Loss of Blood-Brain Barrier Integrity in an In Vitro Model Subjected to Intermittent Hypoxia: Is Reversion Possible with a HIF-1α Pathway Inhibitor?

Several sleep-related breathing disorders provoke repeated hypoxia stresses, which potentially lead to neurological diseases, such as cognitive impairment. Nevertheless, consequences of repeated intermittent hypoxia on the blood-brain barrier (BBB) are less recognized. This study compared two methods of intermittent hypoxia induction on the cerebral endothelium of the BBB: one using hydralazine and the other using a hypoxia chamber. These cycles were performed on an endothelial cell and astrocyte coculture model. Na-Fl permeability, tight junction protein, and ABC transporters (P-gp and MRP-1) content were evaluated with or without HIF-1 inhibitors YC-1. Our results demonstrated that hydralazine as well as intermittent physical hypoxia progressively altered BBB integrity, as shown by an increase in Na-Fl permeability. This alteration was accompanied by a decrease in concentration of tight junction proteins ZO-1 and claudin-5. In turn, microvascular endothelial cells up-regulated the expression of P-gp and MRP-1. An alteration was also found under hydralazine after the third cycle. On the other hand, the third intermittent hypoxia exposure showed a preservation of BBB characteristics. Furthermore, inhibition of HIF-1α with YC-1 prevented BBB dysfunction after hydralazine treatment. In the case of physical intermittent hypoxia, we observed an incomplete reversion suggesting that other biological mechanisms may be involved in BBB dysfunction. In conclusion, intermittent hypoxia led to an alteration of the BBB model with an adaptation observed after the third cycle.

Time Course of Aldehyde Oxidase and Why It Is Nonlinear.

Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-O6-benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [N8-(3-Chloro-4-fluorophenyl)-N2-(1-methyl-4-piperidinyl)-pyrimido[5,4-d]pyrimidine-2,8-diamine dihydrochloride], and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.

N-Acetyltransferase 2 Genotype-Dependent N-Acetylation of Hydralazine in Human Hepatocytes.

Hydralazine is used in the treatment of essential hypertension and is under investigation for epigenetic therapy in the treatment of neoplastic and renal diseases. N-acetyltransferase (NAT) 2 exhibits a common genetic polymorphism in human populations. After recombinant expression in yeast, human NAT2 exhibited an apparent Lineweaver-Burk constant (K-m) value (20.1 ± 8.8 µM) for hydralazine over 20-fold lower than the apparent K-m value (456 ± 57 µM) for recombinant human NAT1 (P = 0.0016). The apparent Vmax value for recombinant human NAT1 (72.2 ± 17.9 nmol acetylated/min/mg protein) was significantly (P = 0.0245) lower than recombinant human NAT2 (153 ± 15 nmol acetylated/min/mg protein), reflecting 50-fold higher clearance for recombinant human NAT2. Hydralazine NAT activities exhibited a robust acetylator gene dose response in cryopreserved human hepatocytes both in vitro and in situ. Hydralazine NAT activities in vitro differed significantly with respect to NAT2 genotype at 1000 (P = 0.0319), 100 (P = 0.002), and 10 µM hydralazine (P = 0.0029). Hydralazine NAT activities differed significantly (P < 0.001) among slow acetylator hepatocytes, (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A). The in situ hydralazine N-acetylation rates differed significantly with respect to NAT2 genotype after incubation with 10 (P = 0.002) or 100 µM (P = 0.0015) hydralazine and were higher after incubation with 100 µM (10-fold) than with 10 µM (4.5-fold) hydralazine. Our results clearly document NAT2 genotype-dependent N-acetylation of hydralazine in human hepatocytes, suggesting that hydralazine efficacy and safety could be improved by NAT2 genotype-dependent dosing strategies.

Lipid Peroxide-Derived Short-Chain Carbonyls Mediate Hydrogen Peroxide-Induced and Salt-Induced Programmed Cell Death in Plants.

Lipid peroxide-derived toxic carbonyl compounds (oxylipin carbonyls), produced downstream of reactive oxygen species (ROS), were recently revealed to mediate abiotic stress-induced damage of plants. Here, we investigated how oxylipin carbonyls cause cell death. When tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells were exposed to hydrogen peroxide, several species of short-chain oxylipin carbonyls [i.e. 4-hydroxy-(E)-2-nonenal and acrolein] accumulated and the cells underwent programmed cell death (PCD), as judged based on DNA fragmentation, an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei, and cytoplasm retraction. These oxylipin carbonyls caused PCD in BY-2 cells and roots of tobacco and Arabidopsis (Arabidopsis thaliana). To test the possibility that oxylipin carbonyls mediate an oxidative signal to cause PCD, we performed pharmacological and genetic experiments. Carnosine and hydralazine, having distinct chemistry for scavenging carbonyls, significantly suppressed the increase in oxylipin carbonyls and blocked PCD in BY-2 cells and Arabidopsis roots, but they did not affect the levels of ROS and lipid peroxides. A transgenic tobacco line that overproduces 2-alkenal reductase, an Arabidopsis enzyme to detoxify α,ß-unsaturated carbonyls, suffered less PCD in root epidermis after hydrogen peroxide or salt treatment than did the wild type, whereas the ROS level increases due to the stress treatments were not different between the lines. From these results, we conclude that oxylipin carbonyls are involved in the PCD process in oxidatively stressed cells. Our comparison of the ability of distinct carbonyls to induce PCD in BY-2 cells revealed that acrolein and 4-hydroxy-(E)-2-nonenal are the most potent carbonyls. The physiological relevance and possible mechanisms of the carbonyl-induced PCD are discussed.

Aldehyde oxidase activity in fresh human skin.

Human aldehyde oxidase (AO) is a molybdoflavoenzyme that commonly oxidizes azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in several drug failures, existing in vitro-in vivo correlations are often poor and the extrahepatic role of AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin, the largest organ of the body, frequently exposed to therapeutic drugs and xenobiotics. Fresh, full-thickness human skin was obtained from 13 individual donors and assayed with two specific AO substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol⋅mg skin(-1)⋅h(-1), resulting in 13 and 2% substrate turnover, respectively, after 24 hours of incubation with 10 µM substrate. Hydroxylation activities for the two substrates were significantly correlated (r(2) = 0.769), with interindividual variability ranging from 3-fold (zoniporide) to 6-fold (carbazeran). Inclusion of hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 µM inhibitor. Reaction rates were linear up to 4 hours and well described by Michaelis-Menten enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan glucuronidation and sulfation and p-toluidine N-acetylation showed that cutaneous AO activity is comparable to tested phase II metabolic reactions, indicating a significant role of AO in cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic activity in human skin.

Hydralazine plays an immunomodulation role of pro-regeneration in a mouse model of spinal cord injury.

Spinal cord injury (SCI) results in severe motor and sensory dysfunction with no effective therapy. Spinal cord debris (sp) from injured spinal cord evokes secondary SCI continuously. We and other researchers have previously clarified that it is mainly bone marrow derived macrophages (BMDMs) infiltrating in the lesion epicenter to clear sp, rather than local microglia. Unfortunately, the pro-inflammatory phenotype of these infiltrating BMDMs is predominant which impairs wound healing. Hydralazine, as a potent vasodilator and scavenger of acrolein, has protective effects in many diseases. Hydralazine is also confirmed to promote motor function and hypersensitivity in SCI rats through scavenging acrolein. However, few studies have explored the effects of hydralazine on immunomodulation, as well as spontaneous pain and emotional response, the important syndromes in clinical patients. It remains unclear whether hydralazine affects infiltrating BMDMs after SCI. In this study, we targeted BMDMs to explore the influence of hydralazine on immune cells in a mouse model of SCI, and also investigated the contribution of polarized BMDMs to hydralazine-induced neurological function recovery after SCI in male mice. The adult male mice underwent T10 spinal cord compression. The results showed that in addition to improving motor function and hypersensitivity, hydralazine relieved SCI-induced spontaneous pain and emotional response, which is a newly discovered function of hydralazine. Hydralazine inhibited the recruitments of pro-inflammatory BMDMs and educated infiltrated BMDMs to a more reparative phenotype involving in multiple biological processes associated with SCI pathology, including immune/inflammation response, neurogenesis, lipid metabolism, oxidative stress, fibrosis formation, and angiogenesis, etc. As an overall effect, hydralazine-treated BMDMs loaden with sp partially rescued neurological function after SCI. It is concluded that hydralazine plays an immunomodulation role of educating pro-inflammatory BMDMs to a more reparative phenotype; and hydralazine-educated BMDMs contribute to hydralazine-induced improvement of neurological function in SCI mice, which provides support for drug and cell treatment options for SCI therapy.

Acrolein plays a culprit role in the pathogenesis of diabetic nephropathy in vitro and in vivo.

Objective: Diabetic nephropathy (DN), also known as diabetic kidney disease (DKD), is a major chronic complication of diabetes and is the most frequent cause of kidney failure globally. A better understanding of the pathophysiology of DN would lead to the development of novel therapeutic options. Acrolein, an α,ß-unsaturated aldehyde, is a common dietary and environmental pollutant. Design: The role of acrolein and the potential protective action of acrolein scavengers in DN were investigated using high-fat diet/ streptozotocin-induced DN mice and in vitro DN cellular models. Methods: Acrolein-protein conjugates (Acr-PCs) in kidney tissues were examined using immunohistochemistry. Renin-angiotensin system (RAS) and downstream signaling pathways were analyzed using quantitative RT-PCR and Western blot analyses. Acr-PCs in DN patients were analyzed using an established Acr-PC ELISA system. Results: We found an increase in Acr-PCs in kidney cells using in vivo and in vitro DN models. Hyperglycemia activated the RAS and downstream MAPK pathways, increasing inflammatory cytokines and cellular apoptosis in two human kidney cell lines (HK2 and HEK293). A similar effect was induced by acrolein. Furthermore, acrolein scavengers such as N-acetylcysteine, hydralazine, and carnosine could ameliorate diabetes-induced kidney injury. Clinically, we also found increased Acr-PCs in serum samples or kidney tissues of DKD patients compared to normal volunteers, and the Acr-PCs were negatively correlated with kidney function. Conclusions: These results together suggest that acrolein plays a role in the pathogenesis of DN and could be a diagnostic marker and effective therapeutic target to ameliorate the development of DN.

Transformation of lupus-inducing drugs to cytotoxic products by activated neutrophils.

Drug-induced lupus is a serious side effect of certain medications, but the chemical features that confer this property and the underlying pathogenesis are puzzling. Prototypes of all six therapeutic classes of lupus-inducing drugs were highly cytotoxic only in the presence of activated neutrophils. Removal of extracellular hydrogen peroxide before, but not after, exposure of the drug to activated neutrophils prevented cytotoxicity. Neutrophil-dependent cytotoxicity required the enzymatic action of myeloperoxidase, resulting in the chemical transformation of the drug to a reactive product. The capacity of drugs to serve as myeloperoxidase substrates in vitro was associated with the ability to induce lupus in vivo.

Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy.

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

Pharmacogenetics, race and global injustice.

This paper discusses the link between pharmacogenetics and race, and the global justice issues that the introduction of pharmacogenetics in pharmaceutical research and clinical practice will raise. First, it briefly outlines the likely impact of pharmacogenetics on pharmaceutical research and clinical practice within the next five to ten years and then explores the link between pharmacogenetic traits and 'race'. It is shown that any link between apparent race and pharmacogenetics is problematic and that race cannot be used as a proxy for pharmacogenetic knowledge. The final section considers the implications of the development of pharmacogenetics for health care systems in low- and middle-income countries.

Tumorigenic effect of 1-hydrazinophthalazine hydrochloride in mice.

A solution of 0.125% 1-hydrazinophthalazine hydrochloride, an antihypertensive drug widely used in humans, was given continuously in drinking water for the life-spans of randomly bred Swiss mice. Consumption of the chemical significantly increased the lung tumor incidence from 36 to 60% in females and from 26 to 46% in males, compared to controls. Histopathologically, the tumors were classified as adenomas and adenocarcinomas of the lungs.

Polymorphic acetylation of hydralazine.

The acetylation of hydralazine has been studied in hypertensive patients undergoing maintenance therapy with the drug. The patients were acetylator phenotyped with sulfamethazine. Using gas-liquid chromatography and high-pressure liquid chromatography, hydralazine and two of its acetylated metabolites, methyltriazolophthalazine (MTP) and 3-hydroxymethyltriazolophthalazine (HOMTP), have been determined in the 0- to 24-hr urine. The excretion of hydralazine and HOMTP but not MTP was found to be related to the acetylator phenotype. The metabolic ratio HOMTP: hydralazine showed a bimodal distribution and the average ratio for slow acetylators (1.6) was lower than the ratio in rapid acetylators (14.9). It is concluded that hydralazine is polymorphically acetylated in man. The acetylated metabolite HOMTP was not, however, the major metabolite reported previously.

Hydralazine kinetics in hypertensive patients after intravenous administration.

Previous studies on intravenous hydralazine kinetics have been performed using nonselective analytical techniques that measure not only hydralazine but also certain hydralazine metabolites such as hydralazine pyruvic acid hydrazone (HPH). We studied the time course of hydralazine and HPH in eight hypertensive patients after 0.3 mg/kg intravenous with selective high-pressure liquid chromatographic assays. "Apparent" hydralazine concentrations were also determined using a nonselective gas-liquid chromatographic procedure. Total plasma clearance, CLT[72.9 +/- 4.9 (SEM) ml . min-1 . kg-1], apparent volume of distribution, Vd area (5.83 +/- 0.30 1 . kg-1), steady-state volume of distribution, Vd ss (1.83 +/- 0.17 . kg-1), and terminal half-life, t1/2 (53.7 min, harmonic mean) were independent of acetylator phenotype. The high ClT is compatible with rapid intravascular conversion of hydralazine to HPH and a high hepatic extraction ratio. Peak HPH concentrations occurred 10 to 60 min after dose; mean HPH t1/2 was 239 min. "Apparent" hydralazine concentrations were usually highest in the 2-min plasma sample and declined with a mean t1/2 of 296 min. Reports based on nonselective assay methods have underestimated CLT, Vd ss, and Vd area and have overestimated the t1/2 of hydralazine.

Hydralazine kinetics after single and repeated oral doses.

In reports on hydralazine kinetics plasma hydralazine levels have been measured with nonspecific assay techniques. The techniques used also include acid-labile hydralazine metabolites and therefore markedly overestimate hydralazine levels. We have developed specific, sensitive assay methods for the measurement of hydralazine and its major plasma metabolite, hydralazine pyruvic acid hydrazone (HPH). By these methods, we determined hydralazine and HPH kinetics after single and repeated oral doses of hydralazine in eight hypertensive patients. Hydralazine bioavailability in the fast acetylator group (9.5% single dose, 6.6% repeated doses) and in the slow acetylator group (31.3% single dose, 39.3% repeated doses) was phenotype dependent. Peak plasma levels were lower than those reported with nonspecific assays: 0.32 microM for the single dose and 0.14 microM for repeated doses in the fast acetylator group and 1.03 microM for the single dose and 0.96 microM repeated doses in the slow acetylator group. There was no alteration in kinetics and no cumulation in plasma on repeated administration. HPH plasma levels were proportional to those of hydralazine in both acetylator groups and were 2.5 to 4 times as high as those of hydralazine. Elimination half-lifes were phenotype independent, ranging from 4 to 6 hr. HPH cumulated in the rapid but not in the slow acetylator group after repeated doses of hydralazine.

Kinetics of hydralazine elimination.

Hydralazine was given orally in single doses of 10, 25, and 50 mg to 2 slow-acetylating subjects, while 2 rapid-acetylating subjects also received 100- and 150-mg doses on different occasions. Administration of the 50-mg dose to the subjects who were slow acetylators and the 150-mg dose to those who were rapid acetylators caused a disproportionately large increase in the amount of unchanged drug appearing in the systemic circulation as judged from the increases in the ratios of areas under concentration-time curves (AUC) to dose. A modification of the gas-liquid chromatographic hydralazine assay allowed the simultaneous determination of hydralazine and its acetylated metabolite, 3-methyl-s-triazolo-3,4,a-phthalazine (MTP), in serum. It was found that the disproportionately large increases in the AUC/dose ratio of hydralazine upon intake of 50 or 150-mg doses by the slow and rapid-acetylating subjects, respectively, were paralleled by a decrease in the ratio AUCMTP/AUChydralazine during a 6-hr observation period. It is concluded that the acetylation of hydralazine in man is a capacity-limited process.

Identification and quantitation of hydrazine in the urine of patients treated with hydralazine.

Hydrazine has been identified by gas chromatography-mass spectrometry in the 0- to 24-hr urine of patients administered hydralazine. With a specific gas chromatographic assay procedure, the amount of hydrazine in the 0- to 24-hr urine was determined in patients treated with various doses of hydralazine. The amount of hydrazine detected in the urine was greater in the slow acetylator phenotype than in the rapid acetylator phenotype. Studies indicated that hydrazine was not produced by chemical breakdown of hydralazine or its known metabolites in urine and therefore was unlikely to be a urinary artefact formed by chemical decomposition in the urine.

Effect of dose on acetylator phenotype distribution of hydralazine.

The effect of dose on acetylator phenotype distribution of hydralazine has been determined, The acetylated metabolites methyltriazolophthalazine (MTP) and 3-hydroxymethyltriazolophthalazine (3-OHMTP) and acid-labile hydralazine (HP) were determined in the 0- to 24-hr urine of patients receiving various doses. The difference between the mean value for the ration 3-OHMTP:HP in the rapid and slow acetylators varied with dose, the greatest difference being after a 200 mg (100 mg twice daily) dose. The distribution of the ratio became less clearly bimodal at lower doses, with overlap between phenotypes occurring at doses of 100 mg (50 mg twice daily) or less. The most effective dose for discriminating between acetylator phenotypes was found to be 200 mg (100 mg twice daily).

Determinants of response to intravenous hydralazine in hypertension.

There is marked interindividual variation in hypotensive response to intravenous hydralazine (H). We examined the determinants of response in patients with hypertension. After a single intravenous dose of 0.3 mg/kg H, response was correlated independently (r = 0.8364) with both predrug blood pressure and acetylator index (AI). Intravenous dose ranging studies showed that response also depended on the amount of H in the systemic circulation. Although plasma H levels depend on AI after oral doses, this is not so after intravenous administration. AI must therefore affect response to H by an alternative, presumably nonmetabolic mechanism which, not related to AI, perhaps indicating specificity of this effect for H. These data reinforce the potential usefulness of determining AI before giving H to a patient.

Hydralazine and furosemide kinetics.

Fursosemide kinetics were studied in 25 patients with congestive heart failure. The elimination half-life (t1/2) was longer and the elimination rate constant and the plasma clearance smaller in patients with advanced (n = 15) than in those with moderate (n = 10) failure. Furosemide kinetics with and without hydralazine were compared in eight patients with advanced heart failure. Furosemide t1/2 patients receiving both drugs fell from 96 +/- 16 to 81 +/- 15 min (P less than 0.02), elimination rate constant increased from 0.0186 +/- 0.0056 to 0.0214 +/- 0.0068 min -1 (P less than 0.05), and plasma clearance rose from 72.6 +/- 18.5 to 88.1 +/- 26.9 ml/min (P less than 0.01). Renal clearance rose from 45.4 +/- 12.0 to 60.9 +/- 19.0 ml/min (P less than 0.01) and creatinine clearance was unchanged. We conclude that hydralazine affects furosemide kinetics.

Prospective study of immunologic effects of hydralazine in hypertensive patients.

Twenty-seven hypertensive patients (23 of whom were black) were treated with hydralazine as their major antihypertensive drug and were followed for evidence of autoimmunity and clinical systemic lupus erythematosus (SLE). Only one patient developed SLE but many, although asymptomatic, had serologic evidence of autoimmunity: antibodies to single- and double-stranded ribonucleic acid (RNA), single-stranded deoxyribonucleic acid (DNA), histones, and lymphocytes. Acetylation phenotype profoundly influenced this response; slow acetylators had a higher incidence and larger amounts of autoantibodies. Antibodies to both types of RNA were a more sensitive index of autoimmunity than antinuclear antibodies. Hydralazine treatment did not alter cell-mediated immune responses. The hydralazine SLE patient had large amounts of autoantibodies that were predominantly IgG, while in the others IgM autoantibodies were predominant. No antibodies, but positive lymphoproliferative responses to hydralazine, were found in half the patients tested.