Patent Application: US-201013387083-A

Abstract:
there is provided insect screening models to determine gastrointestinal absorption of different chemical compounds in vertebrates , and in particular humans , in order to improve the compound screening procedures / processes in the early drug discovery process . this 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 a new methodology for initial assessment and prediction of intestinal uptake of various chemical compounds . the invention is generally particular useful for efficient screening of newly synthesized compounds in the early phase of drug discovery . 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 . hanstrom ; forlagshuset norden a b , malmö , 1964 ): the invention will also relate to the following orders comprising insect species relevant for the intestinal uptake assessment method : the present invention preferably uses large insects , such as the migratory locust , locusta migratoria and the desert locust , schistocera gregaria or cockroach where it &# 39 ; s feasible to administer test compounds and subsequently take hemolymph for analyses of the concentration of the absorbed compound . the locust has been used to develop the model since it &# 39 ; s a large insect and has an alimentary tract and a midgut that is very well characterised . accordingly , the present invention focuses on insect models that are aimed to reflect the vertebrate intestinal uptake of the test compounds . investigations of the intestinal uptake profile are of extreme importance in compound selection during the early phase of drug discovery . in accordance with a preferred embodiment of present invention the migratory locust , locusta migratoria and / or the desert locust , schistocera gregaria , is used since it is easy to breed and it is a relatively large insect with relevant size of the midgut and hemolymph volumes relevant for quantitative measurement of concentrations of compounds taken up . 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 . after decapitation , test compounds are administrated via a catheter or a probe inserted into the midgut of the animal . at various times after administration hemolymph samples are taken for quantitative determination of drug concentration in the hemolymph . the samples are snap - frozen and stored until analyses . drug concentration is analysed by hplc , lc / msms or other methods . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of aminomodified fluorescent polystyrene nano particles ( 100 nm ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration hemolymph samples were collected with a calibrated ( 10 ul ) capillary tube by puncturing the ventral membrane anterior to the thorax . the hemolymph samples were analyzed by fluorescence microscopy and no fluorescent nano particles were detectable in the hemolymph samples . example 1 shows that the insect intestine is a functional barrier , which prevents large polystyrene nano particles administered orally to enter the hemolymph . this reflects the functionality of the vertebrate intestinal barrier which also prevents diffusion of orally administered large molecules into the blood . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of quinidine ( 0 . 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the analysis did not show any detectable quantity of quinidine at 10 or 30 minutes . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of quinidine ( 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average quinidine concentrations measured in the hemolymph after 10 and 30 minutes were 2960 ng / ml and 1465 ng / ml respectively . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of propranolol ( 0 . 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average propranolol concentrations measured in the hemolymph after 10 and 30 minutes were 102 ng / ml and 61 ng / ml respectively . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of propranolol ( 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average propranolol concentrations measured in the hemolymph after 10 and 30 minutes were 1081 ng / ml and 1564 ng / ml respectively . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of caffeine ( 0 . 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average caffeine concentrations measured in the hemolymph after 10 and 30 minutes were 177 ng / ml and 276 ng / ml respectively . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of caffeine ( 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average caffeine concentrations measured in the hemolymph after 10 and 30 minutes were 1154 ng / ml and 1655 ng / ml respectively . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of atenolol ( 0 . 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average atenolol concentrations measured in the hemolymph after 10 and 30 minutes were 458 ng / ml and 318 ng / ml respectively . the head of male locusts ( locusta migratoria ), which have been starving for 12 hours , was cut off and a plastic catheter was inserted under microscope into the esophagus . 20 ul of atenolol ( 1 mg / ml ) was administered via the catheter by using a hamilton syringe . 10 and 30 minutes after administration ( 10 ul ) hemolymph was collected with a calibrated capillary tube by puncturing the ventral membrane anterior to the thorax and immediately blown into a tube containing 40 ul aqua dest and 100 ul of acetonitrile . each sample was centrifuged for 5 minutes ( 10 000 g at 4 ° c .). 100 ul of the supernatants were transferred to new test tubes for lcms analysis . the average atenolol concentrations measured in the hemolymph after 10 and 30 minutes were 336 ng / ml and 121 ng / ml respectively . example 2 - 9 include the compounds : quinidine , a pgp substrate that can be expected to be poorly taken up at low doses due to an efflux mechanism ; caffeine , a compound of high permeability that is transcellularly taken up by passive diffusion ; propranolol and atenolol , two beta blockers with markedly different adme properties . from the examples 2 - 9 it is seen that at low ( 0 . 1 mg / ml ) concentration there was no detectable uptake of quinidine in the hemolymph . however , after caffeine administration the average hemolymph concentration was 177 ng / ml and 276 ng / ml after 10 and 30 minutes respectively . the average hemolymph concentration after 10 minutes of the two beta blockers propranolol and atenolol were 177 ng / ml and 458 ng / ml , respectively . at the high ( 1 . 0 mg / ml ) concentration the average hemolymph concentration of quinidine was 2960 ng / ml after 10 minutes . also after 10 minutes the average caffeine concentration was 1154 ng / ml and for the two beta blockers , propranolol and atenolol the average concentrations were 1080 ng / ml and 336 ng / ml , respectively . thus , as expected there was a marked uptake of caffeine following low dose administration . also as expected there was no detectable uptake of the pgp substrate compound quinidine at the low concentration . however , at the high dose where the efflux transporter is saturated there is a high permeability for quinidine in agreement with the fda classification of this drug . the hemolymph concentration at the low dose ( 0 . 1 mg / ml ) was lower for the high permeability beta blocker propranolol compared to the low permeability blocker atenolol . however , human studies have shown that the longer half life and smaller volume of distribution results also in human in higher plasma atenolol concentrations compared to propranolol ( carmona et al . 2009 ). thus the present data obtained in the locust model highly correlate to data obtained in humans . at the high dose ( 1 . 0 mg / ml ), there was a dose related increase in caffeine and propranolol hemolymph concentrations . however , there was no increase in atenolol uptake . 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