Correcting endogenous concentrations of testosterone influences bioequivalence and shows the superiority of TDS(R)-testosterone versus Androgel(R).

AIM: Circulating concentrations of endogenous compounds such as testosterone, complicate the analysis of pharmacokinetic parameters when these compounds are administered exogenously. This study examines the influence of three correction methods of accounting for endogenous concentrations on the determination of bioequivalence between two testosterone formulations. METHODS: 12 healthy males received 50 mg TDS-testosterone, TDS-placebo, and 50 mg Androgel in a randomized placebo controlled study. Three correction methods (1,2 and 3) to remove the influence of endogenous testosterone from the exogenous blood concentrations data were carried out before the calculation of the AUC and Cmax. The relative bioavailabilities between two treatments were then performed for the AUC and Cmax for all the corrected and uncorrected data. Correction 4 was performed on the AUC and the Cmax values and the average values were calculated for both active treatments. RESULTS: The relative bioavailability comparison of the AUC and Cmax, showed that the TDS-testosterone and Androgel was bioequivalent by using uncorrected data (CI: 93 - 120%; AUC0-12 and 88 - 117%; Cmax). However, they were not bioequivalent when using all the corrections data ((Corr. 1; CI: 52 - 106%; AUC0-12 and 50 - 258%; Cmax), (Corr. 2; CI: 71 - 655%; AUC0-12 and 87 - 286%; Cmax), (Corr. 3; CI: 67 - 315%; AUC0-12 and 88 - 157%; Cmax)). TDS-testosterone also showed the higher AUC0-12 and Cmax compared to Androgel for uncorrected and all the Corrections 1, 2, 3 and 4. CONCLUSIONS: Different results obtained in the relative bioavailability between TDS-testosterone and Androgel for uncorrected data and corrected data, suggests that correcting endogenous concentrations is important for the proper determination of bioequivalent for endogenous compounds such as testosterone.

Rapid analysis of tetracaine for a tape stripping pharmacokinetic study using short-end capillary electrophoresis.

A rapid and simple short-end (reverse) capillary zone electrophoresis method was developed and validated for the separation and quantification of tetracaine in skin using tape samples. The separation was performed in a 485 mm (400 mm to window) x 50 microm internal diameter fused silica capillary using a background electrolyte of phosphoric acid-Tris pH2.5 at -25 kV. The extraction of tetracaine from tape samples was achieved using methanol diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and tetracaine were 1.25 and 1.36 min, respectively. The limit of quantification for tetracaine was 50 microg, with a signal-to-noise ratio greater than 10. The calibration curve was linear from 50 to 1200 microg with r(2) greater than 0.99. The CV for both within- and between-assay imprecision and the percentage inaccuracy for the quality control samples including lower and upper limits of quantitation were <12.1% and <11%, respectively. The absolute mean recovery of tetracaine was >97%. The accuracy and selectivity of this method allowed the rapid measurement of tetracaine in tape samples obtained from a skin tape stripping study of local anaesthetics in healthy subjects.

Validation of high-performance liquid chromatographic-mass spectrometric method for the analysis of lidocaine in human plasma.

A sensitive and simple liquid chromatography-tandem mass spectrometry method is developed and validated for the determination of lidocaine in human plasma. Bupivacaine is used as an internal standard, and the plasma extraction is performed by a simple liquid-liquid extraction. The limit of quantitation (LOQ) is 0.5 ng/mL with a signal-to-noise ratio greater than 5. The calibration curve is linear from 0.5 to 250 ng/mL with an r2 greater than 0.99. The coefficients of variation for within- and between-assay imprecision, including LOQ, are < or = 13% and < or = 8%, respectively. The percentage of inaccuracy for within- and between-assay, including LOQ, are < or = 9% and < or = 5%, respectively. The absolute recovery of lidocaine and bupivacaine are greater than 84% and 82%, respectively. The higher sensitivity and accuracy of this method allow the measurement of low concentrations of lidocaine in plasma from a clinical study of topically applied lidocaine in healthy subjects.

Lack of insulin-like growth factor binding protein-3 variation after follicle-stimulating hormone stimulation in women with polycystic ovary syndrome undergoing in vitro fertilization.

OBJECTIVE: To investigate serum and follicular fluid (FF) insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) behavior in superstimulated cycles in patients with polycystic ovary syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: Department of Obstetrics and Gynecology, University of Naples. PATIENT(S): Thirty-two patients with regular menses and tubal and/or male factor infertility and 21 patients with PCOS undergoing IVF. INTERVENTION(S): The IVF program used leuprolide acetate suppression followed by sequential hMG in the subsequent cycle. After follicular development, hCG administration was followed 34-36 hours later by oocyte retrieval. MAIN OUTCOME MEASURE(S): E2, GH, IGF-I, and IGFBP-3 assayed by RIA and immunoradiometric assay. RESULT(S): The controls and patients with PCOS showed similar increases in E2 and GH titers in response to FSH stimulation. Serum IGF-I did not change in either group and was equivalent in the FF. Patients with PCOS had a higher FF IGFBP-3 titer and did not show the decrease in serum IGFBP-3 levels of the control group after FSH stimulation. CONCLUSION(S): The apparent failure of IGFBP-3 reduction in patients with PCOS alters IGF-I bioavailability. Increased sequestration of IGF-I affects ovarian steroidogenesis and may explain the poor response to gonadotropin stimulation.

Treatment of severe Raynaud's syndrome by injection of autologous blood pretreated by heating, ozonation and exposure to ultraviolet light (H-O-U) therapy.

OBJECTIVE: To determine the effect of re-injection of small samples of autologous blood, pretreated with heat, ozone and ultraviolet light (H-O-U therapy) in patients with severe Raynaud's syndrome. EXPERIMENTAL DESIGN: Open trial in 4 patients. SETTING: Temperature/humidity controlled vascular laboratory. PATIENTS: Severe Raynaud's syndrome of more than 5 years duration and defined as more than 5 attacks daily or 10 attacks in one week, at least half of which were painful and lasting for more than 30 minutes. Three patients were refractory to infusions of Iloprost. INTERVENTIONS: Patients were treated daily or on alternate days for a two to three weeks period by re-injection of citrated autologous blood pre-treated with heat, ozone and ultraviolet light (H-O-U therapy). MEASURES: Clinical observations; mean equilibrated hand temperature (infrared thermography); distributive and microcirculatory blood-flow (venous occlusion strain-gauge plethysmography, infrared photoplethysmography, laser Doppler flowmetry) iontophoresis of acetylcholine and sodium nitroprusside; estimations: serum levels of 6-keto-PGF1alpha and serum levels of anti-hsp65 antibody. RESULTS: Reduction or abolition of Raynaud's attacks for at least three months after treatment. Mean equilibrated hand temperature increased but did not normalise. Blood flow parameters improved but did not reach statistical significance. Iontophoresis of acetylcholine showed an increase in laser Doppler flowmetry which was statistically significant. Serum levels of 6-keto-PGF1alpha, fell significantly in three patients. Serum levels of anti-hsp65 antibody fell in the one patient which was followed sequentially. CONCLUSIONS: H-O-U therapy may prove useful in patients with severe Raynaud's syndrome.

Neuromuscular electrostimulation viathe common peroneal nerve promotes lower limb blood flow in a below-kneecast: A potential for thromboprophylaxis.

OBJECTIVES: We aimed to examine the characteristics of deep venous flow in the leg in a cast and the effects of a wearable neuromuscular stimulator (geko; FirstKind Ltd) and also to explore the participants' tolerance of the stimulator. METHODS: This is an open-label physiological study on ten healthy volunteers. Duplex ultrasonography of the superficial femoral vein measured normal flow and cross-sectional area in the standing and supine positions (with the lower limb initially horizontal and then elevated). Flow measurements were repeated during activation of the geko stimulator placed over the peroneal nerve. The process was repeated after the application of a below-knee cast. Participants evaluated discomfort using a questionnaire (verbal rating score) and a scoring index (visual analogue scale). RESULTS: The geko device was effective in significantly increasing venous blood flow in the lower limb both with a plaster cast (mean difference 11.5 cm/sec(-1); p = 0.001 to 0.13) and without a plaster cast (mean difference 7.7 cm/sec(-1); p = 0.001 to 0.75). Posture also had a significant effect on peak venous blood flow when the cast was on and the geko inactive (p = 0.003 to 0.69), although these differences were less pronounced than the effect of the geko (mean difference 3.1 cm/sec(-1) (-6.5 to 10)). The geko device was well tolerated, with participants generally reporting only mild discomfort using the device. CONCLUSION: The geko device increases venous blood flow in the lower limb, offering a potential mechanical thromboprolylaxis for patients in a cast. Cite this article: Bone Joint Res 2013;2:179-85.

Aspirin has little additional anti-platelet effect in healthy volunteers receiving prasugrel.

BACKGROUND: Strong P2Y(12) blockade, as can be achieved with novel anti-platelet agents such as prasugrel, has been shown in vitro to inhibit both ADP and thromboxane A(2) -mediated pathways of platelet aggregation, calling into question the need for the concomitant use of aspirin. OBJECTIVE: The present study investigated the hypothesis that aspirin provides little additional anti-aggregatory effect in a group of healthy volunteers taking prasugrel. STUDY PARTICIPANTS/METHODS: In all, 9 males, aged 18 to 40 years, enrolled into the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg until day 21. At day 8, aspirin 75 mg was introduced and the dose increased to 300 mg on day 15. On days 0, 7, 14 and 21, platelet function was assessed by aggregometry, response to treatments was determined by VerifyNow and urine samples were collected for quantification of prostanoid metabolites. RESULTS: At day 7, aggregation responses to a range of platelet agonists were reduced and there was only a small further inhibition of aggregation to TRAP-6, collagen and epinephrine at days 14 and 21, when aspirin was included with prasugrel. Urinary prostanoid metabolites were unaffected by prasugrel, and were reduced by the addition of aspirin, independent of dose. CONCLUSIONS: In healthy volunteers, prasugrel produces a strong anti-aggregatory effect, which is little enhanced by the addition of aspirin. The addition of aspirin as a dual-therapy with potent P2Y(12) receptor inhibitors warrants further investigation.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2.

BACKGROUND: Currently, 'aspirin resistance', the anti-platelet effects of non-steroid anti-inflammatory drugs (NSAIDs) and NSAID-aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A(2) formation is non-linear and TXA(2) generation must be inhibited by at least 95% to inhibit TXA(2)-dependent aggregation. This relationship, however, has never been rigorously tested. OBJECTIVES: To characterize, in vitro and ex vivo, the concentration-dependent relationships between TXA(2) generation and platelet activity. METHOD: Platelet aggregation, thrombi adhesion and TXA(2) production in response to arachidonic acid (0.03-1 mmol L(-1)), collagen (0.1-30 microg mL(-1)), epinephrine (0.001-100 micromol L(-1)), ADP, TRAP-6 amide and U46619 (all 0.1-30 micromol L(-1)), in the presence of aspirin or vehicle, were determined in 96-well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. RESULTS: Platelet aggregation, adhesion and TXA(2) production induced by either arachidonic acid or collagen were inhibited in concentration-dependent manners by aspirin, with logIC(50) values that did not differ. A linear relationship existed between aggregation and TXA(2) production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R(2) 0.92; n = 224). The same relationships were seen in combinations of aspirin-treated and naïve platelets, and in blood from individuals taking an anti-thrombotic dose of aspirin. CONCLUSIONS: These studies demonstrate a linear relationship between inhibition of platelet TXA(2) generation and TXA(2)-mediated aggregation. This finding is important for our understanding of the anti-platelet effects of aspirin and NSAIDs, NSAID-aspirin interactions and 'aspirin resistance'.

Validation and application of capillary electrophoresis for the analysis of lidocaine in a skin tape stripping study.

A fast and simple capillary zone electrophoresis method was developed and validated for the determination of lidocaine in skin using tape samples. Separation was performed in a 350 mm (265 mm to window) x 50 microm i.d. fused silica capillary using a background electrolyte of phosphoric acid-Tris pH 2.5. The extraction of lidocaine from tape samples was achieved using methanol, which was diluted to 50% with water before injection. Procaine was the internal standard. The migration times for procaine and lidocaine were 2.9 and 3.2 min, respectively. The limit of quantification for lidocaine was 50 microg, with signal to noise ratio greater than 10. The calibration curve was linear from 50 to 1000 microg with r(2) greater than 0.99. The CV for both within- and between-assay imprecision and the percentage of inaccuracy for the quality control samples including lower and upper limits of quantitation were 97%. The accuracy and selectivity of this method allowed the measurement of lidocaine in tape samples obtained from a skin tape stripping study of local anesthetics in healthy subjects.

Comparison of vibration perception thresholds in individuals with diffuse upper limb pain and carpal tunnel syndrome.

The study objective was to compare vibration perception and patterns of blood flow in outpatients with diffuse upper limb pain disorder (ULPD), carpal tunnel syndrome (CTS) and age and sex matched healthy controls. Vibration perception and discrimination thresholds were compared in subjects with ULPD (n=27), CTS (n=27) and healthy matched controls (n=54). Vibration measurements were taken bilaterally at three sites: (a) over the dorsum of the second and (b) fifth metacarpals and (c) the palmar aspect of the first and second metacarpals, corresponding to the innervation territories of the radial, ulnar and median nerves, respectively. Non-invasive assessments of peripheral blood flow were also performed in both limbs. When compared to healthy controls, subjects with ULPD had widespread elevation of vibration thresholds both ipsilateral and contralateral to the symptomatic limb. Subjects with CTS had similarly elevated vibration thresholds at sites both adjacent to and distant from the site of peripheral nerve injury. The responses to cold pressor testing of the upper limbs were physiologically normal in both the CTS and ULPD patient groups. Furthermore, there were no significant differences in the haemodynamic responses between the patient groups. The global elevation of vibration thresholds in subjects with both ULPD and CTS is consistent with altered central nervous system mechanisms, common to both conditions, which may be either adaptive to or maintaining the perception of pain.

Pharmacokinetics of a new testosterone transdermal delivery system, TDS-testosterone in healthy males.

AIMS: The Transdermal Delivery System (TDS) is a liquid formulation that can be applied to the skin via a metered pump spray to deliver drug to the systemic circulation. The aims of this study were to assess the ability of the TDS preparation to deliver testosterone systemically, and to characterize the pharmacokinetic profiles of the hormone in healthy males. METHODS: An open label, comparative, randomized placebo controlled study involving three treatments and three periods with a minimum of a 1 week washout period was conducted. Twelve healthy males received 50 mg TDS-testosterone, TDS-placebo, and 50 mg of a commercially available topical testosterone preparation (Androgel, 1% topical testosterone gel). RESULTS: The mean AUC(0,12 h) was higher following application of TDS-testosterone (61.8 ng ml-1 h), compared with Androgel (57.7 ng ml-1 h) and TDS-placebo (50.7 ng ml-1 h. The mean Cmax (0,12 h) was similar for TDS-testosterone (6.6 ng ml-1) and Androgel (6.5 ng ml-1) and these values were higher than those for TDS-placebo (5.7 ng ml-1). Analysis of variance showed that the 90% confidence intervals on the relative difference of the ratio for the AUC(0,12 h) and the Cmax (0,12 h) between TDS-testosterone and Androgel, were contained within the bioequivalence limit (80, 125%) (Cmax 89.2, 112.3% and AUC 93.5, 120.5%). Serum testosterone concentrations were lower following TDS-Placebo and were not bioequivalent either to the gel or spray. CONCLUSIONS: The TDS preparation was shown to deliver testosterone systemically to humans and the concentrations of the hormone in the 12 h following TDS administration were bioequivalent to an existing topical delivery gel.

Study of a combined percutaneous local anaesthetic and the TDS system for venepuncture.

Transdermal Delivery System (TDS) is a liquid formulation which can be applied to the skin via a metered pump spray to deliver drug across skin. This placebo controlled, double blind trial compared anaesthetic properties of two TDS systems (TDS alpha and TDS beta) with placebo. The active and placebo treatments were applied to the dorsum of the hands, bilaterally and simultaneously for 5 min on 100 healthy volunteers. Following cannulation, pain perception was measured using the verbal rating score (VRS) and visual analogue score (VAS). Lidocaine plasma levels were assessed at 0 and 2 h. The VRS and VAS results show that TDS beta significantly decreased pain score compared to placebo (p < 0.02). Blood lidocaine at 2 h post application was also higher for TDS beta than for TDS alpha, suggesting that a 5 min application of TDS beta was effective in delivering local anaesthetic and accelerating the onset of skin anaesthesia prior to venous cannulation in adults.

Study of a combined percutaneous local anaesthetic and nitric oxide-generating system for venepuncture.

Nitric oxide (NO) may be generated and delivered into the skin via a novel system of sodium nitrite and ascorbic acid. This placebo-controlled, double-blind trial compared the analgesic properties of this system alone and when supplemented with lidocaine. The pain of dorsal hand vein cannulation was assessed in 100 volunteers. The NO-generating system was prepared by mixing two gels, the first KY jelly and sodium nitrite (10% w/v), the second KY jelly and ascorbic acid (10% w/v). NO-generating gel was the placebo treatment, and when combined with lidocaine (final concentration 5%), formed the active treatment. The gels were applied to the dorsum of the hands bilaterally and simultaneously for 10 min. Following cannulation, pain perception was measured with a verbal rating score (VRS) and a visual analogue score (VAS). The active formulation significantly decreased the VRS (p < 0.0001) and also reduced the mean VAS by > 40% compared with placebo (p < 0.001). This investigation suggests a 10-min topical application of anaesthetic combined with the NO-generation system may provide effective analgesia for venous cannulation in adults.

Characterization of leaf senescence and pod development in soybean explants.

Excised soybean (Glycine max [L.] Merrill) cv Anoka leaf discs tend to remain green even after the corresponding intact leaves have turned yello on fruiting plants. We have found that explants which include a leaf along with a stem segment (below the node) and one or more pods (maintained on distilled H(2)O) show similar but accelerated leaf yellowing and abscission compared with intact plants. In podded explants excised at pre-podfill, the leaves begin to yellow after 16 days, whereas those excised at late podfill begin to yellow after only 6 days. Although stomatal resistances remain low during the first light period after excision, they subsequently increase to levels above those in leaves of intact plants. Explants taken at mid to late podfill with one or more pods per node behave like intact plants in that pod load does not affect the time lag to leaf yellowing. Explant leaf yellowing and abscission are delayed by removal of the pods or seeds or by incubation in complete mineral nutrient solution or in 4.6 micromolar zeatin. Like chorophyll breakdown, protein loss is accelerated in the explants, but minerals or especially zeatin can retard the loss. Pods on explants show rates and patterns of color change (green to yellow to brown) similar to those of pods on intact plants. These changes start earlier in explants on water than in intact plants, but they can be delayed by adding zeatin. Seed dry weight increased in explants, almost as much as in intact plants. Explants appear to be good analogs of the corresponding parts of the intact plant, and they should prove useful for analyzing pod development and mechanisms of foliar senescence. Moreover, our data suggest that the flux of minerals and cytokinin from the roots could influence foliar senescence in soybeans, but increased stomatal resistance does not seem to cause foliar senescence.

Effect of nitric-oxide-generating system on microcirculatory blood flow in skin of patients with severe Raynaud's syndrome: a randomised trial.

BACKGROUND: Patients with Raynaud's syndrome have abnormal digital vasoconstriction, which may be secondary to impaired synthesis of, or impaired sensitivity to, nitric oxide. We studied the effect on microcirculation of a nitric-oxide-generating system applied topically to the finger and forearm of healthy volunteers and patients with primary Raynaud's syndrome. METHODS: We did a single-blind, randomised, placebo controlled, cross-over study of the microcirculatory response to topical application of a nitric-oxidegenerating gel in 20 patients with severe Raynaud's syndrome, and ten healthy volunteers. We prepared the nitric-oxide-generating system by mixing a solution of KY jelly and sodium nitrite (5% weight/volume), with a solution of KY jelly and ascorbic acid (5% weight/volume). About 0.5 mL of each solution was separately applied to the skin of the forearm (3 cm2), and then mixed with a sterile cotton bud. A similar procedure was done simultaneously on the other arm with KY jelly only (placebo). The procedure was then repeated on the finger pulps. Changes in skin microcirculatory volume and flux were measured bilaterally by infrared photoplethysmography and laser doppler fluxmetry, respectively. FINDINGS: In the forearm, blood flow increased significantly after application of the active gel both in patients with Raynaud's syndrome (microcirculatory volume from mean area under the curve 98 [SE 14] to 1024 [130]; microcirculatory flux from 5060 [462] to 74,800 [3940]) and in healthy controls (volume from 85 [19] to 1020 [60]; flux from 4420 [435] to 84,500 [7000]). In the fingers, although baseline blood flow was lower in patients than in controls, both groups showed increases with application of active gel (volume from 1100 [194] to 3280 [672] and 2380 [441] to 6160 [1160], respectively; flux from 33,400 [4200] to 108,000 [13,600] and 52,000 [8950] to 185,000 [19,500]). Increases in blood flow with placebo gel were not significant. No adverse effects were reported. INTERPRETATION: In primary Raynaud's syndrome, topical application of a nitric-oxide-generating system can stimulate an increase in both microcirculatory volume and flux.

A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane.

Nitric oxide (NO) is a mediator of essential biological processes, including vasodilatation, anti-microbial activity and wound healing. A chemical system using sodium nitrite and ascorbic acid has been developed which generates significant amounts of NO. The originally described system was messy and impractical, and the high acidity may cause pain and further tissue damage in ulcerated skin. To overcome this, a selectively permeable, hydrophilic polyester co-polymer membrane system (Sympatex ) has been identified that can be placed between the NO-generating chemicals and the skin. The aim of the present study was to determine whether NO derived from this chemical system was able to diffuse through the membrane and have a measurable vasodilatory effect on forearm skin in healthy volunteers. The Sympatex 10 microm membrane was found to be highly permeable to NO, while preventing passage of the constituents of the NO-generation gel to the skin. The transmembrane NO-generation system had a vasodilatory effect comparable with that resulting from direct topical application. Additionally, the NO generated was effective in killing Staphylococcus aureus and Escherichia coli at doses lower than those required to increase skin blood flow. The vasodilatory and anti-microbial effects of this system may be useful as a patch-based topical therapy for skin ulceration, particularly when there is concomitant ischaemia and infection.

Erythromelalgia: an endothelial disorder responsive to sodium nitroprusside.

Erythromelalgia is an unusual syndrome of painful vasodilatation. Aetiopathology is probably different in children and adults. Presentation can be severe and associated with hypertension. Dramatic benefit from infused nitroprusside suggests the disorder could represent a dysfunctional endothelium.