Compound-carrier systems for assays in nematodes

Abstract

The present invention relates to methods for increasing absorption of compounds of interest by nematode worms through the design of carrier systems containing said compound of interest and expressing chemoattractive tags at their surface.

Claims

1. A method for assessing the toxicity of a compound of interest comprising the steps of: (a) feeding a nematode with a nutrient composition comprising a carrier system, wherein the carrier system comprises a liposome containing said compound, wherein the liposome presents, on its surface, a tag substance that is chemo-attractive to said nematode, wherein the tag substance is selected from the group consisting of i) bacterial autoinducers, amino acids, nucleotides and vitamins; and ii) a whole bacterium linked to the carrier system by a linker substance, wherein ingestion of the compound in the carrier system by the nematode is increased as compared to ingestion of the compound without the carrier system; and (b) assessing whether the compound of interest reduces nematode survival, negatively impacts egg laying behavior or gonad functionality, increases embryonic lethality, or increases neuronal death, wherein a compound of interest which reduces nematode survival, which negatively impacts egg laying behavior or gonad functionality, increases embryonic lethality, or increases neuronal death, is a compound which is toxic, reprotoxic, teratogenic, or neurotoxic, respectively.

2. The method of claim 1, wherein the carrier system further comprises a fluorescent marker encapsulated in said liposome and wherein the fluorescence intensity of the co-absorbed fluorescent marker is measured thereby allowing quantification of the absorbed compound of interest.

3. The method of claim 1, wherein said tag substance is covalently linked to the surface of said liposome.

4. The method of claim 1, wherein said tag substance is selected from the group consisting of AI-2 and acylated homoserine lactones.

5. The method of claim 1, wherein said tag substance is biotin.

6. The method of claim 1, wherein said tag substance is a whole bacterium linked to the carrier system by a linker substance, and wherein said linker substance comprises maltose-based polysaccharides, wherein said maltose-based polysaccharides are polymers of covalently linked repeated units of maltose chosen from the group consisting of linear polymers, cyclic polymers, branched polymers and mixtures thereof.

7. The method of claim 6, wherein said linker substance is a maltodextrin or a maltodextrin analogue with a molecular weight up to 2000 g/mol.

8. The method of claim 5, wherein said tag substance is biotin and wherein said nutrient composition further comprises biotin and a biotin-binding protein which comprises at least two biotin-binding sites.

9. A method for promoting uptake of a compound of interest by a nematode comprising feeding a nematode with a carrier system, wherein the carrier system comprises a liposome containing said compound, wherein the liposome presents, on its surface, a tag substance that is chemo-attractive to said nematode, wherein the tag substance is selected from the group consisting of i) bacterial autoinducers, amino acids, nucleotides and vitamins; and ii) a whole bacterium linked to the carrier system by a linker substance, wherein ingestion of the compound in the carrier system by the nematode is increased as compared to ingestion of the compound without the carrier system.

10. The method of claim 9, wherein said tag substance is covalently linked to the surface of said liposome.

11. The method of claim 9, wherein said tag substance is selected from the group consisting of AI-2 and acylated homoserine lactones.

12. The method of claim 9, wherein said tag substance is biotin.

13. The method of claim 9, wherein said tag substance is a whole bacterium linked to the carrier system by a linker substance, and wherein said linker substance comprises maltose-based polysaccharides, wherein said maltose-based polysaccharides are polymers of covalently linked repeated units of maltose chosen from the group consisting of linear polymers, cyclic polymers, branched polymers and mixtures thereof.

14. The method of claim 13, wherein said linker substance is a maltodextrin or a maltodextrin analogue with a molecular weight up to 2000 g/mol.

15. The method of claim 12, wherein said tag substance is biotin and wherein said nutrient composition further comprises biotin and a biotin-binding protein which comprises at least two biotin-binding sites.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic representation of the different tags designed to be grafted at the carrier surface.

(2) FIG. 2. Succinimidyl N-(8-Carboxy-3-oxooctanoyl)-L-homoserine Lactone that is one of the most worm attractive serine lactones [5].

(3) FIG. 3. N-(8-Carboxy-3-oxooctanoyl)-L-homoserine Lactone grafted on DSPE-PEG-NH2 (chlorhydrate form=—NH3+, Cl—).

EXAMPLES

Materials

(4) Hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phospho ethanolamine-N-[PEG (2000)] conjugate (DSPE-PEG), and DSPE-PEG(2000)Amine (DSPE-PEG-NH2) were obtained from Northern Lipids Inc (Vancouver, BC, Canada). Cholesterol was obtained from Sigma, (St Louis, Mo.).

(5) Drug to be loaded inside liposomes can be selected within the list of 1000 compounds with tested bioaccumulation inside C. elegans previously published by Burns et al in 2010. Other compounds of interest, as described above, can also be used.

(6) In order to evaluate the efficiency of the present invention, two sets of compounds may be tested: those known as easily accumulated in C. elegans to concentrations greater than 50% of that present in the worm's environment, and those that cannot evade the worm's defenses and never penetrate in the animal.

(7) Selection of attracting substances sniffed by the worm in the medium (N-acylhomoserine lactone (AHL)) was done based on the work of Beale et al published in 2006 [5] (FIG. 2). These were synthesized as previously described [Chhabra, 1993; Chhabra, 2003]. They were then grafted on DSPE-PEG-NH2.

(8) 1. Preparation of Liposomes

(9) Liposomes were composed of HSPC, cholesterol, and DSPE-PEG-NH2 in the molar ratio of 65:30:5, respectively.

(10) Accurately weighed amounts of lipids (325 μmol HSPC, 150 μmol cholesterol, and 25 μmol DSPE-PEG) and drug (100 μmol) were dissolved in chloroform:methanol (9:1 vol/vol) in a round-bottom flask.

(11) After mixing, solvent was evaporated under reduced pressure and constant rotation (Rotovapor R-200, Buchi, Flawil, Switzerland) to form a thin lipid film. The lipid film was then hydrated with 50 mM HEPES/150 mM NaCl-buffer pH 6.5 (5 mL) at 62° C. for 2 hours to form large multilamellar vesicles (MLV) at 100 mM total lipid concentration.

(12) The resulting MLV were then sized by repeated extrusion (Lipex extruder, Northern Lipids) through polycarbonate membranes (Nucleopore, Whatman, N.J.) of gradually decreasing pore size (0.8, 0.4, 0.2, and 0.1 μm) to prepare small unilamellar liposomes of ˜100 nm in diameter [Hope, 1985].

(13) Extrusions were performed in a 10-mL size thermobarrel extruder at 62° C. After extrusion, liposomes were stored at 4° C. until used in subsequent experiments.

(14) Liposomes were loaded with fluorescent dye in order to control the success of oral delivery of chemical chemicals into the intestines of C. elegans. Based on the experiments of Shibamura A et al [2009], 25 ml of liposomes containing 50 mg of uranin allow an amount of fluorescent dye absorption in 3 h 100-fold higher as compared with worms administered dye by conventional methods. In addition, fluorescent dye liposome incorporation allows calibrating the amount of drug accumulated by worms.

(15) 2. Preparation of Tagged Liposomes

(16) For preparation of tagged liposomes, AHL derivatives were coupled to the distal end of PEG chains on the liposomes. To enable this ligand coupling, a part of (2 mol %) DSPE-PEG in the liposome formulation was replaced with DSPE-PEG-NH.sub.2 functional lipid. Total lipid concentration of the liposomal dispersion used for the coupling reaction was 100 mM. AHL was dissolved in 50 mM HEPES buffer, pH 6.5 at 2.7 μmol/mL concentration (0.1 mL), were reacted with liposome (4 mL) with NH.sub.2 functional groups on the distal end of PEG chains at pH 6.5 and a molar ratio of 1:30 (AHL) for 12 hours at room temperature (25° C.). A schematic representation of the coupling reaction is given in FIG. 3.

(17) 3. HPLC Determination of AHL Coupling to the Liposomes

(18) Attachment of AHL to the liposome surface was ascertained indirectly by determining non coupled AHL fraction using HPLC.

(19) Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using an analytical column (HiChrom Kromasil KR100-5 C 8; 250 mm×4.6 mm) with a Waters 625 LC system attached to a Waters 996 photodiode array system operating with a Millenium 2010 Chromatograph Manager. Fractions were eluted with a linear gradient of acetonitrile in water (70-100%, v/v) over a 20 min period at a flow rate of 0.7 mL min-1 and monitored at 210 nm. Semi-preparative HPLC was performed with a C8 reverse-phase preparative column (HiChrom Kromasil KR100-5 C 8; 250 mm×8.0 mm) using a Gilson system. Fractions were eluted with an isocratic system, and determined from the analytical HPLC data, at a flow rate of 2.0 mL min-1.

(20) 4. Estimation of Drug Entrapment in Liposome Formulation

(21) Total and free drug “x” in the liposome formulations were determined using HPLC analysis.

(22) In a specific embodiment, drug “x” can be combretastatin A4. Combretastatin is well known for producing quinine derivatives under oxidative metabolism. Quinones bind nucleophiles and enhance oxidative stress by generating radical species [Folkes et al, 2007].

(23) The toxicity of this molecule is measured and characterized with a series of “stress” and “anti-stress” enzymatic activity assays detailed below in paragraph 8.

(24) Total drug was determined after ethanol extraction. An aliquot of the liposome dispersion (50 μL) was diluted to 2 mL with ethanol to release liposome-encapsulated combretastatin A4. Total combretastatin A4 in this clear ethanol extract was determined using HPLC, with a C 18-column (Nova-Pak 3.9×150 mm column, Waters) and methanol:water (50:50) as mobile phase. Flow rate of 0.8 mL/min and UV detection at 295 nm were used. Free combretastatin A4 was separated from the liposome encapsulated part using a Centricon centrifugal filter device (Centricon 10, MWCO 10 kd, Millipore, Bedford, Mass.). An aliquot of the liposome dispersion (100 μL) was diluted to 1 mL with hydration buffer (50 mM HEPES/150 mM NaCl-buffer pH 6.5), and this sample was transferred to the centrifugal filter device. The sample was centrifuged at 10 000 rpm for 15 minutes in a fixed-angle centrifuge. Free combretastatin A4 in the filtrate was then determined using HPLC. Dilution factors were taken into consideration for calculation of total and free drug. Subtraction of free drug from the total drug gave the amount of liposome-entrapped drug. Drug estimations were done in triplicate, and the values were reported as mean±SEM. One can adapt this method with another drug.

(25) 5. Visualization and Size Measurements

(26) Large MLV, before the extrusion process, were visualized using a light microscope (Olympus, CKX41, Tokyo, Japan). Final liposomes were visualized under electron microscope by negative staining technique. A diluted liposome sample was adsorbed onto a formavar- and carbon-coated copper grid, stained with 2% uranyl acetate (pH 7.0) and observed with a JEM1200EX electron microscope (JEOL, Tokyo, Japan) at ×50 000 magnification. Size and size distribution profiles of liposomes were monitored by dynamic light scattering method using the Malvern Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK).

(27) 6. In Vitro Leakage Studies

(28) Liposome encapsulation stability of combretastatin A4 was monitored in vitro, by dialyzing samples for 48 hours against 700 volumes of reverse osmosis water maintained at 37° C.

(29) A 0.5-mL aliquot of the liposome dispersion was placed in a presoaked Pierce dialysis cassette (Slide-A-Lyzer, MWCO 10 kd, Millipore), which was then placed in a beaker containing 350 mL of release medium pre-equilibrated to 37° C. The dialysis cassette was rotated at 100 rpm. The volume of release medium was selected based on careful consideration of sink conditions and sensitivity of the analytical method. At different time points, 0.5-mL samples were taken from the release medium and replaced with an equal volume of fresh release medium. Samples were analyzed for the released drug, combretastatin A4, using the HPLC method of analysis.

(30) From the total drug concentration of the liposome formulation, percentage released at each time point was calculated. Results are reported as mean±SEM (n=3).

(31) 7. High-Throughput Screening of Small Molecules in Caenorhabditis elegans

(32) Culture worms were performed according to established protocols [Lewis and Fleming, 1995; Stiernagle, 1999]. HTS experiments were performed according to Burns et al [2010 and 2006]. Late-stage fourth-larval-stage worms, grown from synchronized hatchlings at 25° C. for 45 h on NA22 Escherichia coli, were used for the accumulation assay. The worms were harvested, washed at least twice and resuspended in enough M9 buffer1 for a final concentration of ˜10 worms per μl. Five hundred microliters of this worm suspension was added to each well of Pall AcropPrep 96-well filter plates (0.45-μm GHP membrane, 1-ml well volume).

(33) Liposomes were added to each well in order to obtain a final concentration of chemicals of 40 μM (0.4% DMSO, v/v). Worms were incubated in the small-molecule solutions at 20° C. for 6 h with aeration, after which the incubation buffer was drained from the wells by vacuum (the filter membranes weaken if incubation is longer than 6 h).

(34) The worms were then washed three times with 500 μl of M9 buffer. After washing, the worms were resuspended in 50 μl of M9 buffer, transferred to new 96-well solid-bottom plates and stored frozen at −20° C. The samples were later lysed by adding 50 μl of a 2× lysis solution (100 mM KCl, 20 mM Tris, pH 8.3, 0.4% SDS, 120 μg ml.sup.−1 proteinase K) to each well and incubating the plates at 60° C. for 1 h with agitation. After lysis, the plates were stored frozen at −80° C. for later processing by HPLC (see Supplementary Methods for the full HPLC methods in Burns et al [2010]).

(35) 8. Post Processing HTS of Small Molecules in Caenorhabditis elegans.sup.2

(36) Caenorhabditis elegans is a versatile in vivo platform for a wide range of applications [Jones et al, 2005; Artal-Sanz, 2006] like screens for human drugs, drug target identification and validation, or mode of action of drugs, screens for ADME (Adsorption, Distribution, Metabolism, and Elimination/Excretion) and Toxicity parameters of pharma hits and leads and xenobiotics for eco- and environment toxicity, cosmetology, study of major cell signaling pathways like apoptosis, inflammation, oxidative stress, respiration, cell energetic, control of water (henodialysis, drinking water, for parenteral injection) contamination by chemicals and bacteria toxins, etc.

(37) Among all these applications, ADME-Tox parameters in the most challenging task since pharmaceutical industry is still confronted to harmful lead drug toxicity lately revealed during the clinical phases.

(38) C. elegans can be very efficient tool for achieving this screening if only coupled with the present invention.

(39) The different field of applications mentioned above need a large panel of experimental techniques for analysis of C. elegans treated with drugs.

(40) Among the available technique the most popular are biochemistry assays (enzymology, proteomics, metabolomics, transcriptomics, miRNA, etc), cell imaging, mutagenesis, gene knock out, etc.

(41) 8.1. Toxicity Screening

(42) Table 1 summarizes toxicity endpoints that can be measured in worms. These endpoints concern either pharmaceutical industry or cosmetology for instance for their lead therapeutic and cosmetic compounds respectively.

(43) Toxicity screening can be easily achieved on frozen worms stored at −80° C. as described in paragraph 7. For instance respiratory chain complexes activities can be automatically measured on hospital biochemistry lab apparatus based on the protocol previously described by Kramer et al in [2005].

(44) In addition, anti-stress enzymes and metabolites can also be measured on the same sample, like SOD [McCord and Fridovich, 1969], GPX [Paglia and Valentine, 1967], GRed, oxidized and reduced glutathione [Beutler and Mitchell, 1968], G6PDH [Krien et al, 1992].

(45) Excess of reactive radical oxygen species, alteration of one or several main cell metabolic pathways, modification of the imbalance between stress and anti stress enzyme activities, can be rapidly and accurately measured on few hundreds worms.

(46) TABLE-US-00001 ENDPOINT ASSAY Survival Dose-response Kinetics of survival Acute/Chronic/Repeated doses toxicity Cytotoxicity Large panel of biomarkers: stress ox; respiratory chain; apoptosis; cell energetic; inflammation; cytochromes function; main cell metabolic pathways like Krebs, glycolysis, β-oxidation, glycogenolysis, glycogenogenesis, miRNA expression, etc In situ fluorescence Acute/Chronic/Repeated doses toxicity Toxicokinetics Assessment of the accumulation of the molecule and its metabolites Changes in the expression of CYPs/GSTs/Transporters Impact of the knock-down of a CYP/GST/transporter Reprotoxicity Assessment of the egg-laying behavior Measurement of the gonad functionality Teratogenicity Embryonic lethality Time of development Analysis of molecular markers specific for each larval stage Neurotoxicity Neuronal death Quantification of neurotransmitters and their metabolites

(47) 8.2. Analysis of Chemicals Metabolites by LC-MS

(48) Metabolites were HPLC-purified from worm lysates and dried using a Savant DNA120 SpeedVac (acid was not added to the HPLC solvents).

(49) Chromatographic separations of the purified metabolites for LC-MS were performed using a nano-AQUITY Ultra Performance Liquid Chromatography (UPLC) system (Waters Corp.) (see Supplementary Methods for full methods in Burns et al (2010)).

(50) Mass spectrometry was performed using a Micromass Quadrupole-Time-of-Flight Premiere instrument (Waters Corp.). The data acquisition software used was MassLynx NT, version 4.0. Mass spectra were acquired in positive ion mode using a nano-ESI with capillary voltage and sample cone voltage set to 3,000 V and 20 V, respectively. The MS acquisition rate was set to 1.0 s, with a 0.1-s interscan delay. Ninety-eight percent argon gas was employed as the collision gas with collision energy varying from 13-46 V for the mass range of 100-1,000 m/z. Ions selected for LC-MS-MS were identified after manual analysis of original LC-MS runs, and a corresponding inclusion list was generated for targeted data-dependent acquisition experiments.

(51) 8.3. Different Strain of C. elegans for ADME Tox Screening

(52) Several wild type (WT) strains of Caenorhabditis elegans are available (see in particular https://www.cbs.umn.edu/cgc/strains (College of Biological Sciences, University of Minnesota), the most common being the strain N2).

(53) By measuring the same biochemical alterations induced by accumulation of a specific chemical in different WT strains, it is possible to validate accurate biomarkers that are associated with a specific toxicity mechanism.

(54) By following the evolution of the toxicity along the time, it is possible to predict impact of such alterations on the worm lifespan.

(55) A large number of mutant strains of C. elegans also exist and are available at the Caenorhabditis Genetics Center of Minneapolis, USA, for instance.

(56) Knock-out strains for one or several molecular actors of the cell metabolic pathways are very useful for anticipating the drug toxicity in mammalians.

(57) Indeed, detoxification mechanisms are very similar in mammalians and C. elegans [Lindblom and Dodd, 2006].

(58) The structure and the function of the mitochondria of C. elegans, particularly in relation to oxidative stress, are very close to those observed in humans [Murfitt et al, 1976].

(59) The main enzymes involved in the metabolism of xenobiotics are found: cytochrome P450 (78 genes), short-chain dehydrogenases—SDR (68 genes), or UDP-glucuronosyl-transferases glycosyl—UGT (63 genes), flavin-monooxydases—FMO (5 genes), glutathione S transferase—GST (48 genes), sulfotranferases, methyltransferases, acetyltransferases, etc.

(60) It is interesting to note also the presence of carriers ATP-binding cassette (60 genes) involved in the transport of xenobiotics.

(61) In addition, the intestinal cells and those forming the excretory system of the nematode assume the equivalent functions of liver and kidney found in humans.

(62) Several comparative studies have established a classification of substances tested for their potential toxicity in the nematode, showing a correlation with measurements made in rodents [Cole et al, 2004; Dengg and van Meel, 2004; Williams et al, 2000]. These studies validate C. elegans as a model organism for assessing the toxicity of a drug.

(63) By comparing the alterations induced by the same chemical in the WT and mutated strains, it is possible to identify and quantify, among the altered cell metabolic pathways, the functional interactions between several molecular actors.

(64) The use of the thermosensitive mutant strain TJ1060, infertile in normal growth conditions at 20-25° C., allows to overcome problems inherent to the rapid fertility cycle of the worm and limit the number of individuals in the plate well. This is a major advantage for the study of chronic toxicity at repeated doses.

(65) A strain like CL2166 expresses some gene coupled to GFP under the control of the glutathion transferase GST-4 promoter. This enzyme is expressed after a xenobiotic stimulus and participates to their metabolization.

(66) In the strain TJ375 GFP expression is controlled by the HSP 16-2 promoter. HSP are sensitive to sepsis, chemical and toxic stress and can be considered as cell stress markers.

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