[Place and role of safety pharmacology in drug development]. / Place et rôle de la pharmacologie de sécurité dans le développement des médicaments.

SUMMARY: Safety pharmacology is a key regulatory step for drug development and approval. Prior to phase I, the effects of a drug candidate should be evaluated and characterized on vital functions (cardiovascular, respiratory and central nervous system) according to good laboratory practice standards. For cardiovascular evaluation, effects on blood pressure and electrocardiogram should be explored with a particular emphasis on ventricular repolarization prolongation, a major risk factor for life-threatening arrhythmias, like "torsades de pointe". Global behaviour, motor activity, reflexes and body temperature should be evaluated in animals. A dedicated study is necessary for respiratory function evaluation. All of these studies should be conducted after single administration of the compound administered by the anticipated clinical route. Dependence potential and abuse liability should be characterized for innovative drugs and/or drugs acting on the central nervous system. Evidence for adverse effects at discovery stage with high throughput systems is becoming a key step of decision-making process for pharmaceutical industry. Therefore, determination of the safety margin, risk/benefit ratio analysis and investigation of adverse effects are major decisional elements for providing safety reassurance to patients. Safety of patients will also be improved through modelling methodologies allowing a safer transposition of experimental pharmacology results to clinical pharmacology.

Modeling thermal influence on animal growth and sex determination in reptiles: being closer to the target gives new views.

Many species of oviparous reptiles, including crocodilians, a majority of turtles, some lizards and the 2 closely related species of Sphenodon have been shown to display temperature-dependent sex determination (TSD). Whereas it has been demonstrated very early that TSD also occurs in natural conditions, the relationship between a time series of changing temperatures and sex ratio remains a challenging problem for reptiles. We describe how a physiological model of embryo growth, gonadal development and aromatase activity can produce outputs that mimic well TSD. We provide an enhancement of a previously published model taking into account direct effect of temperature on aromatase activity. The comparison between the original model and the new one suggests that aromatase expression is controlled by a repressor factor expressed at masculinizing temperatures rather than its enhancement at feminizing temperatures.

Evolution of habitat-dependent sex allocation in plants: superficially similar to, but intrinsically different from animals.

Because pollen disperses and ovules do not, a basic difference in dispersal abilities of male and female gametes exists in plants. With an analytical model, we show that the combination of such sex-biased dispersal of gametes and variation of habitat quality results in two opposite selective forces acting on the evolution of sex allocation in plants: (i) a plant should overproduce pollen in good patches and overproduce ovules in poor patches in order to equilibrate secondary sex ratios of gametes after pollen dispersal; (ii) a plant should overproduce ovules in good patches and overproduce pollen in poor patches in order to increase the likelihood that its progeny establishes in good patches. Our theoretical results indicate that the evolution of habitat-dependent sex allocation should be favoured in plants, in a direction that depends on the relative dispersal ability of pollen and seeds. We also show that superficially similar predictions obtained for habitat-dependent evolutionarily stable sex allocation in animals actually result from a completely different balance between the two underlying evolutionary forces.

Maternal inheritance of chloroplasts in the horsetail Equisetum variegatum (Schleich.).

Reliable data concerning the transmission of chloroplasts in the Pteridophyta are needed both for phylogenies based on chloroplast DNA (cpDNA) sequences and in order to study the evolution of this trait in conjunction with the evolution of the life cycle and the sexual reproduction of land plants. For the first time, this paper describes organelle transmission in the division Sphenophyta, represented by the extant genus Equisetum. By following the fate of polymorphic cpDNA during three intraspecific reciprocal crosses we found no trace of paternal transmission in Equisetum variegatum. The seemingly strict maternal transmission of cpDNA in this species suggests that uniparental chloroplast inheritance preceded the evolution of heterospory in the seed-plant lineage.

Drugs that prolong QT interval as an unwanted effect: assessing their likelihood of inducing hazardous cardiac dysrhythmias.

Medicinal products that, as an unwanted effect, prolong the QT interval of the electrocardiogram (ECG) can trigger episodes of polymorphic ventricular dysrhythmias, called torsades de pointes, which occasionally culminate in sudden death. The accurate measurement of QT interval requires the adoption of appropriate criteria of recording, measurement and data processing. Traditionally, QT interval is standardised to a reference heart rate of 60 beats/min by using the Bazett algorithm. However, this correction method can bias observed QT intervals in either direction. The ECG reflects cardiac electrical currents generated by ions (Na+, K+ and Ca2+) entering and leaving the cytosol mainly via transmembrane channels. Na+ and Ca2+ carry inward depolarising currents (INa, ICa) whereas K+ carries outward repolarising currents (Ito, IKr, IKS and IK1). Sometimes, a prolonged QT interval is a desired drug effect but, more commonly it is not, and reflects abnormalities in cardiac repolarisation heralding torsades de pointes. Furthermore, the potential torsadogenic activity of drugs is favoured by concurrent cardiac risk factors (old age, female gender, bradycardia, electrolyte imbalances, cardiac diseases etc.) which reduce cardiac repolarisation reserve. The evaluation of the cardiac safety of drug candidates can be started by determining their potency as IKr blockers in cloned Human Ether-a-go-go Related Gene (HERG) channels expressed in mammalian cells. Compounds passing successfully this test (desirable cardiac safety index > 30, calculated as ratio of IC50 against IKr over ED50 determined in an efficacy test) should be further investigated in other relevant human cardiac ion currents, in in vitro animal heart preparations and finally in in vivo pharmacodynamic models. The decision as to whether the potential benefit of a new drug outweighs the cardiac risk inherent in its therapeutic use should be made in the light of the condition that it is expected to treat and with reference to alternative drug therapies. If a drug represents a unique therapeutic advance, non-clinical and clinical signals of unsatisfactory cardiac safety may not constitute sufficient grounds to abandon its development. However, if the drug offers only marginal benefits over existing therapies, decisions concerning its possible development should be taken by corporate policy makers.

Preclinical in vitro cardiac electrophysiology: a method of predicting arrhythmogenic potential of antihistamines in humans?

The cardiac action potential results from a dynamic balance between inward depolarising Na+ and Ca2+ currents and outward K+ repolarising currents. During a cardiac cycle, the resultant of repolarisation phase from all ventricular cells is represented by the QT interval of the surface ECG. Congenital long QT syndrome (LQTS) is characterised by polymorphic ventricular tachycardia sometimes with twisting QRS morphology (torsade de pointes) which, although usually self-limiting, can result in sudden cardiac death. Acquired LQTS can be induced by a variety of drugs, including some nonsedative histamine H1 receptor antagonists (astemizole, terfenadine). The Committee for Proprietary Medicinal Products of the European Union has recently proposed studying the action potential in in vitro heart preparations as a preclinical test for predicting the propensity of noncardiovascular drugs to induce malignant QT prolongation in humans. The effects of several histamine H1 receptor antagonists on the electrically evoked action potential have been evaluated in rabbit Purkinje fibres. In this preparation, astemizole (0.3 to 10 micromol/L) prolongs the duration of the action potential measured at the level where repolarisation is 90% complete (APD90). This effect is dependent on drug concentration, incubation time, pacing frequency and K+ or Mg2+ concentration. Astemizole also markedly depresses the rate of rise of the action potential (Vmax). Terfenadine showed qualitatively similar, but quantitatively smaller, effects in this model. The histamine H1 receptor antagonists cetirizine, ebastine, carebastine, loratadine and fexofenadine do not significantly affect APD90 at 1 micromol/L, but cetirizine and carebastine prolong it slightly at 10 micromol/L. In conclusion, in rabbit Purkinje fibres, astemizole and terfenadine produce adverse electrophysiological effects at concentrations which may be achieved in the human myocardium in certain clinical situations. APD90 lengthening induced by carebastine and cetirizine is minor and occurs at concentrations that are very unlikely to be encountered clinically, since these drugs, in contrast to astemizole and terfenadine, do not accumulate in the myocardium. Direct extrapolation of preclinical results to humans requires great caution, since malignant QT prolongations by terfenadine and astemizole are extremely rare clinical events. However, since prolongation of the QT interval often precedes the development of torsade de pointes, any significant delay in cardiac repolarisation produced by noncardiovascular drugs in preclinical, and particularly in clinical, studies should, in general, be considered to indicate a potential cardiac risk in humans. Its significance should subsequently be evaluated in appropriate studies in patients with conditions known to predispose to arrhythmias.

Preferential reduction in vascular responses to endothelin-1 in rats with streptozotocin-induced diabetes.

The effects of vasodepressor (acetylcholine and bradykinin) and pressor [electrical stimulation of the spinal sympathetic outflow, norepinephrine and endothelin-1 (ET-1)] stimuli were determined in rats with 2- and 5-week untreated streptozotocin-induced diabetes (blood glucose 400 and >500 mg/dl, respectively). In pentobarbital-anesthetized animals, the hypotensive response to an intravenous dose of acetylcholine or bradykinin was unaffected in animals treated for 2 weeks with streptozotocin but was significantly reduced (22% and 48%, respectively) after 5 weeks. However, the pressor responses to ET-1 were significantly decreased in animals that had been given streptozotocin 2 (38%) and 5 (45%) weeks previously. In contrast, the vasoconstrictor effects of electrical stimulation of the spinal cord outflow and norepinephrine were significantly inhibited (47% and 30%, respectively) at 5 weeks, but not at 2 weeks, after streptozotocin administration. These results indicate that, in untreated streptozotocin diabetes, a substantial impairment of vascular reactivity to ET-1 appears more rapidly than inhibition of the pressor responses to endogenous and exogenous norepinephrine or to vasodilator substances that require integrity of vascular endothelial cell function to produce their normal effects.

Interplay of methionine tRNAs with translation elongation factor Tu and translation initiation factor 2 in Escherichia coli.

According to their role in translation, tRNAs specifically interact either with elongation factor Tu (EFTu) or with initiation factor 2 (IF2). We here describe the effects of overproducing EFTu and IF2 on the elongator versus initiator activities of various mutant tRNAMet species in vivo. The data obtained indicate that the selection of a tRNA through one or the other pathway of translation depends on the relative amounts of the translational factors. A moderate overexpression of EFTu is enough to lead to a misappropriation of initiator tRNA in the elongation process, whereas overproduced IF2 allows the initiation of translation to occur with unformylated tRNA species. In addition, we report that a strain devoid of formylase activity can be cured by the overproduction of tRNAMetf. The present study brings additional evidence for the importance of formylation in defining tRNAMetf initiator identity, as well as a possible explanation for the residual growth of bacterial strains lacking a functional formylase gene such as observed in Guillon, J. M., Mechulam, Y., Schmitter, J.-M., Blanquet, S., and Fayat, G. (1992) J. Bacteriol. 174, 4294-4301.

Molecular recognition governing the initiation of translation in Escherichia coli. A review.

Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components. These macromolecules interact in a specific and concerted manner to yield the translation initiation complex. This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process. The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA(f)Met formyltransferase are described. In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered. Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process.

Ischemic myocardial cell protection conferred by the opening of ATP-sensitive potassium channels.

The responses of the cardiac myocyte to a potentially injurious ischemic stress are multiple. The opening of the ATP-sensitive K+ channels may constitute one such response. These channels are present in the plasmalemma at very elevated density and have a large unitary conductance. Consequently, the opening of a small fraction (0.01-0.1%) of these channels during ischemia can help to drive the myocyte into an "emergency" state, in which its syncytial functions become rapidly downregulated and strategies appropriate to preserving cell viability are implemented. Thus, ATP-sensitive K+ channels in cardiac myocytes would appear to be an efficient and apparently redundant natural means of defense against metabolic stress. These channels can undergo physiologic modulation, as occurs during cardiac ischemic preconditioning in several species, including humans. The term cardioprotection refers to an endogenous cardioprotective strategy, whereby the myocardium slows its energy demands, produces fewer toxic glycolytic products, and exhibits reduced injury following a potentially lethal ischemic stress. Openers of cardiac ATP-sensitive K+ channels, a class of drugs that includes, in particular, aprikalim and nicorandil, also afford cardioprotection by reducing the functional and biochemical damage produced by ischemia. Hence, these compounds can improve the recovery of cardiac contractility, reduce the extracellular leakage of intracellular enzymes, delay the loss of ATP, and preserve the cell ultrastructure in isolated heart preparations subjected to transient ischemic conditions. Furthermore, when segmental contractility has been strongly depressed by a stunning insult, nicorandil and aprikalim can accelerate recovery at the reperfusion. Finally, nicorandil and aprikalim decrease substantially the size of the necrotic region that results from a prolonged ischemic insult followed by reperfusion. All of these desirable effects of K(+)-channel openers can be abolished by blockers of ATP-sensitive K+ channels, such as glibenclamide. The fundamental mechanism of the myocyte viability protection conferred by K(+)-channel openers is not yet clear. It may exploit some of the same pathways that mediate cardiac ischemic preconditioning. If this suggestion holds true, drugs opening cardiac ATP-sensitive K+ channels would mimic, exploit, or intensify those cardioprotective means that are naturally available to the cardiac myocyte for overcoming metabolic stress.

Late CD8+ lymphocytic alveolitis after allogeneic bone marrow transplantation and chronic graft-versus-host disease.

Late-onset interstitial pneumonitis following allogeneic bone marrow transplantation (BMT) is a rare condition usually caused by a variety of infective agents, although in some cases these are idiopathic. We investigated noninfectious late interstitial pneumonitis with lymphocytic alveolitis in seven allogeneic BMT recipients using bronchoalveolar lavage (BAL), lymphocyte phenotyping analysis, CT lung scans, and pulmonary function tests. The results were compared with those of a control group composed of similar patients with no pulmonary symptoms. Of 65 long-term survivors, seven were included in the study. All had chronic graft-versus-host disease (GVHD) and developed interstitial pneumonitis a median of 210 d (range 120 to 445 d) after BMT. BAL revealed lymphocytosis, with an overall expansion of CD8+ subsets (38 to 90%). Lymphocytic alveolitis was not observed in the control group. Pulmonary function tests revealed a restrictive syndrome, and biopsy samples obtained from 2 patients showed interstitial lymphoid infiltration with fibrosis of the alveolar walls. Of the 7 patients, six were cured by starting immunosuppressive drugs or increasing the dosage with a drastic improvement in respiratory symptoms within 1 mo. These findings suggest that CD8+ alveolitis may be observed in late interstitial pneumonitis in allogeneic BMT recipients and may be a pulmonary manifestation of chronic GVHD.

Cellular pharmacology of potassium channel openers in vascular smooth muscle.

Potassium channel opening is a physiological mechanism which excitable cells exploit to maintain or restore their resting state. Thus drugs that open vascular potassium channels have the potential to restrain or prevent contractile responses to excitatory stimuli or clamp the vessel in a relaxed condition. Hence, potassium channel openers, such as aprikalim and levcromakalim, relax agonist precontracted vascular preparations in vitro and lower systemic and regional vascular resistances in intact animals. Glibenclamide, a blocker of ATP sensitive potassium (KATP) channels, antagonises these effects. The main vasorelaxant mechanism of the potassium channel openers is to increase the potassium efflux through opening plasmalemmal potassium channels, which repolarises and/or hyperpolarises the membrane. This effect lowers the opening probability of voltage dependent calcium channels, restrains agonist induced calcium release from intracellular sources through inhibition of inositol trisphosphate formation, decreases the sensitivity of intracellular contractile elements to calcium, and accelerates the clearance of intracellular calcium via the Na+/Ca+ exchanger. Experimental evidence indicates that mechanisms not linked to potassium channel opening may also contribute to the potassium channel opener induced vasorelaxation; these remain to be clearly defined (for example, inhibition of the refilling of intracellular calcium stores). Potassium channel openers displace the binding of 3H-P1075, a potent potassium channel opener, in myocytes and intact rings from the rat aorta. In patches from vascular myocytes, potassium channel openers primarily open a small conductance (10-20 pS) KATP channel which is gated by [ATP]i and particularly by nucleotide diphosphates and magnesium.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane ion channels and cardiovascular ATP-sensitive K+ channels.

Ion channels are the primary target for a variety of clinically important drugs including local anesthetic, antihypertensive, antianginal, antiarrhythmic, antidiabetic, anticonvulsant hypnotic and anxiolytic agents. Ion channels are specialized proteins inserted in the ion-impermeable cellular membrane, which have a water-filled pore permitting the selective passage of a few biologically important ions (Na+, K+, Ca++ and Cl-) across the membrane. Multiple channels for a given ion can co-exist on the same cell where they have specific functions. The flow of ions through channels produces electrical currents which often act as biological messengers to change and modulate the functional state of the cell. Thus, the influx of Na+ and Ca++ are activating signals whereas the exit of K+ drives the activated cell to a resting state or strengthen the resting state. Interestingly, K+ channels are the most diverse group of ion channels. At least 9 families of K+ channels co-exist in cardiac myocytes where they regulate the heart repolarization and excitability processes under physiological and pathological conditions. ATP-sensitive K+ channels of cardiac myocytes are of particular interest since they have a very high membrane density and are closed under normoxic conditions, becoming operational during ischemic stress when the intracellular levels of ATP decline. Their major function has been proposed to be the preservation of viability of the myocyte during ischemia. ATP-sensitive K+ channels are also present in other tissues, such as the blood vessels, where their opening causes strong relaxing effects. Cardiac and vascular ATP-sensitive K+ channels are the primary target of a novel class of drugs, called K+ channel openers, such as nicorandil and aprikalim.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli.

In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of the fmt gene, encoding the Escherichia coli Met-tRNA(fMet) formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNA(fMet) and tRNA(mMet) species in either initiation or elongation in vivo. The data obtained established that formylation plays a dual role, firstly, by dictating tRNA(fMet) to engage in the initiation of translation, and secondly, by preventing the misappropriation of this tRNA by the elongation apparatus. The importance of formylation in the initiator identity of tRNA(fMet) was further shown by the demonstration that elongator tRNA(fMet) may be used in initiation and no longer in elongation, provided that it is mutated into a formylatable species and is given the three G.C base pairs characteristic of the anticodon stem of initiator tRNAs.

The Escherichia coli fmt gene, encoding methionyl-tRNA(fMet) formyltransferase, escapes metabolic control.

The genetic organization near the recently cloned fmt gene, encoding Escherichia coli methionyl-tRNA(fMet) formyltransferase (J. M. Guillon, Y. Mechulam, J. M. Schmitter, S. Blanquet, and G. Fayat, J. Bacteriol. 174:4294-4301, 1992), has been studied. The fmt gene, which starts at a GUG codon, is cotranscribed with another gene, fms, and the transcription start site of this operon has been precisely mapped. Moreover, the nucleotide sequence of a 1,379-bp fragment upstream from fmt reveals two additional open reading frames, in the opposite polarity. In the range of 0.3 to 2 doublings per h, the intracellular methionyl-tRNA(fMet) formyltransferase concentration remains constant, providing, to our knowledge, the first example of a gene component of the protein synthesis apparatus escaping metabolic control. When the gene fusion technique was used for probing, no effect on fmt expression of the concentrations of methionyl-tRNA(fMet) formyltransferase or tRNA(fMet) could be found. The possibility that the fmt gene, the product of which is present in excess to ensure full N acylation of methionyl-tRNA(fMet), could be expressed in a constitutive manner is discussed.

Long-term results of monthly inhaled pentamidine as primary prophylaxis of Pneumocystis carinii pneumonia in HIV-infected patients.

PURPOSE: To evaluate the long-term efficacy and safety of inhaled pentamidine as primary prophylaxis against Pneumocystis carinii pneumonia (PCP) in patients infected with human immunodeficiency virus (HIV). PATIENTS: Two hundred thirty-two HIV-infected patients with a CD4 cell count below 20% of the total lymphocyte count were given aerosolized pentamidine once every 4 weeks for more than 3 months. Pentamidine aerosols were administered at the hospital under medical supervision. Prevention of bronchospasm was carried out using inhaled salbutamol. RESULTS: Mean duration of prophylaxis was 15.9 months. Eleven patients (4.7%; [95% confidence interval 2% to 7.4%]) developed PCP. Probability to remain free of PCP is 95.6% at 12 months, 94% at 18 months, and 88% at 24 months. Mean delay between the onset of the prophylaxis and the occurrence of PCP for the 11 patients was 12.9 months (range: 4 to 26 months). No major side effect was observed, and minor side effects (cough, acute dyspnea) were infrequent. CONCLUSION: The efficacy and tolerance of aerosolized pentamidine as shown in our study support its use as primary prophylaxis against P. carinii in HIV-infected patients.

[Extrapulmonary intrathoracic pneumocystosis. Apropos of 4 cases in HIV positive patients]. / Pneumocystoses intra-thoraciques extra-pulmonaires.

The authors report 4 cases of intrathoracic extrapulmonary pneumocystosis; 3 of them involved the pleura and 1 the mediastinal lymph nodes. As in other rare but apparently increasingly frequent cases, everything seemed to incriminate the use of prophylactic Pentamidine aerosols, but only a prospective study of systemic versus aerosol prophylaxis can confirm this suspicion.

Minocycline-induced cell-mediated hypersensitivity pneumonitis.

OBJECTIVE: To identify the cause of a hypersensitivity pneumonitis and to determine its pathogenesis. DESIGN: Case study. SETTING: Intensive care unit of a referral hospital. PATIENT: A 51-year-old man with chronic bronchitis who developed a hypersensitivity pneumonitis within 1 month after exposure to minocycline, amoxicillin, and erythromycin. INTERVENTION: Sequential bronchoalveolar lavages after reexposure to minocycline and amoxicillin. MEASUREMENTS: Immunologic analysis of the phenotype and function of alveolar lymphocytes. RESULTS: Reexposure to minocycline but not to amoxicillin was followed by an interstitial pneumonitis. Sequential bronchoalveolar lavages showed a transient rise of eosinophils and neutrophils and a persistent alveolar lymphocytosis. Alveolar lymphocytes consisted predominantly of CD8+ but also CD4+ cells. Two CD8+ lymphocyte subsets were identified: CD8+ D44+ cytotoxic T cells that increased rapidly after the drug was resumed and CD8+ CD57+ suppressor T cells that predominated 11 days after the drug's withdrawal. In-vitro assays showed the presence of a lymphocyte-mediated specific cytotoxicity against minocycline-bearing alveolar macrophages. CONCLUSION: These results support the hypothesis of a central role of T lymphocytes in the pathogenesis of drug-related hypersensitivity pneumonitis.