Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies.

BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.

(67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of (67)Ga-labeled Fabs for low renal radioactivity levels.

Dai nippon printing co., ltd, Medi-Ca AC for enumeration of aerobic bacteria. Performance tested method 041302.

A ready-made dry medium method for aerobic count, the MediCa AC method, was compared to the AOAC Official Method 966.23, Microbiological Methods, for seven different heat-processed meat matrixes: cooked roast beef, Chinese barbecued pork (barbecued pork seasoned with honey-based sauce), bacon, cooked ham, frankfurter (made from beef and pork), and boiled and cooked pork sausage. The 95% confidence interval for the mean difference between the two methods at each contamination level for each matrix fell within the range of -0.50 to 0.50, and no statistical difference was observed at all three contamination levels for five matrixes. These results demonstrate that the Medi-Ca AC method is a reasonable alternative to the AOAC 966.23 method for cooked meat products.

Chemical design of (99m)Tc-labeled probes for targeting osteogenic bone region.

Radionuclide bone imaging using polynuclear (99m)Tc complexes of bisphosphonates is the most common clinical practice in nuclear medicine. However, the improvement in the contrast between normal and osteogenic bone regions has been required. Herein we reported a new (99m)Tc-labeled compound considering the increased vascular permeability of osteogenic region. We selected penta-d-Asp as both a targeting motif to hydroxyapatite (HA) and a molecular size modifier, and two penta-d-Asp molecules were conjugated with the two carboxylate residues of ethylene dicysteine (EC) selected as the (99m)Tc chelating moiety to prepare EC-[(d-Asp)5]2. The molecular size, HA binding, and pharmacokinetics of (99m)Tc-EC-[(d-Asp)5]2 in normal mice and model rats bearing osteogenic tumor were compared to those of (99m)Tc-MDP and (99m)Tc-EC with one (d-Asp)5 motif, (99m)Tc-EC-(d-Asp)5. The molecular size of (99m)Tc-EC-[(d-Asp)5]2 was higher than that of (99m)Tc-MDP and (99m)Tc-EC-(d-Asp)5 when determined by permeability of the (99m)Tc-compounds through a membrane filter (10 kDa). The HA binding of (99m)Tc-EC-[(d-Asp)5]2 was higher than and similar to that of (99m)Tc-EC-(d-Asp)5 and (99m)Tc-MDP. (99m)Tc-EC-[(d-Asp)5]2 exhibited significantly lower accumulation in normal bone of mice than did (99m)Tc-MDP. In osteogenic tumor bearing model rats, (99m)Tc-EC-[(d-Asp)5]2 accumulated in the osteogenic and normal bone region similar to and lower than (99m)Tc-MDP, respectively. Although further studies including the chain length of d-Asp are required, these findings indicated that the present chemical design of (99m)Tc-labeled probe would be applicable to develop (99m)Tc-labeled probes for selective imaging of osteogenic bone region as well as develop therapeutic agents using therapeutic radionuclides such as (90)Y, (177)Lu, (186)Re, or (188)Re and cytotoxic agents to osteogenic bone tumor region.

Two new monoterpene glycosides from Qing Shan Lu Shui tea with inhibitory effects on leukocyte-type 12-lipoxygenase activity.

We evaluated the inhibitory effect of 12 Chinese teas on leukocyte-type 12-lipoxygenase (LOX) activity. Tea catechins such as epigallocatechin gallate have been known to exhibit leukocyte-type 12-LOX inhibition. Qing Shan Lu Shui, which contains lower catechin levels than the other tested teas, suppressed leukocyte-type 12-LOX activity. To characterize the bioactive components of Qing Shan Lu Shui, leukocyte-type 12-LOX inhibitory activity-guided fractionation of the aqueous ethanol extract of the tea was performed, resulting in the isolation of two new monoterpene glycosides: liguroside A (1) and B (2). The structures of compounds 1 and 2 were characterized as (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl)-(1″→3')-(4'″-O-trans-p-coumaroyl)-ß-D-glucopyranoside and (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octa-dienyl- O-(α-L-rhamnopyranosyl)-(1″→3')-(4'″-O-cis-p-coumaroyl)-ß-D-glucopyranoside, respectively, based on spectral and chemical evidence. Ligurosides A (1) and B (2) showed inhibitory effects on leukocyte-type 12-LOX activity, with IC50 values of 1.7 and 0.7 µM, respectively.

Pharmacokinetics of Bictegravir in Older Japanese People Living with HIV-1.

Bictegravir (BIC) is an integrase strand transfer inhibitor widely used in the treatment of HIV-1. Although its potency and safety have been demonstrated in older patients, pharmacokinetics (PK) data remain limited in this patient population. Ten male patients aged 50 years or older with suppressed HIV RNA on other antiretroviral regimens were switched to a single-tablet regimen of BIC, emtricitabine, and tenofovir alafenamide (BIC+FTC+TAF). Four weeks later, plasma samples were collected at 9 time points for PK. Safety and efficacy were also assessed up to 48 weeks. The median age (range) of patients was 57.5 (50 to 75) years. Although 8 (80%) had lifestyle diseases requiring treatment, no participants had renal or liver failure. Nine (90%) were receiving dolutegravir-containing antiretroviral regimens at entry. The trough concentration of BIC was 2,324 (1,438 to 3,756) (geometric mean [95% confidence interval]) ng/mL, which was markedly above the 95% inhibitory concentration of the drug (162 ng/mL). All PK parameters, including area under the blood concentration-time curve and clearance, were similar to those in young HIV-negative Japanese participants in a previous study. No correlations between age and any PK parameters were observed in our study population. No participant experienced virological failure. Body weight, transaminase, renal function, lipid profiles, and bone mineral density were unchanged. Interestingly, urinary albumin was decreased after switching. PK of BIC was not affected by age, indicating that BIC+FTC+TAF may be safely used in older patients. IMPORTANCE BIC is a potent integrase strand transfer inhibitor (INSTI), widely used for the treatment of HIV-1 as part of a once-daily single-tablet regimen that includes emtricitabine and tenofovir alafenamide (BIC+FTC+TAF). Although the safety and efficacy of BIC+FTC+TAF have been confirmed in older patients with HIV-1, PK data in this patient population remain limited. Dolutegravir (DTG), an antiretroviral medication with a similar structural formula to BIC, causes neuropsychiatric adverse events. PK data for DTG have shown a higher maximum concentration (Cmax) among older patients than younger patients and are related to a higher frequency of adverse events. In the present study, we prospectively collected BIC PK data from 10 older HIV-1-infected patients and showed that PK of BIC are not affected by age. Our results support the safe use of this treatment regimen among older patients with HIV-1.

The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4.

The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4 Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4 The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.