Janneke Keemink

Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity

Introduction: The sandwich-cultured hepatocyte (SCH) model has become an invaluable in vitro tool for studying hepatic drug transport, metabolism, biliary excretion and toxicity. The relevant expression of many hepatocyte-specific functions together with the in vivo-like morphology favor SCHs over other preclinical models for evaluating hepatobiliary drug disposition and drug-induced hepatotoxicity. Areas covered: In this review, the authors highlight recommended procedures required for reproducibly culturing hepatocytes in sandwich configuration. It also provides an overview of the SCH model characteristics as a function of culture time. Lastly, the article presents a summary of the most prominent applications of the SCH model, including hepatic drug clearance prediction, drug–drug interaction potential and drug-induced hepatotoxicity. Expert opinion: When human (cryopreserved) hepatocytes are used to establish sandwich cultures, the model appears particularly valuable to quantitatively investigate clinically relevant mechanisms related to in vivo hepatobiliary drug disposition and hepatotoxicity. Nonetheless, the SCH model would largely benefit from better insight into the fundamental cell signaling mechanisms that are critical for long-term in vitro maintenance of the hepatocytic phenotype. Studies systematically exploring improved cell culture conditions (e.g., co-cultures or extracellular matrix modifications), as well as in vitro work identifying key transcription factors involved in hepatocyte differentiation are currently emerging.

Unbound ritonavir concentrations in rat and human hepatocytes

Knowledge regarding intracellular drug exposure is crucial to gain mechanistic understanding of hepatic disposition. This study aims to develop an approach to determine unbound intracellular concentrations (Cu,cell ) of ritonavir. Ritonavir was selected as a model drug as incubations with high ritonavir concentrations inhibited all saturable processes involved in ritonavir disposition including metabolism and transporter-mediated membrane passage. Following this incubation, hepatocytes were re-equilibrated in fresh protein-containing medium before determination of extracellular unbound ritonavir concentrations. In the absence of metabolism and transport, unbound intracellular and unbound extracellular concentrations were identical. In parallel, total intracellular ritonavir concentrations (Ccell ) were determined, enabling the calculation of intracellular free fractions (fu,cell ). Additionally, Ccell was determined after exposing hepatocytes to a therapeutically relevant concentration (0.5 μM). Multiplication of this concentration with fu,cell resulted in Cu,cell . Finally, Kpu,u (intracellular unbound drug accumulation ratio) was calculated. Exposure of rat and human hepatocytes to 0.5 μM ritonavir resulted in Cu,cell of 12 ± 1 and 8 ± 1 nM. Corresponding Kpu,u values were 2.7 ± 0.5 and 1.4 ± 0.2. We present an in vitro method to determine Cu,cell of ritonavir in intact hepatocytes. Cu,cell obtained at clinically relevant extracellular concentrations are in accordance with concentrations known to inhibit cytochrome P450 and are achieved because of ritonavir accumulation in hepatocytes.

Verapamil hepatic clearance in four preclinical rat models: towards activity-based scaling

The current study was designed to cross‐validate rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and the isolated perfused rat liver (IPRL) model against in vivo pharmacokinetic data, using verapamil as a model drug. Michaelis‐Menten constants (Km), for the metabolic disappearance kinetics of verapamil in RLM and SRH (freshly isolated and cryopreserved), were determined and corrected for non‐specific binding. The ‘unbound’ Km determined with RLM (2.8 µ m) was divided by the ‘unbound’ Km determined with fresh and cryopreserved SRH (3.9 µ m and 2.1 µ m, respectively) to calculate the ratio of intracellular to extracellular unbound concentration (Kpu,u). Kpu,u was significantly different between freshly isolated (0.71) and cryopreserved (1.31) SRH, but intracellular capacity for verapamil metabolism was maintained after cryopreservation (200 vs. 191 µl/min/million cells). Direct comparison of intrinsic clearance values (Clint) in RLM versus SRH, yielded an activity‐based scaling factor (SF) of 0.28–0.30 mg microsomal protein/million cells (MPPMC). Merging the IPRL‐derived Clint with the MPPMC and SRH data, resulted in scaling factors for MPPGL (80 and 43 mg microsomal protein/g liver) and HPGL (269 and 153 million cells/g liver), respectively. Likewise, the hepatic blood flow (61 ml/min/kg b.wt) was calculated using IPRL Clint and the in vivo Cl. The scaling factors determined here are consistent with previously reported CYP450‐content based scaling factors. Overall, the results show that integrated interpretation of data obtained with multiple preclinical tools (i.e. RLM, SRH, IPRL) can contribute to more reliable estimates for scaling factors and ultimately to improved in vivo clearance predictions based on in vitro experimentation. Copyright

Primary Hepatocytes in Sandwich Culture

Hepatocytes in sandwich configuration constitute of primary hepatocytes cultured between two layers of extracellular matrix. Sandwich-cultured hepatocytes maintain expression of liver-specific proteins and gradually form intact bile canaliculi with functional biliary excretion of endogenous compounds and xenobiotics. Both freshly isolated and cryopreserved hepatocytes can be used to establish sandwich cultures. Therefore, this preclinical model has become an invaluable in vitro tool to evaluate hepatobiliary drug transport, metabolism, hepatotoxicity, and drug interactions. In this chapter, commonly used procedures to cultivate primary hepatocytes from human and rat in sandwich configuration are described.

Straightforward entry to pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones and their ADME properties.

A straightforward synthesis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones was developed starting from 2-chloropyridine-3-carboxylic acid by esterification, nucleophilic aromatic substitution and amide formation in one step, and ring closure allowing their synthesis with two identical or two different group attached to nitrogen. The structural diversity of these [2,3-d]pyrimidine-2,4(1H,3H)-diones resulted in significant variation in the biopharmaceutical properties. This was reflected by the broad range in fasted state simulated intestinal fluid solubility values (12.6 μM to 13.8 mM), Caco-2 permeability coefficients (1.2 × 10(-6)cm/s to 90.7 × 10(-6)cm/s) and in vitro-predicted human in vivo intrinsic clearance values (0 to 159 ml/min/kg).

In vitro disposition profiling of heterocyclic compounds

Compound libraries that are screened for biological activity commonly contain heterocycles. Besides potency, drug-like properties need to be evaluated to ensure in vivo efficacy of test compounds. In this context, we determined hepatic and intestinal disposition profiles for 17 heterocyclic compounds. All studied compounds showed rapid uptake in suspended rat hepatocytes, whereas metabolism was poor and the rate-limiting step in hepatic elimination. In vitro assays demonstrated a relatively low solubility and high intestinal permeability. Based on these in vitro data, heterocycles were categorized in the biopharmaceutics classification system (BCS) and the biopharmaceutics drug disposition classification system (BDDCS) to predict disposition characteristics before clinical data are available. Our findings emphasized the importance to use hepatocytes in addition to microsomes to study metabolism, since the latter lack non-microsomal enzymes and cellular context. Moreover, intracellular exposure should be considered to gain insight in the relevant fraction of the compound available at the enzymatic site. Finally, the study reveals discrepancies associated with the classification of heterocycles in BCS versus BDDCS. These probably originate from the binary character of both systems.

Caco-2 Cell Conditions Enabling Studies of Drug Absorption from Digestible Lipid-Based Formulations

PurposeTo identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs).MethodsCaco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer.ResultsMost undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion.ConclusionsMost LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.

Impact of Drug Physicochemical Properties on Lipolysis-triggered Drug Supersaturation and Precipitation from Lipid-Based Formulations

In this study we investigated lipolysis-triggered supersaturation and precipitation of a set of model compounds formulated in lipid-based formulations (LBFs). The purpose was to explore the relationship between precipitated solid form and inherent physicochemical properties of the drug. Eight drugs were studied after formulation in three LBFs, representing lipid-rich (extensively digestible) to surfactant-rich (less digestible) formulations. In vitro lipolysis of drug-loaded LBFs were conducted, and the amount of dissolved and precipitated drug was quantified. Solid form of the precipitated drug was characterized with polarized light microscopy (PLM) and Raman spectroscopy. A significant solubility increase for the weak bases in the presence of digestion products was observed, in contrast to the neutral and acidic compounds for which the solubility decreased. The fold-increase in solubility was linked to the degree of ionization of the weak bases and thus their attraction to free fatty acids. A high level of supersaturation was needed to cause precipitation. For the weak bases, the dose number indicated that precipitation would not occur during lipolysis; hence, these compounds were not included in further studies. The solid state analysis proved that danazol and griseofulvin precipitated in a crystalline form, while niclosamide precipitated as a hydrate. Felodipine and indomethacin crystals were visible in the PLM, whereas the Raman spectra showed presence of amorphous drug, indicating amorphous precipitation that quickly crystallized. The solid state analysis was combined with literature data to allow analysis of the relationship between solid form and the physicochemical properties of the drug. It was found that low molecular weight and high melting temperature increases the probability of crystalline precipitation, whereas precipitation in an amorphous form was favored by high molecular weight, low melting temperature, and positive charge.

Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes

Freshly-isolated rat hepatocytes are commonly used as tools for hepatic drug disposition. From an ethical point of view, it is important to maximize the use of isolated hepatocytes by cryopreservation. The present study compared overall hepatocyte functionality as well as activity of the organic anion transporting polypeptide (Oatp), multidrug resistance-associated protein 2 (Mrp2), and UDP-glucuronosyltransferase 1 (Ugt1), in in vitro models established with cryopreserved and freshly-isolated hepatocytes. A similar culture time-dependent decline in cellular functionality, as assessed by urea production, was observed in sandwich-cultured hepatocytes (SCH) obtained from freshly-isolated and cryopreserved cells. Concentration-dependent uptake kinetics of the Oatp substrate sodium fluorescein in suspended hepatocytes (SH) or SCH were not significantly affected by cryopreservation. Mrp2-mediated biliary excretion of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein by SCH was assessed with semi-quantitative fluorescence imaging: biliary excretion index values increased between day 3 and day 4, but did not differ significantly between cryopreserved and freshly-isolated hepatocytes. Finally, telmisartan disposition was evaluated in SCH to simultaneously explore Oatp, Ugt1, and Mrp2 activity. In order to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation, a mechanistic cellular disposition model was developed. Basolateral and canalicular efflux as well as glucuronidation of telmisartan were affected by cryopreservation. In contrast, the disposition parameters of telmisartan-glucuronide were not impacted by cryopreservation. Overall, the relative contribution of the rate-determining processes (uptake, metabolism, efflux) remained unaltered between cryopreserved and freshly-isolated hepatocytes, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes when studying these cellular disposition pathways.