Predictive and Descriptive CoMFA Models: The Effect of Variable Selection.

Aims & Scope: In this research, 8 variable selection approaches were used to investigate the effect of variable selection on the predictive power and stability of CoMFA models. MATERIALS & METHODS: Three data sets including 36 EPAC antagonists, 79 CD38 inhibitors and 57 ATAD2 bromodomain inhibitors were modelled by CoMFA. First of all, for all three data sets, CoMFA models with all CoMFA descriptors were created then by applying each variable selection method a new CoMFA model was developed so for each data set, 9 CoMFA models were built. Obtained results show noisy and uninformative variables affect CoMFA results. Based on created models, applying 5 variable selection approaches including FFD, SRD-FFD, IVE-PLS, SRD-UVEPLS and SPA-jackknife increases the predictive power and stability of CoMFA models significantly. RESULT & CONCLUSION: Among them, SPA-jackknife removes most of the variables while FFD retains most of them. FFD and IVE-PLS are time consuming process while SRD-FFD and SRD-UVE-PLS run need to few seconds. Also applying FFD, SRD-FFD, IVE-PLS, SRD-UVE-PLS protect CoMFA countor maps information for both fields.

Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window".

The cAMP signaling cascade is one of the most frequently targeted pathways for the development of pharmaceutics. A plethora of recent genetic and pharmacological studies suggest that exchange proteins directly activated by cAMP (EPACs) are implicated in multiple pathologies. Selective EPAC inhibitors have been recently developed. One specific inhibitor, ESI-09, has been shown to block EPAC activity and functions, as well as to recapitulate genetic phenotypes of EPAC knockout mice when applied in vivo. However, a recent study raised concern that ESI-09 might act as a non-specific protein denaturant. Herein, we present a detailed biochemical and pharmacological characterization, as well as a structure-activity relationship (SAR) analysis of ESI-09. Our studies show that ESI-09 dose-dependently inhibits activity of both EPAC1 and EPAC2 with apparent IC50 values well below the concentrations shown to induce "protein denaturation". Moreover, the ESI-09's action towards EPAC proteins is highly sensitive to minor modifications of the 3-chlorophenyl moiety. Taken together, these results demonstrate that ESI-09 indeed acts as an EPAC specific antagonist and does not significantly destabilize/denature proteins at pharmacological effective concentrations. This conclusion is further supported by NMR data showing that ESI-09 induces residue-dependent chemical shift changes at low concentrations, while preserving well dispersed peaks.

Mechanism-based inhibition profiles of erythromycin and clarithromycin with cytochrome P450 3A4 genetic variants.

Inhibition of cytochrome P450 (CYP) 3A4 is the major cause of drug-drug interactions (DDI). We have previously reported that the genetic variation of CYP3A4 significantly affected the inhibitory profiles of typical competitive inhibitors. In addition to competitive inhibition, some clinically significant DDI are attributable to mechanism-based inhibition (MBI). However, the differences in the MBI kinetics among CYP3A4 genetic variants remain to be characterized. In this study, we quantitatively investigated the inhibition kinetics of MBI inhibitors, erythromycin and clarithromycin, on the CYP3A4 variants CYP3A4.1, 4.2, 4.7, 4.16, and 4.18. The activity of CYP3A4 was assessed using testosterone 6ß-hydroxylation with recombinant CYP3A4. Both erythromycin and clarithromycin decreased the activity of CYP3A4 in a time-dependent manner. The maximum inactivation rate constants, k(inact,max), of erythromycin for CYP3A4.2 and CYP3A4.7 were 0.5-fold that for CYP3A4.1, while that for CYP3A4.16 and CYP3A4.18 were similar to that for CYP3A4.1. The K(I) values of erythromycin for CYP3A4.2, 4.7, 4.16, and 4.18 were 1.2-, 0.4-, 2.2- and 0.72-fold those of CYP3A4.1, respectively. Similar results were obtained for clarithromycin. In conclusion, the inhibitory profiles of MBI inhibitors, as well as competitive inhibitors, may possibly differ among CYP3A4 variants. This difference may contribute to interindividual differences in the extent of DDI based on MBI.

Urocortins improve dystrophic skeletal muscle structure and function through both PKA- and Epac-dependent pathways.

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Erythromycin inhibited glycinergic inputs to gastric vagal motoneurons in brainstem slices of newborn rats.

BACKGROUND: Motilin has been known to stimulate the motility of digestive organs peripherally via activation of motilin receptors located at gastrointestinal (GI) cholinergic nerve endings and/or smooth muscle cells. Recent studies have indicated that motilin may also promote GI motility via actions in the central nervous system; however the sites of action and the mechanisms are not clear yet. The present study aimed to test the hypothesis that motilin receptor agonist erythromycin alters the synaptic inputs of preganglionic gastric vagal motoneurons (GVMs) located in the dorsal motor nucleus of the vagus (DMV). METHODS: Gastric vagal motoneurons were retrogradely labeled by fluorescent tracer from the stomach wall of newborn rats. Fluorescently labeled GVMs in DMV were recorded using whole-cell patch-clamp in brainstem slices and the effects of motilin receptor agonist erythromycin on the synaptic inputs were examined. KEY RESULTS: Erythromycin (100 nmol L(-1), 1 µmol L(-1), 10 µmol L(-1)) significantly inhibited the frequency of glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) of GVMs and significantly inhibited the amplitude at the concentration of 10 µmol L(-1). These responses were prevented by GM-109, a selective motilin receptor antagonist. In the pre-existence of tetradotoxin (TTX, 1 µmol L(-1)), erythromycin (10 µmol L(-1)) caused significant decreases of the glycinergic miniature inhibitory postsynaptic currents (mIPSCs), in both the frequency and the amplitude. However, erythromycin (10 µmol L(-1)) didn't cause significant changes of the GABAergic sIPSCs. CONCLUSIONS & INFERENCES: Erythromycin selectively inhibits the glycinergic inputs of GVMs.

Developmental etiology for neuroanatomical and cognitive deficits in mice overexpressing Galphas, a G-protein subunit genetically linked to schizophrenia.

Schizophrenia is a widespread psychiatric disorder, affecting 1% of people. Despite this high prevalence, schizophrenia is not well treated because of its enigmatic developmental origin. We explore here the developmental etiology of endophenotypes associated with schizophrenia using a regulated transgenic approach in mice. Recently, a polymorphism that increases mRNA levels of the G-protein subunit Galphas was genetically linked to schizophrenia. Here we show that regulated overexpression of Galphas mRNA in forebrain neurons of mice is sufficient to cause a number of schizophrenia-related phenotypes, as measured in adult mice, including sensorimotor gating deficits (prepulse inhibition of acoustic startle, PPI) that are reversed by haloperidol or the phosphodiesterase inhibitor rolipram, psychomotor agitation (hyperlocomotion), hippocampus-dependent learning and memory retrieval impairments (hidden water maze, contextual fear conditioning), and enlarged ventricles. Interestingly, overexpression of Galphas during development plays a significant role in some (PPI, spatial learning and memory and neuroanatomical deficits) but not all of these adulthood phenotypes. Pharmacological and biochemical studies suggest the Galphas-induced behavioral deficits correlate with compensatory decreases in hippocampal and cortical cyclic AMP (cAMP) levels. These decreases in cAMP may lead to reduced activation of the guanine exchange factor Epac (also known as RapGEF 3/4) as stimulation of Epac with the select agonist 8-pCPT-2'-O-Me-cAMP increases PPI and improves memory in C57BL/6J mice. Thus, we suggest that the developmental impact of a given biochemical insult, such as increased Galphas expression, is phenotype specific and that Epac may prove to be a novel therapeutic target for the treatment of both developmentally regulated and non-developmentally regulated symptoms associated with schizophrenia.

Protective role of dimethyl diphenyl bicarboxylate (DDB) against erythromycin induced hepatotoxicity in male rats.

In this study, dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) was examined to justify its role in the hepatoprotection against erythromycin toxicity in male rats. Oral daily administration of toxic dose of erythromycin stearate (EE, 100 mg/kg body weight) was given to male rats for fourteen days to induce hepatotoxicity. It was found at the end of the experiment (14 days) that the total body weight was markedly decreased in rat treated with erythromycin stearate (EE). Hepatomegaly and splenomegaly were recorded in rats treated with erythromycin stearate (EE). The red blood cells (RBCs) count, haemoglobin content (Hb) and haematocrit value (Hct) were significantly reduced in rats treated with EE. The hepatotoxicities were monitored by increased level of plasma enzymes (aspartate aminotransferase; AST and alanine aminotransferase; ALT), total bilirubin, direct bilirubin, cholesterol, total lipids and glucose. The data obtained showed that oral administration of DDB (100 mg/kg body weight) has significantly prevented the occurrence of EE-induced liver damage. The biochemical data were supplemented by histopathological examination of the liver of control and treated rats. DDB showed a better hepatoprotective effect compared with ursodesoxycholic acid or Silymarin (Sil), as a reference drug.

In vitro antagonism between beta-lactam and macrolide in Streptococcus pneumoniae: how important is the antibiotic order?

We found that the in vitro interaction between penicillin or cefotaxime and erythromycin against Streptococcus pneumoniae varies depending on the order of antibiotic exposure. Time-kill experiments were performed with penicillin, cefotaxime, erythromycin and different order combinations of both beta-lactams with erythromycin. The mean difference between the colony count at 0 and 6h for penicillin, cefotaxime and erythromycin tested separately was 3.5 log cfu/mL, 2.4 and 1.5 respectively for susceptible strains. The mean difference for the combination of beta-lactam and erythromycin studied simultaneously was 1.8 log cfu/mL for these strains. The association of penicillin or cefotaxime with erythromycin added two hours later showed an activity similar to those of beta-lactam alone (mean difference was 3.0 for this association with penicillin and 2.5 with cefotaxime). Therefore, the antagonistic effect of macrolide activity could be less important if erythromycin was administrated after beta-lactam.

Antagonism between penicillin and erythromycin against Streptococcus pneumoniae: does it exist?

Penicillin and erythromycin are commonly used for the treatment of serious infections caused by Streptococcus pneumoniae and combined as empiric therapy of community-acquired pneumonia. A concern about potential antagonism between these drugs prompted a protocol designed to test the hypothesis in timed kill curve experiments with several interpretive criteria applied. Four clinical isolates of S.pneumoniae from the United States referred to the SENTRY Antimicrobial Surveillance Program and one QC strain (ATCC 49619) were tested. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined for each isolate using reference dilution methods (NCCLS). Penicillin MBC results matched very closely to the MIC values. Penicillin and erythromycin were tested at clinically relevant concentrations of 10 and 1 microg/ml, respectively, alone and in combination. Interpretations were calculated comparing the penicillin + erythromycin killing effect versus penicillin or erythromycin rates tested alone. There was consistent bactericidal activity against S. pneumoniae by each drug alone and combined over the monitored five-hour period, except for an erythromycin induced-resistant isolate. Drug interactions ranged from synergy to antagonism, depending on the criteria applied. Antagonism risk of macrolide-penicillin combinations appeared to be minimal and method-dependent by in vitro tests.

Octreotide enhances the accelerating effect of erythromycin on gastric emptying in healthy subjects.

BACKGROUND: Erythromycin exhibits gastrokinetic properties through cholinergic pathways. Reports regarding the action of octreotide on gastric emptying are conflicting. AIM: : To assess: (i) the hypothesis that serotonin receptors are involved in the accelerating effect of erythromycin on gastric emptying; and (ii) any modification of the gastrokinetic action of erythromycin induced by octreotide. SUBJECTS AND METHODS: Gastric emptying of a standard meal was estimated in 20 healthy subjects by scintigraphy on three different occasions in a double-blind, placebo-controlled manner and in random order: (i) after placebo; (ii) after 200 mg of intravenous erythromycin; and (iii) after 200 mg of intravenous erythromycin following pre-treatment with either 4 mg of intravenous ondansetron (10 subjects) or 50 micro g octreotide. RESULTS: Erythromycin significantly accelerated gastric emptying in all subjects by abolishing the lag phase. Pre-treatment with ondansetron abolished the accelerating effect of erythromycin by restoring the emptying times to placebo levels. Octreotide significantly enhanced the accelerating effect of erythromycin by reducing both the lag and post-lag phases of gastric emptying. CONCLUSIONS: Serotonin receptors are involved in the accelerating effect of erythromycin on gastric emptying. This effect seems to be enhanced by pre-treatment with octreotide, possibly as a result of the modification of the gastrointestinal hormonal environment.

Short peptides conferring resistance to macrolide antibiotics.

Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.

Antagonism between penicillin and erythromycin against Streptococcus pneumoniae in vitro and in vivo.

The combination of beta-lactam antibiotics and macrolides is often recommended for the initial empirical treatment of acute pneumonia in order to obtain activity against the most important pathogens. Theoretically, this combination may be inexpedient, as the bacteriostatic agent may antagonize the effect of the bactericidal agent. In this study, the possible interaction between penicillin and erythromycin was investigated in vitro and in vivo against four clinical isolates of Streptococcus pneumoniae with MICs of penicillin ranging from 0.016 to 0.5 mg/L and of erythromycin from 0. 25 to >128 mg/L. In vitro time-kill curves were generated with clinically relevant concentrations of penicillin (10 mg/L) and erythromycin (1 mg/L), either individually or in combination. Antagonism between penicillin and erythromycin was observed for the four isolates. In vivo interaction was investigated in the mouse peritonitis model. After intraperitoneal inoculation, penicillin and erythromycin were given either individually or in combination. For two of the four isolates, mortality was significantly higher in the groups treated with the combination of penicillin and erythromycin than in the groups treated with penicillin alone [32/36 (86%) vs. 3/12 (25%), P<0.05; and 24/36 (67%) vs. 3/12 (25%), P<0.05, respectively]. Using the mouse peritonitis model, in vivo time-kill curves showed that there was antagonism between erythromycin and penicillin for the examined isolate. The antagonism demonstrated in vitro and in vivo between penicillin and erythromycin suggests that ss-lactam antibiotics and macrolides should not be administered together unless pneumococcal infection is ruled out.

In vivo characterization of the colonic prokinetic effect of erythromycin in the rabbit.

The motor effect of erythromycin was characterized in conscious rabbits chronically fitted with electrodes and strain-guage force transducers implanted along the proximal and distal colon. Fecal pellet output was also evaluated as an index of propulsive activity. In order to get an insight into the pathways involved in mediating the effect of erythromycin, the macrolide was also administered after pretreatment with atropine, nifedipine or ondansetron. Furthermore, in vitro experiments with erythromycin alone and in the presence of atropine, nifedipine, tetrodotoxin or ondansetron were carried out with circular muscle strips taken from rabbit distal colon. In vivo, erythromycin (0.087-5.6 mg/kg i.v. bolus) dose-dependently stimulated spike and mechanical activities at both colonic levels, with a more marked effect on the distal colon. Erythromycin also dose-dependently increased the number of aborally migrating long spike bursts and fecal pellet output. The reproducibility of the response to erythromycin was confirmed by experiments with the dose of 2.8 mg/kg i.v. bolus, repeated in five consecutive experiments at 48-hour intervals. Nifedipine, but not atropine or ondansetron, significantly reduced the colonic motor response to erythromycin. In vitro experiments gave results in line with the in vivo data: the concentration-dependent contractile effect of erythromycin was almost suppressed by nifedipine, but resistant to atropine, tetrodotoxin or ondansetron. In conclusion, this study provides evidence that: (1) erythromycin is a prokinetic drug at the colonic level in rabbits, and (2) both in vivo and in vitro, the effects of erythromycin are exerted at the smooth muscle level by mechanisms depending on influx of extracellular calcium, while muscarinic and 5-HT3 receptors are not involved, at least in this model.

Novel mechanism of macrolide resistance in Streptococcus pneumoniae.

The mechanism of macrolide resistance was examined in 73 clinical isolates of Streptococcus pneumoniae. Two distinct resistance phenotypes were observed: high-level macrolides-lincosamides-streptogramin B (MLS) resistance and low-level macrolide resistance with lincosamide susceptibility. High-level MLS resistance was associated with the presence of ermAM. Strains with the low-level resistant phenotype (novel) were negative for ermA, ermC, ermAM, ereA, ereB and msrA by polymerase chain reaction (PCR) amplification with gene-specific primers. Ribosomes isolated from novel strains bound the same amount of [14C]-erythromycin as ribosomes from sensitive strains. These novel strains also did not inactivate the macrolide. The novel mechanism was found in 41% of the erythromycin resistant S. pneumoniae examined.

Erythromycin as a specific substrate for cytochrome P4503A isozymes and identification of a high-affinity erythromycin N-demethylase in adult female rats.

Erythromycin N-demethylation is catalyzed by cytochrome P4503A isozymes. By using [14C]methyl-labeled erythromycin, we were able to develop a N-demethylation assay that is more sensitive and specific than the colorimetric detection of formaldehyde formation. The increased sensitivity allows the use of very low substrate concentration with good sensitivity, 1 microM compared with 400 microM for the colorimetric assay. This 1 microM concentration is within pharmacological blood levels of erythromycin. Using this assay, we detected a high-affinity erythromycin N-demethylase in liver microsomes from untreated adult female rats that was previously unknown. This low KM activity could be inhibited by polyclonal anti-P4503A1 or P4503A2 antibodies to 95%, and these antibodies also detected a band in these microsomes on Western blots that had the same molecular weight (51 kDa) as cytochromes P4503A1/3A2. Monoclonal antibodies specific for P4503A1 or P4503A2, however, did not react with this band. No inhibitory effect was observed with monoclonal antibody P124, which inhibited the erythromycin N-demethylation both in liver microsomes from untreated adult males (P4503A2) and dexamethasone-pretreated adult females (P4503A1). Alternative P4503A substrates (testosterone, troleandomycin, cortisol, corticosterone, cyclosporin A, and 17 alpha-ethinylestradiol) inhibited erythromycin N-demethylation catalyzed by liver microsomes from untreated male, untreated female, and dexamethasone-pretreated female rats, whereas digitoxin and theophylline had no inhibitory effects. Put together, these data suggest that this demethylase in liver microsomes of untreated female rats is not P4503A1 or P4503A2, but P4503A related.

5-hydroxytryptamine 3-receptor antagonist modulates gallbladder emptying and motilin release induced by erythromycin.

In the present study we evaluated the effect of ondansetron (formerly indicated as GR38032F), a potent and selective type-3 5-hydroxytryptamine receptor antagonist, on erythromycin-induced gallbladder emptying and motilin release, as well as gallbladder emptying induced by a regular meal in healthy volunteers. Gallbladder emptying was evaluated by sonography. Ondansetron, at the dose of 0.05 mg/kg, significantly reduced (P < 0.001 by ANOVA) the gallbladder emptying induced by 2 mg/kg/hr erythromycin, but did not increase basal gallbladder volume or inhibit gallbladder emptying induced by a regular meal. Ondansetron also inhibited the motilin release induced by erythromycin (P < 0.001, by ANOVA). These results suggest that serotoninergic mechanisms modulate the effects of erythromycin on the gastrointestinal tract. The exact site of action of ondansetron remains to be identified.

Effect of erythromycin on gallbladder emptying in diabetic patients with and without autonomic neuropathy and high levels of motilin.

A reduction of gallbladder emptying in response to neural or hormonal stimulation has been reported in patients with diabetes mellitus. Decreased gallbladder emptying may be a key factor in the pathogenesis of gallbladder stones. Few drugs, if any, are able to stimulate gallbladder emptying. However, in a previous study we demonstrated that erythromycin, a macrolide antibiotic, stimulates gallbladder emptying and motilin release in healthy human subjects by an atropine-sensitive pathway. Therefore, the present study was designed to evaluate the effect of erythromycin on gallbladder emptying and motilin release in diabetic patients with or without cardiac autonomic neuropathy (AN). Thirteen diabetic patients, six with AN, and 10 healthy subjects were enrolled in the study protocol. Gallbladder emptying was determined by sonography after ingestion of a standard meal and during infusion of erythromycin alone or together with 6 micrograms/kg/hr atropine. We found that 100 mg/hr erythromycin caused a significant reduction in gallbladder volume in both healthy subjects and diabetic patients. The ejection fraction (mean +/- SE) of 45.3 +/- 8.2% and 37.3 +/- 5.0% was similar. The presence of AN had no influence on gallbladder emptying induced by erythromycin. Basal motilin plasma levels were 111.5 +/- 14.5 pmol/liter in diabetic patients and 63.3 +/- 6.0 pmol/liter in healthy subjects (P < 0.01). However, patients with AN had higher (130.0 +/- 11.9 pmol/liter) motilin plasma levels than patients without (74.0 +/- 9.4 pmol/liter, P < 0.01). Erythromycin administration caused an approximately twofold increase in plasma motilin concentrations in healthy subject and patients without AN, but did not stimulate motilin release in neuropathic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythromycin stimulates gallbladder emptying and motilin release by atropine-sensitive pathways.

The effect of administering different doses of erythromycin on gallbladder emptying and plasma concentrations of immunoreactive motilin was investigated in healthy volunteers. Erythromycin was infused for 30 min at four different doses: 20, 50, 100, and 1000 mg/hr. Gallbladder volume was determined by ultrasound scanning every 10 min for 60 min. All doses, except 20 mg/hr, provoked a significant reduction in gallbladder volume (P < 0.01). The gallbladder emptying peak occurred after 20 min infusion. It was approximately 40-45% of basal volume and 60-70% of the emptying observed after a standard meal. At 100 mg/hr, erythromycin caused a 2.5-fold increase in plasma motilin concentration, which reached a peak after 30 min infusion. Plasma motilin peaked following maximum gallbladder emptying in all subjects. To evaluate whether cholinergic pathways were implicated in the action of erythromycin, 100 mg/hr erythromycin was infused together with 6 micrograms/kg/hr atropine. Atropine inhibited both gallbladder emptying and motilin release (P < 0.001). Infusion of 1 microgram/kg/hr somatostatin had the same inhibitory effects (P < 0.001). Our results suggest that atropine acts by inhibiting an erythromycin-activated cholinergic neural mechanism. Somatostatin could exert its inhibitory effect by blocking the release of acetylcholine from neural terminations.