pharmacokinetics of nitroglycerin and its metabolites after administration of sustained-release...

BIOPHARMACEUTICS & DRUG DISPOSITION, VOL. 13, 141-152 (1992)

PHARMACOKINETICS OF NITROGLYCERIN AND ITS METABOLITES AFTER

TABLETS ADMINISTRATION OF SUSTAINED-RELEASE

HAE-RYUN KWON, PRESTON GREEN AND STEPHEN H. CURRY*

Colleges of Pharmacy and Medicine, University of Florida, Gainesville, Florida, 32610. USA

ABSTRACT

Nitroglycerin was administered to eight healthy volunteers in the form of sublingual tablets, oral sustained-release tablets, and an oral solution. Blood samples were collected for measurement of nitroglycerin and its two isomeric glyceryl dinitrate metabolites. Blood pressure and pulse rate were monitored; subjective evaluations of headache, dizziness, facial flushing, skin irritation, and gastrointestinal upset were made. Nitroglycerin itself was virtually undetectable after the solution and tablet preparations; the metabolites were consistently detectable from a few minutes after dosing to 24 h later. Mean total (nitroglycerin plus metabolite) concentrations were comparable in the 15 min following sublingual administration, and the 8 h following tablet administration. The relative bioavailability of the tablets in comparison with the oral solution was 70 per cent based on metabolite concentrations. Nitroglycerin sustained-release tablets appear to exert their beneficial effects in the prolonged prophylaxis of angina through active metabolites.

KEY WORDS Nitroglycerin Sustained release tablets Bioavailability Pharmacokinetics Plasma concentrations

INTRODUCTION

Nitroglycerin is available in a variety of dosage forms.' These include intra- venous solutions, ointments and skin patches for systemic effect, sustained- release tablets and capsules intended to be swallowed, sublingual tablets and aerosols, and buccal tablets. Of these, the sustained-release tablets and capsules are probably the least understood. Designed to administer relatively high doses (typically 6.5 mg) over 6-8 h dosage intervals, they are of proven clinical effi- c a ~ y , ~ ' ~ despite the widespread belief that swallowed nitroglycerin is totally metabolized by a presystemic elimination (first-pass effect) mechanism."'

Several attempts have been made to study the pharmacokinetics of nitroglycerin following administration of swallowed sustained release tablet and capsule doses, but the results have been inconclusive, largely being concerned

* Present address and addressee for correspondence: Dr Stephen H. Curry, Fisons Pharmaceuticals, 755 Jefferson Road, Rochester, New York 14623 USA.

0 142-2782/92/02014 1-1 2$06.00 0 1992 by John Wiley & Sons, Ltd.

Received 5 June 1991 Revised 22 August 1991

142 H.-R. KWON, P. GREEN AND S . H. CURRY

with the argument as to whether nitroglycerin itself is or is not detectable in p l a~rna .~ - '~ Although oral solution doses have been ~tudied, '"~ no published work with tablets or capsules has involved measurement of the putative products of the first-pass effect or involved a comparison of the tablets or capsules with oral solution dosage forms. This paper is the report of a crossover study of sublingual, oral solution, and oral sustained-release nitroglycerin dosage forms in healthy volunteers with measurement of nitroglycerin (GTN) and its two primary metabolites, 1,2-dinitrogIycerin (1,2-GDN) and 1,3-dinitroglycerin (1,3-GDN).

METHODS

Dosage forms

The following three dosage forms were used:

1. Sublingual nitroglycerin tablets, 0.4 mg, from the Parke-Davis company; 2. 6-5 mg Nitrong@ SR tablets (Wharton Laboratories, Inc.); 3. An extemporaneously prepared solution of nitroglycerin in tap water, 6.5 mg

in 162.5 ml, freshly prepared using a 10 per cent lactose adsorbate of nitroglycerin.

Subjects

Eight healthy male volunteers aged 18 to 40 years participated in a crossover study. They were screened by means of medical history, physical examination, blood chemistry analysis, hematology screen with differential, and urine analysis. They were considered free of abnormalities as the result of screening. All subjects were fully informed, and they signed the approved consent form author- ized by the local Institutional Review Board (Ethics Committee). No medica- tions (prescription or over-the-counter) were allowed for 14 days before the study; only aspirin or acetaminophen were allowed during the study.

Method of oral solution administration

The solution was prepared not more than 10 min before the initial dose time. The adsorbate/water mixture was shaken gently to dissolve the nitroglycerin and lactose. Dissolution was virtually instantaneous. Approximately 10 ml of the solution (0.4mg nitroglycerin) was emptied into glass dosing cups and administered every 30 min for 8 h. The dose included a rinsing of the dosing cup with tap water. The dose was immediately followed by ingestion of tap water, with mouth rinsing, to reduce the possibility of nitroglycerin absorption from the mucosal surface of the mouth. The stability of the solution was evaluated by injecting samples of the solution into an HPLC assay system throughout

NITROGLYCERIN ORAL TABLETS 143

the 8 h period. The sustained-release tablet was administered orally with approximately 5-7 ml of water.

Details of study

All subjects received a single sublingual dose of nitroglycerin 0.4 mg at screening. Blood samples were collected for nitroglycerin and nitroglycerin metabolites assay before and at 2, 5 , 10, 20, and 30 min after dosing. Blood pressure and pulse rate were measured prior to and at regular intervals for 30 min after dosing.

After the test dose, the subjects were randomly assigned to two treatment sequence groups and received the 6.5 mg nitroglycerin solution or the 6.5 mg Nitrong@ sustained-release tablet. The treatment periods were separated by a washout phase of at least 3 days. Equal numbers of subjects received the treatments first.

On the morning of the study day, the subjects reported to the Clinical Research Center of Shands Hospital at the University of Florida at 8:OO am, fasting. Baseline blood pressure and pulse rate were recorded. A heparin lock was inserted and a baseline blood sample was collected. The dosing time was 8:30 am, or as close thereto as possible.

Blood samples were obtained at 0 (predose), 10,20,30, and 40 min, at hourly intervals from 1 to 12 h, and at 14, 16, 20, and 24 h after dosing. Supine blood pressure (both diastolic and systolic) and pulse rate were recorded for each subject prior to, and at 30 min, and 1, 2, 4, 6, 8, 10, 12, and 24h after dosing. Subjects were permitted to sit up or walk around except during the 10 rnin prior to blood sampling, or if hypotension developed. The subjects were permitted only water for the first 2 h after dosing; caffeine-free soft drinks were then offered. Lunch was served 4 h after dosing, after which all food and fluid restric- tions were lifted.

All subjects were asked to complete a set of Visual Analogue Scales which graded the common side-effects of nitroglycerin: headache, skin irritation, facial flushing, gastrointestinal upset, and dizziness.

Sample handling

Venous blood samples were collected using a 10 ml Vacutainer tube (Becton- Dickinson) which was chilled in ice for 15 min prior to blood collection. The tube contained 70 pl of 0-4 M p-hydroxymercuribenzoate as an enzyme inhibitor. Blood samples (7 ml) were placed in the tube immediately after withdrawal and then centrifuged. A maximum of 5 min elapsed between withdrawal of blood and storage in the freezer (-20"), so that the potential for nitroglycerin degradation was zero. The p-hydroxymercuribenzoate as an enzyme inhibitor assisted in prevention of degradation - the volume of fluid involved (1 per cent) was insignificant; there was no assay interference. Plasma samples were

144 H . - R . KWON, P. GREEN A N D S. H. CURRY

thawed at room temperature, without application of heat, for 15 min before assay. Each sample was frozen and thawed only once; separate plasma samples were prepared from each blood sample and retained for nitroglycerin and metabolite assays.

Analytical methods

Plasma samples were assayed for nitroglycerin and its primary denitration products (1,ZGDN and 1,3-GDN) content using gas chromatography, with capillary columns and electron capture detection. The methods were those of Noonan and Benet and colleagues with minor modifications to suit local circum- s tance~. '~- '~ The strategy was as follows:

1. Extract the drug and its metabolites from plasma into solvents. 2. Clean and concentrate the solvent extracts. 3. Evaluate drug and metabolite content using gas chromatography with capil-

lary columns and electron-capture detection. 4. Quantitate by comparison of signals from extracts with signals from 'spiked'

known concentrations of the compounds. 5. Subject each plasma sample to two assays, one for unchanged nitroglycerin,

the other for the two dinitroglycerin metabolites, evaluated individually; the metabolites were silylated prior to chromatography.

6. Perform chromatographic evaluations using a Nelson integration system.

Nitroglycerin, 1,2-GDN, and 1,3-GDN were obtained as 10 per cent solutions in ethanol (Radian Corporation, Austin, Texas). The 2,6-dinitrotoluene, pentane, ethylacetate, hexane, dimethyldichlorosilane, 1 -bromonaphthalene, and BSTFA were all of the highest available grade. All glassware was silanized before use.

The gas chromatograph (Varian 3700) was equipped with a Ni-63 electron capture detector and either an on-column injector (J and W Scientific, Inc.) (nitroglycerin assays), or a split injector (Alltech Associates, Inc.) (metabolite assays). The configuration of the column and the detector were optimized as described by Noonan and colleagues. The column was a 30 m X 0.25 mm DB-1 fused silica capillary column (J and W Scientific, Inc.,) (nitroglycerin) or 25 m x 0.25 mm GB-60 Column (Analabs) (metabolites). Helium was used as a car- rier gas at a flow rate of 1.2ml min-I. Nitrogen was used as a make-up gas at a flow rate of 30ml min-I. The column temperature was 130". The detector was at 200".

Dilutions of the nitroglycerin standard were prepared in distilled water (10ngml-I). The dinitrotoluene was prepared as a 50ngml-I solution in distilled water. A 4Opl volume of the internal standard solution was added to each plasma sample before extraction. Nitroglycerin calibration standards (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1-Ongml) were prepared by adding the appropriate aliquot of nitroglycerin standard to 2 ml of blank human plasma in 16 x 150 mm


silanized test tubes with Teflon lined screw caps. Each calibration standard was extracted with lOml pentane by vortexing for 5 min. Each sample was then centrifuged for 10 min at 2300 xg. The pentane layers were transferred to clean test tubes. The residual plasma layers were extracted and centrifuged a second time. The combined pentane extracts were evaporated under a stream of nitrogen and evaporated near to dryness. A 201.11 volume of ethylacetate was added to each test tube and the test tubes were vortexed (5 s). An aliquot (0.5~1) of each extract was injected into the gas chromatograph. The retention time of nitroglycerin in this system was approximately 6 min. Blank plasma showed no peak at this time. A fortified plasma extract, from a plasma solution at 0.25ngml-' of GTN showed a sharp peak approximately 650mm high. Thus no endogenous chemicals interfered with the assay. Specificity was further determined by injecting solutions of nitroglycerin metabolites (1 ,ZGDN) on to the column. It was shown that the retention times for the metabolites were very long and the peaks were very broad in this system. Also, pentane does not extract the metabolites from plasma so the metabolites did not interfere with the nitroglycerin assay. This assay was capable of detecting 50 pg ml-' of nitroglycerin in plasma. At this level, the peak height ratio was approximately 0.1, and the nitroglycerin peak was approximately 17 mm high. For the purpose of calculating means, apparent concentrations below 50 pg ml-' were treated as zero.

Primary stock solutions (100ngml-I each) of 1,ZGDN and 1,3-GDN were prepared by diluting the ethanolic solutions. 1 -Bromonaphathalene was used as an internal standard, diluted with ethyl acetate (1: 10). 1 ,ZGDN and 1,3-GDN calibration standards were prepared by adding the appropriate aliquot of stock solutions to 1 ml of blank human plasma in 16 x 150mm silanized test tubes with Teflon lined screw caps. Each calibration standard was extracted with a 50: 50 mixture of hexane and ethyl acetate by vortexing for 20 min. Each sample was then centrifuged for 10 min at 2300 Xg. The solvent layers were transferred to clean test tubes. The solvent extracts were evaporated under a stream of nitrogen and near to dryness. A 50 pl volume of ethylacetate was added to dissolve the residue. 1,ZGDN and 1,3-GDN were silylated with 10 p1 of BSTFA at room temperature. A 10 pl sample of I-bromonaphthalene was added as an internal standard. An aliquot (0.05 pl) of this solution was injected into the gas chromatograph. No peaks occurred in blank plasma at the retention times of 1,ZGDN and 1,3-GDN. The retention times for 1,ZGDN and 1,3- GDN were 5.0 and 5-5 min, respectively. This assay method was capable of detecting 0.2 ngml-1 of 1,ZGDN and/or 1,3-GDN. Solutions at this concentration gave peak heights for the metabolites of approximately 1 3 m , and peak height ratios in the range 0-14.2. For the purpose of calculating means, apparent concentrations below 0-2 ng ml-I were treated as zero.

In all cases, calibration was linear, with r values in excess of 0-99 and coeffi- cient of variation data below 10 per cent; calibration with spiked standards was conducted daily.

146 H.-R. KWON, P. GREEN AND S. H. CURRY

Pharmacokinetic analysis

centrations using noncompartmental methods after tablet and solution doses: The following pharmacokinetic parameters were estimated from plasma con-

Cmm: peak concentration TmX: time to peak AUC,,,: area under the curve using the trapezoidal rule f: bioavailability from AUC (tablet)/AUC (solution) 1,: elimination rate constant tll2: elimination half-life. k,: absorption rate constant.

The computer program RSTRIP (MicroMath, Inc., Salt Lake City Utah) was used to estimate the absorption rate constant (k,) and elimination rate constant (h,) after sublingual doses. Students t-test was used in data comparisons. A probability level of 0.05 or less was taken as indicating significance. Data are expressed in this paper as means f SD unless otherwise indicated.

RESULTS

Sublingual doses

After sublingual administration, nitroglycerin appeared rapidly in plasma and its levels peaked at 4.13 f 2.80 min and then decreased rapidly with a mean elimination half-life of 2.41 5 0.91 min, becoming barely detectable at 20 min (Figure 1 ) . The mean maximum concentration was 2.31 f 2.06 ngml-'. The mean absorption half-life was 1.45 f 1.10 min; the mean absorption rate constant was 0.78 f 0.64 min. The mean AUC was 0.20 f 0.14ng. h ml-I. 1,2- GDN was detected in plasma at 2 min in most subjects and its levels peaked at 10.00 f 4.64 min. It was eliminated relatively slowly with an apparent mean half-life of 14-74 5 4-84 min. 1,3-GDN levels peaked at 13.75 f 5.18 min and were eliminated with an apparent mean half-life of 21.30 f 9.03 min. The mean maximum concentrations for 1,2- and 1,3-GDN were 3.88 2.38 and 1.16 f 0.62 ng ml-l, respectively. The 1 ,2-GDN concentrations were significantly greater than those of nitroglycerin or 1,3-GDN; the AUC for I,2-GDN was 9.54 f 2.06 times greater than that for nitroglycerin and 2-67 f 0.58 times greater than that for 1,3-GDN.

Oral solutions

The parent compound was undetectable in most plasma samples throughout the 24 h. Only 12 out of 168 samples had detectable concentrations. The highest concentration was 0-27ngml-' in a 2h sample. Most of the concentrations were less than 0.1 ng ml. This supported the widespread belief that nitroglycerin


5

4

1

0 0 5 10 15 20 25 30 35

Time (min)

Figure 1. Mean ( f SEM) concentrations of nitroglycerin (GTN), 1,2-dinitroglycerin (1,2-GDN), and 1,3-dinitroglycerin (I,3-GDN) after sublingual doses (0.4 mg)

is extensively metabolized before reaching the general circulation after swallowed doses.

Both metabolites were detected in plasma at 10 min after initial solution doses in all subjects (Figure 2(a)). The mean concentration of 1,ZGDN at 10minwas 1.20ngml-landthat of l73-GDNwas0-45ngrnl-'. Bothmetabolites were detectable in plasma for 10 to 20 h after administration.

Although the metabolite concentrations within any one subject showed substantial variability, they generally fluctuated about a steady-state value after the first hour after starting administration. This would imply that metabolite production reflected the approximately zero order delivery and subsequent absorption of nitroglycerin. The duration of steady-state varied from subject to subject, but always lasted at least 7 h. In one subject the study state lasted as long as 16 h. The mean concentration of 1,ZGDN at steady-state was 1.66ngml-1 and that of 1,3-GDN was 0*73ngml-'.

Pharmacokinetic parameters, AUC, and elimination half-life for both metabolites, are shown in Table l. The elimination rate constant (A,) for 1,ZGDN

148 H.-R. KWON, P. GREEN AND S. H . CURRY

Table 1 . Mean pharmacokinetic parameters for the two nitroglycerin metabolites after solution and tablet doses

Compound Dose Parameter Value (mean f SEM)

1,2-GDN Solution AUCCL24h 20.06 f 2.90

f l l 2 1.33 f 0.26 1,ZGDN Tablet AUC0-24h 11.26 f 1.84

A z 0.74 f 0.10

1,3-GDN Solution AUClL24h 9.08 f 1.15

tl12 1.46 f 0.26 1,3-GDN Tablet AUC&24h 5.44 f 0.68

4 0.60 f 0.14 t l l2 1.34 f 0.29

1, 0.59 f0.10

f I / Z 1.01 f0.11

1, 0.55 f 0.1 1

Units: AUC,-,,,, ng.h ml-'; A,, h-'; tl12, h.

ranged from 0.30 to 0.88 h for 1,ZGDN (mean 0.59 f 0.22 h-I). The elimination rate constant for 1,3-GDN in five subjects ranged from 0.31 to 0.861 h-' (mean 0.55 f 0.241 h-I). The mean elimination half-life was 1.33 f 0.58 h for I,2-GDN, and 1.46 k 0.59 h for 1,3-GDN.

Headache occurred in four subjects and two subjects took analgesics. Two subjects reported dizziness and two subjects reported facial flushing. One subject reported skin irritation and one subject reported gastrointestinal upset. No significant blood pressure or pulse rate changes occurred.

Sustained-release tablet doses

No nitroglycerin was detected in plasma in any subjects after administration of the sustained-release tablet. This is interpreted as meaning that nitroglycerin was completely metabolized in the liver before reaching the systemic circulation when it was administered in this form.

Both metabolites were detectable in plasma as early as 10 min in all subjects (Figure 2(b)). The mean concentration of 1,2-GDN at 10 min was as high as 0.67ngml-I and that of 1,3-GDN was 0-35ngml-'. In four subjects, the metabolites were detected for 8-9 h after tablet administration. In two subjects, they were detectable for 13-14 h; in one subject, the metabolites were detected for 24 h.

As with the solution doses, although the concentrations within any one subject showed substantial variability, the metabolite concentrations generally fluctuated about a steady-state value after the first 1-2 h following administration. This would again imply that metabolite production reflected approximately zero order delivery of nitroglycerin. The duration of this steady-state varied considerably from subject to subject, ranging from about 4 to 12 h. In one


(a) SOLUTION

4.0 ~

1: '? T T 0 1,2-GDN

2.0 0 1,3-GDN

1.5

1 .o

0.5

0.0 0 5 1 0 1 5 2 0

Time (h)

(b) TABLETS

2.5 t 0 1.2-GDN

0 1,3-GDN !

2.0

1.5

1 .o

0.5

0.0 0 5 1 0 15 20 25

Time (h)

Figure 2. Mean ( f SEM) concentrations of 1,2-dinitroglycerin (1,ZGDN) and 1,3-dinitroglycerin (I,3-GDN) after (a) solution doses (6.5 mg) and (b) sustained-release tablet

150 H.-R. KWON, P. GREEN AND S. H. CURRY

subject the steady-state conditions may have lasted 24 (or more) h. The mean concentration of 1,2-GDN at steady-state was 1.53 ng ml-' and that of 1,3-GDN was 0.65 ng ml-I.

Pharmacokinetic parameters, AUC, and elimination half-life, for both metabolites, are shown in Table l. Because of the shortage of concentration data during the terminal elimination phase, the elimination rate constants (h,) were estimated from only nine of the 16 plasma concentration-time curves. The mean elimination rate constants for 1,2- and 1,3-GDN were 0.74 f 0.23 1 h-' and 0.60 f 0.27 1 h-', respectively. The mean elimination half-life values were 1.10 k 0-30 h for 1,ZGDN and 1.34 f 0.58 h for 1,3-GDN.

Headache occurred in seven subjects and three subjects took analgesics. Two subjects reported dizziness and two subjects reported facial flushing. Two subjects each reported skin irritation and gastrointestinal upset. There were no significant changes in blood pressure or pulse rate.

The relative bioavailability cf) of the tablet formulation in comparison with the solution formulation was assessed by using the AUC values from both 1,ZGDN and 1,3-GDN. The bioavailability was 0.699 f 0.283 (range 0.29 1 to 1.038) based on l,ZGDN, and 0.689 f 0.185 (range 0.385 to 0.909) based on 1,3-GDN.

DISCUSSION

This study was designed to evaluate plasma concentrations of nitroglycerin and its two metabolites after administration of sustained-release nitroglycerin tablets, by comparison with an oral solution dosage form and sublingual tablets. The control sublingual dose data for nitroglycerin were consistent with data obtained by other investigators. After swallowed solution doses, nitroglycerin concentrations were low or undetectable, in agreement with the extensive work of Benet and his colleagues. However, no unchanged nitroglycerin was found after administration of swallowed sustained-release doses.

This agrees with two other relatively recently published ~tudies", '~ and dis- agrees with several other older studies.8-10 For many years, based on animal data, it has been believed that swallowed nitroglycerin undergoes 100 per cent first-pass effect losses. Our data in this study, with the tablets, agree with that concept. Detection of nitroglycerin in plasma in the past following swallowed sustained release doses may have been an artefact of the relatively non-specific assay system used, or because of different conditions prevailing at the time of the clinical investigations. It is our view that the optimized capillary column technique used here, basically developed by Noonan and Benet, is the best one yet applied to this particular problem, and that unchanged nitroglycerin is of no significance to sustained-release tablet or capsule formulations.

Swallowed sustained-release dosage forms of nitroglycerin have been shown to be clinically effective. From animal studies, it appears that the metabolites


are only about 5 per cent as active as the parent compound. However, the pharmacological activity of the metabolites and the contribution of the metabolites to the action of the parent compound have not been adequately studied, and have not been studied at all in humans. It seems probable that the efficacy of oral nitroglycerin results from pharmacological activity of the metabolites, which, though of relatively low potency, are present in relatively high concentrations. There is some evidence that there is a relatively low incidence of unwanted effects as the result of this.

Plasma concentrations of both metabolites were detectable for 8-24 h and 10-20 h after tablet and solution doses. These concentrations appeared to fluc- tuate around a steady state after the first 1-2 h after dosing began. This would imply that the metabolite production reflected the approximately zero order dosing with nitroglycerin. The 6.5 mg sustained-release tablet produced concentration-time profiles similar to those of multiple doses of nitroglycerin solution. The tablets were designed to release nitroglycerin steadily over a 6 8 h period and this they appear to do.

The mean relative bioavailability of the tablet formulation based on the area under the curve measurements of the metabolites was 0.7. Four steps are involved in the appearance and disappearance of the metabolites in and from the plasma-absorption of the parent drug into the portal circulation, conversion of the drug to metabolites in the liver, transfer of the metabolites into plasma, and elimination of the metabolites. It is known that the elimination half-life for nitroglycerin is very short, approximately 2-3 min. Plots of log C,,, (log metabolite concentration) versus time yielded straight line graphs in the terminal elimination phase. The apparent elimination half-life values, estimated from the terminal portions of the plasma metabolite concentration time curves, might be the true elimination half-life values for the metabolites. The elimination half-life values for 1 ,2-GDN were not significantly different across the formulations (p > 0.05, tablet: 1.01 f 0-30 h vs solution: 1-33 0-58 h). The elimination half-life values for 1,3-GDN were not significantly different across the formulations (p > 0.05, tablet: 1-34 f 0.58 h vs solution: 1.46 f 0.59 h).

Finally, it is of interest that solutions, but not sustained release tablets, of nitroglycerin occasionally induced detectable concentrations of nitroglycerin (albeit low) in plasma. This could have reflected an absorption or presystemic elimination phenomenon, or an experimental artefact, such as could occur as a result of nitroglycerin absorption when the oral solution passed through the mouth, in spite of scrupulous care exerted to prevent this happening. Benet and his colleagues have expressed the view that this potential artefact can be avoided, and have reached conclusions regarding the kinetics of intestinal absorption of the small proportion of the oral dose which is believed to be absorbed as unmetabolized nitroglycerin. It may be that, even with pulsed dosing over 8 h, the kinetics of presentation of nitroglycerin to mucous mem- branes are different with solution compared with tablet doses, permitting finite absorption from solutions.

152 H.-R. KWON, P. GREEN AND S . H . CURRY

ACKNOWLEDGEMENTS

We are grateful to the staff of the Clinical Research Center of Shands Hospital, University of Florida for their assistance with this project, and to our subjects. Additionally, we thank Dr Howard Berger and his colleagues for their interest and financial support.

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