Author Correction: Growth hormone regulates neuroendocrine responses to weight loss via AgRP neurons.

The original version of this Article contained an error in the spelling of the author J. Donato Jr, which was incorrectly given as Donato J. Jr. This has now been corrected in both the PDF and HTML versions of the Article.

Growth hormone regulates neuroendocrine responses to weight loss via AgRP neurons.

Weight loss triggers important metabolic responses to conserve energy, especially via the fall in leptin levels. Consequently, weight loss becomes increasingly difficult with weight regain commonly occurring in most dieters. Here we show that central growth hormone (GH) signaling also promotes neuroendocrine adaptations during food deprivation. GH activates agouti-related protein (AgRP) neurons and GH receptor (GHR) ablation in AgRP cells mitigates highly characteristic hypothalamic and metabolic adaptations induced by weight loss. Thus, the capacity of mice carrying an AgRP-specific GHR ablation to save energy during food deprivation is impaired, leading to increased fat loss. Additionally, administration of a clinically available GHR antagonist (pegvisomant) attenuates the fall of whole-body energy expenditure of food-deprived mice, similarly as seen by leptin treatment. Our findings indicate GH as a starvation signal that alerts the brain about energy deficiency, triggering key adaptive responses to conserve limited fuel stores.

Brachial Plexus Blockade Causes Subclinical Neuropathy: A Prospective Observational Study.

Background: The objective of this study is to determine subclinical changes in hand sensation after brachial plexus blocks used for hand surgery procedures. We used Semmes-Weinstein monofilament testing to detect these changes. We hypothesized that patients undergoing brachial plexus nerve blocks would have postoperative subclinical neuropathy detected by monofilament testing when compared with controls. Methods: In total, 115 hand surgery adult patients were prospectively enrolled in this study. All patients undergoing nerve-related procedures were excluded as well as any patients with preoperative clinically apparent nerve deficits. Eighty-four patients underwent brachial plexus blockade preoperatively, and 31 patients underwent general anesthesia (GA). Semmes-Weinstein monofilament testing of the hand was performed preoperatively on both the operative and nonoperative extremities and postoperatively at a mean of 11 days on both hands. Preoperative and postoperative monofilament testing scores were compared between the block hand and the nonoperated hand of the same patient, as well as between the block hands and the GA-operated hands. Results: There were no recorded clinically relevant neurologic complications in the block group or GA group. A statistically significant decrease in sensation in postoperative testing in the operated block hand compared with the nonoperated hand was noted. When comparing the operated block hand with the operated GA hand, there was a decrease in postoperative sensation in the operated block hand that did not reach statistical significance. Conclusions: Brachial plexus blockade causes subtle subclinical decreases in sensibility at short-term follow-up, without any clinically relevant manifestations.

Cdc2-like kinase 2 in the hypothalamus is necessary to maintain energy homeostasis.

OBJECTIVE: To investigate whether the Cdc2-like kinase 2 (CLK2) is expressed in hypothalamic neurons and if it is, whether the hypothalamic CLK2 has a role in the regulation of energy balance. SUBJECTS: Swiss mice on chow or high-fat diet (HFD) and db/db mice on chow diet were used to address the role of CLK2 in the hypothalamus. RESULTS: Hypothalamic CLK2Thr343 phosphorylation, which induces CLK2 activity, is regulated in vivo by refeeding, insulin and leptin, in a PI3K (phosphoinositide 3-kinase)-dependent manner. The reduction of CLK2 expression in the hypothalamus, by chronic pharmacological inhibition with TG003 or by chronic knockdown with small interfering RNA was sufficient to abolish the anorexigenic effect of insulin and leptin, to increase body weight, fat mass, food intake and to decrease energy expenditure in mice on chow. In contrast, CLK2Thr343 phosphorylation in the hypothalamus in response to insulin, leptin or refeeding was impaired in mice on HFD or in db/db mice. Chronic CLK2 inhibition in the hypothalamus was associated with a slight increase in the fasting blood glucose levels, reduction in PEPCK (phosphoenolpyruvate carboxykinase) expression in the liver and enhanced glucose production from pyruvate, suggesting a regulation of hepatic glucose production. Further, overexpressing CLK2 in the mediobasal hypothalami of mice on HFD or in db/db mice by adenovirus partially reversed the obese phenotype. CONCLUSIONS: Thus, our results suggest that protein CLK2 integrates some important hypothalamic pathways, and may be a promising molecule for new therapeutic approaches for obesity and diabetes.

Early Complications Associated with the Thompson Approach to the Proximal Radius.

PURPOSE: The purpose of this study was to investigate the early complications associated with the dorsal approach to the proximal radius. This approach, also called the Thompson approach, is used relatively infrequently for the treatment of forearm fractures. It is primarily reserved for proximal one-third radius fractures where a volar plate may not be placed sufficiently proximal for adequate fixation. METHODS: A retrospective chart review was performed on forearm fractures performed at our institution. Over a period from January 2008 to May 2014 a total of 120 patients underwent fixation for radius shaft fractures either isolated or associated with ulna fractures; of these 120 patients, 11 were found to have utilized the Thompson approach to the proximal radius. Demographic data was collected, along with fracture pattern, and associated complications in the first 2 weeks after surgery. RESULTS: The average age of the patients was 31 years (range: 20 to 46 years). Ten patients were male and one was female. The mean follow-up time was 15 weeks (range: 1 to 52 weeks). The stated indication for the dorsal approach was a proximal location of the radius fracture in 10 cases and presence of dorsal open wounds in one patient. In all cases, the posterior interosseous nerve was identified and protected. The average distance from the fracture to the radial head articular surface was 72 mm (range: 34 mm to 132 mm). Four fractures were open, and seven were closed injuries. There were two postoperative posterior interosseous nerve palsies, along with one compartment syndrome requiring fasciotomy. There were no wound complications. There was an overall complication rate of 27%. CONCLUSION: Postoperative posterior interosseous nerve palsy was the most common complication in this series, occurring in 18% of the patients in spite of identification and protection of the nerve throughout the procedure. High vigilance for compartment syndrome must also be maintained after fixation of any forearm fracture, as it occurred in 1 of 11 patients in this study.

Leptin receptor-positive and leptin receptor-negative proopiomelanocortin neurons innervate an identical set of brain structures.

Neurons that express the prohormone proopiomelanocortin (POMC) in the arcuate hypothalamic nucleus (Arc) are engaged in the regulation of energy balance and glucose homeostasis. Additionally, POMC neurons are considered key first-order cells regulated by leptin. Interestingly, in the Arc, POMC cells that express the leptin receptor (POMC/LepR+ cells) are found side by side with POMC cells not directly responsive to leptin (POMC/LepR- cells). However, it remains unknown whether these distinct populations innervate different target regions. Therefore, the objective of the present study was to compare the projections of POMC/LepR+ and POMC/LepR- neurons. Using genetically modified LepR-reporter mice to identify leptin receptor-expressing cells and immunohistochemistry to stain POMC-derived peptides (α-MSH or ß-endorphin) we confirmed that approximately 80% of Arc ß-endorphin-positive neurons co-expressed leptin receptors. POMC/LepR+ and POMC/LepR- axons were intermingled in all of their target regions. As revealed by confocal microscopy, we found an elevated degree of co-localization between α-MSH+ axons and the reporter protein (tdTomato) in all brain regions analyzed, with co-localization coefficients ranging from 0.889 to 0.701. Thus, these two populations of POMC neurons seem to project to the same set of brain structures, although one of the two subtypes of POMC axons was sometimes found to be more abundant than the other in distinct subregions of the same nucleus. Therefore, POMC/LepR+ and POMC/LepR- cells may target separate neuronal populations and consequently activate distinct neuronal circuits within some target nuclei. These findings contribute to unravel the neuronal circuits involved in the regulation of energy balance and glucose homeostasis.

Brain STAT5 signaling and behavioral control.

Several growth factors and cytokines recruit the signal transducer and activator of transcription 5 (STAT5) signaling pathway to control cell proliferation, differentiation and apoptosis. Nonetheless, the importance of this transcription factor for brain functions is still poorly understood. Because some STAT5-inducing hormones, such as prolactin and leptin, act in the brain to regulate the expression of motivated behaviors, this signaling pathway is likely involved in behavioral modulation. Therefore, the objective of the present review was to summarize and discuss the available data regarding the possible role of central STAT5 signaling in the regulation of brain functions, especially on behavioral control. We discussed studies that investigated the importance of STAT5 signaling in the regulation of maternal and feeding behaviors. Additionally, we highlighted other behaviors that could be potentially affected by STAT5 signaling. This knowledge may help to understand how motivated behaviors are regulated at the cellular level.

Finasteride Quantification in Human Plasma by High-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry. Application to a Comparative Pharmacokinetics Study.

A specific, fast and sensitive LC-MS/MS assay was developed for the determination of finasteride in human plasma using betamethsone dipropionate as the internal standard (IS). The limit of quantification was 1.0 ng/ml and the method was linear in the range of 1.0-25.0 ng/ml. The retention times were 0.75 min for finasteride and 0.85 min for IS. Method intra-batch precision and accuracy ranged from 3.6 to 7.1%, and 96.6 to 103.9%, respectively. Inter-batch precision ranged from 2.5 to 3.4%, while Inter-batch accuracy ranged from 100.3 to 103.5%. The analytical method was applied to evaluate the pharmacokinetic and relative bioavailability of 2 different pharmaceutical formulations containing 1.0 mg of finasteride. This study evaluated 38 volunteers in a randomized, 2-period crossover study with 7 days washout period between doses. The geometric mean and respective 90% CI of finasteride test/reference percent ratios were 95.68% (91.2 - 104.6%) for Cmax, 97.5% (92.1-103.3%) for AUC0-t and 98.1 (92.67-103.8) for AUC0-inf. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for Cmax and AUC0-inf, it was concluded that the test formulation is bioequivalent to the reference one with respect to the rate and extent of absorption of finasteride.

Possible crosstalk between leptin and prolactin during pregnancy.

Rodents exhibit leptin resistance and high levels of prolactin/placental lactogens during pregnancy. A crosstalk between prolactin and leptin signaling has been proposed as a possible mechanism to explain the changes in energy balance during gestation. However, it remains unclear if specific neuronal populations co-express leptin and prolactin receptors. Therefore, our present study was undertaken to identify in the mouse brain prolactin-responsive cells that possibly express the leptin receptor (LepR). In addition, we assessed the leptin response in different brain nuclei of pregnant and nulliparous mice. We used a LepR-reporter mouse to visualize LepR-expressing cells with the tdTomato fluorescent protein. Prolactin-responsive cells were visualized with the immunohistochemical detection of the phosphorylated form of the signal transducer and activator of transcription-5 (pSTAT5-ir). Notably, many neurons that co-expressed tdTomato and pSTAT5-ir were observed in the medial preoptic area (MPA, 27-48% of tdTomato cells), the retrochiasmatic area (34-51%) and the nucleus of the solitary tract (NTS, 16-24%) of prolactin-treated nulliparous mice, pregnant mice and prolactin-treated leptin-deficient (ob/ob) mice. The arcuate nucleus of the hypothalamus (8-22%), the medial tuberal nucleus (11-15%) and the ventral premammillary nucleus (4-10%) showed smaller percentages of double-labeled cells among the groups. Other brain nuclei did not show significant percentages of neurons that co-expressed tdTomato and pSTAT5-ir. Late pregnant mice exhibited a reduced leptin response in the MPA and NTS when compared with nulliparous mice; however, a normal leptin response was observed in other brain nuclei. In conclusion, our findings shed light on how the brain integrates the information conveyed by leptin and prolactin. Our results corroborate the hypothesis that high levels of prolactin or placental lactogens during pregnancy may directly interfere with LepR signaling, possibly predisposing to leptin resistance.

Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey.

The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is composed of multiple cells that function individually as independent oscillators to express the self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding the presence and localization of the proteins and neuroactive substances of the SCN are essential for understanding this nucleus and for its successful manipulation. Although there have been advances in the investigation of the intrinsic organization of the SCN in rodents, little information is available in diurnal species, especially in primates. This study, which explores the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature.

Lesions of the ventral premammillary nucleus disrupt the dynamic changes in Kiss1 and GnRH expression characteristic of the proestrus-estrus transition.

We have recently demonstrated that the ventral premammillary nucleus (PMV) plays a key role in the metabolic control of the female reproductive axis. However, whether PMV neurons modulate the reproductive neural circuitry and/or the expression of sexual behaviors has not been determined. Here, we showed that the expression of estrogen and progesterone receptors in the PMV is modulated by changing levels of sex steroids across the estrous cycle. We also showed that sexual behavior, not the high physiologic levels of sex steroids, induces Fos in PMV neurons. Bilateral lesions of the PMV caused no significant changes in proceptive behavior but a high percentage of PMV-lesioned rats failed to exhibit lordosis behavior when exposed to a sexually experienced male rat (50% vs. 18% in the control group). Notably, lesions of the PMV disrupted the physiologic fluctuations of Kiss1 and GnRH mRNA expression characteristic of the proestrus-to-estrus transition. This neurochemical imbalance may ultimately alter female reproductive behavior. Our findings suggest that the PMV is a component of the neural circuitry that modulates the physiologic fluctuations of key neuroendocrine players (i.e., Kiss1 and GnRH) in the control of the female reproductive physiology.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: application to a pharmacokinetic study.

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0min at a flow-rate of 1.5 mL min⁻¹ into a Phenomenex Gemini® C18, 5 µm analytical column (150 × 4.6 mm i.d.). The calibration curve was linear over the range from 0.2 to 200 ng mL⁻¹ for dextromethorphan and doxylamine and 0.05 to 10 ng mL⁻¹ for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study.

Foot and shoe size mismatch in three different New York City populations.

Proper shoe size is an important element of foot health, especially in the elderly and diabetic populations. An improper fit can lead to pain, functional limitations, and falls. The aim of the present study was to determine the proportion of adults who are unaware of their own shoe size in 3 different New York City populations: a foot specialist private practice, an academic diabetic foot and ankle clinic, and a charity care center, the Bowery clinic, serving the homeless. A shoe size mismatch was defined as a difference of at least 0.5 in size between the measured foot and the shoe size. Demographic data were collected during the examination and retrospectively by chart review. A total of 235 volunteers participated in our study. A significant difference in the prevalence of the measured foot and shoe size mismatch was found between the cohort from the private practice compared with both the diabetic foot and ankle clinic and the Bowery clinic (P < .01 and P < .01, respectively). A significant difference was also detected (P < .05) between the private practice and the Bowery mission cohort when a difference of at least 1.5 sizes was present between the measured foot and the shoe size. Of those with a foot to shoe size mismatch, 60% had a difference of more than 0.5 in the shoe size between their right and left foot. In conclusion, our findings suggest that proper footwear sizing is lacking among a large proportion of our patients and that an adequate shoe size can be achieved with proper counseling.

Characterization of Kiss1 neurons using transgenic mouse models.

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or ß-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Melanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction.

Distribution and neurochemical characterization of protein kinase C-theta and -delta in the rodent hypothalamus.

PKC-theta (PKC-θ), a member of the novel protein kinase C family (nPKC), regulates a wide variety of functions in the periphery. However, its presence and role in the CNS has remained largely unknown. Recently, we demonstrated the presence of PKC-θ in the arcuate hypothalamic nucleus (ARC) and knockdown of PKC-θ from the ARC protected mice from developing diet-induced obesity. Another isoform of the nPKC group, PKC-delta (PKC-δ), is expressed in several non-hypothalamic brain sites including the thalamus and hippocampus. Although PKC-δ has been implicated in regulating hypothalamic glucose homeostasis, its distribution in the hypothalamus has not previously been described. In the current study, we used immunohistochemistry to examine the distribution of PKC-θ and -δ immunoreactivity in rat and mouse hypothalamus. We found PKC-θ immunoreactive neurons in several hypothalamic nuclei including the ARC, lateral hypothalamic area, perifornical area and tuberomammillary nucleus. PKC-δ immunoreactive neurons were found in the paraventricular and supraoptic nuclei. Double-label immunohistochemisty in mice expressing green fluorescent protein either with the long form of leptin receptor (LepR-b) or in orexin (ORX) neurons indicated that PKC-θ is highly colocalized in lateral hypothalamic ORX neurons but not in lateral hypothalamic LepR-b neurons. Double-label immunohistochemistry in oxytocin-enhanced yellow fluorescent protein mice or arginine vasopressin-enhanced green fluorescent protein (AVP-EGFP) transgenic rats revealed a high degree of colocalization of PKC-δ within paraventricular and supraoptic oxytocin neurons but not the vasopressinergic neurons. We conclude that PKC-θ and -δ are expressed in different hypothalamic neuronal populations.

In vitro mutagenicity of anti-inflammatory parsalmide analogues PA7, PA10, and PA31 triggered by biotransformation into hydroxy derivatives.

In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino-N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino-N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino-N-butyl-2-phenoxy-benzamide] and PA31 [5-amino-N-butyl-2-(p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/beta-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2-19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2-14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules.

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251Õ. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251Õ. StreptaseÕ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The UnitinaseÕ and SolustrepÕ formulations were the weakest, showing about 50 percent activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251Õ activity per vial, StreptaseÕ (75.7 ± 5.0 units) and StreptonaseÕ (94.7 ± 4.6 units) had the highest activity, while UnitinaseÕ (31.0 ± 2.4 units) and StrekÕ (32.9 ± 3.3 units) had the weakest activity. SolustrepÕ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay.

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ss-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251. Streptase was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase and Solustrep formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251 activity per vial, Streptase (75.7 +/- 5.0 units) and Streptonase (94.7 +/- 4.6 units) had the highest activity, while Unitinase (31.0 +/- 2.4 units) and Strek (32.9 +/- 3.3 units) had the weakest activity. Solustrep (53.3 +/- 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Cyclosporine bioequivalence study: quantification using fluorescence polarization immunoassay (FPIA) and radioimmunoassay (RIA).

OBJECTIVE: The aim of study was to compare the bioavailability of 2 cyclosporine capsule formulations (100 mg; Sigmasporin Microoral from Novaquímica Divisão Nature's Plus Farmacêutica Ltd., Brazil, as test formulation and Sandimmune Neoral from Novartis Biociências S.A., Brazil, as reference formulation) in 24 healthy male volunteers. METHODS: The study was open, randomized, with a 2-period crossover, a 1-week washout interval between doses. Blood samples were obtained over a 12-hour interval after each oral administration of cyclosporine (2 capsules of 100 mg of each formulation). Cyclosporine blood concentrations were quantified using a fluorescence polarization immunoassay (FPIA) method provided by Abbott Axsym System and Cyclo-Trac SP. Whole-blood radioimmuoassay (RIA) kit was provided by DiaSorin. These assays provided concentration-time curves for cyclosporine in blood concentration from which the following pharmacokinetic parameters were obtained: AUC(last), AUC(inf), Cmax. RESULTS: Geometric mean and 90% confidence intervals (CI) of Microoral/Neoral as percent ratios were 94.5% (90.8-98.4%) for AUC(last), 93.8% (89.7-98.1%) for AUC(inf), and 98.1% (94.5-101.8%) for Cmax when cyclosporine was determined using FPIA and 96.1% (91.9 to 100.6%) for AUC(last), 95.2% (90.2-100.5%) for AUC(inf), and 99.4% (96.4-102.4%) for Cmax using RIA. CONCLUSION: Since the 90% CI for Cmax, AUC(last) and AUC(inf) ratio were within the 80-125% interval proposed by US-FDA, it is concluded that Sigmasporin Microoral 100 mg capsule formulation is bioequivalent to Sandimmune Neoral 100 mg capsule formulation with regard to both rate and the extent of absorption.