Convection-Enhanced Delivery of Macromolecules to the Brain Using Electrokinetic Transport

Electrokinetic transport in brain tissue represents the movement of molecules due to an applied electric field and the interplay between the electrophoretic and electroosmotic velocities that are developed. This dissertation provides a framework for understanding electrokinetic transport and how it may be utilized for short-distance ejections, relevant to capillary iontophoresis, and long-distance infusions, for the clinical management of malignant brain tumors as a novel convection-enhanced drug delivery system.In particular, electrokinetic transport was first analyzed in a series of poly(acrylamide-co-acrylic acid) hydrogels that demonstrated varying electroosmotic velocities. Moreover, a hydrogel was synthesized to mimic the electrokinetic properties of organotypic hippocampal slice cultures (OHSC), as a surrogate for brain tissue. Short- and long-distance capillary infusions of molecules into the hydrogels and OHSC provided a framework to understand the relevant phenomena, such as the effect of varying the capillary tip size, applied electrical current, ζ-potential of the capillary or the outside matrix, infusion time, tortuosity, and properties of the solute (including molecular weight and electrophoretic mobility). Control of the directional transport of molecules was also demonstrated over a distance of several hundred micrometers to millimeters. Finally, electrokinetic infusions were conducted in vivo in the adult rat brain, with results compared to those of pressure-driven infusions.The experiments and results described in this dissertation provide a foundation for further development, by presenting a methodical means to increase the ejection profile and attain clinically relevant penetration distances while minimizing adverse effects to the brain tissue, including from the electric field itself. The rate of electrokinetic transport is greater than the rate of diffusion, and therefore it represents a novel form of convection-enhanced drug delivery system

Study of the pharmacological modification of neuroendocrine mechanisms controlling ovulation in the rat.

The synthetic steroid, RMI 12,936 (17beta-hydroxy-7alpha-methyl-androst-5-en-3-one) inhibits ovulation in the rat. This study investigated the mechanism of action of the drug. The results showed that administration at or before 01:00h on proestrus blocked the preovulatory LH surge, which was restored by LHRH or oestradiol plus progesterone. Administration of oestradiol alone restored ovulation to only 43% of RMI 12,936-treated rats. The negative feedback effect of testosterone on LH release showed similar characteristics. Although RMI 12,936 was shown to be a potent androgen, the peripheral androgenic activity was found not to be correlated with its inhibitory effect on LH release. Instead, it was suggested that RMI 12,936 may act through antioestrogenic and antiprogestational activity. Investigation of the site of action revealed effects: a) at the ovarian level, inhibiting the biosynthesis of oestrogen and progesterone; b) at the adenohypophysial level, preventing full sensitization to LHRH; and c) at the hypothalamic level, inhibiting noradrenergic and tryptaminergic neurotransmission. The first two are not the main sites of antiovulatory activity, since administration of oestradiol plus progesterone - although they restore ovulation - cannot restore full sensitization, and secondly since RMI 12,936 injected into the third cerebral ventricle does not require to be transported to the ovary for effective ovulation blockade. The major site of action was therefore at the hypothalamic level, where RMI 12,936 blocks the neural signal mediated by noradrenaline and triggered by oestrogen. Based on the premise that RMI 12,936 has a similar mechanism of action to that of testosterone, it was proposed that the drug prevents full sensitization of the adenohypophysis by reducing the number of LHRH receptors. During this investigation, adenohypophysial sensitivity to LHRH altered. From observation throughout the year, a pattern emerged indicating the existence of seasonal variation in the mechanisms controlling LH release. This aspect requires further investigation

Adaptations in In Vivo Catecholamine Signaling in Models Of Stress and Addiction

Catecholamine neurotransmission plays a key role in regulating a variety of behavioral and physiological processes, and its dysregulation is implicated in both neurodegenerative and neuropsychiatric disorders. Understanding how catecholamine signaling is regulated in vivo may provide insight into its role in disease states ranging from anxiety and drug addiction to Parkinson’s disease. This work combines rapid, selective, and spatially resolved voltammetric measurements with pharmacology and behavior. We used this approach in divergent animal models to investigate the dynamics of in vivo norepinephrine and dopamine signaling. Our initial investigations focused on norepinephrine release in the ventral bed nucleus of the stria terminalis (vBNST), where we found differential regulation in models of anxiety and depression. When animals were challenged with social-isolation stress and drug-dependence, adaptations in vBNST norepinephrine regulation varied with respect to both stressor and baseline stress-reactivity. We hypothesized that certain stressors elicited catecholamine efflux, and turned to real-time measurements in awake, freely moving animals. To understand how release could produce plasticity in catecholamine regulation mechanisms after drug dependence, we focused on opiate exposure and withdrawal. We found opposing responses from dopamine and norepinephrine: whereas dopamine fluctuations in the nucleus accumbens (NAc) increased during morphine intoxication, they decreased during precipitated withdrawal. Conversely, increased norepinephrine overflow in the vBNST was found only during withdrawal, and was time locked to somatic withdrawal behaviors. While probing real-time catecholamine overflow, we also discovered hemispheric synchrony of NAc dopamine fluctuations, and revealed previously unappreciated cross-hemispheric projections in both the dopaminergic and noradrenergic systems. Our findings of opposing catecholamine responses, combined with genetic differences in response to stressors provide new insight into catecholamine regulation. Future work should continue to address how dopamine and norepinephrine signal in vivo and in real time and contribute to the development of a variety of neuropsychiatric conditions.Doctor of Philosoph

Adrenergic Control of Potassium and Magnesium: Interaction with Drug Therapy

Hypokalaemia is potentially fatal (Chapter 2). The internal regulation of potassium, i. e. the movement of potassium between body compartments, has not been extensively investigated (Chapter 1). Rapid movements of potassium can occur across cell membranes, e. g. in diabetic ketoacidosis the hyperkalaemia can be rapidly reversed by insulin administration, and the existence of a specific membrane enzyme controlling movement of potassium and sodium between the intracellular and extracellular compartments, Na+/K+ ATPase, has been known for 30 years (Chapter 1. 5). Some acutely ill patients have hypokalaemia on admission to hospital which resolves without treatment. This observation led to the hypothesis that increased sympathetic activity, raising circulating adrenaline levels, stimulates a Na+/K+ ATPase linked to a beta2-adrenoreceptor on cell membranes pumping potassium into cells. Animal work by Clausen supported this theory and several studies in humans, some carried out in the Department of Materia Medica, were also supportive, demonstrating that infusing adrenaline resulted in hypokalaemia (Chapter 1). In the studies presented in this thesis both the mechanism and the clinical relevance of adrenaline induced hypokalaemia, with particular emphasis on the effects of a number of widely used drugs, have been studied. Many drugs have been designed to specifically act on receptors in the sympathetic nervous system, either as agonists or antagonists, e. g. beta-blockers and beta2-agonists, and they are frequently administered to patients with cardiovascular disease who are at increased risk of dysrhythmias should hypokalaemia occur. Such patients are at increased risk of suffering acute stress, such as myocardial infarction, which increases circulating adrenaline levels. An infusion regimen of (-)-adrenaline which would safely raise circulating adrenaline to concentrations similar to those seen in acute severe illness was developed (Chapter 3). This regimen consistently raised adrenaline levels seen in normal subjects during supine rest by 10 fold or more. During the infusions adrenaline levels did fluctuate, but they remained in the pathophysiological range. The regimen involved stepwise increases in the rate of adrenaline infusion and proved safe despite the adrenaline infusion being combined with other drugs with sympathomimetic activity. The mechanism of adrenaline induced hypokalaemia in man is unproven. However, the possibility that adrenaline induced hypokalaemia could be the result of B-agonist induced changes in plamsa insulin was excluded (Chapter 4.2). Both plasma insulin and potassium concentrations fell during the adrenaline infusion. Attenuation of adrenaline induced hypokalaemia by beta-adrenoceptor antagonists with varying degrees of cardioselectivity (B1) was studied and demonstrated that adrenaline induced hypokalaemia was mediated via the B2 adrenoceptor (Chapter 4.3 & 4.4). Whether the fact that cardioselective beta-antagonists will be less effective in protecting patients from adrenaline induced hypokalaemia during the acute stress of severe illness is of any clinical significance remains unknown. Salbutamol, a selective beta2-agonist, was also shown to cause hypokalaemia when given intravenously (Chapter 4.2). It is administered in high doses in acute attacks of asthma, where it might be expected that circulating adrenaline levels are raised. An additive hypokalaemic effect of exogenous adrenaline and salbutamol was demonstrated (Chapter 4.2) . Hypokalaemia is a relatively common adverse effect of many diuretics and such hypokalaemia could increase the severity of hypokalaemia during acute stress. No synergistic action on potassium levels was demonstrated (Chapter 5) between adrenaline and any diuretic. However, both frusemide and bendrofulazide lowered plasma potassium and, therefore, during the adrenaline infusion more profound hypokalaemia was observed because baseline potassium was lower. Theophylline, widely used as a bronchodilator, has been reported to increase circulating catecholamine levels and to interact with sympathomimetics (Chapter 6.1 & 6.2). Hypomagnesaemia can occur in situations in which circulating adrenaline levels are known to be raised, such as acute myocardial infarction (Chapter 7.1). The control of internal regulation of magnesium is not understood. The role of adrenaline in the control of magnesium levels was studied, using the same adrenaline infusion regimen, and a small but significant fall in plasma magnesium was observed (Chapter 7.2). This was unaltered by pretreatment with diuretics (Chapter 7.3). The mechanisms and clinical relevance of adrenaline induced hypomagnesaemia require further study but these have not yet been attempted

Contrasting Regulation of Catecholamine Neurotransmission in the Behaving Brain: Pharmacological Insights from an Electrochemical Perspective

Catecholamine neurotransmission plays a key role in regulating a variety of behavioral and physiologic processes, and its dysregulation is implicated in both neurodegenerative and neuropsychiatric disorders. Over the last four decades, in vivo electrochemistry has enabled the discovery of contrasting catecholamine regulation in the brain. These rapid and spatially resolved measurements have been conducted in brain slices, and in anesthetized and freely behaving animals. In this review, we describe the methods enabling in vivo measurements of dopamine and norepinephrine, and subsequent findings regarding their release and regulation in intact animals. We thereafter discuss key studies in awake animals, demonstrating that these catecholamines are not only differentially regulated, but are released in opposition of each other during appetitive and aversive stimuli

Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 182, July 1978

This bibliography lists 165 reports, articles, and other documents introduced into the NASA scientific and technical information system in June 1978

Pharmacokinetics and Therapeutic Uses of Mesna

In the early 1980s, significant advancement in the safety of ifosfamide therapy was achieved by co-administrating mesna (sodium 2-mercaptoethane sulfonate) to prevent dose-limiting hemorrhagic cystitis. Mesna exerts its protective effect within the urine, where its free sulfhydryl group is able to conjugate cytotoxic metabolites. Within the circulation, however, mesna exists primarily as its inactive disulfide, dimesna. Dimesna is currently undergoing clinical development as a prodrug (BNP7787) to treat cisplatin-induced nephrotoxicity. Remarkably, chemoprotection is achieved without attenuation of efficacy of co-administered anti-cancer agents. This is widely attributed to the kidney-specific disposition and stability of dimesna. We sought to evaluate the role of drug transporters in the disposition of dimesna. In vitro screens of uptake and efflux transporters identified putative mechanisms of apical and basolateral uptake of dimesna and subsequent secretion of mesna into renal tubules. Administration of the renal drug transporter inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure while decreasing the renal clearance due to secretion and steady-state volume of distribution. Chemical reduction of dimesna to mesna is essential for the mitigation of ifosfamide- and cisplatin-induced toxicities. In vitro, reduction of dimesna was facilitated by redox enzymes of the thioredoxin and glutaredoxin systems and also by non-enzymatic thiol-disulfide exchange with cysteine and glutathione. These findings supported the further investigation of mesna as a thiol exchange agent to lower the toxic endogenous thiol amino acid homocysteine (Hcy). Increased plasma total homocysteine (tHcy) is a graded, independent risk factor for the development of atherosclerosis and thrombosis. Over 90% of patients with end-stage renal disease (ESRD) have elevated plasma tHcy. Previous studies have expanded the use of mesna to exchange with albumin-bound Hcy, thereby enhancing its dialytic clearance. Although an initial pilot study of 12 mg/kg intravenous mesna administered predialysis caused a significant decrease in plasma tHcy compared to placebo, prolonged treatment had no effect on plasma tHcy. Successful therapeutic uses of mesna and dimesna are likely due to their unique disposition by renal drug transporters and thiol-disulfide redox equilibrium. Loss of renal transporter function due to disease, drug-drug interactions, or genetic variability may decrease their therapeutic efficacy

Prevention of Organ-Specific Doxorubicin Induced Toxicity Using Physiologically-Based Pharmacokinetic Modeling and Therapeutic Drug Monitoring

Physiology-based pharmacokinetic models are mathematical models that characterize the behavior of a drug and have compartmental equations that are representative of specific tissues and physiological processes.[1, 2] Doxorubicin is an anthracycline antibiotic that is effective and widely used in anticancer therapy due to its potent cytotoxicity. Unfortunately, with that potency comes cardiotoxic side effects related to cumulative lifetime dose.[3] Specifically, the toxicity is related to the accumulation of the primary metabolite doxorubicinol (DOXol) in the heart.[4] Since the toxicity is organ-specific, the best way to characterize the behavior is through PBPK modeling.[2] Since PBPK models tend to be large systems of ODEs, several numerical methods were attempted for solving the model before a matrix-based approach was chosen.[5, 6] The eigenvalue/eigenvector solution was evaluated at three time points which were then included in a Composite Simpson’s Rule numerical integration for the length of some time interval.[5, 7] The PBPK model, adapted from a pig model, was fit to mouse data and scaled to predict rat, rabbit, dog, pig, and human data sets using an allometric scaling equation on the blood:plasma partition coefficient B : P .[8, 9, 10] Despite extensive investigation into dose adjustments for DOX, no covariates were consistently found to improve the efficacy and minimize toxicity except dosing schedule – infusion rate and duration.[11] The criterion for decreasing incidence of cardiotoxicity was maintaining a sub-toxic Cmax,heart,DOXol in the heart while maximizing exposure, represented by area under the concentration-time-curve (AUC). Thus, the original mouse data set was ideal since it included both DOX venous blood concentration and DOXol heart concentration.[12] The model was optimized at 10 time points between 1 minute and 72 hours with the goal of (AUC) maximization without exceeding Cmax,heart,DOXol. Using these predictions, therapeutic drug monitoring could be executed by taking the plasma concentration samples during a patient’s first DOX dose, PBPK model predictions could provide AUC and Cmax,heart,DOXol data, which could then inform the infusion parameters for the next dose. Clinical thresholds for Cmax,vb have been established for incidence of adverse effects, and in future work, perhaps a similar threshold for cardiotoxicity could also be established using tissue-specific measures

Evaluation Of Presynaptic Dopamine Dynamics After: Toluene Inhalation Or Trkb Receptor Activation

Dopamine (DA) neurons in the striatum mediate several functions of the brain and have been linked to a host of neurological disorders including Parkinson\u27s disease and addiction, both of which occur as a result of dysfunction in the DA system. In the present study, our first objective was to understand how the striatal DA system adapts to acute and repeated administration of inhalant toluene. The use of toluene as inhalant, like other drugs of abuse, is known to perturb DA neurotransmission in the brain reward pathway. However, the exact mechanism underling toluene\u27s influence on striatal DA neurotransmission is unknown. The current work utilized behavior assays and neurochemical techniques such as slice fast scan cyclic voltammetry (FSCV), in vivo microdialysis, and brain tissue content analysis to examine how toluene inhalation alters the striatal DA system. Overall, both behavior and neurochemical data confirmed that toluene inhalation alters stimulated DA release in striatum. Mechanistically, the neurochemical data indicated that acute toluene inhalation potentiates striatal DA release and catabolism but there is no difference on DA uptake or extracellular DA levels in the caudate putamen (CPu). Furthermore, toluene induced potentiation in DA release is not mediated by DA D3 autoreceptors. Meanwhile, chronic toluene exposure attenuated DA release only in the nucleus accumbens (NAc). Repeated toluene exposure also increased extracellular DA levels in the NAc, which is typical of addictive drugs. However, repeated toluene inhalation had no effect on DA D3 autorecepetors, and DA catabolism. Taken together, the present data suggest that acute or repeated toluene alters the striatal DA system through indirect neuronal action. The second objective was to understand how brain derived neurotrophic factor (BDNF) modulates striatal DA dynamics. Aside from its conventional role as a neurotrophic factor, BDNF has also been implicated in synaptic transmission and neurological disorders. Since BDNF mediates it neurotrophic functions through tyrosine kinase receptor TrkB, the functional effects of tyrosine kinase receptor TrkB on the striatal DA release and uptake rate were examined. This work utilized FSCV to evaluate the effect of exogenous BDNF, TrkB agonist; 7,8-dihydroxyflavone (7,8-DHF), and TrkB antagonists; genistein, tyrphostin 23, and K252a, on DA dynamics in the CPu of brain slices obtain from BDNF deficient (BDNF+/-) mice and their wildtype littermates. Overall, the results obtained highlighted the utility of FSCV to probe the functional effect of Trk receptors on DA dynamics. The results also showed that activation of TrkB receptors with exogenous BDNF and 7,8-DHF potentiated presynaptic DA release in BDNF+/- and wildtype mice respectively, with no effect on DA uptake. However, concentrations greater than 3 μM 7,8-DHF attenuated DA uptake rates in only BDNF+/− mice. In the presence of K252a, the BDNF or 7,8-DHF induced potentiation of DA release was abolished, suggesting that the effect of BDNF or 7,8-DHF on presynaptic DA release is TrkB mediated