Patrice Muret

Assessment of principal parabens used in cosmetics after their passage through human epidermis–dermis layers (ex-vivo study)

Abstract:  Concern is continuously raised about the safety of parabens which are present in most of the cosmetic preparations. In this investigation, methyl‐, ethyl‐, propyl‐ and butyl paraben (MP, EP, PP, BP), in a commercial cosmetic lotion, were deposited on human skin fragments, collected after surgical operations. Permeated parabens were determined after their passage through human epidermis–dermis layers, fixed on Franz diffusion cells. Bovine serum albumin (3%) was employed as receptor fluid. Then, parabens were assessed by liquid chromatography. The objective of this research was to determine the permeation of these molecules through human epidermis–dermis layers, and their possible passage to body tissues and/or accumulation in skin layers. Two groups of experiments were performed. In the first experimental group (G1), unique doses of the cosmetic were deposited on skin fragments fixed on Franz cells (n = 6), at time 0 h, followed with different withdrawn times of the receptor fluid at 12, 24 and 36 h. G1 results demonstrated that parabens penetration was influenced by their lipophilicity: more lipophilic the parabens were (BP > PP > EP > MP), less they crossed the skin layers (BP < PP < EP < MP). The second experimental group (G2) was constituted of three equal deposits on each Franz cell (n = 6) at different hour times 0, 12 and 24 h followed with three withdrawn times of the receptor fluid at 12, 24 and 36 h. The G2 results indicated that investigated parabens had significant increasing permeations in skin layers. This situation provokes the accumulation of these molecules which were considered by some authors as the cause of skin toxicities and carcinogenicity.

Pharmacokinetics, safety and efficacy of a full dose sofosbuvir-based regimen given daily in hemodialysis patients with chronic hepatitis C

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is an independent risk factor for chronic kidney disease and leads to faster liver disease progression in patients requiring hemodialysis than in those with normal renal function. Little is known about the use of a sofosbuvir-containing regimen for infected patients on hemodialysis. We aimed to describe the pharmacokinetics, safety and efficacy of sofosbuvir in 2 dosing regimens and associated antiviral agents in HCV-infected patients requiring hemodialysis. METHODS Multicenter, prospective and observational study of patients receiving sofosbuvir, 400mg once daily (n=7) or 3 times a week (n=5), after hemodialysis with simeprevir, daclatasvir, ledipasvir or ribavirin was conducted. Drug plasma concentrations were determined by liquid chromatography-tandem mass spectrometry before and after a 4h hemodialysis and 1.5h after last drug intake at the end of hemodialysis. RESULTS Plasma concentrations of sofosbuvir or its inactive metabolite sofosbuvir-007 did not accumulate with either regimen between hemodialysis sessions or throughout the treatment course. Sofosbuvir-007 extraction ratio (52%) was consistent with historical data. In one patient receiving the once daily regimen, sofosbuvir-007 half-life was slightly higher (38h) than for patients with normal renal function receiving a full dose. Hemodialysis did not remove any other associated anti-HCV agents. Clinical and biological tolerance was good for all patients. Two relapses occurred with the 3 times a week regimen and none with the once daily. CONCLUSIONS A regimen including sofosbuvir, 400mg once daily, could be proposed for HCV-infected patients requiring hemodialysis and should be associated with close clinical, biological, cardiovascular, and therapeutic drug monitoring. LAY SUMMARY Hepatitis C Virus (HCV) infection in hemodialysis patients is prevalent and aggressive. Effective anti-HCV treatment in these patients may stabilize their renal disease. However, sofosbuvir, the cornerstone of most anti-HCV-containing regimens, should not be administered to these patients until more data is available. In this pharmacokinetic study, sofosbuvir full dose (400mg once daily) administered every day with another direct antiviral agent did not accumulate in hemodialysis patients and was safe and effective.

Ceramide analogue 14S24 selectively recovers perturbed human skin barrier.

Background  Topical ceramide application is an effective therapeutic approach in skin disorders with disturbed barrier function, including atopic dermatitis and psoriasis.

Iron and Ascorbic Acid Concentrations in Human Dermis with Regard to Age and Body Sites

Background: Reactive oxygen species (ROS) contribute to processes relating to cutaneous aging. Iron catalyses ROS formation whereas ascorbic acid (AA) plays a fundamental role in defending the organism against undesirable ROS action. Objective: The aim of this work was to determine the ex vivo iron and AA concentrations in human dermis from different age groups to better understand their role. Methods: Skin fragments were collected from 66 female patients during surgical operations and were grouped according to age: group I (<15 years, before puberty, n = 12), group II (15–50 years, adults, n = 42), and group III (>50 years, advanced age adults, n = 12). Two sites were investigated: the abdomen (unexposed areas) and face (exposed sites). Iron and AA were collected from human dermis by microdialysis and assessed by atomic absorption spectrometry and gas chromatography mass spectrometry, respectively. Results: Iron concentrations in the dermis were significantly higher in group III (27.4 ± 20.9 µg/l) than in group I (13.8 ± 3.3 µg/l; p< 0.05 ). An inverse correlation between AA dermis levels and increasing age was detected. For groups III and I, iron and AA concentrations were significantly different in dermis from the face compared to that of the abdomen (p < 0.05). Conclusion: This study shows for the first time that there is a direct relationship between iron and AA concentrations in the dermis and aging. Moreover, iron and AA concentrations differed according to body site.

Assessment of the recovery of three lipophilic psoralens by microdialysis: an in vitro study

The microdialysis method is extensively developed in neuropharmacological experiments. It is a non-invasive technique with promising applications in biological research. The aim of this work was to establish and to optimize a process to microdialyse chemicals. Three lipophilic molecules used in dermatology were tested: 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and trimethylpsoralen (TMP). These psoralens have the same basic chemical structure (furocoumarin). The different radical substitutions are responsible for their different lipophilicities. In this work, different microdialysis parameters were studied: the relative recovery and the equilibrium time in relation to the perfusion rate and to the drug lipophilicity. The equipment was composed of a CMA/100® microinjection pump, CMA/20® microdialysis probes (membrane length of 10 mm, cut off 20 and 100 kDa) and a CMA/140® microcollector. The perfusate was a phosphate buffer 0.06 M (pH 7.4). The psoralen dialysates were assessed by high performance liquid chromatography. The relative recovery of psoralens increased with the decreasing perfusion rates and with the drug lipophilicity. The equilibrium time decreased with the increasing perfusion rates. TMP was not detected under these experimental conditions. This work defines the optimal parameters for in vivo studies and shows the limit of this technique for the investigation of lipophilic drugs.

Nevirapine-raltegravir combination, an NRTI and PI/r sparing regimen, as maintenance antiretroviral therapy in virologically suppressed HIV-1-infected patients.

BACKGROUND Nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor (PI)/ritonavir sparing regimens may be useful to some HIV-infected patients. Nevirapine (NVP) and raltegravir (RAL) are both potent antiretrovirals with good long-term safety profiles. METHODS We retrospectively identified from our electronic database all patients with HIV RNA<50 copies/ml for >6 months on an NVP-containing regimen and no prior exposure to integrase strand transfer inhibitors who were switched to RAL plus NVP. Data was collected for 36 months or until discontinuation of RAL plus NVP for any reason. RESULTS A total of 39 patients (30 male) were included in this analysis. Median duration of prior antiretroviral therapy was 14 years (IQR 10-17) and median duration with plasma HIV-1 RNA<50 copies/ml prior to switch was 50 months (IQR 22-96). Switched regimens included mainly a boosted PI (n=24) or tenofovir disoproxil fumarate/emtricitabine (n=12). After switching, the percentages of patients with HIV-1 RNA<50 copies/ml were 87.2% (95% CI 76.7, 97.7) and 82.1% (95% CI 70.0, 94.1) at 6 and 12 months, respectively, in the intent-to-treat-exposed analysis, 97.1% (95% CI 91.6, 100) and 94.1% (95% CI 86.2, 100), respectively, in the per-protocol analysis. All patients with follow-up to month 24 (n=22) or month 36 (n=14) had HIV-1 RNA<50 copies/ml. One virological failure was observed (related to archived non-nucleoside reverse transcriptase inhibitor resistance mutation). During follow-up, no patient experienced Grade 3 or 4 adverse events. Median values of serum creatinine and lipids significantly improved after switch. CONCLUSIONS In patients with prolonged HIV-1 RNA<50 copies/ml, switching to NVP-RAL combination maintained virological suppression throughout 36 months. This combination deserves further evaluation in patients unable to tolerate NRTI or PI/ritonavir agents.

An APCI LC-MS/MS method for routine determination of capecitabine and its metabolites in human plasma

The anticancer drug capecitabine and its metabolites [including the active metabolite 5-fluorouracil (5-FU)] display high pharmacokinetic inter-patient variability. Such variability, which may lead to treatment failure or toxicity, could need drug concentration measurement to individualize dosing regimen. However, usual assay methods are often long and fastidious. A simultaneous and cost-effective method was thus developed for the determination of the concentrations of these compounds in human plasma. Compounds were extracted via a classic liquid-liquid extraction. Chromatographic analysis was performed on a C18 reverse phase column with detection by atmosphere pressure chemical ionization LC-MS/MS. Our method allows a good chromatographic separation of the compounds and was fully validated following Food and Drug Administration (FDA) recommendations (good selectivity, no carry-over, linearity of the calibration curves without weighting, deviations from nominal concentrations of standard samples lower than 15%, intra- and inter-assay precision and accuracy lower than 15%). Recovery and stability were also acceptable following the FDA guidelines. A matrix effect impairing the determination of 5-FU was avoided by using a stable isotopic derivative of 5-FU as internal standard. Interestingly, this method allows detection of TetraHydroUridine, an inhibitor of ex vivo degradation of metabolites, which is essential for the stability, the adequate conditioning of blood samples and for good laboratory practice, essential in routine determination. This method seems usable to routinely determine concentrations of capecitabine and its metabolites in blood and may be helpful in further studies aiming at performing therapeutic drug monitoring.

N-Acetyltransferase 2 Acetylation Polymorphism: Prevalence of Slow Acetylators Does Not Differ between Atopic Dermatitis Patients and Healthy Subjects

The genetic polymorphism of human N-acetyltransferase 2 (NAT2) divides the human population into groups with rapid, intermediate and slow acetylator status. Slow acetylator status has been considered a predisposing factor for allergic diseases, lupus erythematosus, toxic epidermal necrolysis or Stevens-Johnson syndrome. The aim of this study was to investigate whether Caucasian patients suffering from atopic dermatitis differed from healthy individuals with regard to the genotype and phenotype of NAT2. Twenty unrelated healthy Caucasian volunteers (9 females and 11 males, aged from 22 to 59 years) and twenty unrelated Caucasian patients suffering from atopic dermatitis (9 females and 11 males, aged between 20 and 54 years) participated in this study. For each one, the NAT2 genotype was determined by polymerase chain reaction with DNA extracted from peripheral blood, using specific primers for the wild-type allele (wt) and the 3 most frequent mutated alleles of NAT2 (C481→T, G590→A and G857→A). The NAT2 phenotype was evaluated with dapsone as a test substrate using high-pressure liquid chromatography. Statistical analysis was performed using the χ2 test. Phenotype and genotype were distributed as follows: (1) of the healthy subjects, 60% were rapid acetylators (RA) and 40% were slow acetylators (SA); 10% of the RA and 15% of the SA were homozygous, 50% of the RA and 25% of the SA were heterozygous; (2) of the patients, 55% were RA, 40% were SA and 5% were intermediate acetylators (IA); 10% of the RA and 10% of the SA were homozygous, 45% of the RA and 35% of the SA were heterozygous. No significant statistical difference was found between the two groups for genotypes (p = 0.75) or phenotypes (p = 0.60). The phenotyping and genotyping results of healthy subjects were comparable to those found in previous studies. The absence of a significant statistical difference between healthy subjects and atopic dermatitis patients is in contrast to the results of previous studies. Some authors considered that allergic patients are mostly SAs. This could be explained by the fact that we only considered patients suffering from atopic dermatitis whereas, in other studies, patients suffered from different (one or several associated) allergic diseases. NAT2 polymorphism does not differ between patients suffering from atopic dermatitis and healthy subjects. The importance attributed to the SA status, which was previously considered a predisposing factor for allergic diseases such as atopic dermatitis, should be reviewed.

Percutaneous absorption of three psoralens commonly used in therapy: Effect of skin occlusion (in vitro study)

Abstract Occlusion is recognised as an enhancer of percutaneous absorption. Some authors suggested that this effect is related to the molecular polarities of applied drugs. To verify this hypothesis, an in vitro study was performed to assess a topical absorption, under occlusion, in relation to the molecular polarity of three furanocoumarins: 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and trimethylpsoralen (TMP). These compounds present the same basic chemical structure but have different degrees of lipophilicity. They are commonly used, orally or topically, in conjunction with UVA irradiations to treat skin diseases such as psoriasis and vitiligo. Ethanolic solutions of the psoralens were deposited onto human abdominal skin fragments (6.3 μg/cm 2 ), mounted on Franz® diffusion cells ( n = 12), which were maintained at 37°C. Occlusion was retained by placing rubber corks on the top of Franz® cells ( n = 6). The receptor fluid was constituted with 1.4% of human serum albumin solution. At each sample point (2, 4, 6, 8, 12 and 24 h), the entire content of the receptor chamber was removed and replaced by fresh albumin solution. Psoralens quantities in the removed solutions were determined by HPLC. The lipophilicity of the psoralens was established via the partition coefficient in an octanol/water system: TMP > 5-MOP > 8-MOP. The outcome of the results illustrates that occlusion does not always have an enhancing effect in percutaneous absorption. Occlusion impact is influenced by the polarity of the drug. It increases the absorption of moderately lipophilic molecules (i.e. 8-MOP, 5-MOP) but is almost ineffective on the absorption rate of molecules highly lipophilic (i.e. TMP).

High iron and low ascorbic acid concentrations in the dermis of atopic dermatitis patients.

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease in which reactive oxygen species (ROS) may be involved. Iron catalyses ROS formation and ascorbic acid (AA) scavenges these species. Objective: The aim of this work was to determine iron and AA levels in AD patients’ dermis and to compare their concentrations with those of healthy volunteers’ dermis. Methods: Five AD patients and 5 healthy subjects (controls) were enrolled in this study. Iron and AA were collected from human dermis by microdialysis and assessed by atomic absorption spectrometry and gas chromatography-mass spectrometry, respectively. Results: The AD dermis demonstrated higher iron concentrations (44.3 ± 4.6 µg/l) compared to controls (21.8 ± 1.2 µg/l) as well as a significantly lower concentration of AA (46.7 ± 0.6 vs. 176.8 ± 14.5 µg/ml, respectively). Conclusion: These results suggest that iron and AA dermis levels could be indicators of inflammatory tissues and might be implicated in dermatological diseases such as AD.