Rainer Alex

Active Apical Secretory Efflux of the HIV Protease Inhibitors Saquinavir and Ritonavir in Caco-2 Cell Monolayers

AbstractPurpose. To investigate in vitro the mechanisms involved in the gastrointestinal absorption of the HIV protease inhibitor, saquinavir mesylate (Invirase®), whose oral bioavailability is low, variable, and significantly increased by co-administration with ritonavir, also an HIV protease inhibitor but with higher oral bioavailability. Methods. Confluent epithelial layers of human Caco-2 cells mimicking the intestinal barrier. Results. Both saquinavir and ritonavir showed polarized transport through Caco-2 cell monolayers in the basolateral to apical direction (secretory pathway), exceeding apical to basolateral transport (absorptive pathway) by factors of 50-70 and 15-25, respectively. Active efflux was temperature dependent, saturable and inhibited by verapamil and cyclosporin A. Saquinavir and ritonavir decreased each others secretory permeability and hence elevated their net transport by the absorptive pathway. Conclusions. Saquinavir and ritonavir are both substrates for an efflux mechanism in the gut, most likely P-glycoprotein, which acts as a counter-transporter for both drugs. Together with sensitivity to gut-wall metabolism by cytochrome P-450 3A, this may partially account for the low and variable oral bioavailability of saquinavir in clinical studies and for its increased bioavailability after co-administration with ritonavir.

Application of in vitro biopharmaceutical methods in development of immediate release oral dosage forms intended for paediatric patients.

Biopharmaceutics is routinely used in the design and development of medicines to generate science based evidence to predict in vivo performance; the application of this knowledge specifically to paediatric medicines development is yet to be explored. The aim of this review is to present the current status of available biopharmaceutical tools and tests including solubility, permeability and dissolution that may be appropriate for use in the development of immediate release oral paediatric medicines. The existing tools used in adults are discussed together with any limitations for their use within paediatric populations. The results of this review highlight several knowledge gaps in current methodologies in paediatric biopharmaceutics. The authors provide recommendations based on existing knowledge to adapt tests to better represent paediatric patient populations and also provide suggestions for future research that may lead to better tools to evaluate paediatric medicines.

Transformation of Oligomers of Lipidated Peptide Induced by Change in pH

Oligomerization of lipidated peptides is of general scientific interest and is important in biomedical and pharmaceutical applications. We investigated the solution properties of a lipidated peptide, Liraglutide, which is one of the glucagon-like peptide-1 (GLP-1) agonists used for the treatment of type II diabetes. Liraglutide can serve as a model system for studying biophysical and biochemical properties of micelle-like self-assemblies of the lipidated peptides. Here, we report a transformation induced in Liraglutide oligomers by changing pH in the vicinity of pH 7. This fully reversible transformation is characterized by changes in the size and aggregation number of the oligomer and an associated change in the secondary structure of the constituent peptides. This transformation has quite slow kinetics: the equilibrium is reached in a course of several days. Interestingly, while the transformation is induced by changing pH, its kinetics is essentially independent of the final pH. We interpreted these findings using a model in which desorption of the monomer from the oligomer is the rate-limiting step in the transformation, and we determined the rate constant of the monomer desorption.

The molecular basis for the prolonged blood circulation of lipidated incretin peptides: Peptide oligomerization or binding to serum albumin?

Hybrid incretin peptides are a new generation of drugs for the treatment of diabetes and obesity. Despite their biological potency, the effectiveness of these peptides as drugs is limited by their short circulation time in blood (typically within minutes). In this work, we show that lipid conjugated forms of a GLP-1/GIP/glucagon hybrid peptides stay in circulation for hours. We studied the oligomerization and albumin-binding of the unconjugated hybrid peptide as well as its lipidated variants. These lipidated peptides differ in the N-terminal mutation, the position of lipidation and the linkage to lipid. We found that these lipidated peptides form stable oligomers at concentrations above 1mg/mL. This concentration range is relevant to formulation and storage of the peptides. We observed no binding between the peptide oligomers and human serum albumin. However, at the expected therapeutic concentration range (~10-100ng/mL), the oligomers dissociate into monomers. The monomers of lipidated peptides bind to albumin. We have determined the dissociation constants of binding between the lipidated peptides and serum albumin. The dissociation constants of albumin-binding of our lipidated peptides are all very close and similar to that of the fatty acid binding of albumin. Our findings suggest that the monomeric lipidated peptides bind to HSA mainly by the fatty acid chain. Therefore, albumin binding is likely to be a universal mechanism of the prolonged circulating duration of lipidated pharmaceutical peptides.

Liquid–Liquid Phase Separation in Oligomeric Peptide Solutions

Oligomeric peptides exist widely in living organisms and play a role in a broad range of biological functions. We report the first observation of liquid-liquid phase separation (LLPS) in peptide solutions, in particular, solutions of peptides consisting of noncovalent oligomers. We determined the binary phase boundary of the oligomeric peptide solution and compared the result to the well-established phase diagram of globular proteins. We also provide simple theoretical interpretations of the similarities and differences between the phase diagrams of peptides and proteins. Finally, by tuning inter-oligomer interactions using a crowding agent, we demonstrated that LLPS is a universal phenomenon that can be observed under different solution conditions for a variety of peptides.

Process and scaling parameters for wet media milling in early phase drug development: A knowledge based approach

Abstract Wet media milling is a well‐established unit operation for the manufacturing of suspension formulations during early phase pharmaceutical development. However, knowledge about the kinetics of particle breakage is limited, although the impact of hydro‐mechanical process parameters on the mean particle size of finished suspensions has been thoroughly investigated. We performed in this work milling trials with two different compounds on two milling devices with different mechanical design and volume scale. We analyzed our data in terms of a kinetic milling model where we included the milling speed as an explicit process parameter in addition to the process duration. We show, that the milling kinetics can be fairly well predicted for a wide range of these operating parameters. The proposed milling model may therefore be useful for rational process planning and scale‐up considerations in the industrial setting. Graphical Abstract Figure. No caption available.