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

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

The ketogenic diet changes metabolite levels in hippocampal extracellular fluid

Despite successful use of the previous termketogenic dietnext term (KD) for the treatment of drug-resistant epilepsy, its mechanism of action is unclear. After KD-feeding, increased plasma beta-hydroxybutyrate (BHB) previous termlevelsnext term appear to be important for protection against seizures. We hypothesized that the KD leads to metabolic previous termchangesnext term in the brain, which are reflected in the previous termhippocampalnext term extracellular fluid (hECF). CD1 mice were fed control or KD for 2-3 weeks since weaning. In-vivo microdialysis of hECF was used to measure the previous termlevelsnext term of glucose, lactate, as well as BHB under basal conditions and during 30 min stimulation with 60 mM K+, which was retrodialysed. The hECF BHB concentration in KD-fed mice was determined as 43.4 ± 10.1 μM using the zero-flow method and 50.7 + 5.5 μM based on in vitro-recovery. The total BHB concentration in brain homogenate from KD-fed mice was 180 nmoles/g. The intracellular BHB concentration is therefore estimated to be about 3-fold higher than the extracellular previous termlevel,next term which suggests that BHB in adolescent mouse brains may not be quickly metabolized. The basal hECF glucose concentration was 30% lower in KD-fed mice, indicating that glucose may be less important as an energy source. Lactate previous termlevelsnext term were similar in control and KD-fed mice. High potassium stimulation elevated lactate by 3-3.5-fold and decreased glucose by 40-50% in both previous termdietnext term groups, consistent with similar anaerobic and aerobic metabolism in both previous termdietnext term groups during high previous termhippocampalnext term activity. Overall, these data 1) defined the BHB concentration in the previous termhippocampalnext term extracellular fluid in KD-fed mice and 2) showed lower glucose metabolism compared to control previous termdietnext term-fed mice. This work will now enable other researchers to mimic the previous termhippocampalnext term extracellular environment in experiments aimed at deciphering the mechanisms of the KD

Anticonvulsant profile of a balanced ketogenic diet in acute mouse seizure models

Anticonvulsant effects of the ketogenic diet (KD) have been reported in the mouse, although previous studies did not control for intake of vitamins, minerals and antioxidants. The aim of this study was to examine the effects of balanced ketogenic and control diets in acute mouse seizure models. The behavior in four mouse seizure models, plasma d-β-hydroxybutyrate (d-BHB) and glucose levels were determined after feeding control diet, 4:1 and 6:1 KDs with matched vitamins, minerals and antioxidants. Feeding 4:1 and 6:1 KDs ad lib to 3-week-old (adolescent) mice resulted in 1.2–2.2 mM d-BHB in plasma, but did not consistently change glucose levels. The 6:1 KD reproducibly elevated the CC50 (current that initiates seizures in 50% mice tested) in the 6-Hz model after 14 days of feeding to adolescent CD1 mice. Higher plasma d-BHB levels correlated with anticonvulsant effects. Despite ketosis, no consistent anticonvulsant effects of KDs were found in the fluorothyl or pentylenetetrazole CD1 mouse models. The 4:1 KD was neither anticonvulsant nor neuroprotective in hippocampus in the C3H mouse kainate model. Taken together, the KD's anticonvulsant effect was limited to the 6-Hz model, required chronic feeding with 6:1 fat content, and was independent from lowering plasma glucose

Concise Total Synthesis of (−)-Erinapyrone B from D-(+)-Malic Acid

<div><p></p><p>A convenient and facile enantioselective synthesis of (−)-erinapyrone B from commercially available D-(+)-malic acid has been achieved in seven steps. One of the key steps in this synthesis was the one-pot reaction of palladium(II)-mediated Wacker-type oxidative cyclization in the presence of a catalytic amount of <i>p</i>-toluenesulphonic acid (<i>p</i>-TsOH) which has been found to be effective for the preparation of enantiopure 2,3-dihydro-4<i>H</i>-pyran-4-one from the corresponding enantiopure β-hydroxyenone via enantio-enriched diketohydroxy intermediate.</p> <p>[Supplementary materials are available for this article. Go to the publisher's online edition of <i>Synthetic Communications®</i> for the following free supplemental resource(s): Full experimental and spectral details.]</p> </div