Systems Biology and Peptide Engineering to Overcome Absorption Barriers for Oral Peptide Delivery: Dosage Form Optimization Case Study Preceding Clinical Translation.

Oral delivery of peptides and biological molecules promises significant benefits to patients as an alternative to daily injections, but the development of these formulations is challenging due to their low bioavailability and high pharmacokinetic variability. Our earlier work focused on the discovery of MEDI7219, a stabilized, lipidated, glucagon-like peptide 1 agonist peptide, and the selection of sodium chenodeoxycholate (Na CDC) and propyl gallate (PG) as permeation enhancer combinations. We hereby describe the development of the MEDI7219 tablet formulations and composition optimization via in vivo studies in dogs.

Development of an orally delivered GLP-1 receptor agonist through peptide engineering and drug delivery to treat chronic disease.

Peptide therapeutics are increasingly used in the treatment of disease, but their administration by injection reduces patient compliance and convenience, especially for chronic diseases. Thus, oral administration of a peptide therapeutic represents a significant advance in medicine, but is challenged by gastrointestinal instability and ineffective uptake into the circulation. Here, we have used glucagon-like peptide-1 (GLP-1) as a model peptide therapeutic for treating obesity-linked type 2 diabetes, a common chronic disease.

Targeted oral peptide delivery using multi-unit particulates: Drug and permeation enhancer layering approach.

Oral delivery of peptides is a challenge due to their instability and their limited transport and absorption characteristics within the gastrointestinal tract. In this work, we used layering techniques in a fluidized bed dryer to create a configuration in which the active peptide, permeation enhancers, and polymers are coated to control the release of the peptide. Formulations were developed to disintegrate at pH values of 5.

Evaluation of Pyrrolobenzodiazepine-Loaded Nanoparticles: A Targeted Drug Delivery Approach.

We describe the development and evaluation of pyrrolobenzodiazepines (PBDs) in poly(dl-lactide-co-glycolide) and lipid nanoparticle drug delivery systems. We have established that the partition coefficient (LogP) of PBD is a key influencer of the encapsulation efficiency in nanoparticle systems, with higher LogP values associated with higher encapsulation efficiencies toward increased drug payload delivery and better antitumor efficacy. Cytotoxicity assays demonstrated that compounds with higher LogP values demonstrated higher 50% inhibitory concentration values than the free drug.

Oral peptide delivery: Translational challenges due to physiological effects.

Oral delivery of peptide therapeutics as a convenient alternate to injections has been an area of research for the pharmaceutical scientific community for the last several decades. However, systemic delivery of therapeutic peptides via the oral route has been a daunting task due to the low pH denaturation of the peptides in the stomach, enzymatic instability, and poor transport across the tight junctions resulting in very low bioavailability. The low bioavailability is accompanied by large intra- and inter-subject variability leading to translational issues, preventing the development of successful peptide therapeutics.

Submicron Aggregation of Chemically Denatured Monoclonal Antibody.

Isothermal chemical denaturation (ICD) has been widely used to evaluate the conformational stability of therapeutic proteins such as monoclonal antibodies. However, the chemical unfolding pathway and the subsequent aggregation of antibodies are not yet well-understood. In the present work, we conducted a systematic study on an ICD-induced aggregation of a pharmaceutical monoclonal antibody.

Metastability Gap in the Phase Diagram of Monoclonal IgG Antibody.

Crystallization of IgG antibodies has important applications in the fields of structural biology, biotechnology, and biopharmaceutics. However, a rational approach to crystallize antibodies is still lacking. In this work, we report a method to estimate the solubility of antibodies at various temperatures.

Improving drug-like properties of insulin and GLP-1 via molecule design and formulation and improving diabetes management with device & drug delivery.

There is an increased incidence of diabetes worldwide. The discovery of insulin revolutionized the management of diabetes, the revelation of glucagon-like peptide-1 (GLP-1) and introduction of GLP-1 receptor agonists to clinical practice was another breakthrough. Continued translational research resulted in better understanding of diabetes, which, in combination with cutting-edge biology, chemistry, and pharmaceutical tools, have allowed for the development of safer, more effective and convenient insulins and GLP-1.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body.

An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.

Novel long-acting crystal formulation of human growth hormone.

Purpose: The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules.

Methods: Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys.

Injectable controlled release formulations incorporating protein crystals.

Development of ready-to-inject in situ formable controlled release gel systems for proteins is extremely challenging due to poor stability of proteins in the organic solvents typically used to fabricate these systems and because of the need of initial drying of proteins. The focus of the present study was to develop and characterize injectable controlled release systems composed of crystals of amylase, a model protein, suspended in solutions of polymeric and non-polymeric matrix materials in organic solvents. In this study, alpha-amylase derived from Aspergillus oryzae was crystallized and crystals were suspended in a poly(DL-lactide-co-glycolide) (PLGA) solution in acetonitrile (PLGA/acetonitrile), or in sucrose acetate isobutyrate (SAIB) plasticized with ethanol (SAIB/ethanol) systems.

Crystalline monoclonal antibodies for subcutaneous delivery.

Therapeutic applications for mAbs have increased dramatically in recent years, but the large quantities required for clinical efficacy have limited the options that might be used for administration and thus have placed certain limitations on the use of these agents. We present an approach that allows for s.c.

Recombinant human insulin IX. Investigation of factors, influencing the folding of fusion protein-S-sulfonates, biotechnological precursors of human insulin.

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency.

Methods for preparation of recombinant cytokine proteins V. mutant analogues of human interferon-gamma with higher stability and activity.

Mutant analogues of recombinant human immune interferon (IFN-gamma) with higher stability and biological activity were prepared. Depending on the analogue, protein structure modification might involve introduction of an intramonomer disulfide bond (through replacements of Glu7Cys and Ser69Cys), C-terminal shortening by 10 amino acid residues, as well as Gln133Leu substitution in truncated variant. Isolation, purification, and renaturation of the IFN-gamma analogues expressed in Escherichia coli as inclusion bodies were performed according to the scheme developed earlier for wild-type protein.

[Methods of preparation of recombinant proteins-cytokines. IV. Renaturation of recombinant human interleukin-3].

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy.

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells.

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide.

Methods of preparation of recombinant cytokine proteins.

Two schemes for efficient and productive isolation for mutant human recombinant tumor necrosis factor-alpha (TNF-alpha R32H) from Escherichia coli cells were developed. The methods include membrane filtration, ion-exchange chromatography and gel filtration, and centrifugation with subsequent free-flow electrophoresis as an alternative procedure. The target product was obtained as active trimer with total yield more than 50% and greater than 98% purity according to PAGE, size-exclusion chromatography, HPLC, and HPCE.

[Preparation of recombinant cytokines. II. Effective method for isolation, purification and renaturation of human recombinant gamma-interferon].

An efficient method for the isolation, purification, and renaturation of human recombinant gamma-interferon from biomass of transformed E. coli cells was developed. It involves the extraction of the protein from the inclusion bodies, preliminary purification of the protein, and three stages of ion-exchange chromatography with an intermediate renaturation between the second and the third stages.