Nitric-Oxide Mediated Effects of Transdermal Capsaicin Patches on the Ischemic Threshold in Patients with Stable Coronary Disease

Abstract

Background 

Capsaicin has been shown to exert direct vasodilating effects through increased calcitonin gene-related peptide (CGRP) release. However, no data exist on its effect following systemic administration in humans.

Methods 

Twelve male patients with stable coronary disease and a persistently positive exercise were selected for study. According to a double blind, placebo-controlled, cross-over study, patients were randomized to placebo or 3 g oleic capsaicin-containing patches, on 2 different days and with a 2-day interval between treatments. Patients performed treadmill exercise testing according to the Bruce protocol. Time to 1 mm ST segment depression and to peak exercise, maximal ST segment depression, and the number of ECG leads showing diagnostic changes were also measured. Blood samples for nitric oxide (NO) and CGRP were drawn at baseline, 2, 6, and 24 hours after exercise.

Results 

On placebo, all patients had a positive ECG during exercise test. Only 1 patient experienced angina, on both treatments. With capsaicin, 1 patient had a negative exercise, while 8 patients significantly increased time to 1 mm ST depression from 328 ± 167 to 401 ± 174 seconds (P = 0.01). Of the remaining patients, 1 did not show any changes and 2 showed a worse ischemic threshold when on capsaicin. CGRP levels were not significantly different between placebo and capsaicin treatment. Conversely, when on capsaicin, NO significantly increased at 6 hours.

Conclusions 

Transdermal capsaicin may improve ischemic threshold in patients with stable coronary disease, probably through arteriolar vasodilation. Increased capsaicin-induced NO availability could represent the principal mechanism of action.

Capsaicin, a natural product of capsicum species (chili pepper–capsicum frutescens), induces reflex coronary vasodilatation by stimulation of vagal C fibers.¹ In experimental studies, capsaicin administration has been shown to evoke the release of calcitonin gene-related peptide (CGRP),^2,3 a principal transmitter in sensory nerves.^4,5 CGRP is one of the most potent endogenous vasodilators yet discovered.⁶ Several endogenous agents and conditions activate cardiac C-fiber afferents, with subsequent local release of CGRP.⁷ During myocardial ischemia, C-fiber afferents not only convey the sensation of pain, but there is also a local efferent release of CGRP in the heart.⁸ After being released, CGRP causes coronary vasodilatation and attenuates the development of myocyte death,⁹ probably through a preconditioning mechanism.¹⁰ In animal studies, capsaicin-induced increase in coronary flow and heart rate¹¹ and hypotensive effects¹² have been shown to be dependent on interplay between CGRP and nitric oxide (NO).

During the years, we have anecdotally and repeatedly observed improved ischemic threshold in patients carrying a capsaicin-containing patch, which in Italy is employed for the relief of low back pain and, at present, is the only available way for administering capsaicin to humans. Based on these serendipity observations, since the effects of systemic administration of capsaicin in patients with coronary disease have not been evaluated yet, we assessed the potential therapeutic benefits of transdermal capsaicin in a group of angina patients on full antianginal treatment.

METHODS

Patients

Twelve consecutive patients (all males, age 66 ± 6 years) with angiographically proven coronary artery disease awaiting myocardial revascularization were selected for study. All patients had a reproducibly positive exercise test. Reproducibility was assessed by exercising patients on 2 consecutive days, before entering the study: a 5% difference in time to 1 mm ST segment depression between the two pre-study exercise tests was considered an exclusion criterion. Patients with a previous myocardial infarction or any cardiac condition potentially interfering with unequivocal interpretation of the 12-lead electrocardiogram (ECG) were excluded from the study. All patients were on maximal tolerated antianginal medications. None of the patients had diabetes mellitus. Three had mild glucose intolerance, controlled by diet only. All patients gave informed consent to the study. Table 1 summarizes main clinical characteristics of study patients.

Table 1: Patients Clinical Characteristics

Protocol

According to a double-blind, placebo-controlled study, patients were randomized to placebo or 3 g oleic capsaicin-containing patches, on 2 different days and with a 2-day interval between treatments. Since 2 days before testing, patients consumed a standard diet without ingredients that could influence endogenous nitric oxide synthesis (sausages, ham, and derivatives). Capsaicin patches are commercially available as over-the-counter preparations for the relief of arthralgias (Kelemata Inc., Turin, Italy). Patches were applied on the back of patients at 9 am and removed 24 hours later. At 4 pm patients performed treadmill exercise testing according to the Bruce protocol. Treadmill exercise tests (Mortara Instruments–X-SCRIBE Stress Exercise System, Milwaukee, WI) was performed in the fasting state. Blood pressure (cuff sphygmomanometer) and the 12-lead ECG were recorded at baseline, during the third minute of each exercise step and throughout recovery. Exercise was terminated for the appearance of ≥2 mm rectilinear or downsloping ST segment depression or for severe angina, fatigue, ventricular tachycardia, or a blood pressure decrease >10 mm Hg. Heart rate, systolic/diastolic blood pressure, and rate-pressure product (RPP) were measured at rest, at the appearance of 1 mm ST segment depression and at peak exercise. Time to 1 mm ST segment depression, to peak exercise and recovery time (time to ST segment return to baseline during recovery after exercise) were recorded. Achieved percentage of age-adjusted maximal predicted heart rate (220-patient age), maximal ST segment depression, and the number of ECG leads showing diagnostic changes were also measured.

Biochemical Testing

Blood sampling for NO and CGRP determination was performed at baseline and after 2, 6, and 24 hours from the application of patches. NO levels were evaluated by measuring the end-products of their metabolism (ie, nitrite and nitrate levels) using enzymatic catalysis coupled with Griess reaction. Specifically, NO₃ was reduced to NO₂ by nitrate reductase 0,1 U, FAD 5 × 10⁻⁶ M and NADPH 250 × 10⁻⁶. Samples were incubated at 37°C for 3 hours, then LDH 8,8 U and pyruvate 10⁻² M was added, and samples incubated for additional 90 minutes at 37°C. Finally, Griess reactive was added to each well and samples read at 540 nm.¹³

For measurement of CGRP, the samples were extracted on a SepPack C18 mini-column (Amprep, Amersham International, Buckingamshire, UK), and the eluate was evaporated in a Speed VAC SC 110 (Savant Instruments, Inc., Farmingdale, NY). The samples were reconstituted with 250 μL of radioimmunoassay (RIA) buffer and assayed with a RIA kit avoid of cross-reactivity with amylin, calcitonin, VIP, substance P, and somatostatin (Peninsula Laboratories Inc., San Carlos, CA).

Statistical Analysis

Data are reported as mean ± 1 standard deviation. To investigate the treatment effect the paired t test was used. The capsicum effect on CGRP and NO at different times was analyzed using the analysis of variance (ANOVA) for repeated measures on log-transformed data because the distribution was not gaussian. Analysis was performed with SAS 6.12 (SAS Institute Inc., Cary, NC).

RESULTS

No significant side effects were recorded during the study. Compared with placebo, when on capsaicin patients showed a non-significant trend toward lower diastolic and systolic blood pressure and higher heart rate and RPP at baseline, at 1 mm ST segment depression, and at peak exercise. RPP was not significantly different compared with placebo at any test stage. Individual results are reported in Table 2.

Table 2: Heart Rate, Systolic and Diastolic Blood Pressure, and Rate-Pressure-Product at Baseline, at 1 mm ST Segment Depression (1 mm) and at Peak Exercise During Placebo and Capsaicin Administration

Reproducibility of Basal Exercise Testing

No significant differences were observed between the two basal tests performed before entering the active study. Additionally, time to 1 mm ST segment depression (359 ± 187 versus 346 ± 159 seconds), total exercise time (484 ± 172 versus 462 ± 139 seconds), recovery time (380 ± 133 versus 374 ± 172 seconds), and cumulative number of ECG leads showing diagnostic changes (3.75 ± 1.76 versus 3.83 ± 1.69) were no significantly different compared with the placebo phase of the active study.

Exercise Testing

On placebo, all patients had a positive ECG during exercise test. Only 1 patient experienced angina, on both treatments. With capsaicin, 1 patient had a negative exercise, while 8 patients, compared with placebo, significantly increased time to 1 mm ST depression from 328 ± 167 to 401 ± 174 seconds (P = 0.01). Of the remaining patients, 1 did not show any changes and 2 showed a worse ischemic threshold when on capsaicin. Considering all patients, when on capsaicin time to 1 mm ST segment depression increased, compared with placebo, from 372 ± 170 to 424 ± 182 seconds (P = 0.027) (Figs. 1 and 2) and the total number of involved ECG leads decreased from 3.75 ± 1.76 to 3.25 ± 2.00 leads (P = 0.05). Accordingly, although the difference did not reach statistical significance, time to peak exercise also tended to be longer (489 ± 189 to 508 ± 202 seconds, P = 0.29, placebo versus capsaicin). Individual results are reported in Table 3.

Table 3: Time (sec) to 1 mm ST Segment Depression (1 mm), to Peak Exercise, Recovery Time, Maximal Exercise-Induced ST Segment Depression, and Number of Involved ECG Leads Showing Diagnostic Changes During Placebo and Capsaicin Administration

FIGURE 1.: Time (sec) to 1 mm ST segment depression and to peak exercise in all study subjects during placebo (white bar) and capsaicin (black bar) administration.

FIGURE 2.: Individual time to 1 mm ST segment depression with placebo (left) and capsaicin (right). Patient n.6, who had a negative exercise test when on capsaicin, is plotted with maximal exercise time achieved with the active drug.

Biochemical Testing

Repeated ANOVA did not reveal a significant effect of time on capsaicin effect for CGRP levels (P = 0.4) (Fig. 3). No significant within-group CGRP changes compared with baseline values after placebo and capsaicin treatment were found. Although the within-subject capsaicin effect on CGRP was increasing with time, the ANOVA was not significant (P = 0.2). Conversely, despite NO within subject differences between capsaicin and placebo were not different compared with baseline values, when on capsaicin, within groups NO significantly increased at 6 hours (Fig. 4). Individual results are reported in Table 4.

Table 4: Nitric Oxide and Calcitonin Gene-Related Peptide Levels at Baseline, at 2, 6, and 24 Hours After Placebo and Capsaicin Administration

FIGURE 3.: CGRP levels at time 0, and 2-6-24 hours after patches application during placebo (continuous line) and capsaicin (dotted line).

FIGURE 4.: NO levels at time 0, and 2, 6, and 24 hours after patches application during placebo (continuous line) and capsaicin (dotted line). The reported significance level refers to the difference between baseline and 6 hours NO levels when patients were on capsaicin.

DISCUSSION

The results of this preliminary study indicate that transdermal administration of capsaicin can improve the ischemic threshold in a significant proportion of patients with coronary artery disease. This improvement might depend on an increased availability of NO, as suggested by the observed augmentation of NO levels at 6 hours from capsaicin patches application. However, the concomitant administration of nitrates in some patients could have hampered the observed capsaicin-induced NO release. In fact, among the 6 patients taking nitrates, 4 showed an increment in NO release at 6 hours. The possibility that the observed increment could be greater in absence of nitrates is not remote. CGRP levels did not change significantly in patients showing improved exercise during capsaicin treatment. RPP was similar between placebo and capsaicin, since the latter determined a decrease of blood pressure balanced by an increase in heart rate.

Role of Nitric Oxide and Calcitonin Gene-Related Peptide

The potent vasodilator calcitonin gene-related peptide (CGRP) is stored in a population of C-fiber afferents that are sensitive to capsaicin. However, the exact mechanism by which capsaicin induces vasodilatation is still controversial. Previous animal studies have evidenced a significant role of NO in capsaicin-induced release of vasodilator quantities of CGRP,^2,14 and consequently, in the dilator effects of capsaicin-sensitive nerves in the coronary,¹⁵ cerebral,¹⁶ and cochlear¹⁷ circulation. Conversely, in rat mesenteric vessels, capsaicin-induced vasodilatation has been shown to be independent from NO-synthesis, but again mediated by CGRP release.¹⁸ Another study has shown that capsaicin-induced vasodilatation involves synthesis of NO in venules, but not in arterioles, where CGRP seems again to be the predominant factor.¹⁹ In isolated perfused rat and guinea pig heart preparations, CGRP administration has evidenced a significant antiarrhythmic and anti-ischemic activity²⁰; accordingly, in human studies CGRP has been shown to be a potent dilator of epicardial coronary arteries²¹ and to improve the ischemia threshold in patients with coronary artery disease.²² Nonetheless, we could not observe any significant changes in CGRP levels following capsaicin in our study, despite the observed augmentation of NO levels at 6 hours from capsaicin patches application. On this ground we cannot exclude a direct capsaicin-mediated activation of NO in our patients, as already hypothesized in a recent paper.²³

To our knowledge, this is the first study aimed at assessing the effects of capsaicin administration in patients with coronary artery disease. The observed beneficial effects were quite striking, since more than two thirds of study patients showed a significant improvement of the ischemic threshold. In these patients, the mechanism involved was probably mediated by increased NO availability following capsaicin administration. In fact, CGRP levels, which has been shown to yield the lowest variability among various neuropeptides measured at frequent sampling intervals,²⁴ were not significantly different between placebo and capsaicin.

Recent human studies have shown that capsaicin administration can induce age-specific cutaneous vasodilatation²⁵ and that this effect may decrease after repeated applications.²⁶ This tolerance-like phenomenon^27,28 could be related to previous experimental observations showing a dose-dependent capsaicin-induced progressive depletion of CGRP immunoreactive nerves in the cardiovascular system,²⁹ with the consequent reduction of its known cardioprotective effects.³⁰ Also, a desensitization of the vanilloid receptors has been hypothesized.³¹ Interestingly, reduced CGRP levels have also been shown to play a major role in the mechanism of nitrate tolerance.³²

Future Developments

A newly synthesized nonivamide derivative, N-[4-O-[2-methoxy, phenoxyethylaminobutyl]-3-methoxy benzyl]-nonamide (VOA), structurally with a vanilloid base of capsaicin, has been shown to exert relaxation effects in rat vascular smooth muscle through the CGRP/KATP channel and the NO/ cGMP pathway.²³ Additionally, VOA did not show any desensitization between 2 doses. Future studies will be aimed at assessing the possibility of desensitization by long-term use of VOA and at differentiating effects on the endothelium and the smooth muscle.

Capsaicin and analogues are also valuable analgesic agents when administered to mammals, including humans, and most research has been focused on the issue of pain. However, their pungency severely limits their use. Recently, structure activity analysis revealed that the initial pungent and general excitatory effects can be prevented by structural modifications in such a way that the analgesic activity is retained. SDZ 249-665 is a potent orally active, analgesic/anti-hyperalgesic agent in mouse, rat, and guinea pig.³³ It lacks the excitatory effects associated with capsaicin and other close analogues, and therefore provides a clear therapeutic window for use in painful conditions. In addition to this favorable profile, no sign of tolerance was detected after a 5-day repeated dose treatment. Possible applications in the cardiovascular field could be attempted.

CONCLUSION

Transdermal capsaicin may improve the ischemic threshold in patients with stable coronary disease, probably through arterial and venous vasodilation. Increased NO availability appears as the principal mechanism of action. Future studies will be aimed at assessing the potential beneficial effects of different ways of administration and dosage regimens of this natural vasodilating substance. The possibility of development of nitrate-like tolerance following its administration should also be investigated. We believe that systemic administration of capsaicin may have a role in the treatment of patients with angina pectoris. These very preliminary observations should prompt further studies.