Biologically active compounds were entrapped in cross-linked serum albumin microbeads. Injection of these drug-impregnated beads into rabbits produced no adverse immunological reactions. Sustained release (20 days) of progesterone was demonstrated in vivo.

Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5.receptor alpha beta complex and signal transduction.

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From extensive circular dichroism measurements of a variety of tetracycline (TC) derivatives in the presence and absence of a variety of chelating ions, it is concluded that 5-hydroxytetracycline possesses an identical solution conformation to that possessed by all fermentation-derived, bioactive TC species in dilute aqueous solutions at pH 7.5 or below. These conditions resemble those encountered under normal physiological circumstances. In alkaline solutions, the conformation of of 5-hydroxytetracycline diverges from the non-5-hydroxylated subclass of TC species. This divergence is rationalized as due to a buttressing effect of colinear perisubstituents at positions 4, 5, and 6 and is assisted by the possibility of hydrogen bonding between the 5 and 12a hydroxyl groups. Chelation, in alkaline solutions, with Mg ions "locks" the molecule into a conformation which has been detected by others in nuclear magnetic resonance studies in concentrated non-aqueous solvents and in X-ray studies with the 5,12a-diacetoxy analogue. Parallel studies with a variety of model TC species and Ca ions provide strong support for the hypothesis that both Ca and Mg ions bind at the BCD juncture and that, above pH 7.5, the Ca ion binds to the A ring between 4-NMe2 and 12a-OH, whereas Mg ion does not do this. If a 5-OH group is present, the Ca ion still binds as before, whereas the Mg ion will bind to the AB rings between 5-OH and 12a-OH. These factors are discussed in conjunction with the various conformations potentially available to the molecule.Earlier papers in this series (9-11) have presented evidence consistent with the concept that all of the common fermentation-derived tetracyclines (TC) possess the same solution conformation in dilute aqueous acidic media. These findings are in agreement with evidence gathered from nuclear magnetic resonance (NMR) (12, 13)and X-ray (1, 2, 4-6, 14, 15) measurements. We have proposed that under neutral conditions the TC species undergo a subtle, but potentially very meaningful, conformational alteration resulting from hydrogen bonding between the 1, 3-diaxially disposed 12a-OH and 4-NMe2 groups. This change results in considerable flattening of the A-ring chromophore.bThere is evidence in the literature, first from NMR work (12, 13) and, quite recently, from X-ray measurements (6, 15), that 5-hydroxytetracycline (OTC) and some of its derivatives may exist in another conformation under cer-I Present address: Parke-Davis and Co., Detroit, Mich. 48232. tain conditions. We have carried out extensive circular dichroism (CD) measurements and conclude that, indeed, under certain conditions, this TC subgroup does behave in a manner distinct from the TC species lacking this function. In this paper, we would like to present results which confirm this X-ray-and NMR-based hypothesis and define some of the conditions under which this conformation is preferred. MATERIALS AND METHODSThe TC derivatives used in this study are the purest samples available and were obtained through the generosity of J. M. Boo...

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The influence of cosolovents on the in-vitro percutaneous penetration of diclofenac sodium from a gel system was studied using a simplex lattice experimental design. Gel formulations were prepared by gelling the vehicle mixture of water, alcohol and propylene glycol with Carbomer 940. The synthetic membrane Durapore and hairless mouse skin were employed as barriers in a Franz-type diffusion cell. It was found that the penetration through the synthetic membrane was well described by the Higuchi model. There existed a better inverse relationship between the penetration rate and the drug solubility in the respective vehicle. It appeared to be a membrane-controlled mechanism when using hairless mouse skin as the barrier. The penetration rates in steady-state for nine formulations were fitted to a polynomial equation based on this simplex lattice method. A three-dimensional plot was constructed in this simplex surface studied. The maximal penetration rate was found to be from the vehicle containing water and ethanol in an exact volume ratio of 3:1 and the minimal penetration rate was observed from the vehicle containing water only.