Measurements of Strong-Interaction Effects in Kaonic-Helium Isotopes at Sub-eV Precision with X-Ray Microcalorimeters.

We have measured the 3d→2p transition x rays of kaonic ^{3}He and ^{4}He atoms using superconducting transition-edge-sensor microcalorimeters with an energy resolution better than 6 eV (FWHM). We determined the energies to be 6224.5±0.4(stat)±0.2(syst) eV and 6463.7±0.3(stat)±0.1(syst) eV, and widths to be 2.5±1.0(stat)±0.4(syst) eV and 1.0±0.6(stat)±0.3(stat) eV, for kaonic ^{3}He and ^{4}He, respectively. These values are nearly 10 times more precise than in previous measurements. Our results exclude the large strong-interaction shifts and widths that are suggested by a coupled-channel approach and agree with calculations based on optical-potential models.

Feasibility and Outcomes of Direct Dual Portal Vein Anastomosis in Living Donor Liver Transplantation Using the Right Liver Graft With Anatomic Portal Vein Variations.

BACKGROUND: Portal vein (PV) reconstruction is a crucial factor in successful living donor liver transplantation (LDLT). In LDLT using the right liver grafts with anatomic PV variations, we sometimes encounter dual PV anastomosis. In this study we describe PV variations of donor liver in detail as well as our experiences with PV reconstruction in right liver grafts with PV variations. METHODS: We performed LDLT in 149 recipients between 2002 and 2016. PV variations of donor liver were classified into 3 major anatomic patterns, and we retrospectively analyzed the procedure and postoperative complications of PV anastomosis. RESULTS: PV variations in donor livers were classified as type A (normal type) in 125 patients, type B (trifurcation type) in 7 (4.7%), and type C (caudal origin of the right posterior branch) in 17 (11.4%). Among 75 right liver grafts, 10 (13.3%) had anatomic PV variations. In 9 of 10 recipients, dual PV of the graft were anastomosed to dual PV branches of the recipient in direct end-to-end fashion. In the remaining recipient, the posterior portal branch of the graft was anastomosed to the recipient portal trunk through the interposed venous graft in end-to-end fashion and the anterior portal branch of the graft was anastomosed to the side wall of the interposed venous graft. These 10 recipients did not develop any postoperative complications associated with PV anastomosis, although 3 of the 149 recipients (2.0%) developed complications associated with PV anastomosis, such as thrombosis and necrosis. CONCLUSION: Dual PV anastomosis of the right liver graft is safe and feasible in LDLT, even in anatomic PV variations.

Intravenous Administration of Tacrolimus Stabilizes Control of Blood Concentration Regardless of CYP3A5 Polymorphism in Living Donor Liver Transplantation: Comparison of Intravenous Infusion and Oral Administration in Early Postoperative Period.

BACKGROUND: We compared achievement rate of sufficient tacrolimus blood concentration in the early postoperative period and incidence of acute cellular rejection within 1 month after living donor liver transplantation (LDLT) between tacrolimus intravenous (IV) and oral administration groups. METHODS: From October 2005 to November 2016, 61 LDLT patients administered tacrolimus, who could be genotyped for CYP3A5*3 and *1, were chosen from the electronic record database. The patients were then divided into the 2 groups (an IV group [n = 38] and an oral group [n = 23]). We defined patients with 1*1 or *1*3 as expressors and those with *3*3 as nonexpressors. Sufficient trough level tacrolimus blood concentration on postoperative day (POD) 3 was defined as 10-20 ng/mL. RESULTS: Comparable concentrations were seen between the 2 groups, with mean blood concentration 13.7 ± 8.5 ng/mL in the oral group and 15.2 ± 4.3 ng/mL in the IV group. Achievement rate of sufficient tacrolimus concentration on POD 3 was significantly higher in the IV group than in oral group: 97% (37 of 38) vs 65% (15 of 23), respectively (P = .001). When we focused on achievement rate in the oral group according to CYP3A5 polymorphism, the frequency of expressors (17%) was significantly lower than that of nonexpressors (82%) (P = .016). However, in the IV group this negative influence was totally eliminated, resulting in high achievement rates regardless of CYP3A5 polymorphism. In terms of incidence of acute cellular rejection, there was no significant difference between the 2 groups (IV 32% vs oral 17%, P = .250). CONCLUSION: IV administration of tacrolimus allowed us to obtain more stable control of blood concentration regardless of CYP3A5 genotype.

Early Enteral Feeding of Daikenchuto Stimulates Early Bowel Movement With Increased Portal Venous Blood Flow After Living Donor Liver Transplantation.

Daikenchuto (DKT), a Japanese Kampo medicine, had been reported to increase small intestinal blood flow after liver resection. The aim of this study was to evaluate the effects of early enteral feeding of DKT on portal venous flow and early bowel movement after living donor liver transplantation (LDLT) in an attempt to clarify whether these effects on bowel motility can prevent bacterial and/or fungal translocation. MATERIALS AND METHODS: Our prospective study included the consecutive 16 LDLT recipients at Mie University Hospital between June 2006 and September 2009. Sixteen patients were divided into the 2 groups according to enteral feeding starting postoperative day (POD) 1: 8 patients in DKT (15 g/d) administration (DKT group, for 1 week) and 8 patients in tepid water administration (non-DKT group, for 1 week). Portal venous flow, portal venous pressure, presence of fungal infection (serum level of ß-D-glucan and fungal polymerase chain reaction assay), time to first food intake, and time to first defecation were serially examined. RESULTS: Portal venous flow (mean [SD] velocity) was significantly increased in DKT group compared with non-DKT group: 47.5 (12.9) vs 31.8 (15.4) (P = .04) on POD 1, 46.8 (11.5) vs 28.8 (12.5) (P = .03) on POD 3, and 42.3 (17.2) vs 25.2 (9.0) (P = .05) on POD 5. However, mean (SD) portal venous pressures did not significantly change between the 2 groups. Between the 2 groups (DKT vs non-DKT), the day of first oral intake was not significantly different: 6.9 (2.5) vs 11.3 (8.7) (P = .061), but the mean (SD) day of first defecation was significantly shorter in the DKT group: 3.9 (1.1) vs 5.5 (2.6) (P = .02). Although fungal polymerase chain reaction assay was not significantly different between the 2 groups (4 vs 4 positive cases), the mean (SD) serum levels of ß-D-glucan were significantly lower in the DKT group than in the non-DKT group: 9.0 (7.4) vs 18.4 (15.9) pg/mL (P = .04). CONCLUSION: Early enteral feeding of DKT after LDLT increased portal vein blood flow without increasing portal vein pressure and stimulated early bowel movement, which in turn might prevent fungal translocation.

De Novo Malignancy Following Adult-to-Adult Living Donor Liver Transplantation Focusing on Posttransplantation Lymphoproliferative Disorder.

OBJECTIVE: In patients with living donor liver transplantation (LDLT), late-onset complications sometimes develop because of long-term use of immunosuppressive drugs. One of the immunosuppressive drug-related complications is de novo malignancies resulting in reduced survival. PATIENTS AND METHODS: Among 153 patients undergoing LDLT, we retrospectively reviewed the medical records of 97 adult recipients (February 2002 to May 2017), who had been followed-up at our hospital for more than one year after LDLT. The median age was 52 years old (20-70) and the median observational period was 6.9 years (2.4-15.3). RESULTS: De novo malignancy after adult LDLT developed in 11.3% (11/97) of patients, including posttransplantation lymphoproliferative disorder (PTLD) (n = 4) (2 in the brain and 2 in abdominal lymph nodes), lung cancer (n = 1), pancreatic cancer (n = 1), gastric cancer (n = 1), laryngeal cancer (n = 1), lower gingival cancer (n = 1), bladder cancer (n = 1), and melanoma (n = 1). Age at cancer diagnosis ranged from 36 to 70 years old with an average age of 61 years. The interval from LDLT to cancer diagnosis was 8.3 years (3.9-12.2). Four patients (36.6%) including PTLD (n = 2), lung cancer (n = 1), and pancreatic cancer (n = 1) died of cancer and all of them were diagnosed with cancer within 10 years after LDLT. Six patients were diagnosed with cancer more than 10 years after LDLT and all of them survived after treatment of cancer. CONCLUSION: De novo malignancy was found in 11.3% of LDLT patients, and more than half of this population subset developed tumors 10 years after LDLT. Long-term close follow-up should be performed by taking any kinds of de novo malignancy into consideration.

Biliary Complications During and After Donor Hepatectomy in Living Donor Liver Transplantation Focusing on Characteristics of Biliary Leakage and Treatment for Intraoperative Bile Duct Injury.

BACKGROUND: Biliary complication is one of the major donor complications during and after hepatectomy in living donor liver transplantation (LDLT). We evaluated risk factors for donor biliary complication in adult-to-adult LDLT. PATIENTS AND METHODS: From March 2002 to November 2016, 126 consecutive patients who underwent donor hepatectomy in adult-to-adult LDLT were divided into 2 groups according to biliary compilations: nonbiliary complication (non-BC) group (n = 114) and biliary complication (BC) group (n = 12). RESULTS: Among 126 donor hepatectomies, 35 patients (28%) experienced perioperative complications, including 10 (7.9%) with Clavien-Dindo classification grade III. Biliary complications occurred in 12 patients (9.5%): bile leakage in 10 and intraoperative bile duct injury in 2. Additional computed tomography- and/or ultrasound-guided drainage or exchange of original drain was required in 7 patients. In comparison between BC and non-BC groups, future remnant liver volume was significantly higher in the BC group than in the non-BC group (63% vs 40%; P = .02). In multivariate analysis, larger future remnant liver volume (P = .005) and shorter operating time (P = .02) were identified as independent risk factors for biliary complications. We had 2 patients with intraoperative bile duct injury: both were successfully treated by duct-to-duct biliary anastomosis with insertion of biliary stent or T-tube. CONCLUSION: Large remnant liver volume was a significant risk factor for biliary complications, especially biliary leakage, after donor hepatectomy. For intraoperative bile duct injury, duct-to-duct anastomosis with biliary stent is a feasible method to recover.

Recombinant Human Soluble Thrombomodulin Attenuates Hepatic Ischemia and/or Reperfusion Injury by Inhibiting Leukocyte Accumulation in Mice With Normal and Fatty Liver.

In an attempt to increase the number of donor livers, there has been an increased use of marginal donor livers, such as steatotic (fatty) livers that increase susceptibility to ischemia and reperfusion injury (IRI). Inflammatory cell accumulation has a greater role in IRI in steatotic liver than in normal liver. Although the recombinant human soluble thrombomodulin (rhsTM) attracts attention as a new treatment for disseminated intravascular coagulation, the therapeutic efficacy of rhsTM in hepatic IRI remains uncertain, especially in fatty livers. We aimed to demonstrate the effect of rhsTM on hepatic IRI using well-established in vivo experimental models with steatotic liver. METHODS: C57/BL6 mice were divided into 2 groups: normal liver (NL) group and fatty liver (FL) group, in which the steatotic liver was induced by high-fat diet for 9 weeks. The mice in the NL and FL groups were premedicated with venous injection of rhsTM (TM) or saline (Control) as control groups. All 4 groups (NL-Control vs NL-TM, FL-Control vs FL-TM) were subjected to partial hepatic warm ischemia followed by reperfusion. RESULTS: rhsTM significantly attenuated liver injury in the FL group as well as the NL group, as evidenced by transaminase levels and histologic finding after hepatic IRI. rhsTM remarkably decreased the accumulation of inflammatory cells, such as macrophages and neutrophils, in both NL and FL tissue after IRI. Furthermore, rhsTM depressed mRNA and protein expressions of adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 in both NL and FL groups after IRI. CONCLUSION: Our results demonstrate that rhsTM has a protective effect on fatty liver as well as normal liver after hepatic IRI. They also suggest that rhsTM contributes to attenuation of leukocyte accumulation caused by depressing expressions of adhesion molecules that facilitate accumulation of leukocytes in liver tissue in hepatic IRI.

Isolated Biliary Fistula After Donor Right Hepatectomy and Its Novel Interventional Treatment: Isolated Liver-Punctured Drainage.

Isolated biliary leakage is difficult to manage, and afflicted patients often develop refractory fistula. The present case was a 43-year-old male donor whose wife developed acute fulminant liver failure. Computed tomography (CT) volumetry showed that the estimated remnant liver volume was only 394 mL (31%) if his right lobe would be harvested. Since remnant liver volume was marginal, our proposed cut line for the right hepatectomy was set in order to preserve branches of the middle hepatic vein draining segments 4b+8 and 5. Right hepatectomy was performed, but on postoperative day 14, the donor developed fever and right back pain, and enhanced CT showed a 6 cm intra-abdominal abscess at the site of cutting, and we diagnosed it as an isolated biliary fistula since the isolated segment 5/8 was receiving arterial blood supply and exhibiting regrowth. A transabdominal abscess drainage was performed, after which the patient lost 30 to 50 mL of bile juice per day in drainage until 2 months after the drainage procedure. Ethanol injection, acetic acid injection, and transarterial or portal embolization for the isolated liver were proposed, but these all were impossible to carry out because there were no accessible routes. Thus, re-abscess drainage with a 7-French drainage catheter was performed through the isolated liver on postoperative day 53, and the isolated functional liver was punctured to induce liver atrophy. After this drainage, the isolated liver started to shrink and bile output had been stopped. In conclusion, our punctured-liver drainage could be effective for the treatment of isolated biliary fistula, allowing physicians to avoid an invasive additional liver resection or other invasive percutaneous approach using chemical reagents.

Pretransplant Serum Hyaluronic Acid Can Be a Biomarker as a Prognostic Predictor in Adult-to-Adult Living Donor Liver Transplantation.

OBJECTIVE: The goal of this study was to evaluate whether pretransplant serum hyaluronic acid (HA) levels can predict outcomes after adult-to-adult living donor liver transplantation (LDLT). METHODS: In study I, 21 patients who underwent LDLT (March 2002-February 2004) were divided into 2 groups: the H-I group (HA ≥500 ng/mL; n = 12) and the L-I group (HA <500 ng/mL; n = 9). The influence of pretransplantation HA levels on short-term surgical outcome was investigated. In study II, 77 LDLT patients (May 2004-December 2014) were also divided into 2 groups: the H-II group (HA ≥500 ng/mL; n = 40) and the L-II group (HA <500 ng/mL; n = 37). We compared long-term survival and investigated prognostic factors. RESULTS: In study I, HA levels significantly decreased after LDLT, and those in the H-I group were significantly higher compared with the L-I group at 1, 3, 5, and 7 days after LDLT. There were significant differences in postoperative peak total bilirubin levels (H-I vs L-I, 17.2 vs 6.2 mg/dL; P = .013), peak ascitic fluid volume (1327 vs 697 mL/d; P = .005), and the hepatocyte growth factor levels at 3 days after LDLT (1879 vs 1092 pg/mL; P = .03). In study II, the 1- and 5-year survival rates were significantly lower in the H-II group than in the L-II group (H-II vs L-II, 65.0% and 48.5% vs 86.5% and 80.8%; P = .004). In multivariate analysis, significant prognostic factors were preoperative HA ≥500 ng/mL (P = .004) and graft to recipient body weight ratio <0.8 (P = .042). CONCLUSIONS: Preoperative HA level can be a prognostic risk factor. Patients with high HA levels are vulnerable and should be carefully managed after LDLT.

Influence of Angiotensin-converting Enzyme Genetic Polymorphism on Late Renal Dysfunction After Adult-to-adult Living-donor Liver Transplantation.

BACKGROUND: Late renal dysfunction (LRD) is known to be one of the most important complications to affect long-term outcome after living-donor liver transplantation (LDLT). The relationship between angiotensin-converting enzyme insertion (I)/deletion (D) gene polymorphism and renal function after LDLT are still unknown. The aim of this study was to elucidate the risk factors for LRD after LDLT, focusing on ACE gene polymorphism. MATERIALS AND METHODS: Among the 94 recipients who underwent adult-to-adult LDLT between March 2002 and September 2009, the total number of subjects who survived more than 1 year after LDLT and in whom angiotensin-converting enzyme genotype could be measured was 64. LRD was defined as estimated glomerular filtration rate level less than 60 mL/min/1.73 m(2) at any point after 1 year from undergoing LDLT. RESULTS: LRD was found in 24 patients (37.5%). The incidence of LRD was significantly higher in D/D type than in I/I or I/D type: 85.7% (6/7) vs. 42.1% (8/19), 35.7% (10/38) (P = .010). Preoperative estimated glomerular filtration rate was significantly lower in D/D type than in I/I, I/D types, and postoperatively they were significantly lower in D/D type at 2, 3, and 4 years after LDLT. By multivariate analysis, age and hypertension were the independent risk factors for LRD. The 10-year survival rate was much lower in the recipients with LRD than in those without LRD at 66.7% versus 87.5%, respectively (P = .053). CONCLUSION: In conclusion, age and hypertension were determined as significant independent risk factors for LRD after adult-to-adult LDLT, and the recipients with D/D genotype should be strictly cared for the development of LRD.

Living Donor Liver Transplantation for Biliary Atresia With Severe Preduodenal Portal Vein Stricture: Success and Pitfall of Portal Vein Reconstruction.

BACKGROUND: We report a rare case of 10-month-old female who underwent living donor liver transplantation (LDLT) for syndromic biliary atresia with preduodenal portal vein (PV) and its severe stricture owing to the previous Kasai portoenterostomy. Because we successfully performed "left at right liver transplantation (LAR-LT) and graft rerotation" in this case, we are present tips and pitfalls for this operation. METHODS: Preoperative computed tomography scan showed that her preduodenal PV was stenotic from the confluence of the superior mesenteric vein and splenic vein to hepatic hilum, which made us consider the necessity of ≥3 cm interposition vein graft to complete a safe PV anastomosis. To reduce a gap between donor and recipient's PV, we decided to put a left lateral section graft at the right subphrenic space called left-at-right liver transplantation. Thus, LDLT was performed with an identical lateral sectional graft from her father. After total hepatectomy, we implanted a graft in her right subphrenic space, and anastomosed the donor left hepatic vein to her inferior vena cava. Then, we anastomosed an interposition graft harvested from her left internal carotid vein to her PV. RESULTS: Even after reflowing PV flow, because the duodenum compressed the interposition vein graft, PV flows were totally insufficient. Therefore, we flipped a liver graft 180° from right to left upper abdominal cavity, which could reduce the gap between PVs and acceptable PV flow was obtained. CONCLUSIONS: In the present case, LAR-LT could reduce the distance of PVs. In addition, our rerotation method could be useful to alleviate tension on the PV anastomosis caused by preduodenal PV.

Impact of Splenectomy Just Before Partial Orthotopic Liver Transplantation Using Small-for-Size Graft in Rats.

BACKGROUND: Portal hypertension is a serious obstacle of partial orthotopic liver transplantation (POLT) with the use of small-for-size liver graft. Several therapeutic strategies including surgical innovations and pharmacological agents to reduce the portal hypertension have been developed. Splenectomy (SP) on POLT is one of surgical procedures to reduce portal pressure. We previously reported a dual cytoprotective mechanism of SP just before POLT, using small-for-size liver graft in a rat model. However, the best timing of SP during POLT has been unclear. We compared liver functions between SP just before and after POLT, using small-for-size rat liver grafts. METHODS: With the use of small-for-size liver grafts (20%) in rats previously reported, the rats were assigned to 2 groups: the pre-SP group (SP just before POLT) and the post-SP group (SP just after POLT). Liver tissues and blood were sampled at 6 and 24 hours after POLT for several liver function tests. RESULTS: The serum alanine aminotransferase levels at 24 hours after POLT were significantly decreased in the pre-SP group compared with the post-SP group (226 ± 78 vs 340 ± 71 IU/L). The infiltrations of neutrophil at 6 hours and ED-1-positive cells at 24 hours were significantly suppressed in the pre-SP group compared with the post-SP group. Serum hyaluronic acid levels, indicating attenuation of endothelial damage, were lower in the pre-SP group than in the post-SP group. CONCLUSIONS: SP before POLT, which directly eliminates splenic inflammatory leukocytes, inhibits inflammatory leukocyte infiltration, which leads to impaired liver function as compared with SP after POLT.

Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model.

BACKGROUND AND PURPOSE: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. EXPERIMENTAL APPROACH: Binding assay with [(3)H]-TAK-242 and nuclear factor-kappaB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. KEY RESULTS: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. CONCLUSIONS AND IMPLICATIONS: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis.

Inhibitory effects of small-molecule CCR5 antagonists on human immunodeficiency virus type 1 envelope-mediated membrane fusion and viral replication.

We established a human immunodeficiency virus type 1 (HIV-1) envelope (Env)-mediated membrane fusion assay and examined the small-molecule CCR5 antagonist TAK-779 and its derivatives for their inhibitory effects on HIV-1 Env-mediated membrane fusion and viral replication. The membrane fusion assay is based on HIV-1 long terminal repeat-directed beta-D-galactosidase reporter gene expression in CD4- and CCR5-expressed HeLa (MAGI-CCR5) cells after cocultivation with effector 293T cells expressing HIV-1 Env. Inhibition of HIV-1 replication was also determined in MAGI-CCR5 cells infected with the corresponding cell-free HIV-1. TAK-779 effectively suppressed R5 HIV-1 (strain JR-FL) Env-mediated membrane fusion as well as viral replication. Its 50% inhibitory concentrations (IC(50)s) for membrane fusion and viral replication were 0.87 +/- 0.11 and 1.4 +/- 0.1 nM, respectively. These values corresponded well to the IC(50) for (125)I-RANTES (regulated on activation, T cell expressed, and secreted) binding to CCR5 (1.4 nM). The inhibitory effects of 18 TAK-779 derivatives on membrane fusion differed from one compound to another. However, there was a close correlation among their inhibitory effects on membrane fusion, viral replication, and RANTES binding. The correlation coefficient between their IC(50)s for membrane fusion and viral replication was 0.881. Furthermore, since this assay depends on Env expressed in the effector cells, it is also applicable to the evaluation of CXCR4 antagonists. These results indicate that the HIV-1 Env-mediated membrane fusion assay is a useful tool for the evaluation of entry inhibitors.

Studies on anti-MRSA parenteral cephalosporins. IV. A novel water-soluble N-phosphono type prodrug for parental administration.

A systematic approach for improving the water-solubility of anti-MRSA (methicillin-resistant Staphylococcus aureus) cephalosporin derivatives is described. We first tried to improve the water-solubility of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-fluoromethoxyiminoacetamido]-3-[(E)-2-(1-methylimidazo[1,2-b]pyridazinium-6-yl)thiovinyl]-3-cephem-4-carboxylate (1a) by substitution of the C-3' pharmacophore. Replacement of the C-3' pharmacophore with a 1-methyl-4-pyridinio group improved the water-solubility without decreasing the anti-MRSA activity. Furthermore, we applied the N-modified prodrug strategy to the C-7 acyl group in order to enhance the water-solubility drastically. Among the compounds prepared, the N-phosphono type prodrugs 2a(1-methylimidazo[1,2-b]pyridazinium derivative) and 2b (1-methyl-4-pyridinio derivative) showed water-solubility appropriate for a product intended for intravenous injection and in vivo anti-MRSA activity comparable to that of vancomycin.

Studies on anti-MRSA parenteral cephalosporins. III. Synthesis and antibacterial activity of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-alkoxyiminoacetamido-3.

In the course of our exploration for a novel cephalosporin derivative having excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), we modified the C-3 linked spacers of cephem derivatives bearing a 1-methylimidazo[1,2-b]pyridazinium-6-yl group at the C-3' position and 2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-cyclopentyloxy-iminoacetyl group at the C-7 position. The optimal spacers were the (E)-2-vinyl and (E)-2-thiovinyl groups seen in 19a and 29aa, respectively. Their anti-MRSA activity was 16 to 32 times as potent as that of cefozopran (CZOP). Focusing on the (E)-2-vinyl and (E)-2-thiovinyl spacers, we further modified the alkoxyimino groups in the C-7 acyl moiety and the 1-alkylimidazo[1,2-b]pyridazinium moieties at the C-3' position and investigated the structure-activity relationships (SAR) of the derivatives. Consequently, we selected 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-fluoromethoxyiminoacetamido]-3-[(E)-2-(1-methylimidazo[1,2-b]pyridazinium-6-yl)thiovinyl]-3-cephem-4-carboxylate (29ca) as a new anti-MRSA parenteral cephalosporin candidate for further biological evaluation. The selected 29ca showed anti-MRSA activity comparable to that of vancomycin (VCM) both in vitro and in vivo, high affinity (IC50)=2.7 microg/ml) for penicillin binding protein 2' (PBP2') of MRSA and potent activity against Gram-negative bacteria as well.

[Prospect for the development of antibacterial agents with new mode of action].

Since there are no antibacterial agents to which there are no resistant bacteria, antibacterial agents with new mode of action are strongly demanded. Here, I describe the recent research for new targets and novel compounds that are active against the new targets. Proteins such as DnaA involved in DNA replication, deformylase and aminoacyl tRNA synthetase involved in protein synthesis, Mur peptide synthetases of peptidoglycan synthesis, FtsZ in cell division, two-component signaling system, quarum sensing system and efflux pumps are targeted and several inhibitors against deformylase, two-component signaling system and efflux pumps are reported. Unknown essential genes in bacteria are also explored in order to find new targets.

Establishment of a CCR5-expressing T-lymphoblastoid cell line highly susceptible to R5 HIV type 1.

The beta-chemokine receptor CCR5 is considered to be an attractive target for inhibition of CCR5-using (R5 or macrophage-tropic) HIV-1. However, R5 HIV-1 cannot replicate in CD4+ T cell or monocyte lines because of the lack of CCR5 expression on their surface, which apparently hampers discovery and development of effective CCR5 antagonists against HIV-1 replication. In this study, we have established the CCR5-expressing T cell line MOLT-4/CCR5, highly permissive to the replication of R5 HIV-1. The cells express a considerable amount of CCR5 on their surface. When the cells were infected with the R5 HIV-1 strains Ba-L and JR-FL, the virus-induced cytopathic effect (syncytium formation) was observed, and the cells produced large amounts of HIV-1 p24 antigen in the culture supernatants. The analyses of progeny viruses for their coreceptor use and gp120 V3 nucleotide sequence revealed that they were R5 HIV-1. The parental cell line MOLT-4 was much less susceptible to Ba-L and totally insusceptible to JR-FL. Furthermore, MOLT-4/CCR5 cells could support the replication of an R5 clinical isolate, but MOLT-4 cells could not. When TAK-779, a novel small-molecule nonpeptide CCR5 antagonist, was examined for its inhibitory effect on R5 HIV-1 replication in MOLT-4/CCR5 cells, the compound displayed potent antiviral activity, as demonstrated in peripheral blood mononuclear cells. These results indicate that the established cell line will be an extremely useful tool for experiments with R5 HIV-1.

Studies on anti-MRSA parenteral cephalosporins. I. Synthesis and antibacterial activity of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)- hydroxyiminoacetamido]-3-(substituted imidaz.

In order to improve the antibacterial activity of cefozopran (CZOP) against methicillin-resistant Staphylococcus aureus (MRSA), we initiated chemical modification to introduce a 2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-hydroxyimino acetyl group at the C-7 position and a 3- or 6-substituted imidazo[1,2-b]pyridazinium or 5-substituted imidazo[1,2-a]pyridinium group at the C-3' position. Although this approach successfully enhanced the anti-MRSA activity of CZOP two to eight times, a slight decrease in the activity against Gram-negative bacteria including Pseudomonas aeruginosa was involved. Among the novel derivatives, 3-(6-aminoimidazo[1,2-b]pyridazinium-1-yl)methyl-7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)hydroxyiminoacetamido]-3-cephem-4-carboxylate (44a) showed an excellent balance of activity against MRSA and Gram-negative bacteria.

Studies on anti-MRSA parenteral cephalosporins. II. Synthesis and antibacterial activity of 7beta-[2-(5-amino-1,2,4- thiadiazol-3-yl)-2(Z)-alkoxyiminoacetamido]-3-(substituted imidaz.

In an effort to discover a novel cefozopran (CZOP) derivative having excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), we performed chemical modification of the alkoxyimino moiety and imidazo[1,2-b]pyridazinium group of CZOP. Among the prepared compounds, the cyclopentyloxyimino derivative 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-cyclopentyloxyiminoacetamido]-3-(3,6-diaminoimidazo[1,2-b]pyridazinium-1-yl)methyl-3-cephem-4-carboxylate (20 g) showed the most potent anti-MRSA activity, reflecting its high affinity (IC50 = 1.6 microg/ml) for penicillin binding protein 2' (PBP2'), although its anti-MRSA activity was slightly inferior to that of vancomycin (VCM). In experimental systemic infection in mice, however, 20 g showed activity comparable to that of VCM against MRSA. In addition, 20 g showed activity similar or slightly inferior to that of CZOP against Pseudomonas aeruginosa both in vitro and in vivo. Considering its favorable antibacterial activity profile, 20 g was considered to be the most promising CZOP derivative for further studies.