Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-10-26
2004-04-20
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S007200, C435S007240, C435S004000, C424S900000
Reexamination Certificate
active
06723527
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of toxicology and more specifically to the toxicity or side effects to therapeutic and/or other compounds. More particularly, it concerns the development of methods for identifying and screening for substances that can ameliorate the toxic-effects of compounds, such as clinical drugs, food additives, and a variety of other chemical compounds, based upon assaying lymphocytes.
2. Description of Related Art
The impact of chemical intervention on the overall health of specific individuals is often uncertain. While a pharmaceutical may remedy the targeted defect, the treatment may be accompanied by serious side effects that can, in some cases be worse than the initial malady. In general, the side effects of a pharmaceutical preparation are known but may often be limited to a small subset of individuals. In such a subset, side effects may often be so severe that alternate therapies are required. In some cases, the severity of side effects in a small subset of those who would be taking a drug may prevent FDA approval.
In one example, a relatively new class of drugs called ‘statins’ function as metabolic inhibitors and are used widely to reduce cholesterol biosynthesis and hence, to reduce levels of cholesterol. The introduction of statins has led to a significant decrease in the mortality and costs associated with ailments involving cholesterol accumulation including atherosclerotic heart disease and stroke. However, a significant number of people who use statins experience side effects. Although the majority of these side effects are mild gasterointestinal ailments several cases of more severe neurological side effects have also been reported. In such cases statin therapy has to be terminated to relieve the side effects. Whether mild or severe side effects in general pose severe deterrents to any drug therapy, especially in the case of drugs that have long-term preventive benefits. Thus, there is an acute need in the art to identify and screen for substances that can counteract side effects of drugs such as statins that cause side effects.
The present inventors have previously developed a specific lymphocyte culture medium and a lymphocyte culture assay which facilitated the assessment of deficiencies or abnormal requirements of nutrients; sensitivity to nutrient imbalances; sensitivity and/or predisposition to side effects of drugs, and/or nutrients, and/or a wide variety of other compounds in an individual (see U.S. Pat. No. 4,499,064, incorporated herein by reference). Although, the inventors previous invention provides methods to predetermine which individuals will experience side effects to a particular compound there is no cost-effective in vitro method in the art to identify substances that can overcome side effects of drugs or other compounds.
SUMMARY OF THE INVENTION
The instant invention overcomes the defects in the art and provides a reliable, safe and effective means of identifying substances that can overcome, ameliorate or at least reduce side effects or toxic-effects or sensitivities to different compounds that an individual may experience.
Provided is a method for testing a substance for the ability of the substance to ameliorate the toxic-effects of a compound, comprising the steps of: a) incubating lymphocytes in the presence of the compound and in the presence and absence of the substance; and b) comparing the toxic-effect of the compound on the lymphocytes incubated in the presence of the substance to the effect of the compound on the lymphocytes in the absence of the substance.
In some aspects the method further comprises incubating the lymphocytes in a chemically-defined media. Growing human lymphocytes or any other cell in a chemically-defined medium allows examination of the effects of addition or removal of different components to the medium on cell growth. Chemically-defined media, specifically designed for the growth of human lymphocytes, is described by the present inventors in U.S. Pat. No. 4,499,064, the entire text of which is incorporated herein by reference.
In some embodiments, it is contemplated that the method will further comprise a control comprising: a) incubating the lymphocytes with a compound with a known toxicity in the presence of a substance known to reverse the known toxicity; and b) analyzing the effect on lymphocytes.
In other embodiments, the method further comprises testing a range of concentrations of a substance identified by the method of the invention to obtain the lowest possible dosage at which the substance can reverse the toxic effect of the compound.
In one embodiment, the toxic-effect of the compound is assessed by assessing its effect on the growth of the lymphocytes. In one such embodiment, the growth rate of the lymphocytes is measured. Methods for measuring the growth rate and/or proliferation of lymphocytes are well known in the art. For example, the most widely used method for measurement of cell growth or cell proliferation is assaying the incorporation of tritiated thymidine (
3
H-thymidine) in cells which is proportional to the rate of DNA synthesis. Other examples include a range of colorimetric procedures such as the MTT assay, the XTT assay, the MTS assay, etc., most of which are based on tetrazolium salts which produce highly colored dyes in response to the metabolic activity of viable cells. The present inventors also intend to use assays that measure the amount and synthesis of ATP in cells using bioluminescence based assays.
In other embodiments, the toxic-effect of the compound is assessed by assessing the effect of such a compound on other responses of lymphocytes. In one such embodiment, the effect of a toxic compound on the cell size of lymphocytes may be measured. In yet another embodiment, a change in the morphology of lymphocytes in response to a toxic compound may be measured. Such measurements may comprise visual identification using microscopic methods. Alternatively, the measurement of a biochemical parameter such as an enzyme activity may be used to access the toxic-effect of the compound on a lymphocyte.
Various types of compounds are known that cause side effects or toxicity. Such compounds include but are not limited to, clinical drugs, teratogenic agents, social drugs, drug additives and stabilizers, food additives, food coloring agents, food components, herbs, herbal extracts, and any other chemical compound that can cause toxicity. The compounds also include agents that are accidentally consumed, for example, certain non-edible mushrooms, non-edible plants, etc.
In one embodiment of the method, the clinical drug is a metabolic inhibitor. In one such aspect the metabolic inhibitor is a statin. The statins are a group of drugs widely used to reduce cholesterol biosynthesis and have proven to be very effective in reducing mortality due to conditions that are caused by cholesterol accumulation in veins such as myocardial infarctions and strokes. Statins are inhibitors of HMG CoA reductase, a liver enzyme involved in cholesterol synthesis. Statins provide a long-term preventive effect on the accumulation of cholesterol. However, a significant percentage of the population suffers from side effects of statins which include a host of mild to severe gasterointestinal effects, muscular inflammation, and peripheral neuropathies. These side effects are remedied by stopping the statin therapy. Given the beneficial effects of statins, termination of statin therapy hinders the long-term health goals sought. The methods of the present invention provide substances that can counteract statin side effects, thereby allowing the continuation of the statin therapy in individuals. In specific aspects, the methods of the invention may be used to identify substances that reverse the toxicity of a statin such as lipitor (Atorvastatin), mevacor (Lovastatin), pravachol (Pravastatin sodium), zocor (Simvastatin), Baychol, Cerivastatin, Fluvastatin and the like.
In another embodiment of the method, the clinical dru
Pettit Flora H.
Shive Gwyn White
Shive William
Board of Regents , The University of Texas System
Fulbright & Jaworski L.L.P.
Leary Louise N.
Shive Gwyn White
LandOfFree
Methods for determining toxicity reversing agents does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for determining toxicity reversing agents, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for determining toxicity reversing agents will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3207284