Encapsulation of the topoisomerase I inhibitor GL147211C in pegylated (STEALTH) liposomes: pharmacokinetics and antitumor activity in HT29 colon tumor xenografts

The topoisomerase I inhibitor GL147211C [7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-campto thecin trifluoroacetate], a camptothecin analogue, has significant activity in tumor cell cytotoxicity assays in vitro and antitumor activity in both animal tumor models and human patients. Its toxicity is significant, however, effectively limiting the amount of drug that can be administered and its clinical utility. To determine whether the therapeutic index of GL147211C could be improved, the drug was encapsulated in long-circulating, pegylated (STEALTH) liposomes (SPI-355). The pharmacokinetics and antitumor activity of SPI-355 were compared to those of nonliposomal GL147211C. The plasma pharmacokinetics of SPI-355 in rats were typical of those of other pegylated liposomal formulations, with significantly increased blood circulation time; the dose-corrected area under the curve and Cmax of SPI-355 (10 mg/kg) were 1250- and 35-fold higher, respectively, than those of nonliposomal GL14711C (8.72 mg/kg). The comparative antitumor activity of SPI-355 and nonliposomal GL1472211C was evaluated in nude mice implanted with HT29 colon carcinoma xenografts. SPI-355 was 20-fold more effective than GL147211C in inhibiting tumor growth (1 mg/kg SPI-355 and 20 mg/kg GL147211C) and produced durable complete remissions of tumors at well-tolerated dose levels that were >5-fold lower than the maximally tolerated dose of GL147211C, which induced no durable complete responses. Signs of toxicity were similar between the two drugs, but liposome encapsulation increased the toxicity of drug approximately 4-fold, with increased weight loss and several deaths with SPI-355 (5 mg/kg SPI-355 versus 20 mg/kg GL147211C). Despite the increased toxicity seen with SPI-355, the therapeutic index of the liposomal formulation was increased approximately 5-fold over that of nonliposomal GL147211C, suggesting that such a pegylated liposomal formulation could demonstrate increased therapeutic index in human patients.     

Determination of mitoxantrone in mouse whole blood and different tissues by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the specific determination of mitoxantrone (MTO) in whole blood and different tissues of mice (liver, heart, spleen, kidneys). MTO was extracted into dichloromethane with ametantrone (AMT) as internal standard. The different tissues were homogenised in citrate buffer (pH 3.0) containing 20% ascorbic acid. Separation of MTO and AMT was carried out using a Nucleosil C18 column. The mobile phase consisted of acetonitrile (33%) and 0.16 M ammonium formate buffer, pH 2.7. UV detection was used at 658 nm. Baseline separation of AMT and MTO was achieved in all matrices. The calibration curves were linear in all matrices (r > 0.999) in the concentration range of 2-200 micrograms/l for whole blood and 2-700 micrograms/l for tissue homogenates, respectively. The within-day and between-day precision studies showed good reproducibility with coefficients of variation below 4.5% for whole blood and below 10% for tissue homogenates, respectively. The extraction efficiencies of MTO are 60% in whole blood and 38% in tissue homogenates. The method described is suitable for pharmacokinetic studies on the distribution of MTO in different tissues of mice.     

Liposomal hamycin in the control of experimental aspergillosis in mice: effect of phosphatidic acid with and without cholesterol

Hamycin incorporated into liposomes containing phosphatidylcholine (SPC) and phosphatidic acid (PA) had reduced toxicity and an enhanced antifungal activity in experimental aspergillosis in balb/c mice. Incorporation of cholesterol into liposomes led to a dose dependent decrease in the toxicity of hamycin. The LD50 (mg/kg) of hamycin contained in SPC/cholesterol/PA (molar ratio 4:5:1) liposomes was 2.8 whereas that in SPC/PA liposomes (molar ratio 9:1) was 0.35. Although the free drug had little or no protective effect on the animals, those administered liposomal hamycin at an equivalent dose (0.1 mg/kg) in the absence of cholesterol (SPC/PA; molar ratio 9:1) showed 90% survival after seven days of therapy. On the other hand the presence of cholesterol in the carrier phosphatidic acid liposomes (SPC/cholesterol/PA; molar ratio 4:5:1) at a similar dose (0.1 mg/kg) led to a 60% survival over the same time period. Hamycin incorporation in phosphatidic acid liposomes both in the presence or absence of cholesterol was found to be effective in reducing the fungal load in lung, liver, spleen and kidney. Studies with distribution of hamycin in various tissues by HPLC showed a significant reduction in the concentration of the liposomal drug in circulation as compared to those seem after administration of free drug.     

Liposomal formulations containing sodium mercaptoundecahydrododecaborate (BSH) for boron neutron capture therapy

Sodium mercaptoundecahydrododecaborate or BSH is a compound most widely used for boron neutron capture therapy (BNCT). Liposome formulations containing BSH, with or without steric stabilization, were prepared as potential agents for delivery of boron compounds for BNCT. Liposomes composed of DPPC/CHOL in a molar ratio 1:1 (PEG concentration: 5 mol%) were prepared having an average diameter in the range of 100-110 nm 200 mu L of liposomes (l.88 mg phospholipid/mouse and 3.5-5.8 mg BSH/kg body weight) were injected in mice via the tail vein. Both types of liposomes resulted in a significant improvement in the circulation time of BSH compared to that obtained previously after injecting free BSH. The mean percent injected BSH remaining in circulation at the end of 24 h was 19% for the PEG-liposomes compared to the corresponding value of 7% for the conventional liposomes. The mean percent uptake by the liver and spleen was not significantly different for the two types of liposomes; the blood/RES ratios were higher for the PEG-liposomes at all time points indicating that a higher fraction of injected BSH was available in circulation. The PEG-liposomes could be further explored as a means of enhance boron drug delivery to tumor cells for BNCT.     

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).     

Comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide, and paromomycin against Cryptosporidium parvum infections in a neonatal mouse model

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1-14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0-10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

Thermoperiodic responses in insects and mites simulated with the double circadian oscillator clock

PURPOSE: To establish the pharmacodynamic relationships between drug biodistribution and drug toxicity/efficacy, a comprehensive preclinical evaluation of sphingomyelin/cholesterol (SM/chol) liposomal vincristine and unencapsulated vincristine in mice was undertaken. METHODS: Pharmaceutically acceptable formulations of unencapsulated vincristine and liposomal vincristine at drug/lipid ratios of 0.05 or 0.10 (wt/wt) were evaluated for toxicity, antitumor activity and pharmacokinetics following intravenous administration. RESULTS: Mice given liposomal vincristine at 2 mg/kg vincristine had concentrations of vincristine in blood and plasma at least two orders of magnitude greater then those achieved after an identical dose of unencapsulated drug. One day after administration of the liposomal vincristine, there were at least tenfold greater drug quantities, relative to unencapsulated vincristine, in the axillary lymph nodes, heart, inguinal lymph nodes, kidney, liver, skin, small intestines and spleen. Increased plasma and tissue exposure to vincristine as a result of encapsulation in SM/chol liposomes was not associated with increased drug toxicities. Treatment of the murine P388 ascitic tumor with a single intravenous dose of unencapsulated drug at 2, 3 and 4 mg/kg, initiated 1 day after tumor cell inoculation, resulted in a 33 to 38% increase in lifespan. In contrast, long-term survival rates of 50% or more were achieved in all groups treated with the SM/chol liposomal vincristine formulations at doses of 2, 3 and 4 mg/kg. At the 4 mg/kg dose, eight of ten and nine of ten animals survived past day 60 when treated with SM/chol liposomal vincristine prepared at the 0.05 and 0.1 drug/lipid ratios, respectively. CONCLUSIONS: Overall, increased and prolonged plasma concentrations of vincristine achieved by liposomal encapsulation were correlated with dramatically increased antitumor activity in comparison with the unencapsulated drug, but no correlations could be established between pharmacokinetic parameters and toxicity.     

HBED: the continuing development of a potential alternative to deferoxamine for iron-chelating therapy

To further examine the potential clinical usefulness of the hexadentate phenolic aminocarboxylate iron chelator N, N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) for the chronic treatment of transfusional iron overload, we performed a subchronic toxicity study of the HBED monosodium salt in rodents and have evaluated the iron excretion in primates induced by HBED. The HBED-induced iron excretion was determined for the monohydrochloride dihydrate that was first dissolved in a 0.1-mmol/L sodium phosphate buffer at pH 7.6 and administered to the primates either orally (PO) at a dose of 324 micromol/kg (149.3 mg/kg, n = 5), subcutaneously (sc) at a dose of 81 micromol/kg (37.3 mg/kg, n = 5), sc at 324 micromol/kg (n = 5), and sc at 162 micromol/kg (74.7 mg/kg) for 2 consecutive days for a total dose of 324 micromol/kg (n = 3). In addition, the monosodium salt of HBED in saline was administered to the monkeys sc at a single dose of 150 micromol/kg (64.9 mg/kg, n = 5) or at a dose of 75 micromol/kg every other day for three doses, for a total dose of 225 micromol/kg (n = 4). For comparative purposes, we have also administered deferoxamine (DFO) PO and sc in aqueous solution at a dose of 300 micromol/kg (200 mg/kg). In the iron-loaded Cebus apella monkey, whereas the PO administration of DFO or HBED even at a dose of 300 to 324 micromol/kg was ineffective, the sc injection of HBED in buffer or its monosodium salt, 75 to 324 micromol/kg, produced a net iron excretion that was nearly three times that observed after similar doses of sc DFO. In patients with transfusional iron overload, sc injections of HBED may provide a much needed alternative to the use of prolonged parenteral infusions of DFO. Note: After the publication of our previous paper (Blood, 91:1446, 1998) and the completion of the studies described here, it was discovered that the HBED obtained from Strem Chemical Co (Newburyport, MA) that was labeled and sold as a dihydrochloride dihydrate was in fact the monohydrochloride dihydrate. Therefore, the actual administered doses were 81, 162, or 324 micromol/kg; not 75, 150, or 300 micromol/kg as was previously reported. The new data have been recalculated accordingly, and the data from our earlier study, corrected where applicable, are shown in parentheses.     

Use of a liposome antigen delivery system to alter immune responses in vivo

It has been reported that a certain peptide encompassing residues 129-140 of the hepatitis B virus core antigen (HBcAg) leads to a Th2-type response in C57BL/10 mice. We postulated that by formulating the peptide in liposomes along with an immune modulator known as MPLA the immune response could be directed toward a Th1-type response. If these liposomes could deliver the peptide along with MPLA to antigen presenting cells, then the immune response generated could be polarized to a Th1 response. The type of immune response initiated after immunization with the peptide HBcAg (126-140) in different formulations was determined by an ex vivo T cell proliferation assay and by analysis of the cytokine profile of the proliferating T cells. A group of C57BL/6 mice immunized with peptide plus MPLA in a liposome formulation displayed a strong T cell proliferative response. The T cell subset was identified as Th1 based on the cytokine profile. The cytokine profiles showed significant production of interferon-gamma (IFN-gamma, a Th1-type cytokine) and extremely low levels of interleukin-4 (IL-4, a Th2-type cytokine). The control group of C57BL/6 mice immunized with peptide plus alum showed a very low level of T cell proliferation, and no increase was seen in IFN-gamma or IL-4 production. These data signify that a Th1-type response occurred in mice treated with peptide in a liposome formulation but not in mice treated with the control formulation.     

Propofol decreases ocular pressure in outpatients undergoing trabeculectomy

OBJECTIVES: The present study was conducted to compare pharmacokinetic behaviors of nicardipine enantiomers given in different doses with different formulations of racemic nicardipine in healthy volunteers. METHODS: One or two 20-mg racemic nicardipine tablets, and a 40-mg sustained-release capsule of nicardipine were administered to eight healthy volunteers in a crossover fashion and pharmacokinetic parameters were evaluated. Enantiomer concentrations were determined by GC-MS combined with chiral stationary phase HPLC. RESULTS AND CONCLUSIONS: Serum concentration of (+)-nicardipine was approximately 2-3 times higher than that of (-)-nicardipine in 20- and 40-mg doses of conventional formulations and a non-linear increase in bioavailability with dose was demonstrated. The value for AUC of (+)-nicardipine was approximately 2.3-2.8 times greater than that of the (-)-nicardipine (P < 0.05) when 20 and 40 mg racemic nicardipine were administered in a conventional preparation. Relative bioavailability of the sustained-release preparation vs the conventional preparation was 28% and 44% for (+)- and (-)-nicardipine, respectively, for the 40-mg dose.     

Efficacy of NS-718, a novel lipid nanosphere-encapsulated amphotericin B, against Cryptococcus neoformans

In vitro and in vivo efficacies of NS-718, a lipid nanosphere-encapsulated amphotericin B (AMPH-B), have been studied. Of the tested AMPH-B formulations, NS-718 had the lowest MIC for Cryptococcus neoformans. In a murine model, low-dose therapy (0.8 mg/kg of body weight) with NS-718 showed higher efficacy than that with AmBisome. High-dose therapy (2.0 mg/kg) with NS-718 was much more effective than those with Fungizone and AmBisome. In mice treated with a high dose of NS-718, only a few yeast cells had grown in lung by 7 days after inoculation. A pharmacokinetic study showed higher concentrations of AMPH-B in lung following administration of NS-718 than after administration of AmBisome. Our results indicated that NS-718, a new AMPH-B formulation, is a promising antifungal agent for treatment of pulmonary cryptococcosis and could be the most effective antifungal agent against C. neoformans infections.     

Disposition of butadiene epoxides in Sprague-Dawley rats

1,2-Epoxybutene (BMO) and diepoxybutane (BDE) are metabolic products of 1,3-butadiene in rodents. Both BMO and BDE are suspect in the development of tumors in rats and mice. To understand the distribution and elimination of these compounds in the absence of the rate-limiting production from butadiene, the pharmacokinetics of BMO and BDE in blood were determined in adult male Sprague-Dawley rats following intravenous administration. All animals were dually cannulated in these studies. For the BMO studies, rats were dosed with 71, 143, or 286 mumol/kg BMO (n = 3 for each dose group). For the BDE studies, rats were dosed with 523 mumol/kg BDE (n = 3). All animals tolerated the BMO and BDE doses without grossly observable adverse effects. Blood was drawn at predetermined time points and extracted in methylene chloride. BDE and BMO concentrations were quantitated by gas chromatography or gas chromatography/mass spectrometry. The BMO distribution half-lives were short and ranged from 1.4 min at the lowest dose to 1.8 min at the highest dose. Volume of distribution at steady state ranged from 0.53 +/- 0.17 to 0.59 +/- 0.31 l/kg. Systemic clearances ranged from 67 +/- 17 to 114 +/- 20 ml/min per kg. The terminal elimination half-lives were also short and ranged from 5.7 to 8.5 min among the doses. The pharmacokinetic parameters after an i.v. dose of 523 mumol/kg BDE were a distribution half-life of 2.7 min, terminal elimination T1/2 of 14 min, volume of distribution at steady state of 0.73 +/- 0.06 l/kg, and systemic clearance of 76 +/- 8 ml/min per kg. These pharmacokinetic parameters demonstrate the similarity between disposition of the two epoxides in rats, that include a rapid distribution after i.v. administration into a small extravascular body compartment as well as a rapid elimination from blood. These pharmacokinetic data provide useful blood clearance information for assessing the critical physiological and biochemical determinants underlying the disposition of butadiene epoxides.     

Stable and monodisperse lipoplex formulations for gene delivery

A stable single vial lipoplex formulation has been developed that can be stored frozen without losing either biological activity or physical stability. This formulation was identified by systematically controlling several formulation variables and without introducing either stabilizers or surfactants. Analytical assays were used to unambiguously characterize the formulations. The critical formulation parameters were: (1) the size of the cationic liposomes; (2) the rate and method of DNA and cationic liposome mixing; and (3) the ionic strength of the suspending vehicle. The mixing conditions were precisely controlled by using a novel, specially designed continuous flow pumping system in which the DNA and liposome solutions were mixed at the junction of a T-connector. Homogenous cationic liposome preparations were prepared by extrusion in two different size ranges of either 400 or 100 nm. Extruded liposomes produced more monodisperse and physically stable lipoplex formulations than unextruded liposomes, but the formulations prepared with 100 nm liposomes were less active in in vitro transfection assays than either the 400 nm or unextruded liposomes. Low ionic strength and 5% sorbitol were required for the lipoplex formulations to survive freezing and thawing. A frozen lipoplex formulation stored for more than a year maintained its biological activity. These results have broad implications for the pharmaceutical development of lipoplex formulations for gene delivery.     

In vivo toxicity, pharmacokinetics, and anticancer activity of Genistein linked to recombinant human epidermal growth factor

Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have vital anti-apoptotic functions in human breast cancer cells. We have shown previously that targeting the naturally occurring PTK inhibitor genistein to the EGFR-associated PTK complexes using the EGF-Genistein (Gen) conjugate triggers rapid apoptotic cell death in human breast cancer cells and abrogates their in vitro clonogenic growth. In the present study, we examined the in vivo toxicity profile, pharmacokinetics, and anticancer activity of EGF-Gen. No toxicities were observed in mice treated with EGF-Gen at dose levels as high as 40 mg/kg administered i.p. as a single dose or 140 mg/kg administered i.p. over 28 consecutive days. EGF-Gen significantly improved tumor-free survival in a severe combined immune deficiency (SCID) mouse xenograft model of human breast cancer, when it was administered 24 h after inoculation of tumor cells. At 100 microg/kg/day x 10 days (1 mg/kg total dose), which is >100-fold less than the highest tested and nontoxic cumulative dose (ie., 140 mg/kg) in mice, EGF-Gen was more effective than cyclophosphamide (50 mg/kg/day x 2 days), Adriamycin (2.5 mg/kg x 1 day), or methotrexate (0.5 mg/kg x 1 day), the most widely used standard chemotherapeutic drugs for breast cancer, and resulted in 60% long-term tumor-free survival. Furthermore, treating SCID mice with established s.c. human breast cancer xenografts of 0.5-cm diameter with EGF-Gen at this dose level resulted in disappearance of the tumors in two of five mice and >50% shrinkage in three of five mice within 10 days, whereas all of the control tumors in five PBS-treated mice as well as five mice treated with unconjugated Gen (1 mg/kg/day x 10 days) showed >200% increase in diameter during the same observation period. EGF-Gen treatment reduced the growth rate of breast cancer xenografts of 1.0-cm diameter, but unlike with tumors of 0.5-cm diameter, it failed to cause shrinkage or disappearance of these larger tumors. The level of EGF-Gen systemic exposure that was effective in SCID mice was achieved in cynomolgus monkeys without any significant side effects detectable by clinical observation, laboratory studies, or histopathological examination of multiple organs. EGF-Gen might be useful in the treatment of breast cancer as well as other EGFR-positive malignancies.     

Pharmacokinetics and tissue distribution of (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine (AZT) in mice

The in-vivo biodistribution and pharmacokinetics in mice of 3'-azido-2',3'-dideoxythymidine (1, AZT), 2-bromomyristic acid (2) and their common prodrug, (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine (3) are reported. The objectives of the work were to enhance the anti-human immunodeficiency virus and anti-fungal effects of 1 and 2 by improving their delivery to the brain and liver. The pharmacokinetics of AZT (beta t1/2 (elimination, or beta-phase, half-life) = 112.5 min; AUC (area under the plot of concentration against time) = 29.1 +/- 2.9 micromol g(-1) min; CL (blood clearance) = 10.5 +/- 1.1 mL min(-1) kg(-1)) and its ester prodrug (3, beta t1/2 = 428.5 min; AUC = 17.3 +/- 4.7 micromol g(-1) min; CL = 17.6 +/- 4.8 mL min(-1) kg(-1) were compared after intravenous injection of equimolar doses (0.3 mmol kg(-1)) via the tail vein of Balb/c mice (25-30 g). The prodrug was rapidly converted to AZT in-vivo, but plasma levels of AZT (peak concentration 0.17 micromol g(-1)) and AUC (12.3 micromol min g(-1)) were lower than observed after AZT administration (peak concentration 0.36 micromol g(-1); AUC 29.1 micromol min g(-1). The prodrug also accumulated rapidly in the liver immediately after injection, resulting in higher concentrations of AZT than observed after administration of AZT itself (respective peak concentrations 1.11 and 0.81 micromol g(-1); respective AUCs 42.5 and 12.7 micromol min g(-1)). Compared with doses of AZT itself, 3 also led to significantly higher brain concentration of AZT (25.7 compared with 9.8 nmol g(-1)) and AUCs (2.8 compared with 1.4 micromol min g(-1)). At the doses used in this study the antifungal agent 2-bromomyristic acid was measurable in plasma and brain within only 2 min of injection. Hepatic concentrations of 2-bromomyristic acid were higher for at least 2 h after dosing with 3 than after dosing with the acid itself. In summary, comparative biodistribution studies of AZT and its prodrug showed that the prodrug led to higher concentrations of AZT in the brain and liver. Although the prodrug did not result in measurably different concentrations of 2-bromomyristic acid in the blood and brain, it did lead to levels in the liver which were higher than those achieved by dosing with the acid itself.     

Epidemiology of oral cavity tumors

In this study we aimed at evaluating the modifications in the pharmacokinetic profile of cyclosporin A (CyA) after conversion from standard formulation (CyA-ST) to a new formulation (CyA-NF, Sandimmun Neoral) in patients with rheumatoid arthritis (RA). It was an open, crossover study that involved 15 RA patients who were on stabilized treatment with CyA-ST. The patient continued receiving CyA-ST (mean dose of 3.0 +/- 0.7 mg/kg per day) for 3 weeks and then converted 1:1 to CyA-NF for a further 3 weeks. CyA pharmacokinetics were established on day 1 (CyA-ST evaluation) and +21 (CyA-NF evaluation). The results showed that the bioavailability of CyA-NF was greater than that of CyA-ST (AUC tau, bss: 3335 +/- 1300 vs 2667 +/- 1155 ng.h/ml, P = 0.0073; AUC tau, bss ratio 1.26 +/- 0.40 vs 1.0 as reference, P < 0.05), with higher and earlier peak blood concentrations (Cmax: 677 +/- 256 vs 475 +/- 213 ng/ml, P = 0.0329; tmax: 1.5 +/- 0.7 vs 2.6 +/- 1.6 h, P = 0.0720). The pharmacokinetic profile of CyA-NF showed greater between-patient reproducibility (lower CV% for all of the considered parameters). In conclusion, when using CyA-NF instead of CyA-ST, greater and more constant exposure to CyA should be expected.     

Pharmacokinetics of liposomal amphotericin B (Ambisome) in critically ill patients

The liposomal formulation of amphotericin B (AmBisome) greatly reduces the acute and chronic side effects of the parent drug. The present study describes the pharmacokinetic characteristics of AmBisome applied to 10 patients at a dose of 2.8 to 3.0 mg/kg of body weight and compares them to the pharmacokinetics observed in 6 patients treated with amphotericin B deoxycholate at the standard dose of 1.0 mg/kg. Interpatient variabilities of amphotericin B peak concentrations (Cmax) and areas under concentration-time curves (AUC) were 8- to 10-fold greater for patients treated with AmBisome than for patients treated with amphotericin B deoxycholate. At the threefold greater dose of AmBisome, median Cmaxs were 8.4-fold higher (14.4 versus 1.7 microg/ml) and median AUCs exceeded those observed with amphotericin B deoxycholate by 9-fold. This was in part explained by a 5.7-fold lower volume of distribution (0.42 liters/kg) in AmBisome-treated patients. The elimination of amphotericin B from serum was biphasic for both formulations. However, the apparent half-life of elimination was twofold shorter for AmBisome (P = 0.03). Neither hemodialysis nor hemofiltration had a significant impact on AmBisome pharmacokinetics as analyzed in one patient. In conclusion, the liposomal formulation of amphotericin B significantly (P = 0.001) reduces the volume of drug distribution, thereby allowing for greater drug concentrations in serum. The low toxicity of AmBisome therefore cannot readily be explained by its serum pharmacokinetics.     

Synthesis and preliminary evaluation of MP-2269: a novel, nonaromatic small-molecule blood-pool MR contrast agent

A nonaromatic, small-molecule, gadolinium(3+)-chelate code named MP-2269 was synthesized and evaluated in animals as a potential MR contrast agent for blood pool. The ligand of MP-2269 was prepared by conjugating a lipophilic, albumin-binding moiety, 4-pentylbicyclo[2.2.2]octane-1-carboxylic acid, to an amino-functionalized DTPA derivative by means of a diaspartic acid linker. Proton relaxometry studies in vitro yielded spin-lattice relaxivities (R1) for MP-2269 of 6.2, 20.0 and 26.1 mM(-1)sec(-1) in water, rabbit blood, and human blood, respectively. The enhanced relaxivities in blood indicate significant binding of the agent to blood proteins. At a dose of 45 micromol/kg, MP-2269 showed a biphasic rabbit blood clearance profile with half-lives of 4.7 and 142 minutes, respectively, for the fast and slow components. In rats, the agent is cleared predominantly through the hepatobiliary pathway (approximately 70% in 24 h by this mode). The LD50 value of MP-2269 is approximately 3.0 mmol/kg in mice. Preliminary MR angiograms obtained in the rabbit showed excellent enhancement of blood vessels. Hence, MP-2269 has potential for future exploitation as a contrast agent for MR angiography.     

In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma

The ability to selectively target liposomal anticancer drugs via specific ligands against antigens expressed on malignant cells could improve the therapeutic effectiveness of the liposomal preparations as well as reduce adverse side effects associated with chemotherapy. Long-circulating formulations of liposomes containing lipid derivatives of poly(ethyleneglycol) [sterically stabilized liposomes (SLs)] have been described previously, and new techniques have recently been developed for coupling monoclonal antibodies (Abs) at the poly(ethyleneglycol) terminus of these liposomes. Ab-targeted SLs [immunoliposomes (SILs)] containing entrapped anticancer drugs are predicted to be useful in the treatment of hematological malignancies such as B-cell lymphomas or multiple myeloma, in which the target cells are present in the vasculature. The specific binding, in vitro cytotoxicity, and in vivo antineoplastic activity of doxorubicin (DXR) encapsulated in SILs coupled to monoclonal Ab anti-CD19 (SIL[anti-CD19]) were investigated against malignant B cells expressing CD19 surface antigens. Binding experiments with SIL[anti-CD19] resulted in a 3-fold higher association of the SILs with a human CD19+ B lymphoma cell line (Namalwa) in comparison with nontargeted SLs. Using flow cytometry, fluorescently labeled SIL[anti-CD19] bound to B cells with no recognition of T cells in a mixture of B cells and T cells in culture. Nontargeted SLs demonstrated significantly lower recognition of either B cells or T cells. Targeted DXR-SIL[anti-CD19] displayed a higher cytotoxicity to B cells relative to DXR entrapped in nontargeted SLs. Therapeutic experiments in severe combined immunodeficient mice implanted with Namalwa cells by the i.v. or i.p. routes resulted in significantly increased effectiveness of DXR-SIL[anti-CD19] compared to similar amounts of free DXR, DXR-SL (no Ab), or isotype-matched nonspecific Abs attached to DXR-SL. Single doses (3 mg/kg) of DXR-SIL[anti-CD19] administered i.v. resulted in a significantly improved therapeutic benefit, including some long-term survivors. From our results, we infer that targeted anti-CD19 liposomes containing the anticancer drug DXR may be selectively cytotoxic for B cells and may be useful in the selective elimination of circulating malignant B cells in vivo.