Synthesis and Comparative Evaluation of Novel Cationic Amphiphile C12-Man-Q as an Efficient DNA Delivery Agent In Vitro.

New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25⁻80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line.

Substituent effects on desferrithiocin and desferrithiocin analogue iron-clearing and toxicity profiles.

Desferrithiocin (DFT, 1) is a very efficient iron chelator when given orally. However, it is severely nephrotoxic. Structure-activity studies with 1 demonstrated that removal of the aromatic nitrogen to provide desazadesferrithiocin (DADFT, 2) and introduction of either a hydroxyl group or a polyether fragment onto the aromatic ring resulted in orally active iron chelators that were much less toxic than 1. The purpose of the current study was to determine if a comparable reduction in renal toxicity could be achieved by performing the same structural manipulations on 1 itself. Accordingly, three DFT analogues were synthesized. The iron-clearing efficiency and ferrokinetics were evaluated in rats and primates; toxicity assessments were carried out in rodents. The resulting DFT ligands demonstrated a reduction in toxicity that was equivalent to that of the DADFT analogues and presented with excellent iron-clearing properties.

Ligand-based design, in silico ADME-Tox filtering, synthesis and biological evaluation to discover new soluble 1,4-DHP-based CFTR activators.

The altered gating of the mutant CFTR chloride channel cystic fibrosis (CF) may be corrected by small molecules called potentiators. We present a molecular scale simulation system for the discovery of ΔF508-CFTR soluble potentiators. Results report the design, ADME-Tox prediction, synthesis, solubility determination and in vitro biological evaluation of two 1,4-dihydropyridines (DHPs). Compound 1 shows a promising ADME-Tox profile and good potency.

Dihydropyridines and multidrug resistance: previous attempts, present state, and future trends.

Multidrug resistance is defined as the resistance of a tumor cell to the cytotoxic action of divergent drugs used in chemotherapy. Dihydropyridines are a class of calcium channel antagonists that were discovered to have a multidrug resistance reversing effect and prompted investigations resulting in the synthesis of hundreds of new derivatives. Most of the investigators tried to achieve two goals: a decrease in Ca²(+) channel-blocking activity and an increase in the multidrug resistance reversing effect. Most of the synthesized compounds failed in the later stages of studies especially in clinical trials because of pharmacokinetic or pharmacodynamic limitations. Therefore, it will be necessary to include new methods, such as combinatorial synthesis, and, more importantly, to apply computational methods based on global structure-activity relationship models that consider all problems. Moreover, some compounds should be synthesized that are effective on several multidrug resistance targets.

SAMe prevents the up regulation of toll-like receptor signaling in Mallory-Denk body forming hepatocytes.

Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug-primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the downstream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-gamma receptors by DDC feeding. INFgamma stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described.

The impact of polyether chain length on the iron clearing efficiency and physiochemical properties of desferrithiocin analogues.

(S)-2-(2,4-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (2) was abandoned in clinical trials as an iron chelator for the treatment of iron overload disease because of its nephrotoxicity. However, subsequent investigations revealed that replacing the 4'-(HO) of 2 with a 3,6,9-trioxadecyloxy group, ligand 4, increased iron clearing efficiency (ICE) and ameliorated the renal toxicity of 2. This compelled a closer look at additional polyether analogues, the subject of this work. The 3,6,9,12-tetraoxatridecyloxy analogue of 4, chelator 5, an oil, had twice the ICE in rodents of 4, although its ICE in primates was reduced relative to 4. The corresponding 3,6-dioxaheptyloxy analogue of 2, 6 (a crystalline solid), had high ICEs in both the rodent and primate models. It significantly decorporated hepatic, renal, and cardiac iron, with no obvious histopathologies. These findings suggest that polyether chain length has a profound effect on ICE, tissue iron decorporation, and ligand physiochemical properties.

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome.

Design and synthesis of new symmetrical derivatives of dihydropyridine containing a pyridyl group on the 3, 5-positions and evaluation of their cytotoxic and multidrug resistance reversal activity.

Today, chemotherapy is an important part in the treatment of several kinds of cancer; however, the development of drug resistance remains one of the major obstacles in successful chemotherapy. Several types of agents have been recognized as multidrug resistance (MDR) inhibitors, among which the 1,4-dihydropyridines (DHPs) have been investigated the most. P-glycoprotein inhibition has been reported as the main MDR reversal mechanism of DHPs, whilst other mechanisms such as inhibition of topoisomerase II have received less attention. Therefore, in this study new derivatives of DHP have been synthesized. Their cytotoxic activity and their effects in reversing atypical MDR have been evaluated. The results confirmed the appropriate effect of these compounds on atypical MDR. Although it was observed that these compounds had a moderate cytotoxic effect, the cytotoxicity of one compound on the K562 cell line (IC50 = 6.61 microM) was comparable with that of doxorubicin (IC50 = 4.17 microM). Finally, the Ca(2+)-channel antagonistic activity, an undesired effect for these compounds, was evaluated.

Fat10 is an epigenetic marker for liver preneoplasia in a drug-primed mouse model of tumorigenesis.

There is clinical evidence that chronic liver diseases in which MDBs (Mallory Denk Bodies) form progress to hepatocellular carcinoma. The present study provides evidence that links MDB formation induced by chronic drug injury, with preneoplasia and later to the formation of tumors, which develop long after drug withdrawal. Evidence indicated that this link was due to an epigenetic cellular memory induced by chronic drug ingestion. Microarray analysis showed that the expressions of many markers of preneoplasia (UBD, Alpha Fetoprotein, KLF6 and glutathione-S-transferase mu2) were increased together when the drug DDC was refed. These changes were suppressed by S-adenosylmethionine feeding, indicating that the drug was affecting DNA and histones methylation in an epigenetic manner. The link between MDB formation and neoplasia formation was likely due to the over expression of UBD (also called FAT10), which is up regulated in 90% of human hepatocellular carcinomas. Immunohistochemical staining of drug-primed mouse livers showed that FAT10 positive liver cells persisted up to 4 months after drug withdrawal and they were still found in the livers of mice, 14 months after drug withdrawal. The refeeding of DDC increased the percent of FAT10 hepatocytes.

Tumour-specific cytotoxicity and MDR-reversal activity of dihydropyridines.

The ability of 41 1,4-diphenyl-1,4-dihydropyridine derivatives to inhibit the transport activity of P-glycoprotein were studied by flow cytometry in a multidrug-resistant human colon cancer cell line (COLO320) and in human mdr1 gene-transfected mouse lymphoma cells (L 5178 Y). The cytotoxicities of these compounds were also examined against human normal and cancer cell lines. The majority of the tested compounds proved to be effective inhibitors of rhodamine 123 outward transport, but their cytotoxicities were not negligible. Some dihydropyridine derivatives displayed cytotoxic activity against four human oral tumour cell lines and against three normal human oral cell lines. There was no clear-cut relationship between the multidrug-resistance activity or cytotoxicity and the chemical structures of the compounds. New ring substituents could prevent the oxidation of the ring of the aromatic compound.

Biological evaluation of bishydroxymethyl-substituted cage dimeric 1,4-dihydropyridines as a novel class of p-glycoprotein modulating agents in cancer cells.

A series of N-substituted cage dimeric 1,4-dihydropyridines 3a-e was evaluated as inhibitors of membrane efflux pump P-glycoprotein (P-gp) in multidrug resistant (mdr) cancer cells. Structure-activity relationships (SAR) and cytotoxic properties are discussed. Effective concentrations for overcoming mdr have been demonstrated in competition studies with the P-gp substrate epirubicin.

New 4-aryl-1,4-dihydropyridines and 4-arylpyridines as P-glycoprotein inhibitors.

Efflux of cytotoxic agents mediated by P-glycoprotein is believed to be an important mechanism of multidrug resistance, which remains a serious limitation to successful chemotherapy in cancers such as metastatic breast cancer. A series of 4-aryl-1,4-dihydropyridines and corresponding aromatized 4-arylpyridines have been synthesized based on structure modifications of niguldipine to enhance multidrug resistance reversal activity, while minimizing calcium channel binding. Thirty new compounds were characterized. [(3)H]Vinblastine accumulation studies indicated that at a concentration level of 3 muM, 15 of 18 4-aryl-1,4-dihydropyridines and all 4-arylpyridines can successfully restore intracellular accumulation of vinblastine in a resistant human breast adenocarcinoma cell line, MCF-7/adr, which overexpresses P-glycoprotein. The most potent compounds led to an approximately 15-fold increase of vinblastine accumulation. All of the test compounds that significantly increased vinblastine accumulation in MCF/adr cells were able to substantially reduce IC(50) values of daunomycin and increase its cytotoxicity in MCF-7/adr-resistant cells, confirming the results of the vinblastine accumulation studies. Calcium channel binding assays for these newly synthesized compounds were conducted using rat cerebral cortex membrane. All but eight compounds demonstrated negligible calcium channel binding over the concentration range from 15 to 2500 nM. The results demonstrate that the newly synthesized series of 1,4-dihydropyridines and pyridines represent P-glycoprotein modulators with negligible calcium channel blocking activity.

Development of tridentate iron chelators: from desferrithiocin to ICL670.

Successful treatment of beta-thalassemia requires two key elements: blood transfusion and iron chelation. Regular blood transfusions considerably expand the lifespan of patients, however, without the removal of the consequential accumulation of body iron, few patients live beyond their second decade. In 1963, the introduction of desferrioxamine (DFO), a hexadentate chelator, marked a breakthrough in the treatment of beta-thalassemia. DFO significantly reduces body iron burden and iron-related morbidity and mortality. DFO is still the only drug for general use in the treatment of transfusion dependent iron overload. However, its very short plasma half-life and poor oral activity necessitate special modes of application (subcutaneous or intravenous infusion) which are inconvenient, can cause local reactions and are difficult to be accepted by many patients. Over the past four decades, many different laboratories have invested major efforts in the identification of orally active iron chelators from several hundreds of molecules of synthetic, microbial or plant origin. The discovery of ferrithiocin in 1980, followed by the synthesis of the tridentate chelator desferrithiocin and proof of its oral activity raised a lot of hope. However, the compound proved to be toxic in animals. Over a period of about fifteen years many desferrithiocin derivatives and molecules with broader alterations led to the discovery of numerous new compounds some of which were much better tolerated and were more efficacious than desferrithiocin in animals, however, none was safe enough to proceed to the clinical use. The discovery of a new chemical class of iron chelators: The bis-hydroxyphenyltriazoles re-energized the search for a safe tridentate chelator. The basic structure of this completely new chemical class of iron chelators was discovered by a combination of rational design, intuition and experience. More than forty derivatives of the triazole series were synthesized at Novartis. These compounds were evaluated, together with more than 700 chelators from various chemical classes. Using vigorous selection criteria with a focus on tolerability, the tridentate chelator 4-[(3,5-Bis-(2-hydroxyphenyl)-1,2,4)triazol-1-yl]-benzoic acid (ICL670) emerged as an entity which best combined high oral potency and tolerability in animals. ICL670 is presently being evaluated in the clinic.

Desferrithiocin is a more potent antineoplastic agent than desferrioxamine.

Desferrithiocin (DFT) is an orally effective Fe chelator, with a similar high affinity and selectivity for Fe to desferrioxamine (DFO), which has been shown clinically to possess antineoplastic activity. In this study, DFT was assessed for antineoplastic potential in hepatocellular carcinoma cell lines (HCC). This was done as there are few treatments for this aggressive neoplasm. The effects of DFT on cell proliferation, cell cycle progression, Fe uptake and toxicity were examined. To establish whether DFT was selective for cancer cells a comparison was made with normal (non-proliferating) hepatocytes and non-tumorigenic (proliferating) fibroblasts (SWISS-3T3). DFT was a potent inhibitor of HCC proliferation (IC(50) approximately 40 microM). DFO also inhibited HCC proliferation under the same conditions, but was much less active (IC(50)=110 - 210 microM). When saturated with Fe, the activity of DFT, like DFO, was greatly diminished, suggesting it may act by depriving the cells of Fe or inactivating essential Fe pool(s). Indeed DFT rapidly decreased Fe uptake from Tf-(59)Fe by hepatoma cells and also by normal hepatocytes. However, DFT (and DFO) had much less effect on cell survival in hepatocytes and fibroblasts than in hepatoma cells. DFT may, like DFO, inhibit the cell cycle in the S phase of DNA synthesis. Both chelators showed low toxicity. These results indicate that DFT has potent antineoplastic activity in HCC. Further investigation into the DFT class of Fe chelators seems warranted, particularly in view of its high activity in relation to DFO, a chelator which is already in clinical trial for neuroblastoma.

The desferrithiocin (DFT) class of iron chelators: potential as antineoplastic agents.

Iron (Fe) is essential for the proliferation of cancer cells. A subgroup of the orthosubstituted phenolate class of Fe chelators, desferrithiocin [2-(3'-hydroxypyrid-2'-yl)4-methyl-delta2-thiazoline-4(S)-carboxylic acid; DFT] and its analogues, have potential application in short-term chemotherapy for cancer by Fe deprivation. Their effects on cell proliferation, cell cycle progression, Fe uptake and toxicity were therefore examined in adult and fetal rat and human hepatocellular carcinoma (HCC) cell lines, as well as in normal cells. DFT was more active than desferrioxamine, in clinical trials as an antineoplastic agent, consistently inhibiting cell proliferation in all cell lines (IC50 = 40 microM). 2-(2'-hydroxyphenyl-2'-yl)-delta2-thiazoline-4(S)-carboxylic acid was the most active analogue (IC50 = 55-90 microM). Inhibition was affected by chelator concentration and ability to prevent Fe uptake. The fetal-cell-derived HCC was more susceptible than adult HCC. Structure-activity studies revealed that thiazol methyl deletion greatly diminished antiproliferative activity of the chelators but stereochemical orientation of COOH around C4 had no effect. Removal of the N from the pyridine ring restored antiproliferative activity. Chelators inhibited DNA synthesis in the S phase. The chelators at their IC50 concentration had little or no effect on Fe uptake in normal cells. This apparent selectivity of these chelators for cancer cells, coupled with their high activity, suggests that further investigation is warranted.

Dexamethasone enhances mallory body formation in drug-primed mouse liver.

In a clinical study in which patients with alcoholic hepatitis were treated with prednisone for 1 month, posttreatment liver biopsies showed diminished inflammation, but Mallory bodies were not diminished. This suggested that steroid treatment may reduce inflammation by inhibiting NFkappaB activation. Sparing of Mallory bodies suggests that NFkappaB activation may not be involved mechanistically in Mallory body formation. To test this idea, we induced Mallory body formation in drug-primed mice with or without dexamethasone treatment. As predicted, dexamethasone decreased NFkappaB activation; however, Mallory body formation was increased. Surprisingly, TNFalpha and iNOS, which normally increase as a result of NFkappaB activation, were upregulated by the dexamethasone treatment. It was concluded that NFkappaB activation is not involved in Mallory body formation. Despite this, induced increases in TNFalpha, iNOS, c-jun/API and c-myc expression indicate that oxidative stress is likely involved in Mallory body formation.

Induction of Leydig cell tumors by lacidipine via up-regulation of the LHRH receptor on Leydig cells in rats.

Long-term (78 weeks) administration of lacidipine, a dihydropyridine calcium antagonist, increased the incidence of Leydig cell tumors (LCTs) in Sprague-Dawley rats. Lacidipine also increased and decreased the plasma luteinizing hormone (LH) and testosterone levels, respectively. Leydig cells from lacidipine-treated rats showed increases in luteinizing hormone-releasing hormone (LHRH) receptor expression, protein kinase C (PKC) activity, expression of proto-oncogenes, and 5-bromodeoxyridine uptake; whereas their calcium level, LH receptors, and testosterone content decreased. These data suggest that LHRH receptors play an important role in the development of rat LCTs induced by lacidipine, which activates a cascade of cell cycle-regulatory genes via PKC. When isolated Leydig cells were cultured with lacidipine or nicardipine, these changes in rat Leydig cells were not demonstrable in mice and monkeys, species having many fewer testicular LHRH receptors than rats. Thus, lacidipine may pharmacologically induce LCTs in rats but not in mice, with the difference depending on the presence or absence of testicular LHRH receptors. The induction of LCTs by lacidipine in rats is unlikely to occur in humans, since their Leydig cells lack LHRH receptors.

In vivo reversibility of multidrug resistance by the MDR-modulator dexniguldipine (niguldipine derivative B859-35) and by verapamil.

The newly synthesized dihydropyridine derivative B859-35 was previously shown in vitro to be highly effective in reversing multidrug resistance (MDR) of P-glycoprotein positive tumor cell lines, such as the adriamycin (ADR) resistant erythroleukemia F4-6RADR cells. In the current study B859-35 was investigated for its efficiency in reversing MDR in an in vivo tumor model for preclinical testing of MDR-modulators. F4-6RADR cells were injected into the right flank of nude mice while the parent cells were injected into the left flank. The animals were treated i.p. with ADR (9.0 mg/kg body weight) combined with B859-35 (5, 10, or 25 mg/kg) or, for comparison and validation, with verapamil (VRP) (75 mg/kg). The effects of ADR and the MDR-modulator combination were evaluated by histological morphometry of the tumors. While ADR alone was shown to be ineffective in resistant cells, the combinations of ADR + B859-35 as well as of ADR + VRP were highly active in reducing the number of viable cells in the resistant tumor nodule by 67 +/- 9% or by 53 +/- 11% of controls. This model provides evidence that even in vivo, MDR modulators can be effective in reversing drug resistance. In addition, it presents a potentially useful and rapid preclinical system for in vivo studies on the modification of drug resistance.

Tolerance, safety, and kinetics of the new antineoplastic compound dexniguldipine-HCl after oral administration: a phase I dose-escalation trial.

Dexniguldipine-HCl is a new dihydropyridine compound that exerts selective antiproliferative activity in a variety of tumor models and, in addition, has a high potency in overcoming multidrug resistance. The purpose of this trial was to determine the toxicity and pharmacokinetics of dexniguldipine and to establish a recommended dose for phase II trials. A total of 37 patients with cancer were treated with oral dexniguldipine in increasing doses for up to 7 days. The main parameters evaluated were subjective tolerance and laboratory and cardiovascular parameters (blood pressure and ECG). Blood samples were drawn for analysis of the drug's pharmacokinetics. Dizziness and nausea were the major adverse events observed in seven patients, but episodes were generally mild and not clearly dose-related. Vomiting occurred in one patient. Hypotensive effects and orthostatic dysregulation were observed in some patients but were not considered to be dose-limiting. Therefore, no dose-limiting toxicity was found and the maximally tolerable dose could not be determined. Pharmacokinetic data showed wide interindividual variation and a dose-dependent increase in steady-state serum concentrations at doses of up to 1,000 mg daily, with no clear further increase being observed at higher doses. Consistently high concentrations were achieved with the 2,500-mg dose. Despite the lack of dose-limiting toxicity, higher doses of dexniguldipine do not appear to be useful for clinical evaluation because of the pharmacokinetic properties of the compound: therefore, 2,500 mg/day is recommended as the daily dose for phase II trials.