WO1999053324A1 - Salivary prolactin test for serotonergic activity - Google Patents

Abstract

Methods of determining serotonergic function are disclosed. The methods include determining the concentration of salivary prolactin in a patient by immunoassay. Such methods are useful as to indicate potential susceptibility to psychiatric disorders such as impaired impulse control, obsessive-compulsive disorder, violent behavior, Type II alcoholism, and suicidal behavior.

Description

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SALIVARY PROLACTIN TEST FOR SEROTONERGIC ACTIVITY

BACKGROUND OF THE INVENTION

Serotonin (5-hydroxytryptamine, or 5-HT) is a monoamine neurotransmitter that influences the secretion of pituitary hormones. Synthesis of 5-HT involves hydroxylation of tryptophan to produce 5-hydroxytryptophan, which is decarboxylated to produce 5-HT (Van de Kar, 1991, Annu. Rev. Pharmacol. Toxicol. 31 :289-320). 5-HT is metabolized by monoamine oxidase to produce 5- hydroxyindole acetic acid (5-HIAA).

Research suggests that a deficit in central serotonin functioning, as measured by cerebrospinal fluid (CSF) 5-HIAA levels, is linked to a number of pyschiatric disorders, including impaired impulse control, obsessive-compulsive disorder, violent behavior, Type II alcoholism, and suicidal behavior (Asberg et al. 1976, Arch. Gen. Psychiatry 33:1193-7; Bastani et al., 1990, Arch. Gen. Psychiatry 47:833-9; Brown et al., 1982, Amer. J. Psychiatry 139:741-6; Coccaro et al. 1995, International Clin. Psychopharmacology 10:177-9; Lidberg et al. 1985, Acta Psychiatrica Scandinavica 71 :230-6; Linnoila et al., 1983, Life Sci. 33:2609-14; Virkkunen and Linnoila, 1993, J. Studies on Alcohol Supp. 11 : 163-9). These studies have shown that there is an inverse relationship between CSF 5-HIAA levels and the listed psychiatric disorders; in other words, that low levels of CSF 5-HIAA (for example, less than 50 pmol/ml in Type II alcoholics) correlate with an elevated susceptibility to these disorders. Similar findings have been reported in studies of non-human primates for impulse control, violent behavior and excessive alcohol consumption (Mehlman et al, 1994, Amer. J. Psychiatry 151 :1485-91; Higley et al., 1996, Biol. Psychiatry 40:1067-82; Higley et al., 1996, Alcoholism: Clin. Exp. Res. 20:629-42). In addition, it has also been established that interindividual differences in CSF 5-HIAA concentrations are stable across time and trait-like (Higley et al., 1992, Biol. Psychiatry 32:127-45; Higley et al., 1993, Arch. Gen. Psychiatry 50:615- 23). Premature mortality resulting from violence has also been linked to abnormal -2-

levels of CSF 5-HIAA in non-human primates (Higley et al., 1996, Arch. Gen. Psychiatry 53:537-43).

These observations suggest that measurement of serotonergic function by a determination of CSF 5-HIAA levels, could be used as a clinical predictor or screening test for patients at risk from such psychiatric disorders. However, measuring CSF 5-HIAA in humans requires taking a CSF sample by lumbar puncture; the invasiveness of this procedure often precludes frequent sampling, and makes it especially difficult to perform such studies in children (see, however, Kruesi et al, 1988, Psychiatry Res. 25:59). Sampling CSF in non-human primates is performed by puncture of the cisterna magna, with the attendant dangers of infection and neurological damage.

Another procedure frequently used to assess central serotonin functioning involves measuring the prolactin response to a fenfluramine challenge (fenfluramine hydrochloride). Prolactin, also known as luteotropic hormone or lactogenic hormone, is a single chain polypeptide hormone of about 23,000 daltons molecular weight. Fenfluramine (FEN) is a presynaptic 5-HT releasing and reuptake-inhibiting agent which, secondary to its activity as a serotonergic agonist, also enhances prolactin secretion in humans (Stoff et al., 1992, Psychiatry Res. 42:65-72; Lozoff et al., 1995, Soc. Biol. Psychiatry 37:4-12; Coccaro et al., 1996, Soc. Biol. Psychiatry 40:157-64). The administration of FEN ultimately results in increased peripheral blood prolactin concentrations. The fenfluramine challenge is believed to assess central nervous system serotonin functioning (Coccaro et al., 1989, Arch. Gen. Psychiatry 46:587-99; Stoff et al., 1992, Psych. Res. 42:65-72) and it has been pharmacologically confirmed that serotonergic neurons stimulate the secretion of prolactin (see Van de Kar, 1991, Annul. Rev. Pharmacol. Toxicol. 31 :289-320).

Hence, an increase in serum prolactin levels is correlated with an increased level of serotonergic activity (i.e., the activity at synapses in the central nervous system (CNS) where serotonin in the neurotransmitter, such as in the frontal cortex region and the dorsal and medial raphe nuclei.) The FEN challenge has been used to assess serotonergic function in the investigation of pyschiatric disorders which are proposed to be related to -3-

abnormalities in serotonergic transmission, such as those discussed above (See Stoff et al., 1992, Psych. Res. 42:65-72). However, it has been difficult to establish a direct numerical relationship between plasma prolactin concentrations and CSF 5- HIAA, perhaps due to the puslatile variations of hormone concentrations that are typically seen in blood. Moreover, measuring the prolactin response after the FEN challenge is also invasive. Typically, blood is sampled every 30 minutes for up to 5 hours post-FEN administration, a technique that is not practical in the clinical setting, especially for children.

Developing a noninvasive means of assessing central serotonin functioning would be of considerable clinical benefit. A noninvasive measure of central serotonin functioning, for example, might be used to determine possible future risk for suicide, violence, or other impulsive behaviors. The three bodily fluids that are conventionally examined to determine effector molecule concentrations (e.g., hormones, neurotransmitters) as an assessment of CNS function are CSF, blood and urine (Coccaro and Kavoussi, 1994, Clin. Chem. 40:319-27). The impracticality of using CSF and blood to assess serotonergic activity are discussed above, and urine, while being the least invasive is also the least informative source for many brain systems, including serotonin (Coccaro and Kavoussi, 1994, Clin. Chem. 40:319-27). There is thus a need for a reliable assay for serotonergic activity that can be performed with a minimum of perturbance to the subject. It is to such an assay method that the present invention is directed.

DESCRIPTION OF THE DRAWING

FIG. 1 is a plot showing a correlation between salivary prolactin and CSF 5-

HIAA levels.

DESCRIPTION OF THE INVENTION

The present invention provides a minimally invasive measure of serotonergic function in a mammal. The invention arises from the discovery that salivary prolactin levels correlate with CSF 5-HIAA. Specifically, it has been discovered that there is a direct relationship between the level of salivary prolactin and the level -4-

of 5-HIAA in CSF. Because of the established correlation between CSF levels of 5- HIAA (the primary metabolite of serotonin) and serotonergic activity, this discovery permits serotonergic activity to be measured without the need for lumbar puncture or repeated phlebotomies for blood. The salivary prolactin assay therefore serves as an indicator of serotonergic activity, and may be used as a first diagnostic test for susceptibility to psychiatric disorders including impaired impulse control, obsessive- compulsive disorder, violent behavior, Type II alcoholism, and suicidal behavior. The assay is also useful in clinical research directed to establishing correlations between serum prolactin levels (concentrations) and CNS serotonin activity. In its simplest form, the invention requires obtaining a salivary sample from a patient and measuring a concentration of prolactin in the sample. The patient may be a human or other animal, and the methods described herein are useful in both human and veterinary medicine. The concentration of prolactin in the sample is then compared to "normal" prolactin levels. Preferably, such "normal" prolactin concentrations are levels found in healthy individuals having similar biological characteristics to the patient, i.e., similar age and sex. Alternatively, when a patient is being monitored through a course of treatment, the measured prolactin concentrations may be compared to levels measured in the same patient at other times in the treatment regimen, to assess response to therapy. Methods of obtaining a saliva sample from an individual are well known to health care workers, and are reviewed in Hold et al. {Int. J. Drug Testing 1 :1, 1996; also available at http://big.stpt.usf.edu/~journal/volumel.html). One suitable and simple method involves placing a sterile cotton swab in the mouth of the patient for between 30 seconds and two minutes. The swab is then removed and stored in a sealed container for subsequent analysis. If necessary, the patient may be given an inert substance to chew in order to stimulate salivation. Commercial devices for obtaining and storing saliva samples are available, and include the ORASURE® device manufactured by Epitope Inc, Beaverton, Oregon U.S.A., described in U.S. Patent Nos. 5,022,409, 5,103,836 and 5,339,829. Alternatively, a patient may simply be asked to expectorate into a suitable vial or other container. If the saliva is not to be assayed immediately, it may either be frozen at -20°C or stored in a -5-

preservative system, such as that described in the aforementioned U.S. Patents. If necessary for subsequent analysis, the saliva may be concentrated using standard methods. Concentrating saliva specimens would be particularly helpful to increase relative prolactin concentrations that are below detectable limits of a particular assay. If the specimen is concentrated, values that correspond to particular levels of CNS serotonergic activity can also be correspondingly adjusted, using the techniques disclosed herein.

Methods of measuring prolactin have been described for the determination of serum prolactin concentrations, and such methods, which are generally immunoassays, are also suitable for use in the present invention. By way of example, a test kit marketed by IMMUNOS (Albany, CA) (catalog no. KH3020) utilizes microtiter wells to which are attached anti-prolactin monoclonal antibodies (the primary antibodies). The assay sample is incubated in the wells at room temperature for 60 minutes with a solution of a second anti-prolactin monoclonal antibody conjugated to horseradish peroxidase. After the incubation period, the wells are rinsed with water to remove unbound labeled antibodies. A solution of the color reagent TMB is added to the wells and incubated for 20 minutes. A blue color develops in those wells in which the second antibody is present. Then, 2N HCL is then added to the wells, and the blue color turns to yellow. The presence of the yellow color is quantified spectrophotometrically at 450nm. The assay is reportedly capable of detecting concentrations of prolactin below 7.5 ng/ml.

It will be recognized that this basic assay approach can be modified in many ways. For example, the primary antibody may be attached to a number of solid surfaces, including petri dishes, columns, magnetic beads and microtiter wells. Alternatively, the primary antibody may be free in solution and the secondary

(labeled) antibody may be immobilized on a suitable surface. The label attached to the secondary antibody may be anything that can be readily detected, such as an enzyme, a radioisotope or an electrochemiluminescent compound.

One such alternative detection system for prolactin is marketed by IGEN International, Inc., Gaithersburg, MD. IGENs ORIGEN™ human prolactin assay uses electrochemiluminescence to produce a sensitive prolactin assay. The assay -6-

sample is incubated with an anti-prolactin antibody labeled with a ruthenium metal chelate (TAG) and a biotinylated anti-prolactin antibody for one hour at room temperature. Streptavidin coated paramagnetic beads are then added to the mixture, which is then incubated for a further 10 minutes. The biotinylated antibodies (bound to prolactin) attach to the beads, and the TAG antibodies attach to the prolactin, creating a sandwich. The mixture is then diluted with an assay buffer containing tripropylamine (TPA), channeled through a flow cell and the beads are captured at an electrode. When voltage is applied, the TAG (in the presence of the TPA) becomes luminescent. Measurement of the luminescence permits quantification of the TAG antibody, and thereby of the prolactin level in the assay sample. The assay is reported to be capable of detecting 0.09 ng/ml prolactin in a sample.

Anti-prolactin antibodies may be purchased commercially from suppliers including Research Diagnostics, Inc., (Flanders, NJ) (catalog nos. RDI-PRO172, RDI-PRO174 and RDI-PRO180) and Biomeda (Foster City, CA) (catalog nos. 016D and Kl 16). Alternatively, anti-prolactin antibodies may be raised using standard methods, for example, those described in Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, ISBN 0-87969-314-2). Purified mammalian prolactin for use in raising antibodies (for example, human and sheep prolactin) may be purchased commercially, from biochemical suppliers, including Sigma (St. Louis, MO) (catalog numbers L 7009 and L 6520), Biomeda (Foster City, CA) (catalog nos. 423M and A18) and Research Diagnostics, Inc., (Flanders, NJ) (catalog no. RDI-4-06-459).

As used herein, the phrase "anti-prolactin antibody" includes prolactin- specific polyclonal and monoclonal antibodies, as well as immunologically active fragments of monoclonal antibodies such as Fab, Fab' F(ab')₂ and Fv fragments which retain the ability to specifically bind to prolactin. As noted above, monoclonal or polyclonal antibodies to prolactin may be prepared by a number of methods, including the classical hybridoma fusion method of Kohler and Milstein (Nature 256:495, 1975) or derivative methods thereof. Briefly, to produce monoclonal antibodies by the hybridoma fusion method, a mouse is repetitively inoculated with a few micrograms of human prolactin over a period of a few weeks. -7-

The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Enzymol. 70:419, 1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, ISBN 0-87969-314-2).

Immunologically effective fragments of monoclonal antibodies may also be used in the methods disclosed herein, including Fab (including Fab without the constrant region), Fab', F(ab')₂ Fabc and Fv portions. Methods of making and using immunologically effective fragments of monoclonal antibodies, are well known and include those described in Better and Horowitz (Meth. Enzymol. 178:476-96, 1989), Better et al. (Advances in Gene Technology: The Molecular Biology of Immune Disease & the Immune Response (ICSU Short Reports), Streilein et al., eds. vol. 10:105, 1990), Glockshuber et al. (Biochemistry 29:1362-7, 1990), and U.S. Patent Nos. 5,648,237 ("Expression of Functional Antibody Fragments"), 4,946,778 ("Single Polypeptide Chain Binding Molecules"), and 5,455,030 ("Immunotherapy Using Single Chain Polypeptide Binding Molecules"), and references cited therein. Optimally, antibodies raised against prolactin will specifically detect prolactin, i.e., they will be prolactin-specific. That is, anti-prolactin antibodies used in the present invention will preferably recognize and bind prolactin and would not substantially recognize or bind to other proteins found in saliva. The determination that an antibody is prolactin-specific is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al., 1989, In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York). For example, to determine that a given mouse antibody preparation -8-

specifically detects prolactin by Western blotting, protein is extracted from saliva and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove non-specifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse Ig antibody conjugated to an enzyme such as alkaline phosphatase; application of the substrate 5-bromo-4- chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a dense blue compound by immuno-localized alkaline phosphatase. Antibodies which specifically detect prolactin will, by this technique, be shown to bind to the prolactin protein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific antibody-prolactin protein binding.

Patients having a salivary prolactin concentration below the range observed for typical, healthy individuals may be considered as having an enhanced susceptibility for psychiatric disorders associated with impaired serotonergic function, such as impaired impulse control, obsessive-compulsive disorder, violent behavior, Type II alcoholism, and suicidal behavior. It will be apparent that the lower prolactin concentrations will be associated with increased susceptibility. It is anticipated that the salivary prolactin assay can be used as a screening test for possible risk (suitable for general clinical use, and in situations where a lumbar puncture is undesirable) and may be followed with confirmatory tests of serotonergic function, including direct CSF measurement of 5-HIAA.

EXAMPLE 1 Comparison of CSF 5-HIAA and Salivary Prolactin Levels in Monkeys Twenty seven adult rhesus macaques (Macaca mulatto) were utilized for the study, 18 females and 9 males, housed at the NIH Animal Center. Subjects ranged -9-

in age from 8 to 13 years, with the mean age for females and males 11.2 and 10.4 years respectively.

All body fluid samples were obtained between 1200 and 1400 hours, on four separate dates. Blood and CSF samples were obtained as soon as possible after subjects had been anesthetized using ketamine hydrochloride (0.15 mg/kg). Blood was collected from the femoral vein, while CSF was collected from the cisterna magna. Saliva was collected from subjects only after all other physiological samples had been obtained, using a pipette to extract the saliva directly from the subject's mouth. Serum and saliva were assayed for concentrations of prolactin, while CSF was assayed for concentrations of 5-HIAA, homovanillic acid (HVA), and 3- methoxy-4-hydroxyphenylglycol (MHPG). Time to obtain the CSF and blood sample were recorded for each subject. The CSF was analyzed for 5-HIAA, HVA, and MHPG concentrations using liquid chromatography with electrochemical detection (Scheinin et al., 1983, Anal. Biochem. 131 :246-53; Seppala et al., 1984, Acta Pharmacol. Toxicol. 55:81-7). Inter- and intra-assay variabilities were less than 10%. Serum and salivary samples were assayed for prolactin concentrations by Covance Biotechnologies using radioimmunoassays (Abraham et al, 1972, Analyt. Lett. 5:757-66), with a lower limit of detection of 3.84 ng/ml. FIG. 1 shows the results obtained when CSF 5-HIAA concentrations were plotted against salivary prolactin concentrations.

Data were analyzed using Pearson Product correlations. Preliminary analyses showed no significant correlations between either salivary prolactin or CSF 5 -HI A A concentrations and any of the variables of weight, time to inject the subjects with ketamine, time to obtain CSF sample, or gender. The assay results showed that CSF 5-HIAA concentrations were positively correlated with salivary prolactin (r= 0.75, df=25, p=.0001), indicating that subjects with low CNS serotonin turnover rates were also low in salivary prolactin concentrations. HVA exhibited a trend level correlation with salivary prolactin (r=.35, df=25, p=.07). However, when 5- HIAA and HVA were entered simultaneously as independent variables in a multiple regression with salivary prolactin as the dependent variable, only 5-HIAA remained statistically correlated (5-HIAA: r= 0.76, df=25, p=.0001; HVA: r=-.02, df=25, -10-

p=.89). Neither of the other two biochemicals significantly correlated with salivary prolactin (MHPG: r=0.21, df=25, p=.30; and serum prolactin: r= 0.13, df=25, p=.51). HVA significantly correlated with serum prolactin (r=.38, df=25, p=.05). However, when one subject, whose serum prolactin concentration value was three standard deviations above the mean, was removed, HVA was no longer significantly correlated (r=.26, df=24, p=.19). 5-HIAA and MHPG did not significantly correlate with serum prolactin (5-HIAA: r=.14, df=25, p= 50; MHPG: r=.03, df=25, p= 89).

The results of this study show that salivary prolactin concentrations correlate positively with CSF 5-HIAA. Previous research has shown that that individuals with a deficit of central serotonin functioning, as measured by low CSF 5-HIAA concentrations, are at risk for many neuro-pathological or psycho-pathological behaviors. Hence, salivary prolactin can be used as an index of CNS serotonin activity, and for identifying individuals at risk for serotonin-related disorders. Since salivary prolactin concentrations are quite low (six subjects had concentrations below 3.86 ng/ml), highly sensitive prolactin assays are preferably employed. The lack of a significant correlation between salivary prolactin concentrations and serum prolactin concentrations is notable and is likely a result of the pulsatile variation in serum prolactin previously reported. This observation suggests that serum prolactin is of questionable utility as a measure of serotonergic activity and further highlights the value of the discovery reported here.

EXAMPLE 2 Determination of Salivary Prolactin Concentrations in Humans

Saliva and blood were collected from ten human subjects, twice, as described in Example 1. Salivary and serum prolactin concentrations in each of the samples were detected (as in Example 1) and prolactin was found to be present in human saliva. Three out of the ten subjects had concentrations of salivary prolactin above 1.0 ng/ml.

As used in this specification, a "psychiatric disorder" is a behavioral disorder, for example a disorder as defined in the DSM-IV (Diagnostic and Statistical Manual IV), which categorizes such disorders by constellations of -11-

symptoms. However, the term "psychiatric disorder" can include recognized behavior disorders that are not included in the DSM-IV.

Having illustrated and described the principles of determining serotonergic activity by means of measuring salivary prolactin concentrations, it will be apparent to one skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications coming within the spirit and scope of the claims presented herein.

Claims

-12-We claim:

1. A method of determining serotonergic activity in a mammal comprising measuring a concentration of prolactin in the saliva of the mammal.

2. The method of claim 1 wherein measuring the concentration of prolactin in the saliva is performed by an immunoassay.

3. The method of claim 1 wherein measuring the concentration of prolactin comprises :

(a) obtaining a sample of saliva from the mammal;

(b) incubating the sample of saliva with a prolactin-specific antibody;

(c) detecting antibody :prolactin conjugates; and

(d) quantifying salivary prolactin.

4. The method of claim 3 wherein the prolactin-specific antibody is linked to a detectable marker.

5. The method of claim 3 wherein the prolactin-specific antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies and immunologically effective fragments thereof.

6. A method for determining the susceptibility of a subject to a psychiatric disorder associated with abnormal serotonergic function, the method comprising determining a concentration of prolactin in saliva of the subject and comparing that concentration with salivary prolactin concentrations found in individuals who do not have the psychiatric disorder.

7. The method of claim 6 wherein the psychiatric disorder is selected from the group consisting of impaired impulse control, obsessive-compulsive disorder, violent behavior, Type II alcoholism, and suicidal behavior. -13-

8. The method of claim 6 wherein measuring the concentration of saliva rmed by an immunoassay.