DNA aptamers block L-selectin function in vivo. Inhibition of human lymphocyte trafficking in SCID mice.

Selectins participate in the initial events leading to leukocyte extravasation from the blood into tissues. Thus the selectins have generated much interest as targets for antiinflammatory agents. Therapeutic molecules based on the monomeric carbohydrate ligand sialyl Lewis X (SLe(X)) have low affinities and are not specific for a given selectin. Using SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technology, we have generated aptamers specific for L-selectin that require divalent cations for binding and have low nanomolar affinity. In vitro, the deoxyoligonucleotides inhibit L-selectin binding to immobilized SLe(X) in static assays and inhibit L-selectin-mediated rolling of human lymphocytes and neutrophils on cytokine-activated endothelial cells in flow-based assays. These aptamers also block L-selectin-dependent lymphocyte trafficking in vivo, indicating their potential utility as therapeutics.

An enzyme-linked oligonucleotide assay.

The recent development of in vitro methods to select high-affinity ligands by combinatorial chemistry methodologies promises unique and theoretically unlimited supplies of novel therapeutic and diagnostic reagents. One such combinatorial chemistry process, systematic evolution of ligands by exponential enrichment (SELEX), allows rapid identification, from large random sequence pools, of the few oligonucleotide sequences that bind to a desired target molecule with high affinity and specificity. We describe an enzyme-linked sandwich assay that uses a SELEX-derived oligonucleotide. This assay demonstrates that these oligonucleotides can be effective and useful analytical reagents.

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.

Bioavailability of a small unilamellar low-clearance liposomal amikacin formulation after extravascular administration.

Amikacin in small, low-clearance liposomes (MiKasome) has prolonged plasma and tissue residence and in vivo activity against extracellular infections, including Klebsiella pneumonia and Pseudomonas endocarditis. Small liposomes may cross endothelial barriers, and enter the systemic circulation after extravascular administration. We compared the systemic bioavailability (F) of low-clearance liposomal amikacin in rats following intravenous (i.v.), intraperitoneal (i.p.), intramuscular (i.m.) and subcutaneous (s.c.) injection (20 mg/kg) and intratracheal (i.t.) instillation (10 mg/kg). Drug-containing liposomes were extensively absorbed after i.p. (F = 87-146%) and i.t. (F = 64%) administration, with maximum amikacin plasma concentrations of 171 micrograms/ml at 9 h and 80 micrograms/ml at 18 h, respectively. Absorption was slower and less extensive following s.c. (plasma Tmax: 20.3 micrograms/ml at 48 h) and i.m. (plasma Tmax: 49.6 micrograms/ml at 19 h) injection, but a significant fraction (12-27%) of the liposomes was absorbed. The plasma AUCs of liposomal amikacin exceeded the AUC of conventional i.v. amikacin by at least 25-fold for all routes. Amikacin AUCs in regional lymph nodes exceeded plasma AUCs by 4-fold after s.c. and i.m. injection of liposomal amikacin. AUCs in tissues surrounding the injection sites were 20- and 191-fold higher than plasma AUCs after i.m. and s.c. injection, respectively. Thus, small low-clearance liposomes produced sustained levels of liposome-encapsulated amikacin in plasma, local tissues and lymph nodes after extravascular administration, suggesting applications in perioperative prophylaxis, pneumonias and intralesional therapy as well as sustained systemic delivery of encapsulated drugs.

Evaluating the efficacy of amikacin in low-clearance unilamellar liposomes in a s. Aureus local infection model.

Traditional therapies for Staphylococcal infections such as osteomyelitis or localized abscesses have a difficult time penetrating into tissue sites. To effectively ameliorate these infections, prolonged therapy and/or high doses of antibiotics are frequently required. Aminoglycosides, such as amikacin, are not routinely utilized for treating local infections due to poor efficacy associated with ineffective tissue penetration, toxicity, and poor penetration in an acid millieu. We postulated that a formulation of amikacin in small unilamellar liposomes might readily be engulfed by inflammatory macrophages facilitating drug delivery to the site of infection. This increased drug load to the site of bacterial infection may result in enhanced bactericidal action compared to conventional aminoglycosides. Tissue drug concentrations were determined for liposomal amikacin (L-AN) and conventional amikacin (AN). Plasma amikacin levels were determined for L-AN. The L-AN was very effective at concentrating at the site of infection compared to AN. Following confirmation of adequate tissue drug levels, a rodent subcutaneous abscess infection using S. aureus as the bacterial challenge agent was evaluated. Sprague-Dawley rats were intravenously administered L-AN every other day due to its prolonged half-life, while the comparator agent, AN, was administered daily. Abscess size, weights, severity, histology, and tissue colony counts were examined. In efficacy studies, L-AN was superior to AN in reducing colony counts.

Altered tissue distribution and elimination of amikacin encapsulated in unilamellar, low-clearance liposomes (MiKasome).

PURPOSE: Amikacin in small unilamellar liposomes (MiKasome) has prolonged plasma residence (half-life > 24hr) and sustained efficacy in Gram-negative infection models. Since low-clearance liposomes may be subject to a lower rate of phagocytic uptake, we hypothesized this formulation may enhance amikacin distribution to tissues outside the mononuclear phagocyte system. METHODS: Rats received one intravenous dose (50 mg/kg) of conventional or liposomal amikacin. Amikacin was measured for ten days in plasma, twelve tissues, urine and bile. RESULTS: Liposomal amikacin increased and prolonged drug exposure in all tissues. Tissue half-lives (63-465 hr) exceeded the plasma half-life (24.5 hr). Peak levels occurred within 4 hours in some tissues, but were delayed 1-3 days in spleen, liver, lungs and duodenum, demonstrating the importance of characterizing the entire tissue concentration vs. time profile for liposomal drugs. Predicted steady-state tissue concentrations for twice weekly dosing were >100 microg/g. Less than half the liposomal amikacin was recovered in tissues and excreta, suggesting metabolism occurred. Amikacin was not detected in plasma ultrafiltrates. Tissue-plasma partition coefficients (0.2-0.8 in most tissues) estimated from tissue-plasma ratios at Tmax were similar to those estimated from tissue AUCs. CONCLUSIONS: Low-clearance liposomal amikacin increased and prolonged drug residence in all tissues compared to conventional amikacin. The long tissue half-lives suggest liposomal amikacin is sequestered within tissues, and that an extended dosing interval is appropriate for chronic or prophylactic therapy with this formulation.

Characterization of a novel insertion of the macrolides-lincosamides-streptogramin B resistance transposon Tn554 in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

Macrolides-lincosamides-streptogramin B resistance in staphylococci can result from a gene, ermA, that comprises part of transposon Tn554. Tn554 is unusual in (i) its high specificity for a primary chromosomal attachment site, att554, and (ii) the variability of its 3'-terminal six or seven nucleotides, which appear to copy the six or seven chromosomal nucleotides 5' to the parent transposon during transposition. We characterized a novel Tn554 insert in the chromosomes of methicillin-resistant Staphylococcus aureus strains involved in a current outbreak. This insert was found to resemble an insert recently discovered in S. epidermidis in its junctional fragment restriction pattern. Sequence analysis of the junctional regions showed that the attachment site, att155, exhibited 78% similarity to att554 (39 of the 50 nucleotides flanking the insertion sites) for both S. aureus and S. epidermidis inserts and that the 3' hexanucleotide of the S. epidermidis transposon (GACATC) resembled the reverse complement (TACATC) of its commonly occurring S. aureus counterpart (GATGTA). Epidemiologic and molecular data indicated that att155 is harbored by extra DNA characteristic of methicillin-resistant strains and absent from methicillin-susceptible ones. Further, Southern hybridization showed that, even in the absence of Tn554 inserts, some methicillin-resistant strains contain DNA related to att155 and Tn554.

Comparison of tube dilution and microtitre methods for the detection of antibiotic tolerance in strains of Staphylococcus aureus.

Comparison of the MICs and MBCs of oxacillin for ten isolates of Staphylococcus aureus determined by tube dilution with those by microtitre dilution tests demonstrated that, whereas the MICs did not differ, the mean MBC by tube dilution was significantly higher (P less than 0.05) than that by microtitre after both 24 and 48 h of incubation. In addition, tolerance (MBC/MIC ratio greater than 32) was seen in four of ten strains with the tube dilution method but in only one strain with the microtitre technique after 24 h of incubation. These results and the poor correlation (r = 0.33) between MBCs by these methods indicate the importance of technical factors in the quantitation of MBCs and resulting identification of tolerance in Staph. aureus, and the need for a reference procedure in determination of MBCs.

Pharmacokinetics and urinary excretion of amikacin in low-clearance unilamellar liposomes after a single or repeated intravenous administration in the rhesus monkey.

Liposomal aminoglycosides have been shown to have activity against intracellular infections, such as those caused by Mycobacterium avium. Amikacin in small, low-clearance liposomes (MiKasome) also has curative and prophylactic efficacies against Pseudomonas aeruginosa and Klebsiella pneumoniae. To develop appropriate dosing regimens for low-clearance liposomal amikacin, we studied the pharmacokinetics of liposomal amikacin in plasma, the level of exposure of plasma to free amikacin, and urinary excretion of amikacin after the administration of single-dose (20 mg/kg of body weight) and repeated-dose (20 mg/kg eight times at 48-h intervals) regimens in rhesus monkeys. The clearance of liposomal amikacin (single-dose regimen, 0.023 +/- 0.003 ml min-1 kg-1; repeated-dose regimen, 0.014 +/- 0.001 ml min-1 kg-1) was over 100-fold lower than the creatinine clearance (an estimate of conventional amikacin clearance). Half-lives in plasma were longer than those reported for other amikacin formulations and declined during the elimination phase following administration of the last dose (from 81.7 +/- 27 to 30.5 +/- 5 h). Peak and trough (48 h) levels after repeated dosing reached 728 +/- 72 and 418 +/- 60 micrograms/ml, respectively. The levels in plasma remained > 180 micrograms/ml for 6 days after the administration of the last dose. The free amikacin concentration in plasma never exceeded 17.4 +/- 1 micrograms/ml and fell rapidly (half-life, 1.47 to 1.85 h) after the administration of each dose of liposomal amikacin. This and the low volume of distribution (45 ml/kg) indicate that the amikacin in plasma largely remained sequestered in long-circulating liposomes. Less than half the amikacin was recovered in the urine, suggesting that the level of renal exposure to filtered free amikacin was reduced, possibly as a result of intracellular uptake or the metabolism of liposomal amikacin. Thus, low-clearance liposomal amikacin could be administered at prolonged (2- to 7-day) intervals to achieve high levels of exposure to liposomal amikacin with minimal exposure to free amikacin.

Molecular epidemiology of macrolides-lincosamides-streptogramin B resistance in Staphylococcus aureus and coagulase-negative staphylococci.

Macrolides-lincosamides-streptogramin B (MLS) resistance is commonly found in Staphylococcus aureus and coagulase-negative staphylococci (22 and 45%, respectively, among isolates from three New Jersey hospitals). We have examined representative subsets of 107 MLS-resistant isolates for the molecular nature of the resistance determinant, the erm gene, by dot blot and Southern hybridization analysis. All of 35 S. aureus isolates examined and 39 of 42 coagulase-negative isolates examined were found to harbor the ermA or ermC evolutionary variant. Genes of the ermC class occurred exclusively on a small plasmid similar to or indistinguishable from one (pNE131) previously described in S. epidermidis. Genes of the ermA class occurred exclusively in the chromosome, and restriction patterns indicated that they were part of a transposon, Tn554, characteristic of the classical S. aureus ermA strain. Unlike S. aureus ermA strains examined previously, which harbor Tn554 at a single specific (primary) site, four of our S. aureus isolates had second inserts at different chromosomal sites. The majority of our coagulase-negative isolates had two or more inserts, neither of which occurred at the classical primary site and many of which differed from one another in location (as inferred from restriction patterns). Coagulase-negative staphylococci constitute a large reservoir of the ermA and ermC class of determinants, with clear potential for interspecies spread.