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. We designed the MEDI7219 immediate-release tablets with the permeation enhancers Na CDC and PG. Immediate-release tablets were coated with an enteric coating that dissolves at pH ≥ 5.5 to target the upper duodenal region of the gastrointestinal tract and sustained-release tablets with a Carbopol bioadhesive polymer were coated with an enteric coating that dissolves at pH ≥ 7.0 to provide a longer presence at the absorption site in the gastrointestinal tract. In addition to immediate- and enteric-coated formulations, we also tested a proprietary delayed release erodible barrier layer tablet (OralogiKTM) to deliver the payload to the target site in the gastrointestinal tract. The design of tablet dosage forms based on the optimization of formulations resulted in up to 10.1% absolute oral bioavailability in dogs with variability as low as 26% for MEDI7219, paving the way for its clinical development.

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. We describe a comprehensive multidisciplinary approach leading to the development of MEDI7219, a GLP-1 receptor agonist (GLP-1RA) specifically engineered for oral delivery. Sites of protease/peptidase vulnerabilities in GLP-1 were removed by amino acid substitution and the peptide backbone was bis-lipidated to promote MEDI7219 reversible plasma protein binding without affecting potency. A combination of sodium chenodeoxycholate and propyl gallate was used to enhance bioavailability of MEDI7219 at the site of maximal gastrointestinal absorption, targeted by enteric-coated tablets. This synergistic approach resulted in MEDI7219 bioavailability of ~ 6% in dogs receiving oral tablets. In a dog model of obesity and insulin resistance, MEDI7219 oral tablets significantly decreased food intake, body weight and glucose excursions, validating the approach. This novel approach to the development of MEDI7219 provides a template for the development of other oral peptide therapeutics.

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 5.5 and 7.0. In addition, sustained-release or mucoadhesive polymers were coated to trigger release at a desired site in the gastrointestinal tract. Dissolution studies with a USP Type I (basket) apparatus confirmed the duration of release. Pharmacokinetic studies were performed in beagle dogs to evaluate bioavailability. A high-disintegration pH was found to be advantageous in enhancing bioavailability.

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. In vivo efficacy studies in mice demonstrated that a single injection of nanoparticle PBD formulations could inhibit tumor growth for nearly 3 weeks, whereas the free drug failed to inhibit growth. Importantly, mice treated with PBD-loaded nanoparticles did not experience significant loss of body weight. These data demonstrate that nanoparticles containing PBD molecules can be used as an alternative to the widely used antibody drug conjugate approach in delivering cytotoxic PBDs.

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. The inter-subject variability leads to large differences in pharmacologic responses in individuals and thus the dose required to produce therapeutic effect could vary between individuals making the development of drug product a very difficult task. A substantial amount of research has been (and continues to be) performed with a focus on getting acceptable absorption and reproducible results. Nonetheless, the high variability and low bioavailability during oral administration of peptides is still a work in progress and under-explored in a systematic way. While there are several review articles and scattered publications that discuss potential technologies for oral peptide delivery, a detailed look into the physiological challenges and absorption barriers which are a hindrance to successful clinical translation, is lacking. Herein, we have analyzed the physiological barriers within the gastrointestinal (GI) tract that are the root causes for the low bioavailability and high variability of oral delivery of peptides in humans. In particular, we have taken a detailed look at the key influencing factors such as the nature of various GI tract parameters, components of the GI tract that influences the uptake, site of absorption, pH of the gastric and intestinal compartments, food effect, and role of peptidases in affecting oral peptide absorption. Lack of in vitro - in vivo correlations and variability in animal models have also been highlighted as key impediments in understanding the challenges. The unique perspective presented herein for overcoming the physiological absorption barriers, will offer better developability approaches and will positively impact clinical translation of future oral peptide therapeutics. A deep understanding of these effects are vital, given the emergence of microbiome and oral biologic drug delivery that are fast emerging as the next wave of personalized patient centric therapies.

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. Using dynamic light scattering, we monitored formation and growth of submicron aggregates in various buffers. Our experiments revealed a nucleation-controlled submicron aggregation of the antibody in the presence of chemical denaturant. After the unfolded protein reached a steady state, we reduced the denaturant concentration by dilution or dialysis to trigger further aggregation after ICD. In this way, we studied the pH effect on aggregation of the stressed protein after removal of denaturant. The ICD-dilution experiment provides a practical means for studying the propensity of unfolded proteins to form aggregates under various formulation conditions. This unique method allows us to control the degree of protein unfolding and the initiation of post-ICD aggregation.

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. Advances in self-administration of insulin and GLP-1 receptor agonist therapies with use of drug-device combination products have further improved the outcomes of diabetes management and quality of life for diabetic patients. The synergies of insulin and GLP-1 receptor agonist actions have led to development of devices that can deliver both molecules simultaneously. New chimeric GLP-1-incretins and insulin-GLP-1-incretin molecules are also being developed. The objective of this review is to summarize molecular designs to improve the drug-like properties of insulin and GLP-1 and to highlight the continued advancement of drug-device combination products to improve diabetes management.

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. We experimentally determined the full phase diagram of an IgG antibody. Using the full diagram, we examined the metastability gaps, i.e., the distance between the crystal solubility line and the liquid-liquid coexistence curve, of IgG antibodies. By comparing our results to the partial phase diagrams of other IgGs reported in literature, we found that IgG antibodies have similar metastability gaps. Thereby, we present an equation with two phenomenological parameters to predict the approximate location of the solubility line of IgG antibodies with respect to their liquid-liquid coexistence curves. We have previously shown that the coexistence curve of an antibody solution can be readily determined by the polyethylene glycol-induced liquid-liquid phase separation method. Combining the polyethylene glycol-induced liquid-liquid phase separation measurements and the phenomenological equation in this article, we provide a general and practical means to predict the thermodynamic conditions for crystallizing IgG antibodies in the solution environments of interest.

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, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

La presente invention concerne des formulations, administrables par voie orale, de proteines, y compris en phase amorphe et cristalline, ainsi que sous forme reticulee. Ces formulations renferment, en particulier, un melange de lipase, de protease et d'amylase, ainsi qu'un agent effervescent. L'invention concerne, en outre, des procedes d'administration de ces formulations, ainsi que leur preparation et leurs conditions d'utilisation. L'invention concerne, plus precisement, des procedes de preparation et d'administration de formulations se revelant interessantes en pediatrie et chez des patients souffrant de troubles associes a la deglutition comme frequemment observe chez les personnes âgees et les personnes handicapees, ou encore pour des patients souffrant d'occlusions oesophagiennes ou de complications atteignant le systeme nerveux central autonome (par exemple dans le cas de la maladie de Parkinson, de la sclerose en plaques ou de la sclerose laterale amyotrophique, etc.).

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.RESULTS:Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance.CONCLUSIONS:The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.

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. The results indicate that the protein crystals could be incorporated in these in situ formable gels without the need for initial drying. The crystals withstand organic solvents and water/organic solvent interfaces, and provide high protein loading (>30%) in these systems. Moreover, changing the morphology of the amylase crystals successfully modulated amylase release profiles. Study of long-term stability at 4 degrees C revealed a greater stability of crystalline protein compared to amorphous amylase. The above-mentioned data suggest that protein crystals might offer greater feasibility in developing sustained release injectable in situ formable protein depot systems.

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. delivery of a small volume of a highly concentrated form of mAbs. Batch crystallization of three Ab-based therapeutics, rituximab, trastuzumab, and infliximab, provided products in high yield, with no detectable alteration to these proteins and with full retention of their biological activity in vitro. Administration s.c. of a crystalline preparation resulted in a remarkably long pharmacokinetic serum profile and a dose-dependent inhibition of tumor growth in nude mice bearing BT-474 xenografts (human breast cancer cells) in vivo. Overall, this approach of generating high-concentration, low-viscosity crystalline preparations of therapeutic Abs should lead to improved ease of administration and patient compliance, thus providing new opportunities for the biotechnology industry.

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. It was thereby found that at pH 8.5, which is the optimal value for renaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 × 10 7 U/mg.

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. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure. This structure causes highly efficient fusion protein folding.