[In vitro and in vivo activities of cefpodoxime proxetil against penicillin-resistant Streptococcus pneumoniae].

We evaluated in vitro and in vivo activities of cefpodoxime proxetil (CPDX-PR) in comparison with other oral beta-lactams, cefdinir (CFDN), cefditoren pivoxil (CDTR-PI), and faropenem (FRPM), against penicillin-susceptible and -resistant Streptococcus pneumoniae. In vitro activities (MICs) of CPDX, CFDN, CDTR, and FRPM against clinical isolates, penicillin-susceptible S. pneumoniae (PSSP: MIC of penicillin G, < or = 0.063 microgram/ml), penicillin-intermediate S. pneumoniae (PISP: MIC of penicillin G, 0.125-1 microgram/ml), and penicillin-resistant S. pneumoniae (PRSP: MIC of penicillin G, > or = 2 micrograms/ml), were tested by an agar dilution method. The MIC80s of CPDX against 27 PSSP strains, 23 PISP strains, and 23 PRSP strains were 0.032, 1, and 8 micrograms/ml, respectively, which were superior to or equal to those of CFDN (0.063, 4, and 8 micrograms/ml) and were inferior to those of CDTR (0.016, 0.5, and 1 microgram/ml) and FRPM (< or = 0.008, 0.25, and 1 microgram/ml). Infection was induced in mice by inoculating with a PRSP clinical isolate, 9605 or 9601 (serotype 6), or 10692 (serotype 19), through the nares of male ddY mice into the lungs. The mice were treated with drugs with doses of 2-50 mg/kg at 18, 26, 42, and 50 hours after the infection. Viable cell numbers in the lungs and blood were assayed at 66 hours after the infection. The efficacy of each drug was dose-dependent. CPDX-PR showed the most potent in vivo efficacy among the drugs tested against the infections caused by the PRSP strains. MICs of the drugs against PRSP 9605, 9601, and 10692 were as follows: CPDX, 4, 4 and 2 micrograms/ml; CFDN, 16, 16, and 4 micrograms/ml; CDTR, 1, 1, and 0.5 microgram/ml; and FRPM, 1, 0.5, and 0.5 microgram/ml, respectively. Thus, CPDX-PR showed a stronger in vivo activity than that expected from the MICs of CPDX. This was probably caused by the pharmacokinetic advantage of CPDX over the other drugs used in this study.

[Combination effect of teicoplanin and panipenem on highly resistant strains of MRSA].

We investigated the in vitro combination effect of teicoplanin (TEIC) and panipenem (PAPM) on highly oxacillin-resistant strains of Staphylococcus aureus (MRSA) isolated from various clinical specimens. Combination of TEIC and PAPM using checkerboard titration technique by agar dilution exhibited an excellent effect with mean fractional inhibitory concentration index of 0.18 +/- 0.07 on 47 MRSA strains, and the effects were judged as synergistic against all of the strains tested. In the combination of TEIC and PAPM at 1/4 MIC each against exponentially growing cells of MRSA, good bactericidal activity was found when TEIC and PAPM were added simultaneously, and PAPM was added at 1 or 2 hours prior to addition of TEIC, although the bactericidal activity was scarcely demonstrated when TEIC was added at 1 or 2 hours prior to addition of PAPM. Bactericidal activity against MRSA was enhanced in the combination of TEIC and PAPM at 1/4 MIC each for MRSA than the bactericidal activity of TEIC at 1 MIC alone. TEIC alone showed no bactericidal activity against P. aeruginosa in the mixed cultures with MRSA, while strong bactericidal activity against P. aeruginosa was induced by PAPM. In vitro bactericidal activities against mixed cultures of MRSA with P. aeruginosa were evaluated under conditions of concentrations of TEIC and PAPM, alone and in combination, whose plasma concentrations in human were simulated by a pharmacokinetic simulation model. Bactericidal activity against MRSA was enhanced by the combination of TEIC at 200 mg twice or once daily with PAPM at 500 mg twice daily in comparison with the bactericidal activity of each antibiotic alone, and P. aeruginosa was killed by the antibacterial activity of PAPM.

Interactions of plaunotol with bacterial membranes.

Plaunotol, a cytoprotective antiulcer agent, has a bactericidal effect against Helicobacter pylori, which may result from interaction of this compound with the bacterial cell membrane. The purpose of the present study was to confirm that plaunotol interacts with the H. pylori membrane. Membrane fluidities were measured using two stearic acid spin labels, namely 5-doxyl-stearic acid (in which the nitroxide group is located in the upper portion of the bacterial cell membrane) and 16-doxyl-stearic acid methyl ester (in which the nitroxide group is located deeper in the bacterial cell membrane), by means of electron spin resonance. The membrane fluidities of plaunotol-treated cells were significantly increased in the measurements made using the two spin labels. We also attempted to isolate plaunotol-resistant H. pylori in vitro by two different methods. To assess the level of resistance that could be reached, H. pylori was passaged five times on an agar plate containing subinhibitory concentrations of plaunotol or metronidazole. To measure the rate of development of resistance, H. pylori was grown with subinhibitory concentrations (0.25 x MIC) of plaunotol or metronidazole, and quantitatively plated on to medium containing 4 x MIC of the compounds. This treatment was repeated once more. No plaunotol-resistant colonies were selected by the two methods. H. pylori developed resistance to metronidazole easily and at a relatively high rate. The mechanism by which plaunotol directly fluidizes and destroys the H. pylori membrane might make it difficult for this organism to develop resistance to plaunotol. It was confirmed that the bactericidal effects of plaunotol were also shown against Staphylococcus aureus, Streptococcus pneumoniae, Neisseria gonorrhoeae, Moraxella catarrhalis and Haemophilus influenzae. No such effect was seen against Escherichia coli and Pseudomonas aeruginosa.

[Combination effect between panipenem and vancomycin on highly methicillin-resistant Staphylococcus aureus].

We investigated the in vitro and in vivo combination effects between panipenem (PAPM) and vancomycin (VCM) on highly methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated from various clinical specimens. Examination of combination between panipenem and vancomycin using checkerboard titration showed a good effect with mean fractional inhibitory index of 0.32 +/- 0.12 on 40 MRSA strains, and the effects were judged as synergistic against 33 strains (83%) and additive against 7 strains (17%). In the combination of PAPM and VCM at 1/4 MIC each against exponentially growing MRSA, bactericidal activity was found when PAPM was added at 1 hour or 2 hours prior to VCM-addition, and PAPM with VCM was added simultaneously, although bactericidal activity was scarcely demonstrated when VCM was added at 1 hour or 2 hours prior to PAPM-addition. Bactericidal activity was enhanced against MRSA in the combination of PAPM and VCM at 1/4 MIC each for MRSA than the bactericidal activity of VCM at 1 MIC alone, and the combination showed a strong bactericidal activity against P. aeruginosa. VCM alone, however, had no bactericidal activity in the in vitro mixed cultures of the two bacteria. Furthermore, the combination of PAPM and VCM induced a marked damage to cell surface and bacteriolysis against MRSA and P. aeruginosa in the mixed cultures, although VCM alone induced only slight morphological alterations. Penicillin-binding proteins (PBPs) including MRSA-specific PBP 2' were decreased greatly in the amounts in MRSA-cells with the increase of VCM-treated concentration. The combination therapy of PAPM and VCM showed a greater efficacy than the therapeutic efficacy of each antibiotic alone against mixed infection in burned mice caused by MRSA and P. aeruginosa, and the activity was judged as synergistic based on the FED index smaller than 0.34.

In vitro and in vivo antibacterial activities of CS-834, a novel oral carbapenem.

CS-834 is a novel oral carbapenem antibiotic. This compound is an ester-type prodrug of the active metabolite R-95867. The antibacterial activity of R-95867 was tested against 1,323 clinical isolates of 35 species and was compared with those of oral cephems, i.e., cefteram, cefpodoxime, cefdinir, and cefditoren, and that of a parenteral carbapenem, imipenem. R-95867 exhibited a broad spectrum of activity covering both gram-positive and -negative aerobes and anaerobes. Its activity was superior to those of the other compounds tested against most of the bacterial species tested. R-95867 showed potent antibacterial activity against clinically significant pathogens: methicillin-susceptible Staphylococcus aureus including ofloxacin-resistant strains, Streptococcus pneumoniae including penicillin-resistant strains, Clostridium perfringens, Neisseria spp., Moraxella catarrhalis, most members of the family Enterobacteriaceae, and Haemophilus influenzae (MIC at which 90% of strains are inhibited, < or =0.006 to 0.78 microg/ml). R-95867 was quite stable to hydrolysis by most of the beta-lactamases tested except the metallo-beta-lactamases from Stenotrophomonas maltophilia and Bacteroides fragilis. R-95867 showed potent bactericidal activity against S. aureus and Escherichia coli. Penicillin-binding proteins 1 and 4 of S. aureus and 1Bs, 2, 3, and 4 of E. coli had high affinities for R-95867. The in vivo efficacy of CS-834 was evaluated in murine systemic infections caused by 16 strains of gram-positive and -negative pathogens. The efficacy of CS-834 was in many cases superior to those of cefteram pivoxil, cefpodoxime proxetil, cefdinir, and cefditoren pivoxil, especially against infections caused by S. aureus, penicillin-resistant S. pneumoniae, E. coli, Citrobacter freundii, and Proteus vulgaris. Among the drugs tested, CS-834 showed the highest efficacy against experimental pneumonia in mice caused by penicillin-resistant S. pneumoniae.

Bactericidal effect of plaunotol, a cytoprotective antiulcer agent, against Helicobacter pylori.

In order to investigate the bactericidal effect of plaunotol, an oily antiulcer agent, against Helicobacter pylori, comparative studies were conducted using its derivatives, M-4, M-5, and M-6, whose hydrophobicity decreased in the order of plaunotol > M-6 > M-5 > M-4 by log P determination. Plaunotol rapidly reduced the viability of H. pylori in vitro, and cell death was associated with cell lysis. In addition, plaunotol showed eightfold stronger bactericidal activity against H. pylori than M-6 and M-5, while the compound with the lowest hydrophobicity, M-4, showed no bactericidal activity. The bactericidal activities of plaunotol and its derivatives were related to the hydrophobicity of these compounds. To investigate a possible interaction between these compounds and the cell membrane of H. pylori, their effects on liposomal membranes prepared from phosphatidylethanolamine and cardiolipin, which are known to be present in the membrane of H. pylori, were determined by detection of glucose release from the liposomes. Plaunotol showed eight-fold higher activity than M-6 and M-5, while M-4 showed no activity. The effects of plaunotol and its derivatives on liposomal membrane were therefore related to their bactericidal activities. In addition, it was confirmed that the bactericidal effect of plaunotol against H. pylori was neutralized by the liposomal membrane, and that plaunotol led to an increase in permeability of the membrane, as evidenced by measurement of the leakage of 260 nm absorbing-material from H. pylori. These results suggest that the bactericidal effect of plaunotol against H. pylori is due to the interaction between this compound and the bacterial cell membrane.

In-vitro and in-vivo antibacterial activity of plaunotol, a cytoprotective antiulcer agent, against Helicobacter pylori.

Recently, some antiulcer agents have been reported to have antibacterial activity against Helicobacter pylori, which is highly associated with gastritis and peptic ulcers. In-vitro and in-vivo activity of plaunotol, a cytoprotective antiulcer agent, against H. pylori was investigated. Antibacterial activity of plaunotol against a standard strain (NCTC 11637) and 14 clinical isolates was compared with those of other cytoprotective antiulcer agents: benexate, sofalcone, teprenone, cetraxate, and gefarnate, by an agar dilution method. The MIC50 and MIC90 of plaunotol against 15 strains were 6.25 and 12.5 mg/L, respectively, making it the most potent of the cytoprotective antiulcer agents. The bactericidal effect of plaunotol was investigated using an in-vitro killing assay. Plaunotol at concentrations of more than 6 mg/L induced a rapid reduction of culture turbidity, with an extensive loss of viability, within 30 min. Observation by scanning electron microscopy revealed that plaunotol caused autolysis and treated cells were deformed. In-vivo activity of plaunotol against H. pylori was examined in a nude mouse gastritis model. Plaunotol significantly decreased the number of H. pylori in the stomach of nude mice. In addition, the antiulcer agent enhanced the antibacterial activity of amoxycillin or clarithromycin in the infection model.

Role of beta-lactamase of methicillin-susceptible Staphylococcus aureus in resistance to first-generation oral cephems both in vitro and in vivo.

Cefaclor, among the oral cephalosporins tested, showed the largest inoculum effect with respect to MIC values for 61 clinical isolates of methicillin-susceptible Staphylococcus aureus, including 39 beta-lactamase producing strains. These 39 strains were divided into eight type A, 29 type B or C, and two type D producers, by comparisons of specific activities to three substrates. Two producers, one each of types A and C, were further studied to investigate the effect of beta-lactamase on staphylococcal resistance to several beta-lactams. Concentrations of cefaclor and cephalexin in cultures of these strains decreased rapidly, whereas hydrolysis of these drugs by the purified beta-lactamases was moderate to low as detected by spectrophotometric assay. Cefaclor showed high affinities for penicillin-binding proteins 1, 2, and 3 of both beta-lactamase producers and their respective penicillinase-non-producing mutants. In experimental intraperitoneal infections in mice, cefaclor was therapeutically effective against both mutants, showing 50% effective doses of less than 10 mg/kg/dose. In contrast, it was not satisfactory against the parent strains, requiring greater-than-10-fold increases in concentration for the same degree of survival. We concluded that resistance to first-generation oral cephems seen both in vitro and in vivo was due mainly to the beta-lactamase production.

Thiomarinol, a new hybrid antimicrobial antibiotic produced by a marine bacterium. Fermentation, isolation, structure, and antimicrobial activity.

Thiomarinol, an antimicrobial antibiotic, was isolated from the culture broth of a marine bacterium, Alteromonas rava sp. nov. SANK 73390. Its structure was deduced as a hybrid composed of a pseudomonic acid analogue and holothin by NMR spectral analysis and chemical degradation. Antimicrobial activity against Gram-positive and Gram-negative bacteria of thiomarinol was stronger than both of pseudomonic acids and pyrrothine antibiotics.

Sequential acquisition of norfloxacin and ofloxacin resistance by methicillin-resistant and -susceptible Staphylococcus aureus.

The acquisition of ofloxacin resistance by a susceptible clinical Staphylococcus aureus strain was found to be achieved in two sequential steps: the first step was accompanied by 4-fold increases in the ofloxacin MIC and 8- to 16-fold increases in the norfloxacin MIC. The second step was accompanied by further increases in both the ofloxacin and the norfloxacin MICs. A mutation of the gyrA gene resulting in an amino acid substitution was found in the second-step but not in the first-step resistant subclone. On the other hand, there was no difference in the accumulation of norfloxacin in the parent strain and the resistant subclones of each step. The rates of mutation to resistance in the steps were (1.58 to 6.81) x 10(-9) and (0.71 to 2.59) x 10(-9), respectively, and did not depend on whether the parent strain was resistant to methicillin. Some implications of these observations for clinical as well as mechanistic aspects of the prevalence of methicillin- and ofloxacin-resistant S. aureus are discussed.

Direct radioimmunoassay for haloperidol in human serum.

A direct radioimmunoassay for the accurate determination of haloperidol in human serum has been developed. Based on recent information about the metabolism of haloperidol, a new haloperidol hapten, in which a (3-carboxypropionyl)methylamino group was attached as a bridge in the place of fluorine atom, was synthesized and coupled to bovine serum albumin through the bridge to provide a new immunogen. Guinea pigs were used for the immunization. Since the antisera obtained by the new immunogen still cross reacted greater than 10% with reduced haloperidol, the immunological tolerance to reduced haloperidol was induced by administration of a copolymer of D-glutamic acid and D-lysine linked with reduced haloperidol. This gave an antiserum in guinea pigs which was highly specific for unchanged haloperidol with negligible cross reactivity (less than or equal to 1.0%) to any haloperidol metabolites including the newly found ones. With the newly developed antiserum and [3H]haloperidol, serum haloperidol levels can be determined over the concentration range from 0.3 to 20 ng/mL, using 0.1 mL of human serum, without an extraction procedure.

In vitro and in vivo antibacterial activities of CS-807, a new oral cephalosporin.

CS-807 is a new oral prodrug of R-3746, a cephalosporin derivative, with potent in vitro and in vivo antibacterial activity against both gram-positive and gram-negative bacteria. The susceptibility of about 1,200 clinical isolates to R-3746 was determined by the agar dilution method. Ninety percent or more of pathogens such as Staphylococcus aureus, streptococci, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, indole-positive and indole-negative Proteus spp., Providencia rettgeri, and Haemophilus influenzae were inhibited at concentrations ranging less than or equal to 0.01 to 1.56 micrograms/ml. Furthermore, at a concentration of 3.13 micrograms/ml, 50% or more of Staphylococcus epidermidis, Morganella morganii, Citrobacter freundii, and Serratia marcescens strains were also inhibited. Pseudomonas aeruginosa and Xanthomonas maltophilia were resistant to R-3746. The activity of R-3746 was scarcely influenced by several growth conditions. R-3746 was highly resistant to hydrolysis by beta-lactamases derived from various species of bacteria. Killing-curve studies demonstrated bactericidal activity of R-3746 at concentrations above the MIC. R-3746 showed high affinity for penicillin-binding proteins 1, 3, and 4 of Staphylococcus aureus and 1A, 1Bs, and 3 of Escherichia coli. Systemic infections in mice caused by various pathogens, including beta-lactamase-producing strains, responded well to therapy with oral doses of CS-807.

Antibacterial activity of cefmetazole alone and in combination with fosfomycin against methicillin- and cephem-resistant Staphylococcus aureus.

In vitro and in vivo antibacterial activities of cefmetazole alone and in combination with fosfomycin against methicillin- and cephem-resistant (MR) strains of Staphylococcus aureus were investigated, and the mechanism of synergistic effect between cefmetazole and fosfomycin was also studied. Cefmetazole inhibited the growth of 71 strains of MR S. aureus at concentrations ranging from 1.56 to 50 micrograms/ml; the antibacterial activity of cefmetazole against these strains was enhanced approximately 4 times with the addition of fosfomycin at a concentration of 1.56 micrograms/ml. The binding affinity of cefmetazole for the penicillin-binding protein 2' fraction specific for MR S. aureus was higher than that of methicillin, cloxacillin, cefazolin, and cefotaxime. A synergy experiment in vitro was performed by checkerboard titration with Mueller-Hinton agar plates containing various concentrations and ratios of cefmetazole and fosfomycin. The fractional inhibitory concentration index ranged from 0.09 to 0.75. Exposure of cefmetazole plus fosfomycin to exponentially growing cultures at a concentration at which both antibiotics had no bactericidal effect when given alone exerted bactericidal action. Combined administration of cefmetazole with fosfomycin at a ratio of 1:1 against systemic MR S. aureus infections with mice showed an excellent therapeutic efficacy as compared with administration of either antibiotic alone. Penicillin-binding protein 2', 2, and 4 fractions were scarcely detectable in MR S. aureus strains grown in the presence of fosfomycin at concentrations of 0.25 MIC and 0.5 MIC, respectively.

Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus.

About 80% of methicillin- and cefazolin-resistant strains of Staphylococcus aureus isolated clinically in Japan in 1982 retained their resistance even after elimination of penicillinase-encoding plasmids. The penicillin-binding proteins (PBPs) of the penicillinase-free, methicillin- and cephem-resistant subclones of Staphylococcus aureus (MRSA) were compared with those of spontaneous susceptible revertants which had been obtained by the replica method after 10 subcultures in drug-free media. A new PBP fraction (PBP2') having a molecular weight of 78,000 and low binding affinities for various beta-lactam antibiotics was found in MRSA exclusively. The levels of resistance of MRSA strains were reduced markedly by culturing them at 43 degrees C or at pH 5.2 or both. We found that the binding capacity of PBP2' for 14C-labeled penicillin G was decreased by preincubation of the membrane fractions of MRSA strains at 43 degrees C for 60 min and that the amount of PBP2' in MRSA strains grown at pH 5.2 was less than that the amount of PBP2' in MRSA strains grown at pH 7.0. Temperature- and pH-dependent expression of resistance in MRSA is likely to reflect the temperature sensitivity and neutral pH-dependent production of the specific PBP fraction (PBP2'). We suggest that MRSA strains can grow in the presence of beta-lactam antibiotics because of the low affinities of the specific PBP2' fraction for various beta-lactam antibiotics.

Cefmetazole: a broad spectrum cephem antibiotic effective on methicillin- and cephem-resistant Staphylococcus aureus.

Antibacterial activity of cefmetazole (CMZ) was investigated by the plate dilution method. Since CMZ is stable to any type of bacterial beta-lactamases, it inhibited growth of Escherichia coli carrying R plasmids, Klebsiella spp., Proteus vulgaris, and Bacteroides fragilis at the concentration of less than 0.78 to 25.0 micrograms/ml. The distinct characteristic of CMZ is its anti-MRSA (methicillin- and cephem-resistant Staphylococcus aureus) activity. Exclusively in MRSA, a new fraction of penicillin binding proteins (PBP) with a relative molecular mass of 78 kd apears, and the 78 kd/PBP possesses low binding affinity to beta-lactam antibiotics. By the competitive binding experiment of beta-lactam drugs to the PBPs of MRSA, it was revealed that CMZ and cephaloridine retain binding affinities to the new 78kd/PBP fraction of MRSA.

[Sensitive determination of haloperidol in human serum by radioimmunoassay].

An improved procedure for the sensitive determination of the neuroleptic drug, haloperidol, in human serum is described. The method is based on a single hexane extraction procedure and a radioimmunoassay. Antiserum was elicited in guinea pigs immunized with haloperidol hemisuccinate derivative coupled to bovine serum albumin. Any appreciable cross-reactivity was observed neither with known metabolites of haloperidol nor with haloperidol decanoate (KD-136). Some devices in the extraction procedure, e.g. the use of 10 ml of extraction solvent, made it possible to measure haloperidol levels as low as 0.05 ng/ml. The accuracy and reproducibility of the method were shown to be sufficient. The present method can be used to study the pharmacokinetics of haloperidol in the phase 1 study of KD-136. In addition, it is simple enough for use in clinical laboratories that are monitoring haloperidol concentrations in the blood of psychiatric patients.

Release of lipoteichoic acid from Staphylococcus aureus by treatment with cefmetazole and other beta-lactam antibiotics.

The effect of cefmetazole on the growth together with the release of cellular lipoteichoic acid from cefazolin-resistant strains of Staphylococcus aureus was compared with that of cefazolin, cefotiam, cefoxitin and cefuroxime. Bacteriolytic actions were measured by turbidity and bactericidal actions were followed by viable cell count. Release of cellular lipoteichoic acid was measured by the radioactivity in the supernatant of the cultures. Cefmetazole exerted more potent effects on the bacterial growth and induced more marked release of cellular lipoteichoic acid from resistant strains as compared with other beta-lactams.

Penem derivatives: beta-lactamase stability and affinity for penicillin-binding proteins in Escherichia coli.

Penem derivatives, a new group of beta-lactam antibiotics with potent activities against a wide range of bacteria, including Pseudomonas aeruginosa, were tested for their stability against hydrolysis by beta-lactamases purified from clinical isolates of Morganella morganii. Proteus vulgaris, and Escherichia coli and by a penicillinase from Bacillus cereus. Penems having 6 alpha substituents, such as hydroxyethyl, hydroxymethyl, and ethyl groups, were very stable against hydrolysis by each of the enzymes. Penems having no 6 alpha substituents were easily hydrolyzed by P. vulgaris and E. coli enzymes, whereas they were rather stable against hydrolysis by M. morganii and B. cereus enzymes, a typical cephalosporinase and penicillinase, respectively. Affinity of the penems for E. coli penicillin-binding proteins (PBPs) was also tested. beta-Lactamase-stable penems having a 6 alpha-hydroxyethyl group showed high affinity for PBP-4, -5, and -6 as well as for PBP-1A, -1Bs, and -2. However, the penems having no 6 alpha substituents showed a far lower affinity for PBP-4, -5, and -6 than that shown by the corresponding 6 alpha-hydroxyethyl penems. Among the penems tested, affinity for PBP-4, -5, and -6 was closely related to their beta-lactamase stability, as was the case among cephamycins and cephalosporins. Effects of the penems on the morphology of a strain of E. coli are also described.

Evidence for a synergistic interaction between phenytoin and phenobarbital in experimental animals.

Combined anticonvulsant effects of phenytoin and phenobarbital in mice and rabbits were studied using the isobolographic method which was based on the plasma concentrations of these drugs. Anticonvulsant activity of test drugs alone or in combination was determined by the method of maximal electroshock seizure test. Response of each animal to drug action was recorded as abolition of the tonic hindlimb extensor component of maximal electroshock seizure. It was demonstrated that the minimal effective plasma concentrations of phenytoin and phenobarbital alone for protection against maximal electroshock seizure was 5.9 +/- 0.2 and 18.1 +/- 0.4 microgram/ml (+/- SE) in mice and 14.7 +/- 0.5 and 17.5 +/- 0.6 microgram/ml (+/-SE) in rabbits, respectively. Isobolograms for mice and rabbits were constructed by identifying and plotting each responder and nonresponder for its corresponding plasma phenytoin and phenobarbital concentrations when these drugs were given in combination. The isoboles in mice and rabbits were concave bowing toward the origin, indicated synergism (potentiation). The apparent synergism between phenytoin and phenobarbital cannot be explained by changes in the amount of these drugs entering into the brain from the blood, since the relationship between brain and plasma concentrations of either drug was not altered by the presence of the other. These results, therefore, suggest that the synergistic action between these two anticonvulsants occurs within the central nervous system; this provides experimental support for the combined use of phenytoin and phenobarbital in the clinical treatment of epilepsy.

Determination of haloperidol in human serum by radioimmunoassay.

A radioimmunoassay has been developed which enables accurate determination of haloperidol in human serum. Antiserum was prepared by immunizing guinea pigs with haloperidol (O-carboxymethyl)oxime derivative (III) coupled with bovine serum albumin. With the antiserum, 3H-haloperidol and dextran-coated charcoal, the assay of haloperidol in serum was possible over a concentration range of 1 to 50 ng/ml, using 0.1 ml of human serum without the need of an extraction procedure. Data obtained by radioimmunoassay are in good agreement with those obtained by gas chromatography. No appreciable cross-reactivity was observed neither with haloperidol metabolites nor with other butyrophenone neuroleptics. Serum levels of haloperidol in schizophrenic patients receiving single oral dosing (6 mg/subject) have also been determined.