Synthesis and study of a cyclic angiotensin II antagonist analogue reveals the role of π*-π* interactions in the C-terminal aromatic residue for agonist activity and its structure resemblance with AT1 non-peptide antagonists

Article abstract of DOI:10.1016/S0968-0896(01)00059-1

The novel amide linked Angiotensin II (ANG II) cyclic analogue cyclo(3, 5)-[Sar¹-Lys³-Glu⁵-Ile⁸] ANG II (18) has been designed, synthesized and bioassayed in anesthetized rabbits. The constrained cyclic analogue with a lactam amide bridge linking a Lys-Glu pair at positions 3 and 5 and possessing Ile at position 8, was synthesized by solution procedure using the maximum protection strategy. This analogue was found to be inhibitor of Angiotensin II. NMR spectroscopy coupled with computational analysis showed clustering between the side chains of the key aminoacids Tyr⁴-His⁶-Ile⁸ similar to that observed with ANG II. The obtained data show that only π*-π* interactions observed in ANG II or its superagonist Sar¹ [ANG II] are missing. Therefore, it can be concluded that these interactions are essential for agonist activity. Conformational analysis comparisons between AT₁ antagonists losartan, eprosartan and irbesartan with C-terminal segment of cyclic compound 18 revealed structural similarities. Copyright

Full text of DOI:10.1016/S0968-0896(01)00059-1

Bioorganic & Medicinal Chemistry 9 (2001) 1639–1647
Synthesis and Study of a Cyclic Angiotensin II Antagonist
Analogue Reveals the Role of ꢀ*–ꢀ* Interactions in the
C-terminal Aromatic Residue for Agonist Activity and Its
Structure Resemblance with AT1 Non-peptide Antagonists
Ludmila Polevaya,a Thomas Mavromoustakos,b,* Panagiotis Zoumboulakis,b
Simona Golic Grdadolnik,c Panagiota Roumelioti,d Nektarios Giatas,d Ilze Mutule,a
Tatjana Keivish,a Demetrios V. Vlahakos,e Efstathios K. Iliodromitis,e
Dimitrios Th. Kremastinose and John Matsoukasd
aLaboratory of Peptide Chemistry, Latvian Institute of Organic Synthesis, Riga, LV-1006, Latvia
bInstitute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48, Athens, Greece
cNational Institute of Chemistry, Hajdrihova 19, P.O.B. 30 SI-1115 Ljubljana, Slovenia
dDepartment of Chemistry, University of Patras, 26500 Patras, Greece
eOnassis Cardiac Surgery Center, 17674 Athens, Greece
Received 5 December 2000; accepted 27 February 2001
Abstract—The novel amide linked Angiotensin II (ANG II) cyclic analogue cyclo(3, 5) -[Sar1-Lys3-Glu5-Ile8] ANG II (18) has been
designed, synthesized and bioassayed in anesthetized rabbits. The constrained cyclic analogue with a lactam amide bridge linking a
Lys-Glu pair at positions 3 and 5 and possessing Ile at position 8, was synthesized by solution procedure using the maximum pro-
tection strategy. This analogue was found to be inhibitor of Angiotensin II. NMR spectroscopy coupled with computational ana-
lysis showed clustering between the side chains of the key aminoacids Tyr4-His6-Ile8 similar to that observed with ANG II. The
obtained data show that only p*–p* interactions observed in ANG II or its superagonist Sar1 [ANG II] are missing. Therefore, it
can be concluded that these interactions are essential for agonist activity. Conformational analysis comparisons between AT1
antagonists losartan, eprosartan and irbesartan with C-terminal segment of cyclic compound 18 revealed structural similarities.
# 2001 Elsevier Science Ltd. All rights reserved.
Introduction
environments has shown proximity of the three key
aminoacid sidechains and the formation of tyrosinate
has been demonstrated by nanosecond time resolved
tyrosinate fluorescence studies.5À11 In addition, the pro-
posed conformation for Sarmesin, an antagonist of
Angiotensin II overlays the recently discovered non-
peptide ANG II receptor antagonist losartan when
molecular modeling techniques and superimposition
studies are applied.10 Furthermore, the ring cluster
conformation was recently supported by the design and
synthesis of a novel constrained ANG II cyclic analo-
gue, [Sar1-Lys3-Glu5] ANG II, which possesses biologi-
cal activity when tested in the rat uterus assay and in
anesthetized rabbits. This potent cyclic analogue was
designed to have as a major molecular feature the
integrity of the ring cluster.7
The octapeptide Angiotensin II (ANG II, Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe) is the main pressure component of
the Renin-Angiotensin System (RAS).1,2 Accumulated
experimental evidence for ANG II supports a bioactive
conformation characterized by a charge relay system
between Tyr hydroxyl, His imidazole and Phe carbox-
ylate, analogous to that found in serine proteases,3 as
well as a ring cluster of the triad key aminoacids Tyr4-
His6-Phe8 which appears to be responsible for activity.
Thus, conformational analysis of ANG II in phospho-
lipid membrane bilayers showed that ANG II adopts a
preferred conformation.4 Conformational analysis using
modern 2D NMR techniques in receptor-simulating
Other structure–activity studies have illustrated the
importance of the C-terminal aromatic residue
*Corresponding author. Tel.: +30-1-7273869; fax: +30-1-7273872;
e-mail: tmavro@eie.gr
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00059-1

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L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
phenylalanine (Phe) for agonist activity. Replacement of
residue Phe at position 8 with an aliphatic one, as Ile,
results in an antagonist [Sar1-Ile8] ANG II (Sar-
ilesin).12,13 Mutation studies have also shown that the
aromaticity of Phe8 position is important for receptor
activation.14
of p*–p* interactions. In addition, an existing cluster
between Tyr4-His6-Ile8 initiated a research activity
which involves the conformational analysis of AT1
antagonists in an attempt to study their stereoelectronic
similarities with such a cluster. Conformational analysis of
losartan, eprosartan and irbesartan (Fig. 1) is performed in
order to examine the possibility of their structural similar-
ity with C-terminal segment of cyclic antagonist.
In this work, the cyclic [Sar1-Lys3-Glu5-Ile8] ANG II
with a Lys-Glu pair at positions 3 and 5 was synthesized
(Fig. 1). The aim of this work was to investigate fur-
thermore the role of a ring cluster receptor conforma-
tion in agonist activity and shed light to intriguing
differences in activity and conformation upon replace-
ment of aromatic residue Phe with aliphatic Ile. Repla-
cement of Phe with Ile at position 8 produces an
antagonist, and the cyclization between 3 and 5 posi-
tions does not affect the antagonist activity. Such a
cyclic analogue is more suitable to study the cluster
system between the key aminoacids Tyr4-His6-Phe8 (or
Ile8) in comparison with the linear ones which adopt
more low energy conformers and prevent the establish-
ment of definite conclusions. A cluster between Tyr4-
His6-Ile8 provides strong evidence that p*–p* interac-
tions between Tyr4-Phe8 must be a key stereoelectronic
element for agonist activity.
Results and Discussion
Rationale for the synthesis of cyclic peptide analogues
The limited stability of peptides often severely restricts
their medical and industrial application. Therefore, the
engineering of stable proteins is of great technological
and economic importance. If designed carefully without
causing drastic changes in the conformation of active
peptides, the rigid geometry of the cyclic peptides
enhances the binding affinity towards a selected target
molecule compared to their linear counterparts..14À16
Furthermore, cyclic analogues are important inter-
mediates in the design and synthesis of non-peptide
mimetics with the potential to be used as drugs.17,18
NMR spectroscopy coupled with computational analy-
sis was performed in order to establish the significance
So far, a limited number of conformationally restricted
via cyclization Angiotensin II analogues have been
Figure 1. Chemical structures of cyclic compound 18 and AT1 antagonists losartan, eprosartan and irbesartan.

L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
1641
reported by us and others. In these studies cyclization
was achieved either by the disulfide method using
cysteine moieties at various locations of the peptide
molecule or by the amide-linkage method.15À22
-COOH side chain groups of Lys and Glu at positions 3
and 5, respectively, which are the least important for
biological activity.
Strategy of the synthesis of c-[Sar1-Lys3-Glu5-Ile8] ANG
II
Our interest in the conformational model of ANG II,
which could be used as a basis for the synthesis of non-
peptide receptor antagonists, prompted us to design and
synthesize the novel cyclic amide linked ANG II analo-
gue c-[Sar1-Lys3-Glu5-Ile8] ANG II (Fig. 1). This allows
to further explore the role of the aromatic residue at
position 8 for agonist activity. Cyclization was achieved
by forming an amide-linkage between the -NH2 and
The 3,5-cyclic analogue of ANG II with lactam amide
bridges linking a Lys3-Glu5 pair was synthesized by the
solution procedure in combination with the maximum
protection strategy as shown in Scheme 1. This analo-
gue was constructed from three major fragments: the
linear N- and C-terminal fragments and the central
Scheme 1.

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L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
cyclic tripeptide part to produce the fully protected
octapeptide. The final unprotected cyclic analogue,
cyclo (3,5)-Sar1-Lys3-Glu5-Ile8-ANG II was obtained
after catalytic hydrogenolysis.
bridgehead residue 9 and by coupling with the linear
precursor 10 via its N-hydroxybenzotriazole active ester
in situ using DIC/HOBt/DIPEA. Cyclic dimer (ꢀ10%)
formed as by-product during the cyclization, was
removed by HPLC chromatography in the last stage of
purification.
The key step in the preparation of this analogue was the
cyclization performed at the tripeptide stage by con-
necting the side chains of lysine and glutamic acid resi-
dues separated by tyrosine residue in the linear
sequences Lys3-Tyr4-Glu5 resulting in the formation of
a cyclic amide (lactam) bond. The fully protected linear
tripeptide 9 was synthesized stepwise using Boc-
Glu(OtBu)-ONb (5) as starting material. The protected
cyclic tripeptide Z-[-cyclo(Lys-Tyr(Bzl)-Glu)]-]-Onb (11)
was prepared after deprotection of the side chain of the
The N- and C-terminal fragments were synthesized step
by step from their respective C-amino acid ester. Z-Sar-
Arg(NO2)-OH (16) was obtained by saponification from
methyl ester 15. The protected tripeptide Boc-His(Bz)-
Pro-Ile-ONb (3) was synthesized with p-nitrobenzyl
ester protection for the C-terminal and Boc group for
the a-amino function. After Boc acidolytic cleavage by
HCl/EtOAc, hydrochloride of C-terminal tripeptide 4
was used for preparation of the protected hexapeptide
13. The hexapeptide 13 was prepared by the azide
method: this method includes hydrazinolysis of the
protected cyclotripeptide p-nitrobenzyl ester 11 leading
to hydrazide 12 and treatment of 12 with tert-butyl
nitrite, and condensation of the obtained azide with H-
His(Bzl)-Pro-Ile-ONb.HCl (4) in the presence of
DIPEA. Further selective cleavage of the Z-protecting
group from 13 was achieved using 30% HBr/AcOH to
afford 14. This compound was then condensed with Z-
Sar-Arg(NO2)-OH (16) by activating the carboxyl func-
tion with DCC in the presence of HOBt resulting finally
in the protected peptide 17. Simultaneous catalytic
hydrogenolysis of all protective groups from 17, further
purification by reversed-phase HPLC, conversion of the
hygroscopic trifluoroacetate salts to the hydrochlorides
and crystallization from iso-propanol provided the
Table 2. Major NOEs that define conformers of AT1 antagonists
eprosartan, irbesartan and losartan
Figure 2. Low energy conformer of c(3,5)-[Sar1-Lys3-Glu5-Ile8] ANG
II.
Sample
NOEs
Intensity
Eprosartan
H 11–H 28
H 19–H 24
H 4–H 24
H 19–H 11
ma
s
m
s
Table 1. Major NOEs that define conformers of antagonist c-[Sar1-
Lys3-Glu5-Ile8] ANG II (2)a
Sample
NOEs
Intensity
m
Irbesartan
Losartan
H 6a,6b,6c—H 13,17,14,16
H 6a,6b,6c—H 10
H 10–H 13,17,14,16
s
m
l
Antagonist
c-[Sar1-Lys3-Glu5-Ile8] ANG II
NH Arg(2)-aHHis(6)
NH Ile(8)-aHArg(2)
NH His(6)-3,5HTyr(4)
4H His(6)-3,5HTyr(4)
4H His(6)-dIle(8)
s
s
s
H 7–H 11
H 6–H 11
H 7–H 13, 17
m
m
m
m
as=strong, m=medium, l=light.
as=strong and m=medium.
Figure 3. Low energy conformers of the three AT1 antagonists losartan, eprosartan and irbesartan derived using a combination of NMR spec-
troscopy and computational analysis.

L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
1643
desired
octapeptide (18).
final-cyclo(lysyl-tyrosyl-glutamyl)-containing
irbesartan and eprosartan were studied using a combi-
nation of computational analysis and NMR spectro-
scopy.23À28 The major NOES observed for these three
analogues and served as NOE constraints are shown in
Table 2.
NMR spectroscopy and computational chemistry
NMR spectroscopy and computational analysis was
performed for the antagonist c-[Sar1-Lys3-Glu5-Ile8]
ANG II. Figure 2 shows a low energy structure for c-
[Sar1-Lys3-Glu5-Ile8] ANG II generated using a combi-
nation of NOE data and minimization procedures. The
most important NOEs that define the conformation of
the cyclic analogue 18 are shown in Table 1.
Low energy conformers for the three AT1 antagonists
under study consistent with NOE constraints are shown
in Figure 3. Detailed conformational analysis of these
molecules will be sought in a subsequent study.
Superimposition of the three AT1 antagonists with the
cyclic analogue were achieved in an attempt to reveal
the similarities of the C-terminal segment with specific
structural features of AT1 antagonists.
NOEs in this molecule show a spatial proximity
between Sar1-Tyr4. It was found in most agonists and
antagonists studied that as a general rule Sar1 or Arg2
protrude to the cluster system, due to energetically favor
rotation through Ca of Arg2. NOEs between Tyr4-His6
and His6-Ile8 were also observed. These results evident a
cluster system between the key amino acids Tyr4-His6-Ile8.
Such superimpositions are very important because (a)
mutation studies point out that C-terminal region of
ANG II and ANG II peptide antagonists act on the
same region of AT1 receptor located in the transmem-
brane region.29À35 In particular, the N-terminal region
of peptides seem to dock on the extracellular part of
AT1 receptor while the C-terminal part as the AT1 non-
peptide antagonists are located in the transmembrane
part of the receptor and especially between TMIII-
TMVII helices; (b) Potent AT1 antagonists such as
losartan were designed based on the C-terminal part of
AII;36À42 (c) Superimposition studies may explain the
different kind of activities between antagonists espe-
cially because their receptor active site is not yet estab-
lished due to not conclusive results obtained by the
mutation studies; (d) Superimposition studies may dis-
cover the structural requirements for agonist and
antagonist activity. These requirements can be tested
with the synthesis of novel synthetic compounds.
The important stereoelectronic characteristics of the
generated model are the following. The cyclic antagonist
adopts a Tyr4-Ile5-His6 bend, a trans amide His6-Pro7
configuration and a side chain aromatic cluster of the
three key aminoacids Tyr4, His6 and Phe8. In cyclic
antagonist peptide c-[Sar1-Lys3-Glu5-Ile8] ANG II with
Ile at position 8, the p*–p* interactions are missing since
this molecule contains the non aromatic aminoacid Ile8
instead of the aromatic Phe8. In recent studies the
importance for such interactions is highlighted.15
To compare the C-terminal region of cyclic antagonist
with AT1 antagonists the conformational analysis of the
well known antihypertensive AT1 antagonists losartan,
Figure 4. Superimposition of losartan with cyclic analogue 18.

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L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
Superimposition of losartan with c-[Sar1-Lys3-Glu5-Ile8]
ANG II
losartan mimics the g-turn formed around Pro7 in c-
[Sar1-Lys3-Glu5-Ile8] ANG II (iv) losartan’s hydroxy-
methyl group with phenolic hydroxyl group of Tyr4
(Fig. 4).
From the different possible overlays, the best super-
imposition between the two molecules was achieved
when the following groups were matched: (i) losartan’s
hydroxymethylimidazole with c-[Sar1-Lys3-Glu5-Ile8]
ANG II His6; (ii) losartan’s tetrazole with c-[Sar1-Lys3-
Glu5-Ile8] ANG II isosteric carboxylate of Phe8; (iii)
losartan’s spacer phenyl ring with c-[Sar1-Lys3-Glu5-
Ile8] ANG II pyrrolidine group of Pro7. Interestingly,
Superimposition of eprosartan with c-[Sar1-Lys3-Glu5-
Ile8] ANG II
From the possible overlays, the best superimposition
between the two molecules was achieved when the fol-
lowing groups were matched: (i) eprosartan’s carbox-
Figure 5. Superimposition of eprosartan with cyclic analogue 18.
Figure 6. Superimposition of irbesartan with cyclic analogue 18.

L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
1645
ylate with c-[Sar1-Lys3-Glu5-Ile8] ANG II carboxylate
of Phe8; (ii) eprosartan’s spacer phenyl ring with c-[Sar1-
Lys3-Glu5-Ile8] ANG II pyrrolidine group of Pro7.
Interestingly, eprosartan as losartan mimics the g-turn
formed around Pro7 in c-[Sar1-Lys3-Glu5-Ile8] ANG II;
(iii) eprosartan’s imidazole with c-[Sar1-Lys3-Glu5-Ile8]
ANG II His6 (Fig. 5).
rabbits43 the cyclic analogue [Sar1, Lys3, Glu5, Ile8]
ANG-II, where an isoleucine was substituted for phe-
nylalanine at position 8 did not preserve any hyperten-
sive action. In contrast, [Sar1-Lys3-Glu5-Ile8] ANG-II
was transformed to a potent ANG- II antagonist and
reduced ANG II-dependent hypertension in a dose-
related manner (Fig. 7).
Superimposition of irbesartan with c-[Sar1-Lys3-Glu5-Ile8]
ANG II
Experimental
Chemistry
Similarly, from the possible overlays, the best super-
imposition between the two molecules was achieved
when the following groups were matched: (a) irbe-
sartan’s tetrazole with c-[Sar1-Lys3-Glu5-Ile8] ANG II
isosteric carboxylate of Phe8; (b) irbesartan’s spacer
phenyl ring with c-[Sar1-Lys3-Glu5-Ile8] ANG II pyrro-
lidine group of Pro7. Interestingly, irbesartan like
losartan and eprosartan mimics the g-turn formed
around Pro7 in c-[Sar1-Lys3-Glu5-Ile8] ANG II; (c)
irbesartan’s substituent 1,3-diazaspiro[4,4]non-1-3en-4-
one with c-[Sar1-Lys3-Glu5-Ile8] ANG II His6 (Fig. 6).
The identity of the cyclic product 18 was established by
FAB/MS and NMR spectroscorpy. FAB spectra were
run on an AEI M29 mass spectrometer modified as
described elsewhere.44 FAB gun was run at 1 mA dis-
charge current and at 8 kV. The FAB matrix used was a
mixture of 6:1 dithiothreitol/dithioerythritol (Cleland
Matrix). The purity of the final cyclic analogue was
checked by thin layer chromatography (TLC). TLC was
carried out with precoated silica gel on glass (Merck
Kieselgel 60 F254) TLC plates. Amino acid analysis of
the cyclic product 18 was performed on a Beckman 6300
high-performance analyzer. Compositional analysis
data were collected from 6 N HCl hydrosylates (100 ꢁC,
18 h) with ninhydrin-based analysis.
The similarities in the superimposition of the three AT1
antagonists suggest a similar way of interacting with
AT1 receptor. Superimposition studies between losartan
with sarmesin10 or c-[Sar1-Lys3-Glu5-Ile8] ANG II His6
reveal identical manner of superimposition. Losartan-
sarmesin superimposition has additional matching
between the butyl chain of losartan with sarmesin’s Ile5
alkyl chain. Due to the lack of Ile5 aminoacid in c-[Sar1-
Lys3-Glu5-Ile8] ANG II such a lipophilic matching is
missing. This result points out that lipophilic segments
of AT1 non-peptide mimetics may not be equivalent
with that of alkyl group of Ile5.
Intermediate peptides and final product 18 were checked
for purity on a Waters HPLC system equipped with a
600E system controller. Fractions were analyzed by ana-
lytical reversed-phase column (Techsil C18, 250Â4.6 mm).
The crude peptide material (18 mg) was dissolved in
methanol (450 mL), clarified by centrifugation and the
solution was injected through a Rheodyne 7125 injector
with a 500 mL sample loop. A Lichrosorb RP-18
reversed-phase preparative column (250Â10 mm) with
7 mm packing material was used. Separations were
achieved with a linear gradient of acetonitrile in 0.1%
aqueous TFA at a flow rate of 3 mL/min. In particular,
the mobile phases used were 0.1% aqueous TFA (A)
and 0.1% TFA in acetonitrile (B) and involved a linear
gradient from 0 to 100% B over 60 min (3 mL/min).
Fractions were manually collected at 0.5 min intervals,
the eluent was monitored at 230 and 254 nm (Waters
996 Photodiode Array Detector) and the elution time of
the major product was typically in the region of 25–
30 min. The amino acid derivatives used in the synthetic
procedures were purchased from Reanal (Hungary) and
Nova Biochem.
Assess of biological activity of [Sar1-Lys3-Glu5-Ile8]
ANG II
As opposed to the c-[Sar1-Lys3-Glu5-Phe8] ANG-II,
which was found capable to induce a dose-related
hypertensive response after infusion into anesthetized
NMR spectroscopy
NMR experiments were carried out using Bruker
300 MHzand Varian 600 MHzNMR spectrometers.
c-[Sar1,Lys3,Glu5] Ile8 ANG II 18, [Sar1] ANG II and
the AT1 antagonists losartan, eprosartan and irbesartan
were studied by dissolving 5 mg of the compounds in
0.5 mL of DMSO. The chemical shifts were reported
relative to the non-deuterated fraction of the methyl
group of DMSO-d6 at 2.50 ppm with respect to TMS.
One-dimensional spectra, and two-dimensional spectra
Figure 7. [Sar1-Lys3-Glu5-Ile8] ANG II causes dose-related reduction
of ANG II induced hypertension in anesthetized rabbits.

1646
L. Polevaya et al. / Bioorg. Med. Chem. 9 (2001) 1639–1647
COSY/TOCSY and NOESY experiments aided in the
assignments of the cyclic peptide, and the AT1 antago-
nists as well as their conformational analysis. Standard
parameters were used for the experiments provided in
the software and installed with the instrument.
entific Program (ZP-10). We wish to acknowledge
pharmaceutical companies Sanofi-Synthelabo, Merch
and Solvay for donating the AT1 antagonists Irbe-
sartan, Losartan and Eprosartan.
References
Molecular dynamics
In the molecular modeling, theoretical calculations were
performed on a Silicon Graphics 02 work station using
QUANTA package of Molecular Simulations and
CHARMm force field. All calculations were run in
DMSO environment (e=45). The lower energy con-
former of the cyclic analogue c-[Sar1-Lys3-Glu5-Ile8]
ANG II was obtained based on the proposed model of
ANG II. Thus, ANG II served as a matrix where the
cyclization between Lys3 and Glu5 was achieved. It was
then minimized using conjugate gradient and adopted
basis Newton Raphson algorithms under NOE con-
straints. The conformational analysis of losartan is
described elsewhere.10 Irbesartan and eprosartan were
manipulated in an identical way. Thus, they were built
and minimized under NOE constraints. Details of the
above techniques have been previously described.45
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Based on previous testing angiotensin II-dependent
hypertension was induced by infusing angiotensin via a
syringe pump (Harvard Apparatus Pump 22, Harvard
Apparatus, Natick, MA, USA) at a constant rate of
0.6 mL/min (3 mg/min). Angiotensin solution was pre-
pared by diluting angiotensin II (Hypertensin, CIBA) in
5% dextrose at a final concentration of 5 mg/mL. Five
min after the establishment of hypertension, boluses of
75, 150 or 300 mg of the novel cyclic angiotensin II
antagonist [Sar1, Lys3, Glu5, Ile8] ANG-II were given via
an ear vein in random sequence and the changes of
blood pressure were recorded.
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Acknowledgements
This work was supported by the Ministry of Develop-
ment, the Secretariat of Research and Technology of
Greece (EPET II/115, PENED 1999), a Joint Research
and Technology Program with Slovenia 1998-1999, a
NATO Linkage Grant (974548), the Latvian Science
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