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Biomedical subjects

T D Gootz

Publications and source records attributed to T D Gootz.

At least 37 records · Page 2Linked to original sources
PubMed1994

Placement of alkyl substituents on the C-7 piperazine ring of fluoroquinolones: dramatic differential effects on mammalian topoisomerase II and DNA gyrase.

Several substituted analogs of 7-(cis-3,5-dimethylpiperazinyl)-6,8-difluoro-5-amino-1-cyclopropyl quinolone were prepared and tested in a DNA cleavage assay with calf thymus topoisomerase II. Positioning of the methyl groups on the C-7 piperazine ring influenced potency against the mammalian enzyme; the cis-3,5-dimethyl configuration did not stimulate cleavage at drug concentrations less than or equal to 2,000 microM, while the trans configuration was active at drug levels as low as 36 microM. Removal of the cis-methyl groups produced a compound that was only sixfold less potent than the antitumor agent etoposide in stimulating enzyme-mediated DNA cleavage. The cis- and trans-methyl substitutions on the piperazine that conferred potency against the mammalian type II enzyme had little effect on bacterial DNA gyrase cleavage activity, suggesting that an asymmetric barrier exists with the mammalian enzyme which influences productive quinolone interaction, favoring the less bulky trans-3,5-dimethylpiperazine substituent at C-7.

Animals↗
PubMed1994

Immunological and molecular characterization of Helicobacter felis urease.

Urease activity has recently been shown to be an important virulence determinant for Helicobacter pylori, allowing it to survive the low pH of the stomach during colonization. Experimental murine infection with Helicobacter felis is now being used as a model for H. pylori infection to study the effects of vaccines, antibiotics, and urease inhibitors on colonization. However, little information comparing the ureases of H. felis and H. pylori is available. Urease was partially purified from the cell surface of H. felis ATCC 49179 by A-5M agarose chromatography, resulting in an eightfold increase in specific activity over that of crude urease. The apparent Km for urea for the partially purified urease was 0.4 mM, and the enzyme was inhibited in a competitive manner by flurofamide (50% inhibitory concentration = 0.12 microM). Antiserum to whole cells of H. pylori recognized both H. pylori and H. felis urease B subunits. Antiserum raised against H. felis whole cells recognized the large and small autologous urease subunits and the cpn60 heat shock molecule in both H. felis and H. pylori. However, this antiserum showed only a weak reaction with the B subunit of H. pylori urease. Two oligomeric DNA sequences were used as probes to evaluate the relatedness of H. felis and H. pylori urease gene sequences. One 30-mer from the ureA sequence, which had been shown previously to be specific for H. pylori, failed to hybridize to H. felis genomic DNA. A probe to the putative coding sequence for the active site of the H. pylori ureB subunit hybridized at low intensity to a 2.8-kb fragment of BamHI-HindIII-digested H. felis DNA, suggesting that the sequences were homologous but not identical, a result confirmed from the recently published sequences of ureA and ureB from H. felis.

Base Sequence↗
PubMed1994

Bisulfite or sulfite inhibits growth of Helicobacter pylori.

Bisulfite or sulfite was found to be inhibitory to Helicobacter pylori growth. A modified version of Brucella broth (BB), bisulfite-less BB (BLBB), supported rapid, robust, and consistent growth of H. pylori. We suggest that BLBB simply be called "Pylori broth" for distinction from Brucella broth.

Bacteriological Techniques↗
PubMed1993

Drug features that contribute to the activity of quinolones against mammalian topoisomerase II and cultured cells: correlation between enhancement of enzyme-mediated DNA cleavage in vitro and cytotoxic potential.

CP-115,953 [6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid] is a novel quinolone that is highly active against topoisomerase II in vitro and in mammalian cells in culture (M. J. Robinson, B. A. Martin, T. D. Gootz, P. R. McGuirk, M. Moynihan, J. A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991). However, the features of the drug that contribute to its activity towards mammalian systems have not been characterized. Therefore, CP-115,953 and a series of related quinolones were examined for their activity against calf thymus topoisomerase II and cultured mammalian cells. CP-115,953 stimulated DNA cleavage mediated by the type II enzyme with a potency that was approximately 600-fold greater than that of the antimicrobial quinolone ciprofloxacin and approximately 50-fold greater than that of the antineoplastic drug etoposide. As determined by the ability to enhance enzyme-mediated DNA cleavage, quinolone activity towards calf thymus topoisomerase II was enhanced by the presence of a cyclopropyl group at the N-1 ring position and by the presence of a fluorine at C-8. Furthermore, the 4'-hydroxyphenyl substituent at the C-7 position was critical for the potency of CP-115,953 towards the mammalian type II enzyme. In this regard, the aromatic nature of the C-7 ring as well as the presence and the position of the 4'-hydroxyl group contributed greatly to drug activity. Finally, the cytotoxicity of quinolones in the CP-115,953 series towards mammalian cells paralleled the in vitro stimulation of DNA cleavage by topoisomerase II rather than the inhibition of enzyme-catalyzed DNA relaxation. This correlation strongly suggests that these quinolones promote cell death by converting topoisomerase II to a cellular poison.

Animals↗
PubMed1993

Novel 1-8-bridged chiral quinolones with activity against topoisomerase II: stereospecificity of the eukaryotic enzyme.

A series of novel C-7 quinolyl-substituted enantiomers of ofloxacin were used to determine the stereospecificity of topoisomerase II for the C-11 methyl group in tricyclic quinolones. In all cases, the S isomer was the most active compound against the eukaryotic enzyme. It was approximately 2.2-fold more potent than the R isomer at inhibiting the overall catalytic activity of topoisomerase II (as monitored by DNA relaxation assays). A markedly greater difference in quinolone activity was observed in enzyme-mediated DNA cleavage reactions. While the S enantiomer stimulated nucleic acid breakage approximately 3.5-fold, the R compound did not enhance and, in fact, decreased initial DNA cleavage levels by approximately 50%. The activity of the racemic mixture more closely resembled that of the R enantiomer. In competition experiments, the DNA cleavage-enhancing effects of the S isomer were attenuated by the R compound. Taken together, these latter results indicate that the R enantiomer is an antagonist of S isomer-promoted topoisomerase II-mediated DNA cleavage. Finally, the cytotoxic potential of quinolyl-substituted ofloxacin analogs correlated with the ability to stimulate enzyme-mediated DNA cleavage. Thus, stereochemistry appears to be a governing factor for the potential development of tricyclic quinolones as topoisomerase II-targeted drugs with antineoplastic activity.

Animals↗
PubMed1992

Synthesis and structure-activity relationships of 7-diazabicycloalkylquinolones, including danofloxacin, a new quinolone antibacterial agent for veterinary medicine.

A series of novel 6-fluoro-7-diazabicycloalkylquinolonecarboxylic acids substituted with various C8 (H, F, Cl, N) and N1 (ethyl, cyclopropyl, vinyl, 2-fluoroethyl, 4-fluorophenyl, 2,4-difluorophenyl) substituents, as well as, 9-fluoro-10-diazabicycloalkylpyridobenzoxazinecarboxylic acids, were prepared and evaluated for antibacterial activity against a range of important veterinary pathogenic bacteria. The diazabicycloalkyl side chains investigated at the 7-position (benzoxazine 10-position) include (1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptane (2), (1S,4S)-2,5-diazabicyclo[2.2.1]heptane (3), (1R,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptane (4), 8-methyl-3,8-diazabicyclo[3.2.1]octane (5), 9-methyl-3,9-diazabicyclo[4.2.1]nonane (6), 1,4-diazabicyclo[3.2.2]nonane (7), 1,4-diazabicyclo[3.3.1]nonane (8), and 9-methyl-3,9-diazabicyclo[3.3.1]nonane (9). Among these side chains, in vitro potency was not highly variable; other properties therefore proved more critical to the selection of possible development candidates. However, the relative potencies observed for several of these compounds in mouse, swine, and cattle infection models correlated well with those seen in vitro. A combination of the N1 cyclopropyl group and the C7 (1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]hept-2-yl appendage conferred the best overall antibacterial, physiochemical, and pharmacodynamic properties. Hence, danofloxacin (Advocin, 2c) (originally CP-76,136, 1-cyclopropyl-6-fluoro-7-[(1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1] hept-2-yl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid) was selected as a candidate for development as a therapeutic antibacterial agent for veterinary medicine.

Actinobacillus Infections↗
PubMed1992

High-level quinolone resistance in clinical isolates of Campylobacter jejuni.

During a recent clinical trial of ciprofloxacin in the therapy of acute diarrhea, two subjects infected with Campylobacter jejuni who received ciprofloxacin failed microbiologically and one also failed clinically. Although both pretreatment isolates were susceptible to ciprofloxacin, the posttreatment isolates were resistant to ciprofloxacin (MIC = 32 micrograms/ml) and to other quinolones. The posttreatment isolates remained susceptible to nonquinolone antimicrobials. DNA gyrase holoenzyme was isolated from one of the resistant posttreatment isolates and was 8- to 16-fold less sensitive to inhibition by ciprofloxacin than was the gyrase from the paired pretreatment susceptible isolate. Ciprofloxacin accumulation was diminished in the two resistant posttreatment isolates. These results show that mutation in C. jejuni can occur in vivo and is associated with clinically significant resistance to the newer quinolones.

Anti-Infective Agents↗
PubMed1992

Effects of novel fluoroquinolones on the catalytic activities of eukaryotic topoisomerase II: Influence of the C-8 fluorine group.

A previous study (M.J. Robinson, B.A. Martin, T.D. Gootz, P.R. McGuirk, M. Moynihan, J.A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991) demonstrated that novel 6,8-difluoroquinolones were potent effectors of eukaryotic topoisomerase II. To determine the contribution of the C-8 fluorine to drug potency, we compared the effects of CP-115,955 [6-fluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] on the enzymatic activities of Drosophila melanogaster topoisomerase II with those of CP-115,953 (the 6,8-difluoro parent compound of CP-115,955). Removal of the C-8 fluoro group decreased the ability of the quinolone to enhance enzyme-mediated DNA cleavage approximately 2.5-fold. Like its difluorinated counterpart, CP-115,955 increased the levels of cleavage intermediates without impairing the DNA religation reaction of the enzyme. Removal of the C-8 fluorine reduced the ability of the quinolone to inhibit topoisomerase II-catalyzed DNA relaxation. In addition, the cytotoxicity of CP-115,955 towards Chinese hamster ovary cells was decreased compared with that of CP-115,953. These results demonstrate that the C-8 fluorine increases the potency of quinolone derivatives against eukaryotic topoisomerase II and mammalian cells. Further comparisons of CP-115,955 with CP-115,953 and CP-67,804 (the N-1 ethyl-substituted derivative of the difluoro parent compound) indicate that the two intrinsic activities of quinolone-based drugs towards topoisomerase II (i.e., enhancement of DNA cleavage and inhibition of catalytic strand passage) can be differentially influenced by alteration of ring substituents. Finally, correlations between the biochemical and cytological activities of these drugs suggest that the ability to inhibit catalytic strand passage enhances the cytotoxic potential of quinolones towards eukaryotic cells.

Animals↗
PubMed1992

Pharmacokinetic studies of CP-74,667, a new quinolone, in laboratory animals.

The pharmacokinetics of CP-74,667 (7-(8'-methyl-3',8'-diazabicyclo[3.2.1]oct-3'-yl)-1-cyclopropyl-6- fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid) were studied following oral or parenteral administration in mice, rats, rabbits, dogs, and cynomolgus monkeys. The mean peak levels of CP-74,667 in serum following a single oral dose of 20 mg/kg of body weight were similar in all species, with a range of 3.7 micrograms/ml in mice to 5.6 micrograms/ml in dogs. In contrast, elimination half-lives were species dependent, with mean values of 2.1, 1.8, 4.5, 7.8, and 13.1 h in mice, rats, rabbits, dogs, and monkeys, respectively. The oral bioavailability of CP-74,667 was 100% in dogs and monkeys, as determined by intravenous-oral crossover experiments. The maximum concentration of drug in serum and area under the concentration-time curve (AUC) of CP-74,667 in dogs were proportional to dose over the range of 5 to 40 mg/kg. Accumulation of drug in serum was observed following the administration of four once-a-day doses of 7.1 mg/kg in monkeys (mimicking a 500-mg human dose), with significant increases in half-life, maximum and minimum concentrations of drug in serum, and AUC. The good tissue penetration of CP-74,667 suggested by a volume of distribution in excess of 2 liters/kg in dogs and monkeys was confirmed by tissue distribution studies with the same species, which demonstrated tissue concentrations (except for those in brain tissue) greater than 1.45 times higher than corresponding levels in serum. The mean urinary recoveries of unchanged drug were 17.7% in rats, 7.8% in monkeys, and 4.9% in dogs. Metabolism studies in dogs, following intravenous dosing, indicated that renal excretion of CP-74,667-related materials accounted for 41.6% of the administered dose, while biliary recoveries accounted for 6.8%. The CP-74,667 N-oxide metabolite was the primary drug-related material eliminated via renal excretion (37.2% of dose). The pharmacokinetics of CP-74,667 describe a quinolone with complete oral absorption, linear pharmacokinetics, a long elimination half-life, and wide distribution into tissues.

Administration, Oral↗
PubMed1991

Effects of quinolone derivatives on eukaryotic topoisomerase II. A novel mechanism for enhancement of enzyme-mediated DNA cleavage.

The effects of two novel quinolone derivatives, CP-67,804 and CP-115,953 (the 1-ethyl and 1-cyclopropyl derivatives of 6,8-difluoro-7-(4-hydroxyphenyl)-4-quinolone-3-carboxylic acid, respectively), on the enzymatic activities of Drosophila melanogaster topoisomerase II were examined. Both drugs enhanced the enzyme's pre- and post-strand passage DNA cleavage activities. CP-67,804 was nearly as potent an enhancer as etoposide, while CP-115,953 was approximately 2 times more potent than this topoisomerase II-targeted antineoplastic drug. In contrast to etoposide, which stabilizes enzyme-DNA cleavage complexes primarily by inhibiting topoisomerase II-mediated DNA religation, neither quinolone impaired the enzyme's ability to religate cleaved DNA. To further assess the characteristics of these unusual quinolone derivatives, the cytotoxic effects of CP-67,804 and CP-115,953 toward wild-type Chinese hamster ovary cells and VpmR-5 cells (an epipodophyllotoxin-resistant Chinese hamster ovary line) were examined. Both quinolones were cytotoxic to the wild-type cells. CP-115,953 was the more potent agent and displayed a level of cytotoxicity similar to that of etoposide. Finally, the VpmR-5 line showed cross-resistance to CP-67,804 (approximately 3.7-fold) and CP-115,953 (approximately 1.3-fold). Although quinolone cross-resistance was less pronounced than observed for etoposide (approximately 12-fold), it indicates that topoisomerase II is a physiological target for CP-67,804 and CP-115,953 in mammalian cells. These findings strongly suggest that these quinolone derivatives represent a novel class of topoisomerase II-targeted drugs which have potential as antineoplastic agents.

Animals↗
PubMed1991

Characterization of high-level quinolone resistance in Campylobacter jejuni.

High-level resistance to quinolones has previously been shown to occur in Campylobacter spp. both in vitro and in patients treated with quinolones. We have selected isolates that are resistant to quinolones by plating cells from a susceptible C. jejuni strain, UA535, on medium containing nalidixic acid at 32 micrograms/ml. Fluctuation analysis indicated that resistance occurred by mutation at a frequency of 5 x 10(-8) per cell plated. Unlike what is observed with other gram-negative organisms, the nalidixic acid-resistant mutants demonstrated high-level cross-resistance (MIC, greater than or equal to 4 micrograms/ml) to newer quinolones, including ciprofloxacin, norfloxacin, and temafloxacin, yet remained susceptible to coumermycin A1 and several other unrelated antibiotics. Mutants with an identical resistance phenotype could also be selected from UA535 with ciprofloxacin and norfloxacin at a similar frequency. To study the mechanism of quinolone resistance, DNA gyrases were purified from C. jejuni UA535 and two resistant mutants by heparin-agarose and novobiocin-Sepharose chromatography. After the respective enzyme concentrations were adjusted to equivalent units of activity in the DNA supercoiling reaction, the DNA gyrases from the resistant mutants were found to be 100-fold less susceptible than the wild-type enzyme to inhibition by quinolones. Subunit switching experiments with purified A and B subunits from the wild type and one of the quinolone-resistant mutants indicated that an alteration in the A subunit was responsible for resistance. These results show that a single-step mutation can occur in vitro in the gene encoding DNA gyrase in C. jejuni, producing clinically relevant levels of resistance to the newer quinolones.

Anti-Infective Agents↗
PubMed1990

Discovery and development of new antimicrobial agents.

The unprecedented growth in the number of new antibiotics over the past two decades has been the result of extensive research efforts that have exploited the growing body of knowledge describing the interactions of antibiotics with their targets in bacterial cells. Information gained from one class of antimicrobial agents has often been used to advance the development of other classes. In the case of beta-lactams, information on structure-activity relationships gleaned from penicillins and cephalosporins was rapidly applied to the cephamycins, monobactams, penems, and carbapenems in order to discover broad-spectrum agents with markedly improved potency. These efforts have led to the introduction of many new antibiotics that demonstrate outstanding clinical efficacy and improved pharmacokinetics in humans. The current review discusses those factors that have influenced the rapid proliferation of new antimicrobial agents, including the discovery of new lead structures from natural products and the impact of bacterial resistance development in the clinical setting. The development process for a new antibiotic is discussed in detail, from the stage of early safety testing in animals through phase I, II, and III clinical trials.

Animals↗
PubMed1989

Use of in vitro topoisomerase II assays for studying quinolone antibacterial agents.

Several quinolones and antitumor compounds were tested as inhibitors of purified calf thymus topoisomerase II in unknotting, catenation, radiolabeled DNA cleavage, and quantitative nonradiolabeled cleavage assays. The antitumor agents VP-16 (demethylepipodophyllotoxin ethylio-beta-D-glucoside) and ellipticine demonstrated drug-enhanced topoisomerase II DNA cleavage (the concentration of drug that induced 50% of the maximal DNA cleavage in the test system [CC50]) at levels of less than or equal to 5 micrograms/ml. Nalidixic acid, norfloxacin, and oxolinic acid did not induce significant topoisomerase II DNA cleavage, whereas ciprofloxacin did induce some cleavage above background levels. CP-67,015, a new 6,8-difluoro-7-pyridyl 4-quinolone which possesses potent antibacterial activity, inhibited bacterial DNA gyrase at 0.125 micrograms/ml in a nonradioactive DNA cleavage assay. Unlike other quinolones characterized to date, CP-67,015 was shown to strongly enhance topoisomerase II-induced radiolabeled DNA cleavage with a CC50 of 33 micrograms/ml and demonstrated cleavage in a nonradiolabeled DNA cleavage assay with a CC50 of 73 micrograms/ml. The topoisomerase II-mediated cleavage of DNA by CP-67,015 is consistent with its reported clastogenic effect on DNA in cell culture and its positive mutagenic response in mouse lymphoma cells. In vitro topoisomerase II catalytic and cleavage assays are useful for gaining preliminary information concerning the possible interaction(s) of some quinolones with eucaryotic topoisomerase II which may relate directly to their safety (mutagenicity, clastogenicity, or both) in human and veterinary medicinal usage.

4-Quinolones↗
PubMed1988

Simple spectrophotometric assay for measuring protein binding of penem antibiotics to human serum.

The binding of antibiotics to plasma (serum) proteins through hydrogen bonding can significantly influence the biological characteristics of these drugs. A rapid spectrophotometric assay has been developed that measures the level of free (unbound) penem antibiotic in serum ultrafiltrates. Whole human serum was adjusted to a standard concentration of antibiotic and then filtered by centrifugation through a Centrifree (Amicon Corp., Lexington, Mass.) filter that retained greater than 99.9% of serum protein. The degree of penem protein binding was determined spectrophotometrically by measuring the level of unbound drug in the ultrafiltrate at 322 nm. At this wavelength, no interfering absorption from residual protein was detected in the ultrafiltrate, and penem absorption was linear over a wide concentration range. The method gave protein-binding values comparable to those obtained by a high-pressure liquid chromatography assay but was more rapid, since it did not require solvent extraction and high-pressure liquid chromatography calibration procedures. The spectrophotometric assay has been used to assay over 100 penems to determine the structure-activity relationships that are involved with the high serum protein binding of these agents. As with penicillins and some cephalosporins, the nonpolar nature of the penem side chain at the C-2 position strongly influenced the degree of penem binding to serum proteins.

Anti-Bacterial Agents↗
PubMed1987

Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution.

Azithromycin (CP-62,993), a new acid-stable 15-membered-ring macrolide, was well absorbed following oral administration in mice, rats, dogs, and cynomolgus monkeys. This compound exhibited a uniformly long elimination half-life and was distributed exceptionally well into all tissues. This extravascular penetration of azithromycin was demonstrated by tissue/plasma area-under-the-curve ratios ranging from 13.6 to 137 compared with ratios for erythromycin of 3.1 to 11.6. The significance of these pharmacokinetic advantages of azithromycin over erythromycin was shown through efficacy in a series of animal infection models. Azithromycin was orally effective in treating middle ear infections induced in gerbils by transbulla challenges with amoxicillin-resistant Haemophilus influenzae or susceptible Streptococcus pneumoniae; erythromycin failed and cefaclor was only marginally active against the H. influenzae challenge. Azithromycin was equivalent to cefaclor and erythromycin against Streptococcus pneumoniae. In mouse models, the new macrolide was 10-fold more potent than erythromycin and four other antibiotics against an anaerobic infection produced by Fusobacterium necrophorum. Similarly, azithromycin was effective against established tissue infections induced by Salmonella enteritidis (liver and spleen) and Staphylococcus aureus (thigh muscle); erythromycin failed against both infections. The oral and subcutaneous activities of azithromycin, erythromycin, and cefaclor were similar against acute systemic infections produced by Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, or S. aureus, whereas azithromycin was more potent than erythromycin and cefaclor against the intracellular pathogen Listeria monocytogenes. The pharmacokinetic advantage of azithromycin over erythromycin in half-life was clearly demonstrated in prophylactic treatment of an acute mouse model of S. aureus infection. These properties of azithromycin strongly support the further evaluation of this new macrolide for use in community-acquired infections of skin or soft tissue and respiratory diseases.

Animals↗