Norito Takamura

The citrus flavonoids hesperetin and naringenin block the lipolytic actions of TNF-α in mouse adipocytes

Obese adipose tissue is characterized by an excessive production of inflammatory adipokines including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates free fatty acid (FFA) secretion through adipocyte lipolysis, and increased plasma levels of FFA promote insulin resistance. In this report, we show that hesperetin and naringenin, two citrus flavonoids, inhibit TNF-alpha-stimulated FFA secretion from mouse adipocytes. These flavonoids block the TNF-alpha-induced activation of the NF-kappaB and ERK pathways. Moreover, hesperetin and naringenin prevent TNF-alpha from downregulating the transcription of two antilipolytic genes, perilipin and PDE3B. These effects are mediated through the inhibition of the ERK pathway. In contrast, the inhibition of the NF-kappaB pathway by hesperetin and naringenin suppresses the transcription of IL-6, which induces FFA secretion in an autocrine manner. Our results provide novel evidence that hesperetin and naringenin directly inhibit TNF-alpha-stimulated FFA secretion. These findings may be useful for ameliorating FFA-induced insulin resistance.

Effect of P-Glycoprotein Modulator, Cyclosporin A, on the Gastrointestinal Excretion of Irinotecan and Its Metabolite SN-38 in Rats

AbstractPurpose. The purpose of this work was to investigate the role of the hepatic and intestinal P-glycoprotein (P-gp) and canalicular multispecific organic anion transporter /multidrug resistance-associated protein 2 (cMOAT/MRP2) on both biliary excretion and intestinal exsorption of irinotecan hydrochloride (CPT-11) and its metabolite, SN-38, in the lactone and carboxylate forms. Cyclosporin A (CsA) was used to modulate P-gp and cMOAT/MRP2. Methods. The transcellular transport of CPT-11 and SN-38 was examined by using LLC-PK1 derivative cell lines transfected with murine mdr1a both in the absence or in the presence of CsA. The excretions of the compounds through the biliary and intestinal membrane routes were investigated by in situ perfusion technique. Results. Basolateral-to-apical transport of CPT-11 lactone in L-mdr1a cells was significantly decreased by CsA (10 μM). The trans- cellular transport of SN-38 lactone showed similar behaviors as those of CPT-11 lactone. The biliary excretion and the intestinal exsorption of both forms of CPT-11 and SN-38 were significantly inhibited when the drug was co-administered with CsA. Conclusions. The transports of CPT-11 and SN-38 via the biliary route seem to be essentially related with cMOAT/MRP2, whereas those of both compounds via the intestinal membrane seem to be related with P-gp.

Mode of Interaction of Loop Diuretics with Human Serum Albumin and Characterization of Binding Site

AbstractPurpose. The purpose of this study was to investigate the binding mechanism of loop diuretics with HSA and to characterize the binding site on HSA. Methods. Quantitative analysis of potential interaction between ligands bound to HSA was performed by equilibrium dialysis and data for binding of the two ligands to HSA were analyzed on the basis of a theoretical model of simultaneous binding of two ligands. Results. The binding of loop diuretics is dependent upon the N-B transition, conformational change of albumin. Furthermore, from the results of binding of the drugs to modified HSA, the lysine residue seems to be involved in the binding of loop diuretics to HSA. Conclusions. Analysis using models describing independent, competitive, cooperative and anti-cooperative binding led to the conclusion that loop diuretics bind to site I, particularly to the warfarin region on HSA.

Interactions of aldosterone antagonist diuretics with human serum proteins

AbstractPurpose. The purpose of this study was to investigate the binding mechanism of aldosterone antagonist diuretics with human serum proteins, human serum albumin (HSA) and α1-acid glycoprotein (AGP), as well as to identify the binding sites of the drugs on these proteins. Methods. Binding activities of spironolactone (SP) and its pharmacologically active metabolite canrenone (CR) to serum and serum protein were examined by ultrafiltration and spectroscopic techniques. The data for the binding of these drugs to HSA were analyzed on the basis of a theoretical model of simultaneous binding of the ligands. Results. The binding percentages of antagonist diuretics SP and CR to human serum proteins were 88.0% and 99.2%, respectively, at therapeutic concentrations. SP bound strongly and almost equally to both HSA and AGP, but CR bound strongly only to HSA. In addition, the displacement results found using fluorescent probes and ultrafiltration methods demonstrated that SP bound to site I, particularly to the warfarin region on HSA, and to the basic binding site on AGP, while CR bound to the warfarin region on HSA. Conclusions. The limited results presented here stress the need for caution on coadministration of acidic drugs which bind to the warfarin region on HSA and basic drugs which bind to AGP with SP and its metabolite CR.

Usefulness of competitive inhibitors of protein binding for improving the pharmacokinetics of 186Re-MAG3-conjugated bisphosphonate (186Re-MAG3-HBP), an agent for treatment of painful bone metastases

PurposeWe have developed a 186Re-mercaptoacetylglycylglycylglycine complex-conjugated bisphosphonate (186Re-MAG3-HBP) for the treatment of painful bone metastases. We assumed competitive inhibitors of protein binding to be useful for procuring a favorable biodistribution of 186Re-MAG3-HBP for the palliation of bone pain because it has been reported that the concurrent administration of 99mTc-MAG3 and drugs with high affinity for serum protein produced competitive displacement at specific binding sites and enhanced total clearance and tissue distribution.MethodsThe displacement effects of several protein-binding inhibitors on the protein binding of 186Re-MAG3-HBP were investigated. Biodistribution experiments were performed by intravenously administering 186Re-MAG3-HBP into rats with ceftriaxone as a competitive protein-binding inhibitor or saline.ResultsThe protein binding of 186Re-MAG3-HBP in rat serum, human serum, and a human serum albumin solution was significantly decreased by the addition of ceftriaxone, which has high affinity for binding site I on serum albumin. In the biodistribution experiments, pretreatment with ceftriaxone enhanced the clearance of the radioactivity of 186Re-MAG3-HBP in blood and nontarget tissues but had no effect on accumulation in bone.ConclusionsThe findings suggested that the use of protein-binding competitive inhibitors would be effective in improving the pharmacokinetics of radiopharmaceuticals with high affinity for serum protein.

Influences of haemodialysis on the binding sites of human serum albumin: possibility of an efficacious administration plan using binding inhibition

BACKGROUND We have studied the possibility that low-dose treatment utilizing the inhibition that may occur between two drugs at the same site of human serum albumin (HSA) improves the pharmacological effects. The purpose is to elucidate the differences in the binding capacities of sites I and II of HSA between pre-haemodialysis (HD) and post-HD in patients with end-stage renal disease. METHODS We evaluated free fractions of site probes, (14)C-warfarin (site I) and (14)C-diazepam (site II), by ultrafiltration in serum between pre-HD and post-HD. To investigate effects on the binding capacities of HSA sites, free fractions of site probes were calculated from the radioactivities measured with a liquid scintillation counter. Endogenous uraemic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulphate (IS) and hippurate (HA), were determined by HPLC. Free fatty acid (FFA) as an endogenous substance was determined with an automatic multi-item simultaneous analyser. RESULTS The concentrations of HSA and FFA increased significantly (post-HD/pre-HD ratio: 1.18 +/- 0.10, 5.46 +/- 4.91), the concentrations of IS and HA decreased significantly (post-HD/pre-HD ratio: 0.69 +/- 0.10, 0.33 +/- 0.15) and CMPF concentrations did not alter significantly (post-HD/pre-HD ratio: 0.97 +/- 0.12, P = 0.471). The free fractions of (14)C-warfarin decreased in all 14 patients at site I at post-HD compared to pre-HD (post-HD/pre-HD ratio: 0.59 +/- 0.13). The free fractions of (14)C-diazepam at site II remarkably decreased in 10 of 14 patients (post-HD/pre-HD ratio: 0.61 +/- 0.17) and unexpectedly increased in 4 (post-HD/pre-HD ratio: 1.08 +/- 0.06) post-HD compared to pre-HD. In these four patients, when we investigated the influences of these variation factors on the reduction of the binding capacities of site II, [FFA]/[HSA] increased significantly post-HD, compared to pre-HD (post-HD/pre-HD ratio: 6.91 +/- 6.58). ([FFA]/[HSA] ratios of the 4 patients were from 1.22 to 3.55, the highest for the 14 patients post-HD, but the ratios of the other 10 were below 1.2 post-HD.) CONCLUSION The binding capacity of site II was unexpectedly decremented by the effects of the remarkable elevation of FFA. Therefore, monitoring the binding capacity of site II in HD is important for patients with end-stage renal disease in the efficacious administration plan using the binding inhibition of HSA.

A diclofenac suppository–nabumetone combination therapy for arthritic pain relief and a monitoring method for the diclofenac binding capacity of HSA site II in rheumatoid arthritis

Diclofenac suppository, a non‐steroidal anti‐inflammatory drug (NSAID), is used widely in rheumatoid arthritis (RA) patients with severe arthritic pain. As the binding percentage of diclofenac to serum proteins is high, its free (unbound) concentration after rectal administration is low. To increase temporarily the free concentration of diclofenac and to enhance its analgesic effect by inhibiting the protein binding of diclofenac, the analgesic effect of diclofenac was examined before and after the start of an inhibitor administration to RA patients with insufficient control of arthritic pain, and the protein binding capacity of diclofenac was evaluated. Binding experiments were performed by ultrafiltration, and arthritic pain was recorded by the face scale. Free fractions of diazepam and diclofenac were augmented by increasing 6‐methoxy‐2‐naphthylacetic acid (6‐MNA; the active metabolite of the NSAID nabumetone) concentrations. The free fraction of diazepam increased after the start of nabumetone administration to RA patients, and arthritic pain relief was observed. These results suggest that 6‐MNA has an inhibitory effect on the protein binding of diclofenac and the free fraction of diazepam can be used to evaluate the binding capacity of diclofenac. It is considered that diclofenac suppository–nabumetone combination therapy and the method for protein binding monitoring by diazepam can positively benefit RA patients with insufficient control of arthritic pain. Copyright

Dosage plan of a flurbiprofen injection product using inhibition of protein binding by lipid emulsion in rats

Flurbiprofen‐axetil (FP‐ax), a bolus injection product of a non‐steroidal anti‐inflammatory drug (NSAID), is a prodrug of flurbiprofen, an NSAID. As flurbiprofen strongly binds to site II of human serum albumin (HSA), the free (unbound) concentration of flurbiprofen after injection of FP‐ax is low. We have examined the inhibitory effect of free fatty acid (FFA), a binding inhibitor for site II of HSA, on the binding of flurbiprofen in‐vitro and in‐vivo by ultrafiltration, to establish an effective dosage of FP‐ax. In‐vitro, fatty acid mixtures (FAs) inhibited the binding of flurbiprofen to rat serum albumin. The free fraction of flurbiprofen was remarkably increased by FAs in rat serum. In‐vivo, FP‐ax was injected into a control group (low FFA concentration in serum) and a lipid emulsion group (high FFA concentration in serum). The area under the curve of the free concentration of flurbiprofen during the alpha phase and the distribution volume of the central compartment of flurbiprofen were significantly higher in the lipid emulsion group than the control group (5.0‐ and 1.2‐times, respectively). When FP‐ax was administered at high FFA concentration, the free concentration of flurbiprofen and distribution of flurbiprofen to tissues increased transiently. This administration method may be useful for patients with cancer pain, having a potent analgesic effect.

Interaction of benzothiadiazides with human serum albumin studied by dialysis and spectroscopic methods

The interaction of a series of benzothiadiazides with human serum albumin (HSA) was investigated by equilibrium dialysis (ED) and spectroscopic methods including circular dichroism (CD). The primary binding site of benzothiadiazides was designated site II, the diazepam site on the HSA molecule, as indicated by displacement experiments using different site-selective probes. Tyrosine and lysine amino acid residues were probably involved in the binding site of these compounds to HSA. Both electrostatic and hydrophobic interactions were found to play a role in the binding of these compounds to HSA. Among the compounds tested, chlorothiazide had the highest affinity (K1 = 5.5 × 104M−1, K2 = 5.8 × 103 M−1).The primary binding affinity of the compounds for HSA was of the order: chlorothiazide > cyclopenthiazide > polythiazide > ethiazide > trichlormethiazide = methyclothiazde > hydrochlorothiazide. Binding was insensitive to the N-B transition of HSA. The binding site is proposed to consist of a cationic site on the surface of the HSA molecule with a hydrophobic crevice to accommodate the aromatic ring of the compounds. Positions 3 and 7 of the benzothiadiazide molecule is thought to affect the binding affinity to HSA.

Competitive Displacement of Serum Protein Binding of Radiopharmaceuticals with Amino Acid Infusion Investigated with N-Isopropyl-p-123I-Iodoamphetamine

When a therapeutic drug is competitively displaced at the binding sites of serum proteins, the free fraction of the drug will be increased, with an increase in the manifestation of pharmacologic properties. In the case of molecular imaging probes, total clearance and tissue distribution are increased in such circumstances. The aim of this study was to observe the increase in cerebral accumulation of N-isopropyl-p-123I-iodoamphetamine (123I-IMP) using the protein-binding displacement method with amino acid infusion. Methods: 123I-IMP binding to human serum was investigated and identified. In addition, protein-binding sites and the specific binding sites of human serum albumin (HSA) and α1-acid glycoprotein (AGP) were examined by ultrafiltration. Then, serum-binding sites and the displacement effects of amino acid infusion, including Proteamin 12X Injection and Kidomin, were confirmed in vitro. Subsequently, displacement of 123I-IMP serum protein binding with Proteamin amino acid infusion was tested in monkeys. A scintigraphic study of 123I-IMP in monkeys loaded with or without Proteamin was performed, and time–activity-curves of 123I-IMP brain accumulation in monkeys were evaluated. Results: 123I-IMP was bound to HSA site II and AGP to nearly equal extents. Compared with control conditions, loading with Proteamin and Kidomin markedly increased free fractions of binding site markers for HSA site II (14C-diazepam: 0.95% ± 0.04% for control, 1.40% ± 0.06% for Proteamin, 1.62% ± 0.05% for Kidomin) and AGP (3H-propranolol: 10.60% ± 0.32% for control, 13.18% ± 0.14% for Proteamin, 13.82% ± 0.72% for Kidomin). Amino acid infusions were thus suitable for use as displacers for binding site II and AGP. With use of Proteamin amino acid infusion to displace protein binding, the free fraction of 125I-IMP (14.95% ± 0.74%) was significantly increased in serum (19.24% ± 0.87%). In a 123I-IMP scintigraphic study of monkeys, average cerebral uptake in 2 monkeys increased by 1.34-fold with Proteamin. Our findings suggested that Proteamin treatment increased the free fraction of 123I-IMP, yielding rapid and pronounced cerebral accumulation in vivo. Conclusion: Amino acid infusion can improve brain accumulation by competitive displacement of serum protein binding in vivo. Further similar studies are needed with other radiopharmaceuticals.