Compounds for treatment of heart failure

Abstract

A combination of: a first tetracycline (TC) component; and a second component capable of releasing nitric oxide (NO) or a nitrate capable of mimicking NO effects in vivo (NO mimetic). The combinations of the invention advantageously act as more effective MMP modulators with selective reductions in circulating MMP-9 levels in-vivo and inhibitory effects on MMP-2 and MMP-9 levels in-vitro. The combinations of the invention also advantageously act as modulators of inflammation mediators. The co-existence of abnormalities of MMP enzymes and inflammation in many diseases make these characteristics advantageous. Therefore, the various combinations of the invention find utility in medical applications where MMPs and/or inflammation is implicated.

Claims

1. A compound selected from: amido-N-[3-methylnitratepiperidinomethy]--6-deoxy-5-oxytetracycline amido-N-[N,N-diethylnitrate-aminomethyl]--6-deoxy-5-oxytetracycline (amido-N-[bis-(-nitrooxyethyl)aminomethyl]--6-deoxy-5-oxytetracycline) amido-N-[(-nitrooxyethyl)aminomethyl]--6-deoxy-5-oxytetracycline amido-N-[3-(nitrooxymethyl)piperidinomethyl]--6-deoxy-5-oxytetracycline amido-N-[3-(nitrooxymethyl)piperidinomethyl]--6-deoxy-5-oxytetracycline amido-N-[4-(nitrooxymethyl)piperidinomethyl]--6-deoxy-5-oxytetracycline amido-N-[4-nitrooxypiperidinomethyl]--6-deoxy-5-oxytetracycline amido-N-[4-nitrooxypiperidinomethyl]-tetracycline amido-N-[bis-(-nitrooxyethyl)methylaminomethyl]--6-deoxy-5-oxytetracyclin amido-N-[bis-(-nitrooxyethyl)methylaminomethyl]--6-deoxy-5-oxytetracyclin amido-N-[bis-(-nitrooxyethyl)ethylaminomethyl]-tetracycline amido-N-[(-nitrooxyethyl)aminomethyl]-tetracycline amido-N-[4-(nitrooxymethyl)piperidinomethyl]-tetracycline or amido-N-[3-(nitrooxymethyl)piperidinomethyl]-tetracycline.

2. The compound according to claim 1 selected from the group consisting of: ##STR00049##

3. A compound selected from the group consisting of: ##STR00050## ##STR00051##

4. A pharmaceutical composition comprising a compound according to any one of claims 1, 2, and 3 and a carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the present invention will be described, with reference to the accompanying drawings, in which

(2) FIG. 1 is a graph depicting MMP-9 activity in response to PMA, 150 M of doxycycline, 450 M of nitro amine and 150 M of MJ3-53 (Manich base dinitrate);

(3) FIG. 2 is a graph depicting MMP-2 activity in response to PMA, 150 M of doxycycline, 450 M of nitro amine and 150 M of MJ3-53 (Manich base dinitrate); and

(4) FIG. 3 is a graph depicting MMP-9 expression in patients with varying degrees of DHF.

(5) FIG. 4 demonstrates the effect of SI1003, SI1004, SI1005 and doxycycline on MMP-9 activity in PMA stimulated breast cancer cells (NC=negative control).

(6) FIG. 5 demonstrates the effect of SI1003, SI1004, SI1005 and doxycycline on pro-MMP-2 activity in PMA stimulated breast cancer cells (NC=negative control).

(7) FIG. 6 demonstrates the effect of SI1003, SI1004, SI1005 and doxycycline on MMP-2 activity in PMA stimulated breast cancer cells (NC=negative control).

(8) FIG. 7 demonstrates inhibition of MMP-2 and MMP-9 activity in response to SI1005 (MJ-169).

(9) FIG. 8 demonstrates inhibition of MMP-2 and MMP-9 activity in response to SI1004 (MJ170).

(10) FIG. 9 demonstrates the change in plasma MMP-9 levels from baseline to 72 hours following the administration of doxycycline hyclate (Group 1), SI1004 (Group 2) and SI1005 (Group 3) to groups of 6 cynomolgus monkeys

(11) FIG. 10. Effect of Doxycycline, SI1004 and SI1005 on colon cancer cell invasiveness (NC=negative control; PC=positive control).

(12) FIG. 11A. Impact of doxycycline hyclate (Doxy) and SI1004 on MMP-9 mRNA in TNF treated human cardiac fibroblasts (n=3 per group). Shaded bar is 0.1% DMSO+TNF; striped bars are 0.1% DMSO+Doxycycline hyclate (concentrations shown) and solid black bars represent 0.1% DMSO+SI1004 (concentrations shown). All values represent mean and SEM. I represents p<0.01 vs TNF, **p<0.01 vs. Doxy. All bars were significantly elevated vs. serum free controls.

(13) FIG. 11B. Impact of doxycycline hyclate (Doxy) and SI1004 on proliferation of human cardiac fibroblasts (n=3 per group) following 72 hours of serum starvation (clear bars) and subsequent exposure to 72 hours of 2% fetal calf serum (FCS) with 0.1% DMSO (shaded bars), 0.1% DMSO+Doxycycline hyclate (concentrations shown, striped bars) and 0.1% DMSO+SI1004 (concentrations shown, black solid bars). All values represent mean and SEM. represents p<0.05 vs. 2% FCS, p<0.01 vs 2% FCS, *p<0.05 vs. Doxy, **p<0.01 vs. Doxy. All bars except SI1004 150 M were significantly elevated vs. serum free controls.

(14) FIG. 12. Impact of doxycycline hyclate (striped bars) and SI1004 (solid bars) on [A] total MMP-9 and [B] total MMP-2 AUC in serum from cynomologus monkeys following daily orogastric gavage dosing for 72 hours (n=6). Doses used were 1.6 mg/kg doxycycline hyclate at time 0 and 4.8 mg/kg doxycycline hyclate at 24 and 48 hours, or the molar equivalents of SI1004.

(15) FIG. 13: Admixtures may be more effective than doxycycline in attenuating fibroblast proliferation, but not as effective as SI1004. In the following study Doxy and nitrate A are significantly better than Doxy at inhibiting Cardiac Fibroblast Proliferation (p=0.011) at 150 uM However, Doxy and nitrate B are not (p=NS) at same concentration. SI1004 is significantly more effective than doxycycline, Doxy and nitrate A, Doxy and nitrate B at 150 uM (all p<0.01).

(16) FIG. 14. Admixtures reduce some inflammatory markers similarly to Doxy, e.g. IL-8. In the following study, Doxy and nitrate A can significantly reduce IL-8 levels in TNFalpha stimulated PBMCs at 150 uM (p<0.01). Doxy alone and Doxy and nitrate B also reduce IL-8 levels compared to controls (p<0.05).

(17) FIG. 15. Admixtures reduce some inflammatory markers more effectively than doxycycline, e.g. IL-1beta. In the following study, Doxy and nitrate A can significantly reduce IL-1 beta levels in TNFalpha stimulated PBMCs (p<0.05). Doxy and nitrate B reduce IL-1 beta levels, but not significantly (p=NS).

(18) FIG. 16. Admixtures reduce some inflammatory markers more effectively than doxycycline, e.g. IL-4. In the following study, Doxy and nitrate A can significantly reduce IL-4 levels in TNFalpha stimulated PBMCs. Doxy and nitrate B admixtures reduce IL-4 levels, but not significantly (p=NS). IL-4 is reduced significantly more (p<0.01) by Doxy and nitrate A than either Doxy or Doxy and nitrate B. In this study, we see that not all NO donors provide similar efficacy.

DETAILED DESCRIPTION OF THE INVENTION

(19) Embodiments of the present invention will now be exemplified, with reference to the following non-limiting examples.

EXAMPLE 1

Preparation of Nitrate-Containing Group, N,N-Diethylnitrate Amine

(20) All materials were purchased from the Sigma-Aldrich chemical company. With reference to Scheme 1, 1.5 mL fuming nitric acid was dissolved in 10 mL DCM at 15 C. Diethanolamine (0.42 g, 4 mmole) dissolved in DCM (3 mL) was added dropwise over 20 minutes. The reaction mixture was then left stirring for a further 30 minutes before acetic anhydride (2 mL) was added to quench the reaction. The reaction was then left stirring for a further 5 minutes to form a precipitate. The precipitate was filtered washed with cold DCM and dried under vacuum to give N,N-diethylnitrate amine as a white solid.

(21) HRMS ESI+ve C4H9N3O6 [M+H] requires 196.0570, found 196.0574. 1H NMR 3.52-3.54 triplet (2CH2-O), 4.81-4.83 multiplet (2CH2-N).

(22) ##STR00034##

EXAMPLE 2

Preparation of Nitrate-Containing Group, 3-Methylnitrate Piperidine

(23) With reference to Scheme 2, 1.5 mL fuming nitric acid was dissolved in 10 mL DCM at 15 C. 3-hydroxymethyl piperidine (0.46 g, 4 mmole) dissolved in DCM (3 mL) was added dropwise over 20 minutes. The reaction mixture was then left stirring for a further 30 minutes before acetic anhydride (2 mL) was added to quench the reaction. The reaction was then left stirring for a further 5 minutes.

(24) The pH of the reaction mixture was then adjusted to 14 with 7M NaOH. The reaction mixture was then extracted with DCM (32o mL) and the combined organic extracts were washed with brine, dried over Na2SO4, filtered and solvent removed in vacuo to give 4-methyl nitrate piperidine as a pale yellow oil.

(25) HRMS ESI+ve C6H12N2O3 [M+H]+ requires: 161.0921; found: 161.0923. 1H NMR: 3.62 multiplet (CH2-ONO2), 3.35-3.20 multiplet (C2, C6 CH2), 2.19 multiplet (C3CH) 2.11-1.90 multiplet (C5CH2).

(26) ##STR00035##

EXAMPLE 3

Preparation of Amido-N[N,N-Diethylnitrate-Aminomethyl]--6-Deoxy-5-Oxytetracycline

(27) Referring to Scheme 3, N,N-di-ethylnitrate amine (0.095 g, 0.487 mmole; as prepared in Example 1) and paraformaldehyde (0.016 g, 0.487 mmole) were suspended in 10 mL isopropyl alcohol and heated to 75 C. under an inert atmosphere for 30 minutes until a clear solution was obtained. The reaction mixture was then cooled to 40 C. and doxycycline hyclate (0.250 g, 0.487 mmole), dissolved in a mixture of 5 mL isopropyl alcohol and 0.5 mL methanol, was added dropwise over 5 minutes. The reaction mixture was stirred at 40 C. for a further two hours. Upon completion of the reaction, the mixture was cooled and solvent removed to give amido-N[N,N-diethylnitrate-aminomethyl]--6-deoxy-5-oxytetracycline as a pale yellow solid.

(28) MS ESI-ve C27H33N5O14 [MH] requires 650.1951, found 650.1938. 1H NMR: 4.05 ppm singlet (Mannich methylene), 7.5, 6.95 ppm triplets and 7.85 ppm doublet (three phenyl protons) 2.9 ppm & 2.8 ppm singlets (dimethylamino, C4), 9.6 ppm singlet (amide), 3.52-3.54, triplet and 4.81-4.83, multiplet (diethyl amino nitrate).

(29) ##STR00036##
or alternative synthesis:

Amido-N-[Bis-(-Nitrooxyethyl)Aminomethyl]--6-Deoxy-5-Oxytetracycline

(30) ##STR00037##

(31) Diethanolamine-dinitrate (234 mg, 1.2 mM, 1.2 eq), doxycycline free base (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion paraformaldehyde (60 mg, 2 mM, 2 eq) were added into the reaction mixture. After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a brown microcrystalline solid (162 mg, 25%). m p.=101-104 C. Calculated for C.sub.27H.sub.32N.sub.5O.sub.14=650.2024; found (MH).sup.=650.1965. .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.2 (1H, s), 11.5 (1H, s), 9.64 (1H, s), 9.1 (1H, s), 7.54 (1H, t, J=8), 6.94 (1H, d, J=8), 6.88 (1H, d, J=8), 5.4 (1H, s), 4.8 (4H, t, J=5), 4.65 (1H, dd, J=12, 7) 4.44 (1H, dd, J=12, 7) 4.17 (1H, s) 3.46-3.44 (5H, m) 2.73-2.65 (7H, m) 2.50-2.52 (1H, m) 1.47 (3H, d, J=7). .sup.13C NMR (400 MHz, d.sub.6-DMSO) ppm: 192.5, 171.6, 161.1, 147.8, 136.6, 115.8, 115.6, 115.5, 107.2, 73.2, 71.6, 68.8, 68.6, 68.0, 67.0, 62.0, 49.8, 45.2, 44.2, 41.3, 31.2; 15.8.

(32) IR (KBr) v (cm.sup.1): 3382; 2969; 1648; 1383; 1283; 849.

EXAMPLE 4

Preparation of Amido-N-[3-Methylnitratepiperidinomethy]--6-Deoxy-5-Oxytetracycline

(33) Referring to Scheme 4, to 6-deoxy-5-oxytetracycline hyclate (0.461 g, 0.899 mmole) in anhydrous THF (10 mL) was added 3-methylnitrate piperidine (as prepared in Example 2) and 0.1 mL 37% formaldehyde solution. The reaction mixture was stirred at 40 C. for 16 hours. The reaction mixture was then cooled and solvent removed under reduced pressure to give amido-N-[4-methylnitratepiperidinomethy]--6-deoxy-5-oxytetracycline as a pale yellow solid. MS APCI C29H36N4O11 [M+NH4] requires 616.2831 found 616.2944. .sup.1H NMR: 4.05 ppm singlet (Mannich methylene), 7.5, 6.95 ppm triplets and 7.85 ppm doublet (three phenyl protons) 2.9 ppm & 2.8 ppm singlets (dimethylamino, C4), 9.6 ppm singlet (amide), 3.62 multiplet (CH.sub.2ONO.sub.2), 3.35-3.20 multiplet (C2, C6CH2), 2.19 multiplet (C3CH) 2.11-1.90 multiplet (C5CH.sub.2)

(34) ##STR00038##
or alternative synthesis:

Amido-N-[3-(Nitrooxymethyl)Piperidinomethyl]--6-Deoxy-5-Oxytetracycline

(35) ##STR00039##

(36) 3-Nitrooxymethyl piperidine (192 mg, 1.2 mM, 1.2 eq), doxycycline free base (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion of paraformaldehyde (60 mg, 2 mM, 2 eq) was added into the reaction mixture. After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a pale yellow microcrystalline solid (153 mg, 25%). Calculated for C.sub.29H.sub.35N.sub.4O.sub.11=615.2308; found (MH).sup.=615.2291. .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.3 (1H, s) 11.6 (1H, s) 10.12 (1H, s) 9.64 (1H, s) 7.54 (1H, t, J=8) 6.94 (1H, d, J=8) 6.88 (1H, d, J=8) 5.72 (1H, s) 4.65 (1H, dd, J=12.7) 4.45-4.30 (3H, m) 4.07 (1H, s) 3.22-3.35 (2H, m) 2.98-2.89 (2H, m) 2.80-2.65 (8H, m) 2.15-1.96 (2H, m) 1.89-1.75 (2H, m) 1.52-1.4 (4H, m).

(37) ##STR00040##

EXAMPLE 5

Preparation of 6-Deoxy-5-Oxytetracycline Nitrate Salt

(38) With reference to Scheme 5, a nitrate salt is prepared by adding silver nitrate (0.50 g, 2.96 mmole) to a solution of doxycycline hydrochloride (1.52 g, 2.96 mmole) in acetonitrile (20 mL). The solution is then stirred at room temperature for 30 minutes.

(39) After 30 minutes, a white precipitate of silver chloride was removed by filtration to leave a pale yellow solution. This solution was added dropwise to cold diethyl ether (100 mL) to form a pale yellow precipitate that was filtered, washed with cold diethyl ether, and dried under vacuum.

(40) HRMS ESI C22H24N2O8 [M]+ requires 445.1605, found 445.1602.

(41) ##STR00041##

EXAMPLE 6

Docycycline-5-Nitrate

(42) A solution of doxycyline (414 mg, 1 mmol) in 5 ml THF was added to the solution of Cu(NO.sub.3).sub.2 (750 mg, 3 mmol) in 15 ml of acetic anhydride, which had been reacted for 2 h at room temperature. After reacted at 10 C. for 3 hour, the reaction mixture was filtered. The solvent of the filtrate was removed and dried under vacuum to give an amber solid (215, 44%). Calculated for C.sub.22H.sub.24N.sub.3O.sub.10=490.1456; found (M+H).sup.+=490.1469.

(43) ##STR00042##

EXAMPLE 7

(44) 4-Nitrooxymethyl piperidine (192 mg, 1.2 mM, 1.2 eq), doxycycline free base (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion of paraformaldehyde (60 mg, 2 mM, 2 eq) was added into the reaction mixture. After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a pale yellow microcrystalline solid (128 mg, 21%). m p.=130-132 C. Calculated for C.sub.29H.sub.35N.sub.4O.sub.11=615.2308; found (MH).sup.=615.2277. .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.3 (1H, s) 11.6 (1H, s) 9.64 (1H, s) 9.1 (1H, s) 7.54 (1H, t, J=8) 6.94 (1H, d, J=8) 6.88 (1H, d, J=8) 5.72 (1H, s) 4.65 (1H, dd, J=12, 7) 4.45-4.30 (3H, m) 4.07 (1H, s) 3.22-3.35 (2H, m) 2.98-2.89 (2H, m) 2.73-2.65 (7H, m) 2.50 (1H, m) 2.15-1.96 (2H, m) 1.89-1.75 (2H, m) 1.52-1.4 (5H, m). .sup.13C NMR (400 MHz, d.sub.6-DMSO) ppm: 192.5, 171.6, 161.1, 147.8, 136.7, 115.8, 115.6, 115.5, 107.1, 76.4, 68.9, 68.2, 66.6, 50.7, 45.3, 41.6, 38.4, 31.2, 26.6, 15.8.

(45) IR (KBr) v (cm.sup.1): 3401; 29769; 1634; 1383; 1279; 867.

EXAMPLE 8

(46) Amido-N-[4-nitrooxypiperidinomethyl]--6-deoxy-5-oxytetracycline

(47) ##STR00043##

(48) 4-nitrooxypiperidine (175 mg, 1.2 mM, 1.2 eq), doxycycline free base (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion of paraformaldehyde (60 mg, 2 mM, 2 eq) was added into the reaction mixture.

(49) After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a pale yellow microcrystalline solid (192 mg, 32%). Calculated for C.sub.28H.sub.33N.sub.4O.sub.11=601.2151; found (MH).sup.=601.2152. .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.3 (1H, s) 11.6 (1H, s) 9.64 (1H, s) 7.54 (1H, t, J=8) 6.94 (1H, d, J=8) 6.88 (1H, d, J=8) 5.72 (1H, s) 5.27-5.30 (m, 1H) 4.65 (1H, dd, J=12, 7) 4.43 (1H, dd, J=12, 7) 4.07 (1H, s) 3.22-3.25 (4H, m) 2.73- 2.65 (7H, m) 2.50 (1H, m), 1.88-1.91 (2H, m) 1.47 (3H, d, J=7).

EXAMPLE 9

(50) Amido-N-[3-(nitrooxymethyl)piperidinomethyl]--6-deoxy-5-oxytetracycline

(51) ##STR00044##

(52) 3-Nitrooxymethyl piperidine (192 mg, 1.2 mM, 1.2 eq), doxycycline free base (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion of paraformaldehyde (60 mg, 2 mM, 2 eq) was added into the reaction mixture. After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a pale yellow microcrystalline solid (171 mg, 28%). Calculated for C.sub.29H.sub.35N.sub.4O.sub.11=615.2308; found (MH).sup.=615.2305.

EXAMPLE 10

(53) Amido-N-[(-nitrooxyethyl)aminomethyl]--6-deoxy-5-oxytetracycline

(54) ##STR00045##

(55) 1-Methylaminoethyl nitrate (200 mg, 1.88 mM, 1.2 eq), doxycycline free base (700 mg, 1.55 mM, 1.0 eq) and paraformaldehyde (93 mg, 3.1 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion paraformaldehyde (93 mg, 3.1 mM, 2 eq) were added into the reaction mixture. After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a pale yellow microcrystalline solid (261 mg, 30%). Calculated for C.sub.26H.sub.31N.sub.4O.sub.11=575.1995; found (MH).sup.=575.2032. .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.2 (1H, s), 11.5 (1H, s), 9.64 (1H, s), 9.64 (1H, s), 7.54 (1H, t, J=8), 6.94 (1H, d, J=8), 6.88 (1H, d, J=8), 5.4 (1H, s), 4.8 (2H, t, J=5), 4.65 (1H, dd, J=12, 7) 4.44 (1H, dd, J=12, 7) 4.17 (1H, s) 3.46-3.44 (3H, m) 2.73-2.65 (7H, m) 2.50-2.52 (1H, m) 1.47 (3H, d, J=7).

EXAMPLE 11

(56) Amido-N-[4-nitrooxypiperidinomethyl]-tetracycline

(57) ##STR00046##

(58) 4-nitrooxypiperidine (175 mg, 1.2 mM, 1.2 eq), tetracycline free base (414 mg, 1 mM, 1.0 eq) and paraformaldehyde (60 mg, 2 mM, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to refluxing for 2 h under nitrogen environment. Then another portion of paraformaldehyde (60 mg, 2 mM, 2 eq) was added into the reaction mixture. After refluxing for another 2 h, the reaction mixture was cooled to room temperature and filtered. The filtrates were collected and the solvent was removed. The resulting solids were dried under vacuum to afford the title compound as a brown microcrystalline solid (216 mg, 36%). Calculated for C.sub.28H.sub.33N.sub.4O.sub.11=601.2151; found (MH).sup.=601.2147. .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.3 (1H, s) 11.6 (1H, s) 9.64 (1H, s) 9.64 (1H, s) 7.54 (1H, t, J=8) 7.1 (1H, d, J=8) 6.93 (1H, d, J=8) 5.27-5.30 (m, 1H) 5.10 (1H, s) 4.65 (1H, dd, J=12, 7) 4.43 (1H, dd, J=12, 7) 4.07 (1H, s) 3.22-3.25 (4H, m) 2.65-2.73 (7H, m) 2.50 (1H, m), 2.04-2.13 (2H, m) 1.88-1.91 (2H, m) 1.53 (3H, s).

EXAMPLE 12

(59) Amido-N-[bis-(-nitrooxyethyl)aminoethyl]--6-deoxy-5-oxytetracycline

(60) ##STR00047##

(61) Diethanolamine dinitrate (195 mg, 1 mmol, 1 eq) doxycycline free base (450 mg, 1 mmol, 1 eq) and acetaldehyde (110 uL, 88 mg, 2 eq) were dissolved in anhydrous tetrahydrofuran (10 ml) and heated to reflux for 2 hours under nitrogen environment. A further 2 equivalents of acetaldehyde were added to the reaction mixture and the reaction continued for a further 2 h. The reaction mixture was then cooled to room temperature and filtered. THF was removed from the filtrate via rotary evaporation and the resultant residue was dried under vacuum to give an amber solid. (235 mg, 35%) .sup.1H NMR (400 MHz, d.sub.6-DMSO) 15.2 (1H, s), 11.5 (1H, s), 9.64 (1H, s), 7.54 (1H, t, J=8), 6.94 (1H, d, J=8), 6.88 (1H, d, J=8), 5.4 (1H, s), 4.57 (4H, t, J=5), 4.44, 1H, dd, J=12, 7) 4.17 (1H, s) 3.46-3.44 (5H, m) 2.73-2.65 (7H, m) 2.50-2.52 (1H, m) 1.78 (3H, d, J=7) 1.47 (3H, d, J=7).

EXAMPLE 13 & 14

(62) Doxycycline-12a-nitrate; and minocycline-12a-nitrate; both of which may be prepared by mild nitration under acidic conditions. In vitro pharmacological evaluation

(63) Cells (CaCo2 cells) were seeded onto a 12-well plate, and allowed to grow to 70% confluence. When cells were 70% confluent, the media on the cells were replaced with serum-free media. Cells were then treated with increasing concentrations of test compound (50 M-250 M), for 3 hours in a 37 C. incubator. After 3 hours, 10 M PMA (Phorbol 12-myristate 13-acetate) was added to the cells to induce production of MMPs. Cells were incubated for 24 hours in a 37 C. incubator. After 24 hours, the media from each well were collected and centrifuged at max speed for 5 minutes to pellet any cellular debris, and the media was removed to fresh microfuge tubes. A Bradford assay was conducted to determine the protein concentration of each media sample. An equal protein concentration of each media sample was loaded onto a zymography gel, which was run for 150V/2 hours. Following this, the zymography gel was washed three times for 20 minutes in 2.5% Triton X Buffer and was washed 2 times in zymography buffer before being incubated in zymography buffer at 37 C. for 48-72 hours to allow any MMP9 and MMP2 present to digest the gelatinase in the gel. Following this, the gels were stained in coomassie blue stain for 3 hours with gentle rocking and destained for 1 hour, resulting in a blue gel with clear bands where MMP's that were present had digested through the gelatine in the zymography gels. Densitometry analysis was performed to quantitate the amount of MMPs present relative to the PMA positive control sample. Referring to FIG. 1, addition of 150 uM of doxycycline did not affect MMP-9 levels. N,N-diethylnitrate amine, at equimolar concentrations to the Mannich base dinitrate (amido-N[N,N-diethylnitrate-aminomethyl]--6-deoxy-5-oxytetracycline) on its own, inhibited MMP-9 by over 50%. However, the combination of doxycycline with the nitrate amine amido-N[N,N-diethylnitrate-aminomethyl]--6-deoxy-5-oxytetracycline] (MJ3-53) suppressed MMP-9 activity by approximately 60%. Referring to FIG. 2, it can be seen that MMP-2 activity was significantly inhibited by doxycycline (80%), and the combination with amido-N[N,N-diethylnitrate-aminomethyl]--6-deoxy-5-oxytetracycline reduced MMP-2 activity by about 40%. These data demonstrate that a compound of the present invention is capable of significantly altering MMP expression, and finds utility in treating or preventing heart failure, optionally heart failure caused by or associated with diastolic dysfunction; where MMP-9 levels are three times higher in the advanced stages compared with mild DHF. In DHF, MMP2 is 40 to 50% higher and MMP9 is 200-300% higher in heart failure patients than in asymptomatic hypertensive patients. In the present example, surprisingly, the amido-N[N,N-diethylnitrate-aminomethyl]--6-deoxy-5-oxytetracycline inhibits MMP9 more than by the constituent doxycycline and N,N-diethylnitrate amine. Moreover, the pattern of MMP 2 and MMP9 inhibition may be more beneficial than doxycycline alone, which, in these examples, did not reduce MMP9.

(64) In-vitro/in-vivo Effects of Doxycycline and SI1004

(65) The purpose of this study was to evaluate the in-vitro/in-vivo effects of doxycycline and SI1004, a novel NO-releasing analogue of doxycycline which could be applied to the treatment of disorders associated with elevated MMP-9 including ALVDD and HFpEF.

(66) Methods

(67) Direct Inhibition of Recombinant MMP-2 and MMP-9 with SI1004 and Doxycycline.

(68) Nitrocycline, SI1004, a dinitroxyethyl conjugate with doxycycline was prepared in-house using conventional chemical approaches and characterised by .sup.1H, .sup.13C NMR, High Resolution Mass Spectroscopy and High Performance Liquid Chromatography. Doxycycline hyclate was obtained from Sigma-Aldrich Ireland. In order to determine the relative direct inhibitory effects of SI1004 and doxycycline on MMP-2 and MMP-9 we used human recombinant enzymes (R&D Systems, Ireland) with the synthetic broad-spectrum fluorogenic substrate (7-methoxycoumarin-4-yl)-acetyl-pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl)-Ala-Arg-NH.sub.2 (R&D Systems, UK) as previously described (34).

(69) Effects of SI1004 and Doxycycline on Human Ventricular Cardiac Fibroblast (HCF) proliferation and on TNF- treated HCF MMP-2 and MMP-9 transcription.

(70) The impact of SI1004 and doxycycline on MMP-2 and MMP-9 transcription was evaluated in primary HCFs purchased from ScienCell Research Laboratories. Cells were cultured in Dulbecco's modified eagles medium (DMEM) (Gibco), supplemented with 10% Fetal Calf Serum (FCS) (Gibco) and penicillin-streptomycin antibiotics (Gibco) in a 5% CO.sub.2 humidified incubator kept at 37 C. To investigate effects of test articles on cell proliferation, HCF cells were serum starved for 72 hours and then treated with either 75 or 150 M of test article in DMSO in 2% FCS for a further 72 hours. Cell viability was measured using the CellTitre-Glo Luminescent Cell Viability Assay (Promega) which measures ATP as an indicator of the number of metabolically active cells. To investigate the relative effects of doxycycline and SI1004 on TNF treated HCF transcription of MMP-2 and MMP-9, cells were treated with 10 ng/mL human recombinant TNF (R&D Systems) for 72 hours in the presence of 75 M or 150 M of test article in DMSO. RNA was isolated using a NucleoSpin RNA II Kit (Macherey-Nagel). First strand cDNA synthesis was carried out using SuperScript II RT (Invitrogen). QPCR primers were designed so that one of each primer pair was exon/exon boundary spanning to ensure only mature mRNA was amplified. The sequences of the gene-specific primers used are as follows; MMP-2, 5-CACGTGACAAGCCCATGGGGCCCC-3 (forward), 5-GCAGCCTAGCCAGTCGGATTTGATG-3 (reverse); MMP-9,5-GTGCTGGGCTGCTGCTTTGCTG-3 (forward), 5-GTCGCCCTCAAAGGTTTGGAAT-3 (reverse). QPCR reactions were normalized by amplifying the same cDNA with GAPDH primers, 5-ACAGTCAGCCGCATCTTCTT-3 (forward), 5-ACGACCAAATCCGTTGACTC-3 (reverse). QPCR was performed using Platinum SYBR Green qPCRSuperMix-UDG (Invitrogen). Amplification and detection were carried out using the Mx3000P System (Stratagene). The PCR cycling program consisted of 40 three-step cycles of 15 seconds/95 C., 30 seconds/TA and 30 seconds/72 C. Each sample was amplified in duplicate. In order to confirm signal specificity, a melting program was carried out after the PCR cycles were completed. The samples were quantified by comparison with a standard calibration curve created at the same time and the data was normalized by an internal control (glyceraldehyde 3-phosphate dehydrogenase).

(71) Effects of SI1004 and Doxycycline on MMPs, TIMP-1 and Inflammatory Markers in Human Peripheral Blood mononuclear cells (PBMC) stimulated with TNF-.

(72) To further explore the relative impact of SI1004 and doxycycline on inflammatory cells (PBMC), venous blood (30 mL) was collected from three healthy volunteers (age 30-37) in 10 mL S-Monovette tubes with anti-coagulant 9NC (Sarstedt). The blood was mixed with an equal volume of D-PBS (Gibco) and two volumes of the mixture were layered over one volume of Lymphoprep gradient solution (Axis-Shield). PBMC were isolated by centrifugation at 400 g for 40 minutes. PBMC were collected from the plasma/lymphoprep interface and washed three times in D-PBS/0.1% BSA/2 mM EDTA. PBMC were suspended at 110.sup.6 cells/mL in pre-warmed RPMI 1640/10% FCS/2 mM L-glutamine/100 g/mL penicillin G/100 g/mL Streptomycin (all from Gibco). Cells (0.210.sup.6) were plated at a concentration of 1.010.sup.6 in 96-well plates in duplicates, stimulated with 10 ng/mL TNF (R&D Systems) with/without doxycycline hyclate or SI1004 (at 75 and 150 M) and incubated for 24 hours at 37 C. On the following day, all samples were centrifuged and supernatants were stored at 80 C. for immunoassays. Percent PBMC viability following drug treatment was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer instructions. The cytokine profile of the cell supernatants was analysed using an ultra-sensitive immunoassay with electrochemiluminescence detection according to the manufacturer's instructions (Meso Scale Discovery). MMP secretion was also quantified using multiplex immunoassays with electrochemiluminescence detection as instructed by the manufacturer (MMP2/10 Duplex and MMP1/3/9 Triplex assaysMesoScale Discovery). Single-plex assays were used for monocyte chemotactic protein (MCP)-1 (Meso Scale Discovery). Plates were analyzed using a Meso Scale Discovery Sector Imager 2400 instrument. Secreted TIMP-1 was quantified using a standard ELISA (Amersham, GE Healthcare). TH1/TH2 10-plex assay was used to study IFN, IL-1, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, and TNF. The sensitivity (lowest level of detection) of the assays was 0.12 ng/mL and 0.1 ng/mL for MMP-2 and MMP-9, respectively. The coefficient of variation of the lower limit of the standard curve for MMP-2 and MMP-9 was 4.9% and 1.2% respectively. Plates were analyzed using a Meso Scale Discovery Sector Imager 2400 instrument.

(73) Relative Effects of SI1004 and Doxycycline on Total MMP-2 and MMP-9 Levels on Acute and Repeated Oral administration over three days with dose titration following day one in non-human primates (NHP).

(74) A total of 12 purpose bred, purpose bred, nave, non-human primates (cynomolgus monkeys, 2.9-4 kg) were sourced and randomly allocated in a parallel group design (n=6 per group) to receive SI1004, SI1005 and equimolar doses of doxycycline daily (1.6 mg/kg doxycycline hyclate equivalents, on day 1 and 4.8 mg/kg doxycycline equivalents on days 2 and 3) by oral gavage in aqueous vehicle over a 3 day period. Studies were carried out consecutively in two contract research organization sites (Charles River, Sparks, Nev., US and Charles River, Shanghai, China). The study protocol was approved by PCS-SHG Institutional Animal Care and Use Committee before conduct. During the study, care and use of animals was conducted in accordance with the guidelines of the USA National Research Council and the Canadian Council on Animal Care. The cynomolgus monkey was chosen for this study in order to maximize the likelihood of identifying responses that are similar to those that may be expected in humans. Each animal was identified by a cage label and body tattoo and was acclimated to orogastric dosing on at least two occasions prior to the initiation of dosing. The vehicle (1% (w/v) tween 80 and 0.5% (w/v) carboxymethylcellulose in deionized water) or 1.6 mg/kg doxycycline hyclate (0 hours) or 4.8 mg/kg doxycycline hyclate (24, 48 hours) or the molar equivalent(s) of SI1004 or SI1005 were administered using an orogastric tube inserted through the mouth and advanced into the stomach. The animals were temporarily restrained (i.e. manually) for dose administration, and were not sedated. Disposable sterile syringes and orogastric tubes were used for each animal/dose. Each dose was followed by a tap water flush of approximately 5 mL. Blood samples and blood pressure measurements were taken at the following timepoints: pre-dose (0 hours) and at 2, 4, 6, 12, 24, 26, 30, 36, 48, 50, 54, 60 and 72 hours after first administration of test article. We have previously demonstrated an acute phase response in this model to repeated venepuncture (3-6 fold increase in high sensitivity C-reactive protein from baseline at 12 and 24 hours post dose respectively, (both p=0.01 vs baseline), data not shown). Blood (300 L) for serum preparation was collected intoBD Vacutainer+Serum SST tubes to accelerate clotting 20 minutes prior to centrifugation to allow complete clotting to occur and centrifuged at 1500-2200 rpm at 2-8 C. for 10-15 minutes. Under these conditions blood cells containing MMP, principally neutrophils and platelets, undergo full degranulation. Since artifactual elevation of MMP-9 was an unavoidable feature of repeated venipuncture in our model, it was logical to stimulate full MMP-9 release during sample collection. This provided greater inter-animal reproducibility and a more dynamic analytical range for assessing the relative effects of the test articles. Subsequent MMP-9 values provide an index of total MMP-9 including circulating enzyme, amplified by repeated venipuncture, along with the cellular load released from storage granules during clotting. The latter is influenced by earlier inflammatory signaling, transcription and storage. The serum was transferred to a cryovial and immediately stored at 70 C. until analyzed for MMP-2 and MMP-9 via a Luminex ELISA (total MMP-2 and MMP-9) within 48 hours of collection. The analysis of each time point was repeated within 5 days. Values that differed by more than 15% were repeated. The primary study endpoint was the change in plasma MMP-2 and MMP-9 levels at 72 hours. Secondary endpoints were area under the curve (AUC) values of MMP-2 and MMP-9 over the following periods: 0-24, 0-48 and 0-72 hours. Additional 0.4 mL aliquots were placed in K.sub.2EDTA tubes and processed to plasma for combined nitrate/nitrate (NO.sub.x) analysis using a modified Greiss assay as previously described (35). Simultaneous blood pressure measurements were made in triplicate using a femur cuff linked to an automated Omron analyzer. Data are presented as meanstandard error of the mean (SEM) for continuous normal variables, median, interquartile range (IQR) with 95% confidence intervals for non-normal continuous variables and frequencies and percents for nominal/categorical variables. Comparisons between doxycycline and SI1004 groups in the NHP study were made on changes over the study period using independent two-sample t-tests for continuous normally distributed data, Mann-Whitney for skewed continuous and chi-squared for categorical data. Within group tests, comparing baseline to 24, 48 and 72 hour values, were conducted using paired sample t-tests and paired sample Wilcoxon tests where appropriate. Analyses were carried out using SPSS V.13 statistical software (Statistical Package for the Social Sciences: SPSS Inc, Chicago, Ill., 2001).

(75) Results

(76) Effects of Doxycycline and SI1004 on Activity of Recombinant Human MMP-2 and MMP-9

(77) Doxycycline and SI1004 had similar direct inhibitory effects on MMP-2 and MMP-9 enzymatic activity. Doxycycline (100 M) inhibited recombinant human MMP-2 (34.03.5%) and MMP-9 (33.33.5%) (p<0.05). Similarly SI1004 (100 M) inhibited MMP-2 and MMP-9 by 29.72.1% and 26.61.7% respectively (p<0.05). However, there was no direct inhibition of either enzyme by the test articles at 10 M. These values suggest weak, non-selective inhibition of both gelatinases at enzyme level and are consistent with doxycycline's low binding affinity for the MMPs.

(78) Effects of Doxycycline and SI1004 on Human Cardiac Fibroblasts

(79) In contrast to doxycycline hyclate, SI1004 significantly inhibited TNF induced upregulation of MMP-9 mRNA (p=0.01, FIG. 1A). MMP-9 protein levels were below the lower limit of quantification in doxycycline hyclate or SI1004 treated cell supernatants. There were no significant effects of doxycycline hyclate or SI1004 on MMP-2 mRNA expression. Also, unlike doxycycline, SI1004 (75-150 M) caused significant inhibition of HCF proliferation in 2% FCS following serum starvation for 72 hours, (p=0.02, FIG. 1B).

(80) Effects of Doxycycline and SI1004 on Markers of Inflammation and Collagen Turnover in Human Peripheral Blood Mononuclear Cells

(81) The effects of doxycycline hyclate and SI1004 on MMPs, TIMP-1, inflammatory cytokines and MCP-1 are presented in Table 1. Both compounds significantly inhibited PBMC supernatant MMP-9, TIMP-1, IFN, IL-8, IL-12p70 and MCP-1 (all p<0.05). SI1004 (150 M) but not doxycycline, inhibited IL-1 production at 150 M (p=0.03). Doxycycline inhibited TIMP-1 to a greater extent than SI1004 at both concentrations (p<0.05) and doxycycline, but not SI1004, inhibited MMP-3 (p<0.02).

(82) Plasma MMP-2 and MMP-9 Levels Over 72 Hours with Daily Dosing of Doxycycline and SI1004 in Cynomolgus Monkeys

(83) Oral administration of SI1004 caused more effective suppression of total serum MMP-9 protein levels than doxycycline (FIG. 12A). Between-group differences were significant by day 2 and remained significant on day 3 in terms of AUC (24-48 and 48-72 hours) and also in terms of MMP-9 change from baseline at 48 and 72 hours (all p<0.05). Total MMP-2 levels were similar over the 3-day treatment period (FIG. 12B). Maximum plasma doxycycline concentration (Cmax) was noted on day 3 of dosing where plasma doxycycline concentration achieved 5.1 M (base equivalents). SI1004 caused an increase in mean plasma nitrite/nitrate (NOx) over the duration of the dosing period, with peaks at 6 hours post-dosing (i.e. at 6, 30 and 54 hours) consistent with activation of the SI1004 nitrate group and NO release. NOx Cmax (g/mL) for SI1004 was 12.12.2, 47.92.2, and 50.412.5, on days 1, 2 and 3 respectively (all at 6 hours post dose). Although the mean systolic blood pressure was higher in the doxycycline hyclate group (109.77.1 mmHg vs 1016.3, p<0.01), there was no difference in diastolic blood pressure (58.66.0 mmHg vs 56.43.8 mmHg, p=NS) and the pattern of NO release was not associated with significant differences in blood pressure (either systolic or diastolic) or heart rate at any time point.

(84) TABLE-US-00001 TABLE 1 Impact of doxycycline hyclate and SI1004 on MMPs, TIMP-1, interleukins and MCP-1 protein levels in supernatants of PBMC treated over 24 hours (n = 3). All values are mean SEM (ng/mL) Doxycycline Doxycycline SI1004 SI1004 Control (150 M) (75 M) (150 M) (75 M) No.(%)/Mean SD No.(%)/Mean SD Interleukin-1 40.8 6.2 30.6 5.0 34.6 5.8 24.6 1.2 27.8 6.6 Interleukin-4 12.0 0.6 9.8 0.8 9.4 1.2 9.6 0.6 10.8 0.2 Interleukin-5 49.8 11.6 31.2 5.8 40.8 9.0 35.6 6.4 35.8 8.4 Interleukin-8 15322 264 6352 1438 8852 2568 8826 1364 10960 484 Interleukin-10 121.2 51.2 92.2 33.8 136.6 74.2 142.0 55.2 125.6 32.2 Interleukin-12p70 19.8 1.0 14.2 2.0 16.2 2.4 15.2 1.0 16.6 1.2 Interleukin-13 106.6 0.6 80.2 3.2 79.6 17.2 89.4 13.2 80.8 24.8 MCP-1 598 338 30.0 8.0 92.0 54.0 40.0 14.0 146 54 Interferon 124.4 6.8 86.6 8.8 99.5 15.0 100.2 7.6 102.8 4.8 MMP-1 262 132 134 48 144.0 80.0 171 66.0 208 102 MMP-2 126.0 104.6 96.8 80.6 61.4 54.2 95.4 52 120.4 86.4 MMP-3 11.3 2.8 2.9 0.4 1.5 1.5 9.1 6.1* 8.7 5.2* MMP-9 29.4 7.6 1.3 0.6 3.4 1.6 8.4 2.1 ** 18.4 7.3 * MMP-10 84.4 25.2 47.6 15.8 36.0 14.0 29.0 14.6 26.6 18.4 TIMP-1 56.0 4.2 6.8 2.4 12.6 1.6 22.0 9.2 * 32.4 9.4 * All values represent mean and SEM. represents p < 0.05 vs. TNF treated controls, p < 0.01 vs TNF treated controls, *p < 0.05 vs. Doxy, **p < 0.01 vs. Doxy. Abbreviations: MCP = monocyte chemotactic protein, MMP = matrix metalloproteinase, TIMP = tissue inhibitor of matrix metalloproteinase.

(85) SI1004 and doxycycline have low binding capacity to MMP-2 and MMP-9 enzymes at concentrations achieved in-vivo. Both compounds inhibit TNF induced MMP-9, TIMP-1, IFN, IL-8, IL-12p70 and MCP-1 expression in PBMC. Unlike doxycycline, SI1004 inhibits IL-1 and also TNF induced MMP-9 mRNA in HCF and HCF proliferation. SI1004 has similar effects on MMP-2 in-vivo and more effectively reduces total plasma MMP-9 (median AUC 4.3 g/mL.hour, IQR 3.1-5.5) than doxycycline (median AUC 8.7 g/mL.hour, IQR 7.3-11.3, p<0.05 vs doxycycline) in NHPs.

(86) Conclusions: This study demonstrates that doxycycline and SI1004 are immunemodulatory MMP inhibitors. SI1004 provides more effective inhibition of inducible MMP-9 than doxycycline.

(87) Discussion

(88) HFpEF accounts for 40-60% of all cases of HF and is set to increase with continued high prevalence of ALVDD driven principally by hypertension and diabetes. Experience to date with renin-angiotensin-aldosterone system (RMS) modifying therapies suggests that novel therapeutic approaches are needed. While RMS modifying therapies have shown anti-fibrotic effects, several lines of in-vitro and in-vivo evidence point to co-existing inflammation and ECM remodeling as key drivers of HFpEF pathophysiology. ECM remodeling is regulated by myocardial MMPs and TIMPs which have been elusive pharmacological targets in the clinic. The present study provides a pharmacological and pathophysiological rationale for further evaluation of immunomodulatory, broad-spectrum MMP inhibitor doxycycline and its novel NO-releasing analogue (SI1004) as components of an anti-remodeling strategy in ALVDD and HFpEF. Furthermore, SI1004 reduces transcription of inducible myocardial MMP-9 and total MMP-9 in-vivo more effectively than doxycycline and this may provide efficacy and safety advantages in chronic therapy. Abnormalities in the cardiac interstitium are central to the pathophysiology of ALVDD and HFpEF. These abnormalities include delayed relaxation, impaired left ventricular filling and/or increased stiffness in the myocardium. Myocardial remodeling is characterized by inflammation, fibrosis (increased collagen production, reduced collagen breakdown, alterations in the relative balance of collagen I/III, changes in the biomechanical properties of myocardial collagen) and alterations in other components of the ECM such as fibronectin, laminin and elastin. Modulation of the cardiac interstitium in pressure/volume overload is partially regulated by MMPs and TIMPs and recent human studies have associated serum and tissue myocardial MMP levels with increased arterial stiffness in patients with hypertension, hypertrophic obstructive cardiomyopathy, diastolic dysfunction and HFpEF. Supporting these observations are animal studies showing MMP-9 and its tissue inhibitor, TIMP-1, are associated with the transition from hypertrophy to HF the development of diastolic dysfunction and HFpEF in models of chronic pressure-overload. MMP-2 and MMP-9 knockout mice develop less marked cardiomyocyte hypertrophy and fibrosis following transverse aortic banding and pharmacological MMP inhibition prevents ventricular remodeling and HF in pressure overload states, including HF induced by inflammatory cytokines. However, direct pharmacological inhibition of MMPs has been unsuccessful as a chronic therapy in the clinic. Over 60 MMP-binding inhibitors have been tested, primarily in cancer and heart disease, with consistently disappointing efficacy or unacceptable side-effect profiles. The 24 human MMPs and their TIMPs also contribute to a large array of important physiological processes. Thus, for example, chronic, direct inhibition of collagenases may actually facilitate myocardial fibrosis in pressure overload states. It is important to note that MMP-2 has collagenase activity and activates other collagenases, unlike MMP-9, suggesting it may have role in the attenuation of excess collagen deposition in the myocardium. Conversely, MMP-9 basal activity is normally low but its gene contains binding sites for AP-1, NF-B, Sp-1, Ets-1 and Egr-1. Global deletion of MMP-9, endows mice with a benign phenotype in the absence of pathophysiological stress. However, following induction of myocardial infarction, MMP-9 knockout mice demonstrate reduced macrophage infiltration, left ventricular dilation and collagen accumulation as well as increased vascularity and perfusion. Taken together, these data indicate that pharmacological attenuation of inducible myocardial MMP-9 and MMP secretion without chronic direct enzyme inhibition could be an effective and/or safer therapeutic approach in patients with ALVDD and HFpEF. Doxycycline is the only therapy licensed for human use as a MMP inhibitor, in the setting of periodontal disease, and is currently under investigation by our group in ALVDD and HFpEF (EudraCT number: 2010-021664-16). As well as direct inhibitory effects on a range of MMPs, doxycycline also inhibits the acute phase MMP-9 release from tertiary granules in neutrophils. The present study suggests that doxycycline has low binding capacity for myocardial MMP at plasma levels achieved in this study and during chronic human dosing (<10 M) and this may be an advantage in terms of long term safety at conventional doses. Furthermore, the effect of doxycycline and SI004 on IFN and IL-12p70 secretion by TNF stimulated PBMCs suggests a reduced capacity to promote T cell activation. Both agents also suppress IL-8 and MCP-1 secretion from activated PBMCs indicating an ability to inhibit neutrophil and monocyte chemotaxis. These data are in accordance with previous in-vivo evidence of doxycycline suppression of neutrophil and cytotoxic T cell accumulation in the aortic wall of patients undergoing elective aneurysmal repair. Given the emerging importance of inflammation in the early phases of HFpEF and the potentially causal role of MCP-1 in the recruitment of monocytes and initiation of interstitial fibrosis in animal models of pressure overload our data suggest a beneficial anti-inflammatory role for doxycycline and SI004 in ALVDD and HFpEF. The additional effects of NO release on pro-inflammatory stimuli and gelatinase activity may amplify doxycycline's inhibitory effect in the setting of HFpEF. Doxycycline reduces NO and peroxynitrite levels in multiple cell types stimulated with inflammatory cytokines, partly through inducible nitric oxide synthase (iNOS) inhibition. It is also known that intracellular NO formation can suppress IL-1 by inhibiting caspase-1, the IL-1 converting enzyme which may explain the significant reduction of SI1004 on this inflammatory cytokine. NO can also affect the cellular distribution and compartmentalization of MMP-9, decrease MMP-9 mRNA stability and inhibit its transcription via effects on AP-1, NFB and PEA3 promoter activity. Furthermore, vascular NO is depleted in hypertension and NO has well-known effects on vascular smooth muscle cells, activating guanylate cyclase and increasing the formation of cyclic guanosine monophosphate (cGMP), causing vasorelaxation, reduced pulse wave reflection and reduced central aortic pressure. NO and cGMP releasing substances are associated with an improvement in diastolic relaxation that suggest a beneficial effect in diastolic HF. A final potential advantage of nitrocycline is that while short and long-term use of doxycycline can cause gastro-eosophageal irritation, NO is gastroprotective and NO donor groups can increase the intestinal tolerability and safety of a number of drugs. The present study identifies a number of key differences between SI1004 and its parent molecule. Of potential importance in myocardial remodeling is that SI1004 has superior efficacy on MMP-9 mRNA in TNF stimulated HCF. SI1004 may have less inhibitory effects on TIMP-1 and MMP-3 which are associated with the attenuation of myocardial remodeling and increased scar volume after myocardial injury. By processing samples to serum with complete clotting, which causes degranulation of PBMCs and platelets, we obtained an index of total MMP-9 protein in-vivo. SI1004 was strikingly more effective than doxycycline hyclate in the inhibition of MMP-9, consistent with inhibitory effects on MMP-9 RNA and a broader anti-inflammatory profile. These effects may make the nitrocycline approach therapeutically relevant in pathologies where there is a strong inflammatory component associated with elevated MMP-9 levels including ALVDD and HFpEF. In conclusion, ALVDD and HFpEF are diseases driven by inflammation, fibrosis and abnormalities of ECM turnover. This study presents in-vitro and in-vivo evidence of efficacy of doxycycline and SI1004, a novel, NO-releasing tetracycline analogue, as immunomodulatory, MMP inhibitors. SI1004 is a more effective inhibitor of MMP-9 transcription and serum MMP-9 in NHPs, than doxycycline. These agents are considered to be useful in treatment of diseases associated with elevated MMP.

(89) Cancer Applications

(90) As discussed in the background section, Matrix Metalloproteinase (MMP) levels in the plasma are known biomarkers of breast, colorectal, renal, pancreas, bladder and lung cancers (see Table 2).

(91) TABLE-US-00002 TABLE 2 Candidate MMP and ADAM Biomarkers of Cancer (Roy, Yang et al. 2009) Type of Cancer and MMPs/ADAMs Detected in Tissue/Body Fluid Breast MMP-13 Tissue MMP-9, TIMP-1 Serum, tissue MMP-9 Urine, serum, plasma, tissue ADAM12 Urine ADAM17 Tissue MMP-1 Tissue, nipple aspirates Pancreas MMP-9 Pancreatic juice, serum MMP-2 Pancreatic juice, tissue MMP-7 Tissue, plasma ADAM9 Tissue Lung VMMP-9, TIMP-1 Serum, bronchial lavage MMP-7 Tissue MMP-1 Tissue Bladder MMP-9 Tissue MMP-9, MMP-2 Urine MMP-9 Urine MMP-9, telomerase Urine Colorectal MMP-2 Tissue, plasma MMP-9 Tissue MMP-2, MMP-9 Plasma MMP-7 Serum MMP-1 Tissue MMP-13 Tissue Ovarian MMP-9 Tissue MMP-9, MMP-14 Tissue MMP-2 Tissue MMP-2, MMP-9, MMP-14 Tissue ADAM17 Tissue Prostate MMP-2, MMP-9 Plasma, tissue MMP-2 Tissue MMP-9 Urine ADAM8 Tissue ADAM9 Tissue Brain MMP-2 Tissue MMP-9 Tissue MMP-2, MMP-9 Tissue, cerebrospinal fluid, urine

(92) MMPs are involved in cancer cell intravasation and extravasation. They effect Extracellular Matrix (ECM) degradation and disrupt cell-cell interactions promoting cell migration. MMP-9 is also involved in endothelial-mesenchymal-transition (EMT) whereby cells acquire migratory characteristics and this is also facilitated by MMP-3 (via interactions with E-cadherin and Rac1b). MMPs modulate growth factors and receptors. MMP-9 modulates vascular endothelial growth factors which promotes tumour growth and angiogenesis. MMP-3 modulates insulin like growth factor binding proteins and basic fibroblast growth factors and is also known to activate MMP-9. MMPs also modulate tumour associated inflammation (e.g. MMP-9 is involved in breast cancer inflammation) via cytokines and their receptors.

(93) Anti Cancer Effect of the Compounds of the Invention

(94) ##STR00048##

(95) SI1004 (MJ-170, Dinitrate MB) is a more effective MMP-9 inhibitor nitrocycline than SI1005 (MJ-169, Piperidine Mono MB), which has been shown to inhibit MMP-3. Accordingly, it may be able to more selectively reduce MMP-9 protein levels than SI1004. Both SI1004 and SI1005 are more potent MMP-9 inhibitors than conventional doxycycline.

(96) In Vitro Data

(97) Using in vitro breast cancer cell models (HT1080 cells), stimulated with a pro-inflammatory insult (PMA) to stimulate the over-production of MMP-9, we see that doxycycline (Doxy), SI1004 (MJ-170, Dinitrate MB) and SI1005 (MJ-169, Piperidine Mono MB) all reduce MMP-9 production at 100 micromolar concentrations. Using the same in vitro breast cancer cell models for examining MMP-2, we see that PMA reduces pro-MMP-2 and doxycycline (Doxy), SI1004 (MJ-170, Dinitrate MB) and to a lesser extent SI1005 (MJ-169, Piperidine Mono MB) all reduce pro-MMP-2 production at 100 micromolar. However, surprisingly, doxycycline (Doxy) also appears to increase the conversion of available pro-MMP-2 to active MMP-2 (FIG. 5). This, potentially, could be a concern for chronic doxycycline therapy in the treatment of cancer. Advantageously, we do not see the same activation of MMP-2 with nitrocyclines.

(98) Using models of direct enzyme inhibition, it is shown below that SI1004 (MJ-170, Dinitrate MB) and SI1005 (MJ-169, Piperidine Mono MB) are more potent inhibitors of MMP-9 than doxycycline. The 1050 value (M) of SI1005 (MJ-169, Piperidine Mono MB) for MMP-2 and MMP-9 are 63 (46-84) and 139 (86-223) respectively. The IC.sub.50 value (M) of SI1004, (MJ-170, Dinitrate MB) for MMP-2 and MMP-9 are 9.4 (8.5-10.4) and 25 (19-32) respectively. These are more potent than doxycycline which has an approximate IC.sub.50 value (M) for MMP-2 and MMP-9 of 129 and 164 respectively

(99) TABLE-US-00003 TABLE 2 IC.sub.50 values for the inhibition of MMP-2 and MMP-9 in response to SI1004, SI1005 and doxycycline. SI1004 (MJ-170) SI1005 (MJ-169) Doxycycline MMP-2 9.4 M 63 M 129 M MMP-9 25 M 139 M 164 M

(100) MMP-8: SI1005 (MJ-169, Piperidine Mono MB) has around 53.8% inhibition at 100 M and 16.9% inhibition at 10 M. SI1004 (MJ-170, MJ-170, Dinitrate MB) has around 60.7% inhibition at 100 M and 26.0% inhibition at 10 M. Doxycyline has around 42.7% inhibition at 100 M. MMP-13: SI1005 (MJ-169, Piperidine Mono MB) has around 28.4% inhibition at 100 M and 6.6% inhibition at 10 M and SI1004 (MJ-170, MJ-170, Dinitrate MB) has around 74.5% inhibition at 1000 and 46.0% inhibition at 10 M. Doxycyline has around 54% inhibition at 100 M. MMP-1: SI1005 (MJ-169, Piperidine Mono MB) has around 22% inhibition at 100 M and 13% inhibition at 10 M while SI1004 (MJ-170, MJ-170, Dinitrate MB) has around 63% inhibition at 100 M and 19% inhibition at 10 M. Doxycyline has around 12% inhibition at 100 M.

(101) In Vivo Data

(102) Nitrocycline compounds SI1004 (MJ-170, Dinitrate MB, Group 2), SI1005 (MJ-169, Piperidine Mono MB, Group 3) and doxycycline hyclate control (Doxy Group 1) were administered to cynomolgus monkeys (n=6 per group) as described in the method below. The test articles were administered by oral gavage once daily for three days (Doxycycline hyclate 1.6 mg/day and equimolar doses of the nitrocyclines were administered on Day 1. These doses were equivalent to 100 mg/day of doxycycline base. The dose of doxycycline hyclate was increased to 4.6 mg/kg on the second and third 20 day. Equimolar doses of nitrocyclines were administered. This dose was equivalent to a 300 mg/day dose of doxycycline base). The primary endpoint of this study was the changes in MMP-9 from baseline to 72 hours. In the high dose doxycycline group (Group 1), MMP-9 levels increase. In the SI1004 (Group 2) MMP-9 levels are significantly reduced compared to doxycycline. In the SI1005 (Group 3) MMP-9 levels are significantly reduced compared to doxycycline and SI1004. These data provide proof-of-concept in vivo support for the use of SI1004 and SI1005 as more potent inhibitors of MMP-9 compared to doxycycline. Furthermore, SI1004 and SI1005 are more potent inhibitors of inflammatory cytokines such as IL-1b, IL-4 and IL-8 compared to doxycycline (data not shown). Finally, in order to provide a functional model of tumour cell invasion, the following data show that that at low dose, doxycycline (Doxy) does not reduce colon cancer cell invasiveness, whereas SI1004 (MJ-170, Dinitrate MB) and SI1005 (MJ-169, Piperidine Mono MB) do (FIG. 10). Overall nitrocyclines SI1004 and SI1005 appear to be more potent inhibitors of MMP enzymes and this may be an advantage in the management of cardiovascular disease and cancer. SI1004 appears to be more MMP-9 specific and does not reduce MMP-3 in in vitro inflammatory cell models. Both nitrocyclines SI1004 and SI1005 are more effective immunomodulatory compounds. They do not appear to activate MMP-2, unlike high concentration (100 M) doxycycline. Finally, they are more effective in reducing tumour cell invasiveness in an in vitro model with human colon cancer cells.

(103) In Vivo, Non-Human Primate Study. Methods

(104) Purpose bred, nave, non-human primates (cynomolgus monkeys, 2.9-4 kg) were sourced and randomly allocated in a parallel group design (n=6 per group) to receive SI1004, SI1005 and equimolar doses of doxycycline daily (1.6 mg/kg doxycycline hyclate equivalents, on day 1 and 4.8 mg/kg doxycycline equivalents on days 2 and 3) by oral gavage in aqueous vehicle over a 3 day period. Studies were carried out consecutively in two contract research organization sites (Charles River, Sparks, Nev., US and Charles River, Shanghai, China). The study protocol was approved by PCS-SHG Institutional Animal Care and Use Committee before conduct. During the study, care and use of animals was conducted in accordance with the guidelines of the USA National Research Council and the Canadian Council on Animal Care. The cynomolgus monkey was chosen for this study in order to maximize the likelihood of identifying responses that are similar to those that may be expected in humans. Each animal was identified by a cage label and body tattoo and was acclimated to orogastric dosing on at least two occasions prior to the initiation of dosing. The vehicle (1% (w/v) tween 80 and 0.5% (w/v) carboxymethylcellulose in deionized water) or 1.6 mg/kg doxycycline hyclate (0 hours) or 4.8 mg/kg doxycycline hyclate (24, 48 hours) or the molar equivalent(s) of SI1004 or SI1005 were administered using an orogastric tube inserted through the mouth and advanced into the stomach. The animals were temporarily restrained (i.e. manually) for dose administration, and were not sedated. Disposable sterile syringes and orogastric tubes were used for each animal/dose. Each dose was followed by a tap water flush of approximately 5 mL. Blood samples and blood pressure measurements were taken at the following timepoints: pre-dose (0 hours) and at 2, 4, 6, 12, 24, 26, 30, 36, 48, 50, 54, 60 and 72 hours after first administration of test article. Blood (300 L) for serum preparation was collected intoBD Vacutainer+Serum SST tubes to accelerate clotting 20 minutes prior to centrifugation to allow complete clotting to occur and centrifuged at 1500-2200 rpm at 2-8 C. for 10-15 minutes. Under these conditions blood cells containing MMP, principally neutrophils and platelets, undergo full degranulation. Since artifactual elevation of MMP-9 was an unavoidable feature of repeated venipuncture in our model, it was logical to stimulate full MMP-9 release during sample collection. This provided greater inter-animal reproducibility and a more dynamic analytical range for assessing the relative effects of the test articles. Subsequent MMP-9 values provide an index of total MMP-9 including circulating enzyme, amplified by repeated venipuncture, along with the cellular load released from storage granules during clotting. The latter is influenced by earlier inflammatory signaling, transcription and storage. The serum was transferred to a cryovial and immediately stored at 70 C. until analyzed for MMP-2 and MMP-9 via a Luminex ELISA (total MMP-2 and MMP-9) within 48 hours of collection. The analysis of each time point was repeated within 5 days. Values that differed by more than 15% were repeated. The primary study endpoint was the change in plasma MMP-2 and MMP-9 levels at 72 hours. Secondary endpoints were area under the curve (AUC) values of MMP-2 and MMP-9 over the following periods: 0-24, 0-48 and 0-72 hours.

(105) Data on Admixtures (FIGS. 13-17)

(106) Admixtures of tetracyclines and nitric oxide donors have benefits in inflammatory and cardiovascular diseases. In FIG. 13, Doxy and nitrate A admixture (Diethanolamine dinitrate, the alkyl nitrate component of SI1004) are significantly better than Doxy at inhibiting cardiac fibroblast proliferation (p=0.011) at 150 micromolar. However, Doxy and nitrate B admixture (Nitroxymethyl piperidine) are not (p=NS) at same concentration. The novel nitrocycline, SI1004, is significantly more effective at inhibiting cardiac fibroblast proliferation than doxycycline, Doxy and nitrate A admixture, Doxy and nitrate B admixture at 150 micromolar (all p<0.01). In some cases, inflammatory cytokines are similarly reduced by Doxy and admixtures with NO donors. In FIG. 14, Doxy and nitrate A admixtures are shown to significantly reduce IL-8 levels in TNFalpha stimulated PBMCs at 150 micromolar (p<0.01). Doxy alone and Doxy and nitrate B admixtures also reduce IL-8 levels compared to controls (p<0.05). However, in some instances, the effects Doxy and nitrate A admixture is more effective than Doxy. In FIG. 15, Doxy and nitrate A (Diethanolamine dinitrate) admixture can significantly reduce IL-1 beta levels in TNFalpha stimulated PBMCs (p<0.05). Doxy and nitrate B (Nitroxymethyl piperidine) admixture reduce IL-1 beta levels, but not significantly (p=NS). Furthermore, in some instances, the choice of NO donor dramatically alters the anti-inflammatory effects. In FIG. 16 it is shown that IL-4 is reduced significantly more (p<0.01) by Doxy and nitrate A (Diethanolamine dinitrate) admixture than either Doxy or Doxy and nitrate B (Nitroxymethyl piperidine) admixture. IL-4 is implicated in inflammatory bowel disease. IL-8 is implicated in invasive bladder cancer, chronic prostatitis, acute pyelpnephritis, non-Hodgkins lymphoma, pulmonary infections and osteomyelitis. IL-1 is implicated in fever, anemia, cryopyrinopathies (hereditary periodic fever syndromes), gout and pseudogout, Septic shock.