In Vivo Evaluation of a Gastro-Resistant Enprotect® Capsule under Postprandial Conditions.

Ready-to-fill enteric hard capsule shells are an evolving field of oral drug and nutraceutical products. Lonza Capsugel® Enprotect® capsules were recently proven to provide reliable release in the small intestine after fasted intake, but robustness against postprandial intake needed to be proven. In this study, the capsules were administered to 16 healthy young subjects after intake of a light meal. The Enprotect® capsules were labelled with 5 mg black iron oxide and 25 mg 13C3-caffeine. Magnetic Resonance Imaging was used to identify the localization and visual dispersion of the capsule filling. The salivary appearance of caffeine was considered a second independent and sensitive marker for the initial release. Whereas the fasted gastric residence time of the capsules amounted to 43 ± 32 min, it was increased to 158 ± 36 min after postprandial intake. Therefore, the mean dispersion time according to MRI and the mean caffeine appearance time were increased to 196 ± 37 min and 189 ± 37 min, respectively. But, similar to fasted administration, no capsule disintegration or leakage was observed in the stomach and 38% of the capsules disintegrated in the jejunum and 62% in the ileum. The mean dispersion time after gastric emptying and the mean caffeine appearance time after gastric emptying amounted to 38 ± 21 min and 31 ± 17 min, respectively. Both did not relevantly change compared to the fasted intake. Only the absolute dispersion time and caffeine appearance were prolonged due to the increased gastric residence and no relevant influence of the light meal was observed on the disintegration or release behavior of Enprotect® capsules after gastric emptying. The capsules also showed robust enteric properties after postprandial administration.

The end of the beginning in understanding SLC22 polyspecificity.

SLC22 transporters involved in drug elimination and organ distribution are polyspecific. Now, the first cryo-EM structure of SLC22A3 (OCT3) is available from the Sitte and Korkhov groups. It paves the way for better understanding OCT3 function and for revealing the exact mechanisms conferring polyspecificity of the whole SLC22 family.

Comparing Salivary Caffeine Kinetics of 13C and 12C Caffeine for Gastric Emptying of 50 mL Water.

Gastric water emptying as a critical parameter for oral drug absorption can be investigated by several imaging techniques or by the interpretation of pharmacokinetics of appropriate substances. Recently introduced salivary caffeine kinetics is a valuable tool, but the required caffeine abstinence limits its applicability. To avoid the caffeine abstinence, stable isotope-labeled caffeine might be used, but the representability and transferability of kinetics for evaluation of gastric emptying must be demonstrated. Thus, salivary caffeine pharmacokinetics were compared for naturally occurring 12C-caffeine and 13C3-caffeine after the administration of water under fasting conditions in six healthy young subjects. For this purpose, an ice capsule containing the two caffeine species was administered with 50 mL tap water. Gastric water emptying was simultaneously quantified using magnetic resonance imaging (MRI). Gastric emptying of 50 mL of water could be successfully evaluated. The salivary caffeine kinetics of 13C3- and 12C-caffeine were nearly congruent and showed good linear correlations in all subjects, with a mean correlation coefficient of 0.96 in pooled data. Thus, the substitution of natural 12C caffeine with stable isotope-labeled 13C3-caffeine offers the opportunity for broader application of the salivary caffeine gastric emptying technique and increases the robustness of the method against environmental contamination with caffeine.

In Vivo Evaluation of a Gastro-Resistant HPMC-Based "Next Generation Enteric" Capsule.

Many orally dosed APIs are bioavailable only when formulated as an enteric dosage form to protect them from the harsh environment of the stomach. However, an enteric formulation is often accompanied with a higher development effort in the first place and the potential degradation of fragile APIs during the coating process. Ready-to-use enteric hard capsules would be an easily available alternative to test and develop APIs in enteric formulations, while decreasing the time and cost of process development. In this regard, Lonza Capsugel® Next Generation Enteric capsules offer a promising approach as functional capsules. The in vivo performance of these capsules was observed with two independent techniques (MRI and caffeine in saliva) in eight human volunteers. No disintegration or content release in the stomach was observed, even after highly variable individual gastric residence times (range 7.5 to 82.5 min), indicating the reliable enteric properties of these capsules. Seven capsules disintegrated in the distal part of the small intestine; one capsule showed an uncommonly fast intestinal transit (15 min) and disintegrated in the colon. The results for this latter capsule by MRI and caffeine appearance differed dramatically, whereas for all other capsules disintegrating in the small intestine, the results were very comparable, which highlights the necessity for reliable and complementary measurement methods. No correlation could be found between the gastric residence time and disintegration after gastric emptying, which confirms the robust enteric formulation of those capsules.

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

Effects of Genetic Polymorphism in CYP2D6, CYP2C19, and the Organic Cation Transporter OCT1 on Amitriptyline Pharmacokinetics in Healthy Volunteers and Depressive Disorder Patients.

The tricyclic antidepressant amitriptyline is frequently prescribed but its use is limited by its narrow therapeutic range and large variation in pharmacokinetics. Apart from interindividual differences in the activity of the metabolising enzymes cytochrome P450 (CYP) 2D6 and 2C19, genetic polymorphism of the hepatic influx transporter organic cation transporter 1 (OCT1) could be contributing to interindividual variation in pharmacokinetics. Here, the impact of OCT1 genetic variation on the pharmacokinetics of amitriptyline and its active metabolite nortriptyline was studied in vitro as well as in healthy volunteers and in depressive disorder patients. Amitriptyline and nortriptyline were found to inhibit OCT1 in recombinant cells with IC50 values of 28.6 and 40.4 µM. Thirty other antidepressant and neuroleptic drugs were also found to be moderate to strong OCT1 inhibitors with IC50 values in the micromolar range. However, in 35 healthy volunteers, preselected for their OCT1 genotypes, who received a single dose of 25 mg amitriptyline, no significant effects on amitriptyline and nortriptyline pharmacokinetics could be attributed to OCT1 genetic polymorphism. In contrast, the strong impact of the CYP2D6 genotype on amitriptyline and nortriptyline pharmacokinetics and of the CYP2C19 genotype on nortriptyline was confirmed. In addition, acylcarnitine derivatives were measured as endogenous biomarkers for OCT1 activity. The mean plasma concentrations of isobutyrylcarnitine and 2-methylbutyrylcarnitine were higher in participants with two active OCT1 alleles compared to those with zero OCT1 activity, further supporting their role as endogenous in vivo biomarkers for OCT1 activity. A moderate reduction in plasma isobutyrylcarnitine concentrations occurred at the time points at which amitriptyline plasma concentrations were the highest. In a second, independent study sample of 50 patients who underwent amitriptyline therapy of 75 mg twice daily, a significant trend of increasing amitriptyline plasma concentrations with decreasing OCT1 activity was observed (p = 0.018), while nortriptyline plasma concentrations were unaffected by the OCT1 genotype. Altogether, this comprehensive study showed that OCT1 activity does not appear to be a major factor determining amitriptyline and nortriptyline pharmacokinetics and that hepatic uptake occurs mainly through other mechanisms.

Inherited and Acquired Determinants of Hepatic CYP3A Activity in Humans.

Human CYP3A enzymes (including CYP3A4 and CYP4A5) metabolize about 40% of all drugs and numerous other environmental and endogenous substances. CYP3A activity is highly variable within and between humans. As a consequence, therapy with standard doses often results in too low or too high blood and tissue concentrations resulting in therapeutic failure or dose-related adverse reactions. It is an unanswered question how much of the big interindividual variation in CYP3A activity is caused by genetic or by environmental factors. This question can be answered by the twin study approach. Using midazolam as CYP3A probe drug, we studied 43 monozygotic and 14 dizygotic twins and measured midazolam and its metabolite 1-OH-midazolam. In addition, endogenous biomarkers of CYP3A activity, 4ß-OH-cholesterol and 6ß-OH-cortisol, were analyzed. Additive genetic effects accounted for only 15% of the variation in midazolam AUC, whereas 48% was attributed to common environmental factors. In contrast, 73, 56, and 31% of 1-OH-midazolam, 4ß-OH-cholesterol and 6ß-OH-cortisol variation was due to genetic effects. There was a low phenotypic correlation between the four CYP3A biomarkers. Only between midazolam and its 1-OH-metabolite, and between midazolam and 6ß-OH-cortisol we found significant bivariate genetic correlations. Midazolam AUC differed depending on the CYP3A4∗22 variant (p = 0.001) whereas plasma 4ß-OH-cholesterol was significantly lower in homozygous carriers of CYP3A5∗3 (p = 0.02). Apparently, non-genomic factors played a dominant role in the inter-individual variation of the CYP3A probe drug midazolam. A small intra-individual pharmacokinetic variation after repeated administration of midazolam was rated earlier as indication of high heritability of CYP3A activity, but according to present data that could also largely be due to constant environmental factors and/or heritability of liver blood flow. The higher heritabilities of 4ß-OH-cholesterol and of 1-OH-midazolam may deserve further research on the underlying factors beyond CYP3A genes. Clinical Trial Registration: ClinicalTrials.gov: NCT01845194 and EUDRA-CT: 2008-006223-31.

Oral Yohimbine as a New Probe Drug to Predict CYP2D6 Activity: Results of a Fixed-Sequence Phase I Trial.

OBJECTIVE: Yohimbine pharmacokinetics were determined after oral administration of a single oral dose of yohimbine 5 mg and a microdose of yohimbine 50 µg in relation to different cytochrome P450 (CYP) 2D6 genotypes. The CYP2D6 inhibitor paroxetine was used to investigate the influence on yohimbine pharmacokinetics. Microdosed midazolam was applied to evaluate a possible impact of yohimbine on CYP3A activity and the possibility of combining microdosed yohimbine and midazolam to simultaneously determine CYP2D6 and CYP3A activity. METHODS: In a fixed-sequence clinical trial, 16 healthy volunteers with a known CYP2D6 genotype [extensive (10), intermediate (2) and poor (4) metaboliser] received an oral dose of yohimbine 50 µg, yohimbine 5 mg at baseline and during paroxetine as a CYP2D6 inhibitor. Midazolam (30 µg) was co-administered to determine CYP3A activity at each occasion. Plasma concentrations of yohimbine, its main metabolite 11-OH-yohimbine, midazolam and paroxetine were quantified using validated liquid chromatography-tandem mass spectrometry assays. RESULTS: Pharmacokinetics of yohimbine were highly variable and a CYP2D6 genotype dependent clearance was observed. After yohimbine 5 mg, the clearance ranged from 25.3 to 15,864 mL/min and after yohimbine 50 µg, the clearance ranged from 39.6 to 38,822 mL/min. A more than fivefold reduction in clearance was caused by paroxetine in CYP2D6 extensive metabolisers, while the clearance in poor metabolisers was not affected. Yohimbine did not alter CYP3A activity as measured by microdosed midazolam. CONCLUSIONS: The pharmacokinetics of yohimbine were highly correlated with CYP2D6, which was further supported by the clearance inhibition caused by the CYP2D6 inhibitor paroxetine. With these data, yohimbine is proposed to be a suitable probe drug to predict CYP2D6 activity. In addition, the microdose can be used in combination with microdosed midazolam to simultaneously evaluate CYP2D6 and CYP3A activity without any interaction between the probe drugs and because the microdoses exert no pharmacological effects. CLINICAL TRIAL REGISTRATION: EudraCT2017-001801-34.

Comparison of In Vitro and In Vivo Results Using the GastroDuo and the Salivary Tracer Technique: Immediate Release Dosage Forms under Fasting Conditions.

The fasted state administration of immediate release (IR) dosage forms is often regarded as uncritical since physiological aspects seem to play a minor role for disintegration and drug release. However, recent in vivo studies in humans have highlighted that fasted state conditions are in fact highly dynamic. It was therefore the aim of this study to investigate the disintegration and drug release behavior of four different IR formulations of the probe drug caffeine under physiologically relevant conditions with the aid of the GastroDuo. One film-coated tablet and three different capsule formulations based on capsule shells either made from hard gelatin or hydroxypropylmethyl cellulose (HPMC) were tested in six different test programs. To evaluate the relevance of the data generated, the four IR formulations were also studied in a four-way cross-over study in 14 healthy volunteers by using the salivary tracer technique (STT). It could be shown that the IR formulations behaved differently in the in vitro test programs. Thereby, the simulated parameters affected the disintegration and dissolution behavior of the four IR formulations in different ways. Whereas drug release from the tablet started early and was barely affected by temperature, pH or motility, the different capsule formulations showed a longer lag time and were sensitive to specific parameters. However, once drug release was initiated, it typically progressed with a higher rate for the capsules compared to the tablet. Interestingly, the results obtained with the STT were not always in line with the in vitro data. This observation was due to the fact that the probability of the different test programs was not equal and that certain scenarios were rather unlikely to occur under the controlled and standardized conditions of clinical studies. Nonetheless, the in vitro data are still valuable as they allowed to discriminate between different formulations.

Effects of genetic polymorphisms on the OCT1 and OCT2-mediated uptake of ranitidine.

BACKGROUND: Ranitidine (Zantac®) is a H2-receptor antagonist commonly used for the treatment of acid-related gastrointestinal diseases. Ranitidine was reported to be a substrate of the organic cation transporters OCT1 and OCT2. The hepatic transporter OCT1 is highly genetically variable. Twelve major alleles confer partial or complete loss of OCT1 activity. The effects of these polymorphisms are highly substrate-specific and therefore difficult to predict. The renal transporter OCT2 has a common polymorphism, Ala270Ser, which was reported to affect OCT2 activity. AIM: In this study we analyzed the effects of genetic polymorphisms in OCT1 and OCT2 on the uptake of ranitidine and on its potency to inhibit uptake of other drugs. METHODS AND RESULTS: We characterized ranitidine uptake using HEK293 and CHO cells stably transfected to overexpress wild type OCT1, OCT2, or their naturally occurring allelic variants. Ranitidine was transported by wild-type OCT1 with a Km of 62.9 µM and a vmax of 1125 pmol/min/mg protein. Alleles OCT1*5, *6, *12, and *13 completely lacked ranitidine uptake. Alleles OCT1*2, *3, *4, and *10 had vmax values decreased by more than 50%. In contrast, OCT1*8 showed an increase of vmax by 25%. The effects of OCT1 alleles on ranitidine uptake strongly correlated with the effects on morphine uptake suggesting common interaction mechanisms of both drugs with OCT1. Ranitidine inhibited the OCT1-mediated uptake of metformin and morphine at clinically relevant concentrations. The inhibitory potency for morphine uptake was affected by the OCT1*2 allele. OCT2 showed only a limited uptake of ranitidine that was not significantly affected by the Ala270Ser polymorphism. CONCLUSIONS: We confirmed ranitidine as an OCT1 substrate and demonstrated that common genetic polymorphisms in OCT1 strongly affect ranitidine uptake and modulate ranitidine's potential to cause drug-drug interactions. The effects of the frequent OCT1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed.

Impact of mineralocorticoid receptor polymorphisms on urinary electrolyte excretion with and without diuretic drugs.

AIM: Polymorphisms in the mineralocorticoid receptor may affect urinary sodium and potassium excretion. We investigated polymorphisms in the MR gene in relation to urinary electrolyte excretion in two separate studies. PATIENTS & METHODS: The genotype-phenotype association was studied in healthy volunteers after single doses of bumetanide, furosemide, torsemide, hydrochlorothiazide, triamterene and after NaCl restriction. RESULTS: High potassium excretion under all conditions except torsemide, and high NaCl excretion after bumetanide and furosemide were associated with the A allele of the intron-3 polymorphism (rs3857080). This polymorphism explained 5-10% of the functional variation and in vitro, rs3857080 affected DNA binding of the transcription factor LHX4. CONCLUSION: rs3857080 may be a promising new candidate for research in cardiac and renal disorders and on antialdosteronergic drugs like spironolactone.

Amelogenin-based sex identification as a strategy to control the identity of DNA samples in genetic association studies.

Misassignment between DNA samples and clinical or epidemiological data may compromise the results of genetic association studies. Genotyping in replicates or controlling for Hardy-Weinberg equilibrium cannot identify misassignments caused by sample mix-ups. DNA-based sex identification (sex typing) is currently the best strategy to identify mix-ups. Here we review the available methods and present validated protocols for sex typing. The protocols are based on single-nucleotide differences between the human amelogenin genes, AMELX and AMELY, and are optimized for real-time PCR (TaqMan), primer-extension (SNaPshot) and PCR-RFLP genotyping platforms. In addition, we review the limitations of the sex-typing strategy, including a limited ability to identify single sample mix-ups, the dependence of the power of this approach on the sex distribution in the study population, and rare genetic conditions. Alternative strategies for mix-up identification and possible consequences of mix-up identification are also discussed.

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines.

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r(2)=0.47, P=0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r(2)=0.56, P=0.03 and r(2)=0.62, P=0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA. LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.

Common genetic variations in human brain-specific tryptophan hydroxylase-2 and response to antidepressant treatment.

OBJECTIVE: Genetic variability within the serotoninergic system may predict the response to antidepressant drugs. Several polymorphisms in the gene coding for the brain-specific tryptophan hydroxylase (TPH2) have been associated with susceptibility to psychiatric diseases. In this study, we analyzed the correlation between TPH2 polymorphisms and response to antidepressant drugs. METHODS: The study included 182 patients who received drug treatment for major depression. To assess the variability in the TPH2 gene, four single nucleotide polymorphisms (SNPs) tagging the common TPH2 haplotypes and six SNPs medically relevant according to data from other studies were analyzed in a multiplex single base primer extension reaction. RESULTS: Two SNPs, rs10897346 and rs1487278, were significantly associated with response to therapy (P=0.003 and 0.007). The rs10897346 variant showed the highest predictive values with carriers of null C alleles showing a 2.6-fold increased risk (95% confidence interval 1.4-4.8) for nonresponse compared with the others. The effect was found in all major types of antidepressant medications administered in this study and was statistically significant in the subgroup on selective serotonin reuptake inhibitors. Multiple logistic regression analyses confirmed the rs10879346 polymorphism as an independent predictor of the antidepressant response (odds ratio: 3.86; 1.75-8.55, P=0.0008). The therapeutically relevant variant rs10897346 is completely linked with the functional Pro312Pro polymorphism, which is known to affect TPH2 expression and may influence serotonin synthesis in the brain. CONCLUSION: The polymorphisms rs10897346 and Pro312Pro in the TPH2 gene might play an important role for TPH2 expression and antidepressant drug response.

Functional characterization of a -100_-102delAAG deletion-insertion polymorphism in the promoter region of the HTR3B gene.

OBJECTIVE: The HTR3B gene encodes the B-subunit of the type 3 serotonin receptor (5-HT3). A -100_-102delAAG deletion in the promoter region has been associated with poor response to antiemetic medication and susceptibility to bipolar affective disorders. The molecular mechanisms underlying these associations, however, remained unclear. METHODS: We performed electrophoretic mobility shift and luciferase reporter gene assays to elucidate the effect of this polymorphism on the HTR3B promoter activity in PC-12 and HEK293 cells. The reporter constructs carried a 2171 bp fragment of the native HTR3B promoter or 30 bp of the polymorphic locus in tandem triplication upstream of the thymidine kinase minimal promoter. RESULTS: Deletion mapping indicated that the sequence around the -100_-102delAAG polymorphism had significant promoter activity. Electrophoretic mobility shift assays indicated differential binding of nuclear proteins to the polymorphic DNA region with stronger binding to the insertion than to the deletion allele. The activity of the native promoter carrying the deletion allele was 25% higher in PC-12 (P=0.016) and 40% higher in HEK cells (P=0.016) compared with the respective insertion construct. Constructs carrying the deletion allele in tandem triplicates showed 43% (PC-12 cells, P=0.002) and 28% (HEK293 cells, P=0.015) higher activity than those carrying the insertion allele. The polymorphism was not linked with known amino acid substitutions in HTR3A and HTR3B. CONCLUSIONS: The -100_-102delAAG 3 bp deletion increases the HTR3B promoter activity in vitro. The consequences of this for the structure and the function of the resulting 5-HT3 receptors remain to be elucidated.

Tissue-specific alternative promoters of the serotonin receptor gene HTR3B in human brain and intestine.

The serotonin receptor type 3 is a pentameric ligand-gated ion channel regulating intestinal motility, nausea, and vomiting in humans. The HTR3B gene codes for the subunit B of this receptor. The HTR3B transcription start site is not unequivocally identified. In the present study we used transcription start site analyses, transcript-specific RT-PCR, and functional promoter analyses to identify the 5' structure of the HTR3B gene. According to these experiments, two alternative promoters control the expression of different HTR3B transcripts in the peripheral and central nervous system. The transcription start sites observed in the intestine corresponded to the current human genome annotation (NCBI Build 36.1, March 2006). The transcription start sites in the brain, however, were localized in a region about 4000 bp downstream. The brain transcripts lacked the coding first exon of the HTR3B structure published earlier but had an upstream-extended exon 2 containing a new potential translational start site. Reporter gene analyses showed significant promoter activity of the genomic region located 1560 bp upstream to 93 bp downstream of the brain-specific transcription start sites. This data suggests a different transcriptional regulation of the HTR3B gene in the peripheral and the central nervous system that leads to the expression of transcripts with variations in the 5' coding sequence. Further studies on the expression, structure and function of therefore expected tissue-specific 5-HT(3B) isoforms are required.

Application of genomewide SNP arrays for detection of simulated susceptibility loci.

The prospect of SNP-based genomewide association analysis has been extensively discussed, but practical experiences remain limited. We performed an association study using a recently developed array of 11,555 SNPs distributed throughout the human genome. A total of 104 DNA samples were hybridized to these chips with an average call rate of 97% (range 85.3-98.6%). The resulting genomewide scans were applied to distinguish between carriers and noncarriers of 37 test variants, used as surrogates for monogenic disease traits. The test variants were not contained in the chip and had been determined by other methods. Without adjustment for multiple testing, the procedure detected 24% of the test variants, but the positive predictive value was low (2%). Adjustment for multiple testing eliminated most false-positive associations, but the share of true positive associations decreased to 10-12%. We also simulated fine-mapping of susceptibility loci by restricting testing to the immediate neighborhood of test variants (+/-5 Mb). This increased the proportion of correctly identified test variants to 22-27%. Simulation of a bigenic inheritance reduced the sensitivity to 1%. Similarly adverse effect had reduction of allelic penetrance. In summary, we demonstrate the feasibility and considerable specificity of SNP array-based association studies to detect variants underlying monogenic, highly penetrant traits. The outcome is affected by allelic frequencies of chip SNPs, by the ratio between simulated "cases" and "controls," and by the degree of linkage disequilibrium. A major improvement is expected from raising the density of the SNP array.