Immunotargeting of daunomycin and adriamycin against avian myeloblastosis virus infection

Abstract

Chemotherapeutic agents have been widely used as an alternative to surgery in the treatment of cancer. An ideal chemotherapeutic agent should be selectively toxic to cancer cells or to infectious viral agents. Most known anticancer agents interfere with the metabolism of the host, resulting in toxic effects. The anthracycline antibiotics daunomycin and adriamycin have been shown to be highly active against several clinical and experimental tumours. However, they are highly toxic when injected into the host. Therefore, it is essential to design strategies to reduce the toxicity of drugs on normal cells. It has been suggested that sitespecific delivery of drugs would solve the problem of nonspecific cytotoxicity. The idea of “drug targeting” to specific areas where an infectious agent multiplies was proposed in 1898 by Dr. Paul Ehrlich. Various targeting strategies have been extensively investigated during the past decade. In the present study, an attempt was made to explore the possibility of targeting antiviral agents against AMV infection in chicks. The anthracycline antibiotics daunomycin and adriamycin were used as antiviral agents. The thesis consists of six chapters.

Chapter I General introduction to the experimental system and an overview of current concepts in cancer chemotherapy.

Chapter II Effects of daunomycin and adriamycin on AMV reverse transcriptase and host DNA polymerases.

At 2 g concentration, both antibiotics inhibited 70% of reverse transcriptase activity. Poly(dA)·(dT)-dependent enzyme activity was inhibited slightly more than poly(rA)·(dT)dependent activity. The drugs inhibited the enzyme competitively with respect to the template. Significant inhibition was observed when AMV genomic RNA was used as a template. Spectroscopic and sucrosegradient analyses indicated drug binding to genomic RNA. AMV reverse transcriptase-associated RNase H and DNA endonuclease activities, as well as host DNA polymerases , and , were significantly inhibited at drug concentrations that inhibited viral polymerase activity.

These results highlighted the nonselective nature of inhibition, indicating the need for targeted delivery.

Chapter III Isolation of AMV envelope glycoprotein gp80, antibody production, and characterization.

Antibodies specifically recognized the virus and a membranebound antigen in virustransformed cells. Antibody was coupled to daunomycin and adriamycin using a dextran bridge. Conjugates contained 40-55 drug molecules per IgG molecule. Conjugates retained 70% of original antibody activity (ELISA and radiolabelled antibody binding assays).

Chapter IV Liposomes as an alternative drugdelivery strategy.

Antibody was palmitoylated to incorporate it into liposomal bilayers.

Palmitoylated antibody: (i) required detergent to remain soluble, (ii) contained ~5 palmitoyl chains per IgG, (iii) showed modification mainly in heavy chains, (iv) retained ~50% biological activity.

Palmitoylated antibody was incorporated into liposomes (detergent dialysis).

Antibody presence was confirmed by fluorescence spectroscopy.

Protease digestion indicated that 50% of antibody molecules projected outward.

Average: 15 antibody molecules per 300 Å liposome.

Integrity evaluated by leakage of encapsulated [³H]adriamycin (25% leakage in 72 h).

Immunoliposomes bound virus and virustransformed myeloblasts significantly better than free liposomes or nonspecific immunoliposomes.

Chapter V Uptake mechanism and biological effects.

Immunoliposome uptake occurred via endocytosis (temperature and metabolismdependent).

Encapsulated drugs were not taken up by nontarget cells and did not inhibit RNA synthesis in them.

Immunoliposomes delivered more drug to target cells and inhibited RNA synthesis more effectively than:

free drug, liposomal drug, drug-IgG conjugates.

Ammonium chloride inhibited drug action, indicating lysosomal involvement.

Immunoliposomal drugs inhibited focus formation and colony formation in virustransformed myeloblasts.

Chapter VI Pharmacokinetics and in vivo antiviral activity.

Free drugs were toxic at 28 mg/kg. Free drugs and drug-IgG conjugates were rapidly cleared from plasma and taken up by the reticuloendothelial system. Cardiac drug levels decreased when drugs were targeted via antibody or liposomes. Immunoliposomes produced a 9.5fold increase in drug levels in bone marrow (target tissue). Drug-IgG conjugates showed little improvement in targeting. Targeted drugs inactivated virus better than free drugs when preincubated with virus. Drugs in immunoliposomes provided better protection than free drugs or liposomal drugs when administered 24 h postinfection, but not at 48 h postinfection. SandwichELISA showed no detectable antibody response against immunoliposomes in chicks.

Summary of Findings

Daunomycin and adriamycin inhibit AMV reverse transcriptase and host DNA polymerases. Antigp80 antibody was raised; drug-IgG conjugates retained ~70% activity with 40-55 drugs per IgG. Immunoliposomes were prepared; 50% of incorporated antibodies were exposed externally and showed enhanced binding to virus and transformed cells. Immunoliposomes were internalized by endocytosis and delivered drugs more efficiently to target cells. Targeted immunoliposomal drugs reached bone marrow efficiently and offered better antiviral protection than free drugs, with no immune response to immunoliposomes.