Exposure to candesartan during the first trimester of pregnancy in type 1 diabetes: experience from the placebo-controlled DIabetic REtinopathy Candesartan Trials.

AIMS/HYPOTHESIS: The teratogenic consequences of angiotensin-converting enzyme inhibitors angiotensin receptor blockers (ARBs) during the second and third trimesters of pregnancy are well described. However, the consequences of exposure during the first trimester are unclear, especially in diabetes. We report the experience from DIRECT (DIabetic REtinopathy and Candesartan Trials), three placebo-controlled studies designed to examine the effects of an ARB, candesartan, on diabetic retinopathy. METHODS: Over 4 years or longer, 178 normotensive women with type 1 diabetes (86 randomised to candesartan, 32 mg once daily, and 92 assigned to placebo) became pregnant (total of 208 pregnancies). RESULTS: More than half of patients were exposed to candesartan or placebo prior to or in early pregnancy, but all discontinued it at an estimated 8 weeks from the last menstrual period. Full-term pregnancies (51 vs 50), premature deliveries (21 vs 27), spontaneous miscarriages (12 vs 15), elective terminations (15 vs 14) and other outcomes (1 vs 2) were similar in the candesartan and placebo groups. There were two stillbirths and two 'sick babies' in the candesartan group, and one stillbirth, eight 'sick babies' and one cardiac malformation in the placebo group. CONCLUSIONS/INTERPRETATION: The risk for fetal consequences of ARBs in type 1 diabetes may not be high if exposure is clearly limited to the first trimester. Long-term studies in fertile women can be conducted with ARBs during pregnancy, provided investigators diligently stop their administration upon planning or detection of pregnancy. TRIAL REGISTRATION: ClinicalTrials.gov DIRECT-Prevent 1 NCT00252733; DIRECT-Protect 1 NCT00252720; DIRECT-Protect 2 NCT00252694. FUNDING: The study was funded jointly by AstraZeneca and Takeda.

Population pharmacodynamics in patients receiving tinzaparin for the prevention and treatment of deep vein thrombosis.

OBJECTIVES: We conducted population anticoagulant pharmacodynamic analysis for patients administered the low-molecular weight heparin tinzaparin. METHODS: Data from 425 patients (2,631 observations) who participated in 2 Phase III clinical studies were utilized in an analysis based on a pharmacodynamic structural model of anti-Xa activity using non-linear mixed effects modeling techniques. Anti-Xa activity from patients participating in a multicenter, randomized, double-blind clinical trial comparing intravenous once daily subcutaneous tinzaparin (175 IU/kg) with heparin for the treatment of deep vein thrombosis (DVT) was first examined using a 2-compartment model with first-order absorption and endogenous anti-Xa activity. Covariates included renal function, body weight, age, gender, race, obesity, concomitant administration of warfarin, and diabetes. RESULTS: The population estimates and 90% confidence intervals (CI) for oral clearance (CL) and apparent volume of distribution of the plasma compartment (Vc) were 0.0176 l/h/kg (CI = 0.012-0.023) and 0.098 l/kg (CI = 0.088 - 0.109), respectively. The elimination half-life was 3.9 h (CI = 2.5-5.2). These estimates are similar to findings in healthy volunteers. The inter-patient variability in clearance was related to plasma creatinine and percent above ideal body weight. Clearance decreased by 22% for patients with severe renal function impairment (creatinine clearance < 30 ml/min), and by about 25% in obese patients (BMI > 30 kg/m2). CONCLUSIONS: Changes of these magnitudes were not clinically important in pooled clinical trial safety and efficacy analyses. Body weight was not a significant covariate in the model supporting the observations in earlier well-defined trials, where tinzaparin was dosed on a weight basis (lU/kg). Clearance was not influenced by age, race or gender. The same model was applied to data obtained from a prospective, randomized, double-blind clinical trial comparing tinzaparin (4,500 IU) to enoxaparin (40 mg) once daily in patients undergoing total hip replacement. Model parameters were similar to those previously obtained supporting the extension of these results across dose and indication. Population analysis in patients with disease and heterogeneity indicated similar pharmacodynamics as in volunteers, supporting weight-based dosing and identified the dependence of clearance on obesity and severe renal function, although the magnitude of these effects are probably not clinically significant.

Pharmacodynamics of intravenous and subcutaneous tinzaparin and heparin in healthy volunteers.

The pharmacodynamics of i.v. and subcutaneous (s.c.) tinzaparin sodium compared with heparin in healthy volunteers were studied. A randomized, open-label, five-treatment, five-period-crossover study with a Latin square design was performed in 30 healthy men to estimate tinzaparin pharmacodynamics (anti-Xa and anti-IIa activities) after single-dose i.v. and s.c. administration, to evaluate absolute bioavailability, to determine the effect of a preservative (benzyl alcohol), to evaluate the dose-activity relationship, and to compare tinzaparin with unfractionated heparin. Treatments were (1) heparin 5,000 units s.c., (2) tinzaparin 4,500 anti-Xa IU without preservative s.c., (3) tinzaparin 4,500 anti-Xa IU without preservative i.v., (4) tinzaparin 12,250 anti-Xa IU with preservative s.c., and (5) tinzaparin 4,500 anti-Xa IU with preservative s.c. Blood samples for the measurement of anti-Xa and anti-IIa activities were drawn over 24 hours. Anti-Xa and anti-IIa activities were determined by chromogenic methods; data were analyzed by using a noncompartmental approach. The clearance of tinzaparin based on anti-Xa activity ranged from 1.14 to 2.04 L/hr. The volume of distribution was 3.1-5.0 L, suggesting that the molecular entities responsible for anti-Xa and anti-IIa activities are confined to the intravascular space. Mean peak anti-Xa activity occurred three to four hours after s.c. injection, independent of the dose. The mean half-life of anti-Xa activity after s.c. injection ranged from 3.41 to 4.13 hours and was independent of the dose. The mean absolute bioavailability of s.c. tinzaparin was 86.7%. Intersubject pharmacodynamic variability was low for tinzaparin compared with heparin. Benzyl alcohol did not affect tinzaparin pharmacodynamics. A clear dose-activity relationship was seen for the two fixed doses of tinzaparin (12,250 and 4,500 IU). Single doses of tinzaparin were safe and well tolerated after administration by either route. The anti-Xa profile of tinzaparin supports the pharmacodynamic superiority of low-molecular-weight heparins over standard i.v. heparin administration. This pharmacodynamic study in healthy volunteers indicates that s.c. tinzaparin sodium was well absorbed; the presence of a preservative, benzyl alcohol, did not affect the activity of tinzaparin; and tinzaparin activity is dose-related.

Anticoagulant pharmacodynamics of tinzaparin following 175 iu/kg subcutaneous administration to healthy volunteers.

Tinzaparin, a sodium salt of a low-molecular-weight heparin (LMWH) produced via heparinase digestion, is used for the treatment of deep vein thrombosis (DVT) and pulmonary embolism in conjunction with warfarin for the prevention of DVT in patients undergoing hip or knee replacement surgery, and as an anticoagulant in hemodialysis circuits. Its average molecular weight ranges between 5500 and 7500 daltons (Da); the percentage of chains with molecular weight lower than 2000 Da is not more than 10% in the marketed tinzaparin formulation. While this fraction is generally considered pharmacologically inactive, this has never been evaluated in vivo. The importance of the < 2000 Da fraction on the anticoagulant pharmacodynamics of tinzaparin assessed by anti-Xa and anti-IIa activity was studied in a two-way crossover trial. In this trial, 30 healthy volunteers received a single 175 IU/kg subcutaneous administration of tinzaparin containing approximately 3.5% of the < 2000 Da fraction and a tinzaparin-like LMWH containing 18.3% of the < 2000 Da fraction. The anti-Xa/anti-IIa ratios of the drug substances were comparable at 1.5 and 1.7 for tinzaparin and the tinzaparin-like LMWH, respectively. Both formulations were safe and well tolerated. Mean maximum plasma anti-Xa activity (A(max)) was approximately 0.818 IU/ml at 4 h following tinzaparin injection. Mean maximum plasma anti-IIa activity was 0.308 IU/ml at 5 h postdose. Intersubject variation was lower (< 18% for both anti-Xa and anti-IIa metrics) than in previous fixed-dose administration studies. There was no correlation between anti-Xa or anti-IIa AUC or A(max) and bodyweight in the present study supporting the weight-adjusted dosing regimen. Individual anti-Xa and anti-IIa profiles following the single 175 IU/kg subcutaneous administration of the tinzaparin-like LMWH were similar to that obtained with tinzaparin. Based on average equivalence criteria, the two LMWH preparations were determined to be bioequivalent using either anti-Xa or anti-IIa activity as biomarkers. The calculated intrasubject variabilities were low (< 14% for anti-Xa activity and < 18% for anti-IIa activity) yielding little evidence for a significant Subject x Formulation interaction. In summary, anti-Xa and anti-IIa activity following a single subcutaneous administration of tinzaparin 175 IU/kg to healthy volunteers yielded activity consistent with targeted therapeutic levels derived from previous trials in adult DVT patients. Weight-based dosing for the treatment of DVT appears rational based on the reduction in anti-Xa and anti-IIa variability consistent with the recommendation derived from earlier fixed-dose pharmacokinetic studies. Furthermore, differences in the percentage of molecules in the < 2000 Da molecular weight fraction of tinzaparin do not translate into differences in anti-Xa and anti-IIa activity in vivo.

Effect of the acyl-CoA:cholesterol acyltransferase inhibitor DuP 128 on cholesterol absorption and serum cholesterol in humans.

Intestinal cholesterol esterification by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is a presumed prerequisite for cholesterol absorption. We evaluated the effect of a potent, poorly absorbed ACAT inhibitor (DuP 128: N'-(2,4-difluorophenyl)-N-[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pe ntyl]- N-heptylurea) on cholesterol absorption in a randomized trial. Thirty subjects received DuP 128 for 7 weeks, 10 each at 900 mg per day, 1800 mg per day, and 3600 mg per day; six subjects received placebo; and nine subjects received 1 gm neomycin twice a day. Cholesterol absorption determinations used a continuous dual isotope 14C-cholesterol and 3H-beta sitosterol method. DuP 128 (pooled doses) induced at 14.4% +/- 11.4% reduction in cholesterol absorption (p < 0.05 versus placebo): 17.6% +/- 8.4% at 900 mg, 9.1% +/- 11.4% at 1800 mg, and 17.1% +/- 12.9% at 3600 mg. Neomycin induced a 26.4% +/- 10.7% reduction (p < 0.01). After 6 weeks, neomycin reduced serum total and low-density lipoprotein cholesterol by 22.4% +/- 9.2% and 24.0% +/- 11.6%, respectively (p < 0.01 versus placebo). DuP 128 induced reductions of 3.9% +/- 11% (difference not significant) and 4.95% +/- 14.3% (p = 0.05). ACAT inhibitors limit cholesterol absorption in humans; however, the magnitude of the effect, as exemplified by DuP 128, is small.

A microtiter plate assay for determining apolipoprotein E genotype and discovery of a rare allele.

Genotype determination using the solid-phase minisequencing method of Syvänen et al. (1990, 1993) has been adapted for use with fluorescein-labeled dideoxynucleotides (F-ddNTPs). PCR is performed using one biotinylated primer and one unbiotinylated primer. The biotinylated products are captured in streptavidin-coated microtiter wells. Following removal of nonbiotinylated strands with NaOH, the bound strands are hybridized with a primer adjacent to the polymorphic site being tested. Using T7 DNA polymerase, the primer is extended using one F-ddNTP in the presence of the other three unlabeled ddNTPs. Incorporation of the F-ddNTP is detected by binding antifluorescein antibody conjugated with alkaline phosphatase followed by incubation with a chromogenic substrate. This assay was used to determine APOE genotypes for 75 subjects. The APOE genotypes were also determined using a method involving the incorporation of mobility-shifting nucleotide analogs (Livak et al., 1992). Investigation of the one discrepancy between the two methods revealed that one subject carries a rare APOE allele that has a 3 bp deletion.