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Antibacterial properties of pipemidic acid
Abstract
Pipemidic acid, 8-ethyl-5, 8-dihydro-5-oxo-2- (1-piperazinyl) -pyrido [2, 3-d] pyrimidine-6-carboxylic acid, is a new derivative of piromidic acid. It was active against gram-negative bacteria including Pseudomonas aeruginosa as well as some gram-positive bacteria. Its potency against gram-negative bacteria was generally greater than that of piromidic acid, nalidixic acid and cephalexin. Crossresistance was not observed between pipemidic acid and various antibiotics, and most of bacteria resistant to piromidic acid and nalidixic acid were moderately susceptible to pipemidic acid.
The activity of pipemidic acid was scarcely affected by the addition of serum, sodium cholate or change of medium pH, but was subject to the influence of inoculum size. Its action was bactericidal above minimal inhibitory concentrations. Additive effect was observed when pipemidic acid was used in combination with carbenicillin or gentamicin. Pipemidic acid was effective by the oral route on various experimental infections in mice. Its in vivo efficacy against gram-negative bacteria was generally better than that of piromidic acid, nalidixic acid, cephalexin, ampicillin and carbenicillin. The infection with bacteria resistant to piromidic acid and nalidixic acid was successfully treated with pipemidic acid.
In Pseudomonas aeruginosa infections, orally administered pipemidic acid was more effective than subcutaneously injected carbenicillin.
The effect of pipemidic acid on the fecal flora was investigated in 10 normal healthy adults receiving the drug for five days. Fecal specimens were cultured quantitatively for aerobic and anaerobic bacteria before, during and after treatment.
The bulk of predominant anaerobic bacteria in the fecal flora were not changed during the pipemidic acid treatment.The count of enterobacteria during the treatment was significantly (P<0.001) lower than that before and after treatment. There was marked suppression of lecithinase-positive clostridia and veillonellae. By one week after the drug treatment the normal fecal flora in all subjects was immediately recovered. No positive culture of Clostridium diffielle was obtained from all the specimens throughout the experimental course.No subjects developed diarrhoea or other side effects.
Laboratory and clinical studies were made on pipemidic acid and the following results were obtained.
1. Peak blood levels were 9.5-20μg/ml 1 hour after oral administration of pipemidic acid at doses of 10 and 25 mg/kg.
2. Pipemidic acid was administered at a daily dose of about 50 mg/kg to 17 cases of urinary tract infections, 1 case of cholecystitis after operation of congenital bile duct atresia and 2 cases of lymphadenitis colli purulenta bearing skin abscess. Clinical results were excellent or good in 14 of 17 cases of urinary tract infections, good in 1 case of cholecystitis and good in 1 of 2 cases of lymphadenitis colli purulenta bearing skin abscess.
The rate of clinical effectiveness was 80% in a total of 20 cases treated. Causative organisms disappeared in 13 cases but were maintained in 3 cases except for 4 unknown cases.
In all cases, there were no untoward side effects regarding symptoms, hematological analysis and renal and hepatic functional tests.
Pipemidic acid (PPA) was well absorbed by the oral route. When administered to mice, rats, dogs, monkeys and men at a single oral dose of about 50 mg/kg, average plasma levels reached peaks ranging between 4.7 and 11.9 µg/ml. About 20% of PPA was bound to proteins in dog plasma and about 30% in human serum. PPA was widely distributed to various organs and tissues at concentrations comparable to or higher than the plasma level. Peak urine levels of PPA were around 1,000 µg/ml in animals and men, and 24 to 88% of administered doses was recovered from urine for 24 hours. An average recovery from feces was about 25% in men receiving a single oral dose of 1 g. A main active principle in plasma and urine was unchanged PPA itself.
In order to evaluate the efficacy, safety and usefulness of pipemidic acid (PPA) in complicated urinary-tract infections, a controlled study using carindacillin (I-CBPC) as a reference standard drug was carried out by a double blind method in collaboration with 48 clinics throughout the country. From the results obtained, the following conclusions were drawn.
1. In total of 586 patients, 55 cases were excluded, 38 cases dropped out and 493 cases which consisted of 249 cases given PPA and 244 cases given I-CBPC.
2. For the background characteristics, there were no statistically significant differences between PPA and I-CBPC, indicating that comparison between these drugs was meaningful.
3. The daily dose for both PPA and I-CBPC was 2g each and medication was continued for 7 days.
As to the overall clinical efficacy, the rate of “excellent” response was found to be 21.3% with PPA and 16.0% with I-CBPC without significant difference between the two groups. The rate of both “excellent” and “good” response was 54.2% with PPA and 35.7% with I-CBPC and the former was significantly higher than the latter.
4. The rate of efficacy on pyuria was found to be 34.1% with PPA and 24.2% with I-CBPC in respect to “cleard” response and 49.8% with PPA and 37.7% with I-CBPC in respect to both “cleared” and “decreased” responses. The rates of PPA were significantly higher than those of I-CBPC.
5. The rate of efficacy on bacteriuria was found to be 43.4% with PPA and 25.4% with I-CBPC in respect to “eliminated” response and 47.0% with PPA and 29.1% with I-CBPC in respect to both “eliminated” and “suppressed” responses. The rates of PPA were also significantly higer than those of I-CBPC.
6. When stratified according to the rate of, infection, it was remarkable that the effective rate of PPA on the prostatic bed infection was significantly higher than that of I-CBPC.
When analyzed according to the type of infection classified by UTI-Committee, the rates of efficacy of PPA on all types of infection except type I and V were more than 50%. In even type I and V, the drop of the rate of efficacy of PPA was less than that of I-CBPC.
The results indicated that PPA was expected to be more or less effective on all types of complicated urinary-tract infections.
7. PPA eliminated more than 40% of causative organisms, suggesting that it might be effective on any organism inducing complicated urinary-tract infections.
It was remarkable that the eradication ratio of PPA for E. coli, indole negative and positive Proteus, and Citrobacter were more than 90%.
No significant difference between PPA and I-CBPC was shown in the rate of disappearance of Pseudomonas.
8. In the global judgement made by attending physicians, the rates of efficacy and usefulness of PPA were significantly higher than those of I-CBPC.
9. Incidence of side effect was 9.4% and 7.6% in PPA and I-CBPC respectively without significant difference between them.
As side effects, symptoms were mainly gastric disturbances with a few allergic responses.
Neither PPA nor I-CBPC showed serious side effect.
From the above results, PPA would be a useful drug for the treatment of complicated urinary-tract infections, which may be applied broadly.
The acute toxicity of pipemidic acid (PPA) was examined in mice, rats, dogs, monkeys and neonatal rats with following results.
animal route sex LD50 (mg/kg)
mouse i. v. male 610
female 649
s. c. male 1, 274
female 1, 516
p. o. male >5, 000
female >5, 000
animal route sex LD50 (mg/kg)
rat i. v. male 575
female 584
s. c. male 1, 635
female 1, 799
p. o. male >5, 000
female >5, 000
animal route sex LD50 (mg/kg)
dog p. o. male >2, 000
female >2, 000
monkey p. o. male >2, 000
female >2, 000
neonatal p. o. male 1, 597
rat female 2, 156
Intravenous administration of PPA near the mean lethal doses caused tonic convulsion, mydriasis, ataxia, muscle relaxation, a decrease in spontaneous locomotion and interrupted respiration.
Subcutaneous administration of PPA near the mean lethal doses also induced sedation, ataxia, ptosis, a decrease in spontaneous locomotion, a drop in body temperature, paralysis of respiration, etc. On the other hand, oral administration of PPA did not cause any toxic signs even at high doses in all the animals examined except for a slight decrease in the body weight gain of neonatal rats.
It seems that there is no significant difference between animal species or sexes in the acute toxicity of PPA.
Subacute and chronic toxicity studies on pipemidic acid (PPA) were performed in male and female rats.
In rats given PPA at daily oral doses of 100, 300, 1, 000, 3, 000 and 5, 000 (male only) mg/kg/day for 30 days, no abnormalities were observed except for a slight decrease in weights of heart, liver and thymus, and the dilatation of cecum at high doses. The decrease in organ weight was not accompanied by histopathological abnormalities, and the dilatation of cecum was similar to that commonly observed after long-term administration of antibiotics.
In rats given PPA at daily oral doses of 50, 200, 800 and 3, 200 mg/kg/day for 6 months, no abnormalities were observed except for a slight variation in body weight gain and the dilatation of cecum. These results indicate that the oral toxicity of PPA is low in rats.
Sensitivity of various clinical isolates to pipemidic acid, a new synthetic antibacterial agent developed at Research Laboratories, Dainippon Pharmaceutical Co., Ltd., was examined in vitro with the following results.
1. Pipemidic acid showed potent antibacterial activity primarily against gram-negative bacteria.
2. The MIC values of pipemidic acid for Pseudomonas aeruginosa ranged around 12.5 μg/ml, and those for nalidixic acid-resistant bacteria around 25 μg/ml.
3. Most of the strains of Escherichia coli and Klebsiella sp. were inhibited by 3.13 μg of pipemidic acid per ml or lower, but a few Klebsiella strains required 25 μg of the drug per ml to be inhibited.
Disposition and biotransformation of pipemidic acid (PPA) were studied in rats and mice orally given a single or multiple doses of 50 mg ¹⁴C-PPA/kg/day with the following results.
1) After a single dosing to rats and mice, ¹⁴C-PPA was absorbed rapidly to give maximum blood levels (5.0 μg Eq/ml in rats and 7. 7 ug Eq/ml in mice) within 1 hour post administration, followed by gradual decreases.
2) In both species, the levels in most of organs and tissues examined changed in parallel with the blood levels. The highest tissue levels were found in liver, gastrointestinal tracts, kidneys and urinary bladder, whereas lower levels as compared with blood levels were found in brain, eye balls, fat and testes. Other tissue levels were found to be higher than blood levels.
3) Twenty-four hours post dosing, tissue levels in both species were so decreased that the recoveries of radioactivity from liver, an organ showing the highest level, were about 0. 1 % of the dose administered.
4) Twenty-four hours, after a single dose, about 40% of the administered dose was recovered from rat urine, about 50% from rat feces, about 10% from rat bile, about 25% from mouse urine and about 60% from mouse feces.
5) When administered to rats once a day for 7 days, the blood level at 1 hour after dosing was gradually elevated to attain a twice higher level on the 7th dosing as that on the 1st dosing.
6) Tissue distribution of ¹⁴C-PPA in rats after 8 consecutive doses was found to be similar to that after a single dose, suggesting no appreciable tissue accumulation of the drug. Daily excretions into urine and feces were also found to be virtually unchanged.
7) Whole-body autoradiographic studies of rats given a single or multiple doses showed tissue distributions being in good agreement with those obtained by radiometric assay.
8) Whole-body autoradiograms of pregnant mice given a single dose showed that fetal tissue levels were similar to maternal blood levels, and elimination rates of the label from fetal tissues were also similar to those of maternal tissues.
9) Both in rats and mice, 90% of either 24-hour urinary or fecal radioactivity was associated with unchanged PPA and the remainder with its glucuronide and an unidentified metabolite.
10) TLC of 6-hour rat bile showed three major peaks together with a minor peak. Major peaks were found to be unchanged PPA, its glucuronide and an unidentified metabolite, respectively.
The fluorimetric characteristics of pipemidic acid (PIPE) have been investigated. It has been proven that the fluorescence emission band of pipemidic acid at 439 nm is significantly intensified in the presence of γ-cyclodextrin. The inclusional complexation between the antibacterial pipemidic acid and γ-cyclodextrin (γ-CD) has been studied. A 1:1 stoichiometry of the complex was established and its association constant was calculated by a nonlinear regression method, monitoring the changes in the fluorescence signal of pipemidic acid in the presence of γ-CD. According to the results obtained, a spectrofluorimetric method for the determination of PIPE has been proposed. The best limits of detection and quantification were obtained in presence of γ-CD, in acidic media. The dynamic range of the method was comprised between 0.18 and 1.40 μg/ml.
Fluorescence-labeled oligonucleotide probes were applied, combined with in situ reduction of the fluorochrome 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to describe the development of bacterial density, phylogenetic diversity and bacterial metabolic activity during the formation of drinking water biofilms. Polyethylene and glass surfaces exposed to drinking water in a modified Robbins device were rapidly colonized by a biofilm community of phylogenetically diverse prokaryotes, and cell density of the biofilm community was strictly controlled by grazing eukaryotic organisms. In situ hybridization with group-specific rRNA-targeted oligonucleotide probes revealed the following: (i) the prevalence of bacteria belonging to the β-subclass of Proteobacteria within the bacterial biofilm populations; (ii) differences in the population composition, assessed by phylogenetic probes, depended on the surface properties of the substrata; (iii) the influence of water retention time on variations in population structure; and (iv) the presence of bacteria belonging to the family Legionellaceae associated with grazing protozoa. The metabolic potential of bacteria was assessed during biofilm formation using fluorescence signals after in situ hybridization and the reduction of the redox dye CTC as an indicator of respiratory activity. Respiratory activity and ribosome content of adherent bacterial cells decreased continuously during the early stages of the biofilm. After 35 days the percentage of CTC-reducing cells stabilized at 30%, and the amount of hybridized cells stabilized at 55%, of the initial cell number. To ascertain the amount of dormant, but potentially active cells, we established a new method, defined as probe active counts (PAC). Biofilms were incubated with a mixture of appropriate carbon sources and an antibiotic preventing bacterial cell division, followed by the determination of metabolic activity by in situ hybridization. By this approach the percentage of hybridized cells could be increased from 50% to 80% of total bacterial cell counts in the oligotrophic drinking water biofilms.
An improved high-pressure liquid chromatography procedure for determining the concentration of pipemidic acid in human plasma
was developed, which uses a normal-phase silica gel column and aqueous mobile solvent system similar to that used in reverse-phase
chromatography. Precolumn derivatization of pipemidic acid was achieved by a methylation reaction with boron trifluoride in
methanol after extraction from plasma with chloroform containing 4% ethanol. The percent recovery of pipemidic acid was 88.3
+/- 7.7, the variation of which became negligible when quinacrine was used as an internal standard for the determination.
High-pressure liquid chromatography analysis was performed by conventional silica gel and mobile solvent mixtures containing
3% of 0.14% HClO4 solution, 19% methanol, and 78% chloroform. The UV detector was set at 265 nm. The detection limit of pipemidic
acid methyl ester was as low as 10 ng of the injection amounts or 0.5 micrograms of the plasma per ml, with 0.01 absorbance
units (full scale) and a signal-to-noise ratio of 3.
AB206 (5,8-dihydro-5-methoxy-8-oxo-2H-1,3-dioxolo-4,5-g quinoline 7-carboxylic acid) is a novel antibacterial agent structurally related to nalidixic acid. AB206 has potent antibacterial activity against clinical isolates of gram-negative bacteria and is 4 to 18 times more active than nalidixic acid. AB206 inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa, which were resistnat to nalidixic acid. Cross-resistance was not observed between AB206 and various antibiotics, and most bacterial strains resistant to nalidixic acid were susceptible to AB206. AB206 showed bactericidal activity against Escherichia coli, Proteus, Klebsiella pneumoniae, and P. aeruginosa. Chemotherapeutic effects after oral administration of AB206 in mice experimentally infected with E. coli, K. pneumoniae, and Proteus morganii showed that AB206 was two to four times more potent than nalidixic acid.
A log-probit graph paper is described by which a simple graphic estimation of the ED50 and its standard error may be quickly made.
The preparation and antibacterial activity of a series of the title compounds (21-73) are described. These compounds were prepared from the 2-methylthio derivatives 2 and 3 via the 2-methylthio-8-substituted compounds 4-20; compounds 4-20 easily underwent displacement reactions with a variety of piperazines to afford 2-(4-substituted or unsubstituted 1-piperazinyl) derivatives 21-56, of which 21, 22, 27 and 51 with unsubstituted piperazinyl group at position 2 are converted subsequently into 57-73 by alkylation, acylation, sulfonylation, or addition of isocyanates to the piperazine nitrogen. The hexahydro-1H-1,4-diazepinyl analog 74 was also prepared. The most active members in this series of compounds were found to be 8-ethyl- and 8-vinyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)pyrido(2,3-d)pyrimidine-6-carboxylic acids (22 and 51), both of which are more active in vitro and in vivo against gram-negative bacteria, including Pseudomonas aeruginosa, than piromidic acid (1). Structure-activity relationships are discussed.
The in vitro activity of 6-[d-alpha-(3 guanylureido)-phenylacetamido]-penicillanic acid (BL-P1654) was evaluated against 117 clinical isolates of Pseudomonas aeruginosa, many of which were known to be resistant to both gentamicin and carbenicillin. BL-P1654 was two to eight times more active than carbenicillin against P. aeruginosa. However, all 28 highly carbenicillin-resistant isolates (minimal inhibitory concentration [MIC] > 500 mug/ml) were also highly resistant to BL-P1654. When an MIC of 32 mug/ml or less and a zone of inhibition of 12 mm or more by a 10-mug disk were used as criteria indicating susceptibility to BL-P1654, the false-resistance rate by the disk test was 10.6% and the false-susceptibility rate was 4.2%. The combination of BL-P1654 and gentamicin was synergistic against 45 of 70 isolates of P. aeruginosa tested, but synergism was demonstrated against only 4 of 24 highly gentamicin-resistant (MIC > 63 mug/ml), 1 of 12 highly BL-P1654-resistant (MIC > 250 mug/ml) isolates, and none of nine isolates highly resistant to both of these antibiotics.