Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Compositions, methods and kits for diagnosis and treatment of Chlamydia pneumoniae infections of the skin and those associated with cutaneous T-cell lymphoma
United States Patent: 
7,282,343
Issued: 
October 16, 2007

Inventors: 
Abrams; J. Todd (Merion, PA), Balin; Brian J. (Paoli, PA)
Assignee:
 Intracell, LLC (Merion, PA)
Appl. No.: 
10/001,067
Filed: 
October 24, 2001


 

Pharm Bus Intell & Healthcare Studies


Abstract

This invention provides methods of diagnosing and treating any C. pneumoniae infection of the skin including C. pneumoniae-associated diseases such as cutaneous T-cell lymphoma (CTCL), mycosis fungoides, Sezary syndrome, lymphomatoid papillosis, Ki-1 lymphoma, exfoliative exematous rash, and digitate parapsoriasis. This invention provides kits that are useful in the methods of the invention as well as for identifying new anti-chlamydial agents in treating skin infections. This invention includes a pharmaceutical vaccine composition comprising an antigen, including a full length antigenic determinant, such as any SAF positive determinant or portion thereof, which produces a detectable immune, humoral and/or cellular response to C. pneumoniae. The invention also includes a method of treating an active CTCL in a living being comprising delivering a therapeutically effective amount of a vaccine, wherein the vaccine comprises an agent, such as an inactivated C. pneumoniae material that produces a detectable immune, humoral and/or cellular response.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of detecting Chlamydia pneumoniae infection in the skin of a mammal having a cutaneous T-cell lymphoma. The method comprises obtaining a sample from a mammal and performing an identification step in the sample for the presence or the absence of a Chlamydia pneumoniae-derived nucleic acid. The identification step further comprises performing one or more of a polymerase chain reaction and a reverse transcription-polymerase chain reaction.

In another aspect, the invention is a method of detecting Chlamydia pneumoniae infection in the skin of a mammal or a human having a cutaneous T-cell lymphoma. The method comprises obtaining a sample from the mammal and performing an identification step the presence or the absence of a Chlamydia pneumoniae-associated antigen wherein the presence of a Chlamydia pneumoniae-associated antigen in the sample constitutes the detection of a Chlamydia pneumoniae infection in the skin of a mammal having a cutaneous T-cell lymphoma. The identification step further comprises performing an analysis, wherein an antibody is contacted with the sample to determine the presence of a Chlamydia pneumoniae-associated antigen.

In another aspect, the invention is a method of treating a Chlamydia pneumoniae infection in a mammal or a human having a cutaneous T-cell lymphoma. The method comprises administering to said mammal or a human having a cutaneous T-cell lymphoma. The method comprises administering to said mammal one or more agents having anti-chlamydial activity in an amount sufficient to treat the Chlamydia pneumoniae infection of the mammal having said cutaneous T-cell lymphoma. The Chlamydia pneumoniae infection can be in the skin of said mammal. The agent having anti-chlamydial activity is selected from the group consisting of fluoroquinolone, a Macrolide.TM., tetracycline, a tetracycline derivative, penicillin, a penicillin derivative, and psoralen in combination with ultraviolet radiation (PUVA).

In another aspect, the invention is a method of treating an active cutaneous T-cell lymphoma in a mammal or a human having a Chlamydia pneumoniae infection. The method comprises administering to a mammal one or more agents having anti-chlamydial activity in an amount sufficient to inhibit or eradicate the Chlamydia pneumoniae infection, thereby treating said active cutaneous T-cell lymphoma in the mammal. The agent having anti-chlamydia activity is selected from the group consisting of fluoroquinolone, a macrolide, tetracycline, a tetracycline derivative, penicillin, a penicillin derivative, and psoralen in combination with ultraviolet radiation (PUVA). The active cutaneous T-cell lymphoma is selected from the group consisting of mycosis fungoides, Sezary syndrome, lymphomatoid papillosis, Ki-1 lymphoma, exfoliative exematous rash, and digitate parapsoriasis. The Chlamydia pneumoniae infection is selected from the group consisting of a skin infection, a blood infection, and a lymph node infection.

In another aspect, the invention is a method of treating Chlamydia pneumoniae infection in the skin of a mammal, where the mammal is a human. The method comprises administering to the mammal a combination of psoralen and ultraviolet radiation A (PUVA). A method of treating a Chlamydia pneumoniae infection can further include agents selected from a group of non-steroidal anti-inflammatory drugs (NSAIDS). PUVA can be used in combination with at least one agent selected from a group of non-steroidal anti-inflammatory drugs (NSAIDS).

In another aspect, the invention is a method of diagnosing an active cutaneous T-cell lymphoma in a mammal or in a human. The method comprises obtaining a sample from said mammal and identifying in the sample one or more of a Chlamydia pneumoniae nucleic acid, a Chlamydia pneumoniae protein and Sezary T-Cell activating factor. Such identification serves as an indication that the analyzed mammal is a mammal with an active cutaneous T-cell lymphoma. The Chlamydia pneumoniae protein can be selected from the group consisting of a lipopolysaccharide antigen and a major outer membrane protein.

In another aspect, the invention is a method of identifying a Chlamydia pneumoniae associated antigen.

In another aspect, the invention is a method of identifying anti-chlamydial agents. The method comprises obtaining keratinocytes from mammals, wherein the mammal is a human. In the method, the cultured keratinocytes are infected with Chlamydia pneumoniae and then cultured with various concentrations of at least on of anti-chlamydial agents (positive control) and agents with undefined anti-chlamydial activity.

In another aspect, the invention is a method of identifying the location of a chlamydial infection in a mammal, wherein said mammal is a human. In this method, a labeled agent capable of detecting a chlamydial infection is placed within the mammal; and the agent's presence is detected using standard imaging techniques. The agent can also be an anti-Chlamydia pneumoniae-associated antigen monoclonal antibody or any fragment or derivative thereof. The agent can also be a humanized anti-Chlamydia pneumoniae-associated antigen monoclonal antibody.

In another aspect, the invention is a method of preventing Chlamydia pneumoniae infection associated skin disorders comprising CTCL in a living being comprising delivering a vaccine. The vaccine comprises at least one agent selected from the group comprising inactivated Chlamydia pneumoniae materials that produce at least one of detectable immune, humoral and cellular response, and a therapeutically acceptable carrier. The vaccine can further comprise Chlamydia pneumoniae OMP, SAF, and other Chlamydia pneumoniae proteins further comprising HSP-60 and type III secretion system proteins (YOP), DNA isolated from Chlamydia pneumoniae.

In another aspect, the invention is a kit for detecting the presence of a C. pneumoniae protein, obtained from the skin of a living being, comprised of at least one antibody to the Chlamydia pneumoniae protein or a portion thereof either bound to a solid support or labeled with an agent to detect the presence of Chlamydia pneumoniae. The antibodies comprise those reacting with chlamydial-associated outer membrane proteins, SAF, and LPS. Kits for detecting the presence of a C. pneumoniae nucleic acids from skin of a living being or cell culture, further comprise a detectably labeled nucleic acid probe or a PCR primer.

In another aspect, the invention is a pharmaceutical vaccine composition comprising at least one anti-chlamydial agent comprising a full length antigenic determinant, a SAF-positive determinant, or any portion thereof, wherein said agent produces a detectable immune response, humoral and/or cellular to Chlamydia; and a therapeutically acceptable carrier.

In another aspect, the invention is a pharmaceutical at least one agent selected from the group of inactivated Chlamydia pneumoniae materials that produce at least one of detectable immune, humoral and cellular response and a therapeutically acceptable carrier.

In another aspect, the invention is a method of treating an active cutaneous T-cell lymphoma in a living being comprising delivering a therapeutically effective amount of a vaccine. The vaccine comprises at least one agent selected from the group of Chlamydia pneumoniae materials that produce at least one detectable immune, humoral and cellular response, said materials being inactivated and a therapeutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods of detecting and treating a Chlamydia pneumoniae infection in the skin of a mammal, preferably, a human. The invention additionally provides methods of diagnosing and treating a cutaneous T-cell lymphoma, such as mycosis fungoides or Sezary syndrome, in a mammal, such as a human. The invention further provides kits that are useful in identifying new agents for treatment of Chlamydial infection in the skin and provides kits that are useful for other method s described herein.

The present invention stems from the discovery that a protein referred to as Sezary T cell Activating Factor (SAF) is a Chlamydia pneumoniae-associated protein. While it is known that SAF stimulates the malignant Sezary T cells involved in the pathogenesis of Sezary syndrome, a type of cutaneous T cell lymphoma (CTCL), until the present discovery, it was not known that SAF is a C. pneumoniae-associated protein. It has further been discovered herein, that SAF is present in the skin of patients with mycosis fungoides, the predominant form of cutaneous T cell lymphoma, that SAF is associated with C. pneumoniae bacteria in the skin, and that SAF is associated with active CTCL disease states.

Accordingly, the present invention includes diagnostic methods, such as a method of detecting a C. pneumoniae infection in a mammal and a method of diagnosing an active cutaneous T-cell lymphoma in a mammal, therapeutic methods, such as a method of treating a C. pneumoniae infection in a mammal and a method of treating an active cutaneous T-cell lymphoma in a mammal, and analytical methods, such as a method of identifying a C. pneumoniae-associated antigen in a mammal.

Methods

A C. pneumoniae infection that is either identified or treated by a method described herein can be located, for example, substantially in the skin (i.e. cutaneous tissue) of a mammal, preferably, a human.

The methods described herein require at least one analysis to be performed on a sample either from a mammal or from a population of cultured cells. A sample from a mammal, that is useful in the methods described herein, includes substantially any type of biological sample obtained from the mammal, preferably, a skin sample, a blood sample, and a lymph node sample. The term "biological sample" is further intended to include tissues, cells and biological fluids isolated or otherwise derived from a mammal, as well as tissues, cells and fluids present within a mammal. That is, the diagnostic, therapeutic, and analytical methods described herein can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. By way of example, a useful biological sample can contain one or more of protein molecules, mRNA molecules, and genomic DNA molecules obtained either from a mammal or from mammalian cells in culture. A preferred biological sample comprises whole cells isolated by conventional means either from a mammal or from mammalian cells in culture.

Several methods described herein include detecting the presence or absence of a C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid in a biological sample obtained from the mammal. These methods can comprise obtaining a biological sample from mammal and contacting the biological sample with a compound or an agent capable of detecting a C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid (e.g., mRNA, genomic DNA) such that the presence of a C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid is detected in the biological sample.

In other embodiments, the methods can further comprise obtaining a control biological sample from a control mammal or from a population of cultured cells that do not have either a C. pneumoniae infection or a C. pneumoniae-associated disorder, such as a CTCL. In these methods, the control sample is contacted with a compound or agent capable of detecting a C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid, such that the presence, or lack thereof, of the C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid is detected in the biological sample. Also in these methods, the level of a C. pneumoniae protein, a C. pneumoniae-associated antigen, or a C. pneumoniae nucleic acid in the control sample is compared with the level of the same C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid in the sample from the mammal to be tested.

Methods described herein that require the detection of nucleic acids in either a sample obtained from a mammal (e.g., a human patient sample) or cultured cells, can include, for example, the steps of obtaining a sample comprising cells from a mammal, isolating a nucleic acid (e.g., DNA, RNA, or both) from the cells of the sample, and contacting the nucleic acid sample with one or more primers that specifically hybridize to a C. pneumoniae nucleic acid or a gene encoding a Chlamydia pneumoniae-associated antigen, under conditions such that hybridization and amplification of the gene (if present) occurs. These methods also comprise either detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the size of the amplification product to the size of the amplification product obtained with a control sample.

Methods for detecting nucleic acids using a PCR or an RT-PCR are well known in the art and are provided in standard references (e.g., Innis et al., ed., 1990, In: PCR Protocols, Academic Press, Inc., San Diego). PCR or RT-PCR may be used as a preliminary amplification step in conjunction with any other well-known techniques used for detecting nucleic acids in a sample that are described herein.

Alternative nucleic acid amplification-detection procedures that may be useful in the methods of the invention include, but are not limited to: self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874 1878), transcriptional amplification system (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173 1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. Other well-known techniques for the detection of nucleic acids, such as Northern hybridizations, in situ hybridizations, and Southern hybridizations, can also be used in the methods of the invention.

A preferred agent for detecting mRNA or genomic DNA comprising a portion of a C. pneumoniae gene sequence or a C. pneumoniae-associated antigen gene sequence is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA comprising a portion of a C. pneumoniae gene sequence or a C. pneumoniae-associated antigen gene sequence. The nucleic acid probe can be complementary to or homologous with a portion of a C. pneumoniae gene sequence or a gene sequence encoding a Chlamydia pneumoniae-associated antigen, and comprising a nucleotide sequence of at least 15, 30, 50, 100, 250 or 500 contiguous nucleotides that is sufficient to specifically hybridize under stringent conditions to a mRNA or DNA encoding a C. pneumoniae protein or a Chlamydia pneumoniae-associated antigen. Preferred probes would be specific for but not limited to MOMP and 16s rRNA genes that will comprise the nucleotide sequences for detection of the presence of C. pneumoniae bacteria.

Methods described herein that include the detection of either a C. pneumoniae protein, such as the major outer membrane protein (MOMP) or the C. pneumoniae lipopolysaccharide (LPS), or a C. pneumoniae-associated antigen, such as SAF, can include substantially any compatible protein detection methods known in the art. Examples of useful protein detection methods include, but are not limited to, in vitro techniques, such as enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence, and, in vivo techniques, such as introducing into a subject a labeled antibody directed against either a C. pneumoniae protein or a C. pneumoniae-associated antigen. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

A preferred agent for detecting either a C. pneumoniae protein or a C. pneumoniae-associated antigen is an antibody capable of binding to either a C. pneumoniae protein or a C. pneumoniae-associated antigen, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. Preferable antibodies would be reactive with either C. pneumoniae heat shock protein 60, C. pneumoniae major outer membrane proteins, SAF, C. pneumoniae lipopolysaccharide, or to Chlamydia type three secretion molecules (YOP). An intact antibody, or a fragment thereof (e.g., Fab or F(ab').sub.2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

Analytical Methods

The invention further includes a method of identifying a C. pneumoniae-associated antigen. This method comprises obtaining a sample from a mammal and contacting the sample with one or more of an isolated nucleic acid having a nucleotide sequence that is homologous with or complementary to a nucleotide sequence derived from C. pneumoniae; an isolated nucleic acid having a nucleotide sequence that is homologous with or complementary to the nucleotide sequence encoding a suspected antigen; and an identifying agent that specifically binds to, adsorbs onto, or chemically reacts with one or more of an isolated nucleic acid that is homologous with or complementary to either of C. pneumoniae and the suspected antigen, and an amino acid sequence of either of C. pneumoniae and the suspected antigen. This method is illustrated herein in the Examples, and further comprises analyzing the sample to determine the presence or absence of a nucleic acid or amino acid sequence associated with C. pneumoniae and the presence or absence of a nucleic acid or amino acid sequence associated with the suspected antigen. Preferred nucleotide primers include but are not limited to those that amplify C. pneumoniae gene sequences as listed in Table 1 (see Original Patent). In addition, the method comprises determining the extent to that the presence of C. pneumoniae is associated with the presence of the suspected antigen; whereby determining a significant association between the presence of C. pneumoniae and the presence of the suspected antigen is an indication that the suspected antigen is a C. pneumoniae associated antigen.

The method of identifying a C. pneumoniae associated antigen can alternatively comprise obtaining a sample from a mammal and contacting the sample with one or more of an antibody specific for a C. pneumoniae protein, such as SAF, or an antibody specific for the suspected antigen that is not a C. pneumoniae protein, and an identifying agent that is capable of specifically binding to, adsorbing onto, or chemically reacting with an amino acid sequence of either a C. pneumoniae protein or the suspected antigen. This alternative method is illustrated herein in the Example, and can further comprise analyzing the sample to determine the presence of an amino acid sequence associated with either a C. pneumoniae or a suspected antigen, and determining the extent to which the presence of C. pneumoniae coincides with the presence of the suspected antigen. According to this method, determining a significant coincidence between the presence of C. pneumoniae and the presence of the suspected antigen is an indication that the suspected antigen is a C. pneumoniae-associated antigen.

One skilled in the art will understand that a C. pneumoniae-associated antigen identified as described above that is associated with or that becomes associated with a disorder, may be useful in the diagnostic methods, treatment methods, and reagent screening methods described herein. By way of example, a C. pneumoniae-associated antigen identified as above, that is found to be associated with or involved in the pathology of a skin disease in a mammal, may be used according to a method described herein to diagnose the associated skin disease in the mammal, and the methods described herein for treating a C. pneumoniae infection in a mammal may further be useful in treating the associated skin disease in the mammal.

Diagnostic Methods

The present invention includes a method of detecting C. pneumoniae infection in the skin of a mammal such as in a mammal having a cutaneous T-cell lymphoma. This method can include subjecting a sample from the mammal to one or more of a PCR and an RT-PCR. The amplification of a C. pneumoniae nucleic acid (e.g. RNA or DNA) in a PCR or RT-PCR comprising the sample from the mammal constitutes the detection of C. pneumoniae infection in the mammal.

Alternatively, the preceding method can comprise contacting the sample from the mammal with an antibody that is specific for either a C. pneumoniae protein or a C. pneumoniae-associated antigen, such as SAF. According to this method, the presence of either a C. pneumoniae or a C. pneumoniae-associated antigen in the sample, is an indication that a C. pneumoniae bacterium is present in the sample, and thus, constitutes the detection of C. pneumoniae infection in the mammal.

One skilled in the art would appreciate, based upon the disclosure herein, that the presence a C. pneumoniae protein or a C. pneumoniae-associated antigen can be assessed in keratinocytes. Thus, the skilled artisan would appreciate that the invention encompasses detection of these proteins and/or antigens in regions of the skin, including but not limited to, stratum spinosum, the stratum granulosum, the stratum germinativum, as well as in histiocytes, and Langerhans dendritic cells, and the like.

According to the above methods, either the identification of a C. pneumoniae nucleic acid in a sample from the mammal or the determination that either a C. pneumoniae protein or a C. pneumoniae-associated antigen is present in the sample constitutes the detection of a C. pneumoniae infection in the skin of a mammal. In this application, the sample that is obtained from the mammal for analysis is a skin (i.e. cutaneous tissue) sample.

In another aspect, the invention includes a method of diagnosing an active cutaneous T-cell lymphoma, such as mycosis fungoides or Sezary syndrome, in a mammal. This method comprises obtaining a sample from the mammal and identifying in the sample one or more of a C. pneumoniae nucleic acid, a C. pneumoniae protein, and SAF. According to this method, an identification in the sample of a C. pneumoniae nucleic acid, a C. pneumoniae protein, or SAF is an indication that the mammal has an active cutaneous T-cell lymphoma. By way of example, this method encompasses diagnosing an active CTCL in a human by identifying in a skin sample from the human a C. pneumoniae nucleic acid using PCR or RT-PCR, or identifying in the skin sample either a C. pneumoniae protein, such as a lipopolysaccharide antigen or a major outer membrane protein, or a C. pneumoniae-associated antigen, such as SAF, using an ELISA.

Therapeutic Methods

Encompassed by the present invention is a method of treating a C. pneumoniae infection in a mammal having a cutaneous T-cell lymphoma. This method comprises administering to a mammal one or more agents having anti-chlamydial activity in an amount sufficient to treat the C. pneumoniae infection. Preferably, the infection is located substantially in the skin of the mammal.

The invention additionally includes a method of treating an active cutaneous T-cell lymphoma (CTCL) in a mammal having a C. pneumoniae infection. In this method, one or more agents having anti-chlamydial activity are administered to the mammal in an amount sufficient to inhibit the C. pneumoniae infection, and thereby treat the active CTCL in the mammal.

For the treatment methods described herein, a CTCL can include, without limitation, mycosis fungoides, Sezary syndrome, lymphomatoid papillosis, Ki-1 lymphoma, exfoliative exematous rash, and digitate parapsoriasis, and the like.

The skilled artisan would appreciate, based upon the disclosure provided herein, that the treatment methods described herein could be applied to treating disorders in addition to CTCL that are associated with C. pneumoniae infection in the skin.

In further regard to the treatment methods of the invention, an agent having anti-chlamydial activity can be any agent that is known or becomes known, that is useful in the methods of the invention. Examples of anti-chlamydial agents that can be used in the methods of the invention include, but are not limited to, fluoroquinolone, macrolides, tetracycline derivatives, and/or the elimination of the active forms of Chlamydia using penicillins and derivatives thereof, as well as psoralen in combination with ultraviolet radiation (PUVA).

The skilled artisan would appreciate, based upon the disclosure provided herein, that the anti-chlamydial agent encompasses a vaccine. That is, a "vaccine," as the term is used herein, means any antigen, including but not limited to, a full length antigenic determinant, including any SAF positive determinant, or any portion thereof, that produces a detectable immune response, humoral and/or cellular, to Chlamydia when administered to a mammal, preferably a human, compared with the immune response in an otherwise identical mammal to that said antigenic determinant is not administered.

The skilled artisan would be able, armed with the teachings of this invention, to readily prepare such vaccines comprising whole or fractionated determinants. Further, useful fractionated determinants that are SAF positive can be readily identified and isolated using the methods disclosed herein or those well-known in the art. For example such a vaccine could be produced by preparing purified bacterial bodies from infected cells cultured through centrifugation, followed by ultraviolet inactivation, and followed by heating to a boil. The preparation would then be sonicated until completely dispersed, after which specific proteins, such as SAF positive determinants, may be purified. Preparations of whole or purified bacterial lysate would be injected in physiologic solution in the presence or absence of rIL-12. Injections would be repeated until a titer of greater than 1:512 is obtained to be considered immunized.

An anti-chlamydial agent (also referred to herein as an "active compound") of the invention can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise the anti-chlamydial agent and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.

It is understood that appropriate doses of anti-chlamydial agents depend upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject (i.e., mammal) or sample being treated, further depending upon the route by that the composition is to be administered, if applicable, and the effect that the practitioner desires the agent to have upon the nucleic acid or polypeptide of the invention. Exemplary doses of an anti-chlamydial agent include milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). It is furthermore understood that appropriate doses of one of the anti-chlamydial agents depends upon the potency of the agent with respect to the infection to be treated. When one or more of these agents is to be administered to a mammal (e.g. a human) in order to treat an infection according to a method of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular mammalian subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of the infection to be treated.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

In view of the number of well-characterized anti-chlamydial agents, the determination of pharmaceutical parameters that are necessary to perform the treatment methods of the invention, such as sufficiently bactericidal dosages and appropriate routes of administration, are considered to be well within the ability of a skilled artisan such as a physician, veterinarian, or researcher.

Kits

The invention encompasses kits for detecting the presence of a C. pneumoniae protein, a C. pneumoniae-associated antigen, or a C. pneumoniae nucleic acid in a biological sample (i.e., a test sample) that is either obtained from a mammal or from cells in culture. The kits described herein can be used to determine if a mammal is infected with C. pneumoniae or if a mammal is suffering from a form of CTCL, such as mycosis fungoides or Sezary syndrome. Alternatively, the kits of the invention can be used to treat a C. pneumoniae infection or a CTCL. For example, the kit can comprise a labeled compound or identifying agent capable of detecting a C. pneumoniae protein, a C. pneumoniae-associated antigen, or a C. pneumoniae nucleic acid in a biological sample and means for determining the amount of the C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae-associated antigen, or a nucleic acid probe that binds to a nucleic acid encoding a C. pneumoniae protein or a C. pneumoniae-associated antigen.

Alternatively, the kits of the invention can be used to screen agents to determine their level of anti-chlamydial activity for skin and or keratinocyte infection and efficacy in treating a CTCL. For example a kit can comprise various known (control) and test agents, and any agents developed in the future for which anti-chlamydial activity for skin infection is to be determined. For an in vitro screening kit, the kit can comprise cultured human keratinocytes grown under appropriate conditions for testing agents, such as under serum free conditions. In addition, the kit could also be comprised of agents for detecting a C. pneumoniae protein, a C. pneumoniae-associated antigen, or a C. pneumoniae nucleic acid in a biological sample and means for determining the amount of the C. pneumoniae protein, C. pneumoniae-associated antigen, or C. pneumoniae nucleic acid (e.g., an antibody that binds a C. pneumoniae protein or a C. pneumoniae-associated antigen, or a nucleic acid probe that binds to a nucleic acid encoding a C. pneumoniae protein or C. pneumoniae-associated antigen in order to determine anti-chlamydial activity of the test agent. The instructional material included in a kit of the invention can be a publication, a recording, a diagram, or any other medium of expression that can be used to communicate one or more of the steps of any analytical, diagnostic, or therapeutic method included in the present invention. The instructional material can also include any medium of expression that is used to communicate the parameters within that to perform the steps of a method of the invention, and troubleshooting indications and precautions as would be obvious to one skilled in the art after reading the present disclosure. The instructional material of a kit included in the invention may comprise a plurality of materials that can be provided with one or more components of the kit. By way of example, a kit that is useful for diagnosing a form of CTCL in human patient may include an instructional material that outlines the method of diagnosing a CTCL in a mammal as described herein. Kits described herein can also include instructions for observing that a mammal to be tested is suffering from or is at risk of developing a disorder associated with aberrant expression fo a C. pneumoniae-associated antigen if the amount of the C. pneumoniae-associated antigen or mRNA encoding the C. pneumoniae-associated antigen is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to either a C. pneumoniae protein or a C. pneumoniae-associated antigen; and, optionally, (2) a second, different antibody that binds to either a C. pneumoniae protein, a C. pneumoniae-associated antigen, or the first antibody, and is conjugated to a detectable agent.

Anti-C. pneumoniae protein antibodies include those reacting with a major outer membrane protein. An antibody reactive with a C. pneumoniae-associated antigen would include those reactive with SAF, which as described herein would be an antibody binding to a 28 to 30 kDa protein that co-localizes with Chlamydial outer membrane protein (OMP) and lipopolysaccharide (LPS) in infected tissue and cell cultures and reacts with bacterial bodies as detected by electron microscopy.

For nucleic acid-based kits, the kit can comprise, for example: (1) a nucleic acid probe, e.g., a detectably labeled nucleic acid probe, that hybridizes to one of a C. pneumoniae nucleic acid sequence and portion of a gene sequence encoding a C. pneumoniae nucleic acid sequence and a portion of a gene sequence encoding C. pneumoniae-associated antigen. The kit can also comprise, e.g., a buffering agent, a preservative, a protein stabilizing agent, or components necessary for detecting the detectable (i.e. identifying) agent, such as an enzyme or a substrate. The kit can further comprise a control sample or a series of control samples that can be tested and compared to a test sample obtained from a mammal or from a cultured cell population. Each component of a kit may be enclosed within an individual container and all of the various containers may be within a single package, or may optionally be shipped separately.

A detectable labeled nucleic acid probe would be an oligonucleotide or PCR product generated using primers described herein, that is labeled with digoxigenin or with a fluorophor, such as fluoroscein isothiocyanate (FITC), well known to one skilled in the art.

The invention also includes a kit comprising an instructional material that outlines, describes, or illustrates a method of the present invention, and a pharmaceutical composition comprising an anti-chlamydial agent along with a delivery device for delivering the composition to a subject. By way of example, the delivery device may be a squeezable spray bottle, a metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, a self-propelling solvent or powder-dispensing device, a syringe, a needle, or a dosage measuring container.

Each of the components of a kit described herein can be promoted, distributed, or sold either as a unit or as separate components to be used cooperatively. By way of example, the instructional material can be affixed to a container that contains one or more analytical, diagnostic, or therapeutic components useful in the methods described herein, or the instructional material can be shipped together with one or more containers that individually contain one or more of these components. By way of alternative example, the instructional material may be shipped separately from the method components with the intention that the instructional material and the components be used cooperatively by the recipient.

A kit provided by the invention can be useful for performing the methods of the invention, such as detecting and treating a C. pneumoniae infection in a mammal, such as a human, and diagnosing and treating a cutaneous T-cell lymphoma, such as mycosis fungoides or Sezary syndrome, in a mammal, such as a human or screening news agents for the treatment of C. pneumoniae infection in the skin of a mammal, such as a human.

 

Claim 1 of 12 Claims

1. A method of treating a Chlamydia pneumoniae infection in skin of a mammal having a cutaneous T-cell lymphoma, said method comprising: administering to said mammal, in amounts sufficient to treat said Chlamydia pneumoniae infection of said mammal having said cutaneous T-cell lymphoma, psoralen ultraviolet A (PUVA) in combination with at least one agent selected from the group consisting of fluoroquinolone, a macrolide, tetracycline, a tetracycline derivative, penicillin, and a penicillin derivative; and examining said mammal during and/or after said administering to monitor treatment of said Chlamydia pneumoniae infection.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]