Formulation and biopharmaceutical evaluation of transdermal patch containing benztropine.

Benztropine (BZ) is a potent muscarinic receptor antagonist that has been used for the treatment of Parkinson disease. However, the oral administration of BZ is often limited because of its many dose-related side effects. In this study, BZ was formulated into drug-in adhesive (DIA) patches in an attempt to overcome these problems. The effects of the formulation factors including pressure-sensitive adhesive (PSA), enhancer, the loading amount of the drug and patch thickness on the skin permeation of the drug were evaluated using excised rat skin. The optimized patch contained 10% BZ in Duro-Tak 2525 as a PSA at a thickness of 100 microm. The pharmacokinetic characteristics of the optimized DIA patch were determined after the transdermal application to rabbits. The calculated relative bioavailability of BZ in the DIA patch was 54% compared to the oral administration of BZ mesylate. This suggests that the transdermal application of BZ in a DIA patch may be used for the treatment of Parkinson disease.

The novel N-substituted benztropine analog GA2-50 possesses pharmacokinetic and pharmacodynamic profiles favorable for a candidate substitute medication for cocaine abuse.

GA2-50 is a novel N-substituted benztropine analog with improved potency and selectivity for the dopamine transporter. The pharmacokinetic and pharmacodynamic properties of GA2-50 were characterized as a part of its preclinical evaluation as a substitute medication for cocaine abuse. In vitro transport and metabolism studies as well as pharmacokinetic studies in rats were conducted. Effect of GA2-50 on the extracelluar nucleus accumbens (NAc) dopamine levels and on cocaine's induced dopamine elevation was evaluated using intracerebral microdialysis. GA2-50 showed high transcellular permeability despite being a P-glycoprotein substrate. GA2-50 was a substrate of human CYP2D6, CYP2C19, CYP2E1, rat CYP2C11, CYP2D1, CYP3A1, and CYP1A2; with low intrinsic clearance values. In vivo, GA2-50 showed high brain uptake (R(i) approximately 10), large volume of distribution (V(ss) = 37 L/kg), and long elimination half-life (t((1/2)) = 19 h). GA2-50 resulted in 1.6- and 2.7-fold dopamine elevation at the 5 and 10 mg/kg i.v. doses. Dopamine elevation induced by GA2-50 was significantly reduced, slower and longer lasting than previously observed for cocaine. GA2-50 had no significant effect on cocaine's induced dopamine elevation upon simultaneous administration. Results from the present study indicate that GA2-50 possesses several attributes sought after for a substitute medication for cocaine abuse.

Population pharmacokinetics, brain distribution, and pharmacodynamics of 2nd generation dopamine transporter selective benztropine analogs developed as potential substitute therapeutics for treatment of cocaine abuse.

A second generation of N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]-tropanes (GA 1-69, JHW 005 and JHW 013) binds with high affinity to the dopamine transporter (DAT) and are highly selective toward DAT compared to muscarinic receptor binding (M1). The objective of this study was to characterize brain distribution, pharmacokinetics, and pharmacodynamics [extracellular brain dopamine (DA) levels] of three novel N-substituted benztropine (BZT) analogs in male Sprague-Dawley rats. The BZT analogs displayed a higher distribution (Vd = 8.69-34.3 vs. 0.9 L/kg) along with longer elimination (t l/2: 4.1-5.4 vs. 0.5 h) than previously reported for cocaine. Brain-to-plasma partition coefficients were 1.3-2.5 vs. 2.1 for cocaine. The effect of the BZT analogs on extracellular brain (DA) levels ranged from minimal effects (GA 1-69) to several fold elevation (approximately 850% of basal DA for JHW 013) at the highest dose evaluated. PK/PD analysis of exposure-response data resulted in lower IC50 values for the BZT analogs compared to cocaine indicating their higher potency to inhibit DA reuptake (0.1-0.3 vs. 0.7 mg/L). These BZT analogs possess significantly different PK and PD profiles as compared to cocaine suggesting that further evaluation as cocaine abuse therapeutics is warranted.

Applicability of the dopamine and rate hypotheses in explaining the differences in behavioral pharmacology of the chloro-benztropine analogs: studies conducted using intracerebral microdialysis and population pharmacodynamic modeling.

Previous studies indicated that the chloro-benztropine analogs differed significantly in their cocaine-like activity, which was not expected based on the similarity in their in vitro binding affinity and functional potency at the dopamine transporter (DAT). The present study was designed to extend the understanding of the involvement of both pharmacokinetic and pharmacodynamic factors in mediating the behavioral differences among these analogs. The pharmacokinetics of 3'-chloro-3alpha-(diphenylmethoxy)tropane (3'-Cl BZT), the analog showing a cocaine-like behavioral profile in rodents, was compared with previously reported pharmacokinetic characteristics of cocaine and 4',4''-dichloro-3alpha-(diphenylmethoxy)tropane (4',4''-diCl BZT), an analog totally devoid of cocaine-like actions. Microdialysis studies in rats were conducted to determine whether 3'-Cl and 4',4''-diCl BZT differed significantly in their effect on nucleus accumbens extracellular dopamine levels, with cocaine serving as a reference. A mechanistic model based on DAT association/dissociation kinetics was used to describe the time delay between the plasma concentrations of the chloro-analogs and their dopaminergic effects. 3'-Cl BZT had plasma elimination half-life of 1.9 h versus 0.5 and 21.1 h for cocaine and 4',4''-diCl BZT, respectively. 4',4''-diCl BZT increased the DA levels at a slower rate and to a significantly lower extent relative to 3'-Cl BZT that were, in turn, lower than cocaine. The duration of dopamine elevation was as follows: 4',4''-diCl BZT > 3'-Cl BZT > cocaine. The model indicated faster association and dissociation with DAT for 3'-Cl BZT relative to 4',4''-diCl BZT. The present results indicate that behavioral differences among the chloro-analogs may be explainable based on both the dopamine and rate hypotheses of drug abuse.

Investigation of the potential pharmacokinetic and pharmaco-dynamic drug interaction between AHN 1-055, a potent benztropine analog used for cocaine abuse, and cocaine after dosing in rats using intracerebral microdialysis.

PURPOSE: AHN 1-055, a benztropine (BZT) analog, binds with high affinity to the dopamine transporter (DAT), possesses behavioral, pharmacokinetic (PK) and brain microdialysate dopamine (DA) profiles distinct from cocaine. Accordingly, the objectives of this study were to evaluate the pharmacokinetics and dopamine release of AHN 1-055, in the presence of cocaine. METHODS: Male Sprague Dawley rats ( approximately 300 g) were administered 5 mg/kg of AHN 1-055 and cocaine i.v. and blood and brain samples were collected over 36 h. In addition, dialysis probes were stereotaxically implanted into the nucleus accumbens and extracellular fluid (ECF) DA levels were measured. PK and PD models were used to describe the relationship between the AHN 1-055, cocaine and DA levels. RESULTS: No significant (p< 0.05) differences were found in the PK parameters of AHN 1-055 alone (V(dss) = 18.7 l/kg, Cl = 1.8 l/h/kg and t(1/2) = 7.69 h) or AHN 1-055 with cocaine (V(dss)=17.4 l/kg, Cl = 1.9 l/h/kg and t(1/2) = 6.82 h). The brain-to-plasma (B/P) ratios (B/P(AHN 1-055) = 4.8 vs B/P(with cocaine) = 4.4) and half-lives (t(1/2(AHN 1-055)) = 6.2 h vs t(1/2(cocaine) = )5.6 h for AHN 1-055 alone and with cocaine were comparable. AHN 1-055 DA profiles were significantly different after co-administration with cocaine. There were no differences in the IC(50) for AHN 1-055, with cocaine, however, the IC(50) for cocaine was significantly reduced with AHN 1-055. CONCLUSIONS: The PK parameters of AHN 1-055 were not changed, however, the effect on DA levels was affected when cocaine was administered with AHNDA profile is affected when dosed with cocaine. This latter effect is a desirable attribute in the development of a medication as a potential substitute therapeutic medication for the treatment of cocaine abuse.

Pharmacodynamic assessment of the benztropine analogues AHN-1055 and AHN-2005 using intracerebral microdialysis to evaluate brain dopamine levels and pharmacokinetic/pharmacodynamic modeling.

PURPOSE: The benztropine (BZT) analogues bind with high affinity to the dopamine transporter (DAT) and demonstrate a behavioral and pharmacokinetic profile unlike that of cocaine. The development of a predictive pharmacokinetic/pharmacodynamic (PK/PD) model to characterize the concentration-effect relationship between the BZT analogues and brain dopamine (DA) levels is an important step in the evaluation of these compounds as potential cocaine abuse pharmacotherapies. Hence, the objective of this study was to mathematically characterize the PD of BZT analogues and cocaine, using appropriate PK/PD models. METHODS: Dialysis probes were stereotaxically implanted into the nucleus accumbens of Sprague-Dawley rats (275-300 g). Extracellular fluid (ECF) DA levels were measured after intravenous administration of the BZT analogues AHN-1055 and AHN-2005, as well as cocaine using high performance liquid chromatography-electrochemical detection (HPLC-ECD). PD models were used to describe the relationship between the BZT analogues or cocaine and brain microdialysate DA, and suitability was based on standard goodness-of-fit criteria. RESULTS: The BZT analogues produced a sustained increase in brain microdialysate DA levels in comparison to cocaine. The time of maximum concentration (T(max)) for brain microdialysate DA was 2 h for AHN-1055 and 1 h for AHN-2005 compared to a T(max) of 10 min for cocaine. The duration of brain microdialysate DA elevation was approximately 12-24 h for the BZTs in comparison to 1 h for cocaine. An indirect model with inhibition of loss of response and a sigmoid E(max) model best described the PK/PD for the BZT analogues and cocaine, respectively. The 50% of maximum inhibition (IC(50)) of the loss of DA was lower for AHN-2005 (226 +/- 27.5 ng/ml) compared to AHN-1055 (321 +/- 19.7 ng/ml). In addition, the EC(50) for cocaine was 215 +/- 11.2 ng/ml. CONCLUSIONS: The slow onset and long duration of BZT analogue-induced DA elevation may avoid the reinforcing effects and craving of cocaine. Further, the developed models will be useful in characterizing the PK/PD of other analogues and aid in the assessment of the therapeutic efficacy of the BZT analogues as substitute medications for cocaine abuse.

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine.

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine.

PET quantification of muscarinic cholinergic receptors with [N-11C-methyl]-benztropine and application to studies of propofol-induced unconsciousness in healthy human volunteers.

This work evaluated kinetic analysis methods for estimation of the receptor availability of the muscarinic receptor using dynamic positron emission tomography (PET) studies with [N-(11)C-methyl]-benztropine. The study also investigated the effect of propofol on central muscarinic receptor availability during general anesthesia. Six volunteers were scanned three times, once for baseline while awake, once during unconsciousness, and once after recovery to conscious level. An irreversible two-tissue compartment model was used to estimate the [N-(11)C-methyl]-benztropine specific binding rate constant k(3), a measure of muscarinic receptor availability. Two different estimation methods were used: 1) optimization with positivity constraints on all the parameters; 2) optimization with additional constraints determined from a one-tissue compartment fit to the cerebellum. In regions with low to middle muscarinic receptor density, the k(3) values from method (2) had lower standard errors than that for method (1) and gave a higher correlation with the density of muscarinic receptors measured in human tissue by in vitro studies (r(2) of 0.98 for Method 2 and r(2) of 0.72 for Method 1). But the k(3) values determined by Method 2 had higher errors for regions with high muscarinic receptor density compared to Method 1. For both methods the mean k(3) values during unconsciousness were generally lower than those during awake for most regions evaluated. Therefore, the method with additional constraints derived from the cerebellum (Method 2) was deemed superior for regions with low to middle muscarinic receptor density, while the method with positivity constraint is the better choice in the regions with high muscarinic receptor density. Our results also suggest the existence of propofol-related reductions in muscarinic receptor availability.

Evaluation of the blood-brain barrier transport, population pharmacokinetics, and brain distribution of benztropine analogs and cocaine using in vitro and in vivo techniques.

The N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropanes (AHN 2-003, AHN 1-055, AHN 2-005, and JHW 007) bind with high affinity to the dopamine transporter and inhibit dopamine uptake more potently than cocaine, but they demonstrate behavioral profiles in animal models of psychostimulant abuse that are unlike that of cocaine. The objective of this study was to characterize the in vitro permeability, brain distribution, and pharmacokinetics of the benztropine (BZT) analogs. Transport studies of cocaine and the BZT analogs (10-4 M) were conducted across bovine brain microvessel endothelial cells. Male Sprague-Dawley rats (approximately 300 g) were administered BZT analogs (10 mg/kg) or cocaine (5 mg/kg) via the tail vein. Blood and brain samples were collected over 36 h and assayed using UV-high-performance liquid chromatography. Transport of both AHN 1-055 (2.15 x 10-4 cm/s) and JHW 007 (2.83 x 10-4 cm/s) was higher (p < 0.05) than that of cocaine (1.63 x 10-4 cm/s). The volume of distribution (12.3-30.5 l/kg) of the analogs was significantly higher than cocaine (0.9 l/kg). The BZT analogs displayed a > or =8-fold higher elimination half-life (4.12-16.49 h) compared with cocaine (0.49 h). The brain-to-plasma partition coefficients were at least two-fold higher for the BZTs versus cocaine, except for AHN 2-003. The BZT analogs are highly permeable across the blood-brain barrier and possess a pharmacokinetic profile different from that of cocaine. These characteristics, in addition to their distinctive behavioral profiles, suggest that the BZT analogs may be promising candidates for the treatment of cocaine abuse.

Determination of the benztropine analog AHN-1055, a dopamine uptake inhibitor, in rat plasma and brain by high-performance liquid chromatography with ultraviolet absorbance detection.

N-Substituted 3alpha-[bis(4'-fluorophenyl)methoxy] tropanes represent a series of novel potential cocaine abuse therapeutics. AHN-1055, a member of this series, has been assessed to be the most suitable analog for pharmacokinetic studies. A sensitive and specific high-performance liquid chromatography method was developed to quantitate AHN-1055 in rat plasma and brain tissue. Reversed-phase chromatography with ultraviolet detection (lambda=220 nm) was utilized to quantitate the eluate. Plasma or brain tissue samples were prepared by liquid-liquid extraction using hexane, followed by evaporation, reconstitution in mobile phase, and injection onto an ABZ+plus column. AHN-1055 and oxprenolol (internal standard) eluted at approximately 9.9 and 5.01 min, respectively, without any interfering peaks. The calibration curves were found to be linear in the range of 25-10000 ng/ml for plasma and 50-5000 ng/g for brain (r2> or =0.999). The intra- and inter-day variabilities were < or =10% whereas the intra- and inter-day errors were < or =8.5%. Plasma and brain recoveries of AHN-1055 were 95 and 79%, respectively. Stability studies showed plasma quality control samples to be stable through at least three freeze-thaw cycles (error<3.5%), for at least 24 h when subjected to room temperature (error<3%) and for at least 30 h after loading the processed samples onto the autosampler (error<3%). AHN-1055 stock solution was found to be stable for at least 4 months when stored at 4 degrees C (error<6%). The validated method accurately quantified AHN-1055 in plasma and brain samples collected from a pharmacokinetic study consisting of an intravenous bolus in the tail vein of adult male Sprague-Dawley rats.

3'- and 4'-chloro-substituted analogs of benztropine: intravenous self-administration and in vitro radioligand binding studies in rhesus monkeys.

RATIONALE: The reinforcing effects of many psychomotor stimulants have been related to increased dopaminergic neurotransmission. Drugs that block dopamine (DA) uptake have generally been found to function as positive reinforcers. Benztropine (BZT) and several of its halogenated analogs have previously been characterized as potent DA-uptake inhibitors with behavioral profiles that indicate diminished psychomotor stimulant effects relative to cocaine. OBJECTIVES: The present experiments were designed to examine, in rhesus monkeys, the reinforcing effects of the DA-uptake inhibitor BZT and two chloro-analogs 3'-Cl-BZT and 4'-Cl-BZT, and to compare self-administration and binding profiles. METHODS: Four rhesus monkeys self-administered cocaine i.v. under a fixed-ratio 10 (FR10) schedule until stable responding was established. Saline, and various doses of cocaine, BZT, and the BZT analogs were then made available for self-administration. Binding of these compounds to monoaminergic and cholinergic sites in monkey brain were determined using standard radioligand binding techniques. RESULTS: Self-administration was maintained by both 3'-Cl-BZT and 4'-Cl-BZT, but not by BZT. Results suggested that 3'-Cl-BZT and 4'-Cl-BZT were weak positive reinforcers. BZT and analogs bound DA transporters (DAT) with affinities higher than that of cocaine and had affinity for muscarinic binding sites. CONCLUSIONS: Surprisingly, high affinity at DATs was associated with weak or no reinforcing effects. The mechanism(s) that may underlie this dissociation between DAT actions and reinforcing effects remains to be established. These data support the proposal that a lead for the discovery of a pharmacotherapeutic agent for cocaine abuse may come from this group of compounds.

Transdermal delivery of antiparkinsonian agent, benztropine. I. Effect of vehicles on skin permeation.

The influence of pH and various lipophilic and hydrophilic vehicles on the epidermal permeation of benztropine (BZ) free base and its mesylate salt were studied in vitro using the hairless mouse (HLM) and human cadaver (HC) skin membranes. The pH-partition behavior of BZ base (pK(a)=10) was examined using n-octanol and Britton-Robinson buffers over the pH range of 5-12. Unexpectedly, the ionized species of BZ yielded a high partition coefficient (log K(octanol/water)=2. 14), which was reflected by its relatively high skin permeability (P=1.6x10(-2)cm h(-1)). BZ base delivered from a lipophilic vehicle with a solubility parameter range of 7.1-10.3 (cal cm(3))(1/2) exhibited a significantly enhanced rate of permeation as compared to that attained from a hydrophilic vehicle of solubility parameter range between 12.5-23.4 (cal cm(3))(1/2). Among the neat solvents examined, a lipophilic carrier, isopropyl myristate (IPM) provided the most enhancing effect on the permeation of BZ base. In addition, the neat IPM carrier offered the maximum BZ base flux of 150 microg per cm(2) h(-1) across HC skin, which was approximately 16 times greater than the target delivery rate of BZ from a 10-cm(2) device. In comparison, BZ base exhibited a 2-60 times greater flux than BZ mesylate when delivered from the neat solvents. However, interestingly enough, the binary cosolvents consisting of IPM and short-chain alkanols such as ethanol (EtOH), isopropanol (iPrOH), and tertiary butanol (tBtOH), in particular a 2:8 combination, produced a marked synergistic enhancement of BZ flux from the mesylate salt, whereas a retarding effect was noticed for the permeation of BZ base. The enhancement potency for the BZ mesylate permeation increased linearly with the carbon number of the branched alcohols present in the binary mixtures. A tBtOH-IPM (2:8) combination produced the highest BZ flux from the mesylate salt, i.e. , 2016 mg per cm(2) h(-1), which was 100-fold greater than from water and 44-540-fold greater than the individual neat solvents, respectively. The observed permeation enhancement of BZ mesylate by the alkanol-IPM mixtures was probably as a result of a combination of decreasing barrier ability of the stratum corneum by the binary vehicles and moderately partitioning BZ mesylate through the viable epidermis/dermis.

Coadministration of fluvoxamine increases serum concentrations of haloperidol.

Four patients with chronic schizophrenia were treated with a combination of fluvoxamine, haloperidol, and benztropine. The combination significantly impaired performance on tests of delayed recall memory and attentional function. Haloperidol concentrations in serum were monitored in three patients and were robustly elevated by fluvoxamine.

Estimation of upper limits on human radiation absorbed doses from carbon-11-labeled compounds.

Radiation absorbed dose estimates for short-lived PET tracers are commonly based on biodistributions in rodents which (because of more rapid distribution and other species differences) may have limited relevance to humans. The initial purpose of this study was to estimate an intravenously injectable quantity of 11C which could not, on a priori grounds, exceed regulatory limits on radiation absorbed doses for individual organs. Upper limits on organ cumulative activities were estimated by assuming that 11C-labeled compounds are instantaneously distributed in the blood plasma, and then transferred solely and irreversibly to a single organ. The rate-constant (min-1) for each organ was taken to be its fractional cardiac output, since the plasma volume of 3 liters is recirculated each minute. The method was extended by using measured time courses of radioactivity in human arterial plasma available from previous PET studies with several 11C compounds in place of the assumption that the injected radioactivity was initially instantaneously distributed throughout the plasma. Calculations for 11C L-deprenyl, cogentin, cocaine, N-methylspiperone, putrescine and 2-deoxy-D-glucose, assuming transfer limited to a single organ, gave the kidneys rather than the thyroid as critical organ in each case. The upper-limit self-doses were 140, 210, 320, 360, 450 and 750 mrad/mCi, respectively, indicating that 34, 24, 15, 14 and 6.5 mCi, respectively, could be administered in a single PET study. These results suggest a strategy for human studies with 11C-labeled compounds: a preliminary study at the 3.5-mCi level would yield 11C arterial plasma data which could in turn be used to give a refined upper limit on radiation absorbed doses. For many 11C compounds, this strategy would demonstrate that sufficient radioactivity could be injected to give acceptable human PET images and would avoid the death of animals for biodistribution studies.