CA1338763C - Compounds and assay for tricyclic antidepressants - Google Patents

Abstract

Reagents, compounds of the general formula:

Where: X=Y=H or taken together to form a bond R=-(CH2)n-NR'

Description

COMPOUND AND ASSAY FOR
TRICYCLrC ANTIDEPRESSAN$S

EIACXGROUND OF THE INVENTION

Technical Field This invention relates to reagents and a method utilizing the reagents, for determining the presence or amount of tricyclic antidepressants, especially Imipramine, Desipramine, Amitriptyline, and Nortriptyline, in ~luids, especially biological fluids such as serum, plasma, spinal and amniotic fluids and the like, and to a method for making the reagents. More particularly, the invention relates to haptens and immunogens used to raise antisera, and to tracers; the haptens, immunogens and tracers are useful as reagents in a fluorescence polarization immunoassay procedure to determine tricyclic antidepressants.

-Background Art The tricyclic antidepressants are a class of compounds which are used to treat chronic depression.
The class is characterized by the general structure:
v- ~ ~ere: V=CH2 or HC=
W--CH2, O or =C~
~~X~~ X=~ or C= H
Y=CH2 or =C~
R=H or CH3 ~NC~,R
Although these compounds have been found to be very effective in treating chronic depression, their concentrations in a patient's blood must be maintained in a therapeutic range. A wide interpatient variation normally exists in the steady-state concentration in human plasma, for a given dose. High doses have been associated with central nervous system disorders, cardiac toxicity, hypertension, seizures, coma and death. S~nce individuals vary greatly in their response, it is essential to monitor the therapy by measuring the serum or plasma levels of these drugs.
Typically, competitive binding immunoassays are used for measuring ligands in a test sample. (For the purposes of this disclosure, a "ligand" is a substance of biological interest to be determined by a competitive binding immunoassay technique) The ligands compete with a labeled reagent, or "ligand analog", or "tracer", for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody:
the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the liqand analog each bind to the antibody in proportion to their respective concentrations.

_ J

In the past, patient serum/plasma levels of tricyelie antidepressants were measured by gas chromatography (GC), liquid ehromatography (LC), or enzyme multiplied immunoassay. However, such assay methods are not without drawbacks. Both GC and ~C are labor intensive, requiring highly skilled personnel to do extractions of the analyte from the biologieal matrix. Enzyme multiplied immunoassays are less labor intensive but are sub~eet to variability due to temperature and matrix effeets which alter enzyme activity.
Fluoreseenee polarization immunoassay procedures provide a guantitative means for measuring the amount of traeer-antibody eonjugate produeed in a competit~ve binding ir~oassay. Sueh fluorescenee polarization teehnigues are based on the prineiple that a fluoreseent labeled eompound, when exeited by ~lane polarized light, will emit fluorescenee having a degree 3f polarization inversely related to its rate of rotation. Aeeordingly, when a traeer-antibody eonjugate having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized ~eeause the fluorophore is eonstrained from rotating between the time that light is absorbed and emitted. In eontrast, when an unbound tracer is exeited by plane pola-rized light, its rotation is much faster than the eorresponding traeer-antibody conjugate and the molecules are more r~omly oriented. As a result, the light emitted from the unbound tracer molecules is depolarized. Such fluorescence polarization technigues are disclosed in U.S. Patent No. 4,420,568 to Wang et al., which is direct~d to the use of a triazinylaminofluoreseein moiety as the fluorophore (tracer).

- ~ - 1 338763 SUMMARY OF ~HE INVENTION
The pr~sent invention offers a~ advance in the art beyond the Wang, et al. patent previously described, particularly in that highly sensitive fluorophores, a method for making the fluorophores, and an assay using th~ fluorophores as tracers are provided specifically for the determination of one or more tricyclic antidepressants with one assay. An assay conducted in accordance with the present invention is particularly accurate, as will be explained infra.
This invention is directed to haptens and immunogens used to raise antibody which can be used ~n an assay for tricyclic antidepressants, to tracers for use in fluorescence polarization immunoassays for tricyclic antidepressants, to methods for conducting such assays, and to methods for makin~ the tracers, haptens and immunogens.
Accordingly, a first aspect of the invention relates to the discovery of novel compounds wh~ch, when covalently attached to protein and used to raise antibodies, provide antisera with high cross reactivities for a number of different tricyclic antidepressants. Thus, accordin~ to the first aspect of the present invent ion, there are provided compounds which are 5-substituted 10,11-dihydro-SH-dibenzo(b,f)azepine (compounds of Formula A), and S-substituted 10,11-dihydro-sH-dibenzo(a,d)cycloheptene (compounds of Formula B):
Where R =~CH2)nNH2 ~N ~ ~ or (CH2)nNHCH3 and I X I when X=Y=H, then Formula A. ~Formula B Y ~ n=O or l; when X
and Y form a bond, R R then n=O
where R=NH2 or NHCH3 ~ 5 ~ 1 338763 A second aspect of the invention relates to the discovery of unique fluorophore tracer compounds having novel structures. Thus, according to the second aspect of the invention, there are provided compounds which are conjugates of 5-substituted-10,11-dihydro-5H-dibenzo(b,f) azepine (Formula A) with 5- or 6-substituted fluorescein (compounds of Formula C), and conjugates of 5-substituted-10,11-dihydro-5H-dibenzo(a,d)cycloheptene (Formula B) with 5- or 6-substituted fluorescein (compounds of Formula D), and biologically acceptable salts thereof:

Y~
X~
Formul- C '~ Formula D '~

~~0 ~0~0 Q O
tn~ere: X=-~CH2)m NR~ Where: R=-~CH )n-NR'-C---and ~=0 or 1 and n=~ or 1 R~H or C~l3 R'=H or C~3 X=Y~H or ta~cen to-gether to form a bond The dotted line in the foregoing structures indicates that the point of attachment to the fluorescein moiety can be at either the 5 or 6 position.
A third aspect of the invention relates to an analytical method, i.e., a method for conducting an assay using as reagents compounds of Formula C or D and antisera raised in response to immunogens prepared from haptens of Formula A or B. According to the third aspect of the invention, an improved fluorescence polarization immunoassay is provided, comprising the steps of contacting a fluid suspected of containing tricyclic antidepressant(s) ~ - 6 - l 338763 with antisera to a tricyclic antidepressant and a fluorescent-labeled tricyclic derivative (e.g., a compound of Formula C or D) capable of producing a detectable fluorescence polarization response to the presence of the antiserum in a homogeneous solution passing plane polarized light through the homogeneous solution, and measuring the fluorescence polarization response therefrom.
According to a fourth aspect of the invention, there are provided synthetic methods, i.e., methods for making the compounds for Formulas A, B, C and D.
Further objects and attendant advantages of the invention will be apparent from the following detailed description taken together with the Examples.

DF~TAITl~n D~.~CRIPTION OF T~ INVFNTION
The present invention is directed to haptens and immunogens (to raise antisera) and to tracers for use in an improved fluorescence polarization immunoassay for tricyclic antidepressants; to synthetic methods, for making the foregoing reagents; and to an analytical method, using the reagents, for detecting the presence or amount of tricyclic antidepressants in fluids, e.g., human body fluids including blood serum, plasma, spinal fluid and the like.
Compounds of Formula B can be prepared by treatment of the corresponding halides with the appropriate amine in alcohols, where R' = halogen, by well-known procedures, for example those described in U.S. Patents 3,981,917 and 3,978,121. The procedure set forth in Example 4, infra, has also been used to prepare immunogens in accordance with the invention.
Specifically, it has been found in accordance with the invention that immunogens prepared from haptens - -of the structures of Formula A or Formula B, when used to raise antisera in animals using techniques well known to those skilled in the art, will yield antisera with unexpected advantageously high cross reactivities to the major tricyclic antidepressants but with low cross reactivities to their ma~or metabolites and other commonly used antidepressants. The relati~e responses of the preferred antisera to tricyclic antidepressants and their metabolities are shown in Table I. The cross reactivities are relative to imipramine which is taken as 100% at each concentration.
The cross reactivity was established by spiking pooled normal human serum with the drugs to be tested up to 1 microgram per milliliter (mcg/ml), and then assaying the samples in the TDxR TCAs system, a commercially a~ailable instrumental analysis system utilizing fluorescence polarization techniques (from Abbott ~aboratories, Abbott Park, Illinois).

TABLE r SE~UM CONCENT~ATION % CROSS
COMPO~ TESTED (nanograms per mi11i!iter) ~EACTIV'TY
A. Maior Tricycl,cs Desipramine~ 100 99 Amitriptyline~ 100 101 Nortriptyline~ 100 9S

B. Minor Tricyclics Doxepin~ 100 58 300 ~S

Trimipramine~ 100 93 300 7~

Clomipramine~ 100 Sl 300 ~6 SOO ~ 1 Protriptyline~ 100 113 C. Metabolites Imipramine N-Oxide 1000 0.7 2-Hydroxyimipramine 300 18 2-Hydroxy desipramine 300 8 10-Hydroxyamitriptyline-CIS 300 9 10-Hydroxynortriptyline-C~S 300 6 D. Other Antidepressants Maprotiline~ lO00 14 Amoxapine~ 1000 2 Trazodone~ 1000 -As previously stated, the tracers of the present invention can be of the structures of Formula C
or D, and biologically acceptable salts thereof. Most preferred are the compounds of Formula D in which Y-Z=H, due to the greater intensity of the fluorescence response produced in these structures which is particularly advantageous in the practice of the method of the present invention to measure the presence or amount of tricyclic antidepressants in biological fluids.
The tracers of the present invention generally exist in an equilibrium between their acid and ionized states, and in the ionized state are effective in the method of the present invention. Therefore, the present invention comprises the tracers in either the acid or ionized state, and for convenience, the tracers of the present invention are structurally represented herein in their acid form. When the tracers of the present invention are present in their ionized state, the tracers exist in the form of biologically acceptable salts. As used herein, the term "biologically acceptable salts" refers to salts such as sodium potassium, ammoniwm and the like which will enable the tracers of the present invention to exist in their ionized state when employed in the method of the present invention. Generally, the tracers of the present invention exist in solution as salts, with the specific salt resulting from the buffer employed. For example, in the presence of a sodium phosphate buffer, the tracers of the present invention will generally exist in their ionized state as a sodium salt.
A tracer which is not complexed to an antibody is free to rotate in less than the time reguired for absorption and re-emission of fluorescent light. As a result, the re-emitted light is relatively randomly oriented so that the fluorescsnce polarization of a tracer not complexed to an antibody i8 low, approaching zero. Upon complexing with a specific antibody, the tracer-antibody complex thus ormed assumes the rotation of the antibody molecule which is slower than that of the relatively small tracer molecule, thereby increasing the ~olarization observed. Therefore, when a ligand competes with the tracer for antibody sites, the observed polarization of fluorescence of the tracer-antibody complex becomes a value somewhere between that of the tracer and tracer-antibody complex.
If a sample contains a high concentration of the ligand, the observed polarization value is closer to that of the free ligand; i.e., low. If the test sample contains a low concentration of the ligand, the polarization value is closer to that of the bound ligand; i.e., high. By sequentially exciting the reaction mixture of an immunoassay with vertically and then horizontally polarized light and analyzing only the vertical component of the emitted light, the polarization of fluorescence in the reaction mixture may be accurately determined. The precise relationship between polarization and concentration of the ligand to be determined can be established by measuring the polarization values of calibrators with known concentrations. The concentration of the ligand can be extrapolated from a standard curve prepared in this nr~er.
The results can be quantified in terms of net millipolarization units, span (in millipolarization units) and intensity. The measurement of millipolarization units indicates the maximum polarization when a maximum amount of the tracer is bound to the antibody in the absence of any tricyclic antidepressant. The higher the net millipolarization, the better the binding of the tracer to the antibody.

The span i8 an indication of the difference between the net millipolarization and the minimum amount of tracer bound to the antibody. A larger span provides for a better numerical analysis of data. The intensity is a measure of the strength of the signal above background.
Thus, a higher intensity will give a more accurate measurement. The intensity is determined, at about a 2 nanomolar concentration of tracer, as the sum of the vertically polarized intensity plus twice the horizontally p~larized intensity.
In accordance with a preferred method of conducting the assay of the present invention, a sample containing tricyclic antidepressant(s) or suspected of containing tricyclic antidepressant(s), is intermixed with a biologically acceptable salt of a tracer, such as the tracers illustrated in Formula C or Formula D, and an antibody specific for one or more tricyclic antidepressants and the tracer. The tricyclic antidepressant(s) and the tracer compete for limited antibody sites, resulting in the formation of tricyclic antidepressant-antibody and tracer-antibody complexes.
By maintaining constant the concentration of tracer and antibody, the ratio of tricyclic antidepressant-antibody complex to tracer-antibody complex that is formed is directly proportional to the amount of tricyclic antidepressant~s) present in the sample. Therefore, upon exciting the mixture with plane polarized light and measuring the polarization of the fluorescence emitted ~y the tracer and the tracer-anti~ody complex, one is able guantitatively to determine the amount of tricyclic antidepressant(s) in the sample.
The pH at which an assay of the present invention is carried out m~st be sufficient to allow the tricyclic antidepressant tracer utilized to exist in its ionized state. The pH may range from about 3 to 12, ~ - 12 - 1 33 87 63 more usually in t~e range of from about S to 10, most preferably from about 6 to 9. Various buffers may be used to achieve and maintain the pH during the assay procedure. Representative well-known buffers include borate, phosphate, carbonate, tris, barbital and the like. The particular buffer employed is not critical to the present invention, but the tris and phosphate buffers are preferred. The cation portion of the bufer will generally determine the cation portion of the tracer salt in solution.
The reagents for the most preferred fluorescence polarization assay of the present invention comprise antibody specific for one or more tricyclic antidepressant(s) raised in response to haptens of Formula A, where R~NH2, which have been conjugated to bov~ne serum albumin and a tracer of Formula D, where 2-CH2 NCH3-C-O, with attachment to the fluorescein moiety being through the 6 position.
Additionally, a pretreatment solution to extract the tricyclic antidepressant(s) being assayed, a dilution buffer, tricyclic antidepressant calibrators and tricyclic antidepressant controls are desirably prepared.
A preferred procedure for the determination of tricyclic antidepressants in accordance with the invention is set forth in the following discussion. All percentages expressed herein are weight/volume unless otherwise indicated.
The tracer formulation presently preferred is a concentration of 82 nanomolar tracer (e.g., compounds of Formulas C and D, suPra) in: 0.1 molar Tris buffer at pH 7.S; 2.0% sodium octyl sulfate; 0.1% sodium azide;
and 0.01% bovine gamma globulin. T~e antiserum formulation com~rises rabbit serum diluted with: 0.1 molar phosphate buffer at pH 6.~; 0.1% sodium azide;
O.01% bovine gamma globulin; 1% human serum (volume/volume); and 2% ethylene glycol (volume/volume). The dilution buffer comprises: 0.1 molar sodium phosphate at pH 7.5; 0.1~ sodium azide; and O.01% bovine gamma globulin. The pretreatment formulation comprises: 0.01% bovine gamma globulin; 0.1 molar tris buffer at pH 7.5; 0.1% sodium azide; and 2.0%
sodium octyl sulfate. Tricyclic antidepressant calibrators comprising imipramine and normal human serum at concentrations of 0, 7S, 150, 300, 600 and 1000 nanograms per milliliter, with 0.1~ sodium azide pressrvative, are useful and preferred. Tricyclic antidepressant controls comprising imipramine and normal human serum at concentrations of 100, 200 and 500 nanograms per milliliter, with 0.1% sodium azide as a preservative, are also useful and preferred.
The preferred procedure is especially designed to be automated and used in conjunction with the Abbott TDxR Fluorescence Polarization Analyzer, commercially available from Abbott Laboratories, Abbott Park, Illinois. In this procedure, fifty microliters of serum or plasma are reguired. The calibrators, controls or unknown samples are pipetted directly into the sample well of the TDx sample cartridge. One of the advantages of this procedure is that the sample does not require any special preparation. For example, if a TDx tricyclic antidepressant assay kit is being used with the TDx analyzer to perform assays in accordance with the present invention, the caps from each of the three reagent containers in the kit are removed and placed into designated wells inside the TDx Analyzer, and the assay procedure from this point is fully automated.
However, if a manual assay is being performed according to the invention, then the sample is prefera~ly mixed with the pretreatment solution in dilution buffer, and a background reading is taken. The t 338763 traeer i8 then mixed with the test solution, and antibody is thereafter mixed into the solution. After incubation, a f luorescence polarization reading is taken.
In instrumental determinations, the fluoreseenee polarization value of each calibrator, eontrol or sample can be determined, and printed on the output tape of an instrument such as the Abbott TDx Analyzer. A standard curve is generated in the instrument, plotting the polarization of eaeh ealibrator versus its eoneentration using a nonlinear regression analysis. The eoneentration of each eontrol or sample is read off the stored ealibration eurve and printed on the output tape.
Complete details and instruetions on proeedural aspeets of the performanee of automated assays using the eompounds of the invention are available in the TDx Analyzer Systems Operation Manual, available from Abbott ~aboratories, Abbott Park, Illinois.
With respeet to the foregoing preferred proeedure, it should be noted that the traeer, antibody, pretreatment solution, ealibrators and eontrols should be stored between about 2 and about 8 degrees C, while the dilution buffer should be stored at ambient temperature.
It is to be appreciated that although preferred embodiments of the invention are herein described with referenee to the determination of speeifie tricyelie antidepressants, or mixtures of tricyelie antidepressants, the concepts of the present invention can also ~e profitably applied not only to measurement of levels of specific ones of these drugs in various biological fluids, sueh as ser~m, plasma, spinal fluid and the like, but also to screen for the presenee or amount of any of ~he broad eategory of drugs represented by the designation "trieyelie antidepressants".

Accordingly, it should be understood that the foregoing detailed description and the following Examples are intended to be illustrative, and not limiting, with respect to the scope of the present invention. Various modifications will become apparent to one skilled in the art, and thus it is intended that the scope of the invention be defined solely by the claims appended hereto and legal equivalents thereof.

EXAMPLES

Example 1: Preparation of N-chloroacetyl iminodibenzyl A solution of iminodibenzyl (6.0 grams (gm)) and chloroacetyl chloride (6.0 gm) was heated at reflux in chloroform (70 milliliters ml)) for 2 hrs. After cooling to room temperature, the solution was washed with water 25 ml and S% sodium bicarbonate (2 X 50 ml).
The organic solution was dried over anhydrous magnesium sulfate, filtered and concentrated to dryness in vacuo to yield 8.3 gm of solid product.

Example 2: Preparation of N-Aminoacetyl iminodibenzyl A solution of N-chloroacetyl iminodibenzyl (2.25 gm) and sodium Iodide (1.85 gm) in acetone ( io ml) was stirred at room temperature for 16 hours. The mixture was diluted with chloroform (150 ml) and washed with water (4 X 25 ml). The orqanic solution was then dried over anhydrous magnesium sulfate, filtered and concentrated to d~yness in vacuo. The residue was dissolved in methanol (sO ml), to which was added tetrabutyl ammonium bisulfate (0.1 gm) and concentrated ammonium hydroxide (50 ml). After stirring at room temperature for 68 hours, the mixture was concentrated to dryness in vacuo. The residue was then triturated with chloroform (100 ml). The organic solution was - ~6 - 1 3 3 8 7 6 3 washed with water (4 X 2S ml), dried over anhydrous magnesium sulfate and filtered. Removal of the solvent in vacuo yielded 1.71 gm of crude product, which was chromatographed on silica gel (120 gm) using 10%
methanol in chloroform (2 liters (L)) followed by 20%
methanol in chloroform (1 ~) to elute the product. The ninhydrin positive fractions were combined and concentrated to dryness in vacuo to yield 0.750 gm of product.

Example 3: Preparation of N-(2-Aminoethyl)-iminodibenzyl To a 1 Molar (M) solution of diborane in tetrahydrofuran (lS ml) at 0C was added N-aminoacetyliminodibenzyl (0.75 gm) in dry methylene chloride (10 ml). The solution was stirred at 0C for 1 hour at room temperature, at reflux for l.S hours and at room temperature for 16 hours. 3 Normal (N) hydrochloric acid (20 ml) was then added cautiously with vigorous evolution of gas. The solution was stirred at room temperature and then concentrated to dryness in vacuo. To the residue was added water (50 ml) and sodium hydroxide until a pH of 12 was reached. The mixture was then extracted with methylene chloride (3 X
25 ml). The combined organic extracts were next washed with saturated brine (2 X 20 ml), dried over anhydrous magnesium sulfate, filtered and concentrated to a viscous oil, in vacuo. The oil was dissolved in methanol (3 ml), filtered and treated with concentrated hydrochloric acid to pH 2. The solvent was removed in vacuo. Recrystallization of the solid from chloroform/diethyl ether yielded 0.20 gm of pure product as the hydrochloride salt.

Example ~: Preparation of Immunogens 50 milligrams (mgm) of an appropriate amine was dissolved in 400 microliters (mcl) of 1 N hydrochloric acid and 400 mcl of dimethylformamide, and diluted to 2 ml with water. The solution was added to a solution of bovine serum albumin in 8 ml of water. After adjustment of the pH to 4.95 with 1 N sodium hydroxide, 52 mgm of ethyldimethylaminopropyl carbodiimide were added with stirring. After 25 minutes the pH was readjusted to 4.9 with 1 N hydrochloric acid and an additional 30 mgm of ethyl dimethylaminopropyl carbodiimide was added. After an additional 25 minutes, the solution was dialyzed against water at room temperature. Dialysis was repeated four times against 1 L of water, each time for 12 hours. This yielded an aqueous solution of purified bovine serum albumin tricyclic conjugate.

Example 5: Preparation of Tracer Compounds A. 6-carboxyfluorescein/10~ dihydroprotriptyline conjuqate A solution of S mgm of 6-carboxyfluorescein, 4.5 mgm of N-hydroxysuccinimide and 7.5 mgm of dicyclohexyl carbodiimide in 0.5 ml of dry pyridine was stirred at room temperature for 2 hours. 5 mgm of 10,11-dihydroprotriptyline hydrochloride were then added. After stirring at room temperature for 18 hours, the solution was streaked onto a silica gel thin layer chromatography plate which was developed with chloroform/methanol/acetic acid, in a ratio of 89/10/1.
The appropriate band was taken and eluted with methanol. The solution was next concentrated and rechromatographed, using the above system. Collection of the appropriate band and a third chromatography on silica gel plates, using diethyl ether/methanol/acetic - 18 - t 3~8763 acid in a ratio of 95/5/0.5, yielded a substantially pure 6-carboxyfluorescein/10,11-dihydroprotriptyline conjugate.

B. 6-carboxyfluorescein/5-rB-~m;noethyll-10 ll-D;hy~ro-5H-d;henzrb.flazep;ne con~ugate A solution of 10.75 mgm of 6-carboxyfluorescein, 4.2 mgm of l-hydroxybenztriazole and 6.45 mgm of dicyclohexylcarbodiimide in 0.4 ml of dry pyridine was stirred at room temperature of 2 hours. 6.86 mgm of 5-[B-aminoethyl]-10,11-dihydro-5H-dibenz[b,f]azepine were added.
After stirring at room temperature for 18 hours, the solution was streaked onto a silica gel thin layer plate and eluted with chloroform/methanol in a ratio of 5/1.
Collection of the appropriate band by extraction from the silica gel with methanol yielded 6-carboxyfluorescein/5-[B-aminoethyl]-10,11-dihydro-5H-dibenz[b,f]azepine as the pyridinium salt.
Substitution of the appropriate amino starting materials yielded the desired tracers, by utilisation of substantially the previously described procedures.
This application is a division of application S.N.
605,277 filed on July 10, 1989 which is itself a division of application S.N. 520,524 filed on October 15, 1986 and which is now C.P. 1,270,819.
X

Claims (2)

1. A compound of the formula:

Where: X=-(CH2)m and m=0 or 1 R = H or CH3 and biologically acceptable salts thereof.

2. A fluorescence polarization immunoassay for determining the presence or amount of a tricyclic antidepressant in a fluid, which assay comprises the steps of contacting the fluid with antisera to the tricyclic antidepressant and a compound of Claim 1 to form a homogeneous solution, passing plane polarized light through the homogeneous solution and measuring the fluorescence polarization response therefrom.