The effect of fatty diacid acylation of human PYY3-36 on Y2 receptor potency and half-life in minipigs.

Peptides are notoriously known to display very short in vivo half-lives often measured in minutes which in many cases greatly reduces or eliminates sufficient in vivo efficacy. To obtain long half-lives allowing for up to once-weekly dosing regimen, fatty acid acylation (lipidation) have been used to non-covalently associate the peptide to serum albumin thus serving as a circulating depot. This approach is generally considered in the scientific and patent community as a standard approach to protract almost any given peptide. However, it is not trivial to prolong the half-life of peptides by lipidation and still maintain high potency and good formulation properties. Here we show that attaching a fatty acid to the obesity-drug relevant peptide PYY3-36 is not sufficient for long pharmacokinetics (PK), since the position in the backbone, but also type of fatty acid and linker strongly influences PK and potency. Furthermore, understanding the proteolytic stability of the backbone is key to obtain long half-lives by lipidation, since backbone cleavage still occurs while associated to albumin. Having identified a PYY analogue with a sufficient half-life, we show that in combination with a GLP-1 analogue, liraglutide, additional weight loss can be achieved in the obese minipig model.

Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide.

Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

Comparison of the pharmacokinetics of three concentrations of insulin aspart during continuous subcutaneous insulin infusion (CSII) in a pig model.

OBJECTIVES: The aim of the study was to investigate the pharmacokinetic properties of insulin aspart (IAsp) in three different concentrations given as a continuous subcutaneous insulin infusion (CSII). METHODS: A randomized cross-over study was performed in pigs, where IAsp U200, U100 or U20 was given for 8 h with the same total dose. Six pigs were included and blood was sampled during the CSII and 3 h after. KEY FINDINGS: The half-life (t(1/2) ) was 24.3 (range 17.3-41.3), 28.8 (range 19.6-54.3) and 23.6 (range 17.4-36.8) min for U200, U100 and U20, respectively. The area under the curve per dose (AUC/D) was determined to be 51.2 ± 19.5, 52.3 ± 12.5 and 51.6 ± 6.7 pm × min/kg for U200, U100 and U20, respectively. The steady state plasma concentration (C(ss) ) was 57.5 ± 27.1, 54.3 ± 10.3 and 55.1 ± 8.0 pm (mean ± SD) for U200, U100 and U20, respectively. Time to steady state (T(ss) ) was 110 ± 36, 98 ± 48 and 90 ± 27 min for U200, U100 and U20, respectively. CONCLUSIONS: In conclusion, no significant difference was found in t(1/2) , AUC/D, C(ss) or T(ss) between the three IAsp concentrations when given at a basal rate in CSII.

In vitro-in vivo correlations of self-emulsifying drug delivery systems combining the dynamic lipolysis model and neuro-fuzzy networks.

The aim of the current study was to evaluate the potential of the dynamic lipolysis model to simulate the absorption of a poorly soluble model drug compound, probucol, from three lipid-based formulations and to predict the in vitro-in vivo correlation (IVIVC) using neuro-fuzzy networks. An oil solution and two self-micro and nano-emulsifying drug delivery systems were tested in the lipolysis model. The release of probucol to the aqueous (micellar) phase was monitored during the progress of lipolysis. These release profiles compared with plasma profiles obtained in a previous bioavailability study conducted in mini-pigs at the same conditions. The release rate and extent of release from the oil formulation were found to be significantly lower than from SMEDDS and SNEDDS. The rank order of probucol released (SMEDDS approximately SNEDDS > oil formulation) was similar to the rank order of bioavailability from the in vivo study. The employed neuro-fuzzy model (AFM-IVIVC) achieved significantly high prediction ability for different data formations (correlation greater than 0.91 and prediction error close to zero), without employing complex configurations. These preliminary results suggest that the dynamic lipolysis model combined with the AFM-IVIVC can be a useful tool in the prediction of the in vivo behavior of lipid-based formulations.

Bioavailability of probucol from lipid and surfactant based formulations in minipigs: influence of droplet size and dietary state.

The influence of droplet size on the absorption from lipid and surfactant based formulations was evaluated from two self-emulsifying formulations, a surfactant solution, and an oil solution. The self-emulsifying formulations was a self-emulsifying (SEDDS) and a self-nanoemulsifying (SNEDDS) formulation containing equal lipid and surfactant load, but exhibiting a large difference (approx 100 times) in the mean particle diameter of the resultant emulsion. The formulations were evaluated in a bioavailability study in fasted and fed Göttingen minipigs using probucol as model drug. In order to determine the bioavailability, an oil-in-water emulsion was included as i.v. formulation. The fasted group was fasted overnight and offered the first daily meal approx 4 h after treatment. The fed group was offered the first daily meal (50% energy from fat) 30 min prior to treatment. In the fasted group the SNEDDS exhibited a slightly faster absorption and higher bioavailability than the SEDDS, though non-significant. Furthermore, the bioavailability from the surfactant solution and the oil solution were slightly lower compared to the SNEDDS, indicating that both small particle size and digestibility are important in ensuring optimal bioavailability. Comparing the absorption in fasted and fed minipigs showed that probucol exhibited no significant food effect, when formulated in a lipid and surfactant based formulations.

Using biorelevant dissolution to obtain IVIVC of solid dosage forms containing a poorly-soluble model compound.

The usefulness of selected biorelevant dissolution media (BDM) to predict in vivo drug absorption was studied. Dissolution profiles of solid formulations of a poorly soluble model compound were compared in BDM simulating fasted and two levels of fed state. A non-physiologically relevant medium containing the cationic surfactant, cetrimide, was also investigated. All the media studied were capable of differentiating between the formulations employed, with formulation A consistently ranking high and formulations C and D ranking low. An in vivo dog study was carried out and an attempt was made to obtain a level A correlation between the plasma absorption curves and in vitro dissolution curves, using non-linear regression software. The in vitro-in vivo correlation (IVIVC) models developed indicated that fed state media (BDM 3) containing high levels of both bile salts (BS) and lipolysis products (LP) were best able to predict in vivo pharmacokinetic parameters (Cmax and AUC) with prediction errors lower than 10%. Overall, design and use of appropriate media for in vitro dissolution is extremely important. This study demonstrates the potential of physiologically relevant media containing both BS and LP for use in formulation and early drug development.

Structural development of self nano emulsifying drug delivery systems (SNEDDS) during in vitro lipid digestion monitored by small-angle X-ray scattering.

PURPOSE: To investigate the structural development of the colloid phases generated during lipolysis of a lipid-based formulation in an in vitro lipolysis model, which simulates digestion in the small intestine. MATERIALS AND METHODS: Small-Angle X-Ray scattering (SAXS) coupled with the in vitro lipolysis model which accurately reproduces the solubilizing environment in the gastrointestinal tract and simulates gastrointestinal lipid digestion through the use of bile and pancreatic extracts. The combined method was used to follow the intermediate digestion products of a self nano emulsified drug delivery system (SNEDDS) under fasted conditions. SNEDDS is developed to facilitate the uptake of poorly soluble drugs. RESULTS: The data revealed that a lamellar phase forms immediately after initiation of lipolysis, whereas a hexagonal phase is formed after 60 min. The change of the relative amounts of these phases clearly demonstrates that lipolysis is a dynamic process. The formation of these phases is driven by the lipase which continuously hydrolyzes triglycerides from the oil-cores of the nanoemulsion droplets into mono- and diglycerides and fatty acids. We propose that this change of the over-all composition of the intestinal fluid with increased fraction of hydrolyzed nanoemulsion induces a change in the composition and effective critical packing parameter of the amphiphilic molecules, which determines the phase behavior of the system. Control experiments (only the digestion medium) or the surfactant (Cremophor RH 40) revealed the formation of a lamellar phase demonstrating that the hexagonal phase is due to the hydrolysis of the SNEDDS formulation. CONCLUSIONS: The current results demonstrate that SAXS measurements combined with the in vitro dynamic lipolysis model may be used to elucidate the processes encountered during the digestion of lipid-based formulations of poorly soluble drugs for oral drug delivery. Thus the combined methods may act as an efficient screening tool.