Preparation and characterization of albumin conjugates of a truncated peptide YY analogue for half-life extension.

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 µmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

Microenvironmental pH and microviscosity inside pH-controlled matrix tablets: an EPR imaging study.

Incorporation of pH modifiers is a commonly used strategy to enhance the dissolution rate of weakly basic drugs from sustained release solid dosage forms. Electron paramagnetic resonance imaging (EPRI) was applied to spatially monitor pH(M) and the rotational correlation time (tau(R)), a parameter which is closely related to the surrounding microviscosity inside HPMC (hydroxypropylmethylcellulose) matrix tablets. Fumaric, citric, and succinic acid were employed as pH modifiers. 4-(methylamino)-2-ethyl-5,5-dimethyl-4-pyridine-2-yl-2,5-dihydro-1H-imidazole-1-oxyl (MEP) was used as spin label. Fumaric and citric acid reduced the pH(M) to equal extents in the initial phase. With the progress of hydration, the more soluble citric acid diffused out from the tablet resulting in an increase in pH(M), originating at the outer layers. In contrast, fumaric acid maintained a constantly reduced pH(M) inside the entire tablet. Due to its lower acidic strength, succinic acid did not reduce the pH(M) as effectively as the other pH modifiers used. The more water-soluble acids stimulated the water penetration into the matrix system, thereby rapidly decreasing tau(R). Once the matrix tablets were hydrated, the included pH modifiers influenced tau(R) insignificantly. EPRI, a novel approach for monitoring pH(M) and tau(R) non-invasively and spatially resolved, was used successfully for the optimization of an pH-controlled formulation.

Strategies for the design of hydrophilic matrix tablets with controlled microenvironmental pH.

Incorporation of weak acids as pH modifiers enhances the release of weakly basic drugs in higher pH environments by reducing the microenvironmental pH (pHM). The objectives of this study were: (a) to investigate the relationship between pHM, drug release, and pH modifier release and (b) to achieve simultaneous release of the drug and the pH modifier over the entire dissolution time (6 h, phosphate buffer, pH 6.8). Using dipyridamole as a model drug, we investigated drug and acid release and determined the average pHM potentiometrically using tablet cryosections. The first approach was based on incorporating different concentrations of pH modifiers in conventional matrix tablets based on hydroxypropylmethylcellulose. Owing to its high acidic strength and low aqueous solubility, fumaric acid resulted in simultaneous release and maintained a constant acidic pHM. Secondly, press-coated matrix tablets, comprising an acidic reservoir, were found to be a valuable approach for retarding the diffusion of more water-soluble acids. Using the power law expression (Mt/Minfinity = ktn) it became evident that the inclusion of acids increased drug release. Higher acid concentrations tended to decrease n standing for the slope, whereas the release constant k increased. Furthermore, the medial check term parameters depended on the type of pH modifier used.

Assessment of tailor-made HPMC-based matrix minitablets comprising a weakly basic drug compound.

Tailor-made, pH-controlled matrix minitablets based on different HPMC types were developed comprising the weakly basic drug dipyridamole. The incorporation of pH modifiers, i.e., fumaric and succinic acid, enhanced the drug release at pH 6.8. Assessing the drug release, acid release, and the microenvironmental pH (pHM) provided detailed understanding of pH-controlled mini-matrices. The extent and duration of pHM alteration was more pronounced in presence of fumaric acid. Minitablets based on the fast dissolving Methocel K100LV (< or = 100 cps) showed simultaneous release rates of dipyridamole and fumaric acid with a constant low average pHM.