Ka-yun Ng

Ultrasound-Induced Mild Hyperthermia as a Novel Approach to Increase Drug Uptake in Brain Microvessel Endothelial Cells

AbstractPurpose. Drug delivery to the central nervous system (CNS) is limited by the blood-brain barrier (BBB). Thus, a noninvasive and reversible method to enhance BBB permeation of drugs is highly desirable. In the present work, we studied if ultrasound-induced mild hyperthermia (USHT, 0.4 watts (W)/cm2 at 41°C) can enhance drug absorption in BBB endothelial cells, and we elucidated the mechanism of USHT on cellular accumulation. Methods. To accomplish these aims, we studied the effects of hyperthermia (41°C), USHT, P-glycoprotein (P-gp) modulator (PSC 833), and combination of USHT and PSC 833 on accumulation of P-gp substrate (R123) and non-P-gp substrates (sucrose, 2-deoxyglucose, and antipyrine) in monolayers of primary bovine brain microvessel endothelial cells (BBMEC). Results. USHT, through its thermal effect, produces a significant (relative to controls; no USHT) and comparable increase in R123 accumulation with PSC 833. We also demonstrate that USHT increases permeability of hydrophobic (R123 and [14C]-antipyrine) and not hydrophilic molecules ([14C]-sucrose and 2-[3H]-deoxy-d-glucose). The enhanced permeability is reversible and size dependent, as USHT produces a much larger effect on cellular accumulation of [14C]-antitpyrine (molecular weight of 188 D) than that of R123 (molecular weight of 380.8 D). Although USHT increases membrane permeability, it did not affect P-gp activity or the activity of glucose transporters. Conclusions. Our results point to the potential use of USHT as a reversible and noninvasive approach to increase BBB permeation of hydrophobic drugs, including P-gp-recognized substrates.

Cell-Mediated Immunotherapy: A New Approach to the Treatment of Malignant Glioma

BACKGROUND The dismal prognosis for patients harboring intracranial gliomas has prompted an intensive search for effective treatment alternatives such as immunotherapy. Our increased knowledge in basic immunology, glioma immunobiology, and molecular biology may lead to the development of effective, rational immunotherapy approaches. METHODS The authors reviewed the literature on glioma immunology, the status of tumor vaccine therapy and on novel techniques to monitor the tumor-specific immune response. RESULTS Experimental conditions currently exist whereby potent antitumor cell-mediated immune responses can be generated. However, clinically, no therapeutic regimen has proven effective. Obstacles to establishing an effective immunotherapy regimen are the lack of a well-defined glioma-specific antigen, the heterogeneity of tumor cells in gliomas, and the modulating effect of the glioma itself on the immune system. Unique strategies to overcome these barriers are being developed. CONCLUSIONS Novel strategies to generate an anti-glioma immune response through use of dendritic cell vaccination, directed cytokine delivery, gene-based immunotherapy, and reversal of tumor-induced immunosuppression are promising. These strategies carry the potential of overcoming the resistance of gliomas to immunotherapeutic manipulation and, undoubtedly, will become a part of our future therapeutic armamentarium.

Competitive Substrates for P-Glycoprotein and Organic Anion Protein Transporters Differentially Reduce Blood Organ Transport of Fentanyl and Loperamide: Pharmacokinetics and Pharmacodynamics in Sprague Dawley Rats

BACKGROUND: Drug transport proteins may be instrumental in controlling the concentration of fentanyl at &mgr; receptors in the brain and may provide potential therapeutic targets for controlling an individual response to opioid administration. P-glycoprotein (P-gp) efflux transporter and organic anion transport protein inward transporters (OATP, human; Oatp, rat) have been implicated in fentanyl and verapamil (only P-gp) transport across the blood–brain barrier. We hypothesized that transport proteins P-gp and Oatp mediate opioid uptake in a drug and organ-specific manner, making them excellent potential targets for therapeutic intervention. METHODS: Opioid (fentanyl or loperamide) was administered by IV infusion to Sprague-Dawley rats alone or in combination with competitive substrates of P-gp (verapamil) or Oatp (pravastatin, naloxone). Plasma, lung, and brain were collected over 10 min and at 60 min after opioid infusion and opioid concentration determined using liquid chromatography/mass spectrometry (LC/LC-MS/MS). Continuous electroencephalogram was used to determine the in vivo response to fentanyl and loperamide in the presence and absence of verapamil. RESULTS: Loperamide brain:plasma (PB) and lung:plasma (PL) partitioning was increased two and fivefold, respectively in the presence of verapamil. Verapamil administration was lethal unless the loperamide dose was reduced by half (0.95–0.475 mg/kg). Fentanyl brain:plasma and lung:plasma were reduced four and sixfold, respectively, by pravastatin and naloxone, whereas verapamil had much less effect. Electroencephalogram results indicated that verapamil reduced the fentanyl-induced central nervous system (CNS) effect and increased the loperamide CNS effect. CONCLUSION: Protein transporters appear to be organ and drug-specific in vivo, affecting first-pass pulmonary uptake and CNS response to opioid administration. Further, data suggest that transport protein inhibition may prove useful for normalizing an individual response to opioids.

In vitro and in vivo evaluation of the effects of PLA microparticle crystallinity on cellular response

Previous research suggests that crystallinity of poly(L-lactide) P(L)LA microparticles can influence surface free energy, which in turn might influence biocompatibility. This work studies the cellular response to P(L)LA microparticles of different crystallinity both in vitro and in vivo. Following incubation with P(L)LA microparticles, the in vitro production of reactive oxygen intermediates (ROI) was measured as a marker of cellular response. In both fluorescence and chemiluminescence experiments to measure ROI, a small effect of microparticle crystallinity on NR8383 AM response was observed. Microparticles of higher crystallinity elicited a smaller inflammatory response compared to lower crystallinity particles. Compared to the elevated inflammatory response induced by zymosan, the response to all P(L)LA microparticles tested was practically negligible. Results from in vivo experiments further supported conclusions that P(L)LA microparticles elicit minimal inflammatory response. Following acute exposure to P(L)LA microparticles in guinea-pig lungs, the inflammatory response was not significantly different from the response observed when sterile saline was administered. In contrast to the in vitro experiments, there were not apparent differences in cellular responses to microparticles of different crystallinity.

Use of circular dichroism spectroscopy in determining the conformation of a monoclonal antibody prior to its incorporation in an immunoliposome

Attachment of antibodies to liposomes endows target specificity to liposomes for a certain cell or organ that express the targeted antigenic determinant. These so-called immunoliposomes hold high promise as targeted drug carriers. One approach of immunoliposome preparation involves conjugating antibodies to hydrophobic anchors (e.g. fatty acids or phospholipid molecules) for incorporation into the liposome membrane. Often, these conjugation reactions are harsh and may result in undesirable chemical and structural changes in the antibody molecule. This necessitates confirmation of the target specificity of the derivatized antibody prior to its incorporation into the liposome. Our approach to this problem is to utilize circular dichroism spectroscopy, which can detect subtle structural differences in proteins with high reproducibility and accuracy in relatively short period of time. In addition, circular dichroism is a non-destructive technique. In this study, we demonstrate the ability of circular dichroism to confirm the conformation of a model antibody, HYB-241, conjugated to N-glutarylphosphatidylethanolamine, prior to its mixing with dioleoylphosphatidylethanolamine/dioleoylphosphatidic acid to form a target-sensitive immunoliposome.