Tissue pharmacokinetics and pharmacodynamics of AmBisome® (L-AmBis) in uninfected and infected animals and their effects on dosing regimens.

By selecting a unique combination of lipids and amphotericin B, the liposome composition for AmBisome® (L-AmBis) has been optimized resulting in a formulation that is minimally toxic, targets to fungal cell walls, and distributes into and remains for days to weeks in various host tissues at drug levels above the MIC for many fungi. Procedures have been standardized to ensure that large scale production of the drug retains the drug's low toxicity profile, favorable pharmacokinetics and antifungal efficacy. Tissue accumulation and clearance with single or multiple intravenous administration is similar in uninfected and infected animal species, with tissue accumulation being dose-dependent and the liver and spleen retaining the most drug. The efficacy in animals appears to be correlated with drug tissue levels although the amount needed in a given organ varies depending upon the type of infection. The long-term tissue retention of bioactive L-AmBis in different organs suggests that for some indications, prophylactic and intermittent drug dosing would be efficacious reducing the cost and possible toxic side-effects. In addition, preliminary preclinical studies using non-intravenous routes of delivery, such as aerosolized L-AmBis, catheter lock therapy, and intravitreal administration, suggest that alternative routes could possibly provide additional therapeutic applications for this antifungal drug.

Differences in efficacy and cytokine profiles following echinocandin or liposomal amphotericin B monotherapy or combination therapy for murine pulmonary or systemic Aspergillus flavus infections.

Given the recent increase in aspergillosis caused by species other than Aspergillus fumigatus, micafungin, caspofungin, and liposomal amphotericin B (L-AmBi) were investigated as monotherapy or combination therapy for murine systemic or pulmonary Aspergillus flavus infection. Treatment for 3 or 6 days was begun at 24 h (intravenous [i.v.], 2.8 × 10(4) conidia) or 2 h (intranasal, 4.1 × 10(6) to 6.75 × 10(6) conidia) postchallenge as follows: 5 or 10 mg/kg L-AmBi, 10 mg/kg caspofungin, 15 mg/kg micafungin, L-AmBi plus echinocandin, L-AmBi on days 1 to 3 and echinocandin on days 4 to 6, or echinocandin on days 1 to 3 and L-AmBi on days 4 to 6. Mice were monitored for survival, fungal burden, serum or tissue cytokines, and lung histopathology. In the systemic infection, micafungin or caspofungin was more effective than L-AmBi in prolonging survival (P < 0.05), and L-AmBi was associated with significantly elevated serum levels of interleukin-6 (IL-6), macrophage inflammatory protein 1α (MIP-1α), and IL-12 (P < 0.05). In contrast, L-AmBi was significantly more effective than the echinocandins in reducing fungal growth in most tissues (P < 0.05). Concomitant therapies produced significantly enhanced survival, reduction in fungal burden, and low levels of proinflammatory cytokines, while antagonism was seen with some sequential regimens. In comparison, in the pulmonary infection, L-AmBi was significantly better (P < 0.05) than caspofungin or the combination of L-AmBi and caspofungin in prolonging survival and reducing lung fungal burden. Caspofungin stimulated high lung levels of IL-1α, tumor necrosis factor alpha (TNF-α), and IL-6, with extensive tissue damage. In summary, systemic A flavus infection was treated effectively with L-AmBi plus micafungin or caspofungin provided that the drugs were administered concomitantly and not sequentially, while pulmonary A. flavus infection responded well to L-AmBi but not to caspofungin.

Differences in tissue drug concentrations following intravenous versus intraperitoneal treatment with amphotericin B deoxycholate or liposomal amphotericin B.

Amphotericin B formulations were compared in preclinical models by using intraperitoneal (ip) and intravenous (iv) delivery of amphotericin B deoxycholate (DAMB) or liposomal amphotericin B. We examined the effects on drug tissue penetration and retention resulting from different routes of drug administration. Mice were treated with equivalent total doses of AmBisome (AmBi) or DAMB (i.e.,15 mg/kg) given ip (3 mg/kg/day for 5 days) or iv (3 mg/kg/day AmBi for 5 days or 1 mg/kg/day DAMB for 15 days), with tissues collected 24 h post-treatment. For drug retention studies, mice were given iv or ip total doses of 30 mg/kg AmBi (10 mg/kg/day 3 x /week) or 60 mg/kg AmBi (20 mg/kg/day 3 x /week) with tissue collection 24 h or 7 days post-treatment. Blood samples were collected at 0.5 h, 2 h, 8 h, 12 h and 24 h after ip or iv drug dosing. A Paecilomyces variottii bioassay was used to determine drug concentrations. AmBi and DAMB were detected in the kidneys following iv, but not ip dosing. Significantly more DAMB than AmBi was detected in the lungs with ip dosing (P = 0.008), and more AmBi than DAMB (P = 0.056) was present with iv dosing. Unlike the lungs, the spleen and liver retained the AmBi for up to one week post-treatment regardless of the route of drug administration. Thus, there are significant differences in AmBi and DAMB tissue distribution depending upon the drug route and these differences could effect how the drugs perform in fungal infection models.

Amphotericin B lipid preparations: what are the differences?

To reduce the in-vivo toxicity of the broad-spectrum antifungal drug amphotericin B, various lipid formulations of amphotericin B, ranging from lipid complexes to small unilamellar liposomes, have been developed and subsequently commercialized. These structurally diverse formulations differ in their serum pharmacokinetics as well as their tissue localisation, tissue retention and toxicity. These differences can affect the choice of formulation for a given infection, the time of initiation of treatment, and the dosing regimen. Although preclinical studies have shown similarities in the in-vitro and in-vivo antifungal activity of the formulations with comparable dosing, their acute and chronic toxicity profiles are not the same, and this has a significant impact on their therapeutic indices, especially in high-risk, immunosuppressed patients. With the recent introduction of new antifungal drugs to treat the increasing numbers of infected patients, the amphotericin B lipid formulations are now being studied to evaluate their potential in combination drug regimens. With proven efficacy demonstrated during the past decade, it is expected that amphotericin B lipid formulations will remain an important part of antifungal drug therapy.

Single-dose AmBisome (Liposomal amphotericin B) as prophylaxis for murine systemic candidiasis and histoplasmosis.

AmBisome is a liposomal formulation of amphotericin B that has broad-spectrum antifungal activity and greatly reduced toxicity compared to the parent drug. In this study, amphotericin B deoxycholate (Fungizone) (1 mg/kg) and AmBisome (1 to 20 mg/kg) were tested as single-dose prophylactic agents in both immunocompetent and immunosuppressed C57BL/6 mice challenged with either Candida albicans or Histoplasma capsulatum. Prophylactic efficacy was based on survival and fungal burden in the target organ (kidneys or spleen). At 9 to 10 days after histoplasma challenge, 80 to 90% of both immunocompetent and immunosuppressed mice in the control and Fungizone groups had died. All AmBisome-treated mice survived, although in the AmBisome groups given 1 mg/kg, the mice became moribund by day 10 to 12. No spleen CFU were detected in the histoplasma-challenged mice given 10 or 20 mg of AmBisome per kg. By 23 to 24 days after histoplasma challenge, fungal growth and/or death had occurred in all immunosuppressed mice except for four mice receiving 20 mg of AmBisome per kg. There were still no detectable fungi in the spleens of immunocompetent mice given 10 or 20 mg of AmBisome per kg. In the C. albicans experiment at 7 days postchallenge, all animals in both untreated and treated groups were alive with culture-positive kidneys. The kidney fungal burdens in AmBisome groups given 5 to 20 mg/kg were at least 1 log unit lower than those in the Fungizone group and significantly lower than those in the untreated control group (P < 0.05). There was a trend toward decreasing fungal growth in the kidneys as the dose of AmBisome was increased. In conclusion, these results show that a single high dose of AmBisome (5 to 20 mg/kg) had prophylactic efficacy in immunocompetent and immunosuppressed murine H. capsulatum and C. albicans models.

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model.

Disseminated Mycobacterium avium complex (MAC) infection has reached epidemic proportions and is a major cause of morbidity and mortality in AIDS patients. We have developed a liposomal preparation of amikacin, VS107, which incorporates the drug in 54-65 nm diameter unilameller phospholipid vesicles and is stable at 4 degrees C for more than 4 months. VS107 exhibits superior microbiological and pharmacological activity over the free amikacin and improves the survival of mice in the established model for MAC infection. The serum half-life of VS107 in mice was 9.1 h and a peak serum level of 730 mg/L was obtained after administering three doses of 160 mg/kg. For the therapeutic study, beige mice infected with 10(7) cfu M. avium complex strain 101 were randomised to be treated with placebo liposomes, buffer, free amikacin or VS107 The drugs were administered via the caudal vein thrice weekly for 1, 3, 5 or 7 weeks beginning 5 days after infection. After 51 days of treatment with VS107, the number of viable M. avium in the liver and spleen was a 100 fold lower than was achieved with conventional amikacin (P < 0.01), and more than six decimal logarithms lower than was found untreated controls (P < 0.001). VS107 was well tolerated and might be a suitable candidate for treating human MAC infections.

A fluorescence digital image microscopy system for quantifying relative cell numbers in tissue culture plates.

Microwell tissue culture plates provide a convenient format for conducting cell growth and cellular cytotoxicity assays, but such assays often have a limited dynamic range. We have developed the digital imaging microscopy scanning system (DIM-SCAN), which is a semiautomated fluorescence digital imaging system for quantifying relative cell numbers in situ in a variety of tissue culture plate formats, especially 96-well plates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, image intensifier, and an 80386 microcomputer with a PC-Vision digitizer. Turbo Pascal software controls the stage and scans the plate taking multiple images per well, calculates total fluorescence per well, provides for daily calibration, and configures easily for a variety of tissue culture plate formats. Thresholding of digital images is used to remove background fluorescence without rinsing. Using cells pretreated with fluorescein diacetate (FDA) and loaded into 96-well plates, linearity was seen over 3.0 logs of cell density (r = 0.996), with similar results in 6-well (r = 0.972), 24-well (r = 0.970), and 48-well plates (r = 0.981). For cells stained directly in plates with Hoechst 33342, linearity was observed over 2.7 logs of cell density (r = 0.975). In low-viability cultures stained with FDA in 96-well plates, excessive background fluorescence completely masked viable cells, but digital thresholding dramatically reduced background fluorescence, producing a linear response (r = 9.333) over 2.7 logs of cell density (from 264 cells/well to 1.35 x 10(5) cells/well). DIM-SCAN is a versatile system for quantifying total or viable cell numbers in tissue culture plates over a wide dynamic range.

A microcomputer program for calculating cell population doubling time in vitro and in vivo.

Determination of the doubling time for a population of cells can involve tedious calculations. We have developed computer software for MS-DOS microcomputers to expedite the analysis of tumor cell growth in vitro and in vivo. This program, DOUBLE-TIME, assists in the collection of cell numbers into a database and calculates the doubling time for a population of cells from the plot of cell growth over time. For experiments where tumor mass is measured in vivo, the software collects measurements of tumor size, calculates tumor volume (mass), generates growth curves for tumor volume change over time, and determines the doubling time of the tumor and the mean for multiple tumors. DOUBLE-TIME plots both total and viable cell numbers over time, calculates standard error of the doubling time, and the doubling time for a selected portion of a growth curve. This software also automates the cell counting process with a software-generated cell counter that allows cell counts to be tallied directly into the computer via a mouse.

Selective in vivo localization of daunorubicin small unilamellar vesicles in solid tumors.

Small unilamellar vesicles (SUVs), consisting of highly purified distearoyl phosphatidylcholine and cholesterol (2:1 mol ratio) selectively increased the delivery of entrapped daunorubicin to solid tumors in vivo. When measured against free drug, SUV-entrapped daunorubicin produced a nearly 10-fold increase in tumor uptake and efficacy when used to treat a murine lymphosarcoma model (P-1798). In a second murine solid tumor model, MA16C mammary adenocarcinoma, the median survival time for daunorubicin SUV treatment at 2 mg/kg (72 days) was equivalent to the median survival time for the free drug optimal dose, 20 mg/kg (70 days), again indicating a 10-fold increased therapeutic efficacy. When compared at maximum efficacious doses in the MA16C model, the proportion of long-term survivors was greater with daunorubicin SUVs: 10 long-term survivors of 10 mice treated with daunorubicin SUVs at 25 mg/kg versus 4 long-term survivors of 10 mice treated with free drug at 20 mg/kg. The lowest toxic doses for MA16C tumor-bearing animals (treatment median survival times less than controls) were 25 mg/kg for free drug and 40 mg/kg for daunorubicin SUVs. The demonstration of enhanced antineoplastic activity and an increased tolerance for daunorubicin suggests that this specific SUV composition may be an effective delivery system for a wide range of chemotherapeutic agents in the treatment of solid tumors.

Pharmacology and toxicology of a liposomal formulation of amphotericin B (AmBisome) in rodents.

AmBisome is a lyophilized preparation of liposomal amphotericin B. The acute intravenous toxicity of AmBisome was evaluated in mice and rats, and the LD50S were found to be greater than 175 and 50 mg/kg, respectively. The corresponding LD50S for conventional amphotericin B were approximately 2.3 and 1.6 mg/kg for mice and rats, respectively. The multiple dose toxicity test confirmed that AmBisome was well tolerated by both species. There were no deaths observed among mice receiving 25 or 50 mg/kg AmBisome for 14 days, and only two deaths among mice receiving 75 mg/kg AmBisome. One rat died in the group receiving 25 mg/kg AmBisome for 30 days. However, five of ten and nine of ten rats died in the groups treated with 50 and 75 mg/kg AmBisome, respectively. Hepatotoxicity was evident by elevation in serum liver enzyme levels for these groups. Initial pharmacokinetic evaluations demonstrated that peak plasma concentrations of 87 and 118 mg/kg, respectively, were attained in mice and rats after injection with 5 mg/kg AmBisome. Terminal plasma half-lives of 3.36 and 7.56 h were calculated for mice and rats, respectively. Tissue accumulations of amphotericin B resulting from multiple dose intravenous administration of either conventional amphotericin B or AmBisome were determined. At equivalent doses of 1 mg/kg, AmBisome treatment resulted in higher liver and spleen uptake of drug, but lower kidney and lung uptake than amphotericin B. At 5 mg/kg, AmBisome treatment resulted in concentrations of drug in the kidney and lungs that were comparable to corresponding tissue levels observed in the group treated with 1 mg/kg conventional amphotericin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of murine candidosis and cryptococcosis with a unilamellar liposomal amphotericin B formulation (AmBisome).

This investigation examined the therapeutic efficacy of AmBisome, a unilamellar (55-75 nm) liposome amphotericin B preparation with a murine LD50 by the intravenous route of greater than 175 mg/kg amphotericin B. Both fungal burden and survival were used to evaluate the drug's efficacy against murine candidosis and cryptococcosis. Single and multiple dose intravenous treatment with AmBisome (2.5, 5.0 and 10.0 mg/kg) reduced the colony forming units/mg kidney in candida-infected mice by 99% and improved survival by at least 40% relative to untreated control mice. Repeated intravenous dosing of candida-infected mice with equivalent amounts (0.75 mg/kg) of conventional amphotericin B (Fungizone) or AmBisome showed comparable reduction of yeasts in the kidneys. When mice were infected systemically with Cryptococcus neoformans, all but one of the 30 mice given AmBisome (5.0, 7.5 or 10.0 mg/kg) survived until the experiment was terminated 35 days after infection. Liver and spleen cultures from AmBisome-treated mice were negative for fungal growth. All the mice given conventional amphotericin B intraperitoneally at 4.5 mg/kg survived and cleared the infection from the livers although some of the mice had infected spleens. The percentage of cultured brains free of cryptococcus was 89% following treatment with 10.0 mg/kg AmBisome, and 80% with 4.5 mg/kg conventional drug. These preclinical studies of systemic candidosis and cryptococcosis demonstrate comparable efficacy of AmBisome and conventional amphotericin B at low doses and improved efficacy with AmBisome at doses higher than can be safely administered of the conventional drug.

Preliminary report: imaging of Kaposi sarcoma and lymphoma in AIDS with indium-111-labelled liposomes.

Kaposi sarcoma and malignant lymphoma were successfully imaged with high purity liposomes containing indium-111 in two patients with AIDS. Gamma camera images of the patient with Kaposi sarcoma showed four discrete lesions along the left foot and calf with no foci along the right foot, and no uptake into clinically enlarged but non-neoplastic lymph nodes. The spread of the Kaposi sarcoma was proximal along the leg. The neoplastic cervical lymph nodes in the second patient who had large-cell diffuse intermediate grade lymphoma were also imaged successfully. Since Kaposi sarcoma and lymphoma frequently involve occult sites, liposome imaging may prove useful in the diagnosis and management of these diseases.

Successful imaging of human cancer with indium-111-labeled phospholipid vesicles.

Twenty-four patients with proven primary and/or metastatic cancer received single intravenous injections of phospholipid vesicles containing 0.5 mCi of Indium-111. Gamma camera scintigraphy 1 to 72 hours later visualized tumors in 22 patients (92%), including carcinomas of breast, lung, colon, prostate, kidney, cervix, thyroid, and soft tissue sarcoma, lymphoma, and melanoma. Tumor sites that were identified included soft tissues, bone, lung, liver, lymph node, and spinal cord. There were only two false-positive images in metastatic sites and four false-negative images in metastatic sites. Overall sensitivity for tumors in 97 individual sites was 85%, whereas specificity was 96%. Unsuspected areas of malignancy were seen in the lumbar subdural space, pleura, liver, thyroid, and lung. Besides tumor accumulations, homogeneous uptake was observed in normal liver and spleen. Radiation doses to these two organs were 2.2 and 2.9 cGy/0.5 mCi In-111, respectively. Whole body radiation dose was 0.3 cGy/0.5 mCi. The use of Indium-111-labeled vesicles permits a wide variety of human tumors in primary and metastatic sites to be imaged without toxicity and with radiation doses comparable to other radionuclide scanning techniques.

In-111-labeled liposomes: dosimetry and tumor depiction.

Neutral phospholipid vesicles (liposomes) were loaded with 0.5 mCi (18.5 MBq) indium-111 and administered to 24 patients with various types of cancer. The median diameter of the liposomes was 77 nm, and lipid dose was 0.78-6.25 mg/kg. Scans obtained 24 and 48 hours after injection of In-111 liposomes showed gradual blood clearance with homogeneous uptake in the normal liver and spleen. Dosimetric estimates for these organs were 2.3 +/- 1.1 and 2.3 +/- 1.4 rad (.02 +/- .01 Gy), respectively, with a whole-body estimate of 0.28 rad (.003 Gy). Radiation dose did not correlate with lipid dose. Total renal excretion of In-111 was less than 2% of the injected dose in all but two patients. Transient eosinophilia occurred in two patients. Tumor was seen in the scans of 22 of 24 patients (unbinded readings). In-111-labeled liposomes may enable the demonstration of suspected or unsuspected sites of tumor.

Tumor uptake as a function of tumor mass: a mathematic model.

Inverse correlations of tumor uptake (u), measured in percent injected dose per gram, with tumor mass (m) are demonstrated for phospholipid vesicle, nonspecific and specific monoclonal antibody tracers. Correlation coefficients implied u = B mA in 11 different animal experiments. Experimental exponent (A) values lay in the range -0.28-0.64 with a mean of -0.43 while intercept (B) values varied from 3 to 18. Spherical and cylindrical tumor models implied exponents of -0.33 and -0.5, respectively. Comparison of three implantation sites of the human LS174T xenograft revealed a narrow range of exponents (-0.38- -0.46) indicating a consistent geometry for this tumor. Blood flow to the lesion site and inside its volume (as dictated by tumor size) are factors in tumor uptake. Our results indicate that biodistribution data should include the variation of tumor uptake with mass. For less than 10 g lesions, we predict that radiation absorbed dose will be highly dependent upon tumor size.

Liposomal blockade of the reticuloendothelial system: improved tumor imaging with small unilamellar vesicles.

The reticuloendothelial system of mice bearing EMT6 tumors was effectively blocked by intravenous injections of small unilamellar vesicles that incorporated a 6-aminomannose derivative of cholesterol in the lipid bilayer. Neutral liposomes loaded with indium-111-nitrilotriacetic acid were then injected. Fifty percent more radioactivity was deposited in tumors of the animals with blocked reticuloendothelial systems than in controls. Twenty-four hours after the injection of radioactive vesicles, well-defined tumor images were observed in whole-body gamma camera scintigraphs. Biodistribution studies showed that tumors from animals with blocked reticuloendothelial systems had more than twice the radioactivity per gram than any other tissue analyzed.

Sensitive radiochemical assay for inosine 5'-monophosphate dehydrogenase and determination of activity in murine tumor and tissue extracts.

Crude tissue or tumor extracts either do not contain sufficient inosine 5'-monophosphate dehydrogenase (IMPD) activity to be measured spectrophotometrically, or interfering enzyme activities prevent the use of a more sensitive radiochemical assay. A modified assay system which incorporates alpha, beta-methylene adenosine 5'-diphosphate, an inhibitor of 5'-nucleotidase; allopurinol, an inhibitor of xanthine oxidase; and ethylenediaminetetraacetate, an inhibitor of alkaline phosphatase, has been developed. [14C]Xanthine monophosphate produced during the assay was separated from [14C]hypoxanthine monophosphate by thin-layer chromatography on flexible diethylaminoethyl-cellulose sheets. Xanthine monophosphate formation was linear for at least 40 min and was inhibited by greater than 95% in the presence of mycophenolic acid, a specific IMPD inhibitor. Partial purified IMPD from murine EMT6 tumors was used to compare assay rates obtained with the radiochemical and spectrophotometric assays under identical conditions. The reaction rate of the radiochemical assay was 0.92 +/- 0.07 (S.E.) of the rate of xanthine monophosphate formation as determined spectrophotometrically at 290 nm, indicating that both assays are measuring product formation with an equal degree of accuracy. The improved radiochemical assay was used to determine IMPD specific activity in supernatants from EMT6 tumors and several normal mouse tissues. The observed activities (nmol/min/mg protein) were: EMT6 tumor, 0.303; spleen, 0.029; brain, 0.022; kidney, 0.015; lung, 0.009; liver, 0.008; and heart and skeletal muscle, less than 0.004.

Tumor-imaging potential of liposomes loaded with In-111-NTA: biodistribution in mice.

EMT6 tumors in BALB/c mice have been successfully imaged with small (less than 0.1 mu), unilamellar lipid vesicles (SUVs) loaded with In-111 nitrilotriacetic acid (In-111 NTA). Neutral SUVs prepared from distearoyl phosphatidylcholine (DSPC) and cholesterol (CH) (ratio 2:1) delivered sufficient radioactivity to allow tumor visualization 24 hr after i.v. injection; so did positively and negatively charged SUVs with the ratio 4:1:1 for DSPC:CH:X, were X was stearylamine or dicetyl phosphate. Other SUVs containing a 6-aminomannose or 6-aminomannitol derivative of cholesterol did not cause significant tumor accumulation of In-111 NTA, and tumor images were not readily discernible. The maximum tumor-associated radioactivity, 18.5% of injected dose per gram of tissue, was achieved with neutral SUVs. This level of tumor-associated In-111 was over 4 times that observed when unencapsulated In-111 NTA was injected. Neutral SUVs also gave the lowest specific activities in the liver and spleen (14.6% and 18.8% of dose respectively).