Bioorganic & Medicinal Chemistry Letters 20 (2010) 5308–5312

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Using an in vitro cytotoxicity assay to aid in compound selectionfor in vivo safety studies

Nigel Greene *, Michael D. Aleo, Shirley Louise-May, David A. Price *, Yvonne WillPfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA

a r t i c l e i n f o

Article history:Received 12 May 2010Revised 19 June 2010Accepted 24 June 2010Available online 1 July 2010

Keywords:c log PToxicologyTPSAAttritionCytotoxicityPhysico chemistry

0960-894X/$ - see front matter � 2010 Elsevier Ltd.doi:10.1016/j.bmcl.2010.06.129

* Corresponding author. Tel.: +1 860 686 9180.E-mail address: david.a.price@pfizer.com (D.A. Pric

a b s t r a c t

Recent publications have demonstrated that using calculated physiochemical properties can help in thedesign of compounds that have a decreased risk of significant findings in rodent toxicology studies. In thisLetter, we extend this concept and incorporate results from a high throughput cytotoxicity assay to helpthe drug discovery community select compounds for progression into in vivo studies. The results are pre-sented in an easily interpretable odds ratio so that teams can readily compare compounds and progresspotential clinical candidates to the necessary rodent in vivo studies.

� 2010 Elsevier Ltd. All rights reserved.

Numerous drugs have been withdrawn from the market placeover the last 20 years for a variety of reasons, however, recent pub-lications suggest that safety concerns are among one of the leadingcauses for attrition.1–3 With the increased investment required forprosecution of projects once they obtain clinical status, pre-clinicalattrition is preferred; however, increasing the likelihood of deliver-ing compounds of high quality to the clinic is the real goal. In re-sponse to this challenge, research groups are increasing the useof both, in vitro and in vivo safety models earlier in the drug dis-covery process to help differentiate compounds and increase thelikelihood of the compound entering the clinic. A recent reviewof safety screening approaches demonstrates that many in vitrosafety assays are used in risk identification mode where specificrelationships exist between well defined mechanisms of toxicityand discrete in vivo end points.4 Examples include the Ames assayas an assessment of genetic toxicity and the potential risk of car-cinogenicity in humans or hERG patch clamp assays and the poten-tial for cardiovascular risk in man. Both Ames and hERG patchclamp assays have become mainstays of safety screening para-digms in the modern drug discovery process.5,6

Many research institutions are now driving the use of shortterm, repeat dose in vivo safety assessment studies as early as pos-sible in a project’s life.6 These exploratory toxicology studies arethen supplemented with the necessary required regulatory studiesto enable the compound to progress safely to clinical studies. It is

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e).

hoped that this aggressive use of early toxicology studies will re-duce overall research costs and cycle times across the industry. Gi-ven the lag time between the discovery cycle and the market therehas not been sufficient time to assess the impact of this new para-digm on the development of new chemical entities (NCEs).

A more difficult decision facing project teams is deciding which,and how many, compounds should be progressed to exploratoryin vivo studies to investigate safety profiles of compounds in ani-mals. These decisions are often poorly informed with littlein vitro safety data to aid compound selection for initial in vivosafety studies. These studies often require relatively large quanti-ties of high purity material to be prepared and so present a signif-icant investment for project teams. The selection of compoundscan be aided by assessments of off-target pharmacology and com-pounds that demonstrate promiscuity are often removed from con-sideration.7 Other non-safety related parameters such as ease ofsynthesis and pharmacokinetics are also taken into considerationbut the decisions made are still highly qualitative rather than datadriven. In this Letter, we describe how an in vitro ATP depletion as-say, that measures a compound’s ability to cause cytotoxicity intransformed human liver epithelial (THLE) cells can be used effec-tively to prioritize compounds for in vivo studies.

To be able to compare a continuous concentration measure-ment in an in vitro cytotoxicity assay to an in vivo measurementof toxicity it is critical to have a measure of the exposure at whichthe toxicity was observed in the in vivo study. Often literature re-ports of toxicology studies do not include information on the druglevels in the circulating blood stream making these types of

Table 1THLE assay result versus in vivo outcome

THLE LC50 bucket # Compd toxicat 10 lM (total: 155)

# Compd cleanat 10 lM (total: 57)

<10 lM 8 010–25 lM 7 325–50 lM 20 050–100 lM 19 3>100 lM 101 51

N. Greene et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5308–5312 5309

comparisons virtually impossible. In this experiment, we used Pfiz-er proprietary compounds, studied in short term exploratory toxi-cology studies, where this detailed information was available. Forthis analysis 290 compounds were selected for screening in theTHLE assay. The compound set represents a diverse spectrum ofchemical structures, intended target pharmacologies and diseaseindications. The purity and correct compound mass for the sampleswere confirmed using NMR and LC–MS. Within the format of thecytotoxicity assay described (see Supplementary data), compoundswere determined to have LC50 values ranging from <10 lM throughto an artificial maximum of >300 lM.

The exploratory in vivo safety studies for this analysis typicallyinvolved dose escalation over three or more dose levels for a dura-tion of four days or longer. No distinction was made regarding thespecies or the sex of the animals assessed in these studies and allcompounds were administered orally. The rationale for combiningspecies was that the in vitro assay was performed in a human cellline and measured cytotoxicity and was considered able to detecttoxicity in all pre-clinical species. In an attempt to differentiate thestructural determinants of toxicity from identified pharmacologicalcauses, we eliminated those in vivo findings where the primarypharmacology of the compound had a well known link to the dis-crete adverse safety outcome. In addition, corresponding exposuredata (Cmax) and plasma protein binding data were also recorded

For the purpose of this exercise two systems for classifyingcompounds according to the observation seen in its exploratorytoxicology study were used. The first system is the binary classifi-cation previously used to look at the influence of physicochemicalproperties on the incidence of adverse findings.8 This classificationscheme uses a 10 lM threshold for Cmax and compounds thatshowed any form of significant adverse finding in the exploratorytoxicology study below this threshold were classified as ‘toxic’and any compound that had no significant findings at Cmax levelsabove this threshold were classified as ‘clean’. For some 78 com-pounds in the original data set, the doses in the in vivo study weresuch that the highest exposure with no significant findings was be-low the 10 lM Cmax threshold but the lowest dose with significantfindings was above the 10 lM Cmax threshold. In these cases it wasimpossible to put these compounds into one or other of the classesand so these were removed from any analysis that used this binaryclassification leaving 212 compounds in the data set.

The second system is a crude way to look at the relative severityof findings in the in vivo study at a particular dose and Cmax con-centration based on a simple scoring system for severity.9 The find-ings for each dose group within a study were broken out into thethree main classes of observations made during the experiment or-gan function, pathological damage and systemic toleration.

Dose groups where significant perturbations in both serumchemistry and hematology parameters were observed were as-signed an organ function score of ‘4’. Dose groups where only per-turbations in serum chemistry parameters or hematologyparameters were found were given an organ function score of ‘2’and those with no compound related changes in either of these cat-egories were given an organ function score of ‘0’. If a dose group hada microscopically observable necrosis in one or more tissues, thenthe dose group was assigned a pathological damage score of ‘‘4”.If the dose group was reported to have any microscopic observationother than necrosis, then it was assigned a pathological damagescore of ‘2’ and if there were no microscopic observations for thedose group then a score of ‘0’ was assigned. The overall score forthe dose group could then be calculated by adding the organ func-tion score and the pathological damage score. However, dosegroups where systemic toleration observations were unscheduleddeaths were given an overall score of ‘12’ since organ functionand pathological damage could not be determined accurately inthese cases.8

For the results of this analysis to be broadly applicable it isimportant that the set of compounds are of diverse chemical struc-ture and representative of the current pharmacological space,(Fig. 1). The molecular property distribution of the cytotoxicitydataset overlaps well with a selection of compounds taken froma diverse subset of the Pfizer file chosen for its coverage of chem-ical space. From analysis of chemical structure alone there did notappear to be any learning around structural motifs that appearedto be higher risk in terms of the THLE assay.

A full listing of all the compound in vitro results and in vivoclassifications are listed in the Supplementary data table. Withinthe dynamic range of the in vitro assay, the data was subdividedinto four categories; compounds with THLE LC50 <10 lM, <25 lM,<50 lM and <100 lM. Of the compounds with THLE LC50 <10 lMnone of them were defined as clean from the in vivo exploratorytoxicology studies, and 8 compounds were classified as toxic fromthe in vivo exploratory toxicology studies. Therefore, the sensitiv-ity of the THLE assay with the cutoff of <10 lM, where sensitivity isequal to the number of correctly identified toxic compounds di-vided by the total number of toxic compounds, was 5% (8:155)with a false positive rate of 0%, where false positive rate equalsthe number of compounds incorrectly called toxic divided by thetotal number of toxic calls, when compared to the gold standardassay (in vivo exploratory toxicology studies). Altering the thresh-old of the THLE assay, we then examined the ability to further dis-criminate between toxic and clean compounds. As the thresholdincreases in the THLE assay the sensitivity and false positive ratesare also modified. The best compromise was to select LC50 <50 lMwhere 35 compounds from the 155 defined as toxic from theexploratory in vivo studies were identified. This gives a sensitivityof 23% (35:155) and a false positive rate of 8% (3:38). These resultsare summarized in Tables 1 and 2.

In summary, the relative odds of being toxic at 10 lM whenTHLE LC50 <50 lM = 35:3 = 11.7 whereas the odds of being toxicat 10 lM when THLE LC50 >50 lM = 120:54 = 2.2 which meanscompounds with a THLE LC50 <50 lM are five times (11.7 dividedby 2.2) more likely to see findings in an exploratory toxicologystudy at a Cmax <10 lM than if it’s LC50 >50 lM.

Comparing the in vitro assay response to the measure of sever-ity presents a greater challenge since the former is a continuousscale and the latter is a categorical measure at a defined Cmax. Sincethe range of Cmax values observed for this data was spread overmultiple orders of magnitude, a log scale conversion was per-formed to give a normal distribution. In addition, we binned eachcompound-dose group combination according to the compound’sLC50 value and the observed score for the dose group. The averagelog (Cmax) was then calculated for each bin and the results areshown in Table 3.

The differences between these bins, either row to row or columnto column are considered statistically significant with approximatep-values <0.001, however, precise calculation of p-values is hin-dered by the categorical nature of the severity scores at the dosegroup level. Nevertheless, average log (Cmax) of compounds elicitingno adverse outcome in the exploratory toxicity studies (i.e., 0) areapproximately 4.7-fold higher on average for compounds with

Table 2Predictive capability using various THLE assay thresholds when compared to in vivo outcomes

In vivo classification (total # compd) # Compd with LC50 <10 lM # Compd with LC50 <25 lM # Compd with LC50 <50 lM # Compd with LC50 <100 lM

Clean at 10 lM (57) 0 3 3 6Toxic at 10 lM (155) 8 15 35 54Relative odds of being toxic at 10 lM 8:0 15:3 = 5:1 35:3 = 11.7:1 54:6 = 9:1Sensitivity (8:155) 5% (15:155) 10% (35:155) 22.5% (54:155) 34.8%False positive rate (0:8) 0% (3:18) 16.6% (3:35) 8% (6:60) 10%

Sensitivity = no. correctly identified toxic compounds/total no. toxic compounds False positive rate = no. incorrect positive predictions/total no. positive predictions.

Table 3THLE assay results versus average log (Cmax) and severity of in vivo outcomes

Dose group severity score

0 2 4 6 or 8 12

THLE assay LC50 bucket 0–50 lM 0.097 0.343 0.590 0.899 0.50150–100 lM 0.473 0.831 0.965 1.186 0.669>100 lM 0.455 0.785 0.921 1.046 1.006

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THLE LC50 >100 lM compared to compounds with THLE LC50 <50lM (0.455 vs 0.097, respectively). Likewise average log Cmax ofcompounds eliciting the most severe outcomes in the exploratorytoxicity studies (i.e., 12) are approximately twofold higher on aver-age for compounds with THLE LC50 >100 lM compared to com-pounds with THLE LC50 <50 lM (1.006 vs 0.501, respectively). Ahigher log Cmax is required to elicit the same dose group categoricalseverity score for compounds with THLE LC50 >100 lM compared tocompounds with THLE LC50 <50 lM.

ATP viability measurements provide a broad and non-mecha-nism specific assessment of a compounds ability to cause celldeath. Mechanisms leading to cytotoxicity can range from simplesurfactant effects causing membrane destabilization and rupture,to interference with key intracellular processes such as apoptosis,ER-stress, mitochondrial function and many others. It would besurprising for a simple, high throughput cytotoxicity assay to behighly predictive of the outcomes of compounds in a system ascomplicated as an in vivo rodent study as it does not account formany of the mechanisms by which toxicity can occur nor does itfactor in the rate of absorption, distribution, metabolism and clear-ance that also play a role in the expression of in vivo toxicity. Com-paring the THLE assay response to broad in vivo outcomes for a setof drug candidates, the level of sensitivity (22.5%) is relatively low,however compounds that have an LC50 650 lM are five times morelikely to see adverse events at lower Cmax thresholds than thosewith an LC50 >50 lM.

This is not to suggest that compounds with an LC50 <50 lMshould necessarily be dropped from further development as thisapproach does not take into consideration the difference betweenthe anticipated efficacious concentration required and the expo-sure at which toxicity is observed. However, if a typical compoundhas a 10 nM affinity (Ki) for its primary target, assuming a threetimes Ki free drug level for >75% receptor occupancy (for antagonistpharmacology), then approximately 30 nM free-drug concentra-tion would be required for efficacy. A compound clean at 1 lMfree-drug concentration would therefore exceed a 30-fold in vivosafety margin, which is often considered to be adequate. Given thisassumption and that the average drug compound is 90% bound toplasma proteins, a 10 lM Cmax threshold is an appropriate thresh-old and consistent with the pharmacological profile of many drugcandidates.

One potential limitation of looking only at the presence or ab-sence of any findings in the in vivo studies in categorization oftoxic versus clean compounds is that there is no consideration of

the severity of the findings observed and hence its likely impacton compound development. In order to address this we created asimple methodology to score the findings seen at each dose leveland hence be able to objectively assess the relative severity ofthe observations across different compounds. Comparing a com-pound’s response in the THLE assay to the average log (Cmax) wherea certain level of toxicity was observed shows a statistically signif-icant (�p <0.001) trend, whereas compounds with higher LC50 val-ues in the cytotoxicity assay have on average higher log (Cmax)values before the same level of toxicity in vivo is observed. The pre-cise p-values for these comparisons cannot be calculated since theseverity score is a categorical scale and the LC50 is a continuousmeasure. Considering that for the typical drug candidate, the min-imum acceptable in vivo safety margin is 30-fold the efficaciousfree drug concentration, a reduction in the Cmax at which signifi-cant toxicity is observed will naturally impact on the ability of thatcompound to progress into the clinic.

Previously published findings have demonstrated a correlationbetween the physicochemical properties of compounds and thelikelihood of seeing an adverse finding in an in vivo toxicologystudy.8 In particular, lipophilicity as assessed by c log P and topo-logical polar surface area (TPSA) were key determinants of risk.In contrast, compounds with c log P>3 and TPSA<75 Å2 were morelikely to have a finding at a Cmax <10 lM than compounds withc log P<3 and TPSA>75 Å2.

When we applied these physicochemical property thresholds tothe THLE data set we found that of the compounds with a c log P >3and TPSA <75 Å2 only four were classified as clean in the in vivoexploratory toxicology study and 71 were classified as toxic givinga sensitivity of 46% and a false positive rate of just 5%. These phys-icochemical parameters are easily calculated during the designphase of the medicinal chemistry contribution and therefore canbe used as simple filters prior to any synthetic efforts and arguablywould provide a more effective measure for prioritization of com-pounds entering into in vivo studies.

However, combining the c log P and TPSA based physicochemi-cal predictions with the THLE assay with a cut off of LC50 <50 lMcould be used to further prioritize compounds for further explor-atory work. In this dataset, 75 compounds would fail the previ-ously published c log P and TPSA property thresholds with afurther 17 compounds being identified as having an LC50 <50 lMin the THLE assay making a total of 92 compounds being predictedto be toxic by one or other of the methods (see Table 4). The sen-sitivity, that is, the number of toxic compounds correctly identified

Figure 1. c log P versus MW plot for test compounds (red dots) compared to a representative set from the Pfizer file (blue dots).

Table 4Comparing compounds THLE responses to the physicochemical properties of the compound

THLE LC50 bucket Compounds classified according to their physicochemical properties total # of compounds (# of toxic at 10 lM compd)

TPSA <75 and c log P >3 75 (71) TPSA >75 and c log P >3 39 (24) TPSA <75 and c log P <3 39 (28) TPSA >75 and c log P <3 59 (32)

<50 lM 21 (20) 8 (7) 1 (1) 8 (7)50–100 lM 16 (15) 2 (0) 1 (1) 3 (3)>100 lM 38 (36) 29 (17) 37 (26) 48 (22)

N. Greene et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5308–5312 5311

divided by the total number of toxic compounds in the data set, hasnow increased to 56% while retaining a false positive rate, that is,the number of negative compounds identified as toxic divided bythe total number of compounds called toxic by this approach, ofless than 10%. In addition, compounds that pass the physicochem-ical property filters and have an LC50 650 lM in the THLE assay arestill five times more likely to give rise to one or more adverse find-ings at a Cmax <10 lM in an in vivo study, suggesting that theseparameters can be used effectively to prioritize chemical matterfor further exploratory development with lower probability of ad-verse safety applicable findings in vivo.

In conclusion, the THLE assay is a high throughput assay, andrequires minimal physical compound matter. It can be effectivelyused to help prioritize compounds that advance into in vivo toxi-cology studies. In addition, readily calculated physicochemicalparameters can be used to help design compounds that are in low-er risk chemical space in terms of their outcomes in an exploratory

in vivo safety study and that, once synthesized, a simple highthroughput cytotoxicity measurement can further enhance theability of drug discovery teams to select compounds with less po-tential for causing adverse findings at low exposures to progress toin vivo safety studies. The combination of improved understandingof chemical space driving high quality drug design and the subse-quent use of low cost, high throughput safety screens to aid com-pound selection for in vivo studies is similar to the currentparadigm used in pharmacokinetic and drug metabolism groupswithin the pharmaceutical industry.10 Development of furthermechanistically refined in vitro assays is clearly the next step for-ward in reducing attrition through safety related findings.

Acknowledgment

Statistical support for this analysis was provided by ShibingDeng and Yao Zhang.

5312 N. Greene et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5308–5312

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bmcl.2010.06.129.

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