Abstract:
There is provided an ex-vivo insect screening model to accurately determine blood-brain barrier penetration of different nanoparticles 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:
FIELD OF THE INVENTION 
       [0001]    The present invention is directed to insect models that are aimed to reflect vertebrate blood-brain barrier (BBB) penetration of nanoparticles. On the other hand BBB penetration of nanoparticles may also cause side effects. Specifically, the present invention relates to the use of insects in screening for BBB penetration of nanoparticles. 
       BACKGROUND OF THE INVENTION 
       [0002]    Although there are anecdotal data indicating a causal relationship between long-term ultrafine particle exposures in ambient air (e.g., traffic related) or at the workplace (e.g., metal fumes) and resultant neurotoxic effects in humans, more studies are needed to test the hypothesis that inhaled nanoparticles (NP) or NPs absorbed via food cause neurodegenerative effects. Some NPs may have a significant environmental safety (hazard) potential, and this will pose a significant risk if there is a sufficient exposure. The challenge is to identify such hazardous NPs and take appropriate measures to prevent exposure. 
         [0003]    It has been shown that certain NPs do permeate the BBB and in this relation it is important that the NPs are readily cleared from the brain such that the NPs do not cause any brain damage. Hindering the NP from entering the brain is not straight forward since the mechanism describing how the NPs permeate the BBB still is under debate. However, it is of utmost importance to identify NPs that permeate the BBB in order to address potential toxicological issues or to use the NP as carrier of a drug. 
         [0004]    In relation to fighting CNS brain disorders NPs have shown promising as carriers of drugs that could not otherwise have passed the blood brain barrier. Despite enormous advances in brain research, CNS brain disorders still remain accountable for a high number of hospitalizations requiring prolonged care. It is estimated that approximately 1.5 billion people worldwide are suffering from various CNS disorders, such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, multiple sclerosis, HIV-dementia and stroke, among others. 
         [0005]    The blood-brain barrier (BBB) has always presented a challenge to scientists for brain drug targeting. The BBB has evolved in such a way that it protects the brain from various foreign substances such as neurotoxins. This mechanism makes the BBB an insurmountable barrier for numerous highly essential drugs, including antibiotics, cytostatics and other CNS-active drugs. 
         [0006]    Many strategies have been developed to overcome the hurdles caused by the BBB. They include both invasive and noninvasive approaches. The invasive approaches include the temporary disruption of the BBB, which allows the entry of drugs to the brain, and direct drug delivery to the brain by means of intraventricular or intracerebral injections, and intracerebral polymeric implants. The noninvasive approaches use colloidal drug carriers. 
         [0007]    Among the non-invasive approaches, polymeric nanoparticles, especially poly(butylcyanoacrylate) (PBCA) nanoparticles coated with polysorbate 80, have recently received much attention from neuroscientists as an attractive and innovative carrier for brain targeting. These nanoparticles may be defined as a submicron drug-carrier system, which are generally polymeric in nature. Since nanoparticles are small in size, they easily penetrate into small capillaries and can be taken up within cells, allowing efficient drug accumulation at targeted sites in the body. The first reported nanoparticles were based on non-biodegradable polymeric systems. Their use for systemic administration, however, could not be considered because of the possibility of chronic toxicity due to the tissue and immunological response towards the non-biodegradable polymer. Hence, nanoparticles prepared with biodegradable polymers such as poly(cyanoacrylate) were exclusively studied. The use of biodegradable materials for nanoparticle preparation allows sustained drug release at the targeted site over a period of days or even weeks after injection. 
         [0008]    Investigation of BBB permeation of nanoparticles is extremely important since there is an increasing use of nanoparticles and the profile for many of these are yet to be understood. Moreover, nanoparticles are important in drug discovery as they have proven to be useful as carriers for potential CNS drugs. On the one hand successful CNS drugs have to cross the BBB. Certain insects may be suitable as model organisms for studying BBB penetration of NPs. Insects are multi cell organisms with complex compartmentalized nervous systems for specialized functions like vision, olfaction, learning, and memory. The nervous systems of the insects respond physiologically in similar ways as in vertebrates with many identical neurohormones and receptors. Insects have avascular nervous systems in which hemolymph bathes all outer surfaces of ganglia and nerves. Therefore, many insects require a sophisticated BBB system to protect their CNS from plant-derived neurotoxins and to maintain an appropriate ionic microenvironment of the neurons. In fact, also in insects a sophisticated BBB system has been an evolutionary advantage. In insects this BBB is mainly based on the glia cell system which certainly shifted to the endothelial system as a response to the increased importance of the microvasculature in the vertebrate brain. In support of this view is the appearance of the glia system in elasmobranch fish and the remnants of their glia barrier in modern mammalian CNS. Thus, insects possess a BBB which is an important component in the ensheathment of the nervous system. The BBBs in insects are highly sophisticated but varies in structure between different insect orders. Thus insects with highly sophisticated brain barriers with complex integrative components that mimic the vertebrate barriers will be excellent models for documentation of penetration of various molecules through this structure. 
         [0009]    Thus, there is a need for efficient screening of NPs permeability of the BBB both in order to address the environmental safety of NP as well as identifying NPs that can be used as carriers of drugs targeting CNS related diseases. This screening is preferentially performed in insect models with intact BBB function and this will contribute to a positive selection of NPs that are non toxic to vertebrates. 
       SUMMARY OF THE INVENTION 
       [0010]    The object of the present invention is to develop an ex-vivo insect screening model to accurately assess blood-brain barrier penetration of different NPs in order to improve the screening procedures/processes. 
         [0011]    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. Finally, it is an object of the present invention that only the penetration trough the blood-brain barrier is determined, without any interference from liver metabolism, accumulation in other tissues etc. 
         [0012]    Accordingly, there is provided a method of conducting blood-brain barrier penetration studies of a NP in an insect, said method comprising the steps:
       optionally anesthetizing the insect;   fixing the head of the insect;   dissecting out the dorsal part of the insect head so as to expose the brain, eyes, antennas, and nerve associations;   treating the brain while in its cuticle shell with a solution of the NP; and   removing the brain from its cuticle;   washing and homogenising the brain; and   determining the concentration of the NP in the homogenised brain material.       
 
         [0020]    In a preferred embodiment of the present invention albumin is added to the solution of the NP to introduce the plasma protein binding and the effect of the plasma protein binding upon the nanoparticle&#39;s BBB penetration (free vs. protein bound nanoparticles). Preferably, hemolymph from the insect is also added to the NP sample either alone or in combination with albumin. It may be necessary to add an anti-coagulation agent to the hemolymph. 
         [0021]    In another preferred embodiment of the present invention hemolymph is added to the solution of the NP to introduce the plasma protein binding and the effect of the plasma protein binding upon the nanoparticle&#39;s BBB penetration (free vs. protein bound nanoparticles). 
         [0022]    In yet another preferred embodiment of the present invention hemolymph and albumin is added to the solution of the NP to introduce the plasma protein binding and the effect of the plasma protein binding upon the nanoparticle&#39;s BBB penetration (free vs. protein bound nanoparticles). 
         [0023]    Preferable the concentration of the NP is determined by LC/MS or ICP-MS. In this respect the determination of the concentration of the NP is performed by homogenizing or ultra sound disintegration (UD) of the dissected brains, and analyzing the concentration of the test agent in the homogenate by liquid chromatography with mass spectrometric detection of the eluted compounds, by ICP-MS or fluorescent microscopy. 
         [0024]    In order to ensure optimum penetration of the NP, the brain is treated with a solution of the NP for a period of 1 min. to 2 weeks, preferably to 1 week, more preferably to 2 days, most preferably to 12 hours In a particularly preferred embodiment of the present invention the neural lamella of the brain is removed before the brain is subjected to the NP. 
         [0025]    In addition to the method of the present invention there is further provided a model for conducting blood-brain barrier penetration studies of a NP, said model obtained by a method comprising the steps:
       optionally anesthetizing an insect having a BBB;   fixing the head of the insect; and   dissecting out the dorsal part of the insect head so as to expose the brain, eyes, antennas, and nerve associations; and   optionally removing the neural lamella, which is surrounding the BBB;       
 
         [0030]    In a preferred embodiment of the present invention the NPs are made less prone to aggregate. Thereby the NPs are also more easily taken up by macrophages (monocytes); this is referred to as opsonization and may be made by mixing the NPs with albumin, such as BSA, possibly in combination with hemolymph. 
         [0031]    The method of the present invention permits the exposure to an insect brain of a NP at a stable concentration during the entire period of exposure. As appears from above the insect model used in the method consists of the dorsal part of the insect head dissected out to consist of the brain, eyes, antennas and the nerve associations between these sense organs. Still in its cuticle shell the brain will be treated with different NPs for various times. The penetration of the test NP over the BBB into the brain is determined as the concentration (amount) of the nanoparticle measured in the isolated brain and preferably determined by LC/MS or ICP-MS. Alternatively the BBB permeability may be determined by fluorescent microscopy. The model is aimed as an early stage test of NPs ability to pass the BBB at a well defined and constant exposure concentration. 
         [0032]    Preferably the dissected brains are homogenized or disintegrated by ultra sound or other methods and eventually lysed in order to obtain a homogeneous liquid reflecting the composition of the brains. The liquid is centrifuged and the supernatant stored until analysis. The further analysis of the liquid may be performed by virtue of liquid chromatography, possibly with mass spectrometric detection of the eluted compounds. 
         [0033]    Alternatively, the presence of NPs in the brain may be indentified and quantified by histochemical methods. 
         [0034]    In various aspects and embodiments the present invention provides the subject-matter set out in the claims below. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0035]    The present invention provides a new methodology for screening nanoparticle&#39;s ability to penetrate the BBB. Certain NPs do permeate the BBB and in this relation it is important that the NPs are readily cleared from the brain such that the NPs do not cause any brain damage. Hindering the NP from entering the brain is not straight forward since the mechanism describing how the NPs permeate the BBB still is debated. However, it is of utmost importance to identify NPs that permeate the BBB in order address potential toxicological issues or to use the NP as carrier of a drug. 
         [0036]    The invention is generally useful for investigating the safety profile of NPs including NPs 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, Aggression and Brain tumors. 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. 
         [0037]    In preferred embodiments the nanoparticles of the present invention is less than 100 nm. 
         [0038]    There are several methods for creating nanoparticles, including both attrition and pyrolysis. In attrition, macro or micro scale particles are ground in a ball mill or other size reducing mechanism. Thermal plasma can also deliver the energy necessary to cause evaporation of small micrometer size particles. Inert-gas condensation is frequently used to make nanoparticles from metals with low melting points. The metal is vaporized in a vacuum chamber and then supercooled with an inert gas stream. The supercooled metal vapor condenses into nanometer-sized particles, which can be entrained in the inert gas stream and deposited on a substrate or studied in situ. 
         [0039]    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 A B, Maölmö, 1964): 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Order 
                 Suborder/family 
                 Comment 
               
               
                   
                   
               
             
             
               
                   
                 Dictyoptera 
                 Blattodea 
                 Cockroach 
               
               
                   
                   
                 Mantoidea 
               
               
                   
                 Orthoptera 
                 Grylloidea 
                 Crickets 
               
               
                   
                   
                 Acridoidea 
                 Grasshoppers 
               
               
                   
                 Cheleutoptera 
                   
                 Stick insects 
               
               
                   
                 Lepidoptera 
                   
                 Moths 
               
               
                   
                 Hymenoptera 
                 Formicoidea 
                 Ants 
               
               
                   
                   
                 Vespoidea 
                 Wasps 
               
               
                   
                   
                 Apoidea 
                 Bee like 
               
               
                   
                   
                   
                 Hymenopterans 
               
               
                   
                   
                 Bombinae 
                 Bumble-bees 
               
               
                   
                   
                 Apine 
                 Proper bees 
               
               
                   
                 Odonata 
                   
                 Dragonflies 
               
               
                   
                 Diptera 
                 Nematocera 
                 Mosquitos 
               
               
                   
                   
                 Brachycera 
                 Flies E.g 
               
               
                   
                   
                   
                 
                   Drosophila 
                 
               
               
                   
                   
               
             
          
         
       
     
         [0040]    In particular the invention relates to insect species selected from  Blattodea, Acridoidea, Cheleutoptera, Brachycera  and  Lepidoptera  and most particular  Acridoidea  ( Locusta migratoria  and  Schistocera gregaria ) are preferred. 
         [0041]    The invention will also relate to the following orders comprising insect species relevant for the method of the present invention: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Order 
                 Suborder/family 
                 Comment 
               
               
                   
                   
               
             
             
               
                   
                 Ephemerida 
                   
                 Mayflies 
               
               
                   
                 Plecoptera 
               
               
                   
                 Dermoptera 
                 Forficuloidea 
                 Earwigs 
               
               
                   
                 Homoptera 
                 Cicadinea 
                 Cicadas 
               
               
                   
                   
                 Aphidine 
                 Plant-louse 
               
               
                   
                 Heteroptera 
                   
                 Hemipteran 
               
               
                   
                 Coleoptera 
                   
                 Beetles 
               
               
                   
                 Trichoptera 
                   
                 Caddis fly 
               
               
                   
                   
               
             
          
         
       
     
         [0042]    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 are preferred. The locust has been used to develop screening models to determine BBB penetration of different NPs and compare this model with existing literature data from conventional in vivo or in situ vertebrate studies. 
         [0043]    In accordance with a preferred embodiment the migratoty 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 (40-60 mm long, weight: approx. 2 g, hemolymph volume: approx. 300 μL, brain weight: approx. 2 mg). 
         [0044]    The application of nanoparticles to insects in a screening method may be as follows, in accordance with a preferred embodiment of the present invention. 
       EXAMPLES 
       [0045]    1. In a preferred embodiment 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°-34° and a 12:12 dark: light photo cycle 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 NP. 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 disintegrated and the sample is frozen until analyses. NP presence is analysed by HPLC, LC/MSMS, ICP-MS or other methods. 
         [0046]    2. In a preferred embodiment 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°-34° and a 12:12 dark: light photo cycle 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 NP. 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 fixated and prepared for histology. The presence of NPs in the brain is analysed by fluorescense microscopy, ICP-MS or other methods. 
         [0047]    3. In a preferred embodiment 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°-34° C. and a 12:12 dark: light photo cycle 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. The neural lamella is removed and the preparation placed in a well of a microtitre plate containing the NP and 4.2% bovine serum albumin. After various times of exposure the preparation is washed in saline. The brain is lysed and the samples are sent for analysis to determine the NP permeability by ICP-MS, microscopy or other methods. 
         [0048]    4. In a preferred embodiment 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°-34° C. and a 12:12 dark: light photo cycle 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. The neural lamella is removed and the preparation placed in a well of a microtitre plate containing the NP in hemolymph and 4.2% bovine serum albumin. After various times of exposure the preparation is washed in saline. The brain is lysed and the samples are sent for analysis to determine the NP permeability by ICP-MS, microscopy or other methods. 
         [0049]    5. In a preferred embodiment 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°-34° C. and a 12:12 dark: light photo cycle 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. The neural lamella is removed and the preparation placed in a well of a microtitre plate containing the NP and hemolymph. After various times of exposure the preparation is washed in saline. The brain is lysed and the samples are sent for analysis to determine the NP permeability by ICP-MS, microscopy or other methods. 
         [0050]    6. In a preferred embodiment 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°-34° and a 12:12 dark: light photo cycle 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. The neural lamella is removed and the preparation placed in a well of a microtitre plate containing the NP. After various times of exposure the preparation is washed in saline. The brain is lysed and the samples are sent for analysis to determine the NP permeability by ICP-MS, microscopy or other methods. 
         [0051]    In the following the present invention is exemplified in further detail. 
       Example A 
       [0052]    A cut was made through the frontal part of the head of female locusts. Each brain in its cuticle was placed in a silver nanoparticle (SNP; 80 nm) solution for 3 hours. The brains were dissected in saline, the neural lamella removed, washed twice in saline and then placed in tubes containing 100 ul nitric acid. The average content of silver (Ag) per brain was 5.61 ng as measured by ICP-MS. Average background content of Ag in control brains was 0.36 ng per brain. 
       Example B 
       [0053]    Locust brains were dissected in saline and the neural lamella removed. The brains were placed in a SNP (80 nm) solution for 3 hours. The brains were washed twice in saline and then placed in tubes containing 100 ul nitric acid. The average content of Ag per brain was 21.7 ng as measured by ICP-MS. 
       Example C 
       [0054]    Locust brains were dissected in saline and the neural lamella removed. The brains were placed in solutions containing a fluorescent polystyrene amino modified 100 nm nanoparticle and exposed for 3 hours. There was a marked uptake in the locust brain barrier and also strong indications of a deterioration of the brain barrier. 
       CONCLUSION 
       [0055]    Ex vivo modeling in the locust clearly indicate the utility of the model in studies of brain permeation with reference to nanoparticles with different chemical properties. The observations of effects on brain barrier integrity are of extraordinary importance since the effects of nanoparticles on brain barrier permeation due to functional deterioration is not fully understood. 
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