Our aim was to develop a new formulation of AmB which is highly water soluble and safer than the conventional AmB-DOC formulation without decreasing its therapeutic effectiveness. This was achieved by conjugating AmB to the polysaccharide AG via an amine or imine bond. Formation of pure oxidized AG was the first step, followed by ion-exchange chromatography to remove excess oxidizing agents. This purification step was essential to avoid oxidative degradation of AmB, a highly oxidation-sensitive drug (2
), during the conjugation step. Omission of this step led to measurable decreases in the antifungal activity and the UV absorption at 405 nm.
The subsequent conjugation step was conducted at 37°C in borate buffer solution at pH 11, at which the drug has some solubility. At pH 10 or below, the yield was low, with most of the AmB powder remaining intact even after 48 h at 37°C. At pH 12, the AmB powder was solubilized within 5 min at room temperature, and the conjugation reaction was complete in 10 min. Although the resulting conjugate was as effective in vitro as AmB in antifungal testing and was not hemolytic, it was toxic to mice.
The use of reaction solutions in which AmB is soluble, such as DMSO and mixtures of DMSO with water or borate buffer solutions, did not improve the reaction yield or decrease the toxicity. It should be noted that previous reports described natural solutions or those with a pH of up to 9 for drug conjugation (17
). In an earlier study (9
), nystatin, a polyene antibiotic with a structure similar to that of AmB, was conjugated to dextran in borate buffer at pH 9 with a high yield, and it had a high degree of efficacy. The reason for the success at this low pH was probably the degradation of dextran at a pH higher than 9. In our study, no change in the MW of AG was observed, which is related to the fact that the main chain of AG does not contain vicinal hydroxyl groups and is thus less susceptible to oxidation.
The reduction step converts the imine and aldehyde bonds in the polymer conjugate to amine and alcohol groups, respectively. Reduction of the excess aldehydes eliminates further binding of the AmB-AG conjugate to proteins and body components in vivo. This reduction step did not affect the polymer’s MW or AmB content.
The antifungal activity of AmB is explained by its preferential binding to ergosterol in fungal cell membranes over cholesterol in mammalian cell membranes. Our studies indicate that covalent binding of AmB to AG does not reduce the antifungal activity of AmB in vitro (with no dependence on the reaction conditions used; Table ). However, the affinity of AmB to cholesterol is thought to be a major cause of its toxicity (4
), so that the hemolytic index of SRBCs is one of the parameters used to detect the in vitro toxicity of AmB (10
). The hemolytic indices of different conjugates synthesized under various reaction conditions were at least 2 orders of magnitude higher than those of free AmB and AmB-DOC (>1 and 0.008 mg/ml, respectively). There were no significant differences in the hemolytic indices between the various conjugates, suggesting that the conjugation itself eliminates the affinity between the SRBC cholesterol and the conjugated AmB.
In contrast to the in vitro toxicity, the pH of the coupling reaction is a major factor that affects the acute toxicity (Fig. ). An increase in the pH of the conjugation reaction from 11 to 12 produced a significant decrease in MTD (from 50 to 10 mg/kg). The chemical reduction of the conjugate also had a considerable impact on the in vivo toxicity. Reduced AmB-AG conjugate 11R was safer than unreduced conjugate 11UR (Fig. ). We hypothesize that the difference in the MTD lies in the strength of the bond between AmB and AG. Under physiological conditions free AmB is possibly released from the conjugate, which was synthesized at a pH of > 11. This in agreement with the high degree of stability of the amine bond, in contrast to the far lower degree of stability of the imine bond. The increased toxicity of the conjugate prepared at pH 12 can be explained either by formation of a physical complex of free toxic AmB molecules with the already conjugated drug molecules or by degradation of AmB into toxic products at high pH. Extraction of the conjugate prepared at pH 12 with DMSO resulted in the isolation of unbound AmB.
AmB-AG conjugate 11R was significantly less toxic than the marketed AmB-DOC formulation (MTDs, 50 and 4 mg/kg, respectively; Fig. ) and, like several lipid-based formulations, was better tolerated in the murine model even at high dosages (more than 1.0 mg/kg/day) (4
). In the mice treated with AmB-DOC, hepatocellular and tubular necroses were observed, whereas no damage was detected in these organs of mice treated with conjugate 11R, even at a much higher dosage of AmB equivalent (8 versus 1 mg/kg/day). This low level of toxicity of AmB-AG, as evinced by the low MTD and the histopathological evaluation, could be partially explained by the reduced hemolytic activities of the conjugates. In addition, there is a possibility that the polymeric matrix prevented the AmB from penetrating the liver and kidney membranes. AmB-AG conjugate 11R was chosen for further therapeutic efficacy studies due to its low level of toxicity and high degree of stability.
Conjugate 11R was as effective as AmB-DOC or AmBisome in mice treated with 1 mg/kg/day in prolonging survival and eradicating yeasts from the kidneys in the murine candidosis model (Fig. ). Moreover, it was significantly (P < 0.01) more effective than AmBisome (4 mg/kg) in reducing the counts of C. albicans in the kidneys (Fig. ). AmB-DOC was toxic at this dose (MTD, 4 mg/kg; Fig. ) and therefore was not tested. Furthermore, due to the higher doses of AmB that could be safely given with the 11R conjugate (4 to 8 mg/kg/day), it was possible to obtain total eradication of C. albicans from the kidneys.
In the murine model of cryptococcosis, the 11R conjugate was as effective as AmB-DOC at the low dosage of 1 mg/kg/day but was less effective than an equal dose of AmBisome in both prolonging the survival of the infected mice (Fig. ) and eradicating yeasts from the target organ (Fig. ). A high dosage of the 11R conjugate (4 mg/kg/day) could be safely given with a high degree of therapeutic effectiveness, which was similar to that of AmBisome. This was probably due to a good penetration through the blood-brain barrier of the conjugate at this high dosage.
The main difference between the conjugated drug and the lipid-based formulations is that the lipid-based formulations are particulate systems, which may present physical and chemical stability problems. In comparison, conjugated AmB is water soluble, is easily sterilized by filtration, and is physically and chemically stable as a lyophilized powder and as a reconstituted solution. The reagents, conjugation reaction, and method of preparation for the conjugate are simple and inexpensive compared to those for the lipid formulations.
The therapeutic activity of AmB-AG is comparable to those described for lipid formulations used in similar murine models of candidosis (5
) and cryptococcosis (5
). However, in our study the AmB-AG conjugate was significantly more effective than the liposomal formulation at equal dosages of 4 mg/kg/day in eradicating the yeast cells from the target organ in murine candidosis (Fig. ) and is as effective in eradicating the yeast cells from the target organ in murine cryptococcosis. Yet, despite the availability of the various AmB lipid formulations, a water-soluble, nontoxic dosage form of AmB remains a highly desirable alternative to the conventional treatment with AmB-DOC. We hypothesize that since AmB-AG is water soluble and differs chemically from the various lipid formulations, it is likely that its pharmaceutical properties (including tissue distribution, organ toxicity, and pharmacokinetics) will be favorably different, making it a useful alternative for the treatment of fungal infections.
The present paper provides evidence that the toxic effects of AmB can be mitigated by conjugating it to AG. In a series of in vitro and in vivo studies with mice, reduced AmB-AG conjugate 11R was found to be highly water soluble, significantly less toxic, and more effective in eradicating C. albicans and C. neoformans than AmB-DOC. This implies that it may have potential therapeutic applications. Pharmacokinetic and organ distribution studies are planned as part of the further investigation of the safety and efficacy of this formulation.