Structural identifiability analyses of candidate models for in vitro Pitavastatin hepatic uptake

Introduction. An in vivo determination of bile acid hepatobiliary transport efficiency can be of use in liver disease and preclinical drug development. Given the increased interest in bile acid Positron Emission Tomography- (PET-) imaging, a further understanding of the impact of 18-fluorine substitution on bile acid handling in vitro and in vivo can be of significance. Methods. A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3-, 7-, or 12-OH function is substituted with a fluorine atom (3α-[18F]FCA; 7β-[18F]FCA; 12β-[18F]FCA); a glycocholic and chenodeoxycholic acid analogue, substituted on the 3-position (3β-[18F]FGCA and 3β-[18F]FCDCA, resp.). Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines and efflux by BSEP in membrane vesicles. PET-scans with the tracers were performed in wild-type mice (n=3 per group): hepatobiliary transport was monitored and compared to a reference tracer, namely, 3β-[18F]FCA. Results. Compounds 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA were synthesized in moderate radiochemical yields (4–10% n.d.c.) and high radiochemical purity (>99%); 7β-[18F]FCA and 12β-[18F]FCA could not be synthesized and included further in this study. In vitro evaluation showed that 3α-FCA, 3β-FGCA, and 3β-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1, and BSEP. In vivo, 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3β-[18F]FCA epimers. Conjugation of 3β-[18F]FCA with glycine had no significant effect in vivo. Compound 3β-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines. Conclusion. A set of 18F labeled bile acids was synthesized that are substrates of the bile acid transporters in vitro and in vivo and can serve as PET-biomarkers for hepatobiliary transport of bile acids.

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Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody–DM1 Drug Conjugates

Cancers ◽

10.3390/cancers11081168 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1168

Author(s):

Haozhong Ding ◽

Mohamed Altai ◽

Sara S. Rinne ◽

Anzhelika Vorobyeva ◽

Vladimir Tolmachev ◽

...

Keyword(s):

Therapeutic Potential ◽

Histopathological Examination ◽

Specific Binding ◽

Hepatic Uptake ◽

In Vitro Cytotoxicity ◽

Drug Conjugates ◽

Biodistribution Studies ◽

Development Approach

Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderate- to high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (ZHER2:2891)2–ABD–MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate–spacer–, (ZHER2:2891)2–ABD–E3–MC-DM1, or a hexaglutamate–spacer–, (ZHER2:2891)2–ABD–E6–MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (ZHER2:2891)2–ABD–MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

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The Phenomenon of Albumin-Mediated Hepatic Uptake of Organic Anion Transport Polypeptide Substrates: Prediction of the In Vivo Uptake Clearance from the In Vitro Uptake by Isolated Hepatocytes Using a Facilitated-Dissociation Model

Drug Metabolism and Disposition ◽

10.1124/dmd.117.077115 ◽

2018 ◽

Vol 46 (3) ◽

pp. 259-267 ◽

Cited By ~ 32

Author(s):

Seiji Miyauchi ◽

Masayuki Masuda ◽

Soo-Jin Kim ◽

Yuudai Tanaka ◽

Kyeong-Ryoon Lee ◽

...

Keyword(s):

Organic Anion ◽

Isolated Hepatocytes ◽

Hepatic Uptake ◽

Anion Transport ◽

Organic Anion Transport Polypeptide ◽

Dissociation Model ◽

In Vitro Uptake ◽

In Vivo Uptake

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FATE OF PREFORMED IMMUNE COMPLEXES IN RABBITS AND RHESUS MONKEYS

Journal of Experimental Medicine ◽

10.1084/jem.134.3.19 ◽

1971 ◽

Vol 134 (3) ◽

pp. 19-31 ◽

Cited By ~ 78

Author(s):

Mart Mannik ◽

William P. Arend

Keyword(s):

Immune Complexes ◽

Rhesus Monkeys ◽

Hepatic Uptake ◽

In Vivo Studies ◽

Complement Components ◽

Immune Adherence ◽

Soluble Immune Complexes ◽

Lattice Formation

Preformed soluble immune complexes injected into rabbits or rhesus monkeys showed similar characteristics of disappearance from circulation. Complexes made with intact γG-antibodies and exceeding the Ag2Ab2 lattice formation were rapidly removed by the hepatic RES. These complexes fixed complement effectively in vitro. Their hepatic uptake was not dependent upon circulating complement components, since their accumulation in the liver was unchanged in complement depleted rabbits. Similar antigen-antibody complexes made with reduced and alkylated γG-antibodies fixed complement ineffectively in vitro. These complexes possessed different disappearance characteristics and were not rapidly taken up by the liver, regardless of their degree of lattice formation. Both in vitro and in vivo studies failed to suggest any role for the immune adherence receptor on primate erythrocytes in the handling of circulating soluble immune complexes composed of BSA and γG-antibodies to this antigen.

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