Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 91/16~5 2 0 ~ PCT/~L91/0
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i Cyclic meptides and their use.
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~ The present inven~ion with a novel class of
- peptides and their application.
It is known that simple oligopeptides maj dis-
. play diverse and potent biological acti-ities including
antibiotic, antitumor, antiviral as well as im~uno-
, suppressive activities. Thus far, cyclic peptides were
mostly of microbial and more in particular of fungus
origin. These so-called c~closporins are known as
immunosuppressive compour.ds and sre used to p.event
.'1 10 grsft rejection after organ trsnsplantation. Disadvant-
sges of cyclospo-ins are the r insolubil~;~ in wacer and
~, their toxicitS, p~rt cula-l- for the k dneys.
Active pep~ides oriE natir, from highe- vl~^,ts
ars ve-y rare. ~ecer.~l~, c~.clic o7~_ape?~ des here
.~ 15 isolated from the roots of Rubia cordifolia and ~. aKane
i~ (Rubiaceae). The cyclic hexapeptide was reportec to
. possess antitumor activity in a mouse leukemia model
(Itokawa, H., Takeya, Koichi, Mori, ~., Kikodoro, S.,
and Ysmamoto, H. ~1984a) Studies on antitumour cyclic
hexapeptides RA obtained from Rubiae radix, Rubiaceae
(IV): Quantitative determination of RA-VII and RA-V in
commercial Rubia radix and collected plants, Plan~a Med.
~1, 313-316 & Ito',:ahD, Y.., TakQ~ ., Mor , ~.,
Hamanaka, T,, Sonobe T,, and Mihara, K, (1984D)
Isolation and antitumour activity o~ cyclic hexapeptides
isolated from Rubiae Radix. Chem. Pharm. Bull. 32 284- :
290). Both cyclosporins snd the Rubia peptides are for
the major part composed of non-proteinogenic amino
.~i acids.
3 Proteins and perhaps also peptides may bind to
the Fc-portion of immunoglobulins. A surface protein
from Staphylococcus aureus (protein A) was shown to bind
. ~ IgG and to enhance complement activation ~ia the
-~ ~ classical pathway (CP) (Masuda, S., Sakurai, S. & Kondo,
:~ 35 I. (1975j Simple and effective method for selecting
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WO91/16~ l 3~ PCT/~L91/0~ ~
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protein A deficient mutants by cosedimentation with
sensitized sheep erythrocytes. Infection and Immunity
12, 24~-251: Van Dijk, H. & Van Bohemen, C.G. (1978)
Indirect plaque-forming cells detected by use of normal
mouse serum I. Normal mouse serum plaque-forming cells
are IgA-producers. Cellular Immunlogy 38, 124-130).
Protein A and an analogue isolated from Streptococcus
; strain G148 (protein G) are used to isolate IgG from
serum and plasma (Bjorck, L & Kronvall, G. (1984) Purif-
ication and some properties of Streptococcal protein G,
a novel IgG-biundine reagent. J. Immunol. 133, 969-973).
Leupeptin (a tripaptide from actinomycete
fermen~ation) and singlQ amino acids can interfere with
complement activation via the CP andior the alternative
pathwa; (AP) (Takada, Y., .~rimoto, Y, Mineda, H. & Taka-
da, .~.. (1978) Inhibition of the classical ar.~ alterna-
~l tive pathwa~s by amino acids and their derivatives.
s~, Immunology 34, 509-515). -
~ It has now been found by us that there are
;, 20 cyclic peptides with IgG-binding properties. This is to
il say that cyclic peptides which are isolated from e.g.
the latex of specific plants or which may be prepared
synthetically or semi-synthetically, were find to bind
to human but also to rabbit and mouse IgG but not to
Ig~7 and IgA in in vitro systems for Is~-binding. The
peptides were isolated and ldentified on the basis of
thelr selective inhibition of complement activation via
the CP (Kosasi, S., Van der Sluis, W.G., Boelens, R., 't
Hart, L.A. & Labadie, R.P. ~1989) Labaditin, a novel
cyclic decapeptide from the latex of Jatropha multifida
1. (Euphorbiaceae) FEBS let~ers Z56, 91-96). AP activ-
ation was not or only s-lightly affected by these
peptides. It was shown that the anticomplemçntary
activity of the peptides is mediated by an interference
with Clq-acceptor sites on the IgG molecules they bind
- to. Tnis means that the cyclic peptides combine protein
A-like IgF-binding activitv with leupeptin- and amino
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acid-like anticomplementary behavior. Such combined
activity has never been described in literature, not for '
proteins but neither for linear peptides and
particularly not for c~clic peptides. The anti-
''.! 5 complementary activity of the novel cyclic peptides is
mechanistically different from that of the linear
peptide AA 27~-290, which represents a major part cf the
Clq-acceptor site on IgG (Prystowski, M.B., Kehoe, J.M., ;~
& Erickson, B.W. (1981) Inhibition cf the classical '
~ 10 complement pathway by synthetic peptides from the second-'i constant comain of the hea~.; chzin Or IgG. Bioche~istry
'~ 21, p, 6349-6358). The latter does not bind to igG but ''
prevents cocple~ent sctivstion by competing hith IsG for
binding to C;. The an~ico~?lementary acti-ity is also
different from the leupep~in-induced 2.-~ amino acid-
induced complement inhibi;ion which is no: 'base~ on
'~ binding to lgG.
'~ An advantage of our novel peptides over other
cyclic peptides, such as cyclosporins, is their extreme
solubility in water (up to 1000 mg per ml) and their
. non-toxic behavior, at least in mice. They also differ
from cyc.osporins and the Rubia peptides in the fact
that they are built up fro~ proteinogenic (= proteinic)
amino acids. '
Therefore, the cyclic peptides all ~o the
invention consist preferably of proteinic a~ino acids.
Thls means that'such amino ~cids do not have to be
modified, e.g. by methyl groups. It should be noted that
the known cyclosporins contain methylated or derived
proteinic amino acids.
' Structural analysis of the cyclic peptides of
the present invention reveals that said cyclic peptides
contain preferably the amino acid Trp and/or His, in
particular the dipeptide groups Trp-Gly and/or His-Gly.
It is preferred tbat the cyclic peptides
- according to the invention contain 8-12, preferably 9-11
amino acid residues.
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~O9l/16~5 2 ~ PCT/~Lgl/o~
In general, the cyclic peptides according to the
invention contain at least 6 amino acid residues which
are preferably selected from the group consis~ing of
Ala, G~y, Val, Trp, Thr, Ile, Ser, and Leu.
Two important examples of cyclic peptides
according to the present invention are characterized by
the sequence Ala-Gly-Val-Trp-Thr-Val-Trp-Gly-Thr-Iie
(Labaditin) and Ala-Ser-Ile-Leu-Gly-Leu-Gly-Trp-.~la-
(Biobollein).
Of course, the cyclic peptides according to the
present invention may be prepared according to classical
peptide synthesis methods. However, they may also be
isolated from plan~ material of the Euphorb aceae
family, in pa-ticuiar the la-~x of Ja~ro?ha species.
The peptides accord r., to tr.e inven~ion ~a~ be
,j, used fo- various purposes such as for the preparation of
pharmaceutical co~positions or for analysis, and/or
:' separation standardization purposes.
,' The use of the cyclic peptides according to the
7 20 invention may - in general - be used for IgG-binding and
~ anticomplementary activity in mammals including human
m beings.
:! .
,~i The present invention also relates to the
~! application mentioned above. The present. invention
further relates to a method for trestlng diseases such
as inflammstory diseases including rheumaticas well as
other systemic or local-auto-immuné, and immune complex-
related diseases including extrinsic allergic alveolitis
in mammals including human beings wherein a cyclic
peptide as defined in the above is used as an active
substance.
The present invention further rçlates to
`i, pharmaceutical compositions for treating diseases such
~;~ as inflammatory diseases including rheumatic as well as
,~j 35 other systemic or local auto-immune, and immune complex-
`~ related diseases including extrinsic allergic alveo-
~ litis, said compositions conta ning a cyclic peptide as
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WO91JI6~5 5 2 0 ~ ~ 312 PCT/~L9t/0~ ~
defined in the above.
In general, the peptides according to the
invention may be applied in composition with an anti-
inflammatory potential and further be used to enrich IgG
from blood serum or plasma to deplete plasma or serum
from IgG, and/or to quantitate IgG levels in e.g. body
liquids.
- With respect to the anticomplementary activity
the following is remarked.
Complement is an important system in the body's
; defense against foreign invaders such as bacteria,
virusses, and other micro-organisms. The activation of
the complement cascsde by foreign materials leads to
inflammation, opsonisation by C3b for phagocytosis, and
l; the lysis of cells b- membrane damage. Complement can
also be activated in diseases such as immune cc~leY.
j and/or auto-immune diseases and immuni ty states ~nere
tissue damage may occur. It is believed that inhibition
. of the complement cascade can prevent tissue injur-. .Abrief review on the C? and AP comple-ent inhibitors is
, given in Ashgar, S,S., ~1984) Pharmalogical ~anipulation
of Complement System, Pharmalogical Reviews 36, 223-224.
Up to now, a limited number of anticomplementory agents
~, are available. Of these agents only cobra venum facto-
~e~ 25 ~CVF) gives rise to efficient complemer.~-de~lst on in
vivo. The complement-depletion brought a~ou~ ~. CVr,
however, is not selective but involves both the CP and
~ the AP complement activation. Therefore, the new C-'~ inhibitors according to the present invention for the
treatment of auto-immune and other immune complex
.,~ diseases are very important. The cyclic peptides
according to the invention have specificity for the CP
and leave the AP unaffected. The latter is essential not
only for the host's general defense potential but also
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ny ~ 35 for the elimination of certain types of immune complexes
(Vogt, W., (i985) Drugs and the complement system.
Trends in Pharmocol. Sciences 6, 114-119). Probabl~. the
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WO91/16~ 2 0 8 ~ 312 PCT/~L91/0~ ~
best CP-inhibitors are substances which interfere with
the binding of Cl to immune aggrc-ates.
Since the cyclic pept ~s according to the
invention cause an inhibition of the classical
complement pathway and leave the AP functionally intact,
it may be assumed that the peptides will not interfere
with the non-specific defense of the body agains-
microbial infections and with processes as the AP-
dependent elimin2tion of immune complexes from the
circulation. This means that the cyclic peptides
; according to the invention are highly interesting sub-
i stances that are suited to t.eat the deleter_ous e~ects
of the CP-acti~ation in vivo as occ~rring in auto-immune
diseases.
1~ It is a ve-y i~portan~ feature of the suDstances
of the invention that no acute toxic effects can be
sho~n in mice in concentrations up to ~ mg per anim21.
Peptides according to the invention could be beneficial
} not only by local application (e.g. in vasculitis) but
may also be of use upon oral or parenteral adm nistr-
ation in the case of diseases such as mentioned above
and in arthr~tis, hepatitis, glomerulo-nephritis etc. It
, is expected that the cyclic peptides according to the
present invention will not show chronic toxicit~,
either.
The cyclic peprides ma- be used in the estim-
ation of complement-activating human IgG's and analogues
in other species by ELISA, the isolation of these
antibodies and analogues by affinity chromatography,
very similar to protein A-sepharose chromatography, and
the selective removal of IgG from the circulating blood
in immune complex diseases and cases of M. Kahler. This
~ could probably be achieved by plasmapheresis and passing
-'~, the plasma over micro-carriers (beads) coated with
cyclic peptides accordin-g to the invention, e.g.
~`~ labatidin or biobollein.
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WO9t/16~; 2 0 i~ 13 ~ ~ PCT/~L9t/000~
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The cyclic peptides according to the invention
may be prepared according to standard procedures for the
s~nthesis of cyclic (oligo) peptides. These procedures
are well known to the man in art.
However, as noted above, the cyclic peptides
according to the invention may be isolated from plant
material, e.g. of the Euphorbiaceae family, in
particular the latex of the genus Jatropha, e.g.
Jatropha multifida L.
The isolation of the cyclic peptides according
to the invention is based on their modulatory effec~s on
specific im~unological parsmeters in vitro. Relevant
experiments are carried out according ~o standard pro-
cedures.
Below an example is given o, the isolz-l~n cf
two important cyclic peptides according to tne
invent on, i.e. labaditin and biobollein.
Immunomodulatory constituents were isolated from
the latex of Jatropha multifida accordin~ to the
following fractonation scheme:
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WO 91/16345 2 0 S ~ 3 i 2 PCI~L91/00066
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J: Me~H:H20 0 100 ! -- 7-- 32 1~0:0
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: rnv~iliCol mullilid~l I
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W091/t6~5 ~ ~g~ 3 ~ 2 PCT/~L91/0
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According to the above scheme the crude latex ~5
ml) was mixed with 25 ml of demineralized water. After
extraction of the supernatant with n-hexane, the super-
natant was lyophillized, yielding a solid (500 mg). The
solid material was dissolved in a small amount of water
and subsequently separated on a polyaaide column. By
elution with 500 ml portions of different methanol-water
mixtures, i.e. (O : lOO), (l : 4), (2 : 3), (3 : 2) and
; (lOO : O) successively, five fractions (I-V) were
obtained. These fractions were tested for modulatory
effects on both CP and AP activation of human
complement, and on the production of rea~ctive oxygen
species (ROS) by zymosan-stimulated human polymorpho-
nuclear neu~rophilis (PM~') monitored as chemi-
l~ luminescence. Fraction IV was found to possess
significant ac~ivity.
; From fraction I~' a novel c~c1i~ aecP?e~ de
(labaditin) and a novel cyclic nonapeptide (biobollein)
, were isolated, both of which show a strong inhibitcry- -
.~, 20 effect on CP acitivity of human complemen~. The
, isolation of labaditin and biobollein respectively is
elucitsted here below.
Isolation of labaditin
The concentrated MeOH : H20 (3 : 2) fraction
(fraction IV) (42 mg) was dissolved in l;. NaHC03. The
solution was exhaustively with ethyl acet&te. E~hyl
- acetate extracts were combined.and the solves1t was e:ap-
j orized under reduced pressure. The residue was dissolved
~ in 2 ml of MeOH and separated-by gel permeation over
i' 3 Sephadex LH-20 ( 40 cm x l cm i.d.) with MeOH as eluting
agent. Per fraction 300 drops were collected. Fractions
4, 5 and 6 showing one single spot on TLC were combined.
The MeOH was evaporated under reduced pressure.
' Subsequently, water was added and the solution was
`~ 35 lyophillized, yielding 14 mg of a white solid.
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W091/16~5 PCT/~L91/O~K~
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Isolation of biobollein
The MeOH : H20 (3 : 2~ fraction (fraction IV)
(42 mg) was directly extracted with ethyl acetate. The
ethyl acetate extracts were combined and the solvent was
evaporated under reduced pressure. The residue was
dissolved in 2 ml of MeOH and by adding 15 ml of acetone
: water (1 : 1), a precipitate was obtained. The
precipitate was dissolved in 2 ml of MeOH and separated
over Sephadex LH-20 (column 40 cm x 1 cm i.d.) with MeOH
ss elutin~ agent. Per frsction 300 drops were collected.
Fractions 4, 5 and 6 showing two spots of TLC were
combined and the solvent was removed under reduced
pressure. The fraction cons ituents were separated by
preparative TLC on silica gel 60 F 254, 1 mm (Merc~
Darmstsdt, FRG) with CHCl3 : MeOH ; H20 (13 10 : 2) as
- eluent (saturated chamber), and were detected unde~
254 nm. The procedure yielded two white solid compounds,
i.e. labsditin (14 mg) and biobollein (11 mg).
The structure of labaditin and biobollein was
determined by means of the following procedures:
Amino acid an21ysis
The amino acid composition wss determined with
sn sutomat~c amino acid analyser (LK~ 4151 Alpha plus,
Na-system 20 cm column) sfter hydrolysis ln 6 N HCL st
110 C for 48 hours and, for tryptophan determination, in
~ 6N HCL with 4X thioglycolic acid at 110 C for 24 hours.
,~ Th~n layer chromatogra~hy (TLC)
Silica gel 60 F-254 TLC plates (Merc~,
~ Darmstadt, FRG) were used with CHCL3 : MeOH : H20 = 13 :
'~ - 30 10 : 2 as solvent system (saturated chamber). Spots were
visualized under UV 254 nm and by spraying with vanilin-
sulphuric acid followed by heating at 110 C for 5 min.
' NMR sPectroscoPY
.
For NMR experiments the purified peptide was
dissolved in DMSO-d6 (conc. 30 mg/ml). For some exper- ~ -
`~ iments 5% D20 was added to exchange amide protons. lH-
`~ NMR spectra were recorded on a Bruker WM-300 spectro-
meter at 303 and 338 K. Two-dimensional lH-NMR spectra
~rj were obtained at 303 X. For the COSY spectrum 257
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W091/16~5 2 ~ ~1312 PCT/~L91/00066
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records of 2K data were recorded at 400 MHz on a Bruke.
MSL-400 apparatus. The phase sensitive NOESY expe.iment
containing 350 records of 2X date was obtained 2- 600
MHz on a Bruker AM-600. The NOES~ data were multiplied
with sinebell windows and Fourier transformed in both
domains. The COSY spectrum was displayed in the absolute
value mode.
FAB-MS measurement -
For the FAB experiments a ZAB-2F VG instrument
was used.
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