704

Effects of Losartan on Blood Pressure, PlasmaRenin Activity, and Angiotensin II in Volunteers

Michael R. Goldberg, Wesley Tanaka, Aaron Barchowsky, Thomas E. Bradstreet,Jacqueline McCrea, Man-Wai Lo, Edward J. McWilliams Jr., and Thorir D. Bjornsson

Losartan is an orally active, nonpeptide angiotensin II (Ang II) (site-1) receptor antagonist. We conducteda multiple-dose study in healthy male volunteers to investigate the tolerability, blood pressure effects, andchanges in plasma renin activity (PRA) and plasma Ang II concentration associated with once-dailyadministration of 100 mg losartan for a week. Subjects were studied on a standardized sodium diet(24-hour urinary sodium excretion, 98±37 [SD] mEq per 24 hours on the placebo run-in day).Measurements of blood pressure, heart rate, PRA, Ang II, and aldosterone were taken during a placeborun-in day and after single and multiple (7 days) daily doses of losartan (100 mg, n = 10) or placebo (n=4).Ang II was measured specifically by high performance liquid chromatography coupled with radioimmu-noassay. In subjects given losartan, respective decreases (systolic/diastolic) from run-in in supine bloodpressure 6 hours after dosing were (mean±SD), compared with the placebo run-in day, first dose:-8.8±9.6/-6.8±5.0, last dose: -11.6±8.9/-7.0±4.8 mm Hg (p<0.05 for all changes). At this 6-hour timepoint, corresponding increases from run-in in PRA were from 1.2±0.6 to 12.0±6.3 (first dose) and 9.6±4.9(last dose) ng angiotensin I per milliliter per hour and in Ang II were from 4.3±1.7 to 72.4±33J and45.7± 14.1 pg/mL. All changes in PRA and Ang II were statistically significant within the losartan-treatedgroup, and the biochemical changes were significantly greater than those in the placebo-treated group.The increment in Ang II was less after the last dose than after the first (p<0.05). The drug was welltolerated by all subjects. These data indicate that, under the conditions of this study, losartanadministration (100 mg/day for eight doses over 9 days) results in treatment-related decreases in bloodpressure and increases in PRA and Ang II octapeptide. (Hypertension 1993;21:704-713)

KEY WORDS • losartan • DuP 753 • angiotensin II • plasma renin activity • receptors,angiotensin

Losartan (Figure 1) is a potent, orally activenonpeptide angiotensin II (Ang II) antago-nist.1"4 The drug is being investigated as ther-

apy for hypertension and heart failure and is antici-pated to be a more specific mechanism for inhibitingthe renin-angiotensin system than angiotensin con-verting enzyme inhibitors. Early clinical investigationof losartan has shown single and multiple doses up to40 mg to be well tolerated and pharmacologicallyactive, with doses of 10 mg or greater blocking pressorresponses to exogenous angiotensin I (Ang I) and AngII.5-6 Also, through blockade of Ang II receptors in thejuxtaglomerular apparatus, which inhibits renin re-lease, losartan administration results in dose-relatedincreases in plasma renin activity (PRA) and immu-noreactive Ang II.56 Studies have also been completedthat demonstrate the tolerability of single doses up to300 mg and more than 90% blockade of responses toexogenous Ang II by oral doses of 80-120 mg (unpub-

From the Merck Research Laboratories (M.R.G., W.T., T.E.B.,M.-W.L., E.J.M.), West Point, and the Division of Clinical Phar-macology (M.R.G., A.B., J.M., T.D.B.), Thomas Jefferson Univer-sity, Philadelphia, Pa.

Supported by a grant from Merck Research Laboratories.Address for correspondence: Michael R. Goldberg, MD, PhD,

Merck Research Laboratories, BL 2-7, West Point, PA 19486.Received July 28, 1992; accepted in revised form January 13,

1993.

lished data, Du Pont Merck Pharmaceutical Corp. andMerck Research Laboratories). In these studies, effectsof losartan on resting blood pressure and heart rate inhealthy subjects have not been readily apparent. Inaddition, pharmacokinetic analyses have indicated that,although losartan has a relatively short half-life (unpub-lished data, Du Pont Merck Pharmaceutical Corp. andMerck Research Laboratories), the predominant circu-lating form of the drug is a carboxylic acid metabolite(E-3174, Figure 1) that is more potent and has a longerhalf-life than losartan.23 In fact, the time course ofincreases in PRA and plasma Ang II concentration andblockade of responses to exogenous angiotensin is bet-ter correlated to levels of the metabolite than to levelsof parent drug (see Reference 5 and unpublished data,Du Pont Merck Pharmaceutical Corp. and Merck Re-search Laboratories).

The general objective of the present study was tofurther investigate the effects of single and multipledoses of losartan in healthy male volunteers. Of interestwere the following specific objectives: 1) confirming thetolerability and absence of hemodynamic effects inhealthy subjects and 2) assessing changes in PRA andAng II concentrations after single and multiple doses,during standardized sodium intake, using a highly spe-cific radioimmunoassay (RIA) for Ang II coupled withhigh performance liquid chromatography (HPLC).

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COOH

FIGURE 1. Drawings show structures of losartan (Dup-753, MK-954) and its active metabolite, E-3174.

N - N

Losartan(Dup-753, MK-954)

E-3174

MethodsStudy Design

This was a double-blind, placebo-controlled, parallelstudy that was divided into three clinical phases: run-in,first-dose, and multiple-dose (Figure 2). In each phase,subjects were carefully studied during an inpatienttesting day when blood pressure and heart rate werecarefully monitored and blood was collected at definedintervals for measurement of PRA, plasma Ang IIconcentration, and plasma aldosterone concentration.The first phase was a single-blind placebo run-in day(first testing day). The second phase (next day) was adouble-blind single-dose day (second testing day) dur-ing which subjects received either a single oral 100-mgdose of losartan (n = 10) or a single dose of placebo(n=4), with treatment assignment according to an un-balanced, random-allocation schedule. Forty-eighthours later, subjects entered the third, double-blindmultiple-dose phase, during which those previously al-

Day

I I Screening Visit (1-2 weeks prior)

i •

i •

Run-in

First-Dose

Placebo

Diet135-165 meq Na+

70-90 meqK+Double-WindTreatment

Losartan (N=10)Placsbo(N=4)

FIGURE 2. Schematic shows outline of study design. Onprocedure days, subjects were sequestered on the research unitthe night before and were fasted overnight. On these days,multiple measurements of blood pressure and heart rate weremade and blood samples collected for measurement of plasmarenin activity, plasma angiotensin II concentration, andplasma aldosterone concentration. On the first-dose day,subjects were randomly allocated to receive losartan, 100mglday (n=10), orplacebo (n=4), for eight doses over 9 days.

located to receive losartan continued to take the drug,100 mg once daily for 7 consecutive days (a total of eightdoses over 9 days), and those allocated to placebocontinued to receive placebo. The third testing daycoincided with the 24-hour period after administrationof the eighth (and last) dose of the double-blind studydrug.

For each of the three testing days, subjects weresequestered at the clinical research unit the eveningbefore dosing and fasted from midnight that night until3 hours after the dose. Subjects were fed 3 and 6.5 hoursafter dosing on the testing days. Subjects remained inthe unit the night after each testing day. On other studydays, all doses of the study drug were administeredunder direct observation, at the same time (±30 min-utes) each day, with 250 mL water and 30 minutesbefore a meal. Beginning 3 days before the run-in dayand continuing through the third testing day (a total of13 days), subjects were provided a diet estimated tocontain 135-165 mEq sodium and 70-90 mEq potas-sium per 24 hours. The purpose of the diet was tostandardize sodium intake to aid in interpretation ofchanges in PRA, Ang II concentration, and bloodpressure. However, we did not attempt to assure thatsubjects were truly in "balance" on the diet beforetesting. To monitor the diet, we made 24-hour urinecollections during the run-in day and after the first andlast dose of losartan. Respectively, on each of thesecollections, urinary sodium excretion (mean±SD) insubjects given losartan/placebo were 98 ±37/93 ±8,66±16/46±11, and 85±21/75±23 mEq per 24 hours.Based on these sodium excretion rates, the subjectsappear to have been moderately sodium restricted, asindicated by the 0.7±0.6 ng Ang I per milliliter per hour(p<0.01) increase in predose PRA from run-in tofirst-dose days in the losartan group (Table 2).

Fourteen male subjects were enrolled, aged 18-31(mean, 24.4) years and weighing 161 ±22 lb (within±20% of ideal body weight). Subjects were in generalgood health, without excessive intake of caffeine (<4cups of coffee per day) or tobacco (<10 cigarettes perday) and with sitting blood pressure < 135/85 mm Hg.Before the study was begun, the protocol and consentform were reviewed and approved by the InstitutionalReview Board of Thomas Jefferson University. All

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subjects provided written, informed consent before en-rollment into the study. Subjects were studied in twogroups of seven subjects each.

Study Procedures and Assay MethodsBlood pressure and heart rate measurements. On each

testing day, supine (average of two measurements after5 minutes in a supine position) and standing (singlemeasurement after 2 minutes of standing) blood pres-sure and heart rate were measured at denned intervalsafter dosing using a Dinamap (Critikon, Tampa, Fla.)automated sphygmomanometer. In evaluating bloodpressure changes after losartan or placebo, the first 8hours of each testing day were analyzed statistically.

Plasma renin activity and aldosterone assays. For mea-surement of PRA and aldosterone, samples were col-lected in precooled syringes containing ethylenedi-aminetetraacetate (EDTA, 1.5 mg/mL whole blood)and quickly centrifuged at 4°C. Plasma was frozen in1-mL aliquots at — 20°C until time of assay.

Plasma aldosterone levels were measured by solid-phase RIA (Coat-a-count, Diagnostic Products, LosAngeles, Calif.). Briefly, 0.2 mL plasma was pipettedinto antibody-impregnated tubes, followed by additionof 1 mL 125I-aldosterone. The tubes were incubated at37°C for 3 hours and decanted, and bound radioactivitywas measured by gamma radiation counting. Samplevalues were calculated from comparison with log-logitcalibration curves created using increasing amounts ofstandards (0-1,200 pg/mL). The sensitivity of the assaywas approximately 16 pg/mL.

PRA was determined by a modification of the meth-ods described by Kodish and Katz.7-9 Plasma aliquots(300 fiL) were added to two separate ice-cold tubescontaining 200 ju,L of 0.12 M maleate buffer, pH 6.0,which contained the protease inhibitors phenylmethyl-sulfonyl fluoride (0.1 mM), soybean trypsin inhibitor (4mg/mL), and benzamidine (1 mM). One assay tuberemained in an ice bath, and the other was placed in a37°C bath for 2 hours. At the end of this period, 200-/xLaliquots from each tube were transferred for RIA ofgenerated Ang I using previously described methods.Specific renin activity was determined by subtracting thevalues obtained in the cooled assay tube from those at37°C. The sensitivity for the assay was approximately 10pg Ang I, and values are reported as nanograms Ang Iper milliliter per hour.

Angiotensin II assay. Ang II concentration in plasmawas measured by modification of published methodol-ogy that couples RIA with a relatively specific antibodyfor Ang II with HPLC separation of angiotensin pep-tides.10-12 For these measurements, blood was placedinto a prechilled tube containing (final concentrations)enalaprilat (3.6 fiM), 1,10-phenanthroline (2.5 mM),and K3EDTA (1.5 mg/mL), cooled thoroughly, centri-fuged at 4°C, and plasma stored frozen at -20°C untilanalysis. Thawed samples (2.0 mL) were spiked with125I-Ang II as internal standard and chromatographedon hexane-prewashed, preconditioned 500-mg C8 dis-posable Bond Elute columns (Analytichem Interna-tional, Harbor City, Calif.). Angiotensin peptides wereeluted with 2 x 1-mL 50% acetonitrile/50% water, con-taining 0.1% trifluoroacetic acid, and were dried, resus-pended in 300 fiL 20% acetonitrile/0.1% trifluoroaceticacid, and filtered.

The filtered samples were chromatographed on a4.6x150 mm Dynamax C8 reversed-phase HPLC col-umn (Rainin Instrument Co., Woburn, Mass.) using aflow rate of 1 mL/min at 45°C. Angiotensin peptideswere separated with a 20-minute linear gradient from20% to 35% acetonitrile containing 0.1% trifluoroaceticacid adjusted to pH 4.0 with NH4OH. Retention times(minutes) for various angiotensin peptides were: Ang I,16.5; [Des-Asp]Ang 1,17.4; Ang II, 11.9; angiotensin III([Des-Asp]Ang II), 13.2; Ang 11(3-8) hexapeptide,13.5; Ang II-(4-8) pentapeptide, 10.8; and 125I-Ang II,16. HPLC fractions (0.5 mL) were dried, reconstitutedwith 200 mL buffer containing 10 mM potassium phos-phate, 1 mM EDTA, 0.25 mM thimerasol, and 0.1%(wt/vol) gelatin, pH 7.3, and an aliquot was counted fordetermination of overall recovery. Ang II was quantita-tively measured by competitive binding RIA using acommercially available Ang II antisera (Amersham No.RPN1771). The RIA reaction mixture was incubatedovernight at room temperature and treated with dex-tran-coated charcoal to separate bound and unboundAng II. The supernatent was counted, and the Ang IIvalues were read off a standard curve made fromsolutions of known (0-96 pg per tube) Ang II concen-tration. Day-to-day precision for plasma control mate-rial yielded coefficient of variation values of 16% at 18.3pg/mL (« = 15) and 13% at 5.1 pg/mL (n = 12). Cross-reactivity of Ang II antisera with angiotensin peptideswas: Ang II, 100%; angiotensin III, 53%; Ang I, 0.1%;Ang II-(3-8), 11%; and Ang II-(4-8), 15%.

Clinical tolerability assessment. The tolerability oflosartan in each subject was monitored by periodicassessment of complete blood counts, chemistry panels,urinalyses, and electrocardiograms. On each testing day,24-hour urine was collected for measurement of elec-trolyte, protein, and creatinine excretion.

Data AnalysisIn planning this study, published data on variability of

PRA and Ang II in healthy volunteers given enalaprilwere used to determine sample size. Based on thesedata10 and assuming a correlation, p, of 0.5 betweenpretreatment and multiple-dosing values, with nine sub-jects completing, the study had 80% power (a=0.05,two-tailed) to detect respective increments in PRA andAng II concentration of 4.5 ng Ang I per milliliter perhour and 4.3 fmol Ang II per milliliter (approximately4.3 pg/mL).

The general approach to the statistical analysis was tocompare measurements collected after the first and lastdose of losartan with measurements at the correspond-ing times after dosing on the placebo run-in day.Multiple-dosing results were then compared with single-dose results. For measurements of PRA, Ang II, andaldosterone, all time points were of interest. For bloodpressure and heart rate, measurements during the first 8hours after dosing on each day were analyzed, withbaseline for supine measurements on each day definedas the average of measurements made at the time ofdosing and 15 minutes before dosing. A single measure-ment of standing blood pressure and heart rate, imme-diately before dose, was baseline for changes in standingblood pressure and heart rate. For the purposes ofreporting the results, run-in refers to the single-blindplacebo run-in day; first dose is the period after the first

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TABLE 1. Supine Blood Pressure and Heart Responses to Losartan and Placebo

Hoursoftp»ralter

dosing0

1

2

3

4

5

6

8

Treatment

LosartanPlaceboLosartanPlaceboLosartanPlaceboLosartanPlaceboLosartanPlaceboLosartanPlaceboLosartanPlaceboLosartanPlacebo

Diastolic

Run-in

60.3 ±8.062.5 ±8.357.1±9.5§57.0±11.358.2±11.060.3±9.560.7±9.660.8±10.860.0+8.262.5±10.159.9±8.459.5 ±7.960.2±7.662.3±11.859.0±9.861.5±6.8

blood pressure (mm Hg)

First dose

1.1±4.2-3.3±5.7

1.6±5.02.3±2.9

-4.2+6.7*-2.0±2.9-6.6±8.9||

1.5±4.7-6.6±4.4t-4.3±5.4-9.9±6.0t

0.5 ±4.4-6.8±5.0t-0.5±4.2-4.6±5.4||-3.5±4.2

Last dose

-3.7±5.6*t-5.9±4.3-3.2±6.4§-1.0±4.7-5.6±7.7||-0.8±7.0

-9.0±5.3*-0.8±6.0-9.1±5.0t-5.8±5.1-9.3±5.6t-3.3±3.4-7.0±4.8t-0.5±5.1-5.9±6.8||-0.3±3.9

Systolic

Run-in

109.2±8.4113.3±17.9108.4±6.6105.0+20.0110.5±9.7105.5±15.4113.9±7.2116.0±14.8114.3±7.0115.0±15.2111.5±7.4107.3±10.1113.8±11.5111.0±13.7114.5±11.3112.3±10.9

blood pressure

First dose

2.5±5.2-4.2±4.3-2.4±10.0-2.3±8.0-5.6±9.2||-1.3±5.5-8.5±8.6||-7.5±2.7-6.3+5.2*-2.8±5.8-9.9+8.4t

0.3±2.1-8.8±9.6||-1.0+6.1

-10.1±11.5||-3.3±8.8

(mm Hg)

Last dose

-6.0±4.6+t-11.5+5.2-6.6±7.2||-6.0±11.7

-10.2± 10.0||0±5.8

-14.9+5.7*-12.8+10.7

-10.8±5.4+t-7.3±5.9

-10.6±6.6*-2.8±1.7

-11.6±8.9t-5.0±5.7-7.3±6.7*-5.8±6.5

Heart rate (bpm)

Run-in

53.3±7.362.5 ±10.250.4±5.259.3±7.653.0+5.162.0±8.753.4±5.460.5 ±5.656.4±5.165.3±8.555.5±6.661.3±5.356.0±6.865.0±4.658.9±10.562.5±3.7

First dose

0.9±3.8-5.9±5.4

2.1±5.5-2.8±1.7

0.3±2.0-4.0±12.0

0.9±8.0-3.3±3.9

5.9±5.6t0±4.3

5.7±7.2||0±1.4

1.2+5.1-1.3±7.7

3.7+11.66.3±8.3

Last dose

-0.3±5.7-6.0±1.8

4.4±6.5*-4.0±1.4-1.6+4.1-7.5±9.9

0.3±5.4-0.3±2.5

3.6+4.2H1.5±8.04.5+5.7H1.5±3.02.2±5.9

-2.5±2.90.6±7.81.5±9.3

bpm, Beats per minute. Values are mean+SD from run-in day to first dose and last dose at corresponding times after dosing. n = 10 forlosartan (100 mg); n=4 for placebo.

*0.05</7<0.10, mean changes from run-in.tpsO.Ol, mean changes from first to last dose (last dose measurement minus first dose measurement).t/><0.01, mean changes from run-in.§/><0.05, mean changes from first to last dose.||p<0.05, mean changes from run-in.

dose of double-blind losartan or placebo; and last doseis the period after the last dose of double-blind losartanor placebo, i.e., the last of seven consecutive once-dailydoses, eight doses total over 9 days.

Mean changes from run-in to first and last dose andfrom first to last dose were evaluated statistically for thelosartan treatment group with a two-tailed paired ttest.13 The Type I error rate was set at a=0.05. Nostatistical evaluation was performed for changes withinthe four subjects in the placebo treatment group.

Differences between the losartan and placebo treat-ment groups in mean changes were evaluated statisticallywith a two-tailed pooled variance Student's t test13 or atwo-tailed Aspin-Welch separate variance t test.13 Theassumption of homogeneity of variances was evaluatedusing the Folded Form F Statistic.13 The Type I error ratewas set at a=0.05 for the / tests and at a=0.10 for theevaluation of homogeneity of variances.

ResultsBlood Pressure and Heart Rate Changes

Supine and standing blood pressures were carefullymonitored to determine whether Ang II blockade wouldalter blood pressure in nonhypertensive subjects. Adepressor effect was noted. Table 1 and Figure 3summarize the changes in supine blood pressure andheart rate from the run-in day to the corresponding timeafter dosing following the first and last dose of double-blind study drug. An average reduction of 9 mm Hg wasapparent. Statistical analysis of these changes within thelosartan group is summarized in Table 1. Because of thesmall number of subjects given placebo, a within-groupanalysis was not performed for this treatment. However,statistical analysis between the two groups showed some

statistically significant (or nearly significant) differencesbetween treatments at the times of apparent maximaleffect: hours 3 (p<0.05 for last dose), 5 (p<0.01 for firstdose,p=0.07 for last dose), and 6 (/?<0.05 for first andlast dose) for supine diastolic blood pressure, and hour5 (p<0.01 for first and last dose) for supine systolicblood pressure. During the 8-hour observation period,particularly after the first dose, supine heart rate (Fig-ure 4) tended to increase more in the losartan thanplacebo group. It was noted that, during the run-in day,supine heart rate was stable in both groups but signifi-cantly higher in the placebo group at 1, 2, 4, and 6 hoursafter single-blind placebo. Together, these data suggesta modest blood pressure-lowering effect of losartan,under the conditions of this study.

Similar trends to those seen for supine blood pressurewere apparent for mean standing blood pressure measure-ments (not shown). For example, the change in standingblood pressure 6 hours after dosing was —14+12/—7±12and -10±10/-6±9 mm Hg for first and last dose, respec-tively, in the losartan group, and 0.3±10/-l±7 and-10±10/- l±10 mmHg at corresponding times in theplacebo group. These effects are generally similar to thechanges in supine blood pressure summarized in Table 1.On the average, standing heart rate changes withingroups, relative to the placebo day, tended to be greater inthe losartan group than in the placebo group (Figure 4),with effects most apparent after the first dose of losartan.These results are consistent with the supine hemodynamicchanges described above (Table 1).

Renin-Angiotensin II-Aldosterone System ResponsesEarlier reports at lower doses56 indicated that losar-

tan administration is associated with sustained, dose-

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Supine Diastolic Blood Pressure

Time ( h o w after dosing) Time (hours after dosing)

Supine Systolic Blood Pressure

T i m (hours after dosing) Time (hows after dosing)

FIGURE 3. Line graphs show mean changes in supine diastolic (top panels) and systolic (bottom panels) blood pressures afterfirst (o—o) and last (•—9) dose of placebo (right panels) and losartan (left panels), 100 mg once daily for eight doses over 9 days.Shown are changes (numbers in parentheses indicate reductions) in measurements from the corresponding time after dosing duringthe placebo run-in day. *y<0.05 for within-group change, losartan-treated subjects only; arrows on placebo graphs indicate wherethe change after losartan was significantly different (p<0.05) from that after placebo, after the first dose (filled arrow) or last dose(open arrow). A meal was served approximately 3 hours after dose.

related increases in PRA and Ang II. However, Ang IIwas measured by RIA without separation of peptides toconfirm that observed increments were in the Ang IIoctapeptide. It was therefore of interest to determinethe level of stimulation of PRA after single- and multi-ple-dose administration of losartan using HPLC toseparate the Ang II octapeptide from Ang I and smallerpeptide fragments. Table 2 summarizes the renin-angio-tensin-aldosterone system measurements during the sin-gle-blind run-in placebo day and the changes from thesemeasurements before ( - 1 hour) and after (6, 6.5, and24 hours) dosing with the first and last doses of double-blind losartan or placebo. As with the analysis of bloodpressure and heart rate, mean change is shown inmeasurements on the "dosing" days from the run-inday. Also included in the table are the differencesbetween first and last doses. Figure 5 depicts thechanges in supine measurements of PRA and Ang II.With respect to both supine and standing measurementsof PRA and Ang II, clinically meaningful1415 and sta-tistically significant (p<0.01) increases from run-inwere noted within the losartan group and between thelosartan and placebo groups. Of note, the significantincrease in predose PRA and Ang II from run-in to firstdose in the losartan group was not different from thesimilar increases in the placebo group. Presumably, asindicated above, this increment is secondary to modestsodium restriction resulting from the diet provided tothe volunteers. Plasma aldosterone levels did notchange appreciably during the study.

Several other observations are relevant to these data.As indicated in Table 2, the 6.5-hour renin-angiotensin-aldosterone system measurements were made in sam-ples collected after 30 minutes of ambulation. Withrespect to PRA, during the run-in day, there was theexpected increase in mean activity of 1-2 ng Ang I permilliliter per hour (approximately doubling) from su-pine to standing positions. As indicated by the changesfrom run-in to treatment days, this absolute posturalincrement in PRA was maintained during the double-blind portion of the study. A similar postural incrementin Ang II was noted during the run-in day and after thelast dose of losartan. Six to 6.5 hours after the first doseof losartan, the postural increment in mean Ang IIconcentration was not apparent.

Six hours after dosing, PRA did not change signifi-cantly from first to last dose (-2.5±6.4 ng Ang I permilliliter per hour,p=NS). In contrast, the mean changein Ang II concentration was significantly less after thelast dose compared with the first dose (-26.7±35.2pg/mL, p<0.05), suggesting some attenuation of thisaspect of the response to losartan after several days ofadministration.

To investigate the relation between PRA and Ang IIduring the study, we initially constructed a scatterplot ofsupine Ang II concentration versus supine PRA 6 and24 hours after single-blind placebo and after the firstand last dose of each double-blind treatment (Figure 6).In general, this plot suggested a relation between Ang II

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Tim (horn altar doaing)

Tim* ( h o n atar 1km (hom

FIGURE 4. Line graphs show mean changes in supine (top panels) and standing (bottom panels) heart rates after first (O—O)and last (m—») dose of placebo (right panels) and losartan (left panels), 100 mg once daily for eight doses over 9 days. Shownare changes (numbers in parentheses indicate reductions) from the corresponding time after dosing during the placebo run-in day(see Table 1 for statistical analysis of supine measurements). *p<0.05 for within-group change, losartan-treated subjects only;arrows on placebo graphs indicate where the change after losartan was significantly different (p<0.05) from that after placebo,after the first dose (filled arrow) or last dose (open arrow), bpm, Beats per minute.

and PRA. It is apparent from this plot that Ang II levelsrelative to PRA in four subjects at 6 hours after the firstdose are elevated from the other points, suggestinggreater increments in Ang II for each unit of PRA. Toexplore this further, we calculated the ratio of Ang II toPRA for each of these points. The geometric meanratios of Ang II to PRA are summarized in Figure 7. Astatistical analysis of the change in these ratios showssignificant increases within the losartan group 6(/?<0.05) and 24 (/?<0.01) hours after the first dose oflosartan. In one extreme, one subject had ratios of 2.9 6hours after placebo, 16.1 after his first dose, and 10.2 6hours after his last dose of losartan. By the last dose oflosartan, on the average, ratios of Ang II to PRA hadreturned toward baseline, being significantly less 6hours after the last dose than 6 hours after the first dose

Tolerability

As in any early study of a new drug, it was alsoimportant to carefully evaluate subjects for untowardconsequences of study drug administration. Subjecttolerability was monitored by regular assessment ofclinical laboratory tests (complete blood count, chemis-try panel, and urinalysis), by electrocardiogram, and byobservation and questioning for clinical adverse events.Analysis of serum chemistry panels showed no signifi-cant (/?<0.05) within-group or between-group changesin any of the measured analyses during the study (the

chemistry panel consisted of measurements of ureanitrogen, creatinine, total bilirubin, alanine and aspar-tate aminotransferases, alkaline phosphatase, lactatedehydrogenase, glucose, total protein, sodium, potas-sium, chloride, bicarbonate, calcium, and inorganicphosphorous). Approximately 600 mL blood was col-lected during the entire 10-day study. Presumably as aresult of this blood loss, at the time of the poststudyevaluation, hemoglobin had decreased approximately 2g/dL and hematocrit had decreased approximately sixpercentage points in subjects given either losartan(p<0.01 within this group) or placebo. Urinalysis re-sults showed no remarkable changes. No clinically sig-nificant changes in electrocardiographic intervals orinterpretation were noted in any subject.

Clinical adverse events were mild and nonspecific,including one episode of loose bowel movements in onesubject, epigastric discomfort in another individual, andseveral occurrences (four subjects) in which blood pres-sure decreases from supine to standing position werenoteworthy but asymptomatic (e.g., supine: blood pres-sure, 102/46 mm Hg; heart rate, 50 beats per minute; 2minutes standing: blood pressure, 70/50 mm Hg; heartrate, 70 beats per minute). One subject reported super-ficial peeling of his palms beginning during the firstseveral days of losartan administration. No local symp-toms (such as burning, itching, or exudate) were asso-ciated with these findings, which resolved over severalweeks. However, this subject also had evidence of

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710 Hypertension Vol 21, No 5 May 1993

TABLE 2. Renin-Angiotensin-Aldosterone Responses to Losartan and Placebo

Variable

Plasma renin activity[(ng Ang I/mL)/hr]

Plasma angiotensin II(pg/mL)

Plasma aldosterone(pg/mL)

Day

Run-in

Run-in tofirst doseRun-in tolast doseFirst tolast dose

Run-in

Run-in tofirst doseRun-in tolast doseFirst tolast dose

Run-in

Run-in tofirst doseRun-in tolast doseFirst tolast dose

Treatment

LosartanPlacebo

LosartanPlaceboLosartanPlaceboLosartanPlacebo

LosartanPlacebo

LosartanPlaceboLosartanPlaceboLosartanPlacebo

LosartanPlacebo

LosartanPlaceboLosartanPlaceboLosartanPlacebo

Hour - 1

1.2±0.61.2±0.6

0.73±0.62*0.43±0.61

3.1±1.9*t-0.05±0.44

2.4±1.8*-0.57±0.78

5.0±2.04.0±2.4

3.3±2.6*1.4±1.9

13.1±7.9*t0.09 ±2.02

9.8±7.1*t-1.4±3.5

151.1±38.7124.6±40.7

24.4 ±38.8*82.9±53.023.2+68.732.8±21.7-1.2±51.2

-50.1±52.4

Parameter at indicated hour after dosing

Hour 6 Hour 6.5

Mean measurement (±SD)1.2±0.6l.l±0.3

Mean

12.0±6.3*t1.0±0.6

9.6±4.9*t0.05 ±0.38-2.5±6.4

-0.95±0.76

2.5±1.23.2±1.9

change (±SD)

12.0±5.6*t2.2±1.3

9.9±5.2*t0.48±1.13-2.2±6.5-2.7±2.3

Mean measurement (±SD)4.3±1.73.7±2.0

Mean

72.4±33.3*t4.5±2.8

45.7±14.1*f1.5±4.3

-26.7±35.2§-3.1±5.3

9.7±4.714.4±13.7

change (±SD)

67.3±34.3*t16.1 ±14.6

66.2±43.1*t-3.0±8.5-1.1±43.8

-19.1+22.5

Mean measurement (±SD)104.1 ±28.9117.5±10.7

Mean10.1 ±49.152.6±62.0

-10.5±15.0$-4.7±28.3

-20.6±53.2-57.3±65.1

160.1 ±50.6234.2+114.1

change (±SD)36.1 ±78.3

132.2±61.8-14.7±67.0-3.7±41.7

-50.9±112.1-135.8±63.1

Hour 24

1.5±0.81.6±0.9

5.1±2.3*t0.95 ±0.37

4.1±3.2*t0.10±0.44

-0.94 ±1.60+-0.85±0.71

5.9±2.46.2+4.3

27.7±10.2*t7.8±5.8

18.1±10.9*t0.19±3.14-9.6±8.8t-8.0±8.7

137.6±49.4168.0±77.8

82.9±44.2*103.8±84.452.6±78.7t

-34.4 ±75.8-30.3 + 64.6

-138.2+151.0

Ang I, angiotensin I. Values are mean±SD measurements on the run-in day and changes from run-in to first dose (day 1) and last dose(day 9) and from first to last dose at corresponding times after dosing. n = 10 for losartan (100 mg); n=4 for placebo. Measurements at Hour6.5 were taken after 30 minutes ambulation.

*p<0.01, mean changes from run-in or first to last dose.tpfiO.01, losartan vs. placebo.$0.05</?£0.10, §p<0.05, mean changes from run-in or first to last dose.

hemolysis (schistocytes, crenated red blood cells) on hisperipheral smear during the treatment period withoutdecreases in haptoglobin, hemoglobinuria, increases inlactate dehydrogenase, or decreases in hemoglobin orhematocrit (other than as expected in such a study).These events were judged to be of uncertain relation toadministration of losartan.

Discussion

In summary, losartan was found to be sufficiently welltolerated at 100 mg once daily to permit use of this dosein future studies. Modest decreases of blood pressurewere noted during losartan administration, which indi-cate that, in healthy men, Ang II can play a role in themaintenance of resting blood pressure. However, theseobservations were made under conditions of modestsodium restriction (based on 24-hour urinary sodium

excretion and small increases in PRA before adminis-tration of double-blind losartan or placebo). The extentto which this study condition influenced our resultscannot be determined from this exploratory study.Blockade of endogenous Ang II was indicated by clearlydenned increases in PRA and concomitant increases inplasma Ang II concentration.

Effects on aldosterone concentration in blood werenot apparent, presumably because of the complex mech-anisms known to control aldosterone release.16-17

Based on prior data5-6 and the known capacity ofendogenous Ang II to act on a feedback receptor on thejuxtaglomerular apparatus,18"20 it was anticipated thatlosartan administration would result in an increase inPRA and Ang II. As indicated above, both supine PRAand plasma Ang II concentration were significantlyincreased 6 and 24 hours after administration of the first

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Goldberg et al Losartan in Healthy Volunteers 711

Supine Plasma Renin Activity

RalDoM

Supine Plasma AngiotenBn Con*

vitiation

FMDOH LMIDOM RntDoM LatfDoa*

FIGURE 5. Bar graphs show mean change from the corresponding time after dosing on the run-in day of plasma renin activity(top panels) and plasma angiotensin II concentration (bottom panels) in subjects allocated to receive losartan 100 mg daily (leftpanels) and placebo (right panels) for eight doses over 9 days. In each panel, changes after the first and last dose of double-blindtherapy are shown. Solid bars indicate changes from 1 hour predose on the run-in day to 1 hour before the first and last dose.Shaded bars indicate corresponding changes 6 hours after dosing. Open bars indicate changes 24 hours after dosing. *p<0.01 vs.change in placebo group. AI, angiotensin I.

and last 100-mg dose of losartan. The methodologyused specifically measures Ang II octapeptide, indicat-ing that observed increments reflect changes in theconcentration of the most biologically active pep-tide.10"12 The magnitude of the change was greater 6hours after dosing, compared with 24 hours. The dataalso indicate that the level of stimulation of PRA andAng II is slightly less after the last than the first dose,consistent with the hypothesis that the magnitude of theinitial response is related to discharge of stored renin

from the juxtaglomerular apparatus, as has been shownin animals given angiotensin converting enzyme inhibi-tors.21 This differs somewhat from prior observations.5

However, study conditions (i.e., sodium balance), losar-tan dose, and methodology for measurement of Ang IIalso differed between the studies.

The implications of these increases in PRA and AngII cannot be determined from this study in healthyvolunteers. Studies in hypertensive patients are re-quired to determine whether similar increases are noted

160 r

•=•120\a.

.£ 80enc

< 40

AAO•

••

Run-in, Hr 6Run-in, Hr 24First Dose. Hr 6First Dose. Hr 24Lost Dose, Hr 6Lost Dose, Hr 24

ffl)

• •

D

D°o

FIGURE 6. Scatterplot shows supine plasmaangiotensin II and plasma renin activity mea-surements in subjects given losartan and pla-cebo (inset).

10 15 20Plasma Renin Activity (ng/ml/hr)

25 30

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712 Hypertension Vol 21, No 5 May 1993

Lasa«w(N==10)

FIGURE 7. Line graph shows ratio of angiotensin II toplasma renin activity (PRA) 6 and 24 hours after placebo(Run-in) and first and last doses of double-blind study drug(see text for statistical analysis of changes in this ratio). AI,angiotensin I.

in this target population. Furthermore, losartan is aselective antagonist of only a single Ang II bindingsite,22-25 which mediates all known physiological effectsof angiotensin. It is not known whether unblockedeffects of increased Ang II at other binding sites are ofany clinical consequence to the safety profile and he-modynamic effects of losartan. In the absence of long-lasting Ang II blockade, increases in plasma Ang IIconcentration of the magnitude demonstrated would beexpected to have hemodynamic activity.14'15

In general, a relation between PRA and Ang II couldbe defined; the ratio of Ang II concentration to PRAwas assumed to be an index of this relation. It was ofinterest that this ratio was higher after the first dose oflosartan, when the greatest increases in Ang II werenoted, than during the run-in day and after the last doseof losartan. The implication is that more Ang II isproduced per unit of PRA at this time than at othertimes. An explanation for this observation is not readilyapparent from the data collected in this study. It ispossible that these differences are a result of greater invitro generation of Ang II associated with initial stimu-lation of renin release.10 Alternatively, several physio-logical hypotheses are possible, including changes inAng II clearance, which seem unlikely, and changes inAng II generation.26 One explanation is that angiotensinconverting enzyme is somehow downregulated, result-ing in less-efficient conversion of Ang I to Ang II. Thisseems unlikely in view of the ubiquity of the enzyme.26

Another potential control point in the system that couldbe rate limiting is the amount of circulating angioten-sinogen, which is under the control of both renin andAng II.27 Thus, it has been demonstrated that (inaddition to consuming angiotensinogen) increases inrenin decrease the production and release of angioten-sinogen, whereas Ang II stimulates its release, an effectapparently antagonized by the peptide Ang II antago-nist saralasin.27 During Ang II blockade with losartan, itis reasonable to postulate that secondary increases inPRA with blockade of Ang II receptors results in netinhibition of angiotensinogen synthesis and release,functionally downregulating the system. This hypothesis

needs to be tested in appropriate preclinical and clinicalmodels.

It was anticipated that Ang II blockade would resultin a decrease in plasma aldosterone concentration. Thiseffect was not observed. However, in the presence of10-fold elevations of Ang II concentrations, it is reason-able to assume that the absence of changes in aldoste-rone concentration is consistent with Ang II block-ade.14-17 Clarification of the effect of Ang II blockade onaldosterone release awaits further study.

The tolerability of losartan for future studies inhealthy subjects and patients was investigated throughevaluation of hemodynamic effects, clinical adverseeffects, and clinical laboratory tests. Hemodynamic ef-fects were of particular interest, as the role of Ang II inthe regulation of blood pressure in healthy volunteersunder the conditions of this study was of physiologicalinterest.

A modest, sustained, and persistent effect of losartanto reduce blood pressure was noted in this study. Thesechanges did not result in apparent clinical symptoms.Also noted were statistically significant increases instanding heart rate, particularly after the first dose oflosartan, but without postural hypotension. Together,these data suggest that, under the conditions of thisstudy (overnight supine rest, moderate sodium deple-tion, limited environmental stimulation), the bloodpressure of healthy male volunteers is somewhat depen-dent on endogenous Ang II.

AcknowledgmentBetty Jean Miller is acknowledged for preparation of the

manuscript.

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McWilliams, Jr and T D BjornssonM R Goldberg, W Tanaka, A Barchowsky, T E Bradstreet, J McCrea, M W Lo, E J

volunteers.Effects of losartan on blood pressure, plasma renin activity, and angiotensin II in

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