Carbazole derivatives for the treatment of fibrotic diseases and related symptoms, and conditions thereof

Abstract

The invention provides carbazole derivatives for the treatment of fibrotic diseases (pathological collagen deposition) in tissues and organs, and related symptoms, and conditions thereof.

Claims

1. A method of treating a disease or disorder associated with excessive collagen deposition in a patient in need thereof, said method comprising administering to said patient a composition comprising at least one active agent having the general formula (I) or any salt thereof: ##STR00012## wherein each of R.sub.1-R.sub.8 is independently selected from the group consisting of H, OH, SH, halogen, nitro, amino, nitrilo, nitroso, acetyl, acetamido, acylamido, alkylamino, straight or branched C.sub.1-C.sub.5 alkyl, straight or branched C.sub.2-C.sub.5alkenyl, straight or branched C.sub.2-C.sub.5alkynyl, amine, straight or branched C.sub.1-C.sub.5alkoxy, straight or branched C.sub.1-C.sub.5 carboxyl, straight or branched C.sub.1-C.sub.5 ester, straight or branched C.sub.1-C.sub.5thioxy, straight or branched C.sub.1-C.sub.5sulfinyl, straight or branched C.sub.1-C.sub.5thionyl; R.sub.9 is selected from straight or branched C.sub.1-C.sub.9 alkyl, straight or branched C.sub.2-C.sub.9alkenyl, straight or branched C.sub.2-C.sub.9alkynyl, piperazinyl, pyridinyl, piperidinyl, morpholinyl and thiomorpholinyl; R.sub.9 is substituted with at least one quaternary amino (ammonium) group or a phosphonium group; wherein said disease or disorder associated with excessive collagen deposition is selected from adhesion-formation following abdominal surgery, organ fibrosis of irradiation-damaged tissues, or over-reacting dermal scars (keloids).

2. A method according to claim 1, wherein R.sub.9 is a straight or branched C.sub.1-C.sub.9 alkyl substituted with at least one quaternary amino group.

3. A method according to claim 1, wherein at least one of R.sub.1-R.sub.4 is a halogen.

4. A method according to claim 1, wherein at least one of R.sub.5-R.sub.8 is a halogen.

5. A method according to claim 1, wherein at least one of R.sub.1-R.sub.4 is a halogen and at least one of R.sub.5-R.sub.8 is a halogen.

6. A method according to claim 1, wherein at least one of R.sub.1-R.sub.4 is OH.

7. A method according to claim 1, wherein at least one of R.sub.5-R.sub.8 is OH.

8. A method according to claim 1, wherein at least one of R.sub.1-R.sub.4 is a nitro and at least one of R.sub.5-R.sub.8 is a nitro.

9. A method according to claim 1, wherein said at least one active agent is being selected from: 3-(3,6-dibromo-9H-carbazol-9-yl)-N,N,N-trimethylpropan-1-aminium 5-(9H-carbazol-9-yl)-N,N,N-trimethylpentan-1-aminium 5-(2-hydroxy-9H-carbazol-9-yl)-N,N,N-trimethylpentan-1-aminium 5-(3,6-dibromo-9H-carbazol-9-yl)-N,N,N-trimethylpentan-1-aminium or any salt thereof.

10. A method according to claim 1, wherein the composition comprises water content of less than 50% of total composition weight.

11. A method according to claim 1, wherein the composition comprises at least 0.1% by weight of said at least one active agent of total composition.

12. A method according to claim 1, wherein the composition is formulated for topical application.

13. A method according to claim 1, wherein the composition is formulated for parenteral injection.

14. A method according to claim 1, wherein said disease or disorder associated with excessive collagen deposition is the irreversible excessive collagen deposition in an organ or a tissue of a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A-1C: Macroscopic view of the scars at time of 2 months sacrifice of a pig treated with RZL-012 of the invention. FIG. 1A is a view of a cross-section cut through the middle of the scar at the non-treated side (L), no internal scar-tissue can be observed. FIG. 1B is an external view of the scar at the treated side (R). FIG. 1C shows the external view of the scar at the non-treated side (L).

(2) FIG. 2A-2C: Macroscopic view of the scars at time of 2 months sacrifice of a pig treated with Kythera. FIG. 2A is a view of a cross-section cut through the middle of the scar at the non-treated side (L). White scar tissue can be clearly see along the cut (marked by a black rectangle). FIG. 2B is an external view of the scar at the treated side (R). FIG. 2C shows the external view of the scar at the non-treated side (L).

(3) FIG. 3A-3D: Scar Histology. H&E staining of cross-sections of the various scare tissues. ×10 objective magnification. FIG. 3A shows the right side RZL-012 treated scar histology. FIG. 3B shows the left side RZL-012 treated scar histology. FIG. 3C shows the right side Kythera treated scar histology. FIG. 3D shows the left side Kythera treated scar histology. While the entire width of the scar-tissue of the RZL-012 treated pig fits into the field of this magnification (FIGS. 3A-B), the scar-tissue of the Kythera treated pig is around five times wider and cannot be contained as a whole under this microscope magnification. Therefore, only small part of it can be seen in this field (FIG. 3C-D).

(4) FIG. 4A-4D: Scar Histology. H&E staining of cross-sections of the various scare tissues. ×20 objective magnification. FIG. 4A shows the right side RZL-012 treated scar histology. FIG. 4B shows the left side RZL-012 treated scar histology. FIG. 4C shows the right side Kythera treated scar histology. FIG. 4D shows the left side Kythera treated scar histology.

(5) FIG. 5A-5D: Scar Histology. H&E staining of cross-sections of the various scare tissues. ×40 objective magnification. FIG. 5A shows the right side RZL-012 treated scar histology. FIG. 5B shows the left side RZL-012 treated scar histology. FIG. 5C shows the right side Kythera treated scar histology. FIG. 5D shows the left side Kythera treated scar histology.

(6) FIG. 6A-6C: Results of Injured arteries analysis of the unilateral ureteral ligation (UUO) kidney fibrosis model of Example 2 below. FIG. 6A and FIG. 6B show the Adventitia/Muscular layers ratio (in percent) for each rat in the example (FIG. 6A) and in average (FIG. 6B). FIG. 6C shows the stained tissue sections (Masson-Trichrome) wherein the muscular layer and an outer layer, excessively rich in collagen, in the kidneys defined as “Adventitia” are visible.

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1

Comparative Experiments Showing Effect of RZL-012 of the Invention and Kybella

(7) The objective of this study is to evaluate the remote effect on local injection of RZL-012 of the invention (5-(3,6-dibromo-9H-carbazol-9-yl)-N,N,N-trimethylpentan-1-aminium) into the s.c. fat, on scar formation in pigs, compared with Kybella (an injectable formulation of Deoxycholate developed by Kythera) an agent that bluntly destroys s.c. fat.

(8) Materials 1. Kythera preparation (according to published formulation): an aqueous solution of 5% Phosphatidylcholine, 4.75% Sodium deoxycholate and 0.9% Benzyl Alcohol. 2. RZL-012 of the invention (5-(3,6-dibromo-9H-carbazol-9-yl)-N,N,N-trimethylpentan-1-aminium) of the invention preparation: 5.0% (w/v) RZL-012 in the below indicated formulation:

(9) TABLE-US-00001 Raw Material % (w/w) (g/vial) RZL-012 5.6 0.28 Tween 80 10 0.5  Propylene glycol 57 2.85 Benzyl alcohol 3 0.15 Water for Injection 24.4 1.22 Total per vial 100 5.0  Note: RZL-012 powder contains also a counter ion and some bound water molecules, amounting to around 10% of the weight.

(10) Experimental Model and Procedures

(11) Eighty kilogram pigs were injected with either Kythera or RZL-012 of the invention preparation, into the s.c. fat (1 cm depth, 27 G needle), at 10 sites, divided into two clusters, on the right flank only. The injection at each site was of a volume of 0.5 ml. In addition, a cut through the s.c. fat was made at the midline of each pig at the right and at the left flank.

(12) Animals were housed in the animal unit of Biotechfarm, Israel and fed on a diet weighing 2% of body weight (B.W.) per day divided to two meals, based on the last effective weekly body weighing.

(13) After 2 months from the day of treatment, the animals were sacrificed; the scars on both sides were photographed before and after making a cross section in the middle of each scar. Thereafter, tissue samples of all cross-sections were formalin-fixed and processed for H&E histology staining.

(14) Results

(15) FIGS. 1 and 2 show a macroscopic view of the scars at time of 2 months sacrifice. As can be seen, the scar at the left, non-treated side, is much finer in RZL-012 of the invention treated pig, compared with Kythera treated pig. Also, after a cross section through the scar, a white scar tissue is clearly evident at the Kythera treated pig (surrounded by black rectangle) while no apparent scare tissue was observed in the carbazole derivative of the invention treated pig. It can be clearly be seen that scar formed at the non-treated side of the RZL-012 treated pig is much finer than the corresponding scar of the Kythera treated pig (FIG. 1C vs. FIG. 2C).

(16) FIG. 3A-3D shows the scar histology (H&E staining of cross-sections of the various scare tissues; ×10 objective magnification). In agreement with the macroscopic view, the width of fibrotic tissue on both sides of the RZL-012 of the invention treated pig are much thinner (FIGS. 3A-3B) than those of the Kythera treated pig (FIGS. 3C-3D). Careful measurement reveals 4-5 times difference in the scar tissue width.

(17) FIG. 4A-4D shows the scar histology of the same preparations as in FIG. 3 but at ×20 objective magnification. It can be clearly seen that the scar-tissue in RZL-012 of the invention treated side (FIGS. 4A-4B) contains less collagen fibers as compared with the scar tissue of FIGS. 4C-4D.

(18) FIG. 5A-5D shows the scar histology of the same preparations as in FIG. 3 but at ×40 objective magnification. It can be clearly seen that the scar-tissue in RZL-012 of the invention treated side (FIGS. 5A-5B) contains less collagen fibers as compared with the scar tissue of FIGS. 5C-5D.

Example 2: Inhibition of Kidney Fibrosis by RZL-012

(19) Animals:

(20) Male SD Rats, 250 g.

(21) Induction of Kidney Fibrosis:

(22) Overnight fasted rats were anesthetized (a mixture of Ketamine 20 mg/kg and Xylazin 2 mg/kg IP). The abdominal cavity was exposed via a midline incision up to 0.5-1 cm before penis about 3 cm along. Then the bladder was lifted out and epididymal fat was remoted. The left ureter was ligated at two points with 4-0 sutures. The wound was closed in layers with the same sutures. Then animals were returned to the cages. This unilateral uretral ligation led to urine accumulation in the affected kidney, resulting in massive tissue fibrosis.

(23) Treatment:

(24) Test (RZL-012 15 mg) and reference (Vehicle) articles were injected at Day 14 and Day 42, following UUO, subcutaneously into the dorsal bilateral inguinal fat layer and the nape-of-the-neck fat, using a 27 G ½″ needle at a dose volume of 0.1 ml/site.

(25) Tissue Processing:

(26) 8 weeks after UUO the rats were sacrificed and the kidneys were removed, fixed and processed for histological examination. The tissue sections were stained with Masson-Trichrome which enable a distinction between fibrotic tissue (staining collagen fiber in Blue) and normal viable tissue (Purple-Red) (see FIG. 6C).

(27) Quantification of Kidney Fibrosis:

(28) In order to quantify, in an unbiased way, the fibrotic response in RZL-treated vs. non-treated rats, the dimensions of the fibrotic layer surrounding aortic blood vessel, were measured. All aortic vessels in each affected kidney were analyzed in the following way: The vessel wall consists of two components, the usual Muscular layer and an outer layer, excessively rich in collagen, in these kidneys, hereby defined as “Adventitia” (FIG. 6C). The net width of the adventitia and that of the Muscular layer were calculated as follows:

(29) $\mathrm{Adventitia}\mathrm{layer}=\frac{\left(AM1-M1\right)+\left(AM2-M2\right)}{2}$$\mathrm{Muscular}\mathrm{layer}=\frac{M1+M2}{2}$

(30) By using the ratio between Adventitia and Muscular layers, the difference among vessels diameter was normalized.

(31) Results:

(32) A total of 6 RZL-012 treated kidneys and 5 Vehicle kidneys were analyzed. The detailed results are depicted in FIG. 6A and the cumulative statistical analysis demonstrated a significant inhibition of kidney fibrosis by RZL-012 (FIG. 6B)