Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-09-24
2001-07-24
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S034000, C435S004000
Reexamination Certificate
active
06265181
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods for determining activity and suitable dosage levels for antimicrobial and/or antiviral drugs to restrict selection of resistant mutants.
BACKGROUND OF THE INVENTION
For any given microbial or viral pathogen (e.g., bacterial, fungal, or viral pathogen), there typically exists a characteristic concentration of a specified antimicrobial or antiviral drug (hereafter “drug”), or combination of drugs, at which recovery of microbial colonies or viral plaques from drug-containing cultures sharply decreases. This concentration is referred to as the “minimum inhibitory concentration” (MIC), and is conventionally defined with reference to a specific percent inhibition of pathogen growth. Thus, for example, the concentration of drug at which 99% of pathogen growth is inhibited is labelled MIC
99
. In spite of the sharp decrease in pathogen growth at the MIC, a small but finite fraction are often able to grow in the presence of the drug. These pathogens are termed “drug-resistant.”
Drug resistant mutants arise spontaneously within pathogen populations. When a pathogen population is treated with a drug for an extended period of time (e.g., one or more days), resistant mutants proliferate while drug-sensitive, wild-type cells do not. Eventually, the pathogen population becomes dominated by the resistant mutants. This process, which is called “selection”, can occur both in vitro and in vivo. The selection process is responsible for the development of resistant mutants in, for example, infected human patients. The mutant pathogens can spread to other persons, resulting in an outbreak of disease unresponsive to the particular drug. It is then necessary to use an alternate drug to treat the disease. The alternate drug will similarly be useful only until mutant pathogens resistant to the alternate drug begin to proliferate and dominate the population.
In many cases, drug-resistant pathogens are resistant to only a single drug or class of drugs. In recent years, however, an alarmingly rapid increase has been observed in the number of pathogens that have become multi-drug resistant, meaning that they are resistant to two or more, and in some cases many, classes of drugs. It may be only a matter of time before some pathogens become resistant to all available drugs. Since it can take many years to develop a new drug, there is an urgent need to obtain reliable, quantitative methods for avoiding spread and further development of drug-resistant pathogens.
The problem of drug resistance is especially acute among immunocompromised patients. In these patients, blocking the growth of pathogens by using doses based on the MIC is not adequate to clear the infection; resistant mutants grow and can be transmitted from the infected person to others. As AIDS has spread through regions of the world where tuberculosis is widespread, for example, drug-resistant tuberculosis strains have emerged and rapidly spread when the drug-resistant bacteria have subsequently infected healthy (i.e., immunocompetent) persons. The diseases caused thereby have proven resistant to traditional treatments.
Drug dosing schedules are often based on a parameter called the area under the curve (AUC), where the curve represents a plot of drug concentration in human serum versus the time after delivery of the antibiotic or other drug into the human. One currently favored approach to dosing within the pharmaceutical industry involves the analysis of an empirical parameter called the AUIC, defined as the ratio of the AUC to minimum inhibitory concentration (MIC). No sound theoretical basis has yet been identified as to why a drug maintained at a particular multiple of the MIC should clear an infection. Moreover, the AUIC concept has not been demonstrated to have any relationship to drug resistance.
SUMMARY OF THE INVENTION
The invention is based on the discovery that, for many drugs (e.g., antiviral or antimicrobial drugs such as antifungal or antibiotic drugs, including bacteriocidal or bacteriostatic drugs) and many pathogen strains (e.g., viral, fungal, or bacterial pathogens), a concentration of drug can be identified at which drug-resistant mutant pathogen strains are not selected. This concentration is herein referred to as the “mutant prevention concentration” (MPC). Maintaining serum concentrations of the drug above the MPC throughout a course of treatment should severely restrict selection of drug-resistant mutants. Additionally, it is discovered that drug-resistant mutant pathogens are selected exclusively within a drug concentration window, termed the “mutant selection window” (MSW; FIG.
1
). A quantitative expression of this window, which we call the “window index” (WI), is defined as the ratio of the MPC to the MIC. The window index is characteristic of a given drug and a given pathogen.
In general, one embodiment of the invention features a method for determining the mutant prevention concentration (MPC) of a drug against a particular pathogen (e.g., a bacterial, fungal, or viral pathogen). The method includes the steps of obtaining a culture of said pathogen grown to high density (e.g., to stationary phase in the cases of bacteria and fungi); dividing at least some portion of the culture among a plurality (e.g., one, five, ten, one hundred, five hundred or more) of containers of a growth medium (e.g., agar plates) containing various concentrations of the drug; incubating the containers; counting the pathogen colonies (i.e., for bacteria or fungi), or plaques or foci (i.e., for viruses), if any, in the containers; plotting the number of counted colonies against drug concentration in each container; and, if necessary, extrapolating the plot to determine the minimum drug concentration corresponding to zero colonies. The minimum drug concentration corresponding to zero colonies is the MPC.
The invention also features a method for determining the mutant prevention concentration (MPC) of a drug against a particular pathogen (e.g., a bacterial, fungal, or viral pathogen). The method includes the steps of obtaining a culture of the pathogen, grown to high density (e.g., stationary phase), dividing at least some portion of the culture among a plurality (e.g., one, five, ten, one hundred, five hundred or more) of containers of a growth medium (e.g., agar plates, or a liquid culture broth) containing various concentrations of the drug; incubating the containers; and identifying the container having the lowest concentration of drug effective to prevent growth of the pathogen. This concentration is the MPC.
In any of the above methods: The culture can be concentrated or diluted, if necessary, prior to dividing among the containers. To refine measurement of the MPC, the method can be repeated with concentrations of drug more closely clustered around the MPC determined in the first iteration of the method.
The various concentrations of drug in the containers can differ from one another by a constant factor (e.g., ten-fold, five-fold, three-fold, or at least about two-fold).
In the dividing step, between about 10
9
and about 10
12
colony-forming units (e.g., between about 10
10
and about 10
12
colony-forming units) of bacteria can be divided among all of the containers that include a single concentration of the drug. The number of containers required for each concentration in order to apply this number of bacteria will be understood to depend on the size of each container, and can vary from one to 100 or more.
In another embodiment, the invention features a method for determining the window index (WI) (i.e., corresponding to the mutant selection window for a drug wherein resistant mutants of a specific pathogen are selected in the presence of the drug). The method includes the steps of determining the minimum inhibitory concentration (MIC) of the drug for the pathogen; determining the mutant prevention concentration (MPC) of the drug for the pathogen; and dividing MPC by MIC to obtain the WI. WI can optionally be added to or multiplied by various constants.
The invention a
Dong Yuzhi
Drlica Karl
Zhao Xilin
Fish & Richardson P.C.
Patten Patricia D
Tate Christopher R.
The Public Health Research Institute of the City of New York, In
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