Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN 192024).

Replacement of the carboxylic acid group of prostaglandin (PG) F(2alpha) with a nonacidic moiety, such as hydroxyl, methoxy, or amido, results in compounds with unique pharmacology. Bimatoprost (AGN 192024) is also a pharmacologically novel PGF(2alpha) analog, where the carboxylic acid is replaced by a neutral ethylamide substituent. Bimatoprost potently contracted the feline lung parenchymal preparation (EC(50) value of 35-55 nM) but exhibited no meaningful activity in a variety of PG-sensitive tissue and cell preparations. Its activity seemed unrelated to FP receptor stimulation according to the following evidence. 1) Bimatoprost exhibited no meaningful activity in tissues and cells containing functional FP receptors. 2) Bimatoprost activity in the cat lung parenchyma is not species-specific because its potent activity in this preparation could not be reproduced in cells stably expressing the feline FP receptor. 3) Radioligand binding studies using feline and human recombinant FP receptors exhibited minimal competition versus [(3)H]17-phenyl PGF(2a) for Bimatoprost. 4) Bimatoprost pretreatment did not attenuate PGF(2alpha)-induced Ca(2+) signals in Swiss 3T3 cells. 5) Regional differences were apparent for Bimatoprost but not FP agonist effects in the cat lung. Bimatoprost reduced intraocular pressure in ocular normotensive and hypertensive monkeys over a 0.001 to 0.1% dose range. A single-dose and multiple-dose ocular distribution/metabolism studies using [(3)H]Bimatoprost (0.1%) were performed. Within the globe, bimatoprost concentrations were 10- to 100-fold higher in anterior segment tissues compared with the aqueous humor. Bimatoprost was overwhelmingly the predominant molecular species identified at all time points in ocular tissues, indicating that the intact molecule reduces intraocular pressure.

The pharmacology of bimatoprost (Lumigan).

Bimatoprost (Lumigan) is a pharmacologically unique and highly efficacious ocular hypotensive agent. It appears to mimic the activity of a newly discovered family of fatty acid amides, termed prostamides. One biosynthetic route to the prostamides involves anandamide as the precursor. Bimatoprost pharmacology has been extensively characterized by binding and functional studies at more than 100 drug targets, which comprise a diverse variety of receptors, ion channels, and transporters. Bimatoprost exhibited no meaningful activity at receptors known to include antiglaucoma drug targets as follows: adenosine (A(1-3)), adrenergic (alpha(1), alpha(2), beta(1), beta(2)), cannabinoid (CB(1), CB(2)), dopamine (D(1-5)), muscarinic (M(1-5)), prostanoid (DP, EP(1-4), FP, IP, TP), and serotonin (5HT(1-7)). Bimatoprost does, however, exhibit potent inherent pharmacological activity in the feline iris sphincter preparation, which is prostamide-sensitive. Bimatoprost also resembles the prostamides in that it is a potent and highly efficacious ocular hypotensive agent. A single dose of bimatoprost markedly reduces intraocular pressure in dogs and laser-induced ocular hypertensive monkeys. Decreases in intraocular pressure are well maintained for at least 24 hr post-dose. Human studies have demonstrated that systemic exposure to bimatoprost is low and that accumulation does not occur. The sclera is the preferred route of accession to the eye. The high scleral permeability coefficient Papp is a likely contributing factor to the rapid onset and long-acting ocular hypotensive profile of bimatoprost.

The M1 muscarinic agonist CI-1017 facilitates trace eyeblink conditioning in aging rabbits and increases the excitability of CA1 pyramidal neurons.

The M1 muscarinic agonist CI-1017 was administered intravenously to aging rabbits on a daily basis before and during hippocampally dependent trace eyeblink conditioning sessions. Circulating levels of CI-1017 were significantly related to the drug dose. The drug was found to significantly increase the rate and amount of learning in a dose-dependent manner with no significant effects on the amplitude, area, or latency of conditioned responses. There was no evidence of pseudoconditioning at the highest drug concentration, and the minimally effective dose produced only mild and temporary hypersalivation as a side effect. CI-1017 (10 microM) was also found to increase the excitability of CA1 pyramidal neurons recorded from hippocampal slices from young and aging naive rabbits as measured by changes in spike-frequency adaptation and the postburst afterhyperpolarization. These biophysical changes were reversed with either atropine (1 microM) or pirenzepine (1 microM). These results suggest that M1 agonists ameliorate age-related learning and memory impairments at least in part by reducing the afterhyperpolarization and spike-frequency adaptation of hippocampal pyramidal neurons and that M1 agonists may be an effective therapy for reducing the cognitive deficits that accompany normal aging and/or Alzheimer's disease.

A Mycobacterium ulcerans toxin, mycolactone, causes apoptosis in guinea pig ulcers and tissue culture cells.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, from M. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.

The neuroprotective effect of the novel AMPA receptor antagonist PD152247 (PNQX) in temporary focal ischemia in the rat.

BACKGROUND AND PURPOSE: Evidence suggests that glutamate contributes to ischemic brain damage through activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor. We tested the novel, selective AMPA receptor antagonist PD152247 (PNQX) in a model of temporary focal ischemia to determine the dose-response relationship and to investigate the contribution of drug-induced hypothermia to the neuroprotective action of AMPA receptor antagonists. METHODS: Temporary focal cerebral ischemia was induced in Sprague-Dawley rats by occluding the middle cerebral artery and both carotid arteries for 3 hours. Body temperature was monitored by telemetry. PNQX was administered intraperitoneally or by intravenous infusion with various doses for 6 hours. Lesion volume was determined after 3 days by stereological methods. RESULTS: PNQX reduced the lesion volume by 51% after intraperitoneal administration. The intravenous dose-response study demonstrated that the lowest effective dose of PNQX was 1.0 mg/kg per hour, which corresponded to a steady state plasma level of 685 ng/mL. Neuroprotection was demonstrated at PNQX plasma concentrations that did not lower body temperature over the entire course of the experiment. CONCLUSIONS: AMPA receptor activation plays an important role in the development of ischemic brain damage. Thus, novel AMPA receptor antagonists may be useful for the treatment of stroke in humans.

Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe human skin disease that occurs primarily in Africa and Australia. Infection with M. ulcerans results in persistent severe necrosis without an acute inflammatory response. The presence of histopathological changes distant from the site of infection suggested that pathogenesis might be toxin mediated. A polyketide-derived macrolide designated mycolactone was isolated that causes cytopathicity and cell cycle arrest in cultured L929 murine fibroblasts. Intradermal inoculation of purified toxin into guinea pigs produced a lesion similar to that of Buruli ulcer in humans. This toxin may represent one of a family of virulence factors associated with pathology in mycobacterial diseases such as leprosy and tuberculosis.

Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis.

Although Mycobacterium ulcerans, M. marinum, and M. haemophilum are closely related, their exact taxonomic placements have not been determined. We performed gas chromatography of fatty acids and alcohols, as well as DNA-DNA hybridization and 16S rRNA gene sequence analysis, to clarify their relationships to each other and to M. tuberculosis. M. ulcerans and M. marinum were most closely related to one another, and each displayed very strong genetic affinities to M. tuberculosis; they are actually the two mycobacterial species outside the M. tuberculosis complex most closely related to M. tuberculosis. M. haemophilum was more distinct from M. ulcerans and M. marinum, and it appeared to be as related to these two species as to M. tuberculosis. These results are important with regard to the development of diagnostic and epidemiological tools such as species-specific DNA probes and PCR assays for M. ulcerans, M. marinum, and M. haemophilum. In addition, the finding that M. ulcerans and M. marinum are more closely related to M. tuberculosis than are other pathogenic mycobacterial species suggests that they may be evaluated as useful models for studying the pathogenesis of M. tuberculosis. M. marinum may be particularly useful in this regard since strains of this species grow much more rapidly than M. tuberculosis and yet can cause systemic disease in immunocompromised hosts.

A summary of mechanistic hypotheses of gabapentin pharmacology.

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

Partial purification and characterization of biological effects of a lipid toxin produced by Mycobacterium ulcerans.

Organisms in the genus Mycobacterium cause a variety of human diseases. One member of the genus, M. ulcerans, causes a necrotizing skin disease called Buruli ulcer. Buruli ulcer is unique among mycobacterial diseases in that the organisms at the site of infection are extracellular and there is little acute inflammatory response. Previous literature reported the presence of a toxin in the culture supernatant of M. ulcerans which causes a cytopathic effect on the mouse fibroblast cell line L929 in which the adherent cells round up and detach from the tissue culture plate. Here we report partial purification of a lipid toxin from the culture supernatant of M. ulcerans which is capable of causing the cytopathic effect on L929 cells. We also show that this cytopathic effect is a result of cytoskeletal rearrangement. The M. ulcerans toxin does not cause cell death but instead arrests cells in the G1 phase of the cell cycle.

Versatile action of Escherichia coli ClpXP as protease or molecular chaperone for bacteriophage Mu transposition.

The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition. As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions. After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly. Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes. Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX. Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes. The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function.

Communication of ClpXP protease hypersensitivity to bacteriophage Mu repressor isoforms.

The immunity repressor (Rep) of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. Although Rep is stable in vivo, an altered immunity repressor (Vir) encoded by virulent, trans-dominant Mu mutants is rapidly degraded by Escherichia coli ClpXP protease. Rep and Vir are degraded at approximately the same maximal velocity (Vmax) by ClpXP, but the Km for Rep (3.6 microM) is over 20-fold higher than the Km for Vir (0.15 microM). Rep is also highly resistant to degradation in the presence of DNA whereas Vir is not. Vir increases the rate of Rep degradation by reducing its Km and imparts to Rep ClpXP sensitivity in the presence of DNA. Vir can drive at an accelerated rate the complete degradation of Rep molecules that outnumber Vir by eightfold or more. So long as Vir is present at a concentration of 0.1 microM or higher, Rep is degraded with a Km that is indistinguishable from that of Vir. These characteristics of repressor may be an important means of transducing physiological signals that induce Mu transposition in response to growth conditions or environmental stress, ClpXP hypersensitivity being disseminated among Rep molecules for the induction of Mu transposition.

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

The simultaneous estimation of the influx and efflux blood-brain barrier permeabilities of gabapentin using a microdialysis-pharmacokinetic approach.

PURPOSE: To determine the apparent bidirectional permeabilities of gabapentin (GBP) across the blood-brain barrier (BBB) using a novel microdialysis-pharmacokinetic approach. METHODS: Rats were administered intravenous infusions of [14C]GBP to achieve clinically relevant steady-state plasma concentrations. Microdialysis was used to monitor GBP concentration in brain extracellular fluid (ECF) in conscious animals. Brain tissue GBP concentration was measured at termination. The BBB influx (CL1) and efflux (CL2) permeabilities of GBP were estimated with a hybrid pharmacokinetic model assuming that transport between intra- and extracellular space was more rapid than transport across the BBB. The time course of GBP concentration in brain tissue was determined independently to validate the model assumption. RESULTS AND CONCLUSIONS: Simulations of the concentration-time course of GBP in brain tissue based on this modeling correlated well with the time-course of brain tissue concentrations determined after intravenous bolus administration and validated this pharmacokinetic-microdialysis approach for estimation of BBB permeabilities. The values for CL1 and CL2 were 0.042 (0.017) and 0.36 (0.16) ml/min.g-brain, respectively, indicating that GBP was more efficiently transported from brain ECF to plasma. The total brain tissue concentration of GBP was significantly higher than the ECF concentration at steady-state due to intracellular accumulation and tissue binding, that if not considered, will lead to underestimated efflux BBB permeability using the tissue homogenate-pharmacokinetic approach.

ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis.

During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes.

Potential treatment of amyotrophic lateral sclerosis with gabapentin: a hypothesis.

OBJECTIVE: To provide the biochemical rationale for the use of the new anticonvulsant agent gabapentin as a treatment for amyotrophic lateral sclerosis (ALS). BACKGROUND: ALS is a neuropathologic disorder of the central nervous system characterized by a progressive loss of upper and lower motor neurons. Although the etiopathology of ALS is incompletely known, it is hypothesized that glutamatergic neurotransmission is related to neuropathology. Glutamate is an excitatory amino acid neurotransmitter that is cytotoxic when overexpressed at synaptic terminals, probably through a calcium-related mechanism. The concentration of glutamate in cerebrospinal fluid is increased in patients with ALS. The increased extracellular concentrations of glutamate may be caused by a decreased capacity of glutamate transport in brain tissue and/or abnormal glutamate metabolism. Recent success with the glutamate release inhibitor riluzole in well-controlled clinical trials supports the excitotoxic mechanism of neuropathology in patients with ALS. POTENTIAL TREATMENT FOR ALS: Gabapentin has demonstrated neuroprotective properties in a model of chronic glutamate toxicity in vitro. Although the neuroprotective mechanism of action of gabapentin is currently unknown, it is hypothesized here that gabapentin decreases the rate of formation of glutamate derived from the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine. The proposed decrease in formation of glutamate from BCAAs may decrease the pool of releasable glutamate and therefore compensate for diminished glutamate uptake capacity and/or abnormal glutamate metabolism in patients with ALS. CONCLUSIONS: Based on this rationale, it is proposed that gabapentin may provide a beneficial effect in the treatment of patients with ALS.

Effects of anticonvulsant drug gabapentin on the enzymes in metabolic pathways of glutamate and GABA.

Gabapentin is a novel anticonvulsant drug. The anticonvulsant mechanism of gabapentin is not known. Based on the amino acid structure of gabapentin we explored its possible effects on glutamate and gamma-aminobutyric acid (GABA) metabolism in brain as they may relate to its anticonvulsant mechanisms of action. Gabapentin was tested for its effects on seven enzymes in the metabolic pathways of these two neurotransmitters: alanine aminotransferase (AL-T), aspartate aminotransferase (AS-T), GABA aminotransferase (GABA-T), branched-chain amino acid aminotransferase (BCAA-T), glutamine synthetase (Gln-S), glutaminase (GLNase), and glutamate dehydrogenase (GDH). In the presence of 10 mM gabapentin, only GABA-T, BCAA-T, and GDH activities were affected by this drug. Inhibition of GABA-T by gabapentin was weak (33%). The Ki values for inhibition of cytosolic and mitochondrial forms of GABA-T (17-20 mM) were much higher than the Km values for GABA (1.5-1.9 mM). It is, therefore, unlikely that inhibition of GABA-T by gabapentin is clinically relevant. As with leucine, gabapentin stimulated GDH activity. The GDH activity in rat brain synaptosomes was activated 6-fold and 3.4-fold, respectively, at saturating concentrations (10 mM) of leucine and gabapentin. The half-maximal stimulation by gabapentin was observed at approximately 1.5 mM. Gabapentin is not a substrate of BCAA-T, but it exhibited a potent competitive inhibition of both cytosolic and mitochondrial forms of brain BCAA-T. Inhibition of BCAA-T by this drug was reversible. The Ki values (0.8-1.4 mM) for inhibition of transamination by gabapentin were close to the apparent Km values for the branched-chain amino acids (BCAA) L-leucine, L-isoleucine, and L-valine (0.6-1.2 mM), suggesting that gabapentin may significantly reduce synthesis of glutamate from BCAA in brain by acting on BCAA-T.

Hepatic and adrenal toxicity of a novel lipid regulator in beagle dogs.

PD 138142-15 is a substituted urea hypolipidemic and potential anti-atherosclerotic agent. To determine the toxicity of PD 138142-15, beagle dogs were given oral doses of 1, 10, 30, and 100 mg/kg daily for 13 weeks. Two animals at 100 mg/kg were euthanized during Week 5 due to poor condition. Clinical findings included decreased serum albumin at > or = 30 mg/kg, and increased ALP (up to 30-fold) and 5'-nucleotidase activities (up to 9-fold) at doses > or = 10 mg/kg. ALT and AST activities were elevated only at 100 mg/kg. There was a two- to threefold increase in cytochrome P450 content of hepatic microsomes from all treated animals and increases in liver weights at 10 mg/kg and above. Hepatic changes included hepatocellular hypertrophy and increased cytoplasmic eosinophilia at > or = 10 mg/kg; single cell necrosis of hepatocytes was noted in moribund animals. ACTH-stimulated cortisol levels were decreased at 30 and 100 mg/kg. Adrenal cholesterol esters were decreased at 10 mg/kg and above, while total adrenal cholesterol was decreased at > or = 30 mg/kg. These changes correlated with adrenal cortical zonal atrophy, principally of the zona fasciculata and zona reticularis, present at 30 and 100 mg/kg. Plasma concentrations of PD 131842-15 increased with increasing dose; plasma levels were significantly lower during Week 12 than those on Day 1, possibly due to autoinduction. Overt hepatotoxicity occurred at 100 mg/kg, whereas hepatic changes at 10 and 30 mg/kg were consistent with cytochrome P450 induction. The hepatic lesions were reversible within 4 weeks, while adrenal lesions were still evident after 4 weeks without treatment.

Hepatic microsomal induction profile of carbamic acid [[2,6-bis(1- methylethyl)phenoxy] sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel lipid regulating agent.

Induction of hepatic microsomal cytochrome P450 produced by carbamic acid [2,6-bis(1-methylethyl)phenoxy]sulfonyl]-2,6-bis(1-methylethyl) phenyl ester, monosodium salt (PD138142-15), a novel water-soluble inhibitor of acyl-CoA: cholesterol acyltransferase, was examined in male and female rats, dogs, and monkeys, and in male guinea pigs. Relative to control, PD138142-15 increased hepatic microsomal total spectral P450 in all species examined. Hepatic microsomal ethoxyresorufin-O-deethylase, pentoxyresorufin-O-dealkylase, and peroxisomal carnitine acetyltransferase activities and cyanide-insensitive Beta-oxidation were affected only marginally. Erythromycin-N-demethylase activity was increased (2- to 6-fold) in all three species in which it was examined (rat, dog and pig). Marked increases in immunoreactive P450 3A were noted in the rats and dogs, while slight increases were seen in monkeys. Pharmacokinetic studies of PD138142-15 in rats and dogs revealed pronounced decreases (80-90%) in plasma Cmax and AUC within 2 weeks of initiation of daily dosing. In spite of the marked decline in plasma drug levels, efficacy in dogs, as determined by serum cholesterol levels, was maintained for up to 6 weeks with continued dosing. Potential acid (gastric) breakdown products of PD 138142-15 were examined for their hepatic cytochrome P450 induction profiles in rats adn were found to differ both quantitatively and qualitatively from profiles produced by the parent compound. This suggested that induction observed in rats was due to parent PD138142-15 and not to any of the known potential acid breakdown products. The cumulative data establish that PD 138142-15 is an inducer of P450 3A in rats and dogs. The results also suggest that P450 3A is induced in monkeys and pigs as well, although the data are less definitive. Decreases in plasma drug levels imply that the compound may be an autoinducer in dogs and rats. The maintenance of efficacy in spite of decreased drugs levels in dogs suggests that the effects on serum cholesterol are due to a metabolite or that cholesterol lowering effects occur before the compound is metabolized by the liver.

The temporal effect of food on tacrine bioavailability.

A four-way cross-over study was performed to assess the temporal effect of food on the rate and extent of tacrine (Cognex, THA) absorption after drug administration to healthy, older volunteers. Each volunteer received four single 40-mg THA doses at 1-week intervals. Doses were administered after an 8-hour overnight fast, 1 hour before a standard breakfast, 15 minutes after beginning a standard breakfast, and 2 hours after completion of a standard breakfast. Gastrointestinal side effects were most frequently reported after drug administration to fasted subjects. Mean Cmax and AUC(0-infinity) values after THA administration during breakfast (9.9 ng/mL and 70.2 ng.hr/mL) and 2 hours after breakfast (11.6 ng/mL and 74.2 ng.hour-1.mL-1) were significantly lower than values determined after administration of THA to fasting subjects (15.8 ng/mL, and 91.8 ng.hour-1.mL-1). Little effect was evident when THA was administered 1 hour before breakfast.

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.