U.S. patent application number 10/757180 was filed with the patent office on 2004-07-29 for methods of determining active levels of drugs in fluid samples.
Invention is credited to Chien, Sue Min, Dunham, Steve H., Stout, Robert L..
Application Number | 20040146852 10/757180 |
Document ID | / |
Family ID | 21896245 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146852 |
Kind Code |
A1 |
Stout, Robert L. ; et
al. |
July 29, 2004 |
Methods of determining active levels of drugs in fluid samples
Abstract
Methods for determining the presence and level of active drugs
in fluid samples are provided. Advantageously, entire families or
classes of drugs can be tested for in one test by identifying the
enzyme or receptor upon which members of that drug family act and
measuring enzyme activity levels or binding activity levels of
receptors. Methods for establishing standard activity levels of
these drugs based upon results from samples having known quantities
of drug therein are also provided.
Inventors: |
Stout, Robert L.; (Overland
Park, KS) ; Chien, Sue Min; (Lenexa, KS) ;
Dunham, Steve H.; (Kansas City, MO) |
Correspondence
Address: |
HOVEY, WILLIAMS, TIMMONS & COLLINS
Suite 400
2405 Grand
Kansas City
MO
64108
US
|
Family ID: |
21896245 |
Appl. No.: |
10/757180 |
Filed: |
January 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10757180 |
Jan 14, 2004 |
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10037772 |
Nov 9, 2001 |
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Current U.S.
Class: |
435/4 |
Current CPC
Class: |
G01N 33/94 20130101;
G01N 33/9453 20130101 |
Class at
Publication: |
435/004 |
International
Class: |
C12Q 001/00 |
Claims
We claim:
1. A method of determining standard enzyme activity levels on a
selected substrate comprising the steps of: providing a sample
containing said enzyme; adding a known quantity of said selected
substrate to said sample; measuring the activity level of said
enzymes on said selected substrate; and using said measured
activity level as said enzyme's standard activity level for said
known quantity of selected substrate.
Description
RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/037,772, filed Nov. 9, 2001, incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is concerned with the screening for
the presence of drugs in fluid samples. More particularly, the
present invention is concerned with screening for the presence of
drugs in fluid samples which may or may not contain any drugs being
screened for. Still more particularly, the present invention is
concerned with the screening for the presence of drugs in fluid
samples when it is known that the individual providing the fluid
sample is on drugs and the screening procedure can help determine
proper medication levels. In the instance where it is not known
whether or not the individual providing the sample is on any drugs,
the present invention is particularly useful in that it identifies
the presence of drug families rather than discreet drugs. The same
is true for when it is known that the individual providing the
sample is supposed to be on a drug that is in a particular family
of drugs whereby the screening procedures can be used to determine
compliance with taking prescribed medications. Even more
particularly, the present invention utilizes a screening technique
which identifies the presence of at least one member of a family of
drugs by the effects of the drugs on specific enzymes or receptors
which are acted upon by the specific families of drugs.
[0004] 2. Description of the Prior Art
[0005] The laboratory screening of drugs, both prescription and
non-prescription, is typically by a form of immunodetection which
relies on the development of a specific antibody to the drug or
compound being screened for. This antibody is then used to detect
the drug in a sample which may or may not contain the drug or
compound of interest. A major hurdle which needs to be overcome to
accomplish immunodetection using this method is the development of
an antibody that is specific for each drug. The antibody must be
produced with a sufficient titer, a measure of concentration, to
efficiently detect the presence of the drug. Additionally, the
antibody must have sufficient specificity to react only with the
drug of interest so that accurate results may be obtained. While
these requirements are normally obtainable, they inherently result
in a system with severe limitations. The greatest of these
limitations is the detection of the drug after it is no longer
biologically active.
[0006] Drugs may be broadly classified into one of two categories.
The first type of drug is active in the form that is present in the
drug as taken by an individual. This category of drugs requires no
structural modification and the drugs in this class are
therapeutically active in the medication. The second category of
drugs requires a structural change before becoming therapeutically
active. In both categories, additional metabolites may also be as
active as the parent compound or its first metabolite while other
metabolites may have little or no activity. This drastically
increases the difficulty in determining the amount of
therapeutically active drug present in any fluid sample taken from
an individual. Due to the similarity of metabolite structures with
the drug as taken or with other metabolites, the metabolites may
also contribute to the immunodetection signal so that, regardless
of prodrug or metabolite, the signal would be related to total drug
exposure. In some instances a metabolite may be therapeutically
inactive while still being detected by the antibody developed to
detect the drug. In this scenario, immunological detection would
overestimate the concentration of active drug present. The opposite
problem may occur where a detected metabolite has an even greater
activity than the parent drug. In this scenario, immunological
detection would underestimate the concentration of active drug
present. Thus, current immunodetection methods cannot differentiate
the biological activity of the drug or its metabolites. The result
is a system which is good at detection of the presence of a drug
but totally ineffective at the more important determination of the
amount of active drug present. Moreover, metabolites which are
similar in structure, regardless of their activity level, may also
be identified and thereby further contribute to an inaccurate
determination of the concentration of active drug present in the
sample tested. Accordingly, one thing needed in the art is a drug
screening test which only determines or detects the presence or
levels of active drugs in fluid samples.
[0007] Many drugs and classes of drugs produce their effect by
activational inhibition of specific receptors or activation or
inhibition of specific enzymes. Often, an entire class of drugs
will produce the same effect on a specific receptor or enzyme,
thereby resulting in the therapeutic effect. In the case of drugs
and classes of drugs effecting receptors, a drug may bind to a
receptor site, thereby inhibiting the binding of the natural
activators or inhibitors. Alternatively, the drug may react in the
receptor site and irreversibly modify the structure or shape of the
receptor, thereby resulting in its inactivation. Other methods of
inhibition include binding to other regulatory sites present on the
receptor, interfering with cofactor binding, or interaction with
other cell surface molecules required for receptor action.
Irrespective of the method of inactivation or inhibition, the
receptor no longer works with its normal efficiency. Accordingly,
another thing needed in the art is a drug screening test which
identifies drug presence by determining effects on specific
receptors. The usefulness of such a test could be greatly increased
if the test could identify the presence of a class of drugs
regardless of which specific drug in that class was actually
present.
[0008] In the case of drugs which effect the activation or
inhibition of specific enzymes, a drug may bind to an enzyme's
catalytic site and inhibit the binding of the natural substrate.
Alternatively, the drug may react in the catalytic site and
irreversibly modify the enzyme, thereby resulting in its
inactivation. Other methods of inhibition include binding to the
regulatory sites present on the enzyme, interfering with cofactor
binding, or interaction with the normal substrate, thereby limiting
its binding to the enzyme. Irrespective of the method of
inactivation or inhibition, the enzyme no longer works with its
normal efficiency. In reality, the drug and/or its metabolites have
reduced the enzyme's catalytic rate. Therefore, another thing
needed in the art is a drug screening test which identifies drug
presence by determining enzyme activity. Again, the usefulness of
such a test could be greatly increased if the test could identify
the presence of an entire class of drugs, regardless of which
specific drug in that class was actually present.
SUMMARY OF THE INVENTION
[0009] The present invention provides a novel approach for
determining the presence of drugs in fluid samples. Advantageously,
entire families of drugs are identified using the present invention
so that one test can provide information on what type of drug is
present in a fluid sample. The methods are based on the effects an
active drug has on either a target enzyme or a receptor. In the
case of drugs which exert their effects on enzymes, the enzymes may
be activated or inhibited by the drug binding to the enzyme's
catalytic site, thereby inhibiting the binding of the natural
substrate. Captopril is a good example of a drug that exhibits this
type of competitive inhibition. Captopril is a member of the drug
family or class known as Angiotensin Converting Enzyme (ACE)
inhibitors which includes the drugs benazepril, captopril,
enalapril, fosinopril, lisinopril, quinapril, moexipril, ramipril,
and trandolapril. This class or family of drugs assists in
regulating blood pressure by inhibiting the conversion of
angiotensin I to angiotensin II which is a powerful vasoconstrictor
that helps regulate blood pressure, renal blood flow, and blood
volume. If there is an excess amount of angiotensin II, which can
be caused by the enzymatic action of ACE, blood pressure increases.
ACE inhibiting drugs prevent the cleavage of angiotensin I to
angiotensin II, thereby reducing blood pressure.
[0010] As noted above, the laboratory screening of drugs is
typically by a form of immunodetection. In the case of the ACE
inhibiting class of drugs, different antibodies must be developed
for nearly every drug as the members of this drug class have
dissimilar structures. Additionally, most ACE inhibiting drugs have
metabolites that also demonstrate varying degrees of activity and,
due to structural similarities, many of these metabolites will be
detected, regardless of their activity level, using an
antibody-based approach. Thus, when screening for use of this type
of drug, one must first know which specific ACE inhibitor is being
used and any results may include a number of false positives which
occur when metabolites which are inactive or have low activity are
identified by the antibody, and thereby contribute to a
determination that the sample tested is positive for the drug. Such
a test does not really provide the needed information of how much
active drug is present in a patient's system. If the specific drug
is not known, a number of different tests may have to be run until
the specific drug is identified.
[0011] However, because all members of the ACE-inhibiting drug
family act on the same enzyme, the present invention can be used
for the detection of the entire family. Advantageously, the active
metabolites will also be identified, thereby providing results of
the amount of active drug present in a patient's system. Thus, the
invention may be used to screen samples for the presence of a class
of drugs including their active metabolites. It may also be used to
monitor patient compliance or to determine why one drug appears to
be more effective in a particular patient. Finally, the present
invention will be useful in emergency type situations where it is
necessary to quickly ascertain what types of drugs a patient is on,
thereby potentially avoiding dangerous drug interactions or
needless dosing of additional medications.
[0012] The present invention is also useful in screening for the
presence of drugs or families of drugs which exert their effects by
reacting in an enzyme's catalytic site, irreversibly modifying and
inactivating the enzyme, binding to other regulatory sites present
on the enzyme, interfering with cofactor binding, or interaction
with the normal substrate and limiting its binding to the
enzyme.
[0013] To test for the presence of a drug or class of drugs, a
sample of fluid is obtained from a patient. The substrate upon
which the enzyme acts is then added to the sample and the activity
of the enzyme is determined. If the activity of the enzyme is
reduced in comparison to a control sample having no drug present,
the sample is deemed positive for that class of drugs. Preferably,
a set of standards will be set up using methods of the present
invention. These standards would be established by testing samples
that have a known quantity of active drug present. Results from
such controlled testing could then be used comparatively to
determine drug presence and levels in samples having unknown
amounts of drug present.
[0014] Other classes of drugs exert their effects on specific
receptors and therefore can also be identified by using methods of
the present invention. The family of drugs commonly called the
"beta-blockers," which includes the drugs atenolol, propranolol,
metoprolol, nadolol, pindolol, timolol, cavediol, and sotalol, are
an example of this class of drug. Members of this class or family
of drugs act as competitive antagonists at the adrenergic beta
receptors and reduce the symptoms connected with hypertension,
cardiac arrhythmias, migraine headaches, and other disorders
related to the sympathetic nervous system. Adrenergic receptors
form the interface between the nerves that serve the heart, blood
vessels and kidneys and the organs themselves. Catecholamines such
as norepinephrine and dopamine are released from sympathetic nerve
terminals and bind to adrenergic receptors on the surface of target
cells, thereby activating receptors, which modify the functions of
these cells. Beta-blocking drugs reduce receptor occupancy by
catecholamines and other beta agonists by competitively binding to
these receptors. Adrenaline (also known as epinephrine) is
classified as a catecholamine hormone and it is mainly the effects
of adrenaline on the body's beta-receptors that are blocked by
beta-blockers.
[0015] Again, because one test can identify the presence of an
active member of a drug class in a sample, time will not have to be
spent developing a specific antibody for each member of a drug
family. Additionally, information regarding patient compliance with
taking medication or efficient detection of active medication in a
patient's system are also possible using the present invention.
[0016] To identify the presence or level of drugs acting on
specific receptors, a sample of fluid is obtained from a patient.
Radiolabeled ligand, which binds to the receptor of interest, is
added to the sample and the mixture is put into a test tube
containing the receptor. If the sample contains a drug, which binds
to the receptor, the drug will compete with the radiolabeled ligand
for the receptor sites during an incubation period. After
incubation, the tubes are centrifuged and decanted, leaving the
membranes with bound drug and radiolabeled ligand in the tubes.
Gamma counter measures the radioactive tracer activity bound to the
receptors in the tube. The amount of activity is inversely
proportional to the amount of unlabeled drug in the sample. Of
course, standards can also be established using methods described
above so that the presence of drugs as well as levels of those
drugs can be determined from any sample.
[0017] It is understood that when the term "active drug" is used
herein, the term encompasses drugs, which are therapeutically
active as taken as well as drugs, which have changed in structure
before becoming therapeutically active. The term also encompasses
metabolites that are therapeutically active. Additionally, the
terms "family" and "class" are used interchangeably when referring
to drugs having similar therapeutic properties.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0018] The following examples set forth preferred embodiments of
the present invention. It is to be understood, however, that these
examples are provided by way of illustration and nothing therein
should be taken as a limitation upon the overall scope of the
invention.
EXAMPLE 1
[0019] This example tested for the presence of an ACE-inhibiting
drug in a fluid sample taken from an individual by measuring enzyme
inhibition in the fluid sample.
[0020] Materials and Methods
[0021] The synthetic pentapeptide substrate,
n-(3(2-furyl)acryloyl)-L-phen- ylanaylglycylglycine(FAPGG) (Sigma
Chemical Company, St Louis, Mo., Cat #305-10) was reconstituted in
a bottle with 5 ml of deionized water and left standing for five
minutes. The bottle was then inverted a few times and then put on a
shaker (Clay Adams CA6000 Centrifuge Becton Dickinson Microbiology
System Sparks, Md.) at speed setting 2 for ten minutes. The ACE
serum used for this Example was derived from a serum pool
consisting of human serum samples which had been tested for ACE
activity. However, it is also commercially available through Sigma
as part of a kit. All samples, which had an ACE activity greater
than 50 units per liter, were combined to form the ACE serum pool.
This serum pool was diluted 1:4 with Tris buffer (pH 8.2, 0.136 M)
and used as the enzyme source. The Tris buffer (Tris
(hydroxymethyl) aminomethane, Sigma Chemical Company, Cat
#25-285-9, Lot #27H5726 m.w.121.1) was prepared by dissolving 1.695
g Tris base in 50 ml deionized water in a 100 milliliter graduated
cylinder. The pH was adjusted to 8.2 with 6 N hydrochloric acid.
The stock solution for the inhibitor was 50 mM Captopril (Sigma
Chemical Company, Cat #4020, Lot #37H120) that was made by
dissolving 10.86 mg Captopril in 1 ml 0.136 mM Tris Buffer. A
positive assay control was prepared by diluting the 50 mM Captopril
stock. Ten (10) microliters of 50 mM Captopril stock was diluted
with 1 milliliter of 0.136M Tris-HCL buffer, pH 8.2. The positive
assay control has a normal concentration of 0.5 mM Captopril. A
cut-off level of control was prepared by dilution of the 0.5 mM
Captopril positive control with Tris buffer. 200 microliters of 0.5
mM Captopril was diluted with 1.8 milliliters of 0.136 M Tris
buffer, pH 8.2. Finally, a negative control of Captopril was
prepared by a 1:10 dilution of the cutoff control. 200 .mu.l of the
0.5 mM Captopril cutoff control was diluted with 1.8 ml of the Tris
Buffer.
[0022] Using the above-described reagents, a Hamilton MicroLab AT
pipetting station was used to transfer 100 .mu.l of the ACE serum
in Tris buffer to each well of a microtiter plate. Next, 25 .mu.l
of the cut-off level calibrator, controls and unknows were added to
their corresponding well locations with the Hamilton MicroLab AT,
as shown in Table 1.
1TABLE 1 Wells A1 B1 C1 D1 E1 F1 G1 H1 Serum Control Negative
Control Cutoff Calibrator Positive Control
[0023] Five different patient samples were then added to five other
wells. The microtiter plate was shaker on Titer Plate Shaker (Lab
Line Instruments, Inc. Melrose, Ill.) at a setting of 2 for at
least five minutes before pipetting 100 .mu.l of the substrate
FAPGG into each well. Next, solution was mixed by shaking the
microtiter plate on the shaker at a speed setting of 2 for one
minute. The microtiter plate was placed in the Spectra Mac Plus
(Molecular Devices, Sunnyvale, Calif.) plate reader and the optical
density of each sample at 340 nanoMeter was determined. The
microtiter plate was then incubated at 37.degree. C. for two hours.
The optical density was again determined on the plate reader. This
two-hour reading of optical density was then subtracted from the
initial reading of optical density and termed the "delta OD
340."
[0024] For quality control purposes the positive control should
have a delta OD 340 less than the delta OD 340 of the cutoff
calibrator. The negative control should have a delta OD 340 which
is greater than the delta OD 340 of the cutoff calibrator.
[0025] Results
[0026] Results for this example are given in Table 2.
2TABLE 2 Sample Initial OD 340 2 HR OD 340 Delta OD 340 ACE Serum
1151 852 299 Negative Control 1141 855 286 Cut-off Calibrator 1148
976 172 Positive Control 1172 1145 27 Unknown 1 1388 1089 299
Unknown 2 1553 1257 296 Unknown 3 1291 975 316 Unknown 4 1554 1512
42 Unknown 5 1322 1026 296
[0027] To interpret these results, the ACE inhibitor activity is
inversely proportional to the delta OD 340. Therefore, a sample
containing unknown amounts of ACE-inhibiting drugs is positive for
ACE inhibitors if the delta OD 340 of the sample is less than the
cutoff calibrator delta OD 340. Conversely, a sample containing
unknown amounts of ACE-inhibiting drugs is negative for ACE
inhibitors if the delta OD 340 of the sample is greater than the
cutoff calibrator delta OD 340. As shown in Table 2, unknown sample
number 4 has a delta OD 340 (42) which is less than the delta OD
340 of the cutoff calibrator (172). This indicates that unknown
sample number 4 is positive for ACE inhibiting drugs and,
therefore, ACE inhibition activity.
EXAMPLE 2
[0028] This example demonstrated that the assay for ACE-inhibiting
drugs identified many different medications from the family of
ACE-inhibiting drugs.
[0029] Materials and Methods
[0030] Fluid samples were obtained from individuals reporting that
they were currently taking an ACE-inhibiting drug. The samples and
controls were assayed as in Example 1. The medications reported by
the patients included eight different medications of the
ACE-inhibiting drug family. Each individual reported taking only
one specific ACE-inhibiting drug. Thus, this example tests the
ability of the assay to identify individuals on ACE-inhibiting
drugs without prior knowledge of the specific drug being taken.
[0031] Results
[0032] Results from this example are given below in Table 3.
3 TABLE 3 DRUG LISTED DETECTED BENAZEPRIL 14 14 CAPTOPRIL 2 2
ENALAPRIL 5 5 FOSINOPRIL 6 5 LISINOPRIL 25 22 MOEXIPRIL 4 3
RAMIPRIL 2 2 QUINAPRIL 15 15 TOTAL 73 68
[0033] In this example, 93.1% of urine samples from individuals
self-reporting ACE use tested positive for ACE-inhibiting drugs.
Thus, the enzyme specific assay for the detection of therapeutic
drugs works and one enzyme assay can detect all members of a drug
class or family. Additionally, the assay is superior to antibody
based amino assays in that no antibody needs to be produced for
each drug to be tested for. In other words, the enzyme-based assay
can detect all members of a drug class while an antibody-based
immunoassay potentially detects only the specific drug that the
antibody was developed against. Of course, the 93.1% identification
rate assumes that all patients that reported taking the medications
had actually taken their prescribed medications as instructed.
EXAMPLE 3
[0034] This example provides a cell receptor assay for B1
adrenergic receptors and tests the accuracy of the assay. Patient
urine that may or may not contain target ligand and a radiolabeled
competitive ligand are added to a test tube containing a limited
concentration of cell membrane containing beta-1-adrenergic
receptors. The unlabeled ligand in the patient's urine competes
with the labeled ligand for the receptor sites during an incubation
period. Following incubation the tubes are centrifuged to
precipitate the cell membrane-receptors. The solution containing
unbound ligand is decanted and the radioactivity retained in the
tubes is detected in a gamma counter. The amount of radioactivity
bound is indirectly proportional to the concentration of unlabeled
ligand present in the patient's urine.
[0035] Materials and Methods
[0036] Tris working buffer (Sigma Chemical) Dissolve 4.55 grams of
Tris base, 1.27 g MgCl2 (hexahydrate), 0.37 g disodium dihydrate
ethylenediaminetetraacetic acid, and 0.5 g ascorbic acid in 450
milliliter of deionized water. Adjust the pH to 7.4 with
concentrated hydrochloric acid and fill to volume with deionized
water.
[0037] Beta-1-adrenergic receptor containing membranes (Sigma
Chemical #RBI B-143). Thaw the stock solution of membrane and
dilute to 30 milliliter with Tris working buffer.
[0038] Radiolabeled 125-iodocyanopindolol (100 microCurries #IM142
Amersham Pharmacia Biotech Piscataway, N.J.) a stock solution of
iodocyanopindolol is prepared by diluting 100 uCi of Amersham
Pharmacia provided stock with 4.9 milliliter of Tris working
buffer. The working solution of radiolabel is prepared by diluting
the stock 1 to 30 with Tris working buffer. Drug free urine is
obtained from UTAK Laboratories, Valencia, Calif. Atenolol,
Propranolol, Metoprolol were from Sigma Chemical Company St. Louis,
Mo. Atenolol, propranolol and metoprolol were diluted with HPLC
grade ethyl alcohol (Aldrich Chemical Company Milwaukee, Wis.) to
produce a 1.0 mg/ml stock solution for each drug. A cut-off control
was prepared by dilution of 100 ul of stock solution of tenolol
with 4.9 milliliter of UTAK drug negative urine, nominal
concentration 20 ug/ml.
[0039] Samples, controls and the cut-off calibrator are diluted 1
to 10 with working buffer prior to assay. 100 microliter of Tris
working buffer, 25 ul of diluted sample, cut-off calibrator, or
control, 25 ul of diluted radiolabeled iodocyanopindolol, and 50 ul
of working membrane solution were added to a 12.times.75 millimeter
test tube. The solution was mixed by vortex and incubated for two
hours at room temperature. After incubation, 1 ml of ice cold Tris
working buffer was added to each tube and then centrifuged at 4,000
rpms in a Clay Adams CA6000, (Becton Dickinson Microbiological
Systems Sparks, Md.) for 10 minutes. The supernate was decanted off
and the tops of the tubes were blotted. The total radioactivity was
detected on Packard Cobra II Auto Gamma counter (Packard Instrument
Company Downers Grove, Ill.). The cut-off was calculated by
multiplying the value for 20 ug/ml of Atenolol times 1.4. The
calculated value is 1.4.times.5117=7164
4TABLE 2 Beta-1-adrenergic-blocker study HPLC Sample Number Counts
per Minute Interpretation (Yes/No) 1 720 Positive 2 5783 Positive 3
2932 Positive 4 2769 Positive 5 5192 Positive 6 6588 Positive 7
1393 Positive 8 3244 Positive 9 5117 Cut-off atenolol 10 6026
Positive 11 1156 Positive 12 11509 Negative 13 9659 Negative 14
13884 Negative 15 7432 Negative 16 12561 Negative 17 13178 Negative
18 10502 Negative 19 6959 Positive 20 9865 Negative 21 7665
Negative 22 8706 Negative 23 8708 Negative 24 12421 Negative
[0040] All positive samples were identified correctly, while one
negative (1/13) also tested positive. The over-all correlation was
calculated to be 95.8%.
* * * * *