Potent Anti-amoebic Effects of Ibogaine, Voacangine and the Root Bark Alkaloid Fraction of Tabernaemontana arborea.

Plants of Tabernaemontana species have several pharmacological activities including antimicrobial effects. Amoebiasis continues to be a public health problem, with increasing evidence of resistance to metronidazole. In this study, we assessed the effect of the alkaloid fraction of T. arborea root bark and the alkaloids ibogaine and voacangine on the viability and infectivity of Entamoeba histolytica trophozoites. Cultures were exposed to 0.1 - 10 µg/mL for 24, 48 and 72 h, and viability was then determined using a tetrazolium dye reduction assay and type of cellular death analyzed by flow cytometry. Results showed that the alkaloid fraction, but mainly ibogaine and voacangine alkaloids, exhibited potent dose-dependent anti-amoebic activity at 24 h post-exposure (IC50 4.5 and 8.1 µM, respectively), comparable to metronidazole (IC50 6.8 µM). However, the effect decreased after 48 and 72 h of exposure to concentrations below 10 µg/mL, suggesting that the alkaloids probably were catabolized to less active derivatives by the trophozoites. The treatment of trophozoites with the IC50 s for 24 h induced significant morphological changes in the trophozoites, slight increase in granularity, and death by apoptonecrosis. The capacity of T. arborea alkaloids to inhibit the development of amoebic liver abscesses in hamsters was evaluated. Results showed that even when the treatments reduced the number of amoebic trophozoites in tissue sections of livers, they were unable to limit the formation of abscesses, suggesting their rapid processing to inactive metabolites. This work leaves open the possibility of using Tabernaemontana alkaloids as a new alternative for amoebiasis control.

Metronidazole removal by means of a combined system coupling an electro-Fenton process and a conventional biological treatment: By-products monitoring and performance enhancement.

In order to mineralize Metronidazole (MTZ), a process coupling an electro-Fenton pretreatment and a biological degradation was implemented. A mono-compartment batch reactor containing a carbon-felt cathode and a platinum anode was employed to carry out the electro-Fenton pretreatment of MTZ. A total degradation of MTZ (100 mg L-1) was observed at 0.07 mA.cm-2 after only 20 min of electrolysis. Yet, after 1 and 2 h of electrolysis, the mineralization level remained low (16.2% and 32% respectively), guaranteeing a significant residual organic content for further biological treatment. LCMS/MS was used to determine the intermediates by-products and hence to propose a plausible degradation pathway. An increase from 0 to 0.44 and 0.6 for 1 and 2 h of electrolysis was observed for the BOD5/COD ratio. Thus, from 1 h of electro-Fenton pretreatment, the electrolysis by-products were considered biodegradable. A biological treatment of the electrolysis by-products after 1 and 2 h was then realized. The mineralization yields reached very close values, about 84% for 1 and 2 h of electrolysis after 504 h of biological treatment, namely close to 89% for the overall process, showing the pertinence of the proposed coupled process.

Synthesis and evaluation of N-nicotinoyl-2-{2-(2-methyl-5-nitroimidazol-1-yl)ethyloxy}-D,L-glycine as a colon-specific prodrug of metronidazole.

Metronidazole (MTZ) is a drug of choice for protozoal infections such as luminal amoebiasis. We designed and synthesized N-nicotinoyl-2-{2-(2-methyl-5-nitroimidazol-1-yl)ethyloxy}-D,L-glycine (NMG) as a colon-specific prodrug of MTZ. The synthetic yield of NMG was about 34%. The apparent partition coefficient of MTZ was greatly reduced by the chemical modification. While (bio)chemically stable in the contents of the upper intestine, NMG was rapidly cleaved to liberate MTZ on incubation with the cecal contents of rats. MTZ metabolized quickly in the cecal contents at least partly by a microbial nitroreductase, suggesting that the metabolism of MTZ is relevant to its bioactivation leading to amoebicidal action. The systemic absorption, analyzed by the blood concentration and urinary recovery of NMG, was very low after oral administration of NMG. In parallel with this, whereas MTZ disappeared mostly during the transit of the proximal small intestine, a substantial amount of NMG remained in the small intestine moving down to the large intestine where it metabolized rapidly. Moreover, comparing systemic absorption of MTZ after oral administration of NMG or MTZ, NMG markedly reduced the systemic absorption. These results suggest that NMG is a potential colon-specific prodrug of MTZ which improves therapeutic and toxicological properties.

The study of drug permeation through natural membranes.

In this study, natural membranes such as the outer membrane of Prunus persica (peach) and Lycopersicon esculentum (tomato), the inner layer of the egg of Gallus domesticus (hen) and the middle membrane of the Allium cepa (onion) were used as controlling barriers for permeation of some model drugs with different MW and lipophilicities. Drug permeation studies were done by using modified Franz diffusion cell. The permeation of drugs through these natural membranes was compared to permeation of them through human skin and synthetic cellophane membrane. Results showed that the rate and amount of diclofenac permeated through onion membrane was not significantly different from that with tomato (p>0.17), egg (p>0.29) and human skin (p>0.93). Permeation of diclofenac through tomato skin and cellophane was not significantly different (p>0.35). Permeation of diclofenac through all studied membranes except for human skin that follows the Fickian kinetic followed non-Fickian mechanism and their permeabilities were not significantly different from each other (p>0.05). Permeation of metronidazole through onion membrane and tomato skin were not significantly different from human skin (p>0.053 and 0.38, respectively). All membranes were significantly different from each other (p<0.0001) for permeation of erythromycin as a relatively large molecular weight and lipohilic molecule through human skin and other studied membranes. Permeation of diclofenac through human skin and metronidazole through egg and tomato skin followed Fick's first law. Diffusion of diclofenac through onion, tomato, egg, cellophane, and peach; metronidazole through onion, peach, cellophane, and human skin, and erythromycin through all studied membranes followed non-Fickian mechanism for diffusion. Statistical analysis showed the most similarity between onion and human skin for diclofenac, tomato and human skin for metronidazole, onion and cellophane for erythromycin.

Novel mucoadhesive buccal formulation containing metronidazole for the treatment of periodontal disease.

Mucoadhesive tablets using different mixture of cellulose and polyacrylic derivatives were prepared in order to obtain new formulations containing metronidazole for periodontal disease treatment. All tablets were characterized by swelling studies, ex vivo and in vivo mucoadhesive time, ex vivo mucoadhesion force, in vitro and in vivo release. The best mucoadhesive performance and the best in vitro drug release profile were achieved by using hydroxyethyl cellulose (HEC) and carbomer 940 2:2 ratio. The chosen tablet, containing 20 mg of metronidazole, performed 12 h drug sustained release with buccal concentrations always higher than its MIC.

Biodegradability of metronidazole, olaquindox, and tylosin and formation of tylosin degradation products in aerobic soil--manure slurries.

The use of veterinary drugs (primarily antibiotics) in animal husbandry harbors the risk that these compounds end up in the farmland when manure is used as fertilizer. The biodegradability of three compounds, olaquindox (OLA), metronidazole (MET), and tylosin (TYL), was simulated in soil--manure slurries with 50 g of soil per liter. Supplemental batch sorption tests revealed that insignificant amounts of OLA and MET were located in the soil phase, whereas only 0.1 to 10% of the added amounts of TYL remained in the liquid phase. This may reduce the bioavailability and thus biodegradation rates of TYL. Unidentified metabolites of OLA and TYL and four known TYL metabolites were detected using HPLC. However, none of these substances were seen to persist in the biodegradation experiments, indicating that OLA and TYL most likely were mineralized in the experiments. Neither the use of sandy or clayey soil nor the use of 0, 1, or 10% (V/V) of manure added to these soils had a significant effect on the degradation rates. Degradation half-lives for the primary degradation were 3.3--8.1 days for TYL, 5.8--8.8 days for OLA, and 13.1--26.9 days for MET. Based on comparisons of results obtained with the benchmark chemical aniline and degradation half-lives of this compound in nature, it was assessed that results obtained with the current test method slightly overestimate real-world biodegradation rates.

Metabolism of metronidazole and antipyrine in isolated rat hepatocytes. Influence of sex and enzyme induction and inhibition.

The metabolism of metronidazole and antipyrine was investigated in freshly isolated hepatocytes from 7 male and 6 female control Wistar rats, 8 males and 5 females pretreated with phenobarbital (PB) and 3 males pretreated with 3-methylcholanthrene (MC). Pretreatment with PB increased the intrinsic clearance (CLi = Vmax/Km) of metronidazole to its acetic acid (MAA) and hydroxy metabolite (HM) 7- and 2.8-fold in the males and 3.2- and 3.0-fold in the females, whereas MC treatment increased the values 9- and 10-fold, respectively (P less than 0.05). The CLi of metronidazole to HM and its glucuronide conjugate was higher in the control and PB treated male than in the corresponding female groups, whereas the rank order was reversed for sulphate formation (P less than 0.05). SKF 525A was a more potent inhibitor of MAA formation than of HM formation, except in the PB treated male group. Pretreatment with MC increased the inhibitory potency of alpha-naphthoflavone and antipyrine toward MAA and HM formation. In male rats PB treatment increased the CLi of antipyrine to 3-hydroxymethyl-(HMAP), nor-(NORAP) and 4-hydroxyantipyrine (OHAP) 2.5-, 2.1- and 4.5-fold, respectively (P less than 0.05). Pretreatment with MC in male and with PB in female rats had no significant effect on antipyrine metabolism. SKF 525A was a more potent inhibitor of HMAP and OHAP formation than of NORAP formation. Treatment with MC increased the inhibitory potency of alpha-naphthoflavone toward the formation of all antipyrine metabolites. Metronidazole increased the formation rate of HMAP, but inhibited the formation of NORAP and OHAP, particularly the latter. The results suggest that the formation of MAA, HM, HMAP, NORAP and OHAP from metronidazole and antipyrine is catalyzed by different cytochrome P-450 isozymes, which may be supplemented or substituted by PB or MC induced species. The involved P-450 isozymes have more or less overlapping substrate and product specificity. Metronidazole appears to be a sensitive probe for detection and identification of PB and MC type induction.

Antipyrine and metronidazole metabolism during occupational exposure to gasoline.

Antipyrine and metronidazole clearance was measured in 18 fuel-filling attendants by the single-sample method while the attendants were being exposed occupationally to gasoline; the measurements were repeated after 2-4 weeks with no exposure. Eighteen office workers were investigated simultaneously. The median concentration of gasoline in the breathing zone of the fuel-filling attendants during filling and cleaning operations was 270 mgm-3 (range 18-1758 mgm-3). Antipyrine clearance was 18% higher during exposure to gasoline than after 2-4 weeks of vacation (P less than 0.01), while antipyrine clearance was unchanged in the office workers. No change was found in metronidazole clearance in either group. Antipyrine clearance was on average 26% higher in the smokers than in the nonsmokers (P less than 0.05), while metronidazole clearance was similar in smokers and nonsmokers. We conclude that gasoline is an inducer of antipyrine elimination, with no impact on metronidazole elimination. This indicates that gasoline has a differential inducing effect on the hepatic drug metabolizing enzymes of man.

[Metronidazole content of normal and tumor tissues in the combined treatment of patients with osteogenic sarcoma]. / Soderzhanie metronidazola v normal'nykh i opukholevykh tkaniakh pri kombinirovannom lechenii bol'nykh s osteogennoi sarkomoi.

A study was made of the time course of the level of metronidazole in the blood and tissues of 97 patients with localized osteogenic sarcoma in several modes of the drug administration (per os, using an enema and their combinations). The maximum drug concentration in the blood was achieved with the combined mode of the drug administration at a dose of 8 g/m2, enhancing local and general effects on a tumor, manifesting themselves in the reduction of sizes of a tumor, its morphological changes and an increase in the average period of metastasis detection. The 3-year survival rates showed no significant differences between groups of patients receiving and not receiving metronidazole.

[Bacteriological, clinical, and pharmacokinetic studies of perioperative antibiotic prophylaxis in head and neck surgery]. / Bakteriologische, klinische und pharmakokinetische Untersuchungen zur perioperativen Antibiotikaprophylaxe in der Kopf-Hals-Chirurgie.

The point of this study was to analyze the possible benefits of perioperative antibiotic prophylaxis in patients from whom oral cavity or throat tumors are removed. The criteria used to judge the efficacy of each treatment included the clinical course of the treatment, the bacterial colonization of the surgical area as well as the growth of bacteria during the postoperative phase. 50 patients were chosen and grouped according to their surgical treatment: laryngectomy (n = 20), partial laryngectomy (n = 22) or tongue, floor of the mouth, soft palate, gum or base of the tongue partial resection (n = 8). Within each surgical group, patients were randomly chosen for antibiotic prophylaxis; others constituted the untreated control group. The antibiotic prophylaxis consisted of 5 g Mezlocillin administered at the time of narcosis for 20 min followed by 0.5 g Metronidazol for 10 min. These medications were given in 8-hour intervals for three days following surgery. Investigation of the first 20 patients (prophylaxis group n = 7, control group n = 13) revealed that the combination of Mezlocillin and Metronidazol positively influenced post-operative recovery (no complications) while the patients without prophylactic antibiotic treatment suffered general or local complication leading to, in 10 cases, the necessity of postoperative therapy. On the basis of these results, the random grouping of the patients was ended and all 30 remaining patients were given the antibiotic prophylaxis. Regardless of antibiotic treatment, the great majority of microbes isolated from throat swabs and tracheal secretions were gram-negative, aerobic bacteria. A prerequisite for efficacious prophylaxis is that the antibiotics be applied before the operation, so that a sufficient concentration is present at the time of pharyngotomy. On the basis of pharmacokinetic investigations, administration of the antibiotic 30 min preoperatively fulfills this requirement. Further, our recommendation, based on our measurement of the spectrum of bacteria present and their growth is that the antibiotics be applied over a period of three days postoperatively. This recommendation is also based on the fact that some patients (those having undergone partial laryngectomy or tongue, floor of the mouth, base of the tongue partial resections) have suffered loss of the swallowing reflex so that there exists a continuous contamination of the surgical area with pathogens or facultative pathogens coming from the nasal or oral cavities.

[Local use of metronidazole in dimethyl sulfoxide in the radiation therapy of cervical cancer]. / Lokal'noe primenenie metronidazola v dimetilsul'fokside pri luchevoi terapii raka sheiki matki.

A method of the local use of metronidazole dissolved in dimethylsulfoxide (DMSO) for cervix uteri cancer patients was worked out. Applications of 1-2 g of metronidazole were well tolerated by the patients. Metronidazole concentrations in cervical tumors were high (about 1000 micrograms/g), in the blood they did not exceed 16 micrograms/ml. Experiments showed that metronidazole in DMSO diffused in the tissue, its concentrations at a distance of 2-3 cm from the surface were 180-260 micrograms/g. The local use of metronidazole in DMSO caused an increase in the rate of tumor radiation regression.

[Accumulation of metronidazole in tumors during whole-body hyperthermia induced by Sulfazin]. / Nakoplenie metronidazola v opukholi pri obshchei gipertermii, vyzvannoi sul'fazinom.

A study was made of metronidazole accumulation in the blood of animals and tumorous tissues of Pliss lymphosarcoma after the total moderate hyperthermia caused by subcutaneous injection of the pyrogenic drug sulfadiazine. Metronidazole concentration in the blood and tumors of hyperthermized animals was 2-3 times as high as in those of the control animals. A time shift of the maximum values of metronidazole accumulation occurred in hyperthermia. Under normal conditions the peak of accumulation was observed 2 h after the administration of the radiosensitizer whereas in hyperthermia depending on its duration it could develop 1 h earlier. Thus the total moderate hyperthermia served as a favorable background for raising metronidazole concentration in tumor opening up prospects for potentiating the radiosensitizing effect of metronidazole and perhaps some other means in the utilization of small nontoxic doses of the drug.

Distinct reduction of nitrofurans and metronidazole to free radical metabolites by Tritrichomonas foetus hydrogenosomal and cytosolic enzymes.

Anaerobic Tritrichomonas foetus hydrogenosomes supplemented with pyruvate and CoA effectively reduce nitrofurans and metronidazole to their respective anion free radicals. Addition of purified ferredoxins from Clostridium pasteurianum or Spinacia oleracea to these preparations causes a great stimulation of metronidazole reduction, but does not affect nitrofuran reduction. A similar stimulatory effect of ferredoxin on metronidazole reduction, but not on nitrofuran reduction, is observed in incubations containing purified NADPH:ferredoxin oxidoreductase from S. oleracea. NADH is less effective than pyruvate as a reducing cofactor for metronidazole and nitrofuran reduction by the hydrogenosomes, and these activities are not modified by the addition of ferredoxins. In contrast to the results observed with hydrogenosomes, the T. foetus soluble fraction supplemented with NADH or NADPH is able to reduce nitrofurans, but not metronidazole. Under aerobic conditions, the anion free radical metabolites generated from metronidazole and nitrofurans are oxidized, resulting in catalytic superoxide anion formation as detected by spin-trapping experiments. Oxygen consumption and H2O2 formation by T. foetus hydrogenosomes and NADPH:ferredoxin oxidoreductase are also stimulated by nitrofurans and high concentrations of metronidazole. Addition of ferredoxin enhances metronidazole-stimulated, but not nitrofuran-stimulated, oxygen consumption and H2O2 formation in both systems. These results support the role of air oxidation as a detoxification reaction of the metronidazole anion radical and the involvement of ferredoxin in its formation. On the other hand, redox cycling of nitrofurans with formation of high steady state concentrations of oxygen-derived radicals might be of toxicological significance.

Treatment of anaerobic infections in children with metronidazole.

The tolerance and efficacy of metronidazole were studied in 15 pediatric patients who had anaerobic infection. 5 had soft tissue abscess, 4 had aspiration pneumonia, 3 had intracranial abscess and 3 had chronic sinusitis. 45 bacterial isolates were recovered (3 isolates per patient). 40 were anaerobes and included 17 Bacteroides sp. (8 sp.), 16 anaerobic cocci and 6 Fusobacterium nucleatum. Metronidazole was given intravenously at the dose of 30 mg/kd/day or orally in the dose of 40-50 mg/kg/day. The length of therapy was between 14 and 52 days (average 26 days). 7 of the patients received initial parenteral therapy for 5-21 days (average 11.6 days), and subsequently received oral therapy. The minimal inhibitory concentration of 38 of the 41 anaerobic isolates (93%) was equal or less than 2 micrograms/ml. The mean peak concentration of metronidazole on the third day of therapy was 24.2 micrograms/ml range 15.2-30 micrograms/ml) and the mean trough was 7.2 micrograms/ml (range 4-11.6 micrograms/ml). No local or systemic adverse reaction as noted. A good response to therapy with a complete cure occurred in 14 of the 15 children. A fair response was achieved in 1 patient. Metronidazole appears to be effective and safe in the treatment of serious anaerobic infection in children.

Metronidazole treatment of Bacteroides fragilis infections.

Seven patients with Bacteroides fragilis infections were treated with intravenous and/or oral metronidazole. Infections treated included endocarditis, osteomyelitis, lung abscess, empyema, peritonitis, septicemia, and pelvic infection. Some patients had failed to respond to therapy with chloramphenicol or clindamycin or both. Metronidazole was used alone or in combination with aminoglycosides. Serum levels of metronidazole several times in excess of the minimal inhibitory concentrations for the organisms were easily achieved and in one patient the CSF metronidazole level was equal to that of the serum. Response to therapy with metronidazole was considered to be excellent. The only serious side effect noted was hypotension, which occurred in the last patient. Therapy was discontinued, and therefore therapeutic results could not be evaluated. Metronidazole appears to be a safe and effective agent in the treatment of B fragilis infections.

Metronidazole in treatment against Haemophilus vaginalis (Corynebacterium vaginale).

The rate of bactericidal activity and inactivation of metronidazole was studied in time-kill curves with Haemophilus vaginalis (Corynebacterium vaginale). The minimum inhibitory concentrations of metronidazole for the eight strains tested ranged from 4 to 16 micrograms/ml. At a concentration of 20 micrograms/ml, metronidazole demonstrated a slow cidal effect against exponential-phase organisms, requiring 24 to 48 h for completion. Inactivation of metronidazole during the time-kill curve was quite variable and averaged 28% of the starting concentration after 48 h. Against stationary-phase organisms (inoculum, 10(10) to 10(11) colony-forming units per ml), a slow cidal effect was also seen, with an average inactivation of metronidazole of 38% after 48 h. At a subinhibitory concentration of 5 micrograms/ml, metronidazole was inactivated to the greatest degree (57% after 48 h). Therefore, in contrast to earlier studies in which metronidazole was rapidly and consistently cidal within 4 h against obligate anaerobes and was almost completely inactivated by 8 h, the bactericidal effect of metronidazole againsts H. vaginalis in this study was much slower and was associated with a variable and slower rate of inactivation.

[Radiosensitization with metronidazole. Pharmacocinetical comparison by means of blood level curves after administration of "metronidazol" in the form of tablets, suppositories, and enemas (author's transl)]. / Strahlensensibilisierung mit Metronidazol. Pharmakokinetischer Vergleich an Hand von Blutspiegelkurven nach Verabreichung von Metronidazol in Form von Tabletten, Suppositorien und Klistier.

Severe acute gastrointestinal side-effects following high oral doses of metronidazole (6 g/m2) could be avoided by a combined oral and rectal application. With tablets and an enema (1 g metronidazole/2 ml) in a ratio 1:4 and an increased dosage of 10 g/m2, maximal serum concentrations of about 200 micrograms/ml are obtained like after an oral dosage of 6 g/m2. The maximal radiosensitizing effect on hypoxic tumor cells is seen five hours after oral application and seven hours after the combined oro-rectal application. No longterm toxicity was found following six high doses of metronidazole.