Discovery of a potent GIPR peptide antagonist that is effective in rodent and human systems.

OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) is one of the two major incretin factors that regulate metabolic homeostasis. Genetic ablation of its receptor (GIPR) in mice confers protection against diet-induced obesity (DIO), while GIPR neutralizing antibodies produce additive weight reduction when combined with GLP-1R agonists in preclinical models and clinical trials. Conversely, GIPR agonists have been shown to promote weight loss in rodents, while dual GLP-1R/GIPR agonists have proven superior to GLP-1R monoagonists for weight reduction in clinical trials. We sought to develop a long-acting, specific GIPR peptide antagonist as a tool compound suitable for investigating GIPR pharmacology in both rodent and human systems. METHODS: We report a structure-activity relationship of GIPR peptide antagonists based on the human and mouse GIP sequences with fatty acid-based protraction. We assessed these compounds in vitro, in vivo in DIO mice, and ex vivo in islets from human donors. RESULTS: We report the discovery of a GIP(5-31) palmitoylated analogue, [Nα-Ac, L14, R18, E21] hGIP(5-31)-K11 (γE-C16), which potently inhibits in vitro GIP-mediated cAMP generation at both the hGIPR and mGIPR. In vivo, this peptide effectively blocks GIP-mediated reductions in glycemia in response to exogenous and endogenous GIP and displays a circulating pharmacokinetic profile amenable for once-daily dosing in rodents. Co-administration with the GLP-1R agonist semaglutide and this GIPR peptide antagonist potentiates weight loss compared to semaglutide alone. Finally, this antagonist inhibits GIP- but not GLP-1-stimulated insulin secretion in intact human islets. CONCLUSIONS: Our work demonstrates the discovery of a potent, specific, and long-acting GIPR peptide antagonist that effectively blocks GIP action in vitro, ex vivo in human islets, and in vivo in mice while producing additive weight-loss when combined with a GLP-1R agonist in DIO mice.

Optimization of Truncated Glucagon Peptides to Achieve Selective, High Potency, Full Antagonists.

Antagonism of glucagon's biological action is a proven strategy for decreasing glucose in diabetic animals and patients. To achieve full, potent, and selective suppression, we chemically optimized N-terminally truncated glucagon fragments for the identification and establishment of the minimum sequence peptide, [Glu9]glucagon(6-29) amide (11) as a full antagonist in cellular signaling and receptor binding (IC50 = 36 nM). Substitution of Phe6 with l-3-phenyllactic acid (Pla) produced [Pla6, Glu9]glucagon(6-29) amide (21), resulting in a 3-fold improvement in receptor binding (IC50 = 12 nM) and enhanced antagonist potency. Further substitution of Glu9 and Asn28 with aspartic acid yielded [Pla6, Asp28]glucagon amide (26), which demonstrated a further increase in inhibitory potency (IC50 = 9 nM), and improved aqueous solubility. Peptide 26 and a palmitoylated analogue, [Pla6, Lys10(γGluγGlu-C16), Asp28]glucagon(6-29) amide (31), displayed sustained duration in vivo action that successfully reversed glucagon-induced glucose elevation in mice.

In vitro and ex-vivo evaluation of topical formulations designed to minimize transdermal absorption of Vitamin K1.

Topical application of Vitamin K1 has been demonstrated to effectively treat papulopustular skin rash, a serious and frequently encountered side effect of Epidermal Growth Factor Inhibitors (EGFRIs). Systemic absorption of vitamin K1 from skin and the resultant consequence of antagonizing EGFRIs anticancer effects jeopardizes the clinical acceptability of this rather effective treatment. The purpose of the present study was to rationally formulate and evaluate the release rate and transdermal absorption of a wide range of Vitamin K1 dermal preparations with a variety of physiochemical properties. A library of 33 formulations with were compounded and tested for Vitamin K1 permeation using hydrophobic membranes and porcine skin mounted in a Fran diffusion cells. Our results demonstrate the lowest diffusion for water-in-oil emulsions, which also demonstrated a negligible transdermal absorption. The statistical analysis showed a significant correlation between in vitro and ex vivo results. While viscosity did not have a significant impact on the diffusion or absorption of vitamin K1, an increase in the lipid content was correlated with an increase in transmembrane diffusion (not with transdermal absorption). Overall, formulation design significantly impacts the release rate and transdermal absorption of vitamin K1, and confirms the possibility of minimal systemic distribution of this vitamin for this specific purpose.

Prolonged monitoring of ethinyl estradiol and levonorgestrel levels confirms an altered pharmacokinetic profile in obese oral contraceptives users.

BACKGROUND: Pharmacokinetic (PK) parameters based on short sampling times (48 h or less) may contain inaccuracies due to their dependency on extrapolated values. This study was designed to measure PK parameters with greater accuracy in obese users of a low-dose oral contraceptive (OC) and to correlate drug levels with assessments of end-organ activity. STUDY DESIGN: Obese [body mass index (BMI) ≥30 kg/m2], ovulatory, otherwise healthy women (n=32) received an OC containing 20 mcg ethinyl estradiol (EE)/100 mcg levonorgestrel (LNG) for two cycles. EE and LNG PK parameters were characterized for 168 h at the end of Cycle 1. During cycle 2, biweekly outpatient visits were performed to assess cervical mucus, monitor ovarian activity with transvaginal ultrasound and obtain serum samples to measure EE, LNG, estradiol and progesterone levels. PK parameters were calculated and correlated with end-organ activity and compared against control samples obtained from normal and obese women sampled up to 48 h in a previous study. Standard determination of PK accuracy was performed, defined by the dependency on extrapolated values ('excess' area under the curve of 25% or less). RESULTS: The mean BMI was 39.4 kg/m2 (SD 6.6) with a range of 30-64 kg/m2. Key LNG PK parameters were as follows: clearance, 0.52 L/h (SD 0.24); half-life, 65 h (SD 40); area under the curve (AUC), 232 h*ng/mL (SD 102); and time to reach steady state, 13.6 days (SD 8.4). The majority of subjects had increased ovarian activity with diameter of follicles ≥8 mm (n=25), but only seven women had follicles ≥10 mm plus cervical mucus scores ≥5. Evidence of poor end-organ suppression did not correlate with the severity of the alterations in PK. As compared to historical normal and obese controls (48-h PK sampling), clearance, half-life, AUC and time to reach steady state were found to be significantly different (p≤.05) in obese women undergoing a longer duration of PK sampling (168 h). Longer sampling also improved PK accuracy for obese women (excess AUC 20%) as compared to both normal and obese controls undergoing shorter sampling times (48 h) with excess AUCs of 25% and 50%, respectively. CONCLUSIONS: Obesity results in significant alterations in OC steroid PK parameters, but the severity of these alterations did not correlate with end-organ suppression. A longer PK sampling interval (168 h vs. 48 h) improved the accuracy of PK testing.