Biological properties of adriamycin bound to biodegradable polymeric carriers

Three different conjugates having adriamycin (ADR) bound to the side chain carboxyl groups of high-molecular weight poly (¿--glutamic acid) (PGA) either directly or by interpolation of GlyGly and GlyGlyGlyLeu spacers, respectively, were compared with respect to immunogenicity and cytotoxicity in mice as well as release of drug by lysosomal enzymes. The cytotoxic efficacy of a single i.p. dose of each conjugate (5 mg ADR-equiv./kg) against L1210 leukemia cells implanted i.p. in DBA2 mice was studied by monitoring the survival time, the body weight and the number of long-term survivors (LTS). PGA-GlyGlyGlyLeu-ADR and PGA-GlyGly-ADR significantly enhanced the mean survival time (MST) of treated animals compared with the untreated control group (T/C 148¿149%) as did free ADR (T/C 147%). The tetrapeptide-spacer containing conjugate effected the presence of LTS at day 50 (2/5) as did free ADR (1/5).\ud \ud The secondary antibody response of the drug conjugates elicited in A/J mice after repeated dosage (125 ¿g/mouse) at day 0, 14 and 28 was evaluated at day 35 using the ELISA technique. IgG titers varied from a very low value (PGA-GlyGlyGlyLeu-ADR) to moderately high levels (PGA-ADR, PGA-GlyGly-ADR) which are 2¿3 orders of magnitude below that obtained for the strong immunogen bovine IgG. The data suggest that certain parts on the surface of the conjugates are immunogenic.\ud \ud The release of extractable low-molecular weight products from the conjugates mediated by lysosomal enzymes was analyzed using reversed-phase HPLC. The release profile of ADR as well as Gly-ADR, Leu-ADR or GlyLeu-ADR was determined. The total amount of ADR released after 77 h was 3.6% for PGA-GlyGlyGly-Leu-ADR, 1.0% for PGA-GlyGly-ADR and 0.5% for PGA-ADR. With all conjugates unidentified products were produced.\ud \ud It is proposed that the mechanism of action of the polymeric conjugates under in vivo conditions may be due to pinocytic capture followed by lysosomal degradation with release of ADR

Biodegradable polymeric prodrugs of naltrexone

The development of a biodegradable polymeric drug delivery system for the narcotic antagonist naltrexone may improve patient compliance in the treatment of opiate addiction. Random copolymers consisting of the ¿-amino acids N5-(3-hydroxypropyl--glutamine and -leucine were synthesized with equimolar initial monomer feeds. The molecular weight of this chemical carrier was determined by viscometry and wide-angle light scattering. In order to get selective covalent coupling of drug to polymer the 3-acetate derivative and the 14-acetate derivative of naltrexone were synthesized and characterized by NMR. Hydrolytic conversion of each monoacetate to parent drug was monitored by HPLC and the rate constant was determined. Both derivatives were coupled via hydrolytically labile carbonate linkages to the polymer hydroxyl groups. The drug conjugates were prepared as particles of various size ranges between 20 and 350 ¿. In vitro studies in phosphate-buffered saline (pH 7.4) demonstrated a release rate dependence on particle size. Nearly constant plasma levels of naltrexone were obtained for one month after subcutaneous injection in rats

The synthesis and characterization of polypeptide-adriamycin conjugates and its complexes with adriamycin. Part I

Poly(α-l-glutamic acid) (PGA) was grafted with amino acid and oligopeptide spacers up to 5 amino acids with the use of N,N'-carbonyldiimidazole and 2,3-dihydro-1,2-benz-isothiazole-3-on-1, 1-dioxide (saccharin) as an additive, and these polypeptides were characterized. The antitumor antibiotic adriamycin was covalently coupled via an amide bond onto PGA and onto the grafted polymers with the use of N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ); these conjugates were characterized. The conjugates containing Gly—Gly—l-Leu spacer arms did yield free adriamycin upon digestion with papain. Adriamycin gave fairly stable complexes with PGA—adriamycin and branched poly peptide—adriamycin conjugates; these complexes were characterized

Optimization of macromolecular prodrugs of the antitumor antibiotic adriamycin

In our earlier work [10] on aminoribosyl-bound prodrugs of adriamycin (ADR) using poly(α-l-glutamic acid) (PGA) grafted in high yield (90–100 mol.%) with various peptide spacers as a plasma-soluble macromolecular carrier we observed rather low cytotoxic activities in L1210 leukemia and B16 melanoma in vitro assays. These results may be tentatively explained by a decreased susceptibility of the spacer-bound adriamycin moiety to hydrolysis by lysosomal enzymes due to the high spacer load. This hypothesis was tested by the study of two conjugates prepared by a different route. Peptide conjugates of adriamycin (Gly-Gly-Leu—ADR and Gly-Gly-Gly-Leu—ADR) were synthesized using the trityl N-protecting group and were coupled to PGA in 4.5 mol.% load according to the method described earlier [11]. However, these conjugates were almost totally devoid of cell growth-inhibiting activity in L1210 and B16 in vitro tests. The data suggest that either the uptake of the polymeric prodrugs into the cell by pinocytosis is highly dependent on spacer load or molecular weight, or that lysosomal digestion is too slow for efficient release of ADR. Possibly, enzymatic degradation of PGA which is known to occur only between pH 4 and 6 is rate-limiting for release of the drug. Current studies include the enzymatic degradation of PGA—peptide spacer—drug systems using p-nitroaniline as a model drug and papain as the enzyme. By variation of the length and load of spacer it can be estimated under which conditions the release of drug (using UV spectrometry) is faster than degradation of the polymer (as determined by viscometry). In addition, the uptake of PGA and derivatives with a fluorescent label into tumor cells is studied using laser flow cytometry and laser microscopy

Synthesis and biodistribution of immunoconjugates of a human IgM and polymeric drug carriers

The synthesis and purification of radiolabelled immunoconjugates, composed of a human IgM monoclonal antibody directed against an intracellular tumour-associated antigen and either poly (alpha-L-glutamic acid) (PGA) or poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG) is described. Coupling of polymers to the antibody was performed through disulfide bond formation involving a single thiol group at the C-terminus of the polymer chain and 2-pyridyldisulfide groups introduced onto the antibody. The antibody was iodinated with 131I before conjugation. The polymers contained tyrosinamide in a low degree of substitution and were radiolabelled with 125I. 125I-labelled PGA and PHEG were found to be stable for at least 3 days in murine and human plasma. The biodistribution in mice of the doubly labelled immunoconjugates was studied and was compared with the pharmacokinetics of the individual components.\ud \ud PHEG showed a relatively slow blood clearance, the half-life being approximately 10 h with low uptake in liver, kidneys and spleen. PGA was rapidly cleared from the circulation and was significantly taken up in liver, kidneys and spleen. The biodistribution of both immunoconjugates was indistinguishable from that of the IgM proper, with plasma half-lives of approximately 6 h, indicating that the pharmacokinetic properties of the immunoconjugates are largely determined by the antibody part

Synthesis and biological evaluation of immunoconjugates of adriamycin and a human IgM linked by poly[N5-(2-hydroxyethyl)-l-glutamine

The synthesis and purification of radiolabelled immunoconjugates, composed of a human IgM monoclonal antibody (IgM 16.88) directed against an intracellular tumour-associated antigen, the drug carrier poly[N5-(2-hydroxyethyl)--glutamine] (PHEG) and the cytostatic drug adriamycin (ADR) are described. The immunoconjugates were constructed to allow selective release of ADR in the putatively acidic environment of the tumour through a novel acid-labile maleamic acid linker. The conjugate of PHEG and the acid-labile ADR derivative effectively released ADR in cytotoxic amounts at a pH of 6.0 as judged from incubation in buffer and from inhibition of the growth of HT-29 colon tumour cells in vitro. Immunoconjugates were prepared by coupling of PHEG-ADR having a hydrolytically stable amide bond with 131I-labelled antibody through thioether bond formation involving a single thiol group at the C-terminus of the polymer chain and maleimido groups introduced onto th