Insects in Drug Discovery

Despite insects are living organisms they can be used in high to medium-throughput screening models and they merge as such the best of in vitro and in vivo ADMET modeling in pre-clinical discovery. N2MO’s solutions provide fast, consistent, predictive and cost-efficient in vivo data.

Insects provide the best of in vitro and in vivo ADME modeling:

  • High to medium-throughput ex vivo screening models.
  • Test on ready-made stock-solutions
  • Low variability, high reproducibility
  • Quality prediction in naturally developed dynamic test system
  • Reduced  laboratory animal testing
The ex vivo BBB permeability model enables BBB permeability studies of compounds at a constant brain exposure level. In this model setting, whole brains, complete with an intact brain barrier, are exposed to a test drug dissolved in buffer at a pre-defined concentration level.

The insect ex vivo BBB permeability model has been tested in an in-house proof of concept study where a number of known drugs were tested.

The Grasshopper: A Novel Model for Assessing Vertebrate Brain Uptake

Olga Andersson, Steen Honoré Hansen, Karin Hellman, Line Rørbæk Olsen, Gunnar Andersson, Lassina Badolo, Niels Svenstrup, and Peter Aadal Nielsen EntomoPharm R&D, Medicon Village, Lund, Sweden (O.A., K.H., G.A., P.A.N.); Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (S.H.H., L.R.O.); and Division of Discovery Chemistry and Drug Metabolism and Pharmacokinetics, H. Lundbeck A/S, Copenhagen, Denmark (L.B., N.S.) Received April 10, 2013; accepted May 10, 2013

ABSTRACT
The aim of the present study was to develop a blood-brain barrier (BBB) permeability model that is applicable in the drug discovery phase. The BBB ensures proper neural function, but it restricts many drugs from entering the brain, and this complicates the development of new drugs against central nervous system diseases. Many in vitro models have been developed to predict BBB permeability, but the permeability characteristics of the human BBB are notoriously complex and hard to predict.

Consequently, one single suitable BBB permeability screening model, which is generally applicable in the early drug discovery phase, does not yet exist. A new refined ex vivo insect-based BBB screening model that uses an intact, viable whole brain under controlled in vitro-like exposure conditions is presented.

This model uses intact brains from desert locusts, which are placed in a well containing the compound solubilized in an insect buffer. After a limited time, the brain is removed and the compound concentration in the brain is measured by conventional liquid chromatography-mass spectrometry. The data presented here include 25 known drugs, and the data show that the ex vivo insect model can be used to measure the brain uptake over the hemolymph-brain barrier of drugs and that the brain uptake shows linear correlation with in situ perfusion data obtainedinvertebrates.Moreover,this study shows that the insect ex vivo model is able to identify P-glycoprotein (Pgp) substrates, and the model allows differentiation between low-permeability compounds and compounds that are Pgp substrates.

Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model

Olga Andersson1, Liesbeth Badisco2, Ane Hakansson Hansen1, Steen Honore Hansen3, Karin Hellman1, Peter Aadal Nielsen1, Line Rørbæk Olsen3, Rik Verdonck2, N. Joan Abbott4, Jozef Vanden Broeck2 & Gunnar Andersson1.

ABSTRACT
In earlier studies insects were proposed as suitable models for vertebrate blood– brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain barrier of the desert locust (Schistocerca gregaria). In an in vivo study on the locust, we found an increased uptake of the two well-known Pgp substrates, rhodamine 123 and loperamide after co-administration with the Pgp inhibitors cyclosporine A or verapamil. Furthermore, ex vivo studies on isolated locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but significantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C and 30°C showed differences in brain uptake between Pgp-effluxed and passively diffusing compounds. The transcriptome data show a high degree of sequence identity of the locust Pgp transporter protein sequences to the human Pgp sequence (37%), as well as the presence of conserved domains. As in vertebrates, the locust brain–barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.


In Vitro P-glycoprotein Assays to Predict the in Vivo Interactions of P-glycoprotein with Drugs in the Central Nervous System

Bo Feng, Jessica B. Mills, Ralph E. Davidson, Rouchelle J. Mireles, John S. Janiszewski, Matthew D. Troutman, and Sonia M. de Morais

Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Global Research and Development, Groton, Connecticut

Received June 26, 2007; accepted October 22, 2007

ABSTRACT

Thirty-one structurally diverse marketed central nervous system (CNS)-active drugs, one active metabolite, and seven non-CNSactive compounds were tested in three P-glycoprotein (P-gp) in vitro assays: transwell assays using MDCK, human MDR1-MDCK, and mouse Mdr1a-MDCK cells, ATPase, and calcein AM inhibition. Additionally, the permeability for these compounds was measured in two in vitro models: parallel artificial membrane permeation assay and apical-to-basolateral apparent permeability in MDCK. The exposure of the same set of compounds in brain and plasma was measured in P-gp knock out (KO) and wild-type (WT) mice after subcutaneous administration. One drug and its metabolite, risperidone and 9-hydroxyrisperidone, of the 32 CNS compounds, and 6 of the 7 non-CNS drugs were determined to have positive efflux using ratio of ratios in MDR1-MDCK versus MDCK transwell as
says. Data from transwell studies correlated well with the brain to-plasma area under the curve ratios between P-gp KO and WT mice for the 32 CNS compounds. In addition, 3300 Pfizer compounds were tested in MDR1-MDCK and Mdr1a-MDCK transwell assays, with a good correlation (R2 0.92) between the efflux ratios in human MDR1-MDCK and mouse Mdr1a-MDCK cells. Permeability data showed that the majority of the 32 CNS compounds have moderate to high passive permeability. This work has demonstrated that in vitro transporter assays help in understanding the role of P-gp-mediated efflux activity in determining the disposition of CNS drugs in vivo, and the transwell assay is a valuable in vitro assay to evaluate human P-gp interaction with compounds for assessing brain penetration of new chemical entities to treat CNS disorders.

An Insect-Based Ex Vivo Blood Brain Barrier Efflux Assay

By Sonia Al-Qadi * Morten Schiott† Steen Honore Hansen‡ Peter Aadal Nielsen + Lassina Badolo•

Drug efflux activity of ABC transporters, at the human blood brain barrier (BBB), constitutes a crucial challenge for central nervous system (CNS) drug development. Accordingly, early screening of CNS drug candidates is pivotal to sort out those whose brain uptake is substantially affected by efflux activity. In this context, affordable, simple, high-throughput and predictive screening models are required. It has recently been proposed that the grasshopper (locust) could be exploited as an ex-vivo model for drug BBB permeability assessment, as it has shown some similarities to vertebrate models. The p-glycoprotein (p-gp), encoded by the ABCB1 gene, is described as the most potent efflux pump that modulates drug brain disposition, so identification and characterization of such a transporter in the locust model is essential to demonstrate its utility and validity for drug development. The present work entails transcriptomic profiling followed by amino acid-based homology analysis of locust genes, in parallel to functional investigations using rhodamine 123 as a selective p-gp substrate. A protein with high sequence similarity to ABCB1 was found in the locust brain transcriptome, which indicates a conserved mechanism of brain efflux activity between insects and vertebrates. Functionally, the developed locust model showed a kinetic behavior comparable to those obtained from in vitro cell models such as the MDCKII cells expressing p-gp. Overall, the locust ex-vivo BBB model holds promise as a cheap model with a high-throughput screening potential in the early discovery phase of CNS drugs.

Parameters like physiochemical properties are known to have an impact on passive diffusion across the BBB both in vivo and in vitro and it is driven by the concentration gradient of the compound across the barrier (Summerfield 2007). In vivo the compound is in equilibrium between bound and unbound states on both sides of the BBB. Thus, it is the concentration gradient of the free fractions in plasma and in the brain, which is responsible for the passive diffusion across the BBB (Figure 1). High brain tissue binding and low plasma protein binding will both lead towards increasing brain uptake. In addition, metabolism and protein mediated influx and efflux affects the concentration gradient and thereby the brain uptake. On the hand, protein mediated transport is affected by the fraction bound since only compound in its free state is expected to undergo protein mediated transport across the BBB. Thus, the brain uptake is influenced by multiple interfering parameters, which makes it hard to develop in vitro models that correlate with in vivo data.

Therefore, rodent models are frequently needed as tool to determine the brain uptake of new chemical entities. The downside by using rodents in the drug discovery phase is the required compound quantity, time and costs, which limits the throughput of the models. To increase the throughput most of the brain uptake studies in the drug discovery process is measured as the brain-plasma (B-P) ratio at single time point after single dosing. The B-P ratio only provides information about the compound brain concentration relative to the plasma concentration, i.e. it tells whether the compound has the ability to enter the brain. Using B-P as optimization parameter is likely to bias the compound design towards higher brain tissue binding, i.e. high lipophilicity (Figure 1). However, the therapeutic effect of a CNS drug depends on the compound affinity towards the target, the presence of the compound in the brain and the fraction unbound in the brain (Udenas). For this reason the brain availability (BA) parameter (1) is frequently used as a primary optimization parameter in the design of new CNS drugs.

BA=(B/P)*(Fub/Fup) (1)

BA includes both the B-P ratio, the free fractions in plasma (Fup) and in brain (Fub). Thus, BA is a simple estimate of the brain to plasma unbound drug concentration ratio (Kpuu), which allows identification of potential CNS drugs, i.e. compounds that permeate the BBB and with free fractions in the brain available for target engagement.

Recently, an ex vivo insect model was introduced to determine the brain uptake of different therapeutic agents (Nielsen 2011, Andersson 2013, Geldenhuys 2012). In the ex vivo insect model the brain is removed from the insect and placed in a well containing the substance of interest. This eliminates the effect of plasma protein binding and metabolism and the exposure concentration is constant. In the model, the brain is intact and all mechanisms and biological events inside the brain having effects on the compound brain uptake are still active. This includes brain tissue binding and protein mediated transport across the BBB, i.e. the measured insect brain concentration corresponds to the total brain concentration measured in rodent B-P studies.

In this study we use the brain uptake measured in insects and we show that the insect brain concentration is comparable to the total brain concentration obtained in rodents. By using the insect data in conjunction with free fractions measured in plasma and brain homogenates from mice we show that the ex vivo model can be used to estimate BA, i.e. an estimate of Kpuu without using rodent in vivo studies.

  • The measured insect brain concentration corresponds to B (in B/P)
  • Brain uptake of passively diffusing compounds is driven by the concentration gradient of the free concentrations (Conc*Fup and Conc*Fub)
  • Kpuu=(B/P)*(Fub/Fup) is used to avoid progression of lipophilic candidates

Using the ex vivo locust model to predict rodent in vivo data

Kpuu in rodents:

Kpuu(rodents)=(B/P)*(Fub/Fup)

As in rodents the brain tissue in insects consists of proteins and fat – thus to estimate Kpuu in insects we can use Fub and Fup obtained in rodent homogenates

Kpuu in Insects can be estimated as:

Kpuu(insects)=(Ctot)*(Fub/Fup)

Identification of a Functional Homolog of the Mammalian CYP3A4 in Locusts

Line Rørbæk Olsen, Charlotte Gabel-Jensen, Peter Aadal Nielsen, Steen Honoré Hansen, and Lassina Badolo

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (L.R.O., C.G.-J., S.H.H.); Department of Discovery ADME, H. Lundbeck A/S, Valby, Denmark (L.B.); and EntomoPharm R&D, Lund, Sweden (P.A.N.)

Received January 31, 2014; accepted April 28, 2014

ABSTRACT

Insects have been proposed as a new tool in early drug development. It was recently demonstrated that locusts have an efflux transporter localized in the blood-brain barrier (BBB) that is functionally similar to the mammalian P-glycoprotein efflux transporter. Two insect BBB models have been put forward, an ex vivo model and an in vivo model.

To use the in vivo model it is necessary to fully characterize the locust as an entire organism with regards to metabolic pathways and excretion rate. In the present study, we have characterized the locust metabolism of terfenadine, a compound that in humans is specific to the cytochrome P450 enzyme 3A4. Using high-resolution mass spectrometry coupled to ultra-high-performance liquid chromatography, we have detected metabolites identical to human metabolites of terfenadine. The formation of human metabolites in locusts was inhibited by ketoconazole, a mammalian CYP3A4 inhibitor, suggesting that the enzyme responsible for the human metabolite formation in locusts is functionally similar to human CYP3A4. Besides the human metabolites of terfenadine, additional metabolites were formed in locusts. These were tentatively identified as phosphate and glucose conjugates.

In conclusion, not only may locusts be a model useful for determining BBB permeation, but possibly insects could be used in metabolism investigation. However, extensive characterization of the insect model is necessary to determine its applicability.

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