An efficient synthesis of the taxane-derived anticancer agent ABT-271.

ABT-271, 1, has been identified as a promising anticancer agent. ABT-271 is a novel taxane possessing a C9-(R)-hydroxyl group as opposed to a C9-ketone which is present in Taxol and Taxotere. To further evaluate ABT-271 as a potential anticancer agent, an efficient synthesis was developed which allows the large scale synthesis of ABT-271. Ketalization of the 7,9-diol of 9-DHAB-III, 2, allows selective removal of the C13-acetate with phenyllithium. The resulting C13-hydroxyl group is then acylated using LiHMDS and beta-lactam 22 to give ABT-271 in protected form. The protecting groups were removed first by acidic hydrolysis followed by basic hydrolysis to provide ABT-271. Application of this synthetic sequence provided over 600 g of ABT-271, 1.

Synthesis of the C-13 side chain precursors of the 9-dihydrotaxane analogue ABT-271.

N-Boc-L-Leucinol was converted to two C-13 side chain precursors of the 9-dihydrotaxane analogue ABT-271. The trans-oxazolidine acid 4 and the cis-Boc-lactam 2b were prepared in 44% and 40% overall yield, respectively, and with excellent (>98%) stereochemical purity.

Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime and diarylvinyl ether derivatives of nipecotic acid and guvacine as anticonvulsant agents.

(3R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid 1 (tiagabine, Gabitril) is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans. This drug, which has a unique mechanism of action among marketed anticonvulsant agents, has been launched for add-on treatment of partial seizures with or without secondary generalization in patients >12 years of age. Using this new agent as a benchmark, we have designed two series of novel GABA uptake inhibitors of remarkable potency, using a putative new model of ligand interaction at the GABA transporter type 1 (GAT-1) uptake site. This model involves the postulated interaction of an electronegative region in the GABA uptake inhibitor with a positively charged domain in the protein structure of the GAT-1 site. These two novel series of anticonvulsant agents contain diaryloxime or diarylvinyl ether functionalities linked to cyclic amino acid moieties and were derived utilizing the new model, via a series of design steps from the known 4,4-diarylbutenyl GABA uptake inhibitors. The new compounds are potent inhibitors of [(3)H]-GABA uptake in rat brain synaptosomes in vitro, and their antiepileptic potential was demonstrated in vivo by their ability to protect against seizures induced by the benzodiazepine receptor inverse agonist methyl 4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) in mice. From structure-activity studies of these new GABA uptake inhibitors, we have shown that insertion of an ether oxygen in conjugation with the double bond in tiagabine (K(i) = 67 nM) improves in vitro potency by 5-fold to 14 nM.

Trypsin-like activity levels of Treponema denticola and Porphyromonas gingivalis in adults with periodontitis.

Treponema denticola (Td) and Porphyromonas gingivalis (Pg) are associated with human moderate and severe adult periodontal diseases. This study quantifies these two anaerobes and their trypsin-like (TL) activities in subgingival plaque collected from both clinically healthy and periodontally diseased sites of human periodontitis patients. Antigen levels of the microorganisms were determined by monoclonal antibodies and TL activities were measured by the fluorescent substrate Z-gly-gly-arg-AFC in a disc format. Significant positive correlations were observed between the antigen levels and the TL activities when the data were subjected to statistical analyses both on a site-specific and on a patient basis. Anaerobe synergism was found between Td and Pg in a continental US population, and positive correlations were found between anaerobe levels (individually and total) and clinical indicators of adult periodontitis.

Treponema denticola and Porphyromonas gingivalis as prognostic markers following periodontal treatment.

Subgingival plaque samples were collected from individuals with advanced periodontitis before and 3 to 11 weeks after scaling and root planing periodontal treatment. The plaque levels of Treponema denticola and Porphyromonas gingivalis antigens were measured before and after treatment by a quantitative immunoassay procedure using monoclonal antibodies specific for these oral bacteria. A decrease in mean levels of T. denticola (P less than .05) and P. gingivalis antigens (P less than .09) were observed following periodontal therapy. Improved health, as measured by a decrease in probing depth, was associated with a decrease in T. denticola antigen (P less than .05). These results suggest that the T. denticola levels of successfully treated sites decreased, while non-responding sites had levels of this microbial marker which were equal to or greater than the pre-treatment levels. These results provide additional evidence that T. denticola is associated with human adult severe periodontal disease, and can serve as a prognostic marker for disease recurrence.

Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population.

Treponema denticola and Porphyromonas gingivalis have been associated with human adult severe periodontitis. In this study, we quantified these putative pathogens in subgingival plaque samples collected from 74 Fijians, 74 Colombians and 73 U.S. Americans stationed at the Multinational Force and Observers encampment in the Sinai Desert, Egypt. A contingency table of T. denticola and P. gingivalis frequency revealed a highly significant synergistic relationship. We discovered that the occurrence of T. denticola apparently requires the presence of P. gingivalis. This represents the first observation of a synergistic relationship between these putative oral pathogens associated with adult severe periodontal disease.

Quantitative immunoassay of Treponema denticola serovar C in adult periodontitis.

Murine monoclonal antibodies specific for Treponema denticola serovar C were produced and characterized in this study. An immunoassay was then developed by using these monoclonal antibodies, and the T. denticola serovar C antigen content of subgingival plaque was quantitated for samples taken from patients with periodontitis and healthy volunteers. The human subgingival plaque samples were grouped by severity of disease and pocket depth measurements at the collection site. The T. denticola serovar C content per milligram of subgingival plaque from deep pockets (greater than 6 mm) of patients with severe periodontitis was found to be twice that of samples collected from deep pockets (4 to 6 mm) of patients with moderate periodontitis or samples collected from healthy subjects (pocket depth, less than 4 mm).

MK886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in ionophore-challenged leukocytes.

Recently, we have shown that ionophore activation of human leukocytes results in leukotriene synthesis and a translocation of 5-lipoxygenase from the cytosol to cellular membrane. This membrane translocation was postulated to be an important early activation step for the enzyme. 3-[1-(p-Chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2- dimethylpropanoic acid (MK886) is a potent and specific inhibitor of leukotriene biosynthesis in vivo and in intact cells, but has no direct effect on 5-lipoxygenase activity in cell-free systems. In this report, we show that MK886 can both prevent and reverse the membrane translocation of 5-lipoxygenase, in conjunction with the inhibition of leukotriene synthesis. Similar compounds of the indole class could also inhibit the membrane translocation of 5-lipoxygenase in a rank order of potency that correlated with their potencies for leukotriene synthesis inhibition. In contrast L-656,224, a direct 5-lipoxygenase inhibitor, had no effect on the translocation of the enzyme. Attempts to demonstrate the effects of MK886 on the association of 5-lipoxygenase with membrane in cell-free preparations failed due to a nonspecific Ca2+-dependent sedimentation of the enzyme. The mechanism of action of MK-886 is therefore to block translocation, prevent subsequent activation of 5-lipoxygenase, and hence block cellular leukotriene biosynthesis.

The affinities of prostaglandin H2 and thromboxane A2 for their receptor are similar in washed human platelets.

Both thromboxane A2 (TXA2) and its precursor prostaglandin H2 (PGH2) are labile and share a common receptor. The affinities of these two compounds for their putative common receptor are unknown. We compared the potencies of TXA2 and PGH2 to aggregate human platelets and bind to the TXA2/PGH2 receptor. TXA2 was more potent than PGH2 in initiating aggregation in platelet-rich plasma, EC50 of 66 +/- 15 nM and 2.5 +/- 1.3 microM, respectively. In washed platelets, however, PGH2 was more potent than TXA2 with EC50 values of 45 +/- 2 nM and 163 +/- 21 nM, respectively. The affinity of these two compounds in washed platelets was determined in radioligand competition binding assays employing [125I]-PTA-OH. The Kd values for PGH2 and TXA2 were 43 nM and 125 nM, respectively. The results demonstrate that the affinity of PGH2 for the platelet TXA2/PGH2 receptor is greater than previously thought. The data raise the possibility that PGH2 may significantly contribute to the responses attributed to TXA2 in vivo.

Quantitative relationship of Treponema denticola to severity of periodontal disease.

The Treponema denticola content of plaque was quantitatively estimated for samples taken from periodontitis patients as well as periodontally healthy subjects among two separate human populations. The populations studied included military volunteers and civilians at a university dental clinic. The plaque samples from each population were grouped according to pocket depth measurements at the collection site. A biotin-avidin enzyme-linked immunosorbent assay procedure was developed with a monoclonal antibody specific for a serovariety of T. denticola. T. denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to the healthy controls and a group with moderate disease. The ratio of T. denticola content per milligram of plaque in the deep pocket groups to that of the other two groups was about 2:1 for both populations. This is the first quantitative evidence of a positive relationship between a specific spirochete species and severe periodontitis.

Effect of hyperbaric oxygen exposure on oxygen tension within the medullary canal in the rabbit tibial osteomyelitis model.

The effect on intramedullary oxygen tension of 100% oxygen exposure at 1, 2, 2.4, and 3 atm pressure was studied in 12 New Zealand white rabbits with chronic right tibial osteomyelitis. The model, modified from that described by others, incorporates a multipuncture silastic closure plug placed transcortically in the proximal tibial metaphysis through which platinum needle, polarographic electrode oxygen tension determinations can be made without repeat surgical exposure. In 40% of the control, left, noninfected tibial metaphyses the baseline oxygen tension with the animals breathing room air at sea level was suboptimal for leukocyte bacterial killing. This oxygen tension was depressed further in the infected right tibia. Medullary canal oxygen tension increased in response to hyperbaric oxygen exposure in both the infected and noninfected tibiae. Whereas the amount of the oxygen tension increase varied with the presence of infection and depth of dive, neither the time for oxygen tension to plateau nor the time required for return to baseline tension after completion of hyperbaric oxygen exposure varied with the presence of infection or depth of dive. After completion of hyperbaric oxygen exposure, the oxygen tension within the medullary canal returned to baseline within 15 min.

Intestinal myoelectric activity in response to live Vibrio cholerae and cholera enterotoxin.

The myoelectric response of the rabbit ileum was studied in response to live Vibrio cholerae culture, a whole cell lysate of cholera, and the purified enterotoxin. Each cholera preparation produced a series of highly organized migrating action potential complexes (MAPC). An MAPC was defined as action potential discharge with a duration of 2.5 s or longer, followed by similar activity on at least one other consecutive electrode site. The mean and modal onset time of MAPC activity occurred 4 h after the infection with live Vibrio cholerae culture, the freeze-dried whole cell lysate preparation, or the purified enterotoxin. After the onset of activity this pattern persisted for the duration of the recording period (up to 12 h). The MAPC had a mean propagation velocity of 0.85+/-0.07 cm/s (mean+/-SEM), which remained constant with time. Direct visual observation of the loop revealed that the MAPC's resulted in contractions that propelled intraluminal contents in an aborad direction. The mean fluid output from the 12-cm ileal loops was 6.4+/-1.1 ml/h (mean+/-SEM). Control experiments consisted of recordings from: (a) a ligated ileal loop into which nothing was placed; (b) a ligated ileal loop into which either uninfected culture broth or 0.9% NaCl solution was injected; (c) a ligated ileal loop infused with 0.9% NaCl solution at a rate of 11.2 ml/h, and (d) rapid injection of 1.0, 2.5, 5.0, or 10.0-ml boluses of 0.9% NaCl into the proximal catheter. MAPC activity was not observed in any of the control experiments. These studies indicate that in addition to a secretory component to cholera, there exists a highly organized MAPC that results in contractions that propel intraluminal contents in an aborad direction.

Stable mycoplasma antigen preparations for indirect hemagglutination tests.

In immunological studies of mycoplasmas, the use of glutaraldehyde for the fixative makes it possible to use erythrocytes from commercially available defibrinated sheep blood. It eliminates the necessity of having to screen blood from individual sheep to obtain a suitable source of erythrocytes, as when employing tannic acid for fixation and sensitization. The chemical bonding of soluble mycoplasma proteins to glutaraladehyde-fixed sheep erythrocytes by bis-diazotized 3,3'dimethoxy derivative, benzidine, yields preparations that are satisfactory antigens for performing the indirect hemagglutination test by the microtiter technique. The antigenic preparations are satisfactory for use after storage at 4 or -10 C for many months. Incorporation of 5% glycerine in the final suspending milieu makes it possible to obtain uniform suspensions of the fixed and sensitized sheep erythrocytes after freezing and after repeated freezing and thawing. Proteins from Mycoplasma arthritidis and M. hominis have been coupled to glutaraldehyde-fixed erythrocytes by diazotization. The last mentioned preparation detected the presence of antibodies in titers greater than 1:10 in 37% of 237 pregnant women whose ages ranged between 20 and 30 years. There was no correlation between the presence of specific antibodies in the blood and the isolation of M. hominis from the cervical canal.

Coagulase and deoxyribonuclease activities of staphylococci isolated from clinical sources.

A total of 504 clinical isolates of the family Micrococcaceae were tested for coagulase, deoxyribonuclease, clumping factor, and phosphatase to determine whether there is a correlation between the results of these tests and the pathogenicity of staphylococci. In the tests for coagulase production, it was found that either human or rabbit plasma could be used with broth cultures, whereas rabbit but not human plasma was satisfactory when microorganisms removed from solid culture medium were used. Deoxyribonuclease production correlated better than the fermentation of mannitol with coagulase production. The use of methyl green, Toluidine Blue O, or acridine orange offered no advantage over the use of HCl for detecting the production of deoxyribonuclease. Neither the presence of clumping factor nor the production of phosphatase correlated well with coagulase production. Strains of staphylococci that did not produce coagulase and deoxyribonuclease were isolated as frequently as, and from a greater variety of clinical sources than, strains which produced these substances. It is concluded that the production of coagulase and deoxyribonuclease are properties of staphylococci which are not necessarily indicative of potential pathogenicity of the organisms for man.

Effect of storage of Mueller-Hinton agar plates on zone sizes for antimicrobial testing.

The length of time Mueller-Hinton agar plates can be stored at 4 C without affecting the size of zones of inhibition in susceptibility testing by the Bauer-Kirby method was studied. It was found that these plates can be stored for 3 weeks at 4 C without an appreciable affect on zone sizes. Storage of plates in sealed plastic bags did not alter the results significantly. The findings indicate that commercially prepared Mueller-Hinton agar plates, which may be several days old when received at the laboratory, are suitable for use in routine susceptibility tests by the Bauer-Kirby method.