Research paper
Reduced hepatic toxicity, enhanced cellular uptake and altered pharmacokinetics of stavudine loaded galactosylated liposomes

https://doi.org/10.1016/j.ejpb.2006.12.019Get rights and content

Abstract

The aim of the present investigation was to reduce the hepatic toxicity, enhance the cellular uptake and alter the pharmacokinetics of stavudine using galactosylated liposomes. β-d-1-Thiogalactopyranoside residues were covalently coupled with dimyristoyl phosphatidylethanolamine, which was then used to form liposomes. The galactosylated liposomal system was assessed for in vitro ligand-specific activity. The drug release from liposomes was studied by dialysis method. Ex vivo cellular uptake study was performed using liver parenchymal cells harvested from male albino rats. Changes in hematological parameters, hepatic enzymes, hepatomegaly, plasma and tissue distribution of the formulations (free stavudine solution, uncoated liposomal and galactosylated liposomes) were determined using albino rats. Percent cumulative drug release in 24 h was low (34.8 ± 2.6%). Enhanced hepatic cellular d4T uptake (27.96 ± 2.41 pg d4T/million cells) was seen in case of galactosylated liposomal d4T. Galactosylated liposomes maintained a significant level of d4T in tissues rich in galactose specific receptors and had a prolonged residence (11.44 ± 1.25 h) in the body resulting in enhanced half-life of d4T (23.07 ± 1.25 h). This formulation did not show either hematological or hepatic toxicity. Galactosylation of liposomes alter the biodistribution of encapsulated drug thereby delivering the drug to cells bearing galactose specific receptors.

Introduction

Targeted drug delivery involves the design and synthesis of carriers displaying ligands that mediate the binding of a drug/carrier complex to a receptor. Subsequent internalisation of the carrier/drug complex leads to accumulation of the drug in the target cells and exclusion from non-target cells that lack the requisite receptor. Ligand coupled liposomes represents a promising approach for improving the selective targeting of drugs to diseased tissues in vivo, leading to reduction in drug toxicity and improved therapeutic outcomes [1], [2]. Cells of macrophage lineage represent a key target of human immunodeficiency virus (HIV) in addition to CD4-lymphocytes. Macrophages possess various receptors such as Fc receptors, mannosyl, galactosyl and many other receptors [3]. Asialoglycoprotein receptor specific for galactose is present on parenchymal cells of hepatocytes. Galactosylated liposomes are selectively removed from circulation by Kupffer cells. Kupffer cells carry on the cell surface a lectin-like receptor with specificity for d-galactose residues [4]. These galactose receptor-bearing cells are the reservoirs of HIV-I. The liposome approach has been used successfully in the treatment of specific diseases in vivo to enhance drug targeting to the cells [5], [6]. Liposomal entrapment is particularly valuable for anti-HIV drugs because liposomes accumulate in the reticulo-endothelial tissues in which HIV infection occurs [7]. Uptake of ligand-incorporated liposomes is significantly higher than liposomes without ligands [8], [9], [10], [11]. The mechanism of sugar recognition that specific cell types possess is well known. The relative hydrophilic glycolipids may reduce immunogenicity; they can be removed from liposomes simply via interaction with plasma lipoproteins or lipids in tissue, resulting in a reduction in cell selectivity after intravenous injection [12], [13]. It is, therefore, desirable to develop superior glycosylating agent that can be securely attached to the liposomal membrane, even under in vivo conditions.

Stavudine (d4T) is one of the important drugs, belonging to the class of reverse transcriptase inhibitors approved by the FDA for the treatment of AIDS. d4T has greater bioavailability (88–99%). However, long-term administration of stavudine over a period of 6 months results in adverse side effects such as a dose limiting peripheral neuropathy, anemia, hypersensitivity, insomnia and malaise. Lactic acidosis, hepatitis or liver failure has been reported with d4T. A liposomal drug delivery system may be ideal in the case of stavudine, as it may alleviate drug toxicity and also deliver the drug directly to the macrophages in a passive manner. Such a reduction in toxicity has been demonstrated in the case of drugs like Doxorubicin and Amphotericin B [14]. This could increase the time-interval between each drug administration during therapy as well as dose of the drug, consequently improving the quality of life of patients with AIDS.

Section snippets

Chemicals

Stavudine was received as a gift sample from M/s Hetero Drugs, Hyderabad, India. Egg phosphatidylcholine (PC), cholesterol (CH), dimyristoyl phosphatidylethanolamine (DMPE), β-d-galactose, Sephadex G-50, Ricinus communis lectin, Dulbecco’s modified Eagle’s medium high glucose (DME-HG), l-glutamine, penicillin, streptomycin, heat-inactivated fetal bovine serum, human AB serum, lactate dehydrogenase diagnostic kit, aspartate aminotransferase diagnostic kit, alanine aminotransferase diagnostic kit

Preparation and characterization of liposomes

Galactosylated dimyristoyl phosphatidylethanolamine was characterized by IR spectrum. O–H stretch (intense broad band) and strong C–O stretch of carbohydrate were obtained around 3350.2 and 1049.1 cm−1, respectively. A characteristic C–H stretching and bending vibration was observed at 2855.3 cm−1 and 1470.8 cm−1, respectively. Strong peaks at 1705.5 cm−1 and 1205.4 cm−1 confirmed the carbonyl (Cdouble bondO) and C–O groups of ester present in dimyristoyl phosphatidylethanolamine, respectively. Weak N–H

Discussion

The larger entrapment efficiency of formulation and lesser particle size of formulation GL4 may be ascribed to the optimized ratio of phosphatidylcholine and cholesterol used in this formulation. The reduced entrapment efficiency and particle size of formulation GL1 may be due to less rigid, leaky and fused vesicles. The larger particle size and lesser entrapment efficiency of formulation GL5 to GL9 may be due to the rigidization of vesicles because of larger cholesterol content. The higher

Conclusion

Conclusively, galactosylation of dimyristoyl phosphatidylethanolamine decreases its more positive charge to lesser positive charge. Galactosylated liposomes alters the biodistribution of encapsulated drug thereby delivering the drug to cells bearing galactose specific receptors. Many of the HIV-I infected cells have these receptors. Liposomes sustain considerable interest to develop ways to fabricate drug delivery systems that would provide a good release without inducing any systemic reactions

References (29)

  • P.M. Girard et al.

    Phase II study of liposomal encapsulated daunorubicin in the treatment of AIDS-associated mucocutaneous Kaposi’s sarcoma

    AIDS

    (1996)
  • Y. Aramaki et al.

    Activation of Fc receptor mediated phagocytosis by mouse peritoneal macrophages following the intraperitoneal administration of liposomes

    Pharm. Res.

    (1994)
  • M. Dutta et al.

    Neogalactosylated liposomes as efficient ligand for the evaluation of specific sugar receptors on macrophages in health and in experimental leishmaniasis

    Parasitology

    (1994)
  • L.A.J.M. Slidregt et al.

    Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor

    J. Med. Chem.

    (1999)
  • Cited by (59)

    View all citing articles on Scopus
    View full text