AU2005332297A1 - Cell nucleus-entering compositions - Google Patents

Description

WO 2006/130171 PCT/US2005/035716 CELL NUCLEUS-ENTERING COMPOSITIONS BACKGROUND OF THE INVENTION CROSS-REFERENCE TO RELATED APPLICATIONS [ooi] The present application claims benefit of U.S. Provisional Application Serial No. 60/684,993, filed May 27, 2005. Field of the Invention [002] The present invention relates to the field of compositions and methods for delivering physiologically active agents. Description of the Background Art [003] There is a need in the art for improved compositions and methods for delivering physiologically active agents. SUMMARY OF THE INVENTION [004] In accordance with the present invention, a pharmaceutically acceptable composition for entering a cell nucleus comprises a cell nucleus entering polypeptide comprising at least one of amino acid sequence LKKTET, amino acid sequence LKKTNT or amino acid sequence KSKLKK, or a conservative variant thereof, linked to a physiologically active agent having at least one of therapeutic or diagnostic application in said cell nucleus. DETAILED DESCRIPTION OF THE INVENTION [005] The present invention provides compositions and methods utilizing actin-sequestering peptides such as thymosin $4 (T14, TB4 or TBeta4) and other actin-sequestering peptides or peptide fragments containing amino acid sequence LKKTET, LKKTNT or KSKLKK, or conservative variants thereof. Included are N- or C-terminal variants such as KLKKTET and LKII'E TQ. These peptides and peptide fragments are useful for entering cell nuclei for treating and/or preventing various conditions, and affecting numerous WO 2006/130171 PCT/US2005/035716 physiological functions. In some preferred embodiments, the cell-entering peptide is Tf4. [oo6] The physiologically active agent can be linked to the cell-entering peptide at any suitable position. For example, the agent can be linked to the N-terminus of T34, or to an amino acid of the cell-entering peptide at another location, most preferably a glutamine residue. The agent can be a drug, chemotherapeutic agent, DNA sequence, RNA sequence, DNA- or RNA activating or deactivating agent, diagnostic agent, or the like. [007] In preferred embodiments, the cell-entering peptide penetrates the nuclear membrane so as to carry the linked agent into the nucleus. The nuclear membrane preferably is mammalian, more preferably human. [008] The invention also is applicable to a method of administering an agent-carrying cell-entering peptide to a mammalian subject. The method may comprise contacting a nuclear membrane or tissue of a subject with a composition as defined herein, preferably a pharmaceutically acceptable composition. In preferred embodiments, the subject is mammalian, more preferably human. [009] Thymosin 14 initially was identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin P4 is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Several roles have been ascribed to this protein including a role in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration and vascularization. [oo1o] Thymosin P4 is a member of the P-thymosin family of highly conserved polar 5-kDa polypeptides found in various tissues and cell types. Originally purified from thymus and regarded as a thymic hormone, thymosin P4 was then found to be involved in multiple biological processes. As the main G-actin sequestering peptide, it plays an important role in regulation of actin assembly during cell proliferation, migration, and differentiation. Numerous studies implicate thymosin P4 in regulation of cancerogenesis, inflammation, angiogenesis, and wound healing. It was found that thymosin -2- WO 2006/130171 PCT/US2005/035716 P4 expression regulated tumorigenicity and metastatic activity in malignant cell lines through actin-based cytoskeletal organization. Thymosin P4 was found to be elevated in tube forming endothelial cells; it increases their attachment, spreading and migration thus promoting angiogenesis. Thymosin P4 was also found in ulcer extracts and wound fluids at high concentrations and was suggested to function as an antibacterial factor. The stimulating role of thymosin P4 in wound healing was demonstrated in several studies with animal models. When added topically or administered intraperitoneally, thymosin P4 enhanced dermal wound healing in a rat full thickness model. The ability to accelerate dermal wound healing has also been observed in db/db diabetic mice, steroid-immunosuppressed mice and in aged mice. Thymosin P4 has also been shown to accelerate healing of the corneal epithelium after burn injuries and to down regulate a number of corneal cytokines and chemokines reducing the inflammatory response. [oi] Activation of the coagulation cascade upon vascular injury results in generation of thrombin which converts fibrinogen into fibrin. Fibrin polymerizes spontaneously to form blood clots which seals damaged places thus preventing the loss of blood. Fibrin also serves as a provisional matrix on which various cell types adhere, migrate and proliferate replacing fibrin with normal tissues during subsequent wound healing processes. [0012] Thymosin P4 serves as a specific substrate for tissue transglutaminase and can be selectively cross-linked by it to collagen, actin, fibrinogen and fibrin, proteins which are also involved in the above mentioned processes. After activation of platelets with thrombin, thymosin P4 is released and cross-linked to fibrin in a time- and calcium-dependent manner. [0013] In preferred embodiments, the cell-entering polypeptide comprises amino acid sequence LKKTET, LKITNT, KSKLIK, KLKKTET, LITETQ, Thymosin 04 (T14), an N-terminal variant of T34, a nucleus-entering C terminal variant of Tr4, an N-terminal fragment of Tp4, an isoform of T$4, a splice-variant of TP4, oxidized TP4, T14 sulfoxide, lymphoid TP4, pegylated -3- WO 2006/130171 PCT/US2005/035716 TP4 or any other actin sequestering or bundling proteins having actin binding domains, or peptide fragments comprising or consisting essentially of the amino acid sequence LKITET, LKKTNT or KSKLK, or conservative variants thereof. International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses isoforms of T14 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET, and conservative variants thereof, which may be utilized with the present invention. International Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Thymosin P4 which may be utilized in accordance with the present invention. Although the present invention is described primarily hereinafter with respect to T$4 and TO4 isoforms, it is to be understood that the following description is intended to be equally applicable to amino acid sequence LKKTET, LKTNT, KSKLKK, KLIKTET, LIKTETQ, peptides and fragments comprising or consisting essentially of LIKTET, LIKTNT, KSKLKK, KLITET, or LIKTETQ, conservative variants thereof, as well as oxidized Thymosin P4, T34 sulfoxide, lymphoid Tf4 and pegylated TP4. [0014] The cell-entering peptide with linked agent may be administered in any suitable effective amount. For example, the cell-entering peptide with linked agent may be administered in dosages within the range of about 0.1-50 micrograms, more preferably in amounts within the range of about 1-30 micrograms. [0015] A composition in accordance with the present invention can be administered once, daily, every other day, every other week, every other month, etc., with a single application or multiple applications per day of administration, such as applications 2, 3, 4 or more times per day of administration. [oo16] TP4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of TP4. Such isoforms include, for example, TP4 a, TP9, TP10, T$11, TP12, TP13, TP14 and T315. Similar to TP4, the TP10 and TP15 isoforms, as well as the Tf4 -4- WO 2006/130171 PCT/US2005/035716 splice-variants, have been shown to sequester actin. T34, T310 and TP15, as well as other isoforms share an amino acid sequence, LKKTET, that appears to be involved in mediating actin sequestration or binding. Although not wishing to be bound to any particular theory, the activity of TP4 isoforms may be due, in part, to the ability to regulate the polymerization of actin. P thymosins appear to depolymerize F-actin by sequestering free G-actin. T34's ability to modulate actin polymerization may therefore be due to all, or in part, its ability to bind to or sequester actin via the LKKTET sequence. Thus, as with T14, other proteins which bind or sequester actin, or modulate actin polymerization, including Tf4 isoforms having the amino acid sequence LKKTET, are likely to be effective, alone or in a combination with T34, as set forth herein, as are cell-entering peptides comprising sequence cell-entering. [0017] Thus, it is specifically contemplated that known T$4 isoforms, such as TP4 a, Tf9, T$1O, T11, T$12, T313, T1p14 and Tf15, as well as Tf4 isoforms and T14 splice-variants not yet identified, will be useful in the methods of the invention. As such Tr4 isoforms are useful in the methods of the invention, including the methods practiced in a subject. The invention therefore further provides pharmaceutical compositions comprising agent-carrying T34, as well as T34 isoforms T$4 a, Tr9, T110, Tr11, T$12, T$13, T114 and T$15, and a pharmaceutically acceptable carrier. [oo18] In addition, other proteins having actin sequestering or binding capability, or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET, LKKTNT or KSKLIKK, for example, can similarly be employed in the methods of the invention. Such proteins include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adsevertin, propomyosin, fincilin, depactin, DnaseI, villin, fragmin, severin, capping protein, P-actinin and acumentin, for example. As such methods include those practiced in a subject, the invention further provides pharmaceutical compositions comprising gelsolin, vitamin D binding protein -5- WO 2006/130171 PCT/US2005/035716 (DBP), profilin, cofilin, depactin, Dnasel, villin, fragmin, severin, capping protein, -actinin and acumentin as set forth herein. Thus, the invention includes compositions and methods utilizing a polypeptide comprising the amino acid sequence LKKTET, LKITNT or KSKLKK, (which may be within its primary amino acid sequence) and conservative variants thereof. [0019] As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. [0020] The actual dosage, formulation or composition utilized may depend on many factors, including the size and health of a subject. However, persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, supra, and the references cited therein, to determine the appropriate dosage to use. [0021] Suitable formulations may include the agent-carrying LKKTET, LKKTNT or KSKLKK peptide in a carrier at a concentration within the range of about 0.0001 - 10% by weight, more preferably within the range of about 0.01 0.1% by weight, most preferably about 0.05% by weight. Any suitable pharmaceutically acceptable carrier may be utilized, such as water for injection. [0022] The invention also relates to methods for delivering a physiologically active agent to a cell nucleus comprising administering to the cell nucleus a pharmaceutically acceptable composition as described herein. The method may involve administering the composition to a cell containing a nucleus, to a nucleus within a cell, to a mammalian subject, or by contacting tissue of a subject with the inventive composition. -6- WO 2006/130171 PCT/US2005/035716 Example 1 [0023] Physiologically active agents having therapeutic and/or diagnostic application in a cell nucleus are linked to T34 as follows. The agents are selected from drugs, chemotherapeutic agents, DNA sequences, RNA sequences, DNA- or RNA-activity or deactivity agents and diagnostic agents. [0024] Agent-linked thymosin B4 is prepared by incubation of 240 pg thymosin B4 (200 pM) with 120 pg agent (1 mM) and 0.2 U guinea pig transglutaminase at room temperature in 240 pl buffer consisting of 10 mM Tris-HC1, pH 7.4, 15 mM CaCl 2 , 3 mM DTT. After 1 and 2 hours, 5 pl of the reaction mixture is subjected to HPLC analysis. The reaction is stopped after 4 hours by addition of 5 p1 trifluoroacetic acid (TFA). Then the reaction mixture is subjected to preparative HPLC. Separated peptides are concentrated in vacuo and then characterised by amino acid analysis and mass spectrometry. [0025] Proteolytic fragments of agent-linked thymosin 134 are prepared by the following procedure: 50 pg of peptide is incubated with 20 pU AsnC endoproteinase in 100 p1 reaction buffer (50 mM sodium acetate, pH 5.0, 0.2 mM DTT, 0.2 mM EDTA) for 16 hours at room temperature. Then the reaction is stopped by adding 5 p1 10% TFA and products are separated by preparative HPLC. Prior to analysis by the samples are concentrated in vacuo. Microinjection experiments [0026] Microinjection is performed with an ECET cell injection system (Eppendorf, Hamburg, Germany) consisting of the micromanipulator 5170 and the microinjector 5242 adapted to an Axiovert 100 inverted microscope (Zeiss, Gbttingen, Germany). Microinjections are visually controlled by a CCD camera on a TV monitor (SSM 121CE, Sony, Tokyo, Japan). Agent-linked thymosin B4 and crosslinked ADP-ribosylated actin:thymosin £34 complex are injected into the cytoplasm at a concentration of 32 pM and 8.27 pM respectively, in 135 mM KCl, 5 mM Na 2

HPO

4 , pH 7.2. The injection pressure is between 65 and 80 hPa (1 hPa=0.1 kPa) and the injection time between 0.5 -7- WO 2006/130171 PCT/US2005/035716 and 0.7 seconds. [0027] Agent-linked thymosin B4 is microinjected into the cytoplasm of cells. Directly after microinjection the linked peptide is evenly distributed throughout the cytoplasm. After incubation for 1 hour a pronounced localization the cell nucleus is detected. [0028] The N-terminal portion of thymosin B4 contains a sequence stretch enriched in lysine residues ( 1 4

KSKLKK

9 ) which may be a functional nuclear localisation signal. An N-terminal fragment (thymosin B "~43 4 ) containing the 1 4

KSKLKKI

9 sequence exhibits a pronounced nuclear localisation. -8-

Claims (20)

1. A pharmaceutically acceptable composition for entering a cell nucleus, comprising a cell nucleus-entering polypeptide comprising at least one of amino acid sequence LKKTET, amino acid sequence LIKTNT or amino acid sequence KSKLKK, or a conservative variant thereof, linked to a physiologically active agent having at least one of therapeutic or diagnostic application in said cell nucleus.

2. The composition of claim 1, wherein said physiologically active agent comprises a drug, chemotherapeutic agent or nucleic acid sequence.

3. The composition of claim 1 wherein said cell nucleus-entering polypeptide comprises amino acid sequence LKXTET or amino acid sequence KSKLKK.

4. The composition of claim 1 wherein said cell nucleus-entering polypeptide comprises thymosin beta 4.

5. The composition of claim 1 wherein said polypeptide comprises an N terminal fragment of thymosin beta 4.

6. The composition of claim 1 wherein said cell-entering polypeptide linked to said agent is present in a pharmaceutically acceptable carrier.

7. The composition of claim 6 wherein said cell nucleus-entering polypeptide linked to said agent is present in said carrier in concentration within a range of about 0.0001-10% by weight of said carrier.

8. A method of delivering a physiologically active agent to a cell nucleus comprising administering to said cell nucleus the composition of claim 1.

9. The method of claim 8 comprising administering said composition to a mammalian subject. -9- WO 2006/130171 PCT/US2005/035716

10. The method of claim 9 comprising contacting a tissue of said subject with said composition.

11. The method of claim 8 wherein said physiologically active agent comprises a drug, chemotherapeutic agent or nucleic acid sequence.

12. The method of claim 8 wherein said cell nucleus-entering polypeptide comprises amino acid sequence LKKTET or amino acid sequence KSKLKK.

13. The method of claim 8 wherein said cell nucleus-entering polypeptide comprises thymosin beta 4.

14. The method of claim 8 wherein said polypeptide comprises an N terminal fragment of thymosin beta 4.

15. The method of claim 8 wherein said cell nucleus-entering polypeptide linked to said agent is present in a pharmaceutically acceptable carrier.

16. The method of claim 15 wherein said cell nucleus-entering polypeptide linked to said agent is present in said carrier in a concentration within a range of about 0.0001 - 10% by weight of said carrier.

17. The composition of claim 4 wherein said physiologically active agent is linked to an N-terminal portion of said thymosin beta 4.

18. The composition of claim 5 wherein said physiologically active agent is linked to an N-terminal portion of said N-terminal fragment.

19. The method of claim 13 wherein said physiologically active agent is linked to an N-terminal portion of said thymosin beta 4.

20. The method of claim 14 wherein said physiologically active agent is linked to an N-terminal portion of said N-terminal fragment. - 10 -