Abstract
ABELCET (ABLC) is a widely used amphotericin B lipid complex formulation that is approved for use in the treatment of invasive fungal infections in patients who are refractory or intolerant of conventional amphotericin B (AmB). The safety profile of ABLC has been characterized in two acute and two repeat-dose toxicity studies in rats. The acute toxicity studies indicated that single intravenous doses of ABLC are at least 20 times less toxic than conventional amphotericin B doses without the lipid formulation, Fungizone. Intravenous doses of 0, 1, 3, or 10 mg/kg/day to groups of rats (10 to 15 rats/sex/group) for 31 days elicited no mortality or overt clinical signs of toxicity, whereas alternate intravenous/intraperitoneal doses (three each per week) for 6 months, produced one death in the control group, one in the intermediate-dose group, and two in the high-dose group. Clinical signs (predominantly piloerection and hunched posture at 10 mg/kg/day) were attributed to granulomatous inflammatory lesions in the abdominal wall, mesentery, and omentum, which were produced by the intraperitoneal injections of ABLC. Feed consumption and body weight gains decreased in high-dose male rats in the one-month study and were significantly lower in male rats at 3 and 10 mg/kg/day in the 6-month study. In contrast, water consumption increased in male and female rats in both studies. Trends of minimal to moderate, dose-related increases in relative kidney, liver and spleen weights, and histological evidence of hypertrophy and hyperplasia of reticuloendothelial cells in the liver and spleen and mild, dose-related impairment of renal function occurred in both the 1- and 6-month studies. Examination of high-dose rats following a recovery period of 28 days after completion of 31 days of dosing suggested that treatment-related changes were reversible. The observed changes for ABLC are similar to those for other amphotericin B lipid formulations, such as AmBisome (LAmB), except for the hepatoxicity, which was observed for LAmB, but not for ABLC.
Amphotericin B (AmB) is a potent, polyene, broad-spectrum antifungal antibiotic derived from a strain of Streptomyces nodosus. Due to its strong affinity for ergosterol in fungal cell membranes, it disrupts the membranes, causes cell death, and therefore is used as a highly effective topical and intravenous treatment of disseminated fungal infections in animals and man. Highly lipophilic and water insoluble, AmB is conventionally formulated with sodium deoxycholate (AmBd; Fungizone), a formulation that is efficacious but restrictive because of a number of side effects, including nephrotoxicity (Bennett 1996). To alleviate the toxicity, amphotericin B lipid complex (ABLC; ABELCET) was developed and in 1995 became the first lipid-formulated AmB product to be approved by the U.S. Food and Drug Administration (Wong-Beringer, Jacobs, and Guglielmo 1998). This formulation provides amphotericin B complexed with two phospholipids in a 1:1 drug-to-lipid molar ratio. The two phospholipids,
This report presents previously unpublished acute and repeat-dose toxicity studies in rats and compares the results to other AmB products on the market. This information is deemed to be important in understanding the toxicity profile of AmB when it is encapsulated/complexed with phospholipids because these formulations exhibit nonlinear pharmacokinetics that may affect the functional properties of the drug product.
All studies, except the initial non-GLP LD50 study, were conducted in compliance with the Good Laboratory Practice (GLP) regulations of the Food and Drug Administration (U.S. FDA 1987).
MATERIALS AND METHODS
Materials
ABLC obtained from Squibb Institute for Medical Research, New Brunswick, NJ, and formulated by The Liposome Co., Princeton, NJ (5 mg amphotericin B/ml in all studies except 6 mg/ml in the acute non-GLP study), and AmBd (Amphotericin B for Injection USP, 50 mg/ml, Squibb Control 7J97300) were stored under refrigeration (2°C to 8°C) and protected from light. Appropriate concentrations of AmBd were made for injection into rats by reconstituting AmBd with 5% dextrose, USP, to a concentration of 0.5 mg/ml. Both formulations had been tested for strength, purity, composition, and stability prior to use and found to conform to specifications. Physiological saline, USP, was used as the control article in the repeat-dose studies.
Animals
Male and female Sprague-Dawley (SD)BR outbred albino rats (Sprague Dawley, Frederick, MD), approximately 5 weeks of age, were used for the non-GLP acute study. Crl:CD(SD)BR outbred albino rats (Charles River Laboratories Wiga GmbH, Sulzfeld, Germany) were used in all other studies (age: 5 weeks in GLP acute study and 6 to 7 weeks for all other studies).
The rats were housed by sex in groups of 5 in stainless-steel cages in the non-GLP acute study. In the second GLP acute study, the rats were acclimated for a period of at least 5 days, then randomized based on body weight and housed individually in Makrolon Type III cages on SAWI Research bedding (Charles River Laboratories Wiga GmbH). Animal rooms were maintained at a target temperature of 22°C, a relative humidity of approximately 60%, with a 12-h light/dark cycle.
For the repeat-dose studies, the rats were acclimated for at least 3 weeks before being assigned to the studies. In the 1-month study, rats were individually housed in Makrolon Type II cages, and in the 6-month study, rats were housed in Makrolon Type IV cages, separated by sex in groups of 5/cage. All cage sizes, housing conditions and animal husbandry procedures were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996). Feed (Ralston Purina Certified Rodent Chow 5002) was available ad libitum in the non-GLP acute study. Altromin bedding, Certified Rodent Chow 1324 (Altromin GmbH, Lage/Lippe, Germany) were available ad libitum in the other studies. Tap water was available ad libitum in all studies.
Procedures
Acute Toxicity Studies (Table 1)
Non-GLP Single-Dose Toxicity Study: Groups of 5 rats/sex/dose received single intravenous (i.v.) doses of 1.25, 2.5, or 5 mg/kg of AmBd (2.5, 5, and 10 ml/kg). Rats were observed daily for 14 days for signs of toxicity and/or mortality. No gross or microscopic pathology evaluations were performed. Groups of 10 male rats then were administered single i.v. doses of 15, 30, 60, 84, or 120 mg/kg of ABLC (2.5, 5, 10, 14, and 20 ml/kg), whereas groups of 10 female rats received 15, 30, 42, 60, or 120 mg/kg of ABLC (2.5, 5, 7, 10, and 20 ml/kg). Different dose levels were used in males and females because of apparent gender differences with AmBd.
GLP Single-Dose Toxicity Study: In the GLP acute study (5 rats/group/sex), rats were administered single i.v. doses of 20, 35, or 50 mg/kg ABLC, then observed daily, sacrificed 14 days after dosing, and gross necropsy examination and histopathology of gross lesions were conducted.
Repeat-Dose Toxicity Studies (Table 1)
One-Month Study: ABLC was administered i.v., 7 days/week for 1 month at doses of 0 (saline) 1, 3, or 10 mg/kg/day (2.0, 0.2, 0.6, or 2.0 ml/kg). A fifth group, treated with 10 mg/kg of ABLC from an earlier test article batch (which was produced at a different facility and used in the 6-month parenteral study), served as a batch comparison group. The 31-day dosing period was followed by a 28-day recovery period. The control- and one of the two high-dose groups were composed of 15 rats/sex/group, 5 rats/sex/group being sacrificed at the conclusion of the recovery period. The remaining groups contained 10 rats/sex/group, all of which were sacrificed at the conclusion of the 31-day dosing period. The investigative parameters included mortality, clinical signs, ophthalmology, feed, water, and body weight changes, clinical pathology parameters, organ weights, gross pathology, and histology of selected tissues from all animals and determination of AmB concentrations of various tissues (blood, brain, kidneys, liver, lungs, and spleen) from 5 rats/sex/group after dose termination and from 5 high-dose rats at the end of the recovery period.
Six-Month Toxicity Study: A chronic 6-month parenteral study was conducted in which alternating i.v. and intrapertoneal (i.p.) doses of 0 (saline), 1, 3, and 10 mg/kg/day were administered on 6 days per week to groups of 15 rats/sex/group. The alternating dosing regimen was considered necessary because i.v. dosing for 6 months was presumed to be impractical due to chronic vascular irritation to tail veins (noted in the pilot and one-month studies). After 3 months of treatment, 5 rats/sex/group (four from female controls) were sacrificed for interim pathological evaluation. Criteria for evaluation included survival, clinical signs, feed, water, and body weight changes, ophthalmoscopy, clinical pathology parameters, organ weights, and gross and histologic evaluation of tissues.
Statistical Analyses
Results of general observations and clinical laboratory tests in treated animals were compared to those in concurrent control animals. Log-transformed values from body weight determinations and clinical laboratory tests and means of the absolute and relative organ weights were compared by Dunnett’s test (Dunnett 1964). The incidences of significant lesions were compared by the Fisher Exact Test (Siegel 1956).
RESULTS
Single-Dose Studies
Mortality rates for the two acute studies are tabulated in Table 2. Results from the first study indicated that ABLC was much less toxic than AmBd. The minimal lethal dose for ABLC was 60 mg/kg (2/10 male and 9/10 female deaths). The LD50 values for male and female rats were estimated to be 68 and 51 mg/kg, respectively. The maximum no-overt-effect dose was found to be 15 mg/kg. In contrast, the minimum lethal dose for AmBd was 2.5 mg/kg (2/5 male and 4/5 female deaths) and the respective LD50 values for male and female rats were estimated to be 3 and 2 mg/kg, respectively.
The number and severity of overt clinical sign of toxicity were reduced with ABLC, as compared with AmBd. Reduced motor activity and respiratory rate, the major overt signs of ABLC toxicity, occurred at 30 mg/kg in both sexes and progressed in severity and duration (from 30 min to 48 h) as the doses increased from 30 to 120 mg/kg. At these doses, transient weight loss was noted on the second day post dose, and all deaths occurred between 6 and 48 h after treatment. In contrast, AmBd toxic signs included ataxia, slower respiratory rates, convulsions, terminal collapse, and all deaths occurred within 30 min post dose.
In the second single-dose GLP study, no deaths and no overt toxic signs occurred at ABLC doses of 20 and 35 mg/kg. At 50 mg/kg, one male (1/5) and one female (1/5) rat died within 33 min post dose. Prior to death, decreased activity and muscle tone, dyspnea, slowed respiration, clonic convulsions, and prostration were observed. Decreased activity, tremors, pale skin color, increased urination, and transient weight loss were noted in surviving high-dose rats. Gross necropsy changes were observed in only one high-dose female rat sacrificed on study day 15 (terminal necropsy). The changes included bilateral, multifocal, irregular, and white discolorations of the kidneys. Microscopically, this finding correlated with moderate segmental fibrosis and edema confined to the kidney cortex and outer medulla and was associated with tubular dilatation and mineralization, atrophy and degeneration/regeneration of tubular epithelium. Clinical pathology parameters were not investigated.
One-Month Repeat-Dose Toxicity Study
Intravenous doses of 0 (saline control), 1, 3, or 10 mg/kg/day produced no mortality in any of the rats during the 31-day treatment period or during the 28-day recovery period. There were no drug-related overt clinical signs or ophthalmological changes in any of the groups. All animals in all groups gained weight, but slight to moderate decreases in feed consumption (both sexes) and male body weight gains (Figure 1) were noted at 10 mg/kg/day throughout the treatment and recovery periods. Water consumption in both sexes also increased significantly at 10 mg/kg/day during the treatment period.
Relevant changes in hematology, serum chemistry and urinalysis parameters are summarized in Table 3. The following trends were noted in hematology parameters: mild, but generally dose-related, decreases in RBCs; hemoglobin and hematocrit values that tended to be statistically significant in both species at 10 mg/kg/day; significant increases in leukocytes in both sexes of rats at 10 mg/kg/day; and differential counts revealed significantly increased numbers of lymphocytes in male rats and significantly increased numbers of segmented neutrophils in female rats. Changes in serum chemistry parameters included significantly increased levels of cholesterol and urea nitrogen in both sexes at 10 mg/kg/day (creatinine remained unchanged); significantly increased alkaline phosphatase levels in female rats at 10 mg/kg/day, and significant decreases in albumin in female rats at 3 mg/kg/day; and in both sexes at 10 mg/kg/day as compared to controls. Changes in urinalyses included a dose-related increase in urine output in male rats, with significant increases occurring in both male and female rats at 10 mg/kg/day; and excretion of urine that was significantly lower in specific gravity by both sexes at 3 and 10 mg/kg/day; and excretion of significant quantities of protein in male and female rats and occult blood (male rats) at 10 mg/kg/day.
At the end of the recovery period (recovery groups included five control and five high-dose animals), all clinical pathology changes returned, or tended to return, to normal values. However, alanine aminotransferase (ALT) values of 52.4 U/L in male rats and aspartate aminotransferase (AST) values of 75.4 U/L in female rats were significantly increased over their respective control values of 34.8 and 51.4 U/L at the end of the recovery period, although they had been equivalent to controls at the end of the treatment period.
At necropsy, treatment-related gross tissue changes were limited to the tail injection sites of all end-of-dose and recovery rats. The changes were characterized by multifocal, irregularly shaped, red and/or gray discolorations. Significant absolute and/or relative changes in organ weights at the end of treatment occurred predominantly in rats from the 10 mg/kg/day groups (Table 4) and included: weight increases of adrenal glands, brain, kidneys, liver, spleen and testes in male rats and increases in kidneys, liver and spleen of female rats. At the conclusion of the 28-day recovery period, only significantly increased spleen weights in both sexes, and decreased heart and thyroid weights in male rats, were observed in the 10 mg/kg/day group.
Analysis of whole blood samples at termination of treatment established that concentrations of AmB increased with dose, but that the increases were not linear. At the end of the 28-day recovery period, the 10 mg/kg high-dose concentrations were still about 35% of those present after 31 days of dosing. There were no differences in concentrations of AmB between the two high-dose groups given different lots of ABLC. Of the six tissues sampled, spleen had the highest concentration of AmB. Mean concentrations were in the order: spleen > liver > lungs > kidneys > blood > brain.
A number of treatment-associated histopathological changes were manifested in various organs of euthanized rats at the end of the treatment period. A dose-dependent hypertrophy and hyperplasia of reticuloendothelial cells, considered to be predominantly Kupffer cells in the liver, and a dose-dependent histiocytic hyperplasia of the spleen were regarded as attributable to the uptake of AmB (and/or the lipid complex) by the reticuloendothelial system (RES). A dose-dependent hyperplasia of the transitional epithelium of the renal pelvis and the urinary bladder was present in rats at 3 and 10 mg/kg/day. In the high-dose rats, there was also an increased incidence of periodic acid–Schiff (PAS)-positive cytoplasmic droplets in the proximal tubules of the kidneys. A dose-related increase in local irritation and inflammation at injection sites was observed in all groups of rats.
Significant morphologic changes in treated recovery group rats (5 rats/sex) included mild chronic inflammation at injection sites; intracytoplasmic PAS-positive droplets in the renal proximal tubules; minimal RES (Kupffer-cell) hypertrophy/hyperplasia in the liver; histiocytic hyperplasia of the spleen; and minimal urothelial hyperplasia of the urinary bladder. These changes were all markedly reduced in severity and/or incidence when compared to the rats from the same group at the end of the treatment period.
Six-Month Repeat-Dose Toxicity Study
Four rats died during the study: two high-dose (10 mg/kg/day) male rats in weeks 11 and 22; one intermediate-dose (3 mg/kg/day) male rat in week 24; and one female control rat in week 13. The three ABLC-treated rats all died within minutes after an intravenous injection, and although necropsy results failed to reveal the cause of death, it is possible that the injections were administered too rapidly (bolus injection within ~1.0 min), which may have produced an embolus. Necropsy of the control rat revealed a large abdominal hematoma that may have been caused by a faulty intraperitoneal injection.
Signs of toxicity included piloerection in some male rats at 3 or 10 mg/kg/day, reduced activity and occasional salivation in male rats at 10 mg/kg/day, and hunched posture in all treated female rats and in many male rats at 10 mg/kg/day during the last 3 months of the study. Water consumption also appeared to increase in all ABLC-treated groups from the third week until at least week 21, and measurement of consumed water during week 12 and 25 confirmed this finding, although results were statistically significant only in the 1 mg/kg/day female and 10 mg/kg/day male rats.
Body weight data are graphed in Figure 2. All animals in all groups gained weight. Compared to controls, there was a dose-related decrease of body weight gains in male rats with statistically significant differences at 3 and 10 mg/kg/day. Female rats given 3 or 10 mg/kg/day had only slight decreases in body weight gains. In parallel with body weights, feed consumption in both male and female rats also decreased slightly in a dose-related fashion, compared to controls.
Clinical pathology results were evaluated in week 13 (during interim sacrifice of 5 rats/sex/group) and in week 26 (terminal sacrifice of 10 rats/sex/group). Hematology parameters (Table 5) revealed that hemoglobin and hematocrit values decreased significantly in male rats at 3 and 10 mg/kg/day at both time periods; decreases also occurred in females, but significance was restricted to week 26. Erythrocyte counts at weeks 13 and 26 were not affected to a significant degree in either sex, but mean corpuscular volume (MCV) of red cells was significantly reduced and reticulocyte percentages were significantly increased in male rats in the 3 and 10 mg/kg/day groups. Mild, but statistically significant, increases in leukocytes in female rats at 3 and 10 mg/kg/day in weeks 13 and 26 were the only other noteworthy changes observed.
Statistically significant and biologically relevant serum chemistry changes (Table 5) were limited to increased urea nitrogen (significant for males at 3 mg/kg/day at week 13; males at 10 mg/kg/day at weeks 13 and 26; females at 10 mg/kg/day at week 13), increased cholesterol (significant for males at 10 mg/kg/day at week 26), and decreased total protein levels (significant for females at 3 mg/kg/day at week 26; males/females at 10 mg/kg/day at week 26). The decreases in total protein were presumably due to decreased albumin levels in male and female rats at 3 and 10 mg/kg/day (significant in females, weeks 13, 26). Globulin increased significantly in males at 3 mg/kg/day, week 13, but then decreased at week 26 in both male and female rats at 3 and 10 mg/kg.
Results of urinalyses (Table 5) revealed that urine volume was increased in both males and females, generally in a dose-related manner, compared to controls in weeks 12 and 25. Specific gravity decreased in both periods to 1.01, but only in the 10 mg/kg/day groups. Other changes (increased protein excretion, reduction of pH, and presence of occult blood) were restricted to 3 and 10 mg/kg/day groups of rats, and were generally more prevalent and/or severe in females and in week 25.
After 12 weeks of treatment, mean relative (% body weight) weights of adrenal glands, kidneys, and spleen were significantly increased in male rats at 10 mg/kg/day; in female rats, significant relative weight increases of kidneys occurred at 3 and 10 mg/kg/day, whereas spleens were increased only at 10 mg/kg/day. At terminal (week 27) necropsy, mean relative brain, heart, liver, and spleen weights were significantly increased and thymus weights were significantly decreased in male rats at 10 mg/kg/day. Significantly increased spleen weights were also evident at 1 and 3 mg/kg/day. In females, relative brain, heart, kidney and spleen weights were significantly increased at 3 and 10 mg/kg/day (Table 6).
Drug-related gross tissue changes observed at interim and terminal necropsy were similar and were relegated to injection sites in the tail and the abdomen of most animals, including controls. The tail changes were characterized by multifocal, irregularly shaped, red and/or gray discolored foci. The changes in the abdominal wall, mesentery/omentum and parietal peritoneum were characterized by discoloration and a granular, nodular appearance, which was more severe in treated than control rats and tended to increase in severity with dose.
Histopathological changes observed in interim and terminally sacrificed rats were primarily due to chronic tissue irritation of repeated intraperitoneal injections, rather than to systemic toxic effects of ABLC. These changes at abdominal injection sites of almost all ABLC-treated rats were characterized as granulomatous inflammatory areas that often contained birefringent intrahistiocytic crystals, presumed to be the test article. The severity of the changes were dose related, and in mid- and high-dose rats the lesions sometimes spread from the mesenteries, omentum, and peritoneum to abdominal organs, especially to the prostate, seminal vesicle, and urinary bladder. Subacute inflammatory foci and/or mononuclear cell infiltrations and hemorrhages were also noted at many tail injection sites. Renal tubular, cytoplasmic, eosinophilic droplets, observed in the kidneys of treated animals, were the only manifestation of systemic toxicity. This change was present in 2/15 male, 2/15 female rats at 1 mg/kg/day, 5/15 male rats at 3 mg/kg/day, and in all male and female rats at 10 mg/kg/day.
DISCUSSION
Results of acute studies established that liposomal encapsulation/incorporation of amphotercin B (AmB) in a lipid complex, as with ABELCET (ABLC), markedly decreased the acute toxicity of AmB. For example, in the LD50 comparison between ABLC and AmBd (amphotericin B for Injection USP, Fungi-zone) in Sprague-Dawley rats, ABLC was at least 20 times less toxic than AmBd, and the no-overt-effect doses for ABLC were 12 to 28 times higher (15 or 35 mg/kg) than those of AmB (<1.25 mg/kg). The female rats were found to be more sensitive to the acute toxicity of ABLC. LD50 values in Sprague-Dawley female rats injected with LAmB (AmBisome), another lipid-formulated AmB product, were essentially identical to those of ABLC.
Doses for the repeat-dose studies were based on a 3-week pilot study in which alternate intravenous and intraperitoneal doses of 5, 10, or 15 mg/kg/day of ABLC were administered to Crl:CD(SD)BR outbred albino rats (5/sex/group). An additional group of 10 rats was injected with 3 ml/kg/day saline. The high dose of 15 mg/kg exceeded the maximum tolerated daily dose (MTD), although there was no mortality. Ten mg/kg/day caused weight loss in male rats, increased urine output, decreased red blood cell (RBC) parameters, and produced inflammation at injection sites and extramedullary hematopoiesis in the spleen. Five mg/kg/day caused mild decreases in RBC parameters and inflammation at injection sites. Based on these data, doses of 1, 3, and 10 mg/kg/day were selected for the subchronic and chronic rat toxicity studies. These doses were intended to characterize the toxicity profile of ABLC in the rat, ranging from a no-adverse-effect dose to an MTD and remain relevant to the indicated human daily dose of 5 mg/kg/day.
Clinical pathology changes observed in the 1- and 6-month studies were almost identical in type, were mild to moderate in degree, and generally did not get more severe with increased length of treatment. Effects tended to be reversible after a 28-day recovery period. Results from hematology determinations suggested that at 3 and 10 mg/kg/day ABLC caused mild, dose-related, hemolytic effects. These effects were not deemed to be toxic to the hematopoietic system because significant increases in reticulocytes and leukocytes attested to an active bone marrow.
Serum urea nitrogen elevations, changes in urinalyses, increased kidney weights, histological evidence of urothelial hyperplasia, and presence of cytoplasmic droplets in proximal renal tubules at doses of 10 mg/kg/day established the kidney as the primary target organ for ABLC toxicity in the 1- and 6-month studies. However, serum creatinine levels remained within normal limits, suggesting that glomerular filtration was not impaired by repeated injections of 10 mg/kg/day ABLC. Similar beneficial results have occurred in clinical trials. In a comparative trial of 231 patients administered equivalent doses of AmBd or ABLC (0.6 to 1.0 mg/kg/day AmBd or 5 mg/kg/day ABLC) for systemic candidiasis, nephrotoxicity was delayed and reduced in the ABLC group. Also, a doubling of baseline serum creatinine values occurred in 28% versus 47% of patients receiving ABLC and AmBd, respectively (Anaissie et al. 1995). These results appear to confirm the studies of Longuet et al. (1991) that demonstrated that AmB encapsulated/complexed with lipids protects the kidneys from the toxic effects of AmB.
Effects on the liver and spleen observed in the present studies were relatively benign and consisted of dose-related increases in reticuloendothelial cell hypertrophy/hyperplasia and an increase in organ weight. There was no evidence of hepatic cell necrosis or consistent elevations of liver enzymes (ALT, AST, AP) and the increased liver and spleen weights were considered to be pharmacokinetic rather than toxic changes. Proffitt, Satorius, and Chiang (1991) and Boswell et al. (1998) demonstrated in studies with LAmB (another AmB-lipid formulation) that as levels of AmB in plasma decrease, those in the spleen and liver increase, suggesting RES uptake. Thus, in the 1-month rat toxicity study, the less-than-linear plasma levels of AmB were presumably due to ABLC uptake by the RES, located predominantly in the spleen and liver (Enzon Pharmaceuticals 2002). The slow release of ABLC from these organs accounted for (1) the increased residence time of AmB in the body and (2) the weight increases of these organs at treatment termination and at the end of the recovery period. The mean tissue concentrations in the order: spleen > liver > lungs > kidneys > brain, found in the 1-month study, were also in the same order in humans (Wong-Beringer, Jacobs, and Guglielmo 1998).
In the 6-month toxicity study, the main clinical observations were reduced activity in male rats at 10 mg/kg/day and hunched posture in many males and in all female rats at 10 mg/kg/day. These effects should not be considered as part of the normal ABLC profile, because they were caused by adverse local effects resulting from intraperitoneal injections of ABLC administered three times per week (alternating with i.v. injections given three times weekly). This conclusion is based on the finding of gross and histological injection-site lesions during the 3- and 6-month necropsies and the absence of overt clinical signs of toxicity at the same dose level in the 1-month study.
In many aspects, the toxicity profile of ABLC appears to be very similar to that of LAmB, with the exception that LAmB exhibited hepatoxicity at doses of 4 to 12 mg/kg/day. For example, the renal toxicity of both drugs was essentially comparable, increased blood urea nitrogen (BUN) levels and caused renal histopathology at similar doses (Bekersky et al. 2000). In addition, both drugs increased circulating neutrophil numbers and cholesterol levels (attributed to the lipids in the formulation). Finally, both drugs caused increased kidney, liver, and spleen organ weights. The organ weight increase is considered to be due to the uptake of the lipids by the reticuloendothial system.
However, the biological effects of all lipid formulations are not identical, and many significant differences in efficacy and toxicity may occur, due to pharmacokinetic and biochemical differences of the lipid preparations (Wong-Beringer, Jacobs, and Guglielmo 1998). Additionally, both the phospholipids:AmB ratio and the type of phospholipids appear to be important determinants of fungicidal activity and toxicity (Joly et al. 1992). Indeed, there are some toxicities observed in one drug, but not in another. Hepatotoxicity was one of the prominent toxicities observed with LAmB, but not with ABLC. At 9 mg/kg, LAmB produced significantly increased ALT and AST levels in female rats (544 IU/L and 758 IU/L, respectively). Microscopic pathology on day 91 indicated that LAmB caused significant hepatic changes, including multifocal hepatoceullar necrosis at all dose levels (Bekersky et al. 2000). In contrast, no hepatotoxicity was observed in any of the studies for ABLC reported here. The slightly increased ALT or AST values observed during the recovery period in the 1-month study is not considered clinically relevant because the magnitude of increase was less than two folds over controls, only one of the enzymes was elevated in either sex, and no histopathologic change was observed in the hepatocytes. Clinical studies also indicate that LAmB is more hepatotoxic than ABLC (Fleming et al. 2001). Conversely, slight decreases in RBC parameters were observed for ABLC, but not for LAmB.
In summary, the reported acute studies indicate that single i.v. doses of ABLC are at least 20 times less toxic than AmBd. Repeated i.v. doses of 1, 3, or 10 mg/kg/day ABLC for 31 days produced no mortality or overt clinical signs of toxicity, although slight to moderate decreases in feed consumption and body weights were observed. Major clinical pathology changes included dose-related slight-moderate decreases in red cell parameters and modest increases in serum urea nitrogen at 10 mg/kg/day. Key organ weight changes and histopathological changes involved the liver, kidney, and spleen. Hypertrophy and hyperplasia of reticuloendothelial cells occurred in the liver and spleen and mild nephrosis was present in the kidneys of rats at 10 mg/kg/day. Effects in the 6-month study were similar to those in the 1-month study, except for local granulomatous inflammatory lesions in the abdominal cavity resulting from three intraperitoneal doses/week that alternated with the three weekly i.v. injections. The observed changes for ABLC are similar to those for other amphotericin B lipid formulations, such as liposomal formulation LAmB, except for the hepatoxicity, which has been observed for LAmB, but not for ABLC.
Footnotes
Figures and Tables
This study and its aspects were funded and conducted by or on behalf of Enzon Pharmaceuticals, Bridgwater, New Jersey.