Supplement to Phosphodiesterase 5 Inhibition Improves Beta Cell

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					Supplement to Phosphodiesterase 5 Inhibition Improves Beta Cell Function in the
Metabolic Syndrome

Supplemental Methods
        Written informed consent was obtained. The study protocol was approved by the
Vanderbilt University Institutional Review Board and conducted in accordance with the
Declaration of Helsinki.
        Tables A1 and A2 provide subject characteristics. The metabolic syndrome was defined
using the National Cholesterol Education Program (NCEP) criteria of 3 or more of the following:
fasting plasma glucose of at least 100 mg/dL, serum triglycerides of at least 150 mg/dL, serum
HDL cholesterol less than 40 mg/dL in men and less than 50 mg/dL in women, blood pressure of
at least 130/85 mm Hg, or waist girth of more than 102 cm in men and 88 cm in women.
Subjects with diabetes (defined by a fasting glucose of ≥ 126 mg/dL or medication use) and
those taking hormone therapy or statins were excluded. Subjects with significant cardiovascular
(other than hypertension), renal, pulmonary, endocrine (other than insulin resistance or
hyperlipidemia), or hematological disease were excluded. Pregnancy was excluded in women of
child bearing potential by measurement of urine ß-hcg at screening and on each study day.

         Figure A1 shows the study protocol.
         During the last week of each treatment, subjects received a nitrate- and sodium-
controlled, weight maintenance diet containing 200mmol/d sodium, 100mmol/d potassium, and
1000mg/d calcium while continuing to take study drug.
         All drugs and placebos were prepared by the Vanderbilt Investigational Pharmacy in
identical appearing capsules. Ramipril was initiated at 2.5mg/d for 3 days and titrated up to
5mg/d for 3 days, followed by 10mg/d for the remainder of the treatment period. Tadalafil was
initially given at a dose of 10mg/day for the first 3 days followed by 10mg every other day. The
daily “loading dose” was eliminated after the first 2 subjects because of side effects (see below).
Frequently Sampled Intravenous Glucose Tolerance Test (FSIVGTT)
         Baseline samples were collected at t=-15 min and t=-5 min for measurement of glucose
and insulin. At t=0, a bolus of 300mg glucose/kg body weight was administered in a 25%
glucose-saline solution administered over 1 minute. At t=20 min, a bolus of 0.02 units/kg body
weight of regular insulin (Actrapid, Novo Nordisk, Princeton, NJ) was given intravenously.
Blood samples were collected for measurement of glucose and insulin at times t=2, 3, 4, 5, 6, 8,
10, 14, 19, 22, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150, 170 and 180 min. Plasma glucose was
measured by the glucose oxidase method with a Beckman glucose analyzer. Plasma insulin
concentrations were determined by radioimmunoassay.

Laboratory and Statistical Analysis
        All plasma or sera were separated and stored at –80oC until the time of assay.
Fibrinolytic parameters
        Blood for measurement of PAI-1 and t-PA was collected in vacutainer tubes containing
acidified 0.105M sodium citrate (Becton Dickinson, Rutherford, NJ). PAI-1 activity was
measured using standardized commercial kits (Chromolize®, Biopool AB, Umea, Sweden), with
results expressed as units/mL (U/mL). Antigen concentrations were determined using a 2-site

enzyme-linked immunosorbent assay (Imulyse®, Biopool AB). Similarly, t-PA activity and
antigen were measured using Chromolize® and Tintelize® assays (Biopool AB).
RAAS parameters
        Blood for PRA and aldosterone were drawn into chilled tubes containing EDTA. PRA
was measured by radioimmunoassay (DiaSorin) for Ang I formation at pH 7.4 and 37oC.
Aldosterone was determined using a radioimmunoassay using 125I-aldosterone (MP
Biomedicals), a primary antibody to aldosterone (National Institute of Diabetes and Digestive
and Kidney Diseases National Hormone and Peptide Program, Torrance, CA), and a secondary
anti-rabbit -globulin antibody (Linco Research, St Charles, MO). Blood for Ang II was collected
in chilled tubes containing a cocktail of protease inhibitors.(1) Ang II was analyzed by
radioimmunoassay (Nichols Institute Diagnostics).(2) Serum ACE activity was determined by a
three-step colorimetric assay in which ACE hydrolyzes the substrate p-hydroxybenzoyl-glycyl-
L-histidyl-L-leucine, and subsequent reactions lead to the formation of quinoneimine dye, which
is measured spectrophotometrically (Fujirebio America Inc., Fairfield, NJ).
Urinary electrolytes
        Urine sodium and potassium concentrations were measured by flame photometry and
creatinine by the sodium picrate method.
NO metabolites, cyclic GMP and amino acids
        NO metabolites were measured using a modified Griess reaction (Oxford Biomedical
Research, Oxford, MI). Plasma cGMP was quantified by radioimmunoassay (Amersham
Pharmacia Biotech AB, Uppsala, Sweden). L-citrulline, L-arginine and alanine were measured
in protein-free extracts of fresh plasma. Amino acids were separated by cation exchange
chromatography on a Beckmann 7300 amino acid analyzer (Beckmann, Palo Alto, CA). After
derivatization with ninhydrin, primary amines were detected at 570 nm and secondary amines at
440 nm. Quantitation was achieved by instrument calibration with standard reference materials
(Sigma, St. Louis, MO).
Statistical Analysis
        Data are presented as means ± standard deviation in text and the tables and as means ±
standard error of the mean in Figures. The effects of treatment on hemodynamic, fibrinolytic
and metabolic parameters were assessed using a general linear model repeated measures
ANOVA in which the within-subject variable was treatment and the between subject variables
were race, gender and/or fasting blood glucose. When there was evidence for an effect of gender
or race, subgroup analyses were completed. To exclude a carryover effect in this crossover
study, we examined the effect of preceding treatment on ACE activity, PRA, aldosterone,
systolic blood pressure, diastolic blood pressure, glucose effectiveness, beta cell function, insulin
resistance, acute insulin response to glucose, and disposition index during placebo. There was
no effect of preceding treatment on responses to placebo (all p>0.15). A 2-tailed P value less
than 0.05 was considered significant. Analyses were performed using SPSS for windows
(version 16.0; SPSS).

Supplemental Results
        In addition to the 18 subjects who completed the study protocol, three additional
participants withdrew. Two withdrew for personal reasons. One female subject withdrew due to
muscle pain, dizziness and a low BP (80/40) after 1 week of treatment. Her treatment was
discontinued and unblinded (tadalafil and ramipril) and her symptoms resolved by the following
day. Serum creatine phosphokinase and aldolase levels were normal. The first 2 female subjects

enrolled also complained of muscle aches during at least one arm of the study but completed the
protocol; the symptoms were subsequently determined to be related to tadalafil use.
        The effect of treatment on DBP differed in the whites and non-whites studied. Tadalafil
reduced DBP (p=0.05) and accentuated the effect of ramipril in whites (p=0.02 for ramipril x
tadalafil interaction), after controlling for gender. There was no significant effect of tadalafil on
DBP in non-whites (Figure A2).
        There was no significant difference in circulating NO metabolites, L-citrulline, L-
arginine, alanine or cGMP concentrations among any of the treatment arms (Table A3).
        Neither ramipril nor tadalafil affected PAI-1 antigen, PAI-1 activity, tPA antigen, tPA
activity or the molar ratio of PAI-1 antigen to tPA antigen. (Table A4).

Supplemental Discussion
        We observed a significant interactive effect of ramipril and tadalafil on diastolic blood
pressure. That this effect was seen in whites, but not in blacks, is consistent with the mechanism
of action of tadalafil in inhibiting the degradation of cGMP. Data from many prior studies
indicate that black Americans are relatively resistant to cGMP-dependent vasodilation .(3-5)
Acute administration of tadalafil alone at a higher dose (20mg) has been reported to lower
ambulatory systolic and diastolic blood pressure,(6) but limited data are available on the
interactive effect of tadalafil and ACE inhibitors. Kloner et al. reported a small effect of a single
dose of 10mg tadalafil, compared to placebo, on supine systolic blood pressure in 16 men taking
        We found no effect of either ACE inhibition or PDE5 inhibition on markers of
fibrinolysis in this study. ACE inhibition decreases circulating PAI-1 concentrations when the
RAAS is activated by low salt intake or diuretic use.(8-11) ACE inhibition does not decrease
PAI-1, however, in salt-replete subjects in whom the RAAS is not activated.(12) Insulin,
glucose, and triglycerides (VLDL) all stimulate PAI-1 expression,(13) and it is possible that
these factors predominate over Ang II and cGMP in the regulation of PAI-1 synthesis in salt-
replete individuals with the metabolic syndrome.

Table A1. Baseline Subject Characteristics

        Characteristic                                                          N=18
        Gender, Women: Men (%)                                            6 (33): 12 (67)
        Race, White: Black: Hispanic (%)                                12 (67): 5 (28): 1(6)
        Body Mass Index (kg/m²)                                                36.5±5.1
        Fasting Blood Glucose (mg/dl)                                          96.4±8.8
        HDL-C (mg/dl)                                                         38.9±13.0
        Hypertensive, N (%)                                                     13 (72)
        SBP (mm Hg)                                                          131.8±10.8
        DBP (mm Hg)                                                           86.8±10.2
        Waist Circumference (cm)                                             118.1±10.1
        Serum Triglycerides (mg/dl)                                         199.7±130.7
       Data are presented as means ± standard deviation of the means. HDL-C indicates high density lipoprotein-
       cholesterol, SBP indicates systolic blood pressure, DBP indicates diastolic blood pressure.

Table A2. Criteria for Metabolic Syndrome among Subjects

          Characteristic                                       Male (N=12)        Female (N=6)
          Hypertensive, N (%)                                     9 (75)             5 (83)
          Elevated Fasting Blood Glucose, N (%)                   3 (25)             4 (66)
          Elevated Triglycerides, N (%)                          10 (83)              0 (0)
          Low HDL-C, N (%)                                        6 (50)             4 (66)
          Central adiposity, N (%)                              12 (100)            6 (100)
               HDL-C indicates high density lipoprotein-cholesterol.

Table A3. Effect of treatment on the renin-angiotensin-aldosterone system and biomarkers of nitric oxide
                                                                           Ramipril +         P               P
                               Placebo        Tadalafil     Ramipril                                                    Ramipril x
                                                                            Tadalafil      Ramipril        Tadalafil
24hr Na excretion, mmols     132.4±54.2     141.7±50.5     147.8±53.6     157.7±37.8        0.12            0.38           0.85
ACE activity, U/L             33.5±20.8      36.1±20.4      18.0±14.3      15.8±11.3      <0.001            0.96           0.26
PRA, ng/mL/hr                   1.4±1.1        1.3±0.9       6.6 ± 8.4       6.9±6.1       0.001            0.99           0.80
Angiotensin II, pg/mL         53.3±26.9      52.8±25.0      43.6±17.0      43.7±16.8        0.04             1.0           0.86
Aldosterone, ng/dL             16.4±3.2       14.8±4.5       14.7±4.8       14.8±3.0        0.13            0.21           0.38
cGMP, pM/mL                   50.1±53.4      37.9±21.4      36.3±18.2      44.5±34.2        0.50            0.57           0.25
NOx, µM                       20.7±11.7      23.8±15.2      21.8±18.7      21.0±14.0        0.66            0.61           0.62
L-citrulline, µM              37.8±21.6      52.5±61.8      38.1±21.4      35.4±18.6        0.27            0.19           0.22
L-arginine, µM                72.0±15.2      80.4±33.2      68.3±11.7      71.4±13.1        0.19            0.17           0.57
Data are presented as means ± standard deviation of the means. Na indicates sodium, ACE activity indicates angiotensin-converting
enzyme activity, PRA indicates plasma renin activity, cGMP indicates cyclic guanosine monophosphate, NOx indicates nitric oxide

Table A4. Effect of treatment on fibrinolytic biomarkers
                                                                          Ramipril +          P               P
Fibrinolytic marker         Placebo         Tadalafil       Ramipril                                                    Ramipril x
                                                                           Tadalafil       Ramipril        Tadalafil
PAI-1 antigen, ng/mL      18.1 ± 12.5      18.4 ± 13.4    21.5 ± 14.4    18.2 ± 11.8       0.32             0.48            0.32
PAI-1 activity, IU/mL     23.0 ± 19.4      20.8 ± 16.5    25.7 ± 17.6    20.8 ± 12.2       0.66             0.22            0.36
tPA antigen, ng/mL         10.7 ± 3.5       10.7 ± 3.5     10.9 ± 3.9     10.7 ± 3.5       0.61             0.43            0.68
tPA activity, IU/mL       0.26 ± 0.27      0.24 ± 0.18    0.22 ± 0.28    0.24 ± 0.23       0.56             0.99            0.67
Molar Ratio PAI-1:t-PA      2.2 ± 1.2        2.6 ± 1.9      2.8 ± 1.9      2.3 ± 1.0       0.50             0.66            0.12
Data are presented as means ± standard deviation of the means, PAI-1 indicates plasminogen activator inhibitor, tPA indicates tissue
       plasminogen activator, Molar ratio indicates PAI-1 antigen: tPA antigen molar ratio

Figure A1. Study protocol.

          Study drugs                Study drugs                  Study drugs              Study drugs

                       Diet                       Diet                    Diet                         Diet

                                             24 hour urine and study day
                        Study drugs: placebo, ramipril, tadalafil, or ramipril + tadalafil

            1      2         3   4     5      6          7    8     9    10      11   12     13   14      15   16

                                                         time (weeks)

Figure A2. Effect of treatment on diastolic blood pressure (DBP) in whites and non-whites. Gender was included as a covariate in the


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