Eur J Clin Pharmacol (1994) 47:231-245 �9 Springer-Verlag 1994

R Mt~ller - T. Cohen �9 M. de Gasparo �9 A. Sioufi A. Racine-Poon �9 H. Howald

Angiotensin II receptor blockade with single doses of valsartan in healthy, normotensive subjects

Received: 22 March 1994 / Accepted in revised form: 23 June 1994

Abstract Valsartan (CGP 48 933), a specific blocker of the angiotensin II (Ang II) receptor subtype 1 (AT 1 re- ceptor) was administered in single, oral doses of 40 mg and 80 mg to six healthy, normotensive male volunteers in a double-blind, placebo-controlled, randomized crossover trial. The aims of the study were a) to assess the extent, time course and dose-dependency of inhibi- tion of the pressor effect of exogenous Ang II; and b) to attempt to correlate AT 1 receptor blockade with the drug levels in plasma and with other markers of biologi- cal activity of the trial drug such as plasma renin activity (PRA).

Using the Finapres device and i. v. bolus injections of exogenous Ang II, AT 1 receptor blockade was assessed by measuring blood pressure (BP) and heart rate (HR) on a beat-by-beat basis. A dose-response curve for Ang II was obtained for each subject before and at 2, 4, 6, 8 and 24 h after administration of placebo and of the two doses of valsartan. PRA was measured with a con- ventional radioimmunoassay method.

Data evaluation included a) descriptive analysis of the changes of the Ang II dose-response curves after valsartan, as compared to the curve on placebo; b) cal- culation of the pressor dose D30 of Ang II at each time- point, using linear regression; c) assessment of the ef- fect of 4 ~tg Ang II on systolic BP and HR and the calcu- lation of the percentage inhibition of these effects after valsartan; d) description of the relationship between drug levels in plasma and the measures of AT 1 block- ade, including pharmacokinetic-pharmacodynamic modeling with an Ema x model for the percentage inhibi- tion of systolic BP and HR.

It is concluded that (a) the methodology used is suit- able to evaluate AT 1 receptor blockade in man; (b) in- stead of using a full dose-response curve for Ang II at each timepoint, an abbreviated approach with only one

R Mfiller ([5~) �9 T. Cohen �9 M. de Gasparo �9 A. Sioufi A. Racine-Poon �9 H. Howald Pharma Division, Ciba-Geigy Limited, Research and Development Departments, CH-4002 Basel, Switzerland

pre-determined Ang II dose may be adopted without substantial loss of information; (c) valsartan is an inhibi- tor of the AT 1 receptor in man, with a mean Ema x of 74 % and a mean ICs0 of 0.53 ~tmol. 1 ~ for the blood pressure response to exogenous Ang II; (d) receptor blockade after single oral doses of 40 mg and 80 mg val- sartan reaches its maximum at 2 h and is detectable up to 24 h after administration; (e) despite a doubling of systemic exposure, the relationship between dose and receptor blockade appears to be rather flat in the dose range 40 to 80 mg; and (f) valsartan is well tolerated by healthy subjects in the dose range tested.

Key words Angiotensin II antagonists; renin-angio- tensin system, angiotensin II challenge, blood pressure, Finapres device, pharmacokinetic-pharmacodynamic modeling

The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and volume homeostasis. Angiotensin II (Ang II), the active princi- ple of the system, acts at various levels (e. g. blood ves- sels, sympathetic nervous system, cortex and medulla of the adrenal gland) to increase vascular resistance and induce volume retention, both resulting in an in- crease in blood pressure (BP). Pharmacologically, the RAS may be influenced at various levels: by inhibition of renin or of angiotensin converting enzyme (ACE), or by blockade at the angiotensin I1 receptor level [1, 2]. ACE inhibitors are widely used in clinical practice, whereas renin inhibitors and Ang II antagonists are cur- rently under development.

The identification of a number of non-peptide Ang II antagonists has led to a new potential approach for the treatment of arterial hypertension, and allowed for the pharmacological characterization of at least 2 distinct subtypes of the Ang II receptor (AT 1 and AT 2 recep- tors) [3-7]. The potential clinical indications for Ang II antagonists are the same as for the ACE inhibitors. The latter exhibit some class-specific adverse effects such as

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cough and, m o r e rarely, ang ioneuro t i c oedema , which appea r to be re la ted to a concomi t an t r educ t ion of the degrada t ion of b radykin in and substance R B lockade of the R A S at the A T r e c e p t o r level should be m o r e spe- cific than inhibi t ion of A C E . Moreove r , it m a y work in- dependen t ly of the pa thway for A n g I I f o rma t ion and of the site where A n g I I is p roduced , i.e. in the b lood or tissues [8].

Valsar tan ( C G P 48933) was charac te r ized in vitro and in vivo as an orally active, potent , and specific com- peti t ive A n g I I antagonis t acting at the A T 1 recep to r sub type [7], which media tes all the k n o w n effects of A n g I I on the cardiovascular sys tem [3, 6]. The com- p o u n d is current ly deve loped as an ant ihyper tens ive drug. Af t e r adminis t ra t ion of single and mult iple oral doses to hea l thy volunteers , valsar tan was systemical ly available and well to lerated. The drug exhibi ted pro- nounced , dose dependen t biological activity such as in- creases in p lasma renin activity and p lasma A n g I I con- centrat ions, as well as a decrease in systolic and dias- tolic BP [9].

The availability of A n g I I for i.v. adminis t ra t ion to humans offers a direct invest igat ion of the AT~ block- ing proper t ies of A n g II antagonists. The effects of exo- genous A n g I I on the no rmotens ive card iovascular sys- t em are easily measurab le as an increase in BP and a de- crease in H R . It m a y be assumed that these effects are inhibi ted or abol ished after b lockade of the receptor . Therefore , A n g I I chal lenge p rocedures in n o r m o t e n - sive subjects m a y be used as a pharmaco log ica l tool to evaluate the basic mechan i sm of act ion of A n g I I an- tagonists in man. Infusions or bolus injections of A n g I I or of the decapep t ide A n g I, which has similar effects to A n g I I because of its very fast convers ion to the octa- pept ide by angiotensin conver t ing enzyme, have been used as pharmacolog ica l tools in h u m a n card iovascular research, e.g. for the eva lua t ion of calc ium antagonis ts [10], A C E inhibi tors [11], and A n g I I inhibi tors [12-14].

The aims of the present trial were (a) to assess the ex- tent, t ime course and dose -de pe nde nc y of inhibi t ion of the pressor effect of exogenous A n g I I after single 40 mg and 80 mg doses of the A T 1 antagonis t va lsar tan and placebo, and (b) to a t t empt to corre la te AT~ recep- tor b lockade with the drug levels in p lasma and with o ther markers of biological activity of the trial drug, such as p lasma renin activity (PRA) .

Subjects and methods Design of the trial

In this double-blind, placebo-controlled, randomized, 3-period crossover trial, six healthy, male subjects were treated with single, oral doses of 40 mg and 80 mg of valsartan or placebo, adminis- tered according to a 6 x 3 complete balanced square. Between per- iods, a wash-out phase of at least 1 week was observed. The cross- over design was chosen to minimize variability of the results across periods and to keep the number of subjects as small as possi- ble.

Trial drug

CGP 48 933 (valsartan), C~4H29NsO3, molecular weight = 435.5, is poorly soluble in acidic media, but has good solubility at neutral to alkaline pH. Capsules containing 40 mg of the active substance and matching placebo capsules have been developed by Pharma- ceutical Development, Ciba-Geigy PLC, Horsham (UK) for clini- cal testing.

One capsule containing 40 mg of valsartan plus one placebo capsule, or two 40-mg capsules, or two placebo capsules were ad- ministered each trial day at around 8.00 a.m., after completion of the baseline (0 h) measurements and blood sampling.

Blood pressure monitoring

For the measurement of the short-lasting effects of Ang II bolus in- jections on BR invasive methods with direct continuous transduc- tion of intravascular pressure would be ideal, A recently devel- oped methodology, the Finapres blood pressure monitoring de- vice (Ohmeda, Englewood CO, USA), offers the advantage of measuring BP beat-by-beat, but in an easy and non-invasive man- ner. Therefore, this specific methodology was used for the present trial. In our laboratory, the equipment has been linked to a perso- nal computer allowing for storage of the beat-to-beat BP and HR values in a spreadsheet file for further evaluation and graphical display.

In the methodological investigations of Christen et al. [11], seri- al bolus injections of Ang I were used to evaluate the suitability of the Finapres system for detecting short lasting pressor responses in man, and of the inhibitory effect of captopril on the pressor re- sponse. These investigations revealed that the device is suitable to monitor BP changes after Ang I bolus injections on a beat-by- beat basis, with a good reproducibility of individual responses to a given dose of Ang I. The authors further demonstrated that the methodology is able to detect dose dependent inhibitory effects of an ACE inhibitor.

Accuracy as compared to conventional BP measurements and to intraarterial pressure recordings and the reproducibility of the measurements with the Finapres device have been tested under various circumstances and were found to be good [15-23],

Angiotensin II challenge

Synthetic angiotensin II, the amide of Ang II (1-L-asparaginyl-5-L- valyl angiotensin octapeptide) was marketed in Switzerland until 1993 under the trade name of Hypertensin CIBA (Ciba-Geigy Limited, Basle). Its intended mode of therapeutic administration in certain states of normovotaemic shock was by slow intravenous infusion at a rate of 1-20 ~tg - min -1.

The effects of i.v. Ang II bolus injections to healthy normoten- sive subjects have been described early after synthetic Ang II be- came available for use in humans [24, 25]. About 30 s after injec- tion of the Ang II bolus, systolic and diastolic BP increase and HR decreases, both effects depending on the dose. The peak ef- fect is reached between 60 and 120 s after injection. The rapid breakdown of Ang II by the enzyme, angiotensinase, results in a very transient effect, with a return of BP back to baseline within less than 5 rain. The individual reaction to a given Ang II dose may vary considerably from one subject to another, but the varia- bility of the effect of a given dose within subjects has been report- ed to be small [24, 25].

After arrival of the volunteers in the laboratory, an indwelling cannula (Venflon 18 gauge, Viggo AB, Sweden) was inserted into a vein at the forearm and was fitted with a three-way lock (Model K69B, Bentley Laboratories, Holland) This system was kept pa- tent by flushing with heparinized saline (125 U Liquemine Roche/ ml) after each challenge test.

For the purpose of the present trial, i.v. bolus doses of Ang II were prepared in the following way:

Solution I: 1 ampoule of Hypertensin CIBA, commercial pre- paration, containing 2.5 mg of dry substance, dis- solved in 50 mlNaC1 0.9 % = 50 btg Ang II per ml.

Solution II: 2 ml of solution I in 50 mlNaC1 = 2 ~*g Ang II per ml. Solution III: 4 ml of solution I in 50 mlNaC1 = 4 ~tg Ang II per ml.

The subjects were kept in supine position for 20 min before the first Ang II dose was injected. The cuff of the Finapres equipment was wrapped around the middle finger of the contralateral hand, and the measurement was started around 10 min before the first challenge dose. The baseline blood samples for the measurement of plasma drug concentration and P R A were taken from the ve- nous cannula at 10 rain before the challenge was started. When stable BP conditions were reached at the end of the 20 min supine rest period, the first Ang II dose was injected. BP was measured and monitored continuously from 10 min before until at least 5 min after the last injection.

The response to a given dose o fAng II was measured as the dif- ference in systolic BP between baseline (mean of all BP values dur- ing the 15 s before injection) and the maximum value observed be- tween 30 and 120 s thereafter. For each challenge test, the maxi- mum increase in systolic BP was then plotted against the dose, re- sulting in a linear dose-response curve. From linear regression, the dose of Ang II which produced an increase in systolic BP by 30 m m H g (D30) was calculated.

The dose increments were adjusted to individual responses, starting at 0.5 btg, followed by doses not higher than double the preceding dose. An injection of saline was inserted between two of the Ang II doses in order to obtain a zero value for the dose-re- sponse curve. After each bolus of Aug II, the cannula was immedi- ately flushed with 10 ml of saline. The volume of Ang II solution injected varied with the dose. For doses from 0.5 to 2.0 btg Aug II, 0.25 to 1.0 ml of solution II were used, whereas for higher doses of Ang II, appropriate volumes of solution III were injected.

The challenge was stopped when one of the following end- points had been reached: a) increase in systolic BP above baseline by 25 to 40 mmHg; b) absolute systolic BP reaching 200 mmHg; c) absence or smaller increase than defined above with an Ang II dose which was 10-fold higher than the baseline D30 of that subject.

After successful performance of the baseline challenge test, the trial drug (valsartan 40 mg or 80 mg, or placebo) was administered and the pharmacodynamic recordings and blood collections were repeated at 2, 4, 6, 8 and 24 h thereafter. Between measurements, the subjects were free to move around on the ward. After comple- tion of the Ang II challenge at 8 h after dosing, the cannula was re- moved and the subjects could leave the laboratory. On the next morning, the final sampling procedures and measurements were performed.

Under the condition of an unblocked RAS (placebo and base- line before treatment), the Ang II dose-response relationship for systolic BP was linear. However, after blockade of AT 1 receptors, this relationship became very flat and - in some but not all cases - appeared to reach a plateau. Therefore, in addition to the evalua- tion of D30 by linear regression, the procedure suggested by Chris- ten et al. [12, 13] and Munafo et al. [14] was adopted. For investiga- tion of the AT~ blockade after losartan, these authors had used only one single (always the same) bolus dose of Ang II for each challenge test. This dose had been established for each individual subject before starting the trial and was defined as the Aug II dose which increased systolic BP by around 30 mmHg. The effect of the A T 1 antagonist was expressed as the percentage inhibition of the effect of this pre-determined dose of Aug II on systolic BP after administration of the antagonist as compared to baseline.

In line with this approach, the data from the present trial were analyzed selecting the 4 ug dose of Ang II, which had been admi- nistered in most of the titration procedures, and which induced an increase of systolic BP by 30-35 m m H g at baseline or on placebo.

233

In those cases where the 4 ug dose of Ang II was not adminis- tered, e.g. when an increase of systolic BP by 3 0 m m H g was reached with 3 ~tg already or when the dose titration had not included the 4 [*g dose of Aug II because of a flat dose-response curve, the effects of the respective nearest dose were used as an ap- proximation. The results of this second method of evaluation are given as the increase of systolic BP to 4 vtg Ang II (mmHg) and the decrease of H R (beats �9 rain<), and as the percentage inhibi- tion of these effects compared to pre-treatment baseline (effect at baseline = 100 %).

Plasma renin activity (PRA)

Blood (3 ml) was collected into E D T A Vacutainer tubes. The tubes were kept chilled in ice-water before and immediately after sam- pling. The blood was centrifuged without delay at 2200 g during 5 rain at 4 ~ and the plasma immediately transferred into polypro- pylene tubes and snap-frozen on dry ice. The samples were stored at -80 ~ until analysis.

Conventional radioimmunoassay (RIA) methods were used for P R A [26] with a limit of detection for angiotensin I of 83 pg �9 m1-1. Since the antibody does not cross-react with Ang II, the blood could be collected through the same indwelling cannula as used for the challenge tests.

Pharmacokinetic measurements

Blood (5 ml) was taken into sodium-heparinized Venoject tubes. The blood was immediately centrifuged at 2200 g during 5 min at 4~ and the plasma transferred into polypropylene tubes. The plasma was frozen and stored at -18 ~ until analysis by means of high-performance liquid chromatography with a limit of quantita- tion (LOQ) of 11.5 nmol . 1 q (5 ng. mlq). The following pharma- cokinetic parameters were determined:

Cm~ x (,amol �9 1-1) highest observed plasma concentration. Cm~xSpec highest observed plasma concentration (gmol - 1-1 �9 gmol <) divided by the dose. tma x (h) time to Cma x. A U C (h- gmol �9 1-1) area under the plasma concentration-time

curve from 0 h to 24 h, calculated by the linear trapezoidal rule. Concentration val- ues not detected (ND) were taken as zero for calculations.

AUCspec Specific A U C = A U C per dose unit (AUC/ (h. btmol. 1 1. btmol-1) dose). tl/2 (h) terminal elimination half-life determined

from the slope of the regression straight line which describes the terminal log-linear segment of the plasma concentration-time curve including 2-5 concentration time points.

Statistical analysis

ANOVA for a 6 x 3 complete balanced design with repeated mea- surements was performed using PC-SAS version 6.06 on the fol- lowing variables: a) plasma renin activity, b) pressor dose D30, c) increase of systolic BP to 4 ~ug of Ang II and d) decrease of H R to 4 btg of Aug II. In addition, Tukey's Studentized Range Test (HSD) was used at each timepoint to compare the two doses and each dose to placebo at a significance level of 0.1. The risk of a type I error was deliberately set at 0.1 in order to decrease the risk of type II errors. This is justified considering the exploratory character and the small sample size of the present phase I trial in which no possible unwanted or wanted effects should be missed.

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An attempt was made to correlate the pharmacokinetic (PK) with the pharmacodynamic (PD) effects of valsartan. Individual and mean values of the drug plasma concentrations were plotted against the individual and mean values of the various PD vari- ables, and were analysed in a descriptive way. The PK-PD relation- ship was explored further for the percent inhibition of the effects of 4 btg Ang II on systolic BP and on H R using formal PK-PD model- ing, since these two parameters were considered to reflect the inhi- bition of the AT receptors by the trial drug most directly.

For a first exploratory data analysis of percentage inhibition of BP and HR, the measured PD effects for each volunteer after both doses of valsartan were plotted along with the measured plas- ma concentration of CGP 48 933 to explore the possible PD-PK re- lationship and the respective time profiles. An Ema x model was used to describe the PD and concentration relationship. The mod- el is given as follows:

Ayi(t]) = Pli(tj) (100 - Emaxi conc i (t]) co~nci (tj) + IC5o~ + Ei]

Where Ayi( 0 denotes the change in BP (or HR) of the ith vo- lunteer at time t during the active treatment periods, pli(t ) denotes the corresponding measurement in the placebo period, cortci(t ) de- notes the corresponding concentration and eij denotes the lack of fit of the model.

The parameters to describe the form of the concentration and PD curve are Emax/and [C50i, that is the maximum inhibition as a percentage of baseline and the concentration which inhibits the maximum response by 50 % for the ith volunteer. These parame- ters were estimated using PROC NLIN in SAS.

Results

Subjects

Six male subjects participated in the trial. They had a mean age of 35 years (range 25-43 kg), a mean weight of 81.3 kg (range 75.0-86.5 kg), a mean height of 180 cm (range 173-184 cm) and a mean body surface area of 2.01 m 2 (range 1.90-2.09 m2).

Evaluation of Ang II challenge methodology

The Finapres equipment and the Ang II challenge methodology had previously been tested in five sub- jects not involved in this study. These investigations led to reliable and reproducible results. As an example of the dose-dependent effects of Ang II on continuous- ly recorded BP and HR, Fig. 1 shows the responses to increasing Ang II bolus injections and repeated doses of 2 ~g Ang II administered to one subject. Figure 2 gives the linear dose-response regression for the maxi- mum increases in systolic BP measured in this subject. The results from all challenges performed during the evaluation of the methodology may be summarized as follows:

The Finapres device and the additional equipment used (personal computer for on-line data registra- tion) are able to register and record BP and H R on a beat-by-beat basis, which is a prerequisite for the use of Ang II bolus injections.

�9 To obtain stable BP readings, it is sufficient that the subjects are in supine position for 20 min and that the cuff is applied 10 min before the first injection of Ang II. A starting dose of 0.5 gg Ang If may be ad- ministered safely in all subjects, followed by step- wise increasing doses as soon as baseline has been reached again, mostly within 5 min.

�9 It appears that D30 is reproducible within the same subject. In four of the five subjects involved in the method evaluation, two or three Ang II challenges were performed on the same day, resulting in similar D30 values. The within-subject variation of D30 in the three subjects who were challenged three times was 12-16 %.

Pharmacokinetics

Method validation

The HPLC method was validated daily by analyzing a series of drug-free human plasma samples spiked with known amounts of CGP 48 933. For the five spiked con- centrations used for validation, mean recovery ranged between 96.6 % (for the highest spiked concentration of 3450 nmol. 1-1) and 99.8 % (for the lowest spiked concentration of 11.5 nmol. 1 <) with coefficients of variation of < 10 %.

Drug plasma concentrations

The mean drug plasma concentrations are shown in Fig.3, and the mean pharmacokinetic parameters are given in Table 1. No active drug was detected in sam- ples taken after administration of placebo. The concen- trations observed at the last sampling point (24 h) were definitely higher than the limit of quantitation (4- to 25-fold LOQ).

The individual AUC values ranged from 10.3 to 28.3h- btmol-1 < with 40mg and from 14.9 to 58.0 h. btmol. 1-1 with 80 mg valsartan. The average be- tween-subject variability as reflected by the coefficient of variation was higher with 80 mg than with 40 mg (49 % and 36 % respectively). The within-subject variability, es- timated from the coefficient of variation of the pure error in an ANOVA applied to AUCspec values was 41%.

1 The AUC ratio (80 mg. 40 mg ) ranged from 0.63 to 3.26 with a mean value of 2.03. The m e a n AUCspec values were similar for both doses (0.20 and 0.18 h. ~tmol �9 1-1 �9 ,umo1-1, respectively). The analysis of variance applied to the log transformed AUCspec v a l u e s did not show any significant differences between sub- jects, periods and treatments. The coefficient of varia- tion of the original data was high (48.4 %), suggesting a low sensitivity of the ANOVA. Therefore, dose propor- tionality could not be established.

Cma x was observed at the first sampling time point at 2 h in all subjects with both doses. The individual Cma x

BP [mmHg] and HR [beats �9 min -I] 200

160

120

80

40

0.5 pg ang II i.v.

JIt BPS

BPD

. . . ' , % - , , . . . - ' - - . . " " , . . . . . _ : ~ . . ~ , . - . . . . , . . . , . - ~ "

HR

0 I I I I I I I I i

-20 0 20 40 60 80 100 120 140 160

Time [s] after injection

BP [mmHg] and HR [beats. min "~] 200

160

120

80

40

1.0 pg ang II i.v.

BPS

BPD

�9 - - . . . . . w , . , - ' " ' , " �9 - , , - . . ,,-

HN

0 I I I I I I I I I

-20 0 20 40 60 80 100 120 140 160

Time [s] after injection

BP [mmHg] and HR [beats- min -1] 200

160

120

80

40

1.5 pg ang II i.v.

BPS

BPD

" - - . . . . . , - ' ' - ' " " ' " . . . . . . .

. . . . , % . - ' - - " ' ' * . . . . . .

HR

0 I I I I I I I I I

-20 0 20 40 60 80 t00 120 140 160

Time [s] after injection

Fig. 1 Responses of systolic blood pressure (BPS), diastolic blood pressure (BPD) and heart rate (HR) to increasing i.v. bolus injec-

BP [mmHg] and HR [beats �9 min "1] 200

235

160

120

80

40

2,0 pg ang II i.v.

BPD

, o ,

- . . . . . . o , - . . . . . . . , , . , - . . - - - " "

" , _ . . . . . . . . . . -

HR

0 I l I I I I I I I

-20 0 20 40 60 80 100 120 140 160

Time [s] after injection

BP [mmHg] and HR [beats �9 min "1] 200

160

120

80

40

2.0 pg ang II i,v. BPS

BPD

p h ~

% , , * ' . . ' - - , ' -

- - , . . . . . . , - - " , . . . . . . . . - -

HR

0 I I I I I I I I I

-20 0 20 40 60 80 100 120 140 160

Time [s] after injection

BP [mmHg] and HR [beats. rain -1] 200

160

120

80

40

2.0 pg ang II i.v.

BPD

. , o , . - ~ , , - . - - - . _ . .

. . . . . . ; . . - ; . . - ' - ' . . " HR

0 I I I I I I I I I

-20 0 20 40 60 80 100 120 140 160

Time [s] after injection

tions of Ang II (0.5-2.0 ~g) and to repeated 2 ~g i.v. bolus doses in one and the same subject

236

Delta BPS [rnmHg] 4 O

30

20

10

r = 0.95

O

I I I " I

0 0.5 1 1.5 2 2.5

Dose [pg Ang II]

Fig.2 Dose-response regression for the maximum increases in sys- tolic blood pressure (BPS) determined in the subject also pre- sented in Fig. 1

p . m o l �9 I -~

6

5 -

4 -

3 -

2 .

1"

Drug p lasma concentrat ion

0 I

0

I

�9 40 mg

I I I I I

4 8 12 16 20 24

n g / A n g l �9 m l "~. h "l

30

Time [h]

values ranged from 1.46-4.40 ~mol. 1-1 with 40 rag, and 25 from 2.68-7.69 ~xmol-1-1 with 80 mg valsartan, with mean values and coefficients of variation of 20 2.94 ~tmol �9 1-1 (36 %) and 4.64 ~mol. 1-1 (39 %), respec- tively. The within-subject variability, estimated from an 15 ANOVA applied to the dose-corrected Cma x values was 41.5 %. 10

The Cm~ x ratio (80 mg. 40 mg -1) ranged from 0.61 to 3.12 with a mean value of 1.80. The m e a n Cmaxs_ec 5 (Cmax/dose) value was slightly higher for 40 mg than f~)r 80 mg valsartan. 0

The elimination h/2 was nearly the same for all sub- jects with both doses, namely around 5 h.

Plasma renin activity

I

j ~ / ' ~ " - - - - - P l a c e b o

I / \ �9 .omg

u " '~'" " " " $ " " " " i~ . . . . . . . . . . . . . . . - T T ~ . . . . . . - - " - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I I [ I I I

4 8 12 16 20 24

Time [h]

Plasma renin activity (PRA)

The mean PRA data are shown in Fig. 3 and Table 2. The mean baseline values of PRA before the three treatments were similar (range 1.88-2.18 ng Ang I- m1-1. h-l), and after placebo there was only a slight increase to 3.45 ng Ang I-m1-1. h -1 at around noon. The variability of PRA within subjects on placebo ran- ged between 12 and 63 % with a mean value of 38 %. The variability between subjects was around 50 %.

After both doses of valsartan, PRA rapidly increased to reach a maximum at 4 h after 40 mg and at 6 h after 80 rag. The peak PRA after both doses was very similar with 23.5 and 23.2 ng Ang I . m1-1. h -1. The AUC (0- 24 h) for PRA was also very similar for both doses: 199 h. ng. m1-1 . h -1 for 40 mg and 211 h �9 �9 m1-1 - h -1 for 80 mg valsartan, with a higher mean inter-subject variability than on placebo (CV > 90 % for both doses).

Between 2 and 8 h after administration, PRAwas sig- nificantly increased above placebo. At 24 h the increase compared to placebo was significant for the 80 mg dose only. PRA was significantly higher with 40 mg valsartan

P r e s s o r d o s e D30 of ang II dose response pg Ang II

"-- +"" Placebo

60 / k l = 40 mg

I "~

20

. . . § . . . . . . . . . . . . . . . . . . 0 I I I I I I

0 4 8 12 16 20 24

Time [hi

Fig.3 Time c~ f~ valsartan plasma c~176 plasma re- nin activities and pressor doses D30 obtained with repeated Ang II challenges after treatment with either 40 mg or 80 mg valsartan or placebo. Data are means with SEM

Table 1 Pharmacokinetic vari- ables: mean values and (stan- dard deviation)

Drug plasma concentration (pmol �9 1 - 1 )

Oh 2h 4h 6h 8h 24h

Valsartan 40 mg 0 2.94 1.80 0.99 0.66 0.07 (0) (1.05) (0.73) (0.4) (0.24) (0.03) Valsartan 80 mg 0 4.64 3.33 2.00 1.37 0.15 (0) (1.82) (1.95) (1.07) (0.74) (0.06)

Pharmacokinetic parameters

AUC AUC/dose AUC Cma x Clnax/dose Cma x (0-24 h) (h. ~tmol �9 Ratio (~xmol �9 1 1) (gmol. Ratio (h. umol. 1 2) 1-2 �9 ~tmo1-2) (80 : 40 mg) 1 -I �9 ~mo1-1) (80 : 40 mg)

Valsartan 40 mg 18.2 0.20 (6.6) (0.07)

Valsartan 80 mg 33.8 0.18 (16.4) (0.09)

2.94 0.032 (1.05) (0.011)

2.03 4.64 0.025 1.80 (1.04) (1.82) (0.010) (0.97)

237

Table 2 Plasma renin activity and pressor dose D30 after treatment with placebo or ac- tive drug: mean values and (standard deviation)

Plasma renin activity (ngAng I . m1-1 . h -2) AUC

0 h 2 h 4 h 6 b 8 h 24 h (0-24 h) (h. ng- ml 2. h-Z)

Placebo 2.18 2.17 3.45 3.01 1.73 2.03 51.2 (1.18) (1.11) (1.27) (1.11) (0.93) (1.37) (23.9) Valsartan 40 mg 1.98 8.10 23.54 17.35 8.37 2.66 196.5 (1.21) (7.67) (14.23) (15.81) (9.88) (2.35) (179.0)

Valsartan 80 mg 1.88 8.92 16.73 23.17 8.74 3.67 207.5 (0.81) (9.27) (17.57) (20.28) (10.03) (1.90) (191.9)

Pressor dose D30 of angiotensin II [~g] AUC

0 h 2 h 4 h 6 h 8 h 24 h (0-24 h) (h. ~g)

Placebo 4.4 5.2 4.1 3.8 4.4 4.7 107.4 (5.4) (4.0) (2.6) (3.6) (5.2) (6.1) (118.9) Valsartan 40 ing 2.9 47.4 45.7 29.2 28.1 7.8 562.2 (0.9) (43.8) (28.5) (13.0) (13.3) (6.6) (300.8)

Valsartan 80 mg 2.9 68.2 50.3 37.2 45.3 13.4 828.8 (2.1) (49.6) (26.0) (22.2) (45.0) (12.5) (653.1)

c o m p a r e d to 80 m g at 4 h a f te r dosing. O n all o the r t imes of de t e rmina t ion , the re was no d i f ference be- tween the two doses of the act ive drug.

A n g I I cha l lenge

Overall effect on A n g H dose-response curves

The m e a n values ob t a ined f r o m p o o l e d da ta of all A n g I I chal lenges ( increase of systolic BP versus A n g I I doses) are shown in Fig. 4. Subject i has b e e n exc luded

f r o m these m e a n values because of his a typica l beha - vior on p l acebo (see below) . In all o the r evaluat ions , this subject was included.

In the absence of r e c e p t o r b lockade , i .e. on p lacebo , the cha l lenge tests with doses ranging f r o m 0.5-4 ~tg A n g II p r o d u c e d effects within the l inear po r t i on of the A n g I I dose - r e sponse curve. For safe ty reasons , injec- t ion of h igher A n g I I doses was s t o p p e d b e f o r e f la t ten- ing of the r e sponse was es tabl ished. A f t e r r e c e p t o r b lockade , the re la t ionsh ip b e t w e e n dose ( l inear scale) and r e sponse was ve ry flat, bu t a l inear regress ion line was still a r e a s o n a b l e fit for the ex t r apo la t i on of D30.

238

A n g II dose - response

Delta BPS [mmHg] 50

4O

3O

20

10

0

0 5 10 15 20 25 30 35 40

Dose [pg Ang II]

45

Delta

40

30

20

10

Increase of systolic BP to 4 IJg of Ang II BPS [mmHg]

0 I I I I I I

0 4 8 12 16 20 24

Time [h]

Delta BPS [mmHg] 50

Ang II log dose - response

40

30

20

10

0

Placebo ---4----- 40 mg �9 80 mg

I [

0.1 1 10 100

Dose [pg Ang II]

Fig.4 Overall changes in systolic blood pressure (BPS) induced by increasing i.v. bolus doses of Ang II after placebo and after single- dose administration of either 40 mg or 80 mg valsartan. Ang II challenge doses plotted on the x-axis are given in absolute (upper panel) and log-transformed (lower panel) values. Data are means with SEM

Decrease of heart rate to 4 pg of Ang II Delta HR [beats. min 1] 0

" ' " = " " Placebo -5 , ~ �9

40 mg

10. / / / I . ~ ~ ~ ~ _ ~ 80mg

1 , , ...tt ............................ 20, , (

25 I I I I I !

0 4 8 12 16 20 24

Time [h]

Fig.5 Time courses for the increase of systolic blood pressure (BPS) and for the decrease in heart rate (HR) after single 4 ~g i. v. bolus injections of angiotensin II to 6 subjects pre-treated with either 40 mg or 80 mg valsartan or placebo. Data are means with SEM

The linear dose-response curves for Ang II were not only flattened but tended to reach a plateau at the up- per end of the Ang II dose range allowed by the proto- col (up to 10-fold the initial D30 , i.e. up to around 40 ~g Ang II), especially after 80 mg of valsartan.

The main effect of valsartan consisted of a shift in the log dose-response curves to the right. This shift was si- milar from 2 to 8 h after dosing and reverted towards baseline at 24 h. The slope of the mean log-linear dose- response curves after the active drug appeared to be slightly less than after placebo, and there was a clear- cut separation between the two doses of valsartan, with a parallel shift of the curve to the right after 80 mg com- pared to 40 mg (Fig. 4).

Pressor dose D30

The mean D30 values are shown in Fig. 3 and Table 2. The mean baseline values of D30 were very similar be- fore the 40 mg and the 80 mg dose of valsartan (2.9 ~g in both cases). Before placebo, the mean baseline D30 was higher (4.4 ~tg) and remained at this level up to 24 h (range 4.1-5.2 ~tg). This was due to the relatively flat dose-response curve observed in subject 1 present during the placebo period only, resulting in individual D30 values ranging from 7.8-17.0 ~tg in this particular subject. In the pre-trial baseline challenge and at base- line before the two doses of valsartan, the dose-re- sponse curves of this volunteer were similar to those of the other subjects. We cannot explain the unusual beha- vior before and after placebo in this case. At least ac- cording to his PRA values, which were in the normal

Table 3 Effect of 4 ~tg angio- tensin II on systolic blood pres- sure and heart rate after treat- ment with placebo or active drug: mean values and (stan- dard deviation)

Increase of systolic blood pressure (mm Hg) AUC 0 h 2 h 4 h 6 h 8 h 24 h (0-24 h)

(h" mmHg)

Placebo 32.7 30.2 32.8 33.5 35.7 32.2 804.0 (8.9) (9.9) (7.8) (7.7) (10.3) (8.1) (178.4) Valsartan 40 mg 35.3 13.7 14.7 17.7 21.3 26.2 528.7 (3.8) (5.3) (4.9) (5.3) (5.9) (5.6) (93.6) Valsartan 80 mg 35.3 11.3 9.0 10.5 18.2 25.3 463.2 (12.1) (4.9) (6.3) (4.8) (10.6) (5.6) (126.7)

239

Decrease in heart rate (beats �9 min -1) AUC

0 h 2 h 4 h 6 h 8 h 24 h (0-24 h) (h- beats, min -1)

Placebo -13.5 -21.0 -16.3 -16.8 -14.8 -17.0 -391.3 (5.1) (1.7) (6.4) (6.7) (5.5) (5.2) (97.1) Valsartan 40 mg -12.3 -6.8 -5.2 -6.5 -7.0 -14.2 -225.7 (5.5) (3.5) (4.0) (4.l) (4.3) (3.5) (82.2)

Valsartan 80 mg -14.2 -7.3 -7.7 -7.2 -9.3 -13.7 -251.8 (8.1) (5.7) (3.3) (3.9) (5.5) (5.1) (103.0)

range, this subject's RAS seemed not to be altered by external factors such as excessive salt intake prior to placebo administration.

The variability of D30 within subjects of the six mea- surements on placebo was between 11% and 54%, with a mean of 37 %. Between-subject variability was around 100 %.

Administrat ion of valsartan was followed by a rapid increase of D30 by a factor of 15 (40 mg) to 25 (80 mg), with a maximum effect at 2 h after dosing. D30 was sig- nificantly increased compared to placebo from 2-24 h after both doses of valsartan, and the effect of 80 mg on D30 was consistently more pronounced, although this difference between doses did not reach statistical significance at any timepoint.

Effect of 4/~g Ang H on systolic blood pressure

The mean values for systolic BP changes to 4 ~g Ang II are shown in Fig. 5 and Table 3. At baseline, mean systo- lic BP increased to injections of 4 ~tg of Ang II by 33- 35 mmHg. During the placebo period, this effect re- mained constant, with a coefficient of variation within subjects of 6 % to 29 % (average 19 %). Between-sub- ject variation was around 20 to 30 %.

After administration of valsartan, the mean response to 4 ~tg of Ang II was drastically reduced, with a maxi- mum suppression at 2 h after 40 mg and at 4 h after 80 mg. At these time points, the response to 4 ~tg of A n g I I had decreased to an average of 13.7 and 9.0 mm Hg, respectively. The effect of the higher dose of valsartan on this variable was clearly more pronounced,

being significantly different from placebo up to 8 h after dosing and different f rom 40 mg at 4 h and 6 h. After 40 mg valsartan, the increase of systolic BP was signifi- cantly inhibited up to 6 h compared to placebo. At 24 h after dosing, there was still a reduction with both doses, but the difference to placebo was no more significant.

Effect of 4/zg Ang H on heart rate

The mean values for the effect of 4 ~tg Ang II on H R are shown in Fig. 5 and Table 3. At baseline, mean H R de- creased to injections of 4~g of A n g I I by 12- 14 beats , min -1. During the placebo period, this de- crease became more pronounced, especially at 2 h after placebo where in all subjects a stronger decrease was observed (mean value 21 beats , min-1). Both within- and between-subject variability was higher (around 30 %) for the decrease in H R as compared to the in- crease in BR

After administration of valsartan, the mean response of H R to 4 ~tg of Ang II was inhibited to about the same degree with both doses, with a significant difference from placebo up to 6 h (80 mg) and 8 h (40 mg), respec- tively.

Descriptive pharmacokinet ic-pharmacodynamic evaluations

The individual and mean values of the drug plasma con- centrations have been plotted against the respective val- ues of the various pharmacodynamic variables to allow

240

for a descriptive analysis of potential relationships be- tween pharmacokinetic (PK) and pharmacodynamic (PD) effects.

Plasma renin activity

PRA was the only PD variable for which the maximum PK values were not associated with the maximum PD effects. The individual values showed a wide scatter. Based on the mean values, a counter-clockwise hyster- esis loop may be assumed (data not shown). This is typi- cal for a lag time between the increase of the drug plas- ma levels and the occurrence of the effect, which is also obvious from Fig. 3.

Inhibition [%]

100

75

50

25

0

-25

-50

-75

-100

A DA A DA A D A

D A l l D

DI~IO [] D D 0 A AD D A D

X~ D O

IA ITI ~ 3 a

a

A A A

A [3

o Placebo [3 40 rng t~ 80 mg

I 1 I

2 4 6

Drug plasma concentration [pmol. I 11

Pressor dose D3o

Both the maximum PK values and the maximum PD ef- fects occurred at 2 h after dosing. Since no measure- ments are available between 0 h and 2 h, it remains un- clear whether the hysteresis suggested by the data is real (not shown). Overall, the mean data point to a hy- perbolic PK-PD relationship for the descending part of the loop.

Increase of systolic BP to 4 #g orAng H

For this variable also, a hyperbolic PK-PD relationship is strongly suggested both by the individual and the mean data (not shown). The two mean curves (40 mg and 80 mg dose) were very similar. The observations made for the increase of systolic BP are also valid for the percentage inhibition of the BP response (Fig. 6). This variable was further evaluated by PK-PD model- ing (see below).

Inhibition [%1 100

75

50

25

0

-25

+.11~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . & . . . . . . . . . . . . . . . .

' / ...... ;;;;;;7 ..................... l i : s y " - - - " ' " '+ "

~ = : : : : : : 2 2 2 ] . . . . . . .

..... * ..... Placebo ..... = ..... 40 mg ...... ~ ..... 80 mg

1 I I I

0 1 2 3 4 5

Drug plasma concentration [pmol-I-]

Fig .6 I n d i v i d u a l (upper panel) and ave rage (lower panel) va lues for p e r c e n t a g e inh ib i t ion of the systol ic b l o o d p ressu re r e sponse in r e l a t i o n to v a l s a r t a n p l a s m a concen t r a t i ons m e a s u r e d in six sub- jects t r e a t e d wi th e i the r 40 mg or 80 mg v a l s a r t a n or p l acebo

Decrease of heart rate to 4 #g orAng H

The scatter of the individual values of the HR effects of 4 btg Ang II was greater than that of the BP effects. Overall, based on the mean data, a hyperbolic PK-PD relationship also appears to be likely for this variable. The percentage inhibition of the effects of 4 btg Ang II on HR were further evaluated by PK-PD modeling (see below).

Increase and percentage inhibition of systolic BP versus PRA

The relationship between the increase of PRA and the inhibition of the AT 1 receptors was hyperbolic with a clear-cut hysteresis. The inhibitory effect of valsartan at the AT~ receptor was already saturated while PRA was still increasing.

Pharmacokinetic-pharmacodynamic Ema x modeling

The measured PD effects for each volunteer after each treatment were plotted along with the measured plas- ma concentration of valsartan in order to explore the possible relationship between the drug plasma concen- trations and the effect of 4 b~g Ang II on systolic BP and on HR.

The highest observed drug plasma concentrations were observed at 2 h post dose for all subjects. Since the PD profiles during the ascending phase of the plasma concentrations were not available, no conclusion con- cerning a possible hysteresis of the effects can be ob- tained. This means that based on the observed data alone we do not know whether the PK-PD relationship is instantaneous. In addition, the available set of data did not allow study of whether there is an effect compart- ment for the PD relationship. On the one hand, the num- ber of samples was not sufficient to estimate the pharma-

Table 4 Summary of statistical analysis of Tukey's Studentized Range Test

* P < 0.1; NS, not significant

Variable Time

Oh 2 h 4 h 6 h 8 h 24h

Plasma renin activity Valsartan 40 mg vs placebo NS * * * * NS Valsartan 80 mg vs placebo NS * * * * * Valsartan 40 mg vs 80 mg NS NS * NS NS NS

Pressor dose D30 Valsartan 40 mg vs placebo NS * * * * * Valsartan 80 mg vs placebo NS * * * * * Valsartan 40 mg vs 80 mg NS NS NS NS NS NS

Increase of systolic blood pressure to 4 pg angiotensin lI Valsartan 40 mg vs placebo NS * * * NS NS Valsartan 80 mg vs placebo NS * * * * NS Valsartan 40 mg vs 80 mg NS NS * * NS NS

Decrease of heart rate to 4 pg angiotensin II Valsartan 40 mg vs placebo NS * * * * NS Valsartan 80 mg vs placebo NS * * * NS NS Valsartan 40 mg vs 80 mg NS NS NS NS NS NS

241

Table S Results from pharma- cokinetic-pharmacodynamic Ernax modeling [E .... maximum inhibition in % of baseline, ICso inhibitory concentration 50 (50 % inhibition of Ema,], indi- vidual values, mean values and (standard deviation)

Subject Estimated parameters for delta blood pressure

no. E ..... 90 % limits ICs0 90 % limits (% inhib.) (~tmol. 1-1)

1 99.9 0.0 100.0 1.13 0.1 2 64.5 16.6 94.3 0.48 0.0 3 80.0 7.7 99.5 1.14 0.1 4 84.0 60.7 94.7 0.25 0.1 5 70.0 47.8 85.6 0.16 0.0 6 44.3 28.9 60.9 0.05 0.0 Mean 73.8 0.53 (SD) (18.9) (0.49)

Subject Estimated parameters for delta heart rate

no. Ema x 90 % limits ICs0 90 % limits (% inhib.) (~tmol. 1-1)

13.6 10.2 14.7 0.8 0.7 0.6

1 39.7 21.4 61.4 0.02 0.0 11.4 2 91.0 0.5 100.0 1.07 0.1 10.6 3 58.6 41.7 73.7 0.20 0.0 1.2 4 81.7 16.3 99.0 0.42 0.0 5.9 5 89.6 72.1 96.6 0.27 0.1 0.5 6 81.6 44.8 96.0 0.20 0.0 1.1 Mean 73.7 0.37 (SD) (20.3) (0.37)

cokinetics profiles, especially during the absorption phase. On the other hand, the lack of PD measurements in the initial phase does not allow to access the rate of ex- change in the effect compartment. Nevertheless, it was assumed that the PK-PD relationship is instantaneous. In addition, there were considerable within- and be- tween-subject variations for the PD measurements.

The estimated parameters to describe the form of the PK-PD c u r v e (Ema x and ICs0), obtained from the Ema x model are listed in Table 5. For Emax the variability be- tween subjects was smaller (average 26 % for BP and 28 % for HR) than for ICs0 (91% and 101%, respective- ly).

The model fitted the data rather poorly, i. e. the 90 % intervals for the estimated parameters were rather wide

for all subjects. This is probably due to the assumption of an instantaneous PK-PD relationship, which may not be appropriate, and because there were few mea- surements available for the estimation. In order to prop- erly describe the PK-PD relationship, more data points in the beginning of the experiment would be necessary.

The inhibition of the effect of 4 btg Ang II on systolic BP and H R fitted using the individual and the mean Ema x and ICs0 values of the 6 subjects is shown in Fig. 7. The variability between subjects of the fitted curves was rather high.

242

Inhibition of effect of 4 pg Inhibition [%]

100

90

80

70

60

50

40

30

20

10

0

Ang II on systolic BP

, .

. , = ' . . . . o . . . . . . . . . . . . . . . . . . . .

. . - " . ' ~~ o . . . . . . . . . . . . . . .

,::~ . . . . . . / : : . , -

0.01 0.1 1 10

Drug plasma concentration [pmol. P]

100

Inhibition of effect of 4 pg ang II on heart rate

Inhibition [%] 100

90

80

70

60

50

4 0

30

20

10

0

. . . . . . . . . . . ~[7"-::::: . . . . . . . .

I i I

0,Ol 0.1 1 10 100

Drug plasma concentration [pmol. I 11

Fig."/ Percentage inhibition of the drug plasma concentration- related effect on systolic blood pressure (upper panel) and heart rate (lower panel) induced by i.v. bolus injections of 4 ~g Ang II to six healthy male volunteers pre-treated with valsartan. Indivi- dual (dotted lines) and mean (solid line) curves were fitted by phar- macokinetic-pharmacodynamic E~.~ modeling

Discussion and conclusions

Methodology

The methodology used in the present study proved to be suitable to assess the AT~ receptor blockade induced by valsartan. Using the Finapres device, the short-lived in- crease in BP and the decrease in H R in response to bo- lus injections of Ang II could easily be monitored and documented on a beat-by-beat basis.

The very strong inhibition of the cardiovascular re- sponse to Ang II injections, regularly detectable at 2 h after dosing with valsartan, led to an unblinding of the trial.

Methodological tests had suggested a linear relation- ship between the Ang II bolus doses and their maximum effect on systolic BR This was confirmed in this trial

especially for the tests at baseline and on placebo, which were performed within the linear portion of the dose-response curve with doses between 0.5 and 4 ~g of Ang II. With higher bolus doses of Ang II up to 10 ~g, the flattening part of the curve might have been reached [24].

For practical reasons, a linear regression was applied to all challenge tests for the evaluation of the data. This allowed calculation of the pressor dose, D30 , for all tests in the same way. The flat dose-response curves after AT 1 blockade, in many cases associated with a relatively large intercept, led to very high (extrapolated) D30 values.

The use of one single bolus dose of Ang II instead of establishing a time-consuming, full dose-response curve would allow measurement of the receptor blockade at shorter intervals, which is desirable if PK-PD modeling is performed. Other authors repeatedly injected the same Ang II bolus doses, which were pre-selected for each individual to produce an increase of systolic BP by 30 mmHg, and used the percentage inhibition of the response to this Ang II dose as a measure of AT 1 block- ade [12, 14]. Selected data from the present trial, those from the 4 ~g doses of Ang II - and where not available from the nearest dose of Ang II to 4 ~g - were used for a similar approach. The aim of this additional evaluation, which was not foreseen in the original protocol, was pri- marily to evaluate the usefulness of the "abbreviated" method compared to the time-consuming titration pro- cedure used in the present trial. The key parameter de- rived from the full dose-response curves (D30) allowed detection of significant changes from placebo up to 24 h without significant differences between the two do- ses of the active compound. On the other hand, the parameters representing the "abbreviated approach" (increase of systolic BP and decrease of H R to 4 ~g Ang II) were slightly better able to distinguish between doses. In addition, the percentage inhibition of the base- line BP response to Ang II allowed determination of an ICs0 value, which is the most common way to express the degree of receptor inhibition. It can be concluded that in future trials the "abbreviated" approach may be used without substantial loss of information, allowing chal- lenge tests to be performed at much shorter time inter- vals than is possible with the lengthy titration proce- dure performed in the present study.

Pharmacokinetics

The AUC values for the plasma drug concentrations ob- served after 40 mg and 80 mg of valsartan were consid- erably higher than those found with similar single doses in earlier trials [9]. This is due to the fact that the latter were performed with a less bioavailable preliminary capsule formulation. The ratio of the AUC values after 80 mg and 40 mg was on the average around 2, but due to the variability, dose proportionality could not be es- tablished statistically. The specific AUC values were

slightly higher than could be expected from the results of a previous multiple-dose phase I trial [9], which was performed with the same oral capsule formulation used in the present study. However, the tma x and the t~/2 val- ues were comparable to those of that earlier investiga- tion.

The within-subject variation of the pharmacokinetics, as estimated from a variance analysis of specific AUC and Cma x values, was of the same order of magnitude as the between-subject variability as expressed by the co- efficient of variation of the AUC values for each dose.

Plasma renin activity (PRA)

PRA increased after administration of valsartan, but unlike in the previous investigations [9] without dose- dependency. Within- and between-subject variability on placebo were similar (around 40-50%), but inter- subject variability increased considerably after valsar- tan. The maximum effect was different with the two do- ses, occurring at 4 h with 40 mg and at 6 h with 80 mg, but the true maximum effect might have been missed because of the rather large spacing between the sam- ples. PRA was the only pharmacodynamic variable which peaked with a lag time behind the peak of the plasma drug concentrations. This is also evident from the hysteresis loop suggested by the PK-PD plot (see below).

The lack of an instantaneous relationship between the increase in PRA and the increase in the drug plas- ma levels has already been observed in previous trials with valsartan and may be explained by the time need- ed to activate the renin angiotensin system (RAS).

The relationship between the increase in PRA and the inhibition of AT1 receptors was not linear, as would be expected for two instantaneously related pharmaco- dynamic parameters, but was hyperbolic with a hyster- esis, indicating that the inhibitory effect of valsartan at the AT 1 receptor was saturated, while PRA still in- creased. The decreasing slope of the hysteresis curve suggests that the receptor blockade disappears more ra- pidly than the stimulation of the RAS. This would indi- cate that the time course and the magnitude of the com- pensatory increase in PRA are not directly linked to the inhibition at the receptor level, but follow with a time lag both for the activation and the deactivation of the RAS.

Ang H dose-response curves

The main effect of valsartan was a parallel shift of the Ang I! log dose-response curves to the right. In addi- tion, there appeared to be a slight decrease in the slope of the log dose-response curves after valsartan and a tendency to flatten at the upper part of the curve, espe- cially after 80 mg of the drug. Since the upper parts of the dose-response curves were not fully established for

243

safety reasons, these data have to be interpreted with caution. Nevertheless, this observation is in line with some of the pre-clinical findings with valsartan [7]. In binding studies in rat aortic smooth muscle cells, valsar- tan was clearly a competitive inhibitor at the AT 1 recep- tor, leading to a parallel shift of the dose-response curves to the right without any decrease in the maxi- mum response. On the other hand, the Ang II log dose- response curves for the contractions of rabbit aortic rings as well as for the blood pressure response in the pithed rat were not only displaced to the right, but showed a slight decrease in the slope and a distinct re- duction in the maximum response. Similar findings were made for other Ang II receptor antagonists [for re- view, see 6], but not for losartan [27]. This type of antag- onism has been termed "insurmountable" due to a slow- er reversibility of the receptor blockade [28]. Such beha- vior has been explained by differences in chemical struc- ture and might be linked to the carboxylic acid moiety [7]. If not losartan, but its active carboxylic acid metabo- lite, EXP3174, is administered, an unsurmountable component in the Ang II dose-response is also ob- served in the conscious rat [29].

Other variables indicative of inhibition of A T 1 receptors

This trial has shown that valsartan blocks AT~ receptors in man. The various parameters used to express the time course and magnitude of receptor blockade differed to some degree in their variability. The highest within-sub- ject variation was observed for D30 (average 37 %). This parameter derived from the full Ang II dose-response curves showed the peak effect at 2 h after dosing, and was able to detect statistically significant receptor blockade up to 24 h after dosing. On the other hand, D30 did not distinguish between the two doses of valsar- tan to a statistically significant degree. Although the ef- fect of the 80 mg dose on D30 was clearly more pro- nounced from 2-24 h after dosing, the between-subject variability was too large to separate the doses statistical- ly. The slope and the angle of the Ang II dose-response curves were practically identical after 40 mg and 80 mg from 2-8 h after administration.

The parameters derived from the "abbreviated" ap- proach (changes of BP and H R to a 4 ~g dose of Ang II and the percentage inhibition of these effects) were, like D30, able to distinguish between doses. For the in- crease of BP the difference was statistically significant at 4 h and 6 h after dosing. These parameters were also mainly influenced by the shift of the Ang II dose-re- sponse curves to the right. Therefore, they are a good measure of the degree of receptor blockade.

Heart rate decreased consistently and dose-depen- dently after Ang II bolus injections. Valsartan-induced inhibition of the effect of Ang II on H R was similar in extent and time course to that on systolic BP. Blockade of the pressor effect of Ang II after valsartan was fol- lowed by a corresponding reduction in the decrease of

244

HR. This is in line with the assumption that the decrease in H R after Ang II is a secondary consequence of reflex vagal activation induced by the direct pressor effect of Ang II [30].

Pharrnacokinetic-pharrnacodynamic evaluations

The relationship between the various parameters used to express A T 1 receptor blockade and drug plasma concen- trations, which was assessed in a descriptive way, did not prove that the relationship is instantaneous. This is be- cause there were no data points on the ascending loop of the concentrat ion-response curves (i. e. between 0 h and 2 h after dosing). For all variables, except for the in- crease in BP and the decrease in H R to 4 pg Ang II, which were slightly more pronounced at 4 h, the maxi- mum effects were reached together with the maximum drug concentrations at 2 h, and only the descending parts of the loops could be established. In previous trials [9] with more data points in the first 4 h after drug admin- istration, the plasma concentrations for valsartan clearly peaked at 2 h, which makes it likely that in the present trial also tma x was really at 2 h. Since the peak effect of most of the variables indicative of receptor blockade did not lag behind the maximum drug concentrations it may be assumed that the ascending parts of the concentra- tion-effect curves were either linear or even followed the hyperbolic course of the descending part.

For all parameters of receptor blockade, the des- cending parts of the concentrat ion-effect curves were hyperbolic, with a very steep slope in the concentrat ion range up to 1-2 ~tmol �9 1-1. At a valsartan concentrat ion of 2 ~mol �9 1-1, the maximum effect on receptor block- ade was almost reached. This was further confirmed by the formal pharmacokinet ic-pharmacodynamic model- ing of the percent inhibition of the BP and H R respon- ses to 4 ~tg Ang II. At 2 ~tmol- 1 -z, 80 % of the maxi- mum attainable effect was reached. The maximum at- tainable effect (Emax) was the same for the inhibition of the BP and the H R response (74 % on average). The be- tween-subject variation for Ema x was less than 30 %. The mean IC50 for the inhibition of BP was slightly higher than that for the inhibition of the H R response (0.5 ~mol. 1-1 and 0.4 btmol �9 1-1, respectively). The be- tween-subject variation of ICs0 was considerable, aver- aging 90-100 %. Based on the very wide 90 % confi- dence intervals for the individual fits it must be con- cluded that the model fitted the data poorly. This is probably due to the assumption of an instantaneous concentration-effect relationship which might not be fully appropriate, and due to the limited number of measurements available for the estimation, especially in the early phase after dosing.

In summary, it may be concluded that:

�9 The methodology used is suitable to evaluate A T 1 re- ceptor blockade in man. Instead of using a full dose-

response curve for Ang II at each timepoint, an ab- breviated approach with only one pre-determined Ang II dose may be adopted without substantial loss of information, but allowing for measuring the de- gree of receptor blockade at shorter intervals, which is desirable if pharmacokinet ic-pharmacodynamic modeling is performed.

�9 Valsartan is an inhibitor of the A T 1 receptor in man, with a m e a n Ema x Of 74% and a mean ICs0 of 0.53 ~mol . 1-2 for the blood pressure response to exo- genous Ang II.

�9 Receptor blockade after single oral doses of 40 and 80 mg valsartan reaches its maximum at 2 h and is de- tectable up to 24 h after administration.

�9 Despite a doubling of systemic exposure, the rela- tionship between dose and receptor blockade ap- pears to be rather flat in the dose range of 40-80 rag. Nevertheless, the receptor blockade after the higher dose is more pronounced.

�9 Valsartan is well tolerated by healthy subjects in the dose range tested.

Acknowledgements The authors are indebted to Mr. Ambroise Habersetzer for the accurate performance of the angiotensin II challenges, to Mrs. Ursula Mtiller, Dr. Frdd6ric Cumin and Mr. Hans-Peter Baum for skillful determination of plasma renin activ- ities, and to Mrs. Fran~oise Marfil for careful analysis of valsartan plasma concentrations. Statistical support by Dr. Georges Preis- werk and fruitful discussions with Dr. Peter Lloyd have greatly contributed to the quality of this paper. Last but not least, the au- thors wish to thank the healthy volunteers for their participation.

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