Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-03-11
2003-10-14
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S009000, C800S013000
Reexamination Certificate
active
06632977
ABSTRACT:
The invention relates to a non-human transgenic animal in which the expression of at least one of the genes which code for opiate receptors is modified.
Opiates—the prototype of which is morphine—are the most potent analgesics available to medicine today. However, their use is limited by a range of secondary effects, including effects on autonomous functions (constipation, respiratory depression, hypotension, diuresis) and psychotropic effects.
The range of actions of opiates is mediated by membrane receptors of the nervous system, which recognize and specifically bind these compounds. 20 years ago, these receptors were discovered by pharmacological studies. Three receptors have been identified: mu, delta and kappa receptors. The genes which code for these three receptors have been cloned and complementary DNA nucleotide sequences which code for the 3 receptors are shown in
FIG. 11
(mu),
FIG. 12
(delta) and
FIG. 13
(kappa). Selective ligands of the three classes of receptors exist at present, and study of the action of these compounds suggests:
that the mu receptor is the privileged target of the prototype opiate morphine, which is the analgesic used the most for treatment of severe pain,
the mu receptor is also the main target of heroin, one of the most feared narcotics in the context of toxicomania,
the delta receptor is also said to be involved in control of pain and the emotional state (well-being), but to a lesser degree,
the kappa receptor, like the other two receptors, is said to play a role in the analgesic action of opiates. On the other hand, and in contrast to mu and delta, it is said to have a dysphorizing psychotropic action, an action which has been regarded as an advantage for the development of potent analgesics lacking a toxicomanogenic potential.
All the strategies devised in the last 20 years by the pharmaceuticals industries to develop an ideal analgesic are based on these pharmacological data. They comprise in vitro and in vivo analysis of the effect of opiate agonists or antagonists. The interpretation of the results is dependent on the mu/delta/kappa selectivity of the products studied and their pharmacokinetic properties for studies in vivo.
A range of pharmacological results seems to indicate the existence of several receptors in each of the classes mu, delta or kappa which could constitute distinct targets for the agonists of each of the classes of receptors. The question of whether only three receptors or several mu (&mgr;), delta (&dgr;) and kappa (&kgr;) receptors exist has not been resolved at present.
Three genes which code for these receptors have been cloned very recently. Each of them corresponds to one of the above classes of receptors defined by pharmacology. Thus a mu gene, a delta gene and a kappa gene have been characterized at the molecular level. The involvement of these genes in the biological action of opiate substances in vivo has not been defined.
The genes which code for the opiate receptors have been cloned very recently (Kieffer B. (1995) Cellular and Molecular Neurobiology 15:615-635).
In the following, the gene of the &mgr; receptor is called MOR, the gene of the 6 receptor is called DOR and the gene of the &kgr; receptor is called KOR.
One of the objects of the invention is to provide an experimental model which enables targeting of medicaments which have potent analgesic properties without having the secondary effects of opiates of the morphine type.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which at least one of the genes of the opiate receptors is no longer expressed.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which the gene of the &mgr; receptor is no longer expressed.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which the gene of the &dgr; receptor is no longer expressed.
One of the objects of the invention is to provide non-human transgenic mammalian animals in which the gene of the &kgr; receptor is no longer expressed.
One of the other objects of the invention is to provide an animal model which is capable of screening medicaments which act on pathologies involving at least one of the opiate receptors.
The invention relates to the use of a non-human transgenic mammalian animal in which the expression of at least one the genes which codes for the opiate receptors is modified, in particular suppressed in the tissues or cells of the brain, with respect to normal expression, in particular in the tissues or cells of the brain, for determination of a medicament which is active on pathologies involving the opiate receptors.
More precisely, the invention relates to the use of a non-human transgenic mammalian animal in which the expression of the gene which codes for an opiate receptor is modified, in particular in the nerve tissues, with respect to normal expression, in particular in the nerve tissues, for determination of a medicament which acts on pathologies involving the opiate receptors, in particular acute or chronic severe pain, toxicomania or the prevention or treatment of transplant rejections.
The term “mammalian” includes all mammals with the exception of humans, advantageously rodents, and in particular mice.
“Transgenic animal” is understood as meaning not only an animal in the genome of which an exogenous gene has been introduced, but also an animal in which expression of an endogenous gene has been deleted, either by interruption of the endogenous gene or by replacement of an endogenous gene or of a fragment thereof by a construction such that it no longer allows expression of the endogenous gene. Such animals will be called “knock-out” animals or those deficient in the said endogenous gene.
Normal expression of one of the opiate receptors can be defined by several methods:
1) Determination of the mRNA corresponding to one of the genes of the opiate receptors: this is possible by the technique of RNA transfer (Northern blot) in which the mRNAs are separated on denaturing agarose gel by electrophoresis; and the RNAs are then transferred and bound to a membrane of the nitrocellulose or nylon type. To reveal the presence of the RNAs corresponding to one of the genes of the opiate receptors, it is possible to use a probe corresponding to all or a fragment of the cDNA of the gene in question.
2) Determination of the amount of protein corresponding to one of the opiate receptors: this is possible by studying the bonding of an opiate ligand (agonist or antagonist), such as diprenorphin, which is non-selective with respect to opiate receptors, DAGO (selective with respect to &mgr;), naltrindole (selective with respect to &dgr;) and labelled CI977 (selective with respect to &kgr;), to receptors present in a tissue (brain) homogenate. As regards the respective definitions of these ligands, these are shown in the legend of FIG.
2
. In particular, the dissociation constant Kd of several specific ligands of one of the opiate receptors is known and is of the order of 1 nanomolar for the ligands generally used. It is furthermore known that the Bmax for the above ligands is of the order of 0.1 picomol/mg membrane protein for &mgr; and &dgr; receptors and 0.02 picomol/mg membrane protein for the &kgr; receptor in respect of the mouse brain. It is thus known that a saturation curve with this ligand on membrane extracts containing the three opiate receptors prepared from the brain and analysis of the results obtained from the saturation curves by the Scatchard method (determination of the number of receptor sites) should give Bmax affinity values divided by two in heterozygotes and zero Bmax values (not measurable) in homozygotes.
This therefore allows quantification of the amount corresponding to one of the opiate receptors.
According to one embodiment, the invention relates to the use of a non-human transgenic mammalian animal which no longer expresses the gene of the &mgr; receptor or of the &kgr; receptor or of the &dgr; receptor.
The modification and absence of expression of
Dierich Andree
Kieffer Brigitte
Lemeur Marianne
Matthes Hans W. D.
Simonin Frederic Herve
Centre National de la Recherche Scientifique
Crouch Deborah
Woitach Joseph
Young & Thompson
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