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Is there correlation between analgesic potency and biodegradation of enkephalin analogs?
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages
October 30, 1978
IS THERE CORRELATION
BETWEEN ANALGESIC
BIODEGRADATION S6ndor
Bajusz,
Andrds
Jcizsef Institute
for
Received
Drug
I.
Patthy,
POTENCY AND
ANALOGS?
Agnes Kenessey,
Szekely
Research,
September
OF ENKEPHALIN
and Andrss
H-1325
1045-1053
Ldszl6
Grbf,
2. R6nai
Budapest,
P.O.Box
82, Hungary
5,1978
Met-enkephalin and its Pro' analogs containing Gly or D amino acids at position 2 were subjected to digestion with aminooeptidase M, rat brain extracts and human sera. The enzyme resistance of these peptides was compared with their analgesic activity determined in tail flick test after central and intravenous administrations. Our data did not reveal an unambigous correlation between the analgesic potency and metabolic stability of the analogs. This suggest that analgesic activity of synthetic peptiues should be due to factors other than enzyme resistance /e.g. receptor binding and transport properties/. The discrepancy sequence like
that
Tyr-Gly-Gly-Phe-Met
activities in vivo
of the
peptides.Prompted
Tyr' -Gly2
bond
have
N-terminus,
lasting
analgesia
that
the
by recent extracts
into
potency
Pro
step
/2,3/
of the Pert
which
et al. has a
profound
rat
The assumption
brain.
is due to
has been
was also
residue
degradation
cleavage
of opioid
were prepared
morphine-
and causes
enzymes
Biodegradation
C-terminal
enzymic
that
of enkephalin
degradation
with
low and transient
I-enkephalinamide
when injected
of enkephalin the
deactivation
peptides
remarkable
by rapid
Tyr '-D-Ala2,
on the
possess
suggestion
ID-Ala',Met' i.e.
studies
two brain
extremely
explained
initial
to degradative
/5,6/.
derivatives Namely
the
low analgesic
susceptibility
cause
by the
synthesized
stable
but
has been
is
/l/,
and -Leu,
in vitro
analgesia
/4/
enkephalins
further peptides
considered in our
was expected
its
high
supported by brain
when Pro5
laboratory to
and long-
retard
171. the
OOOS-291X/78/0844-1045$01.00/O 1045
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 84, No. 4, 1978
action
BIOCHEMICAL
of carboxypeptidases.
residues chain
enkephalin
receptor/s/ ivity
with
to bind
even when injected
141. Despite
ently
of
their
analgesic
the
analogs
sam.e or
between
peptides.
glycine
additional possess
interest
fails
to re-examine
study
if
i.e.
2 were subjected
to hydrolysis
serum,
The stability
resp.
their
analgesic
activity
after
intracerebroventricular
of
of
these
determined
lIATERIALS
/icv/
degradation,
there
Met-enkephalin
/Eth/,
enzyme complex
rat
brain
peptides
act-
are appar-
Thereis
and enzyme resistance
or D amino acids,
of the
anal-
markedly.
D-methionine
with
to elicit
to differ
D-phenylalanine,
ase M /APM/,
site analgesic
to enzymic
potency
side
extra
/D-Ala2,Met5/
D-norleucine/Nle/, at position
an
Tyr-D-Xxx-Gly-Phe-Pro
has been found
analgesic
D amino acid
whereas
resistance
In the present
containing
prepared
the same route
similar
potency
hand,
to a possible
peptides
seemed to be of
correlation these
that
in
RESEARCH COMMUNICATIONS
aim of providing
intravenously
applied
gesia
it
the
171. Pro5 -enkephalins
-enkephalinamide
fore
On the other
were introduced
for
AND BIOPHYSICAL
and its
a real of Pro5
D-alanine, and D-ethionine with
extracts
aminopeptidand of human
was compared with
in the tail or intravenous
flick
test /iv/
in rats
injection.
AND MHTHODS
Met-enkephalin and the analogs Tyr-Xxx-Gly-Phe-Pro-Q, wherein Xxx stands for Gly, D-Ala, D-Nle, D-Phe or D-Eth, and Q stands for OH or NH2 were prepared as described previously /7-g/. For studies on the breakdown of enkephalins in rat brain extracts Marks' approach IlO] was used as reported by Patthy et al. Ill/. Degradation by human serum was studied as follows: 0.6 ml of 0.01 M TRIS buffer 17.61 containing 0.07 mg substrate /about 0.1 umole/ was a i 37oC for 4 h, added to 0.4 ml of fresh human serum and incubated then a 0.5 ml aliquot of the deproteinized supernatant of each sample was subjected to amino acid analysis. APM digestions were performed in 1 ml 0.05 M TRIS buffer /pH 7.61 containing 1 mg substrate /about 1.5 umole/ and 0.1 mg enzyme /Rijhm Gmbl. The during digestions was determined amount of amino acid 4 released in a JEOL /JLC-5AH/ automatic analyzer and expressed in moleThe analgesic activity of peptides percentage of the substrate. was assessed in rats using the tail flick test as described previously 1121.
1046
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION The action first
of APM on Met-enkephalin
since
opioid
peptides
has long cept
the
findings
with
split
off.
In addition, retarded
It
ered
supported /I that
released hours.
longer
side
in significan
the C-terminal
also
seemed to affect
IV,
and IV/.
human sera are given
in Table
even more susceptible
to brain
compound,
i.e.
was averted
II.
rat
somewhat less
Pro'.
Comparing
D-Ala2
in III
extracts
just
like
of
III
and with
proved
to be
the parent
2 in the
in sera.
block
of peptideamides
be observed
the
Interestrelease
IV-VIII
of
minor
in the experiments
APM. Albeit
volved
the enzymes examined in biodegradation
such studies
may provide
in our experiments
of opioid
peptices
estimates
for
1047
the
be
aminopeptidase/s/
than
can completely
acid
3 or more
data
at position
efficiently
hind-
a pentapeptide,
of APM /cf.
of
is
could
for
and serum enzymes than
the degradation
can only
of
of
D-amino itself
Pro5 -enkephalin
residues
enough,
D-Ala
brain
by the D amino acid
ingly
:Jith
with
The action
extracts
of Tyr
was digested
Met-enkephalin.
brain
differences
Moreover,
2
release
than by any other
the action
digestion
the
same
was not
at position
amide function
Data of the
to the
bond in II
liberation
amount when III
Finally,
Hamely
is
It
bond ex-
above notion.
of APX, that
chain.
12-61.
present
residues
that
of
residues.Our
were hydrolyzed
D amino acid
the action
authors
any peptide
the Phe-Pro
also
having
the
and II/
appeared, however, 3 by D-Ala' to a lesser extent
residue
like
partly
the exception
/III-VIII/ Tyr'.
I/
was examined
in deactivation
by several
APM can hydrolyze
and Pro' -enkephalin
extent
out
formed by Pro or D amino acid /Table
analoqs
of aminopeptidases
had been pointed
been known that
those
Met5-
involvement
and its
and those
are not
identical,
susceptibility
of
in-
of
of
Pro-NH2
Pro-NH2
D-Nle
D-Met
D-Eth
D-Phe
digestion
V
VI
VII
VIII
*after
for
Pro-NH2
Pro-NH2
Pro-NH2
D-Ala
IV
Pro-OHX
Pro-OH
D-Ala
GUY
II
Met-OH
III
GUY
changing residues xxx2 Yyy5-Q
I
ccmpounds
No.
Degradation
Tyr
18 h D-Ala
5
5
5
6
27
37
67
65
0.5
percent
enkephalins
74
3h
was
10
10
14
17
55
63
72
I
0
0
0
0
0
+
58
in
0
0
0
0
+
6
64
67
3
h
acids XXX2
66
0.5
amino
released
of
/Tyr-Xxx-Gly-Phe-Yyy-Q/
Table
28%,
Tyr
0
0
0
0
0
+
58
0
0
0
0
+
9
64
67
3h
after
and Gly
GUY
66
0.5
released
in
0
0
0
0
0
0
0
3 h,
?1
WY5
resp.
0
0
0
0
0
0
0
16
9.5
resp.
100 and 35%,
0
0
0
0
0
0
0
71
3h
and Phe
32
0.5
(r),5
by aminopeptidase
0
0
0
0
0
0
0
62
3
h
of
enkephalins
D-Ala
D-Eth
D-Met
D-Nle
D-Phe
IV
VII
VI
V
VIII
GUY
I
D-Ala
Gly
II
III
xxx2
Pro-NH2
Pro-NH2
Pro-NH2
Pro-NH2
Pro-NH2
Pro-OH
Met-OH
Pro-OH
Yyy5-c,
changing residues
of compounds
Number
Degradation
II by brain
extract
0 0
7126 o/15
0
-
12137 8122
11
-
22146 15138
91
99
77188 60/60
C
A/B
o/o
o/o
o/o
Of3
O/6
o/5
25142
50156
A/B
0
0
0
-
0
-
39
71
C
o/o
o/o
o/o
o/o
o/o
o/12
25142
50156
A/B
o/o o/o o/o o/o
0 0 0
o/o
0 -
o/o
51156
62167
A/B
-
39
71
C
01 0 01 0
0
01 0
o/o
01 0
o/3
54174
90/100
A/B
0
0
-
0
-
76
78
C
YYY5
after 4h /C/:
and serum
percent of amino acids released by brain extract 0.5h /A/ and 2h /B/, req., and by serum after 'Tyr xxx 2 Phe GlY
/Tyr-Xxx-Gly-Phe-Yyy-g/
Table
0
0
0
-
0
-
41
100
C
Vol. 84, No. 4, 1978
peptides
BIOCHEMICAL
towards
brain
enzyme resistance
the
ate ation
In case of peptides
central
is
for
any response. "stability"
Beyond values
logical
potencies.
assessed
after
systemic
illustrated
by the practically
ases. the
lack
of
the
for
cannot
of the an analog,
fails
to cause
however,
order
the
for
the bio-
of analgesic
potencies
one must realize value.
is
of
effect
the
clearly III,
ana
to protein-
strongly
with
that
II,
susceptibility
I and II
solely
This
activity
different
analgesic
intravenously
/II/,
identical
the data
of significant
and biodegrad-
information
informatory
compounds of widely
A comparison
applied
less
Data indic-
otherwise
stability,
administration,
of even
together
resistance"
effect
reliable
For explaining
is
three
analgesic
give
"stability"
IV,
a "threshold
threshold
do not
III.
potency
Fro'-enkephalin
this
values
peptide
depends on the way of application.
the
unstable
the
of intact
in Table
analgesic
administration
the extremely
e.g.
strongly
required
percentage
are listed
between
the peptides
To characterize
Such "stability"
potencies
the correlation
of
values
was calculated.
analgesic
that
and serum proteinases.
by numerical
in hydrolysates with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
suggests
that
Met-enkephalin
be due to its
rapid
degradat-
extremely
high
ion. From the bioloqical
above data potency
enkephalinamide or four
of
is
different
we may conclude an enkephalin
a result
of
requirem.ents,
that
analog
namely
to cross
the blood-brain
improved
binding
capacity
and increased
in
the
literature.
ing peptides encountered
latter
undergo yet
that
biodegradation,
to demonstrate
their
1050
barrier,
at least
three
transport
prop-
enhanced
enzyme resistance.
or The
to us to be overestimated
one occurs
Notwithstanding
ID-Met2,Pro51of
favourahle
ability
of the
like
the satisfaction
erties,
significance
the
all
the naturally
no difficulty in vivo
occur-
has been
biological
effect,
D-Phe
VIII
Pro-NH2
Pro-NH2
D-Nle
V loo/as
93174
92178
Pro-NH2
D-Met
VI
88163
Pro-NH2
D-Eth
VII
85162
D-Ala
IV
Pro-NH2
D-Ala
III
40126
101 0
intact in brain extract after 0.5/2h
78154
Met-OH
Gly
I
III of enkephalins
100
100
100
a9
9
0
peptide in human serum after 4h
"STABILITY"
potency
Pro-OH
Pro-OB
Yyy5-*
GUY
xxx2
changing residues
and analgesic
II
pounds
com-
Of
Number
"Stability"
Table
250
7407
24242
5714
1538
250
-1.5
((1
ED50nM/animal
lo3
centrally
POTENCY
204
222
3125
235
a9
a3
0
64
ED50,uM/kg
lo3
intravenously
/Tyr-Xxx-Gly-Phe-Yyy-R/
< 0 P
Vol. 84, No. 4, 1978
whilst if
the
it
analgesic
property
is due to an extreme
physiological note
BIOCHEMICAL
as their
activity
D amino
and IMet2,
two preparations, the
those
the
to the Slet-enkephalin analogous
biological
molecular
interaction
the C-terminal about
all
these
interesting
pig
latters
and the to form
effects
in this
ileum,
do not
between portion
favourable
the
addition
site
/3-endorphin
in the
of the
residues
appear
/residues
6-31
to exert
A particular
intral-4/
1141 might
changes mentioned
two
of D-Met 2 of
/3-endorphin
active
of
/3-
differentiate
populations
regard.
and also
above.
REFERENCES 1. Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A.; and Morris, H.R. /1975/ I!ature 258, 577-579. 2. Chang, J.K., Fong, B.T.W., Pert, A., and Pert, C.B. 119761 Life Sci. 18, 1473-1481. 3. Hambrook, J.M., Morgan, B.A., Rance, M.J., and Smith, C.F.C. 119761 Nature 262, 782-783. 4. Pert, C.B., Pert, A., Chang, J.K., and Fong, B.T.W. 119761 Science 194, 330-332. 5. Marks, N., Grynbaum, A., and Neidle, A. 119771 Biochem. Biophys. Res. Commun. 74, 1552-1559. 6. Grynbaum, A., Kastin, A.J., Coy, D.H., and Marks, N. 119771 Brain Res. Bull. 2, 479-484. 7. Bajusz, S., Ronai, A.Z., Szgkely, J.I., Dunai-Kovacs, ZS., Berzetei, I., and Gr6f, L. 119761 Acta Biochim. Biophys. Acad. Sci. Hung. 11, 305-309. 8. Bajusz, S., Rbnai, A.Z., Szekely, J.I., Grsf, L., Dunai76, 91-92. Kovgcs, Zs., and Berzdtei, I. 119771 FEBS Letters 9. Grdf, L., Ronai, A.Z., Bajusz, S., Cseh, G., and Szekely, 119761 FEES Letters 64, 181-184. J-I.-,
1052
of
possess higher
are equipotent
receptor
moiety
those
and Leu-enkephalins
on guinea
the
than
do. Thus introduction
structure
to
f3-endorphin
analogs
opiate
be of
of
containing
words,
cannot
/D-Vet 2 , Pro'/-enkephalin-
acid
than
- even
of
Yet-
formers
sequence
is
131. While
in other
and Pro-NH2 5 into
it
Pro5/-enkephalinamide
models as the
-
112,
same way the distinct
in vitro
bring
of
on mouse vas deferens
endorphin
context
properties
and Leu-enkephalins
as well
instability
In this
the biological
RESEARCH COMMUNICATIONS
and Leu-enkephalins
metabolic
amide are more reminiscent Met-
of Met-
significance.
that
AND BIOPHYSICAL
Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Peptides, pp. 221-268, 10. Marks, N. 119771 Neurobiology Plenum Press, New York. 11. Patthy, A., Grbf, L., Kenessey, A., Szekely, J.I., and Bajusz, S. 119771 Biochem. Biophys. Res. Commun. 79, 254-259. 12. Szekely, J.I., Rbnai, A.Z., Dunai-Kovacs, Zs., Migli?cz, E., Berzetei, I., Bajusz, S., and Graf, L. 119771 Eur. J. Pharmacol. 43, 293-294. 13. R6nai, A.Z., Berzetei, I., Szekely, J.I., Graf, L., and Bajusz, S. 119781 Pharmacology /in press/. 14. Gr6f, L., Cseh, G., Barat, E., Ronai, A.Z., Szkkely, J.I., Kenessey, A., and Bajusz, S. 119771 Ann. ?l.Y. Acad. Sci. 297, 63-83.
1053
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages
October 30, 1978
IS THERE CORRELATION
BETWEEN ANALGESIC
BIODEGRADATION S6ndor
Bajusz,
Andrds
Jcizsef Institute
for
Received
Drug
I.
Patthy,
POTENCY AND
ANALOGS?
Agnes Kenessey,
Szekely
Research,
September
OF ENKEPHALIN
and Andrss
H-1325
1045-1053
Ldszl6
Grbf,
2. R6nai
Budapest,
P.O.Box
82, Hungary
5,1978
Met-enkephalin and its Pro' analogs containing Gly or D amino acids at position 2 were subjected to digestion with aminooeptidase M, rat brain extracts and human sera. The enzyme resistance of these peptides was compared with their analgesic activity determined in tail flick test after central and intravenous administrations. Our data did not reveal an unambigous correlation between the analgesic potency and metabolic stability of the analogs. This suggest that analgesic activity of synthetic peptiues should be due to factors other than enzyme resistance /e.g. receptor binding and transport properties/. The discrepancy sequence like
that
Tyr-Gly-Gly-Phe-Met
activities in vivo
of the
peptides.Prompted
Tyr' -Gly2
bond
have
N-terminus,
lasting
analgesia
that
the
by recent extracts
into
potency
Pro
step
/2,3/
of the Pert
which
et al. has a
profound
rat
The assumption
brain.
is due to
has been
was also
residue
degradation
cleavage
of opioid
were prepared
morphine-
and causes
enzymes
Biodegradation
C-terminal
enzymic
that
of enkephalin
degradation
with
low and transient
I-enkephalinamide
when injected
of enkephalin the
deactivation
peptides
remarkable
by rapid
Tyr '-D-Ala2,
on the
possess
suggestion
ID-Ala',Met' i.e.
studies
two brain
extremely
explained
initial
to degradative
/5,6/.
derivatives Namely
the
low analgesic
susceptibility
cause
by the
synthesized
stable
but
has been
is
/l/,
and -Leu,
in vitro
analgesia
/4/
enkephalins
further peptides
considered in our
was expected
its
high
supported by brain
when Pro5
laboratory to
and long-
retard
171. the
OOOS-291X/78/0844-1045$01.00/O 1045
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 84, No. 4, 1978
action
BIOCHEMICAL
of carboxypeptidases.
residues chain
enkephalin
receptor/s/ ivity
with
to bind
even when injected
141. Despite
ently
of
their
analgesic
the
analogs
sam.e or
between
peptides.
glycine
additional possess
interest
fails
to re-examine
study
if
i.e.
2 were subjected
to hydrolysis
serum,
The stability
resp.
their
analgesic
activity
after
intracerebroventricular
of
of
these
determined
lIATERIALS
/icv/
degradation,
there
Met-enkephalin
/Eth/,
enzyme complex
rat
brain
peptides
act-
are appar-
Thereis
and enzyme resistance
or D amino acids,
of the
anal-
markedly.
D-methionine
with
to elicit
to differ
D-phenylalanine,
ase M /APM/,
site analgesic
to enzymic
potency
side
extra
/D-Ala2,Met5/
D-norleucine/Nle/, at position
an
Tyr-D-Xxx-Gly-Phe-Pro
has been found
analgesic
D amino acid
whereas
resistance
In the present
containing
prepared
the same route
similar
potency
hand,
to a possible
peptides
seemed to be of
correlation these
that
in
RESEARCH COMMUNICATIONS
aim of providing
intravenously
applied
gesia
it
the
171. Pro5 -enkephalins
-enkephalinamide
fore
On the other
were introduced
for
AND BIOPHYSICAL
and its
a real of Pro5
D-alanine, and D-ethionine with
extracts
aminopeptidand of human
was compared with
in the tail or intravenous
flick
test /iv/
in rats
injection.
AND MHTHODS
Met-enkephalin and the analogs Tyr-Xxx-Gly-Phe-Pro-Q, wherein Xxx stands for Gly, D-Ala, D-Nle, D-Phe or D-Eth, and Q stands for OH or NH2 were prepared as described previously /7-g/. For studies on the breakdown of enkephalins in rat brain extracts Marks' approach IlO] was used as reported by Patthy et al. Ill/. Degradation by human serum was studied as follows: 0.6 ml of 0.01 M TRIS buffer 17.61 containing 0.07 mg substrate /about 0.1 umole/ was a i 37oC for 4 h, added to 0.4 ml of fresh human serum and incubated then a 0.5 ml aliquot of the deproteinized supernatant of each sample was subjected to amino acid analysis. APM digestions were performed in 1 ml 0.05 M TRIS buffer /pH 7.61 containing 1 mg substrate /about 1.5 umole/ and 0.1 mg enzyme /Rijhm Gmbl. The during digestions was determined amount of amino acid 4 released in a JEOL /JLC-5AH/ automatic analyzer and expressed in moleThe analgesic activity of peptides percentage of the substrate. was assessed in rats using the tail flick test as described previously 1121.
1046
BIOCHEMICAL
Vol. 84, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION The action first
of APM on Met-enkephalin
since
opioid
peptides
has long cept
the
findings
with
split
off.
In addition, retarded
It
ered
supported /I that
released hours.
longer
side
in significan
the C-terminal
also
seemed to affect
IV,
and IV/.
human sera are given
in Table
even more susceptible
to brain
compound,
i.e.
was averted
II.
rat
somewhat less
Pro'.
Comparing
D-Ala2
in III
extracts
just
like
of
III
and with
proved
to be
the parent
2 in the
in sera.
block
of peptideamides
be observed
the
Interestrelease
IV-VIII
of
minor
in the experiments
APM. Albeit
volved
the enzymes examined in biodegradation
such studies
may provide
in our experiments
of opioid
peptices
estimates
for
1047
the
be
aminopeptidase/s/
than
can completely
acid
3 or more
data
at position
efficiently
hind-
a pentapeptide,
of APM /cf.
of
is
could
for
and serum enzymes than
the degradation
can only
of
of
D-amino itself
Pro5 -enkephalin
residues
enough,
D-Ala
brain
by the D amino acid
ingly
:Jith
with
The action
extracts
of Tyr
was digested
Met-enkephalin.
brain
differences
Moreover,
2
release
than by any other
the action
digestion
the
same
was not
at position
amide function
Data of the
to the
bond in II
liberation
amount when III
Finally,
Hamely
is
It
bond ex-
above notion.
of APX, that
chain.
12-61.
present
residues
that
of
residues.Our
were hydrolyzed
D amino acid
the action
authors
any peptide
the Phe-Pro
also
having
the
and II/
appeared, however, 3 by D-Ala' to a lesser extent
residue
like
partly
the exception
/III-VIII/ Tyr'.
I/
was examined
in deactivation
by several
APM can hydrolyze
and Pro' -enkephalin
extent
out
formed by Pro or D amino acid /Table
analoqs
of aminopeptidases
had been pointed
been known that
those
Met5-
involvement
and its
and those
are not
identical,
susceptibility
of
in-
of
of
Pro-NH2
Pro-NH2
D-Nle
D-Met
D-Eth
D-Phe
digestion
V
VI
VII
VIII
*after
for
Pro-NH2
Pro-NH2
Pro-NH2
D-Ala
IV
Pro-OHX
Pro-OH
D-Ala
GUY
II
Met-OH
III
GUY
changing residues xxx2 Yyy5-Q
I
ccmpounds
No.
Degradation
Tyr
18 h D-Ala
5
5
5
6
27
37
67
65
0.5
percent
enkephalins
74
3h
was
10
10
14
17
55
63
72
I
0
0
0
0
0
+
58
in
0
0
0
0
+
6
64
67
3
h
acids XXX2
66
0.5
amino
released
of
/Tyr-Xxx-Gly-Phe-Yyy-Q/
Table
28%,
Tyr
0
0
0
0
0
+
58
0
0
0
0
+
9
64
67
3h
after
and Gly
GUY
66
0.5
released
in
0
0
0
0
0
0
0
3 h,
?1
WY5
resp.
0
0
0
0
0
0
0
16
9.5
resp.
100 and 35%,
0
0
0
0
0
0
0
71
3h
and Phe
32
0.5
(r),5
by aminopeptidase
0
0
0
0
0
0
0
62
3
h
of
enkephalins
D-Ala
D-Eth
D-Met
D-Nle
D-Phe
IV
VII
VI
V
VIII
GUY
I
D-Ala
Gly
II
III
xxx2
Pro-NH2
Pro-NH2
Pro-NH2
Pro-NH2
Pro-NH2
Pro-OH
Met-OH
Pro-OH
Yyy5-c,
changing residues
of compounds
Number
Degradation
II by brain
extract
0 0
7126 o/15
0
-
12137 8122
11
-
22146 15138
91
99
77188 60/60
C
A/B
o/o
o/o
o/o
Of3
O/6
o/5
25142
50156
A/B
0
0
0
-
0
-
39
71
C
o/o
o/o
o/o
o/o
o/o
o/12
25142
50156
A/B
o/o o/o o/o o/o
0 0 0
o/o
0 -
o/o
51156
62167
A/B
-
39
71
C
01 0 01 0
0
01 0
o/o
01 0
o/3
54174
90/100
A/B
0
0
-
0
-
76
78
C
YYY5
after 4h /C/:
and serum
percent of amino acids released by brain extract 0.5h /A/ and 2h /B/, req., and by serum after 'Tyr xxx 2 Phe GlY
/Tyr-Xxx-Gly-Phe-Yyy-g/
Table
0
0
0
-
0
-
41
100
C
Vol. 84, No. 4, 1978
peptides
BIOCHEMICAL
towards
brain
enzyme resistance
the
ate ation
In case of peptides
central
is
for
any response. "stability"
Beyond values
logical
potencies.
assessed
after
systemic
illustrated
by the practically
ases. the
lack
of
the
for
cannot
of the an analog,
fails
to cause
however,
order
the
for
the bio-
of analgesic
potencies
one must realize value.
is
of
effect
the
clearly III,
ana
to protein-
strongly
with
that
II,
susceptibility
I and II
solely
This
activity
different
analgesic
intravenously
/II/,
identical
the data
of significant
and biodegrad-
information
informatory
compounds of widely
A comparison
applied
less
Data indic-
otherwise
stability,
administration,
of even
together
resistance"
effect
reliable
For explaining
is
three
analgesic
give
"stability"
IV,
a "threshold
threshold
do not
III.
potency
Fro'-enkephalin
this
values
peptide
depends on the way of application.
the
unstable
the
of intact
in Table
analgesic
administration
the extremely
e.g.
strongly
required
percentage
are listed
between
the peptides
To characterize
Such "stability"
potencies
the correlation
of
values
was calculated.
analgesic
that
and serum proteinases.
by numerical
in hydrolysates with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
suggests
that
Met-enkephalin
be due to its
rapid
degradat-
extremely
high
ion. From the bioloqical
above data potency
enkephalinamide or four
of
is
different
we may conclude an enkephalin
a result
of
requirem.ents,
that
analog
namely
to cross
the blood-brain
improved
binding
capacity
and increased
in
the
literature.
ing peptides encountered
latter
undergo yet
that
biodegradation,
to demonstrate
their
1050
barrier,
at least
three
transport
prop-
enhanced
enzyme resistance.
or The
to us to be overestimated
one occurs
Notwithstanding
ID-Met2,Pro51of
favourahle
ability
of the
like
the satisfaction
erties,
significance
the
all
the naturally
no difficulty in vivo
occur-
has been
biological
effect,
D-Phe
VIII
Pro-NH2
Pro-NH2
D-Nle
V loo/as
93174
92178
Pro-NH2
D-Met
VI
88163
Pro-NH2
D-Eth
VII
85162
D-Ala
IV
Pro-NH2
D-Ala
III
40126
101 0
intact in brain extract after 0.5/2h
78154
Met-OH
Gly
I
III of enkephalins
100
100
100
a9
9
0
peptide in human serum after 4h
"STABILITY"
potency
Pro-OH
Pro-OB
Yyy5-*
GUY
xxx2
changing residues
and analgesic
II
pounds
com-
Of
Number
"Stability"
Table
250
7407
24242
5714
1538
250
-1.5
((1
ED50nM/animal
lo3
centrally
POTENCY
204
222
3125
235
a9
a3
0
64
ED50,uM/kg
lo3
intravenously
/Tyr-Xxx-Gly-Phe-Yyy-R/
< 0 P
Vol. 84, No. 4, 1978
whilst if
the
it
analgesic
property
is due to an extreme
physiological note
BIOCHEMICAL
as their
activity
D amino
and IMet2,
two preparations, the
those
the
to the Slet-enkephalin analogous
biological
molecular
interaction
the C-terminal about
all
these
interesting
pig
latters
and the to form
effects
in this
ileum,
do not
between portion
favourable
the
addition
site
/3-endorphin
in the
of the
residues
appear
/residues
6-31
to exert
A particular
intral-4/
1141 might
changes mentioned
two
of D-Met 2 of
/3-endorphin
active
of
/3-
differentiate
populations
regard.
and also
above.
REFERENCES 1. Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A.; and Morris, H.R. /1975/ I!ature 258, 577-579. 2. Chang, J.K., Fong, B.T.W., Pert, A., and Pert, C.B. 119761 Life Sci. 18, 1473-1481. 3. Hambrook, J.M., Morgan, B.A., Rance, M.J., and Smith, C.F.C. 119761 Nature 262, 782-783. 4. Pert, C.B., Pert, A., Chang, J.K., and Fong, B.T.W. 119761 Science 194, 330-332. 5. Marks, N., Grynbaum, A., and Neidle, A. 119771 Biochem. Biophys. Res. Commun. 74, 1552-1559. 6. Grynbaum, A., Kastin, A.J., Coy, D.H., and Marks, N. 119771 Brain Res. Bull. 2, 479-484. 7. Bajusz, S., Ronai, A.Z., Szgkely, J.I., Dunai-Kovacs, ZS., Berzetei, I., and Gr6f, L. 119761 Acta Biochim. Biophys. Acad. Sci. Hung. 11, 305-309. 8. Bajusz, S., Rbnai, A.Z., Szekely, J.I., Grsf, L., Dunai76, 91-92. Kovgcs, Zs., and Berzdtei, I. 119771 FEBS Letters 9. Grdf, L., Ronai, A.Z., Bajusz, S., Cseh, G., and Szekely, 119761 FEES Letters 64, 181-184. J-I.-,
1052
of
possess higher
are equipotent
receptor
moiety
those
and Leu-enkephalins
on guinea
the
than
do. Thus introduction
structure
to
f3-endorphin
analogs
opiate
be of
of
containing
words,
cannot
/D-Vet 2 , Pro'/-enkephalin-
acid
than
- even
of
Yet-
formers
sequence
is
131. While
in other
and Pro-NH2 5 into
it
Pro5/-enkephalinamide
models as the
-
112,
same way the distinct
in vitro
bring
of
on mouse vas deferens
endorphin
context
properties
and Leu-enkephalins
as well
instability
In this
the biological
RESEARCH COMMUNICATIONS
and Leu-enkephalins
metabolic
amide are more reminiscent Met-
of Met-
significance.
that
AND BIOPHYSICAL
Vol. 84, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of Peptides, pp. 221-268, 10. Marks, N. 119771 Neurobiology Plenum Press, New York. 11. Patthy, A., Grbf, L., Kenessey, A., Szekely, J.I., and Bajusz, S. 119771 Biochem. Biophys. Res. Commun. 79, 254-259. 12. Szekely, J.I., Rbnai, A.Z., Dunai-Kovacs, Zs., Migli?cz, E., Berzetei, I., Bajusz, S., and Graf, L. 119771 Eur. J. Pharmacol. 43, 293-294. 13. R6nai, A.Z., Berzetei, I., Szekely, J.I., Graf, L., and Bajusz, S. 119781 Pharmacology /in press/. 14. Gr6f, L., Cseh, G., Barat, E., Ronai, A.Z., Szkkely, J.I., Kenessey, A., and Bajusz, S. 119771 Ann. ?l.Y. Acad. Sci. 297, 63-83.
1053