparent body burden was calculated to correspondt an actual dose of about 9–10 ng TCDD/kg body wt. Such an actual dose level might be assumed to be reached under steady-state conditions in chronic experiments with daily doses of about 135 pg TCDD/kg body wt (assuming a half-life for TCDD in the marmoset of 6–8 weeks). Er trapolations of the results obtained at higher doses to very low exposures is not justified with respect to the effect induced by TCDD on the immune system of marmosets. A lower doses the effect is clearly reversed.     

In vivo biliary excretion and in vitro cellular accumulation of thyroxine by rats or cultured rat hepatocytes treated with a novel histamine H1-receptor antagonist

Rats treated with temelastine (SK&F 93 944), a novel histamine H₁-receptor antagonist, develop thyroid lesions characterized by hypertrophy and colloid depletion. To investigate the mechanism underlying the lesion the biliary clearance and hepatocellular accumulation of radio-labelled iodothyronines was measured in rats or cultured rat hepatocytes. Treatment with temelastine increased both the biliary clearance (approximately 300% of control) and hepatocellular accumulation (approximately 200%) of thyroxine (T₄) but had little or no effect on tri-iodothyronine (T₃). Chromatographic analysis of bile samples from temelastine — treated rats showed that the majority (approximately 78%) of T₄ was present in the unconjugated form. This contrasted with data from phenobarbitone — treated rats which showed that approximately 80% of T₄ in the bile was present as the glucuronide conjugate. Studies with cultured hepatocytes showed that the hepatocellular accumulation of T₄ was energy dependent. At 4° C the treatment — related increases in accumulation of T₄ were abolished, suggesting that temelastine is specifically affecting the high affinity, energy dependent system which preferentially transports thyroxine into hepatocytes. Because temelastine is metabolized extensively, investigations were undertaken to discover if the hepatic effects were caused by the parent compound or an oxidative metabolite. The results showed that the hepatocellular accumulation of T₄ remained increased in hepatocytes co-incubated with temelastine and 1-aminobenzotriazole (a suicide inhibitor of cytochrome P450), even though no measurable P450 could be found in the cells. Also, in studies with two major rat metabolites of temelastine, i.e. 93944-Met I or 93944-Met VIII, treatments failed to reproduce the responses seen with the parent compound. These data suggest that it is the parent compound and not some oxidative metabolite which is responsible for the hepatic effect. It is concluded that temelastine produces the observed thyroid changes by increasing the hepatic clearance of T4, leading to increases in TSH release resulting in hypertrophy of the gland. The increased hepatic clearance of thyroxine appears to be mediated via a novel mechanism not associated with the induction of thyroxine metabolizing enzyme systems.     

Diclofenac Enables Unprecedented Week-Long Microneedle-Enhanced Delivery of a Skin Impermeable Medication in Humans

Purpose

Microneedles applied to the skin create micropores, allowing transdermal drug delivery of skin-impermeable compounds. The first human study with this technique demonstrated delivery of naltrexone (an opioid antagonist) for two to three days. Rapid micropore closure, however, blunts the delivery window. Application of diclofenac (an anti-inflammatory) allows seven days of naltrexone delivery in animals. The purpose of the current work was to demonstrate delivery of naltrexone for seven days following one microneedle treatment in humans.

Methods

Human subjects were treated with microneedles, diclofenac (or placebo), and naltrexone. Impedance measurements were used as a surrogate marker to measure micropore formation, and plasma naltrexone concentrations were measured for seven days post-microneedle application.

Results

Impedance dropped significantly from baseline to post-microneedle treatment, confirming micropore formation. Naltrexone was detected for seven days in Group 1 (diclofenac + naltrexone, n?=?6), vs. 72 h in Group 2 (placebo + naltrexone, n?=?2). At study completion, a significant difference in impedance was observed between intact and microneedle-treated skin in Group 1 (confirming the presence of micropores).

Conclusion

This is the first study demonstrating week-long drug delivery after one microneedle application, which would increase patient compliance and allow delivery of therapies for chronic diseases.     

Serum P-glycoprotein level: a potential biomarker of DMARD failure in patients with rheumatoid arthritis

Objectives

To evaluate the utility of elevated serum P-glycoprotein (P-gp) as a risk marker of therapeutic response failure in rheumatoid arthritis (RA) patients treated with disease-modifying antirheumatic drugs (DMARDs).

Methods

A cross-sectional study was conducted in 151 RA patients. Patients were classified into two groups according to the response achieved in terms of the disease activity score (DAS)28 after ≥?6 months: (1) patients with a therapeutic response to DMARDs, with DAS28 <?3.2; and (2) patients without a response to DMARDs, with persistent DAS28?≥?3.2. We explored a wide group of clinical factors associated with therapeutic resistance. Serum P-gp levels were measured by ELISA. The risk of P-gp elevation as a marker of failure to achieve a therapeutic response to DMARDs was computed using multivariate logistic regression.

Results

Serum P-gp levels were significantly higher in RA patients (n?=?151) than in the controls (n?=?30) (158.70?±?182.71 ng/mL vs. 14.12?±?8.97 ng/mL, p?<?0.001). The P-gp level was correlated with the DAS28 score (r?=?0.39, p?<?0.001). RA patients with DMARD failure had higher serum P-gp levels than patients with a therapeutic response (206?±?21.47 ng/mL vs 120.60?±?15.70 ng/mL; p?=?0.001). High P-gp levels increased the risk of DMARD failure (OR 3.36, 95% CI 1.54–7.27, p?=?0.001). After adjusting for confounding variables, elevated P-gp remained associated with DMARD failure (OR 2.64, 95% CI 1.29–5.40, p?=?0.01).

Conclusion

Elevated serum P-gp is associated with DMARD failure. The P-gp level can be considered a clinical tool for evaluating the risk of DMARD failure in patients; however, future prospective studies should be performed to evaluate the utility of this marker in predicting long-term responses.

     

Effect of dietary antioxidant on mercuric chloride induced lung toxicity and oxidative stress

This study was conducted to evaluate the effect of environmental contaminant mercuric chloride on levels of trace elements and oxidative parameters in rat lungs and to investigate the efficacy of possible protection by natural antioxidant diallylsulphide (DAS) against lung injury. Twenty-four healthy male rats were randomly divided into four groups: I – control, II – DAS (200?mg/kg), III – HgCl₂ (50?mg/kg), and IV – DAS (200?mg/kg) + HgCl₂ (50?mg/kg). Mercuric chloride induced oxidative stress was indicated by a significant decrease in levels of superoxide dismutase, catalase, and glutathione peroxidase as compared to the control group (p–<0.05). Also, hydroxyproline (HYP) content in lung tissues of mercuric chloride-treated group was significantly increased (p?<?0.05). DAS markedly attenuated mercuric-induced biochemical alterations in lungs by upregulating the activities of antioxidant enzymes. These findings indicated that within the doses selected, DAS can provide significant protection against HgCl₂-induced toxicity.     

The Influence of Different Strains and Age on in Vitro Rat Skin Permeability to Water and Mannitol

Water and mannitol were used as test penetrants to study the effect of age on the skin permeability of the Wistar-derived Alderley Park (AP) rat and Sprague-Dawley (SD) rat. Whole-skin membranes were prepared from rats aged 10 to 120 days, while epidermal membranes were prepared from rats aged 24 to 32 days. The results indicated that the skin permeabilities of the two strains were very similar for either whole-skin or epidermal membranes. The influence of age on skin permeability was found to be negligible for the AP rat, and a small decrease in whole-skin permeability was observed for SD rats above 80 days of age. A statistically derived expression (the separation efficiency factor) was used to determine the optimum age for preparing intact epidermal membranes; these were 26 days for AP rats and 28 days for SD rats. Histological examination of whole-skin membranes for both strains revealed that the stratum corneum and epidermal thickness did not alter significantly with age (10 to 120 days old). Dermal thickness, hair follicle depth, and, to a lesser extent, the surface area occupied by hair follicles all appeared to be influenced by age, although these changes had no detectable effect on skin permeability.     

The effect of DAS on respiration and pulmonary vascular resistance

Summary In stored blood as well as in various organs, a substance appears which causes a marked drop of systemic blood pressure after intravenous injection. The active principle was called depressor active substance (DAS). In order to study the circulatory effects of DAS the systemic and the pulmonary vascular resistance (SVR and PVR) and the ventilation volume were measured. DAS stopped respiratory movements by exciting afferent vagus fibres and led to an abnormal cardiac activity and to bronchoconstriction by stimulation of the vagotonus. Furthermore, DAS showed a direct bronchoconstrictor effect. The application of DAS caused a strong increase of PVR. This effect was strong enough to produce an abrupt drop of the systemic blood pressure because of a reduced diastolic filling of the left ventricle. Higher quantities of DAS led to an acute insufficiency of the right ventricle.Part of these results was presented at the meeting of the German Pharmacological Society, Mainz, March 21st–24th 1971.     

Delivery of Methotrexate and Characterization of Skin Treated by Fabricated PLGA Microneedles and Fractional Ablative Laser

Purpose

This study investigated in vitro transdermal delivery of methotrexate through dermatomed porcine ear and cadaver human skin treated with poly (D,L-lactide-co-glycolide) acid microneedles or fractional ablative laser.

Methods

PLGA microneedles were fabricated and characterized using scanning electron microscopy and mechanical assessment techniques. The integrity of treated skin was evaluated by rheometer, transepidermal water loss, and skin electrical resistance measurements. Successful skin microporation was demonstrated by dye binding, histology, pore uniformity, confocal laser microscopy, and DermaScan studies. In vitro permeation experiment was performed on Franz diffusion cells to determine drug delivery into and across the skin.

Results

Both physical treatments resulted in a considerable decrease in skin resistance and an increase in transepidermal water loss value. The laser-created microchannels were significantly larger than those formed by microneedles (p?<?0.05). An effective force of 41.04?±?18.33 N was required to achieve 100% penetration efficiency of the microneedles. For both porcine ear and human skin, laser ablation provided a significantly higher methotrexate permeability into the receptor chamber and skin layers compared to microneedle poration and untreated skin (p?<?0.05).

Conclusions

Both fractional ablative laser and polymeric microneedles markedly enhanced in vitro transdermal delivery of methotrexate into and across skin.

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Graphical Abstract ?

     

In vitro and in vivo studies on the prenatal toxicity of five virustatic nucleoside analogues in comparison to aciclovir

Several virustatic agents are known to be teratogenic in laboratory animals. Since routinely performed in vivo studies do not always offer the best conditions to detect the teratogenic potential of a drug, we used a combined in vivo/in vitro approach for comparative studies on the prenatal toxicity of five nucleoside analogues. Rat embryos were exposed for 48 h to various concentrations of vidarabine-phosphate (VAP), ganciclovir (GCV), 2,3-dideoxyadenosine (ddA), 2,3-dideoxycytidine (ddC) and zidovudine (= azidothymidine, AZT) in a whole-embryo culture system. The steepness of the concentration-response curves as well as the induced abnormality pattern (head, neural tube, shape) were similar for these compounds. However, a wide range in embryotoxic potency was observed: VAP was the most potent compound (100% abnormal embryos at 25 M) in this in vitro system, while AZT showed the lowest potency to interfere with normal embryonic development (40% abnormal embryos at 3000 M). In addition to these experiments we treated rats on day 10 of gestation with three s.c. injections (8 a.m.; 12 a.m.; 4 p.m.) of 200 mg of each drug/kg body wt. The embryos were evaluated on day 11.5 of gestation, i.e. at a time of development corresponding to the developmental stage at the end of the whole-embryo culture. The same criteria were used as during the in vitro studies for the evaluation of these in vivo exposed embryos. With VAP and GCV we obtained similar results with both exposure routes (in vitro and in vivo), while no abnormalities were detectable with the other compounds after exposure in utero. When the results from the in vitro and in vivo studies are compared with data of similar experiments conducted in our laboratory with the nucleoside analogue aciclovir (ACV) under identical conditions (Klug et al. 1985 a; Stahlmann et al. 1988), the following conclusions can be drawn: under in vitro conditions VAP showed the highest potential of the virustatics to interfere with embryonic development, the toxic potential of AZT was surprisingly low. Under our experimental in vivo conditions ACV reveals the highest teratogenic potential, whereas ddC, ddA, and AZT exhibited an obviously lower toxicity.Dedicated to Professor Gerhard Zbinden on the occasion of his retirement     

In vitro exposure of human lymphocytes to trichothecenes: individual variation in sensitivity and effects of combined exposure on lymphocyte function.

The trichothecenes are mycotoxins produced by fungi of the genus Fusarium, which are commonly present in foods and feed of cereal origin. Owing to the lack of sufficient toxicological data for most of the trichothecenes, in vitro studies may contribute to risk assessments of these toxins. In the present report, human lymphocyte cultures were used to study the individual variation in sensitivity among humans and the effects on in vitro Ig production. Furthermore, proliferative responses of cells exposed to combinations of two of the toxins were studied. Four toxins, T-2 toxin, diacetoxyscirpenol (DAS), nivalenol (NIV) and deoxynivalenol (DON) were included in the study. All four of the tested trichothecenes effectively inhibited mitogen-induced lymphocyte proliferation. There were no statistically significant differences in sensitivity to the toxins between lymphocytes from female and male blood donors. The individual variation in sensitivity, evaluated as the range of IC50 values, was rather limited (within a factor of 3 to 4). Immunoglobulin production by pokeweed-stimulated human lymphocytes was also effectively inhibited with IC50 values similar to the IC50 values in the proliferation tests for DON and NIV. However, IC50 values for Ig synthesis in cultures exposed to T2 were approximately two to three times higher than the corresponding IC50 values found in the proliferation tests. At low levels of exposure, elevated Ig production was observed in lymphocyte cultures from four out of the five blood donors tested. This effect was most pronounced on IgA synthesis. Combinations of NIV with T2, DAS or DON resulted in additive toxicity in the lymphocyte proliferation test, while combinations of DON with T2 or DAS resulted in an inhibition that was slightly lower than what could have been expected from the inhibition produced by the individual toxins. In conclusion, the tested trichothecenes inhibited both proliferation and Ig production in human lymphocytes in a dose-dependent manner with limited variation in sensitivity between individuals. Enhanced Ig production was observed in cell cultures exposed to the lower doses of the toxins. Combined exposure to two of the toxins resulted mainly in additive or antagonistic effects, although synergistic effects cannot be excluded and should be further investigated. These findings indicate that the total intake of type A and B trichothecenes should be taken into account in risk assessments.     

Arsenic dose in patients with cutaneous carcinomata and hepatic haemangio-endothelioma after environmental and occupational exposure

A total of 16 male cases with malignant tumours associated with arsenic-polluted water were observed in Tarapacá and Antofagasta Provinces, northern Chile. Fifteen of them had skin carcinomata and the remaining one a haemangio-endothelioma of liver. The 15 skin cancer cases had latent periods ranging from 12–45 years. Three patients were studied in detail. The first one (skin cancer) had a latent period of 20 years with a weighted mean dose of 1.2 mg/day (total dose for latent period 8.4 g). The second one (skin cancer) had a latent period of 23 years with a weighted mean dose of 1.0 mg/day (total dose for latent period 8.3 g). The third case (liver tumour) exhibited a latent period of 14 years with a weighted mean of 0.6 mg/day (total dose for latent period 3.1 g).Fifteen of the 16 cancer patients were labourers. For normal subjects of different ages and both sexes (n=290) and ingesting arsenic-polluted water (0.60 ppm), the relationship between mean age and mean arsenic dose is expressed by a weighted least square polynomial regression, of second degree: (y) = _o + ₁ ^t + ₂ ^{t 2} where y is mean arsenic dose (mg/person/day) and t is mean age (years). For the general male population and for male labourers, the respective equations are presented.Deceased. National Academy of Medicine, Santiago, Chile