Objective:This study evaluated the safety, tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of eneboparatide (AZP-3601), a novel agonist of the PTH receptor 1 developed for the treatment of hypoparathyroidism. Design:This was a randomized, double-blind, placebo-controlled study. One-hundred four healthy volunteers were recruited into seven single ascending dose (SAD) and five multiple ascending dose (MAD) cohorts. Methods:PK parameters were time to peak, Cmax, area under the curve (AUC) and half-life. PD parameters included albumin-adjusted serum calcium (sCa), serum phosphorus (sPh), serum endogenous PTH, 24 hr urinary excretion of calcium (24 h-uCa), fractional excretion of calcium (FECa) and bone turnover markers (s-CTX and P1NP). Results:There were no serious adverse events. All adverse events were of mild-to-moderate intensity. AUC and Cmax of eneboparatide increased with increasing doses. Time to maximum plasma concentration was 5-20 min. SAD showed a dose-dependent increase of sCa and decrease of sPh associated with a reduction of serum endogenous PTH. MAD demonstrated a rapid access to maximal PD effects and maintained levels of sCa throughout the day. Urinary excretion of calcium did not increase as a function of the dose of eneboparatide. P1NP and s-CTX did not change over the treatment period. Conclusion:The PD effects of eneboparatide were prolonged despite the short half-life. These data suggest that eneboparatide may provide sustained control of serum calcium in patients with hypoparathyroidism with once daily dosing. An open-label phase 2 study in patients with hypoparathyroidism has been recently completed and published and a phase 3 study has been initiated. Clinical Trial Registration Number:NCT05239221.

Abstract Disclosure: G. Ravel: Employee; Self; Amolyt Pharma. R. Datta: Consulting Fee; Self; Amolyt Pharma. S. Milano: Employee; Self; Amolyt Pharma. M. Ovize: Employee; Self; Amolyt Pharma. S. Allas: Employee; Self; Amolyt Pharma. M. Aouadi: None. M.D. Culler: Employee; Self; Amolyt Pharma. Natural parathyroid hormone (PTH) replacement therapy for chronic hypoparathyroidism (cHP) is not ideal due to both short circulating half-life and brief receptor signaling, making the effect too transient. Following agonist binding to the PTH 1 receptor (PTH1R), the magnitude and duration of the receptor signal, and consequently the biological response, appears to depend on the affinity of the ligand for a specific receptor conformation termed R0, which determines the duration of ligand binding. Eneboparatide (Enebo) is a hybrid analog of PTH and PTH-related peptide designed to strongly bind to the R0 conformation while having a short circulating half-life similar to that of PTH. When tested in cHP patients, Enebo eliminated the need for calcium (Ca) (88% patients) and Vitamin D (92% patients) supplementation, while maintaining serum Ca levels over a full 24 hours. Urinary calcium (uCa) was rapidly decreased and was normalized in 92% of patients who were hypercalciuric prior to treatment. To explore the mechanism of the potent effect of Enebo on uCa, a binding assay specific for the R0 conformation of the PTH1R confirmed the high affinity of Enebo versus natural PTH1-34, with IC50 = 1.5nM and 30.9nM, respectively. Postulating that the high-affinity binding of Enebo to R0 should result in longer tissue retention in vivo, we examined the tissue levels of PTH1-34 and Enebo in male, 200-300g rats using quantitative whole-body autoradiography (QWBA) following subcutaneous injection of 3H-labeled Enebo and PTH1-34. Tissue levels of the compounds were normalized based on the concentrations observed in cardiac blood. The highest level observed for both compounds was in the renal cortex, the major site of active PTH-induced Ca reabsorption, with peak concentrations observed 30 minutes post-injection. Six hours after injection, 96.3% of PTH1-34 had disappeared from the renal cortex, as compared with only 61.4% of Enebo, despite both compounds having similar circulating half-lives. With respect to activating the PTH1R, both compounds showed a similar increase in cAMP in human embryonic kidney (HEK293) cells transfected with the PTH1R and the Green Downward cADDis cAMP biosensor, with EC50 of 0.32 and 0.48nM for Enebo and PTH1-34, respectively. To mimic the effect of rapid elimination in vivo, the ligands were added to the cells, washed-off after 15 minutes, and cAMP production followed over the next 16 hours. After washout, the cAMP signal induced by PTH1-34 rapidly returned to baseline in less than 60 minutes, whereas the cAMP signal induced by Enebo gradually decreased and was still evident after 16 hours. Collectively, these data confirm high affinity of Enebo for the R0 conformation of the PTH1R, as well as prolonged receptor signaling and retention by the renal cortex, clearly differentiating Enebo from PTH1-34 and providing strong rationale for the efficacy of Enebo in reducing uCa in cHP patients. Presentation: 6/3/2024

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract Medical treatment of acromegaly is based on either suppressing pituitary GH secretion or inhibiting GH action by preventing interaction with its receptor in order to suppress the elevated levels of IGF1. AZP-3813 is a 16-amino acid, bicyclic peptide antagonist of the GH receptor (GHR) with a KD of 1.9nM for the human GHR, and 18.5nM for the rat GHR. The circulating half-life of AZP-3813 in the rat is 11.2 hours. In previous studies, AZP-3813 was demonstrated to suppress IGF1 secretion in juvenile rats in a dose-related manner, and to maintain IGF1 suppression when given over 4 days. To examine the ability of chronic AZP-3813 treatment to maintain suppression of IGF1 levels, we injected normal, 5-week old (∼150g), male Sprague Dawley rats subcutaneously either with vehicle or with AZP-3813 at doses of either 10 or 30mg/kg QD for 19 days (n=7/group). Blood samples were collected immediately prior to either vehicle or AZP-3813 injection on days 1, 2, 3, 7, 10, 15 and 19, and 24h after the last injection (day 20). Samples were assayed for total IGF1 content by radioimmunoassay, and samples collected on days 2, 10 and 19 were also analyzed for AZP-3813 content by LC-MS/MS. At the initiation of treatment, the average body weight of the rats was 150.3 + 3.0g, and over the 20 days of the study animals treated with vehicle gained an average of 158.4 + 4.9g. In contrast, rats treated QD with 10 or 30mg/kg AZP-3813 gained 23.6 + 2.1 and 27.9 + 2.2% less weight than the vehicle-treated controls, respectively. Accumulation of AZP-3813 levels in the blood was observed with repeated daily injections of both 10 and 30mg/kg, increasing from day 2 levels by 107.1 + 12.5 and 125.2 + 36.3%, respectively, by day 19. In the vehicle-treated control rats, IGF1 levels increased from an initial 395.1 +16.3ng/ml to 674.4 + 19.8ng/ml by day 20. Twenty-four hours after the first injections of 10 and 30mg/kg AZP-3813, IGF1 levels were suppressed by 27.6 + 3.4 and 29.2 + 3.3%, respectively, versus vehicle-treated control levels, and, with continued treatment, a similar magnitude of IGF1 suppression was maintained through day 20 despite the progressively increasing levels of IGF1 observed in the vehicle-treated rats. At study termination on day 20, animals treated with 10 and 30mg/kg AZP-3813 were observed to have 4.1 and 4.6% shorter femur length, 1.1 and 3.6% shorter body length, and 7.7 and 13.3% less liver weight, respectively, as compared with vehicle-treated control rats. These results demonstrate that with continued treatment, the potent GHR antagonist activity exhibited by AZP-3813 translates to sustained in vivo suppression of IGF1 levels and associated parameters, and support its development as a potential therapy for acromegaly. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:05 p.m. - 1:10 p.m.

Abstract Insulin-like growth factor binding protein-2 (IGFBP-2) has been demonstrated to be a key mediator of the peripheral metabolic actions of leptin. The metabolic activity of IGFBP-2 is independent of IGF1 binding, and can be localized to a unique heparin-binding domain (HBD-1) within its structure. AZP-3404 is a 9-amino acid analog of the IGFBP-2 HBD-1 that reproduces the activity of IGFBP-2 on adipocyte and osteoblast differentiation. In addition, AZP-3404 has been demonstrated to increase glucose uptake by differentiated mouse myotubes in vitro, and to increase glucose disposal following an intraperitoneal glucose tolerance test (IPGTT) in leptin-deficient ob/ob mice. In the present study, we hypothesized that AZP-3404 should be able to improve metabolic regulation in the db/db mouse, which is leptin-resistant due to a mutation in the leptin receptor, and, as a result, is also IGFBP-2 deficient. Following pre-treatment with vehicle for 1 week to establish a baseline, 9-week old male db/db mice were treated with either vehicle or AZP-3404 at doses of 1, 3 or 6 mg/kg, sc, bid (n=10/group) for 8 weeks. At the initiation of treatment, the mice weighed an average of 39.7 + 0.3 grams, and after 8 weeks, vehicle-treated mice had gained an average 8.2 + 1.6 grams of body weight. Mice treated with AZP-3404 displayed a progressive decrease in body weight gain that began after 2 weeks and that continued through the 8 weeks of treatment, ultimately resulting in less than 50% of the weight gain observed in the vehicle-treated mice, and without an apparent change in food intake. To assess the impact on glucose disposal, after both 4 and 8 weeks of treatment, and following an overnight fast, the mice were administered an IPGTT (blood glucose measured 0, 30, 60, 90, 120 and 240 minutes post-ip injection of 1 g glucose/kg). By 4 weeks of treatment, a significant increase in glucose disposal was observed in mice treated with the 6 mg/kg dose of AZP-3404 (AUC glucose decreased by 28.7%) versus vehicle-treated controls. By 8 weeks of treatment, all three doses produced similar increases in glucose disposal (AUC glucose decreased by 18.8, 21.4 and 23.1% with 1, 3 and 6 mg/kg AZP-3404, respectively) versus vehicle controls. Correspondingly, 4-hour fasted plasma insulin was decreased by 54, 48 and 52%, and the HOMA measure of insulin resistance was decreased by 55, 52 and 67%, in mice treated for 8 weeks with 1, 3 and 6 mg/kg AZP-3404, respectively, as compared with vehicle-treated mice. These results, demonstrating both improved glucose metabolism and decreased body weight gain in the leptin-resistant db/db mouse, further confirm the ability of AZP-3404 to reproduce the metabolic activity of IGFBP-2, and support the development of AZP-3404 as a novel therapy for disease states characterized by insulin resistance and/or obesity.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Intestinal or mesenteric ischemia generally leads to inflammation and injury, potentially developing hypoxia, causing cell death and tissue necrosis. This in turn can lead to sepsis and shock. Conversely, following shock, the intestinal tract is a main organ to experience ischemic/reperfusion injury. Increased intestinal cell-membrane permeability through mesenteric ischemia provoking bacterial translocation and gut-barrier injury can lead to sepsis and multi-organ failure. Hypotension induced by systemic vasodilation and vascular leak in systemic inflammatory response syndrome and sepsis is countered by immediate fluid resuscitation and vasopressor administration, primarily norepinephrine (NE), with possible arginine vasopressin (AVP) supplementation, an agonist of vasopressin V1A and V2 receptors. Selepressin is a selective V1A-receptor agonist, avoiding potential V2 receptor-associated adverse effects. Selepressin, non-selective AVP, and NE effects on mesenteric blood flow (MBF) and gastric mucosa perfusion (GMP) were compared in control rabbits and a lipopolysaccharide-induced, fluid-resuscitated rabbit endotoxemia model. AVP induced a pronounced decrease in MBF and GMP in non-endotoxemic and endotoxemic rabbits, whereas the reduction after selepressin treatment was significantly less for both indicators in the endotoxemic animals. By contrast, NE increased the MBF and did not affect GMP in both groups. Selepressin and AVP induced a pronounced dose-dependent increase in mesenteric vascular resistance in non-endotoxemic and endotoxemic rabbits, tending to be less in endotoxemic animals, whereas a minor increase in both groups was observed with NE. Therefore, in this safety study, the risk for mesenteric ischemia on selepressin treatment was not inferior to AVP, being less in endotoxemic than in non-endotoxemic animals.

Abstract Background: Prader-Willi syndrome (PWS) is a rare endocrine disorder characterized by hyperphagia and abnormal food-related behaviors that contribute to severe morbidity, early mortality, and significant burdens for patients and caregivers. Dysregulation of acylated ghrelin (AG) and unacylated ghrelin (UAG) in people with PWS and hyperphagia has led to the hypothesis that replacing the relative deficit of UAG observed in this population will reduce hyperphagia. Livoletide is a cyclic, 8 amino acid analogue of UAG being developed as a potential treatment for hyperphagia and food-related behaviors associated with PWS. In a previous Phase 2a study (n=47) in people with PWS, livoletide improved food-related behaviors and hyperphagia scores relative to placebo. An extensive nonclinical safety program to support clinical development of livoletide has been conducted. Here we report the results of study of livoletide in juvenile rats. Methods: Male and female juvenile Wistar rats were administered livoletide (10, 25, or 75 mg/kg/day, subcutaneous) once daily from the age of weaning (postnatal day 21) until 12 weeks of age (n=32/sex/group). Subgroups were sacrificed at the end of the dosing period or after a recovery period. Toxicity was assessed based on local tolerance, clinical observations, body weights, food consumption, tibia length, bone densitometry, behavioral tests, IGF1 (insulin-like growth factor-1), LH (luteinizing hormone), and other endpoints. Macroscopic (necropsy), microscopic/histopathological, and caesarean examinations were also performed. Toxicokinetic (TK) evaluations were performed at various time-points after dosing to assess exposure. Results: Livoletide was not associated with treatment-related clinical signs or any relevant changes in hematological, coagulation, clinical chemistry parameters or urine analysis. Body weight and food consumption were unaffected. Livoletide did not affect systemic concentrations of IGF-1 or LH. No adverse effects of livoletide on bone growth, mating performance, or fertility were observed. Pups delivered by caesarian section did not exhibit abnormalities. Results were consistent with a lack of effect of livoletide on growth hormone signaling. Histological changes were limited to minimal local reversible reactions at injection sites that were non-adverse and observed at low incidence and severity. Conclusions: Livoletide was well-tolerated and not associated with evidence of overt systemic toxicity. The no observed adverse effect level (NOAEL) of 75 mg/kg/day was associated with Cmax and AUC0-24h values of 72.6/83.2 μg/mL and 169/144 μg.h/mL (males/females), respectively. These exposures are >100-fold above the anticipated clinical exposures in the Phase 2b/3 ZEPHYR study, which is enrolling people ages 4 to 65 with PWS.

Despite considerable success in generating human blastocytes with artificial reproductive technologies, embryo implantation remains a challenge for unclear reasons. Excessive uterine movements may cause implantation failure while atosiban (ATO), a mixed vasopressin (VP) V1A/oxytocin (OT) receptor antagonist, is used off-label to normalize uterine movements. In addition to inflammation or iatrogenic factors, poor perfusion can contribute to low implantation rates. It is hypothesized that elevating the uterine blood perfusion may increase the quality of the uterine mucosa and thus implantation success. The objective was to quantify whether ATO or barusiban (BAR), a selective OT antagonist, can improve endometrial blood perfusion in a rabbit model of early pregnancy. In early pregnant rabbits (GD8–10), the endometrial blood flow (EBF) was monitored by laser Doppler flowmetry under propofol anesthesia. Following uterotomy, a laser probe was placed on the luminal site of the endometrium at a blastocyst. Mean arterial pressure (MAP), heart rate (HR) and femoral blood flow (FBF) were monitored. Vehicle or drugs were administered cumulatively via the right jugular vein. Five doses of 10-min duration of 2.22–1390 ng/kg/min OT, 634 pg–407 ng/kg/min VP, 4–40,000 ng/kg/min ATO or 0.4–4000 ng/kg/min BAR or vehicle were tested on EBF and cardiovascular parameters (N=6/group). In the ATO, BAR and vehicle groups, an IV bolus of 4170 ng OT was administered at the end of the infusion to test the antagonistic action of ATO and BAR. EBF at the highest OT infusion dose was markedly reduced by 77% relative to vehicle, and in the VP group by 62% (both P<0.01) with similar potency. By contrast, no changes were apparent after infusion of vehicle, ATO or BAR. Similarly to EBF, a decrease in FBF was observed with VP (or OT), whereas administration of vehicle, ATO or BAR did not result in any change. The administration of an OT bolus to the vehicle group resulted in a decrease of EBF. No such decrease was observed following ATO or BAR pretreatment, indicating that the OT-induced EBF reduction by the bolus was antagonized by either ATO or BAR. Both OT and VP induced a large increase in MAP by 93% and 57%, respectively (P<0.001) with similar potency. MAP did not display any change in the vehicle, ATO or BAR groups. The OT bolus induced a small increase in MAP in the vehicle, ATO and BAR groups and was not blocked. No relevant changes in HR was observed in the vehicle, OT, VP, ATO or BAR groups. ATO or BAR infusions did not influence baseline EBF in the early pregnant rabbit. Indeed, pre-treatment with ATO or BAR prevented OT-induced EBF reduction. This complicated model allowed only a small number of animals. Additionally, it was not possible to elucidate the relative importance of the OT receptor versus the V1A receptor, because no differences were found between the agonists or between the antagonists despite their differences in receptor activity. The study suggests a benefit of OT or VP receptor antagonism in terms of recovery of OT-compromised EBF and thus potentially increases embryo implantation success.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Prader-Willi syndrome (PWS) is a rare and complex endocrine disease characterized by hyperphagia and abnormal food-related behaviors that contribute to severe morbidity and early mortality and to a significant burden on patients and caregivers. Hyperghrelinemia may be involved in the underlying mechanisms of hyperphagia. While ghrelin, the most potent appetite-stimulating hormone, is present at higher concentrations in the plasma of PWS patients compared to age-matched controls, detailed studies of ghrelin and unacylated ghrelin (UAG) reveal a relative deficit of UAG in hyperphagic individuals. UAG is a 28-amino-acid peptide that does not bind the growth hormone secretagogue receptor (GHSR), in contrast to acylated ghrelin. UAG has intrinsic activities that often counteract effects of ghrelin, and exerts its actions through a GHSR-independent mechanism. Livoletide is a cyclic 8-amino-acid analogue of UAG with improved plasma stability and pharmacokinetics. The objective of this nonclinical safety program was to support the clinical development of livoletide including a pivotal Phase 2b/3 clinical trial in patients with PWS. The program was designed to define the safety pharmacology and the chronic toxicologic and toxicokinetic profile, and identify parameters for clinical monitoring of potential adverse effects. Genotoxicity, safety pharmacology, reproductive toxicity, and repeat-dose 13-week toxicology studies were all completed. In the in vivo studies, livoletide was administered subcutaneously consistent with the clinical route of delivery. Livoletide was not cytotoxic or genotoxic. Safety pharmacology studies indicated no treatment-related effects on major physiological systems. Results from preliminary embryo-fetal developmental toxicity studies in rat and rabbit indicated that livoletide at high multiples of the anticipated human exposure is not associated with adverse maternal toxicity, embryo-fetal toxicity or teratogenic potential when administered throughout the period of organogenesis. Repeat-dose toxicity studies of up to 13 weeks’ duration in rats and dogs demonstrated that livoletide is very well-tolerated, with no evidence of systemic toxicity. Cumulative data indicated that livoletide has a wide safety margin relative to planned clinical exposures. The highest chronic doses tested were 75 mg/kg in rat and 30 mg/kg in dog; these were considered to be the NOAELs. These dose levels provided AUC values of ≥50-fold the intended clinical systemic exposure (~1200 ng·h/mL). No anti-livoletide antibodies were detected in any of the toxicology studies. These results confirm the favorable long-term safety profile of livoletide and support the subcutaneous administration of the highest anticipated human clinical dose in the Phase 2b/3 study.

Insulin-like growth factor-binding protein-2 (IGFBP-2), one of six highly-conserved binding proteins that modulate circulating IGF1, has significant regulatory actions on carbohydrate, lipid and bone metabolism that are independent of its IGF-1 binding role. The metabolic activity of IGFBP-2 has been localized to a short peptide sequence within its unique heparin-binding domain, HBD-1, that is able to reproduce the metabolic actions of the full IGFBP-2 on bone and fat. Through structure-activity studies of this sequence, an acylated 9-amino acid peptide, AZP-3404, with enhanced stability and pharmacokinetics, has been developed that retains the activity of IGFBP-2 on fat and bone metabolism. As IGFBP-2 treatment has also been demonstrated to improve glucose homeostasis in animal models of severe insulin resistance, including the leptin-deficient ob/ob mouse, the present study was undertaken to determine whether AZP-3404 is able to reverse insulin resistance and improve glucose homeostasis in the ob/ob mouse. Male B6.Cg-Lepob/J mice were implanted with HD-XG telemetric devices (Data Sciences International), which allows continuous, wireless monitoring of carotid blood glucose under awake, unrestrained conditions. Animals were randomly assigned to groups (n=8) receiving either saline or AZP-3404 at 1 or 3 mg/kg, b.i.d. for four weeks. The mean daily blood glucose level prior to study initiation was 507 ± 102 mg/dL. Treatment with AZP-3404 dose-dependently decreased the average daily glucose concentration, such that by day 27, glucose levels were reduced by 64 ± 29 and 100 ± 16 mg/dL in the 1 and 3 mg/kg AZP-3404-treated groups, respectively, while slightly increased by 14 ± 29 mg/dL in the vehicle-treated group. On days 7, 14, 21 and 28, following a 24-hour fast, the mice were given an intraperitoneal glucose tolerance test (IPGTT: 1g glucose/kg) to evaluate the impact of AZP-3404 on glucose disposal which is impaired in ob/ob mice. By day 28, the fasting glucose level was reduced by 8% and 20% in the 1 and 3 mg/kg AZP-3404-treated groups, respectively, as compared with fasting levels on day 7. The IPGTTs revealed a progressive, dose-dependent reduction in glucose excursion that reached a decrease of 40% after 4 weeks of treatment with 3mg/kg AZP-3404. In related studies, we have observed that AZP-3404, both alone and in an additive manner with insulin, stimulates glucose transport in mouse C2C12 differentiated myotubes through activation of AMP-activated protein kinase (AMPK). These findings suggest that AZP- 3404 stimulates glucose uptake by muscle tissue in ob/ob mice thereby improving glucose homeostasis. The results from these studies confirm that AZP-3404 retains the ability of IGFBP-2 to restore glucose homeostasis and support the development of AZP-3404 as a novel therapeutic approach for syndromes of severe insulin resistance.

Background Septic shock remains associated with significant mortality rates. Arginine vasopressin (AVP) and analogs with V1A receptor agonist activity are increasingly used to treat fluid-resistant vasodilatory hypotension, including catecholamine-refractory septic shock. Clinical studies have been restricted to healthy volunteers and catecholamine-refractory septic shock patients excluding subjects with cardiac co-morbidities because of presumed safety issues. The novel selective V1A receptor agonist selepressin, with short half-life, has been designed to avoid V2 receptor-related complications and long-term V1A receptor activation. Cardiovascular safety of selepressin, AVP, and the septic shock standard of care norepinephrine was investigated in a rabbit model of early-stage atherosclerosis. Methods Atherosclerosis was established in New Zealand White rabbits using a 1% cholesterol-containing diet. Selepressin, AVP, or norepinephrine was administered as cumulative intravenous infusion rates to atherosclerotic and non-atherosclerotic animals. Results Selepressin and AVP induced a slight dose-dependent increase in arterial pressure (AP) associated with a moderate decrease in heart rate, no change in stroke volume, and a moderate decrease in aortic blood flow (ABF). In contrast, norepinephrine induced a marked dose-dependent increase in AP associated with a lesser decrease in the heart rate, an increase in stroke volume, and a moderate increase in ABF. For all three vasopressors, there was no difference in responses between atherosclerotic and non-atherosclerotic animals. Conclusion Further studies should be considered using more advanced atherosclerosis models, including with septic shock, before considering septic shock clinical trials of patients with comorbidities. Here, selepressin and AVP treatments did not display relevant cardiovascular risk in early-stage rabbit atherosclerosis.

BACKGROUND & AIMS:Pancreatic exocrine insufficiency (PEI) reduces pancreatic secretion of digestive enzymes, including lipases. Oral pancreatic enzyme replacement therapy (PERT) with pancreatin produces unsatisfactory results. The lipase 2 produced by the yeast Yarrowia lipolytica (YLLIP2; GenBank: AJ012632) might be used in PERT. We investigated its ability to digest triglycerides in a test meal and its efficacy in reducing fecal fat in an animal model of PEI.METHODS:YLLIP2 was produced by genetically engineered Y lipolytica and purified from culture media. YLLIP2 or other gastric (LIPF) and pancreatic (PNLIPD) lipases were added to a meal paste containing dietary triglycerides, at a range of pH values (pH 2-7), with and without pepsin or human bile and incubated at 37°C. We collected samples at various time points and measured lipase activities and stabilities. To create an animal model of PEI, steatorrhea was induced by embolization of the exocrine pancreas gland and pancreatic duct ligation in minipigs. The animals were given YLLIP2 (1, 4, 8, 40, or 80 mg/d) or pancreatin (100,000 US Pharmacopeia lipase units/d, controls) for 9 days. We then collected stool samples, measured fat levels, and calculated coefficient of fat absorption (CFA) values.RESULTS:YLLIP2 was highly stable and poorly degraded by pepsin, and had the highest activity of all lipases tested on meal triglyceride at pH 4-7 (pH 6 with bile: 94 ± 34 U/mg; pH 4 without bile: 43 ± 13 U/mg). Only gastric lipase was active and stable at pH 3, whereas YLLIP2 was sensitive to pepsin hydrolysis after pH inactivation. From in vitro test meal experiments, the lipase activity of YLLIP2 (10 mg) was estimated to be equivalent to that of pancreatin (1200 mg; 100,000 US Pharmacopeia units) at pH 6. In PEI minipigs, CFA values increased from 60.1% ± 9.3% before surgery to 90.5% ± 3.2% after administration of 1200 mg pancreatin (P < .05); CFA values increased to a range of 84.6% ± 3.0% to 90.0% ± 3.8% after administration of 4-80 mg YLLIP2 (P < .05).CONCLUSIONS:The yeast lipase YLLIP2 is stable and has high levels of activity against test meal triglycerides in a large pH range, with and without bile. Oral administration of milligram amounts of YLLIP2 significantly increased CFA values, similar to that of 1.2 g pancreatin, in a minipig model of PEI.