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Human-driven environmental change has increased the occurrence of harmful cyanobacteria blooms in aquatic ecosystems. Concomitantly, exposure to microcystin (MC), a cyanobacterial toxin that can accumulate in animals, edible plants, and agricultural soils, has become a growing public health concern. For accurate estimation of health risks and timely monitoring, availability of reliable detection methods is imperative. Nonetheless, quantitative analysis of MCs in many types of biological and environmental samples has proven challenging because matrix interferences can hinder sample preparation and extraction procedures, leading to poor MC recovery. Herein, controlled experiments were conducted to enhance the use of ultra-performance liquid-chromatography tandem-mass spectrometry (UPLC–MS/MS) to recover MC-LR and MC-RR at a range of concentrations in seafood (fish), vegetables (lettuce), and environmental (soil) matrices. Although these experiments offer insight into detailed technical aspects of the MC homogenization and extraction process (i.e., sonication duration and centrifugation speed during homogenization; elution solvent to use during the final extraction), they centered on identifying the best (1) solvent system to use during homogenization (2–3 tested per matrix) and (2) single-phase extraction (SPE) column type (3 tested) to use for the final extraction. The best procedure consisted of the following, regardless of sample type: centrifugation speed = 4200 × g; elution volume = 8 mL; elution solvent = 80% methanol; and SPE column type = hydrophilic–lipophilic balance (HLB), with carbon also being satisfactory for fish. For sonication, 2 min, 5 min, and 10 min were optimal for fish, lettuce, and soil matrices, respectively. Using the recommended HLB column, the solvent systems that led to the highest recovery of MCs were methanol:water:butanol for fish, methanol:water for lettuce, and EDTA-Na4P2O7 for soils. Given that the recommended procedures resulted in average MC-LR and MC-RR recoveries that ranged 93 to 98%, their adoption for the preparation of samples with complex matrices before UPLC–MS/MS analysis is encouraged.

Research on the therapeutic applications of the histamine H3 receptor (H3R) has traditionally focused on antagonists/inverse agonists. In contrast, H3R agonists have received less attention despite their potential use in several disease areas. The lower availability of H3R agonists not only hampers their full therapeutic exploration, it also prevents an unequivocal understanding of the structural requirements for H3R activation. In the light of these important issues, we present our findings on 4-benzyl-1H-imidazole-based H3R agonists. Starting from two high throughput screen hits (10 and 11), the benzyl side chain was altered with lipophilic groups using combinatorial and classical chemical approaches (compounds 12-31). Alkyne- or oxazolino-substituents gave excellent affinities and agonist activities up to the single digit nM range. Our findings further substantiate the growing notion that basic ligand sidechains are not necessary for H 3R activation and reveal the oxazolino group as a hitherto unexplored functional group in H3R research.

1: The pharmacokinetics and metabolism of 14C-levetiracetam, a new anti-epileptic agent, in mouse, rat, rabbit and dog after a single oral dose were investigated. Moreover, the in vitro hydrolysis of levetiracetam to its major carboxylic metabolite by rat tissue homogenates was investigated to identify tissues involved in the production of the metabolite. Data are also presented on the induction of the enzyme(s) involved in levetiracetam hydrolysis in the rat. 2: Levetiracetam was rapidly and almost completely absorbed. The unchanged drug accounted for a very high percentage of plasma radioactivity. Levetiracetam did not bind to plasma proteins. Although brain radioactivity concentrations were lower than those of whole blood at early time points, brain-to-blood ratios increased over time. The predominant route of elimination of total 14C was excretion via urine, accounting for about 81, 93, 87 and 89% of the dose in the mouse, rat, rabbit and dog, respectively. Consequently, levetiracetam was poorly metabolized. It was submitted in vivo to hydrolysis and/or oxidation. Hydrolysis of the amide function of levetiracetam produced the corresponding acid. However, levetiracetam could also be oxidized at positions 3 and 4 of the 2-oxopyrrolidine ring. Finally, the compound and the corresponding acid metabolite could be oxidized at position 5 of the 2-oxopyrrolidine ring and then hydrolysed with the opening of the ring. 3: All the investigated rat tissues (liver, kidney, lung, brain, small intestine mucosa) had the potential to produce the acid metabolite. By contrast, the acid was undetectable following incubation of levetiracetam with buffer alone or heat-denaturated liver fractions. 4: No marked species or sex differences were observed in the absorption, disposition and metabolism of levetiracetam. 5:The hydrolysis of levetiracetam is carried out by an enzymatic process characterized by a broad tissue distribution. In the rat, the enzyme system hydrolysing levetiracetam is not induced by phenobarbital, at least under the experimental conditions used herein, whereas the enzyme system(s) involved in the other metabolic pathways is induced.

Levetiracetam and its carboxylic metabolite (AcL) were tested for their potential inhibitory effect on 11 different drug metabolizing enzyme activities using human liver microsomes. The following specific assays were investigated: testosterone 6beta-hydroxylation [cytochrome P-450 3A4 (CYP3A4)], coumarin hydroxylation (CYP2A6), (R)-warfarin hydroxylation (CYP1A2), (S)-mephenytoin hydroxylation (CYP2C19), p-nitrophenol hydroxylation (CYP2E1) tolbutamide hydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), epoxide hydrolase and UDP-glucuronyltransferase (UGT) toward paracetamol (UGT1*6), ethinyloestradiol (UGT1*1), p-nitrophenol (UGT(pl 6.2)), and valproic acid. None of these activities were affected by levetiracetam or AcL added at concentrations up to 1 mM. Additionally, primary cultures of rat hepatocytes were used to assess a potential inducing effect of levetiracetam on CYPs. Phenobarbital (2 mM), beta-naphtoflavone (40 microM), dexamethasone (1 microM), and phenytoin (up to 300 microM) were tested as positive controls. When added to cells for 48 h, all the positive controls increased 7-ethoxycoumarin O-deethylase activity demonstrating the inducibility of CYPs in the present culture conditions. By contrast, levetiracetam did not affect the activity up to 1 mM. The highest levetiracetam concentrations examined in the above in vitro studies are well in excess of those measured in the plasma of patients receiving therapeutic doses. It is thus concluded that levetiracetam is unlikely to produce pharmacokinetic interactions through inhibition of CYPs, UGTs, and epoxide hydrolase. Furthermore, based on the in vitro assays with rat hepatocytes, it could be speculated that levetiracetam does not act as a CYP inducer.

For lack of being able to grow Treponema pallidum, the only possibility that we have to study its biological behavior is to have recourse to experimentation upon animals. The stade comprising primary and secondary syphilis is characterized by visible "warning signals" which are not serious for the patient himself. But all their importance lies in the fact that they reveal the dissemination of T. pallidum inside the whole body. Thus, the healing alone or the disappearance of the lesion does not necessarily mean that it is cured. As a matter of fact, after that phase, infection may proceed "á bas bruit", as a chronic affection, which will come to light later, 10 to 15 years or more after the primary stage, as a visceral phase involving mainly cardiovascular or central nervous system. Those impairments cannot then be cured. Now, during that long period of latency, we are totally without any absolute criterion of "bacteriological sterilization", for we do not know precisely the physio-pathological meaning of these numerous serological reactions which are at out disposal and subsequently we are obliged to interpret them through divergent opinions because these techniques are not established on scientific method but are based upon empiric knowledges.

For lack of being able to grow Treponema pallidum, the only method which allows us to study the biology of this germ and the physiopathology of this infection lies in researches in experimental syphilis. After pointing out the different aspects of Treponema pallidum, either with light microscopy or electron microscopy, the authors review the different kinds of reproduction suggested by syphiligraphs, the recent trials to cultivate the treponema, and the processes of elimination. Then, they examine the biological properties and the antigenic structure of T.p. as it has been established by comparison with cultivable spirochetes. To end with, the authors show that both the TPI test and the FTA test are two very specific reactions; these tests mean nothing but the fact that the patient has been in contact with the antigens of Treponema pallidum and the quantitative tests cannot be considered as expressing the infectious potential capacity.

Symptoms of syphilis have evolved over the ages. In the 16th century, they were essentially cutaneomucous ones. While the intensity of the symptoms decreased localization of the infection in the various organs--mainly those of the cardiovascular and nervous system--gradually appeared. Over a period of years following the introduction of penicillin therapy, the cutaneous and visceral stages became less common. Reminder of the chief serological reactions and of some aspects of the experimental syphilis in the rabbit, similar to certain forms of human syphilis--a strong but late penicillin therapy has been proven ineffective in humans as well as in animals. It is to be regretted that numerous methods of using penicillin (all over the world), had been, from the beginning--except in a few cases--of a purely empirical nature. The authors emphasize--with proof to support their opinion--the failure of antibiotic therapy, which can explain the recent reappearance of hepatic and nervous localizations that had disappeared for 30 years. Despite views to the contrary the authors conclude that penicillin has in no way resolved all the problems raised by the treatment of syphilis.

The aim of our study is to evaluate the activity of antibiotics on Treponema pallidum (Tp) in the early stage of syphilis. Serum elimination kinetics for the antibiotic under study are first determined in rabbits. Four groups of seronegative rabbits are then submitted to intratesticular injection of a calibrated inoculum of Tp (Nichols virulent strain). The antibiotic is given simultaneously (TO), 3, 6 or 10 days after inoculation. Treated animals, as well as a control group of simultaneously infected untreated animals, are monitored for physical and serological changes for three months. Every animal is then tested according to the techniques of experimental rabbit syphilis to provide final conclusions. The technique investigates the direct activity on Tp of given serum concentrations of an antibiotic, and therefore the therapeutical effectiveness of an agent in a selected dosage. It also allows demonstration of sublethal activity on Tp of serum concentrations which are adequate for treating other concurrent infections (including STD) but not syphilis.

Penicillin is undoubtedly the antibiotic most effective on Treponema pallidum, but is it possible to prescribe it according to a standard regimen? Experimentation proves that such a uniform therapeutic plan cannot be determined for the following reasons: 1) Treponema pallidum may divide every 30 to 33 hours, but this concept is only established during the period of exponential growth in the initial lesion. Moreover, if Treponema pallidum are very quickly disseminated throughout the organism, then they do not divide at the same rate. In addition, we take into account a whole series of factors which can interfere with their rate of multiplication. 2) A penicillinemia of 0.03 U/ml may kill all the Treponema pallidum when they divide, but Dr. Eagle's data, although this would be a fairly active serum level, showed that the effective maximal serum concentration should be far higher, about 0.820 U/ml. The experimental data prove it is not possible to point out an accurate correlation between blood and tissue levels; thus, the penicillin levels in the cerebrospinal fluid are about 1/100 lower than those obtained in the serum. Among other factors it is necessary to take into account not only the age of the patient but also the penicillin complex chosen. Although the total of injected penicillin can be the same, the kinetics of serum levels are essentially variable, both regarding the increase of levels and duration according to the drug used. 3) As noted it follows that penicillinotherapy prescribed early with high and prolonged doses may allow a bacteriologic sterilization of primary syphilis.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible activity of thiamphenicol on Treponema pallidum during single-dose treatment of gonococcal infection with 10 tablets of 0.250 mg each was investigated using a new, more accurate method. We found that, under the conditions of our study, thiamphenicol fails to kill T. pallidum, exhibiting only incomplete activity. Thus, thiamphenicol taken during incubation of syphilis may delay or inhibit the emergence of primary manifestations but fails to achieve bacteriologic sterilization of T. pallidum acquired concomitantly with gonococcus. Clinical and serologic evidence of syphilis should therefore be looked for routinely three and six months after treatment.

The problem of immunity in Syphilis is a very complex problem which is tightly bound to healing. But for lack of being able to grow T. pallidum and as we are without any absolute criterion of bacteriological sterilization, the only method we have to be able to consider this study is to have recourse to experimentation upon animals. Two theories had been proposed to give an explanation about this phenomenon occurring during a late treated syphilis: on one side, for Kolle, Evers and Neisser, immunity in syphilis would be due to a premunition given by the only persistence in the organism of T.p. having still kept all their virulence. On the other hand, as far as Chesney and Kemp are concerned it would be a true immunity, obtained before the application of any therapeutic treatment, but which is not due to persistence of T.p. in tissues. However, recent works have proved, and this has been confirmed by several searchers, that a penicillin therapy, even at a high dose, given six months after the infection outset, is no longer able of destroying all T.p. Immunity in syphilis would then be due to a modification of tissular receptivity kept by the persistence of T.p. vegetating in the organism in a commensal state. As far as the nature of this immunity is concerned, all the present research would lead to prove that this refractory state would not be due to tumoral properties, but more likely to tissular properties and probably to cell mediated phenomena. Similar research carried out in man has created an opportunity to demonstrate these facts similar to those observed in experimentations upon animals. They particularly prove that a persistent serology over a long period is not due to a "cicatrice sérologique" (Serological marker) but to the keeping of T.p. in a quiescent state. However, it is a more or less relative immunity which is concerned, depending upon a very large number of factors that are far from being elucidated.

Certain basic treatments of syphilis depend on the following theories: 1) All treponema divide every 30 to 33 hours. Experiments have shown that this idea is only valid during the phase of exponential growth, i.e. it corresponds to the period lyinü between penetration of the germ into the organism and the onset of the initial lesion. One should also be reserved about a whole series of factors which may interfere with the rate of multiplication of treponema. 2) Blood penicillin levels of 0.03 U./ml. would destroy all treponema during cell division. It seems that the efficacy of penicillin mainly depends on levels, at the time of multiplication of the spirochetes. This level of 0.03 U./ml., according to Eagle, should be considered as an average serum concentration, the concentration of maximum efficacy is much higher, about 0.825 U./ml. In any case, it is not possible to obtain with a single dose, equal for all subjects, fixed and certain penicillin levels. For a given quantity injected, serum levels vary from one subject to another and, in the same individual, from one time to another. 3) The epidemiological results do not seem to confirm the optimistic forecasts of certain venereologists who thought that with a standard therapeutic method, it would be possible to eradicate syphilis.

Can penicillin be considered as an infallible treatment for syphilis? Some practitioners have hoped and even believed that this antibiotic would be able to eradicate this infection. To support their thesis, they have put forward an uniform standard therapeutic plan capable of being applied to any stage of the disease and which is based upon the following syllogism: a) all T.p. would divide every 30-33 hours. b) a level of penicillinemia of 0.03 IU/ml would destroy all Treponema when they divide. c) thus, a penicillin therapy administered 3 times giving such levels in serum during a period of 33 hours would produce a bacteriological sterilization of the infected organism. But statistics have shown that these conceptions were wrong. As a matter of fact: 1) every biological research has clearly proved that it is wrong to assert such a rhythm of division for all T.p. Since this multiplication is submitted to a succession of factors that we know more or less well, thus, during the latency phase, T.p. vegetate inside the tissues without apparently multiplying so quickly. 2) For lack of being able to cultivate T.p., it is not possible to state which is the activity level of penicillin on this germ. 3) Finally, the kinetic elimination of penicillin varies extremely, particularly depending on the drug which is used with patients, and on the localization of these germs. All experimental and clinical studies agree and insist on the fact that penicillin therapy must be given early in the infection, at a prolonged and high dose; results having to be estimated on the serological answers, especially with repeated TPI and FTA tests. As far as the late phases of the disease are concerned, for lack of being able to cultivate T. pallidum and because we do not have any absolute criterion of any bacteriological sterilization, the serological answers being often positive, circumspection has always to be a rule: it is better to admit what we do not know rather than to assert without any proof.