Combined Poziotinib with Manidipine Treatment Suppresses Ovarian Cancer Stem-Cell Proliferation and Stemness.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women worldwide, with an overall 5 year survival rate below 30%. The low survival rate is associated with the persistence of cancer stem cells (CSCs) after chemotherapy. Therefore, CSC-targeting strategies are required for successful EOC treatment. Pan-human epidermal growth factor receptor 4 (HER4) and L-type calcium channels are highly expressed in ovarian CSCs, and treatment with the pan-HER inhibitor poziotinib or calcium channel blockers (CCBs) selectively inhibits the growth of ovarian CSCs via distinct molecular mechanisms. In this study, we tested the hypothesis that combination treatment with poziotinib and CCBs can synergistically inhibit the growth of ovarian CSCs. Combined treatment with poziotinib and manidipine (an L-type CCB) synergistically suppressed ovarian CSC sphere formation and viability compared with either drug alone. Moreover, combination treatment synergistically reduced the expression of stemness markers, including CD133, KLF4, and NANOG, and stemness-related signaling molecules, such as phospho-STAT5, phospho-AKT, phospho-ERK, and Wnt/ß-catenin. Moreover, poziotinib with manidipine dramatically induced apoptosis in ovarian CSCs. Our results suggest that the combinatorial use of poziotinib with a CCB can effectively inhibit ovarian CSC survival and function.

Screening of FDA-Approved Drugs for Inhibitors of Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV), an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca2+ channel (VGCC) inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.

Calcium channel blockade blunts the renal effects of acute nitric oxide synthase inhibition in healthy humans.

Our aim was to investigate whether blockade of calcium channels (CCs) or angiotensin II type 1 receptors (AT1R) modulates renal responses to nitric oxide synthesis inhibition (NOSI) in humans. Fourteen sodium-replete, healthy volunteers underwent 90-min infusions of 3.0 µg·kg-1·min-1 NG-nitro-l-arginine methyl ester (l-NAME) on 3 occasions, preceded by 3 days of either placebo (PL), 10 mg of manidipine (MANI), or 50 mg of losartan (LOS). At each phase, mean arterial pressure (MAP), glomerular filtration rate (GFR; inulin), renal blood flow (RBF; p-aminohippurate), urinary sodium (UNaV), and 8-isoprostane (U8-iso-PGF2αV; an oxidative stress marker) were measured. With PL + l -NAME, the following changes were observed: +6% MAP (P < 0.005 vs. baseline), -10% GFR, -20% RBF, -49% UNaV (P < 0.001), and +120% U8-iso-PGF2αV (P < 0.01). In contrast, MAP did not increase during LOS + l-NAME or MANI + l-NAME (P > 0.05 vs. baseline), whereas renal changes were the same during LOS + l-NAME vs. PL + l-NAME (ANOVA, P > 0.05). However, during MANI + l-NAME, changes vs. baseline in GFR (-6%), RBF (-12%), and UNaV (-34%) were blunted vs. PL + l-NAME and LOS + l-NAME (P < 0.005), and the rise in U8-iso-PGF2αV was almost abolished (+37%, P > 0.05 vs. baseline; P < 0.01 vs. PL + l-NAME or LOS + l-NAME). We conclude that, since MANI blunted l-NAME-induced renal hemodynamic changes, CCs participate in the renal responses to NOSI in healthy, sodium-replete humans independent of changes in MAP and without the apparent contribution of the AT1R. Because the rise in U8-iso-PGF2αV was essentially prevented during MANI + l-NAME, CC blockade may oppose the renal effects of NOSI in part by counteracting oxidative stress responses to acutely impaired renal NO bioavailability.

Enantioselective determination of R-(-)-manidipine and S-(+)-manidipine in human plasma by a sensitive and selective chiral LC-MS/MS assay.

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and validated for the enantioselective determination of manidipine in human plasma using isotope-labeled compounds as internal standards. After solid-phase extraction, R-(-)-manidipine and S-(+)-manidipine were chromatographed on a Chiralpack IC-3 C18 column using a isocratic mobile phase composed of 2 mm ammonium bicarbonate and acetonitrile (15:85, v/v). The precursor ion to product ion transitions for the enantiomers and internal standards were monitored in the multiple reaction monitoring and positive ionization mode using an API-4000 mass spectrometer. The method was linear over the concentration range of 0.05-10.2 ng/mL for both enantiomers. The precision and accuracy results over five concentration levels in five different batches were well within the acceptance limits. The mean extraction recovery was >80% for both enantiomers. A variety of stability tests were executed in plasma and in neat samples, which complies with the FDA guidelines. After complete validation, the method was successfully applied to a pharmacokinetic study of a manidipine 20 mg oral dose in 10 healthy South India subjects under fasting conditions. The assay reproducibility is shown through incurred samples reanalysis of 20 subject plasma samples.

Improved stability of solid dispersions of manidipine with polyethylene glycol 4000/copovidone blends: application of ternary phase diagram.

CONTEXT: Manidipine (MDP) is generally used clinically as an antihypertensive agent; however, the bioavailability of orally administered MDP is limited due to their very low water solubility. OBJECTIVE: The objectives of this research were, therefore, to increase the solubility of MDP by the formation of ternary solid dispersions (tSD) with polyethylene glycol 4000 (PEG4000) and copovidone and to improve their stability. METHODS: Solid ternary phase diagram was constructed to find homogeneous solid dispersion region after melting and solidifying at low temperature with different quenching substances. The pulverized powder of solid dispersions was then determined, for their physicochemical properties, by differential scanning calorimetry, powder X-ray diffractometry, Fourier transform infrared (FTIR) spectroscopy and hot stage microscopy. The solubility and dissolution of MDP from the tSD were investigated. The physical stability of tSD was also determined under accelerated condition at 40 °C/75% relative humidity (RH) for 6 months. RESULTS AND DISCUSSION: The results showed that MDP was molecularly dispersed in PEG4000 and copovidone when the tSD was created from homogeneous region of solid ternary phase diagram. FTIR results confirmed that strong hydrogen bonding was presented between MDP and copovidone, leading to a significant increase in the solubility and dissolution of MDP. After storage at accelerated condition (40 °C/75%RH) for 6 months, the tSD still showed a good appearance and high solubility. CONCLUSION: The results of this study suggest that tSD prepared by melting has promising potential for oral administration and may be an efficacious approach for improving the therapeutic potential of MDP.

Enhancement of solubility and oral bioavailability of manidipine by formation of ternary solid dispersion with d-α-tocopherol polyethylene glycol 1000 succinate and copovidone.

CONTEXT: Low bioavailability of oral manidipine (MDP) is due to its low water solubility. OBJECTIVE: The objective of this study was to increase the solubility and bioavailability of MDP by fabricating ternary solid dispersion (tSD) with d-α-tocopherol polyethyleneglycol-1000-succinate and copovidone. METHODS: In this study, solid ternary phase diagram was applied in order to check the homogeneity of tSD prepared by melting and solidifying with dry ice. The physicochemical properties of different formulations were determined by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR) and hot stage microscopy. Their solubility, dissolution, stability and bioavailability were also investigated. RESULTS AND DISCUSSION: The results demonstrated that tSD obtained from ternary phase diagram divided into homogeneous and non-homogeneous regions. In the homogenous region, the transparent characteristics of tSD was observed and considered as a glass solution, which have a higher MDP solubility than that in non-homogenous region. The hot stage microscopy, DSC and PXRD confirmed that solid dispersion was formed in which MDP was molecularly dispersed in the carriers, especially in the homogenous region of phase diagram. FTIR analysis demonstrated strong hydrogen bonding between amine groups of MDP and carbonyl groups of copovidone, which supported a higher solubility and dissolution of tSD. The pharmacokinetic study in Wistar rats showed that the tSD had the greatest effect on oral bioavailability. Immediate hypotensive effect of tSD was also observed in vivo. CONCLUSIONS: The improvement of stability, dissolution and oral bioavailability of MDP could be achieved by using tSD technique.

Photostability evaluation of manidipine tablets and structural determination of its photoproducts.

Manidipine (MP) is a dihydropyridine drug, which is treated for the reduction of high blood pressure. The aim of this study is to clarify the photochemical behavior of MP in the case of ultraviolet light (UV) irradiation for MP tablets (Calslot® tablets). The tablets and its altered forms (powders and suspensions) were UV-irradiated using a black light, and residual amounts of active pharmaceutical ingredients (APIs) were monitored by high-performance liquid chromatography (HPLC). Due to the photoproducts of MP were detected in HPLC chromatograms, the elucidation of their chemical structures was carried out utilizing electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). As a result, APIs in Calslot® tablets were almost completely photodegraded in the case that Calslot® tablets were suspended in an aqueous media along with the generation of some MP photoproducts. LC-ESI-MS/MS analysis clarified the chemical structures of three MP photoproducts, indicating that they were a pyridine analogue, benzophenone and a hydrolysate. Benzophenone was a main MP photoproduct. It was possible that MP might be firstly oxidized to form its pyridine analogue, followed by the oxidation of a dimethyl methylene moiety. This moiety seemed to be eliminated as a benzophenone, and the cleavage of an ester bond of the residual moiety resulted in the generation of a hydrolysate. Finally, toxicological potencies of MP and its photoproducts were predicted in silico toxicity evaluation, suggesting some of biological effects of the photoproducts might be altered compared with MP.

Repurposing drugs with specific activity against L-form bacteria.

Cell wall deficient "L- form" bacteria are of growing medical interest as a possible source of recurrent or persistent infection, largely because of their complete resistance to cell wall active antibiotics such as ß-lactams. Antibiotics that specifically kill L-forms would be of potential interest as therapeutics, but also as reagents with which to explore the role of L-forms in models of recurrent infection. To look for specific anti-L-form antibiotics, we screened a library of several hundred FDA-approved drugs and identified compounds highly selective for L-form killing. Among the compounds identified were representatives of two different classes of calcium channel blockers: dihydropyridines, e.g., manidipine; and diphenylmethylpiperazine, e.g., flunarizine. Mode of action studies suggested that both classes of compound work by decreasing membrane fluidity. This leads to a previously recognized phenotype of L-forms in which the cells can continue to enlarge but fail to divide. We identified a considerable degree of variation in the activity of different representatives of the two classes of compounds, suggesting that it may be possible to modify them for use as drugs for L-form-dependent infections.

Bioenergetics impairment of Trypanosoma cruzi by the antihypertensive manidipine: A drug repurposing strategy.

Considering the lack of effective and safe therapy for the treatment of Chagas disease, the antihypertensive drug manidipine (MDP) was in vitro evaluated against Trypanosoma cruzi. The bioenergetics of trypomastigotes was studied in the presence of the drug using fluorimetric and luminescent assays. Manidipine showed a potent antiparasitic activity, with IC50 values of 0.1 µM (intracellular amastigotes) and 3 µM (trypomastigotes), resulting in a promising selectivity index against the amastigotes (>1459). Using fluorimetric analysis, the drug showed depolarisation of the electric potential of the plasma membrane with no alteration of the permeability. A decrease in ATP levels suggested a bioenergetic alteration of the mitochondria, which was confirmed by the depolarisation of the mitochondrial membrane potential and a slight increase of the ROS levels. This is the first study to show the promising in vitro effectiveness of the antihypertensive MDP against T. cruzi, which may represent a candidate for future investigations in animal models.

Screening and Identification of Lujo Virus Entry Inhibitors From an Food and Drug Administration-Approved Drugs Library.

Lujo virus (LUJV) belongs to the Old World (OW) genus Mammarenavirus (family Arenaviridae). It is categorized as a biosafety level (BSL) 4 agent. Currently, there are no U.S. Food and Drug Administration (FDA)-approved drugs or vaccines specifically for LUJV or other pathogenic OW mammarenaviruses. Here, a high-throughput screening of an FDA-approved drug library was conducted using pseudotype viruses bearing LUJV envelope glycoprotein (GPC) to identify inhibitors of LUJV entry. Three hit compounds, trametinib, manidipine, and lercanidipine, were identified as LUJV entry inhibitors in the micromolar range. Mechanistic studies revealed that trametinib inhibited LUJV GPC-mediated membrane fusion by targeting C410 [located in the transmembrane (TM) domain], while manidipine and lercanidipine inhibited LUJV entry by acting as calcium channel blockers. Meanwhile, all three hits extended their antiviral spectra to the entry of other pathogenic mammarenaviruses. Furthermore, all three could inhibit the authentic prototype mammarenavirus, lymphocytic choriomeningitis virus (LCMV), and could prevent infection at the micromolar level. This study shows that trametinib, manidipine, and lercanidipine are candidates for LUJV therapy and highlights the critical role of calcium in LUJV infection. The presented findings reinforce the notion that the key residue(s) located in the TM domain of GPC provide an entry-targeted platform for designing mammarenavirus inhibitors.

Calcium Channel Blocker-Associated Chyloperitoneum in Patients Receiving Peritoneal Dialysis: A Systematic Review.

Unlike chyloperitoneum associated with clinical conditions including cancer, cirrhosis, and traumatic surgery, calcium channel blocker (CCB)-associated chyloperitoneum is rarely discussed in comprehensive studies on chyloperitoneum. We aimed to investigate the prevalence and characteristics of CCB-associated chyloperitoneum in peritoneal dialysis (PD) patients. The MEDLINE, Embase, CENTRAL, CiNii, and RISS databases were systematically searched for clinical studies on CCB-associated chyloperitoneum in PD patients published up to 31 July 2018. A total of 17 studies (four cohort studies, one case series, and 12 case reports) were selected. Eight CCBs, namely amlodipine, benidipine, diltiazem, lercanidipine, manidipine, nifedipine, nisoldipine, and verapamil, were reported to be associated with chyloperitoneum; manidipine and lercanidipine were the most frequently reported. The average prevalence of chyloperitoneum for lercanidipine was 25.97% in three cohort studies, two of which had a moderate or high risk of bias. Most of the studies revealed chyloperitoneum development within 4 days of initiation of CCB therapy and chyloperitoneum disappearance within 24 h of CCB withdrawal. The results of this study emphasise on the need for awareness among healthcare professionals regarding CCB-associated chyloperitoneum in PD patients. Further studies elucidating the causality and clinical implication of CCB-associated chyloperitoneum are needed.

Repurposing the clinically approved calcium antagonist manidipine dihydrochloride as a new early inhibitor of human cytomegalovirus targeting the Immediate-Early 2 (IE2) protein.

Currently, there are no therapeutic alternatives to DNA polymerase inhibitors to treat human cytomegalovirus (HCMV) infections, a major threat for immunocompromised patients and pregnant women. Here, we explored the potential to repurpose manidipine dihydrochloride (MND), a calcium antagonist clinically approved to treat hypertension, as a new anti-HCMV agent. MND emerged in a previous drug repurposing screen to find early inhibitors of HCMV replication, and now we confirm that it inhibits in the low micromolar range the replication of different HCMV strains, including clinical isolates and viruses resistant to approved DNA polymerase inhibitors. The antiviral activity of MND is specific for HCMV over different both DNA and RNA viruses. Further experiments in HCMV-infected cells testing the effects of MND on viral DNA synthesis and viral proteins expression revealed that it halts the progression of the virus cycle prior to viral DNA replication and E genes expression, but after IE proteins expression. According to these results, we observed that the overall antiviral activity of MND involves a specific interference with the transactivating functions of the viral Immediate-Early 2 (IE-2) protein, an essential viral transcription factor required for the progression of HCMV replication. Given that the inhibitory concentration against HCMV is in the range of clinically relevant concentrations of MND in humans, and the mechanism of action differs from that of the other available therapeutics, this already approved drug is an attractive candidate for repurposing in alternative anti-HCMV therapeutic protocols.

Manidipine: an antihypertensive drug with positive effects on metabolic parameters and adrenergic tone in patients with diabetes.

Antihypertensive treatment of patients with diabetes should include those drugs with a positive effect on metabolic parameters. Most patients with diabetes require at least two antihypertensive agents. Combining a dihydropyridine calcium channel blocker with a renin-angiotensin-aldosterone system inhibitor is a rational approach. However, not all dihydropyridines are equal with respect to their effects on metabolic parameters. Thus, manidipine exerts a positive effect on insulin resistance. However, this effect has not been observed with amlodipine. On the other hand, the excessive activation of sympathetic nervous system has been related with an increase of insulin resistance, pulse pressure, and ankle edema rates. Compared with amlodipine, manidipine activates sympathetic nervous system to a lesser extent. As a result, treatment with manidipine represents a good option in hypertensive patients with diabetes.

Factors associated with the reduction of albumin excretion in diabetic hypertensive patients: differential effect of manidipine versus amlodipine.

AIMS: In AMANDHA trial, the addition of manidipine, but not amlodipine, in diabetic patients with uncontrolled hypertension, microalbuminuria and preserved renal function resulted in a large decrease of urinary albumin excretion (UAE) despite similar blood pressure (BP) reductions. Factors associated with the reduction of UAE were analyzed. METHODS: For this purpose, a multivariable analysis was performed. RESULTS: Although after 6 months of treatment, manidipine and amlodipine decreased BP to a similar extent, reductions of UAE were higher with manidipine. The assigned treatment, changes in mean BP, sympathetic tone and glycemic control were associated with changes in UAE. CONCLUSION: The assigned treatment, changes in mean BP, sympathetic tone and glycemic control were independently associated with changes in UAE. Compared with amlodipine, manidipine reduced UAE to a higher extent, independently of BP reduction.

Treatment of hypertensive patients with diabetes: beyond blood pressure control and focus on manidipine.

Renin-angiotensin system inhibitors should be considered as the first-line therapy in the treatment of patients with hypertension and diabetes. However, most of the diabetic subjects with hypertension require at least two drugs to achieve blood pressure targets. The ACCOMPLISH trial suggested that the best combination in the treatment of high-risk hypertensive patients should include a renin-angiotensin system inhibitor and a dihydropyridine. However, not all dihydropyridines block the same receptors. Those dihydropyridines that block T-type calcium channel blockers may provide additional advantages. A number of studies suggest that compared with amlodipine, manidipine have the same antihypertensive efficacy, but with a lesser risk of ankle edema. In addition, manidipine, but not amlodipine, significantly reduces urinary albumin excretion rates.

Dissolution method for delapril and manidipine combination tablets based on an absorption profile of manidipine.

The present study describes the development and validation of a dissolution method for delapril (DEL) and manidipine (MAN) combination tablets, using a simulated absorption profile based on in vivo data for MAN. The suitable in vitro dissolution profile for this formulation was obtained using 900 mL of citrate buffer pH 3.2 at 37 °C±0.5 °C as dissolution medium and USP apparatus 2 (paddle) at 75 rpm. All samples were analyzed by a liquid chromatography (LC) method. Under these conditions, a significant linear relationship between the absorbed (calculated by deconvolution approach) and dissolved fractions of MAN was obtained (R=0.997) and an in vivo-in vitro (IVIV) correlation for this particular formulation containing MAN can be established. Validation parameters for dissolution methodology such as the specificity, linearity, accuracy and precision were also evaluated according to the international guidelines, giving results within the acceptable range. Therefore, the proposed dissolution conditions can be applied for the simultaneous release analysis of DEL and MAN from the solid dosage form, contributing to the improvement of the quality control of pharmaceutics and minimizing the number of bioavailability studies.

Effects of the fixed combination of manidipine plus delapril in the treatment of hypertension inadequately controlled by monotherapy with either component: a phase III, multicenter, open-label, clinical trial.

BACKGROUND: Failure to achieve good blood pressure (BP) control is probably the most important reason for high rates of morbidity and mortality in patients with hypertension. Combination therapy has been shown to increase the percentage of patients in whom BP control is achieved. One combination is a calcium channel blocker (CCB) and an angiotensin-converting enzyme inhibitor (ACE-I). OBJECTIVE: The aim of this study was to assess the effects of the fixed combination of the CCB manidipine and the ACE-I delapril in the treatment of hypertensive patients already given monotherapy with either component but with poor results (ie, insufficient BP control or adverse events [AEs]). METHODS: In this Phase III, multicenter, open-label, clinical trial, patients with mild to moderate hypertension were assigned to 1 of 2 groups. Group 1 comprised patients whose diastolic BP (DBP) was >90 mm Hg or who experienced AEs with manidipine 20 mg once daily. Group 2 comprised patients who had a DBP >90 mm Hg or who experienced AEs with delapril 30 mg BID. In both groups, patients aged <65 years were to be treated with a fixed combination of manidipine 10 mg plus delapril 30 mg once daily for 12 weeks, whereas patients aged ≥65 years were to be treated with manidipine 5 mg plus delapril 15 mg once daily for 2 weeks and then manidipine 10 mg plus delapril 30 mg once daily for 10 weeks. Patients were assessed at baseline and at 2, 4, 8, and 12 weeks of treatment. At each visit, systolic blood pressure (SBP), DBP, and heart rate were measured 24 hours after dosing, and AEs were recorded. RESULTS: Group 1 included 154 patients (80 men, 74 women; mean [SD] age, 55 [6] years); group 2 included 158 patients (79 men, 79 women; mean [SD] age, 56 [5] years). Mean BP decreased significantly in both groups (P<0.01). Compared with baseline values, mean SBP/DBP decreased 16.2 (3.8)/10.1 (1.9) mm Hg in group 1 and 15.8 (3.1)/11.0 (1.5) mm Hg in group 2 at the last visit. The success rate-rate of normalized DBP (≤90 mm Hg) and responder rate (DBP reduction ≥10 mm Hg)-was 79% in group 1 and 82% in group 2. The rates of treatment-related AEs were 11% in group 1 and 8% in group 2. In group 1, heart rate significantly increased from baseline only at 2 weeks (P<0.05); in group 2, at each visit (P<0.05) except at week 12. However, none of these differences were clinically significant. CONCLUSION: In this study population of patients whose BP was not adequately controlled by monotherapy, the fixed combination of manidipine 10 mg plus delapril 30 mg, once daily, was effective and well tolerated.