Impacts of the 2014 severe drought on the Microcystis bloom in San Francisco Estuary.

The increased frequency and intensity of drought with climate change may cause an increase in the magnitude and toxicity of freshwater cyanobacteria harmful algal blooms (CHABs), including Microcystis blooms, in San Francisco Estuary, California. As the fourth driest year on record in San Francisco Estuary, the 2014 drought provided an opportunity to directly test the impact of severe drought on cyanobacteria blooms in SFE. A field sampling program was conducted between July and December 2014 to sample a suite of physical, chemical, and biological variables at 10 stations in the freshwater and brackish reaches of the estuary. The 2014 Microcystis bloom had the highest biomass and toxin concentration, earliest initiation, and the longest duration, since the blooms began in 1999. Median chlorophyll a concentration increased by 9 and 12 times over previous dry and wet years, respectively. Total microcystin concentration also exceeded that in previous dry and wet years by a factor of 11 and 65, respectively. Cell abundance determined by quantitative PCR indicated the bloom contained multiple potentially toxic cyanobacteria species, toxic Microcystis and relatively high total cyanobacteria abundance. The bloom was associated with extreme nutrient concentrations, including a 20-year high in soluble reactive phosphorus concentration and low to below detection levels of ammonium. Stable isotope analysis suggested the bloom varied with both inorganic and organic nutrient concentration, and used ammonium as the primary nitrogen source. Water temperature was a primary controlling factor for the bloom and was positively correlated with the increase in both total and toxic Microcystis abundance. In addition, the early initiation and persistence of warm water temperature coincided with the increased intensity and duration of the Microcystis bloom from the usual 3 to 4 months to 8 months. Long residence time was also a primary factor controlling the magnitude and persistence of the bloom, and was created by a 66% to 85% reduction in both the water inflow and diversion of water for agriculture during the summer. We concluded that severe drought conditions can lead to a significant increase in the abundance of Microcystis and other cyanobacteria, as well as their associated toxins.

Assessing the bioequivalence of topical retinoid products by pharmacodynamic assay.

PURPOSE: To develop a simple pharmacodynamic (PD) assay for the evaluation of the bioequivalence of topically applied retinoid products. METHODS: Daily applications of products containing tretinoin or adapalene were made to the forearms of human subjects for up to 21 days. Percutaneous absorption was enhanced through the use of polyethylene film occlusion (5 h). Pharmacologic activity was assessed through the daily measurement of three cutaneous responses intimately linked to retinoid-induced changes in epidermal differentiation: (1) erythema; (2) exfoliation (scaling/peeling), and (3) increased transepidermal water loss. RESULTS: The PD model exhibited the sensitivity and specificity required to function as a bioequivalence surrogate. It was possible to differentiate between: (1) three concentrations of tretinoin in a commercial cream product line; (2) two concentrations of tretinoin in a commercial gel product line; (3) different vehicles (gel vs. cream) containing the same concentration of tretinoin, and (4) tretinoin and adapalene at the same concentration. The applicability of this model for bioequivalence testing was established by showing that it had sufficient power to determine that three test tretinoin cream products and two approved generic tretinoin gel products were equivalent to their corresponding reference products. CONCLUSIONS: A surrogate PD model to assess retinoid bioequivalence has been developed.

Challenges obtaining a biowaiver for topical veterinary dosage forms.

Obtaining a biowaiver for topical drugs used in veterinary species faces many of the same challenges associated with human topicals. However, the skin of domestic animals varies anatomically and biochemically and experimental approaches to assess bioequivalence (BE) in veterinary species have challenges that are not often encountered with human skin. This is especially the situation with locally acting drugs. The focus of this paper is to address several of the challenges associated with (i) determining the BE of these locally acting drugs and (ii) critically examine the current technological advances that can act as a surrogate for clinical trials.

Percutaneous absorption in man: in vitro-in vivo correlation.

AIMS: To examine the existing literature to determine the degree to which percutaneous absorption data obtained using the excised human skin model match those obtained from living man. METHODS: The scientific literature was reviewed to collect data on compounds whose percutaneous absorption through human skin had been measured under both in vitro and in vivo conditions. The in vitro-in vivo (IVIV) correlation was evaluated by computing the in vitro/in vivo ratio using total absorption (percent of applied dose) as the metric for comparison. RESULTS: A total of 92 data sets were collected from 30 published studies. The average IVIV ratio across all values was 1.6, though for any single data set there could be a nearly 20-fold difference between the in vitro and in vivo values. In 85% of the cases, however, the difference was less than 3-fold. The correlation was significantly improved when data were excluded from studies in which the protocols for both studies were not fully harmonized. For harmonized data sets the average IVIV ratio was 0.96 and there was a less than 2-fold difference between the in vitro and in vivo results for any one compound, with IVIV ratios ranging from 0.58 to 1.28. The dominant factors leading to exclusion of data were the use of skin from different anatomical sites and vehicles of differing composition. CONCLUSIONS: Percutaneous absorption data obtained from the excised human skin model closely approximate those obtained from living man when the two study protocols are appropriately matched.

Use of excised human skin to assess the bioequivalence of topical products.

BACKGROUND: Establishing the bioequivalence of topical drug products is a costly and time-consuming process since, with few exceptions, clinical efficacy trials are required. OBJECTIVE: To develop a surrogate for clinical bioequivalence testing through evaluation of the kinetics of drug absorption in vitro through excised human skin. METHODS: The percutaneous absorption of seven approved generic topical drug products was compared with their corresponding reference products during preclinical development using the Franz diffusion cell. Thereafter, following the conduct of bioequivalence trials and regulatory approval of these products in the United States, clinical data became available to which the in vitro data were compared. RESULTS: In six of the seven cases the in vitro test:reference ratio for total absorption was close to one and indicated that the products were equivalent, in agreement with the clinical data. Results from the seventh case, in which the test:reference ratio was only 0.63, indicated that the in vitro model actually had greater sensitivity than the clinical method to detect small differences between products. CONCLUSION: These data demonstrate the relevance and predictive power of the in vitro human skin model and strongly support its use as a surrogate for in vivo bioequivalence studies.

Bioavailability of clobetasol propionate in different vehicles.

Topical clobetasol propionate is widely used for the treatment of psoriasis. One formulation of clobetasol propionate, Skin Cap, was thought by some practitioners and patients to be more effective than other formulations. Differences in corticosteroid bioavailability could account for differences in efficacy. The purpose of this study is to compare the relative bioavailability of clobetasol propionate from Skin Cap to two FDA-approved formulations of clobetasol propionate (Olux foam and Temovate scalp application). The bioavailability was assessed by measuring the percutaneous absorption of clobetasol propionate in vitro on scalp skin, using the human cadaver skin model. There was no significant difference in the percutaneous absorption of clobetasol propionate between Olux foam and Skin Cap (2.09 and 1.93% at 48 h, respectively) or between Olux foam and Temovate scalp application (3.70 and 3.46%, respectively) when applied under unoccluded conditions. Under occluded conditions, there was greater absorption of clobetasol propionate from the Olux foam as compared with Skin Cap (4.94 and 1.57%, respectively; p < 0.05). The Skin Cap vehicle does not provide more bioavailable clobetasol propionate than currently available, FDA-approved products. Other differences, including those in patient compliance, could account for the perceived efficacy of the product.

Evaluation of creams and ointments as suitable formulations for peldesine.

In-vitro studies were conducted to study the efficacy of mixed and self-emulsifying creams and hydrophobic ointment formulations in delivering peldesine (BCX-34) into and across cryopreserved human cadaver skin (HCS). Oil-in-water cream formulations, containing 1% w/w of radiolabeled C(14) BCX-34 and propylene glycol (PG), glycerin (GLY), isopropyl myristate (IPM), oleic acid (OA) and capric-caprylic esters (CE) were prepared. Petrolatum and lanolin based ointments were also prepared with PG. Sections of the HCS, 250 microm thick, were fitted to vertical Franz diffusion chambers containing a receptor medium of pH 7.4 phosphate buffer solution maintained at 37 degrees C. Using the finite dose technique, 4-6 mg of a formulation sample was applied to the epidermal surface of each section and drug diffusion was permitted for 12 and 24 h periods. The distribution of drug into the HCS epidermis, dermis and into the receptor medium was measured by scintillation spectroscopy. The results show good correlation of the calculated in-vitro values for flux and skin-vehicle partition coefficients against the observed amounts of drug detected in the HCS. The mixed emulsion cream formulation containing PG delivered higher amounts of drug into the skin when compared to the same formulation containing GLY cream. The self-emulsifying cream formulation containing IPM had a higher skin-vehicle partition coefficient and delivered more drug into the dermis when compared to those formulations that contained OA and CE. The petrolatum ointment delivered six times more drug into the epidermis than the lanolin ointment, and had higher skin-vehicle partition values. In conclusion, creams containing PG and petrolatum-base formulations would be suitable for BCX-34 dermal delivery.

Reoperation for severe pancreatitis: a 10-year experience in a tertiary care center.

OBJECTIVES: To analyze the specific problems encountered in treating patients previously operated on for necrotizing pancreatitis and to determine the benefit of such a complex and demanding procedure. DESIGN AND SETTING: Review of a case series in an academic tertiary care referral center. PATIENTS: Forty-four consecutive patients referred and reoperated on in 10 years. INTERVENTIONS: Reiterative laparotomy with complete debridement of all necrotic sites, followed by Mikulicz packing. Mikulicz packs were replaced by removable drains allowing both local prolonged lavage and open drainage of large solid necrotic debris. Enteral nutrition was performed through a feeding jejunostomy. Associated gastrointestinal tract lesions were simultaneously treated. MAIN OUTCOME MEASURES: Operative findings, bacteriological status of necrosis, in-hospital mortality, length of hospitalization, and surgical complications and their management. RESULTS: Necrosis was infected in 36 (82%) of the 44 cases and associated gastrointestinal tract lesions were found in 20 (45%) of these patients. Mortality was 23%, and was significantly (P = .03) related to the preoperative clinical status. Surgical complications occurred in 31 (70%) of the 44 patients necessitating surgical treatment in 18 (41%) of these patients. Mean (+/- SD) stay in the intensive care unit was 66+/-8 days for survivors. CONCLUSION: This complex and demanding surgical procedure is worthwhile, yielding mortality rates comparable to those observed in de novo severe necrotizing pancreatitis.

Percutaneous absorption of [3H]tretinoin and systemic exposure to mequinol after dermal application of 2% mequinol/0.01% [3H]tretinoin (Solagé) solution in healthy volunteers.

Solagé is a combination product composed of 2% mequinol (4-hydroxyanisole) and 0.01% tretinoin (all-trans-retinoic acid) in an ethanolic solution, which is being studied for its safety and efficacy as a topical treatment for disorders of skin hyperpigmentation. The purpose of this study was to evaluate the extent of percutaneous absorption of [3H]tretinoin and to estimate the systemic exposure to mequinol from this combination product when topically applied to the backs of healthy subjects. Eight subjects received bid topical applications of nonradiolabelled 2% mequinol/0.01% tretinoin solution on a 400 cm2 area of the back for 14 days. The subjects then received a single topical application of 2% mequinol/0.01% [3H]tretinoin solution. After 12 h, the radiolabelled dose was removed and bid treatment with nonradiolabelled 2% mequinol/0.01% tretinoin solution was continued for 7 days. Plasma, urine and faecal samples were analysed for total radioactivity and plasma was analysed for both mequinol and tretinoin by GC/MS procedure. Mean percutaneous absorption of [3H]tretinoin based on the cumulative recoveries of radioactivity in the urine and faeces was about 4.5% (median 2.18%). Tretinoin concentrations in plasma did not increase above endogenous levels. This was consistent with the concentrations of radioactivity in plasma, which showed an average Cmax of 91 pg-eq/mL (median 26 ng/mL). Average Cmax and AUC(0-12 h) values for mequinol were 10 ng/mL and 33 ng h/mL, respectively. Based on the results of this study, systemic toxicity from topical application of tretinoin in this formulation is unlikely, because percutaneous absorption of tretinoin is minimal and because endogenous levels of tretinoin are not increased following bid dosing with this combination formulation. The safety of mequinol in this combination formulation is supported by the low systemic exposures of the subjects in this study compared with the systemic exposures at the highest doses in the dermal toxicity studies in mice (16.6-fold) and rats (34.6-fold).

Retinoic acid-dependent stimulation of 2,2'-azobis(2-amidinopropane)-initiated autoxidation of linoleic acid in sodium dodecyl sulfate micelles: a novel prooxidant effect of retinoic acid.

(E)-Retinoic acid (RA) was shown to stimulate the rate of 2,2'-azobis(2-amidinopropane) (AAPH)-initiated autoxidation of linoleic acid (18:2) in sodium dodecyl sulfate (SDS) micelles. RA-dependent stimulation of 18:2 autoxidation was characterized by enhanced rates of dioxygen uptake which were linear with retinoid concentration. In contrast, 5,6-epoxy-RA, a major oxidation product of RA, failed to affect the rate of dioxygen consumption at all concentrations tested. RA was also shown to stimulate peroxyl radical-dependent oxidation of styrene to the corresponding oxirane when styrene was included in the micellar system as a molecular probe. Furthermore, unequivocal evidence of RA-dependent stimulation of 18:2 autoxidation was obtained by relative quantitation of 13-hydroxy-(9Z, 11E)-octadecadienoic acid (13-HODE) plus 9-hydroxy-(10E,12Z)-octadecadienoic acid (9-HODE) production. In addition, enhanced carbon-centered radical formation was demonstrated in the presence of RA by EPR spectroscopy using alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (4-POBN) as a spin trap. Analysis and quantitation of RA oxidation products indicated that RA was oxidized to one primary product, 5,6-epoxy-RA, which was identified on the basis of cochromatography with synthetic standard (in a reverse-phase HPLC system), electronic absorption spectroscopy, and positive chemical ionization mass spectrometry of the corresponding methyl ester. Other minor oxidation products were also detected but not characterized. In contrast, reaction mixtures devoid of 18:2 failed to demonstrate significant retinoid oxidation. Mechanisms are proposed to account for the prooxidant effects of RA in this system.

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT).

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).

Glucuronidation of all-trans-retinoic acid and 5,6-epoxy-all-trans-retinoic acid. Activation of rat liver microsomal UDP-glucuronosyltransferase activity by alamethicin.

The effects of detergent, alamethicin (a channel-forming peptide), and the inducers phenobarbital and 3-methylcholanthrene on glucuronidation of all-trans-retinoic acid (atRA) and 5,6-epoxy-atRA have been investigated using liver microsomes from Sprague-Dawley and Fischer 344 rats. Conditions for enzymatic glucuronidation were optimized for substrate concentration, protein, and time by using atRA and Sprague-Dawley microsomes. With detergent-activated Sprague-Dawley microsomes, 5,6-epoxy-atRA was shown to be a significantly better substrate than atRA for microsomal glucuronidation (263 vs. 116 pmol/mg/min for 5,6-epoxy-atRA and atRA, respectively). The product of incubation of microsomes with atRA and UDP-glucuronic acid was identified as a glucuronide by beta-glucuronidase hydrolysis and by HPLC analysis. Alamethicin was shown to be a highly effective activator of glucuronidation activity; atRA and 5,6-epoxy-atRA glucuronidation rates were increased 2- and 3-fold, respectively, compared with detergent activation. Alamethicin (but not detergent) significantly increased retinoid glucuronidation by microsomes from Fischer 344 rats treated with phenobarbital and 3-methylcholanthrene, compared with untreated controls. The two compounds were equally effective inducers of activity, although 5,6-epoxy-atRA was again the better substrate. The same control and induced Fischer rat microsomes were photolabeled with [32P]5-azido-UDP-glucuronic acid in the absence or presence of detergent, two concentrations of alamethicin, and a 10-fold molar excess of unlabeled UDP-glucuronic acid. Photoincorporation into microsomal proteins from detergent-disrupted induced microsomes was 2-3 times greater than that of controls. Alamethicin increased photoincorporation of the probe into UDP-glucuronosyltransferase proteins an additional 1.5-2-fold in control and induced microsomes, compared with the respective detergent-activated samples.

Comparative percutaneous absorption of lindane and permethrin.

BACKGROUND AND DESIGN: Because of the concern for potential neurotoxic effects (central nervous system excitation, convulsions) in the treatment of scabies using 1% lindane lotion, 5% permethrin cream has been suggested as an alternative scabicide. Using the finite dose technique, in vitro percutaneous absorption of 5% permethrin cream or 1% lindane lotion was measured in human and guinea pig skin following a single application. In vivo blood and brain levels of the scabicides were measured in guinea pigs following three daily applications of 5% permethrin cream or 1% lindane lotion. Permethrin and lindane levels were quantified by gas chromatography/mass spectroscopy. RESULTS: In vitro percutaneous absorption of the two scabicides was identical in guinea pig skin; however, human skin was 20-fold more permeable to lindane than to permethrin. In vivo guinea pig blood and brain levels of lindane were fourfold greater than permethrin levels. CONCLUSION: The risk for toxic effects, as assessed by systemic exposure during overuse conditions, is projected to be 40 to 400 times lower for 5% permethrin cream than for 1% lindane lotion.

A sensitive high-pressure liquid chromatography/particle beam/mass spectrometry assay for the determination of all-trans-retinoic acid and 13-cis-retinoic acid in human plasma.

A highly sensitive assay for the measurement of all-trans-retinoic acid (tretinoin) and 13-cis-retinoic acid (isotretinoin) has been developed. Collected plasma samples were protein precipitated with 2-propanol followed by solid phase extraction. The retinoic acids were subsequently derivatized to their pentafluorobenzyl esters followed by separation and isolation by reverse phase high-pressure liquid chromatography. The HPLC eluate was directed to a mass spectrometer via a particle beam interface. Selected ion monitoring (299 m/z) for the retinoic acid's carboxylate anion produced by negative chemical ionization using methane reagent gas achieved minimum detection limits of 25 pg injected. Endogenous blood levels in 19 male and 9 female subjects were measured. It was found that females have significantly more all-trans-retinoic acid than males and that both sexes demonstrate significantly more all-trans-retinoic acid then 13-cis-retinoic acid.

Free radical oxidation of (E)-retinoic acid by prostaglandin H synthase.

Cooxidative metabolism of all-trans (E)-retinoic acid (RA) by prostaglandin H synthase was investigated employing ram seminal vesicle microsomes (RSVM) or purified, RSVM-derived enzyme. RA was shown to undergo hydroperoxide [H2O2 or 5-phenyl-4-penten-1-yl hydroperoxide (PPHP)]- or arachidonic acid-dependent cooxidation by microsomal prostaglandin H (PGH) synthase as evidenced by UV spectroscopic analysis of reaction mixtures. Cooxidation of RA by microsomal or purified PGH synthase, using PPHP as substrate, was characterized by uptake of dioxygen which was first order with respect to enzyme concentration. Dioxygen uptake was inhibited by the peroxidase reducing substrate 2-methoxyphenol. In addition, O2 uptake was inhibited by the spin trap nitrosobenzene. ESR spin trapping studies, using alpha-phenyl-N-tert-butylnitrone (PBN) as the spin trap, demonstrated the formation of RA-PBN adducts, characterized by hyperfine coupling constants of alpha H = 3.2 G and alpha N = 15.8 G. Reverse phase HPLC analysis of reaction mixtures demonstrated the formation of 4-hydroxy-RA, 5,6-epoxy-RA, 4-oxo-RA, (13Z)-retinoic acid, and other geometric isomers which were identified on the basis of cochromatography with synthetic standards, UV spectroscopy, and/or mass spectrometry. Mechanisms are proposed for the hydroperoxide-dependent, PGH synthase-catalyzed oxidation of RA that are consistent with these results.

Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.

Although the activation of calcium-independent phospholipase A2 (PLA2) enzymes has been described in the heart, the pathogenetic role of this enzyme(s) in hypoxic cell injury has not been previously examined in any tissue. Therefore, we characterized the time course of activation of calcium-independent PLA2 using both plasmalogen and diacylglycerophospholipid substrates during hypoxia in rabbit proximal tubules and examined whether inhibition of calcium-independent PLA2 activity is associated with a cytoprotective effect. Subjecting rabbit proximal tubules to hypoxia for 5 min resulted in at least a threefold increase in cytosolic calcium-independent PLA2, which was selective for plasmalogen substrates (control 444 +/- 69 vs hypoxia 1,675 +/- 194 pmol.mg protein-1.min-1, n = 5). In contrast, no changes in PLA2 activity were observed in the presence of 4 mM EGTA in the membrane fraction using plasmenylcholine substrates. 20 min of hypoxia resulted in an increase in arachidonate from 3 +/- 1 to 28 +/- 4 ng/mg protein and lactate dehydrogenase release from 7.5 +/- 2% to 38 +/- 5%, n = 4. Pretreatment of proximal tubules with 10 microM Compound I, a specific inhibitor of calcium-independent PLA2, resulted in reduction in the magnitude of both hypoxia-induced arachidonic acid release (11 +/- 3 ng/mg protein) and lactate dehydrogenase release (18 +/- 4%). Our data indicate that a significant fraction of PLA2 activity in the proximal tubule is calcium-independent and selective for plasmalogen substrates. Furthermore, the activation of this enzyme plays an important role in the pathogenesis of membrane injury during hypoxia in the proximal tubule.

Topical lidocaine for anesthesia in patients undergoing pulsed dye laser treatment for vascular malformations.

Pulsed dye laser treatment of vascular malformations is moderately painful. Twenty-five percent lidocaine in 70% dimethyl sulfoxide-ethanol was used to achieve anesthesia in patients undergoing the procedure. Thirteen of 14 patients had some degree of anesthesia (average of 51%) ranging from 10% to 100%, with 8 of them experiencing good results (> 50% anesthesia). Diminishment of pain was assessed by comparison of laser therapy on lidocaine-treated versus untreated sites within the vascular malformation using verbal and visual analog scales. In vitro permeation demonstrated good absorption of lidocaine, peaking at one hour. Permeation of lidocaine was significantly greater then that observed from acid mantle cream or EMLA. Topical lidocaine at 25% concentration in dimethyl sulfoxide 70% was well tolerated and had no side effects except transient mild erythema.

Percutaneous penetration of N-nitrosodiethanolamine through human skin (in vitro): comparison of finite and infinite dose applications from cosmetic vehicles.

N-Nitroso compounds (nitrosamines) have been detected at the parts per billion level in a wide variety of matrices including industrial chemicals, pharmaceuticals, and food. Although N-nitrosodiethanolamine (NDELA) may be detected as an impurity in some cosmetic products, studies on NDELA absorption through human skin have been limited. A study to determine the extent of NDELA absorption following topical application was therefore undertaken to assist in the proper assessment of risk following unintended exposure. NDELA absorption was measured in vitro through human cadaver skin using isopropyl myristate (IPM) and generic prototype personal-care formulations (sunscreen and shampoo) spiked with [14C]NDELA. When applied as a finite dose at a concentration of 0.06% or lower, NDELA absorption was found to be a linear function of concentration. Total absorption at 48 hr ranged from approximately 35 to 65% of the dose and was formulation dependent (IPM > shampoo > or = sunscreen). Absorption occurred relatively rapidly from all formulations and peak rates of absorption were seen within the first 5 hr from the IPM and shampoo formulations. When applied as an infinite dose, total NDELA absorption followed a different rank order (shampoo > or = IPM > sunscreen) and evidence of barrier damage was seen with the shampoo formulation.