Patent Application: US-201013387088-A

Abstract:
there is provided an ex - vivo insect screening model to accurately determine blood - brain barrier penetration of different chemical compounds in order to improve the compound screening procedures / processes in the early drug discovery process . this object offers many advantages relative to prior technologies since insect models are more reliable tools for the decision - making process than the existing in vitro models , and will speed up the drug screening process and reduce the late phase attrition rate . moreover , it will reduce the number of mammals sacrificed during the drug discovery phase .

Description:
the present invention provides new methodology for screening chemical agent &# 39 ; s ability to penetrate the bbb . 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 värld , ed b . hanström ; förlagshuset norden ab , malmö , 1964 ): in particular the invention relates to insect species selected from blattoidea , acridoidea , cheleutoptera , brachycera and lepidoptera and most particular to the acridoidea ( locusta migratoria and schistocera gregaria ). the invention will also relate to the following orders comprising insect species relevant for the method of the present invention : the present invention preferably uses large insects , such as the migratoty locust , locusta migratoria and the desert locust , schistocera gregaria or cockroach where it is feasible to feed and inject drugs and subsequently take hemolymph samples and dissect brain tissues , for analyses . 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 of the present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocera 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 comprising the most frontal parts including the antennae , the compound eyes , the brain and all neural connections between the brain and the antennae and the eyes . this preparation in its cuticle shell 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 of present invention the insects are selected from the order acridoidea and specifically locusta migratoria and schistocera 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 comprising the most frontal parts including the antennae , the compound eyes , the brain and all neural connections between the brain and the antennae and the eyes . this preparation in its cuticle shell is placed in a well of a microtitre plate containing a 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 head of adult female locusta migratoria . each brain in its cuticle shell was placed in a 0 . 12 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell and three brains were placed in each test tube containing 100 ul distilled h2o . 200 ul acn was added to each tube and the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and an average mianserin concentration of 3 ug / ml was measured by lcms . a cut was made through the frontal part of the head of adult female locusta migratoria . each brain in its cuticle shell was placed in a 0 . 12 mg / ml mianserin solution for 15 minutes . the brain was prepared in saline and removed from the cuticle shell and three brains were placed in each test tube containing 100 ul distilled h2o . 200 ul acn was added to each tube and tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and an average mianserin concentration of 2 . 2 ug / ml was measured by lcms . a cut was made through the frontal part of the head of adult female locusta migratoria . each brain in its cuticle shell was placed in a 0 . 12 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell . the brains were washed in saline . after wash three brains were placed in each test tube containing 100 ul distilled h2o . 200 ul acn was added to each tube and the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and an average mianserin concentration of 2 . 4 ug / ml was measured by lcms . a cut was made through the frontal part of the head of adult female locusta migratoria . each brain in its cuticle shell was placed in a 0 . 12 mg / ml mianserin solution for 15 minutes . the brain was prepared in saline and removed from the cuticle shell . the brains were washed in saline . after wash three brains were placed in a test tube containing 100 ul distilled h2o . 200 ul acn was added to each tube and the tubes were placed in each sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 1 . 3 ug / ml was measured by lcms . a cut was made through the frontal part of the head of adult female locusta migratoria . each brain in its cuticle shell was placed in a 0 . 12 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell . to stain the neural lamella evans blue was added and the neural lamella was removed . three brains were placed in each test tube containing 100 ul distilled h2o . 200 ul acn was added to each tube and the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and an average mianserin concentration of 309 ng / ml was measured by lcms . from example a - e it can be concluded that mianserin permeate the grasshopper bbb but it can even be seen that the bbb still works as a regulator preventing free passage of mianserin from the exterior to the brain . moreover , these examples shows that compound material stick to the neural lamella and this cannot be removed by washing but it requires removal of the lamella prior to the compound quantification by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain in its cuticle shell was placed in a 0 . 9 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell . three brains were placed in a test tube containing 100 ul pbs . 200 ul acn was added to each tube . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 1 . 3 ug / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain in its cuticle shell was placed in a 0 . 9 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell . to stain the neural lamella evans blue was added and the neural lamella was removed . three brains were placed in each test tube containing 100 ul pbs . 200 ul acn was added to each tube . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 0 . 6 ug / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain in its cuticle shell was placed in a 0 . 1 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell . three brains were placed in a test tube containing 100 ul pbs . 200 ul acn was added to each tube . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 217 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain in its cuticle shell was placed in a 0 . 1 mg / ml mianserin solution for 5 minutes . the brain was prepared in saline and removed from the cuticle shell . to stain the neural lamella evans blue was added and the neural lamella was removed . three brains were placed in each test tube containing 100 ul pbs . 200 ul acn was added to each tube . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 127 ng / ml was measured by lcms . example f - i show that inclusion of the neural lamella has a large impact on the measured brain concentration . in the 0 . 9 mg / ml mianserin solution the [ with lamella ]/[ without lamella ] ratio is 2 . 3 while the ratio when using the 0 . 1 mg / ml mianserin solution is 1 . 7 , i . e . the ratios are at similar levels . moreover , the results show that the brain concentration increases when the brains are exposed to higher concentrations of the solution . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 95 mg / ml solution of mianserin . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 1439 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 9 mg / ml solution of a trazodone . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average trazodone concentration of 1317 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 67 mg / ml solution of a buspirone . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average buspirone concentration of 909 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 78 mg / ml solution of a caffeine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average caffeine concentration of 1533 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 72 mg / ml solution of a propranolol . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average propranolol concentration of 674 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 93 mg / ml solution of a colchicine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average colchicine concentration of 297 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 2 mg / ml solution of a atenolol . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average atenolol concentration of 23 ng / ml was measured by lcms . example j - p shows that the cns active compounds ( mianserin , caffeine , trazodone , buspirone , and propranolol ) permeate the grasshopper bbb to a much larger extent than peripheral acting drugs ( i . e . colchicine and atenolol ). thus , dissecting the grasshopper brain and placing the brain in a microtiter well containing a solution of a compound of interest is a useful model for determining whether the compound of interest permeate the vertebrate bbb . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 1 mg / ml solution of mianserin . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 105 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 3 mg / ml solution of mianserin . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 174 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 1 mg / ml solution of mianserin . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 100 ul distilled h2o and 200 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average mianserin concentration of 362 ng / ml was measured by lcms . example q - s shows the mianserin brain concentration increases when the brain is exposed to higher mianserin concentrations ( see fig1 ). a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 001 mg / ml solution of quinidine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration measured by lcms was below detection level a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 01 mg / ml solution of quinidine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration measured by lcms was below detection level . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 1 mg / ml solution of quinidine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration was of 47 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well with an approximately 1 . 0 mg / ml solution of quinidine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration was of 812 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well containing a mixture of verapamil ( 0 . 5 mg / ml ) and quinidine ( 0 . 001 mg / ml ). to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration was of 3 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well containing a mixture of verapamil ( 0 . 5 mg / ml ) and quinidine ( 0 . 01 mg / ml ). to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration was of 11 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well containing a mixture of verapamil ( 0 . 5 mg / ml ) and quinidine ( 0 . 1 mg / ml ). to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration was of 52 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well containing a mixture of verapamil ( 0 . 5 mg / ml ) and quinidine ( 1 mg / ml ). to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average quinidine concentration was of 684 ng / ml was measured by lcms . example t - aa shows when the locust brain is exposed to low concentrations of quinidine ( 0 . 001 mg / ml and 0 . 01 ) there is no uptake of quinidine in the locust brain but at higher concentrations there was there is a dose related increase , quinidine is a pgp - efflux substrate and the results shows that the pgp efflux transporter is saturated at high quinidine concentrations resulting in an increased uptake . co - treatment with the pgp inhibitor verapamil and the pgp substrate quinidine , significantly increased the uptake of quinidine at the low dose range while the uptake was unaffected at the high dose where the pgp proteins are expected to be saturated . a cut was made through the frontal part of the head of the blattodea . each brain was placed for 5 minutes in a microtiter well with an approximately 1 . 0 mg / ml solution of trazodone . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average trazodone concentration was of 768 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the blattodea . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 1 mg / ml solution of trazodone . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average trazodone concentration was of 127 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the blattodea . each brain was placed for 5 minutes in a microtiter well with an approximately 1 . 0 mg / ml solution of atenolol . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average atenolol concentration was of 294 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the blattodea . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 1 mg / ml solution of atenolol . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average atenolol concentration was of 27 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the blattodea . each brain was placed for 5 minutes in a microtiter well with an approximately 1 . 0 mg / ml solution of caffeine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average caffeine concentration was of 1028 ng / ml was measured by lcms . a cut was made through the frontal part of the head of the blattodea . each brain was placed for 5 minutes in a microtiter well with an approximately 0 . 1 mg / ml solution of caffeine . to stain the neural lamella evans blue was added and the neural lamella was removed . two brains are placed in each test tube and 50 ul distilled h2o and 100 ul acn is added . the tubes were placed in a sonicator and the brains were disintegrated by approximately 5 seconds of sonication . the samples containing the disintegrated brains were centrifuged for 5 minutes ( 10000 g at 4 ° c .). 100 ul of the supernatant was placed in a new test tube and the average caffeine concentration was of 152 ng / ml was measured by lcms . example ab - ag shows that the cns active compounds ( caffeine and trazodone ) permeate the blattodea bbb to a much larger extent than atenolol , which is a peripheral acting drug . thus , dissecting the blattodea brain and placing the brain in a microtiter well containing a solution of a compound of interest is a useful model for determining whether the compound of interest permeate the vertebrate bbb . moreover , the examples above show that a higher compound concentration exposure increases the compound brain concentration of the three compounds . a cut was made through the frontal part of the head of the adult locusta migratoria . each brain was placed for 5 minutes in a microtiter well containing a mixture of evans blue . the neural lamella was removed from the brain . no visual signs of evans blue were seen in any of the brains , i . e . there is no significant permeation of evans blue through the locust brain barrier . abbott , 2005 dynamics of cns barriers : evolusion , differtentiation , and modulation . 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