Preparation and characterization of liposomes containing a lipophilic cisplatin derivative for clinical use.

Cis-bis-neodecanoato-trans-R,R-1,2 diaminocyclohexane platinum(II) (NDDP) is a lipophilic cisplatin derivative that has been formulated entrapped in multilamellar liposomes composed of dimyristoylphosphatidyl choline (DMPC) and dimyristoylphosphatidyl glycerol (DMPG). A phase I clinical study with liposome-entrapped NDDP (L-NDDP) administered i.v. every 4 weeks has been recently completed. L-NDDP was synthesized, manufactured, and reconstituted for clinical use in our laboratories. L-NDDP was prepared as a lyophilized powder containing the NDDP and the phospholipids (NDDP-lipid weight ratio 1:15; DMPC-DMPG molar ratio 7:3). The liposome suspension was obtained on the day of use just before administration to the patients by adding normal saline (final concentration 1 mg NDDP/ml) and shaking in a water-bath shaker at room temperature according to an established protocol. A total of 54 batches of lyophilized L-NDDP were prepared. Physical appearance, phospholipid content and integrity, and elemental platinum content were determined in all batches and found to be reproducible. All batches contained < 0.24 ng/ml endotoxin. The amount of residual organic solvents was < 0.05 per cent. In all reconstituted doses, drug entrapment was > 90 per cent, and the proportion of liposomes measuring > 5 microns was < 20 per cent. Our results show that reproducible batches of liposomal preparations of new compounds can be prepared in the laboratory facilities of academic institutions, thus allowing for early clinical trials with novel therapeutic agents.

Liposome-encapsulated gentamicin treatment of Mycobacterium avium-Mycobacterium intracellulare complex bacteremia in AIDS patients.

TLC G-65, a liposome-encapsulated gentamicin, was given intravenously twice weekly for 4 weeks to AIDS patients with Mycobacterium avium-M. intracellulare complex (MAC) bacteremia at 1.7 mg of gentamicin per kg of body weight per infusion (4 patients), 3.4 mg/kg (10 patients), and 5.1 mg/kg (7 patients). MAC colony counts in blood fell by 75% or more in all three groups (P < 0.005). Drug resistance did not emerge during the study period. Transient renal insufficiency developed in one patient; no other adverse effects were detected. Liposome-encapsulated gentamicin is a potential therapy for MAC infections in AIDS patients.

Initial clinical (phase I) trial of TLC D-99 (doxorubicin encapsulated in liposomes).

A liposome-encapsulated form of doxorubicin (TLC D-99), which was shown in preclinical toxicology to be less toxic to the gastrointestinal tract and myocardium than free doxorubicin, was administered by constant infusion (1.00-1.80 h) to 38 patients in single doses of 20, 30, 45, 60, 75, and 90 mg/m2 every 3 weeks and daily for 3 days at doses of 20, 25, and 30 mg/m2/day. The dose-limiting toxicity was leucopenia: the maximum tolerated doses were one at 90 mg/m2 and three at 25 mg/m2/day. Nausea, vomiting, and stomatitis were minimal or absent at each dose; alopecia was minor. Fever and chills were noted at most of the doses, and malaise was seen in some patients, especially at the higher doses. No hepatic, renal, or other organ toxicities were observed. Clinical cardiac toxicity was not observed in any patient; however, the cumulative doxorubicin dose was greater than 400 mg/m2 in only one patient. There was large variation among patients in estimated pharmacokinetic parameters and profiles. Higher plasma levels and dose intensities were achieved with TLC D-99 than were predicted for free doxorubicin. Liposome-encapsulated doxorubicin was well tolerated and produced less nausea, vomiting, and stomatitis than would be expected with free doxorubicin administered at equally myelosuppressive doses.

Chromatographic analysis and pharmacokinetics of liposome-encapsulated doxorubicin in non-small-cell lung cancer patients.

A sensitive and specific quantitative assay for total doxorubicin concentrations in plasma containing liposome-encapsulated doxorubicin hydrochloride (TLC D-99) was developed, with solvent extraction and reversed-phase high-performance liquid chromatography (HPLC). Separation of doxorubicin from its metabolites was accomplished with a 15 cm x 3.9 mm i.d., microBondapak phenyl analytical HPLC column. Optimum chromatographic conditions, obtained with a mobile phase gradient from 85 to 50% (v/v) 16 mM ammonium formate buffer in tetrahydrofuran at a flow rate of 2 mL/min, gave a detection limit of 0.3 pmol/injection. Eleven-point standard curves with from 0.00595 to 29.8 microM TLC D-99 and 0.1 microM internal standard in plasma were analyzed on three separate occasions to formally validate this assay. An overall correlation coefficient of 0.9985 was found for the logarithmic transformed data. The pharmacokinetic characteristics of doxorubicin were investigated after administration of TLC D-99 to 12 non-small-cell lung cancer patients as an intravenous infusion at doses of 60 and 75 mg/m2. The data are best described by a three-compartment model with alpha, beta, and gamma elimination half-lives of 0.0721, 2.84, and 25.2 h for the 60-mg/m2 group and 0.103, 2.56, and 14.9 h for the 75-mg/m2 patients. A mean plasma clearance of 9.89 L/h (range: 1.95 to 23.4 L/h) was found for the 60-mg/m2 patients, with that from the 75-mg/m2 group being within these values. Mean area under the plasma concentration versus time curve estimates of 37.1 and 47.9 microM/h were observed for the patients receiving 60 and 75 mg/m2, respectively. The plasma concentration-time course for total doxorubicin following administration of TLC D-99 suggests that the disposition of the liposomal formulation is determined more by the pharmacokinetics of the liposome than the encapsulated drug.

Comparison of the cardiotoxic effects of liposomal doxorubicin (TLC D-99) versus free doxorubicin in beagle dogs.

The cardiotoxic potential of liposome encapsulated doxorubicin (TLC D-99) prepared by a remote-loading technique was compared with that of free doxorubicin (1.5mg/kg administered every 3 weeks for 8 cycles) in beagle dogs. Both agents were equally myelosuppressive, and all dogs completed both treatments. There were no deaths during the study. Experimental animals were killed between 157 and 164 days after the start of the trial. All of the dogs (n = 6) that received free doxorubicin had either moderate (1 animal) or severe (5 animals) vacuolization of myocardial tissue. None of the dogs treated with liposomal doxorubicin had lesions suggestive of cardiomyopathy. Administration of free doxorubicin was associated with transient anorexia, reduced weight gain, alopecia, and gastrointestinal toxicity. Such adverse reactions were either much less severe or absent in animals that received liposomal doxorubicin. The results of this study demonstrate that TLC D-99 significantly decreases both the myocardial toxicity and other adverse reactions of this potent antineoplastic drug. TLC D-99 is now in Phase II clinical trials.

Preclinical toxicology study of liposome encapsulated doxorubicin (TLC D-99): comparison with doxorubicin and empty liposomes in mice and dogs.

A preclinical toxicology study of intravenously administered liposome encapsulated doxorubicin (TLC D-99), free doxorubicin and empty liposomes was carried out in mice and dogs by single and multiple (daily for 5 days) dose schedules. Single dose intravenous injection studies in mice showed the encapsulated form of doxorubicin to be less toxic (LD50 of 32 mg/kg) than free doxorubicin (LD50 of 17 mg/kg). Toxicity in dogs was evaluated by serial serum chemistry, hematology and EKG analysis, urinalysis, clinical observations, necropsy and histopathologic examination. Empty liposomes injected intravenously into dogs were without significant toxicity. The maximally tolerated dose of free doxorubicin in beagles was 1.5 mg/kg; deaths were seen after a 50% escalation to 2.25 mg/kg. The maximally tolerated dose of liposome encapsulated doxorubicin was higher (2.25 mg/kg); deaths were seen after a 50% escalation to 3.37 mg/kg. A toxicity unique to the encapsulated agent was pyexia (as high as 105.6 degrees F) within twenty four hr of single dosage. This was seen in approximately half of the test animals, was not dose-related, and was not observed in animals that received empty liposomes. The organ specific toxicities seen with TLC D-99 were qualitatively similar to those of free doxorubicin, but less severe.

High-pressure liquid chromatographic analysis of aztreonam in sera and urine.

Aztreonam (SQ 26,776) is a new synthetic monocyclic beta-lactam antibiotic which is specifically active against aerobic gram-negative bacteria. High-pressure liquid chromatographic (HPLC) systems were developed for the quantitative analysis of aztreonam in human, monkey, rat, mouse, and rabbit sera and urine. The HPLC conditions employed for these analyses were a muBondapak C18 column, a mobile phase made up of 0.005 M tetrabutylammonium hydrogen sulfate at pH 3.0 and acetonitrile or methanol, UV detection at 293 nm, and a flow rate of 2.0 ml/min. For human sera and urine, the mobile phase was 80% 0.005 M tetrabutylammonium hydrogen sulfate-0.005M (NH4)2SO4 and 20% acetonitrile (vol/vol). For the range of sera and urine, HPLC analyses were shown to have excellent detector linearity of aztreonam over a concentration range of 1.0 mg/ml to 0.5 microgram/ml. Correlation coefficients for plots of aztreonam peak area versus its concentration were greater than or equal to 0.990. The detection limit of aztreonam was 1.0 micrograms/ml in sera and 5.0 micrograms/ml in urine. HPLC and microbiological assays of aztreonam in human sera and urine were in good agreement.

Single-dose pharmacokinetics of the monobactam azthreonam (SQ 26,776) in healthy subjects.

Azthreonam (SQ 26,776) is a new, completely synthetic, monocyclic beta-lactam antimicrobial agent that is highly active in vitro against most gram-negative bacteria. The pharmacokinetics of single intravenous doses of 125 to 4,000 mg, studied in 36 healthy male subjects, were best described by an open, linear, two-compartment kinetic model. The mean peak serum levels at 5 min after completion of 3-min infusions of 500-, 1,000- and 2,000-mg doses were 58, 125, and 242 micrograms/ml, respectively. The mean terminal serum half-life for all doses was 1.66 h, and the apparent volume of distribution was 0.18 liter/kg. The mean serum clearance was 1.27 ml min-1 kg-1, and urinary excretion averaged 68% of the doses administered. The pharmacokinetics of single intramuscular doses of 250 to 1,000 mg, studied in 18 subjects, were best described by a linear, one-compartment model, with first-order absorption and elimination. The mean peak serum levels occurring at 1 h after doses of 250, 500, and 1,000 mg were 12, 22, and 46 micrograms/ml, respectively. Other kinetic parameters were similar to those for intravenous administration. Tolerance of azthreonam was good, with only a mild rash in one subject and with mild to moderate transient elevations in serum transaminases and lactate dehydrogenase in two subjects.