A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

A Co-Processed API Approach for a Shear Sensitive Compound Affording Improved Chemical Stability and Streamlined Drug Product Processing.

The physical properties of active pharmaceutical ingredients (API) are critical to both drug substance (DS) isolation and drying operations, as well as streamlined drug product (DP) processing and the quality of final dosage units. High aspect ratio, low bulk density, API 'needles' in particular are a hindrance to efficient processing, with a low probability that conventional crystallization routes can modify the challenging morphology. The compound evaluated in this manuscript demonstrated this non-ideal morphology, with the added complexity of shear sensitivity. Modest shear exposure resulted in conversion of the thermodynamically stable crystalline phase to the amorphous phase, with the amorphous phase then undergoing accelerated chemical degradation. Slow filtration during DS isolation resulted in uncontrolled and elevated amorphous levels, while subsequent DP operations including blending, densification and compression increased amorphous content still further. A chemically stable final dosage unit would ideally involve a high bulk density, free flowing API that did not require densification in order to be commercialized as an oral dosage form with direct encapsulation of a single dosage unit. Despite every effort to modify the crystallization process, the physical properties of the API could not be improved. Here, an innovative isolation strategy using a thin film evaporation (TFE) process in the presence of a water soluble polymer alleviated filtration and drying risks and consistently achieved a high bulk density, free flowing co-processed API amenable to direct encapsulation. Characterization of the engineered materials suggested the lower amorphous levels and reduced shear sensitivity were achieved by coating surfaces of the API at relatively low polymer loads. This particle engineering route blurred conventional DS/DP boundaries that not only achieved improved chemical stability but also resulted in a optimized material, with simplified and more robust processing operations for both drug substance and drug product.

USP Apparatus 4: a Valuable In Vitro Tool to Enable Formulation Development of Long-Acting Parenteral (LAP) Nanosuspension Formulations of Poorly Water-Soluble Compounds.

Long-acting or extended release parenteral dosage forms have attracted extensive attention due to their ability to maintain therapeutic drug concentrations over long periods of time and reduce administration frequency, thus improving patient compliance. It is essential to have an in vitro release (IVR) testing method that can be used to assure product quality during routine production as well as predict and understand the in vivo performance of a formulation. The purpose of this work was to develop a discriminatory in vitro release method to guide formulation and process development of long-acting parenteral (LAP) nanosuspension formulations composed of poorly water-soluble drugs (BCS class II). Injectable nanosuspension formulations were developed to serve as test articles for method development. Several different IVR methods were evaluated for their application to the formulation screening and process development including (1) USP apparatus 2, (2) dialysis and reverse dialysis sac, and (3) continuous flow-through cell (USP apparatus 4). Preliminary data shows the promising results to support the utilization of USP 4 over more widely accepted USP 2 and dialysis methods. A combination of more representative in vivo hydrodynamics and ease of maintaining sink conditions yields the USP 4 flow-through cell method a more suitable in vitro release method for nanosuspension-based LAP formulations of poorly water-soluble compounds, such as compounds A and B.

New and Evolving Techniques for the Characterization of Peptide Therapeutics.

Advances in technologies related to the design and manufacture of therapeutic peptides have enabled researchers to overcome the biological and technological challenges that have limited their application in the past. As a result, peptides of increasing complexity have become progressively important against a variety of disease targets. Developing peptide drug products brings with it unique scientific challenges consistent with the unique physicochemical properties of peptide molecules. The identification of the proper characterization tools is required in order to develop peptide formulations with the appropriate stability, manufacturability, and bioperformance characteristics. This knowledge supports the build of critical quality attributes and, ultimately, regulatory specifications. The purpose of this review article is to provide an overview of the techniques that are employed for analytical characterization of peptide drug products. The techniques covered are highlighted in the context of peptide drug product understanding and include chemical and biophysical approaches. Emphasis is placed on summarizing the recent literature experience in the field. Finally, the authors provide regulatory perspective on these characterization approaches and discuss some potential areas for further research in the field.

Hypomelanosis of Ito with a trisomy 2 mosaicism: a case report.

INTRODUCTION: Hypomelanosis of Ito is a rare neurocutaneous disorder, characterized by streaks and swirls of hypopigmentation following the lines of Blaschko that may be associated to systemic abnormalities involving the central nervous system and musculoskeletal system. Despite the preponderance of reported sporadic hypomelanosis of Ito, few reports of familial hypomelanosis of Ito have been described. CASE PRESENTATION: A 6-month-old Caucasian girl presented with unilateral areas of hypomelanosis distributed on the left half of her body and her father presented with similar mosaic hypopigmented lesions on his upper chest. Whereas both blood karyotypes obtained from peripheral lymphocyte cultures were normal, a 16% trisomy 2 mosaicism was found in cultured skinfibroblasts derived from a hypopigmented skin area of her father. CONCLUSIONS: Familial cases of hypomelanosis of Ito are very rare and can occur in patients without systemic involvement. Hypomelanosis of Ito constitutes a non-specific diagnostic definition including different clinical entities with a wide phenotypic variability, either sporadic or familial. Unfortunately, a large number of cases remain misdiagnosed due to both diagnostic challenges and controversial issues on cutaneous biopsies in the pediatric population.

Cerebral blood flow in depressed patients with systemic lupus erythematosus.

OBJECTIVE: To characterize the neural circuitry involved in depression associated with systemic lupus erythematosus (SLE), we used single photon emission computed tomography (SPECT) to study regional cerebral blood flow (CBF) in patients with SLE. METHODS: SPECT with (99m)Tc-ethylcysteinate dimer was performed in 30 depressed women patients with SLE, in 14 women patients with SLE and without history of neuropsychiatric disorders, and in 25 healthy women controls. Magnetic resonance imaging was done for all subjects for diagnostic purposes. Analysis of CBF patterns was performed using statistical parametric mapping. Statistical significance was taken at uncorrected p < 0.001 at cluster level. RESULTS: There were no significant differences between depressed and nondepressed patients with SLE for any rheumatologic variable. In comparison to healthy controls, depressed patients with SLE had significantly reduced CBF in bilateral frontal and temporal cortex; global maximum was located in the left precentral gyrus. There were no significant CBF differences between nondepressed patients with SLE and controls. Compared to nondepressed patients with SLE, depressed patients with SLE had significantly lower CBF in 2 clusters that had their local maxima in the right precentral gyrus and in the left superior temporal gyrus. The duration of SLE correlated with decreased perfusion in the left middle and superior frontal gyrus. CONCLUSION: Depressed patients with SLE have CBF reductions in discrete temporal and frontal regions that may account for depressive symptoms.

Modulation of drug release kinetics from hydroxypropyl methyl cellulose matrix tablets using polyvinyl pyrrolidone.

Hydrophilic matrix tablets are widely used to extend the release of a broad range of pharmaceutically active materials. The mechanism and kinetics of drug release are dependent on the solubility of the active moiety and the swelling and erosion properties of the polymer, with water soluble compounds released predominantly by diffusion. The swelling and erosion properties of hydroxypropyl methyl cellulose (HPMC), typically lead to a first order release rate for water soluble compounds as opposed to the more desirable zero-order kinetics. In addition, for compounds with differences in regional absorption within the gastrointestinal tract a dosage form with a bi-modal release profile may be required, which is difficult to achieve with a simple dosage form. The following paper presents a simple, cost effective and elegant solution for achieving a range of predictable release profiles from linear to bi-modal for a water soluble drug (caffeine) from HPMC matrices, through the inclusion of polyvinyl pyrrolidone (PVP). Mechanistic studies using gel rheology, excipient dissolution and near-infrared microscopy (NIR) microscopy are presented which show that the modulation of drug release kinetics is mediated through a reduction in HPMC viscosity in the presence of a critical concentration of PVP, which leads to a break-up of the extended release tablet. A validated mathematical model is also presented which allows drug release profiles to be reliably predicted based on the initial HPMC and PVP content in the tablet.