Safety and efficacy of an extended-release peptide YY analogue for obesity: A randomized, placebo-controlled, phase 1 trial

Aim: To report the results from a Phase 1 trial of an extended-release peptide YY analogue, Y14, developed for the treatment of obesity. Methods: Y14 was evaluated in overweight/obese volunteers in a Phase 1 randomized placebo-controlled trial, conducted in a clinical trial unit in the United Kingdom. Part A was a blinded single-ascending-dose study evaluating doses up to 36 mg. Part B was double-blinded and tested multiple ascending doses between 9 and 36 mg, given at 7-to 14-day intervals, over the course of 28 days, with up to five doses given per participant. The primary outcome was safety and tolerability; the secondary outcome was assessment of pharmacokinetic (PK) characteristics. Exploratory outcomes included food intake, body weight change and glucose tolerance after multiple doses. Results: Between April 11, 2017 and December 24, 2018, 53 participants were enrolled into Part A and 24 into Part B of the trial. The PK characteristics were compatible with administration every 7 to 14 days. The most common adverse events (AEs) were nausea, vomiting or administration site reactions, which were mild in most cases and settled with time. No serious AE occurred. Participants given multiple doses of Y14 lost between − 2.87 and − 3.58 kg body weight compared with placebo ( P <0.0001) at 31 days from the first dose, with profound reductions in food intake of 38% to 55% ( P <0.0001, compared to placebo) and there was no evidence of tachyphylaxis. Conclusions: Our results support the continued development of Y14 as a novel treatment for obesity.

31 days from the first dose, with profound reductions in food intake of 38% to 55% (P <0.0001, compared to placebo) and there was no evidence of tachyphylaxis.
Conclusions: Our results support the continued development of Y14 as a novel treatment for obesity.

| INTRODUCTION
Obesity affects 13% of the world's adult population. 1 It is a key driver of cardiovascular disease, diabetes and cancer, three of the four major noncommunicable diseases identified by the World Health Organization (WHO). Moreover, obesity is an important contributor to mortality from pandemic respiratory infections. 2,3 Although lifestyle change is effective in the short term, most people regain weight to baseline after 1 to 2 years. 4,5 Bariatric surgery leads to sustained weight loss and reductions in mortality, 6 but can have long-term complications such as post-bariatric hypoglycaemia, 7 it is limited in scalability, and patients need to be fit for surgery. 8 Conventional small-molecule drugs including orlistat, rimonabant, lorcaserin, sibutramine and bupropion/naltrexone are modestly effective in reducing weight but have adverse effects: most have been withdrawn from marketing. 9 Biological agents based on satiety hormones such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, peptide YY (PYY) or melanocyte-stimulating hormone have emerged as potential treatments. They exploit natural satiety pathways to reduce food intake and body weight. 10 The GLP-1 analogues have primarily been developed for diabetes and secondarily developed for obesity, with liraglutide now being licensed for obesity therapy. Semaglutide is also currently being developed for this indication. 11 The melanocyte-stimulating hormone analogue setmelanotide has been recently approved for severe obesity caused by the rare genetic conditions of leptin or proopiomelanocortin deficiency. 12 These peptide therapeutics show that satiety hormonebased treatments for obesity are deliverable, safe and practical.
PYY is a peptide hormone that is cosecreted from enteroendocrine L cells with GLP-1 and oxyntomodulin in response to food intake. PYY has two major forms: full length PYY 1-36 that binds to neuropeptide Y1, Y2, Y4 and Y5 receptors, and PYY  , which is derived from PYY  via processing by dipeptidyl peptidase-4. PYY  preferentially activates Y2 and Y5 receptors. It acts as a satiety hormone, suppressing food intake via activation of Y2 in the arcuate nucleus of the hypothalamus. 13 Infusion of PYY  in obese volunteers causes a 30% reduction in food intake 14 and short-term subcutaneous injections increase satiety and reduce hunger. 15 Early studies of nasal delivery of PYY  showed limited tolerability and dose-dependent nausea and vomiting, 16 as there was a rapid "burst" release of PYY to supraphysiological levels. When PYY levels are increased slowly, suppression of food intake is obtained, with better tolerability and less nausea even when combined with other satiety hormones. 13,17 Extended-release pharmacokinetic (PK) profiles are important to mitigate nausea and vomiting 18 and to enable weekly delivery of treatment, for example, Novo Nordisk's PYY analogue 1875, which is currently in development. 19 In the present study, we aimed to evaluate the safety, PK profile and preliminary efficacy of a novel analogue of PYY, formulated for extended release, in a Phase 1 trial.

| Preclinical summary
Y14 is a human PYY analogue based on human PYY  , with the following features: composition wholly of natural L-amino acids without derivatization or cross linking, deletion of the first residue, and substitution of six other residues within the 35-residue peptide, with the receptor active sequence of PYY being unchanged. Y14 is a selective agonist of the human Y2 receptor, with equivalent binding affinity and functional activity to endogenous PYY   On Days 70 to 73 there was a follow-up outpatient visit.
Randomization lists were generated using SAS® by a study centre statistician who was not otherwise involved with the trial. Treatment codes were provided to the study centre pharmacist who prepared labelled syringes containing either placebo or study drug; these were identical in appearance. All clinical and nonclinical staff (except for the PK scientist) remained blinded to treatment assignment until database lock.

| Outcomes
The primary outcomes of the study were to investigate the safety and tolerability of single and multiple doses of Y14. Secondary outcomes were to assess the PK profile of single and multiple ascending doses of Y14. The exploratory outcomes of the study were to investigate the pharmacodynamic (PD) effects of multiple doses of Y14 on food consumption, body weight, enteropancreatic hormone changes and glucose tolerance. The analytical performance of the Imperial College radioimmunoassay for Y14 was also compared with the liquid chromatography/tandem mass spectrometry (LC/MS-MS) assay. Only the exploratory data for body weight, food intake and glucose tolerance are presented in this publication.

| Safety and tolerability assessments
The procedure for recording adverse events (AE) and for their classifica- the cut-off point on screening were taken forward to a confirmation assay. If found positive in the confirmation, the samples were diluted and tested in a titre assay, and the reciprocal highest dilution tested with a response above the threshold was reported as the titre.

| LC-MS/MS method for measurement of Y14
The bioanalytical assay for Y14 was developed by LGC (Fordham, UK).
In brief, an assay, utilizing samples of human plasma (K 3 EDTA) stabilized with aprotinin, based on solid phase extraction and LC/MS-MS, was developed and validated according to applicable European Medicines Agency and US Food and Drug Administration guidance. The lowest limit of quantification (LLOQ) was 0.2 ng/mL and the highest limit of quantification 100 ng/mL. The mean precision (coefficient of variation [CV]) of the assay with quality control samples spanning 0.3 to 80 ng/mL was ≤9.3% and the accuracy was within −1.3% to 5.3% relative error. The selectivity of the assay for Y14 alone was verified prior to approval for bioanalysis. as C max repeat dose/C max single dose or AUC 0-τ repeat dose/AUC 0-τ single dose). Three or more points were required within the terminal phase for λ z and corresponding t ½ to be estimated. Estimates were considered reliable if the period over which λ z was calculated was greater than twice the half-life itself. Actual blood sampling times were used for the PK analysis. Plasma concentrations below the LLOQ of the assay were taken as zero for calculation of concentration summary statistics and all PK parameters. Between-participant variability was based on geometric mean CVs.

| Standardized food intake study
While resident in the clinical unit during 24-hour food intake assessment days, participants were individually provided meals in a designated area. Meals were presented with an excess of food and participants asked to eat until they felt "comfortably full" within a period of 30 minutes. If a volunteer required more time to eat, an extra 15 minutes was to be allowed, and this was recorded as a deviation from protocol. Food was weighed pre-and postmeal to determine consumption and energy intake calculated from summing the energy value of each component of the meal.

| Statistical analysis
The following datasets are presented: the all-participants population (comprising any participants who enrolled in the study and had study assessments recorded in the database as per the protocol), the safety population (consisting of all participants who received study drug, Y14 or placebo), the PK population (consisting of all participants who received Y14 and had data from at least one PK blood draw), and the PD population (consisting of all participants who received study drug, Y14 or placebo, and who had data from at least one PD blood draw or measurement, as appropriate OGTT as a measure of glucose tolerance. Q-Q plots of standardized residuals were used to verify that residuals were normally distributed. No imputation was made for missing data, which were assumed to be "missing at random".

| Participant disposition and characteristics
Supplementary Figure S1 shows the numbers of volunteers screened, enrolled, randomized, followed up and analysed in the trial. Three additional cohorts of six participants each were enrolled in Part A of the trial per protocol to allow for investigation of the PK effect of varying the zinc:peptide ratio. This was to increase the scientific value of the study. One participant in Part A, Cohort A1 discontinued the intervention and was replaced with a back-up volunteer.
One participant in Part B, Cohort B1 (who received placebo) was withdrawn due to a protocol violation (positive test for alcohol during study visit) on Day 4 of the study. This participant's data are included in the all-participants, safety and PD populations. Table 1 summarizes the baseline characteristics of the all-participants populations for Parts A (n = 53) and B (n = 24) of the study, respectively. Of note, only two participants in Part B had impaired glucose tolerance based on the 120 minutes value from their baseline 75-g OGTT and no participants were diabetic. The doses and zinc:peptide ratios of Y14 given to each cohort are also summarized in Table 1.
Although the protocol allowed for maximal doses of up to 96 mg, the maximal dose eventually given in the study was limited to 36 mg, based on the emerging AE profile from Part A (see Safety and adverse events).

| Part A
For the doses given to cohorts A1, A2 and A3, Y14 was below the LLOQ (<0.2 ng/mL) in most samples, so the data for cohort 4 onwards are presented (Supplementary Figure S2 and

| Part B
Supplementary Figure S3 and Table 2

| Safety and adverse events
In general, Y14 was well tolerated, with the most common treatment-

| Y14 reduces body weight and food intake
An exploratory analysis showed dose-related reductions in weight in response to single doses of Y14 (Supplementary Figure S5). When T A B L E 2 Geometric mean (CV%) pharmacokinetic parameters following single doses (Part A) and multiple doses of Y14 (Part B)  participants were given multiple doses of Y14, this was associated with significant weight loss relative to placebo treatment (Figure 1).   (Table 1).

| DISCUSSION
In this Phase 1 trial we have shown that Y14, a novel analogue of PYY, is well tolerated in human subjects. To our knowledge, this is the first publication to report on a clinical trial of an extended-release PYY analogue. The TEAEs recorded were to be anticipated from the mechanism of action, with nausea, vomiting and reduced appetite being reported. Injection site reactions were also reported. Only one participant withdrew due to a TEAE, and no severe AEs were reported.
Although ADAs were detected in 17/24 participants given multipledose Y14 in Part B, these were mostly of low titre. No systemic hypersensitivity was noted in the trial. ADAs against gut hormone analogues are more common in extended-release than immediaterelease formulations, tend to wane with time, and, in most cases, do not seem to affect the desired PD effects nor to cause systemic hypersensitivity reactions as shown with extended-release exenatide. 20 Using a zinc-based extended-release formulation we also saw PK characteristics compatible with weekly/fortnightly administration.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/dom.14358.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.