Blood-brain barrier opening by intracarotid artery hyperosmolar mannitol induces sterile inflammatory and innate immune responses.

Intracarotid arterial hyperosmolar mannitol (ICAHM) blood-brain barrier disruption (BBBD) is effective and safe for delivery of therapeutics for central nervous system malignancies. ICAHM osmotically alters endothelial cells and tight junction integrity to achieve BBBD. However, occurrence of neuroinflammation following hemispheric BBBD by ICAHM remains unknown. Temporal proteomic changes in rat brains following ICAHM included increased damage-associated molecular patterns, cytokines, chemokines, trophic factors, and cell adhesion molecules, indicative of a sterile inflammatory response (SIR). Proteomic changes occurred within 5 min of ICAHM infusion and returned to baseline by 96 h. Transcriptomic analyses following ICAHM BBBD further supported an SIR. Immunohistochemistry revealed activated astrocytes, microglia, and macrophages. Moreover, proinflammatory proteins were elevated in serum, and proteomic and histological findings from the contralateral hemisphere demonstrated a less pronounced SIR, suggesting neuroinflammation beyond regions of ICAHM infusion. Collectively, these results demonstrate ICAHM induces a transient SIR that could potentially be harnessed for neuroimmunomodulation.

High αv Integrin Level of Cancer Cells Is Associated with Development of Brain Metastasis in Athymic Rats.

BACKGROUND/AIM: Brain metastases commonly occur in patients with malignant skin, lung and breast cancers resulting in high morbidity and poor prognosis. Integrins containing an αv subunit are cell adhesion proteins that contribute to cancer cell migration and cancer progression. We hypothesized that high expression of αv integrin cell adhesion protein promoted metastatic phenotypes in cancer cells. MATERIALS AND METHODS: Cancer cells from different origins were used and studied regarding their metastatic ability and intetumumab, anti-αv integrin mAb, sensitivity using in vitro cell migration assay and in vivo brain metastases animal models. RESULTS: The number of brain metastases and the rate of occurrence were positively correlated with cancer cell αv integrin levels. High αv integrin-expressing cancer cells showed significantly faster cell migration rate in vitro than low αv integrin-expressing cells. Intetumumab significantly inhibited cancer cell migration in vitro regardless of αv integrin expression level. Overexpression of αv integrin in cancer cells with low αv integrin level accelerated cell migration in vitro and increased the occurrence of brain metastases in vivo. CONCLUSION: αv integrin promotes brain metastases in cancer cells and may mediate early steps in the metastatic cascade, such as adhesion to brain vasculature. Targeting αv integrin with intetumumab could provide clinical benefit in treating cancer patients who develop metastases.

Intra-arterial administration improves temozolomide delivery and efficacy in a model of intracerebral metastasis, but has unexpected brain toxicity.

We tested the hypothesis that intra-arterial (IA) infusion of temozolomide into the internal carotid artery would safely improve drug delivery to brain and enhance chemotherapy efficacy in a chemosensitive rat brain tumor model. Quantitative autoradiography after 25 µCi (14)C-temozolomide was given by oral, intravenous, or IA route of administration, or IA with osmotic blood-brain barrier disruption (BBBD) (n = 5-7 per group) showed that both IA and IA/BBBD administration increased drug delivery in tumor by over threefold compared to normal brain (P < 0.02), and also significantly elevated delivery throughout the infused right hemisphere. Temozolomide (20 mg/kg; ~150 mg/m(2)) increased median survival when given by oral (25.5 days), intravenous (25.5 days), or IA (33 days) route of administration, compared to 17.5 days in untreated controls (n = 8 per group; overall P < 0.0001). Survival time after IA temozolomide was significantly longer than all other groups (P < 0.01 for all comparisons). BBBD temozolomide was toxic in the efficacy study, but there was no evidence of symptomatic neurotoxicity in rats given IA temozolomide. After these promising animal results, a 49 year old male with glioblastoma multiforme who failed all standard therapy received temozolomide 100 mg/m(2) IA. Upon initiation of the second course of IA infusion the patient had increased heart rate, blood pressure, and rash, and the procedure was terminated without sequelae. Follow up IA infusion of temozolomide diluent in normal rats showed damaged cerebrovasculature as determined by dye leakage. These results demonstrate that IA infusion of temozolomide was toxic, with or without BBBD. We conclude that under the current formulation temozolomide is not safe for IA infusion in patients.

Vascular endothelial growth factor blockade alters magnetic resonance imaging biomarkers of vascular function and decreases barrier permeability in a rat model of lung cancer brain metastasis.

BACKGROUND: Blockade of vascular endothelial growth factor (VEGF) to promote vascular normalization and inhibit angiogenesis has been proposed for the treatment of brain metastases; however, vascular normalization has not been well-characterized in this disease. We investigated the effect of treatment with bevacizumab anti-VEGF antibody on magnetic resonance imaging (MRI) biomarkers of brain tumor vascular characteristics in comparison to small molecule delivery in a rat model of human lung cancer brain metastasis. METHODS: Athymic rats with A549 human lung adenocarcinoma intracerebral xenografts underwent MRI at 11.75 T before and one day after treatment with bevacizumab (n = 8) or saline control (n = 8) to evaluate tumor volume, free water content (edema), blood volume and vascular permeability (Ktrans). One day later, permeability to 14C-aminoisobutyric acid (AIB) was measured in tumor and brain to assess the penetration of a small drug-like molecule. RESULTS: In saline control animals, tumor volume, edema and permeability increased over the two day assessment period. Compared to controls, bevacizumab treatment slowed the rate of tumor growth (P = 0.003) and blocked the increase in edema (P = 0.033), but did not alter tumor blood volume. Bevacizumab also significantly reduced Ktrans (P = 0.033) and AIB passive permeability in tumor (P = 0.04), but not to peritumoral tissue or normal brain. Post-treatment Ktrans correlated with AIB levels in the bevacizumab-treated rats but not in the saline controls. CONCLUSIONS: The correlation of an MRI biomarker for decreased vascular permeability with decreased AIB concentration in tumor after antiangiogenic treatment suggests that bevacizumab partially restored the normal low permeability characteristics of the blood-brain barrier in a model of human lung cancer brain metastasis.

N-acetylcysteine chemoprotection without decreased cisplatin antitumor efficacy in pediatric tumor models.

Decreasing oxidative damage with the antioxidant agent N-acetylcysteine (NAC) can block the side effects of chemotherapy, but may diminish anti-tumor efficacy. We tested the potential for interactions of high dose NAC against a minimally effective cisplatin chemotherapy regimen in rat models of human pediatric cancers. Athymic rats received subcutaneous implantation of human SK-N-AS neuroblastoma cells or intra-cerebellar implantation of human D283-MED medulloblastoma cells. Rats were untreated or treated with cisplatin (3 or 4 mg/kg IV) with or without NAC (1,000 mg/kg IV) 30 min before or 4 h after cisplatin treatment. Blood urea nitrogen (BUN) and tumor volumes were measured. Cisplatin decreased the growth of SK-N-AS neuroblastoma subcutaneous tumors from 17.7 ± 4.9 to 6.4 ± 2.5 fold over baseline 2 weeks after treatment (P < 0.001). Pretreatment with NAC decreased cisplatin efficacy, while 4 h delayed NAC did not significantly affect cisplatin anti-tumor effects (relative tumor volume 6.8 ± 2.0 fold baseline, P < 0.001). In D283-MED medulloblastoma brain tumors, cisplatin decreased final tumor volume to 3.9 ± 2.3 mm(3) compared to untreated tumor volume of 45.9 ± 38.7 (P = 0.008). Delayed NAC did not significantly alter cisplatin efficacy (tumor volume 6.8 ± 8.1 mm(3), P = 0.014 versus control). Cisplatin was minimally nephrotoxic in these models. NAC decreased cisplatin-induced elevations in BUN (P < 0.02). NAC chemoprotection did not alter cisplatin therapy, if delayed until 4 h after chemotherapy. These data support a Phase I/II clinical trial of delayed NAC to reduce ototoxicity in children with localized pediatric cancers.

Imaging and therapy with rituximab anti-CD20 immunotherapy in an animal model of central nervous system lymphoma.

PURPOSE: To evaluate the effect of rituximab monoclonal antibody (mAb) on MRI tumor volumetrics and efficacy in a rat model of central nervous system (CNS) lymphoma when delivery to the brain was optimized with osmotic blood-brain barrier disruption (BBBD). EXPERIMENTAL DESIGN: Female nude rats with intracerebral MC116 human B-cell lymphoma xenografts underwent baseline MRI and were randomized into 5 groups (n = 6 per group): (i) BBBD saline control; (ii) methotrexate with BBBD; (iii) rituximab with BBBD; (iv) rituximab and methotrexate with BBBD; and (v) intravenous rituximab. Tumor volumes were assessed by MRI at 1 week, and rats were followed for survival. RESULTS: BBBD increased delivery of yttrium-90-radiolabeled mAb in the model of CNS lymphoma. Control rats showed 201 ± 102% increase in tumor volume on MRI 1 week after entering the study and median 14-day survival (range: 6-33). Tumor growth on MRI was slowed in the methotrexate treatment group, but survival time (median: 7 days; range: 5-12) was not different from controls. Among 17 evaluable rats treated with rituximab, 10 showed decreased tumor volume on MRI. All rituximab groups had increased survival compared with control, with a combined median of 43 days (range: 20-60, P < 0.001). There were no differences by route of delivery or combination with methotrexate. CONCLUSIONS: Rituximab was effective at decreasing tumor volume and improving survival in a model of CNS lymphoma and was not affected by combination with methotrexate or by BBBD. We suggest that rituximab warrants further study in human primary CNS lymphoma.

Using acetaminophen's toxicity mechanism to enhance cisplatin efficacy in hepatocarcinoma and hepatoblastoma cell lines.

BACKGROUND/AIMS: Acetaminophen overdose causes hepatotoxicity mediated by toxic metabolites generated through the cytochrome P450 enzyme. The objective of this study was to investigate whether acetaminophen (AAP) can enhance cisplatin (CDDP) cytotoxicity against human hepatocarcinoma and hepatoblastoma cells in vitro and whether this effect can be prevented by N-acetylcysteine (NAC). METHODS: In vitro studies (glutathione [GSH] level, cell viability, and immunoblot assays) were performed using human hepatocarcinoma and hepatoblastoma cells cultured in AAP, CDDP, and the combination of both with or without delayed NAC administration. The pharmacology and toxicology of high-dose AAP in rats were also examined. RESULTS: Acetaminophen decreased GSH levels in liver cancer cells in a dose- and time-dependent manner. Acetaminophen combined with CDDP had enhanced cytotoxicity over CDDP alone. The cytotoxicity caused by AAP plus CDDP was decreased by NAC, with the effectiveness being time-dependent. The GSH level was lowered in the liver but not in the blood or the brain in rats treated with a high dose of AAP (1000 mg/kg). The expression of CYP2E1 protein, a key cytochrome P450 enzyme, varies among species but is not correlated to AAP sensitivity in liver cancer cells. CONCLUSIONS: Our results suggest that a chemotherapeutic regimen containing both AAP and CDDP with delayed NAC rescue has the potential to enhance chemotherapeutic efficacy while decreasing adverse effects. This would be a promising approach particularly for hepatoblastomas regardless of cellular CYP2E1 protein level but could also be beneficial in other malignancies.

Bone marrow chemoprotection without compromise of chemotherapy efficacy in a rat brain tumor model.

Thiol chemoprotective agents can reduce chemotherapy side effects, but clinical use is limited due to concerns of impaired chemotherapeutic efficacy. We evaluated whether an optimized bone marrow chemoprotection regimen impaired the efficacy of enhanced chemotherapy against rat brain tumors. Nude rats with intracerebral human lung carcinoma xenografts were treated with carboplatin, melphalan, and etoposide phosphate delivered intra-arterially with osmotic blood-brain barrier disruption (n = 8/group). Thiol chemoprotection was N-acetyl-L-cysteine (1000 mg/kg) 60 min before chemotherapy and/or sodium thiosulfate (8 g/m(2)) 4 and 8 h after chemotherapy, when the blood-brain barrier is reestablished. Blood counts were obtained before treatment on day 3 and at sacrifice on day 9. N-acetylcysteine serum clearance half-life was 9 to 11 min. Pretreatment with N-acetylcysteine combined with delayed administration of sodium thiosulfate protected against toxicity toward total white cells, granulocytes, and platelets (P = 0.0016). Enhanced chemotherapy reduced intracerebral tumor volume to 4.3 +/- 1.0 mm(3) compared with 29.1 +/- 4.1 mm(3) in untreated animals (P < 0.0001). Tumor volume was 3.7 +/- 0.6 mm(3) in rats that received N-acetylcysteine before and sodium thiosulfate after chemotherapy. The data indicate the efficacy of enhanced chemotherapy for rat brain tumors was not affected by thiol chemoprotection that provided excellent protection for hematological toxicity. Negative interactions of thiols with antitumor efficacy were avoided by temporal and spatial separation of chemoprotectants and chemotherapy.