Patent Application
20090305975
1. Field of the Invention
The present invention belongs to the biological pharmacy field. Specifically, the present invention relates to the use of TRAP protein per se as an active ingredient for the manufacture of a medicament for the treatment of Staphylococcus aureus infection.
2. Description of the Relevant Art
Staphylococcus aureus belongs to Staphylococcus and is a major pathogenic bacterium that causes severe infections of pneumonia, endocarditis, burn and war wound, septicemia, and toxic shock, etc. In addition, Staphylococcus aureus is one kind of bacterium that causes food-poisoning. Staphylococcus aureus widely exists in air, soil, water and feeding utensils, and animal and human beings are main vectors. Staphylococcus aureus can be transmitted through spit and contact on a large scale in anthills. There are more than millions of patients suffering with Staphylococcus aureus infections only in hospital per year. Staphylococcus aureus is a major pathogenic bacterium that causes Gram-positive sepsis, infections of burn wound and acute hepatic failure.
The main pathogenic substances of Staphylococcus aureus are exotoxins, including hematoxin, lueocidin, enterotoxin, etc. The recent researches indicate that the synthesis of these virulence factors in Staphylococcus aureus is controlled by a regulatable RNA molecule, i.e., RNA III. RNAIII activates the gene transcription of virulence factors and regulates the translation of virulence factors by base complementarity. The RNAIII level is relatively lower during the early log phase of bacterial growth, but increases 40 times during the late log phase of bacterial growth, while the level of RNA m is regulated by the protein RAP (RNA III activating protein) secreted by Staphylococcus aureus per se, so that RAP is also called as Staphylococcus aureus virulence stimulating factor. Staphylococcus aureus continuously secretes RAP, and RAP activates the production of virulenece factors only when RAP reaches a certain concentration. Staphylococcus aureus without the production of RAP does not cause diseases (Balaban N. et al, Science 1995, 280, 438-440). The recent researches show that RAP activates the transcription of RNA III through the mediation of a 21KD protein TRAP (Target of RNA III-activating protein). When the gene encoding TRAP is inactivated by mutation, RAP cannot activate the transcription of RNAIII.
TRAP consists of 167 amino acids and has His kinase activity. TRAP is phosphorylated during the early phase of the growth of Staphylococcus aureus, and reaches the maximum level during the middle log growth phase. After the RAP action, the signal transduction is performed by autophosphorylation, thereby increasing the intracellular level of RNAIII and accelerating the secretion of Staphylococcus aureus exotoxins (Namomi B. et al, J. Biol. Chem., 2001, 276: 2658-2667). TRAP is a surface protein of Staphylococcus aureus and is highly conserved in Staphylococcus. The main biological function of TRAP is to participate in the regulation of Staphylococcus aureus exotoxins. The level of Staphylococcus aureus exotoxins is up-regulated with the increase of phosphorylation level of TRAP (Gov Y et al, J. Biol. Chem., 2004, 279: 14665-72).
Based on sequence alignment analysis, it has been found at present that TRAP proteins can be divided into three families and they have a homology more than 80% between each other (Yang G. et al, J. Biol. Chem., 2005, 280: 27431-27435). Specifically, it has been found in accordance with the sequence alignment, TRAP proteins are highly conserved in Staphylococcus aureus and Staphylococcus epidermidis. At present, there are TRAP sequences of 10 Staphylococcus aureus strains and 2 Staphylococcus epidermidis strains logged in Gene Bank.
TRAP proteins are divided into following 3 families on the basis of sequence conservation: Family 1 includes 7 TRPA proteins from Staphylococcus aureus which have an identity of 99.49% based on the analysis by DNAMAN (
The recent researches show that the pathogenic effect of Staphylococcus aureus can be effectively reduced by inhibiting the pathways for the production of Staphylococcus aureus exotoxins by using exogenous RAP and TRAP inhibitors. The known RAP and TRAP inhibitors such as small molecular inhibiting peptides (RNAS III inhibiting peptide, hereafter called RIP) and antibodies can effectively reduce the secretion of Staphylococcus aureus exotoxins. The C-terminal of TRAP can be used as an effective peptide vaccine to protect organisms from the Staphylococcus aureus infections (Oleny V. et al, Peptides, 2001, 22: 1621-1627; Yang G. et al, Peptides, 2003, 24, 1823-1828; Yang G. et al, J. Biol. Chem., 2005, 280: 27431-27435).
At present, the Staphylococcus aureus infections are mainly treated by inhibiting the pathways for the production of Staphylococcus aureus toxins described above to reduce the production of Staphylococcus aureus toxin, such as via RIP, RAP antibodies and the like, thereby reducing the pathogenic effect of Staphylococcus aureus.
The DNA sequence and amino acid sequence of a target of RNAIII-activating protein (TRAP) from Staphylococcus aureus RN6390B is known in the art (U.S. Pat. No. 6,689,878). In addition, it has been reported that said TRAP is used to induce the production of an antibody and said antibody can be used as a medicament (U.S. Pat. No. 6,747,129), wherein function of TRAP is inhibited by monoclonal or polyclonal antibodies against TRAP to inhibit the occurrence of Staphylococcus aureus infections.
On the one hand, the drug resistance of pathogenic bacteria including Staphylococcus aureus is continuously generated and increased due to the abuse of antibiotics, and many antibiotics are ineffective to the drug resistant strains, including Methicillin resistant S. aureus (MRSA) that is resistant to β-lactam antibiotics, glycolpeptide resistant S. aureus (GISA) that is resistant to glycolpeptide antibiotics, and vancomycin resistant S. aureus (VRSA), etc. On the other hand, Staphylococcus aureus per se readily develop drug resistance.
To solve the above problems existing in the treatment of Staphylococcus aureus infections at present, it is urgent to develop a method that can effectively control the Staphylococcus aureus infections but cannot make Staphylococcus aureus readily develops drug resistance.
One embodiment is directed to the use of TRAP per se as an active ingredient for the manufacture of a medicament for the treatment of Staphylococcus aureus infection.
Specifically, it is surprisingly found, from an in vitro test (MDBK cell model) and in vivo test (animal model of Staphylococcus aureus infection), that an exogenous TRAP per se can effectively inhibit the production of Staphylococcus aureus exotoxins in the cell model and animal model. The exogenous TRAP per se can be used as an active ingredient to treat Staphylococcus aureus infections and reduce the pathopoiesis, and its therapeutic effect is similar to that of polyclonal antibodies against TRAP.
In the present disclosure, the term “exogenous TRAP” refers to the TRAP proteins added or administrated artificially for the treatment of Staphylococcus aureus infections with respect to endogenous TRAPs produced in said cell or animal model of Staphylococcus aureus infection during the production of Staphylococcus aureus exotoxins after Staphylococcus aureus infection. In the present disclosure, as to an isolated TRAP that is extracted by manual work from Staphylococcus aureus or recombinantly expressed by genetic engineering, whether it directly comes from a specific Staphylococcus aureus strain causing Staphylococcus aureus infections or a strain having a high homology thereto, if only said isolated TRPA is additionally added or administrated for the treatment of the Staphylococcus aureus infection, it is recognized as an exogenous TRAP.
Specifically, in one embodiment, MDBK cell model is employed, and an exogenous TRAP is or is not added to the MDBK cell culture liquid at the time when Staphylococcus aureus is added to said liquid. The results show that the production of Staphylococcus aureus exotoxins can be effectively inhibited by the addition of an exogenous TRAP. In another embodiment, after Staphylococcus aureus is peritoneally injected to mice, then an exogenous TRAP protein is or is not injected, and the survival time and status of each group of mice are observed. The results show that the Staphylococcus aureus infection in mice is reduced by the injection of an exogenous TRAP, thereby increasing the survival rate of mice.
In some embodiments, said TRAP proteins include native TRAP proteins from different kinds of Staphylococcus aureus, recombinant TRAP proteins or fusion TRAP proteins prepared by such as a gene engineering method, and TRAP proteins and/or chemically modified TRAP protein derivatives obtained by such as a chemical synthesis method. The TRAP proteins also include biologically active fragments derived from an intact TRAP protein which retain fully or partially biological active. Said fragments of TRAP proteins can also be used to treat Staphylococcus aureus infections.
U.S. Pat. No. 6,689,878 and U.S. Pat. No. 6,747,129 are incorporated herein by reference in their entirety, wherein the amino acid sequences of TRAP proteins and the polynucleotide sequences encoding them are disclosed in these documents.
In some embodiments, said TRAP proteins refer to the proteins that have a sequence identity equivalent to SEQ ID NO: 2 or SEQ ID NO: 4, or the orthologs thereof, including proteins that have a sequence identity of at least 70%, at least 80%, at least 95%, at least 98% or 100% thereto. Some embodiments include TRAP proteins that have a sequence identity of at least 70%, at least 80%, at least 90%, at least 95% or more then 95% to the amino acid residues from 1 to 167, 30 to 167 or from 50 to 167 of the sequence as shown in SEQ ID NO: 2 or 4.
In one embodiment, said TRAP protein is selected from the sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4, and the TRAP proteins from 12 different origins belonging to 3 different protein families as shown in
The identity percentage between two amino acid sequences can be determined by such as Needelman-Wunsch alignment which can be used in the alignment of protein and DNA. As to the protein alignment, the default scoring matrix used is BLOSUM50 in which the penalty of one residue in spacer is −12, while that of other residues is −2, and the alignment can be carried out by FASTA package, an alignment software of Version v20u6 (W. R. Pearson and D. J. Lipman, “Improved tools for Biological Sequence Analysis”, PNAS, 2005, 85: 2444-2448; and W. R. Pearson, “Rapid and Sensitive Sequence Comparison using FASTP and FASTA”, Methods in Enzymology, 1990, 183: 63-98).
The TRAP proteins may be selected from any known TRAP proteins. With respect to the structure and origin, said TRAP proteins may be the same as or different from the endogenous TRAP proteins produced by Staphylococcus aureus that causes Staphylococcus aureus infection to be treated. The inventors discover that the TRAP proteins from multiple kinds of different Staphylococcus aureus strains have the same or similar effects on the inhibition of the production of Staphylococcus aureus exotoxins and the reduction of pathogenicity of Staphylococcus aureus.
In one embodiment, the TRAP protein as shown in SEQ ID NO: 2 from Staphylococcus aureus RN6390B (which corresponds to the nucleotide sequence AF202641 in GenBank™) is employed as an active ingredient to effectively inhibit the production of Staphylococcus aureus exotoxins in an in vitro test and in vivo test. In another embodiment, the TRAP protein as shown in SEQ ID NO: 4 from Staphylococcus aureus 04018 (which corresponds to the nucleotide sequence AY248703 in GenBank™) as an active ingredient to inhibit the production of Staphylococcus aureus exotoxins in an in vitro test and in vivo test, thereby treating Staphylococcus aureus infections. In another embodiment, a fusion protein consisting of TRAP protein and a polypeptide of 12 amino acids is used to inhibit the production of Staphylococcus aureus exotoxins in an in vitro test and in vivo test, thereby treating Staphylococcus aureus infections.
Another embodiment relates to a pharmaceutical composition including an above said TRAP protein as an active ingredient and an optionally pharmaceutically acceptable carrier or excipient. The TRAP proteins or fragments thereof for the treatment of Staphylococcus aureus infections may be administrated alone in a single dose or multiple doses, or is administrated in combination with a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition including a TRAP protein or an active fragment thereof for the treatment of Staphylococcus aureus infections may be formulated together with a pharmaceutically acceptable carrier or diluent and any known adjuvant or excipient by the traditional techniques as disclosed in the book (Remongton, Science and Practice of Pharmacology, 19th edition, Genearo, Ed., Mack Piblishing Co., Easton, Pa., 1995), and is in a traditional form such as solution, suspension, freeze-dried powder, capsule, tablet or aerosol, etc.
The pharmaceutical composition can be specifically formulated to be administrated in any suitable route, such as parenteral (which includes subcutaneous, intramuscular, intrathecal, intravenous and intradermal), oral, rectal, nasal, pulmonary, topical (which includes buccal and subglossal), cutaneous, intracisternal, endoperitoneal and vaginal routes. It should be understood that the preferred route depends on the general condition and age of the subject who accepts the treatment, the kind of symptom to be treated, and the active ingredient used. The administration route may be any one of routes, if only the active ingredient can be effectively transported to suitable or expected action sites by such route.
The pharmaceutical composition for parenteral administration includes sterile aqueous or non-aqueous injectable solutions, dispersions, suspensions or emulsions, and sterile powders or freeze-dried powders, which are reconstructed in a sterile solution for injection or dispersion before they are used. Storage formulations for injection are also contemplated.
The pharmaceutical composition for oral administration includes solid dosage form, such as hard or soft capsules, tablets, troches, sugar-coated pills, pills, lozenges, powders or granules. If suitable, they can be provided with a coating, such as enteric coating, or formulated by any the known techniques in the art to control the release of active ingredient, such as sustained release or prolonged release.
The liquid dosage form for oral administration includes solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.
Other suitable dosage forms including suppositories, aerosols, ointments, creams, gelatins, inhalants, bandages for skin, and implants, etc.
The typical administration dosage is in a range from about 0.001 to about 100 mg per kg weight per day, such as from about 0.01 to about 50 mg per kg weight per day such as from about 0.05 to 10 mg per kg weight are administrated per day, in one or more doses such as 1 to 3 doses. The exact dosage depends on the status of Staphylococcus aureus infections to be treated, administration frequency and way, the gender, age and general condition of an object who accepts the treatment, the kind and severity of the symptom to be treated, any concomitant disease to be treated and other factors known by a person skilled in the art in the art.
The formulation may expediently exhibit a unit-dose form by a method known in the art. The formulation for parenteral administration, such as intrathecal, intravenous and intramuscular administration and the like is administrated at one or more times a day (such as once to trice a day) in a typical dosage ranging from about 0.001 to about 1000 mg, for example, from about 0.01 to about 500 mg such as from about 0.1 mg to about 100 mg.
The formulation for oral administration is administrated at one or more times a day (such as once to trice a day) in a typical dosage ranging from about 0.05 to about 1000 mg, for example, from about 0.1 to about 500 mg such as from about 0.5 mg to about 200 mg.
The suitable pharmaceutically acceptable carrier includes inert solid diluents or fillers, sterile aqueous solution and multiple organic solvents. For example, the solid carriers include lactose, gypsum powder, sucrose, cyclodextrin, talcum powder, gelatin, agar, pectin, gum Arabic, magnesium stearate, stearic acid and cellulose lower alkyl ether. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acid, fatty acid amide, polyoxyethylene and water. Similarly, carriers or diluents may include any sustained release substance known in the art, such as monostearin or distearin, which is used alone or in combination with a wax. The pharmaceutical composition, which is obtained by the combination of a TRAP protein or a fragment thereof with a pharmaceutically acceptable carrier and used for the treatment of Staphylococcus aureus infections, may be expediently administrated by any disclosed administration routes in a multiple-dose form. The formulation may expediently exhibit a unit-dose form by a method known in the art.
The aqueous suspensions may include a TRAP protein or a fragment thereof which is mixed with an excipient suitable for the production of an aqueous suspension. The excipient is a suspending agent, such as carboxymethyl cellulose sodium, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, bassora gum and gum Arabic; dispersing or wetting agent may be native phospholipids, such as lecithin, or a condensation product of an alkylene oxide and a fatty acid, such as polyoxyethylene stearate, or a condensation product of ethylene oxide and a long chain aliphatic alcohol such as heptadecanylethyleneoxyhexadecanol, or a condensation product of ethylene oxide and a partial ester derived from fatty acid and hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of ethylene oxide and a partial ester derived from fatty acid and hexitol glycoside, such as polyoxyethylene sorbitan monooleate. The aqueous suspension may further comprise one or more toners, one or more flavoring agents and one or more sweetening agents such as sucrose or glucide.
The oily suspension may be prepared by dispersing an active ingredient to vegetable oil (such as peanut oil, olive oil, sesame oil or cocoanut oil) or a mineral oil (such as liquid paraffin). The oily suspension may include a thickening agent such as beeswax, hard paraffin or spermol. The above sweetening agents and flavoring agents may be added to obtain a palatable oral formulation. In addition, an antioxidant (such as ascorbic acid) may be added to the compositions for preservation.
The dispersible powders or granules suitable for preparing an aqueous suspension by adding water are obtained by mixing an active ingredient, dispersing or wetting agent, suspending agent and one or more preservatives. The above-mentioned dispersing or wetting agent may be used as examples of suitable dispersing or wetting agent. Other excipients, toners, flavoring agents and sweetening agents may be added to the suspension.
According to an embodiment, the pharmaceutical composition including a TRAP protein or a fragment thereof for the treatment of Staphylococcus aureus infections may be present in an oil in water emulsion, wherein the oil phase may be vegetable oil (such as olive oil or peanut oil), or a mineral oil (such as liquid paraffin), or a mixture thereof. The suitable emulsifying agent may be a native gum such as gum Arabic, bassora gum, a native phospholipid such as soya and lecithin, an ester or partial ester derived from a fatty acid and hexitol glycoside such as sorbitan monooleate, and a condensation product of said partial ester and ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsifying agent may include sweetening agents and flavoring agents.
The composition may be in present in a suppository form for rectal administration. This composition may be prepared by mixing drugs and a suitable non-irritative excipient, wherein the excipient is a solid at general temperature but is liquefied at rectal temperature, so that it can be thawed in recta to release drugs. Such excipient includes cocoa butter and polyethylene glycol, etc.
As to the external use, the creams, ointments, jellies and suspensions including the compounds disclosed herein are contemplated. For this application purpose, dentilaves and gargarismas are also included therein.
According to an embodiment, TRAP proteins or fragments thereof for the treatment of Staphylococcus aureus infections may be administrated through a liposome delivery system, such as small monolayer vesicles, big monolayer vesicles or multilaminar vesicles, wherein the liposome may consist of different phosphatides such as cholesterol, stearyl amine or phosphatidyl choline.
In addition, according to an embodiment, part of TRAP proteins or fragments thereof for the treatment of Staphylococcus aureus infections may be mixed with water or a general organic solvent to form solvates. Such solvates are involved in the scope of the described embodiments.
If a solid carrier is for oral administration, the formulation may be in form of a tablet, a hard gelatin capsule in which drugs in a powder or pellet form are placed into, a troche or a lozenge. The amount of a solid carrier varies greatly, but it is generally from about 25 mg to about 1 g. If a liquid carrier is used, the formulation may be in form of a syrup, emulsion, soft capsule of gelatin or sterile injectable liquid, such as aqueous or non-aqueous liquid suspensions or solutions.
In an embodiment, TRAP proteins or active fragments thereof for the treatment of Staphylococcus aureus infections may be administrated to a mammalian in need of such treatment, in particular a human being. Said mammalian also includes animals, whether they are tamed ones (e.g. domestic pets) or non-tamed ones (e.g. wild animals).
The pharmaceutical composition including a compound disclosed herein may be administrated one or more times a day or a week. The effective amount of the pharmaceutical composition is one that arrives at the clinically significant effect. Such dosage partially depends on the specific symptom needed to be treated, the age, weight and general health condition of a subject and other factors obviously considered by a person skilled in the art.
Another aspect relates to a method for the treatment of Staphylococcus aureus infections, including administrating a therapeutically effective amount of pharmaceutical composition including said TRAP protein as an active ingredient to a subject suffering from Staphylococcus aureus infection.
The term “treatment” herein refers to the treatment or nursing of a patient to help him against Staphylococcus aureus infections. This term aims to cover the entire scope of the treatment of Staphylococcus aureus infections including the alleviation and abatement of symptoms and complications, and/or cure or elimination of diseases, disorders and symptoms. The subjects who accept such treatment are preferably mammalian, in particular, human beings.
In the present disclosure, the term “therapeutically effective amount” refers to an amount sufficient for the treatment of status of said diseases, and it can vary based on a patient, a disease to be treated and a treatment method used. As to bacterial infections, especially Staphylococcus aureus infections, said therapeutically effective amount refers to an amount sufficient to successfully prevent or eliminate said infections after the occurrence of infections.
According to the embodiments disclosed herein, TRAP proteins or biological active fragments thereof for the treatment of Staphylococcus aureus infections may be administrated alone or in combination with other established therapeutic methods.
It is discovered in some embodiments that TRAP has double effects against Staphylococcus aureus infections, i.e., the protein per se and an inhibitor thereof or a neutralizing antibody thereof have the same effects, which will represent a kind of novel action mode of medicaments.
It is also discovered in some embodiments, when TRAP is used to treat Staphylococcus aureus infections, it certainly induces the production of antibodies against TRAP in a human body, because TRAP is an exogenous protein which is from bacteria. As to a traditional medicament, the production of antibodies means the inhibition of the effects of medicament. However, as to TRAP, the production of antibodies is useful.
Some advantages of the embodiments disclosed herein are as follows.
Advantages of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description of embodiments and upon reference to the accompanying drawings in which:
While the invention may be susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. The drawings may not be to scale. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.
The present application is further illustrated in detail by the following several examples, but they do not limit the present invention in any form.
The preparation of two TRPA proteins of Staphylococcus aureus family (TRAP proteins (GenBank™ Accession Nos.: AF202641 and AY248703)). Two TRAP proteins were separately obtained by designing primers in accordance with base sequence in Genbank by a method disclosed by Yang G. et al (2005), amplifying TRAP gene from genome of strain RN6390B or 04018 by PCR process, respectively and cloning them into PET-28a vector, transforming E. coli, inducing expression via IPTG, and purifying with a Ni2+ affinity resin.
The polyclonal antibodies were prepared with recombinant TRAP proteins. Recombinant TRAP proteins were coupled with Sepherose 4B activated by CNBr to prepare purified antibodies with affinity column. The purified native TRAP proteins having a purity of higher than 95% were obtained by coupling the purified antibodies with Sepherose4B activated by CNBr, purifying TRAP proteins from sonicated supernatant of Staphylococcus aureus RN6390B by affinity purification and further purifying by HPLC.
A fusion protein consisting of a TRAP protein and a polypeptide of 12 amino acids was prepared by selecting an unrelated peptide sequence (TPSIPSLWWTTP, Shen Tong et al., Journal of Cellular and Molecular Immunology, 2002, 18: 623-626), designing primes, obtaining a gene sequence expressing a fusion protein of TRAP-untelated peptide by PCR, cloning it into pET-28a vector, inducing expression via IPTG, transforming E. coli, and purifying with Ni2+ affinity resin.
The concrete steps were as follows:
In
It can be seen from
BALB/c mice: bought from Animal Center of the Academy of Military Medical Sciences; and other materials: being the same as those in preparation examples 1 and 3.
The concrete detecting steps are as follows:
The results show that TRPA proteins can effectively reduce Staphylococcus aureus infections in mice and increase the survival rate of infected mice (see Table 1).
In this patent, certain U.S. patents, U.S. patent applications, and other materials (e.g., articles) have been incorporated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only incorporated by reference to the extent that no conflict exists between such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in such incorporated by reference U.S. patents, U.S. patent applications, and other materials is specifically not incorporated by reference in this patent.
Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 200610075638.7 | Apr 2006 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2007/001246 | 4/16/2007 | WO | 00 | 5/11/2009 |
