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WO2006008775A2 - Methode d'identification d'agents modulant le metabolisme de molecules steroides et agents identifies au moyen de ladite methode - Google Patents

Methode d'identification d'agents modulant le metabolisme de molecules steroides et agents identifies au moyen de ladite methode Download PDF

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WO2006008775A2
WO2006008775A2 PCT/IT2005/000403 IT2005000403W WO2006008775A2 WO 2006008775 A2 WO2006008775 A2 WO 2006008775A2 IT 2005000403 W IT2005000403 W IT 2005000403W WO 2006008775 A2 WO2006008775 A2 WO 2006008775A2
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hpt
agent
peptide
apo
binding
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WO2006008775A3 (fr
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Luca D'andrea
Carlo Pedone
Paolo Abrescia
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ISTITUTO BIOCHIMICO NAZIONALE SAVIO Srl
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ISTITUTO BIOCHIMICO NAZIONALE SAVIO Srl
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the invention relates to a method for detecting/identifying agents/factors modulating the metabolism of steroid molecules.
  • the invention relates to a method for identifying and isolating molecules able to bind haptoglobin (Hpt), without altering its capability of binding haemoglobin, thus avoiding that Hpt binds to HDL, and inhibits the enzyme Lecithin-Cholesterol Acyl Transferase.
  • Hpt haptoglobin
  • Such molecules are candidates as agents for prevention and therapy of cardiovascular diseases, ovarian follicular dysfunction and, in general, pathologies associated to synthesis, metabolism, and transport of steroids.
  • Hpt is an oligomeric plasma protein, which is involved in cardiovascular pathologies, inflammation process, inhibition of prostaglandin synthesis, and immunosuppression. Background
  • Apolipoprotein A-I is a component of the high-density lipoprotein (HDL), which plays a key role in the traffic of cholesterol between liver and peripheral cells. All the cells are supplied with cholesterol (C) and cholesteryl esters (CE) by endocytosis of another major class of lipoproteins, namely the low-density lipoproteins (LDL). On the contrary, some cell types (steroidogenic cells) use receptors to bind just Apo A-I for allowing the transfer of cholesteryl esters from HDL [1-4].
  • Apo A-I is required for normal steroid production in adrenocortical cells [5], and HDL receptors are regulated by C level in luteal cells [6]. Apo A-I is also required for removal of cholesterol excess from the plasma membrane [7, 8], an anti-atherogenic process called "reverse cholesterol transport" [9, 10], which prevents alteration of the membrane properties, and cell death [7, 10-12]. In particular, Apo A-I stimulates the efflux of cholesterol from cell toward HDL [13-16], and the enzyme LCAT (EC 2.3.1.43) to convert, on the HDL surface, cell-derived C into CE, which is then placed into the lipoprotein core and transported through blood circulation to liver for catabolism and bile production [9, 10].
  • LCAT enzyme
  • the ratio of CE with free C in HDL is therefore assumed to reflect the LCAT activity in vivo [17, 18].
  • Mutations in the Apo A-I structure have been reported to be associated with low HDL C tot or CE, and decreased stimulation of the enzyme activity [18].
  • Apo A-I can bind Hpt in blood [19, 20] and follicular fluid [21].
  • Hpt is a plasma oligomeric glycoprotein exhibiting enhanced levels during the acute phase of inflammation, and presenting in humans three distinct phenotypes (determined by genetic polymorphism) with different prevalence in several diseases, including cardiovascular diseases [22]. This binding might influence the role of HDL in the C transport.
  • Hpt inhibits the Apo A-I-dependent LCAT activity in vitro [23], and is associated with low reverse C transport in human ovarian follicular fluid [24]. Also estradiol esterification in the follicle, and ester delivery through HDL-mediated circulation to storage tissue [25, 26] for long-acting hormonal and antioxidant function [27, 28], might be influenced by defective reverse C transport and/or reduced LCAT activity [24]. Hpt is also able of capturing and transporting to the liver free haemoglobin (Hb), in the pathway of iron recycling for erythropoiesis [29].
  • Hb liver free haemoglobin
  • Hb competes with Apo A-I for binding Hpt, although Hb interacts with a Hpt site which is different from that involved in the Apo A-I binding [30].
  • the inhibitory role of Hpt in the regulation of the HDL- dependent removal of C excess from peripheral cells is of great interest in studies on diseases associated with C accumulation.
  • Hpt-dependent masking of the Apo A-I site involved in the LCAT stimulation might be responsible of decreased enzyme activity [23, 30].
  • competition of Hpt with LCAT for the same Apo A-I region might explain the inhibitory role of Hpt on the enzyme activity, and suggests that high Hpt levels, as present in the acute phase of inflammation, play an important role in worsening vascular endothelial dysfunction, and accelerating atherosclerosis.
  • the Authors of the present invention have identified a region of Apo A-I which binds Hpt, by using techniques of protein chemical fragmentation and peptide synthesis, and demonstrated that peptides identical to sequences from this region are able to compete with Apo A-I for the Hpt binding. These peptides are effective in recovering the LCAT activity in the presence of Hpt, and do not interfere with the binding of Hpt to Hb. Therefore, these peptides are an example of molecules which can be used in therapy of diseases associated with decreased reverse C transport, such as cardiovascular diseases, in treatment of altered estradiol function, and improvement of inflammation.
  • Object of the present invention is a method to identify an agent which can modulate the reverse cholesterol transport, or the metabolism of steroid molecules.
  • This method includes the following phases: a) incubating the above defined agent with Hpt, in conditions allowing the formation of a bond between the two molecules, so that a complex "agent-Hpt" can be formed; b) verifying that this complex agent-Hpt is able to bind Hb, with a binding constant essentially comparable to that of free Hpt; c) verifying that the complex agent-Hpt essentially inhibits the binding of complexed Hpt (i.e. agent-linked Hpt) to HDL; d) verifying that, in the complex agent-Hpt, the inhibitory activity of complexed Hpt on the enzyme LCAT is essentially impaired.
  • the part c) is performed by ELISA, but a person skilled in laboratory techniques will appreciate other assay procedures such as, for example, EIA, RIA, calorimetry, Surface Plasmon Resonance, equilibrium dialysis, etc, can be used.
  • the part d) is performed by means of an esterification assay, but a person skilled in the art will appreciate other techniques, such as saponification assay, calorimetry, etc, can be used.
  • the agent modulating the reverse cholesterol transport or the metabolism of steroid molecules, as it is obtained and identified using the method described in the present invention, hi a preferred embodiment , the agent is a peptide, preferably including the amino acid sequence of human Apo A-I, more preferably including at least the amino acid sequence of human Apo A-I from Leul41 to Alal64, most preferably including the amino acid sequence of human ApoA-I from Glul33 to Asnl84.
  • the above mentioned sequences could account, in the peptide structure, even for less than 50% of the total number of amino acid residues.
  • Other protecting group include (but are not limited to) acetyl (Ac), amide, alkyl groups containing 3 to 20 carbon atoms, Fmoc, t-Boc, 9- fluoreneacetyl, 1-fluorenecarboxylic, 9-fluorenecarboxylic, 9-fluorenone-l-carboxylic, benzyloxycarbonyl, Xanthyl (Xan), trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzesulphonyl (Mtr), mesytilene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6- sulphonyl (Pmc), 4-methylbenzyl (MeBzI), 4-
  • the peptides/peptidomimetics (agents) of this invention are typically conjugated with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can contain one or more physiologically acceptable compounds that act, for example, to reduce the clearance or the hydrolysis of the agents, in order to increase or decrease their absorption.
  • physiologically acceptable compounds include, for example, carbohydrates such as dextrans, and plasma proteins such as albumin.
  • Peptides can be used as monomers, dimers, oligomers or polymers.
  • the multimers can include monomers which are associated by means of non covalent forces (such as ionic or hydrophobic interactions) or by covalent linkages (for example, monomers co valently jointed by a linker, branched peptides).
  • Peptides of the present invention can also be peptidomimetic molecule.
  • the agents of the present invention can be used as agents for prevention and therapy of cardiovascular diseases, ovarian follicular dysfunction and, in general, pathologies associated to synthesis, metabolism, and transport of steroids. Description of the figures
  • FIG. 1 Fragmentation of Apo A-I.
  • the amino acid sequence of Apo A-I is reported, and the predicted sites of fragmentation by BrCN or hydroxylamine (HA), Met-X and Asn-Gly respectively, are indicated n (Seq. Id.l).
  • the segments represent Apo A-I and all the peptides which may originate from its fragmentation.
  • the upper numbers indicate the localization of Met sites.
  • the lower number indicates the localization, in the amino acid sequence, of the Asn residue which is involved in the cleavage by HA.
  • Apo A-I was fragmented by CNBr or HA, and the reaction products were fractionated by SDS-PAGE on 20 % or 16.5% polyacrylamide gel respectively.
  • the fragments from CNBr digestion were stained by Coomassie (lane b) or, after blotting onto PVDF membrane and incubation with 0.1 mg/ml Hpt, by rabbit "anti-Hpt IgG” and goat “anti-rabbit IgG linked to horseradish peroxidase" (GAR-HRP) (lane a).
  • Hpt-coated wells were used to bind fragments, which were detected by anti-Apo A-I IgG (solid circles). The molecular weight of the fragments was assessed on the basis of their elution volume, using standard proteins in a calibration chromatography (myoglobin: 17 kd; insulin trimer:16 kd; cytochrome C: 12.3 kd; insulin dimer:10.7 kd; insulin:5.3 kd; glucagone: 3.5 kd).
  • Figure 4 Binding of synthetic peptides to Hpt. Biotinylated peptides, sharing parts of the Apo A-I sequence putatively involved in the Hpt binding (aa 113-184), were synthesised.
  • the aminoacid sequences of Apo A-I matched by the different peptides, are indicated in parentheses.
  • Four solutions of each peptide were prepared (1, 3, 10, and 30 ⁇ M), and samples from each solution were incubated in Hpt-coated wells of a microplate.
  • Avidin conjugated with horseradish peroxidase, was used to detect bound peptides.
  • the samples were analysed in triplicate: the data are expressed as means ⁇ SEM.
  • Figure 5 Competition of the peptide P2a with HDL for binding Hpt.
  • the acetylated form of P2a (matching part of Apo A-I sequence, i.e. aa 141-164) was analysed for its ability to interfere with the Hpt binding to HDL.
  • FIG. 6 Competition of peptide P2a with Hb for binding Hpt.
  • the acetylated form of P2a (aa 141-164 of Apo A-I) was assayed for its ability to compete with Hb for the Hpt binding.
  • Aliquots (50 ⁇ l) of Hpt (1 ⁇ M), previously incubated with different amounts of P2a (3, 5, 10, or 20 ⁇ M) (solid circles) or with 3 ⁇ M Apo A-I (open square) were loaded onto Hb-coated wells of a microtiter plate.
  • Hpt binding to Hb was detected using rabbit anti-Hpt IgG and GAR-P IgG, and monitoring the colour development at 405 nm.
  • the samples were analysed in triplicate. The data are reported as percent of the value obtained by incubation of Hpt alone (open circle), and expressed as means ⁇ SEM.
  • FIG. 7 Effect of peptide P2a on Hpt inhibition in the LCAT assay.
  • the LCAT activity was assayed by incubating a pool of Dextran Sulfate (DS)-treated plasma with a standard reaction mixture, containing a proteoliposome (Apo A-I : lecithin : 3 H- cholesterol, 1.5 : 200 : 18 molar ratio) as substrate.
  • the enzyme activity was measured in the presence of Hpt (0.3 ⁇ M), P2a (0.9 ⁇ M), or both.
  • a control assay was performed without Hpt and P2a.
  • the LCAT activity was expressed as nmoles of C incorporated per hour per ml of plasma. The samples were analysed in triplicate: the data are expressed as means ⁇ SEM.
  • Bovine serum albumin BSA
  • human serum albumin HSA
  • C cholesteryl linoleate
  • human Hpt mixed phenotypes: Hpt 1-1, Hpt 1-2, Hpt 2-2
  • goat anti-rabbit phosphatase-conjugated GA-P
  • p- nitrophenylphosphate p- nitrophenylphosphate
  • o-phenylenediamine horseradish peroxidase-conjugated Avidin, (CNBr)
  • HA horseradish peroxidase-conjugated Avidin
  • Human Apo A-I and rabbit anti-human Apo A-I IgG were from Calbiochem (La Jolla, CA, USA). [l ⁇ ,2 ⁇ - 3 H] Cholesterol (45 Ci/mmol) was obtained from Perkin Elmer (Boston, MA, USA). Organic solvents were purchased from Romil (Cambridge, UK). Polystyrene 96-wells plates were purchased from Nunc (Roskilde, Denmark).
  • the gels were fixed in aqueous solution containing 10% acetic acid and 25% isopropanol, stained with Coomassie R-250 (0.05% in the fixing solution) and destained by shaking the gel in 10% acetic acid. Fixing and staining were omitted when the gel was processed for immunoblotting.
  • the membrane was incubated with Hpt (0.1 mg/ml in T-TBS) overnight at 4°C. After treatment, the membrane was rinsed in T-TBS and, then, incubated in the same buffer, containing rabbit anti-Hpt IgG (1:100 dilution of the commercial stock solution) for 1 h at 37°C. Similarly, after washing in T-TBS, the membrane was incubated with
  • GAR-HRP IgG (1:300 dilution of commercial stock solution) for 1 h at 37°C.
  • the membrane was rinsed again in T-TBS and twice in TBS, and finally treated with TBS containing 0.03% H 2 O 2 and 4 mM 4-chloro-l-naphtol for detecting the immunocomplexes.
  • Peptides were synthesised by solid phase using Fmoc chemistry [34]. The synthesis was performed in a model 348 ⁇ Advanced Chemtech multiple peptide synthesizer using a PAL-PEG-PS resin (Perseptive Biosystem, Hamburg, Germany), which releases the peptide carboxy-terminal end in amide form. Typically, 8 equivalents of a Fmoc amino acid, 7-9 equivalents of HBTU/HOBt, and 16 equivalents of diisopropylethylamine in N-methylpyrrolidone were added to the resin, and the coupling reaction was allowed to proceed for 20 min.
  • ELISA Microtiter plate wells were incubated (overnight, 4°C) with 45 ⁇ l from separated chromatography fractions, or 0.5 ⁇ g of antigen (Hpt, HDL, or Apo A-I) in 50 ⁇ l of 7 niM Na 2 CO 3 , 17 mM NaHCO 3 , 1.5 mM NaN 3 (pH 9.6). Unattached material was removed by two washes with T-TBS buffer, followed by two washes with 0.5 M NaCl, 20 mM Tris-HCl, pH 7.3. Remaining plastic reactive sites were blocked by treatment with 0.5% BSA in TBS (2 h, 37°C).
  • the wells were washed as above, and incubated with 55 ⁇ l of primary antibody, or with 45 ⁇ l of Apo A-I cleavage products (as separated by Sephadex G-50F), or with 55 ⁇ l of biotinylated peptide (1, 3, 10, 30 ⁇ M in T-TBS supplemented with 0.25% BSA).
  • Anti-Hpt IgG (1:1500 working dilution) or anti-ApoAl IgG (1:1000 working dilution) was used as primary antibody.
  • Bound immunocomplexes or peptides were incubated (1 h at 37°C) with 60 ⁇ l of GAR- HRP IgG or HRP-Avidin diluted, as primary antibody, 1:3000 and 1:10000, respectively. Colour development was monitored at 492 nm as previously described [21].
  • CB-TBS buffer (5 mM CaCl 2 , 0.2% BSA, 130 mM NaCl, 20 niM Tris-HCl, pH 7.3) was kept for 2 h at 37°C and, then, incubated in the protein-coated wells (2 h, 37 °C).
  • Hpt subunits were fractionated by electrophoresis, the Coomassie-stained bands were analyzed by densitometry, and the ratio of the subunit D 1 (present in Hpt 1-1 and Hpt 1-2) with the subunit ⁇ 2 (present in Hpt 1-2 and Hpt 2-2) was determined to calculate their molar contribution to the amount of Hpt isoforms [35].
  • the binding of Hpt was detected by anti-Hpt IgG and GAR-HRP IgG, as above described.
  • a pool of plasma samples, treated 0.08% with DS (MW 50 kd) in 0.16 M CaCl 2 to remove very low density lipoprotein and LDL, was used as source of LCAT (DS-treated plasma).
  • 8 ⁇ l of 50 mg/ml egg lecithin in ethanol were mixed with 18 ⁇ l of 1 mg/ml C in ethanol, 40 ⁇ l of [1,2- 3 H(N)]-C (1 ⁇ Ci/ml) into a glass vial.
  • lipids 170 ⁇ l of a suspension medium (85 mM sodium cholate, 150 mM NaCl, 10 mM Tris-HCl, pH 8) were added. After vigorous whirling (3 min, room temperature), the micelle suspension was incubated (90 min, 37°C) and repeatedly shaken every 10 min until clear. Then, 90 ⁇ l of 1.21 mg/ml Apo A-I were added to the lipid suspension, which was further incubated for 1 h at 37° C.
  • a suspension medium 85 mM sodium cholate, 150 mM NaCl, 10 mM Tris-HCl, pH 8) were added. After vigorous whirling (3 min, room temperature), the micelle suspension was incubated (90 min, 37°C) and repeatedly shaken every 10 min until clear. Then, 90 ⁇ l of 1.21 mg/ml Apo A-I were added to the lipid suspension, which was further incubated for 1 h at 37° C.
  • the resulting proteoliposome suspension was extensively dialysed against TBE (140 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.3), at 4 °C, to remove cholate.
  • the dialysate volume was adjusted to 285 ⁇ l using TBE.
  • the reaction mixture (1 ml final volume) was prepared by putting together 697 ⁇ l of TBE containing 5 mM CaCl 2 , 83 ⁇ l of 6% HSA, and 8 ⁇ l of proteoliposome suspension (diluted 1 :20 in TBE) into a screw- capped tube, and heating at 38 0 C for 30 min.
  • the assay was carried out by addition of 2.5 ⁇ l of 2 mM ⁇ -mercaptoethanol and 3.5 ⁇ l of DS-treated plasma to 100 ⁇ l of reaction mixture, which was rapidly divided into three aliquots of 32 ⁇ l and incubated (1 h, 37°C). The reaction was stopped by addition of 130 ⁇ l of ethanol to each aliquot.
  • the lipids were extracted in 600 ⁇ l of hexane, containing 10 ⁇ g/ml C and 10 ⁇ g/ml cholesteryl linoleate. After recovering the organic phase, the aqueous phase was again treated with 500 ⁇ l of the extraction solution (twice), and the three extracts were pooled.
  • ELISA was carried out with single aliquots from chromatography fractions, while at least three replicates were processed in the other cases. Samples in the LCAT assay were analysed in triplicate. The program "Graph Pad Prism 3" (Graph Pad Software, San Diego, CA, USA) was used to obtain trend curves, perform regression analysis or t- test.
  • the apolipoprotein was fragmented with CNBr, and the resulting peptides were analysed by Western blotting for their binding to Hpt.
  • Four peptides are predicted to result from the Apo A-I fragmentation by CNBr (Fig. 1), but more molecular species including the undigested protein were observed by SDS-PAGE (Fig. 2; lane b).
  • the native form of the protein was detected by anti-Apo A-I IgG together with the expected peptides, namely HA-I and the C-terminal fragment (HA-2: aa 185-243, 6400 kd) (Fig. 3; panel B).
  • the binding of HA-I to Hpt was comparable to that of Apo A-I, while complexes of HA-2 with Hpt were not found (Fig. 3; panel B).
  • the analysis of the Apo A-I digestion by HA indicated that the sequence from GIy 185 to GIn 243 is not required for the binding of Apo A-I to Hpt.
  • the peptide P2a was analysed for its ability to interfere with the Hpt binding to HDL in vitro.
  • the acetylated form of the peptide was used.
  • HDL-coated wells of a microplate were incubated with Hpt in the absence or presence of different amounts of P2a.
  • Use of a mixture of Hpt and Apo A-I (molar ratio 1) served as control.
  • the Hpt binding was analysed by anti-Hpt IgG and GAR-HRP IgG.
  • the peptide was able to displace over 78% of Hpt from binding HDL (Fig. 5), that is a value comparable with that produced by Apo A-I. This result confirms previous data on the Hpt property to interact with both free and lipid-embedded Apo A-I [19-21, 30], and demonstrates that the Apo A-I sequence in P2a can effectively prevent the Hpt binding to HDL.
  • Hpt in vitro Hpt was incubated with a 3-fold molar excess of P2a or Apo A-I in Hb- coated wells of a microtiter plate. Incubation of Hpt alone was used as control.
  • the property of the peptide P2a to compete with native Apo A-I for binding Hpt was tested also in the assay of LCAT activity.
  • a standard mixture containing Apo A-I embedded in micelles of phospholipids and 3 H-labelled cholesterol, was incubated with DS-treated plasma (as LCAT source) and 0.3 ⁇ M Hpt, in the absence or presence of 0.9 ⁇ M P2a.
  • the enzyme activity was inhibited by Hpt, but fully restored when also the peptide was present in the reaction mixture (Fig. 7).
  • the peptide, when incubated without Hpt did not significantly increase the C esterification.
  • Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells. Proc Natl Acad Sci USA 94:13600-13605. 5. Plump AS, Erickson SK, Weng W, Partin JS, Breslow JL, Williams DL. 1996. Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for adrenal steroid production. J Clin Invest 97: 2660-2671.
  • Haptoglobin inhibits lecithin-cholesterol acyltransferase in human ovarian follicular fluid. MoI. Reprod. Dev., 59: 186-191. 24. Cigliano L., Spagnuolo MS., Dale B , Balestrieri M., Abrescia P (2001). Estradiol esterification in the human preovulatory follicle. Steroids, 66: 889-896. 25. Hochberg RB. (1998) Biological esterification of steroids. Endocrinol Rev 19, 331-
  • Cigliano L Spagnuolo MS, Abrescia P. Quantitative variations of the isoforms in haptoglobin 1-2 and 2-2 individual phenotypes. 2003. Arch Biochem Biophys, 416: 227-237.

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Abstract

La présente invention concerne une méthode d'identification d'un agent modulant le transport inverse du cholestérol ou le métabolisme de molécules stéroïdes, ladite méthode consistant à faire incuber ledit agent avec de l'haptoglobine (Hpt), dans des conditions assurant la formation d'une liaison entre les deux molécules, de sorte qu'un complexe agent-Hpt puisse être formé; à vérifier que le complexe agent-Hpt se lie à l'hémoglobine (Hb), avec une constante d'association globalement comparable à celle de Hpt libre; à vérifier que le complexe agent-Hpt inhibe globalement Hpt, sous sa forme liée à l'agent, lors de sa liaison à HDL; à vérifier que le complexe agent-Hpt inhibe globalement l'effet négatif de Hpt sur l'enzyme LCAT.
PCT/IT2005/000403 2004-07-16 2005-07-15 Methode d'identification d'agents modulant le metabolisme de molecules steroides et agents identifies au moyen de ladite methode Ceased WO2006008775A2 (fr)

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WO2007149355A3 (fr) * 2006-06-16 2008-12-04 Lipid Sciences Inc Nouveaux peptides qui favorisent un écoulement de lipides

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