Effects of a 5-Lipoxygenase–Activating Protein Inhibitor on Biomarkers Associated With Risk of Myocardial Infarction:  A Randomized Trial FREE

Myocardial infarction (MI) is now the leading cause of death in the world.^1- 2 We recently reported the identification of a gene variant that predisposes patients to MI.³ The gene was mapped with a genome-wide linkage scan without any assumption about the biological pathways contributing to the pathogenesis of MI. The gene encodes the 5-lipoxygenase–activating protein (FLAP) and its risk variant results in an almost 2-fold increased risk of MI.³ The leukotriene pathway, through FLAP, leads to the production of leukotriene B₄, which is one of the most potent chemokine mediators of arterial inflammation.^4- 5

We have shown³ that MI patients produce more leukotriene B₄ than controls, suggesting that the at-risk variant up-regulates the leukotriene pathway. By inhibiting the function of FLAP as a result of down-regulation of the leukotriene pathway, the risk of MI may be decreased in those predisposed to the variant.

This trial was designed to determine whether the genetic defect that predisposes patients to MI through the leukotriene pathway could be compensated by inhibiting FLAP. Of several available inhibitors of FLAP, we tested DG-031, which has been used in asthma clinical trials and has been shown to be safe and well tolerated.^6- 7 In this trial, we examined the influence of DG-031 on biomarkers that have been shown to correlate with risk of cardiovascular events.^8- 12 We also performed an open-label, 1-week follow-up study of 75 patients with the same baseline characteristics and eligibility criteria to further assess the effects of DG-031 on biomarkers that are associated with MI risk.

All patients had a history of MI and were carriers of specific MI-associated variants of FLAP and/or leukotriene A₄ hydrolase. The recruitment process included individuals who had previously participated in a population-based study of the genetics of MI.³ Selection of participants for the study was based on previous haplotype and genotype information from the databases of Decode Genetics Inc (Reykjavik, Iceland). Of the patients enrolled, 87% carried some at-risk variant of FLAP. The at-risk variants A3 and AF include 3 and 2 single nucleotide polymorphism (SNP) markers, respectively, of the at-risk haplotype HapA, which have been previously described.³ Apart from FLAP, we have also observed that allele A of the SNP SG12S25 in the leukotriene A₄ hydrolase gene is associated with MI (H.H., unpublished data, 2004). In particular, among the 13% of individuals who did not carry an at-risk haplotype of FLAP all but one carried the at-risk allele A.

Four SNP markers were genotyped to define the at-risk variants of the study participants (Table 1). The haplotypes carried by each individual were estimated using the Nested Models (NEMO) software program (version 1.01, Decode Genetics)¹³ and 902 in-house population controls. Among more than 900 patients identified as eligible by clinical and genotypic criteria, 640 provided written informed consent granting permission to use their genetic and medical data. The genotypes for the FLAP and leukotriene A₄ hydrolase genes were subsequently reconfirmed and carriers of at-risk variants in the FLAP gene, the leukotriene A₄ hydrolase gene, or both, were eligible for this study if they also met the other inclusion criteria but met none of the exclusion criteria (Box). The study protocol was approved by the National Bioethics Committee of Iceland, the Icelandic Data Protection Agency, and the Icelandic Medicines Control Agency. All patients who participated in the DG-031 trial gave their informed consent.

Inclusion Criteria

Exclusion Criteria

The first patient was enrolled on April 5, 2004, and the study follow-up phase was completed on September 14, 2004. All study participants lived in the metropolitan area or its neighboring townships of Reykjavik, Iceland. All participants were followed up at their outpatient or private clinics by the designated cardiologists from the Landspitali University Hospital (Reykjavik, Iceland). Participants had previously participated in a study on the genetics of MI.³ A medical history was completed, including detailed information on comorbidities, concomitant medications, and cardiovascular history, including current status.

All participants fasted prior to study visits and refrained from taking medications. Cardiologists examined the patients at each study visit and completed case report forms. All blood samples were collected and processed immediately after sampling. The chemistry analyses, hematologic tests, and urine tests were performed at the Icelandic Heart Association (study site), whereas the majority of biomarker measurements were performed at Decode Genetics. Lipoprotein phospholipase A₂ (Lp-PLA₂) was measured at Diadexus (San Francisco, Calif). All blood specimens used for the biomarker studies were processed at Decode Genetics within 2 hours of collection. The trial was double-blinded and treating physicians were blinded to randomization.

The FLAP inhibitor DG-031 used in this study (formerly known as BAY x 1005) was licensed from Bayer Health Care AG (Leverkusen, Germany). DG-031 acts by binding to the FLAP protein and prevents translocation of 5-lipoxygenase from the cytosol to the cell membrane. The drug competes for binding sites on the cell membrane with 5-lipoxygenase, and thus is a functional competitive inhibitor. This drug had been in development for asthma treatment and more than 20 studies have been conducted, including a small phase 3 study (Bay x 1005 study report No. PH-28207, unpublished data, 2005) that lasted for 12 months. The drug was found to be safe and well tolerated in asthma patients. It has not been marketed and there have been no previous studies on DG-031 for use in cardiovascular disease. Bayer had no involvement in the current study.

Patients who met the study eligibility criteria were enrolled and randomized into 6 treatment sequences (Figure 1); a sequence with DG-031 first and then placebo and a sequence with placebo first and then DG-031 for each of the 3 dosages: 250 mg/d (n = 64), 500 mg/d (n = 64), and 750 mg/d (n = 63). All patients received 3 tablets per day.

The treatment periods lasted for 4 weeks and were separated by a 2-week washout period. The placebo tablets were identical to the DG-031 tablets in shape, color, form, and taste. Participants were permitted to take other medications and adhere to the treatment plan prescribed by their cardiologist prior to enrollment. The crossover study design is summarized in Figure 1.

The primary objective was to determine whether the FLAP inhibitor DG-031 has a statistically significant effect compared with placebo on 1 or more biomarkers of MI risk (ionomycin-induced leukotriene B₄ and myeloperoxidase release by neutrophils ex vivo; myeloperoxidase, C-reactive protein [CRP], N-tyrosine, Lp-PLA₂, or serum amyloid A; or leukotriene E₄ in urine). The secondary objective was to determine whether the effect of DG-031 was dose-dependent. The tertiary objective was to assess other biomarkers, including those associated with inflammation. Evaluation of safety and tolerability of DG-031 was also performed.

All data were analyzed according to a preestablished statistical analysis plan and according to the intention-to-treat principle. Each dosage group in the study, as well as pooled sets (combining dosage levels), was considered for the primary analysis. The crossover design contains sets of patients who received treatment first and then placebo and those who received placebo first and then treatment. Biomarkers of MI risk were measured at the end of the treatment periods (visits 4 and 7) and were used as primary response variables. The difference between DG-031 and placebo treatment was the primary outcome and was assessed separately for each biomarker. Treatment effect was tested using a 2-sample t test on the period differences for suitably transformed response variables under an assumption of normality of the transformed data. We report treatment effect as half of the observed mean difference in the 2-sample t test with a 95% confidence interval (CI). Prespecified tests for carryover were performed and are reported separately from the results of the primary analysis. All hypotheses were tested at a 2-sided nominal significance level of .05 and P values based on t tests are reported. The R Statistical System (version 1.9.1, R Foundation for Statistical Computing, http://www.R-project.org) was used for all statistical computations.

For the primary efficacy end points in which the effects of the 2 highest dosage levels on 10 primary variables were studied, a randomization test was performed that corrects for multiple testing and ensures that the key results are not affected by distributional concerns. In particular, 1 million permutations of the patients into the different study tracks were performed, which generated a reference distribution for the maximum of the 10 t statistics of the biomarkers under the null hypothesis of no drug effect. By comparing the observed t statistic of each of the 10 biomarkers to this reference distribution, empirical P values were computed. It was considered likely that experimental manipulations would alter the effect of the drug on 2 of the primary markers; this was indeed the case and we report 8 of the 10 primary markers but all 10 are included in the randomization test.

To cancel out the potential interference from systematic seasonal effects (ie, seasonal allergies that could affect levels of inflammatory biomarkers), carryover effects were also studied in post hoc analyses with t tests that compare measurements of the groups who received DG-031 first and then placebo with the measurements of the groups who received placebo first and then DG-031. To estimate the effect of DG-031 at visit 3 for the groups who received DG-031 first and then placebo after the crossover, measurements taken at visit 2 were subtracted from measurements taken at visit 3. Similarly to estimate the effect of DG-031 at visit 4, measurements taken at visit 2 were subtracted from measurements taken at visit 4. To estimate the effects at visit 5, measurements taken at visit 2 were subtracted from measurements taken at visit 5. For estimating the effect at visit 6, we used the following formula: [(visit 6 − visit 2) + (visit 3 − visit 2)]. Note that visit 6 from the groups who received placebo first includes the effect of DG-031 after 2 weeks that cancels out the effect of DG-031 at visit 3 from the groups who received DG-031 first. Similarly to estimate the effect of DG-031 at visit 7, we used the formula: [(visit 7 − visit 2) + (visit 4 – visit 2)]. Only measurements from the groups who received the 2 higher DG-031 dosages first and then placebo were used for all visits. Measurements from all the groups who received placebo first were used for visits 3, 4, and 5 because each group received the same placebo until visit 5. However, similar to patients who received DG-031 first, only measurements from the patients who received placebo first and were assigned to the 2 higher DG-031 dosage groups were used for visits 6 and 7.

The sample size for this study was chosen so that each of the 3 dosage groups provided at least 80% power (2-sided P = .05) after a dropout rate of up to 5% to detect a relative reduction of 15% for a log-normal response variable, given that an assay for that variable has a coefficient of variation of 20% and the intraperson coefficient of variation is as high as 25%. Based on these assumptions, the recruitment target was 180 patients with randomization into 3 dosage groups.

A study flowchart is shown in Figure 1. At the enrollment visit, an independent study nurse who was blinded to the drug content dispensed medication kits according to a computer-generated randomization list. Randomization of study patients was stratified according to sex. A permuted block design with a block size of 12 was used. All biomarkers were transformed using a shifted log transform (transformed value is natural log of original value plus a shifting constant for each assay). Missing data were filled in using a simple last observation carried forward scheme. If no previous measurement existed, the next observation was carried back. Statistical outliers for data sets were used based on the interquartile range distance from the median prior to unblinding.

The SNPs genotyping within the FLAP and leukotriene A₄ hydrolase genes was performed using the SNP-based Taqman platform (Table 1).³ The enzyme-linked immunosorbent and mass spectrometry assays used are described elsewhere.^14- 31 Apart from measurements in plasma, leukotriene B₄ and myeloperoxidase were also measured in whole blood preparations ex vivo following ionomycin-activation of leukocytes. Both dose- and time-dependent stimulations were performed to determine the maximum leukotriene B₄ and myeloperoxidase output of the cells. Correction was made for white blood cell count because the amount of these mediators produced is proportional to the number of cells in a fixed volume. The adjustment on the log scale was based on a linear model, with coefficients determined empirically at time of blinded review. Several tertiary markers were also measured including: interleukin (IL) 6, IL-12p40, tumor necrosis factor α, matrix metalloproteinase 9, soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, platelet selectin, endothelial selectin, monocyte chemotactic protein 1, and oxidized low-density lipoprotein (LDL) cholesterol.

After completion of the double-blind study reported herein, an open-label randomized study was conducted in an independent cohort of patients with the same eligibility criteria to measure several of the same biomarkers. This study included 75 patients in 3 groups of equal size, each with a distinct dosing regimen of active drug, including the 750 mg/d dose. Each patient received DG-031 for 8 days; there was no placebo group. The primary objective of this study was to determine pharmacokinetic parameters of DG-031 in 3 different doses, as well as to assess the pharmacokinetic/pharmacodynamic relationship between DG-031, leukotriene B₄, and myeloperoxidase.

A total of 191 patients were enrolled and 172 completed all 8 visits. All 191 participants were analyzed as randomized in the intention-to-treat analysis using the last observation carried forward approach. There were no differences in the baseline characteristics of the study participants between the study sequences (Table 2) or in baseline values of the biomarker data (Table 3).

For the primary efficacy end point, 10 variables (Table 4) were considered in the pooled set of patients in the 500 mg/d and 750 mg/d groups (Table 5). The primary efficacy end point of the study was confirmed by showing that DG-031 reduces levels of leukotriene B₄ produced by ionomycin-activated neutrophils ex vivo for the pooled set of the 500 mg/d and 750 mg/d dosage groups by 17% (95% confidence interval [CI], 6%-27%; nominal P = .004), which is statistically significant after correction for multiple testing using the randomization procedure (corrected P = .02; Table 6). Leukotriene E₄ levels in urine were increased by 21% for the pooled set of dosage groups (95% CI, 13%-30%; P<.001 [corrected P<.001]).

For the 750 mg/d of DG-031 group, production of leukotriene B₄ was reduced by 26% (95% CI, 10%-39%; P = .003) and myeloperoxidase production was reduced by 12% (95% CI, 2%-21%; P = .02) (Table 4). Treatment with 750 mg/d of DG-031 also significantly reduced serum soluble intercellular adhesion molecule 1 (P = .03), but no effects were observed on other tertiary markers. Levels of Lp-PLA₂ increased by 9% (95% CI, 3%-16%; P = .006) in response to 750 mg/d of DG-031. Similarly LDL cholesterol increased by 8% (95% CI, 4%-12%; P<.001) with 750 mg/d of DG-031. In contrast, the effects of the 2 lower doses (250 mg/d and 500 mg/d) on Lp-PLA₂ were not significant.

In response to 750 mg/d of DG-031, leukotriene E₄ levels in urine increased by 27% (95% CI, 15%-40%; P<.001). Significant correlation was observed between the change of leukotriene B₄ and myeloperoxidase production (r = 0.62; 95% CI, 0.51-0.70; P<.001). This correlation was also observed when considering only those taking DG-031 when the range of these changes was larger. However, this correlation was also observed for these variables at baseline. The higher 2 DG-031 dosage groups had reductions in CRP by 16% (95% CI, −2% to 31%) at 2 weeks, although this is not significant (P = .07). Reductions in CRP were more pronounced at the end of the washout period (25%; 95% CI, 5%-40%; P = .02) and persisted for another 4 weeks thereafter. All effects on biomarkers were measured at the C_min plasma concentration of DG-031.

A test for the carryover effects from the treatment phase to the placebo phase was performed as a 2-sample t test on the differences between visits 2 and 5 for patients taking DG-031 first and then placebo. The cohort taking DG-031 consists of only patients receiving 500 mg/d and 750 mg/d of DG-031 during the trial and the placebo cohort includes all patients. The resulting P values and 95% CIs for the carryover effect are provided in Table 7 (data were not available for Lp-PLA₂ and N-tyrosine). No carryover effects were observed with leukotriene B₄ and myeloperoxidase. In contrast, significant carryover effects were observed for CRP and serum amyloid A, with a reduction in CRP that was significant at the 5% level (P = .02). Serum amyloid A showed similar carryover effects that were slightly below this significance level (P = .05).

Figure 2 shows the estimated mean effects on CRP and serum amyloid A for the patients receiving the 2 higher dosage levels of DG-031 during the first treatment period. Measurements from patients receiving placebo first also contribute to these estimates to cancel out potential seasonal effects. For visits 3 (after 2 weeks of receiving treatment) and 4 (after 4 weeks of receiving treatment), this constitutes the treatment effect, whereas the carryover effects appear between visits 5 to 7.

Among patients who took DG-031 during the first part of the study, the CRP levels decreased at visits 3 and 4, but not significantly so. The reduction became more pronounced and significant at visit 5, approximately 25% [95% CI, 5%-40%; P = .02). This reduction effect continued until visit 7, during the time the patients were receiving placebo. This reduction effect is part of the reason that the drug effect was not detected in the primary analysis, which did not take this scenario into account. The design of this trial does not have sufficient power for studying such effects, which are reflected in the large SEs in the estimates, particularly for visits 6 and 7. Even though measurements at visits 3 and 6 are not available for serum amyloid A, the observed changes of CRP and amyloid A between visits 2 and 5 are highly correlated (r = 0.68; 95% CI, 0.51-0.80; P<.001). Hence it appears that the drug has similar effects on both biomarkers.

Of the75 patients in the open-label follow-up study who received DG-031 for 8 days, 75 patients completed the study and provided complete data, including 25 patients in the 375 mg/d group and 50 patients in the 750 mg/d group. The 750 mg/d group was split into 2 subcohorts of 25 patients each (375 mg twice daily and 250 mg 3 times daily). Compared with baseline values, CRP levels after 8 days were reduced by 28% (95% CI, 5%-45%) in the 375 mg/d group (P = .02) and by 38% (95% CI, 9%-57%) in the 250 mg 3 times daily group (P = .02). However, no reduction (95% CI, −48% to 32%) was observed in the 375 mg twice daily group. A few individuals demonstrated increases in CRP levels; the lymphocyte counts were also elevated suggesting that viral processes may have been at play possibly increasing sampling variation. While the difference among the effects of the 3 dosage groups is not statistically significant (analysis of variance P = .16), the overall reduction in CRP levels for all 75 patients is significant (23%; 95% CI, 6%-37%; P = .01).

After 8 days of treatment, plasma myeloperoxidase levels were not significantly changed. However, at 6 hours after DG-031 administration on day 8, plasma myeloperoxidase levels were reduced by 3% (95% CI, −40% to 32%) in the 375 mg/d group (P = .88), by 21% (95% CI, −6% to 41%) in the 375 mg twice daily group (P = .11), and by 31% (95% CI, 16%-44%) in the 250 mg 3 times daily group (P<.001). For the full cohort of 75 patients, myeloperoxidase plasma levels measured 6 hours after administration were reduced by 20% (95% CI, 5%-32%; P = .01).

There was no difference in serious adverse events between the dosage groups. In particular, no difference was detected in levels of liver transaminases between the groups receiving active drug or placebo. The only symptom that was reported significantly more often while taking DG-031 was dizziness, which was experienced by 6 patients, compared with no reported events by patients while taking placebo (P = .03). This did not interfere with the daily activities of these patients.

This phase 2 clinical trial was designed to determine whether the effect of the variant of FLAP that predisposes individuals to MI³ could be neutralized, ie, could DG-031 be used to down-regulate FLAP in the leukotriene pathway as shown by a reduction in leukotriene B₄ levels. Furthermore, we examined the influence of DG-031 on biomarkers that have been correlated with risk of MI. Our results demonstrate that in patients with at-risk FLAP and leukotriene A₄ variants, DG-031 has a significant and dose-dependent effect at the cellular, whole blood, and urinary metabolite level: a 26% reduction in leukotriene B₄ production by activated neutrophils (95% CI, 10%-39%; P = .003); a 12% reduction in myeloperoxidase in whole blood (95% CI, 2%-21%; P = .02); and a 27% increase in urinary leukotriene E₄ level (95% CI, 15%-40%; P<.001). Following discontinuation of the FLAP inhibitor DG-031, there was evidence of a persistent effect on high-sensitivity CRP (P = .02) and serum amyloid A (P = .05).

Myocardial infarction occurs as a result of the development of atherosclerotic plaque fissure, erosion, or frank rupture.^32- 34 When such arterial injury manifests, a platelet-thrombosis response is mounted and occlusion of a main epicardial coronary artery leads to myocardial damage. In recent studies,^8- 11,35 many distinct mediators of arterial inflammation have been implicated, and such mediators have been shown to be independently associated with the risk of MI. Such cytokines and chemokines include CRP, serum amyloid A, myeloperoxidase, and intercellular adhesion molecule. Furthermore, at the arterial tissue level, 5-lipoxygenase and FLAP have been shown to track with more complex coronary arterial plaques, reflecting the contribution of leukotrienes to the arterial pathology.^36- 37 Both mRNA and protein expressions of 5-lipoxygenase, FLAP, and the leukotriene A₄ hydrolase genes are increased several fold in resident tissue cells and infiltrating cells of vascular plaques, including cells in the coronary arteries.³⁸ This is the part of the pathway that produces leukotriene B₄, whereas the molecules in the leukotriene C₄ synthase part of the pathway do not show evidence of expression changes. Moreover, unstable vascular plaques show abundant accumulation of activated neutrophils that produce leukotriene B₄, which in turn induces the expression and activation of myeloperoxidase (this is essentially limited to neutrophils themselves) that subsequently generates potent oxidants such as hydrochloric acid and also oxidizes LDL cholesterol and renders them proinflammatory in nature.^39- 41

Myeloperoxidase also inactivates protease inhibitors and consumes nitric oxide, all of which escalate the inflammatory response.⁴² Myeloperoxidase levels have been shown to be elevated in patients with diagnosed coronary artery disease and within atherosclerotic lesions that are prone to rupture.^40,43 Myeloperoxidase is also elevated in patients with chest pain and is predictive of subsequent cardiovascular events at 3 and 6 months.¹⁰ Collectively, the data from various biomarkers of arterial inflammation have reshaped our understanding of the disease process. Lifestyle factors such as weight loss and exercise, and medications including statins, have been shown to reduce the levels of biomarkers of MI risk.^11,44

In this study we show that DG-031 attenuates the capacity of activated neutrophils to generate leukotriene B₄ and myeloperoxidase. Moreover, the FLAP inhibitor DG-031 appears to have an effect on serum CRP and serum amyloid A levels that persists after the drug is discontinued. Our data suggest that DG-031 reduced serum levels of CRP by approximately 25% and amyloid A by 15%. Furthermore, results from the open-label follow-up study demonstrated similar effects on CRP from the highest dose of DG-031 after 1 week of treatment. These results are particularly intriguing because the reduction observed in CRP is in addition to the beneficial effects that may have been achieved with statins, which were taken by 85% of the study participants.^45- 46 While not anticipated in the design of the study, but recognized by systematic sampling, the CRP and serum amyloid A results may reflect the possibility of achieving arterial quiescence. For example, in studies using a brief 12-hour intervention of intravenous platelet glycoprotein IIb/IIIa blockade, there were benefits of a 20% mortality reduction still evident 3 years later.⁴⁷ This has been attributed to the ability to render a disease plaque less “vulnerable” to undergo spontaneous rupture.

The highest dose of DG-031 (750 mg/d) increased plasma Lp-PLA₂ levels by 9% with a corresponding increase in LDL cholesterol of 8%. While the clinical implications of elevated Lp-PLA₂ remain uncertain,⁴⁸ the parallel elevation in LDL cholesterol raises some concern which we have, at least in part, addressed in an animal model of atherosclerotic disease that examined the effects of DG-031 in apolipoprotein E mice that were fed a high-fat diet. The drug reduced atherogenesis in mice suggesting that it may provide protection against atherosclerosis (H.H., unpublished data, 2004). These data are consistent with a study showing that apolipoprotein E knockout mice with the low activity version of the 5-lipoxygenase are less vulnerable to atherogenesis in the aorta than those with the high-activity variant.³⁷ No effects were observed on LDL cholesterol by DG-031 in the follow-up study.

We observed a dose-dependent increase in urinary leukotriene E₄ levels in response to DG-031. While we expected that inhibition of FLAP would reduce urinary leukotriene E₄ levels (measurements of leukotriene C₄, leukotriene D₄, or leukotriene E₄ in plasma are unreliable and were not performed), the concentration of leukotriene E₄ in spot urine samples was increased in this study. While the clinical relevance of the cysteinyl arm of the pathway appears less relevant in relation to MI risk,³⁸ it is noteworthy that urinary leukotriene E₄ excretion showed no correlation with efficacy or pharmacokinetic parameters in asthma patients in previous studies of this FLAP inhibitor (Bay x 1005 study reports No. PH-26825; PH-26199; and MMRR-1296; unpublished data, 2005).

The FLAP inhibitor DG-031 was well tolerated and there were no serious adverse events. The assessment of the inhibitory effects of DG-031 on leukotriene B₄ and myeloperoxidase production was performed at steady state levels of DG-031 (C_min) and we would therefore expect these effects to be greater around the peak level (C_max) of the drug. The follow-up study addresses this relationship in more depth.

Among the primary markers studied, DG-031 significantly attenuated leukotriene B₄ and myeloperoxidase production in activated leukocytes and reduced CRP and serum amyloid A. In the follow-up study, plasma myeloperoxidase was also reduced when measured 6 hours after intake of DG-031 or shortly after maximum inhibition of leukotriene B₄ was reached. In contrast, plasma Lp-PLA₂ and urinary leukotriene E₄ secretion were significantly increased in response to DG-031. We did not observe any effects from DG-031 on N-tyrosine or on any of the tertiary markers studied, apart from the intercellular adhesion molecule (P = .02), suggesting that leukotriene B₄ may be more tightly linked to the regulation of myeloperoxidase and adhesion molecules, but plays little role in the regulation of cytokines such as IL-6 or tumor necrosis factor α or regulation of mediators more tightly regulated by these cytokines, such as matrix metalloproteinase 9 and monocyte chemotactic protein 1.

This study has several limitations. First, we did not collect data on clinical outcomes in this short-term study. A clinical outcome study will have to be completed to determine if the effects of DG-031 on biomarkers of MI risk will translate into decreased risk of MI. Second, the study was conducted at a single site in Iceland and is the first study, to our knowledge, to use at-risk variants in FLAP or leukotriene A₄ hydrolase for the common form of MI as inclusion criteria for a clinical trial. While this design has the potential to improve the power of the study, we do not expect that the effects of the drug observed in this study to be limited only to those patients who carried the specific gene variants used for selection herein. In particular, similar effects are expected for carriers of other at-risk variants in FLAP or in other genes in this pathway that have yet to be identified. Although these variants were uncovered in the founder population of Iceland, the variants of FLAP have now been replicated outside of Iceland.⁴⁹

In our previous study,³ the at-risk FLAP haplotype HapA was found to be carried by 29% of Icelandic MI patients compared with 17% of controls, indicating that the FLAP haplotype confers a risk of almost 2 (for comparison, elevated cholesterol in the top quartile confers an average risk of about 1.6 to MI). We have also replicated the FLAP haplotype in a large US cohort (H.H., unpublished data, 2004) showing that the haplotype is carried by 30% of US whites who have previously sustained a MI.

When taken together, the data from our MI gene-isolation study and the clinical trial reported herein show that DG-031 is a safe and well-tolerated drug that affects a biochemical defect that confers a relative risk of acute cardiovascular events, which is similar to or greater than that conferred by the top quartile of LDL cholesterol. Our data suggest that DG-031 reduces serum levels of CRP, serum amyloid A, and myeloperoxidase and these effects are in addition to any effects attributed to statins. Our hypothesis is that this will cause reduction in the risk of MI. To put these results in a historical context, we believe the promise of the beneficial role of DG-031 in cardiovascular disease may, at least in part, reflect that of statins in the late 1980s when it had been shown that they could lower LDL cholesterol but it had not been shown that lowering LDL cholesterol leads to a decrease in the risk of MI. A study examining clinical outcomes is in the planning stages to determine whether DG-031 does indeed affect the risk of MI.