Journal of Human Hypertension (2008) 22, 401–407 & 2008 Nature Publishing Group All rights reserved 0950-9240/08 $30.00 www.nature.com/jhh
ORIGINAL ARTICLE
Tetrahydrobiopterin: a novel antihypertensive therapy M Porkert1, S Sher1, U Reddy1, F Cheema1, C Niessner1, P Kolm2, DP Jones3, C Hooper4, WR Taylor1, D Harrison1 and AA Quyyumi1 1
Division of Cardiology, Emory University, Atlanta, GA, USA; 2Christiana Care Center for Outcomes Research, Newark, DE, USA; 3General Clinical Research Center, Emory University, Atlanta, GA, USA and 4Centers for Disease Control, Atlanta, GA, USA
Tetrahydrobiopterin (BH4) is a cofactor for the nitric oxide (NO) synthase enzymes, such that its insufficiency results in uncoupling of the enzyme, leading to release of superoxide rather than NO in disease states, including hypertension. We hypothesized that oral BH4 will reduce arterial blood pressure (BP) and improve endothelial function in hypertensive subjects. Oral BH4 was given to subjects with poorly controlled hypertension (BP 4135/85 mm Hg) and weekly measurements of BP and endothelial function made. In Study 1, 5 or 10 mg kg1 day1 of BH4 (n ¼ 8) was administered orally for 8 weeks, and in Study 2, 200 and 400 mg of BH4 (n ¼ 16) was given in divided doses for 4 weeks. Study 1: significant reductions in systolic (P ¼ 0.005) and mean BP (P ¼ 0.01) were observed with both doses of BH4. Systolic BP was 15±15 mm Hg (P ¼ 0.04) lower
after 5 weeks and persisted for the 8-week study period. Study 2: subjects given 400 mg BH4 had decreased systolic (P ¼ 0.03) and mean BP (P ¼ 0.04), with a peak decline of 16±19 mm Hg (P ¼ 0.04) at 3 weeks. BP returned to baseline 4 weeks after discontinuation. Significant improvement in endothelial function was observed in Study 1 subjects and those receiving 400 mg BH4. There was no significant change in subjects given the 200 mg dose. This pilot investigation indicates that oral BH4 at a daily dose of 400 mg or higher has a significant and sustained antihypertensive effect in subjects with poorly controlled hypertension, an effect that is associated with improved endothelial NO bioavailability. Journal of Human Hypertension (2008) 22, 401–407; doi:10.1038/sj.jhh.1002329; published online 6 March 2008
Keywords: tetrahydrobiopterin; endothelium; oxidative stress
Introduction Nitric oxide (NO) is a potent endothelium-derived vasodilator that regulates blood pressure (BP) and regional blood flow.1–6 Decreases in NO production or bioavailability impair endothelium-dependent dilation that can be both a consequence as well as a cause of hypertension. Among other protective roles of the endothelium, mediated in part by NO, are the inhibition of vascular smooth muscle cell proliferation and growth, and inhibition of proinflammatory cytokine expression, platelet aggregation and oxidative modification of low-density lipoprotein.7 Importantly, the presence of endothelial dysfunction heralds increased risk of future adverse cardiovascular events in patients including those with hypertension.8
Correspondence: Dr AA Quyyumi, Division of Cardiology, Emory University, 1364 Clifton Road, Ste D403C, Atlanta, GA 30322, USA. E-mail:
[email protected] Received 25 July 2007; revised 21 November 2007; accepted 1 December 2007; published online 6 March 2008
Nitric oxide is synthesized in the vascular endothelium by endothelial NO synthase (eNOS) and tetrahydrobiopterin (BH4) is an essential cofactor for eNOS. During NO catalysis, oxygen is bound by the ferrous heme in the oxygenase domain of eNOS. BH4 plays a crucial role in donating an electron that leads to scission of the O–O bond, leading to formation of an iron–oxy complex that participates in the hydroxylation of L-arginine, leading to formation of NO. In the absence of BH4, the Fe2 þ O–O complex dissociates to release superoxide (O2 ). This phenomenon is referred to as eNOS uncoupling. Accumulating evidence suggests that hypertension increases vascular production of reactive oxygen species, which can serve to oxidize BH4 and lead to eNOS uncoupling.9,10 Our previous experimental studies have shown that BH4 is oxidized to dihydrobiopterin in the vasculature of the deoxycorticosterone acetate salt hypertensive mouse, and supplementation with oral BH4 can reduce BP and improve oxidative stress in this model.11 Several studies have assessed the effects of BH4 on endothelial function. Sepiapterin, a substrate for BH4 synthesis, improved endothelium-dependent K
Tetrahydrobiopterin for hypertension M Porkert et al 402
vasodilation in coronary arterioles obtained from patients undergoing coronary artery bypass graft surgery (CABG).12 Acute infusion of BH4 improved endothelial function in angiographically normalappearing segments of coronary arteries in subjects with coronary artery disease or hypercholesterolaemia.13,14 A similar improvement was observed in the forearm circulation of diabetics and smokers.15–17 These studies have used either parenteral infusion of BH4 or a single oral dose, and it has not been shown that chronic oral administration of BH4 could cause a sustained improvement in endothelial function. Moreover, no prior study has examined the effect of BH4 in subjects with hypertension. We hypothesized that chronic oral therapy with BH4 in hypertensive patients will result in reduction of arterial BP as a result of improvement in endothelial dysfunction. For this purpose, we conducted two pilot studies where (a) the duration of action of oral BH4 and (b) its dose–response were studied in hypertensive subjects.
uncontrolled hypertension (BP 4180 mm Hg systolic and/or 110 mm Hg diastolic), renal or hepatic dysfunction, and bleeding disorders. All subjects gave informed consent and the study was approved by the Emory University Investigational Review Board.
Study design
Both studies were designed as pilot investigations to (a) evaluate the effects of BH4 on BP and vascular function, (b) the dose–response of this effect. For this reason, they were not placebo controlled, but included a withdrawal phase. After screening, suitable subjects were admitted to the Emory University General Clinical Research Center. All BP and heart rate measurements were made after having the patient rest in a seated position for at least 10 min, by coordinators unaware of study stage. Mean BP was calculated as: (systolic BP þ 2 diastolic BP)/3. Tetrahydrobiopterin (Schircks Laboratories, Jona, Switzerland) was compounded with an equivalent amount of vitamin C to ensure stability of the drug. Subjects were advised to keep the pills in a refrigerator.
Methods Patient selection
Subjects between the ages of 18 and 75 years were recruited if they had uncontrolled hypertension on traditional stable antihypertensive therapy (BP X135/85) or newly diagnosed hypertension (BP X140/90). Patients were continued on their current antihypertensive therapy throughout the study. Exclusion criteria included female subjects with childbearing potential, history of recently symptomatic coronary or peripheral vascular disease, known secondary causes for hypertension, severe
Study 1: investigation of the time of onset and duration of action of oral BH4
Eight hypertensive subjects (Table 1) were assigned to either 5 mg kg1 day1 (n ¼ 4) or 10 mg kg1 day1 (n ¼ 4) of BH4, given in two divided doses orally for 8 weeks. This regimen was based on BH4 dosing in phenylketonuria. Weekly BP measurements were
Table 1 Baseline characteristics Study 1 (n ¼ 8)
Age (years) Males Mean number of antihypertensive medications Treated with ACE-I or ARB Treated with diuretics Treated with calcium antagonists Treated with beta blockade Treated with statins Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg) Past medical history Coronary artery disease High cholesterol (4240 mg per 100 ml) Diabetes Current tobacco Former tobacco
Study 2 (n ¼ 16) 400 mg (n ¼ 8)
200 mg (n ¼ 8)
60.5±11 (48–74) 3 1.4±0.9 (0–3) 4 3 2 2 2 144±6.6 (138–154) 81±14.4 (54–100) 102±9.7 (82–115)
57±9 (43–72) 4 2.0±1.9 (0–6) 4 6 3 2 3 149±10.3 (140–171) 88±11.1 (73–108) 109±10.0 (96–129)
62±8 (45–69) 2 1.9±1.0 (1–3) 7 2 3 3 3 154±12.3 (137–178) 79±13.0 (51–92) 104±9.6 (88–118)
0 2 0 0 2
0 5 2 1 3
2 4 1 1 3
Abbreviations: ACE-I, angiotensin converting enzyme inhibitor; ARB, angiotensin receptor blocker. Continuous values are represented as mean±s.d. with range in parentheses. Journal of Human Hypertension
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Study 1 BH4 0
1
2
3
4
5
6
7
8
9
14
Weeks
BP FMD
Study 2
Vitamin C
-1
-2
BH4
0
1
2
3
4
5
8
Weeks
BP FMD Figure 1 Protocol for Studies 1 and 2. BP, blood pressure, FMD, flow-mediated vasodilation.
after discontinuation of therapy. In Study 2, it was measured at the end of the 2-week vitamin C run-in period, after 4 weeks of BH4 therapy and 4 weeks after discontinuation of BH4. The technique has been described in detail previously.18 Briefly, studies were performed in a temperature-controlled room, and brachial diameter was measured above the antecubital fossa in the non-dominant arm using an 11 MHz high resolution ultrasound transducer (Acuson Inc., Malvern, PA, USA). Flow-mediated vasodilation (FMD) was determined by inflating a BP cuff on the forearm to 4200 mm Hg for 5 min, deflating rapidly, and recording brachial diameter at 1 min after onset of hyperaemia. Three diameter measurements were averaged on end-diastolic frames each time. FMD was calculated as: (average diameter with hyperaemiaaverage baseline diameter)/average baseline diameter 100. Endothelium-independent vasodilator response was measured as brachial artery diameter change 5 min after 0.4 mg sublingual nitroglycerin using the formula: (average diameter post nitroglycerin average baseline diameter)/average baseline diameter 100. Statistical analysis
made throughout the treatment period and at 1 and 6 weeks after discontinuation (Figure 1). Study 2: investigation of dose–response of oral BH4
Of the 16 subjects, 6 were male and mean age was 60±8 years (Table 1). Eight subjects received 100 mg of oral BH4 twice a day and 8 received 200 mg twice daily. There were no significant differences in baseline characteristics between the two groups (Table 1). Because BH4 is compounded in vitamin C in a 1:1 ratio, all subjects received 100 or 200 mg of vitamin C twice a day during the first 2-week run-in period to control for possible changes in BP with vitamin C therapy. After this 2-week run-in period, subjects received BH4 at their assigned dosage for the next 4 weeks. This study was shorter because the peak effect was noted by the third week in the first study. Weekly heart rate and BP measurements were made during the run-in and treatment periods. BH4 was then stopped, and follow-up was performed at 1 and 4 weeks after discontinuation where BP and heart rate measurements were made (Figure 1). A complete metabolic panel and lipid profile was performed prior to initiation of medication, at the end of BH4 therapy, and 1 month after discontinuation.
The primary end point was the change in systolic, mean and diastolic BP during BH4 therapy. Secondary end points included change in FMD with BH4. Linear mixed effects models for repeated measures data were used to analyse the change in BP from baseline to the end of the treatment period and differences in change between 100 and 200 mg dose groups. A quadratic term for week was included in the model to account for nonlinear trends over the treatment period. Dose, week and week squared were the main effect terms in the model; dose by week and dose by week squared were the interaction terms in the model. Significant interaction terms indicate a difference in the pattern of change between the two dose groups. FMD data were analysed using the two-tailed Student’s t-test. All results are expressed as mean±s.d., and P-values o0.05 are considered statistically significant. The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the paper as written.
Results Baseline patient characteristics are included in Table 1. Effect of BH4 on BP
Measurement of endothelial function
Brachial artery endothelium-dependent and -independent function was measured using ultrasound. In Study 1, brachial reactivity was measured at baseline, after 8 weeks of BH4 treatment and 1 week
Study 1. In the combined group, there was a significant reduction in systolic (P ¼ 0.005 quadratic trend) and mean arterial BP (P ¼ 0.01) with BH4 treatment (Figure 2). Systolic BP was lowered by a mean of 13±9 mm Hg after 3 weeks (P ¼ 0.004) and Journal of Human Hypertension
Tetrahydrobiopterin for hypertension M Porkert et al 404
160
160
BH4
BH4
Blood Pressure mmHg
140
p=0.005
130 SBP
120
DBP MBP
110 p=0.01
100 90 80
140 SBP DBP MBP
120
100
80
60 0
70 60 0
1
2
3
4
5 6 Weeks
7
8
9
14
Figure 2 BP response in Study 1. Values are mean±s.e.m. P-value by ANOVA. ANOVA, analysis of variance; BP, blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; SBP, systolic blood pressure.
BH4
160 Blood Pressure mmHg
Blood Pressure mmHg
150
p=0.03
140 SBP DBP
120
p=0.04
MBP
100 80 60 0
1
2
3 Weeks
4
5
8
Figure 3 BP response with 400 mg BH4 in Study 2. Values are mean±s.e.m. P-value by ANOVA. BP, blood pressure; DBP, diastolic blood pressure; SBP, systolic blood pressure, MBP, mean blood pressure.
15±15 mm Hg (P ¼ 0.04) after 5 weeks. This reduction persisted for the 8 weeks of treatment. Mean BP was also significantly reduced by BH4, but the change in diastolic BP did not reach statistical significance (Figure 2). BP returned towards pretreatment levels 6 weeks after discontinuation of therapy (P ¼ NS, compared to baseline). There was no significant change in heart rate with BH4. There were also no statistically significant differences between the 5 and 10 mg kg1 doses of BH4. Study 2. In this study, we aimed to determine whether lower doses of BH4 will have a similar effect on BP. There was no significant change in heart rate or BP during the run-in phase with vitamin C in either group; systolic, diastolic and mean arterial BP in the 16 subjects were 152±11, 84±13, 106±10 mm Hg before, and 148±19, Journal of Human Hypertension
1
2
3 Weeks
4
5
8
Figure 4 BP response with 200 mg BH4 in Study 2. Values are mean±s.e.m. P-value by ANOVA not significant. BP, blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; SBP, systolic blood pressure.
86±11, and 106±12 mm Hg, respectively, after 2 weeks of vitamin C (all P ¼ NS). Subjects given 400 mg of oral BH4 had a significant decrease in systolic (P ¼ 0.03) and mean BP (P ¼ 0.04 by linear trend analysis) (Figure 3). The decrease in diastolic BP did not reach statistical significance (P ¼ 0.08). Mean BP was significantly lower after 1 week (P ¼ 0.02). There was a further significant reduction in BP during the subsequent weeks reaching a nadir after 3 weeks when systolic BP was a mean 16 mm Hg lower; P ¼ 0.04 (Figure 3). A week after termination of therapy, BP remained lower, but 4 weeks after discontinuation of BH4, BP rose back up to baseline levels. No significant change in BP was observed in subjects given 200 mg of BH4 (Figure 4). There was no significant alteration in heart rate with BH4 in either group. Effect of BH4 on endothelial function
Study 1. Flow-mediated vasodilation of the brachial artery increased from a mean of 3.4±1 to 8.2±3.4% after 8 weeks of BH4 (P ¼ 0.05, n ¼ 6) and returned to baseline levels 6 weeks after termination of therapy (3.7±1.3%, P ¼ NS compared to baseline). Nitroglycerin-mediated vasodilation remained unchanged (11.9±3% before and 16.1±5% after BH4, P ¼ 0.1). Study 2. There was a significant improvement in FMD with 400 mg of BH4 (n ¼ 7). FMD improved from 3.7±3% before to 7.1±4.9% after 4 weeks of BH4 therapy (P ¼ 0.016). One month after discontinuation of BH4 therapy, FMD returned to baseline levels (3.2±1.1%, P ¼ 0.6 compared to baseline). There was no significant change in FMD in subjects given 200 mg of BH4 (5.3±2.5 vs 6.2±3.5%; P ¼ 0.55). There was also no significant change in nitroglycerin-mediated, endothelium-independent vasodilation during the study in either group. There
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Table 2 Effects of BH4 on lipid and metabolic markers
Sodium (mequiv l1) Potassium (mequiv l1) BUN (mg per 100 ml) Creatinine (mg per 100 ml) Total cholesterol (mg per 100 ml) LDL cholesterol (mg per 100 ml) HDL cholesterol (mg per 100 ml) Triglycerides (mg per 100 ml) Glucose (mg per 100 ml) Insulin (mU ml1)
Baseline
4 weeks
P-value
139.8±2.3 3.8±0.5 15.1±5.6 1.06±0.28 197.0±51.6
137.5±3.1 4.1±0.3 17.6±5.2 1.07±0.30 195.1±45.6
0.002 0.06 0.12 0.79 0.78
113.9±44.7
119.3±37.9
0.38
54.6±21.6
51.5±16.8
0.12
142.2±92.4
121.1±48.4
0.28
95.9±27.3 91.2±21.1 0.015±0.010 0.019±0.014
0.39 0.36
Abbreviations: BUN, blood urea, nitrogen; LDL, low density lipoprotein; HDL, high density lipoprotein. Values are mean±s.d.
was no change in baseline diameter with BH4 therapy in either group.
Adverse effects of BH4
There were no significant changes in renal function, lipid parameters, glucose and insulin 4 weeks after BH4 therapy in Study 2 subjects except a slight decrease in serum sodium (Table 2). In Study 1, one subject receiving the 10 mg kg1 dose of BH4 complained of worsening heartburn and hot flashes and withdrew after 7 weeks of therapy. These symptoms were present before the patient enrolled in the study. Another patient reported mild nausea. In Study 2, no subject withdrew due to adverse effects. Three subjects complained of mild orthostasis, but it was transient. Two subjects reported mild fatigue, two reported mild ankle oedema, and one reported dysgeusia.
Discussion Our study demonstrates that oral BH4 is effective in lowering arterial BP in subjects with either poorly controlled hypertension or in those who were newly diagnosed. This effect was observed at a dose of 400 mg or higher and was free of any changes in heart rate. Reduction in BP was evident within 1 week of therapy and was sustained for up to 8 weeks of continuous therapy without tachyphylaxis. BP remained lower for at least a week after discontinuation of therapy, but returned towards baseline after 4 weeks. Oral BH4 appeared to be well-tolerated without any serious adverse effects. We found that subjects had significant improvement in endothelial dysfunction after BH4 therapy at the doses that also reduced BP. On the basis of our previous experimental data, we speculate that this improved vascular NO bioavailability and resultant vascular relaxation contributed to the reduction in
BP. Nevertheless, we cannot exclude the possibility that the reduction in BP per se contributed to improvement of vascular endothelial function. Potential mechanisms
Tetrahydrobiopterin is believed to produce its antihypertensive effect via its effect on NO synthesis. NO is a potent endothelium-derived vasodilator that regulates BP and regional blood flow.1–6 A decrease in NO production or bioavailability reduces endothelium-dependent dilation and increases vascular tone. In addition, NO mediates many of the protective functions of the endothelium, including inhibition of vascular smooth muscle proliferation, platelet aggregation and expression of proinflammatory cytokines.19,20 NO is generated from L-arginine in the vascular endothelium by constitutive eNOS in the presence of reduced BH4 that is an essential cofactor of eNOS. Insufficiency of BH4 or its oxidation to dihydrobiopterin by peroxynitrite or other oxidants leads to uncoupling of eNOS that promotes generation of superoxide instead of NO.21 Thus, a deficiency of BH4 produces two additive physiologic consequences: (a) uncoupling of the L-arginine NO pathway resulting in increased formation of oxygen-free radicals by eNOS22 and (b) loss of NO production.23 In a recent study, we found that deoxycorticosterone acetate salt hypertension caused BH4 oxidation in aortas of mice, leading to a marked increase in superoxide production and a significant reduction in NO production. Treatment of these mice with oral BH4 reduced vascular O2 production, increased NO production and blunted the increase in BP due to deoxycorticosterone acetate salt hypertension.11 Similar findings have been reported in other models of hypertension. Aortae of spontaneously hypertensive rats had higher production of superoxide and reduced release of NO compared with normotensive rats, an effect that was blunted in the presence of exogenous BH4.19,24 Our present study confirms these experimental observations in human subjects with essential hypertension in whom oral BH4 reduced BP, likely by reversing the uncoupling of NO synthase and improving NO bioavailability. A previous study by Higashi et al.25 showed that parenteral infusion of BH4 improved endothelial function in hypertensive patients, but this is the first study demonstrating the effects of oral BH4 on endothelial function and BP. BH4 has other potential actions such as tyrosine and tryptophan hydroxylation that may also have contributed to these effects independent of the actions discussed above. K
Study limitations
In the present study, the subjects remained on their previous medications during BH4 therapy. Further studies need to be performed to determine if BH4 Journal of Human Hypertension
Tetrahydrobiopterin for hypertension M Porkert et al 406
will be equally effective in lowering BP when used as monotherapy. Our study was not blinded and did not have a placebo group, but the BP and endothelial function measurements were performed without knowledge of treatment stage. Moreover, withdrawal of therapy resulted in a return of BP and endothelial function to pre-study levels. Vitamin C at higher doses than those used to compound BH4 has been demonstrated in some studies to have a mild antihypertensive effect26 and to improve endothelial function, although this remains a subject of controversy.27–30 To overcome this possibility, we made baseline measurements after a 2-week run-in period with vitamin C alone in Study 2 and found no change in BP. However, a synergistic effect of vitamin C with BH4 cannot be excluded. In fact, recent studies have shown that the immediate product of BH4 oxidation is the BH3 radical and that vitamin C reacts with the BH3 radical and converts it back to BH4.31
6 7 8 9
10
11
K
K
12 What is known about this topic K Nitric oxide bioavailability is reduced in hypertension and is associated with endothelial dysfunction. K This is partly secondary to uncoupling of nitric oxide synthase, which is secondary to reduced availability of tetrahydrobiopterin, a cofactor for this enzyme. What the study adds K Oral therapy with tetrahydrobiopterin can safely lower blood pressure in subjects with poorly controlled hypertension. K This is associated with improvement of endothelial dysfunction.
Acknowledgements Markus Porkert was supported by an American Heart Association training Grant Award Number: 0425402B. Other funding came from a research grant from the Emory University General Clinical Research Center (Grant no. MO1-RR00039).
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