The goal of this dissertation was to investigate three new curve comparison metrics, the Rescigno Index, fl, and the Chinchilli Metric as tools to compare pharmacokinetic profiles for the assessment of assess relative bioavailability (BA) and bioequivalence (BE). The specific objectives were to (1) compare the relative sensitivity of the new metrics to detect differences in AUC and Cmax as a function of the pharmacokinetics of the drug products, and (2) to estimate relative bioavailability and bioequivalence. Methods. Retrospective analysis of experimental data and Monte Carlo simulations of bioequivalence trials were used to evaluate the relative sensitivity of the metrics to detect profile differences. The experimental data study involved determining the degree of discordance with typical criteria when judging individual profiles to be the same or different, and then examining the relationship between the degree of discordance and the pharmacokinetics of the drug product. The simulation studies involved determining the proportion of clinical studies failing bioequivalence under different pharmacokinetic models. Product bioequivalence was estimated using data from 35 typical 2 treatment-2 period bioequivalence study experimental datasets. Three different bioequivalence limits were applied to the curve metrics. Results. The new metrics more effectively detect differences in absorption time lags but less effectively detect differences in Cmax under some conditions. The relative sensitivity to Cmax depends on the shape of the curve, where increasing the ka(ref)/ke(ref) increases the disparity across the metrics. The curve metrics show increased sensitivity to variability in disposition, elimination, and random residual error, but comparable sensitivity to differences in bioavailability. Fourteen of the 35 studies failed typical criteria (AUC and Cmax). Applying bioequivalence limits of 25%, 21 and 26 studies failed the Chinchilli and fl criteria respectively. At the 30% limit, 14 and 20 studies failed the Chinchilli and fl criteria respectively. The specific studies failing each criterion varied. The within-subject variability of the Chinchilli Metric was higher than Cmax. Both the Chinchilli Metric and fl showed a tendency toward extreme values. Conclusions. The metrics differ in pharmacokinetic sensitivities and differ in statistical properties. There are advantages and disadvantages associated with these differences.

Remifentanil (ULTIVA) is an ultra short-acting opioid which has recently been approved for use during surgical procedures requiring opioid analgesia. After i.v. administration, it is rapidly metabolized by non-specific esterases in the blood and other tissues to less active metabolites. Total body clearance of remifentanil in man is 41.2 mL/min/kg, the Vd is 390 mL/kg and the elimination half-life is 10-48 minutes. In dogs, the clearance is 63 mL/min/kg, Vdss is 222 mL/kg and the elimination half-life is 5.7 minutes. Esmolol is an ultra short-acting beta-blocker administered intravenously during surgical procedures to decrease heart rate and blood pressure in patients at risk of cardiovascular complications. It is also metabolized by non-specific esterases in the blood and other tissues to form an inactive acid metabolite and methanol. It is likely both drugs will be administered together in patients with cardiac disease during surgical procedures. The goal of this study was to evaluate a possible drug interaction between remifentanil and esmolol following concomitant administration. This was accomplished by comparing pharmacokinetic and pharmacodynamic parameters of remifentanil after administration alone and in combination with esmolol. In the first study, remifentanil (treatment I, 25 mug/kg/min) and remifentanil plus esmolol (treatment II, (25 mug/kg/min and 200 mug/kg/min, respectively)) were infused for 20 minutes into male Sprague-Dawley rats in a random parallel design. Arterial blood samples were collected over the course of the study, extracted with methylene chloride, and analyzed (off-site) by a validated GC-MS assay. Additionally, cardiovascular measurements were continuously collected from 15 minutes pre-dose until about 20 minutes after end of infusion. Compartmental modeling was performed to determine the pharmacokinetic parameters. In a follow-up study, remifentanil (treatment I, 15 mug/kg/mL) and remifentanil plus esmolol (treatment II, (15 mug/kg/min and 600 mug/kg/min, respectively)) were infused into male Sprague-Dawley rats in a random two-way cross-over design. Blood samples (using limited sampling strategy) were collected and analyzed as before. EEG data were collected continuously over the course of the study. Bayesian estimation was used to determine the pharmacokinetic parameters. Pharmacodynamic parameters were obtained by modeling EEG spectral edge, using a pharmacokinetic-pharmacodynamic link model. Comparison of the pharmacokinetic and pharmacodynamic parameters between treatments I and II in both studies did not detect any significant (p < 0.05) differences. This indicates that at the doses tested, the co-administration of esmolol did not cause a significant change in the pharmacokinetics or pharmacodynamics of remifentanil no drug-drug interaction.

Effective drug delivery to the brain continues to be a challenge to successful therapy of disease states of the central nervous system. Chemotherapeutic molecules such as methotrexate that are used extensively in the treatment of systemic cancers have been found to be ineffective in the management of brain tumors, mainly due to subtherapeutic levels. Similarly, anitretrovirals like zidovudine and saquinavir are effective in significantly reducing systemic viral loads, but fail to reduce viral loads in the central nervous system. After systemic administration, therapeutic agents must permeate the blood brain barrier to reach the brain. The blood brain barrier functions to protect the brain from substances in the systemic circulation. The inter endothelial tight junctions restrict the paracellular diffusion of molecules and the efflux transporters expressed at the blood brain barrier actively transport substrates out of the brain. Thus, the blood brain barrier regulates transport of therapeutic molecules into the central nervous system and by preventing therapeutic levels of drugs from reaching the brain, it hinders effective therapy of these life threatening diseases. This work considered two different approaches in drug delivery to the brain via the transvascular route, namely, tight junction modulation and efflux transport inhibition, so as to enhance the brain distribution of commonly used antiretroviral and anticancer agents. DeltaG is the 12 kD active fragment of Zot, that has been shown to reversibily open tight junctions and enhance the systemic availability of a number of therapeutic agents. Administration of methotrexate in male Sprague Dawley rats along with DeltaG (200 to 600 mug/kg) resulted in an eight to twenty five fold increase in the brain to plasma partitioning of methotrexate. Our results show that DeltaG is capable of tight junctions modulation at the blood brain barrier, similar to its effect on the tight junctions at the intestinal epithelium. Both zidovudine and saquinavir are antiretroviral agents that exhibit limited distribution into the brain and are known to be substrates to efflux transporters expressed at the blood brain barrier. A three fold increase in the brain distribution of zidovudine in vivo in male FVB mice was achieved on treatment with a combination of the MRP1/2 selective inhibitor MK-571 and the non specific MRP inhibitor indomethacin. Similarly, a ten fold increase in the brain concentrations of saquinavir was achieved in vivo in male FVB mice on treatment with a combination of the P-gp inhibitor cyclosporin A and the CYP 3A4 inhibitor ketoconazole. Hence, both tight junction and efflux transport modulation hold considerable potential in enhancing the brain distribution of therapeutic agents.

Drug delivery to the brain presents many challenges to the pharmaceutical scientist in part due to limited drug transport across the blood brain barrier (BBB). This dissertation focused on evaluation of a novel protein, Zonula occludens toxin (Zot) and a P-glycoprotien (P-gp) modulator, itraconazole, to enhance brain drug delivery, and to investigate their potential for drug interactions in the kidney. Zot, a protein elaborated by vibrio cholerae, is known to modulate tight junctions in intestinal epithelial cell models and enhance oral bioavailability. We initially evaluated the ability of Zot to modulate tight junctions in a bovine brain microvessel endothelial cell (BBMEC) model. The results from this study indicate that Zot transiently and reversibly enhances paracellular transport of selected marker and chemotherapeutic compounds. Subsequent in vivo studies in rats were consistent with these findings, where Zot caused a four-fold enhancement in brain uptake of paclitaxel, a poorly permeable compound. Truncation studies on Zot isolated its active fragment (DeltaG). Preliminary studies in rats indicated that DeltaG was susceptible to metabolism by proteases. Use of the protease inhibitors (leupeptin and captopril) with DeltaG improved its activity as measured by brain uptake of sucrose. However, DeltaG did not significantly enhance brain uptake of paclitaxel in rats. Itraconazole, a known P-gp inhibitor has been safely used for many years to treat mycotic infections. We assessed the effect of itraconazole on the brain uptake of paclitaxel in rats. The dosing strategy applied in this study did not significantly enhance brain uptake of paclitaxel suggesting that either higher concentrations (>1 mug/ml) are required or itraconazole may not effectively inhibit P-gp at the BBB. Next, we evaluated the effect of DeltaG and itraconazole on the renal handling of a P-gp substrate, cimetidine, in vitro. In the kidney, P-gp is known to be located in high concentrations in mesangium and proximal tubule where active secretion of drugs occurs. Studies in the renal tubular epithelial cell line MDR1-MDCK indicated that itraconazole decreased P-gp mediated cimetidine efflux. However, DeltaG did not alter cimetidine transport in this model. To further evaluate the effect of itraconazole on renal P-gP in vivo, a Phase I pharmacokinetic drug interaction study was conducted in healthy volunteers. Cimetidine and iothalamate (GFR) clearances were measured at baseline and following itraconazole dosing (200 mg bid x 4 days). Here, itraconazole reduced the tubular secretory clearance of cimetidine by 25%, resulting in a 21% increase in the plasma cimetidine AUC0--4 hrs. This data suggests that the observed renal P-gp interaction may be clinically significant. In conclusion, we anticipate that this research will provide a basis for newer strategies to effectively deliver drugs to the brain and increase awareness of potential drug interactions when using such approaches.

Early Central Nervous System (CNS) discovery generated new pharmaceuticals based on two properties of the compound, lipophilicity and molecular size. Almost all CNS pharmaceuticals presently in clinical trials possess both of these criteria and are capable of crossing the blood-brain barrier (BBB). Currently, modern methods of drug discovery utilize high throughput screening methods that may select molecules that lack both properties and will not undergo transport across the BBB. With the advent of in vitro cell culture models that mimic the BBB in vivo, researchers are now able to evaluate drug interactions at the BBB and to elucidate mechanisms at both the cellular and molecular levels pertinent for drug delivery. The utilization of both in vitro models for relatively rapid screening of permeability and related transport mechanisms, and in vivo models to assess drug pharmacokinetic distribution to the CNS provides a powerful assessment of drug delivery across the BBB to the CNS. Enaminone esters in the carbomethoxy series have previously been evaluated and shown to possess potent oral anticonvulsant activity in the mouse and rat. However, preliminary studies assessing enaminone analogs did not show expected correlations between relevant physiochemical parameters such as lipophilicity and BBB permeability. Therefore, in vitro models were utilized to assess factors associated with drug transport. An in vitro model of the BBB, Bovine Brain Microvessel Endothelial cells (BBMECs) were isolated and used to evaluated permeability and cellular mechanisms influencing enaminone transport. Results demonstrated that a multidrug resistant protein (MDR) influences enaminone permeability at the BBB. Further elucidation of possible mechanisms influencing enaminone distribution and pharmacokinetics were performed in a genetically altered mouse model [mdr1a/ b (--/--)] deficient in the expression of P-glycoprotein. Results comparing the brain distribution and partition coefficients in knockout mdr1a/b (--/--) versus wild type (+/+) counterparts demonstrated a higher accumulation of enaminones in brain tissue of knockout mice. Pharmacokinetic analysis of the tissue disposition of enaminones additionally demonstrated that the lack of P-glycoprotein in the lung and liver influence drug disposition in knockout animals. Lastly, a physiological based pharmacokinetic model was developed and found to be predictive of DM5 [methyl 4-[(4-chlorophenyl) amino]-6-methyl-2-oxocyclohex-3-en-1-oate] tissue distribution in mdr1a/b (--/--) knockout and wild type mice.

The compound 4-aminopyridine (4AP) is a potassium channel blocker under investigation as a potential treatment for a number of neurological conditions. The data resulting from a recent clinical study suggested that the pharmacokinetics of the compound may differ between men and women, with women having significantly higher peak levels than men. The data also suggested that the CNS side effects seen with this compound (mental status changes, convulsions) tended to occur at plasma levels >= 100 ng/mL. The following animal studies were performed in an effort to (1) determine whether there is a gender difference in the pharmacokinetics of 4AP in rats, and (2) determine the relationship between the plasma concentration of 4AP and its CNS toxicity using fast Fourier transform analysis of the EEG and pharmacokinetic/pharmacodynamic (PK/PD) modeling techniques. For the first study, male or female rats were given 4AP either intravenously or orally, and blood samples taken up to 3 hours post-dose. The resulting plasma samples were analyzed for 4AP by a reverse-phase ion-pairing HPLC method developed in preparation for this work. The results of this study indicated that female rats have a significantly decreased mean clearance than male rats (12.0 vs 14 9 mL/min, p < 0.05). This difference is most likely due to differences in body weight. When 4AP was given orally, it was found that female rats had a significantly longer half-life of elimination, and a significantly decreased maximum concentration (Cmax) as compared with male rats. The difference in Cmax persisted when corrected for AUC, indicating a difference in GI absorption between male and female rats. The precise mechanism for this difference is not known, but may be related to female sex hormones. For the second study male rats were implanted with cortical screws for EEG collection. The EEGs of rats dosed with 4AP showed significant increases in high frequencies as compared to rats dosed with saline. The symmetrized percent increase in power from 40-50 Hz was selected as a surrogate marker of CNS toxicity. Using this measurement of drug effect, a PK/PD model of the CNS toxicity of 4AP was developed. Rats were implanted with cortical screws, given either 1 or 0.5 mg 4AP and both EEG data and blood samples were collected over 3 hours. The resulting data were modeled using the Hill equation. The model was validated using Monte Carlo simulation techniques. The model suggests that the CNS toxicity of 4AP is reduced at plasma concentrations below 100 ng/mL, which is similar to what was seen in humans, suggesting that the EEG may be a good cross-species indicator of neurotoxicity.

This work was undertaken to clarify the actions of physostigmine (Phy) and the bridged bis-aminoacridine molecules with various numbers of alkyl carbons, named acridine araphanes, on cholinergic receptors. Electrophysiological studies showed that, in addition to its ability to block cholinesterase, Phy blocks the nicotinic acetylcholine receptor ion-channel complex (AChR) in the open state in a voltage- and concentration-dependent manner. Phy activates the ion channel of the AChR. These results from Phy lead to the discovery of a second pathway of activation of the AChR-channel. Tetrahydro-aminoacridine (THA) and acridine araphanes appeared to be open channel blockers of the AChR. In the acridine araphane series, as the number of carbons (n) in the alkyl chain was decreased from 6 to 2, the ability of these compounds to desensitize the AChR was reduced. The length of the alkyl chain also affected the binding of these compounds to the agonist sites of nicotinic receptors. Functional studies on the atrium, ileum and vas deferens revealed that acridine araphanes antagonize the actions of agonists on several muscarinic receptor sub-types. Binding assays demonstrated that as n increased from 2 to 4, the affinities of the compounds for muscarinic receptors were enhanced by about 10-fold, such that the compound with n = 4 was the most potent in this series. Increasing n to 7 decreased the affinity about 30 to 50-fold. When n = 9 the affinity suddenly returned to a value similar to that of n = 2. Acridine araphanes had 100- to 300-fold higher affinity for muscarinic receptors than did mono-acridines. Protonation of acridine araphanes occurs at the N{dollar}\sb{lcub}\rm ar{rcub}{dollar} (nitrogen in acridine ring). The acridine rings are parallel when n = 2-4 before protonation. After protonation, the rings move apart due to a double bond formation between N{dollar}\sb{lcub}\rm c{rcub}{dollar} (nitrogen in alkyl chain) and C{dollar}\sb9{dollar} of each acridine ring. The acridine rings tend to become coplanar as n increases from 2 to 7 and the inter-N{dollar}\sb{lcub}\rm ar{rcub}{dollar} distance extends from 8.6 to 14 A. Beyond n = 9, the rings fold together. Phy alone can activate the AChR-channel. Phy and acridine derivatives including THA are open channel blockers. Acridine araphanes tend to interact with the ligand-binding site, non-competitive binding site and desensitization site of the AChR. The length of the alkyl chain affects the compounds' interaction with the AChR. The hydrophobic regions are optimal for the specific cholinergic antagonist potency of the acridine araphanes which appear as a new class of cholinergic probes. (Abstract shortened with permission of author.)