Enhanced stability and therapeutic utility of proteins upon conjugation with hydrophilic polymers.

The Pharmacological utility of various exogenous proteins is considerably impeded, since upon infusion, they are liable to rapid systemic clearance, proteolysis, in addition to eliciting severe immunological reactions. Upon conjugation with hydrophilic polymers like polyethylene glycol and dextran, the altered topology of proteins, often hinders interaction with their corresponding complementary surfaces. This is manifest in their increased circulatory-lives, as a fall out of decreased binding to proteases, pre-existing antibodies and other opsonins that mediate clearance by the reticuloendothelial-system as well as receptors mediating specific organ uptake. Further, an increase in conjugate size beyond the threshold limit of 70 kDa, permits small proteins to escape clearance via renal filtration. Upon covalent modification, proteins are generally endowed with an intrinsic inertness which is reflected in their enhanced stability in extremes of pH and temperature, presence of proteases and other denaturing conditions. The enhanced intracellular stability of conjugates which may also be responsible for their altered immunological properties is likely to be a consequence of the changed physiochemical properties of the conjugates. The therapeutic efficacy of these conjugates in clinical trials indicate their tremendous utility in pharmacological procedures.

Liposomal hamycin in the control of experimental aspergillosis in mice: relative toxicity, therapeutic efficacy and tissue distribution of free and liposomal hamycin.

Therapeutic efficacy of liposomal Hamycin has been evaluated in an animal model system for aspergillosis in Balb/c mice. Hamycin was intercalated into soya phosphatidyl choline (SPC), SPC: choline (1:1, vol./vol.) and DMPC liposomes. A single dose of either 0.1 mg/kg, 0.25 mg/kg or 0.5 mg/kg of liposomal Hamycin and 0.1 mg/kg of free Hamycin was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in the survival rate of animals along with decrease in fungal count in various organs was observed with liposomal administration. Incorporation of cholesterol into liposomes decreased the in vivo toxicity of Hamycin in a dose dependent manner. However, antifungal activity both in the presence and absence of cholesterol showed marked variation as compared to that of non-aromatic polyenes, e.g. amphotericin B. Analysis of Hamycin distribution by HPLC in various tissues revealed higher blood concentration of this drug, when given in free form, compared to its liposomised form. These studies suggest that liposomal Hamycin is more effective than free Hamycin in controlling the experimental Aspergillosis.

Macrophages in host defence--an overview.

The importance of macrophages in host defence is well documented. They are distributed in various tissues where they perform functions in normal steady state as well as in diseased condition. Macrophages secrete a number of enzymes, plasma proteins, complement and coagulation factors which regulate the effector functions of the macrophages. Exposure of macrophages to pathogens results in further metabolic changes which activate the former to secrete oxygen metabolites leading to their augmented microbicidal activity. Macrophages respond to the external stimuli by expressing a large repertoire of surface receptors which play an important role in the activation, recognition and endocytosis of foreign microorganisms. A large number of intracellular pathogens are harboured in the macrophages which can reside and replicate in them. A variety of strategem has been employed to target drugs to vacuolar apparatus of the macrophages in order to combat intracellular pathogens. This review covers some of these aspects particularly in relation to hose defence and methods by which therapeutic agents could be specifically delivered to macrophages.

Tissue distribution and antileishmanial activity of liposomised Amphotericin-B in Balb/c mice§.

Antileishmanial activity and organ distribution of the antifungal drug Amphotericin-B in free and liposomised form have been studied in Balb/c mice infected with Leishmania donovani. Results indicate that Amphotericin-B in the liposomised form is significantly more active than the free form. This increase in the activity is perhaps related to the reduced drug toxicity rather than the altered drug distribution at the site of infection.

Conjugation of proteins and enzymes with hydrophilic polymers and their applications.

The efficacy of a number of therapeutically active proteins and peptides is severely limited due to their instability in circulation. Of the various approaches used to stabilise these proteins, the one more successful is covalent modification of the protein or enzyme with some hydrophilic polymers such as dextran or PEG. These conjugates are more stable than the native protein both in vitro as well as in vivo. They exhibit enhanced resistant to proteolytic degradation, have a long-life in circulation and exhibit reduced immunogenicity. The therapeutic efficacy of these conjugates is also greatly enhanced compared to the native protein or enzyme.

Polyene antibiotics in the membrane environment.

The cytotoxic activity of the polyene antibiotics mainly depends on the appearance of the drug species which arises from drug-sterol complexation. The unsaturation and intact macrolide ring of the polyenes are the requirements for the biological activity. All the polyene antibiotics can form the complex with the sterol having 3 beta-OH group, and planar ring and a hydrophobic side chain. Aromatic polyene antibiotics with positively charged head group have been considered as most potential antifungal agents.

Liposomal amphotericin-B in the control of experimental aspergillosis in mice: Part I--Relative therapeutic efficacy of free and liposomal amphotericin-B.

An animal model system for aspergillosis in BALB/c mice has been developed to evaluate the therapeutic efficacy of liposomal Amphotericin-B (Amp-B) and commercial Amp-B (Fungizone). Amp-B was intercalated into liposomes composed of egg phosphatidylcholine, phosphatidylethanolamine and cholesterol in a molar ratio of 6:1:3. A single dose (0.5 mg/kg body wt) of Amp-B both alone as well as in liposomal preparation was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in survival rate of animals and decrease in fungal count in lung, the most affected organ, were observed with liposomal formulation. Inclusion of Amp-B into liposomes also reduced the toxicity of the drug. Tissue distribution analysis of Amp-B by HPLC showed an increase in concentration of the drug in lung for both free and liposomal Amp-B in infected animals as compared to normal. Use of liposomal Amp-B increased the concentration of the drug in the disease affected organs such as lung, spleen. A longer persistence of the drug in the infected organs was also observed. The results suggest that inclusion of Amp-B in liposomes decreases its toxicity and improves therapeutic efficacy which at least in part could be due to more deposition and longer persistence of the drug in infected tissues.

Increased circulatory half-life of liposomes after conjunction with dextran.

Dextran was covalently coupled to neutral unilamellar liposomes. Dextran conjugated liposomes were cleared from the circulation at a much slower rate than unconjugated liposomes. The uptake of dextran conjugated liposomes by liver and spleen was also decreased. The amount of dextran on the surface of liposomes was found to be a determining factor for their stability in circulation. Dextran conjugated liposomes therefore may be a more effective way of controlled drug release.

Fluorescence studies on the interaction of some ligands with carcinoscorpin, the sialic acid specific lectin, from the horseshoe crab, Carcinoscorpius rotundacauda.

The binding affinities of some ligands towards the sialic acid-specific lectin carcinoscorpin, from hemolymph of the horseshoe crab Carcinoscorpius rotundacauda have been determined by protein fluorescence quenching in presence of ligands. Among the ligands studied, the disaccharide O-(N-acetylneuraminyl)-(2→6)-2-acetamido-2-deoxy-Dgalactitol has the highest Ka(l.15 × 106 M–1) for carcinoscorpin. Studies on the effect of pH on Ka values of disaccharide suggests the possible involvement of amino acid residues having pKa values around 6.0 and 9.0 in the binding activity of carcinoscorpin. There were distinct changes in the accessibility of the fluorescent tryptophan residues of carcinoscorpin by ligand-binding as checked through potassium iodide quenching.