Oxidative metabolism of razuprotafib (AKB-9778), a sulfamic acid phosphatase inhibitor, in human microsomes and recombinant human CYP2C8 enzyme.

Razuprotafib, a sulphamic acid-containing phosphatase inhibitor, is shown in vivo to undergo enzymatic oxidation and methylation to form a major metabolite in monkey and human excreta with an m/z- value of 633.LC-MS/MS analysis of samples derived from incubations of razuprotafib with human liver microsomes and recombinant CYP2C8 enzyme has elucidated the metabolic pathway for formation of the thiol precursor to the S-methyl metabolite MS633 (m/z- 633).Under in vitro conditions, the major pathway of razuprotafib metabolism involves extensive oxidation of the thiophene and phenyl rings.A single oxidation takes place at one of the phenyl groups. Multiple oxidations occur at the thiophene moiety: initial oxidation results in the formation of a thiolactone followed by a second oxidation giving rise to an S-oxide of the thiolactone, which is further metabolised to the ring-opened form and ultimate formation of a thiol (m/z- 619).An additional mono-oxidation pathway involves epoxidation of the thiophene followed by hydrolysis to a diol.The thiol and diol metabolites are trapped by the addition of a nucleophilic trapping agent, 3-methoxyphenacyl bromide (MPB), giving adducts with m/z- 767.The thiol is a likely precursor to the major in vivo razuprotafib metabolite, MS633.

The in vivo disposition of subcutaneous injected 14C-razuprotafib (14C-AKB-9778), a sulphamic acid phosphatase inhibitor, in nonclinical species and human.

The disposition of radioactivity following subcutaneous 14C-razuprotafib, a Tie2 activator, was explored in multiple species.The absorption and clearance of razuprotafib and total radioactivity in human plasma are rapid and pharmacokinetics support razuprotafib as primary circulating component. Radioactivity is distributed greater to human plasma than whole blood (B:P = 0.36).In pigmented rats, radioactivity distributes to whole-body tissues rapidly and, within 24 h, is localised to elimination pathway end organs and injection site.Overall recovery of radioactivity across species is >93%, with the majority recovered within 24-48 h, and >80% in faeces.The CYP2C8 enzyme contributes significantly to razuprotafib metabolism.A hydrolysis product of razuprotafib (m/z- 380) is the main component in rat plasma at 2 h (49% peak area radioactivity), while razuprotafib (m/z- 585) is the main component in plasma for dog (58%), monkey (99.3%), and human (100%).Razuprotafib is present in dog, monkey, and human faeces, with the greatest percentage of radioactivity as metabolites. The major metabolite (>25%) in monkey and human, m/z- 633, is an S-methylated oxidised derivative of razuprotafib and is localised in faeces.Overall disposition of 14C-razuprotafib in human is best modelled by monkey over lower order species.

The influence of physicochemical properties on the reactivity and stability of acyl glucuronides †.

1. Formation of 1-O-acyl-ß-d-glucuronide conjugates is a significant pathway in the metabolism of drugs containing a carboxylic acid group. The formation of acyl glucuronides results in an increase in both the aqueous solubility and molecular mass of the conjugate in comparison to the parent drug and thus facilitates excretion in both urine and bile. 2. Acyl glucuronides are effectively esters, which undergo first order decomposition by both hydrolysis and the intra-migration of the acyl group around the glucuronide ring to yield 2-, 3- and 4-O-glucuronic acid esters which, unlike the metabolically formed 1-O-acyl-ß-d-glucuronides, are not substrates for ß-glucuronidase. The first order degradation half-life is therefore a composite value of these two reactions and a useful indicator of chemical reactivity and potential toxicity. 3. Intra-molecular migration is expected to be the predominant pathway due to entropic considerations. 4. Such conjugates, together with their isomeric ester derivatives, react with nucleophilic sites on proteins and small endogenous molecules, such as glutathione, which potentially contributes to the observed toxicity and adverse drug reactions associated with some drugs. 5. Examination of the stability of the 1-O-acyl-ß-d-glucuronides of aryl acetic acid, α-carbon substituted aryl acetic acid, aliphatic and aromatic acids, as determined by their first order degradation half-lives, indicates the significance of electronic and steric features that contribute to conjugate stability under physiological conditions. 6. Examination of the of the electronic properties of the carbonyl carbon atom in acyl glucuronides, as measured by the pKa of the parent acid, together with the steric substituents about the acyl carbonyl provides insight into the reactivity of these conjugates. 7. The investigations reported herein on a large number of 1-O-acyl-ß-d-glucuronides has allowed rationalisation of their physicochemical properties in relation to the structure of the parent drug and has the potential to contribute to the design of carboxylic acid containing drug molecules with increased stability of a major metabolite with potential reduction in toxicity and adverse drug reactions.

First-in-Human Phase 1 Study Evaluating the Safety, Pharmacokinetics, and Pharmacodynamics of DISC-0974, an Anti-Hemojuvelin Antibody, in Healthy Participants.

Pathologic elevations in hepcidin, a key regulator of iron homeostasis, contribute to anemia of inflammation in chronic disease. DISC-0974 is a monoclonal antibody that binds to hemojuvelin and blocks bone morphogenetic protein signaling, thereby suppressing hepcidin production. Reduction of systemic hepcidin levels is predicted to increase iron absorption and mobilize stored iron into circulation, where it may be utilized by red blood cell (RBC) precursors in the bone marrow to improve hemoglobin levels and to potentially alleviate anemia of inflammation. We conducted a first-in-human, double-blind, placebo-controlled, single-ascending dose study to evaluate safety, pharmacokinetics, and pharmacodynamics of DISC-0974 in healthy participants. Overall, 42 participants were enrolled and received a single dose of placebo or DISC-0974 at escalating dose levels (7-56 mg), administered intravenously (IV) or subcutaneously (SC). DISC-0974 was well tolerated, with a safety profile comparable to that of placebo. Pharmacokinetic data was dose and route related, with a terminal half-life of approximately 7 days. The bioavailability of SC dosing was ∼50%. Pharmacodynamic data showed dose-dependent decreases in serum hepcidin, with reductions of nearly 75% relative to baseline at the highest dose level tested, and corresponding increases in serum iron in response to DISC-0974 administration. Dose-dependent changes in serum ferritin and hematology parameters were also observed, indicating mobilization of iron stores and downstream effects of enhanced hemoglobinization and production of RBCs. Altogether, these data are consistent with the mechanism of action of DISC-0974 and support the selection of a biologically active dose range for evaluation in clinical trials for individuals with anemia of inflammation.

Tie2 Activation via VE-PTP Inhibition With Razuprotafib as an Adjunct to Latanoprost in Patients With Open Angle Glaucoma or Ocular Hypertension.

Purpose: To evaluate the ocular hypotensive efficacy and safety of razuprotafib, a novel Tie2 activator, when used as an adjunct to latanoprost in patients with open-angle glaucoma (OAG) or ocular hypertension (OHT). Methods: Subjects with OAG or OHT and an unmedicated IOP from ≥22 mm Hg to <36 mm Hg were randomized to one of three treatment arms: razuprotafib every day (QD) + latanoprost; razuprotafib twice daily (BID) + latanoprost; or latanoprost monotherapy. The primary endpoint was change in mean diurnal IOP from baseline at day 28. Results: A total of 194 subjects were randomized, and 193 (99.5%) completed the study. Razuprotafib BID + latanoprost resulted in a significantly larger reduction in diurnal IOP than latanoprost alone (7.95 ± 0.26 mmHg vs. 7.04 ± 0.26 mm Hg, P < 0.05). A smaller improvement was observed after 14 days of treatment (7.62 ± 0.26 mm Hg vs. 7.03 ± 0.26 mm Hg, P = 0.11). Razuprotafib QD dosing did not demonstrate additional IOP lowering compared to latanoprost alone. Conjunctival hyperemia on Day 28 increased by 1.1 units on the four-point Efron scale two hours post dose from a baseline value of 0.6 units, and decreased thereafter. Conclusions: Topical ocular razuprotafib as an adjunct to latanoprost therapy was well tolerated and significantly reduced IOP in patients with OAG/OHT. Translational Relevance: These data support the IOP lowering efficacy of targeting Tie2 activation in Schlemm's canal in the relevant patient population.

Testosterone reference ranges in normally cycling healthy premenopausal women.

INTRODUCTION: At present, there are no well-accepted reference ranges for serum testosterone concentrations in women. AIM: The aim of this study was to determine the reference ranges for serum testosterone and sex hormone-binding globulin (SHBG) in premenopausal women with normal menstrual cycles. METHODS: We measured serum total, free, and bioavailable testosterone and SHBG concentrations in 161 healthy, normally cycling women (18-49 years). Morning blood samples were collected during follicular, mid-cycle, and luteal phases of the menstrual cycle and analyzed using validated methods. Mean, median, and weighted average hormone levels across menstrual cycle phases as well as percentiles for a typical 30-year-old woman were determined. MAIN OUTCOME MEASURES: Age-related serum levels of total, free, and bioavailable testosterone and SHBG levels in normally cycling premenopausal women. RESULTS: Serum testosterone concentrations exhibited an age-related decline, whereas SHBG remained relatively stable across studied age ranges. Reference ranges for total, free, and bioavailable testosterone and SHBG were established using 5th and 95th percentiles. The estimated 5th and 95th percentiles for a 30-year-old woman were: testosterone, 15-46 ng/dL (520-1595 pmol/L); free testosterone, 1.2-6.4 pg/mL (4.16-22.2 pmol/L); calculated free testosterone, 1.3-5.6 pg/mL (4.5-19.4 pmol/L); bioavailable testosterone, 1.12-7.62 ng/dL (38.8-264.21 pmol/L); and SHBG 18-86 nmol/L. The variations of hormones and SHBG across menstrual cycle were consistent with previous literature. CONCLUSIONS: Reference ranges for free, total, and bioavailable testosterone and SHBG were established in premenopausal women using validated immunoassays and an adequate number of subjects consistent with recommendations by the National Committee for Clinical Laboratory Standards. The increase in testosterone in the mid-cycle period is relatively small compared with the overall variability, so these reference ranges can be applied irrespective of the day in the menstrual cycle the sample has been taken.

VE-PTP inhibition elicits eNOS phosphorylation to blunt endothelial dysfunction and hypertension in diabetes.

AIMS: Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity. METHODS AND RESULTS: In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium-intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases, i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and down-regulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition. CONCLUSIONS: VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension.

Efficacy and safety of a testosterone patch for the treatment of hypoactive sexual desire disorder in surgically menopausal women: a randomized, placebo-controlled trial.

OBJECTIVE: Evaluation of the use of testosterone therapy for hypoactive sexual desire disorder (HSDD) after oophorectomy has mostly involved women treated with oral estrogen preparations. We investigated the efficacy and safety of a testosterone patch in surgically menopausal women receiving concurrent transdermal estrogen. DESIGN: Women with HSDD after oophorectomy, for whom this was a concern, who were using transdermal estrogen, were recruited to a 24-week, randomized, double-blind, placebo-controlled trial in Europe and Australia. Patients were randomly allocated to placebo (n = 40) or testosterone 300 microg/day (n = 37) treatment. Primary endpoints were changes in sexual desire measured by the sexual desire domain of the Profile of Female Sexual Function and the frequency of satisfying sexual activity at 24 weeks. RESULTS: Sixty-one women (79%) completed the trial. All subjects who received at least one application of study medication were included in analysis. The testosterone-treated group experienced a significantly greater change from baseline in the domain sexual desire score compared with placebo (change from baseline, 16.43 versus 5.98; P = 0.02). The domain scores for arousal, orgasm, decreased sexual concerns, responsiveness, and self-image as well as decreased distress were also significantly greater with testosterone therapy than placebo. The frequency of satisfactory sexual events increased but was not statistically different between treatment groups (P = 0.06) Adverse events occurred with similar frequency in both groups, and no serious risks of therapy were observed CONCLUSIONS: In this study, transdermal testosterone therapy via a skin patch improved sexual desire and other sexual function domains. It was well tolerated in these oophorectomized women with HSDD receiving concomitant transdermal estrogen.

Testosterone patch for the treatment of hypoactive sexual desire disorder in naturally menopausal women: results from the INTIMATE NM1 Study.

OBJECTIVE: To evaluate the efficacy and safety of a testosterone patch for the treatment of women with hypoactive sexual desire disorder after natural menopause. DESIGN: A multicenter, randomized, double-blind, placebo-controlled, parallel-group trial was conducted in naturally menopausal women with hypoactive sexual desire disorder receiving a stable dose of oral estrogen with or without progestin (N = 549). Women were randomized to receive testosterone 300 microg/day or placebo patches twice weekly for 24 weeks. The primary efficacy measure was change from baseline in frequency of total satisfying sexual activity over a 4-week period (weeks 21-24). RESULTS: A total of 483 women (88%) were included in the primary analysis population (those with baseline sex hormone binding globulin levels < or = 160 nmol/L). The change from baseline in number of total satisfying sexual episodes was significantly greater for testosterone compared with placebo (participants with baseline sex hormone binding globulin levels < or = 160 nmol/L, mean change of 2.1 +/- 0.28 versus 0.5 +/- 0.23 episodes/4 weeks; P < 0.0001; intent-to-treat population, mean change from baseline of 1.9 +/- 0.26 versus 0.5 +/- 0.21 episodes/4 weeks, P < 0.0001). Testosterone also produced statistically significant improvements compared with placebo in all secondary efficacy measures, including sexual desire and personal distress. The testosterone patch was well tolerated. CONCLUSIONS: Testosterone patch treatment increased the frequency of satisfying sexual activity and sexual desire, decreased personal distress, and was well tolerated in naturally menopausal women with hypoactive sexual desire disorder.

Safety and efficacy of a testosterone patch for the treatment of hypoactive sexual desire disorder in surgically menopausal women: a randomized, placebo-controlled trial.

BACKGROUND: Oophorectomy reduces serum testosterone levels. We studied the efficacy and safety of transdermal testosterone in treating hypoactive sexual desire disorder in surgically menopausal women. METHODS: A 24-week, randomized, double-blind, placebo-controlled, parallel-group, multicenter trial was conducted in women (aged 24-70 years) who developed distressful low sexual desire after bilateral salpingo-oophorectomy and hysterectomy and who were receiving oral estrogen therapy. Women were randomized to receive placebo (n = 119) or testosterone patches in dosages of 150 microg/d (n = 107), 300 microg/d (n = 110), or 450 microg/d (n = 111) twice weekly for 24 weeks. Sexual desire and frequency of satisfying sexual activity were primary efficacy outcome measures. RESULTS: Of the 447 women randomized, 318 (71%) completed the trial. Compared with placebo, women receiving the 300-microg/d testosterone patch had significantly greater increases from baseline in sexual desire (67% vs 48%; P = .05) and in frequency of satisfying sexual activity (79% vs 43%; P = .049). The 150-microg/d group showed no evidence of a treatment effect. The 450-microg/d group also was not statistically different from the 300-microg/d or placebo groups. Marginally significant linear dose-response trends were observed for total satisfying sexual activity and sexual desire at 24 weeks (P = .06 and .06, respectively). Adverse events occurred with similar frequency in both groups; no serious safety concerns were observed. CONCLUSIONS: The 300-microg/d testosterone patch increased sexual desire and frequency of satisfying sexual activity and was well tolerated in women who developed hypoactive sexual desire disorder after surgical menopause.

Testosterone patch increases sexual activity and desire in surgically menopausal women with hypoactive sexual desire disorder.

CONTEXT: Hypoactive sexual desire disorder (HSDD) is one of the most common sexual problems reported by women, but few studies have been conducted to evaluate treatments for this condition. OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of a testosterone patch in surgically menopausal women with HSDD. DESIGN: The design was a randomized, double-blind, parallel-group, placebo-controlled, 24-wk study (the Intimate SM 1 study). SETTING: The study was performed at private or institutional practices. PATIENTS: The subjects studied were women, aged 26-70 yr, with HSDD after bilateral salpingo-oophorectomy who were receiving concomitant estrogen therapy. Placebo (n = 279) or testosterone 300 microg/d (n = 283) was administered. There were 19 patients who withdrew due to adverse events in the placebo group and 24 in the 300 mug/d testosterone group. INTERVENTION: Testosterone (300 microg/d) or placebo patches were applied twice weekly. MAIN OUTCOME MEASURE(S): The primary end point was the change in the frequency of total satisfying sexual activity at 24 wk. Secondary end points included other sexual functioning end points and safety assessments. RESULTS: At 24 wk, there was an increase from baseline in the frequency of total satisfying sexual activity of 2.10 episodes/4 wk in the testosterone group, which was significantly greater than the change of 0.98 episodes/4 wk in the placebo group (P = 0.0003). The testosterone group also experienced statistically significant improvements in sexual desire and a decrease in distress. The overall safety profile was similar in both treatment groups. CONCLUSION: In the Intimate SM 1 study, the testosterone patch improved sexual function and decreased distress in surgically menopausal women with HSDD and was well tolerated in this trial.

Testosterone patch for low sexual desire in surgically menopausal women: a randomized trial.

OBJECTIVE: To assess the efficacy and safety of a 300 mug/d testosterone patch for the treatment of hypoactive sexual desire disorder in surgically menopausal women on concomitant estrogen therapy. METHODS: Five hundred thirty-three women with hypoactive sexual desire disorder who had undergone previous hysterectomy and bilateral oophorectomy were enrolled in a 24-week, multicenter, double-blind, placebo-controlled trial. Patients were randomly assigned to receive placebo or the testosterone patch twice weekly. The primary efficacy endpoint was change from baseline at week 24 in the frequency of total satisfying sexual activity, measured by the Sexual Activity Log. Secondary measures included sexual desire using the Profile of Female Sexual Function and personal distress as measured by the Personal Distress Scale. Hormone levels, adverse events, and clinical laboratory measures were reviewed. RESULTS: Total satisfying sexual activity significantly improved in the testosterone patch group compared with placebo after 24 weeks (mean change from baseline, 1.56 compared with 0.73 episodes per 4 weeks, P = .001). Treatment with the testosterone patch also significantly improved sexual desire (mean change, 10.57 compared with 4.29, P < .001) and decreased personal distress (P = .009). Serum free, total, and bioavailable testosterone concentrations increased from baseline. Overall, adverse events were similar in both groups (P > .05). The incidence of androgenic adverse events was higher in the testosterone group; most androgenic adverse events were mild. CONCLUSION: In surgically menopausal women with hypoactive sexual desire disorder, a 300 mug/d testosterone patch significantly increased satisfying sexual activity and sexual desire, while decreasing personal distress, and was well tolerated through up to 24 weeks of use.

Comparison of salivary versus serum testosterone levels in postmenopausal women receiving transdermal testosterone supplementation versus placebo.

OBJECTIVE: Clinical assessment of androgen action and its correlation to testosterone levels in women has been challenging. The current gold standard for measuring biologically active testosterone (T) is serum free T by equilibrium dialysis. Alternative methods are desirable due to the cost, complexity, and limited availability of the equilibrium dialysis method. Salivary testing has been proposed as a possible substitute for serum testing. This study compared salivary versus serum measurements of total T (TT), bioavailable T (BT; consisting of free T [FT] and albumin-bound T), and FT from samples collected simultaneously in women who were either receiving transdermal T patch supplementation (300 microg/d) or a placebo patch. METHODS: Naturally and surgically postmenopausal women receiving concomitant hormone therapy were recruited to participate in a 24- to 52-week phase III trial of a 300 microg/day transdermal T patch for the treatment of hypoactive sexual desire disorder. RESULTS: Initial analysis demonstrated high correlations between TT, BT, and FT levels (r = 0.776-0.855). However, there was no correlation with salivary T levels for any of the serum T subtypes (r = 0.170-0.261). After log transformation, salivary T correlated modestly with BT (r = 0.436, P < 0.001), FT (r = 0.452, P < 0.001), and TT (r = 0.438, P < 0.001). CONCLUSIONS: Although salivary testing of T concentrations is an appealing alternative because it is inexpensive and noninvasive, our results do not support the routine use of salivary T levels in postmenopausal women.