Biorecognition of HPMA copolymer-adriamycin conjugates by lymphocytes mediated by synthetic receptor binding epitopes.

PURPOSE: The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. METHODS: HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitt's lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC50 doses determined. RESULTS: Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. CONCLUSIONS: The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.

Ethanol: water mutually enhanced transdermal therapeutic system II: skin permeation of ethanol and nitroglycerin.

An optimal concentration range of aqueous ethanol produces 5-10-fold increases in nitroglycerin flux across skin and ethanol skin permeation that are far greater than reported previously. For aqueous ethanol solutions saturated with nitroglycerin with an ethanol volume fraction less than or equal to 0.7, the flux of nitroglycerin across skin is linear with the ethanol flux and is traced to a linear solubility relationship and a constant diffusion coefficient.

Ethanol:water mutually enhanced transdermal therapeutic system. I: Nitroglycerin solution properties and membrane transport.

The solution properties of aqueous ethanol donor solutions were characterized for the particular case of an increased flux nitroglycerin transdermal system. Permeation through porous and nonporous polymer membranes was investigated and modelled. While the permeation of ethanol through the porous membranes is adequately described by theory, clogging of pores occurs in the presence of lactose. Permeation through ethylene vinyl acetate membranes reflects interactions of the solute and solvent with the polymer.

Interaction of antithrombin III with preadsorbed albumin-heparin conjugates.

The adsorption of antithrombin III (AT III) onto polystyrene surfaces preadsorbed with albumin or albumin-heparin conjugates was studied using a two step enzyme immuno assay. When AT III-buffer solutions were used, the highest adsorption values were measured on high affinity albumin-heparin conjugate pretreated surfaces. Less AT III adsorption was found on nonfractionated albumin-heparin conjugate preadsorbed surfaces. AT III adsorption could also be detected on low affinity conjugate and albumin coated surfaces. When AT III was adsorbed from plasma or plasma dilutions with buffer, only AT III on surfaces preadsorbed with high affinity or nonfractionated albumin-heparin conjugate was found. These results demonstrate that the heparin moiety of the conjugate is directed to the solution phase whereas the albumin moiety contacts the polystyrene surface.

Prevention of insulin self-association and surface adsorption.

The self-association of insulin monomers into oligomers and macromolecular aggregates leads to complications in the administration of insulin, both in conventional administration and in the development of long-term insulin delivery systems. These problems are aggravated by the tendency of insulin to adsorb onto the surface of solution containers and infusion devices. Furthermore, with insulin infusion devices, shear rates can be generated which can accelerate the self-association and surface adsorption processes. The effects of urea on shear-induced insulin self-association and surface adsorption were investigated. It was found that the addition of a certain concentration range of urea to insulin solutions greatly reduces both insulin self-association and surface adsorption. Circular dichroic studies established that these concentrations of urea also preserve insulin conformation under high shear rates, where conformations are altered without urea. Higher urea concentrations lead to insulin denaturation and accelerated self-association.

Covalently bound conjugates of albumin and heparin: synthesis, fractionation and characterization.

Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10-16 weight % of heparin) were obtained by a condensation reaction between albumin and heparin using 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Unreacted albumin and heparin were removed by diethyl-aminoethyl (DEAE)-cellulose and Cibacron Blue Sepharose chromatography respectively. The activity of the heparin component incorporated in the albumin-heparin conjugates (Ac) was compared with that of the heparin used for the synthesis of the conjugates (Anat) by thrombin time, inhibition of Factor Xa and the activated partial thromboplastin time (APTT) assays. The Ac/Anat ratio for the above assays was as follows: Thrombin time 1.25, Factor Xa inhibition 0.5. and APTT 0.5. Gel filtration chromatography showed broad-molecular weight distributions. The conjugates were fractionated using immobilized antithrombin III (ATIII). High ATIII and low ATIII affinity conjugate fractions showed the same behavior as ATIII fractionated heparin with respect to thrombin times and Factor Xa inhibition.

The antiplatelet activity of immobilized prostacyclin.

Owing to the chemical instability of prostacyclin, the direct immobilization of this prostaglandin has not been successful. A new procedure is described for the preparation of immobilized prostacyclin based on the conversion of immobilized prostaglandin F2 alpha to immobilized prostaglandin I2-Materials thus prepared show dramatic antiplatelet effects with regard to platelet aggregation and platelet adhesion. Radioimmunoassays of plasmas used in in vitro platelet tests and of buffers used in prostacyclin leakage studies established that these effects are not due to the release of prostacyclin from the respective immobilization substrates.

Immobilized heparin: spacer arm effects on biological interactions.

Heparin immobilized to polymer surfaces via different length diaminoalkane spacer arms was evaluated for anticoagulant activity and for platelet interactions. The anticoagulant activity of the immobilized heparin, as determined by APTT assays, was found to increase with increasing spacer arm length. Variations in spacer arm length produced no affect on platelet retention or PF 4 release for heparin immobilized materials. To investigate immobilized heparin-adsorbed plasma protein interactions, XPS analysis of heparinized surfaces, before and after plasma contact, was conducted. Immobilized heparin was not able to penetrate adsorbed plasma protein layers with any spacer arm length evaluated, indicating that immobilized heparin does not directly interact with platelets.

Prostaglandin releasing polymers - stability and efficacy.

In summary, PGI2 is approximately 3 orders of magnitude more potent than PGE1 in the prevention of platelet aggregation. Similarly PGE1 is an order of magnitude more potent than PGD2 (ID50's = 5.3 x 10(-10) M, 1.3 x 10(-7) M and 1.4 x 10(-6) M for PGI2, PGE1 and PGD2 respectively). Results from biological and infrared stability studies demonstrate that both PGI2 and PGE1 are stable for extended periods of time when dispersed within hydrophobic polymer matrices, an important consideration in the design of nonthrombogenic, prostaglandin controlled release polymers. Finally, both PGE1 and PGI2 controlled release polymers inhibit platelet aggregation in contacting blood, of which PGE1 produced greater platelet aggregation inhibition (90% inhibition) than did PGI2 (75% inhibition). However, PGE1 controlled release polymers significantly reduced platelet adhesion (11.25 +/- 3.68 platelets/mm2) compared to control polymers (50.65 +/- 8.8 platelets/mm2) while PGI2 controlled release polymers demonstrated no improvement in platelet adhesion (25.00 +/- 18.61 platelets/mm2) relative to control polymers (30.43 +/- 7.62 platelets/mm2). One cannot conclude that the lack of reduced platelet adhesion on the PGI2 controlled release surfaces is due to the instability of PGI2. The fact that significant inhibition of platelet aggregation in both blood fractions which contacted the PGI2 controlled release surfaces occurred substantiates that the released PGI2 was active. It must be concluded that PGI2 does not affect platelet adhesion. It is interesting to note that less platelets adhered to the PGI2 control PVC surfaces than on the PGE1 control PVC surfaces. The PGI2 control PVC surfaces were equilibrated with a TRIZMA buffer in 5% dextrose (to provide isotonicity) while the PGE1 control PVC surfaces were equilibrated in isotonic phosphate buffer saline.