Effects of microneedle length,density, insertion time and multiple applications on human skin barrier function: Assessments by transepidermal water loss

Microneedle (MN) arrays have attracted considerable attention in recent years due to their ability to facilitate effective transdermal drug delivery. Despite appreciable research, there is still debate about how different MN dimensions or application modes influence permeabilization. This study aimed to investigate this issue by taking transepidermal water-loss measurements of dermatomed human skin samples following the insertion of solid polymeric MNs. Insertions caused an initial sharp drop in barrier function followed by a slower incomplete recovery – a paradigm consistent with MN-generation of microchannels that subsequently contract due to skin elasticity. While 600 μm-long MNs were more skin-perturbing than 400 μm MNs, insertion of 1000 μm-long MNs caused a smaller initial drop in integrity followed by a degree of long term permeabilization. This is explainable by the longest needles compacting the tissue, which then decompresses over subsequent hours. Multiple insertions had a similar effect as increasing MN length. There was some evidence that increasing MN density suppressed the partial barrier recovery caused by tissue contraction. Leaving MNs embedded in skin seemed to reduce the initial post-insertion drop in barrier function. Our results suggest that this in vitro TEWL approach can be used to rapidly screen MN-effects on skin.     

Fabrication of Dissolving Polymer Microneedles for Controlled Drug Encapsulation and Delivery: Bubble and Pedestal Microneedle Designs

Dissolving microneedle patches offer promise as a simple, minimally invasive method of drug and vaccine delivery to the skin that avoids the need for hypodermic needles. However, it can be difficult to control the amount and localization of drug within microneedles. In this study, we developed novel microneedle designs to improve control of drug encapsulation and delivery using dissolving microneedles by (i) localizing drug in the microneedle tip, (ii) increasing the amount of drug loaded in microneedles while minimizing wastage, and (iii) inserting microneedles more fully into the skin. Localization of our model drug, sulforhodamine B in the microneedle tip by either casting a highly concentrated polymer solution as the needle matrix or incorporating an air bubble at the base of the microneedle achieved approximately 80% delivery within 10 min compared to 20% delivery achieved by the microneedles encapsulating nonlocalized drug. As another approach, a pedestal was introduced to elevate each microneedle for more complete insertion into the skin and to increase its drug loading capacity by threefold from 0.018 to 0.053 μL per needle. Altogether, these novel microneedle designs provide a new set of tools to fabricate dissolving polymer microneedles with improved control over drug encapsulation, loading, and delivery.     

Hydrogel-Forming Microneedles Increase in Volume During Swelling in Skin,but Skin Barrier Function Recovery is Unaffected

We describe, for the first time, quantification of in-skin swelling and fluid uptake by hydrogel-forming microneedle (MN) arrays and skin barrier recovery in human volunteers. Such MN arrays, prepared from aqueous blends of hydrolyzed poly(methylvinylether/maleic anhydride) (15%, w/w) and the cross-linker poly(ethyleneglycol) 10,000 Da (7.5%, w/w), were inserted into the skin of human volunteers (n = 15) to depths of approximately 300 μm by gentle hand pressure. The MN arrays swelled in skin, taking up skin interstitial fluid, such that their mass had increased by approximately 30% after 6 h in skin. Importantly, however, skin barrier function recovered within 24 h after MN removal, regardless of how long the MN had been in skin or how much their volume had increased with swelling. Further research on closure of MN-induced micropores is required because transepidermal water loss measurements suggested micropore closure, whereas optical coherence tomography indicated that MN-induced micropores had not closed over, even 24 h after MN had been removed. There were no complaints of skin reactions, adverse events, or strong views against MN use by any of the volunteers. Only some minor erythema was noted after patch removal, although this always resolved within 48 h, and no adverse events were present on follow-up. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.     

Characterization of chromatin instabilities induced by glyphosate,terbuthylazine and carbofuran using cytome FISH assay

Possible clastogenic and aneugenic effects of pesticides on human lymphocytes at concentrations likely to be encountered in residential and occupational exposure were evaluated with (and without) the use of metabolic activation (S9). To get a better insight into the content of micronuclei (MN) and other chromatin instabilities, lymphocyte preparations were hybridized using pancentromeric DNA probes. Frequency of the MN, nuclear buds (NB) and nucleoplasmic bridges (NPB) in cultures treated with glyphosate slightly increased from 3.5 μg/ml onward. Presence of S9 significantly elevated cytome assay parameters only at 580 μg/ml. No concentration-related increase of centromere (C+) and DAPI signals (DAPI+) was observed for glyphosate treatment. Terbuthylazine treatment showed a dose dependent increase in the number of MN without S9 significant at 0.0008 μg/ml and higher. At concentration lower than 1/16 LD50 occurrence of C+MN was significantly elevated regardless of S9, but not dose related, and in the presence of S9 only NBs containing centromere signals were observed. Carbofuran treatment showed concentration-dependent increase in the number of MN. The frequency of C + MN was significant from 0.008 μg/ml onward regardless of S9. Results suggest that lower concentrations of glyphosate have no hazardous effects on DNA, while terbuthylazine and carbofuran revealed a predominant aneugenic potential.     

Genotoxicity of quinocetone,cyadox and olaquindox in vitro and in vivo

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His⁺ mutants at 6.9 μg/plate in Salmonella typhimurium TA 97, at 18.2 μg/plate in TA 100, TA 1535, TA 1537, and at 50 μg/plate in TA 98. CYA produced His⁺ mutants at 18.2 μg/plate in TA 97, TA 1535, and at 50 μg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ⩾10 μg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.     

Does potassium sorbate induce genotoxic or mutagenic effects in lymphocytes?

The present study evaluates the genotoxic potential of potassium sorbate (PS) in cultured and isolated human lymphocytes. To assess the damage caused by PS in humans, we designed in vitro experiments by measuring chromosomal aberrations (CAs), sister-chromatid exchanges (SCEs), micronucleus (MN) and comet assays. Lymphocytes were treated with negative control (sterile distilled water), positive control (MMC for cultured lymphocytes, and H₂O₂ for isolated lymphocytes) and four concentrations (125, 250, 500, and 1000 μg/ml) of PS. According to the results, PS treatment significantly increases the CAs (with or without gaps at 500 and 1000 μg/ml concentrations) and SCEs (at 250, 500, 1000 μg/ml for 24 h and 125, 250, 500, 1000 μg/ml for 48 h) compared with vehicle control. Following treatment of the isolated lymphocytes for 1 h, significant PS-induced DNA strand breaks were observed, at all concentrations. However, PS failed to significantly affect the MN assay. On the contrary, PS does not cause cell cycle delay as noted by the non-significant decrease in the cytokinesis-block proliferation index (CBPI) and replicative index (RI). Only a slight decrease was observed in the mitotic index (MI) at the highest concentration for both treatment times. From the results, PS is clearly seen to be genotoxic to the human peripheral blood lymphocytes in vitro.     

Levosimendan and its metabolite OR-1896 elicit KATP channel-dependent dilation in resistance arteries in vivo

BackgroundLevosimendan and its long-lived metabolite OR-1896 produce vasodilation in different types of vessels by activating ATP-sensitive (K_ATP) and other potassium channels.MethodsIn the present study we applied intravital videomicroscopy to investigate the in situ effects of levosimendan and OR-1896 on the diameters of real resistance arterioles (rat cremaster muscle arterioles with diameters of ~ 20 μm).ResultsLevosimendan and OR-1896 induced concentration-dependent (1 nM – 100 μM) dilations to similar extents in these arterioles (maximal dilation from 23 ± 2 to 33 ± 2 μm and from 22 ± 1 to 32 ± 1 μm, respectively). The arteriolar dilations induced by the selective K_ATP channel opener pinacidil (1 nM – 10 μM) (maximal dilation from 22 ± 4 μm to 35 ± 3 μm) were diminished in the presence of the selective K_ATP channel blocker – glibenclamide (5 μM) (maximal diameter attained: 22 ± 1 μm). Glibenclamide also counteracted the maximal dilations in response to levosimendan or OR-1896 (to 23 ± 3 μm or 22 ± 5 μm, respectively).ConclusionsIn conclusion, this is the first demonstration that levosimendan and OR-1896 elicit arteriolar dilation in vivo, via activation of K_ATP channels in real resistance vessels in the rat.     

Antidermatitic Perspective of Hydrocortisone as Chitosan Nanocarriers: An Ex Vivo and In Vivo Assessment Using an NC/Nga Mouse Model

The aim of this study to administer hydrocortisone (HC) percutaneously in the form of polymeric nanoparticles (NPs) to alleviate its transcutaneous absorption, and to derive additional wound-healing benefits of chitosan. HC-loaded NPs had varied particle sizes, zeta potentials, and entrapment efficiencies, when drug-to-polymer mass ratios increased from 1:1 to 1:8. Ex vivo permeation analysis showed that the nanoparticulate formulation of HC significantly reduced corresponding flux [~24 μg/(cm² h)] and permeation coefficient (~4.8 × 10^? 3 cm/h) of HC across the full thickness NC/Nga mouse skin. The nanoparticulate formulation also exhibited a higher epidermal (1610 ± 42 μg/g of skin) and dermal (910 ± 46 μg/g of skin) accumulation of HC than those associated with control groups. An in vivo assessment using an NC/Nga mouse model further revealed that mice treated with the nanoparticulate system efficiently controlled transepidermal water loss [15 ± 2 g/(m² h)], erythema intensity (232 ± 12), dermatitis index (mild), and thickness of skin (456 ± 27 μm). Taken together, histopathological examination predicted that the nanoparticulate system showed a proficient anti-inflammatory and antifibrotic activity against atopic dermatitic (AD) lesions. Our results strongly suggest that HC-loaded NPs have promising potential for topical/transdermal delivery of glucocorticoids in the treatment of AD. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1063–1075, 2013     

Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI = 3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI = 150 for CD86 (EC150), and for CD54 (EC200) with LLNA’s EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R = 0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 μg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.     

Evaluation of EpiDerm full thickness-300 (EFT-300) as an in vitro model for skin irritation: Studies on aliphatic hydrocarbons

The aim of this study was to understand the skin irritation effects of saturated aliphatic hydrocarbons (HCs), C9–C16, found jet fuels using in vitro 3-dimensional EpiDerm full thickness-300 (EFT-300) skin cultures. The EFT-300 cultures were treated with 2.5 μl of HCs and the culture medium and skin samples were collected at 24 and 48 h to measure the release of various inflammatory biomarkers (IL-1α, IL-6 and IL-8). To validate the in vitro results, in vivo skin irritation studies were carried out in hairless rats by measuring trans epidermal water loss (TEWL) and erythema following un-occlusive dermal exposure of HCs for 72 h. The MTT tissue viability assay results with the EFT-300 tissue show that 2.5 μl/tissue (≈4.1 μl/cm²) of the HCs did not induce any significant changes in the tissue viability for exposure times up to 48 h of exposure. Microscopic observation of the EFT-300 cross-sections indicated that there were no obvious changes in the tissue morphology of the samples at 24 h, but after 48 h of exposure, tridecane, tetradecane and hexadecane produced a slight thickening and disruption of stratum corneum. Dermal exposures of C12–C16 HCs for 24 h significantly increased the expression of IL-1α in the skin as well as in the culture medium. Similarly, dermal exposure of all HCs for 24 h significantly increased the expression of interleukin-6 (IL-6) and IL-8 in the skin as well as in the culture medium in proportion to the HC chain length. As the exposure time increased to 48 h, IL-6 concentrations increased 2-fold compared to the IL-6 values at 24 h. The in vivo skin irritation data also showed that both TEWL and erythema scores increased with increased HCs chain length (C9–C16). In conclusion, the EFT-300 showed that the skin irritation profile of HCs was in the order of C9 ⩽ C10 ⩽ C11 ⩽ C12 < C13 ≈ C14 ≈ C16 and that the tissue was an excellent in vitro model to predict in vivo irritation and to understand the structural activity relationship of HCs.     

A validated bioanalytical method in mouse,rat, rabbit and human plasma for the quantification of one of the steroid glycosides found in Hoodia gordonii extract

Hoodia gordonii extract contains steroid glycosides, fatty acids, plant sterols and polar organic material. Certain steroid glycosides show appetite suppressant activities following oral ingestion. This study describes the validation of a bioanalytical method for the quantification of one of the steroid glycosides, H.g.-12 (～10% (w/w) of the extract), in mouse, rat, rabbit and human plasma. The method utilises a liquid–liquid extraction with methyl-tert-butyl ether followed by chromatographic separation on a 2.1 × 50 mm C₁₈ Genesis high performance liquid chromatography (HPLC) column and detection on a triple quadrupole mass spectrometer. Detection of H.g.-12 and its stable isotope internal standards is performed using positive TurboIonspray? ionisation in multiple reaction monitoring mode. The validation procedure demonstrated assay sensitivity, linearity, accuracy, precision and selectivity over the calibration range of 0.5–150 ng/mL in human plasma (500 μL sample volume), 1.0–100 ng/mL in rat and rabbit plasma (150 μL sample volume) and 1.0–250 ng/mL in mouse plasma (150 μL sample volume) with good recoveries (≥77%). H.g.-12 was stable in plasma for ≥6 months at ?20 °C, for up to 4 h at ambient temperature (ca 22 °C) and after 3 freeze–thaw cycles. Plasma extracts were stable for up to 24 h at ambient temperature.     

Permeability Enhancement for Transdermal Delivery of Large Molecule Using Low-Frequency Sonophoresis Combined with Microneedles

Transdermal drug delivery is limited by the high resistance of skin towards diffusion of high-molecular-weight drugs. This is mainly because of the fact that the outer layer of the skin, that is the stratum corneum, can prevent diffusion of molecules whose molecular weight is greater than 500 Da. Sonophoresis can be used to enhance the permeability of the skin. However, in the delivery of large molecules, ultrasound alone cannot provide sufficient permeability enhancement. In addressing this issue, we propose optimised ultrasound combined with microneedles to further increase the permeation rates. In this paper, we use porcine ear skin to simulate human skin and treat the skin samples with both ultrasound and microneedles. Further, bovine serum albumin (BSA) is used as a model of larger molecular weight molecule. Our results show that the permeability of BSA is increased to 1 μm/s with the combination of 1.5 mm microneedles patch and 15-W ultrasound output which is about 10 times higher than the permeability obtained in passive diffusion. Diffusion with only microneedles or ultrasound pre-treatment is also tested. The maximum permeability from microneedles and ultrasound treatment reached 0.43 and 0.4 μm/s, respectively. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3614–3622, 2013     

Microneedle-assisted delivery of verapamil hydrochloride and amlodipine besylate

The aim of this project was to study the effect of stainless steel solid microneedles and microneedle rollers on percutaneous penetration of verapamil hydrochloride and amlodipine besylate.Verapamil, 2-(3,4-dimethooxyphenyl)-5-[2-(3,4 dimethoxyphenyl)ethyl-methyl-amino]-2-propan-2-yl-pentanenitrile is a calcium channel blocker agent that regulates high blood pressure by decreasing myocardial contractilty, heart rate and impulse conduction. Amlodipine, (R, S)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1, 4-dihydropyridine, is a calcium channel blocker that is used for the management of hypertension and ischemic heart disease. Passive penetration of verapamil and amlodipine across the skin is low. In vitro studies were performed with microneedle-treated porcine ear skin using vertical static Franz diffusion cells (PermeGear, Hellertown, PA, USA). The receiver chamber contained 5 ml of PBS (pH7.4) and was constantly maintained at 37 °C temperature with a water circulation jacket. The diffusion area of the skin was 1.77 cm². The donor compartment was loaded with 1 ml of the solution containing 2.5 mg/ml of amlodipine besylate. The donor chamber was covered with parafilm to avoid evaporation. Passive diffusion across untreated porcine skin served as control. Aliquots were taken every 2 h for 12 h and analyzed by liquid chromatography–mass spectrometry. Transcutaneous flux of verapamil increased significantly from 8.75 μg/cm²/h to 49.96 μg/cm²/h across microneedle-roller treated porcine skin. Percutaneous flux of amlodipine besylate following the use of stainless steel microneedles was 22.39 μg/cm²/h. Passive flux for the drug was 1.57 μg/cm²/h. This enhancement of amlodipine flux was statistically significant. Transdermal flux of amlodipine with microneedle roller was 1.05 μg/cm²/h in comparison with passive diffusion flux of 0.19 μg/cm²/h. The difference in flux values was also statistically significant. Stainless steel solid microneedles and microneedle rollers increased percutaneous penetration of verapamil hydrochloride and amlodipine besylate. It may be feasible to develop transdermal microneedle patches for these drugs.     

Aerodynamic characteristics of a dry powder inhaler at low inhalation flows using a mixing inlet with an Andersen Cascade Impactor

The aerodynamic characteristics of the dose emitted from a dry powder inhaler (DPI) are inhalation flow dependent but have not been determined for low flows. We have designed novel methodology to measure these at <28.3 l min^?1. The original Andersen Cascade Impactor (ACI) designed for use at 60 l min^?1 was adapted to include a mixing inlet (MIXINLET) which allows inhalation flows through the DPI from 5 to 60 l min^?1. The mean fine particle dose (FPD) from a formoterol Turbuhaler using the MIXINLET method at 10, 20, 28.3, 40 and 60 l min^?1 was 0.55, 1.39, 1.80, 2.88 and 5.86 μg and the mass median aerodynamic diameter (MMAD) was 6.6, 6.0, 5.4, 5.1 and 2.8 μm. Similarly, the FPD using the ACI method was 0.13, 0.69, 1.50, 2.48 and 5.42 μg and MMADs were 12.2, 7.4, 5.5, 4.8 and 2.7 μm. The accuracy of the original ACI <28.3 l min^?1 is unknown. The ACI with the mixing inlet allows the determination of the in vitro dose emission properties of DPIs at flows <28.3 l min^?1 whilst maintaining a constant flow through the ACI. This methodology, therefore, can help to focus attention to the lowest inhalation flow required for a DPI.     

Effects of endocrine disrupting chemicals on in vitro global DNA methylation and adipocyte differentiation

Recent studies suggest that endocrine disrupting chemicals (EDCs) may form a risk factor for obesity by altering energy metabolism through epigenetic gene regulation. The goal of this study is to investigate the effects of a range of EDCs with putative obesogenic properties on global DNA methylation and adipocyte differentiation in vitro. Murine N2A and human SK-N-AS neuroblastoma cells and murine preadipocyte fibroblasts (3T3-L1) were exposed to tributyltin (TBT), diethylstilbestrol (DES), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153), hexachlorobenzene (HCB), hexabromocyclododecane (HBCD), 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47) , perfluorinated octyl acid (PFOA) and perfluorinated octyl sulfonate (PFOS). A modest decrease in global DNA methylation was observed in N2A cells exposed to 10 μM DES, BPA, TCDD, BDE-47, PCB-153 and 1 μM HCB, but no changes were found in the human SK-N-AS cells. We reveal for the first time that BDE-47 increases adipocyte differentiation in a dose-dependent manner (2.5–25 μM). Adipocyte differentiation was also enhanced by TBT (?10 nM) and BPA (>10 μM) and inhibited by TCDD (?0.1 nM). The other chemicals showed either modest or no effects on adipocyte differentiation at the concentrations tested (PFOA, PFOS and HBCD at 10 μM; PCB-153, 3.4 μM and HCB, 1 μM). This study demonstrates that selected EDCs can induce functional changes in murine adipocyte differentiation in vitro which are accompanied by decreased global DNA methylation.     

Safety evaluation of metal exposure from commonly used moisturizing and skin-lightening creams in Nigeria

The concentrations of ten metals (Cd, Pb, Ni, Cr, Cu, Co, Fe, Mn, Zn and Al) were measured in some commonly used moisturizing and skin-lightening creams in Nigeria with a view to providing information on the risk of exposure to metals from the use of these products. The metal concentrations in these products were measured by atomic absorption spectrometry after acid digestion of the samples. The measured concentrations of metals in the skin moisturizing creams ranged from <0.15 to 6.3 μg/g Cd, <0.02 to 17.5 μg/g Cu, 2.25 to 6.25 μg/g Cr, <0.25 to 124.3 μg/g Al, 0.2 to 7.3 μg/g Pb, <0.03 to 10.7 μg/g Ni, 17.3 to 372.0 μg/g Zn, <0.02 to 1.0 μg/g Co, 17.75 to 28.8 μg/g Mn, <0.1 to 89.8 μg/g Fe while the concentrations of metals in the skin-lightening products ranged from <0.15 to 16.5 μg/g Cd, <0.02 to 10.0 μg/g Cu, 4.25 to 8.0 μg/g Cr, <0.25 to 128.0 μg/g Al, 0.5 to 4.5 μg/g Pb, <0.03 to 1.65 μg/g Ni, 24.7 to 267.5 μg/g Zn, <0.02 to 2.5 μg/g for Co, 19.3 to 31.8 μg/g Mn, 9.5 to 211.63 μg/g Fe. In a significant number (>93%) of the samples investigated the concentrations of Pb, Cd, Ni and Co were below the specified limit, or the maximal limit for impurities in colour additives in cosmetics for external use. However, Cr was found at concentrations above the allergenic limit of 1 μg/g. The results also showed that skin-lightening creams contained higher concentrations of the studied metals than the moisturizing creams, except for Ni, which indicates that persons who uses skin-lightening creams in preference to moisturizing ones, are exposed to higher concentrations of metals.     

Development of a Microflow Digital Imaging Assay to Characterize Protein Particulates During Storage of a High Concentration IgG1 Monoclonal Antibody Formulation

The development of a Microflow digital imaging (MDI) method to detect and monitor protein particulates in a high concentration IgG1 monoclonal antibody formulation is presented. The MDI assay was optimized and qualified as a characterization assay in terms of accuracy and precision of particle size and number using polystyrene standards (5–300 μm) and protein particles (2 to > 100 μm). The stability profile of a 90 mg/mL IgG1 formulation stored at 2–8 °C and –70 °C for up to 18 months was then investigated. The MDI assay results showed improved sensitivity to detect subvisible particulates (≥ 5 μm) compared to conventional light obscuration detection, presumably due to the translucent nature of the protein particles. For evaluation of visible protein particles (≥ 70 μm), a good overall correlation was observed for MDI, inverted microscopy and visual assessments. Long-term stability data for a high concentration IgG1 monoclonal antibody formulation demonstrated an accumulation of protein particles in certain size categories with a concomitant increase in the overall particle size distribution over time. The weight amount of protein particulates in the IgG1 formulation was measured experimentally as ~ 0.022% (~ 20 μg/mL) after storage at 2–8 °C for 16 months. Similar results were obtained by calculation from the MDI particle data indicating a low level of protein particulates by weight. The nature and composition of the protein particulates formed during storage were further characterized by a combination of inverted microscopy, FTIR microscopy, and SEM-EDX. Particulates were identified as protein with silicone, although some particles also contained other elements such as aluminum. The combination of MDI results and protein characterization studies have provided an enhanced understanding of protein particulate formation during long-term storage of a high concentration IgG1 monoclonal antibody formulation. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3343-3361, 2010     

Toxicological implications of the delivery of fentanyl from gel extracted from a commercial transdermal reservoir patch

Fentanyl in a rate controlling membrane (RCM) transdermal patch form has been available since the early 1990s for outpatient management of chronic pain. Fatalities associated with misuse or overuse of fentanyl patches have been reported. Concerns have also been raised about the possibility that defects in such patches may result in leaking of the reservoir of the patch onto patients’ skin and consequent overdose. In order to investigate the possibility of fentanyl toxicity arising from leaking of patches, the permeation of fentanyl from the reservoir gel of a commercially available fentanyl transdermal patch was examined in vitro. Finite doses of the formulation were applied to human skin and permeation was monitored, at 32 °C under non-occluded conditions, for 48 h. Similar levels of skin permeation of fentanyl from the 1% gel formulation were obtained for the two skin donor samples tested. After 48 h, the dose of fentanyl that had permeated was 7.4 (±3.6)% and 7.7 (±1.9)% of the respective total amounts applied. At the end of the experiment, most of the drug was found in the residual formulation at the skin surface (i.e. 63–66%). For both the skin samples, a relatively small amount of the fentanyl applied (2–3%) was present in the skin at 48 h after application. The maximum flux from the data generated was between 6 and 24 h over which time frame it was 0.3 μg/cm²/h. Assuming spreading of leaked gel over an area of 100 cm², this would result in a plasma level of 0.6 ng/mL. The anticipated plasma levels from a 100 μg/h patch are known to be approximately 2.5 ng/ml. Thus, the maximum increase in the plasma levels from a patch which leaks gel is calculated to be, at most, about 25%.     

Inhalable tranexamic acid for haemoptysis treatment

PurposeAn inhalable dry powder formulation of tranexamic acid (TA) was developed and tested in a novel high-dose Orbital® multi-breath inhaler. The formulation was specifically intended for the treatment of pulmonary haemorrhage and wound healing associated with haemoptysis.MethodsInhalable TA particles were prepared by spray drying and the powder characterised using laser diffraction, electron microscopy, thermal analysis, moisture sorption and X-ray powder diffraction. The aerosol performance was evaluated using cascade impaction and inline laser diffraction and interaction with epithelia cells and wound healing capacity investigated using Calu-3 air interface model.ResultsThe spray dried TA particles were crystalline and spherical with a D_0.5 of 3.35 μm. The powders were stable and had limited moisture sorption (0.307% w/w at 90% RH). The Orbital device delivered ca. 38 mg powder per ‘inhalation’ at 60 l · min⁻¹ across four sequential shots with an overall fine particle fraction (⩽6.4 μm) of 59.3 ± 3.5% based on the emitted mass of ca. 150 mg. The TA particles were well tolerated by Calu-3 bronchial epithelia cells across a wide range of doses (from 1 nM to 10 nM) and no increase in inflammatory mediators was observed after deposition of the particles (a decrease in IL-1β, IL-8 and INFγ was observed). Time lapse microscopy of a damaged confluent epithelia indicated that wound closure was significantly greater in TA treated cells compared to control.ConclusionA stable, high performance aerosol of TA has been developed in a multi-breath DPI device that can be used for the treatment of pulmonary lesions and haemoptysis.     

Assessment of DNA damage,cytotoxicity, and apoptosis in human hepatoma (HepG2) cells after flurochloridone herbicide exposure

In vitro effects of flurochloridone (FLC) and its formulations Twin Pack Gold® [25% active ingredient (a.i.)] and Rainbow® (25% a.i.) were evaluated in HepG2 cells. Whereas cytokinesis-blocked micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were employed for genotoxicity, MTT, neutral red, and apoptosis detections were used for cytotoxicity evaluation. Activities were tested within the concentration range of 0.25–15 μg/ml FLC. Results demonstrated that neither FLC nor Rainbow® was able to induce MNs. On the other hand, 5 μg/ml Twin Pack Gold® only increased MN frequency. Furthermore, 10 and 15 μg/ml of both formulations resulted in cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. A marked increase in the genetic damage index was observed after treatment with all compounds. SCGE assay appeared to be more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of FLC than did MN. Our results reveal that FLC and its two formulations trigger apoptosis on HepG2 cells. The results represent the first experimental evidence of the in vitro apoptogenic role exerted on mammalian cells by FLC and the FLC-based formulations Rainbow® and Twin Pack Gold®, at least on HepG2 cells.