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WO2002018955A1 - Essai de neuropathie par detection de p75ntr - Google Patents

Essai de neuropathie par detection de p75ntr Download PDF

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Publication number
WO2002018955A1
WO2002018955A1 PCT/GB2001/003859 GB0103859W WO0218955A1 WO 2002018955 A1 WO2002018955 A1 WO 2002018955A1 GB 0103859 W GB0103859 W GB 0103859W WO 0218955 A1 WO0218955 A1 WO 0218955A1
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Prior art keywords
ntr
neuropathy
subject
body fluid
fluid sample
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PCT/GB2001/003859
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English (en)
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David Richard Tomlinson
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The Victoria University Of Manchester
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Priority to AU2001284187A priority Critical patent/AU2001284187A1/en
Publication of WO2002018955A1 publication Critical patent/WO2002018955A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates to a test for identifying, whether or not a subject has, or is likely to develop, a neuropathy.
  • the test is also useful as a method for screening compounds to evaluate their efficacy for treating neuropathy or alternatively to evaluate whether or not a compound may have an undesirable side- effect of causing neuropathy.
  • Neuropathy is a disorder affecting the nervous system and in particular the cranial nerves or the peripheral or autonomic nervous systems.
  • Neuropathies can have serious clinical implications.
  • the peripheral form manifests as a dulling in the feeling of pain, temperature or pressure.
  • This is a feature of various disease states that affect peripheral nerves, and is recognised as a syndrome of symptoms referred to as peripheral neuropathy.
  • This form of neuropathy makes a subject vulnerable to insensible injury.
  • the clinical consequences of such insensibility can be manifold.
  • the insensibility can lead to infections developing which may result in gangrene and subsequently the need for amputation.
  • There is also an increased risk of septicaemia which can have potentially fatal consequences.
  • Symptoms of autonomic system neuropathies can manifest as alternating bouts of diarrhoea and constipation, impotence and reduced cardiac function.
  • Diabetic Neuropathy This disorder is the most commonly occurring chronic complication of diabetes mellitus and can take both peripheral (especially effecting the lower legs and feet) and autonomic fo ⁇ ns.
  • the World Health Organization calculates that there will be 300 million diabetics worldwide.
  • the incidence of neuropathy in the diabetic population lies between 30 and 50%, depending on diagnostic criteria and local demographics. It will therefore be appreciated that there is a serious need to be able to clinically manage neuropathies.
  • a clinician may be able to make an early diagnosis of neuropathy in order that the condition may be treated and/or the subject educated such that they vigorously monitor for insensible injuries. Early or prompt diagnosis may avoid the need for amputation of injured limbs.
  • Such amputations and other clinical complications arising from neuropathy are not only debilitating for the individual but can have grave financial implications for health care providers (in the context that millions of people worldwide may be suffering from neuropathy related clinical problems).
  • an in vitro test for neuropathy comprising detecting for the presence of a 75 kDa Neurotrophin Receptor (p75 NTR ) in a body fluid sample taken from a subject being tested for neuropathy.
  • p75 NTR Neurotrophin Receptor
  • the neurotrophins are a family of proteins that influence the development of the nervous system in the embryo and neonate. In the adult they play a vital role in maintaining the phenotype of specific classes of neurones. This is exemplified by nerve growth factor (NGF) which is considered by many to be the archetypal neurotrophin. NGF maintains the functional identity of a class of sensory neurones known as C fibres. These nerves are responsible for the sensation of temperature from the skin and also radiate low intensity (“background”) pain - sunburn is a typical example.
  • NGF nerve growth factor
  • a neurotrophin receptor of apparent molecular weight of 75kDa (p75 NTR ) is involved in transduction of the neuronal response to NGF.
  • p75 NTR is involved in the internalisation of NGF into the neurone.
  • neurotrophin receptors p75 NTR
  • detection of p75 NTR represents a useful diagnostic aid for clinicians interested in neuropathy.
  • 75kDa represents an apparent molecular mass of the receptor it will be appreciated that the receptor may not have a mass of precisely 75kDa. In fact the mass of the receptor can vary for a variety of reasons not least of all as a result of degradation caused by experimental manipulations or exposure to protease. Therefore the receptor detected according to the invention may be from 70 - 80kDa, preferably 73 - 77kDa and is most preferably 75kDa.
  • Human p75 TR comprises the amino acid sequence disclosed in Figure 1 (SEQ ID No. 1).
  • the receptor is also glycosylated in mature form.
  • the human receptor comprises approximately 427 amino acids (399 amino acids when a signal peptide, amino acids 1 - 28, is removed).
  • the amino acid sequence of p75 ⁇ can vary between individuals of a single species and can also vary between species.
  • the amino acid sequence of rat p75 NTR see Figure 2- SEQ ID No.2
  • the receptor detected according to the present invention may be a functional derivative of the human p75 NTR disclosed in Figure 1.
  • p75 NTR detected according to the present invention may have 75% sequence homology with the receptor of Figure 1, preferably 85% sequence homology and more preferably at least 95% sequence homology.
  • the shedding by cells of receptors for biomolecular ligands hormones, neurotransmitters, cytokines, trophic factors etc) is thought to be a generic phenomenon. Receptor shedding, with consequent appearance (either de novo or in increased titre) in plasma, has been studied in disease states such as neoplasia, osteoarthritis and asthma (each associated with TNF ⁇ receptor shedding). Other cytokine receptors are shed in inflammatory bowel disease and osteoarthritis. The extent to which these observations are suggestive of the mechanism of disease development has not been established.
  • Hruska et al 1993 discloses that a truncated receptor termed p50 or NGFR-t, possibly a truncated form of p75 NTR , may be found in the urine of subjects with neuropathy.
  • Hruska et al. raised polyclonal antibodies against p50 and demonstrated immunoreactivity in the urine of diabetic patients characterised as having nephropathy.
  • the tests performed by Hruska et al. will have no or at least limited value as a clinical test for several reasons. For instance, the observation that p50 may be detected in urine is of little diagnostic use because the phenomenon is likely to be dependent on co-existence of nephropathy and neuropathy.
  • the tests according to the present invention provide reliable information on the health status of a subject or the affect of a compound on the neuropathic condition.
  • neuropathic tissue damage would result in:
  • the extracellular domain of the receptor comprises much less than half of the molecule. Therefore shedding of the extracellular domain would be expected to liberate, into the plasma, a molecule much smaller than 50kD. Accordingly p50 immunoreactive species cannot represent the extracellular domain of a shed receptor and must therefore be a kidney product that has no scientific or clinical relationship to incipient or real nerve damage.
  • the method according to the present invention may be distinguished therefrom for a number of reasons.
  • the prior art relates to a urine based test.
  • the body fluid is a blood-based sample and more preferably a plasma sample.
  • the use of such samples does not cause the sort of restrictions caused by the use of urine samples and offers much greater reliability and sensitivity than testing urine.
  • the method according to the first aspect of the present invention is accurate enough to be used as a quantitative assay of p75 NTR .
  • the intensity of the colour change is directly proportional to the amount of p75 NTR and also the risk of neuropathy developing.
  • the method according to the first aspect of the invention may preferably detects p75 NTR such that there is specificity to p75 NTR over p50.
  • the method may use an agent to detect p75 NTR which is an antibody raised against an epitope found on full length p75 NTR but not on p50.
  • Such an antibody may be polyclonal or monoclonal.
  • the antibody when human p75 NTR is being detected it is preferred that the antibody is raised against an epitope found within amino acids 1 - 140 and more preferably amino acids 29 - 90 of Figure 1 (e.g. -Cys-Lys-Ala-Cys- Asn-Leu-Gly-Glu-Gly-Val-Ala-Gln- SEQ ID No. 3).
  • the antibody may be raised against an epitope found within amino acids 290 - 427 and more preferably amino acids 330- 427 of Figure 1 (e.g. -Glu-Val-Glu-Lys-Leu-Leu-Asn-Gly-Ser-Ala- Gly-Asp- SEQ ID No. 4).
  • the method is based upon the premise that p75 NTR molecules are shed by neurones and/or Schwann cells that have been subjected to cellular stress or injury and that these receptors appear in body fluids such as blood.
  • the method may be performed on venous blood.
  • the method of the invention further comprises the step of isolating and optionally partially purifying the shed receptor (defined as removal of potentially interfering molecules).
  • plasma may be separated from whole blood, the p75 NTR isolated therefrom and semi- purified if required.
  • the plasma extract so produced may be fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
  • p75 is semi-purified by gel electrophoresis. Bands from the gel of molecular mass of about 75 kDa are then transferred to a western blot and bound to a primary antibody raised against p75 NTR . The extent of binding of p75 NTR (the titre) is then determined using a secondary antibody coupled to a visualisation system.
  • the secondary antibody/visualisation system may be conventional, commercially available systems known to those skilled in the art (e.g. Lumiglow®). Fractionation of the proteins by this procedure enables confirmation that the immunoreactivity so detected is associated with a protein fraction of appropriate molecular weight.
  • the preferred protocol referred to in the preceding paragraph is particularly suitable when the method of the first aspect of invention is used to make a detailed analysis, such as a clinical trial of a drug targeted at diabetic neuropathy.
  • the laboratory facilities available to research or clinical trials are often not available for routine clinical use. Under these circumstances it is preferred that the methods of the invention do not require plasma purification or protein fractionation (gel separation) and immunoblotting. In these cases it is preferred that an antibody raised against p75 NTR is used to detect the shed receptor and standard enzyme-linked immunoassay (ELISA) or radioimmunoassay (RIA) may be used for quantitative estimation of receptor titre.
  • ELISA enzyme-linked immunoassay
  • RIA radioimmunoassay
  • the methods of the invention may utilise a plastic test-strip.
  • Such strips may comprise a primary antibody raised against p75 NTR immobilised on a strip. This strip may then be dipped into a body fluid sample and linkage of the antibody-p75 NTR combination may be detected by further dipping the strip into a solution that undergoes a chromogenic reaction in the presence of the antibody-p75 NTR combination.
  • Example 6 the reagents for undergoing the chromogen reaction may be built into the test strip.
  • the method of the invention is suitable as a diagnostic aid.
  • the detection of p75 NTR in body fluids is also useful for testing the efficacy of putative drugs for treating neuropathy and also for testing whether or not a compound may cause neuropathy.
  • a method for screening the efficacy of a putative drug for treating neuropathy comprising administering the putative drug to a subject and detecting for the presence of a 75 kDa Neurotrophin Receptor (p75 NTR ) in a body fluid sample taken from the subject.
  • p75 NTR 75 kDa Neurotrophin Receptor
  • an in vitro test for evaluating whether or not a subject will benefit from a putative drug for treating neuropathy comprising detecting for the presence of a 75 kDa Neurotrophin Receptor (p75 NTR ) in a body fluid sample taken from a subject which has received, or will receive, said putative drug.
  • p75 NTR Neurotrophin Receptor
  • the methods of the third aspect of the invention may also be adapted for testing whether an individual has (or is likely to get) a neuropathy before they are given a prescription drug for treating neuropathies. Such a test is useful for identifying whether or not an individual will benefit from said drug when it is subsequently administered.
  • a method for screening a compound to assess whether or not the compound causes neuropathy comprising administering the compound to a subject and detecting for the presence of a 75 kDa Neurotrophin Receptor (p75 NTR ) in a body fluid sample taken from the subject.
  • p75 NTR 75 kDa Neurotrophin Receptor
  • the methods of the second and fourth aspects of the invention may be performed in suitable animal models (e.g. rodents) for drug or compound testing.
  • suitable animal models e.g. rodents
  • the methods may be adapted for use in ex vivo, cell culture or even in vitro based tests.
  • the methods of the first and second aspects of the present invention are particularly suited for use as a diagnostic aid and a means of monitoring clinical status in subjects with diabetic neuropathy.
  • the methods of the third and fourth aspect of the invention are also particularly suited for screening putative drugs and compounds to assess their effect on diabetic neuropathies.
  • neuropathies include peripheral neuropathies arising post surgically (e.g. postradical nerve dissection, post mastectomy, postlimb amputation), neuropathies caused by tumour infiltration of peripheral nerves, chemotherapy induced neuropathy, radiation induced nerve tumours and cranial neuropathies. These neuropathies cause symptoms such as numbness and/or pain (even from phantom limbs following amputation) and we have found that the methods of the invention may be used to improve the clinical management of these conditions.
  • the methods of the invention may be used over an extended period of time to monitor the clinical status of a subject.
  • a diabetic patient who may be taking a variety of drugs that could either treat or exasperate a neuropathy
  • the methods of the invention e.g. weekly or monthly
  • a method of monitoring the health status of a subject with, or at risk of developing, a neuropathy comprising detecting for the presence of a 75KDa Neurotrophin Receptor (P75 NTR ) in a body fluid sample taken from the subject being monitored.
  • P75 NTR Neurotrophin Receptor
  • a method of testing whether or not a subject is complying with a therapeutic regime for treating diabetes comprising detecting for the presence of a 75 kDa Neurotrophin Receptor (p75 NTR ) in a body fluid sample taken from the subject.
  • Figure 1 illustrates the amino acid sequence of human p75 NTR ;
  • Figure 2 illustrates the amino acid sequence of rat p75 TR ;
  • Figure 3 is a photograph of a Western blot illustrating p75 NTR detected in (a) non-neuropathic patients; (b) neuropathic patients; and (c) sural nerve biopsies;
  • Figure 4 is a graph illustrating the regression of human plasma samples using two preferred antibodies that may be used according to the method of the invention.
  • Figure 5 is a graph illustrating p75 NTR titre in the animal studies of Example 3.
  • Figure 6 is a graph illustrating p50 titre in the animal studies of Example 3.
  • Figure 7 is a graph illustrating p75 NTR titre in the human subjects of Example 4.
  • a test was developed based upon the premise that p75 NTR molecules are shed by neurones and/or Schwann cells that have been subjected to cellular stress or injury and that these receptors appear in plasma.
  • the receptor may be isolated from plasma and semi-purified (defined as removal of potentially interfering molecules) if required.
  • the plasma extract so produced can then be fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), proteins transferred to a western blot and bound to a primary antibody raised against p75 NTR .
  • the extent of binding - the titre - is then determined using a secondary antibody coupled to a commercial visualisation system. Fractionation of the proteins by this procedure enables confirmation that the immunoreactivity so detected is associated with a protein fraction of appropriate molecular weight. This step is suited for use in a detailed analysis, such as a clinical trial of a drug targeted at diabetic neuropathy. Such confirmation is essential during developmental stages.
  • Plasma samples were centrifuged to prepare plasma (approximately 10 minutes at 10,000 g) and plasma pipetted into a clean tube. This was either stored on ice for immediate purification/assay or frozen (-70°C) for storage. Plasma was then diluted 1+4 with 0.02M di-sodium hydrogen orthophosphate and 5 ml loaded onto a Bio-Rad Econo-pac Blue Cartridge (TM), prepared in accordance with the manufacturer's (Bio- Rad) instructions. The sample was left on the column to equilibrate for 5 minutes and then plasma minus albumin washed off the column with 5 ml 0.02M di-sodium hydrogen orthophosphate. Of this eluate, 80 ⁇ l was added to 720 ⁇ l of 0.02M di- sodium hydrogen orthophosphate plus and 200 ⁇ l sodium dodecyl sulphate (SDS) and the mixture boiled for 5 minutes.
  • SDS sodium dodecyl sulphate
  • membranes were incubated at 4°C overnight with anti-human p75 ⁇ TR antibody at 1:5000 dilution in TBS tween + 0.1% sodium azide (20ml). Membranes are washed (3x15 minutes) at room temperature in TBS tween and incubated with secondary antibody - Anti rabbit (New England Biolabs) 1:5000 dilution in TBS tween for 1 hour at room temperature (20ml). This was followed by 3 further washes (each 15 minutes) in TBS tween. Finally, membranes were exposed to standard Lumiglo (New England Biolabs TM) chemiluminescence enhancement and exposed to x-ray film for 10 to 30 seconds.
  • standard Lumiglo New England Biolabs TM
  • the first three left-hand lanes (a) of the gel illustrated in Figure 3 show typical data from 3 patients with no functional signs of neuropathy (sensation and reflexes in the normal range) - controls and the middle three lanes (b) show p75 NTR detected in 3 patients with neuropathy by these criteria.
  • the remaining three lanes (c) show immunoreactivity from 3 sural nerve specimens.
  • the intensity of the bands in the control group (a) were measured as 2.21, 2.17 and 2.50 whereas the bands from patients with neuropathy were 5.34, 5.47 and 4.22. The difference between the two groups was significant.
  • the titre is considerably higher in patients with neuropathy.
  • the neuropathic patients show a titre that is approximately four times (indicated by area x density) that of the non-neuropathic patients.
  • Example 1 The procedure described in Example 1 was refined to develop a preferred full analytical method of testing for neuropathy according to the invention.
  • Plasma purification (see Example 1), which principally involved removal of albumin. This was done because of the similarity in molecular weight between albumin and the full length p75 NTR receptor and the assumption that albumin might interfere with fractionation and blotting (see below). However, the inventors were surprised to discover that the sensitivity of immunodetection was higher than had been suspected. This prompted a comparison between purified (i.e. albumin removed) and native plasma and the finding that there was no difference in read-out. Hence the purification step may be omitted in preferred methods according to the invention.
  • Samples (20 ⁇ l) are then run on 10%> polyarcylamide gels in a running buffer comprising 0.025M Tris, 0.192M glycine, 0.1% (w/v) SDS, pH 8.3 at 120N for45 - 60 minutes. Proteins are then transferred to Hybond Enhanced Chemiluminescence nitrocellulose membrane and blocked for 2 hours at room temperature in Tris buffered saline (TBS) tween (150mM ⁇ aCl lOmM Tris-HCL pH 7.6 , 0.05% Tween 20) containing 5% bovine serum albumin and 5% casein.
  • TBS Tris buffered saline
  • the membranes are incubated at 4°C overnight with anti-human p75 ⁇ TR antibody (see below) at 1:2000 dilution (though optimal dilutions of the primary antibodies listed below range from 1:1000 to 1:5000) in TBS tween + 0.1 % sodium azide (20ml).
  • Membranes are washed (3x15 minutes) at room temperature in TBS tween and incubated with secondary antibody - Anti rabbit (New England Biolabs) 1:5000 dilution in TBS tween for 1 hour at room temperature (20ml). This is followed by 3 further washes (each 15 minutes) in TBS tween.
  • membranes are exposed to standard Lumiglo (New England Biolabs TM) chemiluminescence enhancement and exposed to x-ray film for 10 to 30 seconds.
  • Promega G3231 and Chemicon AB1554 gave a similar result when applied to the same samples (see fig. 4).
  • Promega G3231 binds to the entire intracellular domain of the receptor, whilst Chemicon AB1554 binds to part of the intracellular domain.
  • Antibodies with the same or similar immunoreactivity to these antibodies are preferred antibodies for use according to the invention.
  • the plasma titre of p75 -NTR from diabetic and non-diabetic rats was assessed using the methods described in Example 2.
  • the plasma titres for p75 NTR and p50 were measured in three separate experiments involving rats made diabetic with the drug streptozotocin.
  • Example 3 may be readily adapted to include the administration of a test compound. Therefore this protocol represents a preferred way of carrying out the methods according to the second, third or fourth aspect of the invention.
  • the plasma titre of p75 NTR from patients and healthy volunteers was assessed using the methods described in Example 2.
  • the inventors examined plasma samples from 85 patients and 8 healthy volunteers. The patients were grouped into an initial cohort of 50 and a second cohort of 35 (obtained from two different clinics). All patient samples were assayed blind and the code of the first 50 has been broken; these data are presented here. The code of the second 35 is not broken, because sampling continues from this clinic, and these data are not presented.
  • the titres for the p75 NTR molecular species are presented in Figure 7.
  • the presence of significant p75 immunoreactivity in plasma was associated with diabetes.
  • the titre of p75 was on average greater in diabetic patients with neuropathy compared with diabetics believed to have no neuropathy. Control subjects showed only traces.
  • the presence/absence of neuropathy was based upon a relatively crude clinical diagnosis and the inventors believe a highly significant difference would exist between the two diabetic groups if more sensitive diagnosis is performed. Furthermore, the inventors believe that the test is sensitive enough to detect patients about to develop neuropathy but for whom there were no clinical symptoms at the time the samples were taken.
  • Plasma p50 immunoreactivity was absent from most patient samples and showed no systematic distribution in studies on rats.
  • the protocol for an ELISA based assay was developed.
  • the ELISA procedure measures accurately the titre of p75 NTR immunoreactivity.
  • This embodiment of the invention does not require confirmation of the apparent molecular weight of p75 NTR .
  • 96-well plates were preincubated with anti-human p75 NTR overnight under optimal conditions to coat the plates with antibody. Plasma samples are then prepared and diluted as described in 1.1 or 2.1 and pipetted into the wells. After an optimised incubation period, the wells are buffer-washed and exposed to a conventional anti- rabbit chromogen system. The amount of product is measured in a multi-well plate reader at the wavelength appropriate to the chromogen reaction.
  • a plastic test-strip may be developed by immobilisation of the primary antibody on the strip and linkage of the antibody-p75 TR combination with a chromogenic reaction.
  • Test strips may be impregnated with a known concentration of anti-human p75 NTR linked to an inbuilt chromogen reaction.
  • the chromagen reaction may be based on conventional systems used for assaying blood glucose or Horse Radish peroxidase. Strips are exposed to blood (e.g. from a finger prick) and, after an appropriate incubation time, the strip is blotted to remove the blood and the colour developed compared to a standard provided.

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Abstract

L'invention concerne des essais in vitro de neuropathie et d'évaluation de la question de savoir si le patient bénéficiera ou non d'un médicament présumé destiné au traitement de la neuropathie. Ces essais consistent à détecter la présence d'un récepteur de la neurotrophine 75 kDa (p75NTR) dans un échantillon de liquide organique prélevé chez le patient. L'invention concerne également des procédés de contrôle de l'efficacité d'un médicament présumé pour le traitement de la neuropathie, et d'analyse de composés pour déterminer s'ils provoquent ou non la neuropathie, qui consistent à administrer le médicament ou le composé au patient et à détecter la présence d'un récepteur de la neurotrophine 75 kDa (p75NTR) dans un échantillon de liquide organique prélevé chez le patient.
PCT/GB2001/003859 2000-09-01 2001-08-29 Essai de neuropathie par detection de p75ntr WO2002018955A1 (fr)

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AU2001284187A AU2001284187A1 (en) 2000-09-01 2001-08-29 Test for neuropathy by detection of p75ntr

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GB0021609A GB0021609D0 (en) 2000-09-01 2000-09-01 Test for neuropathy
GB0021609.3 2000-09-01

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003013604A3 (fr) * 2001-08-09 2003-10-09 Genset Sa Traitement des troubles metaboliques par les agonistes et les antagonistes de migenix

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CONTI GIANCARLO ET AL: "P75 neurotrophin receptor induction and macrophage infiltration in peripheral nerve during experimental diabetic neuropathy: Possible relevance on regeneration.", EXPERIMENTAL NEUROLOGY, vol. 146, no. 1, 1997, pages 206 - 211, XP002184863, ISSN: 0014-4886 *
SCHMIDT, R. E. ET AL: "Effect of streptozotocin-induced diabetes on NGF, P75NTR and TrkA content of prevertebral and paravertebral rat sympathetic ganglia", BRAIN RES. (2000), 867(1,2), 149-156, XP001050325 *
TOMLINSON, DAVID R. ET AL: "Neurotrophic factors-regulation of neuronal phenotype", NEUROSCI. RES. COMMUN. (1997), 21(1), 57-66, XP001050381 *
YAMAMOTO, MASAHIKO ET AL: "Expression of low-affinity neurotrophin receptor p75NTR in the peripheral nervous system of human neuropathies", ACTA NEUROPATHOL. (1998), 95(6), 597-604, XP001050385 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003013604A3 (fr) * 2001-08-09 2003-10-09 Genset Sa Traitement des troubles metaboliques par les agonistes et les antagonistes de migenix

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