Pharmacokinetics of monoclonal antibodies locally-applied into the middle ear of guinea pigs.

Countless therapeutic antibodies are currently available for the treatment of a broad range of diseases. Some target molecules of therapeutic antibodies are involved in the pathogenesis of sensorineural hearing loss (SNHL), suggesting that SNHL may be a novel target for monoclonal antibody (mAb) therapy. When considering mAb therapy for SNHL, understanding of the pharmacokinetics of mAbs after local application into the middle ear is crucial. To reveal the fundamental characteristics of mAb pharmacokinetics following local application into the middle ear of guinea pigs, we performed pharmacokinetic analyses of mouse monoclonal antibodies to FLAG-tag (FLAG-mAbs), which have no specific binding sites in the middle and inner ear. FLAG-mAbs were rapidly transferred from the middle ear to the cochlear fluid, indicating high permeability of the round window membrane to mAbs. FLAG-mAbs were eliminated from the cochlear fluid 3 h after application, similar to small molecules. Whole-body autoradiography and quantitative assessments of cerebrospinal fluid and serum demonstrated that the biodistribution of FLAG-mAbs was limited to the middle and inner ear. Altogether, the pharmacokinetics of mAbs are similar to those of small molecules when locally applied into the middle ear, suggesting the necessity of drug delivery systems for appropriate mAb delivery to the cochlear fluid after local application into the middle ear.

A splice-switching oligonucleotide treatment ameliorates glycogen storage disease type 1a in mice with G6PC c.648G>T.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.

A Controlled Release System for Long-Acting Intravitreal Delivery of Small Molecules.

PURPOSE: The short half lives of small molecules in the vitreous requires frequent repeated intravitreal injections that are impractical for treatment of chronic eye diseases. We sought to develop a method for increasing the intravitreal half-life of small-molecule drugs. METHODS: We adapted a technology for controlled release of drugs from macromolecular carriers for use as a long-acting intravitreal delivery system for small molecules. As a prototype, a small molecule complement factor D inhibitor with an intravitreal half-life of 7 hours was covalently attached to a 4-arm PEG40kDa by a self-cleaving ß-eliminative linker with a cleavage half-life of approximately 1 week. RESULTS: After intravitreal injection in rabbits, the drug was slowly released in the vitreous, and equilibrated with the retina and choroid. The intravitreal half-life of the intact PEG-drug conjugate in the rabbit was 7 days, and that of the released drug was 3.6 days. We simulated the anticipated pharmacokinetics of the delivery system in human vitreous, and estimated that the half-life of a 4-arm PEG40kDa conjugate would be approximately 2 weeks, and that of the released drug would be approximately 5 days. CONCLUSIONS: We posit that a linker with a cleavage half life of 2 weeks would confer a half life of approximately 7 days to a released small molecule drug in humans, comparable to the half life of approved intravitreal injected macromolecular drugs. TRANSLATIONAL RELEVANCE: With this technology, a potent small molecule with an appropriate therapeutic window should be administrable by intravitreal injections in the human at once-monthly intervals.

Wide application of a novel topoisomerase I inhibitor-based drug conjugation technology.

To establish a novel and widely applicable payload-linker technology for antibody-drug conjugates (ADCs), we have focused our research on applying exatecan mesylate (DX-8951f), a potent topoisomerase I inhibitor, which exhibits extensive antitumor activity as well as significant myelotoxicity, as the payload part. Through this study, we discovered a promising exatecan derivative (DX-8951 derivative, DXd), that has the characteristics of low membrane permeability and shows considerably less myelotoxicity than that shown by exatecan mesylate in an in vitro human colony forming unit-granulocyte macrophage assay. DXd was further used for drug conjugation by using commercially or clinically useful monoclonal antibodies to evaluate the potency of the ADC. The result revealed that the DXd-ADCs targeting CD30, CD33, and CD70 were effective against each of their respective target-expressing tumor cell lines. Moreover, a novel DXd-ADC targeting B7-H3, which is a new target for ADCs, also showed potent antitumor efficacy both in vitro and in vivo. In conclusion, this study showed that this novel topoisomerase I inhibitor-based ADC technology is widely applicable to a diverse number of antibodies and is expected to mitigate myelotoxicity, thereby possibly resulting in better safety profiles than that of existing ADC technologies.

Synthesis and evaluation of CS-2100, a potent, orally active and S1P(3)- sparing S1P(1) agonist.

Modulators of sphingosine phosphate receptor-1 (S1P(1)) have recently been focused as a suppressant of autoimmunity. We have discovered a 4-ethylthiophene-based S1P(1) agonist 1-({4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-thienyl}methyl)azetidine-3-carboxylic acid (CS-2100, 8) showing potent S1P(1) agonist activity against S1P(3) and an excellent in vivo potency. We report herein the synthesis of CS-2100 (8) and pharmacological effects such as S1P(1) and S1P(3) agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effects on adjuvant-induced arthritis and experimental autoimmune encephalomyelitis (EAE) in animal models. The pharmacokinetic data were also reported. CS-2100 (8) had >5000-fold greater agonist activity for human S1P(1) (EC(50); 4.0 nM) relative to S1P(3) (EC(50); >20,000 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-2100 (8) in rats, lymphocyte counts decreased significantly, with a nadir at 8 and/or 12 h post-dose and recovery to vehicle control levels by 24-48 h post-dose. CS-2100 (8) is efficacious in the adjuvant-induced arthritis model in rats (ID(50); 0.44 mg/kg). In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for 0.3 and 1 mg/kg CS-2100 (8) groups in mice. While CS-2100 (8) showed potent efficacy in various animal disease models, it was also revealed that the central 1,2,4-oxadiazole ring of CS-2100 (8) was decomposed by enterobacteria in intestine of rats and monkeys, implicating the latent concern about an external susceptibility in its metabolic process in the upcoming clinical studies.

Kinetic characterization of rat hepatic uptake of 16 actively transported drugs.

To explore the determinants of hepatic uptake, 16 compounds were investigated with different physicochemical and disposition characteristics, including five statins, three sartans, saquinavir, ritonavir, erythromycin, clarithromycin, nateglinide, repaglinide, fexofenadine, and bosentan. Freshly isolated rat hepatocytes in suspension were used with the oil-spin method to generate kinetic parameters. Clearances, via passive diffusion (P(diff)) and active uptake (CL(active), characterized by maximal uptake rate and K(m)), were estimated from the initial uptake rate data over a 0.01 to 100 µM concentration range. The K(m) values had a range of 15-fold, with 10 of the 16 drugs with K(m) < 10 µM (median 6 µM). Both CL(active) and P(diff) ranged over 100-fold (median 188 and 14 µl/min/106 cells). Assessment of the relative contribution of P(diff) and CL(active) indicated that, at low concentrations (approximately 0.1 µM), the active process contributes >80% to the overall uptake for 13 drugs. Although high P(diff) values were obtained for ritonavir and repaglinide, active process contributed predominantly to uptake; in contrast, high passive permeability dominates over transporter-mediated uptake for saquinavir over the full concentration range. For bosentan and erythromycin, active and passive processes were equally important. Hepatocyte-to-medium unbound concentration ratio was >10 for 9 of the 16 drugs, ranging from 2 to 494 for bosentan and atorvastatin, respectively. Some drugs showed extensive intracellular binding (fraction unbound range 0.01-0.6), which was not correlated with active uptake. LogD7.4 correlated significantly with P(diff) and the extent of intracellular binding but not with active uptake. This study provides systematic assessment of the role of active uptake relative to the passive process; implications of the findings are discussed.

Prediction of human pharmacokinetics of therapeutic monoclonal antibodies from simple allometry of monkey data.

Interspecies allometric scaling is a useful tool for calculating human pharmacokinetic (PK) parameters from data in animals. In this study, in order to determine the scaling exponent in a simple allometric equation that can predict human clearance (CL) and distribution volume at steady state (Vss) of monoclonal antibodies (mAbs) from monkey data alone, PK data of 24 mAbs were collected and analyzed according to the types of targeted antigens (soluble or membrane-bound antigens). Based on the observed PK data in humans (at clinical doses) and monkeys (at >1 mg/kg), where the PK is expected to be linear, the mean scaling exponents in the allometric equation for CL and Vss, respectively, against body weight were calculated to be 0.79 and 1.12 [95% confidence intervals (CIs): 0.69-0.89 and 0.96-1.28] for soluble antigens, and 0.96 and 1.00 (95% CIs: 0.83-1.09 and 0.87-1.13) for membrane-bound antigens. Using these exponents and monkey PK data (at >1 mg/kg) alone, both human CL and Vss of mAbs can be predicted with reasonable accuracy, i.e., within 2-fold of the observed values. Compared with traditional allometric scaling using PK data from three or more preclinical species, this approach is simple, quick, resource-saving, and useful in drug discovery and development.

Basic fibroblast growth factor-binding peptide as a novel targeting ligand of drug carrier to tumor cells.

Drug systems targeting tumor cells using basic fibroblast growth factor (bFGF) have been widely reported. In this study, the peptide KRTGQYKLC (bFGFp), containing cysteine at the carboxyl termination of the bFGF-derived peptide, was applied as a novel ligand targeting tumor cells. bFGFp was conjugated with bovine serum albumin (BSA) and liposomes. The peptide was shown to inhibit the binding of bFGF to FGF receptor-1 (FGFR1). Interestingly, the binding study using surface plasmon resonance (SPR) assay revealed that the bFGFp-BSA was not bound to FGFR1, but was selectively bound to bFGF. Furthermore, the SPR assay showed that bFGFp-BSA is capable of binding to FGFR1 following the pretreatment with bFGF. The confocal microscopy study indicated that the uptake of bFGFp-BSA by NIH3T3 cells, which highly express FGFRs, was significantly enhanced by pretreatment with bFGF. Then, PEGylated liposomes containing bFGFp (bFGFp-liposome) were prepared by conjugating maleimide-PEG-PE with bFGFp. Following the pretreatment of bFGF, the uptake of bFGFp-liposomes by NIH3T3 cells was significantly enhanced. These results suggest that bFGFp-BSA and bFGFp-liposomes are taken by NIH3T3 cells via binding with bFGF. In addition, both bFGFp-BSA and bFGFp-liposomes had no effect on the proliferation of NIH3T3 cells. This strategy can be used as a novel system for targeting tumors highly expressing FGFRs without a proliferation effect.

Role of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution.

To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, (111)In-TNM(6.6)- and (111)In-HNB(2.0)-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than (111)In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.

Intracellular trafficking is the important process that determines the optimal charge ratio on transfection by galactosylated lipoplex in HEPG2 cells.

The purpose of the present study was to gain insight into the major factors affecting transfection efficiency with galactosylated lipoplex in HepG2 cells. In this study, lipoplex and galactosylated lipoplex were examined at different charge ratios (- : +): 1.0 : 1.2, 1.0 : 2.3, 1.0 : 3.1, 1.0 : 4.7, and 1.0 : 7.0. The particle size and zeta potential of the both lipoplexes was dependent on the charge ratio. Cellular uptake was evaluated by using [(32)P]-labeled pCMV-Luc and this showed that the cellular uptake of galactosylated lipoplex was significantly higher than that of lipoplex at a charge ratio ranging from 1.0 : 2.3 to 1.0 : 7.0. As the charge ratio increased in both lipoplexes, the apparent cellular uptake increased. Transfection activity by galactosylated lipoplex was significantly higher than that by lipoplex except at a charge ratio of 1.0 : 7.0. The optimal charge ratio for transfection efficacy was 1.0 : 2.3 and transfection was reduced at higher charge ratios. Both lipoplexes exhibited no significant cytotoxicity at any charge ratio. In conclusion, it is suggested that intracellular trafficking, rather than the degree of uptake and cytotoxicity, is the important process that determines the optimal charge ratio of galactosylated lipoplex in HepG2 cells.

Intravenous administration of mannosylated cationic liposome/NFkappaB decoy complexes effectively prevent LPS-induced cytokine production in a murine liver failure model.

The purpose of this study was to inhibit endotoxin induced cytokines production and liver injury by liver non-parenchymal cell (NPC) selective delivery of nuclear factor kappaB (NFkappaB) decoy using mannosylated cationic liposomes (Man-liposomes). In this study, we examined the distribution, inhibitory effect on cytokines production and ALT/AST of intravenously injected Man-liposome/NFkappaB decoy complex. Man-liposome/[(32)P] NFkappaB decoy complexes mostly accumulated in the liver, preferentially in NPC. In a murine lipopolysaccharide-induced liver failure model, the production of tumor necrosis factor-alpha (TNFalpha), IFNgamma, IL1-beta, ALT and AST were effectively reduced by Man-liposome complexes. However, cationic or galactosylated cationic liposome complexes could not inhibit TNFalpha production.

Inhibition of experimental hepatic metastasis by targeted delivery of catalase in mice.

Bovine liver catalase derivatives possessing diverse tissue distribution properties were synthesized, and their effects on hepatic metastasis of colon carcinoma cells were examined in mice. An intraportal injection of 1 x 10(5) colon 26 cells resulted in the formation of more than 50 metastatic colonies on the surface of the liver at 14 days after injection. An intravenous injection of catalase (CAT; 35000 units/kg of body weight) significantly (P < 0.001) reduced the number of the colonies in the liver. Galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT were also tested in the same system. Of these derivatives, Gal-CAT showed the greatest inhibitory effect on hepatic metastasis, and the number of colonies was significantly (P < 0.001) smaller than following treatment with catalase. High activities of matrix metalloproteinases (MMPs), especially MMP-9, were detected in the liver of mice bearing metastatic tumor tissues, which was significantly (P < 0.05) reduced by Gal-CAT. These results, combined with our previous finding that Gal-CAT can be efficiently delivered to hepatocytes, indicate that the targeted delivery of catalase to the liver by galactosylation is a promising approach to suppress hepatic metastasis. Decreased MMP activity by catalase delivery seems to be involved in its anti-metastatic effect.

Effect of co-administration of cationic macromolecules on the in vivo disposition and in situ renal disposition characteristics of rhIL-11.

The effects of co-administration of cationic proteins on the in vivo disposition characteristics of recombinant human interleukin-11 (rhIL-11) in mice and on the renal disposition in the perfused rat kidney were investigated. Following a bolus intravenous injection of 10 microg/kg (111)In-labeled rhIL-11, along with cationic proteins at high dose (50 mg/kg), the plasma clearance of (111)In-labeled rhIL-11 was significantly decreased mainly due to a reduction in the hepatic clearance of (111)In-labeled rhIL-11. The effect on the renal clearance was relatively small, suggesting that the kidney has a high clearance capacity. The urinary excretion ratio increased by a factor of 2 or 4 with co-administration, suggesting that the cationic character of rhIL-11is involved in tubular re-absorption. An in situ renal disposition study supports these postulations. Thus, the renal and hepatic disposition of rhIL-11 is based on nonspecific cationic interaction. These data suggest that an efficient delivery system for this cytokine would require the reduction of electrostatic interaction of this molecule with these tissues in order to reduce the plasma clearance rate. These findings provide useful information for the construction of an rhIL-11 delivery system.

Pharmacokinetics and preventive effects of targeted catalase derivatives on hydrogen peroxide-induced injury in perfused rat liver.

PURPOSE: To investigate the pharmacokinetics and preventive effects of liver-targeted catalase (CAT) derivatives on hepatic injury caused by reactive oxygen species. METHODS: The hepatic uptake of 111In-CAT, galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT was investigated in isolated perfused rat livers in a single-pass constant infusion mode. Then, pharmacokinetic parameters were obtained by fitting equations derived from a one-organ pharmacokinetic model to the outflow profile. Their effects in preventing hydrogen peroxide-induced injury were determined by lactate dehydrogenase (LDH) release from the perfused liver. RESULTS: The extraction of CAT derivatives by the liver was dose-dependent, and increased by the chemical modifications described. After being bound to the cell surface, chemically modified CAT derivatives were internalized by the liver faster than CAT. Preperfusion of a CAT derivative significantly reduced LDH release by hydrogen peroxide at least for 30 min, and Man-CAT and Suc-CAT effectively inhibited this release. CONCLUSIONS: Internalized CAT derivatives are also effective in degrading hydrogen peroxide and targeted delivery of CAT to liver nonparenchymal cells by mannosylation or succinylation is a useful method for the prevention of hepatic injury caused by reactive oxygen species.