Biomedical subjects

S J Milstein

Publications and source records attributed to S J Milstein.

4 recordsLinked to original sources

PubMed1998

Partially unfolded proteins efficiently penetrate cell membranes--implications for oral drug delivery.

We have previously reported on the biological activity of members of a library of low molecular weight compounds (carriers) that enable the oral delivery of proteins (Milstein, Proceedings of the 1995 Miami Bio/Technology Winter Symposium on Protein Engineering and Structural Biology, IRL Press at Oxford University Press, 1995, p. 13; Leone-Bay et al., J. Med. Chem. 38 (1995) 4263-4269; Leone-Bay et al., J. Med. Chem. 39 (1996) 2571-2578; [1-3]). When rats or primates are orally administered a solution of carrier and either recombinant human alpha-interferon (rhIFN), insulin or recombinant human growth hormone (rhGH) significant serum concentrations of the proteins are detectable. The transport activity of these compounds is positively correlated with their structural effects on the protein molecules. Direct measurement of the interaction of these carrier molecules with the proteins indicates that they reversibly destabilize the native state of the molecule favoring a partially unfolded conformation. Apparently these intermediate protein conformations are transport competent and are able to be absorbed through the intestinal tissue and into the bloodstream. Since the measured binding of the carriers to the partially unfolded proteins is relatively weak (Kb = 100 M(-1)) and the systemic activity of the proteins appears to be unaffected, the changes in the structure of the proteins are manifestly reversible.

Administration, Oral↗

PubMed1998

The conformational equilibrium of human growth hormone.

The structural stability of recombinant human growth hormone (rhGH) has been studied by differential scanning calorimetry, circular dichroism and by following the tyrosine and histidine chemical shifts in the 1H NMR spectrum. These studies demonstrate that the folding/unfolding equilibrium of rhGH involves a partially folded dimeric intermediate. The formation of this dimeric intermediate is a reversible process. At acid pH (pH 3) the conformational equilibrium is reversible even at high protein concentrations (10 mg/ml). At neutral pH reversibility is observed only at low protein concentrations (<0.5 mg/ml). The free energy of this intermediate conformation is only approximately 3 kcal/mol apart from the native state indicating that the conformational equilibrium can be effectively modulated by changes in solvent composition or physical conditions. According to the spectroscopic and thermodynamic results, the formation of the dimeric intermediate occurs without a major loss in helical content and is driven by the formation of substantial hydrophobic contacts between two partially folded molecules. A thermodynamic model that accounts quantitatively for the experimental data has been developed. These studies demonstrate that partially folded conformations of certain proteins are able to form stoichiometric complexes, and that the formation of these complexes provide a significant source of stabilizing Gibbs energy for conformational states that, otherwise, will be characterized by extremely unfavorable free energies.

Calorimetry, Differential Scanning↗

PubMed1994

Stability study of drug-loaded proteinoid microsphere formulations during freeze-drying.

Drug-loaded proteinoid microspheres were freeze-dried to facilitate shipping and handling and to enable long term storage. Heparin was chosen as the model drug in developing the optimum lyophilization process. The factors influencing the integrity of either heparin-loaded or unloaded ('empty') proteinoid microspheres during freeze-drying were determined, with emphasis on: selecting an optimum freezing and resuspending temperature; choosing an appropriate cryoprotectant and its optimum concentration in the formulation; and, designing a suitable method for formulating the microspheres. Freezing at/below -70 degrees C was found to minimize damage to the microspheres. Addition of sugars, such as trehalose and lactose, as cryoprotectants, further increased the stability of the heparin-loaded microspheres during freeze-drying. The optimum trehalose or lactose concentrations were determined to be 5% (w/v). Using the optimumized lyophilization process described in this manuscript, microspheres remained intact during freeze-drying. The freeze-dried microspheres were stable for at least three months post-lyophilization.

Cryoprotective Agents↗

PubMedDate not supplied

pH-dependent microspheres from modified soybean protein hydrolysate.

Soybean hydrolysate is a hydrophilic mixture of amino acids and low molecular peptides which are soluble over the whole pH range. Chemical modification of soybean hydrolysate with aromatic acyl chlorides resulted in a product that yielded pH-dependent microspheres. Investigation into the physiochemical properties of the microsphere forming material indicated that acylation had alerted the hydrophobic/hydrophilic ratio as evidenced by an increased column retention time on reverse phase HPLC. This was further confirmed by analysis of the amino acid composition of the modified material. The data indicated that the hydrophobic/hydrophilic ratio and low molecular were critical factors in the formation of this supramolecular complex. An estimation based on sedimentation rate revealed an average molecular weight of these microspheres as 10(7)-10(8) Daltons. Light scattering experiments indicated that the microspheres have discrete chambers in the interior. Included among the properties of the microspheres that have been determined are the pH range of the phase transition, size distribution and zeta-potential. The physiochemical characteristics of the microspheres prepared from modified soybean protein are similar to the microsphere forming material produced by thermal condensation of amino acids. Formation of microspheres in solution containing either porcine insulin or salmon calcitonin resulted in the encapsulation of nearly 60% of the dissolved proteins. Oral gavage of encapsulated porcine insulin or salmon calcitonin into the stomach of rats resulted in significant titers of either protein in the systematic circulation.

Amino Acids↗