Patent Application: US-201214367997-A

Abstract:
there is provided an ex - vivo insect screening model to determine blood - brain barrier penetration of different chemical compounds . the method comprises the steps : • optionally anesthetizing the insect • fixing the head of the insect • dissecting out the brain of the insect head thereby removing the brain from its cuticle • optinally removing the neural lamella of the brain • treating the brain with a solution of the chemical compound • washing anf homogenising or ultra sound disintegrating the brain • determining the concentration of the chemical compound in the homogenised brain material and • calculating the penetration of the chemical compound through the blood - brain barrier the concentration of the chemical compound is determined by lc / ms and the chemical compound can be a cns drug .

Description:
the present invention provides new methodology for screening chemical agent &# 39 ; s ability to penetrate the bbb also at low exposure concentrations . the invention is generally particular useful for high throughput screening for agents developed in drug discovery programs targeting a variety of diseases and disorders , specifically degenerative disorders , including : parkinson &# 39 ; s disease , alzheimer &# 39 ; s disease , huntington &# 39 ; s disease , diseases with motor neuron inclusions , tauopathies , corticobasal degeneration neuropsychiatric disorders , including : depression bipolar disease , schizophrenia , anxiety , and aggression . moreover , the invention is applicable for drug discovery programs targeting peripherical targets where no cns driven side effect can be tolerated . moreover , the present invention is applicable in the screening of agents developed in drug discovery programs targeting eating disorders and sleep disorders etc . a drug in accordance with the present invention is defined in its broadest scope as a chemical compound that , when absorbed into the body of a living organism , alters normal bodily function . more specifically , a drug in accordance with the present invention is a chemical compound that may be used in the treatment , cure , prevention , or diagnosis of disease or used to otherwise to enhance physical or mental well - being . of particular interest in accordance with the present invention are psychoactive drugs , which are chemical compounds that cross the bbb and acts primarily upon the central nervous system where it alters brain function , resulting in changes in perception , mood , consciousness , cognition and behavior . the present invention relates to but is not restricted to the use of insects selected from the following orders : ( taxonomy according to : djurens värid , ed b . hanström ; förlagshuset norden ab , malmö , 1964 ): in particular insect species selected from blattoidea , acridoidea , cheleutoptera , brachycera and lepidoptera and most particular acridoidea ( locusta migratoria and schistocerca gregaria ) are preferred . the invention will also relate to the following orders comprising insect species relevant for the method of the present invention : large insects , such as the migratory locust , locusta migratoria and the desert locust , schistocerca gregaria or cockroach where it is feasible to feed and inject drugs and subsequently take hemolymph samples and dissect brain tissues , for analyses , are preferred . the locust has been used to develop screening models to determine bbb penetration of different therapeutic drugs and compare this model with existing literature data from conventional in vivo or in situ vertebrate studies . 1 . in a preferred embodiment the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from local suppliers or bred in house . the grasshoppers were reared under crowded conditions at 28 ° c . and a 12 : 12 dark : light photocycle and fed fresh grass and bran . before experiments the grasshoppers were fed ecologically cultivated wheat for two weeks . animals used are adult males ( in some experiments females ) between two to four weeks after adult emergence . a cut is made through the frontal part of the locust head and the brain is dissected out . the brain is placed in a well of a microtitre plate containing the test solution . after various times of exposure the preparation is washed in saline and the brain is dissected under microscope with fine forceps . the neural lamella surrounding the brain is removed in saline and the brain is then ud in saline , centrifuged and the supernatant frozen until analyses . drug concentration is analysed by hplc , lc / msms or other methods . 2 . in a preferred embodiment the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from local suppliers or bred in house . the grasshoppers were reared under crowded conditions at 28 ° c . and a 12 : 12 dark : light photocycle and fed fresh grass and bran . before experiments the grasshoppers were fed ecologically cultivated wheat for two weeks . animals used are adult males ( in some experiments females ) between two to four weeks after adult emergence . a cut is made through the frontal part of the locust head and the brain is dissected out . the neural lamella is removed from the brain and the brain is placed in a well of a microtitre plate containing the test solution . after various times of exposure the preparation is washed in saline and the brain is dissected under microscope with fine forceps . the brain is then ud in saline , centrifuged and the supernatant frozen until analyses . drug concentration is analysed by hplc , lc / msms or other methods . 3 . in a preferred embodiment the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from local suppliers or bred in house . the grasshoppers were reared under crowded conditions at 28 ° c . and a 12 : 12 dark : light photocycle and fed fresh grass and bran . before experiments the grasshoppers were fed ecologically cultivated wheat for two weeks . animals used are adult males ( in some experiments females ) between two to four weeks after adult emergence . a cut is made through the frontal part of the locust head and the brain is dissected out . the brain is placed in a well of a microtitre plate containing the test solution comprising the substance of interest and 4 . 2 % bovine serum albumin . after various times of exposure the preparation is washed in saline and the brain is dissected under microscope with fine forceps . the neural lamella surrounding the brain is removed in saline and the brain is then ud in saline , centrifuged and the supernatant frozen until analyses . drug concentration is analysed by hplc , lc / msms or other methods . 4 . in a preferred embodiment the insects are selected from the order acridoidea and specifically locusta migratoria and schistocerca gregaria are used . the insects may be obtained from local suppliers or bred in house . the grasshoppers were reared under crowded conditions at 28 ° c . and a 12 : 12 dark : light photocycle and fed fresh grass and bran . before experiments the grasshoppers were fed ecologically cultivated wheat for two weeks . animals used are adult males ( in some experiments females ) between two to four weeks after adult emergence . a cut is made through the frontal part of the locust head and the brain is dissected out . the neural lamella is removed from the brain and the brain is placed in a well of a microtitre plate containing the test solution comprising the substance of interest and 4 . 2 % bovine serum albumin . after various times of exposure the preparation is washed in saline and the brain is dissected under microscope with fine forceps . the neural lamella surrounding the brain is removed in saline and the brain is then ud in saline , centrifuged and the supernatant frozen until analyses . drug concentration is analysed by hplc , lc / msms or other methods . a cut was made through the frontal part of the locust head . each brain in its cuticle was placed in a well of a microtitre plate containing a 30 um buffered atenolol test solution . after a five minute exposure at 30 ° c . the preparation was washed in ice cold buffer and the brain was dissected under microscope with fine forceps . the neural lamella surrounding the brain was removed in buffer and the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of atenolol was 0 . 39 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 30 um buffered atenolol test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of atenolol was 0 . 74 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . the neural lamella was removed from the brain in buffer and each brain was placed in a well of a microtitre plate containing a 30 um buffered atenolol test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of atenolol was 3 . 74 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 30 um buffered carbamazepine test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of carbamazepine was 40 . 3 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 30 um buffered carbamazepine and 100 um verapamil test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of carbamazepine was 40 . 0 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 30 um buffered quinidine solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of quinidine was 6 . 9 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 30 um buffered quinidine and 100 um verapamil test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of quinidine was 21 . 4 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 3 um buffered quinidine test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of quinidine was 0 . 33 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . each brain was placed in a well of a microtitre plate containing a 3 um buffered quinidine and 100 um verapamil test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and the neural lamella surrounding the brain was removed . the brain was then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of quinidine was 1 . 52 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . the neural lamella was removed from the brain in buffer and each brain was placed in a well of a microtitre plate containing a 3 um buffered quinidine test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for drug concentration by , lc / ms . the average uptake of quinidine was 2 . 67 pmol / brain . a cut was made through the frontal part of the locust head and the brain was dissected out under microscope with fine forceps . the neural lamella was removed from the brain in buffer and each brain was placed in a well of a microtitre plate containing a 3 um buffered quinidine and 100 um verapamil test solution . after a five minute exposure at 30 ° c . the brain was washed in ice cold buffer and then ultra sound disintegrated in buffer , centrifuged for 5 minutes ( 10000 × g at 4 ° c .) and the supernatant analyzed for 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