Biodistribution of NX211, liposomal lurtotecan, in tumor-bearing mice.

Prolonging tumor exposure to topoisomerase I inhibitors has been correlated to enhance the efficacy of those agents. Lurtotecan, a water-soluble camptothecin analog, was formulated as a liposomal drug, NX211, to enhance the delivery of drug to tumors. Tumor-bearing mice were treated with either [14C]NX211 containing [14C]lurtotecan, [3H]NX211 containing [3H]phosphatidylcholine or [14C]lurtotecan, euthanized at selected times post-injection, and tissues, plasma, urine and feces were collected. These studies demonstrated that KB tumors of [14C]NX211-treated mice had approximately 70-fold greater concentrations of [14C]lurtotecan at 24 h, respectively, compared to concentrations of [14C]lurtotecan of the KB tumors of [14C]lurtotecan-treated mice. The area under curve (AUC) from 0 to 48 h of [14C]lurtotecan for the KB tumors of [14C]NX211-treated animals was over 17-fold greater than the AUC of [14C]lurtotecan for the tumors of [14C]lurtotecan-treated animals. Treatment with [3H]NX211 demonstrated that the lipid component continually accumulated over 24 h in the tissues. HPLC analysis of extracted material from tumors of [14C]NX211-treated mice showed that more than 95% of the radioactive material was intact [14C]lurtotecan. These findings are one of the keys justifying the development of a liposomal formulation of lurtotecan, which has the intent to increase tumor exposure and increase antitumor efficacy.

Antitumor efficacy, pharmacokinetics, and biodistribution of NX 211: a low-clearance liposomal formulation of lurtotecan.

Lurtotecan is a clinically active water-soluble camptothecin analogue that has been formulated into a low-clearance unilamellar liposome, NX 211. Comparative studies between free drug and NX 211 have been performed assessing pharmacokinetics in nude mice, tissue distribution in tumor-bearing mice, and antitumor efficacy in xenografts. Compared with lurtotecan, NX 211 demonstrated a significant increase in plasma residence time and a subsequent 1500-fold increase in the plasma area under the drug concentration curve. The volume of distribution was also greatly restricted, suggesting altered tissue distribution. Evaluation of tissues 24 h after administration of either [14C]NX 211 or [14C]lurtotecan to ES-2 tumor-bearing mice demonstrated a 40-fold increase in radiolabeled compound in the tumors of NX 211-treated mice compared with mice treated with lurtotecan. In single-dose efficacy studies, NX 211 produced a consistent 3-fold or greater increase in therapeutic index compared with lurtotecan in both the KB and ES-2 xenograft models. When compared at equitoxic levels in repeat-dose efficacy studies, NX 211 generated durable cures lasting >60 days and a 2-8-fold increase in log10 cell kill, compared with lurtotecan and topotecan, respectively. Together, these data demonstrate that NX 211 has significant therapeutic advantage over lurtotecan and that the improved antitumor activity is consistent with increased exposure and enhanced drug delivery to tumor sites.

Transcriptional activity of quinone methides derived from the tumor promoter butylated hydroxytoluene in HepG2 cells.

Butylated hydroxytoluene (BHT) is a pulmonary toxin and tumor promoter in mice presumably due to the formation of two quinone methides (QMs) that alkylate cellular nucleophiles. The activation of stress genes by these electrophilic metabolites was investigated with an assay system consisting of 14 recombinant cell lines derived from the human hepatoma line HepG2, each carrying a unique promoter or response element construct fused to the reporter gene for chloramphenicol acetyl transferase (CAT). The largest responses to QMs occurred in cells containing either the metallothionein IIA, glutathione S-transferase Ya, or 70 kDa heat shock protein promoter, or the xenobiotic response element. The other cell lines exhibited only small or no effects. These results are consistent with transcriptional activities reported for several other electrophiles known to undergo covalent interactions with proteins.

In vivo properties of an in situ forming gel for parenteral delivery of macromolecular drugs.

PURPOSE: This study characterizes the in vivo properties of an in situ forming gel, comprising of IPC of water-soluble polymers, PMA and PEG, for sustained release of macromolecular drugs. METHODS: 40, 50, or 60% w/v formulations were injected subcutaneously in a rat model either alone, or containing model macromolecules, 3A2-ATG-psODN or REV-psODN, to (i) determine the approximate gelling and residence time of the gel at the site of injection (ii) assess the biological efficacy of the formulation using a MZ sleep time model and (iii) demonstrate specificity of the sequence and selectivity of the psODNs by measuring changes in microsomal enzyme levels and urine volumes. RESULTS: A sol to gel transition requires 15 min in vivo, and the 60% w/v IPC gel remains at the site of injection for up to 72 hr. The MZ sleep times and CYP3A2 expression due to 3A2-ATG-psODNs released from the gel are significantly different compared to that of REV-psODNs. CONCLUSIONS: The IPC solutions exhibit phase transformation in vivo. and demonstrate no evidence of toxicity. The pharmacological effects observed from the of release of 3A2-ATG-psODNs suggest that the formulation can entrap, protect, and sustain the delivery of macromolecules. .

Pharmacokinetics of a synthetic, chemically modified hammerhead ribozyme against the rat cytochrome P-450 3A2 mRNA after single intravenous injections.

Modulation of gene expression via nucleic acid sequence-specific intervention represents a new paradigm for drug discovery and development. Ribozymes are small RNA structures capable of cleaving RNA target molecules in a catalytic fashion. A 2'-O-allyl-modified hammerhead ribozyme designed to cleave the messenger RNA of cytochrome P-450 3A2 was administered to rats via 0.25 mg intravenous injections to investigate the disposition of this compound. The chemically modified ribozyme binds to serum albumin and can be displaced by phosphorothioate oligonucleotides. A biphasic plasma clearance with a distribution half-life of 12 min and an elimination half-life of 6.5 h was observed. A volume of distribution of 2.1 l/kg indicates perfusion into tissues well beyond the vascular system. The chemically modified ribozyme can be detected intact in the plasma up to 48 h after injection. Metabolic degradation of the chemically modified ribozyme occurs at unmodified ribonucleotides, leaving the 2'-O-allyl-modified sites intact. Recovery of intact chemically modified ribozyme was 1.9% of the administered dose at 12 h along with significant metabolites. The renal clearance of the intact ribozyme is an average 34.3 ml/h. The tissue distribution of the chemically modified ribozyme at 48 h is primarily to kidney and liver but the only detected material is a single 27-mer metabolite that has been cut in the unmodified GAAA region. The brain concentration of the prominent 27-mer metabolite is greater than that observed in the lung or spleen. Examination of tissues reveals no morphological evidence of toxicity. These data strongly support the potential utility of synthetic, 2'-O-allyl-modified hammerhead ribozymes as therapeutic agents in vivo.

Inhibition of the rat cytochrome P450 3A2 by an antisense phosphorothioate oligodeoxynucleotide in vivo.

CYP3A2 is one of the most abundantly expressed cytochrome P450s (CYPs) in the rat liver and metabolizes numerous clinically important drugs. Studies were designed to examine efficacy, potency and specificity of antisense inhibition of CYP3A2 in vivo. Three phosphorothioate ODNs were used: 3A2-ATG, antisense to the CYP3A2 mRNA translational start site; 3A2-REV, 5' to 3' reverse sequence of 3A2-ATG; and C-MYC, antisense to the C-MYC mRNA translational start site. Midazolam (MZ) sleep times were used as CYP3A2-specific in vivo marker in male Sprague-Dawley rats. Administration of 1 mg/day 3A2-ATG for 2 days i.p. significantly increased MZ sleep times from 22.4 +/- 0.4 (saline) to 35.3 +/- 1.5 min. Administration of equivalent doses of noncomplementary 3A2-REV or C-MYC produced no significant changes in MZ sleep times (22.4 +/- 0.6 and 22.8 +/- 1.3, respectively). Liver microsomal erythromycin demethylase activity, a specific CYP3A2 assay, was significantly decreased from 124 +/- 13 mumol/mg per min in saline controls to 63.8 +/- 8 in 3A2-ATG-treated rats. Enzyme activities for CYPs 2E1, 1A1/2 and 2B1/2 were not significantly different between saline controls and 3A2-ATG-treated animals. The control ODNs 3A2-REV and C-MYC had no significant changes in enzymatic activities compared to saline. Western blot analysis revealed decreases in CYP3A2 protein but not CYP2B1 protein in 3A2-ATG rat microsomes compared to controls. These studies demonstrate for the first time that antisense ODNs can effectively, potently, and specifically inhibit CYP3A2 in vivo.

Dermatologic problems in amputees.

Improvements in surgical techniques and prosthetic devices and the establishment of prosthetic clinics have altered the outlook for the amputee. A cooperative effect on the part of professionals looking after amputee patients, as carried out in these clinics, offers the best means of recognition and treatment of difficulties as they arise. Until the "bionic man" becomes a reality, amputees will continue to have skin problems. In describing a number of illustrative cases, we have attempted to: (a) renew interest in these problems; (b) demonstrate the value of the group approach; and (c) encourage the participation of interested dermatologists in the prosthetic clinic team to facilitate earlier recognition and treatment of troublesome skin disorders in the amputee.