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1.  Introduction: A welcome to the first special animal health issue of AAPS PharmSci 
AAPS PharmSci  2002;4(4):189-192.
The goal of this special volume is to provide veterinary scientists with state-of-the art reviews in animal health and to inform human health scientists of the various challenges and collaborative opportunities associated with their animal health counterparts. The contributors are highly respected experts, providing invaluable insights into current issues and state-of-the-art advances within veterinary medicine.
PMCID: PMC2751328  PMID: 12647740
Veterinary medicine; harmonization
2.  Comparison of the formulation requirements of dosator and dosing disc automatic capsule filling machines 
AAPS PharmSci  2002;4(3):45-60.
The overall objective of this study was to provide ‘semi-quantitative’ or ‘rigorous’ definitions of the fluidity, lubricity and compactibility requirements of formulation for representative dosator and dosing disc capsule filling machines. To that end, model formulations were developed for those properties using Carr's compressibility index, ejection force, and plug breaking force at a specified compression force to gauge fluidity, lubricity, and compactibility, respectively. These formulations were each encapsulated on an Hofliger-Karg GKF-400 dosing disc machine and a Zanasi LZ-64 dosator machine. Each machine was instrumented to measure plug compression and ejection forces. The encapsulation process was evaluated for %CV of fill-weight, ejection force, plug breaking force and the dissolution of marker drugs incorporated in the formulations. The f2 metric was used to compare dissolution profiles. The results suggest: (1) formulations should meet different flow criteria for successful encapsulation on the two machines, (2) a relatively lower level of lubricant may be sufficient for the dosing disc machine, (3) a higher degree of formulation compactibility is needed for the dosator machine, and (4) transferring formulations between these machine types (same class, different subclass per FDA's SUPAC-IR/MR Manufacturing Equipment Addendum) could be challenging. In certain cases dissolution profiles for the same formulation filled on the two machines with equivalent compression force were different based on f2<50. Overall, the results of this study suggest a range of formulation characteristics appropriate for transferring formulations between these two types of machines.
PMCID: PMC2751356  PMID: 12423066
Capsules; Formulation; Flow; Compactibility; Lubrication; Filling Machines
3.  Chitosan and sodium alginate—Based bioadhesive vaginal tablets 
AAPS PharmSci  2002;4(4):224-230.
Metronidazole was formulated in mucoadhesive vaginal tablets by directly compressing the natural cationic polymer chitosan, loosely cross-linked with glutaraldehyde, together with sodium alginate with or ine cellulose (MCC). Sodium carboxymethylcellulose (CMC) was added to some of the formulations. The drug content in tablets was 20%. Drug dissolution rate studies from tablets were carried out in buffer pH 4.8 and distilled water. Swelling indices and adhesion forces were also measured for all formulations. The formula (FIII) containing 6% chitosan, 24% sodium alginate, 30% sodium CMC, and 20% MCC showed adequate release properties in both media and gave lower values of swelling index compared with the other examined formulations. FIII also proved to have good adhesion properties with minimum applied weights. Moreover, its release properties (% dissolution efficiency, DE) in buffer pH 4.8, as well as release mechanism (n values), were negligibly affected by aging. Thus, this formula may be considered a good candidate for vaginal mucoadhesive dosage forms.
PMCID: PMC2751333  PMID: 12646014
metronidazole; chitosan; sodium alginate; mucoadhesion; swelling; release study
4.  Evaluation of USP apparatus 3 for dissolution testing of immediate-release products 
AAPS PharmSci  2002;4(1):1-5.
We sought to evaluate whether U.S. Pharmacopeia (USP) apparatus 3 can be used as an alternative to USP apparatus 2 for dissolution testing of immediate-release (IR) dosage forms. Highly soluble drugs, metoprolol and ranitidine, and poorly soluble drugs, acyclovir and furosemide, were chosen as model drugs. The dissolution profiles of both innovator and generic IR products were determined using USP apparatus 2 at 50 rpm and apparatus 3 at 5, 15, and 25 dips per minute (dpm). The dissolution profiles from USP apparatus 3 were compared to those from USP apparatus 2 using the f2 similarity test. The dissolution profile from USP apparatus 3 generally depends on the agitation rate, with a faster agitation rate producing a faster dissolution rate. It was found that USP apparatus 3 at the extreme low end of the possible agitation range, such as 5 dpm, gave hydrodynamic conditions equivalent to USP apparatus 2 at 50 rpm. With appropriate agitation rate, USP apparatus 3 can produce similar dissolution profiles to USP apparatus 2 or distinguish dissolution characteristics for the IR products of metoprolol, ranitidine, and acyclovir. Incomplete dissolution was observed for the furosemide tablets using USP apparatus 3. Although it is primarily designed for the release testing of extended-release products, USP apparatus 3 may be used for the dissolution testing of IR products of highly soluble drugs, such as metoprolol and ranitidine, and some IR products of poorly soluble drugs, such as acyclovir. USP apparatus 3 offers the advantages of avoiding cone formation and mimicking the changes in physiochemical conditions and mechanical forces experienced by products in the gastrointestinal tract.
PMCID: PMC2751286  PMID: 12049485
Dissolution; USP apparatus 2; USP apparatus 3; Immediate-Release; and Product
5.  Pharmacodynamic modeling of chemotherapeutic effects: Application of a transit compartment model to characterize methotrexate effects in vitro 
AAPS PharmSci  2002;4(4):212-222.
The time course of chemotherapeutic effect is often delayed relative to the time course of chemotherapeutic exposure. In many cases, this delay is difficult to characterize mathematically through the use of standard pharmacodynamic models. In the present work, we investigated the relationship between methotrexate (MTX) exposure and the time course of MTX effects on tumor cell growth in culture. Two cancer cell lines, Ehrlich ascites cells and sarcoma 180 cells, were exposed for 24 hours to MTX concentrations that varied more than 700-fold (0.19–140 μg/mL). Viable cells were counted on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 20, 22, and 24 for Ehrlich ascites cells and on days 1, 2, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, and 21 for sarcoma 180 cells, through the use of a tetrazolium assay. Although MTX was removed 24 hours after application, cell numbers reached nadir values more than 100 hours after MTX exposure. Data from each cell line were fitted to 3 pharmacodynamic models of chemotherapeutic cell killing: a cell cycle phase-specific model, a phase-nonspecific model, and a transit compartment model (based on the general model recently reported by Mager and Jusko, Clin Pharmacol Ther. 70:210–216, 2001). The transit compartment model captured the data much more accurately than the standard pharmacodynamic models, with correlation coefficients ranging from 0.86 to 0.999. This report shows the successful application of a transit compartment model for characterization of the complex time course of chemotherapeutic effects; such models may be very useful in the development of optimization strategies for cancer chemotherapy.
PMCID: PMC2751331  PMID: 12646013
methotrexate; cell growth inhibition; modeling; chemotherapeutic effect; transit compartment model
6.  Role of plasma lipoproteins in modifying the toxic effects of water-insoluble drugs: Studies with cyclosporine A 
AAPS PharmSci  2002;4(4):95-101.
Lipoproteins are a heterogeneous population of macromolecular aggregates of lipids and proteins that are responsible for the transport of lipids through the vascular and extravascular fluids from their site of synthesis or absorption to peripheral tissues. Lipoproteins are involved in other biological processes as well, including coagulation and tissue repair, and serve as carriers of a number of hydrophobic compounds within the systemic circulation. It has been well documented that disease states (eg, AIDS, diabetes, cancer) significantly influence circulating lipoprotein content and composition. Therefore, it appears possible that changes in the lipoprotein profile would affect not only the ability of a compound to associate with lipoproteins but also the distribution of the compound within the lipoprotein subclasses. Such an effect could alter the pharmacokinetics and pharmacological action of the drug. This paper reviews the factors that influence the interaction of one model hydrophobic compound, cyclosporine A, with lipoproteins and the implications of altered plasma lipoprotein concentrations on the pharmacological behavior of this compound.
PMCID: PMC2751319  PMID: 12646002
7.  Novel system to investigate the effects of inhaled volume and rates of rise in simulated inspiratory air flow on fine particle output from a dry powder inhaler 
AAPS PharmSci  2002;4(2):7-12.
This study evaluated the effect of inhaled volume and simulated inspiratory flow rate ramps on fine particle output from dry powder inhalers (DPIs). A simple, robust system was developed to account for “rate of rise” (ramp) effects while maintaining a constant air flow through a multi-stage liquid impinger (MSLI), used for sizing the emitted particles. Ramps were programmed to reach 30 and 60 L/min over 100 milliseconds; 500 milliseconds; and 1, 2, and 3 seconds. Rotahaler was chosen as the test DPI. Testing was done with simulated inhalation volumes of 2 L and 4 L. Testing was also carried out using the USP apparatus 4. At 30 L/min, for a 2 L volume, the amount of drug exiting the device in fine particle fraction (FPF) increased from 2.33 μg to 6.04 μg from the 3-second ramp to the 100-millisecond ramp, with 11.64 μg in FPF for the USP (no ramp) method. At the same flow rate, for a 4 L volume, FPF increased from 2.23 μg to 8.45 μg, with 10.25 μg for the USP method. At 60 L/min, similar trends were observed. In general, at both flow rates, an increase in FPF was noted going from the shallowest to the steepest ramp. However, there were no significant differences in FPF when a 2 L inhaled volume was compared with a 4 L volume at each flow rate. Overall, these data suggest that the existing USP apparatus may overestimate FPF at flow rates lower than those recommended by the USP.
PMCID: PMC2751291  PMID: 12102620
Dry Powder Inhalers; Ramps In Vitro Testing; Fine Particle Output; Inhaled Volume
8.  Deposition of nanoparticles in the arterial vessel by porous balloon catheters: Localization by confocal laser scanning microscopy and transmission electron microscopy 
AAPS PharmSci  2002;4(4):206-211.
Restenosis remains the major limitation of percutaneous transluminal angloplasty (PTA) and stenting in the treatment of patients with atherosclerotic disease. Catheter-based local delivery of pharmacologic agents offers a potential therapeutic approach to reducing restenosis and minimizing undesirable systemic side effects. However, the intramural retention of liquid agents is low. Therefore, to achieve a sustained and regional release of the therapeutic agent it must be encapsulated in nanoparticle carrier systems. The purpose of this study was to investigate the size dependence of the penetration of nanoparticles after local delivery into the vessel wall of the aorta abdominalis of New Zealand white rabbits. Two milliliters of a 0.025% fluorescence-labeled polystyrene nanoparticle suspension with diameters ranging from 110 to 514 nm were infused at 2 atm and at constant PTA pressure of 8 atm into the aorta abdominalis. After the infused segments were removed, the location of nanoparticles was visualized using confocal laser scanning microscopy and transmission electron microscopy. The study demonstrates a size-dependent nanoparticle penetration into the intact vessel wall. While nanoparticles of about 100 and 200 nm were deposited in the inner regions of the vessel wall, 514-nm nanoparticles accumulated primarily at the luminal surgace of the aorta. The observations confirm that size plays a critical role in the distribution of particles in the arterial vessel wall. It is additionally influenced by the formation of pressure-induced infusion channels, as well as by the existence of anatomic barriers, such as plaques, at the luminal surface of the aorta or the connective elastic tissue.
PMCID: PMC2751330  PMID: 12646012
angioplasty; restenosis; local deposition; nanoparticles
9.  An in vitro examination of the impact of polyethylene glycol 400, pluronic P85, and vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate on P-glycoprotein efflux and enterocyte-based metabolism in excised rat intestine 
AAPS PharmSci  2002;4(4):193-205.
The potential inhibitory effects of 3 excipients (polyethylene glycol [PEG] 400, Pluronic P85, and vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate [TPGS]) on the P-glycoprotein (P-gp)-mediated efflux of digoxin (DIG) and cytochrome P450 3A (CYP3A)-mediated metabolism of verapamil (VRP) have been examined in an in vitro permeability model.
Experiments were conducted utilizing rat jejunal tissue mounted in diffusion chambers and included assessment of the serosal to mucosal (s to m) transport of DIG and the formation of norverapamil (NOR) during the mucosal to serosal transport of VRP, as measures of P-gp efflux and CYP3A metabolism, respectively.
The presence of PEG at 1%, 5%, and 20% (wt/vol) reduced both the s to m flux of DIG (by 47%, 57%, and 64%, respectively, when compared to control) and the metabolism of VRP (by 54%, 78%, and 100%) in a concentration-dependent manner. P85 (0.1% wt/vol) significantly reduced s to m DIG flux by 47% and inhibited VRP metabolism by 42%. TPGS had insignificant effects on both metabolism and efflux at a concentration of 0.01% (wt/vol). The P-gp inhibitory effects of PEG and P85 were evident regardless of whether the excipient was added to the mucosal side, the serosal side, or both sides of the tissue.
The current data suggest that inclusion of PEG and P85 as solubilizing agents during in vitro permeability assessment may have a significant impact on both drug metabolism and efflux processes. These compounds appear to exert their effects on P-gp primarily via direct transporter inhibition-or indirectly, through effects on buffer osmolarity, membrane fluidity, and/or mitochondrial toxicity and subsequent adenosine triphosphate (ATP) depletion.
PMCID: PMC2751329  PMID: 12646011
P-glycoprotein; CYP3A; intestinal; metabolism; permeability; PEG
10.  Interspecies considerations in the evaluation of human food safety for veterinary drugs 
AAPS PharmSci  2002;4(4):126-136.
Residues are composed of the parent drug and metabolites, and therefore interspecies comparisons must involve a consideration of comparative xenobiotic metabolism. The focus of this article will be the residue studies that are required to establish human food safety, and the interspecies pharmacokinetic differences and similarities that impact drug residues in animal- derived foods. To illustrate the factors that can complicate and assist these comparisons, 2 drugs will be examined in detail: ivermectin and fenbendazole. In addition, the activities of 2 US programs, the Food Animal Residue Avoidance Databank (FARAD) and the NRSP-7 (National Research Support Project Number 7) Minor Use Animal Drug Program will be presented, along with strategies that may be employed in the study of species differences.
PMCID: PMC2751323  PMID: 12646006
food safety; residues; comparative metabolism
11.  Evolving importance of biologics and novel delivery systems in the face of microbial resistance 
AAPS PharmSci  2002;4(4):119-125.
Methods to control infectious diseases in livestock are growing in importance. As the size of the average farm increases-for poultry, dairy and beef cattle, swine, and fish-the risk of rapid spread of infectious diseases increases as well. This increases the need for alternative methods of control of infectious agents. Improvements in specific immunogens, adjuvants, and delivery systems are needed to meet the demand for vaccines to ensure a healthy and safe meat supply. This article explores the challenges, trends, and recent advances in the control of infectious diseases through the use of biologics.
PMCID: PMC2751322  PMID: 12646005
biologics; vaccines; animal health; novel delivery systems
12.  Effects of obesity on the pharmacodynamics of nitroglycerin in conscious rats 
AAPS PharmSci  2002;4(4):80-88.
Literature reports have suggested that hemodynamic response toward organic nitrates may be reduced in obese patients, but this effect has not been studied. We compared the mean arterial pressure (MAP) responses toward single doses of nitroglycerin (NTG), 0.5–50μg) in conscious Zucker obese (ZOB), Zucker lean (ZL), and Sprague-Dawley (SD) rats. NTG tolerance development in these animal groups was separately examined. Rats received 1 and 10μg/min of NTG or vehicle infusion, and the maximal MAP response to an hourly 30μg NTG IVchallenge dose (CD) was measured. Steady-state NTG plasma concentrations were measured during 10μg/min NTG infusion. The Emax and ED50 values obtained were 33.9± 3.6 and 3.5±1.7μg for SD rats, 33.2±4.1 and 3.0±1.4μg for ZL rats, and 34.8±3.9 and 5.3±2.8μg for ZOB rats, respectively. No difference was found in the dose-response curves among these 3 groups (p>.05, 2-way ANOVA). Neither the dynamics of NTG tolerance development, nor the steady-state NTG plasma concentrations, were found to differ among these 3 animal groups. These results showed that ZOB rats are not more resistant to the hemodynamic effects of organic nitrates compared with their lean controls. Thus, the acute and chronic hemodynamic effects by the presence of obesity in a conscious animal model of genetic obesity.
PMCID: PMC2751317  PMID: 12646000
nitrate tolerance; Zucker; noninsulin dependent diabetes; mellitus; nitric oxide; obesity
13.  Pharmacogenomics: The implementation phase 
AAPS PharmSci  2002;4(2):1-6.
Pharmacogenomics makes use of genetic and genomic principles to facilitate drug discovery and development, and to improve drug therapy. Its goal is to attain optimal therapy for the individual patient. This article analyzes current trends in pharmacogenomics and asks how this new science affects drug development in the pharmaceutical industry and the clinical use of drugs.
PMCID: PMC2751290  PMID: 12102619
14.  Ocular tolerance of absorption enhancers in ophthalmic preparations 
AAPS PharmSci  2002;4(1):6-10.
The use of absorption promoters is a way to improve the bioavailability and therapeutic response of topically applied ophthalmic drugs. The ocular tolerance of 9 potential absorption promoters was investigated as well as the influence of the enhancers' concentration on the ocular tolerance. The substances tested were instillated repetitively (4 times per day, during 3 days, and once just before examination) as aqueous solutions onto rabbit corneas. Fluorescein dyeing enabled us to specifically mark corneal damage that was observed by confocal microscopy. The degree of corneal injury was assessed with an image-processing system that calculated the total fluorescent areas. Confocal microscopy results showed the relatively good tolerance of permeation enhancers like dimethyl sulfoxide (DMSO), decamethonium, edetate, glycocholate, and cholate in contrast to the poorly tolerated saponin and fusidate. Increasing the promoters' concentration led generally to an increase in corneal lesions.
PMCID: PMC2751287  PMID: 12049486
ocular absorption; permeation enhancers; confocal microscopy; toxicity
15.  The prediction of plasma and brain levels of 2,3,5,6-tetramethylpyrazine following transdermal application 
AAPS PharmSci  2002;4(4):243-250.
The purpose of this study was to construct a pharmacokinetic (PK) model and to determine PK parameters of 2,3,5,6-tetramethylpyrazine (TMP) after application of TMP transdermal delivery system. Data were obtained in Sprague-Dawley (SD) rats following a single dose of TMP transdermal delivery system. Blood samples were obtained at 0, 0.25, 0.5, 1, 2, 4, 6, 16, and 24 hours after the transdermal application. In the brain level study, 18 SD rats were divided into 6 groups. Three SD rats before and after transdermal application were culled and sacrificed at each of the following time intervals: 2, 4, 6, 16, and 24 hours after the TMP-TTS application. TMP concentrations in plasma and brain tissues were determined using high performance liquid chromatography and data were fitted using a zero-order absorption and a firstorder-elimination 3-compartment PK model. Fitted parameters included 2 volumes of distribution (V1, V2) and 2 elimination rate constants (k10, k20). The elimination half-life for TMP in plasma and brain was 26.5 and 31.2 minutes, respectively. The proposed PK model fit observed concentrations of TMP very well. This model is useful for predicting drug concentrations in plasma and brain and for assisting in the development of transdermal systems.
PMCID: PMC2751335  PMID: 12646016
tetramethylpyrazine; percutaneous absorption; transdermal drug delivery system; in vitro/in vivo; pharmacokinetic model
16.  A thermogravimetric analysis of non-polymeric pharmaceutical plasticizers: Kinetic analysis, method validation, and thermal stability evaluation 
AAPS PharmSci  2002;4(4):231-242.
Four non-polymeric plasticizers, propylene glycol, diethyl phthalate, triacetin, and glycerin have been subjected to rising temperature thermogravimetry for kinetic analysis and vaporization-based thermal stability evaluation. Since volatile loss of a substance is a function of its vapor pressure, the thermal stability of these plasticizers has been analyzed by generating vapor pressure curves using the Antoine and Langmuir equations. Unknown Antoine constants for the sample compounds, triacetin and glycerin have been derived by subjecting the vapor pressure curves to nonlinear regression. For the first time, the entire process of obtaining the unknown Antoine constants through thermogravimetry has been validated by developing an approach called the double reference method. Based on this method, it has been possible to show that this technique is accurate even for structurally diverse compounds. Kinetic analysis on the volatilization of compounds revealed a predominant zero order process. The activation energy values for vaporization of propylene glycol, diethyl phthalate, triacetin, and glycerin, as deduced from the Arrhenius plots, have been determined to be 55.80, 66.45, 65.12, and 67.54 kJ/mol, respectively. The enthalpies of vaporization of the compounds have been determined from the Clausius-Clapeyron plots. Rising temperature thermogravimetry coupled with nonlinear regression analysis has been shown to be an effective and rapid technique for accurately predicting the vapor pressure behavior and thermal stability evaluation of valatile compounds.
PMCID: PMC2751334  PMID: 12646015
plasticizers, vapor pressure plots; thermal stability; Antoine and Langmuir equations; thermogravimetry
17.  Introduction: Pharmaceutics and drug delivery 
AAPS PharmSci  2002;4(4):223.
PMCID: PMC2751332  PMID: 12647743
18.  Impact of antimicrobial resistance on regulatory policies in veterinary medicine: Status report 
AAPS PharmSci  2002;4(4):150-159.
Increasing resistance to antimicrobial agents is of growing concern to public health officials worldwide. The concern includes infections acquired in hospitals, community infections acquired in outpatient care settings, and resistant foodborne disease associated with drug use in food-producing animals. In the United States, a significant source of antimicrobial-resistant foodborne infections in humans is the acquisition of resistant bacteria originating from animals. The US Food and Drug Administration's (FDA's) goal in resolving the public health impact arising from the use of antimicrobial drugs in food-producing animals is to ensure that significant human antimicrobial therapies are not compromised or lost while providing for the safe use of antimicrobials in food animals. The FDA's approach to the problem is multipronged and innovative. The strategy includes revision of the pre-approval safety assessment for new animal drug applications, use of risk assessment to determine the human health effect resulting from the use of antimicrobials in food animals, robust monitoring for changes in susceptibilities among foodborne pathogens to drugs that are important both in human and veterinary medicine, research, and risk management.
PMCID: PMC2751326  PMID: 12646009
Antimicrobial resistance; animal drugs; foodborne disease; veterinary medicine
19.  The role of the clinical pharmacologist in animal health 
AAPS PharmSci  2002;4(4):145-149.
Like most scientific disciplines, pharmacology is replete with subspecialties. Certainly most scientists recognize the value of animal studies in drug development for human pharmaceuticals. However, animals as the target species also represent a major focus of investigation. According to recent estimates, in the United States for the year 2000, 98.1 million cattle, 59.8 million pigs, and 1.5 billion chickens existed. Added to that estimate were companion animals, including 4 million horses, 59 million cats, and 52.9 million dogs1,2. The estimate does not include the so?called “minor” species, such as 7 million sheep and 320 000 acres of freshwater fish production1,3. In most respects, the medical needs of these animals are addressed in a manner parallel to that of human medicine. One such parallel, with certain distinct differences from its human counterpart, is veterinary clinical pharmacology.
PMCID: PMC2751325  PMID: 12646008
veterinary clinical; pharmacology; species differences; industry; academia; regulatory
20.  Dissolution test development for complex veterinary dosage forms: Oral boluses 
AAPS PharmSci  2002;4(4):137-144.
Fundamental aspects of electrolyte chemistry were used to design an appropriate dissolution medium with the capacity to maintain sink conditions throughout the test. Dissolution of various bolus dosage forms was studied using USP Apparatus II at various stirring speeds. Complete dissolution of each drug in the designed medium was achieved, and there is evidence that such a dissolution test could be discriminating. This review details the development of potentially discriminating in vitro dissolution tests for veterinary boluses using USP Apparatus II and examines the potential role of such testing during product quality assessments, in the evaluation of postapproval manufacturing changes and for the establishment of the generic equivalence of veterinary products.
PMCID: PMC2751324  PMID: 12646007
dissolution; veterinary products; bolus
21.  Linking human and veterinary health: Trends, directions and initiatives 
AAPS PharmSci  2002;4(4):112-118.
The objective of this brief article is to provide an overview of some of the important harmonization efforts that are currently under way within the animal health community. Topics include: scientific networks and interdisciplinary communication: organizations that address animal-related public health concerns; the role of the veterinary pharmaceutical scientist within human health-oriented professional organizations; recent publications pertaining to veterinary pharmacology, pharmaceutics and therapeutics; and the role of global networking in veterinary product research and development.
PMCID: PMC2751321  PMID: 12646004
communication; globalization; veterinary medicine
22.  Quantitative comparison of functional screening by measuring intracellular Ca2+ with radioligand binding at recombinant human dopamine receptors 
AAPS PharmSci  2002;4(4):102-111.
The purpose of this study was to test whether screening at dopamine receptors performed with a recently described functional assay for G-protein coupled receptors (GPCRs) provides data that correlate significantly with radioligand binding data in the literature, thus possibly allowing researchers to replace radioligand binding with nonradioactive functional screening. Human dopamine receptors hD1 and hD2L (representing Gs [hD1] or G [hD2L] coupled GPCRs) were recombinantly expressed in human embryonic kidney (HEK293) cells. Cells were loaded with Oregon Green 488 BAPTA-1/AM and evenly distributed in 384 well plates. Seventeen test compounds were screened for agonistic activity by injection into the cell suspension and monitoringH of intracellular Ca2+ with a fluorescence microplate reader. Then, standard agonists (100nM SKF38393 for hD1, 30nM quinpirole for hD2L) were injected into wells preincubated with test compounds (screening for antagonism). Injection of various agonists resulted in a concentration-dependent increase in fluorescence. Further, preincubation of antagonists with dopamine receptor expressing cells inhibits concentration-dependent the agonist-induced increase in fluorescence. Calculated apparent functional Ki values correlate with radioligand binding data in the literature (r2=0.7796 for D1, r2=0.7743 for D2). The correlation between apparent functional K values and radioligand binding data for the 17 tested compounds suggests that screening of test compounds at dopamine receptors with the functional Ca2+ assay can replace radioligand binding studies. Furthermore, besides apparent K values, information about agonistic or antagonistic properties of a test compound can be obtained with the functional Ca2+ assay.
PMCID: PMC2751320  PMID: 12646003
dopamine receptor; functional screening; Ca2+ assay; microplate reader; radioligand binding
23.  Comparison of stepwise covariate model building strategies in population pharmacokinetic-pharmacodynamic analysis 
AAPS PharmSci  2002;4(4):68-79.
The aim of this study was to compare 2 stepwise covariate model-building strategies, frequently used in the analysis of pharmacokinetic-pharmacodynamic (PK-PD) data using nonlinear mixed-effects models, with respect to included covariates and predictive performance. In addition, the effects of stepwise regression on the estimated covariate coefficients wise regression on the estimated covariate coefficients were assessed. Using simulated and real PK data, covariate models were built applying (1) stepwise generalized additive models (GAM) for identifying potential covariates, followed by backward elimination in the computer program NONMEM, and (2) stepwise forward inclusion and backward elimination in NONMEM. Different versions of these procedures were tried (eg, treating different study occasions as separate individuals in the GAM, or fixing a part of the parameters when the NONMEM procedure was used). The final covariate models were compared, including their ability to predict a separate data set or their performance in cross-validation. The bias in the estimated coefficients (selection bias) was assessed. The model-building procedures performed similarly in the data sets explored. No major differences in the resulting covariate models were seen, and the predictive performances overlapped. Therefore, the choice of model-building procedure in these examples could be based on other aspects such as analyst-and computer-time efficiency. There was a tendency to selection bias in the estimates, although this was small relative to the overall variability in the estimates. The predictive performances of the stepwise models were also reasonably good. Thus, selection bias seems to be a minor problem in this typical PK covariate analysis.
PMCID: PMC2751316  PMID: 12645999
stepwise model building; covariate analysis; GAM; NONMEM; selection bias
24.  Which concentration of the inhibitor should be used to predict in vivo drug interactions from in vitro data? 
AAPS PharmSci  2002;4(4):53-60.
When the metabolism of a drug is competitively or noncompetitively inhibited by another drug, the degree of in vivo interaction can be evaluated from the [I]u/Ki ratio, where [I]u is the unbound concentration around the enzyme and Ki is the inhibition constant of the inhibitor. In the present study, we evaluated the metabolic inhibition potential of drugs known to be inhibitors or substrates of cytochrome P450 by estimating their [I]u/Ki ratio using literature data.
The maximum concentration of the inhibitor in the circulating blood ([I]max), its maximum unbound concentration in the circulating blood ([I]max,u), and its maximum unbound concentration at the inlet to the liver ([I]in,max,u) were used as [I]u, and the results were compared with each other. In order to calculate the [I]u/Ki ratios, the pharmacokinetic parameters of each drug were obtained from the literature, together with their reported Ki values determined in in vitro studies using human liver microsomes.
For most of the drugs with a calculated [I]in,max,u/Ki ratio less than 0.25, which applied to about half of the drugs investigated, no in vivo interactions had been reported or “no interaction” was reported in clinical studies. In contrast, the [I]max,u/Ki and [I]max/Ki ratio was calculated to be less than 0.25 for about 90% and 65% of the drugs, respectively, and more than a 1.25-fold increase was reported in the area under the concentration-time curve of the co-administered drug for about 30% of such drugs. These findings indicate that the possibility of underestimation of in vivo interactions (possibility of false-negative prediction) is greater when [I]max,u or [I]max values are used compared with using [I]in,max,u values.
PMCID: PMC2751314  PMID: 12645997
Drug interaction; metabolism; quantitative prediction
25.  Pharmacokinetics, mass balance, and tissue distribution of a novel DNA alkylating agent, VNP40101M, in rats 
AAPS PharmSci  2002;4(4):46-52.
VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2 methylamino)carbonyl] hydrazine), a novel DNA alkylating agent, is currently under clinical development for the treatment of cancer in Phase I clinical trials. This study investigated the pharmacokinetics, mass balance, and tissue distribution of [14C]-VNP40101M in rats following a single intravenous dose of 10 mg/kg. After 7 days, the total recovery of radioactivity was 85% for males and 79% for females. Most of the radioactivity was eliminated within 48 hours through urine (70%), with less excreted in feces (6%). Tissue contained relatively high radioactive residues with the highest concentrations in kidneys, liver, lung, and spleen. After 7 days, tissue still contained 9% of the dose. At both 5 minutes and 1 hour post-dose, brain contained relatively high radioactivity (5.9 and 3.3 μg equivalence/g and 50% and 30% of the blood concentration, respectively), suggesting that VNP40101M penetrated the blood-brain barrier. The elimination half-life of VNP40101M was approximately 20 minutes, the peak plasma concentration (Cmax) averaged 11.3 μg/mL, the volume of distribution (Vss) averaged 0.91 L/kg, and the total body clearance (CI) averaged 33.5 mL/min/kg. The metabolite profile in urine was complex, indicating VNP40101M was extensively metabolized. There were no apparent sex differences in pharmacokinetic parameters of VNP40101M in the rat.
PMCID: PMC2751313  PMID: 12645996
VNP40101M; Mass balance; Pharmacokinetics; Tissue distribution

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