Interplay of OATP1A/1B/2B1 uptake transporters and ABCB1 and ABCG2 efflux transporters in the handling of bilirubin and drugs.

Transmembrane drug transporters can be important determinants of the pharmacokinetics, efficacy, and safety profiles of drugs. To investigate the potential cooperative and/or counteracting interplay of OATP1A/1B/2B1 uptake transporters and ABCB1 and ABCG2 efflux transporters in physiology and pharmacology, we generated a new mouse model (Bab12), deficient for Slco1a/1b, Slco2b1, Abcb1a/1b and Abcg2. Bab12 mice were viable and fertile. We compared wild-type, Slco1a/1b/2b1-/-, Abcb1a/1b;Abcg2-/- and Bab12 strains. Endogenous plasma conjugated bilirubin levels ranked as follows: wild-type = Abcb1a/1b;Abcg2-/- << Slco1a/1b/2b1-/- < Bab12 mice. Plasma levels of rosuvastatin and fexofenadine were elevated in Slco1a/1b/2b1-/- and Abcb1a/1b;Abcg2-/- mice compared to wild-type, and dramatically increased in Bab12 mice. Although systemic exposure of larotrectinib and repotrectinib was substantially increased in the separate multidrug transporter knockout strains, no additive effects were observed in the combination Bab12 mice. Significantly higher plasma exposure of fluvastatin and pravastatin was only found in Slco1a/1b/2b1-deficient mice. However, noticeable transport by Slco1a/1b/2b1 and Abcb1a/1b and Abcg2 across the BBB was observed for fluvastatin and pravastatin, respectively, by comparing Bab12 mice with Abcb1a/1b;Abcg2-/- or Slco1a/1b/2b1-/- mice. Quite varying behavior in plasma exposure of erlotinib and its metabolites was observed among these strains. Bab12 mice revealed that Abcb1a/1b and/or Abcg2 can contribute to conjugated bilirubin elimination when Slco1a/1b/2b1 are absent. Our results suggest that the interplay of Slco1a/1b/2b1, Abcb1a/1b, and Abcg2 could markedly affect the pharmacokinetics of some, but not all drugs and metabolites. The Bab12 mouse model will represent a useful tool for optimizing drug development and clinical application, including efficacy and safety.

Pharmacokinetics of the KRASG12C inhibitor adagrasib is limited by CYP3A and ABCB1, and influenced by binding to mouse plasma carboxylesterase 1c.

Adagrasib (Krazati™) is the second FDA-approved specific KRASG12C inhibitor for non-small cell lung cancer (NSCLC) patients harboring this mutation. The impact of the drug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing enzymes CYP3A and carboxylesterase 1 (CES1) on the pharmacokinetics of oral adagrasib were studied using genetically modified mouse models. Adagrasib was potently transported by human ABCB1 and modestly by mouse Abcg2 in vitro. In Abcb1a/b-/- and Abcb1a/b;Abcg2-/- mice, the brain-to-plasma ratios were enhanced by 33- and 55-fold, respectively, compared to wild-type mice, whereas ratios in Abcg2-/- mice remained unchanged. The influence of ABC transporters was completely reversed by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, increasing the brain penetration in wild-type mice by 41-fold while no signs of acute CNS toxicity were observed. Tumor ABCB1 overexpression may thus confer adagrasib resistance. Whereas the ABC transporters did not affect adagrasib plasma exposure, CYP3A and Ces1 strongly impacted its apparent oral availability. The plasma AUC0-8 h was significantly enhanced by 2.3-fold in Cyp3a-/- compared to wild-type mice, and subsequently 4.3-fold reduced in transgenic CYP3A4 mice, indicating substantial CYP3A-mediated metabolism. Adagrasib plasma exposure was strongly reduced in Ces1-/- compared to wild-type mice, but tissue exposure was slightly increased, suggesting that adagrasib binds to plasma Ces1c in mice and is perhaps metabolized by Ces1. This binding could complicate interpretation of mouse studies, especially since humans lack circulating CES1 enzyme(s). Our results may be useful to further optimize the clinical safety and efficacy of adagrasib, and give more insight into potential drug-drug interactions risks.

Natural deletion of mouse carboxylesterases Ces1c/d/e impacts drug metabolism and metabolic syndrome development.

Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis. Ces1c/d/e-/- mice showed strongly impaired conversion of the anticancer prodrug irinotecan to its active metabolite SN-38 in plasma, spleen and lung. Plasma hydrolysis of the oral anticancer prodrug capecitabine to 5-DFCR was also profoundly reduced in Ces1c/d/e-/- mice. Our findings resolved previously unexplained FVB/NKI pharmacokinetic anomalies. On a medium-fat diet, Ces1c/d/e-/- female mice exhibited moderately higher body weight, mild inflammation in gonadal white adipose tissue (gWAT), and increased lipid load in brown adipose tissue (BAT). Ces1c/d/e-/- males showed more pronounced inflammation in gWAT and an increased lipid load in BAT. On a 5-week high-fat diet exposure, Ces1c/d/e deficiency predisposed to developing obesity, enlarged and fatty liver, glucose intolerance and insulin resistance, with severe inflammation in gWAT and increased lipid load in BAT. Hepatic proteomics analysis revealed that the acute phase response, involved in the dynamic cycle of immunometabolism, was activated in these Ces1c/d/e-/- mice. This may contribute to the obesity-related chronic inflammation and adverse metabolic disease in this strain. While Ces1c/d/e deficiency clearly exacerbated metabolic syndrome development, long-term (18-week) high-fat diet exposure overwhelmed many, albeit not all, observed phenotypic differences.

Carboxylesterase 1 family knockout alters drug disposition and lipid metabolism.

The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated Ces1 cluster knockout (Ces1 -/- ) mice, and a hepatic human CES1 transgenic model in the Ces1 -/- background (TgCES1). Ces1 -/- mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues. TgCES1 mice exhibited enhanced metabolism of irinotecan to SN-38 in liver and kidney. Ces1 and hCES1 activity increased irinotecan toxicity, likely by enhancing the formation of pharmacodynamically active SN-38. Ces1 -/- mice also showed markedly increased capecitabine plasma exposure, which was moderately decreased in TgCES1 mice. Ces1 -/- mice were overweight with increased adipose tissue, white adipose tissue inflammation (in males), a higher lipid load in brown adipose tissue, and impaired blood glucose tolerance (in males). These phenotypes were mostly reversed in TgCES1 mice. TgCES1 mice displayed increased triglyceride secretion from liver to plasma, together with higher triglyceride levels in the male liver. These results indicate that the carboxylesterase 1 family plays essential roles in drug and lipid metabolism and detoxification. Ces1 -/- and TgCES1 mice will provide excellent tools for further study of the in vivo functions of Ces1/CES1 enzymes.

Organic anion-transporting polypeptide 2B1 knockout and humanized mice; insights into the handling of bilirubin and drugs.

Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.

Rifampin and ritonavir increase oral availability and elacridar enhances overall exposure and brain accumulation of the NTRK inhibitor larotrectinib.

INTRODUCTION: Larotrectinib is an FDA-approved oral small-molecule inhibitor for neurotrophic tropomyosin receptor kinase (NTRK) fusion-positive cancer treatment. Here larotrectinib pharmacokinetic behavior upon co-administration with prototypical inhibitors of the efflux transporters ABCB1/ABCG2 (elacridar), the SLCO1A/1B (OATP1A/1B) uptake transporters (rifampin), and the drug-metabolizing enzyme CYP3A (ritonavir), respectively, was investigated. METHODS: Inhibitors were orally administered prior to oral larotrectinib (10 mg/kg) to relevant genetically modified mouse models. Larotrectinib plasma and tissue homogenate concentrations were measured by a liquid chromatography-tandem mass spectrometric assay. RESULTS: Elacridar increased oral availability (2.7-fold) and markedly improved brain-to-plasma ratios (5.0-fold) of larotrectinib in wild-type mice. Mouse (m)Oatp1a/1b but not hepatic transgenic human (h)OATP1B1 or -1B3 restricted larotrectinib oral availability and affected its tissue distribution. Rifampin enhanced larotrectinib oral availability not only in wild-type mice (1.9-fold), but surprisingly also in Slco1a/1b-/- mice (1.7-fold). Similarly, ritonavir increased the larotrectinib plasma exposure in both wild-type (1.5-fold) and Cyp3a-/- mice (1.7-fold). Intriguingly, both rifampin and ritonavir decreased liver and/or intestinal larotrectinib levels in all related experimental groups, suggesting additional inhibition of enterohepatic Abcb1a/1b activity. CONCLUSIONS: Elacridar enhances both larotrectinib plasma and tissue exposure and especially relative brain penetration, which might be therapeutically relevant. Hepatic mOatp1a/1b but not hOATP1B1 or -1B3 transported larotrectinib. Additionally, rifampin enhances larotrectinib systemic exposure, most likely by inhibiting mOatp1a/1b, but probably also hepatic and/or intestinal mAbcb1. Similar to rifampin, dual-inhibition functions of ritonavir affecting both CYP3A enzymes and enterohepatic Abcb1 transporters enhanced larotrectinib oral availability. The obtained insights may be used to further optimize the clinical-therapeutic application of larotrectinib.

P-Glycoprotein (ABCB1/MDR1) and BCRP (ABCG2) Limit Brain Accumulation and Cytochrome P450-3A (CYP3A) Restricts Oral Exposure of the RET Inhibitor Selpercatinib (RETEVMO).

Selpercatinib is a targeted, FDA-approved, oral, small-molecule inhibitor for the treatment of rearranged during transfection (RET) proto-oncogene mutation-positive cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A complex in selpercatinib pharmacokinetics. Selpercatinib was efficiently transported by hABCB1 and mAbcg2, but not hABCG2, and was not a substrate of human OATP1A2, -1B1 or -1B3 in vitro. In vivo, brain and testis penetration were increased by 3.0- and 2.7-fold in Abcb1a/1b-/- mice and by 6.2- and 6.4-fold in Abcb1a/1b;Abcg2-/- mice, respectively. Oatp1a/1b deficiency did not alter selpercatinib pharmacokinetics. The ABCB1/ABCG2 inhibitor elacridar boosted selpercatinib brain penetration in wild-type mice to the levels seen in Abcb1a/1b;Abcg2-/- mice. Cyp3a-/- mice showed a 1.4-fold higher plasma AUC0-4h than wild-type mice, which was then 1.6-fold decreased upon transgenic overexpression of human CYP3A4 in liver and intestine. In summary, ABCG2, and especially ABCB1, limit brain and testis penetration of selpercatinib. Elacridar coadministration could mostly reverse these effects, without causing acute toxicity. CYP3A-mediated metabolism can limit selpercatinib oral exposure and hence its tissue concentrations. These insights may be useful in the further clinical development of selpercatinib.

ABCB1 and ABCG2, but not CYP3A4 limit oral availability and brain accumulation of the RET inhibitor pralsetinib.

BACKGROUND AND PURPOSE: Pralsetinib is an FDA-approved oral small-molecule inhibitor for treatment of rearranged during transfection (RET) proto-oncogene fusion-positive non-small cell lung cancer. We investigated how the efflux transporters ABCB1 and ABCG2, the SLCO1A/1B uptake transporters and the drug-metabolizing enzyme CYP3A influence pralsetinib pharmacokinetics. EXPERIMENTAL APPROACH: In vitro, transepithelial pralsetinib transport was assessed. In vivo, pralsetinib (10 mg/kg) was administered orally to relevant genetically modified mouse models. Pralsetinib concentrations in cell medium, plasma samples and organ homogenates were measured using liquid chromatography-tandem mass spectrometry. KEY RESULTS: Pralsetinib was efficiently transported by human (h)ABCB1 and mouse (m)Abcg2, but not hACBG2. In vivo, mAbcb1a/1b markedly and mAbcg2 slightly limited pralsetinib brain penetration (6.3-and 1.8-fold, respectively). Testis distribution showed similar results. Abcb1a/1b;Abcg2-/- mice showed 1.5-fold higher plasma exposure, 23-fold increased brain penetration, and 4-fold reduced recovery of pralsetinib in the small intestinal content. mSlco1a/1b deficiency did not affect pralsetinib oral availability or tissue exposure. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted pralsetinib plasma exposure (1.3-fold) and brain penetration (19.6-fold) in wild-type mice. Additionally, pralsetinib was a modest substrate of mCYP3A, but not of hCYP3A4, which did not noticeably restrict the oral availability or tissue distribution of pralsetinib. CONCLUSIONS AND IMPLICATIONS: SLCO1A/1B and CYP3A4 are unlikely to affect the pharmacokinetics of pralsetinib, but ABCG2 and especially ABCB1 markedly limit its brain and testis penetration, as well as oral availability. These effects are mostly reversed by oral coadministration of the ABCB1/ABCG2 inhibitor elacridar. These insights may be useful in the further clinical development of pralsetinib.

Bioanalytical method for the simultaneous quantification of irinotecan and its active metabolite SN-38 in mouse plasma and tissue homogenates using HPLC-fluorescence.

A simple and rapid bioanalytical method was developed for the simultaneous quantification of irinotecan and SN-38 in mouse plasma and tissue homogenates using High-Performance Liquid Chromatography with Fluorescence detection (HPLC-FL). Camptothecin was used as internal standard and protein precipitation with acetonitrile-methanol (1:1, v/v) followed by acidification with 0.5 M hydrochloric acid was used for sample pre-treatment. The analytes and the internal standard were detected using an excitation and emission wavelength of 368 and 515 nm, respectively. The linearity, selectivity, accuracy and precision, carry-over, limit of detection and lower limit of quantification of the method are described. The method was linear from 7.5 to 1500 ng/mL for irinotecan and from 5 to 1000 ng/mL for SN-38. For all matrices, the accuracy bias and precision variation were within ±15% and ≤15%, respectively. This method was successfully applied to study the pharmacokinetics of irinotecan and SN-38 using in vivo mouse models.

Quantitative bioanalytical assay for the selective RET inhibitors selpercatinib and pralsetinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Selpercatinib and pralsetinib are potent and selective tyrosine kinase inhibitors targeting the rearranged during transfection (RET) receptor in various types of cancer. In this study, a bioanalytical assay was developed and fully validated for selpercatinib and pralsetinib in mouse plasma and partially in eight mouse tissue homogenates using liquid chromatograph-tandem mass spectrometry. Samples were pre-treated by protein precipitation with acetonitrile using erlotinib as internal standard. Separation of the analytes was performed on an ethylene bridged octadecyl silica C18 column by gradient elution using ammonium hydroxide (in water) and methanol. Analytes were detected by positive electrospray ionization in selected reaction monitoring mode. A linear concentration range of 2-2000 ng/ml was used for the validation of the assay for both inhibitors. The precision values (within-day and between-day) ranged between 3.4 and 10.2% for selpercatinib and 3.1-14.6% for pralsetinib in all matrices. Furthermore, data obtained for accuracy were between 91.7 and 109.3% and 85.1-114.1% for selpercatinib and pralsetinib, respectively. No significant matrix effects or extraction losses were observed and both analytes were stable under all investigated conditions. Finally, a pilot study for selpercatinib in mice was conducted employing this method, followed by a successful incurred sample reanalysis.

OATP1A/1B, CYP3A, ABCB1, and ABCG2 limit oral availability of the NTRK inhibitor larotrectinib, while ABCB1 and ABCG2 also restrict its brain accumulation.

BACKGROUND AND PURPOSE: Larotrectinib is a FDA-approved oral small-molecule inhibitor for treatment of neurotrophic tropomyosin receptor kinase fusion-positive cancer. We here investigated the functions of the multidrug efflux transporters ABCB1 and ABCG2, the SLCO1A/1B (OATP1A/1B) uptake transporters, and the multispecific drug-metabolizing enzyme CYP3A in larotrectinib pharmacokinetic behaviour. EXPERIMENTAL APPROACH: In vitro, transepithelial drug transport and uptake assays were performed. In vivo, larotrectinib (10 mg·kg-1 ) was administered orally to relevant genetically modified mouse models. Cell medium, plasma samples, and organ homogenates were measured by a sensitive and specific LC-MS/MS larotrectinib assay. KEY RESULTS: In vitro, larotrectinib was avidly transported by human (h) ABCB1 and mouse (m) Abcg2 efficiently by hABCG2 and modestly by hOATP1A2. In vivo, both mAbcb1a/1b and mAbcg2 markedly limited larotrectinib oral availability and brain and testis accumulation (by 2.1-fold, 10.4-fold, and 2.7-fold, respectively), with mAbcb1a/1b playing a more prominent role. mOatp1a/1b also restricted larotrectinib oral availability (by 3.8-fold) and overall tissue exposure, apparently by mediating substantial uptake into the liver, thus likely facilitating hepatobiliary excretion. Additionally, larotrectinib is an excellent substrate of CYP3A, which restricts the oral availability of larotrectinib and hence its tissue exposure. CONCLUSIONS AND IMPLICATIONS: ABCG2 and especially ABCB1 limit the oral availability and brain and testis penetration of larotrectinib, while OATP1A/1B transporters restrict its systemic exposure by mediating hepatic uptake, thus allowing hepatobiliary excretion. CYP3A-mediated metabolism can strongly limit larotrectinib oral availability and hence its tissue concentrations. These insights may be useful in the further clinical development of larotrectinib.

P-glycoprotein (MDR1/ABCB1) controls brain accumulation and intestinal disposition of the novel TGF-ß signaling pathway inhibitor galunisertib.

Galunisertib (LY2157299), a promising small-molecule inhibitor of the transforming growth factor-beta (TGF-ß) receptor, is currently in mono- and combination therapy trials for various cancers including glioblastoma, hepatocellular carcinoma and breast cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters and the drug-metabolizing CYP3A complex in galunisertib pharmacokinetics. In vitro, galunisertib was vigorously transported by human ABCB1, and moderately by mouse Abcg2. Orally administered galunisertib (20 mg/kg) was very rapidly absorbed. Galunisertib brain-to-plasma ratios were increased by ~24-fold in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice compared to wild-type mice, but not in single Abcg2-/- mice, whereas galunisertib oral availability was not markedly affected. However, recovery of galunisertib in the small intestinal lumen was strongly reduced in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted galunisertib brain accumulation in wild-type mice to equal the levels seen in Abcb1a/1b;Abcg2-/- mice. Oatp1a/1b deficiency did not alter oral galunisertib pharmacokinetics or liver distribution. Cyp3a-/- mice showed a 1.9-fold higher plasma AUC0-1 hr than wild-type mice, but this difference disappeared over 8 hr. Also, transgenic human CYP3A4 overexpression did not significantly alter oral galunisertib pharmacokinetics. Abcb1 thus markedly restricts galunisertib brain penetration and affects its intestinal disposition, possibly through biliary excretion. Elacridar coadministration could fully inhibit both processes, without causing acute toxicity. Moreover, mouse Cyp3a, but not human CYP3A4, may eliminate galunisertib at high plasma concentrations. These insights may help to guide the further clinical development and application of galunisertib.

P-glycoprotein (ABCB1/MDR1) limits brain accumulation and Cytochrome P450-3A (CYP3A) restricts oral availability of the novel FGFR4 inhibitor fisogatinib (BLU-554).

Fisogatinib (BLU-554) is a highly selective and potent oral fibroblast growth factor receptor 4 (FGFR4) inhibitor currently in Phase I clinical trials for treatment of hepatocellular carcinoma (HCC). Using (male) genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A complex in fisogatinib pharmacokinetics. In vitro, fisogatinib was modestly transported by hABCB1. Upon oral administration of 10 mg/kg fisogatinib, its brain accumulation was substantially increased in Abcb1a/1b-/- (6.3-fold) and Abcb1a/1b;Abcg2-/- mice (7.2-fold) compared to wild-type mice, but not in single Abcg2-/- mice. The oral plasma pharmacokinetics and liver distribution of fisogatinib were not significantly affected by the absence of Oatp1a/1b drug uptake transporters. We further found that plasma exposure of fisogatinib in Cyp3a-/- mice increased by 1.4-fold, and was subsequently 1.6-fold decreased upon transgenic overexpression of human CYP3A4 in liver and intestine. However, the relative tissue distribution of fisogatinib remained unaltered. In summary, in mice, fisogatinib brain accumulation is substantially limited by ABCB1 P-glycoprotein in the blood-brain barrier, and oral availability of fisogatinib is markedly restricted by CYP3A activity. The obtained insights may be useful for optimizing the clinical efficacy and safety of fisogatinib.

Oral coadministration of elacridar and ritonavir enhances brain accumulation and oral availability of the novel ALK/ROS1 inhibitor lorlatinib.

Lorlatinib, a novel generation oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor with high membrane and blood-brain barrier permeability, recently received accelerated approval for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), and its further clinical development is ongoing. We previously found that the efflux transporter P-glycoprotein (MDR1/ABCB1) restricts lorlatinib brain accumulation and that the drug-metabolizing enzyme cytochrome P450-3A (CYP3A) limits its oral availability. Using genetically modified mouse models, we investigated the impact of targeted pharmacological inhibitors on lorlatinib pharmacokinetics and bioavailability. Upon oral administration of lorlatinib, the plasma AUC0-8h in CYP3A4-humanized mice was ∼1.8-fold lower than in wild-type and Cyp3a-/- mice. Oral coadministration of the CYP3A inhibitor ritonavir caused reversion to the AUC0-8h levels seen in wild-type and Cyp3a-/- mice, without altering the relative tissue distribution of lorlatinib. Moreover, simultaneous pharmacological inhibition of P-glycoprotein and CYP3A4 with oral elacridar and ritonavir in CYP3A4-humanized mice profoundly increased lorlatinib brain concentrations, but not its oral availability or other relative tissue distribution. Oral lorlatinib pharmacokinetics was not significantly affected by absence of the multispecific Oatp1a/1b drug uptake transporters. The absolute oral bioavailability of lorlatinib over 8 h in wild-type, Cyp3a-/-, and CYP3A4-humanized mice was 81.6%, 72.9%, and 58.5%, respectively. Lorlatinib thus has good oral bioavailability, which is markedly restricted by human CYP3A4 but not by mouse Cyp3a. Pharmacological inhibition of CYP3A4 reversed these effects, and simultaneous P-gp inhibition with elacridar boosted absolute brain levels of lorlatinib by 16-fold without obvious toxicity. These insights may help to optimize the clinical application of lorlatinib.

Quantitative bioanalytical assay for the tropomyosin receptor kinase inhibitor larotrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Larotrectinib is a promising tyrosine kinase inhibitor for solid tumors harboring tropomyosin receptor kinase gene fusions. A bioanalytical assay was developed for this drug in small volume samples using a 96-well format to efficiently support multiple mouse studies. The assay was completely validated for mouse plasma and partially for homogenates of eight different tissues: brain, heart, kidneys, liver, lungs, small intestine, spleen, and testes. Proteins in 10-µl samples were precipitated using acetonitrile containing momelotinib as internal standard. Chromatographic separation of analyte and internal standard from endogenous interferences was performed on an ethylene bridged octadecyl silica column using 0.1% (v/v) formic acid (in water) and methanol for gradient elution. Electrospray ionization and selected reaction monitoring on a triple quadrupole mass spectrometer were used for detection. In the range 1-2000 ng/ml the drug could be quantified in all 9 matrices with precisions (within-day and between-day) in the range 2.7-11.1% and accuracies in the range 87.4-101.4%. Compounds were sufficiently stable under all investigated conditions except for kidney homogenate. A pilot pharmacokinetic and tissue distribution study in mice demonstrated the applicability of the new presented assay for larotrectinib.

P-glycoprotein and breast cancer resistance protein restrict brigatinib brain accumulation and toxicity, and, alongside CYP3A, limit its oral availability.

Brigatinib is an FDA-approved oral anaplastic lymphoma kinase (ALK) inhibitor for treatment of metastatic non-small cell lung cancer (NSCLC). Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug-metabolizing enzyme CYP3 A in plasma pharmacokinetics and tissue distribution of brigatinib. In vitro, brigatinib was exceptionally well transported by human ABCB1 and mouse Abcg2, and efficiently by human ABCG2. Following oral brigatinib administration (10 mg/kg), brain accumulation was dramatically increased in Abcb1a/1b-/- (19.3-fold) and Abcb1a/1b;Abcg2-/-(41.8-fold), but not in single Abcg2-/- mice compared to wild-type mice. Brigatinib testis accumulation showed qualitatively similar behavior. mAbcb1a/1b and mAbcg2 together restricted systemic exposure of brigatinib: with both systems absent oral availability increased 1.9-fold. Coadministration of elacridar, an ABCB1/ABCG2 inhibitor, caused a pronounced increase (36-fold) in brain-to-plasma ratios of brigatinib, approaching the levels seen in Abcb1a/1b;Abcg2-/- mice. Unexpectedly, lethal toxicity of oral brigatinib was observed in mice with genetic knockout or pharmacological inhibition of mAbcb1a/1b and mAbcg2, indicating a pronounced protective role for these transporters. In Cyp3a-/- mice, brigatinib plasma exposure increased 1.3-fold, and was subsequently 1.8-fold reduced by transgenic overexpression of human CYP3 A4 in liver and intestine. The relative tissue distribution of brigatinib, however, remained unaltered. ABCB1 and ABCG2 thus limit brain accumulation, toxicity, and systemic exposure of brigatinib, whereas CYP3 A also markedly restricts its oral availability. Unexpected toxicities should therefore be carefully monitored when brigatinib is coadministered with ABCB1/ABCG2 inhibitors in patients. Collectively, these insights may support the clinical application of brigatinib.

P-glycoprotein (MDR1/ABCB1) restricts brain accumulation and cytochrome P450-3A (CYP3A) limits oral availability of the novel ALK/ROS1 inhibitor lorlatinib.

Lorlatinib (PF-06463922) is a promising oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor currently in Phase III clinical trials for treatment of non-small-cell lung cancer (NSCLC) containing an ALK rearrangement. With therapy-resistant brain metastases a major concern in NSCLC, lorlatinib was designed to have high membrane and blood-brain barrier permeability. We investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug-metabolizing enzyme CYP3A in plasma pharmacokinetics and tissue distribution of lorlatinib using genetically modified mouse strains. In vitro, human ABCB1 and mouse Abcg2 modestly transported lorlatinib. Following oral lorlatinib administration (at 10 mg/kg), brain accumulation of lorlatinib, while relatively high in wild-type mice, was still fourfold increased in Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice, but not in single Abcg2-/- mice. Lorlatinib plasma levels were not altered. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar increased the brain accumulation of lorlatinib in wild-type mice fourfold, that is, to the same level as in Abcb1a/1b;Abcg2-/- mice, without altering plasma exposure. Similar results were obtained for lorlatinib testis accumulation. In Cyp3a-/- mice, the plasma exposure of lorlatinib was increased 1.3-fold, but was then twofold reduced upon transgenic overexpression of human CYP3A4 in liver and intestine, whereas relative tissue distribution of lorlatinib remained unaltered. Our data indicate that lorlatinib brain accumulation is substantially limited by P-glycoprotein/ABCB1 in the blood-brain barrier, but this can be effectively reversed by elacridar coadministration. Moreover, oral availability of lorlatinib is markedly restricted by CYP3A4 activity. These insights may be used in optimizing the therapeutic application of lorlatinib.

The Impact of Different Preservation Conditions and Freezing-Thawing Cycles on Quality of RNA, DNA, and Proteins in Cancer Tissue.

BACKGROUND: High-quality cancer tissues are essential for future research, especially molecular research. For the sake of better quality of tissues, some storage methods are chosen according to lab conditions. But the impact of different storing conditions on the quality of RNA, DNA (especially the degree of DNA methylation), and protein of tissues that have undergone a thawing process, is not clear. METHODS: We analyzed the influence of different storage conditions including in RNALater solution, normal saline, Opti-mum Cutting Temperature compound (OCT), and snap frozen with no protective reagent (as control) in paired tissue samples on the quality of RNA (RNA Integrity Number value and mRNA expression), DNA quality (DNA amplification and DNA methylation degree of gene RASSF1a), and protein quality. Further, we analyzed the RNA quality of tissues that underwent three freeze-thaw cycles. RESULTS: The RNALater-treated group retained good RNA quality as expected on three repeated freeze-thaw cycles (RIN>8), but the snap-frozen tissues showed relatively poor results after one freeze-thaw cycle (RIN<7) and three times repeated freeze-thaw cycles (RIN<6). RNA from saline- and OCT-treated groups also yielded good results when we repeated freezing and thawing one time (RIN>7) and two times (RIN>6). The impact of different storing conditions on DNA amplification is small. However, DNA methylation and protein quality are different with different storing conditions. OCT seems to be more secure and stable compared with other two experimental groups, and show a similar trend with control group. CONCLUSIONS: In consideration of budget and efficiency, we suggest OCT as the best storing method that not only preserves RNA quality during the freezing-thawing process well, but also ensures more secure and stable DNA and protein.

Membrane-bound complement regulatory proteins are prognostic factors of operable breast cancer treated with adjuvant trastuzumab: a retrospective study.

Complement-dependent cytotoxicity (CDC) is an important antitumor mechanism of monoclonal antibodies (mAbs). However, trastuzumab, an anti-HER2 mAb, exerts only minor CDC. Overexpression of membrane-bound complement regulatory proteins (mCRPs), which suppress CDC, have been implicated in various malignant tumors. Here, we explored the predictive role of the expression levels of three mCRPs (CD55, CD59 and CD46) in the prognosis of breast cancer cases that underwent adjuvant trastuzumab treatment. We also studied the effect of mCRP downregulation on trastuzumab-induced CDC in vitro. Sixty-five HER2-positive breast cancer patients who received adjuvant therapy containing trastuzumab, were retrospectively analyzed. Levels of CD55, CD59 and CD46 expression were detected by immunohistochemistry. Chi-square test, Kaplan­Meier survival analysis and a Cox proportional hazards model were used to analyze the association between CD55, CD59 and CD46 expression and prognosis. HER2-positive SK-Br3 and BT-474 breast cancer cells were pretreated with various drugs to reduce mCRP expression. Afterwards, trastuzumab­mediated cytolytic effects were measured. Among the 65 patients, 46.2% had high expression of CD55, 44.6% had high expression of CD59 and 44.6% had high expression of CD46. The median follow-up duration was 47 months (range from 24 to 75 months). Patients with CD55 or CD59 overexpression had a higher relapse rate than those with low expression of CD55 (33.3 vs. 8.6%; P=0.013) or CD59 (31.0 vs. 11.1%; P=0.046). Similarly, mean disease-free survival of patients with CD55 or CD59 overexpression was significantly shorter than those with a low expression of CD55 (56 vs. 70 months; log-rank test, P=0.008) or CD59 (56 vs. 69 months; log-rank test, P=0.033). Multivariate analysis confirmed that CD55, but not CD59, was an independent risk factor of recurrence (HR=4.757; 95% CI, 0.985-22.974; P=0.05). In vitro, we found that tamoxifen inhibited both the protein and mRNA expression levels of CD55, but not CD59 or CD46 in SK-Br3 and BT-474 cells. After pretreatment of tamoxifen, trastuzumab-induced cytolysis was enhanced through CD55 downregulation. In conclusion, CD55 overexpression is an independent risk factor for recurrence in breast cancer patients receiving postoperative adjuvant therapy containing trastuzumab. Combined use of tamoxifen and trastuzumab for HER2-positive breast cancer treatment may enhance the antitumor effects of trastuzumab by elevated CDC, which warrants further study.

CD55 limits sensitivity to complement-dependent cytolysis triggered by heterologous expression of α-gal xenoantigen in colon tumor cells.

Engineering cancer cells to express heterologous antigen α-gal and induce the destruction of tumor cells depending on the complement cascade may be a promising strategy of tumor therapy. However, the feasibility and effect of using α-gal to induce colorectal adenocarcinoma cell line cytolysis is not yet known. In this study, we evaluated α-gal expression's ability to sensitize human colorectal adenocarcinoma cell lines to complement attack in cell lines LoVo, SW620, and Ls-174T. Nearly all α-gal-expressing LoVo and SW620 cells were killed by normal human serum (NHS), but α-gal-expressing Ls-174T cells showed no significant lysis. We analyzed the expression levels of membrane-bound complement regulatory proteins (mCRPs) on the three cell lines, and their protective role in α-gal-mediated activation of the complement. LoVo showed no expression of any of the three proteins. CD59 was strongly expressed by SW620 and Ls-174T. CD46 and CD55 varied between the two cell lines. CD46 on SW620 was only half the intensity of CD46 on Ls-174T. Ls-174T showed a notable expression of CD55, while expression of CD55 on SW620 was not detected. The sensitivity of Ls-174T expressing α-gal to NHS greatly increased following the downregulation of CD46 and CD55 with short hairpin RNA (shRNA). However, there is no increase in cell killing when CD59 expression was diminished. Our findings suggest that the use of α-gal as antigen to induce tumor cell killing may be a potential therapeutic strategy in colon cancer and that CD55 plays a primary role in conferring resistance to lysis.