Title: Methods and treatment of multiple sclerosis
United States Patent: 6,710,033
Issued: March 23, 2004
Inventors: Stratton; Charles W. (Nashville, TN); Mitchell; William M. (Nashville, TN); Sriram; Subramaniam (Nashville, TN)
Assignee: Vanderbilt University (Nashville, TN)
Appl. No.: 528348
Filed: March 17, 2000
The invention features methods and reagents for the diagnosis, monitoring, and treatment of multiple sclerosis. The invention is based in part on the discovery that Chlamydia is present in patients with multiple sclerosis, and that anti-chlamydial agents improve or sustain neurological function in these patients.
SUMMARY OF THE INVENTION
In a first aspect, the present invention features a method of diagnosing or monitoring multiple sclerosis in an individual, including assaying a test sample from the individual for the presence of Chlamydia, wherein the presence of Chlamydia in the sample indicates the presence of multiple sclerosis.
In preferred embodiments, the Chlamydia is selected from the group consisting of Chlamydia pneumoniae, Chlamydia pecorum, Chlamydia psittacci, and Chlamydia trachomatis, and the test sample is selected from the group consisting of blood, serum, peripheral blood mononuclear cells, cerebrospinal fluid, urine, nasal secretion, and saliva.
In one embodiment, the test sample is assayed for the presence of Chiamydia by contacting cultured chlamydia-free indicator cells (e.g., HL cells, H292 cells, HeLa cells, or Hep-2 cells) with the test sample; and then detecting the presence of Chlamydia in the cultured indicator cells. The presence of Chlamydia in the cultured indicator cells is indicative of the presence of Chlamydia in the test sample.
The presence of Chlamydia in the cultured indicator cells can be detected by detecting an antibody to Chlamydia (e.g, an antibody to a Chlamydia elementary body antigen), a Chlamydia gene, or a Chlamydia protein in the test sample. The presence of the antibody, gene, or protein is indicative of the presence of Chlamydia in the test sample. In one embodiment, the test sample is incubated under disulfide reducing conditions (e.g., incubating a disulfide reducing agent such as 2,3-dimercaptosuccinic acid, penicillamine, .beta.-lactams, dithiotreitol, mercaptoethylamine, or N-acetylcysteine) prior to detecting the presence of Chlamydia.
In another aspect, the invention features a method of isolating elementary bodies from a receptacle containing elementary bodies. The method includes treating the receptable with trypsin/EDTA to release elementary bodies adhered to the receptacle; and then concentrating the elementary bodies by centrifugation or filtration.
In still another aspect, the invention features a method of releasing DNA from elementary bodies, the method including incubating the elementary bodies under disulfide reducing conditions and digesting the elementary bodies with a protease.
In yet another aspect, the invention features a method of treating an individual diagnosed to have multiple sclerosis, including administering to the individual an effective amount of at least one anti-chlamydial agent. In one embodiment, the individual is administered the anti-chlamydial agent until the individual tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia. In another aspect, the individual is administered the anti-chlamydial agent for at least 45 days. The adminstration can be continued for longer periods, and it may be preferable to continue the treatment for at least 90 days, at least 180 days, or even for one year or more.
Preferable anti-chlamydial agents include rifamycins, azalides, macrolides, ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles, nitrofurans, quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, and tetracyclines.
In one embodiment, the individual is also administered an effective amount of an agent that increases inducible nitric oxide synthase (iNOS) activity, such as a type-1 interferon (e.g., .alpha.-interferon or .beta.-interferon), a synthetic type-1 interferon analog, or a hybrid type-1 interferon. Preferably, the type-1 interferon analog or hybrid binds to the same receptor as a naturally-occurring type-1 interferon. In another embodiment, the individual is administered at least two anti-chlamydial agents.
In yet another aspect, the invention features a method of treating an individual diagnosed to have multiple sclerosis, including administering to the individual (i) a rifamycin; and (ii) a compound selected from the group consisting of azalides, macrolides, ketolides, and streptogramins. In addition, the individual can optionally be administered ampicillin, amoxicillin, probenecid, a nitroimidazole, a nitrofuran, or any combination thereof.
In another aspect, the invention features a method of treating an individual diagnosed to have multiple sclerosis, including administering to the individual one of the following combinations: a rifamycin, ampicillin or amoxicillin, and probenecid; a quinolone or a fluoroquinolone and a rifamycin; a rifamycin, a sulfonamide, and an isonitotinic congener; or a rifamycin and a tetracycline. The individual can also be administered an effective amount of a compound that increases iNOS activity (e.g., .beta.-interferon).
The administration is preferably continued until the individual tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia, or for at least 45 days.
In still another aspect, the invention features a pharmaceutical composition that includes one of the following combinations: a rifamycin, ampicillin or amoxicillin, and probenecid; a quinolone or a fluoroquinolone and a rifamycin; a rifamycin, a sulfonamide, and an isonitotinic congener; or a rifamycin and a tetracycline. The composition can optionally include a compound that increases iNOS activity (e.g., .beta.-interferon).
In yet another aspect, the invention features a kit that includes an anti-chlamydial agent and a compound that increases iNOS activity. In a preferred embodiment, the compound that increases iNOS activity is a type-1 interferon (e.g., .beta.-interferon), a synthetic type-1 interferon analog, or a hybrid type-1 interferon, wherein the type-1 interferon analog or hybrid binds to the same receptor as a naturally-occurring type-1 interferon. In another preferred embodiment, the anti-chlamydial agent is selected from the group consisting of rifamycins, azalides, macrolides, ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles, quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, and tetracyclines.
In still another aspect, the invention features a method for determining whether a candidate compound is a potential drug for the treatment of a disease caused or exacerbated by chlamydial infection, the method including the steps of: (a) infecting a non-human animal (e.g., a non-human mammal) with Chiamydia; (b) administering a candidate compound to the animal; and (c) assaying for the presence of a chlamydial infection in a test sample from the mammal. A decrease in the level of infection, relative to the level of infection of a control animal infected with chlamydia but not administered a candidate compound, identifies the candidate compound as a potential drug for the treatment of disease caused or exacerbated by a chlamydial infection. Preferably, the animal is a non-human mammal and brain of the mammal is infected with Chlamydia.
In preferred embodiments, the Chlamydia is selected from the group consisting of Chlamydia pneumoniae, Chlamydia pecorum, Chlamydia psittacci, and Chlamydia trachomatis, and the test sample is selected from the group consisting of blood, serum, cerebrospinal fluid, urine, nasal secretion, and saliva. In another preferred embodiment, the disease is multiple sclerosis. The animal can be, for example, a mouse, rat, rabbit, or amoeba.
In one embodiment, the test sample is assayed for the presence of Chlamydia by contacting cultured chlamydia-free indicator cells (e.g., HL cells, H292 cells, HeLa cells, or Hep-2 cells) with the test sample; and then detecting the presence of Chlamydia in the cultured indicator cells. The presence of Chlamydia in the cultured indicator cells is indicative of the presence of Chlamydia in the test sample.
The presence of Chlamydia in the cultured indicator cells can also be detected by detecting an antibody to Chlamydia (e.g, an antibody to a Chlamydia elementary body antigen), a Chlamydia gene, or a Chlamydia protein in the test sample. The presence of the antibody, gene, or protein is indicative of the presence of Chlamydia in the test sample. In one embodiment, the test sample is incubated under disulfide reducing conditions (e.g., incubating a disulfide reducing agent such as 2,3-dimercaptosuccinic acid, penicillamine, .beta.-lactams, dithiotreitol, mercaptoethylamine, or N-acetylcysteine) prior to detecting the presence of Chlamydia.
In a related aspect, the invention features a second method for determining whether a candidate compound is a potential drug for the treatment of multiple sclerosis. This method includes the steps of: (a) infecting the brain of a non-human mammal (e.g., a rat, mouse, or rabbit) with Chlamydia; (b) administering a candidate compound to the mammal; and (c) assaying for the loss of white matter in the brain of the mammal, wherein a decrease in the loss of white matter, relative to the loss of white matter in a control mammal infected with chlamydia but not administered any candidate compound, identifies the candidate compound as a potential drug for the treatment of multiple sclerosis.
By "Chlamydia" or "chlamydial cell" is meant any organism of the order Chlamydiales. Examples include, but are not limited to, C. psittacci, C. trachomatis, C. pecorum, C. abortus, C. caviae, C. felis, C. suis, C. muridarum, WSU-86-1044, Parachlamydia acanthamoebae, and Simkania negevensis. By "chlamydial infection" is meant an infection of a cell by a chlamydial cell.
By "indicator cell" is meant a cell capable of being infected by a Chlamydia cell. Preferred indicator cells include HL cells, H292 cells, HeLa cells, and Hep-2 cells, which have been shown to be free of chlamydial infection.
By "long-term therapy" is meant the treatment of a disease (e.g., MS) for at least 45 days, more preferably for at least 60 days or even 90 days, and most preferably for at least 120 days, 180 days, or for a year or more. The long-term therapy can be continued for a given length, or can be stopped when a patient tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia (e.g., by PCR of a disulfide reducing agent-treated sample from the patient).
It may be desirable to change one or all of the drugs in the middle of the long-term therapy. Changes in drug combinations may be for many reasons, such as to reduce side effects or cost to the patient, or in response to a change in the patient's condition or degree of infection. Moreover, while it is preferable that the therapy is continuous, it is understood that interruption for as much as two weeks or even a month may be desirable or necessary. For example, an individual may take drug combination A for 30 days, stop therapy for two weeks, and then resume therapy (switching to drug combination B) for an additional 30 days. Interrupted therapy and therapy in which one or more drugs are added or removed are each considered to be long-term therapy if the number of days of therapy (i.e., excluding the days in which no drugs for the treatment of MS were administered) is at least 45.
By "anti-chlamydial agent" is meant an agent that results in a decrease in the viability or replication of chlamydial cells at a concentration that would not be substantially detrimental to the cells in which the chlamydial cells were contained. Preferably, the anti-chlamydial agent decreases the viability or replication of chlamydial cells by at least 50%, more preferably by at least 75% and most preferably by at least 90% or even 95%. Preferred anti-chlamydial agents include, without limitation, rifamycins, azalides, macrolides, ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles, quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, and tetracyclines.
The present invention provides methods for the diagnosis of MS with a significant reduction in cost. In addition, these diagnostic assays provide objective data concerning the course of the disease and, thus, the ability to monitor disease progress and the effectiveness of therapy. The invention also provides methods and reagents for the treatment of a patient diagnosed with MS, as well as methods for identifying new drugs for the such treatment.
DETAILED DESCRIPTION OF THE INVENTION
C. pneumoniae belongs to the order Chlamydiales (the members of which are herein referred to collectively as Chlamydia). Members of this order are obligately intracellular pathogens that are infectious to humans and other vertebrates. Other species currently recognized include C. psittacci, C. trachomatis, and C. pecorum. C. psittacci is known to infect microglial cells, while C. pecorum in cattle causes a syndrome known as sporadic bovine encephalomyelitis, for which detailed neuropathologic data are lacking (Storz J., Chlamydia and Chlamydial Induced Diseases. Springfield, Ill.: Charles C. Springer, 1971: 358). C. trachomatis and C. pneumoniae are pathogenic primarily to humans and are recognized to cause latent disease. Meningoencephalitis and other neurological complications have been described in patients with infections due to C. psittaci and C. trachomatis (Korman et al., Clin. Infect. Dis. 25:847-851, 1997). In addition, the Chlamydiales order includes C. abortus, C. caviae, C. felis, C. suis, C. muridarum, WSU-86-1044, Parachlamydia acanthamoebae, and Simkania negevensis (Everett et al. Intl. J. System. Bacteriol. 49:415-440, 1999).
We have demonstrated a strong correlation between the presence of C. pneumoniae in the CSF of patients with MS by cell culture, polymerase chain reaction (PCR), and immunological methods. C. pneumoniae was isolated from CSF cultures and also was identified in CSF by PCR amplification of the ompA gene of C. pneumoniae. Moreover, CSF titers of IgM and IgG against C. pneumoniae EB antigens were elevated as measured by ELISA methodologies. The specificity of this antibody response for C. pneumoniae was shown by Western blot assays. PCR data in which CSF samples from MS patients and other neurologic diseases (OND) controls were analyzed for the 16S rRNA gene of C. pneumoniae using a nested PCR procedure followed by Southern hybridization with a digoxigenin labeled specific probe also established a link between MS and C. pneumoniae infection. Moreover, IEF/affinity-driven immunoblot assays show that the cationic antibodies in MS patients react to C. pneumoniae EB antigens.
As the presence of Chlamydia correlates with the presence of MS, the invention features a method for diagnosing a patient with MS. In the methods of the invention, a test sample from an individual, such as an individual who is suspected of having MS, is used. The test sample can include blood, serum, cerebrospinal fluid, urine, nasal secretion, saliva, or any other bodily fluid or tissue, or antibodies or nucleic acids isolated from one of the foregoing samples.
The test sample can be assayed for the presence or absence of Chlamydia by culturing the test sample with indicator cells. The indicator cells can be any cells which are capable of being infected by Chlamydia, and which preferably have been shown to be free of infection by Chlamydia and free of elementary bodies of Chlamydia. Representative indicator cells include HL cells, H292 cells, HeLa cells, Hep-2 cells, or any other cell line capable of supporting replication of Chlamydia. The indicator cells are cultured in the presence of the test sample and then assayed for the presence or absence of Chlamydia by an appropriate method, such as by exposing the cultured indicator cells to a detectable antibody that is specific for Chlamydia. The presence of Chlamydia in the cultured indicator cells indicates the presence of Chlamydia in the test sample.
The test sample can also be assayed for the presence or absence of Chlamydia by detecting the presence or absence of a Chlamydia gene (e.g., a gene encoding MOMP, OMP-B, GRO-ES, GRO-EL, DNAK, 16S RNA, 23S RNA, ribonuclease-P, the 76 kD attachment protein, or a KDO-transferase) in the test sample. For example, the test sample can be assayed for the presence or absence of the Chlamydia gene by Southern hybridization using a detectable probe for the appropriate gene. Alternatively, the test sample can be assayed using quantitative PCR or RT-PCR (e.g., by using a LightCycler.TM. (Idaho Technology Inc., Idaho Falls, Id.) and fluorescent LightCycler.TM. probes). The presence of the Chlamydia gene in the test sample is indicative of the presence of Chlamydia in the test sample. To facilitate assaying a test sample for the presence or absence of Chlamydia by detecting the presence or absence of a Chlamydia gene, the test sample can be subjected to methods to enhance isolation of Chlamydia elementary bodies from the test sample and to release DNA from the elementary bodies. For example, elementary bodies have a tendency to adhere to the walls of a receptacle containing them; the elementary bodies can be removed from the receptacle by treating the receptacle containing the elementary bodies with trypsin/EDTA, thereby releasing elementary bodies that adhered to the receptacle; and then concentrating the released elementary bodies, such as by centrifugation or filtration. To release DNA from elementary bodies, the elementary bodies are incubated under disulfide reducing conditions, such as incubating the elementary bodies with a disulfide reducing agent such as dithiothreitol (DTT) or 2-mercaptoethanol; and digesting the elementary bodies with a protease.
The test sample can also be assayed for the presence of Chlamydia by detecting the presence of a protein from Chlamydia. For example, the presence of a MOMP protein in the test sample can be detected through the use of ELISA methodologies with an antibody that specifically recognizes the MOMP protein. Alternatively, the test sample may be assayed for the presence of Chlamydia by detecting the presence of antibodies to Chlamydia, or to Chlamydia EB antigens, in the test sample. The presence of Chlamydia protein or antibodies to Chlamydia or Chlamydia EB antigens in the test sample is indicative of the presence of Chlamydia in the test sample. In either of these methods, Chlamydia EB antigens can be prepared by incubating Chlamydia EBs under disulfide reducing conditions, such as in the presence of at least one disulfide reducing agent such as DTT or 2-mercaptoethanol, or another disulfide reducing agent. The presence of proteins or antibodies may be detected by appropriate methods such as by ELISA, Western blot, or isoelectric focusing.
The diagnostic methods described herein are useful for detecting or confirming the disease in a patient, as well as for monitoring the progress of the disease. Disease monitoring is useful, for example, for determining the efficacy of a particular therapy.
The invention also provides a diagnostic reagent kit including one or more containers filled with one or more of the ingredients used in the assays of the invention. Optionally associated with such a kit can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of diagnostic products, which reflects approval by the agency of manufacture, use or sale for human administration. The kit can be labeled with information regarding mode of administration, sequence of execution (e.g., separately, sequentially, or concurrently), or the like. The kit can be a single unit assay or it can be a plurality of unit assays. In particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. For the purpose of this invention, a unit assay is intended to mean material sufficient to perform only a single assay.
In addition to demonstrating that C. pneumoniae infection correlates with MS, we have also found that patients with MS that were treated with anti-chlamydial agents showed improved Expanded Disability Status Scale (EDSS; Kurtzke, Neurology 33:1444-1152, 1983) scores. Thus, it is highly likely that chlamydial infection causes or exacerbates MS. We have also identified combination therapy regimens that, because of the phase of Chlamydia targeted by each drug, are particularly suited for the treatment of MS.
A) Anti-chlamydial Agents
Chlamydia are obligate intracellular bacterial parasites of eukaryotic cells. Members of this order have a unique biphasic development cycle with distinct morphological and functional forms. This developmental growth cycle alternates between (i) intracellular life forms of which two are currently recognized: an intracellular form which can exist as a metabolically-active, replicating organism known as the reticulate body (RB) or a persistent, nonreplicating form known as the cryptic body; and (ii) an extracellular EB form that is infectious and metabolically-inactive.
EBs are small (300 to 400 nm) infectious spore-like forms which are resistant to a variety of physical insults such as enzyme degradation, sonication, and osmotic pressure. This physical stability is likely a result of extensive disulfide cross-linking of the cysteine-rich MOMP. Under the oxidizing conditions of the extracellular milieu of the host, the outer membrane of EBs is relatively impermeable and indestructible.
A number of effective agents that are specifically directed against the initial phase of chlamydial infection (i.e., the transition of the chlamydial EB to a reticulate body (RB)) have been identified. These include compounds in the rifamycin class and act against DNA-dependent RNA polymerase, which is present when the EB begins to transform into the RB phase. Inhibition of this chlamydial DNA-dependent RNA polymerase prevents this transition.
A number of effective agents that are specifically directed against the cryptic growth phase have also been identified. This cryptic growth phase, unlike that of the replicating chlamydial microorganism, which uses host cell energy, involves electrons and electron transfer proteins, as well as nitroreductases. Accordingly, the initial phase of Chlamydia infection is susceptible to the antimicrobial effects of nitroimidazoles, nitrofurans, and other agents directed against anaerobic metabolism in bacteria. Nitroimidazoles and nitrofurans are synthetic antimicrobial agents that are grouped together because both are nitro (NO2 --) containing ringed structures and have similar antimicrobial effects. These effects require degradation of the agent within the microbial cell such that electrophilic radicals are formed. These reactive electrophilic intermediates then damage nucleophilic protein sites including ribosomes, DNA, and RNA. Nitroimidazoles and nitrofurans were not previously considered to possess antimicrobial activity against Chlamydia. This apparent lack of antimicrobial activity, however, is due to the fact that conventional susceptibility testing methods only test for effect on the replicating form of Chlamydia, and do not measure the presence of other forms of Chlamydia.
Examples of suitable nitroimidazoles include, but are not limited to, metronidazole, tinidazole, bamnidazole, benznidazole, flunidazole, ipronidazole, misonidazole, moxnidazole, ronidazole, sulnidazole, and their metabolites, analogs, and derivatives thereof. Metronidazole is most preferred. Examples of nitrofurans that can be used include, but are not limited to, nitrofurantoin, nitrofurazone, nifurtimox, nifuratel, nifuradene, nifurdazil, nifurpirinol, nifuratrone, furazolidone, and their metabolites, analogs, and derivatives thereof. Nitrofurantoin is preferred within the class of nitrofurans. Throughout this application and for purposes of this invention, "metabolites" are intended to embrace products of cellular metabolism of a drug in the host (e.g., human or animal) including, but not limited to, the activated forms of prodrugs. The terms "analogs" and "derivatives" are intended to embrace isomers, optically active compounds, and any chemical or physical modification of an agent, such that the modification results in an agent having similar or increased, but not significantly decreased, effectiveness against Chlamydia, compared to the effectiveness of the parent agent from which the analog or derivative is obtained. This comparison can be ascertained using susceptability testing. Cells to be treated can already be cryptically infected or they can be subjected to stringent metabolic or environmental conditions which cause or induce the replicating phase to enter the cryptic phase. Such stringent conditions can include changing environmental/culturing conditions in the instance where the infected cells are exposed to .gamma.-interferon; or by exposing cells to conventional antimicrobial agents (such as macrolides and tetracyclines) which induce this cryptic phase of chlamydial infection in human host cells.
A class of anti-chlamydial agents that is effective against the replicating and cryptic stationary phases of Chlamydia (and possibly against some other stages of the cryptic phase) have been identified. This class of agents includes ethambutol and isonicotinic acid congeners, which include isoniazid (INH), isonicotinic acid (also known as niacin), nicotinic acid, pyrazinamide, ethionamide, and aconiazide. INH is the most preferred compound in this class. Although these compounds were previously considered effective only for mycobacterial infections, we have discovered that these agents, in combination with other antibiotics, are effective against Chlamydia. It is believed that the isonicotinic acid congeners target the constitutive production of catalase and peroxidase, which is a characteristic of microorganisms, such as mycobacteria, that infect monocytes and macrophages. Chlamydia can also successfully infect monocytes and macrophages.
Using INH to eradicate Chlamydia from macrophages and monocytes subsequently assists these cells in their role of fighting infection. These agents appear to be less effective in vitro against the cryptic phase. Thus, ethambutol, INH, and other isonicotinic acid congeners ideally should be used in combination with agents that target other phases of the chlamydial life cycle. These isonicotinic acid congeners are nevertheless excellent agents for the long term therapy of chronic/systemic chlamydial infection.
Adverse conditions, such as limited nutrients, antimicrobial agents, and the host immune response, produce a stringent response in Chlamydia. This stringent response alters the morphological state of the intracellular microorganism and creates dormant forms, including the intracellular EB, which then can cryptically persist until its developmental cycle is reactivated. Conversely, the host cell may lyse and allow the EBs to reach the extracellular milieu. Thus, it is necessary to utilize a combination of agents directed toward the various life stages of Chlamydia and, in particular, against the elementary body for successful management of infection.
During the chlamydial life cycle, it is known that metabolically-inactive spore-like EBs are released into the extracellular milieu. Although these released EBs are infectious, they may not immediately infect nearby susceptible host cells until appropriate conditions for EB infectivity are present. The result of this delay in infection is the extracellular accumulation of metabolically-inactive, yet infectious, EBs. This produces a second type of chlamydial persistance referred to herein as EB "tissue/blood load." This term is similar in concept to HIV load and is defined herein as the number of infectious EBs that reside in the extracellular milieu. Direct microscopic visualization techniques, tissue cell cultures, and polymerase chain reaction test methods have demonstrated that infectious EBs are frequently found in the blood of apparently healthy animals, including humans. This phenomenon is clearly of great clinical importance in chlamydial infections as these metabolically-inactive EBs escape the action of current anti-chlamydial therapy which is directed only against the replicating intracellular forms of Chlamydia. The presence of infectious extracellular EBs after the completion of short term, anti-replicating phase therapy for chlamydial infections has been shown to result in intracellular infection relapse. Thus, the duration and nature of anti-chlamydial therapy required for management of chlamydial infections is, in part, dictated by the extracellular load of EBs. For purposes of this invention, short term therapy can be approximately two to three weeks; long-term therapy, in contrast, may continue for one or several months (see below).
It is also believed that persistance of chlamydial infections may be due in part to the presence of cryptic forms of Chlamydia within the cells. This cryptic intracellular chlamydial form apparently can be activated by certain host factors such as cortisone (Yang et al., Infect. and Immun., 39:655-658, 1983; Malinverni et al., J. Infect. Dis., 172:593-594, 1995). Anti-chlamydial therapy for chronic Chlamydia infections must be continued until any intracellular EBs or other intracellular cryptic forms have been activated and extracellular EBs have infected host cells. This reactivation/reinfection by chlamydial EBs clearly is undesirable as it prolongs the therapy of chlamydial infections, as well as increases the opportunity for antimicrobial resistance to occur.
Physiochemical agents have been identified that can inactivate chiamydial EBs in their respective hosts by reducing disulfide bonds which maintain the integrity of the outer membrane proteins of the EBs. For Chlamydia, disruption of the outer membrane proteins of EBs thereby initiates the transition of the EB form to the RB form. When this occurs in the acellular milieu where there is no available energy source, the nascent RB perishes or falls victim to the immune system. Thus, disulfide reducing agents that can interfere with this process are suitable as compounds for eliminating EBs.
One such class of disulfide reducing agents are thiol-disulfide exchange agents. Examples of these include, but are not limited to, 2,3-dimercaptosuccinic acid (DMSA; also referred to herein as "succimer"); D,L,-.beta.,.beta.-dimethylcysteine (also known as penicillamine); .beta.-lactam agents (e.g., penicillins, penicillin G, ampicillin and amoxicillin, which produce penicillamine as a degradation product), cycloserine, DTT, mercaptoethylamine (e.g., mesna, cysteiamine, dimercaptol), N-acetylcysteine, tiopronin, and glutathione. A particularly effective extracellular anti-chlamydial agent within this class is DMSA, which is a chelating agent having four ionizable hydrogens and two highly charged carboxyl groups which prevent its relative passage through human cell membranes. DMSA thus remains in the extracellular fluid where it can readily encounter extracellular EBs. The two thiol (sulfhydryl) groups on the succimer molecule (DMSA) are able to reduce disulfide bonds in the MOMP of EBs located in the extracellular milieu. Penicillamine can also be used as a disulfide reducing agent to eliminate chlamydial EBs. The use of penicillamine, however, may cause undesirable side effects. Thus, as an alternative, those .beta.-lactam agents which are metabolized or otherwise converted to penicillamine-like agents in vivo (i.e., these agents possess a reducing group) can be orally administered to the human or animal as a means of providing a controlled release of derivative penicillamine, by non-enzymatic acid hydrolysis of the penicillin, under physiologic conditions. Clavulonic acid is not required for this hydrolysis or for using .beta.-lactam agents to create penicillamine in vivo.
As chlamydial RBs transform into EBs, they begin to utilize active transcription of chlamydial DNA and translation of the resulting mRNA. As such, these forms of Chlamydia are susceptible to currently used antimicrobial agents. The anti-chlamydial effectiveness of these agents can be significantly improved by using them in combination with other agents directed at different stages of Chlamydia life cycle, as discussed herein.
Classes of suitable antimicrobial agents include, but are not limited to, rifamycins (also known as ansamacrolides), quinolones, fluoroquinolones, chloramphenicol, sulfonamides/sulfides, azalides, cycloserine, macrolides, ketolides, and tetracyclines. Examples of these agents which are members of these classes, as well as those which are preferred, are illustrated below in Table 1.
TABLE 1 Drug Class Examples Preferred Quinolones/ Ofloxacin Levofloxacin Fluoroquinolones Levofloxacin Trovafloxacin Sparfloxacin Norfloxacin Lomefloxacin Cinoxacin Enoxacin Nalidixic Acid Fleroxacin Ciprofloxacin Sulfonamides Sulfamethoxazole Sulfamethoxazole/ Trimethoprim Azalides Azithromycin Azithromycin Macrolides Erythromycin Clarithromycin Clarithromycin Lincosamides Lincomycin Clindamycin Clindamycin Tetracyclines Tetracycline Minocycline Doxycycline Minocycline Methacycline Oxytetracyline Rifamycins Rifampin Rifampin (Ansamacrolides) Rifabutin
Members of Chlamydia, including C. pneumoniae, were previously considered to be inhibited, and some killed, by the use of a single agent selected from currently used antimicrobial agents such as those described above. We have found, however, that complete eradication of Chlamydia cannot be achieved by the use of any one of these agents alone, unless the administration is of sufficient length (see below), because none are efficacious against all phases of the Chlamydia life cycle and appear to induce a stringent response in Chlamydia, causing the replicating phase to transform into cryptic forms and resulting in a persistent infection that can be demonstrated by PCR techniques which assess the presence or absence of chlamydial DNA. Nevertheless, one or more of these currently used agents, or another agent directed against the replicating phase of Chlamydia, should be included as one of the chlamydial agents in a combination therapy in order to slow or halt the transition of the EB to the RB as well as to inhibit chlamydial replication.
For the treatment of MS, the combinations of anti-chlamydial agents shown in Table 2 are preferred.
TABLE 2 Combination Drug Class Preferred 1 Rifamycin Rifampin Azalide Azithromycin Macrolide Ketolide Streptogramin 2 Rifamycin Rifampin Ampicillin or Amoxicillin Probenecid 3 Rifamycin Rifampin Azalide Macrolide Ketolide Ampicillin or Amoxicillin Azithromycin Probenecid 4 Rifamycin Rifampin Azalide Azithromycin Macrolide Ketolide Streptogramin Ampicillin or Amoxicillin Probenecid Nitroimidazole Metronidazole 5 Fluoroquinolone Ofloxacin Levoflozacin Rifamycin Rifampin 6 Sulfonamide Sulfamethoxazole/ Trimethoprim Rifamycin Rifampin Isonicotinic congener INH 7 Rifamycin Rifampin Tetracycline Minocycline
To any of the drug combinations, any or all of the following compounds can also be added: probenecid, disulfide reducing agents (e.g., penicillamine), statins (e.g., dantolene), type-1 interferons (e.g., .alpha.-IFN or .beta.-IFN), and activators of iNOS activity.
B) Compounds That Increase iNOS Expression or Activity
Nitric oxide (NO) is a relatively unstable free radical synthesized from L-arginine by inducible nitric oxide synthase (iNOS) and is considered to play a role in containing and/or eradicating intracellular pathogens. NO is implicated in a number of in vitro and in vivo models of host resistance to intracellular pathogens such as Leishmania major, Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis. iNOS may also play a role in inhibiting replication of C. trachomatis in epithelial cells. Moreover, disruption of the iNOS gene in mice leads to dissemination of C. trachomatis-infected macrophages and delays the clearance of C. pneumoniae infections.
We have discovered that heat-killed EBs from C. pneumoniae increase iNOS expression, which, as described above, likely helps eradicate intracellular pathogens. Thus, any compound that increases iNOS activity will likely reduce chlamydial infection and improve or maintain neurological function in patients with MS. iNOS activity may be measured, for example, by measuring NO production, nitrate levels, or the level of iNOS mRNA. Preferably, the increase in iNOS activity is by at least 10%, more preferably by at least 25%, and most preferably by 50%, 100%, or more.
C) Type-1 Interferons
We have discovered that .beta.-IFN increases iNOS activity. Based on these findings, it is likely that any type-1 interferon would also increase iNOS activity and, thus, be useful for the treatment of MS.
In accordance with the present invention, a type-1 interferon may be a purified, naturally-occurring, or recombinant subtype, or it may be a hybrid of two or more subtypes or an analog thereof. Further, mixtures containing any two or more of the above may be used in accordance with the present invention. Many variations of the .alpha.-IFN and/or .beta.-IFN subtypes, hybrids, and/or analogs may be used. Furthermore, in accordance with the present invention, the .alpha.-IFN and/or .beta.-IFN may originate from any mammalian species. Thus, for example, bovine .beta.-IFN subtypes may be used in human therapy.
First, .alpha.-IFN and/or .beta.-IFN subtypes may be used which have a length of 166 amino acid units, and which have at least 60% of the consensus sequence shown in Tables 1 and 2 of U.S. Pat. No. 5,780,021, respectively. The remaining portion of the consensus sequence and any portion of or all of the non-consensus portions of any .alpha.-IFN or .beta.-IFN may be substituted by any other amino acid, whether naturally occurring or not. By the term "non-consensus" portion or "non-consensus" amino acids is meant those amino acids which do not fall within the amino acids which are sequentially common to .alpha.-IFN and/or .beta.-IFNs as shown in Tables 1 and 2 of U.S. Pat. No. 5,780,021. Thus, for example, any .alpha.-IFN subtype from Table 1 and/or any .beta.-IFN from Table 2 may be used as a starting model, and up to 40% of the consensus sequence may be substituted and up to 100% of the non-consensus sequence may be substituted by amino acids, such as, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, hydroxylysine, histidine, arginine, phenylalanine, tyrosine, and tryptophan, or even arnithine or citrulline.
Second, .alpha.-IFN and/or .beta.-IFN subtypes, hybrids, and/or analogs may be used which are fewer than 166 amino acid residues. In accordance with the present invention, the same rules will apply here as with the first variation above, except that the overall sequence length may be abbreviated to at least 70%, preferably at least 80% (132 or 133 units), and more preferably still to at least 90% (149 or 150 units).
Third, the .alpha.-IFN and/or .beta.-IFN subtypes, hybrids, and/or analogs or mixtures thereof may be incorporated as an "active portion" into a larger polypeptide or protein of the formula:
wherein .gamma. is the "active portion" as defined above, and .epsilon. and .omega. each independently represent from 0 to up to about 10,000 amino acids as defined above, with the proviso that the polypeptide or protein has the active portion, .gamma., topologically available at the surface of the polypeptide or protein in the event that it is folded in a three-dimensional structure. The design of such structures, such that a particular portion is available at the surface of the structure is within the skill of one in the art. Further, in reference to type-1 interferons, the term "analog" means any active portion or sequence described herein having at least 60% of the same amino acids in the same sequence as any sequence described in Table 1 or Table 2 of U.S. Pat. No. 5,780,021.
Generally, the term "interferon" refers to a family of proteins that confer non-specific resistance to a broad range of viral infections, affect cell proliferation, and modulate immune responses. Three major interferons, .alpha.-, .beta.- and .gamma.- have been identified based upon antigenic and physico-chemical properties, the nature of the inducer, and the cellular source from which they are derived. .alpha.-IFN and .beta.-IFN (known collectively as type-1 interferons), are structurally related and compete for the same cell surface receptor. .gamma.-IFN, known as type-2 interferon, is structurally unrelated to type-1 IFNs and is acid labile and has a different cell surface receptor.
.alpha.-IFN refers to a family of highly homologous proteins that inhibit viral replication and cellular proliferation and which modulate immune responses. .alpha.-IFN is produced by many cells in the body, including peripheral blood leukocytes or lymphoblastoid cells upon exposure to live or inactivated virus, double-stranded RNA, or bacterial products. Moreover, there are multiple subtypes of .alpha.-IFN which contain 165-166 amino acids and which have molecular weights of about 18,000 to 20,000 daltons. .beta.-IFN is a cytokine having antiviral, antiproliferative, and immunomodulatory activities. Generally, .beta.-IFN is a glycoprotein containing 166 amino acids having a molecular weight of about 20,000 daltons.
The amount of single subtype of .alpha.-IFN or .beta.-IFN, hybrids, analogs or mixtures thereof administered per dose either prior to or after onset of disease is about 1x105 units to about 7.5x107 units with administrations being given from once per day to once per week. Amounts may be used, however, which are less than 1x105 units, such as 5x104 units or lower, or which are more than 7.5x107 units, such as 1x108 units or higher. Of course, the precise amount used will vary, depending upon the judgment of the attending physician, considering such factors as the age, weight, and condition of the patient.
By "consensus sequence" is meant that sequence which is common to all .alpha.-IFN or .beta.-IFN subtypes (see Tables 1 and 2 of U.S. Pat. No. 5,780,021).
Table 1 of U.S. Pat. No. 5,780,021 provides a detailed sequence listing of various .alpha.-IFN subtypes, showing a consensus sequence for all. In accordance with the present invention, any .alpha.-IFN subtype may be used singly or in admixture with others or as hybrids and/or analogs or mixtures thereof as long as it contains at least 60% of the consensus sequence shown in Table 1 as described above or a sequence which exhibits substantially the same .alpha.-IFN activity against autoimmune disease as a sequence having at least that portion of the consensus sequence.
Table 2 of U.S. Pat. No. 5,780,021 provides a comparison of detailed sequence listings for .beta.-IFN of human, murine, and bovine origin. In accordance with the present invention, any .beta.-IFN subtype may be used as long as it contains at least 60% of the consensus sequence shown in Table 2 as described above or a sequence which exhibits substantially the same .beta.-IFN activity against autoimmune disease as a sequence having at least the consensus sequence.
Further, hybrid interferons may be constructed and used. Such hybrid interferons are well known (see, for example, Pestka et al., J. Biol. Chem. 257:11497-11502, 1982).
Modes of Administration
The agents of the present invention can be formulated in a physiologically acceptable vehicle in a form which will be dependent upon the method by which it is administered. In one aspect, the invention pertains to a combination of agents, each of which is targeted against a different phase of the chlamydial life cycle or enhances the anti-chlamydial activity of other agents. The combination of agents can be used in the management of chlamydial infection or prophylaxis thereof to prevent recurrent infection. The combination of agents can be in the form of an admixture, as a kit, or individually, and/or by virtue of the instruction to produce such a combination. It is understood that combination therapy can include multiple agents that are effective within a particular phase of the chlamydial life cycle. The combination of agents can also include immunosuppressants, anti-inflammatory agents, vitamin C, or combinations thereof.
The therapeutic methods described herein can be used to ameliorate or stabilize conditions/symptoms associated with MS, when the disease is caused or aggravated by chlamydial infection. Compounds and agents described herein can be administered to an individual using standard methods and modes which are typically routine for the disease state. While any mammal may be treated, such as dogs, cats, cows, pigs, horses, or poultry, it is particularly desirable that the mammal treated be human.
Combinations of agents of this invention can be used for the manufacture of a medicament for simultaneous, separate, or sequential use in managing chlamydial infection or prophylaxis thereof. The agents can also be used for the manufacture of a medicament for the treatment of MS. The agents can be administered subcutaneously, intravenously, parenterally, intraperitoneally, intradermally, intramuscularly, topically, enteral (e.g., orally), sublingually, rectally, nasally, buccally, vaginally, by inhalation spray, by drug pump or via an implanted reservoir in dosage formulations containing conventional non-toxic, physiologically acceptable carriers or vehicles. The preferred method of administration is by oral delivery. The form in which it is administered (e.g., syrup, elixir, capsule, tablet, solution, foams, emulsion, gel, sol) will depend in part on the route by which it is administered. For example, for mucosal (e.g., oral mucosa, rectal, intestinal mucosa, bronchial mucosa) administration, nose drops, aerosols, inhalants, nebulizers, eye drops, or suppositories can be used. The compounds and agents of this invention can be administered together with other biologically active agents.
In a specific embodiment, it may be desirable to administer the agents of the invention to the brain; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant (e.g., a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes or fibers). When it is desirable to direct the drug to the central nervous system, techniques which can opportunistically open the blood brain barrier for a time adequate to deliver the drug there through can be used. For example, a composition of 5% mannitose and water can be used.
The present invention also provides pharmaceutical compositions. Such compositions include a therapeutically (or prophylactically) effective amount of the agent, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.
Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrolidone. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Agents described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of agents which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions and/or adjunct therapies of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. Agents assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
Claim 1 of 18 Claims
What is claimed is:
1. A method of treating an individual diagnosed to have multiple sclerosis, comprising administering to the individual an effective amount of at least one anti-chlamydial agent and an agent that increases iNOS activity, wherein the individual is administered both agents until the individual tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia.