Development of a physiologically based pharmacokinetic model of fostemsavir and its pivotal application to support dosing in pregnancy.

It is critical to understand the impact of significant physiological changes during pregnancy on the extent of maternal and fetal drug exposure. Fostemsavir (FTR) is a prodrug of temsavir (TMR) and is approved in combination with other antiretrovirals for multi-drug-resistant human immunodeficiency virus (HIV) infections. This physiologically based pharmacokinetic model (PBPK) study was used to estimate TMR PK in pregnant populations during each trimester of pregnancy to inform FTR dosing. A PBPK model was developed and validated for TMR using PK data collected following intravenous TMR and oral FTR dosing (immediate-release and extended-release tablets) in healthy volunteers. Predicted TMR concentration-time profiles accurately predicted the reported clinical data and variability in healthy (dense data) and pregnant (sparse data) populations. Predicted versus observed TMR geometric mean (CV%) clearance following intravenous administration was 18.01 (29) versus 17 (21) (L/h). Predicted versus observed TMR AUC0-inf (ng.h/mL) in healthy volunteers following FTR administration of the extended-release tablet were 9542 (66) versus 7339 (33). The validated TMR PBPK model was then applied to predict TMR PK in a population of pregnant individuals during each trimester. Simulations showed TMR AUC in pregnant individuals receiving FTR 600 mg twice daily was decreased by 25% and 38% in the second and third trimesters, respectively. However, TMR exposure remained within the range observed in nonpregnant adults with no need for dose adjustment. The current PBPK model can also be applied for the prediction of local tissue concentrations and drug-drug interactions in pregnancy.

Physiologically-based pharmacokinetic modeling of prominent oral contraceptive agents and applications in drug-drug interactions.

Considerable interest remains across the pharmaceutical industry and regulatory landscape in capabilities to model oral contraceptives (OCs), whether combined (COCs) with ethinyl estradiol (EE) or progestin-only pill. Acceptance of COC drug-drug interaction (DDI) assessment using physiologically-based pharmacokinetic (PBPK) is often limited to the estrogen component (EE), requiring further verification, with extrapolation from EE to progestins discouraged. There is a paucity of published progestin component PBPK models to support the regulatory DDI guidance for industry to evaluate a new chemical entity's (NCE's) DDI potential with COCs. Guidance recommends a clinical interaction study to be considered if an investigational drug is a weak or moderate inducer, or a moderate/strong inhibitor, of CYP3A4. Therefore, availability of validated OC PBPK models within one software platform, will be useful in predicting the DDI potential with NCEs earlier in the clinical development. Thus, this work was focused on developing and validating PBPK models for progestins, DNG, DRSP, LNG, and NET, within Simcyp, and assessing the DDI potential with known CYP3A4 inhibitors (e.g., ketoconazole) and inducers (e.g., rifampicin) with published clinical data. In addition, this work demonstrated confidence in the Simcyp EE model for regulatory and clinical applications by extensive verification in 70+ clinical PK and CYP3A4 interaction studies. The results provide greater capability to prospectively model clinical CYP3A4 DDI with COCs using Simcyp PBPK to interrogate the regulatory decision-tree to contextualize the potential interaction by known perpetrators and NCEs, enabling model-informed decision making, clinical study designs, and delivering potential alternative COC options for women of childbearing potential.

Physiologically based pharmacokinetic modeling for development and applications of a virtual celiac disease population using felodipine as a model drug.

In celiac disease (CeD), gastrointestinal CYP3A4 abundance and morphology is affected by the severity of disease. Therefore, exposure to CYP3A4 substrates and extent of drug interactions is altered. A physiologically-based pharmacokinetic (PBPK) population for different severities of CeD was developed. Gastrointestinal physiology parameters, such as luminal pH, transit times, morphology, P-gp, and CYP3A4 expression were included in development of the CeD population. Data on physiological difference between healthy and CeD subjects were incorporated into the model as the ratio of celiac to healthy. A PBPK model was developed and verified for felodipine extended-release tablet in healthy volunteers (HVs) and then utilized to verify the CeD populations. Plasma concentration-time profile and PK parameters were predicted and compared against those observed in both groups. Sensitivity analysis was carried out on key system parameters in CeD to understand their impact on drug exposure. For felodipine, the predicted mean concentration-time profiles and 5th and 95th percentile intervals captured the observed profile and variability in the HV and CeD populations. Predicted and observed clearance was 56.9 versus 56.1 (L/h) in HVs. Predicted versus observed mean ± SD area under the curve for extended release felodipine in different severities of CeD were values of 14.5 ± 9.6 versus 14.4 ± 2.1, 14.6 ± 9.0 versus 17.2 ± 2.8, and 28.1 ± 13.5 versus 25.7 ± 5.0 (ng.h/mL), respectively. Accounting for physiology differences in a CeD population accurately predicted the PK of felodipine. The developed CeD population can be applied for determining the drug concentration of CYP3A substrates in the gut as well as for systemic levels, and for application in drug-drug interaction studies.

Clinical Relevance of Hepatic and Renal P-gp/BCRP Inhibition of Drugs: An International Transporter Consortium Perspective.

The role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-drug interactions (DDIs) and limiting drug absorption as well as restricting the brain penetration of drugs with certain physicochemical properties is well known. P-gp/BCRP inhibition by drugs in the gut has been reported to increase the systemic exposure to substrate drugs. A previous International Transporter Consortium (ITC) perspective discussed the feasibility of P-gp/BCRP inhibition at the blood-brain barrier and its implications. This ITC perspective elaborates and discusses specifically the hepatic and renal P-gp/BCRP (referred as systemic) inhibition of drugs and whether there is any consequence for substrate drug disposition. This perspective summarizes the clinical evidence-based recommendations regarding systemic P-gp and BCRP inhibition of drugs with a focus on biliary and active renal excretion pathways. Approaches to assess the clinical relevance of systemic P-gp and BCRP inhibition in the liver and kidneys included (i) curation of DDIs involving intravenously administered substrates or inhibitors; (ii) in vitro-to-in vivo extrapolation of P-gp-mediated DDIs at the systemic level; and (iii) curation of drugs with information available about the contribution of biliary excretion and related DDIs. Based on the totality of evidence reported to date, this perspective supports limited clinical DDI risk upon P-gp or BCRP inhibition in the liver or kidneys.

Robust physiologically based pharmacokinetic model of rifampicin for predicting drug-drug interactions via P-glycoprotein induction and inhibition in the intestine, liver, and kidney.

P-glycoprotein (P-gp) is an efflux transporter that plays an important role in the pharmacokinetics of its substrate, and P-gp activities can be altered by induction and inhibition effects of rifampicin. This study aimed to establish a physiologically based pharmacokinetic (PBPK) model of rifampicin to predict the P-gp-mediated drug-drug interactions (DDIs) and assess the DDI impact in the intestine, liver, and kidney. The induction and inhibition parameters of rifampicin for P-gp were estimated using two of seven DDI cases of rifampicin and digoxin and incorporated into our previously constructed PBPK model of rifampicin. The constructed rifampicin model was verified using the remaining five DDI cases with digoxin and five DDI cases with other P-gp substrates (talinolol and quinidine). Based on the established PBPK model, following repeated dosing of 600 mg rifampicin, the deduced net effect was an approximately threefold induction in P-gp activities in the intestine, liver, and kidney. Furthermore, in all 12 cases the predicted area under the plasma concentration-time curve ratios of the P-gp substrates were within the predefined acceptance criteria with various dosing regimens. Intestinal effects of P-gp-mediated DDIs had their greatest impact on the pharmacokinetics of digoxin and talinolol, with a minimal impact on the liver and kidney. For quinidine, predicted intestinal P-gp/cytochrome P450 3A-mediated DDIs were slightly underestimated because of the complexity of nonlinearity and transporter-enzyme interplay. These findings demonstrate that our rifampicin model can be applicable to quantitatively predict the net impact of P-gp induction and/or inhibition on diverse P-gp substrates and investigate the magnitude of DDIs in each tissue.

When special populations intersect with drug-drug interactions: Application of physiologically-based pharmacokinetic modeling in pregnant populations.

Pregnancy results in significant physiological changes that vary across trimesters and into the postpartum period, and may result in altered disposition of endogenous substances and drug pharmacokinetics. Pregnancy represents a unique special population where physiologically-based pharmacokinetic modeling (PBPK) is well suited to mechanistically explore pharmacokinetics and dosing paradigms without subjecting pregnant women or their fetuses to extensive clinical studies. A critical review of applications of pregnancy PBPK models (pPBPK) was conducted to understand its current status for prediction of drug exposure in pregnant populations and to identify areas of further expansion. Evaluation of existing pPBPK modeling efforts highlighted improved understanding of cytochrome P450 (CYP)-mediated changes during pregnancy and identified knowledge gaps for non-CYP enzymes and the physiological changes of the postpartum period. Examples of the application of pPBPK beyond simple dose regimen recommendations are limited, particularly for prediction of drug-drug interactions (DDI) or differences between genotypes for polymorphic drug metabolizing enzymes. A raltegravir pPBPK model implementing UGT1A1 induction during the second and third trimesters of pregnancy was developed in the current work and verified against clinical data. Subsequently, the model was used to explore UGT1A1-related DDI risk with atazanavir and rifampicin along with the effect of enzyme genotype on raltegravir apparent clearance. Simulations of pregnancy-related induction of UGT1A1 either exacerbated UGT1A1 induction by rifampicin or negated atazanavir UGT1A1 inhibition. This example illustrated the advantages of pPBPK modeling for mechanistic evaluation of complex interplays of pregnancy- and drug-related effects in support of model-informed approaches in drug development.

Physiologically-based Pharmacokinetic (PBPK) Modelling of Transporter Mediated Drug Absorption, Clearance and Drug-drug Interactions.

Membrane transporters play an important role in intestinal absorption, distribution and clearance of drugs. Additionally transporters along with enzymes regulate tissue exposures (e.g. liver, kidney and brain), which are important for safety and efficacy considerations. Early identification of transporters involved guides generation of in vitro and in vivo data needed to gain mechanistic understanding on the role of transporters in organ clearance, tissue exposures and enables development of physiological-based pharmacokinetic (PBPK) models. A lot of progress has been made in developing several in vitro assay systems and mechanistic in silico models to determine kinetic parameters for transporters, which are incorporated into PBPK models. Although, intrinsic clearance and inhibition data from in vitro systems generally tend to underpredict in vivo clearance and magnitude of drug-drug interactions (DDIs), empirical scaling factors derived from a sizable dataset are often used to offset underpredictions. PBPK models are increasing used to predict the impact of transporters on intestinal absorption, clearance, victim and perpetrator DDIs prior to first in human clinical trials. The models are often refined when clinical data is available and are used to predict pharmacokinetics in untested scenarios such as the impact of polymorphisms, ontogeny, ethnicity, disease states and DDIs with other perpetrator drugs. The aim of this review is to provide an overview of (i) regulatory requirements around transporters, (ii) in vitro systems and their limitations in predicting transporter mediated drug disposition and DDIs, (iii) PBPK modelling tactics and case studies used for internal decision making and/or for regulatory submissions.

Intestinal P-gp and Putative Hepatic OATP1B Induction: International Transporter Consortium Perspective on Drug Development Implications.

There is an increasing interest in transporter induction (i.e., decreased systemic drug exposure due to increased efflux-limited absorption or transporter-mediated clearance) as a mechanism of drug-drug interactions (DDIs), although evidence of clinical relevance is still evolving. Intestinal P-glycoprotein (P-gp) and hepatic organic anion transporting polypeptides 1B (OATP1B) can be important determinants of drug absorption and disposition, as well as targets for DDIs. Current data indicate that intestinal P-gp protein levels can be induced up to threefold to fourfold in humans primarily with pregnane X receptor (PXR) activators, and that this induction can decrease the systemic exposure of drugs with P-gp efflux-limited absorption (e.g., ≤ 67% decrease in the exposure of total dabigatran following rifampin multiple oral dosing). Evaluation of the clinical relevance of P-gp induction as a DDI mechanism must consider the induction potential of the perpetrator drug for P-gp and attenuation of exposure of the victim drug in the context of its therapeutic window. Practical drug development recommendations are provided herein. Reports are contradictory on OATP1B induction by PXR activators in human hepatocytes and liver biopsies. Some clinical investigations demonstrated that rifampin pretreatment decreased exposure of OATP1B substrates, while other studies found no differences, and the potential involvement of other mechanisms in these observed DDIs cannot be definitively ruled out. Thus, further studies are needed to understand hepatic OATP1B induction and potential involvement of other mechanisms contributing to reduced exposure of OATP1B substrates. This review critically summarizes the state-of-the-art on intestinal P-gp and hepatic OATP1B induction, and highlights implications for drug development.

Drug Interactions for Low-Dose Inhaled Nemiralisib: A Case Study Integrating Modeling, In Vitro, and Clinical Investigations.

In vitro data for low-dose inhaled phosphoinositide 3-kinase delta inhibitor nemiralisib revealed that it was a substrate and a potent metabolism-dependent inhibitor of cytochrome P450 (P450) 3A4 and a P-glycoprotein (P-gp) substrate. An integrated in silico, in vitro, and clinical approach including a clinical drug interaction study as well as a bespoke physiologically based pharmacokinetic (PBPK) model was used to assess the drug-drug interaction (DDI) risk. Inhaled nemiralisib (100 µg, single dose) was coadministered with itraconazole, a potent P4503A4/P-gp inhibitor, following 200 mg daily administrations for 10 days in 20 male healthy subjects. Systemic exposure to nemiralisib (AUC0-inf) increased by 2.01-fold versus nemiralisib alone. To extrapolate the clinical data to other P4503A4 inhibitors, an inhaled PBPK model was developed using Simcyp software. Retrospective simulation of the victim risk showed good agreement between simulated and observed data (AUC0-inf ratio 2.3 vs. 2.01, respectively). Prospective DDI simulations predicted a weak but manageable drug interaction when nemiralisib was coadministered with other P4503A4 inhibitors, such as the macrolides clarithromycin and erythromycin (simulated AUC0-inf ratio of 1.7), both common comedications in the intended patient populations. PBPK and static mechanistic models were also used to predict a negligible perpetrator DDI effect for nemiralisib on other P4503A4 substrates, including midazolam (a sensitive probe substrate of P4503A4) and theophylline (a narrow therapeutic index drug and another common comedication). In summary, an integrated in silico, in vitro, and clinical approach including an inhalation PBPK model has successfully discharged any potential patient DDI risks in future nemiralisib clinical trials. SIGNIFICANCE STATEMENT: This paper describes the integration of in silico, in vitro, and clinical data to successfully discharge potential drug-drug interaction risks for a low-dose inhaled drug. This work featured assessment of victim and perpetrator risks of drug transporters and cytochrome P450 enzymes, utilizing empirical and mechanistic approaches combined with clinical data (drug interaction and human absorption, metabolism, and pharmacokinetics) and physiologically based pharmacokinetic modeling approaches to facilitate bespoke risk assessment in target patient populations.

Physiologically-Based Pharmacokinetic Models for Evaluating Membrane Transporter Mediated Drug-Drug Interactions: Current Capabilities, Case Studies, Future Opportunities, and Recommendations.

Physiologically-based pharmacokinetic (PBPK) modeling has been extensively used to quantitatively translate in vitro data and evaluate temporal effects from drug-drug interactions (DDIs), arising due to reversible enzyme and transporter inhibition, irreversible time-dependent inhibition, enzyme induction, and/or suppression. PBPK modeling has now gained reasonable acceptance with the regulatory authorities for the cytochrome-P450-mediated DDIs and is routinely used. However, the application of PBPK for transporter-mediated DDIs (tDDI) in drug development is relatively uncommon. Because the predictive performance of PBPK models for tDDI is not well established, here, we represent and discuss examples of PBPK analyses included in regulatory submission (the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Pharmaceuticals and Medical Devices Agency (PMDA)) across various tDDIs. The goal of this collaborative effort (involving scientists representing 17 pharmaceutical companies in the Consortium and from academia) is to reflect on the use of current databases and models to address tDDIs. This challenges the common perceptions on applications of PBPK for tDDIs and further delves into the requirements to improve such PBPK predictions. This review provides a reflection on the current trends in PBPK modeling for tDDIs and provides a framework to promote continuous use, verification, and improvement in industrialization of the transporter PBPK modeling.

Clinical Extrapolation of the Effects of Dolutegravir and Other HIV Integrase Inhibitors on Folate Transport Pathways.

Preliminary analysis of ongoing birth surveillance study identified evidence of potential increased risk for neural tube defects (NTDs) in newborns associated with exposure to dolutegravir at the time of conception. Folate deficiency is a common cause of NTDs. Dolutegravir and other HIV integrase inhibitor drugs were evaluated in vitro for inhibition of folate transport pathways: proton-coupled folate transporter (PCFT), reduced folate carrier (RFC), and folate receptor α (FRα)-mediated endocytosis. Inhibition of folate transport was extrapolated to the clinic by using established approaches for transporters in intestine, distribution tissues, and basolateral and apical membranes of renal proximal tubules (2017 FDA Guidance). The positive controls, methotrexate and pemetrexed, demonstrated clinically relevant inhibition of PCFT, RFC, and FRα in folate absorption, distribution, and renal sparing. Valproic acid was used as a negative control that elicits folate-independent NTDs; valproic acid did not inhibit PCFT, RFC, or FRα At clinical doses and exposures, the observed in vitro inhibition of FRα by dolutegravir and cabotegravir was not flagged as clinically relevant; PCFT and RFC inhibition was not observed in vitro. Bictegravir inhibited both PCFT and FRα, but the observed inhibition did not reach the criteria for clinical relevance. Elvitegravir and raltegravir inhibited PCFT, but only raltegravir inhibition of intestinal PCFT was flagged as potentially clinically relevant at the highest 1.2-g dose (not the 400-mg dose). These studies showed that dolutegravir is not a clinical inhibitor of folate transport pathways, and it is not predicted to elicit clinical decreases in maternal and fetal folate levels. Clinically relevant HIV integrase inhibitor drug class effect on folate transport pathways was not observed. SIGNIFICANCE STATEMENT: Preliminary analysis of ongoing birth surveillance study identified evidence of potential increased risk for neural tube defects (NTDs) in newborns associated with exposure to the HIV integrase inhibitor dolutegravir at the time of conception; folate deficiency is a common cause of NTDs. Dolutegravir and other HIV integrase inhibitor drugs were evaluated in vitro for inhibition of the major folate transport pathways: proton-coupled folate transporter, reduced folate carrier, and folate receptor α-mediated endocytosis. The present studies showed that dolutegravir is not a clinical inhibitor of folate transport pathways, and it is not predicted to elicit clinical decreases in maternal and fetal folate levels. Furthermore, clinically relevant HIV integrase inhibitor drug class effect on folate transport pathways was not observed.

Can Bile Salt Export Pump Inhibition Testing in Drug Discovery and Development Reduce Liver Injury Risk? An International Transporter Consortium Perspective.

Bile salt export pump (BSEP) inhibition has emerged as an important mechanism that may contribute to the initiation of human drug-induced liver injury (DILI). Proactive evaluation and understanding of BSEP inhibition is recommended in drug discovery and development to aid internal decision making on DILI risk. BSEP inhibition can be quantified using in vitro assays. When interpreting assay data, it is important to consider in vivo drug exposure. Currently, this can be undertaken most effectively by consideration of total plasma steady state drug concentrations (Css,plasma ). However, because total drug concentrations are not predictive of pharmacological effect, the relationship between total exposure and BSEP inhibition is not causal. Various follow-up studies can aid interpretation of in vitro BSEP inhibition data and may be undertaken on a case-by-case basis. BSEP inhibition is one of several mechanisms by which drugs may cause DILI, therefore, it should be considered alongside other mechanisms when evaluating possible DILI risk.

Unmasking the Role of Uptake Transporters for Digoxin Uptake Across the Barriers of the Central Nervous System in Rat.

The role of uptake transporter (organic anion-transporting polypeptide [Oatp]) in the disposition of a P-glycoprotein (P-gp) substrate (digoxin) at the barriers of central nervous system, namely, the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and brain-cerebrospinal fluid barrier (BCSFB), was studied using rat as a preclinical species. In vivo chemical inhibition of P-gp and Oatp was achieved using elacridar and rifampicin, respectively. Our findings show that (1) digoxin had a low brain-to-plasma concentration ratio (B/P) (0.07) in rat; (2) in the presence of elacridar, the B/P of digoxin increased by about 12-fold; (3) rifampicin administration alone did not change the digoxin B/P significantly when compared with digoxin B/P alone; (4) rifampicin administration along with elacridar resulted only in 6-fold increase in the B/P of digoxin; (5) similar fold changes and trends were seen with the spinal cord-to-plasma concentration ratio of digoxin, indicating the similarity between BBB and the BSCB; and (6) unlike BBB and BSCB, the presence of rifampicin further increased the cerebrospinal fluid-to-plasma concentration ratio (CSF/P) for digoxin, suggesting a differential orientation of the uptake transporters at the BCSFB (CSF to blood) compared with the BBB (blood to brain). The observations for digoxin uptake, at least at the BBB and the BSCB, advocate the importance of uptake transporters (Oatps). However, the activity of such uptake transporters became evident only after inhibition of the efflux transporter (P-gp).

Importance of Hepatic Transporters in Clinical Disposition of Drugs and Their Metabolites.

This review provides a practical clinical perspective on the relevance of hepatic transporters in pharmacokinetics and drug-drug interactions (DDIs). Special emphasis is placed on transporters with clear relevance to clinical DDIs, efficacy, and safety. Basolateral OATP1B1 and 1B3 emerged as important hepatic drug uptake pathways, sites for systemic DDIs, and sources of pharmacogenetic variability. As the first step in hepatic drug removal from the circulation, OATPs are an important determinant of systemic pharmacokinetics, specifically influencing systemic absorption, clearance, and hepatic distribution for subsequent metabolism and/or excretion. Biliary excretion of parent drugs is a less prevalent clearance pathway than metabolism or urinary excretion, but BCRP and MRP2 are critically important to biliary/fecal elimination of drug metabolites. Inhibition of biliary excretion is typically not apparent at the level of systemic pharmacokinetics but can markedly increase liver exposure. Basolateral efflux transporters MRP3 and MRP4 mediate excretion of parent drugs and, more commonly, polar metabolites from hepatocytes into blood. Basolateral excretion is an area in need of further clinical investigation, which will necessitate studies more complex than just systemic pharmacokinetics. Clinical relevance of hepatic uptake is relatively well appreciated, and clinical consequences of hepatic excretion (biliary and basolateral) modulation remain an active research area.

Estimation of the unbound brain concentration of P-glycoprotein substrates or nonsubstrates by a serial cerebrospinal fluid sampling technique in rats.

The unbound concentration in plasma drives the transport of the drug into the brain, and the unbound drug concentration in the central nervous system (CNS) drives the interaction with the target eliciting the pharmacological effect. Delivery of the drug to the CNS is a challenge because of the unique neurovascular unit, which restricts the passage of drugs into the brain. The efflux transporters [especially P-glycoprotein (P-gp)] present at the blood-brain barrier (BBB) act as one of the major detractors for keeping drugs outside the CNS. The cerebrospinal fluid (CSF) drug concentration has been used as a surrogate for unbound brain concentrations and has proven to be a good indicator to relate to CNS activity. Herein, we have established a serial CSF sampling technique in rats, which allowed CSF sampling from a single animal and reduced the number of animals required, as well as the interanimal variance associated with a composite/terminal study design. Concentrations in the CSF sampled from the cisterna magna serially from the same rat were compared with the concentrations obtained from discrete CSF sampling and with brain concentrations. The serial CSF sampling technique was also authenticated by ensuring no change in the barrier without any indication of damage caused by the repeated puncture of cisterna magna. This technique was corroborated using three passively permeable compounds (carbamazepine, theophylline, and propranolol), three P-gp substrates (quinidine, verapamil, and digoxin), and one l-amino acid uptake transporter substrate (gabapentin). The P-gp substrates were also used in separate studies with the P-gp inhibitor elacridar to assess the effect on CSF concentration versus brain concentration on P-gp inhibition. The CSF concentration and unbound brain concentration were comparable (within 3-fold) for all compounds, including P-gp substrates even in the presence of elacridar. Therefore, this technique can prove to be beneficial for predicting the unbound drug concentrations in the brain from the CSF concentrations and reduce the cost incurred in preclinical animal models. Chemical inhibition by elacridar and prediction of the brain unbound concentrations from the serial CSF sampling of P-gp substrates in the rat may be an attractive alternative to the use of genetically knocked out rodents.

Lapatinib distribution in HER2 overexpressing experimental brain metastases of breast cancer.

PURPOSE: Lapatinib, a small molecule EGFR/HER2 inhibitor, partially inhibits the outgrowth of HER2+ brain metastases in preclinical models and in a subset of CNS lesions in clinical trials of HER2+ breast cancer. We investigated the ability of lapatinib to reach therapeutic concentrations in the CNS following (14)C-lapatinib administration (100 mg/kg p.o. or 10 mg/kg, i.v.) to mice with MDA-MD-231-BR-HER2 brain metastases of breast cancer. METHODS: Drug concentrations were determined at differing times after administration by quantitative autoradiography and chromatography. RESULTS: (14)C-Lapatinib concentration varied among brain metastases and correlated with altered blood-tumor barrier permeability. On average, brain metastasis concentration was 7-9-fold greater than surrounding brain tissue at 2 and 12 h after oral administration. However, average lapatinib concentration in brain metastases was still only 10-20% of those in peripheral metastases. Only in a subset of brain lesions (17%) did lapatinib concentration approach that of systemic metastases. No evidence was found of lapatinib resistance in tumor cells cultured ex vivo from treated brains. CONCLUSIONS: Results show that lapatinib distribution to brain metastases of breast cancer is partially restricted and blood-tumor barrier permeability is a key component of lapatinib therapeutic efficacy which varies between tumors.

Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer.

PURPOSE: Brain metastases of breast cancer appear to be increasing in incidence, confer significant morbidity, and threaten to compromise gains made in systemic chemotherapy. The blood-tumor barrier (BTB) is compromised in many brain metastases; however, the extent to which this influences chemotherapeutic delivery and efficacy is unknown. Herein, we answer this question by measuring BTB passive integrity, chemotherapeutic drug uptake, and anticancer efficacy in vivo in two breast cancer models that metastasize preferentially to brain. EXPERIMENTAL DESIGN: Experimental brain metastasis drug uptake and BTB permeability were simultaneously measured using novel fluorescent and phosphorescent imaging techniques in immune-compromised mice. Drug-induced apoptosis and vascular characteristics were assessed using immunofluorescent microscopy. RESULTS: Analysis of over 2,000 brain metastases from two models (human 231-BR-Her2 and murine 4T1-BR5) showed partial BTB permeability compromise in greater than 89% of lesions, varying in magnitude within and between metastases. Brain metastasis uptake of ¹4C-paclitaxel and ¹4C-doxorubicin was generally greater than normal brain but less than 15% of that of other tissues or peripheral metastases, and only reached cytotoxic concentrations in a small subset (∼10%) of the most permeable metastases. Neither drug significantly decreased the experimental brain metastatic ability of 231-BR-Her2 tumor cells. BTB permeability was associated with vascular remodeling and correlated with overexpression of the pericyte protein desmin. CONCLUSIONS: This work shows that the BTB remains a significant impediment to standard chemotherapeutic delivery and efficacy in experimental brain metastases of breast cancer. New brain permeable drugs will be needed. Evidence is presented for vascular remodeling in BTB permeability alterations.

Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA double-strand breaks.

PURPOSE: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer. EXPERIMENTAL DESIGN: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo. RESULTS: Following systemic administration, uptake of [(14)C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52. CONCLUSIONS: We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation.