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.

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.

The inhibition of neutrophil-endothelial cell adhesion by hyaluronan independent of CD44.

OBJECTIVE: To study the effect of hyaluronan on cell adhesion and recruitment both in vitro and in vivo, since hyaluronan both inhibits restenosis and is anti-inflammatory. When administered to animals undergoing angioplasty the recruitment of cells into the restenotic plaque is inhibited, as well as into inflammatory lesions. The recent discovery that ICAM-1 binds hyaluronan and exhibits the B(X(7))B HA binding motif, led us also to investigate whether cell adhesion could be modulated by hyaluronan. MATERIALS AND METHODS: Human neutrophils were adhered to human umbilical vein (HUVEC) or Ea.hy.926 HUVEC cells stimulated with phorbol myristate acetate (PMA) or tumour necrosis factor (TNFalpha). Neutrophil binding in vivo utilized FMLP-stimulated hamster cheek pouch post-capillary venules. RESULTS: Hyaluronan inhibited human neutrophil adhesion to both PMA and TNFalpha-stimulated HUVEC. Ea.hy.926 human immortal HUVECs expressed ICAM-1 in response to TNFalpha and PMA. E-selectin was also upregulated by 6 h with TNFalpha but not significantly with PMA. TNFalpha induced CD44 expression within 4 h, but PMA not significantly up to 6 h. However, specific binding of [125I]hyaluronan to Ea.hy.926 cells was increased by PMA-stimulation at 4 h. Neutrophil adhesion to PMA-stimulated Ea.hy.926 HUVECs was inhibited in a concentration dependent fashion by both anti-ICAM-1 and hyaluronan (1 ng/ml-10 microg/ml) at 4 h. At 1 mg/ml adhesion was stimulated by hyaluronan. Hyaluronan had no effect on neutrophil adhesion to resting Ea.hy.926 cells. Hyaluronan (25 mg/kg, i.v.) inhibited cell adhesion to FMLP-stimulated post capillary venules of the hamster cheek pouch, whilst leaving cell rolling unaffected. CONCLUSIONS: These results show that hyaluronan, at concentrations below those where intra-molecular associations occur, binds selectively to stimulated endothelial cells and inhibits neutrophil adhesion in vitro and in vivo via a mechanism which may involve molecules other than CD44, such as ICAM-1.

Effects of low dose ionizing radiation on murine chronic granulomatous tissue.

PURPOSE: Substantial clinical evidence shows the efficacy of low-dose radiotherapy in the treatment of a wide variety of benign conditions. However, experimental investigations into these empirically clinical observations remain scarce. We investigated in vivo low-dose radiation effects on chronic granulomatous tissue by using the air pouch model in mice. MATERIAL AND METHODS: Chronic granulomatous air pouches were induced in mice and dosed according to 4 protocols: group I: sham control; group II: 2 Gy on day 2; group III: 2 Gy on day 6; group IV: 5 daily doses of 0.5 Gy from day 2 to 6. On day 7 after granuloma induction the granuloma wet and dry weight was estimated, the vascular content was assessed by the formation of vascular casts incorporating carmine, the inducible nitric oxide synthase (iNOS)- and heme oxygenase 1 (HO-1)-expression in tissue homogenates was assessed by Western blot analysis, and the immunohistochemical localization of iNOS was carried out in cryostat sections of the granulomatous tissue. RESULTS: We did not observe any significant reduction in granulomatous tissue wet weight or dry weight following the different radiation treatments, which indicates that anti-proliferative effects in response to the low radiation doses used, are probably not involved in the effects of anti-inflammatory radiotherapy. A single dose of 2 Gy on day 2, as well as fractionated treatment with 5 x 0.5 Gy lead to an increase in vascularity. iNOS-expression in the homogenized granulomatous tissue was decreased, being most pronounced after single-dose irradiation with 2 Gy on day 2, early on in the acute phase of inflammation. In contrast, the HO-1-expression was increased in all irradiated groups. CONCLUSION: Low doses of radiation interfere with the NO- and the HO-1 pathway. Since NO contributes to several aspects of inflammation such as oedema formation and inflammatory pain, we put forward the hypothesis, that the inhibitory effect of low doses of ionizing radiation on the NO pathway is one radiobiological mechanism underlying the clinically observed efficacy of anti-inflammatory radiotherapy and might result in the reduction of swelling as well as relief of pain. Furthermore, the suppression of iNOS activity could be due to the increase in the stress protein HO-1 by low dose radiotherapy.

Mechanisms of the anti-inflammatory activity of low-dose radiation therapy.

PURPOSE: To investigate the hypothesis that modulation of the function of activated macrophages is one of the mechanisms of the clinically observed anti-inflammatory and analgesic efficacy of low-dose radiotherapy in the treatment of a variety of painful joint diseases with total doses between 1 and 6 Gy. MATERIALS AND METHODS: Metabolic activity, cell proliferation, reproductive integrity, nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by unstimulated or [LPS/gamma-IFN-stimulated macrophages in vitro was investigated at different times after radiation doses ranging from 0.3 Gy to 10 Gy. In vivo, chronic granulomatous air pouches were induced in mice and either sham treated or irradiated with 2 Gy on day 2 or day 6, or with five daily doses of 0.5 Gy. On day 7, the iNOS expression was assessed by Western blot and localized by immuno-histochemistry in cryostat sections. RESULTS: In stimulated macrophages, metabolic activity, proliferation and reproductive integrity were not affected by radiation doses up to 10 Gy since they are apparently irreversible post-mitotic cells. However, a dose-dependent modulation of the NO pathway was observed with significant inhibition by the low radiation doses used in anti-inflammatory radiotherapy but with super-stimulation by the high radiation doses used in cancer therapy. CONCLUSIONS: The empirically based anti-inflammatory radiotherapy of benign diseases appears to act through specific modulation of different pathways of inflammatory reactions such as the nitric oxide pathway in stimulated macrophages.

Effects of diacerhein on granuloma induced cartilage breakdown in the mouse.

OBJECTIVE: Diacerhein, an anti-osteoarthritic agent, was tested for its ability to suppress synthesis of proinflammatory cytokines in a model of granuloma-induced cartilage breakdown. DESIGN: 50 TO mice received a subcutaneous implant of cotton-wrapped rat femoral head cartilage for a period of 2 weeks. Animals (N = 10/group) were dosed daily with either 6 mg/kg p.o. diclofenac or diacetylrhein at 5, 15 or 50 mg/kg p.o. in 0.1.ml 1% gum tragacanth which served as a control. Implanted cartilages were assayed for glycosaminoglycan (GAG) and hydroxyproline content. The surrounding granulomas were assayed for interleukin-1 alpha (IL-1 alpha), tumour necrosis factor-alpha (TNF-alpha) and IL-6. Statistical analysis was by Mann-Whitney U test. RESULTS: Diclofenac had no significant effect on GAG or hydroxyproline content of implanted cartilage or on granuloma cytokine concentrations. Diacerhein protected implanted cartilages against hydroxyproline loss, implanted control cartilages contained 220 micrograms hydroxyproline compared with diacerhein at 5, 15 and 50 mg/kg which produced a 21, 16 and 59% decrease in hydroxyproline loss compared with non-implanted controls (P < 0.05, 0.05 and 0.001) respectively. Diacerhein also protected against GAG loss at 5 mg/kg and 50 mg/kg, control cartilages contained 134 micrograms GAG compared with diacerhein at 5 mg/kg and 50 mg/kg which produced a 24 and 38% decrease in GAG loss respectively (P < 0.05 for both). Diacerhein significantly reduced granuloma interleukin-1 alpha content at 5 mg/kg (control level of 2.4 micrograms/ml reduced by 58%; P < 0.05), reduced TNF-alpha at 5 mg/kg and 15 mg/kg (reduced by 61%: P < 0.01 and 49%: P < 0.05 respectively; control level of 469 pg/ml) and reduced IL-6 at 15 mg/kg and 50 mg/kg (control level of 537 pg/ml reduced by 60 and 51%, respectively; P < 0.01 for both). CONCLUSIONS: The mechanism of the chondroprotective effects of diacerhein is not understood but may be explained by a reduction in the concentrations of proinflammatory cytokines.

The pharmacological modulation of angiogenesis in chronic granulomatous inflammation.

Angiogenesis is required for the progression of chronic inflammation, and agents that alter it can affect the development of inflammation and the consequent tissue destruction. However, in vivo quantification of neovascularization and its modulation by angiostatic and angiogenic agents is difficult. Studies have relied on reported effects of drugs on embryonic and tumor vasculature to infer angiomodulatory actions. We have characterized a vascular casting method that incorporates carmine in gelatin. Vascularity expressed as micrograms dye/mg dry tissue (vascularity index, V.I.) was studied in the murine chronic granulomatous air pouch. Carmine was retained within the vasculature by gelatin, and its content increased before the granulomatous tissue, resulting in a V.I. peak at 5 days, regression and a second peak over 14 to 28 days. The modulation of prostaglandin synthesis, plasma exudation and vasomotor tone showed that the carmine V.I. remained unaffected, unlike Evans blue, illustrating independence from acute inflammatory processes such as vasomotor tone and plasma exudation. The angiogenic stimulus p.o. heparin increased the V.I., whereas a sub-anti-inflammatory dose of cortisone with 1000 U heparin reduced it. Higher doses of heparin overcame this. The potent angiostatic steroid tetrahydrocortisol significantly reduced the V.I. in the absence of heparin. Cortisone exhibited independence from heparin on topical administration in hyaluronan. Dexamethasone inhibited granulomatous tissue development with a resulting increase in V.I. These observations indicated the differential effects of angiostatic and anti-inflammatory steroid activity. The pharmacological modulation of angiogenesis in inflammation can therefore be quantified.

Angiostasis and vascular regression in chronic granulomatous inflammation induced by diclofenac in combination with hyaluronan in mice.

Angiostasis and vascular regression in chronic granulomatous inflammation was assessed in mice induced with diclofenac in combination with hyaluronan. The local injection of 0.1 mL HYAL EX-0001 (0.18% diclofenac in 2.5% hyaluronan) reduced granulomatous development after six days treatment from 150.4 +/- 13.8 (0.18 saline) to 117.1 +/- 17.8 mg (dry weight, n = 10), but not significantly when compared with 0.1 mL 2.5% hyaluronan alone or diclofenac in 0.1 mL saline. Diclofenac administered in saline had no significant effect when compared with saline control. The vascular density, expressed as carmine content per mg dry weight tissue, in those animals treated with HYAL EX-0001 was also significantly reduced to 5.27 +/- 0.55 micrograms mg-1 (P < 0.1, n = 10) when compared with saline control (7.99 +/- 1.0), hyaluronan alone (7.20 +/- 1.0), and diclofenac in saline. (7.36 +/- 1.28). A similar profile of activity was seen on topical application except that all treatments did not affect granulomatous tissue development. On therapeutic dosing of mice daily with HYAL EX-0001 from day 7 after induction of the granulomatous tissue, the granulomatous tissue development was dramatically reduced from 111.67 +/- 4.40 mg (n = 14 on day 7) to 60.23 +/- 7.22 (P < 0.001, n = 8 on day 14) and 54.98 +/- 7.88 (P < 0.001, n = 8 on day 21). HYAL EX-0001 after 14 days of application significantly reduced granulomatous tissue mass when compared with the hyaluronan-dosed control on day 21 (89.58 +/- 7.49, P = 0.01, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

The modulation of granulomatous tissue and tumour angiogenesis by diclofenac in combination with hyaluronan (HYAL EX-0001).

In a novel application, hyaluronan has been utilized as a delivery system for topical and i.v. therapeutics. Clinical trials and case reports show that topical diclofenac delivered in hyaluronan (HYAL CT-1101) is effective against basal-cell carcinoma and actinic keratosis. The effect of this drug formulation on tumour growth and angiogenesis, as well as granulomatous tissue angiogenesis, has been investigated experimentally. The evidence that hyaluronan has a permissive effect on the inhibition of granulomatous tissue angiogenesis by diclofenac (as assessed by the carminel/gelatin vascular casting method) when injected into the lesion or applied topically is reviewed. Topical diclofenac in hyaluronan also induces a regression of the existing neo-vasculature of granulomatous tissue when applied therapeutically. The diclofenac formulated in hyaluronan was also found to be profoundly effective against the development of subcutaneous Colon-26 tumours in syngeneic balb/c mice (T/C ratio after 12 days topical application of 0.174, p < 0.0001). Analysis of the tumour vasculature showed that vascular development was retarded by 12 days. This was shown by the reduction in the tumour density of carmine in the vascular casts, as well as reduced blood-vessel density visualized by rat anti-mouse CD31 immunohistology. Hyaluronan alone had a significant effect on tumour development with a 50% inhibition of tumour growth and only a transient reduction in vascularity. The effects noted when diclofenac is formulated in hyaluronan, and applied topically, could be related to trans-dermal delivery and deposition properties of hyaluronan, and to the binding properties of hyaluronan to areas of pathology with high expression of hyaluronan receptors such as RHAMM, ICAM-1, and CD44.