Pilot Study of Losartan and N-acetylcysteine as Inhibitors of Muscle Oxidative Stress in Elderly

Condition(s) targeted: Aging

Intervention: N-acetylcysteine (Drug); Losartan (Drug); PLacebo losartan and placebo NAC (Other)

Phase: Phase 1/Phase 2

Status: Not yet recruiting

Sponsored by: The University of Texas, Galveston

Official(s) and/or principal investigator(s):
Melinda Sheffield-Moore, PhD, Principal Investigator, Affiliation: UTMB
Astrid M Horstman, PhD, Study Director, Affiliation: UTMB

Overall contact:
Melinda Sheffield-Moore, PhD, Phone: 409-772-8707, Email: melmoore@utmb.edu

The general hypothesis is that elderly have diminished nutritive flow to skeletal muscle and impaired capacity for building of muscles. In aging populations, this decreased ability to build muscles may represent a tipping point in the progression towards chronic physical frailty and disability. The goal is to examine whether novel pharmacologic therapies can improve nutritive blood flow to the muscles and muscle building in the elderly.

The purpose of this study is 1) to determine if losartan administration will enhance the building of muscles via proteins and suppress muscle breakdown 2) to determine if N-acetylcysteine (NAC) will enhance blood flow to muscles and the building of muscles via proteins.

The investigators will study community dwelling, healthy older men and women (60-85 years). Subjects will be randomized to one of three groups:

Experimental Group 1: Placebo losartan and placebo N-acetylcysteine (NAC). Experimental Group 2: losartan (25mg/dose) and placebo N-acetylcysteine (NAC). Experimental Group 3: N-acetylcysteine (NAC) (50 mg/kg/dose) and placebo

Subjects will admit the night before and get their first dose of NAC/ losartan/ placebo with dinner. Subjects will be fasted after 10 pm. The next morning at 6 am blood samples will be taken and leg blood flow (LBF) will be measured. Subjects will receive their second dose of NAC/ losartan/ placebo. As from 6 am every hour blood will be drawn until 1 pm. At 8am, the second biopsy is taken and LFB and CEU will be measured. At 11am they get their third dose of NAC/ losartan/ placebo together with leucine (oral) and again a muscle biopsy is taken and LBF is measured. Leucine is anabolic to skeletal muscle of elderly and so the investigators will use it to stimulate muscle building.

Official title: Pilot Study of Losartan and N-acetylcysteine as Inhibitors of Muscle Oxidative Stress in Elderly

Study design: Allocation: Randomized, Endpoint Classification: Efficacy Study, Intervention Model: Parallel Assignment, Masking: Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor), Primary Purpose: Treatment

Primary outcome:

Lean body mass

Fatigue

Fractional synthetic rate (FSR)

Secondary outcome:

Blood Flow and Muscle Perfusion

Signaling pathways involved in muscle protein synthesis and degradation

Muscle morphology and satellite cell activation.

Detailed description: The overall goals of this project are to 1) provide control (ambulatory) data for a subsequent grant proposal in which losartan will be tested for its ability to reduce loss of muscle mass and function during simulated hospitalization (SH) in older individuals, 2) determine the effect of losartan on basal and meal-stimulated muscle protein synthesis, in older individuals, and 3) determine the effect of N-acetylcysteine (NAC) on basal and meal-stimulated muscle protein synthesis in older individuals.

Theoretical Framework The investigators have determined that skeletal muscle tissue perfusion in healthy elderly is blunted compared to young. Additionally, local skeletal muscle vasodilation with sodium nitroprusside or exercise increased muscle perfusion in the elderly to more youthful levels, and when performed in combination with increased plasma amino acid availability, greatly amplified muscle protein synthesis. In the absence of physical activity, protein ingestion via traditional mixed nutrient meals is the primary means of stimulating muscle protein anabolism via an increase in precursor availability. However, anabolic efficiency and protein synthetic response to a mixed nutrient meal is often blunted in elderly. Therefore, they propose that a common vasodilatory drug, losartan, newly discovered to block TGF-β activation and signaling in skeletal muscle by antagonizing the angiotensin II type 1 receptor (AT1), will restore skeletal muscle nutritive flow via its vasodilatory actions and concomitantly prevent angiotensin II-induced muscle wasting. Notably, the vasodilatory effects of losartan are thought to be at least partially due to alleviation of oxidative stress (1) and the investigators previously found evidence for age-related oxidative stress in older individuals who exhibited resistance to the anabolic effects of amino acids (2). This latter finding is consistent with a previous report that antioxidant administration alleviates age-related anabolic resistance to leucine in rats (3). Accordingly, in the current study the investigators postulate that administration of the antioxidant N-acetylcysteine (NAC) will restore skeletal muscle nutritive blood flow and sensitivity to the anabolic effects of leucine. Thus, in the current study they propose to administer placebo, losartan or NAC in a group of healthy elderly to target key mechanisms responsible for inactivity-induced losses of lean body mass and function.

Hypotheses and Specific Aims

The investigators will test the following hypotheses in older men and women (60-85 y):

1. Losartan therapy will enhance skeletal muscle nutritive blood flow and nutritive flow-mediated muscle protein synthesis, will improve skeletal muscle redox homeostasis, and will suppress skeletal muscle TGF-β activation/signaling.

2. NAC therapy will enhance skeletal muscle blood flow, nutritive flow-mediated muscle protein synthesis, and will improve skeletal muscle redox homeostasis

The following specific aims will be tested:

1. To determine if losartan administration will enhance nutritive flow-mediated muscle protein synthesis and suppress activation of TGF-β.

2. To determine if NAC will enhance skeletal muscle blood flow, nutritive flow-mediated muscle protein synthesis, and improve skeletal muscle redox homeostasis.

RESEARCH DESIGN AND METHODS

Subjects. The investigators will conduct a randomized, double-blind, placebo-controlled intervention study designed to clinically and mechanistically assess novel therapies targeted at minimizing or preventing diminished nutritive flow to skeletal muscle and impaired capacity for net muscle protein anabolism in elderly. Subjects will be randomized to one of three groups:

Group 1: Placebo losartan and placebo N-acetylcysteine Group 2: Losartan (25mg/dose) and placebo N-acetylcysteine Group 3: N-acetylcysteine (50 mg/kg/dose) and placebo losartan Recruitment. They will recruit and study 24 (n=8 per group) elderly men and women representative of the ethnic makeup of the United States (see Targeted/Planned Enrollment). The investigators will screen 48 people to achieve a total of 24 completed studies Subjects will be recruited through the Sealy Center on Aging Volunteer Registry at the University of Texas Medical Branch (UTMB). This registry provides a contact point for more than 600 older volunteers. Recruitment coordinators within the Center are trained to telephone screen individuals for past medical history, current health status and medications to determine if they will "fit" into a particular study. Recruitment and study coordinators will then arrange to meet the volunteer at the Clinical Research Center (CRC) to discuss the study and review the consent form. If consented, volunteers are screened and deemed eligible if they meet all inclusion criteria.

Rationale for Subject Selection. The rationale for studying healthy elderly is that elderly have diminished nutritive flow to skeletal muscle and impaired capacity for net muscle protein anabolism. While it is understood that older subjects will fall along a continuum for any measurement of physical frailty, the investigators will perform this study in freely living subjects whose daily life does not depend on the direct assistance of others.

Methods. Subjects will admit the night before and get their first dose of NAC/ losartan/ placebo with dinner. Subjects will be fasted after 10 pm. The next morning at 6 am blood samples will be taken and leg blood flow (LBF) will be measured. Subjects will receive their second dose of NAC/ losartan/ placebo. As from 6 am (t = 0) every hour blood will be drawn until t = 7. At t = 2, the second biopsy is taken and LFB and CEU will be measured. At t = 5 they get their third dose of NAC/ losartan/ placebo together with leucine (oral) and again a muscle biopsy is taken and LBF is measured. Leucine is anabolic to skeletal muscle of elderly and so they will use it to stimulate anabolism.

Screening Visits. All procedures and risks associated with the study will be explained to the volunteer while obtaining informed consent. To determine eligibility for participation, all subjects will be screened with a battery of tests, including: 1) a physical examination and medical history (including history of asthma); 2) blood tests (25 ml blood), including cell count and liver function, hepatitis B and C, HIV, cholesterol, and electrolytes; coagulation panel; Urea Nitrogen (BUN), creatinine 3) urinalysis for kidney function; 4) an electrocardiogram (ECG); 5) an oral glucose tolerance test (OGTT) and 6) a 2D echo with agitated saline.

- Oral Glucose Tolerance Test: On the Screening Visit all subjects will undergo a blood

test for fasting blood glucose and then undergo a standard 2 hour oral glucose tolerance test. This test is important because losartan therapy has been suggested to improve insulin sensitivity in patients with type 2 diabetic nephropathy and reactive oxygen species have been reported to improve insulin sensitivity, an effect blocked by NAC.

- Baseline plasma creatinine and electrolytes will be measured. Once all screening test

reports have been generated, the study team physicians and the PI will meet to determine subject eligibility. Eligible subjects will be notified by phone and scheduled for second screening visit.

- Second screening visit: 2D echo Doppler with agitated saline will be performed to rule

out right to left shunt for administration of Definity® microbubbles for assessment of muscle microvascular perfusion.

Randomization. All subjects will undergo block randomization based upon age and sex to ensure balanced experimental groups. Randomization will be performed by our biostatistician.

Enrolled subjects will arrive at 3pm the day before measurement day at the CRC. Measurement of Muscle Mass and Volume with DEXA (Night before). Muscle mass will be determined using a DEXA scan (GE Lunar iDXA). The patient will be placed supine on the table and a whole body DEXA performed. The procedure takes ~15 minutes and radiation exposure is comparable to the dose received in a 2-hour airplane flight (~0. 04 mrem). The same CRC operator will perform all DEXA analyses. The body will be divided in subregions: Truncal area, android, gynoid, right and left arm, and right and left leg. The investigators will be able to measure lean and fat mass individually for each subregion. The investigators will also analyze specific regions of interest for lean body mass, including the thigh and calf. The coefficient of variation for repeated measures of lean tissue is <1%.

Femoral Doppler Blood Flow and Heart Rate (t = 0, 2, 5, 6, 7) Subjects will be evaluated via Doppler ultrasound after placement of three ECG leads for heart rate and R wave monitoring. For the two-dimensional (2-D) and Doppler ultrasound measurements, an ultrasound system (HDI-5000; Philips Medical Systems, Bothell, WA) with a linear array transducer (L7-4) will be used with a transmit frequency of 12MHz. 2-D imaging of the common femoral artery will be performed in the long axis. Images will be triggered to the R wave of the cardiac cycle, and the femoral artery diameter will be measured using online video calipers. A pulsed-wave Doppler sample blood volume will be placed at the same location in the center of the artery, and the mean blood velocity will be measured using online angle correction and analysis software. Femoral artery mean blood flow will be calculated from 2-D and Doppler ultrasound data using the equation: Q = vπ ∙ (d/2)2, where Q is femoral blood flow, v is mean femoral artery blood flow velocity, and d is femoral artery diameter. The investigators have used this technique extensively in the first cycle of this grant.

Contrast-enhanced Ultrasound (Night before and at t = 2, t = 7) All subjects will undergo imaging of the vastus lateralis muscle using an HDI-5000 and a P4-2 transducer. Imaging will be performed in a transaxial plane over the lateral portion of the vastus lateralis muscle group using power Doppler imaging after the continuous infusion of Definity® microbubbles. A mechanical index of 1. 3 will be used. Depth, focus, and gain will be optimized and held constant throughout each study. Intermittent imaging will be performed using an internal timer. Data will be recorded on an SVHS videotape. A suspension of octafluoropropane gas-filled albumin microbubbles (Definity®, Lantheus Medical) will be infused intravenously at a rate of 3. 0 mL/min for 8 min. Once systemic microbubble concentrations reach steady-state (~1. 5 min), background images will be obtained at a frame rate of 1 second. Intermittent imaging will then be performed at a pulsing interval (PI) ranging from 1-25 seconds, thus allowing progressively greater replenishment of the ultrasound beam elevation between destructive pulses. At least three images will be acquired at each PI. Videotape images will be digitized to an offline image analysis system. Several background frames will be averaged and digitally subtracted from similarly averaged frames at each PI. Background-subtracted video intensity (VI) at each PI will be measured from a region of interest (ROI) placed around the vastus lateralis muscle. PI vs. VI data will be fitted to

the function: y = A (1 - e-βt), where y is video intensity at PI t, A is plateau video

intensity (an index of microvascular blood volume) and β is the rate constant, which provides a measure of microvascular flow velocity. The investigators have used this technique extensively in past studies.

Losartan Administration (Night before and t = 0, t = 5) Losartan is a novel orally available FDA-approved drug that pharmacologically inhibits the renin-angiotensin-aldosterone system. In particular, its primary mechanism of action is to inhibit production of the vasoconstrictor angiotensin II. It has a strong record of safety and efficacy and benefits patients with all grades of heart failure. losartan dosing is not based on individual body weight. The max dosing regimen proposed (25 mg losartan/day) is predicted to drop systolic blood pressure by approximately 5-12 mmHg and diastolic by 4-8 mmHg. The response is expected to be most profound among individuals with the highest systolic blood pressure. For safety purposes the investigators will administer losartan at 25 mg BID.

N-acetylcysteine (NAC) Administration (Night before and t = 0, t = 5) NAC is a cysteine donor and antioxidant approved for use in humans, orally or intravenously. When given to treat acetaminophen overdose, NAC is typically given as a large bolus (~120-150 mg/kg over 15-60 minutes), followed by continued administration over the next 20-36 hours. NAC has also been used as a cysteine donor for malnourished children, HIV infected individuals, and patients with pulmonary disease. When administered orally, the sulfur content of NAC reduces palatability. As a result, it is typically mixed with soda, juice, or a flavored drink mix. A recent study found that NAC, administered as a 75mg/kg oral bolus mixed with flavored drink mix, was well tolerated and markedly influenced 3-methylhistidine levels. The investigators will follow this strategy, administering 50 mg/kg doses mixed with flavored drink.

With both oral and intravenous administration, most side effects are associated with the initial bolus loading period. With intravenous infusion, "anaphylactoid" reactions are more common and potentially more serious than with oral administration. As a result, an intravenous loading protocol was developed by McKenna's research group specifically to safely study acute effects of NAC in skeletal muscle. In this protocol, subjects receive an initial bolus infusion of 125 mg/kg/h NAC for 15 minutes, followed by constant infusion at 25 mg/kg/h thereafter; this protocol is well-tolerated with minimal side effects. As no established protocol exists for the chronic infusion of NAC with the aim of improving skeletal muscle redox homeostasis and anabolic sensitivity during hospitalization in older adults, the investigators propose to administer NAC orally, to reduce the likelihood of anaphylactoid reactions, in 50 mg/kg boluses administered 3 times each day. Our rationale is as follows: 1) As opposed to the setting of acute acetaminophen toxicity, oxidative stress is not expected to develop suddenly during hospitalization-induced inactivity; thus, a more gradual approach to a plateau in systemic NAC levels should be satisfactory and should minimize the likelihood of NAC-induced side effects, 2) based on a recent study of NAC effects on exercise-induced oxidative stress, such an infusion should affect redox status in vivo, 3) the total NAC dose per day will be 150 mg/kg, which is approximately half of the total amount administered over the first 24 hours following acetaminophen overdose or in a previous study of NAC effects in healthy young controls (~320 mg/kg).

Although not expected based on the literature (i. e. in the absence of bolus dosing), subjects will be monitored for side effects as described previously following NAC administration to healthy volunteers: peripheral effects (swelling, sweating, erythema, pruritis, conjunctivitis), altered sensations (lightheadedness, drowsiness, dysphoria, metallic taste), gastrointestinal disturbances (nausea, dyspepsia, flatulence, diarrhea), and hypotension. Side effects will be assessed and recorded by CRC nursing staff as well as by the investigators. Our intent is to avoid side effects and, if they should occur, manage them with reduced dosing or, if necessary, diphenhydramine administration, as directed by the study physician(s). Subjects with a history of asthma will be excluded, as they have been reported to experience more severe anaphylactoid reactions to NAC.

Any uneaten food items will be recorded. Water will be provided ad libitum. Metabolic Studies for Fractional Synthesis Rate of Muscle Protein (Night before, t=2, t=5, t=6 t=7) At night subjects will be given a standardized dinner, and allowed only ad libitum water thereafter. The next morning, fasting anthropometric indices (height, weight, leg volume) will be recorded. The subject will then undergo a stable isotopic study to measure muscle protein turnover.

For the tracer study, a forearm catheter will be placed for tracer infusion, and a retrograde catheter will be placed in the opposite hand for arterialized blood sampling, with the hand heated thereafter. After collecting a background blood sample, a primed-continuous infusion of L-[ring-13C6] Phenylalanine (priming dose=2 umol/kg; infusion rate = 0. 05 umol/kg/min) will be started to measure muscle protein synthesis and boluses of [methyl d3] methionine (17. 5umol/kg) and [methyl d3] 3-methylhistidine (0. 5umol/kg) will be infused to measure myofibrillar protein synthesis and breakdown using the pulse-bolus method when the leucine is taken. At t=2 the first muscle biopsy will be taken from the vastus lateralis using aseptic procedure and local anesthesia with lidocaine to measure basal muscle protein synthesis rate with the precursor-product model (see E. Human Subjects for details on protections against risks), cell signaling, and redox homeostasis (reduced and total glutathione). Between t=5 and t=7, frequent arterialized blood samples will be taken from the hand vein to measure phenylalanine, methylhistidine and methionine enrichments and concentrations; additionally, blood flow and muscle perfusion will be measured by real-time Doppler and CEU (see Methods). At the end of basal period (t=5) a second, muscle biopsy will be taken to measure muscle protein synthesis.

After the second biopsy, subjects will consume a test meal, with 10 grams of leucine, within 10 minutes. To avoid disruption of the phenylalanine isotopic steady state due to ingestion of unlabeled amino acids, L-[ring-13C6]Phenylalanine, [methyl d3] 3-methylhistidine and [methyl d3] methionine will be added to the meal to enrich it to the expected arterial enrichments. The investigators have shown that this approach can adequately prevent changes in the arterial isotopic enrichment during bolus ingestion of an amino acid supplement, thus allowing for steady state equations to calculate muscle protein synthesis. After meal intake the investigators will follow the blood amino acid concentrations and enrichments with frequent blood sampling. At t=6 and t=7 a third and fourth muscle biopsy will be taken to measure muscle protein synthesis, cell signaling, and redox homeostasis during treatment and in response to the meal. Between t=6 and t=7 Doppler blood flow and muscle perfusion (CEU) will be measured again to determine the effect of treatment and meal on these parameters. After the last muscle biopsy, the tracer infusion will be stopped and the subject will be made comfortable and fed.

Fatigue (Night before, t = 3, t = 7) Handgrip strength and skeletal muscle fatigue will be assessed using handgrip dynamometry. Strength (maximal voluntary contraction) will determined from 3 maximal efforts, separated by 1 minute. Performance fatigue will be measured as the total number of successful 3 second contractions at 50% maximal voluntary contraction, with a 50% duty cycle, as described in a previous study of NAC effects on handgrip fatigue. To assess perceptual fatigue, the investigators will administer two questionnaires: the Brief Fatigue Inventory Score and Multidimensional Fatigue Symptom Inventory-Short Form (MFSI-SF). In addition, perceptual fatigue following the handgrip fatigue protocol will be assessed using a 0-10 scale (0 = no fatigue, 10 = worst fatigue imaginable).

Methods detailed in this section are common to all groups. Analytical methods and procedures are presented below in translational context, from molecular to functional.

Sample storage and processing. Blood samples to measure amino acid enrichments and concentrations will be collected in an ice-cold sulfosalicylic acid solution containing

appropriate internal standards, spun, and the supernatant stored in cryovials at - 80 Celsius

until further analysis. Blood samples for hormonal and cytokine assays will be collected in clotting-activated tubes, immediately spun and the supernatant serum will be stored in

cryovials at - 80 Celsius. Muscle samples for measurement of amino acid free enrichment and

incorporation in contractile proteins, and for molecular biology assays will be immediately rinsed with ice-cold saline, blotted and quickly (less than 5 seconds) frozen in liquid

nitrogen and stored in cryovials at - 80 Celsius until further analyses. Immediately after

collection, muscle samples for microscopic and morphologic analysis will be mounted and snap

frozen at - 80 Celsius until later analysis.

Measurement of intracellular signals. Muscle tissue samples (100-200 mg) will be immediately quick-frozen in liquid nitrogen following biopsy, and kept under liquid nitrogen until analyzed. Expression of NF-κB fragment will be measured using an ELISA kit (TransAM, Active Motif, ), Total protein abundance of matrix metalloproteinase (MMP) and FKBP12 will be measured using Western blotting. Phosphorylation of Akt, AMPKα, mTOR, S6K1, FoxO, eIF-2β, eIF-4, 4E-BP1, Smad2/3 and GSK-3β will be measured with phospho-Akt (Ser473) antibody, phospho-AMPKα (Thr172), phospho-mTOR (Ser2448) antibody, phosphor-p70 S6 kinase (Thr389) antibody, phospho-FoxO1 (Ser256), phosphor-FoxO3a (Ser318/321) antibody, phospho-FoxO3a (Ser192) antibody, phospho-eIF-2β (Ser 540) antibody, phospho-eIF-4E (Ser 209) antibody, phosphor-4E-BP1 (Thr37/46) antibody, phospho-Smad2 (Ser465/467) antibody, phospho-Smad3 (Ser423/425) antibody and phospho-GSK-3β (Ser9) antibody (Cell Signaling Technology, Inc., Beverly, MA), respectively, and chemiluminescence autoradiography as previously described (47). Western blot signals will be analyzed with a BioRad ChemiDoc Chemiluminescence imaging system (BioRad, Hercules, CA), which detects ECL signals over 5 orders of magnitude, and its sensitivity range far exceeds that of conventional densitometry.

Total abundance of GSH/GSSG will be measured using commercial kits (Cayman Chemical, Ann Arbor, MI). MnSOD and CuZnSOD will be evaluated using an activity assays. Notably, mice lacking CuZnSOD are a model for age-related muscle loss and exhibit increased production of reactive oxygen species. Likewise, MnSOD activity has been suggested to play a role in age-related muscle loss and dysfunction.

Measurement of muscle morphology and satellite cell activation. Muscle morphology will be determined using immunohistochemical techniques. Muscle fiber cross sectional area (CSA), proportion, and assessment of intramuscular oxidative enzyme content, glycolytic enzyme content, and lipid content will be determined using standard methods. Tissue sections will be cut at 10 μm thickness in a cryostat maintained at 25ºC, mounted on Fisherbrand Superfrost®/Plus microscope slides (Fisher Scientific, USA). Satellite cell content will be determined using anti-CD56 antibody directed against the neural cell adhesion molecule (NCAM) (identical to the Leu 19 antigen) [Clone MOC-1, (DakoCytomation, Carpinteria, CA) and Pax7. For muscle cell boundary polyclonal rabbit anti-laminin antibody directed against laminin (extracellular matrix protein) (DakoCytomation) will be utilized. Fiber typing will be accomplished with anti-Myosin Heavy Chain-slow (MyHC-slow, Sigma, St. Louis, MO). Nuclear marker 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI; Molecular Probes) will be used to localize DNA (nuclei). Digital images will be captured on a Zeiss Axio-Cam Epi-Fluoroscent microscope and analyzed using the Zeiss AV4 software.

Femoral Doppler Blood Flow. Contrast-enhanced Ultrasound. Immunoassay of Serum and Muscle Inflammatory Biomarkers. For both experimental groups, the investigators will assess various serum and muscle cytokines including TNF-a, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, GM-CSF, and IFN-γ. Samples will be measured by immunoassay using the Millipore high sensitivity Human Cytokine-13plex kit (Millipore, Billerica, MA). TGF-β will be determined using the Millipore Single Plex kit.

Hormone Assays. Serum concentrations of CRP, IGF-I, IGFBP-3, insulin, hGH, C-peptide, and urinary cortisol will be measured using a high-throughput, continuous random access immunoassay analyzer, Immulite 2000. Briefly, after the sample is incubated with an alkaline phosphatase-labeled reagent, the reaction mixture is separated from the bead by spinning the reaction tube at high speed along its vertical axis. The bound label specific to each analyte is then quantified using the dioxetane substrate to produce light. Amount of light emitted is proportional to the amount of analyte in the sample. Losartan, angiotensin II, renin, and aldosterone will be measured by UTMB Clinical Services.

GCMS Analysis. The enrichment of free L-[ring-13C6]Phenylalanine and 3-Methylhistidine in blood and tissue fluid will be measured by GCMS after addition of appropriate internal standard U-13C9-15N Phenylalanine , precipitation of blood and tissue proteins with sulfosalicylic acid, extraction with cation exchange chromatography, and tert-butyldimethylsilyl derivatization (t-BDMS). Correction for skewed isotopomer distribution and overlapping spectra will be performed as previously described. The incorporation of L-[ring-13C6]Phenylalanine, [methyl d3] methionine and [methyl d3] 3-methylhistidine in the mixed and myofibrillar muscle proteins will be measured after protein extraction and hydrolysis, amino acid extraction with cation exchange chromatography, t-BDMS derivatization, and GCMS analysis.

Calculation of muscle protein synthesis and myofibrillar protein breakdown. The fractional synthesis rate (FSR) of mixed muscle proteins will be calculated from the incorporation rate of L-[ring-13C6]Phenylalanine into the mixed and myofibrillar muscle proteins, and the free-tissue phenylalanine enrichment:

(1) where EP/t is the slope of the straight line that fits the protein-bound phenylalanine enrichment across two sequential biopsies, t is the time interval encompassing the two biopsies, EM(1), and EM(2) are the phenylalanine enrichments (tracer/tracee) in the free muscle pool in the two biopsies. The results are presented as %.h-1. Myofibrillar protein synthesis and breakdown will be calculated using the pulse-bolus method.

D. 5. Statistical Considerations Analyses for all aims will primarily be statistical models to assess the effects of the two main treatment variables of losartan (Yes vs. No) and NAC (Yes vs. No). These two factors along with the control (placebo losartan plus placebo NAC) condition form the three groups of interest for the statistical models.

Statistical Analysis. Data for most outcomes will be collected at baseline and during the performance of the metabolic study. Our general approach to address the specific and secondary aims is to develop an analysis of variance (ANOVA) model for each outcome of interest. Only losartan and NAC effects will be tested in these models, as a combined (losartan + NAC) treatment group is not proposed. Each outcome will have a different ANOVA model.

The main outcomes of FSR will have 3 measures. The statistical models will be fit with a maximum likelihood based class of models called mixed models, which allow some dropout to occur without introduction of bias. Common software is available such as MIXED procedure in SAS. Main effects will be assessed. A covariance structure will be chosen according to suggestions by Grady and Helms. Mean differences in outcomes and 95% confidence intervals will be reported.

Gender differences will be assessed by including a gender covariate in the model. The

investigators will also examine the (gender - treatment) interaction to assess if the

treatment is equally effective for both genders. If that term is not significant, the investigators will initially leave gender in the model for potential adjustments. If the gender term is not significant, it will imply that gender does not add information to the model and it will be dropped.

Sample Size and Power. A power analysis was conducted to assess the ability of the study design to detect statistical significance and to avoid type II errors. Estimates of variation are from data that originated in the PI's lab as well as published studies. Conservative assumptions are made so as to not overestimate power. Power is based on change across two time points or at the end of the study.

The strategy is to demonstrate power for each outcome using ANOVA models, which address all specific aims simultaneously.

FSR Extensive preliminary data are available from our research team regarding changes in protein synthesis. The primary statistical approach is ANOVA and the study design described above for Aim 1. Preliminary data from the PI's lab were available for the outcome of FSR. The mean increase in FSR with vasodilator (SNP) infusion was 0. 02, with a SD of the change score of 0. 026. Based on a previous animal study which found that antioxidant administration restored amino acid stimulation of muscle protein synthesis, along with the fact that losartan and NAC may exert non-perfusion related effects that could augment anabolic responses, the investigators estimate a change of 0. 07 with each treatment, with a SD of the change score of 0. 026. Power calculations from the SAS procedure GLMPOWER indicate with N=8 per group (24 total) will provide >99% power to detect the treatment effects, using a 2-sided alpha level of significance of 0. 05.

Summary: Sample size calculations demonstrate that N=8 subjects per group (N=24 total) will provide sufficient power to assess each of our specific aims, across two main outcomes for assessing lean body mass and functional assessment. The investigators also show that they can study FSR with this design. As in all human intervention studies, the investigators expect attrition. To account for an expected attrition rate of 20% the investigators will randomize 1. 2x8=10 subjects per group (N=30 total). The investigators will screen 48 people to achieve a total of 24 completed studies.

Future Studies Many potential avenues may be explored following these experiments. If our hypothesis is correct, and findings that losartan is capable of significantly improving nutritive flow to skeletal muscle and capacity for net muscle protein anabolism, the investigators will pursue longer-term outcome studies with this novel vasodilator, perhaps during a simulated hospitalization period. Potential future studies include looking further into the amino acid signaling pathways and determining if suppression of TGF-β blocks transcriptional upregulation of the ubiquitin E3 ligases. However, if findings that losartan does not promote nutritive flow to skeletal muscle or the capacity for net muscle protein anabolism, then the investigators will further examine the mechanisms behind the apparent lack in nutritive flow mediated stimulation of muscle protein synthesis (e. g., studies with amino acids to determine where the defect lies in the intracellular signaling pathway). If losartan or NAC can successfully improve nutritive flow to skeletal muscle and capacity for net muscle protein anabolism in healthy older subjects, the investigators will pursue additional outcome studies during and following inactivity, like common elective surgeries such as hip and knee replacement.

Minimum age: 60 Years. Maximum age: 85 Years. Gender(s): Both.

Criteria:

Inclusion Criteria:

1. Age between 60-85 years.

2. Ability to sign informed consent.

3. Ability to sign consent form.

4. Ability to pass a mini-mental status exam (score >23 on the 30-item Mini Mental State Examination, MMSE).

5. Free-living, prior to admission.

Exclusion Criteria:

1. Subjects with cardiac abnormalities considered exclusionary by the study physicians (e. g., unstable angina or a cardiology-confirmed ECG that demonstrates cardiac abnormalities such as > 0. 2 mV horizontal or downsloping ST segment depression, frequent arrhythmia's (> 10 PVC/min), or valvular disease).

2. Subjects with uncontrolled metabolic disease, including liver or renal disease.

3. Subjects with vascular disease characterized by a combination of risk factors of peripheral atherosclerosis. (e. g., uncontrolled hypertension, obesity, uncontrolled diabetes, hypercholesterolemia > 250 mg/dl, claudication or evidence of venous or arterial insufficiency upon palpitation of femoral, popliteal, and pedal arteries.

4. Any history of hypo- or hyper-coagulation disorders. (e. g., Coumadin use or history of DVT or PE).

5. Subjects with chronically elevated systolic pressure >170 or a diastolic blood pressure > 100. Subjects may be included if they are taking medication and have a blood pressure below these criteria.

6. Subjects with cancer or recently (6 months) treated cancer other than basal cell carcinoma.

7. Any subject currently on a weight-loss diet or a body mass index > 33 kg/m2.

8. Inability to abstain from smoking for duration of study.

9. A history of > 20 pack per year smoking.

10. Subjects with atrial fibrillation, history of syncope, angina, or congestive heart failure.

11. Any subject that is HIV-seropositive or has active hepatitis.

12. Recent anabolic or corticosteroids use (within 3 months).

13. Subjects with low hemoglobin or hematocrit (i. e., lower than accepted lab values).

14. Agitation/aggression disorder.

15. Dementia.

16. History of stroke with motor disability.

17. A recent history (<12 months) of GI bleed.

18. Alcohol (more than 3 drinks per day) or drug abuse.

19. Polycystic ovary syndrome (PCOS) and/or hyperthecosis.

20. Non-classical adrenal hyperplasia.

21. Cushing's syndrome.

22. Pregnancy.

23. Hyperprolactinoma, hypothyroidism.

24. Lactose intolerance.

25. Subjects with coronary heart or mitral valvular rheumatic heart disease.

26. Subjects with impaired renal function and/or renal artery stenosis.

27. Subjects with pulmonary hypertension.

28. Subjects on any medications known to vasodilate the peripheral arteries.

29. Subjects taking NSAIDs

30. Physical dependence or frailty (impairment of activities of daily living, ADLs).

31. History of falls (>2/year).

32. Depression (>5 of the 15 items on the Geriatric Depression Scale (GDS).

33. Subjects suffering malnutrition or with a BMI < 20 kg/m2 with low albumin or transferrin.

34. Asthma

35. Any other condition or event considered exclusionary by the PI and covering faculty physician.

Melinda Sheffield-Moore, PhD, Phone: 409-772-8707, Email: melmoore@utmb.edu

University of Texas Medical Branch, Galveston, Texas 77555, United States

Starting date: July 2011
Last updated: June 27, 2011