WO2005120580A2

WO2005120580A2 - Compositions and methods for treatment of fibro-proliferative disorders

Info

Publication number: WO2005120580A2
Application number: PCT/US2005/019372
Authority: WO
Inventors: Richard E. Fitzpatrick; Frank Litvack
Original assignee: Immusol Incorporated
Priority date: 2004-06-03
Filing date: 2005-06-01
Publication date: 2005-12-22
Also published as: WO2005120580A3

Abstract

The invention provides novel compositions and methods for the treatment of fibro-proliferative disorders, such as keloids and hypertrophic scars. The compositions and methods comprise the use in combination of 5-FU and a ribozyme.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF FIBRO-PROLIFERATIVE DISORDERS

Field of the Invention The present invention relates generally to therapeutic compositions and methods of treatment. More specifically, the present invention provides a novel combination therapy for the treatment of fibro-proliferative disorders, such as keloids and hypertrophic scars.

Background of the Invention Fibro-proliferative diseases and conditions, such as keloids and hypertrophic scars, are exceedingly common, affecting millions of people; and yet, relatively few therapies have been developed that provide long-lasting and consistent relief. Both keloids and hypertrophic scars result from abnormal wound response to a variety of cutaneous injuries, including inflammatory skin diseases such as acne and chicken pox, trauma to the skin such as burns, abrasions, contusions and lacerations, as well as surgical procedures. Keloids are generally defined as an overgrowth of dense fibrous tissue that extends beyond the borders of the original wound, does not regress spontaneously, and tends to recur after excision. Hypertrophic scars are defined by their confinement to the borders of the original wound and a greater tendency to regress spontaneously. However, many consider keloids and hypertrophic scars to be parts of the same spectrum of fibro-proliferative disease, and it is often difficult to distinguish whether a particular lesion is a keloid or a hypertrophic scar. Both keloids and hypertrophic scars may be unsightly, but they are also typically characterized by pain, pruritus, and restriction of motion. For these reasons, treatment is undertaken not only for cosmetic reasons, but also to relieve the pain and restriction of movement that are often associated with these conditions. The basic cause of the over-exuberant wound healing that characterizes such fibro-proliferative conditions remains a mystery, although genetic predisposition appears to play a part. Thus, keloids are more common among the darker pigmented races and have been reported in as many as 16% of blacks. Most keloids occur in patients between the ages of 10 and 30 and are more likely to develop during puberty or pregnancy, indicating a hormonal influence. A variety of treatment regimens have been used and are currently being used to treat fibro-proliferative conditions such as keloids and hypertrophic scars. These include surgical excision or revision of scar, cryosurgery, radiation without excision, pressure therapy, topical silicone gel sheeting, pulsed dye laser, intralesional injections of steroids, 5-fluorouracil (5-FU), and interferon gamma (IFN-δ). However, most of these involve multiple treatments over a relatively long period of time, and none of these approaches, alone or in combination, have achieved consistently successful results. Thus, there is a definite need for new and improved methods for the treatment of fibro-proliferative conditions such as keloids and hypertrophic scars. Definitions As used herein, the term "ribozyme" refers to a molecule comprising RNA that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including Group I ribozymes, hammerhead ribozymes, hairpin ribozymes and RNAse P. Synthetic oligonucleotides comprising chimeric or hybrid ribozymes have also been described, in which some of the RNA is replaced with DNA. As used herein, the term "5-FU" or "5-fluorouracil" refers to the compound 5-fluoro-2,4(lH ,3H )-pyrimidinedione, having the following chemical structure:

In its pure form, 5-FU is a white, crystalline powder which is sparingly soluble in water and slightly soluble in alcohol. One gram of 5-FU is soluble in 100 mL of propylene glycol. Its molecular weight is 130.08. 5-FU has been used for many years, alone or in combination with other drugs, for the treatment of a wide range of indications, including various forms of cancer, as well as skin lesions such as keloids and hypertrophic scars and psoriasis. See: W. Manuskiatti and R. E. Fitzpatrick, "Treatment Response of Keloidal and Hypertrophic Sternotomy Scars," Arch. Dermatol. 138:1149-1155 (2002). 5-FU may be administered to a patient in a variety of forms, including topically, parenterally, and via interlesional injection. Detailed Description of the Invention WO 01/30362, assigned to the Assignee hereof, describes compositions and methods for the treatment of inflammation that accompany or are a result of proliferative skin diseases and conditions, such as psoriasis, atopic dermatitis, actinic keratosis, squamous or basal cell carcinoma, and viral or sebhorrheic warts. Also described are compositions and methods for the treatment of injuries to the skin that involve scarring, such as keloids, adhesions, hypertrophic burn scars and trauma. The compositions and methods described in this application encompass ribozymes that cleave RNA encoding a cytokine involved in the inflammation process, a matrix metalloproteinase, a cyclin, cell-cycle dependent kinase, a growth factor, or a reductase. Preferred ribozymes are those that target the mRNA of proliferating cell nuclear antigen (PCNA). The inventors hereof have discovered, quite unexpectedly, that the use of a ribozyme in combination with 5-FU, results in a relatively rapid and sustained reduction of the scarring and other symptoms associated with fibro-proliferative conditions such as keloids and hypertrophic scars. Patients having multiple lesions may be treated with the combination therapy of the present invention comprising both 5-FU and a ribozyme. In this combination therapy, one or more lesions are injected with a therapeutic amount of 5-FU, and shortly before or shortly thereafter an additional intra-lesional injection of a therapeutic amount of the ribozyme is administered. The intra-lesional injection of the ribozyme may be at a different site . When 5-FU alone is used in the treatment of hypertrophic scars, a patient typically must be treated 5-10 times, with the injections administered once to twice weekly or bi-weekly. Within 2-6 months, the lesions become less tender, soften and gradually flatten to about 50% of their original size. When keloids are treated with a similar protocol, a larger number of treatment sessions are generally necessary, and the overall response is less satisfactory. In contrast, when the combination therapy of the present invention is administered, improvement is noticeable after a relatively short period of time (as little as a week or two), the degree of improvement is quite marked, and fewer treatments are necessary. Keloid lesions subjected to the combination therapy will flatten up to 90% of their size, and will remain flat and asymptomatic for many months thereafter. In addition, they will tend to form a superficial dry crust and show some hyperpigmentation, which typically do not occur when 5-FU alone is used. A typical therapeutic dosage amount of 5-FU in accordance with the present invention is 5-50 mg/lesion/treatment, up to a maximum of 100 mg/treatment. A typical therapeutic dosage amount of the ribozyme in accordance with the present invention is 4mg/treatment. When administering 5-FU in accordance with the present invention, a corticosteroid such as TAC (triamcinilone acetonide) may optionally be administered at the same time, typically in a dosage amount of 0.1-1.0 mg/ml/treatment. In the practice of the present invention, any of the ribozymes described in WO 01/30362 may be utilized. As mentioned, preferred ribozymes are those that target PCNA mRNA. A particularly preferred PCNA ribozyme has the following sequence (the nucleotides that bind to the complementary PCNA mRNA, i.e. the "binding arms" of the ribozyme, are marked in bold and with a double underline): 5'-ag£££Ugcugaugaggccguaaggccgaaac£agg£g£-3' (SEQ ID NO: 1) The nucleotide sequences of the ribozyme may be chemically modified in any of a number of ways, to achieve a balance between in vivo stability on the one hand, and catalytic activity on the other. Thus, a chimeric (RNA/DNA hybrid) may be synthesized, in which any of a number of the RNA bases may be replaced with DNA. For example, the above sequence may be modified as follows (RNA marked in lower case and DNA in upper case): 5'-AGCCCugcugaugagGCCGTAAGGCcgaa £cAGGCα£- 3' (SEQIDNO:2) Further possible modifications to enhance stability and/or catalytic activity include 2' O methylation of some of the RNA bases, and replacement of the stem loop of the catalytic core with propanediol linkers (see: US Patent 6,379,931). For example, the above sequence may be modified as follows ( 2' O methylated RNA marked with a box; Pd indicates propanediol): 5'- AG£ ££0§J3 u g a@g @§Pd Pd Pd Pd@ g a@aggAGα £G£ -3' (SEQ ID NO:3). It will be appreciated that in the practice of the present invention, each of the ribozyme and the 5-FU may comprise additional components, such as carriers, excipients and the like, as are known and typically provided for in the composition of pharmaceutical products. The present invention also encompasses a pharmaceutical product for the administration of the combination therapy of the present invention comprising one or more therapeutic dosage amounts of 5-FU and one or more therapeutic dosage amounts of a ribozyme. The dosage amounts of 5-FU and the ribozyme may be suitably packaged for single and/or multiple administrations of the therapy described herein.

Examples

Example 1 (Patient #04/N-M) Five keloids treated. This patient had been previously treated for keloids on at least two occasions only with 5-FU (or 5-FU combined with TAC) and typically her keloids would improve about 50% after 10 or 12 injections. However, after a single administration of the combination of the PCNA ribozyme and 5-FU (the lesions injected with 5-FU were also injected with TAC 10 mg/ml), all of the keloids treated with 5-FU were virtually flat and also had developed a dry, crusty surface with a brownish discoloration and had softened dramatically. Six months after the initial treatment, the keloids were observed to have remained virtually flat and asymptomatic.

Example 2 (Patient #11/P-W) Two keloids treated. Two keloid scars located on the upper and lower mid abdomen of this patient were treated with 5-FU at weeks 1, 2, 3 and 4, after initial administration of the PCNA ribozyme. After week 16, the subject stated," the ... scars did real well and they are both flat". The subject also said that at the beginning of the study his self- assessment of the scars for overall improvement was 0 on a scale of 0-4 and by the end of the study his assessment improved to a 3 with a 70% improvement.

Example 3 (Patient #14 VJD) Four hypertrophic scars treated. After administration of the PCNA ribozyme, four hypertrophic scars located right peri-aerola, left peri-aerola, left mid inferior breast and right lateral inferior breast of this patient were injected with 5-FU and TAC. After week 16, the subject stated that all of the scars improved 75%.

Example 4 (Patient #15/MAR) Two keloids treated. After initial administration of the therapy, this subject refused injections of 5- FU at weeks 1 and 2, due to increased pain from study scar injections, but accepted injections at weeks 3 and 4 for two keloid scars located left superior jaw line and left inferior jaw line. Assessment to measure progress of scars was made at weeks 4, 8 and lό.The scars were about 50% decreased at each assessment.

Example 5 (Patient #16/AMF) Nine hypertrophic scars treated. This patient had multiple hypertrophic scars. Nine located on the left deltoid were injected with 5-FU and TAC at weeks 2, 3 and 4, after initial injection of the PCNA ribozyme. At week 16, it was observed that the treated scars "improved 75%... [were] flatter, more supple, more yielding, more improved". At week 16, the patient filled out the Lesion Symptom Assessment-Patient Query table and the Patient Self-Assessment table, and marked in all categories a 1 at pretreatment and a 4 in all categories at week 16, with a 75% overall improvement to all treated scars.

Example 6 (Patient #19/L-A) Two hypertrophic scars treated. This patient had two hypertrophic scars located superior and inferior mid abdomen. He was treated at week 1 only, finding the injections too painful. At the week 8 visit the subject stated, '"both scars are 75% overall improved and much more flat."

Claims

1. A pharmaceutical composition for the treatment of fibro-proliferative lesions comprising a therapeutically effective dosage amount of 5-FU and a therapeutically effective dosage amount of a ribozyme targeting PCNA mRNA.

2. The pharmaceutical composition of claim 1 wherein the fibro-proliferative lesions are keloids or hypertrophic scars.

3. The pharmaceutical composition of claim 1 wherein the dosage amount of 5- FU is 10-50 mg/lesion, and the dosage amount of the ribozyme is 4 mg/ml.

4. The pharmaceutical composition of claim 1 wherein the ribozyme comprises a nucleotide sequence selected from SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3.

5. Use of a therapeutic dosage amount of 5-FU and a therapeutic dosage amount of a ribozyme targeting PCNA mRNA in the manufacture of a pharmaceutical composition for the treatment of fibro-proliferative lesions.

6. The use of claim 5 wherein the fibro-proliferative lesions are keloids or hypertrophic scars.

7. The use of claim 5 wherein the dosage amount of 5-FU is 10-50 mg/lesion/treatment and the dosage amount of the ribozyme is 4 mg/ml.

8. The use of claim 5 wherein the ribozyme comprises a sequence selected from SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3.

9. A composition comprising 5-FU and a ribozyme.

10. The composition of claim 9, wherein the ribozyme targets PCNA mRNA.

11. The composition of claim 9, wherein the ribozyme comprises a nucleotide sequence selected from SEQ ID NO:l, SEQ ID NO:2 and SEQ ED NO:3.

12. A method for treating fibro-proliferative lesions in a patient, the method comprising administering to the patient at the lesions a therapeutic dosage amount of 5-FU and also administering to the patient a therapeutic dosage amount of a ribozyme. targeting PCNA mRNA

13. The method of claim 12 wherein the administration is intralesional.

14. The method of claim 12 wherein the ribozyme is administered at a site different from the site of the lesions treated with 5-FU.

15. The method of claim 12 wherein the dosage amount of 5-FU is 5-50 mg/lesion/treatment.

16. The method of claim 12 wherein the dosage amount of the ribozyme is 4 mg/treatment.

17. The method of claim 12 wherein the fibro-proliferative lesions are keloids.

18. The method of claim 12 wherein the fibro-proliferative lesions are hypertrophic scars.

19. The method of claim 12 wherein the ribozyme comprises a nucleotide sequence selected from SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3.