Patent Grant
7727958
This invention relates to pharmaceutical formulations, particularly to formulations of amino acids, peptides and small proteins, and specifically to formulations for PKC peptide/transporter conjugates.
Protein kinase C (“PKC”) is a key enzyme in signal transduction involved in a variety of cellular functions, including cell growth, regulation of gene expression, and ion channel activity. The PKC family of isozymes includes at least 10 different protein kinases that can be divided into at least three subfamilies based on their homology and sensitivity to activators. (See
Individual isozymes of PKC have been implicated in the mechanisms of various disease states, including the following: cancer (alpha and delta PKC); cardiac hypertrophy and heart failure (beta I and beta II PKC) nociception (gamma and epsilon PKC); ischemia including myocardial infarction (delta and epsilon PKC); immune response, particularly T-cell mediated (theta PKC); and fibroblast growth and memory (zeta PKC).
In accordance with the objects outlined above, the disclosed invention provides a pharmaceutical formulation for a protein kinase C modulatory peptide and a cationic (i.e., positively charged) transport peptide and an anti-aggregant. A preferred anti-aggregant is a sugar characterized by having a sufficient number stereochemically aligned hydroxyl moieties to interact with the modulatory peptide and/or the transport peptide hydrophobic and/or positively charged portions so as to favor their organization with the anti-aggregant, rather than aggregation with each other. PKC modulatory peptides, such as peptides derived from various PKC variable regions, comprise preferred embodiments. Cationic transport moieties useful in the invention include cationic peptides, such as poly-arginine and HIV-tat. A particularly preferred embodiment comprises a PKC inhibitory peptide and a HIV-tat derived transport peptide. An example of such an embodiment is KAI-9803 (SEQ ID NO:1).
In one of the particular aspects of the above-described pharmaceutical formulation, the ratio of anti-aggregant to peptide/transporter conjugate ranges from about 100:1 to about 1:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, and 1:1.
Another aspect of the invention provides a stable pharmaceutical product for shipping and storing prior to use, including a lyophilized cake of KAI-9803 and an anti-aggregant in a sealed container. The lyophilized product is preferably obtained from a solution of KAI-9803 plus acetate counterion. The ratio of KAI-9803 to anti-aggregant is from about 1:5 to about 1:100, particularly about 1:80 and especially about 1:8. The anti-aggregant is preferably a sugar. One specific such product is 5 mg KAI-9803 and 40 mg mannitol in a stoppered glass vial. Instructions for reconstitution are preferably incorporated on the container or its attached label, outer packaging and/or package insert.
Another aspect of the invention provides a formulation for parenteral (particularly intracoronary) administration that is about 2.5 mg/mL KAI-9803 and about 20 mg/mL mannitol reconstituted from a lyophilized cake using sodium chloride for injection, USP (preferably 0.9%) to a concentration ranging from about 0.001 to 2.5 mg/mL, preferably about 0.01 to 1.0 mg/mL. To reconstitute the lyophilized formulation for administration, a sealed container of product is first warmed to about room temperature, after which a pharmaceutically acceptable solvent (such as saline, preferably 9% saline) is added in an amount sufficient to solubilize the lyophilized cake, followed by the addition of such additional quantity of solvent as is necessary to obtain a desired concentration for administration.
Still another aspect of the invention is a method of manufacture, including the steps:
Other aspects and embodiments will be apparent to those skilled in the art form the following detailed description.
The presently described invention relates to pharmaceutical formulations of peptides which modulate the activity of one or more protein kinase C isozymes. In certain embodiments, the peptides discussed herein are coupled to a carrier moiety to facilitate transport of the modulatory peptide to a target cell. Typically, preferred embodiments of the disclosed pharmaceutical formulations further comprise an anti-aggregant and one or more excipients. The pharmaceutical formulations comprising the modulatory peptides provide advantages in the handling of the active pharmaceutical ingredients, in formulation manufacture, stability, concentration and ease of use. These and other advantages are described in greater detail below.
As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.
A “PKC modulatory peptide” or “a peptide which modulate the activity of one or more protein kinase C isozymes” refer to a peptide that can promote, enhance or activate one or more PKC isozymes, or alternatively the peptide can also inhibit or inactivate one or more PKC isozymes.
The term “API” means active pharmaceutical ingredient, which as used herein refers to a PKC modulatory peptide and a transport moiety, covalently bound to one another, and/or one or more active agents.
The term “disorder” or” disease state” means any mammalian disease, condition, symptom, or indication, preferably arising in a human patient.
The term “effective amount” refers to that amount of an API that is sufficient to effect treatment, as defined below, when administered to a mammal in need of such treatment.
The term “KAI-9803” refers to an peptide derived from the first variable region of δPKC conjugated via a Cys-Cys disulfide linkage to a HIV Tat-derived transporter peptide, and can be represented as follows:
The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where such event or circumstance occurs and instances in which it does not.
As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.
The term “pharmaceutically acceptable salt” or “counterion” refers to salts which retain the biological effectiveness and properties of the API and which are not biologically or otherwise undesirable. In many cases, the API will be capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable base addition salts can be prepared from inorganic and/or organic bases. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and/or organic acids. For example, inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.
The term “pharmaceutical product” refers to an API, formulated and filled into a container for storage, transportation, or administration.
The term “PKC-derived peptide” refers to a PKC isozyme- and/or variable region-specific peptides as described, for example, in U.S. Patents and Publications Nos. U.S. Pat. No. 5,783,405, U.S. Pat. No. 6,165,977, US2002/0150984, US2002/0168354, US2002/057413, US2003/0223981, US2004/0009922 and in copending U.S. provisional application Ser. No. 60/550,755, filed Mar. 5, 2004, all of which are hereby incorporated by reference in their entirety.
The term “transporter moiety” means a component of an API that facilitates cellular uptake, such as cationic polymers, peptides and antibody sequences, including polylysine, polyarginine, Antennapedia-derived peptides, HIV Tat-derived peptides and the like. An example of a transporter moiety is a “transporter peptide”, which is a peptide which facilitates cellular uptake of a PKC modulating peptide which is chemically associated or bonded to the transporter peptide.
The term “treatment” or “treating” means any treatment of a disease or disorder in a mammal, including: preventing or protecting against the disease or disorder, that is, causing the clinical symptoms not to develop; inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and/or relieving the disease or disorder, that is, causing the regression of clinical symptoms.
The term “prophylaxis” is intended as an element of “treatment” to encompass both “preventing” and “suppressing” as defined herein. It will be understood by those skilled in the art that in human medicine it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events.
Protein Kinase C Modulatory Peptides
Various PKC isozyme—and variable region-specific peptides have been described and can be used with the presently disclosed invention. Preferably, the PKC modulatory peptide is a V1, V3 or V5-derived peptide. The following US patents or patent applications describe a variety of suitable peptides that can be used with the presently disclosed invention: U.S. Pat. Nos. 5,783,405, 6,165,977, 6,855,693, US2004/0204364, US2002/0150984, US2002/0168354, US2002/057413, US2003/0223981, US2004/0009922 and U.S patent application Ser. No. 10/428,280, each of which are incorporated herein by reference in their entirety. Table 1 provides a listing of preferred PKC modulatory peptides for use with the present invention.
As discussed more fully below, it is preferable that the PKC modulatory peptide be chemically associated with a transport peptide. In a particularly preferred embodiment, the modulatory peptide and the transport peptide are linked via a disulfide bond. In the case of the forming a disulfide bond, it may be advantageous to add Cys residue to the PKC modulatory peptide sequence, preferably at the amino terminus of the peptide. Alternatively, an endogenous Cys residue can be exploited to link the modulatory peptide with the transport peptide or moiety. Methods of forming disulfide bonds are well known to those of ordinary skill in the art, for example mixing components in a reducing environment and then introducing the components to an oxidizing environment.
Transport Peptide
A wide variety of molecules (particularly macromolecules such as peptides) intended for cellular uptake were found to be transported poorly across cell membranes. Among the solutions proposed to facilitate cellular uptake have been the use of transporter moieties such as cationic (i.e., positively charged) polymers, peptides and antibody sequences, including polylysine, polyarginine, Antennapedia-derived peptides, HIV Tat-derived peptides and the like. (See, for example, US patents and Publications Nos. U.S. Pat. Nos. 4,847,240, 5,652,122, 5,670,617, 5674,980, 5,747,641, 5,804,604, 5,888,762, 6,316,003, 6,593,292, US2003/0104622, US2003/0199677 and US2003/0206900, all of which are hereby incorporated by reference in their entirety.)
A particular example of a peptide/transporter conjugate is KAI-9803 (SEQ ID NO: 1), which is made up of a δPKC-derived peptide and a HIV Tat-derived transporter peptide. It is currently being developed for human therapeutic use in the treatment of reperfusion injury. As with most pharmaceutical active agents, KAI-9803 is prepared as a pharmaceutical formulation with certain stability, tolerability and bioavailability requirements.
Excipients and Anti-Aggregants
Pharmaceutically acceptable excipients suitable for use as carriers or diluents are well known in the art, and may be used in a variety of formulations. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990); Remington: The Science and Practice of Pharmacy, 20th Edition, A. R. Gennaro, Editor, Lippincott Williams & Wilkins (2000); Handbook of Pharmaceutical Excipients, 3rd Edition, A. H. Kibbe, Editor, American Pharmaceutical Association, and Pharmaceutical Press (2000); and Handbook of Pharmaceutical Additives, compiled by Michael and Irene Ash, Gower (1995).
Lyophilized formulations are typically prepared from an active agent dissolved in a pharmaceutically acceptable solvent, optionally including excipients such as bulking agents, solubility enhancers, pH buffers and the like. The solution is subjected to reduced temperatures and pressure to drive off the liquids, leaving a solid cake that can be stored for future use.
The lyophilized formulations of the disclosed invention advantageously include an anti-aggregant, such as a sugar, where such sugars are sufficient to interact with the active agents' hydrophobic and/or positively charged portions to favor their organization with the sugar, rather than aggregation with each other. Suitable anti-aggregant sugars include fructose, lactose, glycerol, mannitol and D-mannose, preferably mannitol.
The transport moieties used to facilitate cellular uptake of peptides (such as the δPKC sequence portion of KAI-9803) share certain attributes (generally being cationic) that contribute to their functionality in vivo, but have been discovered to give rise to the formation of aggregates under lyophilized storage conditions. The modulatory peptides may also possess structural features which facilitate the formation of aggregates. While not wishing to be bound to any particular theory, such aggregation may result from peptide dimerization and a tendency for the peptides to “organize” into aggregates.
The formation of a detrimental level of aggregates interferes with re-dissolution of peptides and peptide conjugates, in turn interfering with administration where the possibility of particulates would be unacceptable for certain routes of administration (e.g., intracoronary). Notwithstanding these drawbacks, the creation of a detrimental level of aggregates in the formulation complicates determining final concentration in that the precise amount of peptide dissolved per unit of liquid. Such a determination cannot be accurately calculated without first determining and then subtracting the weight of undissolved material. Unacceptable levels pf aggregation can result in from 0.1 to 50% aggregation of the peptide conjugates. Thus, another aspect of the present invention pertains to the incorporation of an anti-aggregant in lyophilized formulations of peptides or peptide/transporter conjugates. In addition to suppressing the formation of aggregants in the lyophilized product, certain anti-aggregants can enhance the stabilization of the pharmaceutical formulation.
Administration
Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly, intraperitoneal, intravenously, and in the case of the present invention via intracoronary injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid (e.g., dried or lyophilized) forms suitable for reconstitution into solution or suspension in liquid prior to injection, or as emulsions. Generally, suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, minor amounts of non-toxic auxiliary substances can be employed, such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, tonicifiers and the like including, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc. Dosage forms for intravenous (IV) administration generally comprise an active agent incorporated into a sterile solution of simple chemicals such as sugars, amino acids or electrolytes, which can be easily carried by the circulatory system and assimilated. Such solutions are typically prepared with saline or buffer. The pH of such IV fluids may vary, and will typically be from 3.5 to 8.0, as known in the art.
The intracoronary injection formulations of the disclosed invention are typically prepared using sodium chloride for injection, USP (preferably 0.9%) for reconstitution of the lyophilized API into solution at concentrations ranging from 0.001 to 2.5 mg/mL, preferably 0.01 to 1.0 mg/mL.
Exemplary Formulations
The pharmaceutical formulations of the disclosed invention preferably include a PKC modulatory peptide, a transport peptide, and an anti-aggregant agent. Typically, the PKC modulatory peptide and the transport peptide are chemically associated with one another. For example, it is preferred that the modulatory peptide and the transport peptide are covalently bonded to one another. In a preferred embodiment, the PKC modulatory peptide and the transport peptide are linked via a disulfide bond.
In a pre-lyophilized embodiment of the invention, the formulation further includes a sufficient amount of a pharmaceutically acceptable solvent (preferably water for injection, USP) to solubilize the foregoing components. A lyophilized embodiment of the invention includes components described above in the form of a solid cake.
The ratio of peptides to anti-aggregant in the disclosed formulations ranges from about 100:1 to about 5:1, preferably from about 80:1 to about 5:1, more preferably from about 80:1 to about 8:1; the actual ratio will depend upon the identity of the components and the concentration desired for the lyophilized and/or reconstituted drug products.
One aspect of the present invention provides a pre-lyophilized formulation for a peptide or peptide/transporter conjugate, as follows:
and the lyophilized product therefrom.
Another aspect of the preferred peptide or peptide/transporter conjugate formulation can be obtained by lyophilization of the following:
A preferred peptide or peptide/transporter conjugate formulation can be obtained by lyophilization of the following:
Another preferred peptide or peptide/transporter conjugate formulation can be obtained by lyophilization of the following:
Still another preferred peptide or peptide/transporter conjugate formulation can be obtained by lyophilization of the following:
A further preferred peptide/transporter conjugate formulation can be obtained by lyophilization of the following:
Alternatively, aqueous parenteral solutions of KAI-9803 can be prepared substantially free of sugars, at concentrations ranging from about 0.01 to about 10.0 mg/mL, preferably about 0.1 to about 5.0 mg/mL, and most preferably about 0.1 to about 1.0 mg/mL. The pH of such aqueous solutions is adjusted to between about 2.0 and 4.0, preferably between about 2.5 and 3.5.
Methods of Manufacture and Use
The PKC modulatory peptides and the transporter peptides can be synthesized according to conventional (e.g., solid phase) procedures. After activation of the Cys on one of the PKC modulatory and transporter peptides [e.g., using 2,2′-dithiobis(5-nitropyridine) to activate the carrier peptide] the two peptides are coupled, isolated and then purified [e.g., by preparative RP-HPLC using acetonitrile elution in a TEAP buffer (triethylamine and phosphoric acid) giving rise to the purified phosphate salt]. The fractions from the HPLC containing the purified and coupled peptides are then pooled. A pharmaceutically acceptable salt can be exchanged by repeat RP-HPLC eluting with acetonitrile and the desired organic or inorganic acid counter-ion donor (such as acetic acid, hydrochloric acid, tartaric acid and the like, preferably acetic acid). The desired end product is pooled, divided into lyophilization flasks, lyophilized, and transferred to suitable containers for storage prior to formulation (preferably in sealed amber glass containers at reduced temperature, e.g., −20° C.).
The counterion employed during the production of KAI-9803 has a positive effect on solubility and stability. An acetate counterion is used in the preferred embodiment. Other counterions, such as a chloride counterion are also contemplated. And while use of an acetate counterion is a preferred embodiment of the disclosed invention, it is not required.
The pharmaceutical formulations of the present invention can be manufactured according to most accepted practices, for example, as follows:
It is recommended that such pharmaceutical products be stored at or below room temperature, preferably at about 2-8° C. (more preferably 5° C.), which instructions should be displayed on the container or its attached label, its outer packaging and any package insert included therein.
To reconstitute the lyophilized formulation for administration, the product is first warm to about room temperature before opening. Shortly prior to use, the sealed container is accessed via a needle through the rubber stopper and a pharmaceutically acceptable solvent (such as saline, preferably 9% saline) is added in an amount sufficient to solubilize the lyophilized cake and provide the desired concentration for administration. Such instructions for reconstitution can be provided in a pharmacy manual, on dosing cards, or can be incorporated on the container or its attached label, outer packaging and/or package insert.
Testing
Testing of the pharmaceutical formulations of the present invention can be accomplished by procedures well known in the art, for example, including: determination of active pharmaceutical ingredient identity and concentration by HPLC-UV (measuring absorbance at 206, 220 and/or 280 nm) e.g., before and after lyophilization and reconstitution; determination of water content in lyophilized product; pH of pre-lyophilized solution and reconstituted solution; and appearance of lyophilized cake.
The following examples serve to describe more fully the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are incorporated by reference in their entirety.
A. Peptide Fragment Synthesis
Merrifield resin is pre-swelled in dichloromethane (DCM) for at least 2 hours. The DCM is drained. Transporter and δ-PKC peptides are prepared by solid phase synthesis as follows:
Deprotection and coupling are monitored by performance of a ninhydrin test. After incorporation of the final amino acid residue on each peptide (Cys), the resin peptide is deprotected and washed (steps 1, 2 and 4, above). The peptide-resin bond and side chain protecting groups are cleaved by treatment with HF/anisole and precipitated by ethyl ether.
B. Peptide Fragment Purification and Isolation
Transporter and δ-PKC peptides obtained, e.g., as described in Example IA are subjected to preparative RP-HPLC on a C-18 column, using an acetonitrile gradient in trifluoroacetic acid solution. The acceptance criterion for purity of these intermediate peptides is not less than 90.0%.
C. Coupling
Transporter peptide obtained, e.g., as described in Example IB is activated by contact with 2,2′-dithiobis(5-nitropyridine) and then contacted with the δ-PKC peptide from Example IB to afford the coupled peptide conjugate KAI-9803.
D. Purification, Salt Exchange and Isolation
Crude KAI-9803 obtained, e.g., as described in Example IC is purified by preparative RP-HPLC using an acetonitrile elution in a TEAP buffer (triethylamine and phosphoric acid) on a YMC C-18 column. The fractions resulting from the purification are analyzed by an analytical RP-HPLC in-process method. Those fractions that meet the purity criterion (not less than 95%) are pooled, loaded back onto the same C-18 column and eluted with acetonitrile in an acetic acid buffer to give the corresponding KAI-9803 acetate salt. The thus-purified KAI-9803 acetate salt is pooled, divided into lyophilization flasks and frozen. The frozen flasks are connected to a lyophilizer manifold and the lyophilization is performed. Upon completion of lyophilization, the resulting KAI-9803 acetate powder is weighed, samples are taken for testing, and the remainder transferred into 50 mL amber glass containers. The containers are closed with 20 mm, (grey)butyl, snap-on stoppers, and stored at −20° C.
KAI-9803 acetate powder (50.0 mg) obtained, e.g., as described in Example ID and mannitol USP (400.0 mg) are dissolved in about 14.0 mL of WFI, followed by the addition of WFI as necessary to total 20 ml (˜6 ml) to give a clear, colorless solution. Clarity, color and complete dissolution of solids are confirmed by visual examination. The solution is aseptically filtered through two serial 0.22 μm filters into a class 100 aseptic filling suite. Two mL of the filtered solution are aseptically dispensed into each of ten pre-sterilized 20 mL vials. Each vial is capped with a slotted lyophilization stopper and loaded onto shelves pre-chilled to −50° C. A primary drying cycle is performed at a shelf temperature of 5° C. for not less than 20 hours, followed by a secondary drying step with a shelf temperature of 25° C. for not less than 3 hours. Upon completion of the lyophilization cycle, the vials are stoppered under nitrogen with a partial vacuum and sealed. The stoppered vials are crimped and inspected in a class 10,000 processing suite. The vials are labeled and then moved to 2-8° C. storage under quarantine.
A vial containing a lyophilized pharmaceutical formulation of 5 mg KAI-9803 and 40 mg mannitol (obtained, e.g., as described in Examples 1 and 2) is injected with 20 mL of 0.9% sodium chloride for injection, USP, is added to the vial and the contents are dissolved with gentle swirling to yield a clear solution. To a sterile, empty IV bag is added 18 mL of 0.9% sodium chloride for injection, USP, followed by the addition of 2 mL of KAI-9803 solution (taken from the vial) to yield a total volume of 20 mL of a 0.1 mg/mL solution of KAI-9803 in the IV bag. The solution is stored at room temperature and used within 4 hours of preparation.
Formulations of KAI-9803 are prepared, for example as described in Examples 1 and 2, using mannitol and substituting mannitol with fructose and sucrose as the anti-aggregant sugar for the formulating procedure of Example 2. All of the solutions are visually inspected for clarity, color and complete dissolution of solids, and an aliquot is removed from each. Each aliquot is analyzed for KAI-9803 concentration using HPLC-UV, measuring absorbance at 206 and 280 nm. The remaining solutions are filtered, filled, lyophilized and finished, e.g., as described in Example 2. One set of vials representing each of the formulations is separated for immediate reconstitution and testing (HPLC-UV @ 206/280 nm) to confirm KAI-9803 concentration in the reconstituted product. The remaining vials are divided into groups for storage at reduced temperature (e.g., 2-8° C.), at room temperature, and at elevated temperature (e.g., 35° C.). Sets of vials representing each of the formulations are withdrawn at selected time points (e.g., 1 day, 1 week, 1 month, 3 months, 6 months), are reconstituted, visually inspected and tested for KAI-9803 concentration (HPLC-UV @ 206/280 nm).
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. All patents and publications cited above are hereby incorporated by reference.
This application is a divisional of U.S. patent application Ser. No. 11/240,962, filed on Sep. 30, 2005, now U.S. Pat. No. 7,265,092, which claims the benefit of priority of U.S. Provisional Patent Application No. 60/615,486 filed Sep. 30, 2004, both of which are hereby incorporated by reference in its entirety. The entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows: File NameDate of CreationSize (bytes)578422000110Seqlist.txtAug. 25, 200989,109 bytes
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| Number | Date | Country | |
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| Number | Date | Country | |
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| Parent | 11240962 | Sep 2005 | US |
| Child | 11849929 | US |
