II receptor
`Dul? 532, an angiotensin
`antagonist: First administration and
`comparison with losartan
`
`in healthy male
`of oral Dul? 532
`activity
`II antagonist
`and angiotensin
`tolerability
`the
`We investigated
`or heart
`rate.
`no effect on blood
`pressure
`(1
`to 200 mg) was well
`tolerated,
`with
`subjects. DuI? 532
`responses
`to
`Compared
`with
`losartan
`(100 mg), Dul? 532
`(200 mg) was a weak antagonist
`of pressor
`intravenous
`angiotensin
`II. Maximum
`inhibition
`of diastolic
`pressor
`response was 86%
`(95% confidence
`interval
`[CI],
`84%, 88%) approximately
`4.6 hours after
`losartan
`and 48% (95% CI, 38%, 56%) 8.7 hours
`after Dul? 532. Twenty-four
`hours after dosing,
`inhibition
`by losartan
`and DuP 532 was similar
`(40%
`to
`45%). DUP
`532
`is extensively
`bound
`in human
`plasma, with an in vitro
`free
`fraction
`of 0.06. Althaugh
`Dul? 532 and EXP3174
`(losartan’s
`active metabolite)
`have similar
`AT,-receptor
`potency,
`and plasma
`concentrations
`of DuP 532 were much greater
`than
`losartan/EXP3174,
`the level of antagonism
`was much
`less for DuP 532. These
`results
`indicate
`that multiple
`factors determine
`the in vivo potency of angiotensin
`II antagonists,
`including
`alfinity
`for and distribution
`to
`the receptor
`as modulated
`by plasma binding.
`(Clm Pharmacol
`Ther 1997;61:59-69.)
`
`Lo, PhD, David D. Christ, PhD,
`MD, PhD, Man-Wai
`Michael R Goldberg,
`Rita Chiou, MS, Christine
`I. Furtek,
`BS, Ohad Amit, MS,
`Alexandra
`Carides, MS, Jerome Biollaz, MD, Valerie P&pet, MD,
`Juerg Nussberger,
`MD, PhD, and Hans R Brunner, MD
`West Point,
`Pa., Wilnzin~ton, Del., and Lamanne, Switzerland
`
`Losartan was the first orally active, nonpeptide
`AT,-selective
`angiotensin
`II antagonist
`to be eval-
`uated
`in humans.re5 This article describes
`the ini-
`tial clinical experience with DuP 532,6,7 a struc-
`turally
`related angiotensin
`II antagonist. Unlike
`losartan,2.5 DuP 532 is a carboxylic acid that does
`not have an active metabolite
`(Fig. 1)”
`In preclin-
`ical studies, compared with
`losartan, DuP 532
`showed similar potency and duration of angioten-
`sin II antagonist activity.6 Like EXP3174,
`the ac-
`tive metabolite
`of losartan, DuP 532 is an insur-
`antagonist. 6,7*9 Losartan,
`EXP3174,
`mountable
`
`From Merck Research Laboratories, West Point; DuPont Merck
`Pharmaceutical Company, Wilmington;
`and Centre Hospi-
`talier Universitaire Vaudois, Lausanne.
`Supported by a clinical grant from Merck Research Laboratories,
`West Point, Pa.
`Received for publication April 1, 1996; accepted Aug. 26, 1996.
`Reprint
`requests: Michael R. Goldberg, MD, PhD, Merck Re-
`search Laboratories, BL 2-7, West Point, PA 19486.
`Copyright 0 1997 by Mosby-Year Book, Inc.
`0009-9236/97/$5.00 + 0 13/l/77568
`
`to be extensively
`532 were all shown
`and Duf
`the greatest bind-
`protein bound
`(?.99%),i’ with
`ing evident
`for DuP 532.
`In vitro, such binding
`manifests pharmacologically
`as a reduction
`in the
`apparent potency at the AT,-receptor
`when
`inhi-
`bition
`is determined
`in the presence of human
`serum albumin. Thus, k values increased
`from 1.2
`to 5300 nmol/L
`for DuP 532,
`from 24 to 400
`nmol/L
`for losartan, and from 1 to 200 nmol/L
`for
`EXP3174.7 Further, DuP 532 was not metabolized
`in human and
`rat microsomal
`preparations
`but
`had a low apparent
`oral bioavailability
`in ani-
`mals8 Because of these pharmacokinetic
`differ-
`ences, it was of interest
`to compare
`this compound
`to losartan as an angiotensin
`II antagonist
`in the
`same clinical paradigm used to establish the activity
`of losartan. This article summarizes
`the tolerability
`results from a rising-dose phase I study, a crossover
`study of the antagonism by DUP 532 and losartan of
`pressor responses of angiotensin
`II, and results of in
`vitro protein binding studies in plasma from several
`species, including humans.
`
`59
`
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`

`
`60 Goldbeg et al.
`
`CLINICAL
`
`P HARMACOLOGY
`
`&THERAPEUTICS
`JANUARY 1997
`
`days, with
`Each panel was studied on alternating
`dose advancement between panels based on clinical
`tolerability. The dose was advanced until a maxi-
`mum dose of 200 mg had been received or until
`symptoms, blood pressure effects, or abnormal
`lab-
`oratory
`tests precluded a further
`increase in dose.
`The first panel received doses of 0.1,5, 25, and 100
`mg; the second panel received doses of 1, 10,50, and
`200 mg. Within each panel,
`the allocation schedule
`was designed so that all subjects received all doses of
`DuP 532 for
`that panel, as well as placebo. One
`week separated each treatment period
`for each sub-
`ject.
`The second study was a two-part study (A and B)
`designed to investigate
`the ability of maximally
`tol-
`erated doses of DuP 532 from
`the
`first study to
`antagonize pressor responses to exogenous angio-
`tensin
`II. The methods used were similar to those
`previously used to study losartan.192 Part A of the
`study was an exploratory, single-blind, nonrandom-
`ized rising-dose study of four healthy volunteers.
`Oral doses of DuP 532 (10, 50, 100 and 200 mg)
`were advanced at weekly intervals to confirm safety
`results of the rising-dose
`tolerability
`study and to
`select doses for the second part of the study.
`Part B of this study was to be a double-blind,
`placebo-controlled,
`four-period
`crossover study of
`eight additional
`healthy volunteers
`receiving oral
`doses of placebo, 100 mg losartan, and two doses of
`DuP 532 to compare
`the duration
`and extent of
`angiotensin antagonism by the two angiotensin
`II
`antagonists. However, preliminary
`data
`from
`the
`tolerability
`study and antagonism
`results of part A
`(not shown)
`indicated
`that DuP 532 might be less
`active than anticipated and had a very long half-life,
`so that a l-week
`interval after a single dose might
`not be sufficient
`to ensure washout of drug effect.
`Therefore
`part B was modified
`to a three-period
`study in six subjects with
`losartan and placebo
`in
`random sequence in the first two periods, preceding
`the 200 mg dose of DuP 532.
`The study protocols were reviewed and approved
`by the ethical review committee of the Centre Hos-
`pitalier Universitaire Vaudois
`(Lausanne, Switzer-
`land). All subjects provided written
`informed con-
`sent for their participation
`in the study.
`Subjects andprocedures. Healthy male subjects be-
`tween the ages of 18 and 40 years were enrolled
`in
`these studies. For the first (i.e., tolerability)
`study,
`subjects were provided a diet that contained 130 to
`150 mmol sodium and 80 to 100 mmol potassium
`every 24 hours for 4 days before each dosing day and
`
`Fig. 1. Chemical structures of DUP 532 (A), losartan (B),
`and EXP3174 (C). Asterisk
`(*) indicates ring location of
`14C for protein binding studies.
`
`METHODS
`to in-
`Study designs. Two studies were conducted
`vestigate the tolerability and activity of oral DuP 532
`in healthy male subjects. The first study was a single-
`rising-dose, placebo-controlled,
`double-blind
`five-
`period study in two alternating panels of 10 subjects.
`
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`

`
`CLINICAL
`VOLUME
`
`PHARMACOLOGY
`61, NUMBER 1
`
`& THERAPEUTICS
`
`Goldbeg et al. 61
`
`on the dosing days. For the second (i.e., antagonism)
`study, subjects were instructed
`to follow a diet that
`contained approximately 180 mmol sodium every 24
`hours. Before
`the start of the second study, an in-
`travenous bolus dose of angiotensin
`II
`that
`in-
`creased reclining systolic blood pressure approxi-
`mately 25 mmHg, was identified
`for each subject by
`monitoring
`the blood pressure response to angioten-
`sin II at the finger with a Finapres
`recorder.”
`The subjects fasted
`from 8 pm the night before
`until 4 (tolerability
`study) or 6 (antagonism study)
`hours after dosing on each study day. For the an-
`tagonism study, individual
`responses to the selected
`angiotensin
`II dose were confirmed
`before each
`dose. Study drug was then given with 200 ml water.
`In the tolerability
`study, blood pressure and heart
`rate were measured with an automatic sphygmoma-
`nometer
`(Dinamap)
`and recorded
`frequently
`from
`predose until 12 hours after dosing.
`In the second
`study,
`resting blood pressure and
`the onset and
`duration of angiotensin
`II blockade were assessed by
`continuous monitoring of supine blood pressure and
`heart rate and responses to the selected angiotensin
`II dose with
`the Finapres machine.’ Eight bolus
`doses of angiotensin
`II were given at OS- to 2-hour
`intervals during
`the first 12 hours after administra-
`tion of the study drug and then again 24 hours after
`study drug. Two angiotensin
`II responses were av-
`eraged 24 hours after dosing.
`Material. DuP 532 (4-pentafluoroethyl-2-propyl-l-
`[(2’-(lH-tetrazol-5-yl)biphenyl-4-yl)methyl]
`imidazole-
`5-carboxylic acid; Fig.
`l),
`losartan, and EXP3174
`were synthesized by the DuPont Merck Pharmaceu-
`tical Co. (Wilmington, Del.), 14C-DuP 532 (specific
`activity
`38.9 mCi/mmole;
`radiochemical
`purity
`>99%
`[stated]) was synthesized by New England
`Nuclear Co. (Boston, Mass.). Human serum albu-
`min and a,-acid glycoprotein were bought
`from Cal-
`biochem Corp.
`(San Diego, Calif.). Frozen human
`plasma
`from male, nonsmoking white donors was
`bought
`from Biological Specialty Corp.
`(Lansdale,
`Pa.). All buffer salts and solvents were obtained
`from commercial
`sources and were
`the highest
`grade available. The micropartition
`devices were
`bought
`from Amicon
`(Centrifree, Danvers, Conn.)
`and were used after pretreatment
`according
`to the
`manufacturer’s
`recommendation.
`Plasma protein binding determinations. Plasma
`protein binding and binding
`to purified
`albumin
`or ol,-acid glycoprotein
`was determined
`as de-
`scribedI with use of ultrafiltration.
`All determi-
`nations were done
`in triplicate. Plasma binding
`
`rats, rhesus monkeys,
`
`was also determined with
`and beagle dogs.
`ratio determinations. The
`Blood/plasma distribution
`in vitro blood/plasma
`ratio
`for 14C-DuP 532 was
`determined
`as described” with use of fresh beagle
`dog and rat blood.
`Plasma drug levels: Plasma renin activity (PRA) and
`In both studies, blood
`angiotensin
`II concentration.
`was drawn at specified
`times
`for measurement
`of
`plasma drug levels, PRA, and plasma angiotensin
`II
`concentration
`up
`to 24 hours after dosing. Total
`concentrations of DuP 532, losartan, and the active
`metabolite
`of losartan
`(EXP3174) were measured
`by HPLC with ultraviolet detection).13,14 PRA and
`angiotensin
`II were measured according
`to estab-
`lished methods.15,16
`II were
`to angiotensin
`Data analysis. Responses
`determined
`from the Finapres tracing by subtracting
`the systolic or diastolic blood pressure value imme-
`diately preceding
`the
`injection of angiotensin
`II
`from
`the respective maximum systolic or diastolic
`blood pressure value after
`the agonist
`injection
`(noted within 1 to 3 minutes). Baseline
`for assess-
`ment of treatment effects was defined on each study
`day as the angiotensin
`II response before adminis-
`tration of study drug. Heart
`rate responses were
`expressed as the difference between heart rate be-
`fore angiotensin
`II and
`the minimum
`heart
`rate
`after agonist injection. Antagonism of angiotensin
`II
`responses was assessed from
`the ratios of the post-
`treatment
`angiotensin
`II responses to the baseline
`response, with the smallest ratio indicating
`the max-
`imum
`treatment effect (i.e., greatest inhibition). To
`assess angiotensin
`II antagonism,
`four parameters
`were derived
`from
`these measurements: maximum
`inhibition,
`inhibition at 24 hours after dosing, area
`under
`the inhibition versus time curve (AUC), and
`time to maximum
`inhibition.
`measurement-to-
`To
`account
`for
`potential
`measurement
`fluctuations
`in the observed
`level of
`inhibition of responses to angiotensin
`II, maximum
`inhibition was defined based on a moving average
`through
`the 1Zhour postdose measurements. For
`heart rate, the maximum drug effect was defined as
`the greatest decrease after the angiotensin
`II infu-
`sion after ingestion of study medication, also based
`on moving averages. AUC
`from 0 to 24 hours was
`derived
`for both blood pressure and heart rate re-
`sponses to angiotensin
`II. AUC was calculated with
`use of the trapezoidal
`rule and was based on the
`proportion
`of response relative to baseline (defined
`as the ratio of posttreatment
`angiotensin
`II response
`
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`

`
`62 Goldbeg et al.
`
`CLINICAL
`
`PHARhtACOLOGY
`
`& THERAPEUTICS
`JANUARY 1997
`
`II response). This method
`to baseline angiotensin
`results in a greater numerical area when there is no
`inhibition
`(i.e., ratio approaches 1) compared
`to
`inhibition
`(ratio
`less than 1). Time
`to maximum
`inhibition was also determined, defined as the aver-
`age of the
`two
`time points used to calculate
`the
`moving average defining
`the maximum effect. Geo-
`metric means were calculated
`for ratios of maximum
`and 24-hour
`inhibition.
`Treatment
`effects were tested with an ANOVA
`model17; data from all three periods were included.
`Ninety-five percent confidence
`intervals were calcu-
`lated for the mean of each treatment group with the
`mean square error from the ANOVA model on the
`basis of the logs of ratios (when calculated),
`then
`exponentiated. Pairwise differences between
`treat-
`ments were assessed with the 95% confidence
`inter-
`vals on
`the difference
`in treatment mean values.
`These
`intervals also used the mean square error
`from
`the ANOVA model.
`to be “statistically signif-
`A result was determined
`test yielded a two-tailed
`icant” when
`the statistical
`probability of 0.05 or less. Statistical analyses were
`restricted
`to the PRA data from
`the first study and
`angiotensin
`II antagonism data from part B of the
`second study.
`
`RESULTS
`Subject characteristics and clinical results
`A total of 35 subjects (age range, 22 to 45 years)
`were evaluated
`in both studies. The study treat-
`ments were generally well tolerated. Adverse events
`were mild and self-limited, although one subject was
`discontinued
`from
`the
`first study for a migraine
`headache during placebo, another was discontinued
`for orthostatic hypotension
`considered
`to be unre-
`lated to DuP 532 (0.1 mg), and a third was discon-
`tinued
`for faintness after
`the 100 mg dose of DuP
`532. No subjects in the second study discontinued
`dosing because of adverse events.
`Fig. 2 summarizes supine blood pressure and
`heart rate changes after the 100 and 200 mg doses of
`DuP 532 in the tolerability study. Clinically apparent
`mean changes in supine and standing blood pressure
`and heart
`rate were not
`identified
`in the healthy
`subjects in this study.
`
`Plasma drug concentrations
`Study 1. In the first study, total plasma concen-
`trations of DuP 532 were measured at defined
`intervals after dosing on most study days. Fig. 3
`shows
`the concentrations
`of DuP 532
`for
`two
`
`subjects who had
`representative
`in two
`periods
`received 50 mg DuP 532 1 week before
`these
`measurements
`(i.e., period 3): 200 mg at 0 hours
`and placebo 1 week
`later. Measurable
`levels of
`DuP 532 were present before dosing on both sam-
`pling days. The drug appeared
`to be absorbed
`slowly because maximum measured
`concentra-
`tions were achieved at 6 and 30 hours after
`the 200
`mg dose in the two subjects. Plasma levels of DuP
`532 could be detected
`throughout
`the sampling
`period 1 week after administration
`of the 200 mg
`dose, after administration
`of placebo on that day.
`These data show an extremely
`long half-life
`for
`DuP 532 in humans after a single dose.
`Study 2. In the second study, respective plasma
`concentrations
`of losartan,
`its active metabolite
`(EXP3174),
`and DuP 532 were measured before
`and 2, 4, 8, and 24 hours after the 100 mg dose of
`losartan and the 200 mg dose of DuP 532. Fig. 4
`shows the mean plasma concentrations
`achieved
`in
`this study. Plasma concentration
`profiles
`of
`DuP 532 were clearly different
`from
`losartan, with
`much higher drug concentrations
`and a very long
`half-life
`(half-life
`of
`losartan
`averages 1 to 2
`hours; half-life
`of EXP3174
`averages 6
`to 9
`hours).18
`
`In vitro protein binding and blood/plasma
`ratio studies
`to plasma,
`in vitro
`The binding of DuP 532
`purified
`albumin,
`and ar,-acid glycoprotein
`(oro-
`somucoid) was measured
`by ultrafiltration
`with
`14C-DuP 532. DuP 532 was extensively bound
`in
`the plasma of all species examined, with 0.06% 2
`0.02%, 0.21%
`rt O.Ol%, 0.56% +- 0.05%, and
`0.17% + 0.09%
`free (unbound, mean + SD, n =
`4 samples analyzed
`in triplicate)
`at 1.0 Fg/ml
`in
`human,
`rhesus monkey, beagle dog, and Sprague
`Dawley
`rat plasma,
`respectively. Binding was sig-
`nificantly greater
`in human plasma than
`in plasma
`from dogs and rhesus monkeys
`(p < 0.001) and
`nearly significantly
`greater
`in human compared
`with rat plasma
`(p = 0.066). This extreme degree
`of plasma binding
`(i.e., >99.4%)
`results primarily
`from binding
`to albumin because physiologic con-
`centrations
`(4.5 gm/dl) of rat and human albumin
`bind 99.82%
`(0.18% + 0.01%
`free) and 99.62%
`(0.38%
`t 0.08%
`free) of added DuP 532, respec-
`tively. Binding
`to orosomucoid was insignificant,
`with 73.18% ? 1.51% and 80.99%
`t 1.36% free at
`physiologic concentrations
`(1 mg/ml). The blood/
`plasma distribution
`ratio, assessed with
`fresh dog
`
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`
`

`
`CLINICAL
`VOLUME
`
`PHABMACOLOGY
`61, NUMBER 1
`
`&THERAPEUTICS
`
`Goldbeg et al. 63
`
`Supine Systolic Blood Pressure
`
`s ‘0,
`sl
`4
`
`-10 .
`
`-15
`
`4
`
`0
`
`--C Placebo
`+lOO
`mg OuP 532
`+200
`mg OuP 532
`
`.
`
`6
`6
`Time after Oosing(Hours)
`
`10
`
`12
`
`.
`
`2
`
`.
`
`4
`
`Supine Diastolic Blood Pressure
`
`.
`
`.
`
`4
`6
`6
`Time after Oosing(Hours)
`
`10
`
`Supine Heart Rate
`
`0
`
`2
`
`4
`6
`6
`Time after Oosing(Hours)
`
`10
`
`12
`
`Fig. 2. Mean changes from predose values in supine systolic blood pressure (top panel),
`diastolic blood pressure (middle panel), and heart rate (bottom panel) in healthy male subjects
`given 100 mg DuP 532 (n = 9), 200 mg DuP 532 (n = lo), and placebo (n = 19).
`
`from 0.44 2 0.01
`and rat blood, was low, ranging
`to 0.49 t 0.03 for the rat and 0.56 t 0.02 to 0.61 2
`0.03
`for
`the dog at DuP 532 concentrations
`of
`0.050 to 2.500 pg/ml.
`
`II infusions
`Responses to angiotensin
`II
`Responses
`to bolus
`infusions of angiotensin
`in the’ crossover phase
`(part B) of study 2 are
`summarized
`in Fig. 5, which shows the mean sys-
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1030 - Page 5
`
`

`
`64 Goldbeg et al.
`
`CLINICAL P HARMACOLOGY
`
`&THERAPEUTICS
`JANUARY 1997
`
`100 100
`
`0 0
`
`50 50
`
`100 100
`
`150 150
`
`200 200
`
`
`
`Time Time
`
`
`
`after Dosing after Dosing
`
`
`
`(Hours) (Hours)
`
`
`Fig. 3. Plasma concentrations of DuP 532 during periods 4 (200 mg at hour 0) and 5 Fig. 3. Plasma concentrations of DuP 532 during periods 4 (200 mg at hour 0) and 5
`
`(placebo at hour 168) in two subjects from the tolerability study who received 50 mg DuP (placebo at hour 168) in two subjects from the tolerability study who received 50 mg DuP
`532 in period 3.
`
`rate
`tolic and diastolic blood pressure and heart
`responses
`to angiotensin
`II at each
`time point.
`Table I summarizes
`the statistical analysis of these
`data,
`including
`95% confidence
`intervals about
`treatment
`effect parameters. Angiotensin
`II doses
`that produced approximately
`25 mm Hg increases
`in systolic blood pressure averaged 30 rig/kg; four
`subjects received
`this dose and one each received
`20 and 40 rig/kg. The average blood pressure
`in-
`crease in response
`to angiotensin
`II after placebo
`was about 28125 mm Hg, with a 14 beatslmin
`decrease in heart rate. The smallest mean pressor
`response
`to angiotensin
`II-15/13
`mm Hg,
`-8.3
`beats/min-occurred
`8 to 9 hours after 200 mg
`DuP 532, whereas
`the smallest mean response
`to
`angiotensin
`II-5.5/4.3
`mm Hg, -6.7 beats/min-
`occurred 4 to 6 hours 100 mg after
`losartan. As
`seen in Fig. 5, apparent
`inhibition
`produced
`by
`losartan and DuP 532 was similar 24 hours after
`dosing.
`II challenges were
`Responses to the angiotensin
`analyzed statistically by comparison of four param-
`eters derived
`from
`the individual
`time courses of
`responses to angiotensin
`II: maximum and 24-hour
`effects (as ratios), area about
`the response curve
`(with a greater area indicating
`less inhibition),
`and
`
`to maximum effect. Results of the statistical
`time
`analysis of these parameters are summarized
`in Ta-
`ble I. Both losartan and DuP 532 significantly
`inhib-
`ited responses to angiotensin
`II (Fig. 5); however,
`the
`time course and extent of inhibition
`differed
`significantly between
`the two antagonists. The level
`of inhibition was greater
`for losartan
`than for DuP
`532, as shown by the analysis of both maximum
`(86% versus 48%) level of inhibition and integrated
`effects (9.2 versus 15.1 mm Hg * hr). Further,
`the
`mean time of maximal
`inhibition was delayed about
`4 hours for DuP 532 relative to losartan (4.6 versus
`8.7 hours for inhibition of diastolic responses, p <
`0.05). Interestingly,
`the 24-hour effect was similar
`for the two treatments.
`
`II concentrations
`PRA and angiotensin
`Study 1. In the tolerability study, PRA was mea-
`sured frequently after dosing with placebo and DuP
`532 doses of 25 to 200 mg. However,
`in view of the
`plasma concentration
`data cited earlier, potential
`effects of the previous dose of DuP 532 on PRA
`must be considered. Nonetheless, PRA appeared
`to
`increase relative to placebo after the 200 mg dose of
`DuP 532. The difference between 200 mg DuP 532
`and placebo was most apparent
`from 6 to 24 hours
`
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`
`CLINICAL
`VOLUME
`
`P HARMACOLOGY
`61, NUMBER 1
`
`& THERAPEUTIC5
`
`GoldbeT et al. 65
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`10000
`
`-
`
`loo0
`
`-
`
`#fro
`
`100
`
`-
`
`IO-l
`
`T
`
`5
`5
`5
`5
`2
`5
`E
`s
`
`1
`
`0
`
`I
`4
`
`I
`a
`Time
`
`+
`-I+
`+
`
`I
`
`Losartan
`E-31 74
`Dup 532
`I
`
`16
`
`12
`Dosing(Hours)
`
`after
`
`I
`
`20
`
`1
`
`24
`
`Fig. 4. Mean plasma concentrations of DuP 532, losartan, and EXP3174 after administration
`of 200 mg DuP 532 in period 3 and 100 mg losartan in period 1 or 2 of the antagonism study
`(n = 6).
`
`after the dose. These data were analyzed statistically
`by calculating an area under
`the PRA versus time
`curve
`(ng angiotensin
`I
`[AI]/ml/hr
`* hr). Respec-
`tively, geometric mean areas were 61.7 (95% CI,
`50.0, 76.1) and 42.2 (95% CI, 34.2, 52.2) for 200 mg
`DuP 532 and placebo (p < 0.01).
`II
`Study 2. Analysis of PRA and angiotensin
`changes in the antagonism study was confounded by
`the presence of carryover effects
`(not shown) so
`that, with the small sample size, a formal statistical
`analysis of these data could not be done. However,
`8 hours after dosing, losartan
`(100 mg) administra-
`tion resulted
`in mean + SD increases in angiotensin
`II from 2.5 + 1.9 to 10.8 -t 4.3 fmol/ml. After 200 mg
`DuP 532, there was a mean increase from 2.8 ? 1.7
`to 7.4 + 6.8 fmol/ml. Corresponding
`changes
`in
`PRA were from 0.5 2 0.2 to 5.5 ? 3.7 ng AI/ml/hr
`for 100 mg losartan and 0.5 t 0.2 to 2.0 * 2.2 ng
`AI/ml/hr
`for 200 mg DuP 532.
`
`DISCUSSION
`DuP 532 is a nonpeptide AT,-selective angioten-
`sin II receptor antagonist6,7 considered
`for study for
`its antihypertensive
`efficacy. The studies summa-
`rized in this article examined
`the clinical tolerability
`of DuP 532 in healthy male subjects and compared
`
`losartan
`DuP 532 to the prototypical AT,-antagonist
`with respect to the extent and duration of blockade
`of responses to exogenous angiotensin
`II. DuP 532
`(1 to 200 mg) and 100 mg losartan
`treatments were
`well tolerated. Single doses of DuP 532 did not alter
`blood pressure or heart rate in healthy male volun-
`teers maintained on a sodium replete diet. Changes
`in PRA and plasma angiotensin
`II concentrations
`were also measured
`to provide complementary
`bio-
`chemical data on receptor blockade of the feedback
`loop controlling
`renin release.1,4,5 DuP 532 differs
`from
`losartan in that no active metabolite
`is formed
`because
`it
`is a carboxylic
`acid,
`similar
`to
`EXP3174.6-s On the basis of preclinical data,6,7 we
`anticipated
`that DuP 532 would be similar in phar-
`macodynamic profile
`to losartan
`in humans. How-
`ever, as the results of these studies indicate, phar-
`macodynamic
`effects of single doses of DuP 532
`differed considerably
`from
`those of losartan.
`Preclinical studies of DuP 532 showed the com-
`pound
`to be of similar or greater potency to losartan
`in a variety of in vitro and in vivo models of angio-
`tensin II antagonism. 6,7 Notable differences between
`the compounds were relative shifts in potency
`in the
`presence of bovine serum albumin and extent of
`binding
`in human plasma.
`In the absence of albu-
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1030 - Page 7
`
`

`
`66 Goldberg et al.
`
`CLINICAL P HARMACOLOGY
`
`& THERAPEUTICS
`JANUARY 1997
`
`Systolic Blood Pressure
`
`2
`
`35
`
`30
`25
`20
`15
`10
`5
`
`35
`30
`25
`20
`15
`10
`5
`
`0
`
`0
`
`5
`
`15
`10
`Time after Dosing(Hours)
`
`20
`
`Diastolic Blood Pressure
`
`5
`
`15
`10
`Time after Dosing(Hours)
`
`20
`
`Heart Rate
`
`0
`
`5
`
`15
`10
`Time after Dosing(Hours)
`
`20
`
`panel), and
`Fig. 5. Mean systolic blood pressure (top panel), diastolic blood pressure (middle
`heart rate (bottom panel) responses to bolus injections of angiotensin II (20 to 40 rig/kg) before
`and after placebo (open squares), 100 mg 1osarta.n (solicl circles), and 200 mg DuP 532 (open circles)
`in the antagonism study (n = 6). For statistical analysis of these data, see Table I.
`
`IC,, values for inhibition of angio-
`min, respective
`to rat adrenal cortical microsomes
`tensin II binding
`for
`losartan, EXP3174
`(active carboxylic acid me-
`tabolite of losartan), and DuP 532 were 5.5, 1.3, and
`3.1 nmol/L.?
`In the presence of 0.25% bovine serum
`
`IC,, values were 13, 11, and
`respective
`albumin,
`4700 nmol/L, showing a much more dramatic effect
`of binding on DuP 532 compared with losartan and
`EXP3174 and suggesting a mechanism
`for reduced
`potency in vivo. Similar (or greater) shifts in in vitro
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1030 - Page 8
`
`

`
`CLINICAL
`VOLUME
`
`P HARMACOLOGY
`61, NUMBER 1
`
`& THERAPEUTICS
`
`Goldberg et al. 67
`
`Table I. Statistical analysis of responses to angiotensin
`Treatment
`
`II (n = 6)
`Placebo
`
`Losartan,
`
`100 mg
`
`DuP 532, 200 mg
`
`0.17 (0.12,0.23)?
`0.74 (0.64,0.88)
`12.5 (9.2, 15.7)‘f
`4.0 (1.2, 6.9)
`
`0.54 (0.40,0.73)$
`0.69 (0.59,0.81)?
`17.1 (13.8,20.3)?$
`7.4 (4.5, 10.3)
`
`0.14 (0.12,0.16)?
`0.61 (0.54,0.68)?
`9.2 (7.9, 10.4)“r
`4.6 (2.7,6.6)
`
`0.52 (0.44,0.62)?$
`0.55 (0.49,0.62)1
`15.1(13.9,16.3)?$
`8.7 (6.7, 10.6)$
`
`0.21 (0.16,0.27)-f
`0.86 (0.61,1.21)
`14.1(11.0,17.3)~
`5.0 (2.4,7.6)
`
`0.59 (0.44,0.78)$
`0.61 (0.43,0.87)
`18.6 (15.5,21.8)7$
`7.6 (5.0, 10.2)t
`
`to angiotensin
`
`II
`
`to angiotensin
`
`II
`
`II
`
`response
`Systolic blood pressure
`Ratio (maximum effect)
`Ratio (24 hr)
`AUC (ratio * hr)
`La
`(W
`response
`Diastolic blood pressure
`Ratio (maximum effect)
`Ratio (24 hr)
`AUC (ratio * hr)
`t,,
`(W
`to angiotensin
`Heart
`rate response
`Ratio (maximum effect)
`Ratio (24 hr)
`AUC (ratio * hr)
`t,, W)
`inhibition.
`to maximum
`time
`t,,,
`time curve;
`versus
`inhibition
`the
`AUC, Area under
`*Values
`shown are means
`(geometric
`for
`ratios)
`and 95% confidence
`intervals.
`TSignificantly
`different
`from placebo, p < 0.05.
`ZSignificantly
`different DuP 532 versus
`losartan, p < 0.05.
`
`0.77 (0.57, 1.05>*
`0.92 (0.79, 1.09)
`22.6 (19.4,25.9)
`6.5 (3.6,9.3)
`
`0.74 (0.62,0.88)
`0.90 (0.80,l.Ol)
`21.7 (20.5,22.9)
`6.6 (4.7,8.6)
`
`0.69 (0.52,0.92)
`1.00 (0.71,1.41)
`25.2 (22.1,28.3)
`3.5 (0.9, 6.1)
`
`induced by human serum albumin were
`potency
`in functional models.7 However,
`in rats
`observed
`and dogs, the in vivo potency of DuP 532 was com-
`parable
`to
`losartan, with doses of 1 to 3 mg/kg
`showing activity in hypertension models and as an
`angiotensin
`II antagonist.6
`In addition, DuP 532
`does not appear to be metabolized by rat or human
`microsomes
`in vitro.’
`Plasma binding of DuP 532 in vitro was greater in
`human plasma compared with other species. The
`free fraction was lower
`in human plasma
`(0.06%)
`than in plasma from dogs (0.56%)
`rats (0.17%), or
`rhesus monkeys
`(0.21%). Although
`the
`levels of
`bound
`radioactivity detected
`in the binding studies
`were always greater
`than twice the background,
`the
`possibility
`that the true extent of binding
`is under-
`estimated cannot be ruled out. Thus although DuP
`532 and
`losartan have similar pharmacodynamic
`profiles
`in animals,
`the effects of
`the extensive
`plasma binding
`for DuP 532 on the pharmacody-
`namics in humans may be greater, consistent with
`the greater effect of albumin on the in vitro binding
`and antagonism of DuP 532.7
`At the doses studied and plasma concentrations
`achieved, DuP 532 appeared
`to be a relatively weak
`angiotensin
`II antagonist
`in humans. The onset of
`effect of DuP 532 was clearly slower than
`that of
`losartan. The 100 mg dose of losartan
`rapidly and
`significantly
`inhibited
`the pressor response to angio-
`tensin II
`in comparison
`to placebo.
`Inhibition was
`
`observed by 1 hour after administration, with mean
`maximum
`inhibition observed at 4 to 5 hours. Mean
`maximal
`inhibition by 100 mg losartan
`(moving av-
`erage) was 83%, with
`levels of inhibition
`24 hours
`after dosing of 26% for systolic and 39%
`for dia-
`stolic blood pressure responses. These results are
`comparable
`to those reported earlier that emphasize
`the important
`contribution
`of EXP3174
`to the ac-
`tivity of losartan.2 DuP 532 was also an active an-
`giotensin
`II antagonist
`in this study. As a single
`dose, its onset of action and peak effects were ob-
`served later (2 to 3 hours and 8 to 9 hours, respec-
`tively). Maximum
`blockade was less than
`that of
`losartan, about 50% inhibition
`(compared
`to 80% to
`90%). However, at 24 hours, effects of the two an-
`tagonists were similar. Because multiple doses of
`DuP 532 were not studied,
`it is unclear whether
`the
`two antagonists would differ as much in their activ-
`ities in this model at steady state compared with
`single doses.
`Effects of DuP 532 and losartan on PRA and
`plasma angiotensin
`II concentration
`also differed.
`Both seem to have been increased by DuP 532, with
`less robust effects than those observed with losartan
`here and in previous studies.1,2,4 Time courses and
`levels of stimulation of PRA by the two antagonists
`paralleled
`the antagonism
`results for DUP 532, with
`smaller effects appearing
`later
`that persisted
`for a
`longer duration.
`These results are consistent with a lower-than-
`
`Petitioner Mylan Pharmaceuticals Inc. - Exhibit 1030 - Page 9
`
`

`
`68 Goldberg et al.
`
`CLINICAL PHARMACOLOGY
`
`& THERAPEUTICS
`JANUARY 1997
`
`level of receptor occupancy by DuP 532 or
`predicted
`with
`functional
`differences
`in
`the human AT,-
`receptor compared
`to other species. Although
`the
`bioavailability of DuP 532 may be low (e.g., less than
`10% in animals8), the doses used in the clinical study
`produced maximum plasma concentrations
`(free)
`that exceeded the IC,,
`for receptor binding, assum-
`ing a free drug concentration of 0.06%. These con-
`centrations will be expected to result in greater
`lev-
`els of blockade
`than we observed.
`If the degree of
`plasma binding
`in humans
`is underestimated,
`the
`actual free plasma concentrations may be less; how-
`ever, even a fivefold
`to
`tenfold
`underestimation
`would still be expected
`to produce greater antago-
`nism than was observed.
`In rats, DuP 532 produces
`plasma concentrations
`that effectively block exoge-
`nous AT1-responses6 and that are equal to or exceed
`the ICSo determined
`in vitro when expressed on a
`free or unbound basis. Further,
`it has been shown
`that in dogs the degree of AT,-blockade
`in vivo, as
`determined by the blockade of exogenous vasopres-
`sor responses, also correlates very well with the free
`fraction of losartan available for receptor occupan-
`cy.l’ A similar relationship has been shown between
`free drug concentrations
`and AT,-receptor
`block-
`ade produced
`in rats by EXP3174,
`the carboxylic
`acid metabolite of losartan, which is also extensively
`bound to plasma proteins.”
`In contrast, Beauchamp
`et a1.20 have demonstrated
`that in rats, for a series of
`AT-antagonists
`including DuP 532, the ED,,
`for
`antagonism of responses to a single dose of angio-
`tensi