Effect of proton pump inhibitors on the development of hypomagnesemia induced by panitumumab.

Panitumumab, a therapeutic agent for unresectable advanced/recurrent colorectal cancer, is a human IgG2 monoclonal antibody that binds to and inhibits the activity of the epidermal growth factor receptor (EGFR). The onset of hypomagnesemia is a known side effect of anti-EGFR inhibitors, including panitumumab, and it is thought that inhibition of reabsorption of Mg in renal tubules is one of the causes. In addition, recent reports have shown that long-term administration of proton pump inhibitors (PPIs) reduces serum magnesium levels. Therefore, in this study, 102 patients who received oral PPIs treated with panitumumab were classified into a PPI combination group and a PPI non-combination group, and the effect of PPIs on the development of grade 2 or higher hypomagnesemia was investigated. The incidence of hypomagnesemia in the PPI combination group (46.9%, 15/32) was higher than that in the PPI non-combination group (25.7%, 18/70). A comparison of the backgrounds of the two groups of patients showed a significant difference in serum albumin levels. PPI administration was significantly associated with panitumumab-induced hypomagnesemia development when adjusted for known risk factors, serum albumin level, renal function, and oral magnesium oxide tablets in Cox proportional hazards regression analysis (hazard ratio 2.09; 95% confidence interval 1.03-4.22; P =0.040). These results indicate that detailed monitoring of serum magnesium levels is recommended for patients treated with panitumumab and co-administration of PPIs.

Calcium channel blocking and vasodilating actions of the novel dihydropyridine derivative AE0047.

1. The pharmacological characteristics of AE0047, a newly synthesized dihydropyridine (DHP) derivative, were investigated in vitro. 2. In bovine aortic membrane, AE0047 and other DHP calcium channel blockers (nitrendipine, nicardipine) displayed concentration-dependent antagonism to specific [3H]-PN200-110 binding sites with the following values for inhibition constants (Ki) obtained: 20.8 +/- 8.9, 12.3 +/- 4.5 and 3.9 +/- 1.0 nmol/L for AE0047, nitrendipine and nicardipine, respectively. 3. In guinea-pig ventricular myocytes, AE0047 blocked the L-type calcium current, with values for the dissociation constant (Kd) and Hill coefficient of 11.4 +/- 5.7 nmol/L and 0.852 +/- 0.061, respectively, indicating in the terms of Hill's hypothesis that one drug molecule blocks one calcium channel molecule. 4. In rat aorta, AE0047 inhibited 45Ca uptake induced by high K+ (100 mmol/L) by 55%. 5. AE0047 and nitrendipine concentration dependently relaxed rat aortic strips contracted with 30 mmol/L KCl. The response to nitrendipine reached a plateau within 60 min and disappeared after drug washing. Interestingly, AE0047 required 5 h or more to produce a plateau of response, with no effect of drug washing. This confirmed the slow onset and long duration of its vasodilating action. 6. With AE0047, tissue content in rat aorta increased more slowly than with nitrendipine and release of AE0047 from tissue was also slower. 7. The data suggest that AE0047 is incorporated slowly into smooth muscle membranes, approaches receptors slowly through the membrane bilayer and accumulates in the membrane because of its high lipophilicity, resulting in an anti-hypertensive action that is slow in onset and of long duration.

Possible mechanism for the anti-atherosclerotic action of the calcium channel blocker AE0047 in cholesterol-fed rabbits.

1. The present study was designed to investigate the anti-atherosclerotic effect of AE0047, a calcium channel blocker, and to compare it with that of nilvadipine in cholesterol-fed rabbits. Furthermore, the effects of AE0047 on low-density lipoprotein (LDL) oxidation were studied in vitro. 2. A 7 week treatment period with AE0047 (3 and 10 mg/kg, p.o.) led to a dose-dependent reduction in the lipid deposition area by Oil Red-O staining (surface index) without affecting serum lipid levels. There was no reduction in the surface index following treatment with the same dose of nilvadipine (10 mg/kg). 3. In a vehicle-administered high-fat diet group of rabbits, levels of total cholesterol (TC) and esterified cholesterol (EC) and calcium content in the aorta were increased approximately two- to three-fold over those of the normal diet group. Increased levels of TC and EC and calcium content were reduced to the same levels as the normal diet group by AE0047 treatment, whereas nilvadipine did not affect TC and EC levels. 4. In an in vitro study, AE0047 (10 micromol/L) inhibited LDL oxidation and the aggregation of apolipoprotein (Apo) B-100 induced by Cu2+. Furthermore, AE0047 inhibited the degradation of oxidized LDL by macrophages. In contrast, the same dose of nilvadipine (10 micromol/L) did not inhibit either LDL oxidation or the aggregation of ApoB-100. 5. In summary, AE0047 inhibited LDL oxidation, resulting in a decrease of its uptake into macrophages and an inhibition of cholesterol esterification. This leads to an anti-atherosclerotic effect of AE0047. Thus, AE0047 may have therapeutic potential in preventing cardiovascular disease in hypertensive patients.

Possible mechanism of action of AE0047, a calcium antagonist, on triglyceride metabolism.

We evaluated the effect of AE0047, a dihydropyridine-type calcium antagonist, on the plasma lipid levels of obese Zucker rats. In rats treated orally with 3 to 10 mg/kg/day AE0047 for 7 days, plasma triglyceride (TG) and TG-rich lipoprotein levels dose-dependently decreased, whereas those of high-density lipoprotein cholesterol increased. Total cholesterol and low-density lipoprotein levels did not change. To elucidate the mechanism by which AE0047 decreases plasma TG levels, we examined the effect of AE0047 on the synthesis and secretion of TG-rich lipoproteins in human intestinal cell line Caco-2, as well as on the association and degradation of very low density lipoprotein (VLDL) in human hepatoblastoma cells HepG2. When Caco-2 cells were grown on a membrane filter and 14C-oleic acid was added to the apical side, 10(-5) and 10(-6) M AE0047 inhibited basolateral secretion of 14C-TG. AE0047 also suppressed the basolateral secretion of apolipoprotein B. In HepG2 cells, AE0047 increased the cellular uptake of 125I-VLDL. These results suggested that AE0047 decreased plasma TG level by the inhibition of intestinal chylomicron secretion and the enhancement of hepatic uptake of VLDL. AE0047 may be beneficial for the treatment of hypertensive patients with hypertriglyceridemia to reduce the risk factors of coronary heart disease.

Inhibition of cholesterol biosynthesis by squalene epoxidase inhibitor avoids apoptotic cell death in L6 myoblasts.

The relationship between the inhibition of cholesterol biosynthesis and occurrence of myopathy was studied in L6 myoblasts using two lines of cholesterol biosynthesis inhibitors, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (simvastatin) and squalene epoxidase inhibitors (TU-2078 and NB-598). All inhibitors completely inhibited the cholesterol synthesis in L6 myoblasts at doses of 1 and 3 microM. Simvastatin (3 microM) inhibited the fusion reaction of L6 myoblasts followed by the severe cellular damage. The myoblasts also had failed actin fiber formation and creatinine phosphokinase (CPK) production. Additionally, this agent also caused apoptotic cell death in differentiated L6 muscle fiber, indicating that skeletal myopathy by HMG-CoA reductase inhibitors seems to occur not only in differentiating immature myoblasts but also in matured skeletal myotubes. In contrast, TU-2078 and NB-598 had no effect on the fusion reaction of differentiating myoblasts or on the cellular viability of muscle fiber at 3 microM, enough to completely inhibit cholesterol biosynthesis. It is conceivable that the mevalonate depletion and subsequent failure of ras farnesylation induced by simvastatin might cause the defects in differentiation and maintenance of the muscle fiber. Squalene epoxidase inhibitors did not show this adverse effect presumably because of the enzyme inhibition downstream of farnesyl synthesis. The present findings suggest the safe use of squalene epoxidase inhibitors in lipid-lowering therapy.

AL0671, a new potassium channel opener, inhibits nonenzymatic glycation of protein and LDL oxidation.

1. The effects of AL0671, a novel potassium channel opener, on protein glycation and low-density lipoprotein (LDL) oxidation were tested. 2. AL0671 dose-dependently inhibited both fluorescence development of bovine serum albumin and cross-linking of lysozyme. These inhibitory effects for glycation were no less potent than aminoguanidine. 3. AL0671 dose-dependently inhibited both increase in negative charge and apo B-100 fragmentation during incubation of LDL with Cu2+. In addition, AL0671 significantly decreased the LDL degradation in rat peritoneal macrophages. 4. Neither pinacidil nor levcromakalim inhibited protein glycation and LDL oxidation. 5. Antioxidant properties of AL0671 might be due to its potent electron-donating ability, and this agent is expected to be useful for hypertensive diabetes.

Synthesis and pharmacological evaluation of N-(6-functionalized-amino-3-pyridyl)-N'-bicycloalkyl-N''-cyanoguanidine s as antihypertensive agents.

A series of amino acid conjugates of N-(6-amino-3-pyridyl)-N'-[exo-bicyclo[2.2.1]hept-2-yl]-N''- cyanoguanidine (4) were prepared and evaluated as antihypertensive agents. The parent compound 4 showed potent potassium channel-opening and antihypertensive activities, but with undesirable changes of the urinary balance of electrolytes. However, alanine and histidine congeners (9,19) reduced this undesirable side effect of 4 through improved pharmacokinetics without loss of antihypertensive activity. They also provided additional information on the structural requirements for pinacidil-type potassium channel openers.

The effect of AL0671, a novel potassium channel opener, on potassium current in rat aortic smooth muscle cells.

1. We evaluated the mechanism of activation by AL0671, a novel potassium channel opener, of potassium current in rat aortic smooth muscle cells. 2. Under conditions of whole cell recording, AL0671 (1-1000 microM) markedly increased potassium current with a Hill coefficient of 2 and dissociation constant of 1.5 x 10(-4) M. This activation was completely inhibited by intracellular ATP. 3. Under inside-out patch conditions, the ATP-sensitive K+ channels (KATP) treated with AL0671 (100 microM) showed prolongation of the slower open time component and shortening of the slower closed time component without modification of channel conductance.

A possible mechanism of action of a new potassium channel opener, AL0671, on lipid metabolism in obese Zucker rats.

Antihypertensive drugs are expected to have a lipid-lowering effect for use in treating ischemic heart disease. We evaluated the effect of (+)-N-(6-amino-3-pyridil)-N'-[(1S,2R,4R)-bicyclo-[2.2.1]hept-2-yl] -N"- cyanoguanidine hydrochloride (AL0671), a newly synthesized cyanoguanidine-derivative potassium channel opener, on serum lipid and lipoprotein levels in obese Zucker rats, a genetically engineered model of type IV hyperlipidemia. AL0671 dose-dependently decreased systolic blood pressure in obese Zucker rats. Serial administration (for 1 or 2 weeks) of AL0671 (5 mg/kg/day) significantly decreased serum total triglyceride, chylomicron and very-low-density lipoprotein levels with increasing high-density lipoprotein cholesterol, whereas low-density lipoprotein levels did not change. AL0671 (5 mg/kg/day) increased lipoprotein lipase activities 4-fold and hepatic triglyceride lipase activities 3-fold in postheparin plasma. Another urea-derivative compound, AL0674, whose potassium channel-opening activity is diminished, did not affect serum lipid and lipoprotein levels. These results suggested that AL0671 activates both lipoprotein lipase and hepatic triglyceride lipase activities through its potassium channel-opening activity followed by decreasing triglyceride-rich lipoproteins in genetically obese hyperlipemic rats. Therefore, AL0671 might be beneficial in the treatment of hypertensive patients with hypertriglyceridemia (probably with insulin resistance).

Novel potassium-channel openers: preparation and pharmacological evaluation of racemic and optically active N-(6-amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine derivatives.

The previous paper reported on the synthesis and pharmacological evaluation of N-(6-amino-3-pyridyl)-N'-bicycloalkyl-N"-cyanoguanidine derivatives, from among which three compounds were selected as potent potassium-channel openers. In the present study, selected compounds were tested for antagonism of potassium-induced contraction of rat aorta, hypotensive activity in normotensive rats, and diuretic activity in spontaneously hypertensive rats. This led to further evaluation of compound (+/-)-10 and selection of (+)-N-(6-amino-3-pyridyl)-N'- [(1S,2R,4R)-bicyclo- [2.2.1]hept-2-yl]-N"-cyanoguanidine ((+)-10) (AL0670) for development as an antihypertensive agent. Although AL0670 is regarded as a pinacidil-type K(+)-channel opener, it showed different pharmacological and conformational profiles from pinacidil.

Novel potassium channel openers: synthesis and pharmacological evaluation of new N-(substituted-3-pyridyl)-N'-alkylthioureas and related compounds.

This report describes the synthesis and pharmacological evaluation of a series of novel potassium channel openers related to the pinacidil-type compounds. Thioureas, cyanoguanidines, and pyridine N-oxides were systematically evaluated for their effects on both the inhibition of spontaneous mechanical activity in rat portal vein (in vitro) and their antihypertensive activity (in vivo), and the structure-activity relationship for this series of compounds was discussed. Good correlation between in vitro and iv antihypertensive activity was observed for these compounds. Among them, cyanoguanidines bearing a conformationally rigid unit such as a norbornyl group generally possessed potent activity in both in vitro and in vivo studies. Especially, N-(6-amino-3-pyridyl)-N'-cyano-N"-(1-methyl-2-norbornyl)guanidine (23d) was identified as a more potent potassium channel opener in vitro (EC100 = 3 x 10(-8) M) than pinacidil (EC100 = 10(-7) M).

Collection of acrosome-reacted human sperm using monoclonal antibody-coated paramagnetic beads.

A monoclonal antibody (MAb) against human acrosome-reacted sperm was attached to paramagnetic polystyrene beads. Human sperm prepared by the swim-up method were 1) incubated in m-BWW, 2) incubated and ionophore treated, or 3) incubated with 5% seminal fluid. After treatment, sperm were mixed with the beads and incubated for 1 hr. Variously treated sperm showed different binding abilities to the beads. Sperm bound to the beads were collected by a magnet and subjected to triple staining. Most of the collected sperm were acrosome reacted. The results suggested that the beads can be used to estimate the acrosomal status of sperm, and that the use of antibody-coated paramagnetic beads provides a convenient way of collecting acrosome-reacted sperm. The acrosomal status detected by the beads was also compared with the ability of sperm to fuse with zona-free hamster eggs. It was found that greater bead-binding ability correlated with more sperm fusing with zona-free hamster eggs.

A human sperm antigen possibly involved in binding and/or fusion with zona-free hamster eggs.

A monoclonal antibody to human sperm (MH61) was established. The antibody did not attach to ejaculated sperm but to a head region of some sperm that was incubated in medium for 4 to 12 hours. A23187 treatment significantly increased the number of sperm that were reactive to the antibody. When sperm that bound to the zona-free hamster egg were subjected to the indirect immunofluorescence staining, all the sperm reacted to MH61 with their entire head region. The addition of the antibody to the medium before sperm addition reduced the fusion rate of human sperm to zona-free hamster egg.

Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody OBF13.

The variable reactivity of OBF13 monoclonal antibody to mouse sperm was studied using a fluorescein activated cell sorter. Sperm from cauda epididymis were incubated with ionophore A23187, subjected to indirect immunostaining and analyzed by a cell sorter. Three peaks showing different fluorescence intensities were observed. These peaks contained (i) not stained (N), (ii) acrosomal cap or anterior region stained (A) and (iii) head stained (H) sperm, respectively. H type sperm showed more intense integral fluorescence than A type sperm. It was also noted that the H type were observed when cauda epididymal sperm were incubated with A23187, but not among non-incubated, or A23187 treated caput epididymal sperm. When sperm were pre-categorized as "dead" or "alive" by propidium iodide staining, no A type were observed in the "live" population. These results suggest that the sperm exhibiting the OBF13 antigen in the acrosome region lost their viability before they accomplished a "true" acrosome reaction.

Glucosamine enhanced sperm-egg binding but inhibited sperm-egg fusion in mouse.

In order to study the sperm-egg recognition mechanism on the surface of the plasma membrane, zonae were removed from mouse eggs by exposure to acidic conditions. Sperm binding to denuded eggs was then observed in the presence of various sugars. Among several carbohydrates tested, only glucosamine (GlcN) was found to increase the number of sperm bound to eggs while inhibiting sperm-egg fusion. The inhibition was reversible; when denuded eggs were transferred to a GlcN free medium, a high rate of polyspermy was observed.

Effect of a monoclonal anti-mouse sperm antibody (OBF13) on the interaction of mouse sperm with zona-free mouse and hamster eggs.

Mouse eggs freed from zonae by chymotrypsin were mixed with sperm and pronuclear formation was observed. When anti-mouse sperm monoclonal antibody (OBF 13) from ascites fluid was added to the medium (at a final concentration of 0.05%), fertilization was significantly inhibited (9.7 +/- 4.3% compared to control 56.7 +/- 7.4%, P less than 0.01). This was based on the inhibition of sperm binding to the egg. However, when similar experiments were performed using zona-free hamster eggs, addition of the OBF 13 antibody caused no significant reduction in fertilization rate (91 +/- 7.1% compared to control 97 +/- 3.2%). It was also observed that binding of mouse sperm to hamster eggs was not inhibited by the antibody. It is therefore suggested that mouse sperm and mouse egg recognize each other in a species-specific manner.