NOVEL COMBINATION AND USE

Abstract

The present invention relates to the use of one or more compounds selected from the following: caffeic acid, thymol, aspirin, benzydamine hydrochloride, diclofenac sodium, flurbiprofen, ibuprofen, indomethacin, trifluoperazine hydrochloride, chlorprothixene hydrochloride, triflupromazine hydrochloride, suloctidil, thioridazine hydrochloride, dichlorophen, saccharin and piroxicam, in combination with a polymyxin selected from colistin or polymyxin B or a pharmaceutically acceptable derivative thereof, for use in the treatment of a microbial infection, and in particular for killing clinically latent microorganisms associated with microbial infections. The invention also provides a combination comprising suloctidil or a pharmaceutically acceptable derivative or prodrug thereof, and a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof. This combination is particularly useful for the treatment and/or prevention of microbial infections.

Claims

1. A combination comprising suloctidil or a pharmaceutically acceptable derivative or prodrug thereof, and a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof.

2. A combination according to claim 1, wherein the polymyxin is polymyxin E or a pharmaceutically acceptable derivative thereof.

3. A combination according to claim 1 for use in the prevention and/or treatment of a microbial infection.

4. A combination according to claim 3 for use in killing multiplying, non-multiplying or clinically latent microorganisms associated with a microbial infection.

5. A pharmaceutical composition comprising suloctidil or a pharmaceutically acceptable derivative or prodrug thereof, a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable adjuvant, diluent or carrier.

6. Use of suloctidil or a pharmaceutically acceptable derivative or prodrug thereof in combination with a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the prevention and/or treatment of a microbial infection, in particular for killing multiplying, non-multiplying and/or clinically latent microorganisms associated with such an infection.

7. Use of one or more compounds selected from the following: caffeic acid, thymol, aspirin, benzydamine hydrochloride, diclofenac sodium, flurbiprofen, ibuprofen, indomethacin, trifluoperazine hydrochloride, chlorprothixene hydrochloride, triflupromazine hydrochloride, suloctidil or a pharmaceutically acceptable derivative or prodrug thereof, thioridazine hydrochloride, dichlorophen, saccharin and piroxicam, in combination with a polymyxin selected from colistin or polymyxin B or a pharmaceutically acceptable derivative thereof, for treating a microbial infection, preferably killing multiplying, non-multiplying and/or clinically latent microorganisms associated with a microbial infection.

8. Use according to claim 6, wherein the polymyxin is colistin or a pharmaceutically acceptable derivative thereof.

9. Use according to claim 6, wherein the infection is a bacterial infection.

10. Use according to claim 9, wherein the microbial infection is caused by E. coli, Enterobacteriaceae, Haemophilus influenzae, Mycobacteria or Klebsiella.

11. Use according to claim 10 wherein the infection is caused by E. coli or Klebsiella.

12. Use according to claim 10, wherein the infection is caused by a drug-resistant strain.

13. Use according to claim 12, wherein the infection is caused by a carbapenemase-resistant strain or “extended spectrum β-lactamase” (ESPL) strain, such as New Delhi Metallo-beta-lactamase-1 (NDM-1) resistant Klebs. Pneumonia or NDM-1 E. coli.

14. Use according to claim 6 for the treatment of tuberculosis, anthrax, abscesses, acne vulgaris, actinomycosis, asthma, bacilliary dysentry, bacterial conjunctivitis, bacterial keratitis, bacterial vaginosis, botulism, Buruli ulcer, bone and joint infections, bronchitis (acute or chronic), brucellosis, burn wounds, cat scratch fever, cellulitis, chancroid, cholangitis, cholecystitis, cutaneous diphtheria, cystic fibrosis, cystitis, diffuse panbronchiolitis, diphtheria, dental caries, diseases of the upper respiratory tract, eczema, empymea, endocarditis, endometritis, enteric fever, enteritis, epididymitis, epiglottitis, erysipelis, erysipclas, erysipeloid, erythrasma, eye infections, furuncles, gardnerella vaginitis, gastrointestinal infections (gastroenteritis), genital infections, gingivitis, gonorrhoea, granuloma inguinale, Haverhill fever, infected burns, infections following dental operations, infections in the oral region, infections associated with prostheses, intraabdominal abscesses, Legionnaire's disease, leprosy, leptospirosis, listeriosis, liver abscesses, Lyme disease, lymphogranuloma venerium, mastitis, mastoiditis, meningitis and infections of the nervous system, mycetoma, nocardiosis, non-specific urethritis, opthalmia, osteomyelitis, otitis, orchitis, pancreatitis, paronychia, pelveoperitonitis, peritonitis, peritonitis with appendicitis, pharyngitis, phlegmons, pinta, plague, pleural effusion, pneumonia, postoperative wound infections, postoperative gas gangrene, prostatitis, pseudo-membranous colitis, psittacosis, pulmonary emphysema, pyelonephritis, pyoderma, Q fever, rat-bite fever, reticulosis, ricin poisoning, Ritter's disease, salmonellosis, salpingitis, septic arthritis, septic infections, septicameia, sinusitis, skin infections, syphilis, systemic infections, tonsillitis, toxic shock syndrome, trachoma, tularaemia, typhoid, typhus, urethritis, wound infections, yaws, aspergillosis, candidiasis, cryptococcosis, favus, histoplasmosis, intertrigo, mucormycosis, tinea, onychomycosis, pityriasis versicolor, ringworm and sporotrichosis; or infections with MSSA, MRSA, Staph. epidermidis, Strept. agalactiae, Strept. pyogenes, Escherichia coli, Klebs. pneumoniae, Klebs. oxytoca, Pr. mirabilis, Pr. rettgeri, Pr. vulgaris, Haemophilus influenzae, Enterococcus faecalis and Enterococcus faecium.

15. A pharmaceutical composition comprising one or more compounds selected from the following: caffeic acid, thymol, aspirin, benzydamine hydrochloride, diclofenac sodium, flurbiprofen, ibuprofen, indomethacin, trifluoperazine hydrochloride, chlorprothixene hydrochloride, triflupromazine hydrochloride, suloctidil, thioridazine hydrochloride, dichlorophen, saccharin and piroxicam, in combination with a polymyxin selected from colistin or polymyxin B or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable adjuvant, diluent or carrier for use in treating microbial infection, preferably killing clinically latent microorganisms associated with a microbial infection.

16. A product comprising one or more compounds selected from the following: caffeic acid, thymol, aspirin, benzydamine hydrochloride, diclofenac sodium, flurbiprofen, ibuprofen, indomethacin, trifluoperazine hydrochloride, chlorprothixene hydrochloride, triflupromazine hydrochloride, suloctidil, thioridazine hydrochloride, dichlorophen, saccharin and piroxicam, in combination with a polymyxin selected from colistin or polymyxin B or a pharmaceutically acceptable derivative thereof, as a combined preparation for simultaneous, separate or sequential use in treating a microbial infection, preferably in killing clinically latent microorganisms associated with a microbial infection.

17. A product comprising suloctidil or a pharmaceutically acceptable derivative or prodrug thereof and a polymyxin selected from polymyxin E and polymyxin B or a pharmaceutically acceptable derivative thereof, as a combined preparation for simultaneous, separate or sequential use in the treatment of a microbial infection.

Description

EXAMPLES

[0125] The chequerboard method and Time kill experiments are described below and in Antimicrob Chemo (2013) 68, 374-384.

BRIEF DESCRIPTION OF THE DRAWINGS

[0126] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Example 1: Time Kill Experiments

[0127] (a) Thymol (HT013015) and Colistin Against NDM-1 Klebsiella pneumoniae

[0128] FIG. 1 is the time kill curve showing combination of colistin and HT013015 against NDM-1 K. pneumoniae.

[0129] (b): Trifluoperazine Hydrochloride (HT0120566) and Colistin Against NDM-1 Klebsiella pneumoniae

[0130] FIG. 2 is the time kill curve showing combination of colistin and HT0120566 against NDM-1 Klebsiella pneumoniae.

[0131] (c): Chlorprothixene Hydrochloride (HT0120707) and Colistin Against NDM-1 E. coli

[0132] FIG. 3 is the time kill curve showing combination of colistin and HT0120707 against NDM-1 E. coli.

[0133] (d): Triflupromazine Hydrochloride (HT0120700) and Colistin Against NDM-1 E. coli

[0134] FIG. 4 is the time kill curve showing combination of colistin and HT0120700 against NDM-1 E. coli.

[0135] (e): Suloctidil (HT0120093) and Colistin Against NDM-1 E. coli

[0136] FIG. 5 is the time kill curve showing combination of colistin and HT0120093 against NDM-1 E. coli.

[0137] (f): Thioridazine Hydrochloride (HT0120553) and Colistin Against NDM-1 Klebsiella pneumoniae

[0138] FIG. 6 is the time kill curve showing combination of colistin and HT0120553 against NDM-1 Klebsiella pneumoniae.

[0139] (g): Dichlorophen (HT0121567) and Colistin Against NDM-1 K. pneumoniae

[0140] FIG. 7 is the time kill curve showing combination of colistin and HT0121567 against NDM-1 K. pneumoniae.

[0141] (h): Aspirin (HT0121219) and Colistin Against NDM-1 Klebsiella pneumoniae

[0142] FIG. 8 is the time kill curve showing combination of colistin and HT0121219 against NDM-1 K. pneumoniae

[0143] (i): Indomethacin (HT0120451) and Colistin Against NDM-1 K. pneumoniae

[0144] FIGS. 9 and 10 are the time kill curves showing combination of colistin and HT0120451 against NDM-1 K. pneumoniae.

[0145] (j): Piroxicam (HT0120492) and Colistin Against NDM-1 E. coli

[0146] FIG. 11 is the time kill curve showing combination of colistin and HT0120492 against NDM-1 E. coli.

[0147] (k): Benzydamine Hydrochloride (HT0120969) and Colistin Against NDM-1 E. coli

[0148] FIG. 12 is the time kill curve showing combination of colistin and HT0120969 against NDM-1 E. coli.

[0149] (l): Ibuprofen (HT0120448) and Colistin Against NDM-1 K. pneumoniae

[0150] FIG. 13 is the time kill curve showing combination of colistin and HT0120448 against NDM-1 Klebsiella pneumoniae.

[0151] (m): Diclofenac Sodium (HT0120365) and Colistin Against NDM-1 E. coli

[0152] FIG. 14 is the time kill curve showing combination of colistin and HT0120365 against NDM-1 E. coli.

[0153] (n): Flurbiprofen (HT0120417) and Colistin Against NDM-1 E. coli

[0154] FIG. 15 is the time kill curve showing combination of colistin and HT0120417 against NDM-1 E. coli

[0155] (o): Caffeic Acid (HT013001) and Colistin Against NDM-1 E. coli

[0156] FIG. 16 is the time kill curve showing combination of colistin and HT013001 against NDM-1 E. coli

[0157] (p): Saccharin (HT0120098) and Colistin Against NDM-1 E. coli

[0158] FIG. 17 is the time kill curve showing combination of colistin and HT0120098 against NDM-1 E. coli.

Example 2: Chequerboard Method

[0159] (a) In Vitro Synergy Effect of Colistin and Each of HT013015 (Thymol), HT0121219 (Aspirin), HT0120448 (Ibuprofen), HT0120451 (Indomethacin), HT0120566 (Trifluoperazine Hydrochloride), and HT0121567 (Dichlorophen) Against Log Phase NDM-1 Klebsiella pneumoniae Using the Chequerboard Method

[0160] Growth of Bacteria

[0161] Log phase growth of NDM-1 Klebsiella pneumonia was carried out as described in the art.

[0162] The effects of each combination of the present invention were examined by calculating the fractional inhibitory concentration index (FICI) of each combination, as follows: (MIC of drug A, tested in combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested in combination)/(MIC of drug B, tested alone). The interaction of the combination was defined as showing synergy if the FICI was ≦0.5, no interaction if the FICI was >0.5 but <4.0 and antagonism if the FICI was >4.0.

TABLE-US-00001 HT013015 256 128 64 32 16 8 4 2 1 0.5 0.25 0 Colistin 16 0.40 0.41 0.42 0.42 0.70 0.73 0.74 0.91 0.91 0.41 0.90 0.41 8 0.41 0.42 0.42 0.45 0.45 1.00 0.94 1.07 1.05 1.09 1.09 1.13 4 0.42 0.42 0.60 0.92 1.04 1.05 1.12 1.07 1.07 1.13 1.11 1.17 2 0.42 0.43 0.76 0.99 1.03 1.12 1.15 1.14 1.10 1.11 1.17 1.20 1 0.42 0.68 0.93 1.00 1.10 1.13 1.15 1.16 1.11 1.17 1.17 1.23 0.5 0.42 0.84 0.98 1.07 1.14 1.17 1.19 1.16 1.15 1.19 1.22 1.26 0.25 0.42 0.83 1.00 1.09 1.16 1.18 1.20 1.20 1.21 1.22 1.22 1.30 0 0.42 0.91 1.10 1.17 1.22 1.24 1.26 1.28 1.27 1.29 1.26 1.32 Colistin 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0 HT0121219 256 0.05 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.05 0.47 0.50 0.53 128 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.42 0.51 0.51 0.52 64 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.09 0.51 0.57 0.52 0.52 32 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.40 0.51 0.53 0.53 0.50 16 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.50 0.52 0.56 0.52 0.49 8 0.04 0.04 0.04 0.04 0.04 0.04 0.43 0.52 0.52 0.57 0.52 0.50 4 0.05 0.04 0.04 0.04 0.04 0.23 0.44 0.50 0.52 0.53 0.49 0.52 0 0.05 0.04 0.13 0.13 0.45 0.50 0.51 0.51 0.51 0.51 0.51 0.53 HT0120448 256 0.05 0.05 0.05 0.05 0.05 0.14 0.42 0.44 0.49 0.48 0.51 0.56 128 0.05 0.04 0.04 0.05 0.05 0.30 0.45 0.49 0.47 0.50 0.50 0.54 64 0.05 0.04 0.04 0.07 0.05 0.34 0.49 0.51 0.51 0.53 0.53 0.54 32 0.04 0.04 0.04 0.05 0.05 0.44 0.51 0.53 0.53 0.55 0.57 0.55 16 0.04 0.04 0.04 0.07 0.27 0.49 0.53 0.51 0.54 0.56 0.54 0.57 8 0.05 0.04 0.05 0.11 0.37 0.54 0.50 0.52 0.58 0.55 0.57 0.54 4 0.05 0.05 0.05 0.11 0.46 0.52 0.54 0.52 0.53 0.58 0.55 0.55 0 0.14 0.14 0.16 0.34 0.48 0.52 0.53 0.56 0.54 0.60 0.54 0.58 HT0120451 256 0.05 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.42 0.49 0.55 0.55 128 0.05 0.04 0.04 0.04 0.04 0.04 0.04 0.35 0.47 0.52 0.51 0.52 64 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.45 0.53 0.54 0.50 0.50 32 0.04 0.04 0.04 0.04 0.04 0.04 0.39 0.48 0.53 0.52 0.52 0.48 16 0.04 0.04 0.04 0.04 0.04 0.04 0.48 0.51 0.54 0.56 0.53 0.51 8 0.04 0.04 0.04 0.04 0.04 0.42 0.49 0.58 0.54 0.58 0.50 0.51 4 0.04 0.04 0.04 0.04 0.04 0.47 0.49 0.48 0.52 0.46 0.48 0.49 .49 256 128 64 32 16 8 4 2 1 0.5 0.25 0 HT0120566 Colistin 32 0.58 0.51 0.52 0.52 0.52 0.52 0.52 0.52 0.53 0.53 0.53 0.54 16 0.58 0.50 0.52 0.51 0.52 0.52 0.53 0.53 1.33 1.44 1.56 1.60 8 0.58 0.53 0.52 0.52 0.52 0.53 0.59 1.63 1.65 1.62 1.61 1.66 4 0.58 0.52 0.52 0.52 0.53 0.53 1.73 1.71 1.72 1.68 1.63 1.75 2 0.58 0.52 0.52 0.52 0.53 1.68 1.75 1.74 1.77 1.72 1.69 1.75 1 0.58 0.53 0.52 0.53 1.70 1.71 1.72 1.73 1.75 1.68 1.72 1.73 0.5 0.58 0.52 0.51 1.70 1.72 1.73 1.74 1.77 1.75 1.76 1.74 1.76 0 1.81 1.76 1.74 1.77 1.79 1.79 1.76 1.77 1.76 1.77 1.76 1.77 HT0121567 Colistin 32 0.39 0.39 0.39 0.38 0.38 0.38 0.38 0.38 0.39 0.40 0.45 0.38 16 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.38 0.40 0.45 0.47 0.38 8 0.39 0.39 0.39 0.38 0.39 0.38 0.39 0.39 0.39 0.44 0.53 0.52 4 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.40 0.47 0.64 0.95 2 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.77 0.74 0.74 0.72 1 0.39 0.39 0.39 0.39 0.72 0.71 0.81 0.92 0.89 0.85 0.83 1.08 0.5 0.39 0.39 0.39 0.60 0.62 0.72 0.81 0.84 0.99 0.85 0.90 1.03 0 0.39 0.39 0.39 0.74 0.67 0.74 0.91 0.99 1.04 1.06 1.07 1.15 indicates data missing or illegible when filed

[0163] (b) In Vitro Synergy Effect of Colistin and Each of HT0120969 (Benzydamine Hydrochloride), HT0120365 (Diclofenac Sodium), HT0120417 (Flurbiprofen), HT0120707 (Chlorprothixene Hydrochloride), HT0120700 (Triflupromazine Hydrochloride), HT0120093 (Suloctidil), HT0120492 (Piroxicam) and HT0120098 (Saccharin) Against Log Phase NDM-1 E. coli Using the Chequerboard Method

[0164] Growth of Bacteria

[0165] Log phase growth of NDM-1 E. coli was carried out as described in the art.

[0166] The effects of each combination of the present invention were examined by calculating the fractional inhibitory concentration index (FICI) of each combination, as follows: (MIC of drug A, tested in combination)/(MIC of drug A, tested alone)+(MIC of drug B, tested in combination)/(MIC of drug B, tested alone).

[0167] The interaction of the combination was defined as showing synergy if the FICI was ≦0.5, no interaction if the FICI was >0.5 but <4.0 and antagonism if the FICI was >4.0.

TABLE-US-00002 Colistin 64 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 0 HT0120969 256 0.04 0.04 0.04 0.04 0.04 0.04 0.24 0.25 0.26 0.25 0.26 0.26 128 0.04 0.04 0.04 0.04 0.09 0.28 0.35 0.46 0.49 0.50 0.51 0.48 64 0.04 0.10 0.06 0.09 0.24 0.44 0.50 0.57 0.54 0.56 0.60 0.67 32 0.04 0.04 0.16 0.21 0.35 0.43 0.46 0.53 0.53 0.57 0.62 0.72 16 0.04 0.07 0.21 0.29 0.40 0.53 0.55 0.53 0.55 0.54 0.62 0.72 8 0.05 0.04 0.28 0.27 0.44 0.49 0.52 0.55 0.64 0.54 0.63 0.69 4 0.05 0.09 0.34 0.41 0.51 0.54 0.58 0.57 0.57 0.56 0.67 0.72 0 0.05 0.50 0.42 0.57 0.62 0.70 0.66 0.76 0.71 0.76 0.73 0.72 HT0120365 256 0.05 0.05 0.05 0.05 0.57 0.64 0.66 0.72 0.72 0.66 0.71 0.62 128 0.04 0.04 0.04 0.04 0.42 0.55 0.59 0.67 0.64 0.62 0.65 0.72 64 0.04 0.04 0.04 0.04 0.45 0.50 0.54 0.61 0.60 0.60 0.64 0.82 32 0.05 0.04 0.38 0.29 0.50 0.52 0.55 0.60 0.57 0.62 0.64 0.76 16 0.04 0.04 0.16 0.44 0.50 0.56 0.56 0.56 0.58 0.58 0.63 0.76 8 0.34 0.04 0.44 0.44 0.54 0.53 0.55 0.57 0.66 0.56 0.69 0.73 4 0.05 0.04 0.31 0.49 0.55 0.57 0.56 0.61 0.63 0.60 0.71 0.73 0 0.05 0.24 0.59 0.68 0.69 0.72 0.71 0.78 0.74 0.79 0.72 0.73 Colistin 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0 HT0120417 256 0.04 0.04 0.04 0.05 0.05 0.20 0.32 0.42 0.47 0.48 0.49 0.50 128 0.04 0.04 0.04 0.05 0.05 0.28 0.42 0.50 0.50 0.56 0.51 0.49 64 0.05 0.04 0.04 0.05 0.05 0.34 0.51 0.53 0.54 0.60 0.54 0.54 32 0.04 0.04 0.04 0.05 0.06 0.44 0.50 0.54 0.55 0.52 0.56 0.51 16 0.04 0.04 0.05 0.06 0.27 0.47 0.51 0.54 0.53 0.56 0.57 0.51 8 0.04 0.04 0.05 0.06 0.35 0.50 0.51 0.53 0.51 0.58 0.56 0.51 4 0.04 0.04 0.05 0.12 0.44 0.51 0.54 0.56 0.57 0.57 0.57 0.53 0 0.05 0.06 0.12 0.17 0.46 0.51 0.53 0.53 0.53 0.53 0.58 0.55 256 128 64 32 16 8 4 2 1 0.5 0.25 0 HT0120707 Colistin 32 0.40 0.40 0.40 0.39 0.39 0.65 0.65 0.52 0.40 0.59 0.39 0.39 16 0.41 0.39 0.40 0.41 0.40 0.40 0.40 0.39 0.40 0.40 0.40 0.40 8 0.40 0.40 0.40 0.41 0.59 0.67 0.47 0.76 0.76 0.72 0.72 0.68 4 0.39 0.39 0.40 0.45 0.53 0.58 0.60 0.67 0.69 0.65 0.77 0.80 2 0.40 0.39 0.40 0.46 0.77 0.81 0.87 0.83 0.87 0.81 0.90 1.07 1 0.40 0.40 0.40 0.78 0.91 0.93 0.93 0.95 0.85 0.89 0.98 1.13 0.5 0.40 0.39 0.62 0.81 0.90 0.94 0.94 0.94 0.97 0.95 0.94 1.12 0 0.40 0.62 0.70 1.05 1.10 1.13 1.12 1.12 1.13 1.15 1.14 1.18 HT0120700 Colistin 32 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 16 0.57 0.43 0.40 0.51 0.40 0.40 0.39 0.40 0.43 0.40 0.50 0.69 8 0.40 0.40 0.40 0.49 0.40 0.46 0.46 0.60 0.67 0.63 0.62 0.63 4 0.40 0.40 0.40 0.56 0.63 0.75 0.65 0.77 0.70 0.68 0.86 0.89 2 0.40 0.39 0.40 0.58 0.79 0.83 0.85 0.86 0.83 0.88 0.94 1.09 1 0.40 0.40 0.48 0.78 0.86 0.86 0.87 0.89 0.86 0.90 0.99 1.12 0.5 0.40 0.39 0.58 0.83 0.90 0.93 0.94 0.94 0.95 0.98 0.97 1.14 0 0.41 0.76 0.73 1.08 1.11 1.17 1.14 1.13 1.14 1.13 1.11 1.15 HT0120093 Colistin 32 0.39 0.38 0.36 0.36 0.35 0.36 0.36 0.36 0.36 0.35 0.36 0.36 16 0.39 0.38 0.37 0.36 0.36 0.36 0.36 0.36 0.53 0.36 0.46 0.53 8 0.42 0.38 0.36 0.35 0.36 0.36 0.36 0.37 0.54 0.41 0.51 0.44 4 0.42 0.36 0.37 0.36 0.36 0.36 0.36 0.49 0.68 0.66 0.78 0.87 2 0.41 0.37 0.37 0.36 0.36 0.63 0.62 0.67 0.80 0.80 0.78 0.95 1 0.37 0.37 0.36 0.37 0.55 0.63 0.74 0.92 0.86 0.84 0.83 1.02 0.5 0.37 0.36 0.37 0.53 0.57 0.68 0.81 0.85 0.89 0.82 0.95 0.98 0 0.61 0.44 0.38 0.60 0.60 0.70 0.94 0.93 0.98 1.03 0.97 1.03 Colistin 16 0.43 0.41 0.41 0.39 0.40 0.40 0.40 0.41 0.93 0.90 0.88 0.83 8 0.45 0.42 0.41 0.40 0.40 0.40 0.41 0.97 0.98 1.03 1.11 0.98 4 0.43 0.42 0.41 0.40 0.41 0.80 0.79 0.93 0.98 1.10 1.16 1.04 2 0.42 0.42 0.42 0.42 0.63 0.76 0.90 0.96 1.03 1.16 1.15 1.23 1 0.42 0.41 0.43 0.64 0.68 0.87 1.02 1.09 1.05 1.10 1.18 1.20 0.5 0.44 0.41 0.42 0.68 0.78 0.96 1.12 1.17 1.09 1.09 1.23 1.26 0.25 0.44 0.42 0.47 0.69 0.74 1.01 1.17 1.24 1.19 1.21 1.25 1.34 0 0.83 0.77 0.67 0.81 0.84 1.09 1.18 1.24 1.25 1.27 1.28 1.33 Colistin 64 32 16 8 4 2 1 0.5 0.25 0.125 0.0625 0 HT0120492 256 0.05 0.04 0.04 0.04 0.05 0.05 0.05 0.20 0.27 0.47 0.51 0.53 128 0.05 0.04 0.06 0.07 0.04 0.04 0.08 0.31 0.39 0.37 0.39 0.50 64 0.04 0.05 0.09 0.06 0.10 0.06 0.26 0.47 0.42 0.44 0.45 0.51 32 0.05 0.05 0.04 0.06 0.04 0.07 0.37 0.40 0.41 0.47 0.45 0.54 16 0.05 0.04 0.04 0.05 0.07 0.08 0.38 0.42 0.45 0.45 0.44 0.52 8 0.05 0.09 0.10 0.09 0.15 0.34 0.42 0.46 0.48 0.45 0.45 0.53 4 0.05 0.12 0.07 0.07 0.17 0.40 0.44 0.48 0.44 0.44 0.42 0.55 0 0.07 0.05 0.04 0.18 0.45 0.51 0.52 0.53 0.51 0.53 0.54 0.52 colistin 16 8 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.015625 0 HT0120098 256 0.05 0.04 0.04 0.04 0.04 0.19 0.34 0.44 0.51 0.53 0.56 0.72 128 0.05 0.04 0.05 0.04 0.04 0.07 0.32 0.43 0.46 0.45 0.56 0.63 64 0.04 0.04 0.06 0.04 0.04 0.06 0.36 0.44 0.48 0.64 0.54 0.60 32 0.06 0.05 0.04 0.04 0.17 0.28 0.35 0.40 0.47 0.48 0.59 0.61 16 0.04 0.04 0.04 0.08 0.14 0.10 0.37 0.41 0.47 0.47 0.55 0.61 8 0.04 0.05 0.04 0.05 0.05 0.23 0.37 0.48 0.46 0.49 0.63 0.62 4 0.30 0.04 0.04 0.04 0.08 0.33 0.40 0.42 0.53 0.60 0.65 0.61 0 0.04 0.05 0.05 0.38 0.60 0.58 0.60 0.60 0.66 0.66 0.65 0.71

Example 3

[0168] In Vitro Synergy Effect of Suloctidil in Combination with Colistin Against Log Phase NDM-1 Klebsiella pneumoniae

[0169] The synergistic effect of suloctidil in combination with colistin was tested against log phase NDM-1 Klebsiella pneumoniae using time-kill methods over a period of 24 hours.

[0170] Materials and Methods [0171] Bacterial strain used: NCTC 13443 strain of NDM-1 Klebsiella pneumoniae [0172] Growth of bacteria: Log phase growth of the bacteria was carried out according to methods known in the art.

[0173] Compounds and Preparation:

(i) Suloctidil was obtained from a commercial source and dissolved in DMSO to make a stock concentration of 10 mg/ml.
(ii) Colistin was obtained from a commercial source at a concentration of 20 mg/ml.

[0174] Both suloctidil and colistin were then added to 96 well plates either alone or in the combinations shown below in Table 1.

TABLE-US-00003 TABLE 1 Agent (Concentration) Number/Letter Combination Combination Colistin (32 μg/ml) 1 1&A 1&C Colistin (16 μg/ml) 2 2&A 2&C Colistin (8 μg/ml) 3 3&A 3&C Colistin (4 μg/ml) 4 4&A 4&C Suloctidil (32 μg/ml) A 1&B 1&D Suloctidil (16 μg/ml) B 2&B 2&D Suloctidil (8 μg/ml) C 3&B 3&D Suloctidil (4 μg/ml) D 4&B 4&D

[0175] The overnight culture was diluted with nutrient broth (Oxoid) to 10.sup.7 CFU/ml and 280 μl and 290 μl of the culture was added to each combination well and single agent well, respectively, to make a final concentration of 300 μl. Incubation of the compounds with the bacterial suspension was carried out for 24 hours. At 0, 2, 4, 7 and 25 hours, CFU counts were performed to measure the kill effects of the drug combination.

[0176] Results

[0177] The time-kill curves are shown in FIGS. 18 to 27, where suloctidil is referenced as “93”.

[0178] Summary and Conclusion [0179] 1. It can be seen from FIGS. 18 to 27 that suloctidil (i.e. “93”) had no antimicrobial effect against log phase NDM-1 K. pneumoniae when used alone at concentrations of 32, 16, 8 and 4 μg/ml. [0180] 2. It can be seen from FIGS. 20, 21, 24, 25 and 27 that colistin also had no antimicrobial effect against log phase NDM-1 K. pneumoniae when used alone at concentrations of 8 and 4 μg/ml. [0181] 3. FIGS. 18, 19, 22, 23 and 26 then demonstrate that colistin had an antimicrobial effect against log phase NDM-1 K. pneumoniae at concentrations of 32 and 16 μg/ml, but that this effect was not long term. [0182] 4. FIG. 19 for example shows that colistin (16 μg/ml) caused complete kill after 7 hours and bacteria re-growth after 25 hours. [0183] 5. In each of FIGS. 18 to 27, it can, however, be seen that there was a significant synergistic effect against log phase NDM-1 K. pneumoniae when suloctidil and colistin were used in combination. This synergistic effect resulted in both a faster and longer term kill of the bacteria.

Example 4: In Vitro Synergy Effect of Suloctidil in Combination with Polymyxin E (Colistin) Against Log Phase NDM-1 E. coli

[0184] The synergistic effect of suloctidil in combination with colistin was tested against log phase NDM-1 E. coli using time-kill methods over a period of 24 hours.

[0185] Materials and Methods [0186] Bacterial strain used: BAA2469 strain of NDM-1 E. coli [0187] Growth of bacteria: Log phase growth of bacteria was carried out according to methods known in the art.

[0188] Compounds and Preparation:

(i) Suloctidil was obtained from a commercial source and dissolved in DMSO to make a stock concentration of 10 mg/ml.
(ii) Colistin was obtained from a commercial source at a concentration of 20 mg/ml.

[0189] Both suloctidil and colistin were then added to 96 well plates either alone or in the combinations shown below in Table 2.

TABLE-US-00004 TABLE 2 Agent (Concentration) Number/Letter Combination Combination Colistin (8 μg/ml) 1 1&A 1&C Colistin (4 μg/ml) 2 2&A 2&C Colistin (2 μg/ml) 3 3&A 3&C Colistin (1 μg/ml) 4 4&A 4&C Suloctidil (32 μg/ml) A 1&B 1&D Suloctidil (16 μg/ml) B 2&B 2&D Suloctidil (8 μg/ml) C 3&B 3&D Suloctidil (4 μg/ml) D 4&B 4&D

[0190] The overnight culture was diluted with nutrient broth (Oxoid) to 10.sup.7 CFU/ml and 280 μl and 290 μl of the culture was added to each combination well and single agent well, respectively, to make a final concentration of 300 μl. Incubation of the compounds with the bacterial suspension was carried out for 24 hours. At 0, 2, 4, 7 and 25 hours, CFU counts were performed to measure the kill effects of the drug combination.

[0191] Results

[0192] The time-kill curves are shown in FIGS. 28 to 34, where suloctidil is referenced as “93”.

[0193] Summary and Conclusion [0194] 1. It can be seen from FIGS. 28 to 34 that suloctidil (i.e. “93”) had no antimicrobial effect against log phase NDM-1 E. coli when used alone at concentrations of 16, 8 and 4 μg/ml. [0195] 2. It can be seen from FIGS. 28 to 34 that colistin also had no antimicrobial effect against log phase NDM-1 E. coli when used alone at concentrations of 8, 4, 2 and 1 μg/ml. [0196] 3. In each of FIGS. 28 to 34, it can, however, be seen that there was a significant synergistic effect against log phase NDM-1 E. coli when suloctidil and colistin were used in combination. This synergistic effect resulted in a faster time-kill of the bacteria.