Clinical pharmacology considerations for first-in-human clinical trials for enzyme replacement therapy.

Inborn errors of metabolism (IEM) such as lysosomal storage disorders (LSDs) are conditions caused by deficiency of one or more key enzymes, cofactors, or transporters involved in a specific metabolic pathway. Enzyme replacement therapy (ERT) provides an exogenous source of the affected enzyme and is one of the most effective treatment options for IEMs. In this paper, we review the first-in-human (FIH) protocols for ERT drug development programs supporting 20 Biologic License Applications (BLA) approved by the Center for Drug Evaluation and Research (CDER) at the US Food and Drug Administration (FDA) in the period of May 1994 to September 2023. We surveyed study design elements across these FIH protocols including study population, dosage form, dose selection, treatment duration, immunogenicity, biomarkers, and study follow-up. A total of 18 FIH trials from 20 BLAs were identified and of those, 72% (13/18) used single ascending dose (SAD) and/or multiple ascending dose (MAD) study design, 83% (15/18) had a primary objective of assessing the safety and tolerability, 72% (13/18) included clinical endpoint assessments, and 94% (17/18) included biomarker assessments as secondary or exploratory endpoints. Notably, the majority of ERT products tested the approved route of administration and the approved dose was tested in 83% (15/18) of FIH trials. At last, we offer considerations for the design of FIH studies.

Dose selection for biological enzyme replacement therapy indicated for inborn errors of metabolism.

This paper summarizes key features of the dose-finding strategies used in the development of 11 approved new molecular entities that are first-in-class enzyme replacement therapy (ERT), with a goal to gain insight into the dose exploration approaches to inform efficient dose-finding in future development of biological products for Inborn Errors of Metabolism (IEM). Dose exploration should preferably begin in in vitro studies, followed by testing multiple doses in an appropriate animal disease model, when available, which can provide important information for dose assessment in humans. Performing adequate dose-finding in early phase clinical studies in a well-defined study population, including pediatric subjects, is generally critical to inform dose selection for pivotal trials; alternatively, additional dose exploration can be incorporated as part of a pivotal trial. Two important considerations for successful dose selection include (1) identifying appropriate disease-specific endpoints, including pharmacodynamic (PD) end points and intermediate clinical end points or clinical end points, and (2) designing a study with adequate treatment durations for evaluating these end points. Appropriately selected PD biomarkers is useful for dose selection, and early development of these biomarkers can facilitate the overall clinical development program. Optimization of ERT doses, as well as evaluations of patient intrinsic factors and/or immune tolerance strategies may be necessary to overcome antibody responses or increase efficacy in IEM.

Impact of Organ Impairment on the Pharmacokinetics of Therapeutic Peptides and Proteins.

The kidneys and liver are major organs involved in eliminating small-molecule drugs from the body. Characterization of the effects of renal impairment (RI) and hepatic impairment (HI) on pharmacokinetics (PK) have informed dosing in patients with these organ impairments. However, the knowledge about the impact of organ impairment on therapeutic peptides and proteins is still evolving. In this study, we reviewed how often therapeutic peptides and proteins were assessed for the effect of RI and HI on PK, the findings, and the resulting labeling recommendations. RI effects were reported in labeling for 30 (57%) peptides and 98 (39%) proteins and HI effects for 20 (38%) peptides and 55 (22%) proteins. Dose adjustments were recommended for RI in 11 of the 30 (37%) peptides and 10 of the 98 (10%) proteins and for HI in 7 of the 20 (35%) peptides and 3 of the 55 (5%) proteins. Additional actionable labeling includes risk mitigation strategies; for example, some product labels have recommended avoid use or monitor toxicities in patients with HI. Over time, there is an increasing structural diversity of therapeutic peptides and proteins, including the use of non-natural amino acids and conjugation technologies, which suggests a potential need for reassessing the need to evaluate the effect of RI and HI. Herein, we discuss scientific considerations for weighing the risk of PK alteration due to RI or HI for peptide and protein products. We briefly discuss other organs that may affect the PK of peptides and proteins administered via other delivery routes.

Physiologically Based Pharmacokinetic Modeling of Metformin in Children and Adolescents With Obesity.

Childhood obesity continues to rise in the United States and, with it, the off-label use of metformin for weight loss. The influence of age and obesity on the drug's disposition and exposure has not previously been studied using a mechanistic framework. Here, an adult physiologically based pharmacokinetic (PBPK) model of metformin was scaled to pediatric populations without obesity, with overweight/obesity, and with severe obesity; a published virtual population of children and adolescents with obesity was leveraged during model evaluation. When the pediatric model was simulated in groups aged 10 to 18 years, oral clearance following 1000 mg of metformin was higher (≈1200 mL/min) in those with obesity and severe obesity compared to the groups without and with overweight (≈1000 mL/min). In addition, simulated area under the concentration-time curve in older children and adolescents with obesity and severe obesity was comparable to that in adults with a similar dose-exposure relationship. Overall, simulations using the pediatric PBPK model support the use of adult doses of metformin in older children and adolescents with obesity. Moreover, the virtual population of children and adolescents with obesity offers a valuable tool to facilitate development of pediatric PBPK models for studying populations with obesity and, in turn, contribute information to inform drug labeling in this special population.

Effects of SLC22A1 Polymorphisms on Metformin-Induced Reductions in Adiposity and Metformin Pharmacokinetics in Obese Children With Insulin Resistance.

Steady-state population pharmacokinetics of a noncommercial immediate-release metformin (hydrochloride) drug product were characterized in 28 severely obese children with insulin resistance. The concentration-time profiles with double peaks were well described by a 1-compartment model with 2 absorption sites. Mean population apparent clearance (CL/F) was 68.1 L/h, and mean apparent volume of distribution (V/F) was 28.8 L. Body weight was a covariate of CL/F and V/F. Estimated glomerular filtration rate was a significant covariate of CL/F (P < .001). SLC22A1 genotype did not significantly affect metformin pharmacokinetics. The response to 6 months of metformin treatment (HbA1c , homeostasis model assessment for insulin resistance, fasting insulin, and glucose changes) did not differ between SLC22A1 wild-type subjects and carriers of presumably low-activity SLC22A1 alleles. However, SLC22A1 variant carriers had smaller reductions in percentage of total trunk fat after metformin therapy, although the percentage reduction in trunk fat was small. The median % change in trunk fat was -2.20% (-9.00% to 0.900%) and -1.20% (-2.40% to 7.30%) for the SLC22A1 wild-type subjects and variant carriers, respectively. Future study is needed to evaluate the effects of SLC22A1 polymorphisms on metformin-mediated weight reduction in obese children.

Evaluating and Reporting the Immunogenicity Impacts for Biological Products--a Clinical Pharmacology Perspective.

Immunogenicity assessment is important for biological products due to potential impacts of immunogenicity on safety and efficacy. We reviewed the prescribing information and the FDA's clinical pharmacology review of 121 approved biological products for evaluating and reporting of immunogenicity data. Of the 121 products, 89% (n = 108) reported the incidence of immunogenicity and 49% (n = 59) reported immunogenicity impact on efficacy. However, only 26% (n = 31) reported whether the immunogenicity affected pharmacokinetics. A subset of 16 products reported effects of anti-drug antibodies (ADA) on both systemic clearance and efficacy; 8 of 16 products had increased systemic clearance coinciding with reduced efficacy, and 6 of 16 products had no changes in either clearance or efficacy. Factors contributing to infrequent reporting of the ADA effect on exposure and methods for determining the effect of ADA on exposure are summarized. Measuring ADA and drug concentrations concurrently over time enables the evaluation of ADA impact on pharmacokinetics. Within-subject comparison of concentration data (before vs. after ADA formation) is a useful alternative to between-subject (ADA+ vs. ADA-) comparison when sample size is limited or when the majority of subjects developed ADA. The biological complexity of immune responses presents challenges to quantifying the ADA impact on pharmacokinetics using model-based methods. Our findings support that pharmacokinetic exposure is more sensitive than efficacy endpoints for evaluating ADA effects. A decrease in drug concentration due to formation of ADA during treatment can serve as an early indicator for potential reduced efficacy occurring at a later time.

Increased consumption and vasodilatory effect of nitrite during exercise.

This study investigated the effects of aerobic-to-anaerobic exercise on nitrite stores in the human circulation and evaluated the effects of systemic nitrite infusion on aerobic and anaerobic exercise capacity and hemodynamics. Six healthy volunteers were randomized to receive sodium nitrite or saline for 70 min in two separate occasions in an exercise study. Subjects cycled on an upright electronically braked cycle ergometer 30 min into the infusion according to a ramp protocol designed to attain exhaustion in 10 min. They were allowed to recover for 30 min thereafter. The changes of whole blood nitrite concentrations over the 70-min study period were analyzed by pharmacokinetic modeling. Longitudinal measurements of hemodynamic and clinical variables were analyzed by fitting nonparametric regression spline models. During exercise, nitrite consumption/elimination rate was increased by ∼137%. Cardiac output (CO), mean arterial pressure (MAP), and pulmonary artery pressure (PAP) were increased, but smaller elevation of MAP and larger increases of CO and PAP were found during nitrite infusion compared with placebo control. The higher CO and lower MAP during nitrite infusion were likely attributed to vasodilation and a trend toward decrease in systemic vascular resistance. In contrast, there were no significant changes in mean pulmonary artery pressures and pulmonary vascular resistance. These findings, together with the increased consumption of nitrite and production of iron-nitrosyl-hemoglobin during exercise, support the notion of nitrite conversion to release NO resulting in systemic vasodilatation. However, at the dosing used in this protocol achieving micromolar plasma concentrations of nitrite, exercise capacity was not enhanced, as opposed to other reports using lower dosing.

Topical sodium nitrite for chronic leg ulcers in patients with sickle cell anaemia: a phase 1 dose-finding safety and tolerability trial.

BACKGROUND: Well-tolerated and effective treatments are needed for chronic leg ulcers in sickle cell anaemia. Topical sodium nitrite, a known nitric oxide donor, enhances blood flow in ulcers and has known bacteriostatic effects. We aimed to assess the safety, tolerability, and pharmacokinetics of topical sodium nitrite in patients with sickle cell disease and chronic leg ulcers. METHODS: We enrolled adult patients from an ambulatory clinic at the National Institutes of Health (Bethesda, MD, USA) with sickle cell anaemia with leg ulcers (with a surface area of 2.5-100 cm2) persisting for at least 4 weeks into a safety and tolerability phase 1 dose-escalation trial of topical sodium nitrite. Increasing concentrations of sodium nitrite cream were applied twice weekly for 4 weeks to one ulcer per patient at five dose levels (0.5%, 1%, 1.5%, 1.8%, and 2%). The primary endpoints were safety and tolerability, with secondary endpoints of pharmacokinetics, blood flow, and wound healing. Pain relief was analysed post hoc. Endpoints were analysed over time for the whole study population and according to dose level. This study is registered with ClinicalTrials.gov, number NCT01316796. FINDINGS: Between April 4, 2011, and March 19, 2013, we enrolled 18 adult patients with sickle cell anaemia and leg ulcers into our trial. We assigned three patients into each cohort, and each cohort was treated with a different concentration of sodium nitrite cream (cohort 1: 0.5%, cohort 2: 1.0%, cohort 3: 1.5%, and cohort 4: 2.0%). Patients were not enrolled into the next cohort dose until we were able to establish that no dose-limiting toxicities were observed. An additional six patients were enrolled to cohort 3a: 1.8%, after two patients in cohort 4 had asymptomatic drops in diastolic blood pressure. No grade 3-4 adverse events were observed, and there were no serious adverse events or dose-limiting side-effects. Pharmacokinetic analysis showed that systemic absorption of sodium nitrite was very low. Application of topical sodium nitrite was associated with a significant increase in peri-wound cutaneous blood flow measured by laser speckle contrast imaging (p=0.0002), corroborated by increased peri-wound skin temperature by infrared thermography (p=0.0119). We recorded a dose-dependent decrease in leg ulcer size (p=0.0012) and pain (p<0.0001). Ulcers healed completely in three patients who received the highest concentrations of topical sodium nitrite (the 1.8% and 2% cream). In our post-hoc analysis of pain, brief pain inventory scores improved in pain severity (p=0.0048) and pain interference (p=0.0013). INTERPRETATION: Our results indicate that topical sodium nitrite 2% cream is suitable for additional clinical trials in adults with sickle cell anaemia to promote healing of leg ulcers. FUNDING: National Heart, Lung and Blood Institute Division of Intramural Research (National Institutes of Health).

Development of a chiral micellar electrokinetic chromatography-tandem mass spectrometry assay for simultaneous analysis of warfarin and hydroxywarfarin metabolites: application to the analysis of patients serum samples.

The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE-MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-L,L-leucylvalinate (poly-L,L-SULV) as a chiral pseudophase in MEKC-MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45 min. This is in comparison to 100 min required for separation of the same mixture with packed column CEC-MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC-MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000 ng/mL for R- and S-warfarin, 5 to 1000 ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000 ng/mL for R and S-4'-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC-MS/MS were found to be at levels of 2 and 5 ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC-MS/MS method to accurately quantitate with high sensitivity.

Associations of ABCB1 and IL-10 genetic polymorphisms with sirolimus-induced dyslipidemia in renal transplant recipients.

BACKGROUND: Hyperlipidemia is a common adverse effect of sirolimus (SRL). We previously showed significant associations of ABCB1 3435C>T and IL-10 -1082G>A with log-transformed SRL dose-adjusted weighted-normalized trough. We further examined to see whether these polymorphisms were also associated with SRL-induced dyslipidemia. METHODS: Genotyping was performed for ABCB1 1236C>T, 2677 G>T/A, and 3435C>T; CYP3A4 -392A>G; CYP3A5 6986A>G and 14690G>A; IL-10 -1082G>A; TNF -308G>A; and ApoE ε2, ε3, and ε4 alleles. The longitudinal changes of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels after SRL treatment before statin therapy were analyzed by a linear mixed-effects model, with adjustments for selected covariates for each lipid. RESULTS: Under the dominant genetic model, ABCB1 3435C>T was associated with TC (P=0.0001) and LDL-C (P<0.0001) values after SRL administration. Mean TC and LDL-C levels were 26.9 and 24.9 mg/dL higher, respectively, in ABCB1 3435T carriers than 3435CC homozygotes at an average SRL trough concentration of 4 ng/mL without concomitant medication. ABCB1 1236C>T under the recessive model and IL-10 -1082G>A under the dominant model were associated with log-transformed TG values (P=0.0051 and 0.0436, respectively). Mean TG value was 25.1% higher in ABCB1 1236TT homozygotes compared with ABCB1 1236C carriers and was 12.4% higher in IL-10 -1082AA homozygotes than -1082G carriers. CONCLUSIONS: ABCB1 polymorphisms were found to be associated with lipid responses to SRL treatment, confirming the role of ABCB1 gene in SRL pharmacokinetics and pharmacodynamics. Further studies are necessary to define the role of ABCB1 and IL-10 polymorphisms on SRL-induced dyslipidemia in renal transplantation.

Associations of ABCB1 3435C>T and IL-10-1082G>A polymorphisms with long-term sirolimus dose requirements in renal transplant patients.

BACKGROUNDS: Sirolimus (SRL) absorption and metabolism are affected by p-glycoprotein-mediated transport and CYP3A enzyme activity, which are further under the influences of cytokine concentrations. This retrospective study determined the associations of adenosine triphosphate-binding cassette, subfamily B, member 1 (ABCB1) 1236C>T, 2677 G>T/A, and 3435C>T, cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4) -392A>G, cytochrome P450, family 3, subfamily A, polypeptide 5 (CYP3A5) 6986A>G and 14690G>A, interleukin (IL)-10 -1082G>A, and tumor necrosis factor (TNF) -308G>A polymorphisms with SRL dose-adjusted, weight-normalized trough concentrations (C/D) at 7 days, and at 1, 3, 6, and 12 months after initiation of SRL. METHODS: Genotypes for 86 renal transplant patients who received SRL-based maintenance immunosuppressive therapy were determined using polymerase chain reaction followed by chip-based mass spectrometry. The changes of log-transformed C/D over the days posttransplantation were analyzed using a linear mixed-effects model, with adjustments for body mass index and weight-normalized doses of tacrolimus, prednisone, clotrimazole, and statins. RESULTS: ABCB1 3435C>T and IL-10 -1082G>A were significantly associated with log C/D (P=0.0016 and 0.0394, respectively). Mean SRL C/D was 48% higher in patients with ABCB1 3435CT/TT genotype than those with 3435CC genotype, and was 24% higher in IL-10 -1082GG compared with -1082AG/AA. CONCLUSIONS: ABCB1 3435C>T and IL-10 -1082G>A were significantly associated with long-term SRL dose requirements. Genetics can play a significant role in SRL dosing and may be useful in therapeutic monitoring of SRL in renal transplantation. Future replication studies are needed to confirm these associations.

Characterization of erythrocytic uptake and release and disposition pathways of nitrite, nitrate, methemoglobin, and iron-nitrosyl hemoglobin in the human circulation.

Nitrite-hemoglobin reactions have been studied extensively in vitro, but there is a lack of information on the kinetics of nitrite and its metabolites in humans. In this study, we developed a nine-compartment physiological pharmacokinetic model to describe the in vivo erythrocytic uptake and release and disposition pathways of nitrite, nitrate, methemoglobin, and iron-nitrosyl hemoglobin in the human circulation. Our model revealed that nitrite entered erythrocytes rapidly with a rate constant of 0.256 min(-1) (i.e., half-life = 2.71 min). The formation of iron-nitrosyl hemoglobin from nitrite, which involves the reduction of nitrite by deoxyhemoglobin to generate nitric oxide (NO) and reaction of NO with deoxyhemoglobin to form iron-nitrosyl hemoglobin, occurred rapidly as well (k = 2.02 min(-1); half-life = 0.343 min = 21 s). The disposition kinetics of methemoglobin was complex. Nitrate formation occurred primarily in erythrocytes through the nitrite-oxyhemoglobin reaction and was higher when nitrite was administered intra-arterially than intravenously. Nitrate reduction was an insignificant metabolic pathway. This study is the first to comprehensively evaluate the kinetics of nitrite and its metabolites in humans and provides unique insights into the rapid equilibrium of nitrite into erythrocytes and conversion to NO in the red cell, which is kinetically associated with vasodilation.

Evaluation of tacrolimus abbreviated area-under-the-curve monitoring in renal transplant patients who are potentially at risk for adverse events.

In a cohort of 32 renal transplant patients who are potentially at risk for adverse events, we compared tacrolimus (TAC) abbreviated AUC values calculated by a method developed in Asians (AUCw) with those derived for Caucasians (AUCa). The relationships between TAC trough (C0), abbreviated AUC, and biopsy results were also assessed. Forty-eight AUCs and 15 associated biopsies were evaluated. For AUCs obtained only from Caucasian patients, median AUCw value was lower than that of AUCa (104 vs. 115 ng×h/mL, n=29, p<0.0001). AUCs obtained from the two methods for all patients correlated with C0 (rs>0.72, n=48, p<0.0001). Median AUCw (72.9 vs. 174 ng×h/mL, p=0.043) and AUCa (81.0 vs. 203 ng×h/mL, p=0.043) were lower in patients experiencing biopsy-proven acute rejection (AR) than those with normal histology. C0 tended to be lower in biopsies showing AR>6 months post-transplant (5.80 vs. 11.0 ng/mL, p=0.110). Thus, lower abbreviated AUCs were obtained for Caucasians using a method developed in Asians. C0 correlated well with abbreviated AUCs. Lower C0 and AUC appeared to be associated with biopsy-proven AR>6 months post-transplant. Further prospective evaluation of TAC AUC and C0 monitoring in a larger cohort of patients is warranted.

Nitrite anion provides potent cytoprotective and antiapoptotic effects as adjunctive therapy to reperfusion for acute myocardial infarction.

BACKGROUND: Accumulating evidence suggests that the ubiquitous anion nitrite (NO2-) is a physiological signaling molecule, with roles in intravascular endocrine nitric oxide transport, hypoxic vasodilation, signaling, and cytoprotection. Thus, nitrite could enhance the efficacy of reperfusion therapy for acute myocardial infarction. The specific aims of this study were (1) to assess the efficacy of nitrite in reducing necrosis and apoptosis in canine myocardial infarction and (2) to determine the relative role of nitrite versus chemical intermediates, such as S-nitrosothiols. METHODS AND RESULTS: We evaluated infarct size, microvascular perfusion, and left ventricular function by histopathology, microspheres, and magnetic resonance imaging in 27 canines subjected to 120 minutes of coronary artery occlusion. This was a blinded, prospective study comparing a saline control group (n=9) with intravenous nitrite during the last 60 minutes of ischemia (n=9) and during the last 5 minutes of ischemia (n=9). In saline-treated control animals, 70+/-10% of the area at risk was infarcted compared with 23+/-5% in animals treated with a 60-minute nitrite infusion. Remarkably, a nitrite infusion in the last 5 minutes of ischemia also limited the extent of infarction (36+/-8% of area at risk). Nitrite improved microvascular perfusion, reduced apoptosis, and improved contractile function. S-Nitrosothiol and iron-nitrosyl-protein adducts did not accumulate in the 5-minute nitrite infusion, suggesting that nitrite is the bioactive intravascular nitric oxide species accounting for cardioprotection. CONCLUSIONS: Nitrite has significant potential as adjunctive therapy to enhance the efficacy of reperfusion therapy for acute myocardial infarction.

Variant CYP2C9 alleles and warfarin concentrations in patients receiving low-dose versus average-dose warfarin therapy.

This study compared the frequency of variant cytochrome P450 2C9 (CYP2C9) alleles and warfarin S/R concentration ratio in patients who required low-dose (<2.5 mg/day) and average-dose (5+/-0.5 mg/day) warfarin. Patients who achieved a therapeutic international normalized ratio were recruited from the Atlanta Veterans Affairs Medical Center anticoagulation clinic. CYP2C9*2 and *3 alleles were determined by validated Taqman allelic discrimination assays. Warfarin S and R concentrations were determined by chiral capillary electrochromatography with electrospray ionization mass spectrometry. At least 1 variant allele was found in 66.7% and 22.2% of patients in the low-dose and average-dose groups, respectively (P= .001, chi(2)). The warfarin S/R concentration ratio was 0.665 (range, 0.162-3.58) and 0.452 (range, 0.159-2.36) for patients receiving low-dose and average-dose therapy, respectively (P= .097). A warfarin requirement of <2.5 mg/day and an elevated warfarin S/R concentration ratio were each associated with a higher frequency of variant CYP2C9 alleles.

Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation.

BACKGROUND: The recent discovery that nitrite is an intrinsic vasodilator and signaling molecule at near-physiological concentrations has raised the possibility that nitrite contributes to hypoxic vasodilation and to the bioactivity of nitroglycerin and mediates the cardiovascular protective effects of nitrate in the Mediterranean diet. However, important questions of potency, kinetics, mechanism of action, and possible induction of tolerance remain unanswered. METHODS AND RESULTS: In the present study, we performed biochemical, physiological, and pharmacological studies using nitrite infusion protocols in 20 normal human volunteers and in nonhuman primates to answer these questions, and we specifically tested 3 proposed mechanisms of bioactivation: reduction to nitric oxide by xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin. We found that (1) nitrite is a relatively potent and fast vasodilator at near-physiological concentrations; (2) nitrite functions as an endocrine reservoir of nitric oxide, producing remote vasodilation during first-pass perfusion of the opposite limb; (3) nitrite is reduced to nitric oxide by intravascular reactions with hemoglobin and with intravascular reductants (ie, ascorbate); (4) inhibition of xanthine oxidoreductase with oxypurinol does not inhibit nitrite-dependent vasodilation but potentiates it; and (5) nitrite does not induce tolerance as observed with the organic nitrates. CONCLUSIONS: We propose that nitrite functions as a physiological regulator of vascular function and endocrine nitric oxide homeostasis and suggest that it is an active metabolite of the organic nitrates that can be used therapeutically to bypass enzymatic tolerance.

Endogenous histamine and cortisol levels in subjects with different histamine N-methyltransferase C314T genotypes : a pilot study.

BACKGROUND: Histamine N-methyltransferase (HNMT) catalyzes the methylation of histamine and plays an important role in histamine biotransformation in bronchial epithelium. Enzymatic activity of HNMT has been shown to be regulated by genetic factors, including polymorphisms in the HNMT gene. In this pilot study we determined endogenous levels of histamine and cortisol in plasma and whole blood samples from subjects with different genotypes for the HNMT C314T polymorphism, and investigated whether these parameters differed between individuals with the HNMT CC genotype and those with the CT genotype. METHODS: Blood samples were collected from 48 unrelated volunteers (36 males, 12 females), aged 21-40 years, who participated in the study. PCR-restriction fragment length polymorphism analysis was used to determine HNMT C314T genotypes. Erythrocyte HNMT activity was determined as well as plasma and whole blood levels of histamine and cortisol. Two-group comparisons of the various parameters were analyzed by Blocked Wilcoxon test and Wilcoxon Rank Sum test as appropriate. RESULTS: Thirty-seven subjects (24 Caucasians, three African Americans, one Middle Eastern, five Indians, three Chinese, and one Filipino) were found to have the homozygous CC genotype. Ten subjects (eight Caucasians, one Middle Eastern, and one Chinese) were heterozygous and one individual (Pakistani) was homozygous for the variant 314T allele. The frequency of HNMT CT heterozygotes in the small Caucasian cohort was 0.125. Median enzyme activity was significantly lower in subjects with the heterozygous CT genotype than in those with the homozygous CC genotype (485 vs 631 U/mL of red blood cells; p=0.023). A broad range of histamine levels in plasma and whole blood was observed for all subjects. Whereas the median plasma histamine level was found to be higher in heterozygotes for the wild-type 314C allele than homozygotes (3.32 vs 2.30 nmol/L; p=0.021), there was no difference between the two groups in histamine levels in whole blood. Cortisol levels were similar between individuals with the homozygous CC genotype and those with the heterozygous CT genotype. CONCLUSION: Wide variability of plasma and whole-blood histamine levels was observed in subjects with different HNMT C314T genotypes. Endogenous levels of histamine are likely to be affected by various genes and polymorphisms.

Reduced methylprednisolone clearance causing prolonged pharmacodynamics in a healthy subject was not associated with CYP3A5*3 allele or a change in diet composition.

The influence of diet and genetics was investigated in a healthy white person who had distinctly low methylprednisolone clearance. Pharmacokinetic and pharmacodynamic parameter values were similar on 2 occasions during the consumption of a low-carbohydrate diet and a Weight Watchers diet, indicating that the decreased clearance was unlikely attributable to a change in diet composition. Although the subject was found to be homozygous for CYP3A5*3, genetic findings were not significant for a number of other CYP3A4 and CYP3A5 allelic variants. Because of the high prevalence of CYP3A5*3/*3 in whites and because 5 of 7 white control subjects are also homozygous for CYP3A5*3, this genotype cannot fully explain the reduced metabolism of the drug. Other genetic or contributing factors might have been involved. New polymerase chain reaction-based genotyping methods for functionally defective CYP3A5*6, *8, *9, and *10 alleles were developed in this study. These assays will be useful for CYP3A5 genotype analysis in future clinical studies.

Lack of pharmacokinetic interaction between lamotrigine and olanzapine in healthy volunteers.

STUDY OBJECTIVE: To investigate the potential drug-drug interaction between lamotrigine, an antiepileptic agent used to treat bipolar disorders, and olanzapine, an atypical antipsychotic drug also used to treat bipolar disorders, both of which are metabolized by the uridine diphosphate glucuronosyltransferase system. DESIGN: Prospective cohort study. SETTING: University center for clinical research. SUBJECTS: Fourteen nonsmoking, healthy volunteers. INTERVENTION: Subjects received lamotrigine 25 mg/day for 5 days, then 50 mg/day for 10 days to achieve steady-state concentrations. On day 15, blood samples were obtained before and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours after the dose. Lamotrigine 50 mg/day was then given for an additional 3 days. On the next day, lamotrigine 50 mg and olanzapine 5 mg were coadministered. Blood samples were obtained at the same times as before and at 48, 72, and 96 hours after dosing. MEASUREMENTS AND MAIN RESULTS: Blood samples were assayed for lamotrigine and olanzapine concentrations by means of high-performance liquid chromatography. Olanzapine did not significantly affect lamotrigine disposition, as we observed no differences in the area under the concentration-time curve from 0-24 hours or in lamotrigine plasma concentrations at baseline or at 24 hours. For lamotrigine, the mean time to reach maximum concentration was significantly prolonged during olanzapine coadministration (mean +/- SD 1.9 +/- 1.3 vs 4.0 +/- 3.0 hrs, p = 0.025), possibly because of the anticholinergic properties associated with olanzapine. Mild sedation was the only adverse effect that occurred during lamotrigine and olanzapine coadministration. CONCLUSION: Lamotrigine and olanzapine can safely be combined in healthy volunteers at the low doses studied, without a clinically significant interaction. When prescribing high doses of olanzapine and lamotrigine for bipolar disorder, patients must be carefully monitored.