Rapid method to characterize lactococcal bacteriophage genomes.

We present a rapid method to isolate and analyze bacteriophage DNA. Cells are infected and phage replication is allowed to proceed normally for 30 to 60 min. Prior to DNA packaging and cell bursts, the infected cells (1 ml) are harvested and lysed by using a combination of lysozyme and sodium dodecyl sulfate treatments. The total DNA recovered is enriched for phage genomes, and restriction fragments of the phage DNA can be readily visualized on agarose gels. This method was used to grossly compare the genomes of nine lactococcal phages isolated from different cheese plants at different times. The method was also used to visualize the inhibitory effects of pTR2030-induced abortive infection on the replication of phage nck202.31 in its homologous host, Lactococcus lactis NCK203.

Inhibitors of cyclic AMP phosphodiesterase. 4. Synthesis and evaluation of potential prodrugs of lixazinone (N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro-2- oxoimidazo[2,1-b]quinazolin-7-yl)-oxy]butyramide, RS-82856).

The cyclic AMP phosphodiesterase (cAMP PDE) inhibitor and cardiotonic agent lixazinone (N-cyclohexyl-N-methyl-4-[(1,2,3,5-tetrahydro-2- oxoimidazo[2,1-b]quinazolin-7-yl)oxy]butyramide, RS-82856, 1) and its acid and base addition salts were found to be insufficiently soluble in formulations suitable for intravenous administration. These results prompted an investigation into potential prodrugs with enhanced aqueous solubility designed to deliver 1 by three distinct mechanisms: (1) decarboxylation of alpha-carboxamides; (2) hydrolytic loss of a solubilizing N-1-(acyloxy)methyl or (N,N-dialkylamino)methyl moiety; or (3) intramolecular closure of a guanidino ester or amide. The target compounds were evaluated as delivery systems for 1 by three criteria: (1) chemical conversion rate to 1 under physiological conditions; (2) inhibition of type IV cAMP PDE at a fixed time point; and (3) in vivo inotropic activity in anesthetized dogs by both intravenous and oral administration. Release of 1 from 4a (series 1) was found to be too slow to be of value as a prodrug of 1, since decarboxylation could be induced only by strong acid, conditions under which hydrolytic ring opening was found to severely compete. Conversely, 1 was released too readily on exposure of (N,N-dialkylamino)methyl derivatives such as 8d (series 2) to physiological conditions, although no large increase in aqueous solubility was realized. Finally, both the physicochemical and in vitro studies indicated that ring closure of the guanidinium esters and amides 17a-k (series 3) to 1 was quantitative and pH- and time-dependent, suggesting the possibility of delivery of the open, water-soluble prodrug form, followed by closure to 1 in plasma. Detailed examination of these agents in vivo, however, demonstrated that only those compounds that rapidly cyclized to 1, as measured by plasma levels of 1, exhibited inotropic activity, indicating that the open prodrug form was not efficiently absorbed upon oral administration.

Single intravenous dose and steady-state oral dose pharmacokinetics of nicardipine in healthy subjects.

Nicardipine HCl oral doses (10-40 mg) were administered sequentially to six healthy subjects. For each regimen the capsule dose was administered every 8 hours (q 8 h) for 3 days and the plasma profiles of nicardipine and its pyridine analogue (M5) were determined following the last dose on day 4. Steady-state plasma concentrations of nicardipine for each subject were fitted very well by the Michaelis-Menten equation. An intravenous tracer dose (0.885 mg nicardipine HCl) was administered simultaneously with the final oral dose on the fourth day of the 30 mg q 8 h regimen. The steady-state bioavailability of nicardipine was shown to be dose-dependent and averaged 19 per cent (10 mg), 22 per cent (20 mg), 28 per cent (30 mg), and 38 per cent (40 mg). Nicardipine undergoes linear first-pass metabolism to M5. Other metabolic pathways are responsible for the saturable first-pass metabolism observed for nicardipine.

Capillary column gas chromatographic method using electron-capture detection for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma.

A rapid, specific and direct method based on capillary column gas chromatography with electron-capture detection is described for the simultaneous determination of nicardipine, a new calcium antagonist, and its pyridine metabolite II in human plasma. In this method, the nicardipine, its pyridine metabolite II and internal standard are extracted from the plasma and then partially purified by acid-base partitioning prior to the final injection onto the capillary column gas chromatograph for quantification by means of an electron-capture detector. The quantification limit of the method is 1 ng/ml of plasma for both nicardipine and its pyridine metabolite II. The coefficients of variation for nicardipine and the pyridine metabolite II at concentrations of 1-50 ng/ml are less than 7% and less than 9% (n = 4), respectively. The method has been validated against a previously developed high-performance liquid chromatographic method (sensitivity 5 ng/ml).

A high-performance liquid chromatographic method for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma.

A rapid and specific method in which reverse-phase high-performance liquid chromatography (HPLC) with UV detection was used for the simultaneous determination of nicardipine and its pyridine metabolite II in human plasma is described. Nicardipine, its pyridine metabolite II, and the internal standard were extracted from plasma and partially purified by acid-base partitioning. Final purification and quantitation were achieved by HPLC by using a reverse-phase column and a UV detector (254 nm). The extraction efficiencies for nicardipine and its pyridine metabolite II from 1 mL of plasma were 77.4 and 81.1%, respectively. The sensitivity of the assay was 5 ng/mL for both nicardipine and its pyridine metabolite II, and the linear concentration range of the assay was 5-150 ng/mL for both compounds. The low coefficients of variation (less than or equal to 5%) for samples spiked with nicardipine and its pyridine metabolite II in this concentration range demonstrate good reliability and reproducibility of the assay. The HPLC procedure has been validated by comparison with a GC-electron-capture detection (ECD) procedure, which gives the combined concentration of nicardipine-its pyridine metabolite II (total) and with an HPLC/GC-ECD procedure, which gives the concentration of its pyridine metabolite II. All three methods, which were developed in our laboratory, were used to analyze nicardipine and its pyridine metabolite II in specimens of plasma from subjects treated with nicardipine hydrochloride. Good correlations were found for concentrations of nicardipine, its pyridine metabolite II, and nicardipine plus the metabolite determined by these three procedures. The HPLC procedure is suitable for use in pharmacokinetic studies following administration of nicardipine hydrochloride to humans.

Antibacterial activity of N-(beta-styryl) formamides related to tuberin.

A series of para-substituted N-(beta-styryl)formamides, analogues of tuberin (4a), has been prepared and assayed for antibacterial activity. The methylthio, ethoxy, and methyl analogues 4e, 4j, and 4t were about twice as active as tuberin against Mycobacterium phlei. Although tuberin lacks activity against Staphylococcus aureus, several of the analogues described were found to inhibit this organism. The phenyl group of tuberin is not a prerequisite for activity since analogues based on naphthyl or ferrocenyl groups were also active. A quantitative structure-activity relationship further implied that an aromatic group need not be present, suggesting the synthesis of the cyclohexyl and n-amyl analogues which were found to possess high activity.