For the phase I and II clinical studies described here, the protocols, informed consent forms, and assent forms for subjects under legal adult age (phase I study only) were submitted to an institutional review board or independent ethics committee for review and approval before the start of the study. The studies were conducted in compliance with the principles of the International Conference on Harmonisation (E6 1997) Good Clinical Practice, US Food and Drug Administration guidelines, and the Declaration of Helsinki, 1964 (as amended in Edinburgh [2000]), and informed consent was obtained.
Phase I PK Study
Study Design and Subjects
Seven study centers in the USA screened and enrolled subjects (six sites enrolled subjects in the GT arm and one site enrolled subjects in the oral glycopyrrolate arm). The first subject enrolled on 2 May, 2016, and the last subject completed on 20 July, 2016.
In the phase I, open-label study, GT 2.4% cloth was applied topically once daily for 5 days to both axillae of patients aged 9–65 years with primary axillary hyperhidrosis, while three doses of oral glycopyrrolate solution (Cuvposa®) were administered to healthy adult volunteers aged 18–65 years every 8 h for 5 days for each dose (Fig. 1). GT was applied by study staff to control for application method and prevent non-axillary exposure, and GT was administered by wiping each axilla once using the same GT cloth. Oral glycopyrrolate was administered based on the prescribing information for Cuvposa® [16] starting at 1 mg/8 h on day 1 and titrated in 1-mg increments every 5 days to a maximum dose of 3 mg/8 h if no dose-limiting side effects occurred. Subjects in the oral glycopyrrolate arm were required to fast for ≥ 8 h prior to the first daily dose, ≥ 2 h prior to the second and third daily doses, and ≥ 3 h following each dose; water was withheld for ≥ 2 h prior to and following all doses. Subjects in the GT arm returned to the study center on each treatment day (days 1–5) and on day 6 (study exit); a safety telephone follow-up call visit was conducted on day 7 or 2 days after early termination. Subjects in the oral glycopyrrolate arm were housed in a study unit from day − 1 through day 16 (study exit) or early termination; a safety follow-up telephone call was conducted on day 17 or 2 days after the subject stopped oral glycopyrrolate dosing.
Patients eligible for the GT arm were male or non-pregnant, non-lactating female patients with primary axillary hyperhidrosis for ≥ 6 months, gravimetrically measured sweat production of ≥ 50 mg/5 min in each axilla, Axillary Sweating Daily Diary [17] Item 2 (severity) score ≥ 4 (0–10 numeric rating scale), and Hyperhidrosis Disease Severity Scale (HDSS) grade 3 or 4. Pediatric patients (ages 9 to < 18 years) who were participating in a phase III, open-label extension trial of GT (NCT02553798) were allowed to participate concurrently in this phase I study; these patients underwent a 7-day wash-out of GT prior to day 1 of this study. The study drug from the phase III, open-label extension trial of GT was withheld throughout the subject’s participation in the phase I study and resumed following the day 6 study exit visit. Patient exclusion criteria for the GT arm included prior surgical procedure for hyperhidrosis, prior axillary treatment with an anti-hyperhidrosis medical device, prior treatment with axillary iontophoresis within 4 weeks of day 1 or botulinum toxin (e.g., Botox®) for axillary hyperhidrosis within 1 year of day 1, axillary use of nonprescription antiperspirants within 1 week or prescription antiperspirants within 2 weeks of day 1, secondary axillary hyperhidrosis or presence of a condition that may cause secondary hyperhidrosis (e.g., lymphoma, malaria, severe anxiety not controlled by medication, carcinoid syndrome, substance abuse, hyperthyroidism), or history of Sjögren’s syndrome, Sicca syndrome, or glaucoma.
Patients eligible for the oral glycopyrrolate arm were healthy male or non-pregnant, non-lactating female patients as assessed by history, physical exam, vital signs, electrocardiography, and clinical laboratory tests at screening and day − 1, with weight of ≥ 48 kg and a body mass index ≥ 18.0 kg/m2 and ≤ 33.0 kg/m2 at screening and day − 1. Exclusion criteria included history or manifestations of clinically significant urological, gastrointestinal, renal, hepatic, neurological, hematological, metabolic, oncologic, pulmonary, immunologic, psychiatric, or cardiovascular disease or disorder, history of any medical or surgical conditions with potential to interfere with or alter the gastrointestinal absorption, distribution, metabolism, or excretion of the study drug, history of Sjögren’s syndrome or Sicca syndrome, history of glaucoma, inflammatory bowel disease, toxic megacolon, or recent febrile illness (within 14 days and > 100.0 °F), male patients with a history of urinary retention requiring catheterization because of prostatic hypertrophy or severe obstructive symptoms of prostatic hypertrophy, evidence of urinary obstruction or difficulty in voiding at screening, and taking any drug containing glycopyrrolate within 4 weeks of study entry.
Blood Sample Collection and Analysis
For all samples, approximately 3 mL and 2 mL of blood was collected from adults and pediatric patients, respectively. In patients treated with GT, blood samples were collected on days 1–5 (Fig. 1). Differences in sample collection between adult and pediatric patients because of guidelines regarding safe volumes of blood sampling in pediatric patients (9 to < 18 years of age) are noted, where applicable, below. All GT-treated patients underwent intensive PK sampling on days 1 and 5: samples were collected prior to the application of the study drug (pre-dose) and 30 min, 1, 1.5, 2, 2.5, 3, 4, and 6 h post-application; additional samples were collected in adults at 8, 10, and 24 h post-application. Pre-dose samples were collected from adults on days 3 and 4, and no samples were collected from pediatric patients on days 2, 3, and 4. Oral glycopyrrolate-treated subjects underwent intensive PK sampling on days 1, 5, 10, and 15 (Fig. 1). On these days, blood samples were collected pre-dose and 30 min, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, and 24 h post-dose.
Plasma concentrations of glycopyrronium were determined using a validated liquid chromatography-tandem mass spectrometry method. Assays were performed by PPD Laboratories (Richmond, VA, USA). The validated lower limit of quantification (LLOQ) of the plasma assay for glycopyrronium was 0.01 ng/mL. The PK evaluable population included subjects who received the study drug and had one or more PK samples collected. As specified in the statistical analysis plan, samples were excluded from the analysis if there were plasma concentration values > 3 standard deviations from the mean value for a given timepoint. Pre-dose samples on day 1 were set to 0. Noncompartmental PK analysis was conducted using WinNonlin (version 6.4, Pharsight Corp., Mountain View, CA, USA). The maximum plasma concentration (Cmax), time to Cmax (Tmax), area under the plasma concentration vs time curve (AUC) from 0 to 24 h (AUC0–24h), AUC from 0 to 6 h, and elimination (terminal) half-life were determined.
Comparisons of exposure metrics (Cmax, AUC) for topical GT vs oral glycopyrrolate were evaluated using the ln-transformed parameters of test (GT) to reference (oral glycopyrrolate). Data from the two treatments were analyzed using an analysis of variance model with a fixed-effect term for formulation (topical vs oral) using R (programming language, version 3.2.0 [https://www.r-project.org/]). Least-squares mean differences and their 90% confidence intervals (CIs) in the logarithmic scale were back transformed to produce geometric mean ratios (point estimates) and associated 90% confidence intervals (CIs) for each ratio. A similar comparison of exposure metrics for pediatric patients treated with GT (test) and adult patients treated with GT (reference) was performed.
Safety
Safety assessments included treatment-emergent adverse events (TEAEs) and local skin reactions (GT arm only). Adverse events were collected after the first dose of study drug was applied. Subjects were instructed to inform the investigator and/or study staff of any AE. At each visit, subjects were asked about AEs in a nonspecific manner using open-ended questions so as not to bias the response (e.g., “How have you been since the last visit?”). Adverse events were also assessed during the safety telephone follow-up call visit (day 7 for GT and day 17 for oral glycopyrrolate, or 2 days after early termination). Anticholinergic AEs were identified by medical review of reported events without knowledge of the patients in which these events occurred. The safety population included all enrolled subjects who received one or more confirmed dose of the study drug. Local skin reactions included burning/stinging, pruritus, edema, erythema, dryness, and scaling and were assessed pre-dose on day 1, post-dose following each GT application, and day 6.
Population PK Study
Study Design and Subjects
The Pop PK analysis was performed with data from two randomized, double-blind, vehicle-controlled, phase II studies conducted in adults (aged ≥ 18 years) with primary axillary hyperhidrosis (DRM04-HH01 [NCT02016885] and DRM04-HH02 [NCT02129660]); Fig. 1 of the Electronic Supplementary Material [ESM]). Patients were randomized at 20 study centers in the USA for DRM04-HH01 (first subject enrolled 10 December, 2013; last subject completed 15 July, 2014) and at 15 study centers in the USA and Canada for DRM04-HH02 (first subject enrolled 9 April, 2014; last subject completed 5 November, 2014). In both trials, eligible patients had primary axillary hyperhidrosis for ≥ 6 months, gravimetrically measured sweat production of ≥ 50 mg/5 min in each axilla, and HDSS grade 3 or 4. Results from a prior proof-of-concept study (n = 38) with glycopyrronium bromide 2% and 4% informed the doses selected for study in the two phase II trials. In DRM04-HH01, patients were randomized 1:1:1:1:1 to one of four doses of topical glycopyrronium bromide (0.8%, 1.6%, 2.4%, 3.2%) or vehicle. Patients in DRM04-HH02 were randomized 1:1:1:1:1 to one of two doses of GT (1.6%, 2.4%), one of two doses of glycopyrronium bromide (1.6%, 2.4%), or vehicle. Both glycopyrronium bromide and GT contain the glycopyrronium cation, which is the active anticholinergic moiety [18, 19]. In both trials, patients were to apply the study drug to the axillae once daily in the evening (wiping each axilla once using the same cloth) for 4 weeks with a 2-week off-drug follow-up. Subjects returned to the study center at weeks 1, 2, 3, 4 (end of treatment), 5, and 6 (study exit).
Blood Sample Collection and Analysis
For all samples, at least 500 μL of plasma (at least 1 mL of blood) was collected. For DRM04-HH01, blood samples were collected pre-dose and at approximately 30 min, 1, 2, 3, 4, and 24 h after application on day 1. Trough blood samples were collected pre-dose at the end of weeks 1, 2, 3, and 4. For DRM04-HH02, approximately 10 subjects were to provide PK samples at the baseline/day 1 visit (pre-dose and 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, and 24 h post-dose) and pre-dose samples at weeks 4, 5, and 6. Approximately 20 subjects were to provide PK samples at the baseline/day 1 visit (pre-dose samples), at the week 2 visit (pre-dose and 30 min, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, and 24 h post-dose), and pre-dose samples at weeks 4, 5, and 6. All other patients in DRM04-HH02 were to provide pre-dose blood samples on day 1 and at the weekly visits through week 6. Plasma concentrations of glycopyrronium were determined using a sensitive and selective liquid chromatography-tandem mass spectrometry method. Assays were performed by PPD Laboratories. The validated LLOQ of the plasma assay for glycopyrronium was 0.02 ng/mL. The PK population was defined as subjects who had blood collected for a PK analysis pre-dose and at least once post-dose.
Population PK Model Development
All glycopyrronium concentration data from the active treatment arms of DRM04-HH01 and DRM04-HH02 were pooled into a single database (data from vehicle arms were not included in the Pop PK model development). Pre-dose samples were excluded from the database as were samples for which no results were reported, observations were missing, or the conditional weighted residual was > 5 and the key parameter estimates differed by more than 15% or impacted on the convergence of the models. For missing data, imputation strategies were implemented; for example, if the covariate value was missing at a given time/visit, it was imputed using the nearest assessment. The database was used to create a Pop PK model for glycopyrronium administered topically using NONMEM (Version 7.2.0, Icon PLC, Dublin, Ireland). The Pop PK model was developed in steps; a base model for description of the structural components of the model, a full model including all of the pre-specified covariate effects of interest, then the final model chosen by retaining only the statistically significant covariate effects. A one-compartment model with first-order input and elimination was used as the base Pop PK model. Given the number of plasma samples from subjects in the PK database that were below the limit of detection of the assay, a mixture-of-models approach was used, whereby the Pop PK model classified subjects as “absorbers” or “non-absorbers.” In the final model that provided the best fit of all the data, 71 subjects were classified as “absorbers” and 37 were classified as “non-absorbers.” Values below the limit of quantitation were assigned a value of ½ the LLOQ. Then, a full covariate model was developed using pre-specified covariates, including age, body mass index, weight, sex, race, and formulation. The full model underwent backward elimination to identify the most important variables in a stepwise manner, leading to a parsimonious final model that contained covariates that were statistically significant. After model development and qualification were complete, the final Pop PK model was used to generate metrics of exposure (Cmax, AUC) for patients who had been randomized to receive active treatment. For subjects in the “absorber” subpopulation, peak concentration values (Cmax) were taken directly from the observed data and AUC was calculated as the administered dose divided by clearance. For the “non-absorber” subpopulation, Cmax was set to the LLOQ, and the AUC was computed assuming concentrations over the dose interval were at LLOQ. These exposure metrics were then used for subsequent exploration of exposure–response relationships in terms of AEs and efficacy. Additional methodological details are included in the ESM.
Safety and Efficacy
Adverse events were monitored throughout the phase II studies. Patients were instructed to inform the investigator and/or study staff of any AE, and at each visit, patients were asked about AEs in a non-specific manner using open-ended questions so as not to bias the response (e.g., “How have you been since the last visit?”). The severity of an AE was designated as mild, moderate, or severe by the investigator based on the subject’s description and, when applicable, by direct observation on the part of the investigator. Efficacy was assessed at each visit by gravimetric measurement of sweat production and HDSS.
For the Pop PK analysis, exposure metrics were used to assess the relationship between topical glycopyrronium pharmacokinetics and anticholinergic-related AEs or efficacy. Patient data from the vehicle treatment arms were included in these analyses (exposure metrics for vehicle patients were set to 0). The Pop PK-AE model database included information on glycopyrronium exposure and the most severe grade of AEs that could be due to anticholinergic activity. The AEs included in the analysis were dry mouth, vision blurred, urinary retention, dry eye, mydriasis, urinary hesitation, urine flow decreased, dry tongue, constipation, nasal dryness, and vulvovaginal dryness. Level of severity was captured using a numerical code (0 for no events, 1 for mild, 2 for moderate, and 3 for severe). For the analysis of efficacy, a Pop PK-pharmacodynamic model database included information on glycopyrronium exposure and multiple assessments of gravimetric sweat production and HDSS scores.