Rajesh B. Gandhi

Oral cavity as a site for bioadhesive drug delivery

Abstract Accessibility of the oral cavity makes it a potentially attractive route for drug delivery. However, rapid removal of conventional delivery systems, primarily through copious salivary flow, and the relative impermeability of the buccal tissue are clear impediments to successful use of this route. Bioadhesive polymers can overcome the removal issue and can be coupled with penetration enhancers to generate a novel and successful delivery system for both local and systemic drug delivery. The present review examines factors influencing the disposition of drugs and delivery systems in the oral cavity as well as pertinent biological barriers. It also provides the theoretical and practical basis of bioadhesives in oral mucosal drug delivery systems, as well as the limited studies on penetration enhancers for this tissue. Finally, future trends are discussed.

Enhancement of oral bioavailability of an HIV-attachment inhibitor by nanosizing and amorphous formulation approaches

BMS-488043 is an HIV-attachment inhibitor that exhibited suboptimal oral bioavailability upon using conventional dosage forms prepared utilizing micronized crystalline drug substance. BMS-488043 is classified as a Biopharmaceutics Classification System (BCS) Class-II compound with a poor aqueous solubility of 0.04mg/mL and an acceptable permeability of 178nm/s in the Caco2 cell-line model. Two strategies were evaluated to potentially enhance the oral bioavailability of BMS-488043. The first strategy targeted particle size reduction through nanosizing the crystalline drug substance. The second strategy aimed at altering the drugs physical form by producing an amorphous drug. Both strategies provided an enhancement in oral bioavailability in dogs as compared to a conventional formulation containing the micronized crystalline drug substance. BMS-488043 oral bioavailability enhancement was approximately 5- and 9-folds for nanosizing and amorphous formulation approaches, respectively. The stability of the amorphous coprecipitated drug prepared at different compositions of BMS-488043/polyvinylpyrrolidone (PVP) was evaluated upon exposure to stressed stability conditions of temperature and humidity. The drastic effect of exposure to humidity on conversion of the amorphous drug to crystalline form was observed. Additionally, the dissolution behavior of coprecipitated drug was evaluated under discriminatory conditions of different pH values to optimize the BMS-488043/PVP composition and produce a stabilized, amorphous BMS-488043/PVP (40/60, w/w) spray-dried intermediate (SDI), which was formulated into an oral dosage form for further development and evaluation.

Pharmaceutical relationships of three solid state forms of stavudine

Three solid state forms of stavudine designated forms I, II and III have been identified and characterized. Forms I and II are anhydrous polymorphs whereas form III is hydrated and is pseudopolymorphic with forms I and II. Physico-chemical and thermodynamic properties of the three solid state forms have been characterized. Solid-state stability and potential for interconversion of the forms to aid in the selection of preferred form for development and commercialization has been studied. Conditions of recrystallization governing the formation of thermodynamically most stable polymorphic form I devoid of other forms was identified.

The effect of PEGylation on the stability of small therapeutic proteins

The influence of PEGylation on the thermal stability of small therapeutic proteins was evaluated using two model proteins. Changes in the midpoint of thermal unfolding and the ability to properly refold after thermal denaturation were monitored by differential scanning calorimetry (DSC) as a function of PEGylation and pH. The results showed that PEGylation increased the thermal stability of both model proteins as well as their ability to refold properly after thermal denaturation. The DSC results were compared to traditional accelerated stability data that were collected using size exclusion high performance liquid chromatography (SE-HPLC). The DSC data agreed reasonably well with those from SE-HPLC indicating that microcalorimetry can be an efficient screening tool for PEGylated proteins.

Use of Microcalorimetry and Its Correlation with Size Exclusion Chromatography for Rapid Screening of the Physical Stability of Large Pharmaceutical Proteins in Solution

The utility of microcalorimetry as a rapid screening tool for assessing the solution stability of high molecular weight pharmaceutical proteins was evaluated by using model recombinant antibodies, Protein I and Protein II. Changes in the transition midpoint, Tm, were monitored as a function of pH and/or in the presence of excipients, and results were compared with traditional accelerated stability data from samples that were analyzed by size exclusion chromatography (SEC). The data from microcalorimetry were well correlated with those from SEC for predicting both optimal solution pH as well as excipient effects on solution stability. These results indicate that microcalorimetry can be an efficient screening tool useful in identifying optimal pH conditions and excipients to stabilize pharmaceutical proteins in solution formulations.

Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE.

Fragmentation of monoclonal antibodies has been routinely observed in non-reducing SDS-PAGE, mainly due to disulfide-bond scrambling catalyzed by free sulfhydryl groups, resulting in a method induced artifact. To minimize this artifact, alkylating agents like iodoacetamide (IAM) and N-ethylmaleimide (NEM) were commonly included in SDS sample buffer to block free sulfhydryls. However, the selection of agents and the applied concentrations differ from study to study. In addition, there is no direct comparison of these agents thus far, resulting in difficulties in selecting the suitable agent. To address these questions, we have tested the activities of IAM and NEM in inhibiting the fragment-band artifact of IgG4 monoclonal antibodies. Our data suggest that the inhibition activity of both agents is concentration dependent. Interestingly, 5mM NEM can achieve the same inhibition effect as 40 mM IAM. In addition, NEM still retained strong activity after prolonged sample heating, whereas IAM lost most of its activity. Overall, NEM appears to have a better inhibition effect than IAM on all tested IgG4 proteins, either with SDS-PAGE or CE-SDS methods. These observations demonstrate that NEM has stronger fragmentation inhibition activity than IAM, and thus is a more suitable alkylating agent for both SDS-PAGE and CE-SDS method to reduce this fragmentation artifact.

Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B22*, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.

Development of a precipitation-resistant solution formulation to increase in vivo exposure of a poorly water-soluble compound

In vitro and in vivo investigations were conducted to develop a suitable formulation for early toxicology and clinical studies of ((R)-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-fluorophenyl)pyrrolidin-1-yl)methanone (Compound A), a nonionizable and poorly water-soluble compound that selectively inhibits the ultrarapid potassium current (IKur) and is intended for the treatment of arrhythmia. Various nonaqueous solution formulations were evaluated, in vitro, for ability to prevent or delay precipitation of Compound A from solution following dilution with water. The plasma exposures of precipitation-resistant solutions, non precipitation-resistant solutions, and aqueous suspensions were then compared in rats, dogs, and/or humans. The data indicated that a solubilized, precipitation-resistant formulation achieved the highest plasma concentrations in all species and also improved dose proportionality, particularly in rats. Development of such formulations may be highly valuable for achieving in vivo blood levels often required for successful toxicological and early clinical evaluation of poorly soluble compounds.

Scale Considerations for Selection of Saccharide Excipients for Liquid Formulations

Formulated bulk drug substances (DS) are often frozen to prolong shelf life, allow operational flexibility, and maintain quality of poorly stable products. Aside from the active construct, biologic formulations typically include a variety of functional excipients. To this end, saccharides are commonly used as stabilizers and/or tonicity adjustors. During manufacture of a drug product containing 5% (w/v) mannitol, precipitation was observed in 2 L bottles containing 1.8 L of DS that had been frozen at −80◦C and then thawed at 5◦C. The precipitate dissolved upon mixing at room temperature and had an X-ray powder diffraction pattern consistent with the original form of mannitol, indicating that crystalline mannitol precipitated from the bulk. No phase transformation was observed. Precipitation had not been observed in laboratory-scale freeze–thaw studies. Hence, the relationship between scale and mannitol precipitation from bulk solution was investigated along with the freeze–thaw behavior of alternate saccharides (sucrose, sorbitol, and trehalose). Bottles of 2 and 1 L, and 125 and 10 mL capacity were filled 90% full with isotonic solutions and then frozen at −80◦C. Upon thawing 5% (w/v) mannitol solutions at 5◦C, precipitation was observed in 1 and 2 L bottles but not in 125 and 10 mL bottles. Precipitation was not observed upon thawing 5% (w/v) sorbitol, 9.4% (w/v) sucrose, or 9.4% (w/v) trehalose solutions at any scale studied. To further investigate the impact of scale on mannitol precipitation, blocks of 5% (w/v) mannitol solution frozen at −80◦C in 2 L, 125 and 50 mL

A Fluorescence-Based High-Throughput Coupled Enzymatic Assay for Quantitation of Isoaspartate in Proteins and Peptides.

Formation of isoaspartate (IsoAsp) from spontaneous asparagine (Asn) deamidation or aspartate (Asp) isomerization is one of the most common non-enzymatic pathways of chemical degradation of protein and peptide pharmaceuticals. Rapid quantitation of IsoAsp formation can enable rank-ordering of potential drug candidates, mutants, and formulations as well as support shelf life prediction and stability requirements. A coupled enzymatic fluorescence-based IsoAsp assay (CEFIA) was developed as a high-throughput method for quantitation of IsoAsp in peptides and proteins. In this note, application of this method to two therapeutic candidate proteins with distinct structural scaffolds is described. In addition, the results obtained with this method are compared to those from conventional assays.