Osmotic Blood-brain Barrier Opening Research Articles

Enhancing Antibody Exposure in the Central Nervous System: Mechanisms of Uptake, Clearance, and Strategies for Improved Brain Delivery

Antibodies (mAbs) are attractive molecules for their application as a diagnostic and therapeutic agent for diseases of the central nervous system (CNS). mAbs can be generated to have high affinity and specificity to target molecules in the CNS. Unfortunately, only a very small number of mAbs have been specifically developed and approved for neurological indications. This is primarily attributed to their low exposure within the CNS, hindering their ability to reach and effectively engage their potential targets in the brain. This review discusses aspects of various barriers such as the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier (BCSFB) that regulate the entry and clearance of mAbs into and from the brain. The roles of the glymphatic system on brain exposure and clearance are being described. We also discuss the proposed mechanisms of the uptake of mAbs into the brain and for clearance. Finally, several methods of enhancing the exposure of mAbs in the CNS were discussed, including receptor-mediated transcytosis, osmotic BBB opening, focused ultrasound (FUS), BBB-modulating peptides, and enhancement of mAb brain retention.

Label-Free Assessment of Mannitol Accumulation Following Osmotic Blood-Brain Barrier Opening Using Chemical Exchange Saturation Transfer Magnetic Resonance Imaging.

Mannitol is a hyperosmolar agent for reducing intracranial pressure and inducing osmotic blood-brain barrier opening (OBBBO). There is a great clinical need for a non-invasive method to optimize the safety of mannitol dosing. The aim of this study was to develop a label-free Chemical Exchange Saturation Transfer (CEST)-based MRI approach for detecting intracranial accumulation of mannitol following OBBBO. In vitro MRI was conducted to measure the CEST properties of D-mannitol of different concentrations and pH. In vivo MRI and MRS measurements were conducted on Sprague-Dawley rats using a Biospec 11.7T horizontal MRI scanner. Rats were catheterized at the internal carotid artery (ICA) and randomly grouped to receive either 1 mL or 3 mL D-mannitol. CEST MR images were acquired before and at 20 min after the infusion. In vitro MRI showed that mannitol has a strong, broad CEST contrast at around 0.8 ppm with a mM CEST MRI detectability. In vivo studies showed that CEST MRI could effectively detect mannitol in the brain. The low dose mannitol treatment led to OBBBO but no significant mannitol accumulation, whereas the high dose regimen resulted in both OBBBO and mannitol accumulation. The CEST MRI findings were consistent with 1H-MRS and Gd-enhanced MRI assessments. We demonstrated that CEST MRI can be used for non-invasive, label-free detection of mannitol accumulation in the brain following BBBO treatment. This method may be useful as a rapid imaging tool to optimize the dosing of mannitol-based OBBBO and improve its safety and efficacy.

Hyperosmolar blood-brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance.

The blood-brain barrier (BBB) is the main obstacle to the effective delivery of therapeutic agents to the brain, compromising treatment efficacy for a variety of neurological disorders. Intra-arterial (IA) injection of hyperosmotic mannitol has been used to permeabilize the BBB and improve parenchymal entry of therapeutic agents following IA delivery in preclinical and clinical studies. However, the reproducibility of IA BBB manipulation is low and therapeutic outcomes are variable. We demonstrated that this variability could be highly reduced or eliminated when the procedure of osmotic BBB opening is performed under the guidance of interventional MRI. Studies have reported the utility and applicability of this technique in several species. Here we describe a protocol to open the BBB by IA injection of hyperosmotic mannitol under the guidance of MRI in mice. The procedures (from preoperative preparation to postoperative care) can be completed within ~1.5 h, and the skill level required is on par with the induction of middle cerebral artery occlusion in small animals. This MRI-guided BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain.

Optimization of osmotic blood-brain barrier opening to enable intravital microscopy studies on drug delivery in mouse cortex

Intra-arterial (IA) infusion of mannitol induces osmotic blood-brain barrier opening (OBBBO) and that method has been used for decades to improve drug delivery to the brain. However, high variability of outcomes prevented vast clinical adoption. Studies on dynamic multi-scale imaging of OBBBO as well as extravasation of IA injected therapeutic agents are essential to develop strategies assuring precision and reproducibility of drug delivery. Intravital microscopy is increasingly used to capture the dynamics of biological processes at the molecular level in convenient mouse models. However, until now OBBBO has been achieved safely in subcortical structures, which prevented direct insight into the process of extravasation through the skull window. Here, we used our previously developed real-time MRI to adjust the procedure to achieve robust cortical OBBBO. We found that catheter-mediated delivery to the cortex from the ipsilateral carotid artery can be improved by temporarily occluding the contralateral carotid artery. The reproducibility and safety of the method were validated by MRI and histology. This experimental platform was further exploited for studying with intravital microscopy the extravasation of 0.58 kDa rhodamine and 153 kDa anti-VEGF monoclonal antibody (bevacizumab) upon IA injection. Dynamic imaging during IA infusion captured the spatiotemporal dynamic of infiltration for each molecule into the brain parenchyma upon OBBBO. Small-sized rhodamine exhibited faster and higher penetration than the antibody. Histological analysis showed some uptake of the monoclonal antibody after IA delivery, and OBBBO significantly amplified the extent of its uptake. For quantitative assessment of cortical uptake, bevacizumab was radiolabeled with zirconium-89 and infused intraarterially. As expected, OBBBO potentiated brain accumulation, providing 33.90 ± 9.06% of injected dose per gram of brain tissue (%ID/g) in the cortex and 17.09 ± 7.22%ID/g in subcortical structures. In contrast IA infusion with an intact BBB resulted in 3.56 ± 1.06%ID/g and 3.57 ± 0.59%ID/g in the same brain regions, respectively. This study established reproducible cortical OBBBO in mice, which enabled multi-photon microscopy studies on OBBBO and drug targeting. This approach helped demonstrate in a dynamic fashion extravasation of fluorescently-tagged antibodies and their effective delivery into the brain across an osmotically opened BBB.

PET imaging of distinct brain uptake of a nanobody and similarly-sized PAMAM dendrimers after intra-arterial administration.

We have recently shown that intracerebral delivery of an anti-VEGF monoclonal antibody bevacizumab using an intra-arterial (IA) infusion is more effective than intravenous administration. While antibodies are quickly emerging as therapeutics, their disadvantages such as large size, production logistics and immunogenicity motivate search for alternatives. Thus we have studied brain uptake of nanobodies and polyamidoamine (PAMAM) dendrimers. Nanobodies were conjugated with deferoxamine (DFO) to generate NB(DFO)2. Generation-4 PAMAM dendrimers were conjugated with DFO, and subsequently primary amines were capped with butane-1,2-diol functionalities to generate G4(DFO)3(Bdiol)110. Resulting conjugates were radiolabeled with zirconium-89. Brain uptake of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 upon carotid artery vs tail vein infusions with intact BBB or osmotic blood-brain barrier opening (OBBBO) with mannitol in mice was monitored by dynamic positron emission tomography (PET) over 30min to assess brain uptake and clearance, followed by whole-body PET-CT (computed tomography) imaging at 1h and 24h post-infusion (pi). Imaging results were subsequently validated by ex-vivo biodistribution. Intravenous administration of 89ZrNB(DFO)2 and 89ZrG4(DFO)3(Bdiol)110 resulted in their negligible brain accumulation regardless of BBB status and timing of OBBBO. Intra-arterial (IA) administration of 89ZrNB(DFO)2 dramatically increased its brain uptake, which was further potentiated with prior OBBBO. Half of the initial brain uptake was retained after 24h. In contrast, IA infusion of 89ZrG4(DFO)3(Bdiol)110 resulted in poor initial accumulation in the brain, with complete clearance within 1h of administration. Ex-vivo biodistribution results reflected those on PET-CT. IA delivery of nanobodies might be an attractive therapeutic platform for CNS disorders where prolonged intracranial retention is necessary.

Primary Central Nervous System Lymphoma and the Blood-Brain Barrier

Recent reports propose high-dose (HD) methotrexate (MTX)-based cytoreduction followed by consolidation with HD chemotherapy (HDC) with or without autologous stem-cell transplantation (ASCT) as first-line treatment for primary central nervous system lymphoma (PCNSL). The main rationale is that HDC-ASCT may improve disease control by achieving higher therapeutic concentrations of cytotoxic chemotherapy in the CNS, thus circumventing the blood-brain barrier (BBB). We have employed an alternative approach to treat PCNSL patients effectively. This first-line treatment enhances delivery of standard dose (SD) MTX-based chemotherapy administered intra-arterially (i.a.) with osmotic BBB opening plus the anti-CD20 monoclonal antibody (mAb) rituximab, followed in complete responders by anti-CD20 mAb maintenance immunotherapy. The main features that distinguish our approach from HDC-ASCT consolidation are to utilize BBB properties to maximize delivery of SD chemotherapy to the CNS sanctuary, and maintenance immunotherapy to delay recurrence. A multi-center study of 149 PCNSL patients (2,079 i.a. BBB treatments) resulted in one death within 48 hrs after treatment, from pulmonary embolism. Prior to routine use of granulocyte colony stimulating factor, granulocytopenic fever occurred in 3% of BBB treatments (Angelov, J Clin Oncol 2009). This contrasts with HDC-ASCT consolidation which carries substantial morbidity and treatment-related mortality (6% to 9%) (Ferrari, Lancet Hemat 2016; Omuro, Blood 2015). Further, HDC-ASCT is used primarily in younger PCNSL patients (< 60-65 yrs) with good performance status and is not feasible in many PCNSL patients given the median age at diagnosis is 60-65 yrs.First-line enhanced BBB delivery of SD MTX-based chemotherapy in 149 patients provided an overall response rate (ORR) of 82% (58% CR rate) and median overall survival (OS) of 37 mo. In low risk patients (age < 60 yrs and KPS ≥ 70) median OS was approximately 14 yrs. Survivors in CR a minimum of 2 yrs after diagnosis (n = 24) were evaluated with standard neuropsychological tests. Median follow-up was 12 yrs (range 2 to 26 yrs) and results indicated long-term preserved or improved cognitive function (Doolittle, J Clin Oncol 2013). Recently we compared efficacy from two first-line MTX-based regimens: HD MTX (intravenous, 4gr/m2) (Thiel, Lancet Oncol 2010), and enhanced BBB delivery of MTX (i.a., 3.5gr/m2). After adjusting for patient characteristics, CR rate and progression free survival were higher in patients < 65 yrs treated with enhanced delivery of MTX (p < 0.001). When first-line rituximab was added to the i.a. enhanced delivery regimen in 24 patients (median age 64 yrs; median KPS 65), the ORR improved to 92% (75% CR) and OS to 61 mo.To prevent recurrence, Ney (Leuk Lymphoma 2009) treated a small group of patients in CR after standard PCNSL treatment, with maintenance rituximab. The addition of maintenance immunotherapy resulted in average disease control of 22 mo or longer. Consistent with the Ney report, we noted prolonged median CR duration of 38 mo (range: 10 to 85 mo) in a small subset treated with maintenance rituximab (n = 9). Increased survival was also seen in translational laboratory studies of a rodent model of intracerebrally implanted MC116 human B-cell lymphoma cells, using single agent rituximab, whether or not delivered with BBB disruption and whether or not MTX was included (Muldoon, Clin Cancer Res 2011). These results suggest that rituximab slowly leaked into main CNS tumor mass even in absence of BBB opening; and given the long half-life, was trapped by binding to CD20+ on tumor cells, attaining sufficient concentration for antitumor efficacy.Our goal is to maximize first-line enhanced BBB delivery of SD chemotherapy to enable less treatment-related morbidity and mortality than is associated with HDC-ASCT in PCNSL, and increase CR duration. Neurocognitive safety has been demonstrated in long-term survivors treated with BBB delivery. Based on the encouraging results from Ney et al, our pilot data using maintenance rituximab, and translational lab studies of mAb delivery to brain, a new randomized multi-center trial is underway to study the effect of maintenance obinutuzumab on CR duration, neurocognition and quality of life in PCNSL patients who achieve CR following first-line MTX-based chemotherapy (NCT02498951). A trial update will be presented during the ASH 2016 meeting. DisclosuresNo relevant conflicts of interest to declare.

CT imaging and spontaneous behavior analysis after osmotic blood-brain barrier opening in Wistar rat.

In our previous experiments we demonstrated that osmotic opening of the blood brain barrier (BBB) in rats by administration of mannitol into the internal carotid artery leads to cerebral edema. The aim of this study was to confirm objectively the development of brain edema and determine whether it affects spontaneous locomotor activity in rats (SLA). Brain edema was verified by computer tomography (CT) examination of the brain and SLA was observed during open field test. Twenty four adult male rats were divided into four groups of six: (1) control animals (C), (2) controls with anesthesia (CA), (3) controls with sham surgery (CS), (4) experimental - osmotic opening of the BBB (MA). Osmotic BBB disruption manifested by reducing the density of brain tissue (hypodensity), suggesting a higher water content in the brain tissue. SLA was compared between C, CA, CS and MA groups and between MA and CA groups. Significant difference was found only between the control group and MA group. In the first 30 min of the examination, rats after the mannitol administration revealed a marked limitation of spontaneous locomotor activity. Experimental results demonstrated reduction of spontaneous locomotor activity in rats with induced brain edema.

Hypertonic saline solutions for treatment of intracranial hypertension

This review aims to provide an update on recent knowledge gained on hypertonic saline solutions for the treatment of intracranial hypertension. Explanatory approaches to the mechanisms underlying the edema-reducing effects of the solutions are outlined, practical aspects of use are presented, and trials that assessed their clinical utility are highlighted. With an established trauma system, hypertonic saline added to conventional fluid resuscitation did not improve long-term outcome in multiple injury with hypotension and brain trauma. In intensive care, hypertonic saline reduced intracranial hypertension after subarachnoid haemorrhage, brain trauma, and a variety of other brain diseases, including cerebral edema in acute liver failure. Hypertonic saline solutions have evolved as an alternative to mannitol or may be used in otherwise refractory intracranial hypertension to treat raised intracranial pressure. With high osmolar loads, the efficacy of the solution is enhanced, but no simple relationship between the saline concentration and the clinical effects of a solution is established. Caution is advised with high osmolar loads because they carry increased risks for potentially deleterious consequences of hypernatremia or may induce osmotic blood-brain barrier opening with possibly harmful extravasation of the hypertonic solution into the brain tissue.

Complexities in the association of human blood brain barrier disruption with seizures: importance of patient population and method of disruption

In “Blood–brain barrier leakage may lead to progression of temporal lobe epilepsy” (van Vliet et al ., 2007), the authors found that osmotic opening of the blood–brain barrier (BBB) resulted in transient or permanent increased seizure frequency in chronically epileptic rats. They relate these results to findings in humans that osmotic BBB opening using mannitol induces seizures in patients with brain …

Imaging, Distribution, and Toxicity of Superparamagnetic Iron Oxide Magnetic Resonance Nanoparticles in the Rat Brain and Intracerebral Tumor

Superparamagnetic iron oxide nanoparticle magnetic resonance imaging (MRI) contrast agents are gaining use in the central nervous system. The purpose of this study was to evaluate the imaging characteristics, distribution, time course, and neurotoxicity of the clinical agents ferumoxtran-10, ferumoxides, and ferumoxytol, and the laboratory preparation MION-46 in rat brain. Iron oxide agents were administered by intracerebral inoculation or intraarterially after osmotic blood-brain barrier opening in normal rats and intravenously in nude rats with intracerebral tumor xenografts. Rat brains were imaged by MRI at multiple time points and then were assessed for iron histochemistry and pathological features. After intracerebral injection, MRI signal changes declined slowly over weeks to months. After transvascular delivery, transient (3 d) enhancement was seen with ferumoxtran-10 or ferumoxytol, whereas ferumoxides induced long-term (28 d) signal dropout. No pathological brain cell or myelin changes were detected after delivery of the clinical iron oxide agents to normal brains. In tumor models, ferumoxtran-10 enhanced one small-cell lung carcinoma intracerebral tumor, which correlated with iron staining in cells with macrophage morphological features at the tumor margin. Little enhancement was seen in two other models. These studies demonstrate the safety and efficacy of iron oxide-based MRI contrast agents in the brain and provide imaging parameters and time course data for future studies in brain tumors and neurological lesions.

Sequelae of the osmotic blood-brain barrier opening in rats

Histopathological sequelae of the osmotic blood-brain barrier opening were studied in 69 adult Wistar rats sacrificed between 2 minutes and 6 days after infusion of 1.6 M mannitol into the unilateral internal carotid artery. The results were correlated with immunohistochemical localization of autologous albumin in the brain parenchyma on paraffin sections. Extravasation of serum albumin was evident in all rats, and the albumin immunoreactivity, commonly localized to the territories of the ipsilateral anterior, middle, and posterior cerebral arteries and contralateral anterior cerebral artery, showed maximum intensity in the rats sacrificed 30 minutes after infusion. The albumin immunoreactivity remained macroscopically visible in the brain parenchyma for 24 to 48 hours, and then gradually faded out. Serum extravasation was accompanied by widening of the perivascular space and focal edema, which largely subsided within 48 hours as the albumin immunoreactivity of the tissue diminished. Although no overt neurological sequelae were seen in the present experiment, minute but definite foci of infarction with focal accumulation of albumin were found in 23 (38%) of 61 rats surviving more than 30 minutes. In addition, ischemic neuronal death of delayed onset was encountered among neurons in the CA-1 region of the hippocampus, in the cerebellum, and in the thalamus in five (25%) of 20 rats sacrificed between Days 4 and 6. Thus, care should be exercised in the practice of this procedure.

Reversible osmotic opening of the blood-brain barrier in mice.

Unilateral reversible osmotic opening of the blood-brain barrier can be produced in mice. Infusion of 1.8 molal arabinose in water at a rate of 0.64 ml/min for 30 seconds into the internal carotid artery consistently results in ipsilateral brain staining by intravascular Evans blue dye. Osmotic opening is concentration-dependent (threshold, 1.6 molal arabinose) and reversible within 4 hours. No long-term neurologic deficit occurs. These results suggest that reversible osmotic blood-brain barrier opening can be applied to disease models in mice.

Increased Delivery of Tumor-specific Monoclonal Antibodies to Brain after Osmotic Blood-Brain Barrier Modification in Patients with Melanoma Metastatic to the Central Nervous System

We evaluated the delivery of melanoma-specific radiolabeled monoclonal antibody (MAb) Fab fragments in a pilot study of three patients with melanoma metastatic to the central nervous system. Tumor samples demonstrated excellent immunohistochemical reactivity with Fab 96.5, specific for a 97,000-molecular-weight melanoma antigen (p97), or Fab 48.7, specific for a melanoma-associated proteoglycan antigen. All three patients received 131I-labeled tumor-specific Fab (5 to 7 mg, 1 mCi/mg) intravenously. On a separate occasion, two patients received 131I-labeled nonspecific Fab (5 to 7 mg, 1 mCi/mg). There was no uptake of either antibody into the region of the tumor (as documented by gamma camera brain images). However, there was increased uptake in the blood-brain barrier (BBB)-modified areas in all three patients when radiolabeled tumor-specific MAb was administered intravenously in conjunction with osmotic BBB opening. In one patient, the estimated cerebrovascular permeability X capillary surface area (PA) for the tumor-bearing hemisphere 3 hours after disruption was 1.16 X 10(-6) sec-1 compared to the PA of 0.395 X 10(-6) sec-1 in the nondisrupted hemisphere. Serial brain scans showed that greater than 90% of the radiolabeled antibody cleared from the brain by 72 hours. The highest radiation doses (rads) calculated per 7 mCi injection were: left brain (barrier-modified hemisphere), 5.46; right brain (non-barrier modified hemisphere), 1.68; thyroid, 98; stomach, 9.1; kidney, 39.9; and total body, 1.33. There seemed to be increased uptake of antibody in the tumor region after barrier modification in one patient, but antibody clearance from that region occurred at the same rate as from surrounding and apparently tumor-free brain. In one patient who had carcinomatous meningitis, we demonstrated antibody bound to only a fraction of the antigen binding sites on tumor cells in the cerebrospinal fluid after BBB modification. We have not shown distinct, persistent localization of antibody in brain tumor; studies investigating MAb dose and other parameters as the basis for this problem are under way.

Tight‐Junctional Modification as the Basis of Osmotic Opening of the Blood‐Brain Barriera

Osmotic opening of the blood-brain barrier (BBB) most likely is mediated by modification of interendothelial tight junctions, subsequent to shrinkage of cerebrovascular endothelial cells, and not by stimulation of transendothelial vesicular transport or by channel formation. This paper summarizes evidence for this conclusion: osmotic BBB opening is mediated by endothelial cell shrinkage, electron microscopy, with single or serial thin sections, demonstrates penetration of intravascular tracer into brain via tight junctional complexes, the BBB remains open after the brain is fixed, osmotic BBB opening is rapidly reversible, and is insensitive to phenothiazines, and BBB closure following osmotic treatment is size-dependent, indicative of a sieve (pore) mechanism with bulk flow. The entire mechanism of vesicular transport in normal tissue is, furthermore, in doubt, because vesicles that are found in tissue layers connected by tight junctions (e.g., frog nerve perineurium and capillary endoneurium) do not support macromolecular transport.

Therapeutic Efficacy of Multiagent Chemotherapy with Drug Delivery Enhancement by Blood-Brain Barrier Modification in Glioblastoma

Reversible osmotic blood-brain barrier (BBB) modification was used in 38 patients with glioblastoma to enhance the delivery of chemotherapeutic agents. The patients ranged in age from 14 to 70 years (mean, 43), and all had prior surgery and radiation; 5 had also received systemic chemotherapy. Karnofsky Performance Status (KPS) scores ranged from 60 to 100% (mean, 79) on admission to the treatment program. Barrier modification was achieved by intracarotid or intravertebral artery infusion of mannitol, and a chemotherapy regimen of methotrexate, cytoxan, and procarbazine was given in conjunction with barrier modification. The 38 glioblastoma patients were compared to two control groups of patients with glioblastoma; these encompassed 14 patients treated with surgery and radiation and 8 treated with surgery, radiation, and systemic chemotherapy. Survival analysis using the Cox Proportional Hazards Regression Model (corrected for age, sex, presence or absence of necrosis, and functional status) showed that patients receiving chemotherapy with BBB modification had a statistically significant (P = 0.0006) longer expected survival (17.5 months) than the control groups (12.8 and 11.4 months, respectively). Presently 16 patients of the barrier-enhanced treatment group are alive at 5 to 42 months from diagnosis (median, 20) with KPS scores ranging from 40 to 90% (median, 65). The neurological complications seen included a stroke-like syndrome in 3 patients (1 with decreased motor movement in the hand, 1 with marked hemiparesis, and 1 with hemiplegia), transient exacerbation of preexisting neurological deficits lasting 2 to 3 days, and a 15% incidence of seizures during or within 24 hours of the BBB modification. In 2 of the 38 patients, radiographic documentation of central nervous system tumor regression concurrent with the development of new tumor nodule(s) in portions of the brain distant from the region of osmotic BBB opening was seen. These studies indicate that chemotherapeutic drug delivery to tumors (as well as surrounding brain) can be augmented by osmotic BBB modification and that such therapy can result in a prolongation of survival.

Osmotic blood-brain barrier opening to IgM monoclonal antibody in the rat.

Pharmacokinetic parameters of iodinated monoclonal antibody (MAb) delivery to normal rat brain were examined. The mean cerebrovascular permeability-surface area (PA; permeability X capillary surface area) to immunoglobulin M (IgM) MAb (mol wt 1,000,000) 10 min after infusion was 0.40 X 10(-6) S-1. When osmotic blood-brain barrier (BBB) disruption is utilized, the PA increased to 8.36 X 10(-6) S-1. Neither intravenous nor intracarotid MAb administration significantly affected delivery to brain. However, osmotic BBB opening significantly (P less than 0.0005) increased MAb uptake independent of the route of administration. After BBB opening and intracarotid MAb the maximum concentration in brain at 1 h was 0.72% per gram of the total administered dose. For 6 h postdisruption, ipsilateral brain levels were 25- to 100-fold greater than in the contralateral hemisphere or nonbarrier-disrupted controls. MAb concentration in brain slightly decreased over 72 h (P less than 0.05). Antibody recovered from disrupted brain retained 90% of its immunological reactivity for at least 24 h. MAb delivery to ipsilateral brain after BBB disruption was linear over a dose of 0.5-5.0 micrograms IgM, whereas the percentage of the total administered dose remained unchanged. After osmotic treatment, barrier opening was maximal to MAb delivery for 1 min with delivery declining rapidly thereafter. The type of anesthesia used and the administration of a thyroid-blocking agent were found to affect brain MAb levels after BBB disruption.

Osmotic Blood-Brain Barrier Modification and Combination Chemotherapy: Concurrent Tumor Regression in Areas of Barrier Opening and Progression in Brain Regions Distant to Barrier Opening

Chemotherapeutic drug delivery can be enhanced by administering drugs into the internal carotid or vertebral artery circulation after osmotic opening of the blood-brain barrier (BBB). As evidence of the clinical implications of this technique, radiographic documentation of central nervous system (CNS) tumor regression was observed in three patients concurrent with the development of new tumor nodule(s) in portions of the brain distant from the region of osmotic blood-brain barrier opening. These three patients, one with metastatic carcinoma of the breast, one with glioblastoma, and one with primary CNS lymphoma, highlight the importance of drug delivery to CNS malignancies.

Chemotherapeutic Agent Permeability to Normal Brain and Delivery to Avian Sarcoma Virus-induced Brain Tumors in the Rodent: Observations on Problems of Drug Delivery

Four chemotherapeutic agents (cyclophosphamide, 5-fluorouracil (5-FU), methotrexate (MTX), and bleomycin) were given intravenously to rats harboring the avian sarcoma virus-induced glioma. Drug content in brain, tumor, and systemic tissue was measured. The uptake of drug and the consistency of drug levels in normal brain and tumor varied widely among these agents. [14C]Cyclophosphamide and its metabolites penetrated normal brain and, to a greater extent, brain tumor. [14C]5-FU and its metabolites also entered normal brain and brain tumor, but to lesser extent than [14C] cyclophosphamide and its metabolites. Immunoreactive MTX entry into tumor was variable, ranging from 30 to 1080 ng/g of tissue, with only minimal concentrations in normal brain. After conventional doses, immunoreactive bleomycin levels in tumor were also variable, ranging from 24 to 164 mu units/g of tissue, and little if any drug entered surrounding brain. In addition, the cerebrovascular permeability of 5-FU and MTX in normal rats was measured. Utilizing the method of Rapoport, the PA (product of permeability and capillary surface area) of 5-FU was found to be 6- to 12-fold greater than that of MTX. The PA of both drugs was increased 4- to 7-fold after osmotic blood-brain barrier opening. The variable "leakiness" of glial tumors, both experimentally and clinically, as well as the varied permeabilities of different water-soluble chemotherapeutic agents, makes the drug delivery problem in brain tumors a very complex issue.

Neurotoxicity of chemotherapeutic agents after blood-brain barrier modification: neuropathological studies.

We examined in 47 dogs the effects of 5-fluorouracil, Adriamycin (doxorubicin hydrochloride), cis-platinum (cis-diamminedichloroplatinum) cyclophosphamide, and bleomycin given in association with osmotic blood-brain barrier modification. The dose of drug ranged from 100% to as little as 5 to 10% of the conventional systemic dosage. Serial neurological observation and subsequent postmortem neuropathological evaluation at times varying from 2.5 hours to 52 days after drug administration showed that cis-platinum and Adriamycin were highly neurotoxic, as evidenced by neurological deficits and pathological changes in the central nervous system parenchyma; 5-fluorouracil and bleomycin had much less, but consequential neurotoxicity; and cyclophosphamide was not associated with substantial toxicity. Intracarotid cis-platinum, unlike the other drugs, damaged the blood-brain barrier and resulted in marked neurotoxicity in the absence of osmotic blood-brain barrier opening. The neural lesions produced by these agents were not specific but were manifested as foci of hemorrhagic necrosis and edema. In addition, secondary brainstem hemorrhage was observed in animals that developed transtentorial herniation. On the basis of these studies, of five drugs studied at a wide range of doses, only cyclophosphamide appears to be safe enough to evaluate in clinical trials that utilize blood-brain barrier modification to enhance drug delivery. These studies also suggest that the lack of neurotoxicity associated with the usual administration of most chemotherapeutic agents probably stems from limited entry of drug into the brain through an intact blood-brain barrier.

Successful Treatment of Primary Central Nervous System Lymphomas with Chemotherapy after Osmotic Blood-Brain Barrier Opening

Three patients with primary central nervous system (CNS) lymphoma have had major tumor regression with multiagent chemotherapy given in association with reversible blood-brain barrier opening used to enhance drug delivery to the tumor. In addition, in one patient barrier modification was carried out in the posterior fossa by mannitol infusion into the vertebral artery without untoward effects, an approach not heretofore accomplished. Computed tomographic (CT) studies documented that discontinuation of steroids rapidly effected an increase in the delivery of contrast agent to the tumor. CT monitoring of the degree of barrier modification showed tumor nodules and tumor size not apparent on the control scans, thereby providing additional evidence of the existence of a blood-brain barrier in CNS tumors. These studies further show that drug (contrast) delivery to the tumor, as well as to the surrounding barrier, is enhanced after reversible blood-brain barrier modification. Finally, chemotherapy administered by this approach resulted in defined, objective tumor responses in these three patients.