Vinorelbine distribution to brain metastases of breast cancer and factors limiting in vivo efficacy



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Vinorelbine is a tubulin-binding chemotherapeutic drug which is increasingly used in the first-line treatment of metastatic breast cancer due to its positive activity and reduced toxicity. Brain metastases are an increasing problem in metastatic breast cancer which shows limited response to most chemotherapeutic agents. In this dissertation, the brain and brain metastasis distribution of vinorelbine was determined using the human MDA-MB-231BR "brain seeking" breast cancer cell line in immune compromised female mice. Breast cancer cells were injected into the systemic circulation and allowed to form metastases in brain and other organs over a period of 4-6 weeks. Once neurologic symptoms developed, mice were taken and administered [3H]vinorelbine (12 mg/kg, i.v.) which was allowed to circulate for 0.5 - 8 h. At differing times, the concentrations of total, free, and bound [3H]vinorelbine were determined in brain and brain metastases by autoradiography, and compared to matching total concentrations in peripheral metastases and other organs. Of all the tissues examined, brain had the lowest distribution of [3H]vinorelbine with an integrated area-under-the-curve total drug distribution Kp value (0.30 ± 0.1) relative to plasma, which was 50-80 times less than non-barrier brain regions and most systemic tissues (Kp 15-40). [3H]Vinorelbine distribution to brain metastases was highly heterogeneous with most (77%) showing moderate elevations (average 4.4 fold), a small subset showing large elevations average (13 fold), and the remainder (14%) showing no difference from surrounding normal brain. Brain metastasis distribution appeared to be restricted as average measured [3H]vinorelbine concentrations was 17 fold less than matching systemic metastases without a barrier and the extent of vinorelbine distribution in brain metastases correlated with the extent of blood-brain barrier compromise in passive permeability (r2 =0.577, P<0.001). The measured brain distribution of [3H]vinorelbine was low even though brain showed one of the highest levels of vinorelbine binding protein, tubulin. To further evaluate the factors accounting for limited vinorelbine distribution in the nervous system, the time course of free vinorelbine exposure was determined in brain and brain metastases and compared to signs of in vivo drug activity as measured by TUNEL staining. [3H]Vinorelbine was found to bind highly in brain and brain metastases with free fraction of 1-2%. Calculation of the free drug Kp,uu indicated that brain vascular barrier restriction played an important role as integrated brain free vinorelbine exposure was only 2.5% of that of plasma free exposure, with brain metastasis values either matching that or increasing up to 23%. Thus, the results are consistent with a blood-brain barrier active efflux transport component that limits free vinorelbine concentration in brain to a concentration 40 fold less than systemic exposure and this barrier is fully or partially retained in brain metastases. Consistent with this, significant TUNEL apoptosis staining was only seen in subset of brain metastases with the highest vinorelbine concentrations, with 42% of brain metastases showing little or no staining at all. The estimated ICso of free vinorelbine for apoptosis was 56 nM, which fell within 4 fold of that obtained from in vitro exposure in cell culture (14.3 + 1.4 nM). Extrapolation of in vivo data suggested that 15-20 fold elevation in vinorelbine delivery would be required to produce >87.5% cytotoxicity in the vast majority of brain tumors. Because drug was being administered at a dose close to the minimal toxic level, the mechanisms restricting vinorelbine uptake were investigated using in situ brain perfusion. Evidence was found for a joint role of p-glycoprotein and MRP7 in mediating active efflux transport of vinorelbine at the blood-brain barrier. Genetic knock out and chemical inhibition of these two blood-brain barrier transporters raised brain vinorelbine uptake by up to 4 fold. Evidence for a possible third transporter was obtained as chemical inhibition with tariquidar or lapatinib raised brain vinorelbine uptake even further ( 10-15 fold) without compromising barrier integrity. The results suggest that chemical inhibition of blood-brain barrier active efflux transport may allow entry of sufficient vinorelbine to obtain free drug levels in the range to produce significant cytotoxicity to brain metastases of breast cancer. Further, the results demonstrate for the first time important role of the active efflux transporter, MRP7 at the blood-brain barrier. Overall, the study demonstrates the critical role of restricted bloodbrain barrier transport in limiting anticancer drug activity against brain metastases of breast cancer and suggests that this restriction may be overcome either via development of new improved agents with minimal affinity for barrier active efflux transporters or by short term chemical inhibition of these transporters to allow delivery at a level expected to produce therapeutic effect.



Vinorelbine distribution, Brain metastases, Breast cancer, In vivo efficacy