U.S. patent application number 09/779703 was filed with the patent office on 2003-06-05 for tnf-derived peptides for use in treating oedema.
Invention is credited to Bloc, Alain, De Baetselier, Patrick, Fransen, Lucie, Lucas, Rudolf, Pugin, Jerome.
Application Number | 20030105021 09/779703 |
Document ID | / |
Family ID | 27239778 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030105021 |
Kind Code |
A1 |
Lucas, Rudolf ; et
al. |
June 5, 2003 |
TNF-derived peptides for use in treating oedema
Abstract
The present invention relates to the finding that peptides
derived from a specific domain of tumor necrosis factor-alpha
(TNF-.alpha.) can efficiently be used to treat oedema. More
specifically, the present invention relates to the usage of
peptides derived from the region of human TNF-.alpha. from
Ser.sup.100 to Glu.sup.116 to treat pulmonary oedema. Moreover, the
present invention concerns a circularized peptide having amino acid
sequence CGQRETPEGAEAKPWYC which is very efficient in inducing
oedema resorption.
Inventors: |
Lucas, Rudolf; (Aartselaar,
BE) ; De Baetselier, Patrick; (Berchem, BE) ;
Pugin, Jerome; (Puplinge, CH) ; Bloc, Alain;
(Bas-Monthoux, FR) ; Fransen, Lucie; (Hertsberge,
BE) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201
US
|
Family ID: |
27239778 |
Appl. No.: |
09/779703 |
Filed: |
February 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09779703 |
Feb 9, 2001 |
|
|
|
PCT/EP99/05806 |
Aug 10, 1999 |
|
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Current U.S.
Class: |
424/85.1 ;
514/1.5; 514/17.4 |
Current CPC
Class: |
C07K 14/525 20130101;
A61P 7/00 20180101; A61P 7/10 20180101; A61P 11/00 20180101; A61P
3/00 20180101; A61P 43/00 20180101; A61K 38/191 20130101 |
Class at
Publication: |
514/13 ; 514/14;
514/15; 514/16; 514/9 |
International
Class: |
A61K 038/12; A61K
038/10; A61K 038/08 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 14, 1998 |
EP |
98870180.1 |
Sep 18, 1998 |
EP |
98870198.3 |
Oct 21, 1998 |
EP |
98870222.1 |
Claims
1. Use of a peptide comprising a chain of 7 to 17 contiguous amino
acids derived from the region of human TNF-.alpha. from Ser.sup.100
to Glu.sup.116 or from the region of mouse TNF-.alpha. from
Ser.sup.99 to Glu.sup.115 for the manufacture of a medicament for
treating oedema.
2. Use of a peptide according to claim 1, wherein said peptide
comprises a chain of 11 to 16 contiguous amino acids.
3. Use of a peptide according to claim 1, wherein said peptide
comprises a chain of 13 to 15 contiguous amino acids.
4. Use of a peptide according to claim 1, wherein said peptide
comprises a chain of 14 contiguous amino acids.
5. Use of a peptide according to claim 4, wherein said chain of 14
contiguous amino acids are chosen from the group consisting, of the
contiguous amino acid sequences QRETPEGAEAKPWY and
PKDTPEGAELKPWY.
6. Use of a peptide according to any of claims 1 to 5, wherein said
peptide is circularized.
7. Use of a peptide according to claim 6, wherein said peptide is
circularized by replacing the NH.sub.2-- and COOH-terminal amino
acids by cysteine so that a disulfide bridge is formed between the
latter cysteines.
8. Use of a peptide according to claim 7, wherein said circularized
peptides are chosen from the group consisting of the circularized
peptides CGQRETPEGAEAKPWYC and CGPKDTPEGAELKPWYC.
9. Use of a peptide according to any of claims 1 to 8, wherein said
oedema is pulmonary oedema
10. A pharmaceutical composition for treating oedema comprising a
peptide according to any of claims 1 to 9.
Description
FIELD OF THE INVENTION
[0001] The present invention is based on the finding that peptides
derived from a specific domain of tumor necrosis factor-alpha
(TNF-.alpha.) can efficiently be used to treat oedema. More
specifically, the present invention relates to the usage of
peptides derived from the region of human TNT-.alpha. from
Ser.sup.100 to Glu.sup.116 to treat pulmonary oedema. For example,
the circularized peptide having amino acid sequence
CGQRETPEGAEAKPWYC is shown to be very efficient in inducing oedema
resorption.
BACKGROUND OF THE INVENTION
[0002] Pulmonary transplantation is shown to be successful in the
treatment of patients with end-stage pulmonary disease. However,
pulmonary oedema or edema (both terms can be used interchangeably)
following reperfusion of the transplant is a major clinical problem
for which no efficient drug exists at this moment. In addition,
recent evidence indicates that the endothelium plays an essential
role in regulating the dynamic interaction between pulmonary
vasodilatation and vasoconstriction and is a major target during
ischemia/reperfusion and acute respiratory distress syndrome
(ARDS)-related lung injury. Thus, given that pulmonary edema often
results in lung transplant rejection and that there is a persistent
shortage of lungs available for transplantation, there is an urgent
need to efficiently prevent or treat pulmonary edema.
[0003] During ischemia and reperfusion (I/R), a typical induction
of inflammatory cytokines like tumor necrosis factor-alpha (TNF)
occurs. TNF is a pleiotropic cytokine, mainly produced by activated
macrophages, that is synthesized as a transmembrane molecule that
can be released by metalloproteinases from the cell surface into
the circulation (Gearing et al., 1994). TNF has been shown to bind
to at least two types of membrane-bound receptors, TNF receptor 1
(55 kD) and TNF receptor 2 (75 kD), that are expressed on most
somatic cells, with the exception of erythrocytes and unstimulated
T lymphocytes. TNF can be considered as a two-edged sword: indeed,
when overproduced, TNF has been shown to be implicated in the
pathology of various infectious diseases, such as LPS-induced
sepsis (Beutler et al., 1985), cerebral malaria (Grau et al.,
1987), as well as treatment-associated mortality in African
trypanosomiasis (Lucas et al., 1993). In contrast, TNF was shown to
be one of the most efficient protective agents against cecal
ligation and puncture-induced septic peritonitis in mice and rats
(Echtenacher et al., 1990, Alexander et al., 1991; Lucas et al.,
1997) and to be implicated in host defense during pneumococcal
pneumonia in mice (van der Poll et al., 1997). Moreover, mice
deficient in TNF receptor 1 were shown to be significantly more
sensitive to Listeria monocytogenes (Rothe et al., 1993; Pfeffer et
al., 1993) and Mycobacterium tuberculosis infection Flynn et al.,
1995) as well as against fungal (Steinshamn et al., 1996) and
Toxoplasma infections (Deckert-Schluter et al., 1998). Therefore,
it becomes clear that apart from its detrimental effects during
overproduction or during prolonged chronic secretion, TNF is also
one of the most potent protective agents against infections by
various pathogens. In this regard, peptides derived from TNF have
been suggested to be used as treatment against disease (DE 3841759
to Bohm et al.)
[0004] Apart from exerting a plethora of effects mediated by the
activation of its two types of receptors (TNF receptor 1, 55 kD,
and TNF receptor 2, 75 kD), TNF can also mediate
receptor-independent activities. The tip domain of TNF is located
on the top of its bell-shaped structure and is spatially distinct
from its receptor binding sites, that are localized at the base of
the trimeric molecule (Lucas et al., 1994). This domain has
lectin-like affinity for specific oligosaccharides, such as
trimannose and diacetylchitobiose. Both TNF and the tip peptide of
TNF are capable of mediating a trypanolytic activity by interfering
with the lysosomal integrity of the trypanosome, a pH-dependent
effect probably involving the insertion of TNF into the lysosomal
membrane (Magez et al., 1997). Moreover, mutants of the tip peptide
in which three critical amino acids (T(105): E(107); E(110)) were
replaced by A, were completely unable to mediate this activity
(Lucas et al., 1994). A mouse TNF (mTNF) triple mutant,
T105A-E107A-E110A (referred to hereafter as triple mTNF), lacks the
trypanolytic and lectin-like affinity to oligosaccharides as
compared to wild type TNF. The triple mTNF has significantly
reduced systemic toxicity as compared to wild-type mTNF in vivo,
but retains its penrtonitis-protective effect in a murine model
(Lucas et al., 1997).
[0005] Another receptor-independent activity of TNF is its
membrane-inserting and sodium channel forming capacity (Baldwin et
al. 1996). Indeed, others have shown that TNF forms a
Na.sup.+-channel in an artificial lipid bilayer model, an activity
that is pH-dependent, probably because it requires the "cracking"
of the trimer, thus exposing hydrophobic residues to the membrane
(Kagan et al., 1992).
[0006] Recent observations have indicated that instillation of
anti-TNF-neutralizing antibody into the lungs of rats 5 min before
bacterial infection inhibits the increase in alveolar liquid
clearance, which is known to be driven by a change in intracellular
sodium content in the alveolar epithelial cells. Moreover,
instillation of TNF in normal rats increases alveolar liquid
clearance by 43% over 1 hour (Rezaiguia et al., 1997). Although the
latter findings indicate that TNF might be used to induce alveolar
liquid clearance, wild type TNF cannot be used therapeutically due
to its high systemic toxicity. The present invention relates to the
usage of a selected group of TNF-derived peptides which can, to our
surprise, efficiently be used to induce edema resorption and which
have, compared to wild type TNF, lost systemic toxicity.
AIMS OF THE INVENTION
[0007] It is clear that there is an urgent need to efficiently
prevent or treat pulmonary edema. Although some data demonstrate
that TNF might be involved in oedema resorption, it is clear that
this pleiotropic and potentially toxic molecule can not be used to
treat oedema.
[0008] In this repect, the present invention aims at providing a
non-toxic molecule with the same oedema resorption-inducing
capacity as TNF. More specifically, the present invention aims at
providing non-toxic peptides derived from TNF which can be used to
prevent or treat oedema. Moreover, the present invention aims at
providing a pharmaceutical composition comprising TNF-derived
peptides which induce oedema resorption. In essence, the present
invention aims at providing a new medical use of the TNF-derived,
trypanocidal peptides as described by Lucas et al. (1994) and
fragments and variants thereof.
[0009] All the aims of the present invention are considered to have
been met by the embodiments as set out below.
BRIEF DESCRIPTION OF DRAWINGS
[0010] FIG. 1: (A) Current-voltage relationship in murine lung
microvascular endothelial cells, preincubation for 30 min with wt
mTNF (100 ng/ml) or NES buffer at pH 6 and at pH 7.3. The values
represent the means of .gtoreq.5 cells.+-.SEM (*:P.ltoreq.0.05).
(B) Characteristic current traces of a lung MVEC pretreated with
medium (top) or with 100 ng/ml of TNF (bottom) at pH 6.0.
[0011] FIG. 2: Current-voltage relationship in resident peritoneal
macrophages isolated from (A) control and (B) TNFR 1/2.sup.0/
C57BL/6 mice. cells were pretreated for 30 min with medium, wt mTNF
(100 ng/ml) or Ltip peptide (100 .mu.g/ml). The values indicate the
means of .gtoreq.5 cells.+-.SEM (*:P.ltoreq.0.05).
[0012] FIG. 3: Effect of amiloride (100 .mu.M), added for 30 min
during the preincubation step, on wt mTNF-induced increase in
membrane conductance in MVEC. Comparison of the effect of triple
mTNF (100 ng/ml) and wt mTNF (100 ng/ml), upon 30 min preincubation
with lung MVEC. Values indicate the means of .gtoreq.5 cells.+-.SEM
(*:P<0.05).
[0013] FIG. 4: (A) Effect of Ltip (100 .mu.g/ml) versus controls in
CBA lung MVEC at pH 6 and pH 7.3. (B) Comparison of the effect of
30 min preincubation of MVEC with Ltip peptide, mutTip peptide, and
scramblTip peptide at pH 6. Effect of amiloride (100 .mu.M) added
during the preincubation, on Ltip peptide-induced increase in
membrane conductance in MVEC. Values indicate the means of
.gtoreq.5 cells.+-.SEM (*:P<0.05).
[0014] FIG. 5: Effect of mTNF tip peptide (1 mg/lung) on lung
weight change (in g) during an isolated lung perfusion experiment
lasting 150 min.
[0015] FIG. 6: Effect of wild type mTNF (.circle-solid.,1
.mu.g/lung) or mTNF tip peptide (.tangle-solidup.,1 mg/lung) versus
controls [.largecircle., NaCl] on lung weight change (in % versus
baseline lung weight at 30 min) during isolated lung perfusion
experiments after 150 min. Each symbol [.largecircle.,
.circle-solid. or .tangle-solidup.] represents one lung.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The invention described herein draws on previously published
work and pending patent applications. By way of example, such work
consists of scientific papers, patents or pending patent
applications. All these publications and applications, cited
previously or below are hereby incorporated by reference.
[0017] The present invention relates to the use of a peptide
comprising a chain of 7 to 17, preferably a chain of 11 to 16, more
preferably a chain of 13 to 15 and most preferably a chain of 14
contiguous amino acids derived from the region of human TNF-.alpha.
from Ser.sup.100 to Glu.sup.116 or from the region of mouse
TNF-.alpha. from Ser.sup.99 to Glu.sup.115 for the manufacture of a
medicament for treating oedema. More specifically the present
invention relates to the use of a peptide as described above
wherein said chain of 14 contiguous amino acids are chosen from the
group consisting of the contiguous amino acid sequences
QRETPEGAEAKPWY and PKDTPEGAELKPWY as described by Lucas et al.
(1994). The latter sequences are given in the well-known one-letter
code for amino acids (the three-letter code is sometimes used
further).
[0018] The term "peptide" refers to a polymer of amino acids (aa)
derived from the trypanolytic TNF domain having lectin-like
affinity as described by Lucas et al. (1994). Moreover, the latter
term relates to a polymer of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or
17 contiguous amino acids derived from the region of human
TNF-.alpha. from Ser.sup.100 to Glu.sup.116 or from the region of
mouse TNF-.alpha. from Ser.sup.99 to Glu.sup.115. The latter TNF
regions also refer to the regions shown in FIG. 5, p. 172 of
Pennica and Goeddel in Webb and Goeddel, eds. (1987). However, it
should be clear that the region of human TNF-.alpha. from
Ser.sup.100 to Glu.sup.116 is identical to human TNF-.alpha. from
Ser.sup.99 to Glu.sup.116 in FIG. 5, p. 172 of Pennica and Goeddel
in Webb and Goeddel, eds. (1987) and that the region of mouse
TNF-.alpha. from Ser.sup.99 to Glu.sup.115 is identical to mouse
TNF-.alpha. from Ser.sup.98 to Glu.sup.115 in FIG. 5, p. 172 of
Pennica and Goeddel in Webb and Goeddel, eds. (1987). The term
"peptide" more specifically relates to a peptide comprising the
hexamer TPEGAE of the latter TNF regions or any peptide comprising
the corresponding amino acids T, E and E of the latter hexamer
which were shown to be three critical amino acids by Lucas et al.
(1994). It should be clear that the present invention relates to
any peptide derived from the latter TNF regions and does not
exclude post-translational modifications of the peptides such as
glycosylation, acetylation, phosphorylation, modifications with
fatty acids and the like. Included within the present invention
are, for example, peptides containing one or more analogues of an
aa (including unnatural aa's), peptides with substituted linkages,
mutated versions or natural sequence variations of the peptides,
peptides containing disulfide bounds between cysteine residues, as
well as other modifications known in the art. The peptides of the
present invention are also defined functionally, that is, the
present invention relates to any peptide which can be used to treat
oedema or which can be used for the manufacture of a medicament for
treating oedema. In essence, the present invention relates to any
molecule, obtained by any method known in the art, with the same or
very similar characteristics as the trypanolytic peptides defined
by Lucas et al. (1994).
[0019] The peptides of the present invention can be prepared by any
method known in the art such as classical chemical synthesis, as
described by Houbenweyl (1974) and Atherton & Shepard (1989),
or by means of recombinant DNA techniques as described by Maniatis
et al. (1982) and, more specifically, by Lucas et al. (1994).
[0020] The term oedema (or edema) relates to any abnormal excess
accumulation of (serous) fluid in connective tissue or in a serous
cavity. In particular, the latter term relates to pulmonary oedema
(see also Examples section).
[0021] Furthermore, the present invention concerns the use of a
peptide as described above wherein said peptide is circularized.
More specifically, the present invention relates to the use of a
peptide as described above, wherein said peptide is circularized by
replacing the NH.sub.2-- and COOH-terminal amino acids by cysteine
so that a disulfide bridge is formed between the latter cysteines.
In this regard, the present invention concerns the use of a peptide
as described above wherein said circularized peptides are chosen
from the group consisting of the circularized peptides
CGQRETPEGAEAKPWYC and CGPKDTPEGAELIPWYC as described by Lucas et
al. (1994).
[0022] The present invention finally relates to a pharmaceutical
composition for treating oedema comprising a peptide as described
above. The terms "a pharmaceutical composition for treating oedema"
relates to any composition comprising a peptide as defined above
which prevents, ameliorates or cures oedema, in particular
pulmonary oedema. More specifically, the terms "a pharmaceutical
composition for treating oedema" or "a drug or medicament for
treating oedema" (both terms can be used interchangeably) relate to
a composition comprising a peptide as described above and a
pharmaceutically acceptable carrier or excipient (both terms can be
used interchangeably) to treat oedema Suitable carriers or
excipients known to the skilled man are saline, Ringer's solution,
dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5%
dextrose in saline, substances that enhance isotonicity and
chemical stability, buffers and preservatives. Other suitable
carriers include any carrier that does not itself induce the
production of antibodies harmful to the individual receiving the
composition such as proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids and amino acid
copolymers. The "medicament" may be administered by any suitable
method within the knowledge of the skilled man. The preferred route
of administration is parenterally. In parenteral administration,
the medicament of this invention will be formulated in a unit
dosage injectable form such as a solution, suspension or emulsion,
in association with the pharmaceutically acceptable excipients as
defined above. However, the dosage and mode of administration will
depend on the individual. Generally, the medicament is administered
so that the peptide of the present invention is given at a dose
between 1 .mu.g/kg and 10 mg/kg, more preferably between 10
.mu.g/kg and 5 mg/kg, most preferably between 0.1 and 2 mg/kg.
Preferably, it is given as a bolus dose. Continuous infusion may
also be used. If so, the medicament may be infused at a dose
between 5 and 20 .mu.g/kg/minute, more preferably between 7 and 15
.mu.g/kg/minute.
[0023] The present invention will now be illustrated by reference
to the following examples which set forth particularly advantageous
embodiments. However, it should be noted that these embodiments are
illustrative and can not be construed as to restrict the invention
in any way.
EXAMPLES
Example 1
[0024] Material and Methods
[0025] Animals, cells and reagents. Male CBA/J or C57BL/6 mice, as
well as male TNFR 1/2.sup.0/0 C57BL/6 mice deficient in TNF
receptors (Bruce et al., 1996 ) provided by H. Bluethmann, F.
Hoffmann-La Roche, Basel, Switzerland, were used at the age of 8-10
weeks. Their care was in accordance with institutional guidelines.
Lung microvascular endothelial cells were isolated from CBA/J mice
and characterized as described (Jackson et al., 1990) using
magnetic beads (Dynabeads M-450, Dynal, Oslo, Norway), covalently
bound to a purified rat-anti-mouse PECAM-1 monoclonal antibody
(donated by B. Imhof, University of Geneva). Microvascular lung
endothelial cells were resuspended in DMEM containing 2 mM
L-glutamine, 100 U/ml penicillin, 10 mg/ml streptomycin, 20% FCS,
40 U/ml heparin and 100 mg/ml endothelial cell growth supplement
(Brunschwig Chemie, Basel, Switzerland). For patch clamp
experiments, cells were plated onto 35.times.10 mm easy grip Petri
dishes (Beckton Dickinson, Plymouth, UK), pre-coated with 0.2%
gelatin (Sigma, Buchs, Switzerland). Resident peritoneal
macrophages, isolated in ice cold RPMI containing antibiotics and
10 U/ml Heparin, were left to adhere onto 35.times.10 mm easy grip
Petri dishes for 4 h, after which the non-adherent cells were
removed. Cells were grown in RPMI 1640 containing 2 mM L-glutamine,
100 U/ml penicillin, 10 .mu.g/ml streptomycin and 10% fetal bovine
serum (all from Gibco). For patch clamp, the macrophages were used
24 h after isolation.
[0026] TNF and peptides. E.coli-derived recombinant murine TNF
(further referred as TNF in the text) and an E.coli-derived
recombinant (T104A-E106A-E109A) triple TNF mutant (mutTNF) were
synthesized as described elsewhere (Lucas et al., 1997).
TNF-derived peptides were synthesized with the use of Fmoc-a-amino
group protection (Fields et al. 1990), and purified with a C18
reversed-phase high-performance liquid chromatography column.
[0027] The following TNF-derived peptides were synthesized:
1 Long tip peptide 99-115 (LTip) GG-CGPKDTPEGAELKPWYC (SEQ ID NO 6)
Mutated tip peptide 99-115 (mut- GG-CGPKDAPAGAALKPWYC (SEQ ID NO 7)
Tip) Scrambled tip peptide (scamblTip) GG-CGTKPWELGPDEKPAYC (SEQ ID
NO 8) Short tip peptide (STip) CTPEGAEC (SEQ ID NO 9)
[0028] To theoretically retain the original TNF conformation as
much as possible, Ltip, MutTip and ScamblTip peptides were
circularized. Ser.sup.99 of the TNF sequence was replaced by Cys,
and Cys.sup.100 by Gly so that the disulfide bridge could be formed
between Cys.sup.99 and Cys.sup.115 in the peptides. The STip
peptide could not be circularized. The peptides were
NH.sub.2-biotinylated.
[0029] Electrophysiology. Cells were pretreated for 30 min with
TNF, mutTNF and tip peptides at 37.degree. C. in a buffer
consisting of 145 mM NaCl, 3 mM KCl, 2 mM CaCl.sub.2, 2 mM
MgCl.sub.2, 10 mM D-glucose, and 10 mM Hepes, and pH-adjusted with
NaOH to required value. Cells were then washed with the same buffer
pH-adjusted at 7.3, and experiments were performed using the
tight-seal, whole-cell recording technique. Currents were recorded
with an Axopatch-200A amplifier (Axon Instrument Inc, Foster City,
Calif. USA), low pass-filtered at 1 kHz. Digitalization and
off-line analysis was performed using the WCP program (J. Dempster,
Strathclyde Electrophysiology Software, Glasgow, UK). Patch
pipettes were pulled from borosilicate glass and fire polished to
have an open resistance of 3-5 MW with an internal solution
containing 130 mM CsCl, 2 m I MgCl.sub.2, 10 mM EGTA, 20 mM TEA-Cl,
10 mM D-glucose, 10 mM Hepes, pH-adjusted to 7.3 with CsOH. Series
resistances were kept under 10 MW. Capacitance and series
resistance compensation were applied and set to 70%. All
experiments were done at room temperature. Results are given as
mean.+-.SEM, unless otherwise indicated. Analysis of variance was
performed on currents and membrane conductance values, with
post-hoc Dunn-Bonferroni test for significance of differences
observed between two groups. A P value of 0.05 was considered
significant.
[0030] Tryptophan fluorescence. Fluorescence measurements were made
with a PTI spectrofluorimeter. The excitation wavelength was 295 nm
and slit widths were 5 nm and 2.5 nm for excitation and emission
respectively. For each recorded spectrum, the Raman scatter
contribution was removed by subtraction of a buffer blank. All
buffers contained 150 mM NaCl, and 20 mM of N-[2-morpholino]
ethane-sulfonic acid (MES) buffer at the desired pH. The samples
were allowed to incubate for 1 h 30 at the desired pH before
measuring the emission spectrum. The wild type and mutant TNF
concentrations were 6 .mu.g/ml.
[0031] Preparation of liposomes. Large unilamellar liposomes were
prepared by reverse phase evaporation as previsouly described
(Vecsey-Semjen et al, 1996). Liposomes were prepared of either 100%
egg phosphatidylglycerol (EPG) or a mixture of EPC and EPG (1:1
W/W) in a buffer containing 100 mM KCl, 20 mM N-[2-Hydroxyethyl]
piperazine-N'-[2-ethane-sulfonic acid] (HEPES), pH 7.4 and 1.5
mg/ml of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ).
[0032] Choride efflux measurements. All fluorescence experiments
were carried out using a PTI spectrofluorometer equipped with a
thermostated cell holder (37.degree. C.). The dye was excited at
350 nm and emission was recorded at 422 nm, both excitation and
emission band widths were set to 5 nm. Liposomes were diluted to a
final concentration of 50 .mu.g/ml in a solution containing 100 mM
KNO.sub.3 and 20 mM MES pH 6.1 or 20 mM HEPES, pH 7.4. Wild type
and mutant TNF were added to a final concentration of 3
.mu.g/ml.
[0033] Proinflammatory activity of TNFs and TNF tip peptides.
Proinflammatory activity of TNF and derived peptides was tested
using a bioassay measuring their capacity to induce the surface
upregulation of intercellular adhesion molecule (ICAM)-1 in
alveolar type II-like epithelial A549 (Pugin et al, 1996). Briefly,
A549 cells were plated at confluence in a microtiter plate, and
incubated with the various concentrations of TNF, mutTNF, and
peptides for 18 hrs at 37.degree. C. Surface upregulation of ICAM-1
was detected by direct ELISA on cells using a first anti-ICAM-1
antibody (R&D systems, Abdington, UK), a second donkey-anti
mouse IgG-peroxidase conjugated antibody (Jackson), revealed by
o-phenylenediamine (Sigma), and stopped by H.sub.2SO.sub.4. Optical
densities (O.D.) were read at 490 nm, with subtraction of 620 nm
O.D. readings.
RESULTS
Example 1.1
Effect of TNF on Membrane Conductance in Murine Cells
[0034] We first investigated whether TNF modified the whole cell
current in primary murine cells. A 30 min preincubation of resident
peritoneal macrophages and lung microvascular endothelial cells
with 100 ng/ml of TNF resulted in a significant increase in outward
and, to a lesser extent, inward current in the case of
microvascular endothelial cells, as measured by means of whole-cell
patch clamp, as compared to cells unexposed to TNF (endothelial
cells, FIG. 1A; and macrophages, FIG. 2). A reduction in
preincubation time (down to 5 min) or in dose of TNF (down to 10
ng/ml) gave similar results (data not shown). This effect required
acidic preincubation conditions, since it did not occur when the
preincubation was performed at pH 7.3 (FIG. 1). The conductance
induced by TNF was voltage-independent and showed a reversal
potential of about 0 mV in the case of endothelial cells. In order
to investigate whether the ion current increase induced by TNF was
TNF receptor-dependent, resident peritoneal macrophages were
isolated from mice deficient in both TNF receptor-1 and -2
(TNFR1/2.sup.0/0), and tested in the whole cell patch clamp assay.
TNF induced a voltage-dependent current in cells lacking TNF
receptors (FIG. 2B). This critical experiment showed that the
TNF-induced conductance in mammalian cells occurred in a
TNT-receptor independent manner. These results also indicated that
the TNF-induced current is not cell type specific.
[0035] Since the lectin-like domain of TNF is spatially and
functionally distinct from its receptor binding sites, we next
investigated whether it was implicated in the observed ion channel
activating effect of TNF in mammalian cells. Therefore, the effect
of a TNF mutant (mutTNF), in which the three critical residues for
the lectin-like activity of TNF were replaced by an alanin, was
compared with TNF in endothelial cells. As shown in FIG. 3, mutTNF
completely lacked the conductance activating effect of TNF, even at
a 100-fold higher dose (1 .mu.g/ml mutTNF versus 10 ng/ml of TNF,
data not shown). In contrast, the native and the mutated TNF
molecules showed similar potencies in the induction of ICAM-1 in
A549 epithelial cells (FIG. 4). This indicated that despite a
conserved TNF receptor-mediated activity, mutTNF was unable to
increase ion permeability. In order to test the hypothesis that TNF
gated a sodium channel, we performed additional experiments in the
presence of amiloride, an epithelial sodium channel blocker. One
hundred .mu.M amiloride added during the pretreatment at pH 6.0
abrogated the TNF-induced increase in conductance FIG. 3).
Example 1.2
The Tip Domain of TNF Mediates its Membrane Conductance Increasing
Effect
[0036] Since the tip domain of TNF seemed to be critical for its
activation of ion permeability, we next tested whether a peptide
mimicking this region was sufficient for increasing membrane
conductance, as observed with native TNF. Treatment of endothelial
cells and macrophages with the 17 amino acid (aa) circularized long
tip peptide (Ltip peptide), that mimics the lectin-like domain of
TNF, resulted at acidic pH in increased outward, and inward
currents in the case of microvascular endothelial cells. In
contrast to TNF, the effect persisted at neutral pH, although less
pronounced (FIGS. 2A and 4A). Similarly to TNF, the effect was
blocked by 100 .mu.M amiloride (FIG. 4B). A mutant
(T104A-E106A-E109A) 17 aa circularized peptide (mutTip peptide) and
a 17 aa circularized peptide containing the same aa as Ltip peptide
in a random sequence (scramblTip peptide) were inactive with regard
to the ion channel activity (FIG. 4B). These results indicated that
the tip domain of TNF was mediating its membrane conductance
increasing activity, and confirmed that residues T104, E106 and
E109 were essential for this effect. Ltip peptide was also active
in cells deficient in both TNFR-1 and -2 receptors (FIG. 2B).
However, a short tip hexapeptide containing the 3 critical aa
failed to induce a voltage-dependent current in microvascular
endothelial cells (data not shown), suggesting that this peptide
was below the minimal structure carrying the ion channel effect.
Importantly, none of the peptides induced ICAM-1 in A549 cells,
indicating that they lacked a TNF receptor-mediated activity.
Example 1.3
Native and Mutated TNF Undergo Partial Unfolding at Acidic pH
[0037] It was previously shown that TNF interacted with lipids in a
pH dependent manner and that this membrane interaction correlated
with partial unfolding of the protein (Hlodan et al., 1994)
(Baldwin et al., (1996). We therefore investigated whether the lack
of activity of mutTNF on lung MVEC at acidic pH was due to its
inability to undergo partial unfolding and to interact with
membranes. The conformation of mutTNF at various pH values was
followed by measuring the intrinsic tryptophan fluorescence of the
molecule. The fluorescence intensity dropped upon acidification of
the medium, and the maximum emission underwent a red shift from 318
nm at pH 6 to 339 nm at pH 4.6. These observations indicated that
the initially buried tryptophan residues became exposed to the
solvent. The protein was however not fully unfolded since the
spectrum at pH 4.6 was not as red shifted as that of mutTNF in 6 M
GuHCl. These results show that mutTNF was able to undergo acidic
unfolding. Acidic unfolding of mutTNF was in fact more rapid and
slightly more extended than that of wild type TNF.
Example 1.4
Both Native and Mutated TNF Interact with Membranes at Acidic
pH
[0038] We next investigated whether mutTNF was able to interact
with membranes at acidic pH by following its ability to induce
chloride leakage from liposomes containing the chloride sensitive
dye SPQ. These experiments were performed using liposomes
containing 100% egg phosphatidyl glycerol (EPG). Native TNF induced
chloride efflux at pH 6.1. MutTNF was still folded at pH 6; we have
however previously shown that the pH at the surface of 100% EPG
vesicles was far lower than that of the bulk pH, and more
specifically that at a bulk pH of 6, the surface pH was 4.35.
Therefore, mutTNF is likely to have undergone partial unfolding at
the surface of the EPG vesicles. The effect of mutTNF on SPQ
fluorescence was even more pronounced than that of wild type TNF,
in agreement with the fact that its acidic unfolding was more rapid
than that of wild type TNF. As previously observed for native TNF
(Baldwin et al., 1996), mutTNF did not interact with membranes at
neutral pH.
[0039] In order to investigate whether chloride efflux was due to
membrane binding or membrane insertion of TNF, we have analyzed
whether brominated lipids were able to quench the intrinsic
fluorescence of TNF and mutTNF upon membrane interaction.
Brominated lipids have been useful in determining the topology of
membrane proteins (Bolen et al., 1990) (Markello et al., 1985) as
well as studying the membrane interaction of pore-forming toxins
(Gonzalez-Manas et al., 1992) (Van der Goot et al., 1991)
(Vecsey-Semjen et al., 1997). TNF contains two tryptophan residues,
one at the top of the receptor binding domain and one at the top of
the so called tip domain. If the tip of the TNF trimer were to
insert into the lipid bilayer, the fluorescence of Trp-113 should
be quenched upon insertion into liposomes composed of
dioleoylphosphatidylglycerol that had bromines attached at
positions 9 and 10 of the acyl chains. We have indeed previously
observed that tryptophans located at the boundary between the lipid
head groups and the acyl chains were succeptible to bromide
quenching. We were however unable to see any fluorescence quenching
when adding either TNF or mutTNF at acidic pH to vesicles formed of
brominated lipids.
[0040] The observations described above show that mutTNF undergoes
partial unfolding at acidic pH and is then able to interact with
membranes. The lack of quenching by brominated lipids however
2 Long tip peptide 99-115 (LTip) GG-CGPKDTPEGAELKPWYC Mutated tip
peptide 99-115 (mut- GG-CGPKDAPAGAALKPWYC Tip) Scrambled tip
peptide (scrablTip) GG-CCTKPWELGPDEKPAYC Short tip peptide (STip)
CTPEGAEC
[0041] suggests that chloride release was due to binding of the
partially unfolded TNF molecules to the lipid bilayer rather then
to membrane insertion of the molecule.
[0042] We next tested whether the TNF tip peptides were able to
induce chloride efflux from SPQ containing vesicles and whether
tryptophan quenching could be observed upon interaction with
bromninated lipids. Liposomes containing either 100% neutral
lipids, 100% acidic lipids or a 1:1 mixture of both were used. For
none of the lipid compositions and for peptide concentrations up to
300 .mu.g/ml could we observe any change in SPQ fluorescence nor
any quenching by brominated lipids, and this with all 4 peptides.
These experiments suggested that the LTip as well as the modified
tip peptides were unable to interact with membranes.
Example 2
[0043] Isolated Lung Perfusion Experiments
[0044] Lungs of female Whistar rats weighing about 300 g were
isolated as described in DeCampos et al. (1993). The lungs were
injected intratracheally with either 500 .mu.l of sterile 9% NaCl,
wild type murine TNF (1 .mu.g/lung) or mTNF tip peptide (Ltip, see
above; 1 mg/lung). Subsequently, the lungs were perfused with blood
isolated from the same rat. Thirty minutes later, the lungs were
injected intratracheally with 2 ml of sterile 9% NaCl solution
which leads to a weight increase of about 2 g (FIG. 5). The weight
evolution was then followed continuously for 150 min (FIG. 5).
[0045] The weight of control lungs (pretreated with NaCl) did not
decrease with time whereas, in contrast, the lungs that had been
pretreated with either wt TNF or tip peptide showed a significant
decrease of weight of 25 to 50% after 150 min (FIG. 5 & 6)
which corresponds with a diminished presence of hydrostatic oedema.
In the case of the TNF tip peptide, the weight loss started
immediately upon injection of the 2 ml of Na Cl solution (FIG.
5).
[0046] These experiments demonstrate that the tip peptide of mTNF,
like the wild type molecule, can lead to oedema resorption.
However, the tip peptide, in contrast to wt mTNF, does not interact
with the TNF receptors and does not lead to an increased expression
of adhesion molecules in lung endothelial- and epithelial cells.
Consequently, the tip peptide induces less lung toxicity if
compared to wt mTNF.
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Sequence CWU 1
1
9 1 14 PRT Homo sapiens 1 Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala
Lys Pro Trp Tyr 1 5 10 2 14 PRT Mus musculus 2 Pro Lys Asp Thr Pro
Glu Gly Ala Glu Leu Lys Pro Trp Tyr 1 5 10 3 6 PRT Homo sapiens 3
Thr Pro Glu Gly Ala Glu 1 5 4 17 PRT Homo sapiens 4 Cys Gly Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr 1 5 10 15 Cys 5 17
PRT Mus musculus 5 Cys Gly Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu
Lys Pro Trp Tyr 1 5 10 15 Cys 6 19 PRT Mus musculus 6 Gly Gly Cys
Gly Pro Lys Asp Thr Pro Glu Gly Ala Glu Leu Lys Pro 1 5 10 15 Trp
Tyr Cys 7 19 PRT Mus musculus 7 Gly Gly Cys Gly Pro Lys Asp Ala Pro
Ala Gly Ala Ala Leu Lys Pro 1 5 10 15 Trp Tyr Cys 8 19 PRT Mus
musculus 8 Gly Gly Cys Gly Thr Lys Pro Trp Glu Leu Gly Pro Asp Glu
Lys Pro 1 5 10 15 Ala Tyr Cys 9 8 PRT Mus musculus 9 Cys Thr Pro
Glu Gly Ala Glu Cys 1 5
* * * * *