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Procainamide (PA) and its in vivo metabolite, N-acetylprocainamide (NAPA), display some pharmacological differences. Although it is agreed that PA is a class IA antiarrhythmic, it has been reported that NAPA is a pure class III antiarrhythmic that affects only the repolarizing phase of the cardiac action potential. This last concept, observed exclusively in dogs, gained wide acceptance, appearing in classic pharmacology textbooks. However, evidence in species such as mice and rats indicates that NAPA can affect cardiac Na+ channels, which is unexpected for a pure class III antiarrhythmic drug. To further clarify this issue, the effects of PA (used as a reference drug) and NAPA on the maximum upstroke velocity (Vmax) and half-decay time (HDT) of the cardiac action potential were examined in the isolated right papillaris magnus of the guinea pig heart. Both PA and NAPA affected Vmax at lower concentrations than required to affect HDT, and NAPA had weaker effects on both variables. Thus, NAPA displayed typical class IA antiarrhythmic behavior. Therefore, the concept that NAPA is a pure class III antiarrhythmic drug is more species-dependent than previously envisioned. In addition, we demonstrated that the differential pharmacology of PA and NAPA is explainable, in molecular terms, by steric hindrance of the effects of NAPA and the greater number of potent aromatic-aromatic and cation π interactions with Na+ or K+ cardiac channels for PA.

Epigenetic post-translational modifications are essential for human malaria parasite survival and progression through its life cycle. Here, we present new functionalized suberoylanilide hydroxamic acid (SAHA) derivatives that chemically combine the pan-histone deacetylase inhibitor SAHA with the DNA methyltransferase inhibitor procainamide. A three- or four-step chemical synthesis was designed starting from cheap raw materials. Compared to the single drugs, the combined molecules showed a superior activity in Plasmodium and a potent inhibition against human HDAC6, exerting no cytotoxicity in human cell lines. These new compounds are fully active in multidrug-resistant Plasmodium falciparum Cambodian isolates. They target transmission of the parasite by inducing irreversible morphological changes in gametocytes and inhibiting exflagellation. The compounds are slow-acting and have an additive antimalarial effect in combination with fast-acting epidrugs and dihydroartemisinin. The lead compound decreases parasitemia in mice in a severe malaria model. Taken together, this novel fused molecule offers an affordable alternative to current failing antimalarial therapy.

CHARGE syndrome is an autosomal dominant congenital disorder caused primarily by mutations in the CHD7 gene. Using a small molecule screen in a zebrafish model of CHARGE syndrome, we identified 4 compounds that rescue embryos from disease-like phenotypes. Our screen yielded DAPT, a Notch signaling inhibitor that could ameliorate the craniofacial, cranial neuronal and myelination defects in chd7 morphant zebrafish embryos. We discovered that Procainamide, an inhibitor of DNA methyltransferase 1, was able to recover the pattern of expression of isl2a, a cranial neuronal marker while also reducing the effect on craniofacial cartilage and myelination. M344, an inhibitor of Histone deacetylases had a strong recovery effect on craniofacial cartilage defects and could also modestly revert the myelination defects in zebrafish embryos. CHIC-35, a SIRT1 inhibitor partially restored the expression of isl2a in cranial neurons while causing a partial reversion of myelination and craniofacial cartilage defects. Our results suggest that a modular approach to phenotypic rescue in multi-organ syndromes might be a more successful approach to treat these disorders. Our findings also open up the possibility of using these compounds for other disorders with shared phenotypes., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Objectives: The authors studied the response rates and relative sensitivity of the most common agents used in the sodium-channel blocker (SCB) challenge., Background: A type 1 Brugada electrocardiographic pattern precipitated by an SCB challenge confers a diagnosis of Brugada syndrome., Methods: Patients undergoing an SCB challenge were prospectively enrolled across Canada and the United Kingdom. Patients with no prior cardiac arrest and family histories of sudden cardiac death or Brugada syndrome were included., Results: Four hundred twenty-five subjects underwent SCB challenge (ajmaline, n = 331 [78%]; procainamide, n = 94 [22%]), with a mean age of 39 ± 15 years (54% men). Baseline non-type 1 Brugada ST-segment elevation was present in 10%. A total of 154 patients (36%) underwent signal-averaged electrocardiography, with 41% having late potentials. Positive results were seen more often with ajmaline than procainamide infusion (26% vs. 4%, p < 0.001). On multivariate analysis, baseline non-type 1 Brugada ST-segment elevation (odds ratio [OR]: 6.92; 95% confidence interval [CI]: 3.15 to 15.2; p < 0.001) and ajmaline use (OR: 8.76; 95% CI: 2.62 to 29.2; p < 0.001) were independent predictors of positive results to SCB challenge. In the subgroup undergoing signal-averaged electrocardiography, non-type 1 Brugada ST-segment elevation (OR: 9.28; 95% CI: 2.22 to 38.8; p = 0.002), late potentials on signal-averaged electrocardiography (OR: 4.32; 95% CI: 1.50 to 12.5; p = 0.007), and ajmaline use (OR: 12.0; 95% CI: 2.45 to 59.1; p = 0.002) were strong predictors of SCB outcome., Conclusions: The outcome of SCB challenge was significantly affected by the drug used, with ajmaline more likely to provoke a type 1 Brugada electrocardiographic pattern compared with procainamide. Patients undergoing SCB challenge may have contrasting results depending on the drug used, with potential clinical, psychosocial, and socioeconomic implications., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: This study sought to compare the differences between procainamide and flecainide to stress the His-Purkinje system during electrophysiological study (EPS) in patients with syncope and bundle branch block (BBB)., Background: Patients with syncope and BBB are at risk of developing atrioventricular block. EPS is recommended including class I drug challenge to unmask His-Purkinje disease in cases with baseline normal His-ventricular interval. There is little data on differences between different class I drugs., Methods: This was a prospective study of all consecutive patients undergoing EPS for syncope and BBB at a single center (January 1, 2012 to June 30, 2017). Of those patients with negative baseline EPS, 2 cohorts were compared: group A (historical cohort: procainamide) and group B (flecainide)., Results: During the study, 271 patients (age 73.9 ± 12.1 years, 64.9% male, QRS duration: 139.4 ± 13.9 ms) underwent EPS. In 166, baseline EPS was negative and class I drug challenge was performed (90 procainamide, 76 flecainide). The final value and percentage increase in the His-ventricular interval (76 ± 16 ms vs. 64 ± 10 ms and 22.5 ± 6.2% vs. 11.8 ± 5.3%; p < 0.001) and diagnostic yield (14.5% vs. 7.8%, p = 0.04) were higher with flecainide. No differences were found in baseline characteristics. During follow-up (25.8 ± 6.3 months), 39 patients (24.8%) with negative EPS (19.2% with flecainide vs. 30.1% with procainamide: relative risk: 5.1; 95% confidence interval: 2.6 to 10.2; p < 0. 001) received a pacemaker., Conclusions: Flecainide has a higher diagnostic yield than does procainamide in patients with BBB, syncope, and negative baseline EPS due to a greater increase of the His-ventricular interval. Additionally, there is a lesser need for pacemaker implantation in patients in whom the class I drug test using flecainide was negative., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

We have previously reported the enhancement of the antiproliferative and apoptotic activities of cis-diamminedichloroplatinum(II) (DDP) when it is coadministered with a class I antiarrhythmic drug procainamide hydrochloride (PA). Here, we determined the antiproliferative activity of DDP, either in solution or loaded in liposomes, in the presence of PA, in the bulk solution, or directly embedded in liposomes together with DDP. Our results show that PA potentiates the activity of DDP-liposomes and that this effect is maintained at least in some of the investigated cell types when both drugs were mixed and loaded together into liposomes.

To improve the anticolitic efficacy of 5-aminosalicylic acid (5-ASA), a colon-specific mutual prodrug of 5-ASA was designed. 5-ASA was coupled to procainamide (PA), a local anesthetic, via an azo bond to prepare 5-(4-{[2-(diethylamino)ethyl]carbamoyl}phenylazo)salicylic acid (5-ASA-azo-PA). 5-ASA-azo-PA was cleaved to 5-ASA and PA up to about 76% at 10 h in the cecal contents while remaining stable in the small intestinal contents. Oral gavage of 5-ASA-azo-PA and sulfasalazine, a colon-specific prodrug currently used in clinic, to rats showed similar efficiency in delivery of 5-ASA to the large intestine, and PA was not detectable in the blood after 5-ASA-azo-PA administration. Oral gavage of 5-ASA-azo-PA alleviated 2,4,6-trinitrobenzenesulfonic acid-induced rat colitis. Moreover, combined intracolonic treatment with 5-ASA and PA elicited an additive ameliorative effect. Furthermore, combined treatment with 5-ASA and PA additively inhibited nuclear factor-kappaB (NFκB) activity in human colon carcinoma cells and inflamed colonic tissues. Finally, 5-ASA-azo-PA administered orally was able to reduce inflammatory mediators, NFκB target gene products, in the inflamed colon. 5-ASA-azo-PA may be a colon-specific mutual prodrug acting against colitis, and the mutual anticolitic effects occurred at least partly through the cooperative inhibition of NFκB activity.

Background: We describe the potentiation of antiproliferative and apoptotic activities triggered by cis-diamminedichloroplatinum(II) (DDP), and obtained in vitro by the co-administration of procainamide hydrochloride (PdHCl) in murine P388, and human A2780 and A549 cells., Methods: We determined the antiproliferative and apoptotic activities of DDP and PdHCl combinations by different techniques. Moreover, cell cycle analysis, restriction enzyme inhibition followed by agarose gel electrophoresis, and TUNEL analysis of tumour cells in vivo were also used to strengthen our hypothesis., Results: Our results show that PdHCl may significantly increase the inhibition of cell proliferation and apoptosis. Experiments in vivo showed that the co-administration of DDP and PdHCl increased the percentage of apoptotic cells compared to DDP alone treatment, both in subcutaneous (sc) and intraperitoneal (ip) P388 tumours. We finally demonstrated that the co-administration of PdHCl prevents DNA digestion accounting for a restriction enzyme inhibition that in some cases was greater than that obtained by DDP alone. Moreover, when PdHCl was mixed with the reaction products (RP) of DDP (RP-PdHCl) we obtained a restriction enzyme inhibition greater for some enzymes (Bsp1407I, Hin1II, and Psp1406I) than that obtained by the DDP-PdHCl solution., Conclusions: On the whole our data demonstrate that the class I antiarrhythmic drug PdHCl may increase the antiproliferative activity of DDP by improving its triggering of apoptosis, and that this phenomenon may be likely linked to the formation of a new Pt compound., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Overt systemic inflammatory response is a predisposing mechanism for infection-induced skeletal muscle damage and rhabdomyolysis. Aberrant DNA methylation plays a crucial role in the pathophysiology of excessive inflammatory response. The antiarrhythmic drug procainamide is a non-nucleoside inhibitor of DNA methyltransferase 1 (DNMT1) used to alleviate DNA hypermethylation. Therefore, we evaluated the effects of procainamide on the syndromes and complications of rhabdomyolysis rats induced by lipopolysaccharide (LPS). Rhabdomyolysis animal model was established by intravenous infusion of LPS (5 mg/kg) accompanied by procainamide therapy (50 mg/kg). During the experimental period, the changes of hemodynamics, muscle injury index, kidney function, blood gas, blood electrolytes, blood glucose, and plasma interleukin-6 (IL-6) levels were examined. Kidneys and lungs were exercised to analyze superoxide production, neutrophil infiltration, and DNMTs expression. The rats in this model showed similar clinical syndromes and complications of rhabdomyolysis including high levels of plasma creatine kinase, acute kidney injury, hyperkalemia, hypocalcemia, metabolic acidosis, hypotension, tachycardia, and hypoglycemia. The increases of lung DNMT1 expression and plasma IL-6 concentration were also observed in rhabdomyolysis animals induced by LPS. Treatment with procainamide not only inhibited the overexpression of DNMT1 but also diminished the overproduction of IL-6 in rhabdomyolysis rats. In addition, procainamide improved muscle damage, renal dysfunction, electrolytes disturbance, metabolic acidosis, hypotension, and hypoglycemia in the rats with rhabdomyolysis. Moreover, another DNMT inhibitor hydralazine mitigated hypoglycemia, muscle damage, and renal dysfunction in rhabdomyolysis rats. These findings reveal that therapeutic effects of procainamide could be based on the suppression of DNMT1 and pro-inflammatory cytokine in endotoxin-induced rhabdomyolysis.

Procainamide is class Ia Na(+) channel blocker that may prolong ventricular repolarization secondary to inhibition of IK r , the rapid component of the delayed rectifier K(+) current. In contrast to selective IN a blockers such as lidocaine, procainamide was shown to produce arrhythmogenic effects in the clinical setting. This study examined whether pro-arrhythmic responses to procainamide may be accounted for by drug-induced repolarization abnormalities including impaired electrical restitution kinetics, spatial gradients in action potential duration (APD), and activation-to-repolarization coupling. In perfused guinea-pig hearts, procainamide was found to prolong the QT interval on ECG and left ventricular (LV) epicardial monophasic APD, increased the maximum slope of electrical restitution, enhanced transepicardial APD variability, and eliminated the inverse correlation between the local APD and activation time values determined at distinct epicardial recording sites prior to drug infusion. In contrast, lidocaine had no effect on electrical restitution, the degree of transepicardial repolarization heterogeneities, and activation-to-repolarization coupling. Spontaneous episodes of monomorphic ventricular tachycardia were observed in 57% of procainamide-treated heart preparations. No arrhythmia was induced by lidocaine. In summary, this study suggests that abnormal changes in repolarization may contribute to pro-arrhythmic effects of procainamide., (© 2013 Société Française de Pharmacologie et de Thérapeutique. Published by John Wiley & Sons Ltd.)

Background: Targeting abnormal DNA methylation represents a therapeutically relevant strategy for cancer treatment as demonstrated by the US Food and Drug Administration approval of the DNA methyltransferase inhibitors azacytidine and 5-aza-2'-deoxycytidine for the treatment of myelodysplastic syndromes. But their use is associated with increased incidences of bone marrow suppression. Alternatively, procainamide has emerged as a potential DNA demethylating agent for clinical translation. While procainamide is much safer than 5-aza-2'-deoxycytidine, it requires high concentrations to be effective in DNA demethylation in suppressing cancer cell growth. Thus, our laboratories have embarked on the pharmacological exploitation of procainamide to develop potent DNA methylation inhibitors through lead optimization., Methods: We report the use of a DNA methylation two-component enhanced green fluorescent protein reporter system as a screening platform to identify novel DNA methylation inhibitors from a compound library containing procainamide derivatives., Results: A lead agent IM25, which exhibits substantially higher potency in GSTp1 DNA demethylation with lower cytotoxicity in MCF7 cells relative to procainamide and 5-aza-2'-deoxycytidine, was identified by the screening platform., Conclusions: Our data provide a proof-of-concept that procainamide could be pharmacologically exploited to develop novel DNA methylation inhibitors, of which the translational potential in cancer therapy/prevention is currently under investigation.

Secreted Wingless type (Wnt) ligands have previously been shown to be involved in tumor developmental processes and oncogenesis. Aberrant promoter methylation of Wnt inhibitory factor-1 (WIF-1) is a fundamental mechanism of epigenetic silencing in human cancers. Procaine, a local anesthetic drug, and procainamide, a drug for the treatment of cardiac arrhythmias, have been reported as inhibitors of DNA methylation, causing demethylation and reactivation of methylation-silenced genes such as RARbeta and GSTP1. The promoter demethylation of WIF-1 has not previously been reported on. We demonstrated previously that WIF-1 is silenced due to promoter hypermethylation in lung cancer cell lines. In this study, we demonstrate promoter demethylation of WIF-1; restoration of WIF-1 expression, and underexpression of cytosolic beta-catenin protein and TCF reporter activity, after procaine and procainamide treatment in H460 and A549 cell lines. Our results provide the first evidence that procaine and procainamide reactivate WIF-1 in these cancer cells and downregulate the Wnt canonical pathway. These results further suggest that procaine and procainamide may have a potential use for preventing the development of lung cancer.

Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent KM 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (> or =24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking.

Procainamide (PA) is a drug that is used to treat tachycardia in postoperative patients or for long-term maintenance of cardiac arrythmias. Unfortunately, its use has also been associated with agranulocytosis. Here, we have investigated the metabolism of PA by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that PA oxidation by MPO/H 2O 2 would produce a PA cation radical that, in the absence of a biochemical reductant, would lead to the free radical oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms by the reaction of DMPO with a protein free radical. We found that PA metabolism by MPO/H 2O 2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. N-acetyl-PA did not cause DMPO-MPO formation, indicating that the unsubstituted aromatic amine was more oxidizable. PA had a lower calculated ionization potential than N-acetyl-PA. The DMPO adducts of MPO metabolism, as analyzed by electron spin resonance spectroscopy, included a nitrogen-centered radical and a phenyl radical derived from PA, either of which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with PA/H 2O 2 and was found to contain DMPO using the anti-DMPO antibody. Mass spectrometry analysis confirmed the identity of the protein as human MPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by free radical metabolites of PA, which we characterized by spin trapping. We propose that drug-induced free radical formation on MPO may play a role in the origin of agranulocytosis.

Atrial electrical remodeling (ER) after spontaneous or pacing-induced atrial fibrillation has been previously described in humans. We investigated atrial ER induced by a 5-minute period of rapid atrial pacing and the pharmacologic effects of verapamil and procainamide on this atrial ER phenomenon. The atrial effective refractory periods (ERPs) at drive cycle lengths of 400 (ERP 400 ) and 600 (ERP 600 ) ms, at five representative atrial sites (high right atrium [HRA]; proximal, middle and distal coronary sinus; interatrial septum), were determined in 20 patients at baseline and immediately after cessation of a 5-minute period of rapid pacing from the HRA at a rate of 150 bpm. The degrees of atrial ERP 400 and ERP 600 shortening after pacing were calculated as acute atrial ER. The same protocol was repeated in another 15 patients after intravenous administration of verapamil (0.15 mg/kg) and in another 15 patients after intravenous administration of procainamide (15 mg/kg). The results demonstrated that, in the control state acute atrial ER can be significantly demonstrated at each atrial representative site ( p < 0.001). The mean ERP 400 and ERP 600 shortenings were 9 +/- 4% and 8 +/- 4%, respectively. After procainamide infusion, but not after verapamil, baseline ERP 400 and ERP 600 values were significantly prolonged at the five representative atrial sites ( p < 0.01). Acute atrial ER could still be demonstrated at each atrial site after procainamide or verapamil infusion ( p < 0.001). In conclusion, acute atrial ER can be demonstrated after only a 5-minute period of rapid atrial pacing in humans. Intravenous verapamil or procainamide does not abolish this ER process.

Cisplatin is one of the most widely utilized anticancer drugs; nevertheless its use is often hampered by the onset of serious side effects. In spite of its tight binding to DNA, great teratogenic effects do not characterize cisplatin, although its embryolethal and growth retardation activities are quite remarkable. On the basis of our previous observations, demonstrating the usefulness of procainamide hydrochloride for the protection against cisplatin toxic effects in adult mice and rats, we now analyze the feasibility of the combined treatment with cisplatin and the antiarrhythmic drug procainamide hydrochloride in pregnant mice and the possible protective action of procainamide against the embryotoxic activity of cisplatin. Our data, obtained in CD-1 dams after treatment with 8 or 12 mg/kg cisplatin ip, with or without 50 mg/kg procainamide hydrochloride iv, confirm the embryotoxic effects of cisplatin. We also demonstrate that procainamide may be administered with cisplatin without causing an increase in its embryotoxic effects, but slightly improving some embryotoxicity parameters in living embryos such as the fetal weight, the percentage of fetuses with skeletal anomalies, and the number of ossification centres. The mechanism of action of this partially protective activity seems to be linked in part to the lower cisplatin accumulation in fetal tissue, probably due to an interaction of drugs at the level of placenta, in part to the protection of procainamide against maternal toxicity of cisplatin. A relevant result of this research is the suggestion that procainamide hydrochloride might be administered in case of pregnancy to protect against the maternal toxic effects of cisplatin without an increased embryotoxic/teratogenic risk for the offspring.

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CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2'-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2'-deoxycytidine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer.

Procainamide and related triethylamine-substituted 4-aminobenzamides, such as metoclopramide and declopramide, exert cellular effects potentially exploitable in oncology at millimolar concentrations (DNA demethylation, nuclear factor-kappaB inhibition, apoptosis) and display anti-inflammatory properties. However, these drugs induce massive cell vacuolization at similar concentrations, a response initiated by vacuolar (V-) ATPase-dependent ion trapping into and osmotic swelling of acidic organelles. We have examined whether this overlooked response might be related to the effects on cell proliferation and viability using cultured vascular smooth muscle cells and tumor-derived cell lines (Morris 7777 hepatoma, HT-1080 fibrosarcoma). Giant vacuole formation, of confirmed trans-Golgi origin (labeled with C5-ceramide, p230, golgin-97), is a cellular response to all tested amines in the series (> or = 2.5 mM), including triethylamine. These drugs and the V-ATPase inhibitor bafilomycin A1 inhibited smooth muscle cell proliferation, suggesting that acidification of a cellular compartment is essential to cell division. The cytotoxicity was maximal with metoclopramide, and this effect was minimally influenced by bafilomycin A1; furthermore, metoclopramide (2.5 mM) induced apoptosis in tumor cells as judged by poly(ADP-ribose)polymerase (PARP) cleavage. Triethylamine and procainamide exhibit a low level of cytotoxicity variably reduced by bafilomycin co-treatment. In Morris cells, the secretion of alpha-fetoprotein is inhibited by amines, consistent with the impairment of the secretory pathway. The most highly substituted 4-aminobenzamides are significant NF-kappaB inhibitors in smooth muscle cells. Although some effects of 4-aminobenzamides are independent of V-ATPase-driven ion trapping (inhibition of NF-kappaB nuclear translocation, agent-specific cytotoxicity, PARP cleavage), other effects are dependent on this phenomenon (vacuolization, a component of the cytotoxicity, inhibition of secretion).

Background: The class 1 antiarrhythmic drug procainamide hydrochloride might protect against acute cisplatin-induced nephrotoxicity and hepatotoxicity in mice and rats. In this report, the protective activity of procainamide hydrochloride against renal and hepatic tissue damage induced by repeated administration of low doses of cisplatin was analyzed morphologically and histochemically., Materials and Methods: Light microscopy observations were performed on liver, renal and heart samples obtained from female Wistar rats treated twice a week for 10 weeks with 1 mg/kg cisplatin (cumulative dose: 20 mg/kg), with or without 100 mg/kg procainamide hydrochloride (cumulative dose: 2 g). Samples were then submitted to histochemical stainings [i.e. H & E, periodic acid Schiff (PAS) and Sudan Black]., Results: Light microscopy analysis revealed that the coadministration of cisplatin and procainamide hydrochloride significantly reduced tissue alterations both in the kidneys and liver, while in the heart, neither cisplatin nor the combination of cisplatin and procainamide hydrochloride caused any evident tissue damage., Conclusion: The morphological and histochemical data confirm that procainamide hydrochloride is able to protect not only from acute cisplatin-induced toxicities, but also from tissue alterations induced in the liver and kidneys by the administration of repeated low doses of cisplatin.

Antiarrhythmic properties of taurhythman were demonstrated on experimental models of ventricular (early occlusion and calcium chloride-induced) and atrioventricular (aconitine-induced) arrhythmias. The preparation reduced or prevented episodes of paroxysmal tachycardia and ventricular fibrillation, decreased the incidence of arrhythmias, and increased the lethal dose (LD) of arrhythmogenic agents. By its efficiency, taurhythman was superior to procainamide and comparable to lidocaine.

Unlabelled: A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined., Objective: To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL., Methods: Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis., Results: Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen., Conclusion: Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.

The cardiac arrhythmic suppression trial (CAST) reported that antiarrhythmic treatments in post-myocardial infarction (MI) patients resulted in poor outcome and decreased in heart rate variability indices (HRV). The goal of the present study was to determine whether aprindine and procainamide, antiarrhythmic agents that increase HRV, result in beneficial effects in post-MI rabbits. Four weeks before experiment, MI was induced in four rabbits by ligating the major branch of left coronary artery. A total of eight rabbits (four post-MI and four normal rabbits) were randomly assigned to treatment with either intravenous aprindine (1 mg/kg) or intravenous procainamide (15 mg/kg). Frequency domain HRV (low frequency spectra, LF, 0.04-0.15 Hz; high frequency spectra, HF, 0.15-0.40 Hz) were assessed by MemCalc software. Aprindine significantly increased HF and LF in both MI and normal rabbits, whereas procainamide tended to decrease HF and LF in MI and normal rabbits (in total rabbits; aprindine, LF, from 6.3 +/- 7.9 to 16.5 +/- 15.0 ms(2)/Hz, P < 0.05; HF, from 8.0 +/- 11.7 to 17.5 +/- 15.0 ms(2)/Hz, P < 0.05; procainamide, LF, from 4.9 +/- 7.4 to 4.8 +/- 8.5 ms(2)/Hz, NS; HF, from 11.1 +/- 23.0 to 5.1 +/- 10.6 ms(2)/Hz, NS). Under pharmacological denervation with propranolol (0.1 mg/kg) and atropine (0.04 mg/kg), aprindine increased LF and HF (LF, from 0.2 +/- 0.2 to 0.8 +/- 0.7 ms(2)/Hz, P < 0.05; HF, from 0.1 +/- 0.0 to 0.2 +/- 0.0 ms(2)/Hz, P < 0.05). These data suggest that aprindine can increase HRV in post-MI rabbits. Further experiments in human subjects would be of benefit.

DNA cytosine methylation plays a considerable role in normal development, gene regulation, and carcinogenesis. Hypermethylation of the promoters of some tumor suppressor genes and the associated silencing of these genes often occur in certain cancer types. The reversal of this process by DNA methylation inhibitors is a promising new strategy for cancer therapy. In addition to the four well-characterized nucleoside analogue methylation inhibitors, 5-azacytidine, 5-aza-2'-deoxycytidine (5-Aza-CdR), 5-fluoro-2'-deoxycytidine, and zebularine, there is a growing list of non-nucleoside inhibitors. However, a systemic study comparing these potential demethylating agents has not been done. In this study, we examined three non-nucleoside demethylating agents, (-)-epigallocatechin-3-gallate, hydralazine, and procainamide, and compared their effects and potencies with 5-Aza-CdR, the most potent DNA methylation inhibitor. We found that 5-Aza-CdR is far more effective in DNA methylation inhibition as well as in reactivating genes, compared with non-nucleoside inhibitors.

It has been observed that decrease of DNA methyltransferase 1 (DNMT1) activity is associated with low content of the CD3-zeta (zeta) chain in T cell receptor (TCR)/CD3 complex of T cells in systemic lupus erythematosus (SLE) patients. The CD3-zeta chain plays a pivotal role in intracellular signal transmission between TCR/CD3 complex and nuclei. The compounds 5'-azacytidine (AZC) and procainamide (PCA) belong to inhibitors of DNMT1, whose low activity correlates with increase in transcription of various genes. Using the reverse-transcription and real-time quantitative PCR (RQ-PCR) analysis, we indicated that AZC and PCA did not profoundly affect on CD3-zeta chain transcription in Jurkat T leukemia cells clone E6-1. However, the flowcytometric analysis revealed that AZC and PCA decreased intracellular contents of CD3-zeta chain in these cells in dose dependent manner. Our results suggest that decrease of DNMT1 activity may alter intracellular signal transmission without effect on transcription level of CD3-zeta chain.

The aim of the study was to investigate the effect of class IA antiarrhythmic drug procainamide and its new derivatives PA20, PA23, PA28 and PA53 on carbachol-induced action potential duration and contraction force in guinea pig atrial muscles. Experiments were carried out using standard method of registration of myocardium electromechanical activity. Under control conditions (perfusion of atrial strips with Tyrode solution), mean action potential duration measured at 90% (AP90) and 50% (AP50) of repolarization were 92.5+/-4.5 ms and 44.4+/-2.9 ms (n=18), respectively, and contraction force was of 2.7+/-0.5 mN (n=12). Carbachol (10(-6) M), an agonist of muscarinic acetylcholine receptor and activator of K(Ach) channels, markedly decreased AP90 to 32.4+/-2.4%, AP50 - to 25.4+/-2.2% (n=18) (p<0.001) and contraction force - to 24.2+/-5.8% (n=16) (p<0.05), vs. control. Procainamide and its new sulfonylcarbamide derivatives PA20, PA23, PA28 (10(-5)-3x10(-3) M), and PA53 (10(-5)-10(-3) M) reversed the carbachol-induced action potential duration shortening at different extent: procainamide derivative PA20 (N-cyclohexylsulfonylcarbamide fragment is linked up to benzene ring) had the most potent anticholinergic effect on action potential duration and contraction force of guinea pig atrial muscles. N-ethylsulfonylcarbamide fragment in PA23 or permanent positive charge of aliphatic nitrogen in PA28 had a weaker anticholinergic effect (similar to procainamide action) on action potential duration and contraction force than PA20. The weakest anticholinergic effect was induced by procainamide derivative PA53 with isosteric form of sulfonylcarbamide.

Procaine and some other basic drugs reportedly induce vacuolization of various cell types. We addressed the concentration-effect and structure-activity relationships as well as the mechanism of this effect using three cell lines. Massive vacuolization occurs over several hours in primary cultures of rabbit pulmonary artery smooth muscle cells (SMCs) and COS-1 cells in response to procaine and loosely related amine compounds (procainamide, N-acetyl-procainamide, metoclopramide, lidocaine, triethylamine, nicotine) used at 2.5 mM. Furthermore, chloroquine, propranolol, diphenhydramine, and neutral red are active in this respect at 100 to 250 microM in SMCs and COS-1 cells. Human embryonic kidney 293 cells mildly responded to triethylamine, nicotine, and propranolol only. Tetraethylammonium was uniformly inactive, as well as many other drugs in all three cell types (concentrations up to 2.5 mM). Procainamide does not induce apoptosis in SMCs treated for up to 48 h, although the vacuolization is sustained and proliferation and migration are reduced during this period. Procainamide-induced vacuolization is reversible on drug washing, largely prevented by bafilomycin A1 cotreatment, and has a tentatively identified Golgi origin (uptake of ceramide-C5). Procainamide and neutral red are concentrated in SMCs in a bafilomycin A1-sensitive manner. The preventive effect of bafilomycin A1 suggests that the vacuoles originate from the osmotic swelling of acidic organelles in which the charged basic drugs are trapped at low pH. Drug transport at the plasma membrane may be limiting for this type of response, as suggested by the cell type selectivity of agents and the inhibitory effect of some drugs such as quinidine.

Focal discharges (FDs) are present in thoracic veins during atrial fibrillation (AF). We hypothesize that procainamide exerts its anti-AF action by suppressing FDs in the thoracic veins. We studied six mongrel dogs (22-27 kg) with sustained (>6 h) AF induced by 47 +/- 20 days of chronic rapid LA appendage (LAA) or pulmonary vein (PV) pacing. Procainamide was infused intravenously until AF was terminated or a cumulative dose of 20 mg/kg was reached. High-resolution mapping during AF showed FDs in the vein of Marshall, PVs, and the LAA. Procainamide significantly (P < 0.05) reduced the frequency of these FDs and suppressed the interactions of wave fronts between PVs and LA. The cumulative dose of PA needed to terminate AF correlated negatively (r =-0.9, P < 0.05) with the baseline effective refractory period (ERP) of PV and positively (r = 0.8, P < 0.05) with the baseline maximum dominant frequency (DF) of AF. In four of five dogs, AF converted to atrial tachycardia originating from the PVs before termination. Attempts to reinduce sustained AF were unsuccessful in these five dogs. AF was resistant to procainamide in the sixth dog. In conclusion, procainamide reduced the rate of FDs in the thoracic veins and the LA and suppressed the interaction between PVs and LA. Second, FDs in the PV are more resistant to procainamide's action than FDs in the atria. Third, inherent PV ERP is important in determining the antifibrillatory efficacy of procainamide.

We have synthesized a series of hybrid compounds combining the pharmacophoric redox moieties of vitamin E and key features responsible for the antiarrhythmic properties of the class I antiarrhythmics procainamide and lidocaine. Procainamide analogue (2a) and lidocaine analogues (14a) and (14b) are very strong inhibitors of lipid peroxidation. All analogues tested at 100 or 30 microM enhanced the post ischemic recovery without inducing ventricular fibrillations while there was no evidence in our experiments for drug-induced pro-arrhythmia. In addition, they induced a widening of the QRS intervals. Our data suggest that the efficacy of the new compounds in preventing reperfusion arrhythmias could be attributed to their combined effects involving inhibition of free radical mediated damage coupled with antiarrhythmic properties.

Class Ia antiarrhythmic drugs, including procainamide (PROC), are associated with cardiac sodium channel blockade, delayed ventricular repolarisation and with a risk of ventricular pro-arrhythmia. The HERG K(+) channel is frequently linked to drug-induced pro-arrhythmia. Therefore, in this study, interactions between PROC and HERG K(+) channels were investigated, with particular reference to potency and mechanism of drug action. Whole-cell patch-clamp recordings of HERG current (I(HERG)) were made at 37 degrees C from human embryonic kidney (HEK 293) cells stably expressing the HERG channel. Following activating pulses to +20 mV, I(HERG) tails were inhibited by PROC with an IC(50) value of approximately 139 microM. I(HERG) blockade was found to be both time- and voltage-dependent, demonstrating contingency upon HERG channel gating. However, I(HERG) inhibition by PROC was relieved by depolarisation to a highly positive membrane potential (+80 mV) that favoured HERG channel inactivation. These data suggest that PROC inhibits the HERG K(+) channel by a primarily 'open' or 'activated' channel state blocking mechanism and that avidity of drug-binding is decreased by extensive I(HERG) inactivation. The potency of I(HERG) blockade by PROC is much lower than for other Class Ia agents that have been studied previously under analogous conditions (quinidine and disopyramide), although the blocking mechanism appears similar. Thus, differences between the chemical structure of PROC and other Class Ia antiarrhythmic drugs may help provide insight into chemical determinants of blocking potency for agents that bind to open/activated HERG channels.

The role of cardiac surface QT intervals (QTI) in assessing ventricular repolarisation dispersion was investigated in 10 pentobarbital-anesthetized, open-chest sheep. Activation-recovery interval (ARI) and QTI were measured from multiple ECGs acquired from left ventricular epicardium, before and after procainamide administration (15 mg/min i.v. for 20 min). Procainamide therapy resulted in a significant prolongation in ARI and corrected QT interval (cQTI). A correlation between QTI, cQTI and ARI was found in only one animal (r = 0.78 and 0.79 respectively, p = 0.01) before procainamide therapy. No correlation was found between pooled QTI and ARI dispersion from the 10 animals at baseline or after procainamide administration (p > 0.05). There was a moderate correlation between pooled cQTI and ARI dispersion (r = 0.69, p = 0.02) before procainamide therapy. In conclusion, epicardial QTI have no significant correlation with the duration of ventricular repolarisation. Epicardial QTI dispersion is not a useful index of repolarisation heterogeneity.

Purpose: The purpose of this study is to evaluate the demethylating and tumor suppressor-reactivating activity of hydralazine and procainamide., Experimental Design: MDA-231, MCF-7, and T24 cell lines were treated for 5 days with 10 micro M hydralazine or 10 micro M procainamide. 5-aza-deoxycytidine at 0.75 micro M was used as positive control. BALB/c nu/nu mice xenografted with MDA-231 cells were treated with these drugs for 7 days by i.p. route. Methylation was assessed by PCR after digestion with methylation-sensitive enzymes for the ER gene and with methylation-specific PCR for retinoic acid receptor (RAR)beta and p16 genes. Gene expression was evaluated by reverse transcription-PCR and Western blot. The duration of the gene re-expressing effect of hydralazine was analyzed on T24 cells. Functionality of the re-expressed proteins was evaluated by the induction of the estrogen-responsive gene PS2 on MDA-231 cells and by the induction of G(1) arrest on T24 cells. The gene demethylating and re-expressing ability of hydralazine was tested in two patients with cervical and head and neck carcinomas, respectively., Results: Hydralazine and procainamide induced de-methylation and re-expression of the ER, RARbeta, and p16 genes in cultured cells. Both drugs also demethylated and re-expressed the ER gene in mice. Hydralazine re-expressed the p16 gene longer as compared with 5-aza-deoxycytidine. The re-expressed genes were functional. In addition, the treatment with oral hydralazine demethylated and re-expressed the RARbeta and p16 genes in the cervical and head and cancer patients., Conclusions: These cardiovascular drugs have a promising tumor suppressor-reactivating action and could potentially be used in clinic as an anticancer treatment, most likely to increase the efficacy of current biological or chemotherapeutic treatments.

In this study the membrane fluidity of fibroblasts under different pharmacological treatment was investigated. Two drugs, hydralazine and procainamide, were used to treat the immortalized mouse NIH 3T3 and hamster B14 fibroblasts. Membrane lipid dynamics was measured by fluorescence spectroscopy and electron spin resonance techniques. Two kinds of fluorescent probes (TMA-DPH and 12-(9-anthroyloxy)-stearic acid (12-AS)) and two spin labels (5-doxylstearic acid (5-DS) and 12-doxylstearic acid (12-DS)) were used to monitor fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. The drugs influenced the membrane hydrophobic core, of which hydralazine induced fluidization and procainamide increased the rigidity. The membrane fluidity at the surface of the lipid bilayer was not modified by the drugs which indicates that both drugs intercalated mainly into the inner core of the cell membrane.

Procainamide and quinidine inhibition of the degradation of meperidine in human liver was investigated by incubation of two concentrations of either drug with meperidine in homogenates of human liver over 24 and 36 h. Meperidine concentrations declined by 26% after incubation for 24 h and by 42% after incubation for 36 h. In the presence of procainamide, however, they decreased by only 15% to 18% at 24 h and by only 26% to 28% at 36 h. In the presence of quinidine, they declined by only 18% to 19% at 24 h and by only 27% to 28% at 36 h. Procainamide and quinidine may inhibit human hepatic carboxylesterase hCE-1, which is responsible for catalyzing the hydrolysis of meperidine. This inhibition may prolong the biological half-life of meperidine in patients receiving the drug together with either procainamide or quinidine.

Background: Our previous studies showed that procainamide hydrochloride may be an important modulator of cisplatin toxicity and antitumour activity. This study was performed in order to investigate if procainamide hydrochloride may influence the therapeutic index of cisplatin by inducing modifications of its pharmacokinetics and pharmacodymanics in vivo., Materials and Methods: The pharmacokinetic profile of cisplatin administered either in the presence or absence of procainamide hydrochloride was investigated in BDF1 female mice bearing 6-day P388 leukemia. Procainamide hydrochloride was administered i.v. at the dose of 50 mg/kg, immediately before cisplatin which, in turn, was administered i.p. at the dose of 8 mg/kg., Results: The combined administration of the antiarrhythmic drug and cisplatin caused significant differences in the pharmacokinetic profiles of Pt in plasma, ascites fluid and tissues. Filterable Pt was significantly increased both in plasma and ascites fluid in animals given the combined treatment. Similarly, a small increase was also found for total plasma Pt. These differences caused some changes of the pharmacokinetic parameters of filterable (plasma: AUC0-1 h = +16%, t1/2 alpha = +29%, t1/2b = +14%, K2p = -32%; ascites fluid: AUC0-1 h = +23%, t1/2 alpha = +78%, t1/2 beta = -49%, and total Pt (plasma: AUC0-1 h = +19%, t1/2 alpha = +27%, t1/2 beta = -22%; ascites fluid: AUC0-1 h = +6%, AUC0-infinity = +43%, t1/2 alpha = +30%). The analysis of tissue Pt content showed the general increase of Pt concentration in the main organs of animals treated with cisplatin and procainamide hydrochloride, with AUC0-24 h increased by 95%, 22%, 90% and 28% in kidney, liver, spleen and lung, respectively. The analysis of binding of Pt to DNA and percent interstrand cross-links (%ISCL) in P388 tumour cells showed that the % ISCL (10.44 +/- 3.81% vs. 3.51 +/- 0.01%) and the efficiency of ISCL formation (0.51 +/- 0.14 vs. 0.17 +/- 0.02 %ISCL.microgram DNA/pg Pt) were significantly greater when cisplatin was administered in association with procainamide hydrochloride., Conclusion: Our results show that procainamide hydrochloride may alter the pharmacodynamics and the pharmacokinetics and distribution of Pt in tumored mice treated with cisplatin.

In this study we use the N-substituted benzamides declopramide (3-CPA) and N-acetyl declopramide (Na-3-CPA) to investigate the involvement of the transcription factor NF-kappaB in the induction of apoptosis and surface immunoglobulin kappa (Igkappa) expression in the mouse pre-B cell line 70Z/3. We first showed that 3-CPA-induced apoptosis at doses around 500 microM and that the 3-CPA-induced apoptosis could be suppressed by over-expression of the Bcl-2 protein. Na-3-CPA was shown to be non-apoptotic at doses up to 1-2 mM. On the other hand, Na-3-CPA inhibited LPS-induced Igkappa expression while 3-CPA had no effect. Further analysis showed that while 3-CPA inhibited breakdown of IkappaBalpha, Na-3-CPA inhibited breakdown of IkappaBbeta. In addition, we used a 70Z/3 cell line expressing a dominant negative IkappaBalpha (70Z/3(deltaNIkappaBalpha)). The 70Z/3(deltaNIkappaBalpha) cell line was shown to be more sensitive to apoptosis and cytotoxicity induced by 3-CPA as well as by LPS, probably due to a defect in NF-kappaB rescue mechanism. Taken together, our data implicate distinct roles for IkappaBalpha and IkappaBbeta in regulating various NF-kappaB activities.

To investigate the pharmacological effect of procainamide on transmural ventricular repolarisation in normal heart, the transmural activation-recovery intervals (ARI) and their responses to procainamide (20 mg/min i.v. for 20 min) were studied in 6 open-chest, pentobarbitone-anaesthetised sheep. ARI was measured from the unipolar ECGs acquired with 4 plunge needles inserted into the basal and apical parts of the left ventricular wall. During sinus rhythm (cycle length 500-700 ms), there was no significant difference in the pooled ARI between the epicardium (266.0 +/- 30.5 ms), midmyocardium (265.0 +/- 28.9 ms) and endocardium (265.7 +/- 28.1 ms) (p > 0.05). Procainamide prolonged ARI in all myocardial layers. The pooled ARI prolongation from the epicardium, midmyocardium and endocardium of the 6 animals was 66.8 +/- 18.3, 70.3 +/- 14.7 and 65.3 +/- 15.7 ms (p > 0.05), respectively. In conclusion, sodium channel blocker procainamide results in a similar repolarisation prolongation in the left ventricular epicardium, midmyocardium and endocardium of a healthy heart.

In preceding papers, we proposed that procainamide hydrochloride, a class I antiarrhythmic agent, was able to protect mice and rats from cisplatin-induced nephrotoxicity and that it could exert its action through accumulation in kidneys followed by coordination with cisplatin (or its hydrolysis metabolites) and formation of a less toxic platinum compound similar to the new platinum(II) triamine complex cis-diamminechloro-[2-(diethylamino)ethyl 4-amino-benzoate, N4]-chlorideplatinum(II) monohydrochloride monohydrate, obtained by the reaction of cisplatin with procaine hydrochloride. Hepatotoxicity is not considered as a dose-limiting toxicity for cisplatin, but liver toxicity can occur when the antineoplastic drug is administered at high doses. Here, we report that procainamide hydrochloride, at an i.p. dose of 100 mg/kg, reduces cisplatin-induced hepatotoxicity, as evidenced by the normalization of plasma activity of glutamic oxalacetic transaminase and gamma-glutamyl transpeptidase, as well as by histological examination of the liver tissue. Twenty-four hours after i.p. treatment with the combination of 7.5 mg/kg cisplatin and 100 mg/kg procainamide, a significant increase of procainamide (+56%, P<0.05), total platinum (+31%, P<0.05), platinum-DNA adducts (+31%, P<0.05) and percent DNA-DNA interstrand cross-links (+69%, P<0.02) was found in liver tissue, as compared to animals treated with cisplatin alone. Moreover, in accordance with these findings, we also observed a slightly lower concentration and cumulative excretion of platinum in the feces. Since mitochondrial injury is considered a central event in the early stages of the nephrotoxic effect of cisplatin, the distribution of platinum in these subcellular organelles obtained from hepatocytes was determined after treatment with cisplatin with or without procainamide hydrochloride, together with platinum concentration in their cytosolic fraction. Our data show that the coadministration of procainamide hydrochloride produced a rearrangement of subcellular platinum distribution in hepatocytes with a slight decrease in mitochondria (-15%, P<0.10) and a slight increase in the cytosolic fraction (+40%, P<0.10) of platinum content, compared to the treatment with cisplatin alone. In analogy with our previous results in the kidney, confirmed here by our data in vitro, we suggest that the hepatoprotective activity of procainamide hydrochloride is linked to the formation of a less toxic platinum complex, which leads to inactivation of cisplatin itself and/or its highly toxic hydrolysis metabolites and to a different subcellular distribution of platinum.

We have analysed the mechanism of action for induction of apoptosis by N-substituted benzamides using declopramide as a lead compound. We show here that declopramide at doses above 250 microM in the mouse 70Z/3 pre-B cell line or in the human promyeolocytic cancer cell line HL60 induced cytochrome c release into the cytosol and caspase-9 activation. The broad spectrum caspase inhibitor zVADfmk and caspase-9 inhibitor zLEDHfmk inhibited apoptosis and improved cell viability when administrated to cells 1 h before exposure to declopramide, whereas the caspase-8 inhibitor zIEDHfmk had less effect. Also, the over expression of Bcl-2 by transfection in 70Z/3 cells inhibited declopramide-induced apoptosis. Prior to the induction of apoptosis, a G(2)/M cell cycle block was induced by declopramide. The cell cycle block was also observed in the presence of broad spectrum caspase inhibitor zVADfmk and in a transfectant expressing high levels of Bcl-2. Furthermore, while p53 was induced in 70Z/3 cells by declopramide, neither the apoptotic mechanism nor the G(2)/M cell cycle block were dependent on p53 activation since both effects were also seen in p53 deficient HL60 cells after addition of declopramide., (Copyright 2002 Cancer Research UK)

Among the many somatic genome alterations present in cancer cells, changes in DNA methylation may represent reversible "epigenetic" lesions, rather than irreversible "genetic" alterations. Cancer cell DNA is typically characterized by increases in the methylation of CpG dinucleotides clustered into CpG islands, near the transcriptional regulatory regions of critical genes, and by an overall reduction in CpG dinucleotide methylation. The transcriptional "silencing" of gene expression associated with such CpG island DNA hypermethylation presents an attractive therapeutic target: restoration of "silenced" gene expression may be possible via therapeutic reversal of CpG island hypermethylation. 5-Aza-cytidine (5-aza-C) and 5-aza-deoxycytidine (5-aza-dC), nucleoside analogue inhibitors of DNA methyltransferases, have been widely used in attempts to reverse abnormal DNA hypermethylation in cancer cells and restore "silenced" gene expression. However, clinical utility of the nucleoside analogue DNA methyltransferase inhibitors has been limited somewhat by myelosuppression and other side effects. Many of these side effects are characteristic of nucleoside analogues that are not DNA methyltransferase inhibitors, offering the possibility that nonnucleoside analogue DNA methyltransferase inhibitors might not possess such side effects. Human prostate cancer (PCA) cells characteristically contain hypermethylated CpG island sequences encompassing the transcriptional regulatory region of GSTP1, the gene encoding the pi-class glutathione S-transferase (GSTP1), and fail to express GSTP1 as a consequence of transcriptional "silencing." Inactivation of GSTP1 by CpG island hypermethylation, the most common somatic genome alteration yet reported for human PCAs, occurs early during human prostatic carcinogenesis and results in a loss of GSTP1 "caretaker" function, leaving prostate cells with inadequate defenses against oxidant and electrophile carcinogens. We report here that the drug procainamide, a nonnucleoside inhibitor of DNA methyltransferases, reversed GSTP1 CpG island hypermethylation and restored GSTP1 expression in LNCaP human PCA cells propagated in vitro or in vivo as xenograft tumors in athymic nude mice.

N-substituted benzamides are compounds that have recently been reported to inhibit nuclear factor-kappaB (NF-kappaB) activity and induce apoptosis in a pre-B cell line. In this study, we focused on the effects of N-substituted benzamides on transcriptional regulation in Jurkat T cells. We used a model system where the cells can be stimulated either through TCR/CD28 or by treatment of the cells with PMA and ionomycin to induce transcription factors typical for T lymphocyte activation. Treatment of the Jurkat cells with procainamide did not influence the transcription factor profile of stimulated cells, while treatment with a derivative having an acetyl group in position 4 of the aromatic ring inhibited NF-kappaB and nuclear factor of activated T cells (NFAT) activity. Declopramide, which contains a chloride in position 3 of the aromatic ring, was inactive in this system, whereas also the acetylated derivative of this compound inhibited NF-kappaB and NFAT activity. In contrast, the transcriptional activity and nuclear expression of activator protein 1 induced by TCR/CD28 stimulation or PMA and ionomycin treatment was enhanced by the acetylated variants of the N-substituted benzamides. Finally, we investigated the effect of N-substituted benzamides on intact promoters for two genes central in immune regulation; the CD40 ligand (CD40L) and IL-2 promoters. The transcriptional activity of the CD40L promoter as well as surface expression of the CD40L induced by signaling through TCR/CD28 was inhibited by addition of acetylated N-substituted benzamides, while the transcriptional activity of the IL-2 promoter was enhanced. Taken together, these data indicate that derivatives of N-substituted benzamides are potential drug candidates for quantitative as well as qualitative modulation of immune functions.

The post-repolarization refractoriness (PRR) is an important factor to determine the conduction block in cardiac muscle. Recently, we proposed the block coupling interval (BCI) as an useful electrophysiological index for evaluating the PRR. In the present study, the effect of procainamide on PRR was evaluated using the BCI and the effective refractory period (ERP). In five beagle dogs, radiofrequency linear ablation was performed on the right atrial surface parallel to the AV groove, forming an artificial isthmus (8-10 mm width and 15-20 mm length). Bipolar recordings were performed in the isthmus at a resolution of 1.2 mm and single extrastimuli with eight basic drive trains were delivered to cause conduction blocks in the isthmus. When a conduction block occurred, the recorded coupling interval at the recording site just proximal to the site of block was defined as BCI. At the site of the block, the ERP and duration of the monophasic action potential (MAP) at each drive cycle length was measured. The PRR was calculated using two different formulas: (1) [ERP-MAP] and (2) [BCI-MAP]. Procainamide was administrated intravenously at a dose of 15 mg/kg after the control study and the whole study protocol was repeated. The site of the block in an individual dog was always the same. BCI, ERP, and MAP were all shortened in accordance with the shortening of the basic drive cycle length, and the BCI was always the longest, ERP the middle, and the MAP was the shortest. The administration of procainamide prolonged each parameter, but the order of BCI > ERP > MAP remained unchanged. The PRR calculated as [BCI-MAP] was prolonged from 15 +/- 10 ms to 29 +/- 8 ms by the administration of procainamide (P = 0.048), but [ERP-MAP] was unchanged (8 +/- 10 ms vs 8 +/- 4 ms). In the conduction block model in the canine right atrium, procainamide prolonged the [BCI-MAP], but did not change the [ERP-MAP]. The procainamide effect of prolonging the PRR might be expressed better by the change in the BCI than the ERP.

To be positively selected, immature thymocytes must receive signaling through their T-cell receptor (TCR), and engagement of relatively low-affinity self-peptides permits further T-cell maturation. However, mature T cells no longer overtly respond to such low-affinity antigens, indicating that T cells acquire a higher threshold for activation during thymopoiesis. We wondered whether partial interference in positive selection could produce T cells that respond to the selecting self-peptide. This possibility was tested by injecting procainamide-hydroxylamine (PAHA), a lupus-inducing drug, into the thymus of adult normal mice. Three weeks after the second injection, IgG antichromatin antibodies appeared in the circulation and remained for several months. The murine antichromatin antibodies reacted with the (H2A-H2B)-DNA subnucleosome complex, the predominant specificity in patients with procainamide-induced lupus. In thymus organ and reaggregate cultures, PAHA had no effect on negative selection of T cells with high affinity for a co-present antigen, but acted on CD4+ CD8+ immature T cells as they underwent positive selection. TCR transgenic T cells specific to cytochrome c peptide 88-104 acquired the capacity to respond to the low-affinity analogue at position 99 (lys-->ala) if PAHA was present during their development. PAHA also blocked the capacity of a T-cell line to become anergic after anti-CD3 treatment, suggesting that PAHA prevents the production of negative regulators that accumulate in response to partial signaling through the TCR. These results are consistent with the view that T cells acquire self-tolerance during positive selection, and disruption of this process can result in autoreactive T cells and systemic autoimmunity.

The relaxant effect of procainamide, a class Ia antiarrhythmic agent, was examined in bovine tracheal smooth muscle. Procainamide produced concentration-dependent decreases in tension and full relaxation in the preparations contracted with methacholine (0.3 microM). By comparison, in preparations contracted with 40 mM K(+), procainamide had only slight relaxant effects. The relaxant effects of cromakalim and salbutamol on 40 mM K(+)-contracted preparations were significantly (P<0.01) smaller than those on 0.3 microM methacholine-contracted ones. On the other hand, the concentration-response relationships for quinidine, lidocaine, mexiletine and propafenone were not so dramatically different between 0.3 microM methacholine- and 40 mM K(+)-contracted preparations. Tetraethylammonium (300 microM), iberiotoxin (30 nM) and Ba(2+) (1 mM) significantly (P<0.05) attenuated the relaxant effects of procainamide on methacholine-induced contractions, whereas apamin (100 nM), 4-aminopyridine (300 microM), and glibenclamide (10 microM) did not affect them. The inhibitory effect of a combination of iberiotoxin and Ba(2+) was greater than that of iberiotoxin or Ba(2+) alone (P<0.01). These results suggest that the activation of at least two types of K(+) (maxi-K(+) and inward rectifier K(+)) channels contributes to the procainamide-induced relaxation of bovine tracheal smooth muscle.

Procainamide protects mice bearing P388 leukemic cells against the toxicity of cisplatin without diminishing antitumor activity. The mechanism of action of procainamide protection was investigated both in vitro and in vivo. HPLC studies showed that procainamide forms a complex with cisplatin in vitro that has a UV spectrum similar to that of DPR, a triamine platinum complex that contains procaine as ligand. We report here the effect of the reaction product of cisplatin and procainamide on both cisplatin-induced DNA interstrand cross-links (ISCLs) and on the total DNA platination of isolated DNA. Total DNA platination in vitro of isolated DNA was increased by 113% (P <.01) and 17% (P <.05) after incubation times of 1.75 and 6 h, respectively, compared with products from the reaction of cisplatin with water. Furthermore, the reaction product of cisplatin and procainamide was bound to DNA to a significantly greater extent than was cisplatin itself. ISCLs were decreased by 41% when this drug combination was incubated with DNA for 1.75 h, but no changes were observed after incubation for 6 h. We also examined the influence of the time interval between administration of cisplatin and procainamide on normal kidney injury, the renal distribution and urinary excretion of platinum, and the formation of cisplatin-DNA adducts in renal tissue of Sprague-Dawley rats after i.p. administration of 7.5 mg/kg cisplatin either with or without procainamide. The plasma concentrations of urea and creatinine and kidney histology demonstrated that procainamide provided effective protection in vivo in the rat when administered either simultaneously or at 0.5 and 1 h before or after cisplatin. The protection was accompanied by both higher renal levels of platinum and cisplatin-DNA adducts and by an increase in the formation of ISCLs. Moreover, a dose-dependent reduction of urinary excretion and concentration of platinum was also observed. We propose that procainamide, after accumulation in the kidney, may coordinate with cisplatin to form a less toxic DPR-like complex that renders rats less susceptible to cisplatin-induced toxicity.

Background: Patients with inducible sustained ventricular tachycardia (VT) sometimes receive intravenous procainamide during electrophysiologic testing. Unfortunately, the responses to intravenous and subsequent oral drug therapy are variable and may be discordant., Hypothesis: It was the aim of this study to determine whether this variability might be explained by heterogeneity in the electropharmacologic response, even in a homogeneous population., Methods: We studied 42 patients who had spontaneous malignant ventricular arrhythmia and were inducible to sustained monomorphous VT during electrophysiologic testing. Each received 15 mg/kg of intravenous procainamide followed by a 2 mg/min infusion. Serum levels were drawn immediately following programmed stimulation. The mean procainamide level was 6.7 +/- 1.4 mcg/ml with an N-acetyl procainamide level of 1.0 +/- 0.5 mcg/ml. The 14 procainamide responders (5 of whom were noninducible and 9 whose VT cycle length increased > 100 ms) and the 28 nonresponders had similar procainamide and NAPA levels (6.5 +/- 1.4 vs. 6.7 +/- 1.4 mcg/ml)., Results: There was no significant difference in baseline clinical parameters, His to ventricular electrogram (HV) interval, effective refractory period, or VT cycle length. Prolongation of the effective refractory period and infra His conduction time occurred to a similar extent in responders and nonresponders., Conclusion: We conclude that procainamide has a consistent dose-response relationship with respect to refractoriness and conduction in patients with malignant arrhythmias. However, acute antiarrhythmic efficacy of procainamide cannot be predicted by clinical factors, drug levels, or drug-induced changes in common electrophysiologic parameters.

Benzamides have been in clinical use for many years in treatment against various disorders. A recent application is that as a sensitizer for radio- or chemotherapies. We have here analysed the mechanism of action of N-substituted benzamides using an in vitro system. We found that while procainamide was biologically inert in our system, the addition of a chloride in the 3' position of the benzamide ring created a compound (declopramide) that induced rapid apoptosis. Furthermore, declopramide also inhibited NFkappaB activation by inhibition of IkappaBbeta breakdown. An acetylated variant of declopramide, N-acetyl declopramide, showed no effect with regard to rapid apoptosis induction but was a potent inhibitor of NFkappaB activation. In fact, the addition of an acetyl group to procainamide in the 4' position was sufficient to convert this biologically inactive substance to a potent inhibitor of NFkappaB activation. These findings suggest two potential mechanisms, induction of early apoptosis and inhibition of NFkappaB mediated salvage from apoptosis, for the biological effect of N-substituted benzamides as radio- and chemo-sensitizers. In addition it suggests that N-substituted benzamides are potential candidates for the development of anti-inflammatory compounds using NFkappaB as a drug target.

Background: Ventricular fibrillation (VF) is maintained by 2 mechanisms: first by reentry formation and second by spontaneous wave break or wave splitting. We hypothesized that spontaneous wave break results from a critical shortening of the action potential duration (APD) during VF and that its prevention by procainamide eliminates spontaneous wave break., Methods and Results: The endocardial surfaces of 7 isolated, perfused swine right ventricles were mapped with a 3.2x3.8 cm plaque with 477 bipolar electrodes. Activation pattern during VF was visualized dynamically while simultaneously recording epicardial action potentials with a glass microelectrode. APD restitution curves were constructed during VF (dynamic) and during S(1)S(2) protocols. At baseline, VF was maintained by 5.3+/-1 wavelets. Procainamide (PA) at 10 microgram/mL decreased the number of wavelets to 3.5+/-1 (P<0.05). At baseline VF was maintained by spontaneous wave break and by new reentrant wave front formation. PA eliminated spontaneous wave break during VF while having no effect on reentry formation. PA increased the cycle length of the VF (148.5+/-41.2 ms vs 81+/-10 ms, P<0.01) and the core area of the reentry from 5.8 to 14.5 mm(2) (P<0.05). Dynamic APD restitution curve during VF showed that PA eliminated the initiation of activation with APDs shorter than 30 ms. The effects of PA on cellular properties and wave front dynamics were reversed during 60 minutes of drug-free perfusion., Conclusions: Critically short APDs during VF promote spontaneous wave break. Their elimination with PA, however, maintains VF by generating new reentrant wave front.

Lidocaine, procainamide and quinidine binding to human placenta was investigated in vitro. Pooled whole human placental homogenate supplemented with either non-radiolabelled lidocaine, procainamide or quinidine over the concentration range 50-5,000 x 10(-7) mol/L was submitted to equilibrium dialysis against phosphate buffer, pH 7.4, 0.1 mol/L. Post-dialysis drug concentrations were measured by enzyme immunoassay. Data were analyzed by the method of Scatchard. No binding to placenta was noted for either lidocaine or procainamide. In contrast, up to 22 percent of quinidine was bound. Two binders were defined as follows: #1 (Ka, 7.37 x 10(5) L/mol; Bo, 1.55 x 10(-7) mol/L) and #2 (Ka, 7.11 x 10(4) L/mol; Bo, 4.05 x 10(-6) mol/L). The concentrations of quinidine binding sites in moles per gram of placenta were 1.55 x 10(-9) and 4.05 x 10(-8), respectively. These data suggest that quinidine may be accumulated in human placenta.