Synthesis and evaluation of hedgehog signaling inhibitor with novel core system.

As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next focused on modification of the lower part including core skeletons to discover new Hh signaling inhibitors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure analysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1 expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441) because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical candidate with good structural and pharmacokinetic advantages.

TAK-441, a novel investigational smoothened antagonist, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling.

Hedgehog (Hh) signaling is a highly conserved intercellular and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up-regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen-deprived and castration-resistant prostate cancer (CRPC). In a cohort of therapy naive, short- and long-term neoadjuvant hormone therapy-treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long-term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh-producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration-induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK-441, could impair castration-resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. Although TAK-441 or cyclopamine did not affect androgen withdrawal-induced Shh up-regulation or viability of LNCaP cells, castration-resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK-441. In TAK-441-treated xenografts, expression of murine orthologs of the Hh-activated genes, Gli1, Gli2 and Ptch1, was substantially suppressed, while expression of the corresponding human orthologs was unaffected. As androgen-deprived LNCaP cells up-regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK-441 leads to delayed castration-resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression.

Pharmacokinetic and pharmacodynamic modeling of hedgehog inhibitor TAK-441 for the inhibition of Gli1 messenger RNA expression and antitumor efficacy in xenografted tumor model mice.

6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (TAK-441) is a potent, selective hedgehog signaling pathway inhibitor that binds to Smo and is being developed for the treatment of cancer. The objectives of these studies were to explore the possibility of establishing of a link between the pharmacokinetics of TAK-441 and the responses of Gli1 mRNA in tumor-associated stromal or skin cells and the antitumor effect of hedgehog inhibition. To this end, we built pharmacokinetic and pharmacodynamic models that describe the relationship of the concentrations of TAK-441 plasma to the responses of Gli1 mRNA in the tumor (target) and skin (surrogate) and to tumor growth inhibition in mice bearing xenografts of human pancreatic tumors (PAN-04). The responses of Gli1 mRNA and tumor growth were described by an indirect response model and an exponential tumor growth model, respectively. The IC50 values for Gli1 mRNA inhibition in the tumor and skin by TAK-441 were estimated to be 0.0457 and 0.113 µg/ml, respectively. The IC90 value for tumor growth inhibition was estimated to be 0.68 µg/ml. These results suggest that a >83% inhibition of Gli1 mRNA expression in the skin or a >94% inhibition of Gli1 mRNA expression in the tumor would be required to sufficiently inhibit (>90%) hedgehog-related tumor growth in the xenografted model mice. We conclude that Gli1 mRNA expression in the tumor and skin could be a useful biomarker for predicting the antitumor effect of hedgehog inhibitors.

Chymase inhibition and cardiovascular protection.

Human chymase, an angiotensin II-forming chymotrypsin-like serine proteinase, posses various biological actions mediating through local angiotensin II formation in the tissue level of many cardiovascular organs. Our previous experimental data have shown that chymase inhibitor increased a survival rate of the hamster post-myocardial infarction model with concomitant improvements of the cardiac function and hypertrophy, decreased hamster aortic atherosclerotic lesion induced by a high fat diet and improved hamster diabetic nephropathy decreasing the proteinuria and increased renal antiotensin II levels. Although chymase inhibitor has not yet been applied for clinical use, clinical cardiovascular diseases above mentioned appear to be the target of chymase inhibitor. The related basal and clinical circumstances are discussed in this review article for chymase inhibitor.

Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine derivative, as a highly potent and orally active hedgehog signaling inhibitor: modification of the core skeleton for improved solubility.

We recently reported the discovery of the novel pyrrolo[3,2-c]quinoline-4-one derivative 1 as a potent inhibitor of Hedgehog (Hh) pathway signaling. However, the PK evaluation of 1 at high dosage (100 mg/kg) revealed the C(max) value 3.63 µg/mL, likely due to poor solubility of this compound. Efforts to improve solubility by reducing the aromatic ring count of the core system led to N-methylpyrrolo[3,2-c]pyridine derivative 11. Further optimization of the 3-alkoxy group led to compound 11d with acceptable solubility and potent Hh inhibitory activity. Compound 11d suppressed transcription factor Gli1 mRNA expression in tumor-associated stromal tissue and inhibited tumor growth (treatment/control ratio, 3%) in a mouse medulloblastoma allograft model owing to the improved PK profile based on increased solubility. Compound 11d (TAK-441) is currently in clinical trials for the treatment of advanced solid tumors.

Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog signaling inhibitors.

The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimization. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allograft model.

Bacteriostatic activity of Whey Acidic Protein (WAP).

We have previously reported the action of whey acidic protein (WAP) inhibiting the proliferation of mouse mammary epithelial cells in the experiments utilizing in vivo and in vitro systems. We report herein the bacteriostatic activity of WAP. Western blot analysis demonstrated successful isolation of WAP from whey fractions of rat milk by column chromatography. The WAP fraction inhibited the growth of Staphylococcus aureus JCM2413 in a dose-dependent manner, but did not inhibit the growth of Escherichia coli. The bacteriostatic activity of WAP was highest at pH 6.6 and was not affected by the presence of 150 mM NaCl. A scanning electron micrograph of bacteria treated with WAP exhibited the disruption of the bacterial cell walls.

Whey acidic protein (WAP) regulates the proliferation of mammary epithelial cells by preventing serine protease from degrading laminin.

Whey acidic protein (WAP) is a major whey protein in milk that has structural similarity to the family of serine protease inhibitors with WAP motif domains characterized by a four-disulfide core. We previously reported that enforced expression of the mouse WAP transgene in mammary epithelial cells inhibits their proliferation in vitro and in vivo by means of suppressing cyclin D1 expression (Nukumi et al., 2004, Dev Biol 274: 31-44). This study was conducted in order to clarify the molecular mechanism of the inhibitory function of WAP in HC11 cells, a mammary epithelial cell line. The assembly of laminin, a component in the extracellular matrix, was much more prominent around WAP-clonal HC11 cells that stably expressed the WAP transgene than around mock-clonal HC11 cells, and the proliferation of WAP-clonal HC11 cells was particularly inhibited in the presence of laminin. A laminin degradation assay demonstrated that WAP inhibited the activity of the pancreatic elastase-mediated cleavage of laminin B1 and the phosphorylation of ERK1/2. ERK1/2 phosphorylation was blocked by an inhibitor of the epidermal growth factor (EGF) receptor AG1478. Treatment with pancreatic elastase was found to enhance the proliferation of mock-clonal HC11 cells, but had no effect on that of WAP-clonal HC11 cells. The proliferation of WAP-clonal HC11 cells was recovered by the addition of exogenous EGF. We concluded that WAP plays some role in regulating the proliferation of mammary epithelial cells by preventing elastase-type serine protease from carrying out laminin degradation and thereby suppressing the MAP kinase signal pathway.

Reduction of tumorigenesis and invasion of human breast cancer cells by whey acidic protein (WAP).

Whey acidic protein (WAP) is a major component of whey, which has two or three WAP motif domains characterized by a four-disulfide core (4-DSC) structure similar to the serine protease inhibitor. We have previously found that WAP inhibits the proliferation of mammary epithelial cells in vitro and in vivo [N. Nukumi, K. Ikeda, M. Osawa, T. Iwamori, K. Naito, H. Tojo, Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro, Dev. Biol. 274 (2004) 31-44]. We report herein that WAP also reduces the progression of human breast cancer cells (MCF-7 and MDA-MB-453 cells). We have demonstrated that the forced expression of WAP in MCF-7 cells reduces the proliferation in either the presence or absence of estrogen. The tumor progression of WAP-expressing MCF-7 cells in nude mice is significantly suppressed more than that of mock-MCF-7 cells following the reduced expression of angiopoietin-2 gene. We have confirmed that the invasive activity of breast cancer cells is reduced to approximately 30% of that of mock cells by the forced expression of exogenous WAP through its inhibition of degradation of laminin. These data suggest that WAP has a protease-inhibitory function on the progression of breast cancer cells. It is therefore possible to utilize WAP as therapeutic protein against tumorigenesis of breast cancer.

Pilsicainide for atrial fibrillation.

Pilsicainide is a class IC antiarrhythmic drug, which has a pure sodium channel blocking action with slow recovery pharmacokinetics. In experimental studies, pilsicainide has a depressant effect on intra-atrial conduction and a prolonging effect on the atrial effective refractory period (ERP). In patients with paroxysmal atrial fibrillation (AF), pilsicainide significantly prolonged the ERP of the distal pulmonary vein (PV), PV-left atrium (LA) junction and LA, and the conduction time from the distal PV to the PV-LA junction. In some patients, PV-LA conduction block has been observed just before pilsicainide-induced termination of AF; this isolation of the PV may provide a new insight into the mechanism of pharmacological conversion of AF. Hybrid therapy with pilsicainide and PV isolation (by radiofrequency catheter ablation) appears to be an effective therapeutic approach for AF. The pharmacological PV isolation by pilsicainide and its suppression of focal discharges from atrial tissue may prevent the development of AF after unsuccessful ablation.

Analysis of gender difference of cardiac risk biomarkers using hGH-transgenic mice.

We investigated gender difference in the effects of chronic exposure to human growth hormone (hGH) on cardiac risk biomarkers using transgenic mice with non-pulsatile circulating hGH. Blood plasma was obtained from transgenic and control mice at 8, 12, and 16 weeks of age, and was used for the measurement of hGH and the following cardiac risk biomarkers: total cholesterol (CHO), triglyceride (TG), HDL cholesterol (HDL), LDL cholesterol (LDL), non esterified free fatty acids (NEFA), and lipid peroxides (LPO). The hearts and the livers of transgenic mice were weighed and histopathologically examined, and the results were compared with those of control mice. Transgenic males exhibited higher levels of LDL at 8 and 12 weeks of age and higher levels of LPO at every week of age examined, as compared to those of the control males, while transgenic females exhibited somewhat lower levels of LDL and LPO from 8 to 16 weeks of age, as compared to the control females. The relative heart weight in males increased with aging and was significantly higher in the 16-week-old transgenic males compared to those of the control mice. The present results demonstrate that transgenic males had cardiac risk potential caused by chronic-exposure to hGH as compared to females. The results also show that the present transgenic mouse line is a useful model for the study of gender difference in cardiac disorders caused by hGH.

Evaluation of H-FABP as a marker of ongoing myocardial damage using hGH transgenic mice.

BACKGROUND: There are few heart-specific and highly sensitive biomarkers of cardiac disorders in experimental animals. To evaluate ongoing myocardial damage in experimental mice, available and reliable biomarkers are needed. We investigated whether or not heart-type fatty acid-binding protein (H-FABP) is useful as a biomarker for predicting ongoing myocardial disorders, by using CAG/EGFP-WAP/hGH transgenic male mice with heart disease induced by overexpression of human growth hormone (hGH). METHODS: Blood samples were collected from transgenic and control male mice at 8, 12, 16, and 36 weeks of age and were measured for aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), and H-FABP. The hearts of the transgenic mice were examined histopathologicaly and the results were compared with those of control mice. RESULTS: At 36 weeks of age, significant increases in AST, CK, and LDH values were observed in the transgenic mice compared to the control mice. Minute histological changes along with focal and slight degeneration of cardiomyocytes were observed in the transgenic hearts at 12 weeks of age, but the only chemical value to change was that of H-FABP, which increased significantly. CONCLUSIONS: H-FABP is available as a biomarker for predicting ongoing cardiomyocyte damage in the mouse model.

The Formin family protein, formin homolog overexpressed in spleen, interacts with the insulin-responsive aminopeptidase and profilin IIa.

Insulin stimulates translocation of glucose transporter isoform type 4 (GLUT4) and the insulin-responsive aminopeptidase (IRAP) from an intracellular storage pool to the plasma membrane in muscle and fat cells. A role for the cytoskeleton in insulin action has been postulated, and the insulin signaling pathway has been well investigated; however, the molecular mechanism by which GLUT4/IRAP-containing vesicles move from an interior location to the cell surface in response to insulin is incompletely understood. Here, we have screened for IRAP-binding proteins using a yeast two-hybrid system and have found that the C-terminal domain of FHOS (formin homolog overexpressed in spleen) interacts with the N-terminal cytoplasmic domain of IRAP. FHOS is a member of the Formin/Diaphanous family of proteins that is expressed most abundantly in skeletal muscle. In addition, there are two novel types of FHOS transcripts generated by alternative mRNA splicing. FHOS78 has a 78-bp insertion and it is expressed mainly in skeletal muscle where it may be the most abundant isoform in humans. The ubiquitously expressed FHOS24 has a 24-bp insertion encoding an in-frame stop codon that results in a truncated polypeptide. It is known that some formin family proteins interact with the actin-binding profilin proteins. Both FHOS and FHOS78 bound to profilin IIa via their formin homology 1 domains, but neither bound profilin I or IIb. Overexpression of FHOS and FHOS78 resulted in enhanced insulin-stimulated glucose uptake in L6 cells to similar levels. However, overexpression of FHOS24, lacking the IRAP-binding domain, did not affect insulin-stimulated glucose uptake. These findings suggest that FHOS mediates an interaction between GLUT4/IRAP-containing vesicles and the cytoskeleton and may participate in exocytosis and/or retention of this membrane compartment.

Valsartan, an angiotensin II type-I receptor blocker, and left ventricular diastolic function--a case report.

Impaired diastolic function is related to subjective symptoms, reduced exercise capacity, and poor prognosis in patients with congestive heart failure, and an angiotensin II type-I receptor blocker might have a beneficial effect on diastolic function in such patients with heart failure. A 53-year-old woman underwent valvuloplasty of the mitral valve and later presented with heart failure symptoms, including exertional dyspnea and easy fatigue. Although no pathological changes could be identified by radiography of the chest, electrocardiography, or routine echocardiography, the assessment of diastolic function with Doppler echocardiography revealed left ventricular diastolic dysfunction. Her neurohumoral parameters and left ventricular diastolic dysfunction improved after 1 month of treatment with Valsartan, an angiotensin II type-I receptor blocker, accompanied by improvement of her subjective symptoms. This case implies that angiotensin II type-I receptor blocker could improve left ventricular diastolic dysfunction and that Doppler echocardiography might be useful for detecting diastolic dysfunction in high-risk patients undergoing cardiac surgery.

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein.

It is known that a characteristic of the mammalian preimplantation-embryo-specific cell cycle is the substantially shortened G1-phase, although the regulation mechanisms of the unique cell cycle remain unclear. In the present study, we first examined the presence of retinoblastoma (RB) tumor suppressor gene product throughout mouse preimplantation embryo development and found that the RB expression was down-regulated between the four-cell and morula stages. Furthermore, the overexpression of RB protein in the mouse embryos during this phase inhibited their development significantly. These results suggest that the absence of RB protein contributes to the preimplantation-embryo-specific cell cycle.

Maturation/M-phase promoting factor regulates aging of porcine oocytes matured in vitro.

Control of oocyte aging during manipulation of matured oocytes should have advantages for recently developed reproductive technologies, such as cloning after nuclear transfer. We have shown that the enhanced activation ability and fragmentation of porcine in vitro matured and aged oocytes bore a close relationship to the gradual decrease in maturation/M-phase promoting factor (MPF) activity and that porcine aged oocytes contained plenty of MPF, but it was in an inactive form, pre-MPF, as a result of phosphorylation of its catalytic subunit p34(cdc2) and, therefore, had low MPF activity. We incubated porcine oocytes with vanadate and caffeine, which affected the phosphorylation status and MPF activity, and evaluated their activation abilities and fragmentation frequencies. Incubation of nonaged oocytes with vanadate increased p34(cdc2) phosphorylation and reduced MPF activity to levels similar to those of aged oocytes and increased their parthenogenetic activation and fragmentation rates compared with those of the control oocytes. Conversely, treating aged oocytes with caffeine reduced p34(cdc2) phosphorylation and increased MPF activity. These oocytes showed significantly lower parthenogenetic activation and fragmentation rates than aged mature oocytes. These results suggest that MPF activity is a key mechanism of oocyte aging and controlling MPF activity by altering p34(cdc2) phosphorylation with these chemicals may enable oocyte aging to be manipulated in vitro. We expect those ideas will be applied practically to pig cloning.

Effect of Bachmann's bundle pacing on atrial fibrillation: electrophysiologic assessment.

BACKGROUND: It has recently been reported that simultaneous multisite atrial pacing, Bachmann's bundle (BB) pacing, and coronary sinus (CS) pacing are useful for preventing the induction of atrial fibrillation (AF). HYPOTHESIS: We investigated whether a simple pacing approach via BB could reduce the induction of AF by extrastimuli (S2) from the right atrial appendage (RAA). METHODS: Programmed electrical stimulation was performed from the RAA and the area of BB at the superior aspect of the atrial septum, and bipolar recordings were obtained from the RAA, BB, and CS in 14 patients. RESULTS: In five patients, AF was induced with critically timed RAA-S2 delivered during RAA pacing. However, AF was not induced in any patient when RAA-S2 was delivered during BB pacing. The duration of the P wave during BB pacing was significantly shorter than that during RAA pacing and sinus rhythm (BB 80 +/- 16 ms vs. RAA 106 +/- 36 ms vs. sinus rhythm 100 +/- 24 ms, p < 0.05). The intra-atrial conduction time to the distal coronary sinus (CSd) caused by early S2 at the RAA was significantly reduced by BB pacing (BB 114 +/- 22 ms vs. RAA 157 +/- 35 ms, p < 0.001). CONCLUSION: Bachmann's bundle pacing reduces atrial conduction time caused by RAA-S2 and may be useful for preventing the induction of AF.

Novel development of mammary glands in the nursing transgenic mouse ubiquitously expressing WAP gene.

Although whey acidic protein (WAP) has been suggested to have some biological functions, its true function has not yet been clearly elucidated. We have generated transgenic mice ubiquitously and highly expressing the WAP gene. The pups born from one female among these transgenic mice showed low growth or died during nursing. This transgenic founder showed novel development of the mammary glands, and demonstrated normal parturition and nursing behavior. The mammary glands showed low-distended ductal structures, and poor development of lobulo-alveolar and acinous formations despite normal nursing, while mammary ducts were rather large in comparison with those of normal lactating females. Although this founder was found to be mosaic for transgenesis, it was shown to be a useful animal model for investigating WAP function.

Continuous measurement of oxygen consumption using the reversed fick method.

We developed a continuous oxygen consumption (Vo2) measurement system employed the reversed Fick method, in which Vo2 in computed from continuously measured sured arterial and mixed venous oxygen saturation assed by pulse oximetry and mixed venous oximetry, respectively, and cardiac output by the heat deprivation technique. This system was compared with the conventional intermittent reversed fick method in 7 patients during surgery and with indirect calorimetry in 4 intensive care unit (ICU) patients. The Vo2 measured by the continuous reversed Fick method showed a high correlation with those simultaneously measured by the intermittent Fick method (r=0.97,P<0.01) and by indirect calorimetry (r=0.74,P<0.01). The 95% confidence limits (bias±2 SD) of the continuous reversed Fick method were -0.6±45 ml·min-1 with the intermittent Fick method and -31±56 ml·min-1 with indirect calorimetry. The continuous Fick method is in satisfactory agreement with the conventional methods for the measured of Vo2 and potentially allows for convenient assessment of Vo2 in critically ill patients.

A case of primary aldosteronism who experienced cardiopulmonary arrest, was resuscitated and cured.

A 45-year-old female went into cardiopulmonary arrest. She was in ventricular fibrillation (VF) and was defibrillated using an automated external defibrillator. After arrival at our hospital, electrocardiography monitoring showed QT prolongation. Serum potassium was low at 2.2 mEq/L, and hypokalemia-induced long QT syndrome was considered to be the cause of this patient's VF. An intravenous infusion of potassium and magnesium sulphate was started, which normalized her serum potassium and QTc interval, with no recurrence of ventricular arrhythmias. Endocrinological investigations showed a plasma renin activity of <0.1 ng/(mL h) and a plasma aldosterone concentration 258 pg/mL. Computed tomography scanning revealed a low signal area 16 mm × 20 mm in size of the right adrenal gland. From the above findings, this patient was diagnosed with a right adrenal tumor and primary aldosteronism. We concluded that the right adrenal tumor was excreting excess amounts of aldosterone from adrenal vein sampling, and performed laparascopic right adrenalectomy. Serum potassium levels rose immediately to normal levels postoperatively. We were able to withdraw her antihypertensive medication 3 months after adrenalectomy. We report a case of primary aldosteronism who experienced cardiopulmonary arrest, was resuscitated, and cured. .