US 2020/0046856 A1Published application

Methods for identifying mblac1-dependent molecular networks

OwnerRandy D. Blakely, Maureen K. Hahninventors

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Abstract

Based on the discovery that MBLAC1 is a specific, high-affinity target for Ceftriaxone (Cef), MBLAC1 may be used for identifying molecules and molecular networks associated with MBLAC1's role in the actions of substances of abuse, e.g., molecules and networks of molecules modulated by Cef in an animal that expresses MBLAC1 and is administered a substance of abuse and not in an animal that does not express MBLAC although administered the same substance of abuse. Methods for identifying MBLAC1-dependent molecules and molecular networks include analyzing and comparing levels of molecules (nucleic acids, neurotransmitters, proteins, and metabolites) in MBLAC1 knock-out animals and wild-type MBLAC1 animals. These methods and MBLAC1 knock-out animals can also be used for identifying therapeutic drug targets for treatment of addiction to, or withdrawal from, a substance of abuse.

Claims26

Dependent claims

A method of identifying molecular networks in an animal that are modulated by Cef in the presence of MBLAC1 but not in the absence of MBLAC1, the method comprising the steps of:

(a) providing a group of test MBLAC1 knock-out (KO) animals, a group of control MBLAC-1 KO animals, a group of test wild-type (WT) MBLAC1 animals and a group of control WT MBLAC1 animals, or cells from each group of animals;
(b) administering a dose of Cef to the group of test MBLAC1 KO animals and to the group of test WT MBLAC1 animals, or to cells from the group of test MBLAC1 KO animals and to cells from the group of test WT MBLAC1 animals at least once under conditions known to reduce effects of a substance of abuse in an animal;
(c) collecting biological samples from all the groups of animals and analyzing levels of nucleic acids, neurotransmitters, proteins, and metabolites in the biological samples or analyzing levels of nucleic acids, neurotransmitters, proteins, and metabolites in the cells from all the groups of animals, resulting in a plurality of test MBLAC1 KO molecular networks, a plurality of control MBLAC1 KO molecular networks, a plurality of test WT MBLAC1 molecular networks, and a plurality of control WT MBLAC1 molecular networks;
(d) comparing the plurality of test WT MBLAC1 molecular networks to the plurality of control WT MBLAC1 molecular networks and identifying any molecular networks present in the test WT MBLAC1 animals but not in the control WT MBLAC1 animals as Cef-responsive molecular networks;
(e) comparing the Cef-responsive molecular networks to the plurality of test MBLAC1 KO molecular networks and to the plurality of control MBLAC1 KO molecular networks and identifying any Cef-responsive molecular networks that are not present in the plurality of test MBLAC1 KO molecular networks or in the plurality of control MBLAC1 KO molecular networks, or that are overrepresented relative to the plurality of test MBLAC1 KO molecular networks, as molecular networks that are modulated by Cef in the presence of MBLAC1 but not in the absence of MBLAC1.

2 more elements

A method of identifying molecules and/or molecular networks thereof modulated by a substance of abuse and Cef in an MBLAC1 dependent manner, the method comprising the steps of:

(a) providing a group of control WT MBLAC1 animals, a first test group of WT MBLAC1 animals, a second test group of WT MBLAC1 animals, a third test group of WT MBLAC1 animals, a group of control MBLAC1 KO animals, a first group of test MBLAC1 KO animals, a second test group of MBLAC1 KO animals and a third test group of MBLAC1 KO animals;
(b) administering a substance of abuse to the first test group of WT MBLAC1 animals and collecting biological samples from the group of control WT MBLAC1 animals and from the first test group of WT MBLAC1 animals;
(c) analyzing levels of molecules selected from the group consisting of:
nucleic acids, neurotransmitters, proteins, and metabolites in the biological samples and determining differences in the levels of the molecules between the group of control WT MBLAC1 animals and the first test group of WT MBLAC1 animals;
(d) administering a dose of Cef to the second test group of WT MBLAC1 animals and administering the same dose of Cef and the substance of abuse to the third test group of WT MBLAC1 animals, wherein the dose of Cef is administered at least once under conditions known to reduce effects of the substance of abuse in an animal;
(e) collecting biological samples from the second test group of WT MBLAC1 animals and the third test group of WT MBLAC1 animals after administration of the dose of Cef and the substance of abuse;
(f) analyzing levels of molecules selected from the group consisting of:
nucleic acids, neurotransmitters, proteins, and metabolites in the biological samples of step (e) and determining differences in the levels of the molecules between the second test group of WT MBLAC1 animals and the third test group of WT MBLAC1 animals;
(g) comparing the differences in the levels of step (c) to the differences in the levels of step (f) to identify any molecules or one or more molecular networks thereof whose levels are modulated by Cef and by the substance of abuse;
(h) administering a substance of abuse to the first test group of MBLAC1 KO animals and collecting biological samples from the group of control MBLAC1 KO animals and from the first test group of MBLAC1 KO animals;
(i) analyzing levels of molecules selected from the group consisting of:
nucleic acids, neurotransmitters, proteins, and metabolites in the biological samples of step (h) and determining differences in the levels of the molecules between the group of control MBLAC1 KO animals and the first test group of MBLAC1 KO animals;
(j) administering a dose of Cef to the second test group of MBLAC1 KO animals and administering the same dose of Cef and the substance of abuse to the third test group of MBLAC1 KO animals, wherein the dose of Cef is administered at least once under conditions known to reduce effects of the substance of abuse in an animal;
(k) collecting biological samples from the second test group of MBLAC1 KO animals and the third test group of MBLAC1 KO animals after administration of the dose of Cef and the substance of abuse;
(l) analyzing levels of molecules selected from the group consisting of:
nucleic acids, neurotransmitters, proteins, and metabolites in the biological samples of step (k) and determining differences in the levels of the molecules between the second test group of MBLAC1 KO animals and the third test group of MBLAC1 KO animals;
(m) comparing the differences in the levels of step (i) to the differences in the levels of step (l) to identify any molecules or one or more molecular networks thereof that modulate actions of the substance of abuse in any MBLAC1 KO animal that has been administered the substance of abuse;
and
(n) comparing the molecules or one or more molecular networks thereof whose levels are modulated by Cef and by the substance of abuse of step (g) to the molecules or one or more molecular networks thereof that modulate actions of the substance of abuse in any MBLAC1 KO animal of step (m) and identifying molecules or one or more molecular networks thereof whose levels are modulated by Cef and by the substance of abuse in an MBLAC1-dependent manner.

16 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/057,013Filed 7 Aug 2018
Published: 13 Feb 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification8 codes

A61K 49/0008
Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for…:…
A01K 67/0276
Genetically modified vertebrates, e.g. transgenic: knock-out vertebrates
A01K 2207/05
Modified animals: animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
A01K 2217/075
Animals genetically altered by homologous recombination: inducing loss of function, i.e. knock out