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Incubation of human neutrophils (PIN) in vitro with recombinant human tumor necrosis factor (rhTNF), decreased PMN non-directed and directed migration. The alkylxanthines, rentoxifylline (RTX)

High concentrations of free human neutrophil elastase in bronchial epithelial fluid are believed to be a major factor in the evolution of pulmonary injury in cystic fibrosis (CF). To test this hypothesis, we studied pentoxifylline, a compound that inhibits tumor necrosis factor alpha transcription and its stimulatory effect on polymorphonuclear neutrophils, in patients with CF who had chronic Pseudomonas bronchitis. Subjects older than 11 years of age randomly received placebo or pentoxifylline (1600 mg/day) orally, in a double-blind fashion, for 6 months. Pulmonary function and sputum elastase concentrations were determined before therapy and bimonthly during therapy; compliance was determined by measuring serum drug concentrations. Of the 16 patients who completed the study, 9 received pentoxifylline. The sputum elastase concentrations among placebo recipients were significantly increased from baseline at 4 and 6 months (F = 3.44; p0.05); the values remained unchanged in the treatment group. The mean forced vital capacity for the placebo group decreased from 59.2% +/- 15.4% predicted at baseline to 52.0% +/- 12.9% predicted at 6 months; the values in the treatment group remained largely unchanged. The forced vital capacity improved between baseline and 6 months for four of nine pentoxifylline recipients and none of the seven control patients (p = 0.09). During the study, four of seven placebo recipients experienced a significant pulmonary exacerbation compared with one of nine treated patients (p = 0.077). These findings support the hypothesis that polymorphonuclear neutrophil elastase is a factor in the evolution of CF lung disease; further studies are needed to define the role of pentoxifylline in the treatment of CF.

We have recently established a rhesus monkey model of chronic Pseudomonas aeruginosa (PA) endobronchitis by bronchoscopic instillation of PA-embedded agar beads. All experimental animals developed chronic neutrophilic endobronchitis similar to chronic PA endobronchitis in cystic fibrosis (CF). Histopathologic studies further confirmed similarities to chronic PA endobronchitis in CF, including marked peribronchial inflammation, epithelial damage, presence of degraded cilia and ciliary abnormalities, appearance of PA bacterial clusters, mucosal hyperplasia, goblet cell hypertrophy/hypersecretion, airway obstruction, alveolar abnormalities, bronchiectasis, and fibrosis.

This guideline summarizes recommendations for (1) developing cogent procedures for diagnosis and antimicrobial susceptibility testing; (2) developing quality-control parameters for the microbiological components of clinical trials; (3) continually updating U.S. Food and Drug Administration (FDA) guidelines; (4) reviewing microbiological recommendations from other groups, such as Microbiology Subcommittees of the National Committee for Clinical Laboratory Standards; and (5) improving the microbiological aspects of FDA package inserts for antimicrobial drugs. Sensitive and specific methods for isolation and identification of pathogens are essential to the proper conduct of clinical trials. Susceptibility tests should be performed in an accurate and reproducible fashion. Verification of results in a reference laboratory is encouraged to monitor quality control.

Host defense abnormalities in cystic fibrosis (CF) against Pseudomonas aeruginosa (PA) lead to excessive neutrophil influx into the infected lungs, resulting in pulmonary complications. We have developed a rhesus monkey model of chronic PA endobronchitis by intrabronchial instillation of PA-embedded agar beads, utilizing flexible fiberoptic bronchoscopy. Treatment of infected monkeys with pentoxifylline suppressed neutrophil influx and ameliorated pulmonary damage. The results suggest a method by which neutrophil influx and pulmonary damage in CF patients can be managed or prevented.

Cefpirome, a so-called fourth-generation cephalosporin, was tested alone and in combination with the sulfone beta-lactamase inhibitor, tazobactam, against 63 members of the Bacteroides fragilis group. The cefpirome MIC50 was only 64 micrograms/ml, but the MIC was reduced eightfold with tazobactam (2:1 ratio). The addition of tazobactam to cefpirome or the use of metronidazole as a codrug appear to be alternative choices to enhance the antianaerobic spectrum. Over 98% of strains had cefpirome-tazobactam MICs of less than or equal to 32 micrograms/ml.

Cytokines have been implicated in the pathogenesis of gram-negative bacterial meningitis. The effects of pentoxifylline and dexamethasone on the release of tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6 from primary murine microglial cell cultures were explored using bioassays. When added concomitantly with lipopolysaccharide, pentoxifylline blocked the release of TNF and IL-1 but not IL-6, while dexamethasone inhibited the release of TNF and IL-6. After a 2-h exposure of microglia to lipopolysaccharide, pentoxifylline but not dexamethasone still inhibited the release of TNF. Release of TNF was enhanced 20-fold by priming of the microglia with interferon-gamma; only pentoxifylline blocked the priming effect of interferon-gamma on TNF release. These results demonstrate that pentoxifylline and dexamethasone differentially regulate the release of cytokines in microglial cell cultures and provide potential insight into their role in the treatment of gram-negative bacterial meningitis.

Polymorphonuclear (PMN) overactivation plays a critical role in microcirculation as well as in conditions such as multiorgan failure (MOF). Pentoxifylline has been shown to prevent PMN activation by endotoxin and cytokines such as TNF alpha and IL-1. In addition, MOF induced by IL-2 in animals can be prevented by pentoxifylline. The present studies evaluated two aspects of PMN activation and pentoxifylline interaction. The first was the time sequence for pentoxifylline prevention of TNF alpha activation and the second was the activity of pentoxifylline on amphotericin B activation of PMNs. TNF alpha activation of PMNs is blocked by pentoxifylline when cells are exposed to pentoxifylline prior to TNF alpha or after TNF alpha. Amphotericin B activation of PMNs was demonstrated by a decreased chemotaxis, increased chemiluminescence, and increased PMN spreading. In all conditions, pentoxifylline decreased amphotericin B activation of PMNs. These results suggest that pentoxifylline can reverse cytokine activation of PMNs and that pentoxifylline may alter some of the toxic effects of amphotericin.

An extensive series of carboxyarylindoles has been evaluated for antiinflammatory activity in the carrageenin paw edema assay. The requirements for optimal antiinflammatory activity in this series are relatively specific: a central pyrrole nucleus with (a) a 3-carboxy-4-hydroxyphenyl moiety substituted directly on the nitrogen, (b) a 2-phenyl group (R2) with a substituent of low electronegativity, (c) absence of a substituent in the 3 position (R3), and (d) a system fused across the 4,5 positions (X), which is lipophilic, quasiplanar, and does not interact sterically with the N-phenyl group. One derivative, 3-(3-carboxy-4-hydroxyphenyl)-2-phenyl-4,5-dihydro-3H-benz[e]indole (42), has been selected for further study.

The synthesis and antihypertensive and diuretic activity of several N-sulfur derivatives of 3-phenylspiro[isobenzofuran-1(3H),4'-piperidine] are reported. Benzenesulfenamide 3 possessed marked, species-specific diuretic and antihypertensive activity in rats.

A series of [(3-aryl-1,2-benzisoxazol-6-yl)oxy]acetic acids was synthesized and tested for diuretic activity in saline-loaded mice and in conscious, water-loaded dogs. The structural requirements for good diuretic activity in both mice and dogs were found to be very specific. In summary, the compounds with the best diuretic activity (13i, 13q, and 13ff) were substituted with a 2-fluorophenyl group at the 3 position and chlorine or bromine at the 7 position. Compound 13ff, [(7-bromo-3-(2-fluorophenyl)-1,2-benzisoxazol-6-yl]oxy]acetic acid (HP 522), was found to be moderately uricosuric in chimpanzees and was selected for further development.

Apomorphine administered at a dose of 1.5 mg/kg SC induces an intense climbing behavior in mice lasting for approximately 30 min. It is known that neuroleptics attenuate apomorphine climbing behavior (AC) but the influence of GABA agonists and antagonists on this behavior is unclear. The GABA agonist, muscimol, blocked AC with an ED50=0.9 mg/kg IP. Pretreatment (5 hr) with aminooxyacetic acid (50 mg/kg IP), a GABA-T inhibitor, produced a 65% inhibition of AC. The GABA antagonists, picrotoxin and bicuculline, had little effect on AC. Baclofen had no effect on AC from 0.5 to 4 mg/kg IP. Interaction studies, combining muscimol and haloperidol were conducted to evaluate the interaction between GABA-ergic and dopaminergic systems on AC. In the first studies mice were treated with haloperidol (0.04-0.15 mg/kg SC) then challenged with inactive (0.2 mg/kg IP) and active (0.4 mg/kg IP) doses of muscimol. In the second series of studies mice were treated with muscimol (0.25-1 mg/kg IP) then challenged with inactive (0.04 mg/kg SC) and active (0.08 mg/kg SC) doses of haloperidol. In all of these experiments, there was an enhanced suppression of AC. Our studies suggest that in addition to dopamine antagonists, GABA agonists are also capable of blocking AC. Our results further suggest that the effect of GABA agonists in inhibiting AC may be mediated through dopaminergic neurons.

Fluradoline (HP 494), a tricyclic dibenz (b, f) oxepine derivative with an analgesic profile, was tested for antidepressant activity. After oral administration, fluradoline was twice as potent as imipramine and similar in potency to desmethylimipramine in blocking tetrabenazine-induced ptosis. Like standard antidepressants, fluradoline selectively increased response rates for electrical stimulation of the median forebrain bundle using internal capsule-lesioned rats. Response rates in nonlesioned rats were unaffected. There was partial protection against yohimbine toxicity and no potentiation of 5-hydroxytryptophan-induced seizures in mice. When administered to squirrel monkeys, the EEG profile from cortically-placed electrodes resembled that found for imipramine. Using in vivo and in vitro techniques, it was shown that fluradoline was not a monoamine oxidase inhibitor; however, the compound did block the reuptake of norepinephrine, serotonin and dopamine in brain homogenates. These results suggest that in addition to the analgesic profile, there is a concomitant antidepressant profile which may enhance the spectrum of activity of fluradoline.

This presentation has attempted to describe some of the lesser recognized expertise needed to bring an antimicrobial agent to the hands of the physician. It should be remembered that the scale-up chemist, microbiologist, toxicologist, research pharmacist, and pharmacokinetic expert all contribute in an indispensable way in this process. In addition, we should appreciate the time and cost for this development. The scaling-up from milligram quantities to thousands of kilograms often requires investment in new chemical microbiology and production facilities. Preparation for marketing the new agent must begin years in advance. As mentioned previously, a 3-month toxicity study can take 6 to 9 months to complete. The cost of a 3-month rat toxicity study is approximately $60,000. If one adds a dog or monkey study, or extends the “in-life” phase to 6 months or 1 year, the costs increase accordingly. Carcinogenicity studies in mice or rats can have dosing periods of 18 months to 2 years and require close to 3 years to finish. The cost of these studies can reach $400,000 each. Development of sensitivity test systems (disks or MICs) is an ongoing procedure that is often completed about the time clinical trials are completed. After the basic paper disks and microdilution procedures are established, it may be necessary for development of the automated systems for the new agent. In most cases, these more elaborate systems are not ready at the time of market introduction and may follow by 1 year or more. We have all heard or viewed the papers at the Interscience Conference on Antimicrobial Agents and Chemotherapy and American Society for Microbiology on compounds that seem to then disappear. In many cases, it is because of unexpected toxicity, uneconomical scale-up costs, or a poor pharmacokinetic profile. In each case, considerable effort was expended before the decision to discontinue development. In the final analysis the value of an antimicrobial rests in its ability to be effective and safe in the clinical situation. When we listen to presentations of clinical results or read articles of clinical efficacy or see the attractive advertisement for a new antimicrobial, we need also to remember the “Other Side of Early Antimicrobial Development” and those who made it possible.

A model of sequentially arranged pharmacologic assays was developed to evaluate myocardial-depressant properties of various drugs. The sequence of assays consisted of the following: hemodynamics in normal dogs, hemodynamics in ganglion-blocked dogs, isolated-stimulated guinea pig left atria, spontaneously beating guinea pig atria, and an optional follow-up experiment consisting of hemodynamics in ganglion-blocked dogs with cardiac pacing. Four reference compounds (clonidine, verapamil, hydralazine, and mixidine) plus a test compound (P79-4058) were subjected to the test model with the goal of being able to discriminate between various types of myocardial depression. Results from this study indicate that the model was able to separate out three classes of actions: drugs with no depressant activity (hydralazine), drugs that cause myocardial depression indirectly via the nervous system (clonidine), and a third group that elicit myocardial depression directly (verapamil and P79-4058). Mixidine produced variable results in this model. The proposed model appeared to be adequate in that enough information can be obtained so that new pharmacologic compounds may be evaluated and decisions as to further development can be made.

Recent findings have suggested that the 1-methyltetrazole-5-thiol (MTT) group contained in several beta-lactam antibiotics may be responsible for the hypoprothrombinaemia associated with these drugs. In order to determine if the hypoprothrombinaemia associated with cefazolin is due to the presence of the structurally related 2-methyl-1,3,4,-thiadiazole-5-thiol (MTD) group which it possesses, the ability of MTD and cefazolin to produce hypoprothrombinaemia in rats was examined. Female rats maintained on a vitamin K-deficient diet for ten days developed hypoprothrombinaemia after the intravenous administration of cefazolin for two subsequent days. Hypoprothrombinaemia was also produced by the oral administration to vitamin K-deficient rats of either cefazolin or MTD, while the oral administration of cefotaxime, which does not contain a thiol group, had no effect. In a rat liver microsomal system, MTD was found to be a much more potent inhibitor than cefazolin of the vitamin K-dependent step in clotting factor synthesis, the gamma-carboxylation of glutamic acid. These results suggest that the hypoprothrombinaemia associated with cefazolin may be due to the MTD group.

HRP 913 {1-[3-(6-fluoro-1,2-benzisoxazole-3-yl)propyl]-4-(2-oxo-1-benzimidazolinyl)} piperidine demonstrated preclinical antipsychotic activity with features that may provide a clinical advantage over current therapy. It was effective in blocking amphetamine stereotypy in rats (ED50 = 0.4 mg/kg, i.p.), amphetamine circling in SN-lesioned rats (ED50 = 0.3 mg/kg, i.p.), apomorphine stereotypy in rats (ED50 = 0.8 mg/kg, i.p.), but not apomorphine circling in SN-lesioned rats (up to 10 mg/kg, i.p.). It also blocked Sidman avoidance in rats (ED50 = 0.17 mg/kg, i.p.) and monkeys (ED50 = 0.2 mg/kg, p.o.) and blocked intracranial self-stimulation in rats (0.09 mg/kg, i.p.). A unique biphasic effect on catalepsy was found. Monkey EPS studies demonstrate a potential for EPS that is lower than some existing clinical standards. HRP 913 displaced 3H-spiroperidol from rat striatal sites (IC50 = 6.0 × 10−9 M) and inhibited WB-4101 binding (IC50 = 2.6 × 10−8 M) with only slight effect on QNB binding. HRP 913 does not appear to have marked α-blocking properties in vivo. HRP 913 is a potent dopamine antagonist and is predicted to have less side effects than current therapy.

Increased oxygen tension increases the activity of monoamine oxidase (MAO) obtained from various tissues of rats, mice, and guinea pigs. The increase varies with tissue, species, and substrate. Per cent inhibition of MAO by iproniazid, d , l -amphetamine, and trans-2-phenylcyclopropylamine is not changed by altered oxygen tension. Finally, the response of the isolated right atrium of the rat to tyramine is altered by the oxygen tension of the bath. This effect can be explained on the basis of the change in intracellular MAO activity.

It is well recognized that the susceptibility interpretive values published by NCCLS play an important role in the clinical use of any given antimicrobial agent. The major objective is to provide meaningful breakpoints that allow prediction of clinical efficacy and quality-control values that truly reflect the quality of susceptibility test results generated in a clinical laboratory. To reach these goals, the NCCLS subcommittee requires manufacturers to present data ranging from pharmacokinetics in healthy subjects and patients to clinical verification of proposed breakpoints. It is obvious that this complex area cannot exist without some unpredicted problems. The NCCLS Subcommittee has an ongoing review process to respond to questions from the field on older drugs. These questions are encouraged, as it is often the only way the Subcommittee can know of a particular problem. The Subcommittee attempts to generate semiannual supplemts of its reference tables to reflect the addition of new antimicrobial agents and to correct data for older drugs. The guiding principle is to produce meaningful and usable breakpoints and quality control values.

The present studies indicate that in the dog chlorpromazine, prochlorperazine, and trifluoperazine are rapidly absorbed from the intestine and that there is a rapid hepatic clearance of the phenothiazines. The biliary and urinary excretions of these compounds are greatly affected by the side chain of the phenothiazine nucleus. The “methylpiperazinylpropyl” side chain (prochlorperazine and trifluoperazine) markedly increases the rate of biliary excretion and reduces the urinary excretion of the parent compounds and/or their metabolites compared with the excretion of chlorpromazine and/or its metabolites (dimethylaminopropyl side chain). Moreover, chlorpromazine and/or its metabolites excreted in bile are reabsorbed from the intestine, resulting in an entero-hepatic recirculation.

Cefotaxime is a third-generation cephalosporin with a broad spectrum of activity. Concentrations of cefotaxime in serum and urine are sufficient for clinical efficacy. This report reviews the available data on the penetration of cefotaxime into other body fluids and tissues. Therapeutic doses of cefotaxime result in significant levels in bile (20 micrograms/ml), in cerebrospinal fluid of patients with meningitis (5-10 micrograms/ml), in pleural fluid (2-7 micrograms/ml), and in otitis media exudate (2-10 micrograms/ml). Lower levels of cefotaxime are obtained in uninflamed aqueous humor (1 microgram/ml), in breast milk (0.1-0.5 microgram/ml), and in cerebrospinal fluid of patients without meningitis (0.2 microgram/ml). Cefotaxime levels in tissues are generally well within the range required for clinical efficacy--i.e., 2-5 micrograms/g, with some higher levels reported in testis, prostate, ureter, skin, and gallbladder wall. Drug levels of less than 2 micrograms/g have been reported in fat, muscle, and uterus. All body fluids and tissues, including bone and skin, are penetrated easily by therapeutic doses of cefotaxime.

The activity of monoamine oxidase was studied in the heart mitochondria of normal and hyperthyroid rats using manometric techniques. A definite age effect was observed in normal rats with an increase from 0-trace activity in post-natal to approximately 1000 μl. O2 consumed/gram/hour in 5–600 gram rats. Desiccated thyroid diet (2%) and l-triiodothyronine produced an increase in heart monoamine oxidase activity of from 70 to 200% depending upon the age group of the animals used. d-Triiodothyronine did not have this stimulating effect when used at equimolar doses. The effect of oxygen tension on the oxidation rate of tyramine is different for the heart and liver enzymes.

‘Department of Microbiology, Ramon y Cajal Hospital, Madrid, Spain; 2Bristol Myers Squibb, Wallingford, Connecticut, USA; 3Antibacterial Agents Unit, Institut Pasteur, Paris, France; 4Department of Biosciences, University of Hertfordshire, Hatfield, Herts, UK; 5Antimicrobial Agents Research, Department of Infection, University of Birmingham, Birmingham, UK; 63 Bartles Road, Lebanon, New Jersey, USA