Immortalized Human Hepatocytes: A New …

1 137 Human hepatocytes support numerouspreclinical drug developmentdisciplines, includingassessment of the likely clinical effectsof drugs and chemicals on the liver and,conversely, assessment of possibleeffects of the liver on candidatecompounds. Cryopreserved or platedhuman hepatocytes offer manyadvantages, but their utility is hamperedby unpredictable supply and significantinter-individual differences in theexpression of Drug MetabolizingEnzymes (DMEs) and responses totoxicants. New, Immortalized humanhepatocytes with near-normalmorphology and function are availableand should dramatically improveefficiency, reproducibility andpredictive value of Human Bioavailability and efficacy of aco-administered drug is reducedby virtue of increased activity ofthe DMEs involved in It can cause pharmacokinetictolerance (or auto-induction): acompound inducing its ownhepatic Recent evidence implicatesenzyme induction as an importantinfluence in drug-inducedhepatotoxicity (1).

2 Thecorrelation between toxicity andinduction may increase regulatoryagencies' expectations forintensified scrutiny of compoundsthat demonstrate moderate topotent induction potential inorder to ensure a low risk ofproduction of reactiveintermediates of the candidate orof concurrently Increased activity of effluxtransporters may be important,suggesting that a candidatecompound poses risk of reducingthe efficacy of other co-administered the risk of clinicalinduction among various candidatecompounds should provide for reducedexpenses by elimination of potentialproblem compounds early indevelopment, or by improvements inplanning, and study and trial designs, asinducers progress (3) studied both theinduction of mRNA, using a differenttechnology from that employed byMills, and the metabolic activity ofCYPs 1A2, 2C9, 3A4, and 3A5, as wellas transporters MDR-1 and commented that: Appropriate induction wasobserved, , Rifampin andPhenobarbital induced both CYP2C9and CYP3A4, whereas Omeprazoleinduced CYP1A2.

3 Comparableinduction was obtained in the 6-welland 96-well formats. (emphasis added).They also assessed the specificity ofresponses, using PCR (TaqMan)Primers and Probes, and demonstratedonly 3A4 positivity with 3A4 plasmidDNA, only 3A5 positivity with 3A5plasmid DNA, and strong 3A7positivity with a slight 3A5 responseusing 3A7 plasmid recently published asummary of the results obtained frommore than 60 enzyme induction studiesconducted with primary cultures ofhuman hepatocytes (4). Using thatinformation as a reference, we analyzedin vitroet Technologies ofProvidence, RI used their patentedtechnique to produce a line ofimmortalized Human hepatocytes ,which was recently licensed byXenoTech, headquartered near KansasCity. Evaluations of the cells byscientists from XenoTech and twomajor pharmaceutical companies havedemonstrated the presence and activityof numerous DMEs; responses totoxicants; production of metabolitesand adult proteins; and induction ofPhase I and II DMEs and oftransporters.

4 The results arereproducible between experiments andacross different passages, and themagnitude and specificity of responsescorrespond to those seen in typicalpreparations of normal humanhepatocytes. The morphologies ofimmortalized cells and of a preparationof high-quality, Human hepatocytes aredepicted in. The morphology ofimmortalized hepatocytes resemblesthat of many preparations of normalhepatocytes, which is a significantfinding because hepatocellularmorphology is tied to hepatocellularfunction, reflecting expression ofhighly differentiated properties induction ofcytochrome P450 (CYP) and otherDMEs, or of efflux transporters, is awell-recognized cause of clinicallysignificant drug interactions throughthe following mechanisms:The suitability of the immortalizedhepatocytes as a substitute for primaryhuman hepatocytes was studied byMills(2), using prototypicalinducers of expression of the mRNA ofthe major inducible cytochrome P450 DMEs; the Phase II DME, UGT1A; andthe efflux transporter, MDR1.

5 Theyfound that the Fa2N-4 cells responded as expected using a 24-well Types of StudiesUsing Immortalized HumanHepatocytesInduction of Drug MetabolizingEnzymes and Transporterset (line) cellsperformed much like fresh humanhepatocytesDavid 16825 West 116th Street, Lenexa, Kansas 66219 Immortalized Human Hepatocytes: a new Advance in Convenience and PerformanceCurrent Separations 20:4 (2004)CYP2B6 - BupropionRifampinFold InductionExp 1 Exp 2 Exp 3543210 CYP1A2 - PhenacetinRifampinFold InductionExp 1 Exp 2 Exp 3403020100 CYP3A4 - MidazolamRifampinFold InductionExp 1 Exp 2 Exp 386420 CYP2C9 - DiclofenacRifampinFold InductionExp 1 Exp 2 Exp of enzymatic activity of theimmortalized cells treated withprototypical inducers, to compare themwith primary cultures. Whileexperimental conditions were notidentical (some older referencemethods have been changed), they weresufficiently similar to determine if theresponse of the Immortalized cellssupports their use as a replacement forprimary cultures.

6 Results of thecomparison are depicted inOurconclusion is that the immortalizedhepatocytes respond appropriately toenzyme inducers, with a magnitude ofresponse equaling or exceeding that ofmost preparations of fresh results are listed in general orderof importance of these CYPs. Thesignificant difference among modal,median, and mean numbers reflects theinfluence of a very small number ofindividuals. Using fold induction ofCYP3A4 as an example, 65% of allpreparations of primary hepatocytecultures will demonstrate a maximumof 5-fold induction when treated with20 M of Rifampin for three days; a verylimited number (less than 5%) willexceed 50-fold. Thus, the meaninduction of 3A4 activity reflects thedramatic and unpredictablecontribution of a few outlier individuals and is not a useful index ofexpected induction, the distribution ofresults being non-Gaussian.

7 Inter-individual variation may also bereflected in the fold induction of 1A2 inthe Immortalized hepatocytes , becauseit is higher than most primary overall conclusion is that theimmortalized cells perform as well asthe majority of preparations of primarycultures of Human hepatocytes instudying the induction of major,inducible scientists have also investigatedother components of the immortalizedcells response to inducers and found: depicts the inter-experimentalreproducibility achieved withimmortalized hepatocytes ; this isfar less variance than we haveobserved in studies with primarycultures. The time-course of induction(days of treatment with inducer)is similar to that with primarycultures. The responses in 6-, 12-, 24- of Human hepatocytes (left) and Fa2N-4 cells (right) at the light microscopy level. Notethat Fa2N-4 cells look remarkably similar to Human of CYP enzyme induction in Fa2N4 cells and in primary cultures of Human of CYP enzyme induction in Fa2N-4 Median MeanEnzymeFold induction in F cellsFold inductionin primary Human cultures5341020n/a8132 (Rifampin)(Omeprazole in F cells,(Rifampin) (Inducer used)(Mean value, 96-well)BNF in primary cells)(Rifampin; F cell studies useddifferent endpoint than primary)13996-well formats are similar.

8 Cryopreservation of the cells,followed by thawing and plating,has no noticeable effect on theirinduction responses. Concentration response curves aresimilar to those of primarycultures. Magnitude of induction responseis essentially the same across 10passages, as shown in. The specificity of CYP3A4induction is as expected; noinduction is measured withcompounds not known to induce3A4 studies are planned or ongoing toexamine induction of these cells relativeto other responses. Overall, we aresatisfied with their performance to theextent that we now use them in contractinduction cell line can offer some utility inanalysis of candidate compounds potential for cytotoxicity, one beingavailability of cells. The immortalizedhepatocytes may provide the best ofboth worlds retention of somemetabolic and transporter function, toallow for potentially superior predictivevalue in toxicity screening, as well asunlimited availability.

9 XenoTechscientists measured loss of theintracellular protein GST (kit fromBiotrin, Inc., Dublin, Ireland) by theimmortalized hepatocytes followingexposure to compounds whosetoxicity risks are well established ().The results compared favorably withexpectations. Compounds known tohave relatively low risk ofhepatotoxicity (rifampin, omeprazole,felbamate, probenecid, andacetaminophen) all caused release ofaGST from the Fa2N4 cellsapproximating that of vehicle control(DMSO, ). It bears mentionthat, while felbamate andacetaminophen are associated withreports of hepatotoxicity, felbamatetoxicity is idiosyncratic in nature, andtoxicity in both appears to require dosesmuch higher than the screening onesused here. For instance, acetaminophentoxicity is associated with dosesexceeding 4 g/day as well as otherconcurrent environmental of the immortalizedhepatocytes to the known toxicants 3-methylcholranthene, methotrexate,menadione, rotenone, and troglitazoneproduced significantly greater releaseof GST from the conclusion, these studies lead us toexpect that these immortalizedhepatocytes will be suitable for specifictoxicity screens.

10 We also expectthat some characteristics of theimmortalized hepatocytes response totoxicants may differ from some primaryhuman hepatocytes , but theimmortalized hepatocytes offer thedistinct advantages of reproducibilityand access. Their basal metabolism islower than the average in normalhepatocytes, so experiments wouldneed to be optimized for detection ofmetabolism-dependent induction, toxicity, and metabolismstudies with these Immortalized humanhepatocytes have yielded verypromising results as described above,and we anticipate completion of allremaining work in early 2004. Cells(cryopreserved, plated, or both) can beobtained for evaluation under a limiteduse license, along with MFE medium,which we recommend for their support,and you can email the author at:for additionalinformation. We should mention thatwe do not currently recommend the useof these Immortalized hepatocytes instudies submitted to regulatoryagencies, particularly in absence of anysupporting data from normal humanpreparations.