Evaluation of the distribution and excretion of [14C]-inclisiran following single subcutaneous administration in cynomolgus monkeys.

Inclisiran is a small interfering RNA molecule that was designed to reduce plasma low-density lipoprotein cholesterol (LDL-C) levels by inhibiting proprotein convertase subtilisin/kexin type 9 synthesis in the liver. This study aimed to characterize the tissue distribution and excretion of inclisiran after dosing in monkeys. A single 20 mg/kg subcutaneous injection of [14C]-inclisiran was administered to 12 male cynomolgus monkeys. Plasma concentrations and tissue binding parameters for inclisiran were assessed up to 42 days after injection using liquid scintillation of blood samples and tissue homogenates, as well as quantitative whole-body autoradiography. Radioactivity was highest in the liver at all time points from 24 h onward and remained elevated throughout the entire study period. Radioactivity was also detected in the kidneys and bladder wall, returning to low levels by 24 h. The concentration of radioactivity in the liver (402.97 µg equivalent/g) was 15.7-fold higher than in the kidneys (25.70 µg equivalent/g). Very low amounts of radioactivity were detected in all other tissues examined. The highest radioactivity in tissue homogenates was in the liver and kidney pyramid (327 and 351 µg equivalent/g, respectively). This study confirmed the selective uptake of inclisiran by the liver, indicating that the N-acetylgalactosamine linker allows for selective uptake via the asialoglycoprotein receptors expressed on hepatocytes compared with other tissues that lack asialoglycoprotein receptors. The long tissue retention in the liver supports the infrequent, biannual dosing schedule for inclisiran in the clinic and the temporal disconnect between short-term systemic exposure and sustained lowering of LDL-C.

Oritavancin Pharmacokinetics and Bone Penetration in Rabbits.

The pharmacokinetics and bone concentrations of oritavancin were investigated after a single intravenous dose was administered to rabbits. The pharmacokinetic profile of oritavancin in rabbits showed that it is rapidly distributed to bone tissues, with concentrations remaining stable for up to 168 h, the last measured time point. Based on these findings, further evaluation of oritavancin for the treatment of infections in bone tissues is warranted.

In vitro characterization of oritavancin clearance from human blood by low-flux, high-flux, and continuous renal replacement therapy dialyzers.

BACKGROUND: Oritavancin is an investigational lipoglycopeptide antibiotic under clinical development for the treatment of gram-positive bacterial infections. The impact of hemodialysis on plasma concentrations of oritavancin is unknown and may be important in making dosage adjustments in such patients. The present study sought to determine the clearance of oritavancin from human blood by various commercially available dialyzers in an in vitro hemodialysis model. METHODS: Three types each of low-flux (Dicea 130, F6, and Polyflux 14L) and high-flux (Revaclear, Exeltra 150, and Optiflux F160NR) dialyzers and one type of continuous renal replacement therapy (CRRT) dialyzer (Prismaflex M100) were studied. Heparinized human blood containing oritavancin (200 mg/L) was circulated from a reservoir to the dialyzers and back to the reservoir. Fresh dialysate was pumped through the dialyzers in a countercurrent manner. Blood samples from each side of the dialyzers and contaminated dialysate samples were collected at periodic intervals. Oritavancin levels were analyzed by a liquid chromatography/mass spectrometry method with a limit of quantification of 12.5 ng/mL and plasma clearances of oritavancin were calculated. RESULTS: The mean dialytic clearance of oritavancin was insignificant for each of the low-flux, high-flux and CRRT dialyzers. Clinically significant amounts of oritavancin were not detected in dialysate during any of the experimental dialysis sessions. CONCLUSIONS: The clearance of oritavancin from human blood by the dialyzers used in this study is insignificant. Further clinical studies would be required before making changes in dosage of oritavancin in hemodialysis patients.

Synthesis and in vitro evaluation of bisphosphonated glycopeptide prodrugs for the treatment of osteomyelitis.

As therapeutic agents of choice in the treatment of complicated infections, glycopeptide antibiotics are often preferentially used in cases of osteomyelitis, an infection located in bone and notoriously difficult to successfully manage. Yet frequent and heavy doses of these systemically administered antibiotics are conventionally prescribed to obtain higher antibiotic levels in the bone and reduce the high recurrence rates. Targeting antibiotics to the bone after systemic administration would present at least three potential advantages: (i) greater efficacy, by concentrating the therapeutic agent in bone; (ii) greater convenience, through a reduction in the frequency of administration; and (iii) greater safety, by reducing the levels of systemic drug exposure. We present here the design, synthesis and in vitro evaluation of eight prodrugs of the glycopeptide antibacterial agents vancomycin and oritavancin taking advantage of the affinity of the bisphosphonate group for bone for delivery to osseous tissues.

Vancomycin and oritavancin have different modes of action in Enterococcus faecium.

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-(13)C]lysine and complexed with desleucyl [(19)F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala-d-Ala binding pocket still binds to E. faecium peptidoglycan. The (13)C{(19)F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.

Linking bisphosphonates to the free amino groups in fluoroquinolones: preparation of osteotropic prodrugs for the prevention of osteomyelitis.

Osteomyelitis is an infection located in bone and a notoriously difficult disease to manage, requiring frequent and heavy doses of systemically administered antibiotics. Targeting antibiotics to the bone after systemic administration may provide both greater efficacy of treatment and less frequent administration. By taking advantage of the affinity of the bisphosphonate group for bone mineral, we have prepared a set of 13 bisphosphonated antibacterial prodrugs based on eight different linkers tethered to the free amino functionality on fluoroquinolone antibiotics. While all but one of the prodrugs were shown in vitro to be effective and rapid bone binders (over 90% in 1 h), only eight of them demonstrated the capacity to significantly regenerate the parent drug. In a rat model of the disease, a selected group of agents demonstrated their ability to prevent osteomyelitis when used in circumstances under which the parent drug had already been cleared and is thus inactive.

Oritavancin exhibits dual mode of action to inhibit cell-wall biosynthesis in Staphylococcus aureus.

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the D-Ala-D-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan D-Ala-D-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.

A new class of small molecule RNA polymerase inhibitors with activity against rifampicin-resistant Staphylococcus aureus.

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.

Triaminotriazine DNA helicase inhibitors with antibacterial activity.

Screening of a chemical library in a DNA helicase assay involving the Pseudomonas aeruginosa DnaB helicase provided a triaminotriazine inhibitor with good antibacterial activity but associated cytotoxicity toward mammalian cells. Synthesis of analogs provided a few inhibitors that retained antibacterial activity and demonstrated a significant reduction in cytotoxicity. The impact of serum and initial investigations toward a mode of action highlight several features of this class of compounds as antibacterials.

Novel non-nucleobase inhibitors of Staphylococcus aureus DNA polymerase IIIC.

The preparation and biological evaluation of 5-substituted-6-hydroxy-2-(anilino)pyrimidinones as a new class of DNA polymerase IIIC inhibitors, required for the replication of chromosomal DNA in Gram-positive bacteria, are described. These new dGTP competitive inhibitors displayed good levels of in vitro inhibition and antibacterial activity against Staphylococcus aureus. A new class of dATP competitive inhibitors, 6-substituted-2-amino-5-alkyl-pyrimidin-4-ones, whose antibacterial activity was unaffected by serum, were identified.

A synthetic receptor for choline and carnitine.

A synthetic receptor is described with the appropriate shape and size for alkylated trimethylammonium ions such as choline and carnitine. The structure features a deep, concave binding site, lined with aromatic walls that provide cation-pi interactions between host and guest. Molecular mechanic calculations suggest that the host's shape is maintained through intermolecular hydrogen bonding with DMSO solvent molecules. The cavity is too small to accommodate larger ions. Choline and carnitine are recognized and bound with high affinity even though no complementary charges are involved in the process.

Rigid tetranitroresorcinarenes.

[reaction: see text]. o-alkylation of C2V-symmetrical resorcinarene tetraesters 2 with 2 equiv of 1,3-difluoro-4,6-dinitrobenzene readily affords conformationally rigid octanitro resorcinarene 3, which is a potential scaffold for the design of supramolecular structures.

Water-stabilized cavitands.

Tetrabenzimidazole cavitands 4 were prepared by condensation of ortho esters with octaamino cavitand 3 in 70-80% yield. Molecular modeling predicted that no intramolecular hydrogen bonds are possible between the imidazole fragments in the vase conformation of 4. Instead, this conformation provides four perfect binding sites for hydroxyl-containing molecules through an N-H---O-H---N pattern. Such interactions provide the means for sealing the cavitand's cavity. Accordingly, dry compounds 4 are not soluble in dry CDCl3 but readily dissolve upon addition of small amounts of alcohols or by saturation of the solution with water. 1H NMR spectroscopy revealed that in these solutions molecules 4 adopt a vase conformation while 1D GOESY experiments revealed their monomeric nature. In water-saturated CDCl3, these cavitands 4 form kinetically stable 1:1 inclusion complexes with tetramethylphosphonium bromide and triethylammonium chloride in which the cation is incorporated into the pi-basic cavity. Thus, cavitands 4 are a novel class of open-ended molecular containers capable of the formation of highly kinetically stable complexes upon assistance by hydrogen-bonding water molecules.