Link:  Pharm/Biotech Resources

Title:  Animal models and methods for sepsis

United States Patent:  6,964,856

Issued:  November 15, 2005

Inventors:  Bellinger-Kawahara; Carole (Redwood City, CA); Contag; Pamela R. (San Jose, CA); Hubbard; Alan (Berkeley, CA)

Assignee:  Xenogen Corporation (Alameda, CA)

Appl. No.:  439902

Filed:  May 15, 2003

Methods for selecting a candidate drug for treating sepsis are disclosed. The methods involve labeling a sepsis-causing pathogen with a reporter and monitoring the progress of infection by detecting levels of the reporter in animals treated with test compounds or drugs. The comparisons may be made between experimental and control animals, as well as within a single animal or group of animals. Also disclosed is a method for predicting an expected time of death of an experimental animal in a model system of sepsis using data generated in the initial part of the experiment.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method for selecting a candidate drug for treating sepsis. The method includes the steps of (i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis in the animal species, in which model system a critical rate of pathogen load increase has been ascertained; (ii) infecting an experimental animal of the animal species with a dose of reporter-labeled pathogen of the pathogen species, where the dose is sufficient to result in a rate of pathogen load increase exceeding the critical rate; (iii) administering a test drug to the experimental animal; (iv) measuring the level of the reporter in the experimental animal; and (v) selecting the test drug as a candidate drug for treating sepsis if the test drug is effective to decrease the rate of pathogen load increase in the experimental animal below the critical rate of pathogen load increase.

In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.

In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.

In another aspect, the invention includes a method for selecting a candidate drug for treating sepsis. The method includes the steps of (i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis in the animal species, in which animal species (a) a time of onset of terminal sepsis in response to a selected dose of the pathogen species, and (b) a critical infection level of the pathogen species, have been ascertained; (ii) infecting an experimental animal of the animal species with a dose of reporter-labeled pathogen of the pathogen species, where the dose is sufficient to cause the onset of terminal sepsis in an untreated animal; (iii) administering a test drug to the experimental animal; (iv) measuring the level of the reporter in the experimental animal at a selected time after onset of terminal sepsis, where the level of reporter corresponds to the level of infection in the experimental animal; and (v) selecting the test drug as a candidate drug for treating sepsis if the test drug is effective to drop the level of infection below the critical infection level.

In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.

In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.

In another aspect, the invention includes a method for selecting a candidate drug for treating sepsis. The method includes the steps of: (i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis in the animal species, in which animal species a time of onset of terminal sepsis, in response to a selected dose of the pathogen species, has been ascertained; (ii) infecting experimental and control animals of the animal species with a reporter-labeled pathogen of the pathogen species; (iii) administering a test drug to the experimental animals; (iv) measuring the level of reporter in the experimental and the control animals at a selected time after onset of terminal sepsis; and (v) selecting the test drug as a candidate drug for treating sepsis if the test drug is effective to cause a statistically-significant reduction in the level of reporter in the experimental animals as compared with the control animals.

In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.

In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.

The invention further includes a method for predicting an expected time of death of an experimental animal in a model system of sepsis. The method includes the steps of:

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Ausubel, F. M., et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Media Pa.; and Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989).

Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

2.1 Models of Sepsis

The present invention may be applied in connection with any animal model of sepsis which utilizes the progress of infection by the sepsis-causing pathogen as a basis for the model. Exemplary animal models of sepsis include rodent, rabbit and monkey models. Rodent models are particularly suitable, as they can be carried out economically without need for specialized primate centers. Suitable rodents include mice, rats, hamsters, gerbils and guinea pigs.

For use in connection with the invention, the animal model of sepsis is adapted for use with a particular sepsis-causing organism, such as a eukaryotic parasite or a bacterium. As is recognized in the art, a number of different types of bacteria are capable of causing sepsis. They include, but are not limited to, the following species: Gram positive organisms, such as members of Enterococcus spp. (e.g., E. faecalis), Staphylococcus spp. (e.g., S. aureus), and Streptococcus spp. (e.g., S. agalactiae); and Gram negative organisms, such as members of the Enterobacteriacae family (e.g., Escherichia coli), Providencia spp. and Pseudomonas spp. (e.g., P. aeruginosa)

In practicing the present invention, the pathogen causing sepsis is labeled with a reporter, preferably a reporter that can be detected in a living animal. Exemplary reporters with such properties include light-emitting reporters, such as fluorescent and luminescent reporters. A further desirable characteristic of the reporter is that it does not become diluted as the labeled pathogen reproduces. Accordingly, preferred reporters suitable for use with the present invention comprise polypeptides expressed by the pathogen. Polynucleotide cassettes encoding such polypeptides are typically transfected into the pathogen as extra-chromosomal genetic elements (e.g., plasmids) or are stably incorporated into the pathogen genome (e.g., "hopped" in using a transposon).

Polypeptides which result in the generation of light in a living organism (bioluminescence) include, but are not limited to, various luciferases, green fluorescent protein (GFP), yellow fluorescent protein and aequorin (Wilson and Hastings, 1998, Annu. Rev. Cell Dev. Biol. 14:197-230). Luciferase is a particularly suitable reporter, since it is a luminescent molecule, and thus does not require excitation in order to generate light. It does, however, typically require a substrate (e.g., luciferin, an aldehyde or coelenterazine), an energy source (e.g., ATP) and oxygen. In the case of bacterial luciferases, the genes encoding the substrate can be supplied the same vector as the gene(s) encoding the luciferase enzyme, thus eliminating the need for exogenously-supplied substrate (see, e.g., U.S. Pat. No. 5,650,135).

In a preferred embodiment of the present invention, the pathogen is transfected with a vector encoding a light-generating protein in order to label the pathogen. Vectors suitable for such transformation are known in the art, and include the vector described in U.S. Pat. No. 5,650,135, as well as the E. coli-P. aeruginosa shuttle vector 4027 2-11 described in the Materials and Methods, below. Of course, other transformation methods, plasmids, vectors, or methods of integrating polynucleotides into the genome, known in the art, may be used by one of skill in the art to label a selected sepsis-causing pathogen with a selected polypeptide-based reporter.

After the pathogen is labeled with a suitable reporter, it is introduced into a selected animal model of sepsis for use with the present invention. If the reporter is a light-generating reporter, it may be imaged within the living host animal as described, e.g., in U.S. Pat. No. 5,650,135, and related publications (e.g., Contag, et al., 1998, Nature Medicine 4(2):245-247; and Contag, et al., 1995, Molecular Microbiology 18(4):593-603).

It is desirable to calibrate the experimental system to ascertain variables and parameters useful in adapting the methods of the invention to different model systems of sepsis. This process is illustrated for the model system comprising a mouse Pseudomonas aeruginosa model of sepsis in the section titled "Calibration Process". In those cases where the parameters determined in the calibration process (e.g., LD₅₀, the critical rate of pathogen load increase, time of onset of terminal sepsis, critical infection level and/or death expectation curve) are already known (e.g., from previous experiments or publications), the calibration process may, of course, be bypassed and the invention practiced (e.g., methods for screening drugs; methods for prediction of time of death based on a reporter signal at a selected time) using the known parameters.

One of the factors typically determined in the calibration process is a suitable dose of pathogen for the selected model system of sepsis. This dose is preferably based on the LD₅₀ of the pathogen in the particular animal model of sepsis being used. The LD₅₀, or "Lethal Dose 50", is a measure for quantifying the effects of a perturbation (e.g., a test compound, procedure, or bacterial infection) on a biological system. It represents the 50% mortality point—that is, the concentration or level of a particular perturbation at which half of the animals die by the end of the experiment.

The LD₅₀ may change depending on the experimental parameters. The measure is therefore expressed as an LD₅₀ for a given experimental system. By way of example, it was discovered in the course of experiments performed in support of the present invention that the type of anesthesia used to prepare the animals for imaging can have a significant effect on the LD₅₀. Specifically, it was discovered that anesthesia induced by injectable Ketamine may potentiate the virulence of some pathogens. In one set of experiments, luminescent Pseudomonas aeruginosa had an LD₅₀ of approximately 2.5×10⁶ CFU in non-anesthetized mice versus 4.6×10⁵ CFU in Ketamine-sedated mice.

No surprisingly, the LD₅₀ is also affected by treatment with compounds which affect the host immune system. For example, it was found during experiments performed in support of the present invention that pathogen virulence is boosted by administration of 5% (by volume) hog gastric mucin to the host. This common adjuvant impairs local macrophage function for 2-3 hours after administration (Comber, et al., 1975, Antimicrob. Agents Chemother. 7:179-185).

Although different experimental manipulations, such as types of anesthesia or the presence of mucin, may alter the initial input dose needed to establish terminal sepsis, they do not impact the predictive nature of the methods herein, so long as the calibrations (described below) are carried out under experimental conditions similar to those used in subsequent screening studies.

The LD₅₀ is a reliable measure of the ultimate effect of a particular perturbation, but is a very crude readout of the state of the biological system (e.g., animal) since it reduces an underlying graded response in the animal to an all or nothing event (the survival or death of the animal). The LD₅₀ provides no information about the state of the animals before they die (e.g., the progress of an infection by the pathogen under study) or after they die (e.g., identification of the underlying pathology, such as identification of specific organ system(s) which failed).

As will be appreciated in view of the following sections, the present invention provides a read-out of the processes underlying a sepsis infection in any given model system of sepsis, and provides methods by which this read-out can be quantitatively related to the traditional LD₅₀ in that model system. In this way, it provides a method for studying the progress of sepsis in living animals, and for predicting the time of death for a particular animal, so the effects of a particular compound or treatment can be assessed the without waiting for the animal to die.

Applications

General Considerations

The methods followed in the following applications are similar to those carried out during the calibration process. Typically, a model system of sepsis is calibrated using the same procedure as will be used during the subsequent application. For example, in cases where the investigator wishes to use the "rate of pathogen load increase" method, both the calibration and the subsequent application (e.g., drug screening) will employ a calculation of the rate of pathogen load increase in the data analysis. Similarly, if the investigator wishes to use adjustment and trend periods, terminal sepsis, death expectation curves, and/or critical infection level parameters in the calibration, data from subsequent applications (e.g., drug screening) will be analyzed in the context of the same parameters.

Selection of Candidate Drugs for Treating Sepsis

The invention described herein may be applied in a number of ways readily apparent to one of skill in the art. For example, the invention includes methods for selecting a candidate drug for treating sepsis. In the methods, a suitable model system of sepsis is selected. Selecting such a model system includes selecting the host animal for the model, as well as a pathogen capable of causing sepsis in that host animal. Examples of suitable animals and sepsis-causing pathogens are provided above. Preferred animal species include mammals, especially rodents such as rats, hamsters, gerbils and guinea pigs. Examples of applicable pathogens include bacteria, such as Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. It will be understood that the preceding host animals and pathogens are merely illustrative—a variety of sepsis models and sepsis-causing pathogens are known in the art, and may be used in connection with the practice of the present invention.

Once a model system is selected, parameters such as the critical rate of pathogen load increase, or a time of onset of terminal sepsis in response to a selected dose of the pathogen and a critical infection level of the pathogen, are typically ascertained. The parameters may be ascertained in several ways, e.g., by using a calibration process such as is described above, or from previously-performed calibration experiments or published data.

In one method of selecting a candidate drug, an experimental animal is infected with a dose of reporter-labeled pathogen of the appropriate pathogen species, where the dose is sufficient to cause the onset of terminal sepsis in an untreated animal. The pathogen (e.g., bacterium) is preferably labeled with a light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. A test drug is then administered to the host or experimental animal. The drug may be administered at any suitable time, e.g., before infection, or at a selected time after infection, depending on what type of effect is being screened for (prophylactic or therapeutic).

The level of the reporter is then measured, preferably at a selected time after onset of terminal sepsis (or after the time by which an untreated animal would exhibit onset of terminal sepsis with the particular dose of pathogen administered). If the reporter is a light-emitting reporter, a preferred method of measurement is using a photon detection device, such as an intensified CCD camera, a cooled CCD camera, or any other photon detection device with a high sensitivity. However, other methods may of course be used. For example, a light-emitting reporter may also be detected using a sensitive luminometer; a radioactive reporter may be detected by counts, X-ray imaging or scintillation. Since the reporter labels the pathogen, the level of reporter corresponds to the level of infection by the pathogen of the experimental or host animal.

A test drug which is effective to reduce the level of reporter is then selected as a suitable candidate drug for treating sepsis. If the model system of sepsis is calibrated using the rate of pathogen load increase method, the suitable drug for treating sepsis reduces the rate of pathogen load increase relative to untreated animals exposed to the same dose of pathogen. In a preferred embodiment, the suitable drug for treating sepsis reduces the rate of pathogen load increase below the critical rate of pathogen load increase, under conditions where most of the untreated animals exhibit a rate of pathogen load increase greater than the critical rate. Alternatively, if the model system of sepsis is calibrated in the context of a critical infection level, the suitable drug for treating sepsis preferably reduces the level of reporter detected below the critical infection level.

Candidate drugs for treating sepsis may be evaluated further. Examples of such further evaluation may include additional studies (e.g., different drug dosages, times of administration, different pathogen dosages, etc.) in the present model system of sepsis, as well as other studies, such as toxicology studies, pharmacology studies, and clinical trials. Candidate drugs which prove effective in such follow-up studies may then be commercially produced for treatment of sepsis according to standard production methods known in the art. In this respect, the invention includes methods of screening for such candidate drugs which further include the subsequent manufacture or sale of the candidate drug for treatment of sepsis.

Another method which may be used to select a candidate drug for treating sepsis employs groups of control and experimental animals. The method is performed as described above, except that the pathogen, e.g., bacterium, is used to infect both experimental and control animals with the reporter-labeled pathogen. The test drug is then administered only to the experimental animals, and reporter levels in the experimental animals are compared to reporter levels in controls by any of the preceding methods. Of course, the test drug may be administered according to any suitable dosage or administration protocol, prophylactically or therapeutically.

In one embodiment, the level of reporter in the experimental and the control animals is measured at a selected time after onset of terminal sepsis. A candidate drug may be identified by looking for a statistically-significant reduction in the level of reporter in the experimental animals as compared with the control animals.

In another embodiment, a series of measurement are made over the time interval used for generating a critical rate of pathogen load increase. A candidate drug may be identified by looking for a statistically-significant reduction in the rate of pathogen load increase in the experimental animals as compared with the control animals.

Predicting an Expected Time of Death of a Host Animal Using a Death Expectation Curve

Another application of the present invention involves a method for predicting an expected time of death of an experimental animal in a model system of sepsis. A model system of sepsis is selected as described above. A death expectation curve is then constructed at a selected time after onset of terminal sepsis for all doses administered in the experiment. The death expectation may be constructed using any of the methods previously mentioned herein. An experimental animal is then infected with a reporter-labeled pathogen of the appropriate pathogen species, and the level of reporter is measured at about the same time after infection as the data used to construct the death expectation curve. The level of reporter is then used to predict the expected time of death using the death expectation curve.

It can be appreciated that this method provides a sensitive measure of the state of the animal during the course of the sepsis-causing infection (which a death-as-an-endpoint model does not), as well as a measure of expected time of death, which can be compared and/or correlated with data generated using traditional death-as-an-endpoint LD₅₀ studies.
 

Claim 1 of 36 Claims

1. A method for selecting a candidate drug for treating sepsis, comprising:

(i) selecting a model system of sepsis, said model system comprising an animal species and a pathogen species that causes sepsis in said animal species, in which model system a critical rate or pathogen load increase has been ascertained,

(ii) infecting an experimental animal of said animal species with a dose of reporter-labeled pathogen of said pathogen species, where said dose is sufficient to result in a rate of pathogen load increase exceeding said critical rate;

(iii) administering a test drug to said experimental animal;

(iv) measuring the level of said reporter in said experimental animal; and

(v) selecting said test drug as a candidate drug for treating sepsis if said test drug is effective to decrease the rate of pathogen load increase in the experimental animal below said critical rate of pathogen load increase.

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