Pamela Snodgrass

Controlled Modulation of BBB Permeability Using the Bradykinin Agonist, RMP-7

Previous studies have shown that the bradykinin agonist, RMP-7, can safely permeabilize the blood brain barrier (BBB) by activation of constitutive B2 receptors on endothelial cells. The paper describes a series of studies using quantitative autoradiography and intracarotid infusions of RMP-7 to further elucidate the effect on BBB permeability. Because earlier studies also demonstrated that even greater effects of RMP-7 were observed in the BBB associated with brain tumors, animal models were employed so that comparisons could be made between the effects of RMP-7 within tumor, brain tissue proximal to tumor, and brain tissue distal from tumor. In the first study, the effect of RMP-7 on enhancing the BBB permeation of three compounds of different physical characteristics was directly compared ([14C]carboplatin, small, hydrophilic; [14C]dextran, large, hydrophilic; [14C]BCNU, small, lipophilic). RMP-7 increased permeability of the vascular barriers to both hydrophilic compounds, carboplatin and dextran. While the effects of RMP-7 were observed on nontumor BBB, the greatest and most consistent effects were observed on the blood brain tumor barrier. This was true for both carboplatin and dextran, with progressively less effect seen as the distance from tumor boundary increased. This topographic effect was more pronounced with the larger molecular weight compound, dextran. No effect of RMP-7 was seen in permeabilizing the BBB or the blood brain tumor barrier for the lipophilic drug, BCNU. In a second study, the generality of RMP-7s effects was established by demonstrating similar increases in permeability to carboplatin in both inbred (Fischer 344) and outbred (Wistar) rat strains, implanted with varying tumor cell lines. Finally, several additional studies were performed to gain greater insight into the dynamics involved with eventual restoration of the BBB following RMP-7 administration. In one series, it was demonstrated that the BBB begins to close nearly immediately upon withdrawal of RMP-7, with complete restoration occurring within minutes. In another series, tachyphylaxis or desensitization resulting from continuous RMP-7 infusion was studied. These studies demonstrated that 60 min of continuous RMP-7 infusion resulted in complete, spontaneous restoration of the barrier to both carboplatin and dextran. Moreover, the desensitization appears to be linked to the initial activation of the receptors in a way which suggests that obligatory desensitization may exist as part of a more complete response. These data are discussed as they relate to practical issues to enhance delivery of drugs across the BBB, as well as more fundamental issues involving the function of the BBB and its interaction with the brain.

Central analgesic actions of loperamide following transient permeation of the blood brain barrier with Cereport (RMP-7).

The bradykinin analog, Cereport (RMP-7), was designed to increase permeability of the blood brain barrier (BBB). Over the past several years it has been developed primarily as a means of increasing permeability of the blood brain tumor barrier, where early evidence indicated a particularly robust and reliable effect. The present series of experiments were intended to determine whether Cereport might also be used to increase delivery of pharmacological agents across the normal (i.e., non-tumor) BBB. This was accomplished by testing the ability of Cereport to enhance delivery of the peripherally acting opiate agonist, loperamide, to the brain, as evidenced by induction of a centrally mediated analgesic effect. Intravenous administration of a combination of Cereport and loperamide produced a significant analgesic effect (2-fold increase in response times) when animals were tested on a hotplate apparatus. Loperamide alone did not produce analgesia. An analysis of the time course of analgesia revealed a graded onset of analgesia which peaked at 30 min, maintained asymptote at 60 min, and began to diminish by 120 min following Cereport and loperamide administration. Finally, the analgesic effects of combining Cereport and loperamide were completely blocked when animals were pre-treated with the opiate antagonist naloxone, demonstrating that the analgesia was mediated through opiate receptors. Collectively, these results suggest that Cereport was able to increase delivery of loperamide across the BBB, allowing it to gain access to opiate receptors in the CNS to produce a centrally mediated analgesic effect. They therefore provide clear evidence that safe and well-tolerated doses of Cereport can increase permeability of the normal (i.e., non-tumor) BBB. Moreover, they provide the first evidence of a pharmacological effect specifically enabled by controlled (i.e., receptor-mediated) modulation of the BBB.

Injectable chemotherapeutic microspheres and glioma I: enhanced survival following implantation into the cavity wall of debulked tumors.

AbstractPurpose. Implantation of biodegradable polymers provides a powerfulmethod to deliver high, sustained concentrations of chemotherapeuticsto brain tumors. The present studies examined the ability of injectablepolymeric microspheres, formulated to release carboplatin or BCNUfor 2–3 weeks, to enhance survival in a rodent model ofsurgically-resected glioma. Methods. Rat glioma (RG2) cells were implanted into the cortex ofrats and allowed to grow for 10 days prior to surgical resection. Ratswere given either surgical resection only, bolus injection (100 μg) ormicrospheres containing 10, 50, or 100 μg of carboplatin or BCNU.The microspheres were implanted, via hypodermic injection, eitherdirectly into the surgical cavity or into the tissue along the perimeterof the cavity. Results. The order of survival among treatment groups was: noresection < resection only < bolus chemotherapy < sustained releasechemotherapy. Carboplatin and BCNU did not differ in this respectand in each case, the enhanced survival achieved with sustained releasewas dose-related. However, the enhanced survival achieved withcarboplatin was substantially greater when the microspheres wereimplanted into the perimeter wall of the resection cavity, compared toimplantation into the cavity itself. The enhanced survival produced bycarboplatin implants along the resection perimeter was associated witha significant attenuation of regrowth of the tumor. Finally, in a separatestudy in non-tumor brain, atomic absorption spectrophotometryrevealed that while the microspheres produced significantly prolongedtissue levels of carboplatin relative to a bolus injection, carboplatindiffusion was limited to brain tissue extending primarily 0.5 mm fromthe injection site. Conclusions. These data demonstrate: (1) that sustained delivery ofchemotherapy is superior to equipotent bolus doses following tumorresection, and (2) that direct injection of sustained release microspheresinto the tissue surrounding a growing tumor mass may provide superioreffects over injections into the surgical cavity. They also suggest thatsuccessful implementation of this approach in humans may requiremeasures or circumstances that improve upon the limited spatial drugdiffusion from the implantation site.

Injectable chemotherapeutic microspheres and glioma II: enhanced survival following implantation into deep inoperable tumors.

AbstractPurpose. Delivery of chemotherapeutics using implantable,biodegradable polymers provides a potentially powerful method of treating braintumors. The present studies examined the ability of injectablemicrospheres, formulated to release carboplatin or BCNU for 2–3 weeks,to enhance survival in a rodent model of deep, inoperable glioma. Methods. Rat glioma (RG2) cells were implanted into the striatum ofrats. In a first experiment, the tumors were allowed to grow for 3 days,followed by either no treatment, bolus chemotherapy (100 μg), orimplantation of microspheres containing 10, 50, or 100 μg ofcarboplatin. The microspheres were implanted, via hypodermic injection,directly into the center of the small, 3-day-old tumors. In a secondexperiment, tumors grew for 8 days prior to treatment with eithercarboplatin- or BCNU-loaded microspheres. The microspheres werethen injected either directly into the center of these larger tumors orinto three sites along the perimeter of the tumor. Separate sets ofanimals received bolus chemotherapy (100 μg) into either the tumorcenter or around the tumor perimeter. Results. Injection of carboplatin-loaded microspheres into the centerof the small 3 day old, tumors produced dose-related increases insurvival. When injections of carboplatin- or BCNU-loadedmicrospheres were made into the center of the larger, 8-day-old tumors,survival was not enhanced. However, when the microspheres wereinjected along the perimeter of the larger tumors, sustained-releasechemotherapy did significantly prolong survival. Bolus chemotherapywas less effective than sustained release chemotherapy. Conclusions. Together, these data: (1) demonstrate that sustaineddelivery of chemotherapy in or near the tumor site is superior toequipotent bolus doses in inoperable tumors, (2) demonstrate thatinjection of sustained release microspheres into the tissue surroundinga growing tumor may provide superior effects over injections directlyinto the tumor mass, and (3) suggest that this approach may providea useful means of selectively delivering chemotherapeutics to tumorsor portions of tumors that cannot otherwise be treated with conventionalsurgical approaches.

Sustained release chemotherapeutic microspheres provide superior efficacy over systemic therapy and local bolus infusions.

AbstractPurpose. The present studies evaluated the ability of injectable, biodegradable microspheres releasing carboplatin, doxorubicin, or 5-fluorouracil to suppress the growth of solid tumors implanted subcutaneously or intramuscularly. Methods. Seven to 10 days after implantation of MATB-III cells, rats received systemic chemotherapy, intratumoral bolus chemotherapy, or injections of chemotherapeutic microspheres into the tumor center or multiple sites along the outer perimeter of the tumor. Results. A single treatment with carboplatin, doxorubicin, or 5-fluorouracil microspheres along the perimeter of the tumors produced a significant, dose-related suppression in tumor growth, relative to injections directly into the tumor center. Moreover, five temporally-spaced microsphere treatments along the tumor perimeter (with either doxorubicin or 5-fluorouracil microspheres) completely eradicated 100% of the subcutaneous tumors and 40-53% of the intramuscular tumors. Polypharmacy, accomplished by blending doxorubicin- and 5-fluorouracil-loaded microspheres and injecting them into the tumors was even more efficacious than sustained delivery of either drug alone. Comparable doses of systemic chemotherapy or intratumoral bolus chemotherapy were ineffective. Conclusions. Injectable microspheres might be ideal for local, sustained delivery of chemotherapeutic agents to solid tumors. However, attention must be paid to the placement of the microspheres, for injections around the tumor perimeter may be required for efficacy.

Intravenous Cereport (RMP-7) enhances delivery of hydrophilic chemotherapeutics and increases survival in rats with metastatic tumors in the brain

AbstractPurpose. The following experiments determined whether intravenous infusions of Cereport enhance delivery of chemotherapeutics and prolong survival in rats with metastatic tumors in the brain. Methods. Autoradiography and scintillation were used to examine uptake of the lipophilic (paclitaxel and carmustine) and the hydrophilic (carboplatin) chemotherapeutic agents, as well as the large hydrophilic marker, 70 kDa dextran. Cereport was also tested in combination with the chemotherapeutic drugs carboplatin, vinorelbine, gemcitabine and carmustine to determine if Cereport could enhance the survival benefit beyond that provided by chemotherapy alone. Results. Cereport enhanced the uptake of carboplatin and dextran, but not paclitaxel or carmustine. The pattern of Cereports uptake effect with carboplatin revealed that Cereport selectively increased the proportion of highly permeable regions. Survival was significantly enhanced when Cereport was combined with either carboplatin, vinorelbine, or gemcitabine, but not carmustine, compared to each chemotherapeutic agent alone. Conclusions. These data provide the first evidence that Cereport, or any receptor-mediated approach intended to enhance the permeability of the blood-brain tumor barrier, can increase the delivery hydrophilic drugs to metastatic tumors in the brain, increasing survival in tumor-bearing rats.

Evidence that Cereport's Ability to Increase Permeability of Rat Gliomas Is Dependent Upon Extent of Tumor Growth: Implications for Treating Newly Emerging Tumor Colonies

Cereport (RMP-7) enhances delivery of chemotherapeutics into brain tumors by increasing the permeability of the glioma vasculature (i.e. , the blood-brain tumor barrier; BBTB). Its effect on brain tumors has consistently been more robust than that on normal brain. The present experiments tested the hypothesis that the ability of Cereport to increase the permeability of infiltrating glioma colonies increases as the glioma colonies develop, in situ. In an initial preliminary experiment, the significant and selective effects of Cereport in tumor tissue and brain surrounding tumor were verified using [(14)C]carboplatin as a marker, 8 days after implantation of 50,000 RG2 cells. A second preliminary experiment established that the number of tumor cells initially seeded influences the growth rate of the tumor mass. Tumors seeded with 50,000 cells were larger than those seeded with 25,000 cells 3, 5, and 8 days after implantation. Next, the hypothesis that the extent of tumor growth increases Cereports effects on the BBTB was tested by measuring the concentration of radiolabeled carboplatin in the tumor when 50,000 cells were implanted 3, 8, or 13 days prior to the experiment. While a reliable, approximately twofold increase in carboplatin concentration was seen in the 8- and 13-day-old tumors, no significant effect of Cereport was observed in the tumors that developed only 3 days, in situ. Finally, another test of the hypothesis was made by comparing Cereports effects on 8-day-old tumors initially seeded with either 50,000 or 25,000 cells (the latter producing a smaller, more slowly developing, tumor mass). Again, significantly higher carboplatin concentrations were seen with Cereport in the 50,000 cell tumors (greater than two-fold increase), compared to the smaller, more slowly developing, 25,000 cell tumors (<30% increase). The tumor and its vasculature were characterized in additional rats implanted with RG2 cells using CD-31, laminin, and bradykinin B(2) receptor immunocytochemistry. Intense B(2) receptor staining was observed on cells within the parenchyma of normal brain and tumor but not on the vasculature of tumor or brain. An extensive network of CD-31 and laminin staining was seen within and around the tumors in all groups, indicating relatively rapid and robust changes in vascularity in response to the gliomas. However, no consistent difference in vascularity between groups was observed to account for the uptake differences seen with Cereport. Collectively, these data offer initial preclinical empirical support for the hypothesis that Cereports effects on tumor permeability increase as the tumor grows, which we further hypothesize is likely related to features of vascular development within the tumor independent of numbers or general morphology of vessels. If a similar phenomenon is shown to occur with infiltrating colonies from spontaneously forming gliomas in humans or from newly emerging metastases in brain, these data could impact the design and conduct of future trials using approaches intended to enhance delivery of chemotherapeutics through increased permeability of the tumor vascular barrier.

Use of CereportTM (RMP-7) to Increase Delivery of Carboplatin to Gliomas: Insight and Parameters for Intracarotid Infusion Via a Single-Lumen Cannula

CereportTM (RMP-7) is a selective bradykinin B receptor ago2 nist that increases the permeability of the blood-brain tumor barrier (BBTB) to increase delivery of chemotherapeutic agents to brain tumors. Two experiments were performed in a rat glioma model to evaluate and refine intra-arterial dosing parameters using Cereport. The first experiment investigated whether desensitization or tachyphylaxis to the effects of Cereport on vascular permeability can be avoided during the course of super-selective, intracarotid drug administration. Super-selective administration may be used when two different arterial branches supply a single tumor in order to ensure that enough of the drug reaches the entire tumor. In this instance, the chemotherapeutic drug is infused separately and sequentially into each branch. This procedure necessitates an infusion duration that is sufficiently long to raise concerns that tachyphylaxis to B2 receptor stimulation might occur during the Cereport infusion. To study this possibility...