Efflux Liability Research Articles

Target-Agnostic P-Glycoprotein Assessment Yields Strategies to Evade Efflux, Leading to a BRAF Inhibitor with Intracranial Efficacy

The blood-brain barrier (BBB) presents a major hurdle in the development of central nervous system (CNS) active therapeutics, and expression of the P-glycoprotein (P-gp) efflux transporter at the blood-brain interface further impedes BBB penetrance of most small molecules. Designing efflux liabilities out of compounds can be laborious, and there is currently no generalizable approach to directly transform periphery-limited agents to ones active in the CNS. Here, we describe a target-agnostic, prospective assessment of P-gp efflux using diverse compounds. Our results demonstrate that reducing the molecular size or appending a carboxylic acid in many cases enables evasion of P-gp efflux in cell-based experiments and in mice. These strategies were then applied to transform a periphery-limited V600EBRAF inhibitor, dabrafenib, into versions that possess potent and selective anti-cancer activity but now also evade P-gp-mediated efflux. When compared to dabrafenib, the compound developed herein (everafenib) has superior BBB penetrance and superior efficacy in an intracranial mouse model of metastatic melanoma, suggesting it as a lead candidate for the treatment of melanoma metastases to the brain and gliomas with BRAF mutation. More generally, the results described herein suggest the actionability of the trends observed in these target-agnostic efflux studies and provide guidance for the conversion of non-BBB-penetrant drugs into versions that are BBB-penetrant and efficacious.

Evaluation of a Conformationally Constrained Indole Carboxamide as a Potential Efflux Pump Inhibitor in Pseudomonas aeruginosa

Efflux pumps in Gram-negative bacteria such as Pseudomonas aeruginosa provide intrinsic antimicrobial resistance by facilitating the extrusion of a wide range of antimicrobials. Approaches for combating efflux-mediated multidrug resistance involve, in part, developing indirect antimicrobial agents capable of inhibiting efflux, thus rescuing the activity of antimicrobials previously rendered inactive by efflux. Herein, TXA09155 is presented as a novel efflux pump inhibitor (EPI) formed by conformationally constraining our previously reported EPI TXA01182. TXA09155 demonstrates strong potentiation in combination with multiple antibiotics with efflux liabilities against wild-type and multidrug-resistant (MDR) P. aeruginosa. At 6.25 µg/mL, TXA09155, showed ≥8-fold potentiation of levofloxacin, moxifloxacin, doxycycline, minocycline, cefpirome, chloramphenicol, and cotrimoxazole. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA09155. TXA09155 was determined to lower the frequency of resistance (FoR) to levofloxacin and enhance the killing kinetics of moxifloxacin. Most importantly, TXA09155 outperformed the levofloxacin-potentiation activity of EPIs TXA01182 and MC-04,124 against a CDC/FDA panel of MDR clinical isolates of P. aeruginosa. TXA09155 possesses favorable physiochemical and ADME properties that warrant its optimization and further development.

EXTH-01. INHIBITION OF DNA-PKcs BY M3814 POTENTIATES EFFICACY OF IONIZING RADIATION IN PATIENT-DERIVED XENOGRAFTS OF MELANOMA BRAIN METASTASES

Abstract Radio-resistance mechanisms limit the benefit of radiation therapy (RT) for melanoma brain metastases. A key pathway for radiation-induced DNA double-strand break repair is non-homologous end joining, which is critically mediated by DNA-dependent protein kinase (DNA-PKcs). Here we evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells at concentrations of ≥300 nM, and a minimum of 16 h exposure with ~300 nM M3814 was required for effective sensitization. M3814 inhibited RT-induced (5 Gy) auto-phosphorylation of serine-2056 of DNA-PKcs in primary cultures of M12, M15 and M27 PDX lines. Interestingly, inhibition of RT-induced DNA-PKcs by M3814 coincided with increased KAP1 phosphorylation, a DNA damage signaling regulated via ATM. Persistent γH2AX foci were observed in 28% M12 cells at 24 hours after co-treatment with M3814 and RT as compared to 12% cells following RT alone. In vivo pharmacokinetic analysis after single oral dose of 20 mg/kg M3814, showed reasonably short half-life (~2.44 hours) and poor brain distribution in wild-type FBV mice (Kpuu, 0.027). Consistent with an efflux liability, brain distribution of M3814 in triple knockout mice for BCRP/MDR1A/B was ~11 fold higher (Kpuu, 0.215). Compared to normal brain, much higher M3814 concentrations were detected in intracranially implanted M12 tumors (~23 fold and ~20 fold) 2 and 6 hours after a single oral dose of 50mg/kg respectively. The relative exclusion of M3814 from normal brain as compared to brain metastases suggests that this drug may have a favorable toxicity profile when combined with radiation for treatment of melanoma brain metastases, and this hypothesis is being tested in ongoing efficacy studies.

Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Preclinical Lead Optimization of a 1,2,4-Triazole Based Tankyrase Inhibitor.

Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans-cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC50 inhibition in WNT/β-catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.

Abstract 3067: OHM-581, a dual JAK2-BET inhibitor for the treatment of myelofibrosis and other hematologic malignancies

Abstract Ruxolitinib is the only JAK2 inhibitor approved for myelofibrosis (MF). Although JAK2 inhibitor therapy improves splenomegaly and systemic symptoms, it does not significantly reduce the MF clone or alter the natural history and survival in most patients. The novel small molecule OHM-581 not only inhibits JAK2, but also BET (bromodomain and extra terminal) proteins such as BRD4. Dual JAK2 and BET inhibition using other molecules has shown superior efficacy in murine MF models compared with JAK2 inhibition alone including amelioration of fibrosis, synergistic induction of apoptosis of primary patient, post-MF secondary acute myeloid leukemia (sAML) blasts, and significant improvement of the survival of mice engrafted with human sAML cells. OHM-581 is being developed for the treatment of MF and other hematologic malignancies to leverage these advantages of dual JAK2-BET inhibition. Results: In vitro biochemical assays show OHM-581 to inhibit BRD4 BD1 and BD2 with IC50s of 189 and 116 nM, respectively, and JAK2 with an IC50 of 7.1 nM (JAK1=244 nM; JAK3=76 nM). No significant hERG liability was seen. OHM-581 displays significant anti-proliferative activity against multiple liquid cancer cell lines. Notably, the GI50 values for MV4-11 (AML, MLL fusion+), HEL 92.1.7 (AML, JAK2V617F+) and UKE-1 (AML, JAK2V617F+) are 32, 87 and 140 nM, respectively. Consistent with its mechanism of action, OHM-581 robustly down-regulates cMYC expression (cMYC mRNA down-regulation IC50=18.6 nM compared with 41.4 nM for BET inhibitor JQ1) and JAK2 signaling. Moreover, the compound triggers apoptosis in MV4-11 cells (~7-fold increase in Caspase 3/7 activity at 24-h). OHM-581 does not reflect an efflux liability, the efflux ratio being nearly 1.8. OHM-581 is soluble in PBS pH7.4 at 26 µM and metabolically stable with nearly 51%, 67% and 88% remaining in mouse, rat and human liver microsomes, respectively, after 30 min. OHM-581 exhibits an oral bioavailability of ~40%. As compared to the small molecule fedratinib, which is also being developed for MF, OHM-581 does not inhibit thiamine uptake. Dose proportionality with a greater than proportional increase in both Cmax and AUC at doses up to 60 mg/kg, and a saturation of Cmax at doses of 60 mg/kg and higher, are observed for OHM-581. In vivo efficacy studies using an MV4-11 xenograft model demonstrate robust tumor inhibition (~75-90%) following doses of 30 and 60 mg/kg po BID for 20 days. Conclusion: OHM-581 is a novel dual inhibitor of JAK2 and BET that has potential as a therapeutic agent for MF and other hematologic malignancies. In addition to IND-enabling studies, current OHM-581 development activities are focused on further proof-of-concept analyses using preclinical models of MF and other myeloproliferative neoplasms, with results to be reported. Citation Format: Ram S. Upadhayaya, Raghava Reddy Kethiri, Stephan W. Morris, H. Michael Shepard, Samuel R. Saks, Michael J. Weickert. OHM-581, a dual JAK2-BET inhibitor for the treatment of myelofibrosis and other hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3067.

Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators.

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

Nitrothiophene carboxamides, a novel narrow spectrum antibacterial series: Mechanism of action and Efficacy

The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.

In vitro assessment of the interactions of dopamine β-hydroxylase inhibitors with human P-glycoprotein and Breast Cancer Resistance Protein

Inhibition of the biosynthesis of noradrenaline is a currently explored strategy for the treatment of hypertension, congestive heart failure and pulmonary arterial hypertension. While some dopamine β-hydroxylase (DBH) inhibitors cross the blood-brain barrier (BBB) and cause central as well as peripheral effects (nepicastat), others have limited access to the brain (etamicastat, zamicastat). In this context, peripheral selectivity is clinically advantageous, in order to prevent alterations of noradrenaline levels in the CNS and the occurrence of adverse central effects. A limited brain exposure results from the combination of several factors, such as a reduced passive permeability or affinity for efflux transporters, but efflux liabilities may also lead to unwanted drug-drug interactions (DDIs) in the presence of co-administered substrates or inhibitors.Thus, the purpose of the study herein presented was to explore the interaction of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), the two major efflux transporters of the BBB that hamper the entry of several drugs to the brain, with the DBH inhibitors, etamicastat, nepicastat and zamicastat. Madin-Darby canine kidney cells (MDCK II) and transfected lines with human MDR1 (MDCK-MDR1) and ABCG2 (MDCK-BCRP) genes were used as a BBB surrogate model. P-gp and BCRP substrates and/or inhibitors were identified through intracellular accumulation and bidirectional permeability assays. The obtained data revealed that zamicastat is a concentration-dependent dual P-gp and BCRP inhibitor with IC50 values of 73.8 ± 7.2 μM and 17.0 ± 2.7 μM, while etamicastat and nepicastat inhibited BCRP to greater extent than P-gp, with IC50 values of 47.7 ± 1.8 μM and 59.2 ± 9.4 μM, respectively. Additionally, etamicastat was identified as P-gp and BCRP dual substrate, as demonstrated by net flux ratios of 5.84 and 3.87 and decreased >50% by verapamil and Ko143. Conversely, nepicastat revealed to be a P-gp-only substrate, with a net flux ratio of 2.01, reduced to 0.92 in the presence of verapamil. Furthermore, nepicastat displayed a consistently higher apparent permeability (>8.49 × 10−6 cm s−1) than etamicastat (<0.58 × 10−6 cm s−1).The identification of etamicastat as a dual efflux substrate suggests that P-gp and BCRP may be partially responsible for the limited central exposure of this compound, in association with its low passive permeability. Moreover, the weak efflux inhibitory potencies of etamicastat and nepicastat revealed a low DDI risk, while the dual P-gp/BCRP inhibition of zamicastat could be studied in the future with synergically effluxed compounds, for which BBB penetration is severely impaired.

Optimization of CoaD Inhibitors against Gram-Negative Organisms through Targeted Metabolomics.

Drug-resistant Gram-negative bacteria are of increasing concern worldwide. Novel antibiotics are needed, but their development is complicated by the requirement to simultaneously optimize molecules for target affinity and cellular potency, which can result in divergent structure-activity relationships (SARs). These challenges were exemplified during our attempts to optimize inhibitors of the bacterial enzyme CoaD originally identified through a biochemical screen. To facilitate lead optimization, we developed mass spectroscopy assays based on the hypothesis that levels of CoA metabolites would reflect the cellular enzymatic activity of CoaD. Using these methods, we were able to monitor the effects of cellular enzyme inhibition at compound concentrations up to 100-fold below the minimum inhibitory concentration (MIC), a common metric of growth inhibition. Furthermore, we generated a panel of efflux pump mutants to dissect the susceptibility of a representative CoaD inhibitor to efflux. These approaches allowed for a nuanced understanding of the permeability and efflux liabilities of the series and helped guide optimization efforts to achieve measurable MICs against wild-type E.coli.

Restricted Delivery of Talazoparib Across the Blood–Brain Barrier Limits the Sensitizing Effects of PARP Inhibition on Temozolomide Therapy in Glioblastoma

Poly ADP-ribose polymerase (PARP) inhibitors, including talazoparib, potentiate temozolomide efficacy in multiple tumor types; however, talazoparib-mediated sensitization has not been evaluated in orthotopic glioblastoma (GBM) models. This study evaluates talazoparib ± temozolomide in clinically relevant GBM models. Talazoparib at 1-3 nmol/L sensitized T98G, U251, and GBM12 cells to temozolomide, and enhanced DNA damage signaling and G2-M arrest in vitroIn vivo cyclical therapy with talazoparib (0.15 mg/kg twice daily) combined with low-dose temozolomide (5 mg/kg daily) was well tolerated. This talazoparib/temozolomide regimen prolonged tumor stasis more than temozolomide alone in heterotopic GBM12 xenografts [median time to endpoint: 76 days versus 50 days temozolomide (P = 0.005), 11 days placebo (P < 0.001)]. However, talazoparib/temozolomide did not accentuate survival beyond that of temozolomide alone in corresponding orthotopic xenografts [median survival 37 vs. 30 days with temozolomide (P = 0.93), 14 days with placebo, P < 0.001]. Average brain and plasma talazoparib concentrations at 2 hours after a single dose (0.15 mg/kg) were 0.49 ± 0.07 ng/g and 25.5±4.1 ng/mL, respectively. The brain/plasma distribution of talazoparib in Bcrp-/- versus wild-type (WT) mice did not differ, whereas the brain/plasma ratio in Mdr1a/b-/- mice was higher than WT mice (0.23 vs. 0.02, P < 0.001). Consistent with the in vivo brain distribution, overexpression of MDR1 decreased talazoparib accumulation in MDCKII cells. These results indicate that talazoparib has significant MDR1 efflux liability that may restrict delivery across the blood-brain barrier, and this may explain the loss of talazoparib-mediated temozolomide sensitization in orthotopic versus heterotopic GBM xenografts. Mol Cancer Ther; 16(12); 2735-46. ©2017 AACR.

TTI-281, an Orally Bioavailable Bromodomain and Extraterminal Domain (BET) Inhibitor, Is a Promising Candidate for the Treatment of Multiple Myeloma

Although recent advances in the development of multiple myeloma (MM) therapies such as proteasome inhibitors and immunomodulatory agents have improved patient outcomes, MM remains incurable. Additional therapeutic agents with high efficacy, low toxicity and the convenience of oral administration are in high demand. BET inhibitors, such as JQ-1, have been considered as potential therapeutic agents for MM. In the present study, we report that TTI-281, an orally bioavailable BET inhibitor, displays anti-MM activity with a low toxicity profile in preclinical studies.First, TTI-281 was tested for binding and anti-tumor activity in vitro. BROMOscan and AlphaScreen assays demonstrated that TTI-281 bound to bromodomains of BRD2/BRD3/BRD4 with Kd values less than 10 nM. In MTS assays, TTI-281 inhibited the growth of MM cell lines (MM.1s, NCIH929, and RPMI-8826) with cell growth-inhibition (IC50) values less than 300 nM.Next, in vitro ADME screening and in vivo PK studies were conducted. Permeability assays using murine gastrointestinal epithelial cells indicated that TTI-281 had good permeability with little efflux liability (efflux ratio <1), suggesting favorable properties for oral absorption. Indeed, TTI-281 displayed excellent oral bioavailability in both mice and rats (93.1% and 91.8%, respectively). In addition, TTI-281 did not interfere with the metabolism of representative CYP isozyme substrates at concentrations up to 50 μM in pooled human liver microsomes. Data also suggested minimal potential for drug-drug interactions, allowing for the possible combination with first-line therapy to improve therapeutic and survival outcomes.Finally, TTI-281 was tested for anti-myeloma efficacy and tolerability in vivo. NOD-SCID mice (n=10/group) subcutaneously engrafted with the human myeloma cell line MM.1S were treated orally once daily for 21 days with different doses of TTI-281, vehicle control or the benchmark drug carfilzomib. TTI-281 reduced tumor growth in a dose-dependent manner in this MM xenograft model. At 30 mg/kg/day, TTI-281 led to a statistically significant decrease in tumor growth compared with the vehicle control and carfilzomib (reduced tumor volume: 67% after TTI-281 treatment vs 33% after carfilzomib treatment, p<0.0003). Furthermore, TTI-281 treatment was well tolerated, with no effect on body weight or other obvious toxicity.In summary, our preclinical data suggest that the orally available BET inhibitor TTI-281 has an excellent efficacy and safety profile, highlighting its potential as a promising drug candidate for myeloma therapy. DisclosuresWang:Trillium Therapeutics: Employment, Patents & Royalties. Choi:Trillium Therapeutics: Employment. Dove:Trillium Therapeutics: Employment, Patents & Royalties. Wang:Trillium Therapeutics: Employment. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Design, Optimization, and Biological Evaluation of Novel Keto-Benzimidazoles as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A)

Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

De novo prediction of p-glycoprotein-mediated efflux liability for druglike compounds.

P-glycoprotein (Pgp) is capable of recognizing and transporting a wide range of chemically diverse compounds in vivo. Overcoming Pgp-mediated efflux can represent a significant challenge when penetration into the central nervous system is required or within the context of developing anticancer therapies. While numerous in silico models have been developed to predict Pgp-mediated efflux, these models rely on training sets and are best suited to make interpolations. Therefore, it is desirable to develop ab initio models that can be used to predict efflux liabilities. Herein, we present a de novo method that can be used to predict Pgp-mediated efflux potential for druglike compounds. A model, which correlates the computed solvation free energy differences obtained in water and chloroform with Pgp-mediated efflux (in logarithmic scale), was successful in predicting Pgp efflux ratios for a wide range of chemically diverse compounds with a R(2) and root-mean-square error of 0.65 and 0.29, respectively.

Strategies to lower the Pgp efflux liability in a series of potent indole azetidine MCHR1 antagonists

A series of potent indolyl azetidine rMCHR1 antagonists were found to show poor CNS penetration due to Pgp efflux. We envisioned a strategy which included: lowering basicity; changing the conformational flexibility motif; and removal of a hydrogen bond donor, in an attempt to optimize this property while maintaining target receptor efficacy. This work resulted in mitigation of Pgp efflux, and led us to identify 1-dihydroindolyl azetidine derivatives with CNS penetration and excellent rMCHR1 binding affinity.

Permeability for Intestinal Absorption: Caco-2 Assay and Related Issues

In vitro permeability assays remain a valuable tool of screening scientists for lead compound optimization. As a majority of discovery projects are focused on the development of orally bioavailable drugs, the need for predictability and correlation of in vitro permeability data to in vivo absorption results has never been greater. For more than a decade, the Caco-2 screening assay has remained a popular, in vitro system to test compounds for intestinal permeability and efflux liability. Despite advances in artificial membrane technology and in silico modeling systems, drug compounds still benefit from testing in cell-based epithelial monolayer assays for lead optimization and SAR. This review discusses the strengths and limitations of the Caco-2 permeability assay, and puts into context the power of combining multiple assays and approaches to improve predictability and rank-ordering for lead compound optimization. Technical information for dealing with some of the most pressing issues with in vitro permeability assays (i.e. low aqueous solubility and low post-assay recovery) is also discussed. Insights are offered to help researchers avoid common pitfalls in the interpretation of in vitro permeability data, which can often lead to the perception of misleading results for correlation to in vivo data. In addition, the advantages of addressing the issue of efflux liability early in the drug development process is discussed, detailing the usefulness of Caco-2 cells for this type of screening paradigm.