Patent Application
20130210167
This application is a continuation-in-part of UK application No. 1202223.2 filed Feb. 9, 2012.
The invention relates to the field of analytical detection of pyrrolidinophenones. The pyrrolidinophenones, typically classified as a sub-set of the synthetic cathinones because of their structural similarities, represent a recent class of psychoactive drug comprising the following structure (also referred to as a ‘sub-structure’).
Several pyrrolidinophenones have been described which possess psychoactive properties including (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)pentan-1-one (commonly referred to as 3,4-methylenedioxypyrovalerone or MDPV, molecule 25 of
Yohannan J. C. and Bozenko J. S. (2010). Microgram J., 1:12-15.
Strano-Rossi S. et al. (2010). Rapid Commun. Mass Spectrom., 24:2706-2714.
Fitzgerald S. P. et al. (2005). Clin. Chem., 51: 1165-1176.
Described herein is the first known immunoassay for the detection of pyrrolidinophenones. The immunoassay is an inexpensive, relatively simplistic analytical technique. The invention describes antibodies, derived from novel haptens (pre-immunogenic molecules) and immunogens, whose binding properties enable the detection of several psychoactive pyrrolidinophenones. The haptens, in a given embodiment, before conjugation to a crosslinking group, are uniquely in possession of a hydroxyl group at the 3-position of the pyrrolidone ring. This hydroxyl group can be attached, using standard reagents and techniques, initially to a crosslinking molecule prior to attachment to the antigenicity conferring carrier material (accm) to form the immunogens of the invention. Also described are novel methods, kits and uses each comprising antibodies of the invention. What is particularly surprising and beneficial about this immunoassay is the high number of psychoactive pyrrolidinophenones of diverse structure that the antibodies are able to bind, providing for an immunoassay of great scope and utility.
In a first aspect the invention describes haptens and immunogens of Formula I:
wherein,
for the haptens n=0; Q is C1-C4 alkyl; X is N, O or S and m=0 or 1; Y is a C1-C10, more preferably a C1-C6, most preferably a C1-C3 substituted or unsubstituted straight chain alkylene moiety; Z is selected from a carboxyl, a dithiopyridyl, a maleimidyl, amino, hydroxyl, thiol or an aldehyde moiety,
and for the immunogens n=1; Q is C1-C4 alkyl; X is N, O or S and m=0 or 1; Y is a C1-C10, more preferably a C1-C6, most preferably a C1-C3 substituted or unsubstituted straight chain alkylene moiety; Z, before connection to the accm, is selected from a carboxyl, a dithiopyridyl, a maleimidyl, amino, hydroxyl, thiol or an aldehyde moiety; the accm is an antigenicity-conferring carrier material.
According to a first aspect the present invention, there is provided a hapten having the general Formula IA:
wherein,
X is an atom selected from N, O, and S and m=0 or 1;
According to a further aspect of the present invention, there is provided an immunogen comprising a hapten according to the first aspect of the present invention and an antigenicity-conferring carrier material.
Optionally, the accm is attached to Z of the hapten.
Optionally, the immunogen has the general Formula IB:
wherein,
It is understood that, when the accm is attached to Z, Z forms at least one bond with each of Y and the accm.
Optionally, Q is a C2-C3 alkyl moiety, which can be substituted or unsubstituted, linear or branched. Further optionally, Q is a linear C2-C3 alkyl moiety, which can be substituted or unsubstituted. Still further optionally, Q is an unsubstituted, linear C2-C3 alkyl moiety.
Optionally, X is an O atom and m=1.
Optionally, Y is a C1-C10, more preferably a C1-C6, most preferably a C1-C3 straight chain alkylene moiety, substituted with an oxygen atom. Further optionally, Y is a butanonyl moiety.
Optionally, Z is a carboxyl group.
For both the haptens and immunogens it is preferable that X is O, Y is —C(O)—CH2—CH2—, Z is carboxy or amino (for the immunogen, Z is carboxy or amino before connection to the accm) and Q is preferably a straight chain alkyl most preferably either ethyl or propyl. The immunogens are prepared by coupling a hapten (a pre-immunogenic molecule) to an antigenicity-conferring carrier material (accm) usually by way of a crosslinker. The accm and the crosslinkers are standard reagents in the field of antibody development. The accm comprises polyamino acid segments and is preferably bovine thyroglobulin (BTG), bovine serum albumin (BSA) or keyhole limpit haemocyanin (KLH). Alternatively, if it has a suitable functional group, the hapten may be attached directly to the accm without the use of a crosslinker. An example of a crosslinker described herein is succinic anhydride, activated by N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide.
A second aspect of the invention is antibodies raised to immunogens of Formula I, the antibodies being capable of binding to at least one structural epitope of a molecule of Formula II:
wherein
Q1 is C1-C4 alkyl; R3 is —CH2— or —C(O)—; R1 and R2 are H, C1-C4 alkyl,C1-C4 alkoxyl, hydroxyl-substituted C1-C4 alkyl, carboxyl or hydroxyl, or together form substituted or unsubstituted
to form a fused bicyclic ring with the benzene moiety of Formula II. In preferred embodiments the at least one structural epitope of Formula II bound by the antibodies are when the fused bicyclic ring is unsubstituted, Q1 is methyl, ethyl or propyl and R3 is —CH2—. The antibodies are obtained using standard methods; immunogens of the invention are administered to a non-human mammalian host, preferably a sheep, to elicit antibody production after which harvested polyclonal or monoclonal antibodies are used to develop immunoassays. Other suitable immunoglobulin-derived molecules such as short-chain or single chain variable fragments are readily applied alternatives known to the skilled person. Examples of pyrrolidinophenones which are bound by the antibodies of the invention are shown in
A further aspect of the invention is a method of detecting or determining one or more pyrrolidinophenones in an in vitro sample or in a solution comprising a substance to be tested, optionally a substance suspected of containing one or more pyrrolidinophenones (the substance optionally having been pre-treated to attain a suitable state for analysis) the method comprising contacting the sample or solution with at least one antibody of the invention, optionally with at least one detecting agent; detecting or determining the at least one antibody of the invention, optionally detecting or determining the detecting agent(s); and deducing from a calibration curve the presence of, or amount of, pyrrolidinophenones in the sample or solution. Preferably the pyrrolidinophenones to be detected or determined are listed in
A further aspect of the invention is a compound of Formula III:
in which X is NH2, OH or SH and Q is C1-C4 alkyl.
In preferred embodiments X is OH and Q is ethyl or propyl. It is also preferable that the asymmetric carbon atom of the heterocyclic ring of Formula III is stereospecifically of the R configuration. Compounds of Formula III are representative haptenic intermediate molecules which can be conjugated to crosslinking reagents prior to immunogen formation.
Another aspect of the invention is a kit for detecting or determining pyrrolidinophenones the kit comprising at least one antibody of the invention. The kit preferably detects or determines one or more pyrrolidinophenones listed in
General Methods, Examples and Results
Preparation of Haptens, Immunogens and Detecting Agents
Although haptens provide defined structural epitopes, they are not in themselves immunogenic and therefore need to be conjugated to an antigenicity conferring carrier material (accm), which will elicit an immunogenic response when administered to a host animal. Appropriate accms commonly contain poly(amino acid) segments and include polypeptides, proteins and protein fragments. Illustrative examples of useful carrier materials are bovine serum albumin (BSA), egg ovalbumin, bovine gamma globulin, BTG, keyhole limpet haemocyanin (KLH) etc. Alternatively, synthetic poly(amino acids) having a sufficient number of available amino groups, such as lysine, may be employed, as may other synthetic or natural polymeric materials bearing reactive functional groups. Also, carbohydrates, yeasts or polysaccharides may be conjugated to the hapten to produce an immunogen. The haptens can also be coupled to a detectable labelling agent such as an enzyme (for example, horseradish peroxidase), a substance having fluorescent properties or a radioactive label for the preparation of detecting agents for use in the immunoassays. The fluorescent substance may be, for example, a monovalent residue of fluorescein or a derivative thereof. Immunogen formation for the invention described herein involves conventional conjugation chemistry. In order to confirm that adequate conjugation of hapten to carrier material has been achieved, prior to immunisation, each immunogen is evaluated using matrix-assisted UV laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS).
General Procedure for MALDI-TOF Analysis of Immunogens
MALDI-TOF mass spectrometry was performed using a Voyager STR Biospectrometry Research Station laser-desorption mass spectrometer coupled with delayed extraction. An aliquot of each sample to be analysed was diluted in 0.1% aqueous trifluoroacetic acid (TFA) to create 1 mg/ml sample solutions. Aliquots (1 μl) were analysed using a matrix of sinapinic acid and bovine serum albumin (Fluka) was used as an external calibrant.
Preparation of Antisera
In order to generate polyclonal antisera, 2 mgs of the immunogen of the present invention is prepared in PBS, mixed at a ratio of 50% immunogen in PBS to 50% Freund's Complete adjuvant (Sigma, Product Number F5881) and emulsified by repeatedly passing the mixture through a tip on the end of a 1 ml syringe, until it reaches the required semi-solid consistency. 1 ml of the emulsified mixture is injected intramuscularly into each sheep. Various host animals could be used, such as rabbit, sheep, mouse, guinea pig or horse. Sheep are the preferred host animal. Further injections (boosts) are made at monthly intervals, (1 mg of immunogen is prepared in PBS and mixed at a ratio of 50% Immunogen in PBS/50% Freunds Incomplete adjuvant, Sigma, Product Number—F5506) and serum is sampled monthly by collection from the jugular vein for evaluation of the antibody titre. When the optimal titre (i.e. maximum titre achieved with no further increase after subsequent boosts) has been attained, the host animal is bled to yield a suitable volume (e.g. approximately 250 ml per bleed per sheep) of specific antiserum. The degree of antibody purification required depends on the intended application. For many purposes, there is no requirement for purification, however, in other cases, such as where the antibody is to be immobilised on a solid support, purification steps (such as caprylic acid/ammonium sulphate precipitation) can be taken to remove undesired material and eliminate non-specific binding.
Immunoassay Development
The process of developing an immunoassay is well known to the person skilled in the art. Briefly, for a competitive immunoassay in which the target analyte is a non-immunogenic molecule such as a hapten, the following process is conducted: antibodies are produced by immunising an animal, preferably a mammalian animal, by repeated administration of an immunogen. The serum from the immunised animal is collected when the antibody titre is sufficiently high. A detecting agent (e.g. appropriate hapten conjugated to HRP) is added to a sample containing the target analyte and the raised antibodies, and the detecting agent and analyte compete for binding to the antibodies. The process may comprise fixing (e.g. dilution of antibodies in coating buffer and incubation at 37° C. for 2 hours to allow antibody binding to surface) said serum antibodies to a backing substrate such as a polystyrene solid support or a ceramic chip. The antibodies can be polyclonal or monoclonal. The signal emitted in the immunoassay is proportionate to the amount of detecting agent bound to the antibodies which in turn is inversely proportionate to the analyte concentration. The signal can be detected or quantified by comparison with a calibrator with known levels of target analyte.
To a solution of 1-(benzo[d][1,3]dioxol-5-yl)butanone (25 g, 0.13 mole) in acetic acid (100 ml) was added dropwise a solution of bromine (21.8 g, 0.137 mole) in acetic acid (100 ml). The reaction mixture was then stirred at room temperature for two hours. Acetic acid was removed under high vacuum. Water (200 ml) was added to the mixture and the solution was extracted with dichloromethane (2×200 ml). The combined organic layers were washed with saturated NaHCO3 solution (100 ml), water (100 ml) and brine (100 ml). The dichloromethane solution was dried over Na2SO4, filtered and concentrated to dryness under vacuum. The crude product obtained was purified by chromatography on silica gel using 5% ethyl acetate in hexane to give the title compound (29.6 g, 84%).
To a solution (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-bromobutanone (7.92 g, 27.8 mmol) in acetonitrile (100 ml) was added potassium carbonate (7.93 g, 57.4 mmol) and (R)-(+)-3-pyrrolidinol (5.0 g, 57.4 mmol) and the mixture stirred under nitrogen overnight at room temperature. The mixture was filtered and the solution evaporated to dryness. The crude obtained was purified by chromatography on silica gel using 50% ethyl acetate in hexane to give the title compound as a brown oil (5.5 g, 69.2%).
To a solution of 1-(Benzo[d][1,3]dioxol-5-A-2-((R)-3-hydroxypyrrolidin-1-yl)butanone (4.5 g, 16.25 mmol) in anhydrous pyridine (100 ml) was added succinic anhydride (3.25 g, 32.5 mmol) and the mixture stirred overnight at room temperature. The pyridine was removed under high vacuum and the dark brown crude obtained purified by chromatography on silica gel using 20% methanol in chloroform to give Hapten-A (
To a solution of Hapten-A (42.62 mg, 0.113 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (25.64 mg, 0.125 mmol) and N-hydroxysuccinimide (14.3 mg, 0.16 mM) and the mixture stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BSA (150 mg, 2.3 μmol) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml). The mixture was then stirred overnight at 4° C. The solution was dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give Immunogen I.
MALDI results showed 16.58 molecules of Hapten-A had been conjugated to one molecule of BSA.
To a solution of Hapten-A (50.94 mg, 0.135 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (30.64 mg, 0.150 mmol) and N-hydroxysuccinimide (17.1 mg, 0.15 mmol) and the mixture stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BTG (150 mg, 2.25 μmol) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml). The mixture was then stirred overnight at 4° C. The solution was dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give Immunogen II.
EDC hydrochloride (10 mg) was dissolved in water (0.5 ml) and immediately added to a solution of hapten-A (2 mg) in DMF (0.2 ml). After mixing, the solution was added dropwise to a solution of HRP (20 mg) in water (1 ml). Sulfo-NHS (5 mg) was added and the reaction mixture incubated in the dark at room temperature overnight. Excess hapten was removed with double PD-10 columns (Pharmacia) in series, pre-equilibrated with PBS at pH 7.2. The hapten-HRP conjugate was then dialysed overnight against 10 L of PBS at pH 7.2 at 4° C.
To a solution of 1-(benzo[d][1,3]dioxol-5-yl)pentanone (25 g, 0.12 mole) in acetic acid (100 ml) was added dropwise a solution of bromine (25.2 g, 0.158 mole) in acetic acid (100 ml). The reaction mixture was then stirred at room temperature for two hours. Acetic acid was removed under high vacuum. Water (200 ml) was added to the mixture and the solution extracted with dichloromethane (2×200 ml). The combined organic layers were washed with saturated NaHCO3 solution (100 ml), water (100 ml) and brine (100 ml). The dichloromethane solution was dried over Na2SO4, filtered and concentrated to dryness under vacuum. The crude product obtained was purified by chromatography on silica gel using 5% ethyl acetate in hexane to give the title compound (30.5 g, 89%).
To a solution (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-bromopentanone (10.0 g, 35.07 mmol) in acetonitrile (100 ml) was added potassium carbonate (10.0 g, 72.41 mmol) and (R)-(+)-3-pyrrolidinol (6.3 g, 72.4 mmol) and the mixture stirred under nitrogen overnight at room temperature. The mixture was then filtered and the solution evaporated to dryness. The crude obtained was purified by chromatography on silica gel using 50% ethyl acetate in hexane to give the title compound as a brown foamy solid (6.1 g, 60.0%).
To a solution of (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-((R)-3-hydroxypyrrolidin-1-yl)pentanone (5.8 g, 20.0 mmol) in anhydrous pyridine (100 ml) was added succinic anhydride (3.0 g, 30.0 mmol) and the mixture was stirred overnight at room temperature. The pyridine was removed under high vacuum and the dark brown crude obtained was purified by chromatography on silica gel using 20% methanol in chloroform to give the pure Hapten-B as a tan solid (5.3 g, 68.0%).
To a solution of Hapten-B (35.22 mg, 0.09 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (20.42 mg, 0.099 mmol) and N-hydroxysuccinimide (11.39 mg, 0.099 mmol) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BSA (150 mg, 2.3 μmol) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml). The mixture was then stirred overnight at 4° C. The solution was dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give Immunogen-III. MALDI results showed 19.63 molecules of Hapten-B had been conjugated to one molecule of BSA.
To a solution of Hapten-B (44.22 mg, 0.113 mmol) in DMF (1.0 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (25.64 mg, 0.13 mmol) and N-hydroxysuccinimide (14.7 mg, 0.13 mmol) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BTG (150 mg, 2.25 μmol) in 50 mM sodium bicarbonate solution (pH 8.5) (10 ml). The mixture was then stirred overnight at 4° C. The solution was dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried to give Immunogen-IV.
EDC hydrochloride (10 mg) was dissolved in water (0.5 ml) and immediately added to a solution of hapten-B (2 mg) in DMF (0.2 ml). After mixing, this solution was added dropwise to a solution of HRP (20 mg) in water (1 ml). Sulfo-NHS (5 mg) was added and the reaction mixture was incubated in the dark at room temperature overnight. Excess hapten was removed with double PD-10 columns (Pharmacia) in series, pre-equilibrated with PBS at pH 7.2. The hapten-HRP conjugate was then dialysed overnight against 10 L of PBS at pH 7.2 at 4° C.
A semi-automated Evidence Investigator analyser (Randox Laboratories Ltd. Crumlin, UK) was used as the platform for a biochip assay for the detection of pyrrolidinophenones Immunogen IV was administered to adult sheep on a monthly basis to provide target-specific polyclonal antisera. IgG was extracted from the antisera, and the purified antibody was immobilised on a biochip (9 mm×9 mm) (Fitzgerald et al 2005). The assay is based on competition for binding sites of a polyclonal antibody between Hapten-A conjugate (Example 6) and pyrrolidinophenones and potential cross-reactants. The antibody was immobilised and stabilised onto the biochip surface as previously described (Fitzgerald et al., 2005). Assay diluent (155 μL), calibrator/pyrrolidinophenone or potential cross-reactant (see the Tables) (25 μL) followed by Hapten-A conjugate (120 μL) were added to the appropriate biochip. The biochips were then incubated for 30 minutes at 30° C. on a thermoshaker set at 370 rpm. The biochips were then subjected to 2 quick wash cycles using the wash buffer provided, followed by 4×2 minute wash cycles. 250 μL of signal (1:1 luminol+peroxide, v/v) was then added to each biochip, and after 2 minutes the biochip carrier was imaged in the Evidence Investigator analyser. Calibration curves were generated and these were used to determine the sensitivity and specificity of the immunoassay for pyrrolidinophenones and potential cross-reactants. The results of this study are presented in Table 1, cross-reactivity being calculated according to the following formula:
% CR=IC50 MDPBP/IC50 CR×100
where % CR is the percentage cross-reactivity, IC50 MDPBP is the concentration of MDPBP which causes 50% displacement of signal and IC50 CR is the concentration of pyrrolidinophenone/potential cross-reactant that causes 50% displacement of signal.
Chemicals
MDPBP HCl, MDPV HCl, (RS)-1-(4-methylphenyl)-2-(pyrrolidin-1-yl)butanone HCl, naphyrone HCL, and MDPPP HCl were obtained from the Australian Government National Measurement Institute (LGC stds); MDMA and MDA were obtained from Cerilliant; amphetamine, methamphetamine HCl and JWH-018 were obtained from Sigma Chemicals; mescaline HCl and (+)-pseudoephedrine were obtained from Sigma Aldrich; MDMA and MDA were obtained from Cerilliant; 1-(3-chlorophenyl)piperazine was obtained from Alfa Aesar; salvinorin A and 1-benzylpiperazine were obtained from Aaron Chemistry; JWH-250 was obtained from Cayman Chemicals; mephedrone HCl was synthesised at Randox Laboratories.
Results
The data in Table1 confirm that antibodies of the invention bind to pyrrolidinophenones incorporating the sub-structure depicted in Structure I while not binding to molecules lacking this sub-structure.
The antibodies of the invention are also able to bind to an epitope of molecules of Formula IV in which R1 and R2 are independently methyl, ethyl or propyl. Two such molecules are butylone and pentylone (Table 2).
The invention further describes a kit comprising antibodies of the invention, the kit used to detect or determine molecules comprising either Formula II or Formula IV; preferably the kit is used to detect one or more of (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)butanone, (RS)-1-(2-naphthyl)-2-(pyrrolidin-1-yl)pentanone, (RS)-1-(4-methylphenyl)-2-(pyrrolidin-1-yl)butanone, (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)pentanone, (RS)-1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)propanone, (RS)-1-(4-Methylphenyl)-2-(pyrrolidin-1-yl)hexanone, pyrovalerone, butylone and pentylone; the skilled person would recognize that the antibody incorporated in the kit is able to bind further molecules not described herein if such molecules incorporate a structure depicted by either Formulas II or IV.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1202223.2 | Feb 2012 | GB | national |
