Reduced elimination of IgG antibodies by engineering the variable region.

Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.

Quantitative structure-intestinal permeability relationship of benzamidine analogue thrombin inhibitor.

The intestinal permeability of benzamidine analogue thrombin inhibitor is correlated with molecular volume, lipophilicity (calculated log P and IAM column capacity factor), hydrogen bond acidity/basicity and dipolarity.

Transport of N(G)-nitro-L-arginine across intestinal brush border membranes by Na+ -dependent and Na+-independent amino acid transporters.

PURPOSE: To clarify the transport mechanism of NG-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). METHODS: Dog intestinal BBM vesicles were used. RESULTS: The time course of L-NNA uptake showed a Na+ -dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na+, indicating participation of Na+ -dependent and Na+ -independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na+. N(G)-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. CONCLUSIONS: The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na+ differently from that across plasma membranes of nonepithelial cells, because B0,+ and b0,+ amino acid transporters function partly in L-NNA transport across intestinal BBM.

Na(+)-dependent and Na(+)-independent transport of L-arginine and L-alanine across dog intestinal brush border membrane vesicles.

We prepared intestinal brush border membrane vesicles (BBMVs) from beagle dogs fed a commercial diet (protein content: 24-26%), and investigated the characteristics of transport for basic and neutral amino acids across the intestinal BBMVs. To determined the kinetic parameters for L-arginine and L-alanine uptake, their total uptake was resolved into three routes: (1) Na(+)-dependent carrier-mediated transport; (2) Na(+)-independent carrier-mediated transport; and (3) simple diffusion. We could observe subtle, but clear-cut, Na(+)-dependent basic amino acid transport for the first time among studies with intestinal BBMVs prepared from mammals fed a normal diet. The Na(+)-dependent system for L-arginine transport can be best characterized as 'low affinity, low capacity', in contrast to that for L-alanine transport, which is 'low affinity, high capacity'. Maximal velocities of the Na(+)-dependent carrier-mediated transport are estimated to be higher for both L-arginine and L-alanine in dog intestinal BBMVs than in rabbit intestinal BBMVs reported previously. These results suggest that food habit of mammals is an important factor to decide the characteristic of system B0,+, a Na(+)-dependent carrier-mediated transport system common to basic and neutral amino acids across intestinal brush border membranes, as is protein content of the diet.

Antiarrhythmic effects of a novel class III drug, KCB-328, on canine ventricular arrhythmia models.

KCB-328 is a newly synthesized class III drug. To determine whether this drug has antiarrhythmic or proarrhythmic effects, we used canine ventricular arrhythmia models induced by coronary ligation and reperfusion, programmed electrical stimulation (PES), two-stage coronary ligation, digitalis, or epinephrine. KCB-328, in an intravenous infusion of 0.5 mg/kg/30 min, prolonged the QTc interval only 11%, but had antiarrhythmic effects on the reentry arrhythmias induced by PES (12 of 12 dogs with old myocardial infarction; p < 0.05). KCB-328, in an infusion of 1 mg/kg/h, suppressed the occurrence of fatal ventricular fibrillation (VF) induced by coronary ligation and reperfusion under either halothane anesthesia (p < 0.05) or pentobarbital anesthesia (p < 0.05). Under the halothane anesthesia, KCB-328 alone showed proarrhythmic effects [i.e., induction of ventricular premature contractions (VPCs)], but it did not induce a more severe effect such as torsades de pointes-type ventricular tachycardia (VT). In addition, KCB-328 had weak antiarrhythmic effects on the automaticity arrhythmias induced by 24-h coronary ligation but was effective neither on 48-h coronary ligation arrhythmias nor on the digitalis- and epinephrine-induced arrhythmias. Our results indicate that KCB-328 has powerful antiarrhythmic effects with fewer proarrhythmic potencies.

The stability and degradation pathway of recombinant human parathyroid hormone: deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues.

PURPOSE: The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products. METHODS: Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60 degrees C. The approximate molecular mass and pI values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC. RESULTS: At pH2, eight degradation products were found: 1-30rhPTH, 1-74rhPTH, 1-71rhPTH, 1-56rhPTH, 1-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Asp16-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5. CONCLUSIONS: Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent, rhPTH is most stable at pH5.

Oxidation of recombinant human parathyroid hormone: effect of oxidized position on the biological activity.

PURPOSE: To determine the oxidation products of recombinant human parathyroid hormone (rhPTH) treated with H2O2, the amino acid residue oxidized, and the biological activity of the oxidation products. METHODS: Oxidized residues were determined by CNBr cleavage, trypsin digestion and subsequent fast atom bombardment mass spectrometry. The biological activity of each oxidized rhPTH was examined in rat osteosarcoma cell adenylate cyclase assay. RESULTS: Three oxidized products were isolated, namely, Met at position 8 (Met8) sulfoxide, Met at position 18 (Met18) sulfoxide and both positions Met sulfoxide. It appears that the Met8 and Met18 oxidized forms are intermediates in the generation of the Met doubly oxidized form. All oxidized forms possessed reduced biological activity, more so for oxidation at Met8 than at Met18. CONCLUSIONS: The region around Met8 is important for the activity of the parathyroid hormone.

Peptide mapping of recombinant human parathyroid hormone by enzymatic digestion and subsequent fast-atom bombardment mass spectrometry.

Peptide maps of recombinant human parathyroid hormone (rhPTH) were determined by both trypsin and V-8 protease digestion with subsequent fast-atom bombardment mass spectrometry (FAB-MS). Coverage of the sequence was 85% when using trypsin and 90% when using V-8 protease. Five rhPTH variants that were recombinantly produced as models of Asn deamidated type degradation products were measured, and molecular weight differences between their respective deamidated peptide fragments were completely detected. In the V-8 protease digests of some variants, characteristic peptide ions caused by the deamidation were observed and this greatly facilitated the assignment and recognition of the deamidated position. Our data suggest that FAB-mapping of rhPTH via the protease digestion methods used, appears to have great potential for structural investigations of the peptide.