Patent Application
20220026427
The present invention relates generally to the measurement of cholinergic activity. More specifically, the present invention relates to methods and kits for assessing cholinergic and anticholinergic activity of a sample.
Cholinergic receptor blockade in the central nervous system (CNS) is associated with impaired cognitive function and for this reason medications with anticholinergic activity are often carefully prescribed and dosed. However, many prescription and non-prescription drugs have varying degrees of anticholinergic activity, and when these drugs are combined, a significant amount of anticholinergic activity may result.5 The elderly, who very often take multiple medications for various different types of health issues, are particularly vulnerable in this respect particularly since CNS cholinergic function diminishes with aging. To assess the total burden of anticholinergic activity, a serum anticholinergic activity assay (SAA) was introduced in the early 1980s by Tune and Coyle1, 2 and has since been used as a putative marker of cognitive dysfunction in several conditions, albeit not always with consistent results3-12. The original assay was based on the displacement of 3H-QNB binding to rat brain homogenates by anticholinergics in human serum. Subsequently, however, questions were raised concerning the basic validity of the SAA protocol and several potential limitations have been identified.9 Among these is a potential role for large serum proteins which may significantly mask or distort SAA values.13 Another issue refers to the fact that the original protocol did not discriminate between various subtypes of muscarinic receptors.14 This may be particularly relevant for studies correlating SAA with cognitive status, given that only two of the five known muscarinic receptor subtypes (M1 and M2) have been shown to be involved in cognitive functions.14 These and other issues have highlighted the need for an alternative protocol to assess total anticholinergic activity in human serum.
According to an embodiment of the present invention there is provided a method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) anticholinergic activity in a blood serum sample, the method comprising:
In a further embodiment, there is provided a method as described herein wherein the M1 receptor ligand is [3H] quinuclidinyl benzilate (3H-QNB), [3H] N-methyl-scopolamine (3H-NMS) or [3H] pirenzepine (3H-PZP). Any other isotopic label which permits efficient detection also may be used as may be any other suitable receptor ligand. In a preferred embodiment the M1 receptor ligand is 3H-QNB or 3H-NMS.
There is also provided a method as described herein, wherein the blood serum sample is derived from a patient or subject that exhibits one or more signs or symptoms of elevated M1 receptor anticholinergic activity, is suspected of having elevated blood levels of M1 receptor anticholinergic activity, exhibits no symptoms of elevated M1 receptor anticholinergic activity, or wherein the level of anticholinergic activity is unknown.
Also provided by the present invention is a method as described herein wherein the standard is atropine and wherein displacement by atropine of ligand binding to the M1 receptor is performed to generate one or more standard curves. The binding of atropine to the M1 receptor may be performed under essentially the same conditions as the M1 receptor binding to the M1 ligand. In an embodiment of the present invention, the level of M1 receptor anticholinergic activity may be expressed as atropine equivalents wherein the level of M1 receptor anticholinergic activity in the blood serum sample is calculated or estimated on the basis of the amount of atropine that would provide a substantially similar or identical degree of inhibition of the specific binding of the M1 receptor ligand to the M1 receptor.
In a further embodiment of the present invention, there is provided a method as described herein, wherein an elevated M1 receptor anticholinergic activity is equivalent to or higher than about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or 140 pmol/mL atropine, and optionally associated with an age, a minimum age or an age range.
The present invention also contemplates a method as described herein wherein the M1 receptor is a rat receptor or human M1 receptor. Receptors from other species may be employed, particularly those which exhibit 100% identity to the rat or human M1 receptor.
In a further embodiment, there is provided a method as described herein which employs about 20 to 35 μg of membrane preparation, for example, but not limited to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or about 35 μg of membrane preparation. In a preferred embodiment about 27 μg of membrane preparation is employed in a 96 multiwell plate.
Also provided herein is a method wherein a volume of serum is deproteinized to perform the method.
For example, but not to be considered limiting in any manner, about 1.5 mL of serum may be deproteinized and then an aliquot of that deproteinized solution, for example, but not limited to 100 μL may be employed in the method. The manufacturer of the protein deproteinization kit employed herein considers the deproteinized serum samples to be diluted to about 76% of the original concentration.
Also provided is a method as described herein wherein the ratio of the membrane preparation:treated serum sample is about 0.1 g:1 L to 0.4 g:1 L, for example, but not limited to about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 or 0.4 g:1 L. In a preferred embodiment, which is not meant to be limiting in any manner, the method employs about 100 μl deproteinized serum with about 27 μg M1WT3 protein. In a further embodiment the method employs about 10, 11, 12, 13, 14, 15, 16, 17, 18, 29, 20, 21, or 22 nM of ligand.
In a preferred embodiment, the method employs about 16 nM of ligand which is close to the Kd of the ligand with the receptor, which is preferred. The nature of the ligand and its concentration may be changed or varied as needed or required as would be understood by a person of skill in the art.
In a further embodiment of the present invention, there is provided a method as described herein, wherein binding of the M1 receptor to M1 ligand employs a buffer comprising about 20 mM HEPES, about 100 mM NaCl and about 10 mM MgCl2 adjusted to a pH of about 7.4. Other suitable buffers also may be used, for example, but not limited to about 50 mM Na2PO4 pH 7.7 and about 10 mM KNaPO4 pH 7.4.
Also provided is a method as described herein, wherein the PCA-treated serum sample, membrane preparation, M1 ligand and buffer are mixed at about 0° C. and the incubating step is performed at about 20 to 25 degrees, for example, but not limited to 24° C. for between about 30 and 120 minutes, for example, but not limited to 60 min.
In a further embodiment, after the step of incubating and before the step of detecting, an unbound M1 ligand is removed by filtering the membrane preparation on a filter with a pore size suitable for filtering unbound M1 ligand and retaining the M1 receptor followed by rinsing the membrane preparation.
Also provided herein is a method for assessing anticholinergic activity of a sample, the method comprising:
There is also provided a method for assessing anticholinergic activity of a serum sample, the method comprising:
In a further embodiment there is provided a method of modulating serum anticholinergic activity in a patient or subject about to receive medication or that is currently receiving medication, the method comprising:
Also provided is a method as described herein that further comprises repeating the steps before the modulating step to determine if the modulating changed the serum anticholinergic activity of the patient or subject. In a preferred embodiment, the modulating reduces serum anticholinergic activity of the patient or subject.
Also provided herein is a method of modulating serum anticholinergic activity of a patient receiving medication and exhibiting one or more signs of cognitive side effects, the method comprising: obtaining a serum sample from the patient;
In a further embodiment, there is provided a method that further comprises repeating the steps before the modulating step to determine if the modulating decreased the serum anticholinergic activity.
In still a further embodiment, there is provided a method as described above and herein throughout, wherein one or more signs or symptoms comprise one or more cognitive side effects or non-cognitive side effects such as, but not limited to dementia, memory loss, cognitive decline, decrease in global cognitive functioning, psychomotor speed, decrease in visual and/or declarative memory, implicit learning or communication ability, confusion, disorientation, agitation, euphoria or dysphoria, respiratory depression, inability to concentrate, inability to sustain a train of thought, incoherent speech, irritability, wakeful myoclonic jerking, unusual sensitivity to sudden sounds, illogical thinking, photophobia, visual disturbances, visual, auditory, or other sensory hallucinations, orthostatic hypotension, urinary problems and/or kidney failure, salivary problems such as dry mouth, blurred vision, constipation, hypohydrosis, dizziness and the like.
The present invention also provides a kit for assessing or determining anticholinergic activity comprising one or more of the following components in any combination: cells expressing an M1 receptor, one or more cell culture media, one or more cell wash media, one or more buffers, protein concentration assay determination reagent(s), one or more anticholinergic compounds or compositions, atropine, one or more multi-well plates, M1 receptor membrane preparations adhered to a plate, filter or other substrate, one or more filtration membranes, scintillation fluid, one or more M1 ligands, deproteinization solution, perchloric acid, perchloric acid neutralization solution, data analysis software, serum containing one or more anticholinergic compounds or compositions, glassware, centrifuge tubes, instructions for performing the anticholinergic assay or any combination thereof.
In a preferred embodiment, which is not meant to be limiting in any manner, the kit comprises:
Optionally the kit as described herein may further comprise one or more multiwell plates, one or more multi-well plates comprising a filter with a pore size suitable for filtering unbound M1 ligand and retaining the M1 receptor, buffer, scintillation fluid or any combination thereof.
In still a further embodiment there is provided a kit for assessing anticholinergic activity of a serum sample, the kit comprising:
one or more multiwell plates comprising, in each well, a membrane preparation from cultured cells expressing rat muscarinic receptor subtype 1 (M1 receptor), or one or more multiwell plates comprising a filter with a pore size capable of filtering unbound M1 ligand and retaining the M1 receptor. The membrane preparation expressing rat muscarinic subtype 1 (M1 receptor) may be bound to the filter through which unbound M1 ligand in the assay may be washed away, or it may be provided separately.
In another embodiment, there is provided herein a method for identifying a subject as being at risk of having or developing cognitive impairment, the method comprising:
In yet another embodiment, the method may further comprise a step of:
In still another embodiment, the subject may be a subject being treated with at least one drug having anticholinergic properties.
In yet another embodiment, the cognitive impairment may be in the spatial working memory cognitive domain.
As will be understood, while the above embodiments pertain to determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) anticholinergic activity in a blood serum sample, similar methods may be employed to determine the level of other muscarinic receptor cholinergic activity in a sample using a suitable receptor ligand and suitable membrane preparation expressing the other muscarinic receptor.
In a further embodiment of the present invention, there is provided a non-radioactive method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) cholinergic and anticholinergic activity in a blood serum sample, the method comprising:
The cultured cells used in the methods as described herein may be any suitable cells known in the art. In a preferred embodiment, the cells are human or rat cells. In a preferred embodiment, which is not meant to be limiting, the cells are CHO cells expressing human or rat M1 Muscarinic receptors.
The non-radioactive methods as described herein contains different phases, described generally herein, but not wishing to be limiting, as a pre-carbachol phase and a post-carbachol phase. It is important to note that each phase provides useful and interesting information and can be practiced alone or in combination. Accordingly, the present invention provides a non-radioactive method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) cholinergic and anticholinergic activity in a blood serum sample, the method comprising either A) or B):
A) removing protein from the blood serum sample by treatment with perchloric acid (PCA) to produce a PCA-treated serum sample;
incubating the PCA-treated serum sample with a membrane preparation from cultured cells expressing the M1 receptor, the M1 receptor loaded with a calcium sensitive fluorophore or dye;
collecting fluorescence measurements from the cells for a first period of time;
comparing acetylcholine receptor agonist activity of the blood serum sample to a control blood serum sample, wherein the control blood serum sample is known to contain no cholinergic agonists or other drugs with cholinergic activity;
wherein comparing the activity of the blood serum sample to a control blood sample provides a measure of pure agonist cholinergic properties of a subject's serum;
or;
B) removing protein from the blood serum sample by treatment with perchloric acid (PCA) to produce a PCA-treated serum sample;
incubating the PCA-treated serum sample with a membrane preparation from cultured cells expressing the M1 receptor, the M1 receptor loaded with a calcium sensitive fluorophore or dye;
adding an aliquot of carbachol solution or another acetylcholine receptor agonist to produce maximal or near maximal fluorescence of the cells from release of calcium;
collecting fluorescence measurements from the cells for a period of time;
comparing post-carbachol, or other acetyl choline receptor agonist, activity of the blood serum sample to post-carbachol activity, or other acetylcholine receptor agonist, to a control blood serum sample, wherein the control blood serum sample is known to contain no cholinergic agonists or other drugs with cholinergic activity;
wherein comparing post-carbachol activity provides a measure of the subject serum's net agonist and antagonistic cholinergic properties.
In a further embodiment of the present invention, there is provided a method as described above and herein, wherein the blood serum sample is derived from a patient or subject that exhibits one or more signs or symptoms of high or elevated M1 receptor cholinergic or anticholinergic activity, is suspected of having high or elevated blood levels of M1 receptor cholinergic or anticholinergic activity, exhibits no symptoms of high M1 receptor cholinergic or anticholinergic activity, or wherein the level of cholinergic or anticholinergic activity is unknown. The subject may exhibit one or more signs or symptoms, such as, but without limitation, eye miosis, or blurry vision, nausea, vomiting, diarrhea, bronchoconstriction, bronchorrheal or increased secretions in the tracheobronchial and/or gastrointestinal system, bradycardia, increased urinary frequency and/or urgency. Other conditions are contemplated as would be understood by a person of skill in the art.
Also provided herein are kits for assessing or determining cholinergic and/or anticholinergic activity comprising one or more of the following components in any combination: cells expressing an M1 receptor, one or more cell culture media, one or more cell wash media, one or more buffers, protein concentration assay determination reagent(s), one or more cholinergic compounds or compositions, atropine, one or more multiwell plates, M1 membrane preparations adhered to a plate or other substrate, one or more filtration membranes, scintillation fluid, one or more M1 ligands, deproteinization solution, perchloric acid, perchloric acid neutralization solution, data analysis software, serum containing one or more cholinergic compounds or compositions, a calcium sensitive dye or fluorophore, glassware, centrifuge tubes, instructions for performing an cholinergic assay or any combination thereof. In a preferred embodiment, the kit comprises any one or all components to conduct and/or perform one or more fluorescence-based tests to determine cholinergic activity of a blood or serum sample.
Described herein are embodiments illustrative of compositions, kits and methods for assessing anticholinergic and cholinergic activity of a sample. It will be appreciated that the embodiments and examples described herein are for illustrative purposes intended for those skilled in the art and are not meant to be limiting in any way. All references to embodiments or examples throughout the disclosure should be considered a reference to an illustrative and non-limiting embodiment or an illustrative and non-limiting example.
In the methods recited herein, assessing anticholinergic activity of a serum sample involves assessing the capacity of drugs and/or other compounds in the serum to bind to muscarinic receptors thereby reducing the binding of a test radioligand to, for example, but not limited to, the muscarinic M1 receptor. The procedure thus determines the total burden of anticholinergic activity at the receptor, irrespective of the type and amounts of individual anticholinergic compounds that may be present in serum. This is especially important when the serum may contain a combination of drugs and/or compounds that can result in high or elevated anticholinergic activity, possibly leading to negative cognitive or other side effects. The method is based on the competitive binding between specific ligands and anticholinergic drugs and/or compounds in the blood serum that interact with the muscarinic receptors, for example, but not limited to the M1 receptor. In an embodiment, which is not meant to be limiting in any manner, binding of the M1 ligand to the M1 receptor is reduced in a proportion to the concentration and potency of the anticholinergic drugs and/or compounds in the serum.
According to an embodiment of the present invention, there is provided a method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) anticholinergic activity in a blood serum sample, the method comprising:
According to an alternate embodiment of the present invention, there is provided a method for assessing anticholinergic activity of a serum sample, the method comprising:
In the context of the present invention, the term “membrane preparation” is meant to encompass any suitable cellular preparation that comprises membrane proteins, including the muscarinic receptors, for example, but not limited to the M1 muscarinic receptor. Any suitable technique known in the art may be utilized to obtain the membrane preparation. Particularly suitable techniques substantially maintain the structure and/or activity of the receptor as would be appreciated by a person of skill in the art. For example, and without being limiting, the membrane preparation may be obtained by a modified method of Lazareno et al.15 Briefly, cells may be homogenized on ice for 30 seconds, centrifuged at 40,000×g for 90 min at 4° C., rinsed with a solution comprising 20 mM HEPES and 0.1 mM EDTA adjusted to pH 7.4, centrifuged again at 40,000×g for 10 min, reconstituted, and stored at −80° C. Other suitable procedures known in the art may also be used to obtain the membrane preparation.
The membrane preparation may be obtained from cultured cells that express the M1 receptor. To obtain cultured cells that express the M1 receptor, molecular biology techniques commonly known in the field may be used. For example, an appropriate cell may be transformed with a vector comprising the necessary nucleic acid information to result in the expression of the M1 receptor. To confirm the presence of the M1 receptor on the transformed cells, the M1 receptor may be detected with the use of antibodies, or any other biochemical technique known in the art. In an embodiment of the present invention, ATCC® M1WT3 cells, which express rat muscarinic receptor subtype 1, may be used. In an embodiment of the present invention the M1 receptor is a rat M1 receptor. In a further embodiment, the M1 receptor is a human M1 receptor. In a further embodiment the cultured cells and the M1 receptor are from different species. In a further embodiment, the cultured cells and the M1 receptor are from the same species. In an embodiment the M1 receptor is a rat M1 receptor and the cultured cells are CHO cells.
The use of cell lines has distinct advantages over the use of rat brain tissue. Cells can be commercially purchased and grown in the laboratory as needed, thereby obviating the need to sacrifice experimental animals for the assays. Tests suggest that with the M1WT3 cell line, there is no appreciable loss of binding after 60 cycles of cell growth. In addition, the fact that much smaller quantities of material are needed makes it possible to achieve large-scale simultaneous processing, thereby increasing precision and reliability. A related advantage is the reduction in the total amount of M1 ligand needed per assay, which contributes significantly to reducing overall costs.
The removal of proteins, for example, large proteins, from serum may aid in removing interference caused by proteins in the method. Perchloric acid (PCL) deproteinization is one method that may be used in the removal of serum proteins. Briefly, the serum sample may be mixed with ice-cold PCA, incubated on ice for about 5 minutes and then centrifuged at about 13,000×g for 2 min. The supernatant may be removed and neutralized, after which the precipitated PCA may be removed to produce the treated serum sample.
After incubating the serum with the M1 ligand in the presence of the membrane preparation, the amount of binding of the M1 ligand to the M1 receptor is detected and quantified. Detection can be performed using any technology known in the art. For example, the M1 ligand may be a radioligand or may be labeled with a fluorescent dye, moiety or group where the radioactivity or fluorescence of the bound M1 ligand may be detected. Alternatively, the M1 ligand bound to the M1 receptor may be detected with the use of immunolabeling. In a preferred embodiment of the present invention, the M1 ligand is a radioligand. In a further embodiment of the present invention, the M1 ligand is [3H]quinuclidinyl benzilate ([3H] QNB), [3H] N-methyl-scopolamine ([3H] NMS) or [3H] pirenzepine ([3H] PZP). In a preferred embodiment, the M1 ligand is [3H] QNB or [3H] NMS.
The binding of the M1 ligand to the M1 receptor is usually compared to a standard or reference. For example, one or more standard curves for the displacement of increasing concentrations of a particular anticholinergic drug, for example and without being limiting, atropine, may be performed with one or more concentrations of the M1 ligand that is used in the method. A standard curve may be constructed by plotting the reduction of M1 binding induced by increasing amounts of atropine. A subject's SAA level is estimated by the reduction that serum from this subject induces in M1 binding, and it is expressed in terms of the amount of atropine that would be necessary to achieve the same effect. The result may be expressed as atropine equivalents. The standard curves may be performed with concentrations of atropine ranging from, for example, but not limited to 0.0 nmol/mL to 100 nmol/mL, or more in serum. The results obtained from the method may be calculated on the basis of the amount of atropine (atropine equivalent in pmol/mL, or any other suitable units value) that would provide a similar or identical degree of inhibition. Examples of standard curves are shown in
In an embodiment of the present invention, the blood serum sample may be derived from a patient or subject that exhibits one or more signs or symptoms of high or elevated M1 receptor anticholinergic activity, is suspected of having high or elevated blood levels of M1 receptor anticholinergic activity, exhibits no symptoms of high or elevated M1 receptor anticholinergic activity, or wherein the level of anticholinergic activity is unknown.
By the term “high” or “elevated” M1 receptor activity, it is meant an anticholinergic activity that is equal to or greater than a specific activity level, which may be recited in terms of pmol/mL atropine or any other appropriate unit as would be known in the art, for example, but not limited to 50, 55, 60, 65, 70, 75, 80, 85 90, 95, 100, 105, 110, 115, 120, 130, 135, 140 pmol/mL atropine or higher. Further the term may be defined by a range of any two values recited or any two values recited therein between. Signs or symptoms of elevated anticholinergic activity may include one or more cognitive side effects or non-cognitive side effects such as, but not limited to dementia, memory loss, cognitive decline, decrease in global cognitive functioning, psychomotor speed, decrease in visual and/or declarative memory, implicit learning or communication ability, confusion, disorientation, agitation, euphoria or dysphoria, respiratory depression, inability to concentrate, inability to sustain a train of thought, incoherent speech, irritability, wakeful myoclonic jerking, unusual sensitivity to sudden sounds, illogical thinking, photophobia, visual disturbances, visual, auditory, or other sensory hallucinations, orthostatic hypotension, urinary problems and/or kidney failure, salivary problems such as dry mouth, blurred vision, constipation, hypohydrosis, dizziness and the like. In a preferred embodiment, the signs include one or more cognitive side effects. In a further embodiment, the signs include one or more non-cognitive side effects.
In an embodiment of the method, after the step of incubating and before the step of detecting, unbound M1 ligand is removed, for example, but not limited to filtering the membrane preparation on a filter with a pore size suitable for removing unbound M1 ligand and retaining the M1 receptor/M1 ligand complex, followed by rinsing the membrane preparation. For example, but without wishing to be limiting, a GF/B filter, which has a pore size of about 1 μm, that is presoaked in 0.1% poly(ethyleneimine) may be used. The membrane preparation may be rinsed with any suitable physiological buffer, for example, a buffer comprising about 50 mM Tris HCl, about 150 mM NaCl adjusted to a pH of about 7.4 at about 0° C. Other buffers may be employed under other conditions as would be understood by a person of skill in the art.
It is contemplated that the method or methods of the present invention can be performed in a multiwell plate, for example, but not limited to a plate comprising 6, 24, or 96 wells. Plates comprising other numbers of wells also may be used. Similarly other suitable devices and/or systems may be employed as would be understood by a person of skill in the art.
The present invention also provides for a method for assessing anticholinergic activity of a serum sample, the method comprising:
In some instances, it may be desirable to test samples other than serum samples to assess anticholinergic activity. Such a sample may comprise compounds, compositions, drugs or medicaments which exhibit anticholinergic activity dissolved in a solvent such as, but not limited to water or the like.
In such cases, removing protein from samples may not be required, particularly if it is known in advance that the samples do not comprise protein or other components which should be removed by deproteinization. Thus, according to a further embodiment of the present invention, there is provided a method for assessing anticholinergic activity of a sample, the method comprising:
The methods presented herein are useful for determining, assessing and understanding the level of anticholinergic components in a sample such as a patient's blood sample. This in turn may assist in determining, assessing and/or understanding the effects of one or more medications administered to a patient on serum anticholinergic activity, allowing for the ability to modulate the type of medication(s), dosage, dosage form, delivery route and/or dosage regimen of medication administered to the patient depending on the result.
Therefore, in one embodiment, the present invention provides for a method of modulating serum anticholinergic activity in a patient receiving medication, the method comprising:
Modulating the type of medication may encompass stopping administration of a medication, changing a medication, adding one or more new medications or any combination thereof. Modulating the dosage of medication may comprise lowering or increasing the dosage of a medication as desired and/or necessary. Further, the dosage form or delivery route of a medication or combination of medications may be changed depending on the result of the assay. For example, but without wishing to be limiting in any manner, it may be desirable to change medications from a quick release dosage form to a sustained delivery dosage form. Similarly it may be desirable to change from oral delivery to intravenous delivery of the medicament. Further, it may be desirable to change from a once a day delivery regimen to smaller doses multiple times a day, for example.
The present invention further contemplates assessing the serum anticholinergic activity after the medication has been modulated to determine if the change in the medication had the intended outcome.
The present invention also provides for a method of modulating serum anticholinergic activity of a patient receiving medication and exhibiting one or more cognitive side effects, the method comprising:
The methods described herein also may comprise a step of monitoring a subject for one or more signs or symptoms of high or elevated serum anticholinergic activity. Signs or symptoms of high anticholinergic activity include, may include, without limitation a variety of cognitive side effects.
The present invention further contemplates assessing the serum anticholinergic activity after the medication has been modulated to determine if the change in the medication decreased serum anticholinergic activity, thereby possibly decreasing the cognitive side effects.
The present invention also provides for a kit for assessing anticholinergic activity of a serum sample, the kit comprising:
The kit may further comprise a perchloric acid solution to remove protein from the serum sample to produce a treated serum sample, perchloric acid neutralization solution, one or more multi-well plates, one or more multiwell plate comprising a filter with a pore size suitable for removing unbound M1 ligand while retaining the M1 receptor-M1 ligand complex intact, one or more buffers, cell culture medium, scintillation fluid, instructions for assessing anticholinergic activity in a sample or any combination thereof.
In a further embodiment, the kit further comprises one or more multiwell plates, or one or more multiwell plates comprising a filter with a pore size suitable of filtering unbound M1 ligand and retaining the M1 receptor, and buffer. In an even further embodiment, the kit further comprises scintillation fluid.
Another embodiment of a kit of the present invention comprises:
In a further embodiment there is also contemplated a method as described above and herein throughout, wherein the muscarinic acetylcholine receptor is the M2 receptor or both the M1 and M2 receptors. Preferably the M1 or M2 or both M1 and M2 receptors are expressed on the surface of cultured cells and maintain native or near native conformation and/or activity as compared to the same receptor(s) in their natural environment. In still a further embodiment, the cultured cells express one or more other muscarinic acetylcholine receptors, such as, but not limited to the M3, M4 or M5 receptors, alone or in combination with the M1 receptor, M2 receptor or both M1 and M2 receptors and preferably under the same conditions as described above and herein throughout. Also contemplated herein are methods which separately employ a cell line expressing the M1 receptor and separate cell lines expressing individual muscarinic receptors. Such methods may be preferred in situations to determine anticholinergic activity in samples which may be relevant to multiple muscarinic receptor types, for example, but not limited to M1 and M2; M1, M2 and M3; M1 and M4; M1 and M5, M1, M4 and M5; M1, M2, M3, M4, and M5, and the like. In an embodiment wherein the method employs cells expressing the M2/M4 receptors, the ligand 3H-AFDX-384 may be employed. In an embodiment wherein the method employs cells expressing the M1/M3 receptors, the radioligand 3H-4-DAMP may be employed. Other suitable ligands may be used for other receptors as would be understood by a person of skill in the art.
The method described herein provides improvements over the original SAA assay described by Tune and Coyle.1,2 The use of cultured cells expressing the M1 receptor offers advantages over the use of rat brain tissue as the latter rat brain tissue expresses several types of muscarinic receptors, all of which may not be involved in human cognitive function. Further, the use of cultured cells, compared to rat brain cells, reduces costs, provides ease of maintenance and obviates the need to sacrifice animals. In addition, more precise results may be obtained by PCA pre-treatment to neutralize potential effects of endogenous proteins in serum samples. The method described herein addresses defects in the prior art to assaying serum anticholinergic activity, for example, but not limited to requiring less sample volumes, requiring lesser amounts of M1 ligand and obviating the need to use animal brain tissue.
In still another embodiment, there is provided herein a method for identifying a subject as being at risk of having or developing cognitive impairment, the method comprising:
In another embodiment, the subject may be an aged or elderly subject. In certain embodiments, for example, the subject may be an older subject, such as a subject between about 59-86 years old.
As will be understood, the healthy control level may be any suitable reference level (i.e. a threshold, or range) determined as being indicative of a healthy state in which no significant cognitive impairment is experienced. By way of example, the healthy control level may be a reference level determined for an aged or older control group of subjects being cognitively intact with good medical health. In certain embodiments, the control group may comprise subjects of about 59-86 years old, for example. In certain embodiments, the healthy control level may be a reference level determined as being indicative of a healthy state in which no significant cognitive impairment is experienced, as indicated by a MoCA result of higher than about 24, and a score of about 0 in the CDR. In certain embodiments, the healthy control level may be a pre-determined level derived by performing the method on a group of healthy non-cognitively impaired control subjects. In certain embodiments, the healthy control level may be a level previously measured in the subject while the subject was not cognitively impaired, or may be a level previously measured in the subject prior to treating the subject with a drug with anticholinergic properties, for example.
In yet another embodiment, the method may further comprise a step of:
In still another embodiment, the subject may be a subject being treated with at least one drug having anticholinergic properties.
In yet another embodiment, the cognitive impairment may be in the spatial working memory cognitive domain.
In a further embodiment of the instant invention there is provided a non-radioactive method for determining the level of muscarinic acetylcholine receptor subtype-1 (M1 receptor) cholinergic activity in a blood serum sample, the method comprising:
removing protein from the blood serum sample by treatment with perchloric acid (PCA) to produce a PCA-treated serum sample;
incubating the PCA-treated serum sample with a membrane preparation from cultured cells expressing the M1 receptor, the M1 receptor loaded with a calcium sensitive fluorophore or dye;
collecting fluorescence measurements from the cells for a first period of time;
optionally, adding an aliquot of carbachol solution or another acetylcholine receptor agonist to produce maximal or near maximal fluorescence of the cells from release of calcium;
optionally, collecting fluorescence measurements from the cells for a second period of time;
comparing pre-carbachol (or another acetylcholine receptor agonist) activity of the blood serum sample to pre-carbachol activity of a control blood serum sample, wherein the control blood serum sample is known to contain no cholinergic agonists or other drugs with cholinergic activity;
optionally comparing post-carbachol (or other acetyl choline receptor agonist) activity of the blood serum sample to post-carbachol activity (or other acetylcholine receptor agonist) of the blood serum sample,
wherein comparing pre-carbachol activity provides a measure of pure agonist properties of a subject's serum and optionally, comparing post-carbachol activity provides a measure of the subject serums's net agonist and antagonistic properties.
Also provided is a method wherein only pre-carbachol activity is measured. In a further embodiment, both pre- and post-carbachol activity are measured. In still a further embodiment, only post-carbachol activity is measured. The activity may be defined as a ratio of a subject's serum's activity relative to a control serum's activity. Other ways of presenting the level of muscarinic acetylcholine receptor subtype-1 anticholineric activity, for example, in absolute units, a ratio compared to a control, or the like, may be provided as would be understood by a person of skill in the art.
In still a further embodiment, a standard curve may be generated under equivalent conditions using a cholinergic agent, for example, but not limited to, clozapine or other cholinergic agent known in the art and that assay results may be expressed in, for example, clozapine equivalent units or units of the other cholinergic agent. In a preferred embodiment, the cholinergic agent produces monotonic activity curves which are generally easier to fit sample values.
Other aspects, characteristics, steps and the like provided for the radio-active methods described above and herein may be employed in the context of the non-radioactive methods described herein and below.
1. Cell Culture and Obtaining Membrane Preparations
Chinese hamster ovary (CHO) cells stably expressing rat M1 muscarinic receptors (M1WT3; American Type Culture Collection, ATCC, Manassas, Va.) were grown to 90% confluence in 25 mL F-12K medium (ATCC) supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin and 100 μg/mL geneticin in a T175 flask at 37° C. in humidified air and 5% C02. Cells were harvested using 8 mL Accutase® (Sigma-Aldrich, Oakville, ON), rinsed with magnesium- and calcium-free Dulbecco's PBS, and stored at −80° C. in 20 mM HEPES, 10 mM EDTA, pH 7.4. Membrane preparations were obtained according to Lazareno et al,15 with minor modifications. Briefly, cells were homogenized for 30 seconds on ice with a polytron (Brinkmann, Canada), centrifuged at 40,000×g for 90 min at 4° C., rinsed with 20 mM HEPES, 0.1 mM EDTA pH 7.4, centrifuged again at 40,000×g for 10 min, reconstituted, and stored at −80° C. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Nepean, ON).
2. Serum Test Solutions
To ascertain the effects of anticholinergics in serum and to simulate patient samples containing varying levels of anticholinergic medications, normal human serum (Millipore, Billerica, Mass.) was spiked with a test solution containing known amounts of compounds with varying degrees of anticholinergic activity5. The test serum solution consisted of: Clozapine 1000 ng/ml, Amitriptyline 100 ng/ml, Scopolamine 0.05 ng/ml, Chlorpromazine 50 ng/ml, and Paroxetine 100 ng/ml. The test serum solution was then diluted with normal serum in 20% steps from 100% down to 0% in order to obtain varying amounts of anticholinergic drugs in serum samples.
3. Binding Assay Protocol in M1WT3 Cells
The binding assay consisted of 100 μL of serum, 27 μg of M1WT3 protein and nanomolar concentrations of the appropriate M1 ligand in buffer (20 mM HEPES, 100 mM NaCl and 10 mM MgCl2, pH 7.4) in a total volume of 500 μL, assembled on ice in a 96-well Whatman™ uniplate (GE Healthcare, Mississauga, ON). Receptor concentration was estimated to be approximately 713 fmol/mg membrane protein. The microplate was then incubated at 24° C. for 60 min. In all cases nonspecific binding was defined in the presence of 10 μM atropine. Membranes were collected by filtration on GF/B filtermats presoaked in 0.1% PEI, using a Filtermate Harvester (Perkin Elmer, Waltham, Mass.), then rinsed with 50 mM Tris HCl, 150 mM NaCl pH 7.4 at 0° C. Filtermats were dried, soaked in scintillation fluid (Microscint-PS, Perkin Elmer) and sealed. Bound radioactivity was counted in a Microbeta-2 microplate scintillation counter (Perkin Elmer).
4. Deproteinization
4.1 Protein Filtration Experiments
4.2 Protein Removal by Precipitation
Perchloric acid deproteinization experiments were performed with a deproteinizing kit (catalog #K808-200; BioVision, Milpitas, Calif.) according to manufacturer's instructions. Briefly, 400 μL of sample were mixed with 100 μL of ice-cold PCA, placed on ice for 5 min and the centrifuged at 13,000×g for 2 min; 380 μL of the supernatant was transferred to a fresh tube to which was added 20 μL of ice-cold Neutralization Solution. The precipitate was resuspended and mixed to neutralize the sample and precipitate excess PCA. Samples were placed on ice for 5 min and spun briefly prior to use.
5. M1 Ligands
[3H] quinuclidinyl benzilate ([3H] QNB, 30-60 Ci/mmol), [3H] N-methyl-scopolamine ([3H] NMS, 70-90 Ci/mmol) and [3H] pirenzepine ([3H] PZP, 70-90 Ci/mmol) were obtained from PerkinElmer (Waltham, Mass.).
6. Binding Assay in Rat Brain Homogenates
Binding of [3H] QNB to rat brain homogenates followed established protocols (Cox et al., 2009). Briefly, 27 μg of rat brain tissue (frontal cortex and caudate-putamen) was used in a total volume of 500 μL, consisting of phosphate-buffered saline, 0.16 nM [3H] QNB and 20 μL of the spiked serum test solution diluted to various concentrations as described above.
7. SAA Assays
Standard curves were constructed by measuring the displacement of radioligand binding by 13 different concentrations of atropine (0.0 pmol/mL to 100 pmol/mL) in serum. Standard curves were repeated on 6 separate occasions. Point-by-point coefficients of variability ranged from 9% to 20% for the 13 points in the standard curve (mean=16.75%, sem=0.97%). All measurements were performed in triplicate. Data is expressed as percentage inhibition of radioligand binding in pmol/mL atropine equivalents.
8. Data Analyses
Curve fitting, estimation of receptor binding parameters and other statistical analyses were performed with GraphPad Prism v. 7 (La Jolla, Calif.). Multiple comparisons following ANOVAs used Sidak-adjusted P levels.
9. Binding Parameters in M1WT3 Cells
Binding parameters for the standard muscarinic ligand [3H] QNB in cell membranes were determined in buffer. Two other M1 ligands were used for comparison and further validation: [3H] NMS was chosen as a pan-muscarinic receptor ligand similar to [3H] QNB in most respects; [3H] PZP was chosen as an M1-selective ligand, which might potentially provide greater sensitivity. For each of the three M1 ligands, saturation experiments showed that binding to M1WT3 cells was saturable and displaceable by 10 μM atropine (
As can be seen in
Effects of Serum Proteins on [3H] QNB and [3H] NMS Binding in M1WT3 Cells
To examine a possible effect of large serum proteins on the cell assay13 two different approaches were taken. First, serum filtration experiments were performed by using a 50 kDa membrane filter as previously done by Cox and colleagues and confirmed a substantial loss of binding in the filtered samples, as reported by these authors.13 A second approach involved precipitating serum proteins by perchloric acid pre-treatment. Results are shown in
In the course of testing spiked serum it was also noted that normal serum containing no added anticholinergics induced a decrease in binding. This in itself suggested that a component endogenous to normal serum has some ability to decrease binding counts in the absence of any exogenous compounds, possibly by sequestering some of the test radioligand as had been previously suggested.13 To address this possibility in the specific case of M1WT3 cells, binding was compared in buffer vs. normal serum containing no known anticholinergic compounds, with or without PCA-treatment. As shown in
Assessments of total serum anticholinergic activity (SAA) may be of considerable interest for potential involvement in cognitive impairment associated with, for example, polydrug states in the elderly and other populations. Over the past three decades, SAA assay has been used in efforts to quantify drugs that possess anticholinergic activity in vitro and to document elevated serum anticholinergic levels in community-dwelling and hospitalized patients with delirium and dementia18-21. To assess the total burden of anticholinergic activity, a serum anticholinergic activity assay (SAA) was introduced in the early 1980s by Tune and Coyle22-23 and has since been used as a putative marker of cognitive dysfunction in several conditions, albeit not always with consistent results (for reviews see references 18, 24, and 25). The original Tune and Coyle assay was based on the displacement by human serum of [3H]QNB binding to rat brain homogenates22. Subsequently, however, questions were raised concerning the basic validity of this SAA protocol and several potential limitations have been identified18, 24. Among these is a potential role for large serum proteins, which may significantly mask or distort SAA values26. A second potential source of variability in the original SAA protocol is that it did not discriminate between various subtypes of muscarinic receptors, which may limit applicability in studies that are interested in observing the role of anticholinergic burden on cognitive status, as this is a potential predictor of cognitive decline. Only two of the five known muscarinic receptor subtypes (M1 and M2) have been shown to be involved in cognitive functions37,38, where M1 is the most abundant muscarinic receptor in brain39 and the one that has been most clearly implicated in cognitive functions40. These and other issues, including the fact that alternatives, such as anticholinergic risk scales, also show lack of uniformity and variability of outcomes41-43 indicate a need for improved, additional, and/or alternative methods for assessing anticholinergic activity.
Furthermore, as discussed above, cholinergic receptor blockade in the central nervous system (CNS) is associated with impaired cognitive function, and for this reason medications with anticholinergic activity are often carefully prescribed and dosed. However, many prescription and non-prescription drugs have varying degrees of anticholinergic activity, and a significant amount of anticholinergic activity may result when these drugs are combined27-32. Since CNS cholinergic function diminishes with aging33, the elderly, who very often take multiple medications for various different types of health issues, are particularly vulnerable in this respect20, 30, 34-36.
As described hereinabove, methods and kits for assessing anticholinergic activity in a sample have now been developed. In Example 1 above, such methods and kits were studied, and used to assess anticholinergic activity in serum samples. In the present Example, methods and kits described herein are used to assess serum anticholinergic activity levels of human serum taken from subjects before and after treatment with scopolamine, a known anticholinergic drug, and treated subjects were further subjected to CANTAB testing to determine cognitive changes following treatment with the scopolamine.
In this Example, subjects were healthy, cognitively intact individuals under well controlled conditions. For this study a sample of 10 individuals were subjected to cognitive tests before and 30 min after an intravenous injection of the cholinergic blocker scopolamine. Serum was likewise collected before and after scopolamine. It was hypothesized that SAA would be increased after single dose scopolamine, and that scores on cognitive measures would correspondingly decline after the treatment.
Methods
1. Cell Culture, Membrane Preparation and 3H-QNB Binding Assay.
a) Chinese hamster ovary (CHO) cells stably expressing rat M1 muscarinic receptors (M1WT3; American Type Culture Collection) were grown in T175 flasks at 37° C. in humidified air and 5% CO2. Cells were then harvested using 8 mL Accutase solution, rinsed with magnesium- and calcium-free Dulbecco's PBS, and stored at −80° C.
Membranes were prepared according to Lazareno et al. (1998)45 with minor modifications. Briefly, cells were homogenized for 30 sec on ice, centrifuged at 40,000×g for 90 min at 4° C., rinsed with 20 mM HEPES, centrifuged again at 40,000×g for 10 min, reconstituted, and stored at −80° C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit.
The binding assay used 100 μL of serum, 27 μg of M1WT3 protein, and 0.16 nM [3H]QNB in HEPES buffer in a total volume of 500 μL, assembled on ice in a 96-well Whatman™ uniplate. The microplate was then incubated at 24° C. for 60 min. Membranes were collected by filtration on GF/B filtermats presoaked in 0.1% PEI, then rinsed with 50 mM Tris HCl, dried, and sealed. Bound radioactivity was counted in a Microbeta-2 microplate scintillation counter.
b) Protein Removal by Precipitation
Prior to their use in the binding assay, serum samples underwent deproteinization, which was performed with a commercial kit (BioVision, Milpitas, Calif.; catalog #K808-200) according to manufacturer's instructions.
c) SAA Assays
Standard curves were constructed for each assay by measuring the displacement of 0.16 nM [3H]QNB binding by atropine (0.0 pmol/mL to 100 pmol/mL) in normal human serum. Standard curves were fitted to a competitive inhibition model which was then used to express test sample anticholinergic activity as inhibition of [3H]QNB binding in pmol/mL atropine equivalents—i.e. the atropine concentration that would induce a comparable reduction in radioligand binding. All measurements were performed in triplicate.
2. Clinical Cognitive Measures
Structured Clinical Interview for the DSM-IV (SCID): Participants were screened for any psychiatric disorder including a Neurocognitive Disorder using the Structured Clinical Interview for the DSM IV to determine study eligibility. The SCID-IV assesses current and lifetime depression and other psychiatric disorders. It was used to clarify psychiatric inclusion and exclusion criteria.
The Montreal Cognitive Assessment (MoCA): is a validated, brief cognitive screening tool for detecting mild cognitive impairment (MCI) with high sensitivity and specificity.
Clinical Dementia Rating Scale (CDR): This scale is useful in quantifying the severity of dementia based on six domains of cognitive and functional ability: memory, orientation, judgment and problem solving, community affairs, homes and hobbies, and personal care. Each item is rated on a 5-point scale through a semi-structured interview with the participant or reliable informant.
The 3 tests listed above were used to screen subjects in visit one, to ensure the absence of significant neuropsychiatric impairments. The following tests were administered before and after anticholinergic intervention during visit 2:
Cambridge Neuropsychological Test Automated Battery (CANTAB-AD):
Two main challenges are often encountered with many of the newer neurocognitive tasks. First is the lack of standardization in administering such tasks. Second is the lack of information on their psychometric properties. The Cambridge Neuropsychological Test Automated Battery (CANTAB) system addresses both of these challenges (www.cambridgecognition.com). The design of the CANTAB is based on well-established neurocognitive experimental paradigms. It was designed initially to provide componential analysis of cognitive functions in the elderly and individuals with dementia. The CANTAB Eclipse consists of 22 tasks that assess neurocognitive processes within a wide range of relatively independent cognitive domains, including visual memory, attention, working memory and problem solving. Each of the tasks is graded, allowing the assessment of patients with varying level of impairment. Increasing the difficulty of a task avoids a ceiling effect. Conversely, decreasing the difficulty of a task avoids a floor effect and allows the distinction between specific and generalized deficits (a concern discussed above). The tasks are automated and therefore testing is given in a standardized manner with a standardized feedback about accuracy and speed. The CANTAB has also a large normative database based on over 2000 normal control subjects, aged 4-90 years. Estimates of test-retest reliability and of practice effects are available for many of the CANTAB tasks based on an elderly sample with an age range of 60-80 years. The CANTAB has been used in a variety of clinical populations providing an opportunity to compare findings related to different disorders, including findings examining cognition in late-life bipolar disorder. Finally, the CANTAB tasks are independent of language and culture and can be used in population for whom English is not a primary language46.
Administration time is approximately 55 minutes.
Clinical Samples and Study Medication Biomarkers: Blood samples were collected for evaluating SAA. Assays will be performed in the laboratory. Total amount of blood to be drawn was four 10 ml blood tubes.
Scopolamine Hydrobromide: Scopolamine is a naturally occurring alkaloid of the belladonna plant. Scopolamine, like atropine, is an antimuscarinic agent antagonizing the action of acetylcholine at muscarinic receptors. The anticholinergic properties of scopolamine and atropine differ in that scopolamine has more pronounced sedative, antisecretory and antiemetic activity while atropine has stronger effects on the heart, intestine and bronchial muscle and a more prolonged duration of action47. Scopolamine has many uses including the prevention of motion sickness, treatment of excessive salivation, colicky abdominal pain, sialorrhoea, diverticulitis, irritable bowel syndrome and motion sickness and also postoperative nausea and vomiting48.
The variability of absorption and poor bioavailability of oral Scopolamine (Scopace®) indicate that this route of administration may not be reliable and effective49 for the purposes of the present assay testing as well as testing the cognitive effects induced by Scopolamine administration. Oral Scopolamine will introduce large variability and may confound any anticipated findings. The advantages of administering it using IV route include rapid onset of action (5-10 minutes), known pharmacokinetics (plasma levels peak at 30 minutes) and shorter half-life (approximately 1 hour)47-50. Another reason is that oral form of Scopolamine is not available in Canada, while the IV form can be supplied by Canadian manufacturers. Accordingly, IV administration of Scopolamine was used.
Participants and Recruitment
10 cognitively intact healthy participants were recruited. Cognitive assessments were performed on participants who did not meet DSM IV criteria for any diagnosis.
Study Design
Ten cognitively intact healthy participants underwent cognitive testing prior to and following a single 0.4 mg dose of IV scopolamine. Participants attended the Centre for Addiction and Mental Health on two separate occasions:
Visit 1: Study visit 1 was an assessment of eligibility criteria. Information was collected regarding any current or past mental health (Structured Clinical Interview for DSM-IV) or medical issues.
Visit 2: Upon arrival at CAMH, participants were administered the CANTAB. An IV line was then set up by a research nurse and the first blood sample was taken. Following this, the nurse injected 0.4 mg scopolamine. After a 30 minute interval (the time at which scopolamine reaches peak plasma concentration) a second blood sample was drawn. The CANTAB was completed once more. Participants remained on site for observation for 3 hours after scopolamine administration. The qualified investigator or designate was available for the entirety of the study visit.
A schematic of the study design is shown in
Inclusion/Exclusion Criteria:
No exclusion criteria were based on race, ethnicity, gender, or HIV status.
Inclusion:
Exclusion:
Results
1. Subjects
One of the 10 recruited subjects did not return for the second visit and thus 9 of the 10 recruited subjects completed the study. The sample consisted of 2 males and 7 females with ages ranging from 59 to 86 years (mean=69.88, median=71). All subjects were white, non-Hispanic, and on the screening visit did not display evidence of neuropsychiatric deficits on the SCID. They were free of known potent anticholinergic medications and showed no indications of significant cognitive impairment. As shown in Table 2, MoCA scores ranged from 24 to 30 (scores on the MoCA range from 0 to 30, with a score of 26 and higher generally considered normal). For the Clinical Dementia Rating (CDR™) test one subject had a score of 0.5 and all others had a score of zero (Table 2), which is considered normal in this 5-point scale.
2. SAA Results
Administration of a single i.v. dose of 0.4 mg scopolamine resulted in a significant increase in SAA activity as measured 30 min later.
3. CANTAB Tests
A description of each of the CANTAB variables assessed is provided in Table 1. As shown in
To probe potential associations between SAA changes and changes in cognitive measures, correlations were computed between SAA difference scores (pre-post scopolamine) and CANTAB difference scores (pre-post scopolamine). Despite the low sample size, associations emerged between SAA difference scores and CANTAB difference scores, a difference score referring to pre- vs. post-scopolamine scores in each case. The two highest correlations referred to the positive association between SAA changes and spatial working memory deficits (swm_be, r=0.69, p<0.05) and the positive association between SAA changes and poor use of an appropriate strategy in the spatial working memory test (swm_strat, r=0.41). Scatterplots for these two variables are shown in
Discussion
The aim of this study was to further investigate the effectiveness of the new cell-based SAA methods described herein in detecting serum anticholinergic activity induced under well-controlled circumstances in a within-subject design. A single i.v. dose of 0.4 mg scopolamine in aged but cognitively intact and drug-free subjects resulted in a strong increase in SAA as measured with the cell assay described above (
These findings indicate that experimental introduction of a common anticholinergic drug at a dose that is well-within commonly used clinical dosage range led to readily measurable anticholinergic changes using the cell-based assays described herein, which is of particular interest as it demonstrates the effectiveness of the methods and kits described herein for detecting anticholinergic load in a human sample (see
The fact that the acute treatment also resulted in cognitive changes using well-validated procedures reinforces the importance of accurately assessing anticholinergic load in a number of clinical conditions, particularly in the elderly. The results of this experimental study demonstrates that the presence of anticholinergic drugs in the blood of human subjects may be readily measured by the cell-based assays, methods, and kits described herein.
a Values are mean differences post-pre scoplamine ± sem.
b paired t tests
Cell culture and preparation are as described herein and also as described previously72. Briefly, Chinese hamster ovary (CHO) cells stably expressing human or rat M1 muscarinic receptors (M1WT3; American Type Culture Collection, ATCC, Manassas, Va.) were grown to 90% confluence in 25 mL F-12K medium (ATCC) supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin and 100 μg/mL geneticin in a T175 flask at 37° C. in humidified air and 5% C02. Cells were harvested using 8 mL Accutase® (Sigma-Aldrich, Oakville, ON), rinsed with magnesium- and calcium-free Dulbecco's PBS, and stored at −80° C. in 20 mM HEPES, 10 mM EDTA, pH 7.4.
Calcium mobilization was assessed with the FluoForte™ Calcium mobilization kit (Enzo Life Sciences; https://www.enzolifesciences.com/ENZ-51016/fluoforte-calcium-assay-kit/) according to manufacturer's instructions. Test compounds carbachol, clozapine, N-desmethlyclozapine, amitriptyline, scopolamine and paroxetine were purchased from Sigma-Aldrich (St. Louis, Mo., USA). [3H]quinuclidinyl benzilate ([3H]QNB, 30-60 Ci/mmol70-90 Ci/mmol) was purchased from PerkinElmer (Waltham, Mass.).
The basic protocol involves loading cells with dye that fluoresce when exposed to free calcium. Activation of membrane-bound receptors induce changes in the intracellular G protein-IP3 pathway leading to release of bound calcium from the endoplasmic reticulum75. Free (mobilized) calcium then binds the preloaded dye and the resulting transient fluorescence change can be detected and quantified in real time by suitable equipment, as described below.
For the assay cells were grown overnight in a black walled clear bottom 96-well microplate at a density of 20,000 cells per well. Medium was replaced with 100 μL of loading dye (FluoForte™). After a 2 hr incubation 20 μL of human serum (Sigma-Aldrich) was added, which contained (or not) clinical compounds of interest, and mixed for 8 seconds. Fluorescence values were collected every 4 sec for 2.5 min on a fluorescence plate reader (Thermo Fisher Scientific, ON). Immediately thereafter 20 μL of the cholinergic agonist carbachol was added (to a final concentration of 500 nM) and fluorescence readings were collected for another 2.5 minutes. Calcium mobilization was initially expressed in relative fluorescence units (RFU, defined as % change from stable baseline fluorescence readings in plate wells before any materials were added to the cells). When solutions were added separate peaks were computed for the 2.5 min prior to carbachol and the corresponding period after carbachol was introduced (
All assays involving serum were preceded by deproteinization with perchloric acid using a deproteinizing kit (BioVision, Milpitas, Calif.; catalog #K808-200) as described herein and according to manufacturer's instructions.
Assessments of serum anticholinergic activity (SAA) using [3H]QNB as a radioligand in M1 expressing cells were performed as described herein and as recently described72. Briefly, cells were grown and prepared as described above. For the binding assay they were homogenized for 30 sec on ice, centrifuged at 40,000×g for 90 min at 4° C., rinsed with 20 mM HEPES, centrifuged again at 40,000×g for 10 min, reconstituted, and stored at −80° C. Protein concentration were determined using the Pierce BCA Protein Assay Kit.
The binding medium consisted of 100 μL of serum, 27 μg of M1WT3 protein and 0.16 nM [3H]QNB in HEPES buffer in a total volume of 500 μL, assembled on ice in a 96-well Whatman™ uniplate. The microplate was then incubated at 24° C. for 60 min. Membranes were collected by filtration on GF/B filtermats presoaked in 0.1% PEI, rinsed with 50 mM Tris HCl, dried, and sealed. Bound radioactivity was counted in a Microbeta-2 microplate scintillation counter.
A standard curve was constructed by measuring the displacement of 0.16 nM [3H]QNB binding by atropine (0.0 pmol/mL to 100 pmol/mL) in normal human serum. The standard curve was fitted to a competitive inhibition model which was then used to express test sample serum anticholinergic activity as inhibition of [3H]QNB binding in atropine equivalents—i.e. the atropine concentration (pmol/mL) that would induce a comparable reduction in radioligand binding. All measurements were performed in triplicate.
Curve fitting, estimation of receptor binding parameters and all other statistical analyses were performed with GraphPad Prism v. 9.1.2 (La Jolla, Calif.). Goodness of fit values (R2) were used as a criterion for selecting appropriate functions in each case.
We first determined the concentration of carbachol which induced near optimal calcium mobilization, defined as a concentration achieving 80% of maximal response. Ten carbachol concentrations were tested in the range 0 to 50,000 nM. Near optimal Ca2+ mobilization was observed with 500 nM carbachol and this concentration was then used in all subsequent tests.
As an initial test of the new protocol we chose to examine the effects of the clinical compound clozapine (CLZ) and its metabolite N-desmethylclozapine (NDMC), both of which are active at muscarinic receptors. Nine concentrations in the range of 0 to 50,000 nM were examined for both compounds. As can be seen in
Since the clozapine metabolite NDMC is also active on cholinergic receptors68 we next tested the effects of various NDMC concentrations on Ca2+ mobilization. As shown in
As a final test we examined the effects of test solutions containing different ratios of CLZ/NDMC, with decreasing CLZ concentrations corresponding to increasing NDMC concentrations in serum. The effects CLZ and NDMC mixed at 15 different ratios were examined, while keeping a constant total concentration.
To simulate conditions that we might obtain in the clinic we followed a procedure and prepared a test solution in commercial human serum, consisting of the following clinical compounds: chlorpromazine (50 ng/mL), paroxetine (100 ng/mL), amitriptyline (100 ng/mL), clozapine (1,000 ng/mL), and scopolamine (0.05 ng/mL). The test solution was then diluted in 20% steps to obtain 6 decreasing concentrations of the mixture and each was tested on the assay. Results were expressed as % of maximal Ca2+ mobilization responses induced by drug-free serum. As shown in
The same clinical drug solution at various dilutions was used to compare effects of the Ca2+ assay to those of conventional serum anticholinergic activity (SAA) based on displacement of [3H]QNB binding by test solutions, as had been previously done herein and reported72. For both pre- and post-carbachol phases near perfect correlations were observed between Ca2+ fluorescence changes and [3H]QNB binding values (
We describe a cell-based fluorescence assay for assessing cholinergic receptor activity that targets intracellular calcium mobilization rather than inhibition of radioligand binding to membrane receptors. The approach, which was chosen for its ability to detect and quantify changes in upstream receptor activity with high temporal resolution, seemed reliably sensitive to varying concentrations of clinical test compounds with different levels of cholinergic receptor activity, an issue of continuing high clinical interest70. In vitro results with the new assay show excellent correlations with the existing radioligand binding protocol. The new assay allows—possibly for the first time—an assessment of pure agonistic effects of clinical compounds in serum as well as their net effects on cholinergic receptor activation status. Insofar as intracellular calcium mobilization is a dynamic process, the assay may indeed be the first to effectively tag receptor functioning rather than a static binding event.
It may be reasonable to assume that Ca2+ changes during the initial pre-carbachol phase reflect “pure” agonist properties of serum compounds whether exogenous (e.g. medications) or endogenous in nature. It is suggested that Ca2+ mobilization changes during the pre-carbachol phase may reflect potential effects of compounds during steady-state conditions of the cholinergic system, whereas the changes during the carbachol phase might reflect net effects to be expected during endogenous activation of the cholinergic receptor system.
The observation that drug-free serum in itself causes an initial transient increase in Ca2+ mobilization was not totally unexpected, as we had previously observed a inhibition of [3H]QNB binding by serum alone in cells or brain homogenates72. This serum effect is consistently observed, in human as well as rodent serum, from commercial as well as from living sources. It may signal the presence of an otherwise unidentified serum factor that survives deproteinization, is insensitive to atropine and interacts with the fluorogenic dye. Attempts to identify possible mechanical, temperature- or pH-related variables involved in this effect have not so far yielded evidence of artifacts, although of course additional possibilities exist.
In the case of the clinical compounds tested in the present study, initial pre-carbachol effects generally followed a curvilinear relationship, with strongest effects induced by intermediate concentrations of test compounds, best described by second or third degree polynomial functions. While we do not have a simple explanation for this pattern of serum effects, additional experiments with other agonists (e.g. pilocarpine) and antagonists (e.g. atropine) confirmed the agonist nature of this serum peak: pure antagonists such as atropine completely blocked this agonist response while having no effect on their own (
More detailed analyses focused on clozapine, a major psychiatric drug, and its metabolite NDMC. Both of these are active at cholinergic receptors and are often thought to have opposing effects at M1 receptors, CLZ acting as an antagonist and NDMC as a partial agonist68, 77, 79, 78. We found that virtually all CLZ and NDMC concentrations tested resulted in increases in Ca2+ mobilization during the pre-carbachol phase, which would be consistent with a pure agonist effect, while attenuating the subsequent carbachol responses, consistent with an antagonistic effect78. This was observed when CLZ and NDMC were examined separately and also when they were combined in different proportions. Without wishing to be bound by theory or limiting in any manner, it is possible to speculate that both CLZ and NDMC have agonist properties at M1 receptors in the resting state, while competing with acetylcholine (or carbachol) when receptors are actively engaged, thereby inhibiting the strength of the carbachol signal. Whether or not this speculation is correct, in no case did we observe an opposing relationship between CLZ and NDMC concentrations, which suggests that both compounds have qualitatively similar effects on M1 receptors.
The effects of different CLZ/NDMC ratios were reliable and reproducible across various final concentrations, supporting the notion of synergistic effects of the parent compound and its major metabolite in this case. However, it should be noted that with actual human samples it would not necessarily be realistic to assume perfectly reciprocal relationships between CLZ and NDMC concentrations as done here, since in vivo it is of course possible for the two compounds to be metabolized independently and at different rates. Nevertheless, further modeling of CLZ/NDMC ratios may be well worth pursuing as ratios appear to be clearly relevant to functional effects74 (Rajji et al., 2015.
The observation that Ca2+ assay results correlated well with [3H]QNB binding results in vitro contributes to an initial validation of the assay. A lower correlation might have been expected, given that the [3H]QNB assay targets static receptor binding while the Ca2+ mobilization assay targets a dynamic process of receptor activation. It is important however to note that in vitro exogenous addition of various compounds does not include metabolic products or effects that might result from in vivo administration.
In summary, we describe a further new approach to quantifying cholinergic receptor activation in human serum samples. Among advantages of the new assay are the high efficiency of cell-based assays and the fact that the assay obviates the need to work with radioactivity as is the case with existing radioreceptor binding assays. The described Ca2+ mobilization assay relies instead on fluorometric measures which are much more widely accessible than radioactivity-based measurements. The assay allows for a separate assessment of agonist properties of compounds, presumably during steady-state conditions, as well as net effects (agonist+antagonist) during active receptor engagement. Both pre- and post-carbachol measures correlate well with binding measures in vitro. By targeting a time-dependent process that reflects consequences of changes in activation of membrane cholinergic receptors, the assay may constitute a viable and useful addition to efforts to quantify anticholinergic as well as agonistic cholinergic receptor activity in human serum.
Various embodiments of compounds, composition and methods for assessing cholinergic and anticholinergic levels in a sample have been described. The above-described embodiments are intended to be examples, and alterations and modifications may be effected thereto by those of ordinary skill in the art without departing from the spirit and scope of the teachings.
All references are herein incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 62466651 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16490680 | Sep 2019 | US |
| Child | 17396030 | US |
