HK1104460B

HK1104460B - Use for the manufacture of a medicament for treatment or prophylaxis of infectious diseases

Info

Publication number: HK1104460B
Application number: HK07109654.1A
Authority: HK
Inventors: kJældgaard Giwercman BIRGIT
Original assignee: Bkg Pharma Aps
Priority date: 2004-04-30
Filing date: 2005-04-29
Publication date: 2011-05-13

Description

Application of medicine for treating or preventing infectious diseases

Technical Field

The present invention relates to the use of chemosensitising compounds, in particular thioxanthene derivatives and phenothiazine derivatives, in combination with an anti-infective agent for the treatment of infectious diseases.

Background

Resistance to chemotherapy is a common clinical problem in patients with infectious diseases. In the treatment of infections, it is often found that drug targeting of prokaryotic or eukaryotic microbial cells is resistant to a variety of drugs with different structures and functions. This phenomenon is called multidrug resistance (MDR). Organisms ranging from bacteria to humans have transmembrane transporters that make them resistant to toxic compounds. Based on their biological significance, the structure and function of prokaryotic and eukaryotic multidrug transporters are very similar. In addition, other types of important resistance mechanisms can work in conjunction with the efflux pump, thereby generating high levels of resistance. There is an urgent need for drugs that can inhibit (reverse) or circumvent the drug resistance mechanism and enhance the efficacy of commonly used anti-infective agents.

The incidence of multiple antimicrobial resistance of bacteria causing infections in hospitals/intensive care units is increasing and it is not uncommon to find microorganisms that are not sensitive to more than 10 different antibiotics. Examples of such drug-resistant bacteria include methicillin-resistant and methicillin-resistant vancomycin-resistant Staphylococcus aureus (Staphylococcus aureus); vancomycin-resistant enterococci such as Enterococcus faecalis (Enterococcus faecium) and Enterococcus faecium (Enterococcus faecium); penicillin-resistant Streptococcus pneumoniae (Streptococcus pneumoniae), and gram-negative bacilli (escherichia coli) resistant to cephalosporins and quinolones, such as escherichia coli (e.coli), Salmonella (Salmonella), Klebsiella pneumoniae (Klebsiella pneumoniae), Pseudomonas (Pseudomonas) and Enterobacter (Enterobacter). Recently, gram-negative and gram-positive bacilli have emerged that are resistant to all antibiotics.

The rapidity of the emergence of these multiple antibiotic resistances is not reflected by the same rate of development of new antibiotics, and it is therefore conceivable that patients with severe infections will soon be no longer treated with currently available anti-infective agents. Several international reports have highlighted the potential problems associated with the emergence of antimicrobial resistance in many areas of medicine, and also outline the difficulties in treating patients with infections caused by these microorganisms.

Although most of the more resistant microorganisms are present in hospitals, multidrug resistant strains such as streptococcus pneumoniae and Mycobacterium tuberculosis (Mycobacterium tuberculosis) also cause serious community-acquired infections. Since 1980, the incidence of drug-resistant streptococcus pneumoniae has increased 60-fold, with 51% and 8% of isolates showing moderate or high resistance to penicillins or third-generation cephalosporins, respectively. Thus, treatment of pneumococcal pneumonia with first-line anti-infective agents is becoming more difficult. Drug-resistant bacteria from hospitals can be introduced into the community by discharging patients carrying them for continued treatment at home, such as multidrug-resistant staphylococcus aureus and vancomycin-resistant enterococci.

The resistance mechanism of bacteria can be mediated by chromosomes or plasmids. Studies of emerging resistance have shown that bacterial resistance can progress from low to high unless plasmids are obtained on which there is already substantial resistance developed. For example, penicillin-resistant pneumococci that initially appear have a slightly reduced sensitivity to antibiotics, but develop a high degree of resistance over time. Penicillin and tetracycline resistance in gonococci occurs in a similar manner. This phenomenon has also been observed in all antibiotic resistant E.coli where multiple steps are required to achieve clinically relevant levels of resistance.

Anti-infective agents can cause failure through three main mechanisms: i) destruction or alteration of the antibiotic (e.g., by production of beta-lactamases and aminoglycoside-inactivating enzymes), ii) alteration of the target site, and iii) prevention of access to the target site (e.g., alteration of permeability or efflux).

All three of these major resistance mechanisms, alone or in combination, are clinically important and can lead to treatment failure. However, recently, efflux-related multidrug resistance (MDR) has become a significant complication in the chemotherapy of bacterial infections.

Efflux mechanisms that explain resistance to a variety of anti-infective agents are often found in a wide range of bacteria. The term MDR system refers to a group of transporters capable of excreting a wide range of completely different substrates. Although such systems were described in eukaryotic cells first in the late 80 s of the 20 th century, the presence of MDR efflux pumps in bacteria that could indicate resistance to several drugs has been reported in the literature more and more. MDR cell lines are often associated with reduced drug accumulation due to increased efflux and decreased influx of chemotherapeutic drugs.

Overexpression of MDR efflux pumps following induction or due to mutations in their regulatory elements is a major mechanism for acquired bacterial resistance to multiple anti-infective agents. The main two groups of outflow systems are known: specific exporters and transporters conferring multidrug resistance (MDR). MDR systems are capable of removing different classes of anti-infective agents from bacterial cells and sometimes play a role in the inherent resistance of some bacteria to certain anti-infective agents. Their genes are often located on the chromosome of the bacteria. The mechanism leading to MDR is usually caused by transmembrane foreign transporter molecules belonging to the ATP-binding cassette (ABC) transporter superfamily. MDR efflux resistant to compounds such as tetracyclines, fluoroquinolones, macrolides, lincosamides, rifampin, chloramphenicol, and aminoglycosides has been generally identified. In contrast, genes encoding specific efflux systems are often associated with altered genetic components that can be readily interchanged between bacteria. Specific efflux systems resistant to compounds such as macrolides, lincosamides and/or streptogramins, tetracyclines and chloramphenicol in gram-positive and gram-negative bacteria have been generally identified.

Resistance of various viruses, such as HIV and herpes viruses in AIDS patients, to antiviral agents is a global problem. Drug resistance is not limited to well-known viral mutations, but it can also develop at the cellular level in humans. It has been shown in the prior art that resistance of HIV-1 to AZT (3 '-azido-3' -deoxythymidine) is caused by cellular mechanisms of resistance to AZT-. The outflow of AZT from cells is significantly increased, resulting in drug resistance. Thus, extracellular efflux resistance mechanisms, inhibiting which can reverse resistance and enhance the action of antiviral compounds, are an important factor in limiting the efficacy of antiviral chemotherapeutic agents for treating virally infected patients, such as HIV infected patients.

Prokaryotic and eukaryotic multidrug transporters are very similar in structure and function. For eukaryotic cells, drug efflux pumps have been considered by many authors as complement enzyme-based detoxification systems.

In prokaryotic cells, several resistance mechanisms can work in conjunction with efflux pumps, resulting in high drug resistance and therapeutic failure.

Inhibition of the resistance mechanisms restores the activity of these mechanisms as substrates against infectious agents. Recent elucidation and elucidation of the phenomenon of developing resistance has led to the search for drugs that can improve the effectiveness of anti-infective agents.

Two possible groups of MDR inhibitors are phenothiazines and thioxanthenes.

Phenothiazines and thioxanthenes are used clinically as neuroleptic and antiemetic agents. Phenothiazines and structurally related antipsychotics inhibit several cellular enzymes and block the function of key cellular receptors. Extrapyramidal side effects associated with antipsychotic therapy are caused by dopamine receptor binding. Generally, these extrapyramidal side effects have been demonstrated to be dose limiting in clinical trials using phenothiazines and thioxanthenes in non-psychiatric fields such as anticancer therapy.

Phenothiazines and thioxanthenes have been shown to inhibit the function of eukaryotic MDR efflux pumps and certain prokaryotic MDR efflux pumps.

Phenothiazines have been shown to belong to the class of drugs known to alter the resistance of certain bacteria to one or more antibacterial agents. Although the mechanism by which phenothiazines and other drugs modulate MDR is not known, it has been shown that their pharmacological properties can be mediated at least in part by the inhibition of efflux pumps. In addition, promethazine has been considered as an effective anti-granulating agent in cultures containing bacterial species such as Escherichia coli, Yersinia enterocolitica (Yersinia enterocolitica), Staphylococcus aureus, and Agrobacterium tumefaciens (Agrobacterium tumefaciens). However, the concentrations used are generally higher than clinically relevant concentrations.

Kaatz et al (2003) emphasize the fact that the effective concentration is higher than the clinically relevant concentration. Although it was shown that the chosen phenothiazines and the two geometric stereoisomers cis and trans flupentixol could inhibit the efflux pump of certain types of staphylococcus aureus, the authors still concluded that: unfortunately, inhibitors for the efflux of EtBr, acridine yellow and pyronin YIC of₅₀Higher value than IC used in clinical practice₅₀Value ".

Phenothiazines and thioxanthenes have moderate but extensive antimicrobial activity. MIC is usually higher than clinically relevant concentrations because the minimum effective concentration in vitro is about 20mg/l to several hundred mg/l, and the relevant serum levels are about 0.3. mu.g/l to 0.5mg/l (0.3ng/ml to 0.5. mu.g/ml).

Thioxanthenes exhibit geometric stereoisomerism. It has been previously shown that the cis and trans forms have roughly the same moderate antimicrobial efficacy. The MIC is usually much higher than the clinically relevant concentrations. Kristiansen et al in 1998 suggested a synergistic effect between penicillin and trans-clopenthixol in a study using a non-resistant mouse pathogen isolate of Streptococcus pneumoniae highly sensitive to penicillin (MIC 0.02. mu.g/ml penicillin). However, the authors did not show significant differences in MIC values for penicillin alone and penicillin in combination with trans-clopenthixol and did not perform concentration/response/time related studies, i.e. the concentration of drug in infected mice was unknown. Furthermore, the response of the infected bacteria in the mice is also unknown.

Johnstone et al in clinical trials of the antipsychotic effect of cis-flupentixol on trans-flupentixol on placebo showed that cis-flupentixol is a potent neuroleptic agent (especially for "positive" symptoms), whereas trans-flupentixol is not active as an antipsychotic. Trans-flupentixol is free of these side effects because it is a dopamine antagonist with very low potency and the extrapyramidal side effects associated with antipsychotic therapy are caused by dopamine receptor binding.

The trans forms such as trans-flupentixol and trans-clopenthixol are clearly free of antipsychotic activity or extrapyramidal side effects, which makes them of particular interest for use as anti-drug resistance agents.

U.S. patent 6,569,853 discloses several phenothiazine and thioxanthene derivatives.

British patent 925,538 discloses flupentixol as a tranquilizer, antiemetic, antihistamine, antispasmodic and general central nervous system inhibitor having efficacy. But does not mention any anti-infective or anti-drug resistant activity.

British patent 863,699 discloses several thioxanthene derivatives as tranquilizers. But does not mention any anti-infective or anti-drug resistant activity.

From the above discussion, it is clear that: increased resistance to anti-infective agents such as antibiotics is a major obstacle to the treatment of infections. Therefore, there is an urgent need for drugs that can inhibit or circumvent the mechanism of drug resistance and increase the efficacy of currently available anti-infective agents.

It is an object of the present invention to provide chemosensitizing compounds that are capable of sensitizing drug-resistant, including multi-drug resistant, cells or microorganisms to anti-infective agents by administering clinically relevant amounts of these chemosensitizing compounds to a subject in need thereof. It is another, but related, object of the invention to improve the efficacy of anti-infective agents in the treatment of infectious diseases, particularly when these infectious diseases are caused by drug-resistant, including multidrug-resistant, microorganisms.

Summary of The Invention

It will be appreciated from the above discussion that the prior art has heretofore considered thioxanthenes and phenothiazines to be unsuitable for use in combination with anti-infective agents in the treatment of infectious diseases, since the necessary therapeutic amounts of these chemosensitising compounds can lead to serious side effects.

The present inventors have realised that the above conclusions are generally based on studies conducted on artificially resistant or multi-resistant microorganisms or on microorganisms selected in vitro. Thus, despite the following prejudices in the art: in order to combat drug-resistant or multi-resistant microorganisms, it would be necessary for the concentration of thioxanthenes and phenothiazines to be well above clinically relevant levels, but the inventors decided to investigate the effect of these chemosensitising compounds in combination with anti-infective agents on clinically relevant resistant and multi-resistant isolates by using clinically relevant amounts of the chemosensitising compounds described herein, and thus investigate this problem in more detail.

Surprisingly, it was found that clinically relevant isolates of drug resistance and multidrug resistance can be effectively killed by the use of a clinically relevant amount of a chemosensitizing compound described herein in combination with an anti-infective agent. Contrary to what was previously believed, this unexpected finding opens up the possibility of effectively combating resistant and multiply resistant microorganisms through the combination of the chemosensitizing compounds described herein with commonly used anti-infective agents.

Accordingly, in a first aspect the present invention relates to the use of a compound of formula (I), or a metabolite or salt thereof, in the manufacture of a medicament for use in combination with an anti-infective agent in the treatment or prevention of an infectious disease,

wherein

V is selected from S, SO₂SO, O and NH;

w is N- (CHX)_n-N(R₁₀)(R₁₁) Or W is C ═ CH- (CHX)_m-N(R₁₀)(R₁₁)；

n is an integer of 2 to 6;

m is an integer of 1 to 5;

each X is independently selected from hydrogen, halogen, hydroxy, amino, nitro, optionally substituted C_1-6Alkyl and optionally substituted C_1-6An alkoxy group;

R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈and R₉Each independently selected from hydrogen, halogen, hydroxy, aminoNitro, optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl and optionally substituted C_1-6Alkoxy, optionally substituted C_2-6Alkenyloxy, carboxy, optionally substituted C_1-6Alkoxycarbonyl, optionally substituted C_1-6Alkylcarbonyl, formyl, optionally substituted C_1-6An alkylsulfonylamino group, an optionally substituted aryl group, an optionally substituted aryloxycarbonyl group, an optionally substituted aryloxy group, an optionally substituted arylcarbonyl group, an optionally substituted arylamino group, an arylsulfonylamino group, an optionally substituted heteroaryl group, an optionally substituted heteroaryloxycarbonyl group, an optionally substituted heteroaryloxy group, an optionally substituted heteroarylcarbonyl group, an optionally substituted heteroarylamino group, a heteroarylsulfonylamino group, an optionally substituted heterocyclic oxycarbonyl group, an optionally substituted heterocyclic oxy group, an optionally substituted heterocyclic carbonyl group, an optionally substituted heterocyclic amino group, a heterocyclic sulfonylamino group, a mono-and di- (C)_1-6Alkyl) amino, carbamoyl, mono-and di- (C)_1-6Alkyl) aminocarbonyl, amino-C_1-6Alkyl-aminocarbonyl, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-aminocarbonyl, C_1-6Alkylcarbonylamino, amino-C_1-6Alkyl-carbonylamino, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-carbonylamino, amino-C_1-6Alkyl-amino, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-amino, cyano, guanidino, ureido, C_1-6Alkanoyloxy group, C_1-6Alkylsulfonyl radical, C_1-6Alkylsulfinyl radical, C_1-6Alkylsulfonyloxy, aminosulfonyl, mono-and di- (C)_1-6Alkyl) aminosulfonyl and optionally substituted C_1-6An alkylthio group; and is

R₁₀And R₁₁Each independently selected from hydrogen, optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl, optionally substituted C_1-6Alkoxycarbonyl radicalOptionally substituted C_1-6Alkylcarbonyl, optionally substituted aryl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyl, optionally substituted heteroaryl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyl, aminocarbonyl, mono-and di- (C)_1-6Alkyl) aminocarbonyl; or R₁₀And R₁₁Together with the nitrogen atom to which they are attached form an optionally substituted nitrogen-containing heteroaryl group or an optionally substituted heterocyclyl group.

Other aspects of the invention will be apparent from the following description and the appended claims.

Brief Description of Drawings

FIG. 1 shows a series of bars depicting the effect of chemosensitising compounds against drug-resistant and multi-resistant microorganisms (values from tables 1-4 below). The Y-axis represents the DR ratio, defined as the ratio of the MIC of the anti-infective agent alone divided by the MIC of the anti-infective agent in the presence of the chemosensitizing compound. This ratio represents the apparent increase in the efficacy of the anti-infective agent produced by each chemosensitizing compound. Anti-infective agents: ciprofloxacin. Chemosensitizing compounds: 1. promethazine; 2.1-chlorpromazine; 3. chlorpromazine; 4.7-hydroxy chlorpromazine; 5.7, 8-dihydroxychlorpromazine; 6. methylthio-promazine; 7. trifluoropropylamine; 8. oxychloroprozine; 9. demethyl chlorpromazine; 10. perphenazine; 11. prochlorperazine; 12. fluphenazine; 13. trifluoperazine; 14.2-chloro-10- (2-dimethylaminoethyl) phenothiazine; 15. promethazine; 16. cis-flupentixol; 17. trans-flupentixol; 18. trans-clopenthixol.

Figures 2A and 2B show a series of bars depicting the strong synergistic effect of cis-, trans-and trans-clopenthixol on ciprofloxacin, gentamicin and piperacillin, respectively (values from table 5 below). The tested microorganisms were escherichia coli 331ME (fig. 2A) and pseudomonas aeruginosa (p.aeruginosa)432B (fig. 2B). The Y-axis represents the Fractional Inhibitory Concentration (FIC) index. For FIC indices less than 0.5, synergy is defined.

Figure 3 shows the enhancement of ciprofloxacin effect by trans-clopenthixol in a mouse peritonitis model.

FIG. 4 shows the serum levels of trans-clopenthixol in mice after a single dose administration of 0.3mg per mouse.

Detailed Description

Definition of

Herein, the term "C_1-6Alkyl "is intended to mean a straight-chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl.

Herein, the term "C_3-6-cycloalkyl "is intended to comprise 3, 4, 5 and 6 membered rings containing only carbon atoms, while the term" heterocyclyl "is intended to denote 3, 4, 5 and 6 membered rings in which carbon atoms together with 1 to 3 heteroatoms form said ring. The heteroatoms are independently selected from oxygen, sulfur and nitrogen. C_3-6The cycloalkyl and heterocyclyl rings may optionally contain one or more unsaturated bonds arranged in such a way that no aromatic pi-electron system is present.

“C_3-6Illustrative examples of "cycloalkyl" are the carbocyclic cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1, 3-cyclohexadiene and 1, 4-cyclohexadiene.

Illustrative examples of "heterocyclyl" are nitrogen-containing heterocyclic 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 2-imidazolinyl, imidazolidinyl, 2-pyrazolinyl, 3-pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, and piperazinyl. The bond to the heterocycle may be at the position of the heteroatom or through a carbon atom of the heterocycle.

Herein, the term "C_2-6Alkenyl radical"is intended to denote a linear or branched hydrocarbon radical having from 2 to 6 carbon atoms and containing one or more double bonds. C_2-6Illustrative examples of alkenyl groups include allyl, homo-allyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. C with multiple double bonds_2-6Illustrative examples of alkenyl groups include butadienyl, pentadienyl, and hexadienyl. The position of the double bond may be anywhere along the carbon chain.

Herein, the term "C_2-6Alkynyl "is intended to denote a straight-chain or branched hydrocarbon radical containing from 2 to 6 carbon atoms and containing one or more triple bonds. C_2-6Illustrative examples of alkynyl groups include acetylene, propynyl, butynyl, pentynyl, and hexynyl. The triple bond may be located at any position along the carbon chain. There may be more than one bond that is unsaturated, so that "C" is_2-6Alkynyl "is a di-or enediyne, as is known to those skilled in the art.

As used herein, the term "C" is used_1-6Alkoxy "is intended to denote C_1-6Alkyl-oxy, for example methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy and n-hexoxy.

The term "halogen" includes fluorine, chlorine, bromine and iodine.

As used herein, the term "aryl" is intended to mean a carbocyclic aromatic ring or ring system. Furthermore, the term "aryl" includes groups in which at least two aromatic rings or at least one aryl group and at least one C_3-6-a fused ring system wherein the cycloalkyl group or at least one aryl group and at least one heterocyclyl group share at least one chemical bond. Illustrative examples of "aryl" rings include phenyl, naphthyl, phenanthryl, anthracenyl, acenaphthenyl, tetrahydronaphthyl, fluorenyl, indenyl, indolyl, coumaranyl, coumarinyl, chromanyl, isochromanyl, and azulenyl.

As used herein, the term "heteroaryl" is intended to mean a group in which one of the aromatic rings is presentAryl in which one or more carbon atoms have been replaced by one or more heteroatoms selected from nitrogen, sulfur, phosphorus and oxygen. Further, as used herein, the term "heteroaryl" includes where at least one aromatic ring and at least one heteroaromatic ring, at least two heteroaryl groups, at least one heteroaryl group and at least one heterocyclic group, or at least one heteroaryl group and at least one C_3-6-fused ring systems in which the cycloalkyl groups share at least one chemical bond.

Illustrative examples of heteroaryl groups include furyl, thienyl, pyrrolyl, thiophenylOxazinonyl (phenoxazonyl),Azolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, isothiazolylAzolyl, imidazolyl, isothiazolyl,Oxadiazolyl, furazanyl, triazolyl, thiadiazolyl, piperidinyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl and triazinyl, isoindolyl, indolinyl, benzofuranyl, benzothienyl, benzopyrazolyl, indazolyl, benzimidazolyl, benzothiazolyl, purinyl, quinolizinyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl thienofuranyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinylOxazinyl and thianthryl.

Herein, the term "optionally substituted" is intended to indicate that said group may be substituted one or several times, such as 1 to 5 times, preferably 1 to 3 times, most preferably 1 to 2 times, by one or more groups selected from: c_1-6Alkyl radical, C_1-6Alkoxy, oxo (which may be represented in the tautomeric enol form), carboxy, amino, hydroxy (which may be represented in the tautomeric keto form when present in the enol system), nitro, sulphono, sulfanyl, C_1-6Carboxy, C_1-6Alkoxycarbonyl group, C_1-6Alkylcarbonyl, formyl, aryl, aryloxy, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono-and di- (C)_1-6Alkyl) amino, carbamoyl, mono-and di- (C)_1-6Alkyl) aminocarbonyl, amino-C_1-6Alkyl-aminocarbonyl, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-aminocarbonyl, C_1-6Alkylcarbonylamino, cyano, guanidino, ureido, C_1-6Alkanoyloxy group, C_1-6Alkylsulfonyloxy, dihalo-C_1-6Alkyl, trihalo-C_1-6Alkyl and halogen, where the aryl and heteroaryl substituents may themselves be substituted by C_1-6Alkyl radical, C_1-6Alkoxy, nitro, cyano, hydroxy, amino or halogen 1-3 times. Typically, the above substituents may allow further optional substitution.

The term "infectious agent" is intended to mean pathogenic microorganisms, such as bacteria, viruses, fungi and intracellular or extracellular parasites.

In a particular embodiment of the invention, the term "infectious agent" does not denote an infectious agent selected from the group consisting of Staphylococcus aureus strain ATCC 2593 and strains SA-1199, SA-1199B, SA-K1712, SA-K1748, SA 8325-4 and SA-K2068 derived therefrom.

In another embodiment of the invention, the term "infectious agent" does not denote Plasmodium falciparum (Plasmodium falciparum).

Similarly, the term "infectious disease" is used to refer to a disease caused by an infectious agent.

As used herein, the term "anti-infective agent" includes compounds that are capable of killing, inhibiting or retarding the growth of an infectious agent, such as commercially available antibiotics.

Specific examples of commonly used antibiotics for the treatment of bacterial and fungal infections include, but are not limited to, aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin; cabecephyms, such as chlorocarbacephem; carbapenems such as ertapenem, imipenem/cilastatin and betaergine; cephalosporins, such as cefadroxil, cefazolin, cephalexin, cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefbutam, ceftizoxime, ceftriaxone and cefepime; macrolides such as azithromycin, clarithromycin, dirithromycin, erythromycin and oleandomycin; a monamine ring; penicillins, such as amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin and ticarcillin; polypeptides such as bacitracin, polymyxin E and polymyxin B; quinolones such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin and trovafloxacin; sulfonamides, e.g. mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisomAzole and trimethoprim-sulfamethoxazoleAzole; tetracyclines, such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline.

Specific examples of antiviral compounds commonly used to treat viral infections include, but are not limited to, acyclovir, amantadine, cidofovir, famciclovir, fomivirsen, foscarnet, ganciclovir, interferon alpha, oseltamivir, penciclovir, ribavirin, rimantadine, trifluridine, valganciclovir, vidarabine, and zanamivir.

Specific examples of antifungal compounds commonly used to treat severe fungal infections include, but are not limited to, amphotericin B, caspofungin, fluconazole, flucytosine, itraconazole, ketoconazole, and voriconazole.

An infectious agent is referred to herein as "resistant" if it has undergone a change that reduces or eliminates the efficacy of an anti-infective agent commonly used to treat infections caused by the infectious agent. Similarly, the term "drug resistance" refers to the condition when a disease, such as an infectious disease, is not responsive to a therapeutic agent, such as an anti-infective agent. Resistance may be intrinsic, which means that the disease has never responded to the therapeutic, or acquired, which means that the disease ceases responding to the therapeutic to which the disease previously responded.

An infectious agent is referred to herein as "multidrug resistant" if it has undergone a change that reduces or eliminates the efficacy of two or more anti-infective agents commonly used to treat infections caused by the infectious agent. Similarly, "multi-drug resistance" is a type of resistance in which a disease, such as an infectious disease, is resistant to multiple drugs, such as multiple anti-infective agents.

The term "clinically relevant amount" is intended to mean that the chemosensitizing compound is administered to a patient in an amount that on the one hand is capable of reducing the symptoms of the infectious disease or curing the infectious disease to which the patient is being treated, and on the other hand is non-toxic to the patient and does not cause unacceptable side effects. As indicated above, many if not all of the chemosensitising compounds described herein are known to cause serious side effects in patients when administered at too high a concentration, i.e. in amounts which are not "clinically relevant".

Herein, when the term "naturally occurring" is used in conjunction with the term "infectious agent", i.e., with a pathogenic microorganism, it means that the infectious agent that produces the infectious disease is a microorganism that can be found in nature, including in humans. It is to be understood that infectious agents such as genetically manipulated laboratory strains or infectious agents that have been altered by other methods and/or manipulated by human intervention are not to be considered encompassed by the term "naturally occurring".

The term "serum" is used in its normal meaning, i.e. plasma free of fibrinogen and other coagulation factors.

In order to combat or prevent infectious diseases, the chemosensitising compounds disclosed herein should be administered together or in combination with an anti-infective agent. When the terms "together" and "combination" are used herein, they should not be construed narrowly as meaning that the chemosensitizing compound and the anti-infective agent should necessarily be administered simultaneously and/or form part of the same pharmaceutical composition, although this is an embodiment of the present invention. Thus, it will be understood that the terms "together" and "in combination" mean that the dosage (including dosage form) and frequency of administration of each compound, i.e., the chemosensitizing compound and the anti-infective agent, can be individually controlled. For example, one compound may be administered orally three times daily during treatment, while another compound may be administered intravenously once daily during treatment. Likewise, one compound may be administered daily during the treatment period, while another compound may be administered only once or for several days during the treatment period. As explained above, the chemosensitising compound and the anti-infective compound may be administered simultaneously and they may be comprised in the same pharmaceutical composition. Thus, the terms "together" and "in combination" mean that the chemosensitizing compound is administered to the patient at least once during the treatment period, and the anti-infective agent is also administered to the patient at least once during the treatment period.

The term "steady state serum concentration" of a (chemosensitizing compound) is defined herein as those values that recur with each dose and represent the state of equilibrium between the amount of the chemosensitizing compound administered and the amount being eliminated within a given time interval.

As used herein, the term "treatment" refers to administering a drug to a subject and includes i) preventing an infectious disease (i.e.: rendering clinical symptoms of infectious disease non-existent), ii) inhibiting infectious disease (i.e.: arresting the development of clinical symptoms of infectious disease) and iii) alleviating the disease (i.e.: regressing clinical symptoms of infectious diseases) and combinations thereof.

The term "prevention" or "prophylactic treatment" refers to the treatment of a subject who has not yet been infected, but who may be susceptible to or at risk of infection.

The term "subject" as used herein means a living vertebrate, e.g., a mammal such as a human.

"pharmaceutically acceptable" means suitable for use in mammals, particularly in humans.

The term "chemosensitizing compound" as used herein is intended to mean a drug that reverses the resistance of a microorganism or cell to a given anti-infective agent. Thus, a "chemosensitizing compound" as used herein can sensitize a drug-resistant or multi-resistant microorganism or cell to the action of an anti-infective agent.

Chemosensitizing compounds

With respect to the above general formula (I), the substituent R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈And R₉Each independently selected from hydrogen, halogen, hydroxy, amino, nitro, optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl and optionally substituted C_1-6Alkoxy, optionally substituted C_2-6Alkenyloxy, carboxy, optionally substituted C_1-6Alkoxycarbonyl, optionally substituted C_1-6Alkylcarbonyl, formyl, optionally substituted C_1-6Alkylsulfonylamino, optionally substituted aryl, optionally substituted aryloxycarbonyl, optionally substituted aryloxy, optionally substituted arylcarbonyl, optionally substituted arylamino, arylsulfonylamino, optionally substituted heteroaryl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroaryloxy, optionally substituted heteroarylcarbonyl, optionally substituted heteroarylaminoA group, a heteroarylsulfonylamino group, an optionally substituted heterocyclic group, an optionally substituted heterocyclyloxycarbonyl group, an optionally substituted heterocyclyloxy group, an optionally substituted heterocyclylcarbonyl group, an optionally substituted heterocyclylamino group, a heterocyclylsulfonylamino group, a mono-and di- (C)_1-6Alkyl) amino, carbamoyl, mono-and di- (C)_1-6Alkyl) aminocarbonyl, amino-C_1-6Alkyl-aminocarbonyl, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-aminocarbonyl, C_1-6Alkylcarbonylamino, amino-C_1-6Alkyl-carbonylamino, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-carbonylamino, amino-C_1-6Alkyl-amino, mono-and di- (C)_1-6Alkyl) amino-C_1-6Alkyl-amino, cyano, guanidino, ureido, C_1-6Alkanoyloxy group, C_1-6Alkylsulfonyl radical, C_1-6Alkylsulfinyl radical, C_1-6Alkylsulfonyloxy, aminosulfonyl, mono-and di- (C)_1-6Alkyl) aminosulfonyl and optionally substituted C_1-6An alkylthio group.

In a preferred embodiment of the invention, R₂The substituents being electron-withdrawing groups, e.g. halogen, nitro or halogen-substituted C_1-6An alkyl group. More preferably, R₂Selected from F, Cl, Br, I, CH₂Y、CHY₂And CY₃(wherein Y represents a halogen atom), e.g. CH₂Cl、CH₂F、CHCl₂、CHF₂、CCl₃Or CF₃In particular CCl₃Or CF₃. Most preferably, R₂Is Cl or CF₃。

Substituent R₁、R₃、R₄、R₅、R₆、R₇、R₈And R₉Preferably each independently selected from hydrogen, optionally substituted C_1-6Alkyl and optionally substituted C_1-6An alkoxy group. More preferably, all R₁、R₃、R₄、R₅、R₆、R₇、R₈And R₉Are all hydrogen.

Thus, in a very preferred embodiment of the invention, R₂Is Cl or CF₃And R is₁、R₃、R₄、R₅、R₆、R₇、R₈And R₉Each is hydrogen.

As mentioned above, V is selected from S, SO₂SO, O and NH, for example S or SO. In a very preferred embodiment of the invention, V is S.

It should be understood that when W is N- (CHX)_n-N(R₁₀)(R₁₁) And V is S, the chemosensitizing compound of the general formula (I) is changed into phenothiazine of the general formula (II),

wherein n is an integer from 2 to 6, such as 2, 3, 4, 5 or 6, and each X is independently selected from hydrogen, halogen, hydroxy, amino, nitro, optionally substituted C_1-6Alkyl and optionally substituted C_1-6An alkoxy group. R₁₀And R₁₁Each independently selected from hydrogen, optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl, optionally substituted C_1-6Alkoxycarbonyl, optionally substituted C_1-6Alkylcarbonyl, optionally substituted aryl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyl, optionally substituted heteroaryl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyl, aminocarbonyl, mono-and di- (C)_1-6Alkyl) aminocarbonyl; or R₁₀And R₁₁Together with the nitrogen atom to which they are attached form an optionally substituted nitrogen-containing heteroaryl group or an optionally substituted heterocyclyl group.

In a preferred embodiment of the invention, n is 2 or 3 and X is hydrogen or CH₃. Accordingly, in the present inventionIn a preferred embodiment, W is N- (CH)₂)₃-N(R₁₀)(R₁₁) Or N-CH₂-CH(CH₃)-N(R₁₀)(R₁₁). Particularly preferably wherein W is N- (CH)₂)₃-N(R₁₀)(R₁₁) The structure of (1).

In one target embodiment of the invention, R₁₀And R₁₁Each independently selected from hydrogen and optionally substituted C_1-6An alkyl group. According to this embodiment, R is preferred₁₀And R₁₁Are all optionally substituted C_1-6An alkyl group. Most preferred is R₁₀And R₁₁Are all CH₃。

In another target embodiment of the present invention, R₁₀And R₁₁Together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic group, such as an optionally substituted 2-pyrrolinyl group, an optionally substituted 3-pyrrolinyl group, an optionally substituted pyrrolidinyl group, an optionally substituted 2-imidazolinyl group, an optionally substituted imidazolidinyl group, an optionally substituted 2-pyrazolinyl group, an optionally substituted 3-pyrazolinyl group, an optionally substituted pyrazolidinyl group, an optionally substituted piperidyl group, an optionally substituted morpholinyl group, an optionally substituted thiomorpholinyl group, or an optionally substituted piperazinyl group. According to this embodiment, R is preferred₁₀And R₁₁Together with the nitrogen atom to which they are attached form an optionally substituted piperidinyl group or an optionally substituted piperazinyl group, in particular an optionally substituted piperazinyl group. The piperazinyl ring may be unsubstituted, but is preferably optionally substituted C_1-6Alkyl substitution, in particular C substituted in the para position, i.e. optionally substituted_1-6The alkyl group is covalently attached to the second nitrogen atom of the piperazinyl ring. In a very preferred embodiment of the invention, C, optionally substituted_1-6Alkyl is selected from-CH₃、-CH₂OH、-CH₂-CH₃and-CH₂-CH₂OH, e.g. -CH₃or-CH₂-CH₂OH, especially-CH₂-CH₂OH。

Specific examples of phenothiazines mentioned above include promazine, 1-chlorpromazine, 7-hydroxychloropromazine, 7, 8-dihydroxychlorpromazine, methylthiopromazine, trifluoropromazine, demethylchlorpromazine, perphenazine, prochlorperazine and 2-chloro-10- (2-dimethylaminoethyl) phenothiazine.

It is also understood that when W is C ═ CH- (CHX)_m-N(R₁₀)(R₁₁) And V is S, the chemosensitizing compound of formula (I) is changed to thioxanthene of formula (III),

the thioxanthenes of general formula (III) give rise to cis and trans isomerism. Herein, the compounds of formula (IIIa) are referred to as cis configuration, while the compounds of formula (IIIb) are referred to as trans configuration:

wherein m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5, and each X is independently selected from hydrogen, halogen, hydroxy, amino, nitro, optionally substituted C_1-6Alkyl and optionally substituted C_1-6An alkoxy group. R₁₀And R₁₁Each independently selected from hydrogen, optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted C_2-6Alkynyl, optionally substituted C_1-6Alkoxycarbonyl, optionally substituted C_1-6Alkylcarbonyl, optionally substituted aryl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyl, optionally substituted heteroaryl, optionally substituted heteroaryloxycarbonyl, optionally substituted heteroarylcarbonyl, aminocarbonyl, mono-and di- (C)_1-6Alkyl) aminocarbonyl; or R₁₀And R₁₁Together with the nitrogen atom to which they are attached formAn optionally substituted nitrogen-containing heteroaryl group or an optionally substituted heterocyclic group.

It is generally preferred that the compound of formula (III) has a trans configuration, i.e., a structure represented by formula (IIIb).

In a preferred embodiment, X is hydrogen and m is 2 or 3, in particular 3. Thus, in a preferred embodiment of the invention, W has the structure C ═ CH- (CH)₂)₂-N(R₁₀)(R₁₁)。

Specific examples of the phenothiazines mentioned above include trans-haloperidol, cis-haloperidol, trans-clopenthixol, and cis-clopenthixol. Particularly preferred chemosensitising compounds for use in the present invention are trans-flupenthixol and trans-clopenthixol. Most preferred is trans-clopenthixol.

It is apparent from the general formulae shown herein and the definitions relating thereto that certain chemosensitising compounds described herein are chiral. Furthermore, the presence of certain unsaturated or cyclic fragments or multiple stereogenic atoms provides the presence of diastereomeric forms for some chemosensitizing compounds. The invention is intended to include all stereoisomers, including optical isomers and mixtures thereof, as well as pure, partially enriched or (where appropriate) racemic forms. In particular, the various chemosensitising compounds described herein may be in the form of the E-or Z-stereoisomers or mixtures of such isomers.

Furthermore, it will also be appreciated that the chemosensitising compounds described herein include possible salts thereof, the pharmaceutically acceptable salts of which are of course particularly suitable for therapeutic use. Salts include acid addition salts and base salts. Examples of acid addition salts are hydrochloride, fumarate, oxalate and the like. Examples of base salts are those wherein the (remaining) counter-ion is selected from alkali metals such as sodium and potassium, alkaline earth metals such as calcium, potassium and ammonium ions (A), (B), (C), (⁺N(R′)₄Wherein R' independently means optionally substituted C_1-6Alkyl, optionally substituted C_2-6Alkenyl, optionally substituted aryl or optionally substituted heteroaryl). Pharmaceutically acceptable salts are, for example, Remington's-The Science and Practice of Pharmacy (20 th edition, Alfonso R.Gennaro (eds.), Lippincott, Williams&Wilkins; ISBN: 0683306472, 2000) and the full manual of Pharmaceutical Technology (Encyclopedia of Pharmaceutical Technology).

The effect of a chemosensitizing compound in reversing drug resistance or multidrug resistance can be determined as described herein and the potency of a chemosensitizing compound in combination with a selected anti-infective agent against a selected microorganism can be expressed in MIC values, DR ratios, and/or FIC indices.

The Minimum Inhibitory Concentration (MIC) is defined as the minimum inhibitory concentration that shows no visible growth according to NCCLS guidelines.

The resistance (DR) ratio is defined as the ratio of the MIC value of the anti-infective agent alone divided by the MIC of the anti-infective agent in the presence of the chemosensitizing compound. This ratio represents the increase in the apparent potency of the anti-infective agent caused by the chemosensitizing compound, which can be expressed as:

DR ratio (MIC)_{Anti-infective agents})/(MIC_{Anti-infective + chemosensitizing compounds})

The Fractional Inhibitory Concentration (FIC) index for each anti-infective agent alone and in combination with a chemosensitizing compound can be calculated according to the following formula:

FIC＝FIC_{chemosensitizing compounds}+FIC_{Anti-infective agents}

Wherein:

FIC_{chemosensitizing compounds}＝(MIC_{Chemosensitizing compound + anti-infective agent})/(MIC_{Chemosensitizing compounds})

FIC_{Anti-infective agents}＝(MIC_{Anti-infective + chemosensitizing compounds})/(MIC_{Anti-infective agents})

The synergistic effect of the chemosensitising compounds described herein, i.e. their ability to reverse the resistance or multidrug resistance of a microorganism, can be assessed by any method available to the skilled person, including the in vitro assays described in the examples herein. In a preferred embodiment of the invention, the chemosensitising compound, the anti-infective agent and the infectious agent (and hence the infectious disease to be treated) exhibit a FIC index of at most 0.5, when determined according to the method described in the examples herein. More preferably, the FIC index is at most 0.4, such as at most 0.3, such as at most 0.2. Even more preferably, the FIC index is at most 0.1, e.g. at most 0.075, at most 0.05 or even at most 0.025.

As previously mentioned, the chemosensitising compounds described herein generally have high MIC values. For a chemosensitising compound which is a potent inhibitor, this represents the ratio (MIC)_{Chemosensitizing compound + anti-infective agent})/(MIC_{Chemosensitizing compounds}) Close to 0, which thus represents FIC_{Chemosensitizing compounds}0. This also means that FIC ≈ FIC_{Anti-infective agents} ＝(MIC_{Anti-infective + chemosensitizing compounds})/(MIC_{Anti-infective agents})≈1/DR。

Thus, in another preferred embodiment of the invention, the chemosensitising compound, the anti-infective agent and the infectious agent (and hence the infectious disease to be treated) exhibit a DR ratio of at least 2 when determined according to the method described in the examples herein. More preferably the DR ratio is at least 5, such as at least 10, such as at least 20. Even more preferably the MIC value is at least 30, such as at least 50, at least 75 or even at least 100.

Treatments, pharmaceutical compositions and dosages

As explained above, the chemosensitising compounds described herein may be used in combination with an anti-infective agent for the treatment of infectious diseases. Thus, the chemosensitising compounds described herein may be used in the preparation of a medicament for use in combination with an anti-infective agent in the treatment of an infectious disease.

Furthermore, the chemosensitizing compounds described herein in combination with an anti-infective agent can also be used for the prophylactic treatment of infectious diseases. This is of particular interest in cases where the human is at high risk of infection, such as immunosuppressed patients or patients who have undergone surgery. Thus, the chemosensitising compounds described herein may also be used in the preparation of a medicament for the prophylactic treatment of infectious diseases in combination with an anti-infective agent.

Furthermore, the chemosensitizing compounds described herein in combination with an anti-infective agent may also be used for the preparation of a medicament for the treatment or prevention of an infectious disease.

In another aspect, the invention relates to a chemosensitizing compound as described herein for use as a medicament in combination with an anti-infective agent or to a chemosensitizing compound as described herein for use as a medicament in combination with an anti-infective agent.

In another related aspect, the chemosensitizing compounds described herein can be used to reduce resistance, particularly multi-resistance, to an infectious agent. Accordingly, the chemosensitising compounds described herein may be used in the preparation of medicaments for reducing the resistance of infectious agents to anti-infectious agents.

In another related aspect, the chemosensitizing compounds described herein can be used to sensitize sensitive, drug-resistant, or multi-drug resistant cells, preferably drug-resistant and multi-drug resistant cells, more preferably multi-drug resistant cells, to an infectious agent. Thus, the chemosensitising compounds described herein may be used in the preparation of a medicament for sensitizing sensitive, drug-resistant or multi-drug resistant cells, preferably drug-resistant and multi-drug resistant cells, more preferably multi-drug resistant cells, to an anti-infective agent.

Another aspect of the invention relates to a method of treating or preventing an infectious disease in a subject, said method comprising administering to said subject a chemosensitizing compound as described herein in combination with an anti-infective agent.

Another aspect of the invention relates to a method of reducing drug resistance, particularly multidrug resistance, of an infectious agent.

Another aspect of the invention relates to a method of sensitizing a susceptible, drug-resistant, or multi-drug resistant microorganism or cell, preferably a resistant and multi-drug resistant microorganism or cell, more preferably a multi-drug resistant microorganism or cell, to an anti-infective agent.

Another aspect of the invention relates to a method of preventing the development of drug resistance or multi-drug resistance in an infectious agent, said method comprising administering to a subject a chemosensitising compound as described herein.

Yet another aspect of the invention relates to a kit comprising a first dosage unit comprising a chemosensitising compound as described herein and a further dosage unit comprising an antimicrobial agent.

Treatment of

It will be understood from the disclosure herein that the infectious disease to be treated is one that is commonly treated with anti-infective agents. Infectious diseases are usually caused by infectious agents such as bacteria, viruses, fungi or intracellular or extracellular parasites. The infectious agent is typically naturally occurring, i.e., a naturally occurring bacterium, a naturally occurring virus, a naturally occurring fungus, or a naturally occurring intracellular or extracellular parasite.

More specifically, the infectious agent may be a gram-negative or gram-positive bacterium.

Specific examples of gram-negative bacteria include bacteria selected from the group consisting of Escherichia (Escherichia), Proteus (Proteus), Salmonella (Salmonella), Klebsiella (Klebsiella), Providencia (Providencia), Enterobacter (Enterobacter), Burkholderia (Burkholderia), Pseudomonas (Pseudomonas), Acinetobacter (Acinetobacter), Aeromonas (Aeromonas), Haemophilus (Haemophilus), Yersinia (Yersinia), Neisseria (Neisseria), Erwinia (Erwinia), Rhodopseudomonas (Rhodoperudeomomas), and Burkholderia (Burkholderia).

Specific examples of gram-positive bacteria include bacteria selected from the group consisting of Lactobacillus (Lactobacillus), Azorhizobium (Azorhizobium), Streptococcus (Streptococcus), Pediococcus (Pediococcus), Photobacterium (Photobacterium), Bacillus (Bacillus), Enterococcus (Enterococcus), Staphylococcus (Staphylococcus), Clostridium (Clostridium), vibrio (Butyrivibrio), Sphingomonas (Sphingomonas), Rhodococcus (Rhodococcus), and Streptomyces (Streptomyces).

In other embodiments, the infectious agent is, for example, a genus selected from the group consisting of methanobacterium (methanobasidium), Sulfolobus (Sulfolobus), archaebacteria (archaeogenbu), Rhodobacter (Rhodobacter) and Sinorhizobium (Sinorhizobium).

In other embodiments, the infectious agent is, for example, a fungus from the genus Mucor (Mucor) or Candida (Candida) such as Mucor racemosus or Candida albicans, from the genus cryptococcus (cryptococcus) such as cryptococcus neoformans (cr. neoformans) or from the genus Aspergillus (Aspergillus) such as Aspergillus fumigatus (a. fumago).

In further embodiments, the infectious agent is a virus, such as a picornaviridae, reoviridae, orthomyxoviridae, paramyxoviridae, adenoviridae, coronaviridae, human immunodeficiency virus, hepatitis virus, herpesviridae, oncovirus, cytomegalovirus, papovaviridae, or a prion.

In other embodiments, the infectious agent is a protozoan, such as malaria or a cryptosporidium parasite.

In a particular embodiment of the invention, the infectious agent is not a Staphylococcus aureus strain selected from ATCC 2593 and strains SA-1199, SA-1199B, SA-K1712, SA-K1748, SA 8325-4 and SA-K2068 derived therefrom.

In another embodiment of the invention, the infectious agent is not plasmodium falciparum.

Toxicity and therapeutic efficacy of the chemosensitizing compounds described herein can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., by determining LD₅₀(lethal dose for 50% of the population) and ED₅₀(therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which may be in LD₅₀And ED₅₀Ratio (LD) between₅₀/ED₅₀) To indicate. Chemosensitising compounds that exhibit a large therapeutic index are preferred. The data obtained from these cell culture assays or animal studies can be used to formulate a range of dosage for use in human subjects. The dose of these chemosensitising compounds is preferably at a dose which includes the ED₅₀And circulating concentrations with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

Pharmaceutical composition

The chemosensitizing compounds described herein are generally formulated into pharmaceutical compositions prior to use as a medicament.

Thus, another aspect of the present invention relates to a pharmaceutical composition comprising a chemosensitising compound as described herein and at least one pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition may contain one or two or more chemosensitising compounds as described herein. In an object embodiment of the invention, the pharmaceutical composition comprises one or more anti-infective agents in addition to the chemosensitizing compound.

The route of administration of the chemosensitising compounds described herein may be any suitable route which allows concentrations in the blood or in the tissues to be comparable to clinically relevant concentrations. Thus, for example, the following routes of administration are applicable, although the invention is not limited thereto: oral, parenteral, dermal, transdermal, nasal, topical, rectal, vaginal, and ocular routes. It will be clear to the skilled person that the route of administration will depend on the particular chemosensitising compound in question, and in particular the choice of route of administration will depend on the physicochemical properties of the chemosensitising compound, as well as the age and weight of the patient and the particular disease or condition and its severity. However, the oral and parenteral routes are generally preferred.

The chemosensitizing compound described herein can be included in the pharmaceutical composition in any suitable amount, typically in an amount of from about 0.1 to about 95% by weight based on the total weight of the composition. The compositions may be presented in dosage forms, e.g., unit dosage forms, suitable for oral, parenteral, rectal, dermal, transdermal, nasal, topical, vaginal and/or ocular routes of administration. Thus, the compositions may be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injections, implants, sprays, aerosols, and other suitable forms.

Pharmaceutical compositions may be formulated in accordance with conventional Pharmaceutical practice, see, for example, "Remington's Pharmaceutical Sciences" and "the Pharmaceutical technical complete book" by Swarbrick, J. & j.c. boylan (Marcel Dekker, new york, 1988). Generally, the chemosensitizing compounds described herein are formulated with (at least) a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients are those known to those skilled in the art.

Oral preparation

Pharmaceutical compositions for oral use include tablets containing a chemosensitizing compound as described herein, optionally in combination with at least one anti-infective agent, and non-toxic pharmaceutically acceptable excipients. These excipients may be, for example:

inert diluents or fillers such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate;

granulating and disintegrating agents, such as cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginates or alginic acid;

binders, such as sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol; and

lubricants, including glidants and antiadherents, such as magnesium stearate, zinc stearate, stearic acid, silicates, hydrogenated vegetable oils or talc.

Other pharmaceutically acceptable excipients may be colorants, flavors, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, to obtain a controlled release formulation, the coating may be adjusted to release the chemosensitizing compound in a predetermined pattern (see below), or the coating may be adjusted so that the active drug is not released until after passage through the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g. based on hydroxypropyl methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers (Eudragit E)) Polyethylene glycol and/or polyvinylpyrrolidone) or enteric coatings (e.g. based on methacrylic acid copolymers (Eudragit)L and S), cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethyl cellulose).

In addition, time delay materials such as glyceryl monostearate or glyceryl distearate may also be employed.

Furthermore, the solid tablet composition as mentioned above may also be provided with a coating adapted to protect the composition from adverse chemical changes such as chemical degradation prior to release of the chemosensitising compound.

Coatings can be applied to solid dosage forms in a manner similar to that described by James A.Seitz, "aqueous film coating" in the pharmaceutical technical Instructions (Vol.1, pp. 337-349, Swarbrick, J. & J.C. Boylan, Marcel Dekker, N.Y., 1988).

Formulations for oral use may also be presented as chewable tablets or hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin, or soft gelatin capsules wherein the active ingredient is mixed with water or an oil base such as peanut oil, liquid paraffin or olive oil.

Powders and granules can be prepared in a conventional manner using the ingredients mentioned above in tablets and capsules using, for example, a mixer, a fluidized bed apparatus or a spray-drying apparatus.

Controlled release compositions for oral use, for example, can be formulated to release the active agent by controlling the dissolution and/or diffusion of the active agent.

Controlled release of dissolution or diffusion can be obtained by suitable coating of tablets, capsules, pellets or granules of the chemosensitising compound or by adding the chemosensitising compound to, for example, a suitable matrix.

The controlled release coating may comprise one or more of the coating substances mentioned above and/or for example shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, palmitoyl stearyl glyceride, ethylcellulose, acrylic resins, racemic polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylates, methyl methacrylate, 2-hydroxy methacrylate, methacrylate hydrogels, 1, 3-butylene glycol, ethylene glycol methacrylate and/or polyethylene glycol.

In controlled release matrix formulations of the chemosensitising compounds, the matrix material may comprise, for example, hydrated methyl cellulose, carnauba wax and stearyl alcohol, carbopol 934, polysiloxane, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene and/or halogenated fluorocarbons.

The controlled release composition of the chemosensitising compound described herein may also be in the form of a floating tablet or capsule, i.e. a tablet or capsule that floats on top of the gastric contents for a period of time after oral administration. The floating tablets of the chemosensitising compound may be prepared by granulating a mixture of the chemosensitising compound, excipients and 20-75% w/w of a hydrocolloid, such as hydroxyethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose. The resulting granules can then be compressed into tablets. Upon contact with the gastric fluid, the tablet may form a substantially water-impermeable gel barrier around its surface. This gel barrier is involved in maintaining a density of less than 1, thereby keeping the tablet floating in the gastric juice.

Fluid/liquid composition for oral use

Powders, dispersible powders or granules suitable for preparation of aqueous suspensions by the addition of water are also convenient dosage forms. Formulations as suspensions, emulsions, or dispersions may be presented as the active ingredient in admixture with a dispersing or wetting agent, suspending agent, and/or one or more preservatives. These formulations may also be suitable for use in which the active substance is applied to, for example, a mucosal membrane such as the gastrointestinal, oral, nasal, rectal or vaginal mucosa, or for application to intact or damaged skin or wounds.

Suitable dispersing or wetting agents are, for example, naturally occurring phosphatides, for example lecithin or soya lecithin; condensation products of ethylene oxide with, for example, fatty acids, long-chain fatty alcohols, or partial esters derived from fatty acids and hexitols or hexitol anhydrides, such as polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate, and the like.

Suitable suspending agents are, for example, naturalGums present, such as gum arabic, xanthan gum or tragacanth; cellulose, e.g. sodium carboxymethylcellulose, microcrystalline cellulose (e.g. Avicel)RC 591), methyl cellulose; alginates such as sodium alginate and the like.

Suitable examples of preservatives for use in the formulations of the present invention are parabens, such as methyl or propyl paraben and benzalkonium chloride.

Rectal and/or vaginal formulation

For application to the rectal or vaginal mucosa, formulations suitable for use in the present invention include suppositories (emulsion or suspension dosage forms), enemas, and rectal gelatin capsules (solutions or suspensions). Suitable pharmaceutically acceptable suppository bases include cocoa butter, esterified fatty acids, glycerogelatin, and various water-soluble or dispersible bases such as polyethylene glycols and polyoxyethylene sorbitan fatty acid esters.

Various additives such as accelerators or surfactants may be added.

Nasal preparation

For application to the nasal mucosa, nasal sprays and aerosols for inhalation are applied to the compositions of the invention. In typical nasal formulations, the active substance is present in the form of a particulate formulation optionally dispersed in a suitable vehicle. The pharmaceutically acceptable vehicles and excipients present in the compositions, and optionally other pharmaceutically acceptable materials such as diluents, promoters, flavoring agents, preservatives and the like, are selected in accordance with conventional pharmaceutical practice in a manner understood by those skilled in the art of pharmaceutical formulation.

Nasal administration can be used in situations where immediate action is desired. Furthermore, the active substance can be adsorbed on the nasal mucosa after administration of the nasal preparation of the invention. Adsorption to the mucosa is believed to produce less irritation than with liquid vehicles containing, for example, permeation enhancers or propellants.

Topical formulations

For application to the skin, the formulations of the present invention may contain conventional, non-toxic pharmaceutically acceptable carriers and excipients, including microspheres and liposomes. Formulations include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other kinds of transdermal drug delivery systems. Pharmaceutically acceptable excipients may include emulsifiers, antioxidants, buffers, preservatives, humectants, penetration enhancers, chelating agents, gelling agents, ointment bases, fragrances, and skin protectants.

Examples of emulsifying agents are naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean lecithin, and sorbitan monooleate derivatives.

Examples of antioxidants are Butylated Hydroxyanisole (BHA), vitamin C and its derivatives, vitamin E and its derivatives, butylated hydroxyanisole and cysteine.

Examples of preservatives are parabens (e.g. methyl or propyl paraben) and benzalkonium chloride.

Examples of humectants are glycerol, propylene glycol, sorbitol and urea.

Examples of permeation enhancers are propylene glycol, DMSO, triethanolamine, N-dimethylacetamide, N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofuryl alcohol and lauryl nitrogenKetones。

Examples of chelating agents are EDTA-Na, citric acid and phosphoric acid.

Examples of other excipients are edible oils such as almond oil, castor oil, cocoa butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil and tea oil; examples of polymers are, for example, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, chitosan, pectin, xanthan gum, carrageenan, locust bean gum, gum arabic, gelatin and alginates. Examples of ointment bases are beeswax, paraffin, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span), polyethylene glycol and condensation products between sorbitan esters of fatty acids and ethylene oxide such as polyoxyethylene sorbitan monooleate (Tween).

The above-mentioned topical formulations may also be applied to wounds, or they may be suitable for direct application or insertion into body-related openings such as the rectal, urethral, vaginal or oral cavity. The formulation may simply be applied directly to the part to be treated, e.g. the mucosa.

Parenteral formulation

The pharmaceutical compositions may also be administered parenterally, by injection, infusion or implantation (intravenously, intramuscularly, intra-articularly, subcutaneously, etc.), in dosage forms, formulations or, for example, suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.

The formulation and preparation of such compositions is well known to those skilled in the art of pharmaceutical formulation. Specific formulations can be found in textbooks entitled "Remington's Pharmaceutical Sciences".

Compositions for parenteral use may be presented in unit dosage form, e.g. in ampoules or vials containing several doses and to which a suitable preservative may be added (see below). The compositions may be in the form of solutions, suspensions, emulsions, infusion devices or implantable delivery devices, or they may be presented as a dry powder for reconstitution with water or other suitable vehicle just prior to use. In addition to the chemosensitizing compounds described herein, the compositions can also contain suitable parenterally acceptable carriers and/or excipients, or the active agent can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, and the like, for controlled release. In addition, the compositions may conveniently contain suspending agents, solubilising agents, stabilising agents, pH-adjusting agents and/or dispersing agents.

In another object embodiment of the present invention, the pharmaceutical composition is a solid dosage form such as a tablet prepared from the particulate material described in WO 03/004001 and WO 2004/062643.

As indicated above, the pharmaceutical composition may contain the chemosensitising compound in a sterile injectable form. To prepare such a composition, the chemosensitising compound may be dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that may be employed include water, water adjusted to an appropriate pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or an appropriate buffer, 1, 3-butanediol, Ringer's solution and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives, such as methyl, ethyl or n-propyl paraben. In case the chemosensitising compound is only sparingly soluble or slightly soluble in water, a solubility promoter or solubilizer may be added, or the solvent may contain 10-60% w/w of propylene glycol or the like in addition to water.

Dosage form

As discussed in detail above, an important aspect of the present invention is the following discovery: the chemosensitising compounds described herein are capable of reversing drug resistance or multidrug resistance when administered in clinically relevant amounts, i.e. amounts small enough to avoid the serious side effects normally associated with the chemosensitising compounds described herein.

It will be appreciated that the dose to be administered may depend on the form of administration (see below). Independent of the form of administration, the chemosensitising compounds should be administered in a clinically relevant amount, i.e. in an amount which on the one hand produces the relevant therapeutic effect and on the other hand does not lead to serious side effects.

Preferably the chemosensitising compound as described herein is administered in a clinically relevant amount that results in a steady state serum concentration of less than 8.0 mg/l. More preferably the chemosensitising compound is administered in a clinically relevant amount that results in a steady state serum concentration of less than 7.0mg/l, such as less than 6.0mg/l, such as less than 5.0 mg/l. Even more preferably the chemosensitising compound is administered in a clinically relevant amount resulting in a steady state serum concentration of less than 4.0mg/l, such as less than 3.0mg/l, such as less than 2.0 mg/l. Most preferably, the chemosensitising compound is administered in a clinically relevant amount that results in a steady state serum concentration of less than 1.5mg/l, for example about 1.0mg/l or about 0.5 mg/l.

In other words, the chemosensitising compound is preferably administered in a clinically relevant amount that results in a steady state serum concentration of from 0.01. mu.g/l to less than 8.0mg/l, such as from 0.02. mu.g/l to 7.0mg/l, such as from 0.04. mu.g/l to 6.0 mg/l. More preferably, the steady state serum concentration of the chemosensitising compound is in the range of from 0.06. mu.g/l to 5.0mg/l, for example from 0.08. mu.g/l to 4.0mg/l, for example from 0.1. mu.g/l to 3.0 mg/l. Even more preferably the steady state serum concentration of the chemosensitising compound is in the range of from 0.2. mu.g/l to 2.0mg/l, such as from 0.4. mu.g/l to 2.0mg/l, such as from 0.5. mu.g/l to 2.0 mg/l. It is still more preferred that the steady state serum concentration of the chemosensitising compound is from 0.6. mu.g/l to 2.0mg/l, such as from 0.8. mu.g/l to 2.0mg/l, such as from 0.9. mu.g/l to 2.0 mg/l. Most preferably the steady state serum concentration of the chemosensitising compound is from 1.0. mu.g/l to 2.0mg/l, such as from 1.5. mu.g/l to 1.5 mg/l.

The chemosensitising compound is preferably administered in an amount of about 0.1 to 3000 mg/day, for example about 0.5 to 2000 mg/day. The skilled person will understand that the actual amount to be administered may also depend inter alia on the route of administration, i.e. whether the chemosensitising compound is administered orally, intravenously or intramuscularly etc.

For compositions suitable for oral administration for systemic use, the dose is from 0.1mg to 3g per dose, for example from 0.1mg to 1000mg, preferably from 1mg to 600mg, more preferably from 2mg to 400mg, administered 1-10 times daily, for example 1-4 times daily, for a total of 1 day to 12 months, depending on the infectious disease to be treated. The dosage of the composition administered by inhalation is within these same ranges.

For parenteral administration, particularly intravenous administration, a dose of about 0.1 to about 2000mg per day is convenient. For intravenous administration, a dosage of about 0.1 to about 2000 mg/day and from 1 day to 12 months is convenient.

For transdermal and topical administration, the dose is 0.1mg to 5 g/dose, administered 1 to 10 times daily for a total of 1 day to 12 months. For rectal administration, the dose is typically 0.1 to 2000mg per dose, administered 1 to 10 times daily for a total of 1 day to 12 months.

The steady state serum concentrations and dosages mentioned above may produce the desired clinical effect and at the same time avoid the serious side effects normally associated with the chemosensitizing compounds described herein. However, some of the chemosensitising compounds described herein, in particular the chemosensitising compounds of general formula IIIb, can be administered in higher amounts, thereby resulting in steady state serum concentrations above the levels indicated above. This is due to the fact that: these chemosensitising compounds are not expected to show serious side effects, even when administered in higher amounts.

The invention is further illustrated by the following non-limiting examples.

Materials and methods

Bacteria

The bacteria were subcultured on Mueller-Hinton agar plates and incubated overnight at 37 ℃. Colonies from agar plates were inoculated into pre-warmed Mueller-Hinton broth and incubated for about 3 hours to give a log phase culture. The log phase cultures of the bacteria were diluted with fresh pre-warmed medium and adjusted to the specified Optical Density (OD) at 600nm to give a final assay concentration of 1X 10⁴Bacteria/ml medium.

DR cells are approximately 10 to 100 times more resistant than sensitive cell lines and maintain a stable DR phenotype when grown in drug-free media. The only exception was the enterococcus faecalis F84 isolate. The strain was cultured in the presence of 4. mu.g/ml vancomycin to maintain the MIC value at 4. mu.g/ml.

Strains were obtained from Statens Serominstinit (Denmark), technical university of Denmark and the clinical microbiology line of Sonderberg Hospital (Denmark).

Isolate bacteria

Escherichia coli LN 3164. Acr AB-TolC MDR in selected mutants in vitro. Resistant to tetracyclines, beta-lactams, fluoroquinolones, chloramphenicol, and aminoglycosides.

Escherichia coli 331 ME. Selected multi-drug resistant clinical isolates of E.coli in vivo. The strain was isolated from patients with severe cystitis/urinary sepsis. Resistant to tetracyclines, beta-lactams, fluoroquinolones, chloramphenicol, and aminoglycosides.

Pseudomonas aeruginosa 432 b. Clinical isolates of multiple drug resistance. Resistant to tetracyclines, beta-lactams, fluoroquinolones, and aminoglycosides. Beta-lactamases cause changes in penicillin binding proteins and changes in outer membrane proteins.

Staphylococcus aureus E45 MRSA. And (5) clinically separating bacteria. Is resistant to methicillin. Sensitive to teicoplanin, chloramphenicol, fosfomycin, netilmicin, and vancomycin.

Staphylococcus aureus O11. Clinical isolates resistant to penicillin. Sensitive to methicillin, tetracycline, beta-lactams, fluoroquinolones, chloramphenicol, and aminoglycosides.

Enterococcus faecalis F84. Clinical isolates of multiple drug resistance. The drug resistance to ampicillin, ciprofloxacin and gentamicin is reduced. As a major resistance mechanism, changes in expression (VanA gene expression) at cell wall precursor targets.

Enterococcus faecalis F86. Sensitive clinical isolates. Drug resistance did not develop.

Medicine

Drugs were dissolved in small amounts of water or 1% DMSO (final culture concentration of DMSO less than 0.05%) before dilution with culture medium. Solutions for each experiment were prepared fresh.

Chlorpromazine, promazine, promethazine, prochlorperazine, trifluoperazine, fluphenazine, thioridazine, chlorprothixene, trans-clopenthixol, cis-and trans-flupentixol were obtained from h.lundbeck (copenhagen, denmark) and british pharmacopoeia committee laboratory (Middlesex, uk). 7-Hydroxypropylazine, 7, 8-monohydroxypropylazine, demethylchlorpromazine hydrochloride, trifluoropropylazine, oxychloroprozine, 1-chlorpromazine, 2-chloro-10- (2-dimethylaminoethyl) -phenothiazine hydrochloride were obtained from the national institute for mental health (USA). Methylthiopromazine is obtained from Statens Serominstintt (Copenhagen, Denmark). Perphenazine was obtained from Sigma (copenhagen, denmark). Fusidic acid (fusidic acid) was obtained from LeoPharma AS (copenhagen, denmark). Aztreonam is available from Bristol Meyers (Bromma, sweden). Vancomycin is obtained from Alpharma AS (copenhagen, denmark). Ciprofloxacin was obtained from 1APharma (Copenhagen, Denmark) and gentamicin was obtained from Schering-Plough Europe (Brussel, Belgium).

Effect of drugs on growth and DR of microbial cells

The growth of the cells was determined by MIC sensitivity test using microdilution broth method according to NCCLS guidelines (NCCLS guidelines, dilution antimicrobial sensitivity test for aerobically growing bacteria; approved standards, sixth edition, Vol.23; phase 2). 100% Mueller-Hinton broth and 1X 10 were used⁴Bacterial concentration per ml.

The log phase cultures of bacteria were diluted with fresh pre-warmed medium and adjusted to the specified OD at 600nm to give a final concentration of 1X 10 per well⁴Bacteria/ml medium. Each chemosensitising compound was added to the bacterial culture in the wells at a double dilution to give a final concentration of 8 to 500 μ g/ml. The plates were incubated at 37 ℃ for 16 hours. The Minimum Inhibitory Concentration (MIC) is defined as the lowest MIC that does not show visible growth according to NCCLS guidelinesAnd (4) degree.

The effect of the chemosensitizing compound on DR was investigated by contacting cells with 0-64 μ g/ml of an anti-infective drug in the presence or absence of the chemosensitizing compound via the above described mini-escalation assay. Each experiment was repeated 3 times. MIC values represent the average of two separate experiments.

The log phase cultures of bacteria were diluted with fresh pre-warmed medium and adjusted to the specified OD at 600nm to give a final concentration of 1X 10 per well⁴Bacteria/ml medium. The chemosensitising compound was added to the bacterial culture in the wells so that the final concentration was 1/4 which is the MIC value of the chemosensitising compound. Anti-infective agents were added to bacterial cultures in wells at a double dilution to give a final concentration of 0 to 64 μ g/ml. The plates were incubated at 37 ℃ for 16 hours. The DR ratio is defined as the ratio of the MIC value of the anti-infective agent alone divided by the MIC of the anti-infective agent in the presence of the chemosensitizing compound. This ratio represents the increase in the apparent potency of the anti-infective agent caused by the chemosensitizing compound, which can be expressed as:

Examples

Example 1 Effect of modified promazine

Table 1 shows the results with different R₁、R₂、R₇And R₈Structure, MIC values and DR ratios of a series of substituted promazine derivatives.

The anti-infective agent is ciprofloxacin, and the strain is Escherichia coli LN 3164.

The chemosensitising compounds (promazine and its derivatives) have the following general structure:

the results obtained are compiled in table 1 below:

TABLE 1

It can be seen that the unsubstituted chemosensitizing compound (promazine) inhibited cell growth and sensitized 100% of ciprofloxacin resistant e.coli cells (DR ═ 2). However, the introduction of chlorine atoms at the 1-or 2-position increases the efficacy against drug resistance. Specifically, the introduction of a chlorine atom at the 2-position has the greatest effect and sensitizes the drug-resistant cells by 220%. Similarly, CF is introduced at the 2-position₃The groups also increase the efficacy against cell growth and drug resistance. The oxygen chlorine promazine generated by oxidizing the episulfide atom has the same anti-drug resistance activity as chlorpromazine.

To determine the effect of the amino side chain, a chemosensitizing compound having the following general structure was determined as described above:

the results obtained are compiled in table 2 below:

TABLE 2

Table 2 above shows that phenothiazines containing a tertiary amine (e.g., chlorpromazine) and a secondary amine (demethylchlorpromazine) have similar cell growth inhibitory activity (MIC of 32. mu.g/ml). However, tertiary amine containing phenothiazines such as chlorpromazine are more potent DR antagonists than secondary amine containing phenothiazines such as demethylchlorpromazine, which increases the anti-DR activity by 1.6 fold. Other changes in amino type may also affect anti-DR activityAnd (4) sex. For example, piperazinyl derivatives may increase anti-DR potency. Thus, the DR ratio of trifluoperazine and fluphenazine compounds is greater than that of trifluoroperazine compounds having the same ring substitution pattern but having an aliphatic side chain. Similarly, perphenazine and prochlorperazine are more potent DR antagonists than chlorpromazine. This series of experiments also showed a CF at the 2-position₃Importance of substitutions for anti-DR activity. For example, the DR ratio of trifluoperazine is greater than that of prochlorperazine. These phenothiazines have the same structure except that the former has a CF at the 2-position₃Groups other than chlorine atoms. A similar relationship can be seen by comparing fluphenazine and perphenazine. Finally, para-methyl substitution on the piperazinyl ring appears more potent than ethanol substitution, as can be seen by comparing the DR ratios of prochlorperazine and perphenazine or trifluoperazine and fluphenazine.

To determine the effect of the length of the amino-containing side chain, a chemosensitizing compound having the following general structure was determined as described above:

the results obtained are compiled in table 3 below:

TABLE 3

Table 3 above demonstrates the effect of a series of dimethylaminophenothiazines in which the length of the amino-containing side chain is different on cell growth and DR. It can be seen that changing from a two-carbon alkyl bridge to a three-carbon alkyl bridge increases the anti-DR effect of these chemosensitising compounds. For example, chlorpromazine has a greater DR than 2-chloro-10- (2-dimethylaminoethyl) phenothiazine. Promethazine with isopropyl side chains is a more potent DR inhibitor than promethazine with a three carbon alkyl straight chain.

Example 2 Effect of stereochemistry

To investigate the effect of cis and trans stereochemistry, a series of thioxanthenes were determined as described above. Table 4 shows the MIC values and DR ratios for the thioxanthenes tested.

TABLE 4

The above results indicate that stereoisomeric configurations are necessary for optimal anti-DR activity. For example, trans-flupentixol is a more potent anti-DR agent than cis-flupentixol, whereas trans-clopenthixol is the most potent anti-DR agent. Thus, the orientation of the side chain amines relative to the tricyclic core appears to be an important determinant of anti-DR activity.

Example 3 Effect of hydrophobicity

To determine whether the difference in anti-DR potency in the side chain altered chemosensitising compounds was also due to a change in overall hydrophobicity, octanol for each drug in tables 1, 2, 3 and 4 was: the buffer partition coefficients were compared to their DR ratios. No statistically significant correlation was found between hydrophobicity and anti-DR activity (P > 0.5) (data not shown).

Example 4 synergistic Effect of thioxanthene derivatives

The synergistic effect of trans-flupentixol, cis-flupentixol and trans-clopenthixol on a group of sensitive and resistant bacterial isolates representing different species and resistance mechanisms was investigated for a variety of anti-infective agents.

The synergy was studied by a checkerboard combination test by contacting cells with 0-64 μ g/ml anti-infective in the presence or absence of trans-flupentixol, cis-flupentixol or trans-clopenthixol. Each experiment was repeated 3 times. MIC values represent the average of two separate experiments.

The log phase cultures of bacteria were diluted with fresh pre-warmed medium and adjusted to 600nmThe OD was specified so that the final concentration in each well was 1X 10⁴Bacteria/ml medium. Each chemosensitising compound was added to the bacterial culture in the wells at a double dilution to give a final concentration of 0 to 8. mu.g/ml. Anti-infective agents were added to bacterial cultures in wells at a double dilution to give a final concentration of 0 to 64 μ g/ml. The plates were incubated at 37 ℃ for 16 hours. The growth of the pores was visually evaluated. The Fractional Inhibitory Concentration (FIC) of each anti-infective agent alone and in combination with trans-flupenthixol, cis-flupenthixol or trans-clopenthixol was calculated. The FIC index is calculated using the formula:

FIC＝FIC_{chemosensitizing compounds}+FIC_{Anti-infective agents}Wherein:

Synergy is defined as FIC index < 0.5. The calculated FIC index is shown in table 5 below:

table 5.^*R ═ isolate resistant to the anti-infective agent; s ═ isolate sensitive to anti-infective agents; MDR is a multi-drug resistant isolate.

The FIC indices of trans-clopenthixol and cis-and trans-halopenthixol indicate that these compounds can strongly synergistically enhance the bacteriostatic effect of anti-infective agents in drug resistant cells. The majority of the FIC indices for chemosensitising compounds measured on resistant cells are < 0.5. Trans-clopenthixol is the most potent of all the chemosensitising compounds tested, i.e. the trans form is more potent than the cis form. Thus, clinical use of, for example, trans-clopenthixol or trans-haloperidol in combination with an anti-infective agent would likely cause the MIC of the anti-infective agent for DR cells to become significantly lower than clinically achievable concentrations, showing an effective concentration < 500ng/ml (effective concentration of chemosensitising compound is 0.32. mu.g/ml to 4. mu.g/ml). The anti-DR effect was most potent in resistant cells. However, significant antibiotic potentiation was also shown in sensitive cells, strongly suggesting that the anti-DR effect of these chemosensitising compounds is not restricted to cells overexpressing efflux pumps and that the anti-DR mechanism is not restricted to this target either. The FIC index of antibiotic-sensitive cells is 0.25 to 0.5.

Furthermore, the results also show that cis and trans flupentixol and trans clopenthixol enhance the anti-infective activity of the β -lactam antibiotic piperacillin against pseudomonas aeruginosa cells expressing a β -lactamase capable of inhibiting piperacillin (gibercman B et al, 1992 and 1990), indicating that the anti-DR mechanism of the compounds is not limited to inhibition of MDR efflux pumps. The antiproliferative effect of chemosensitising compounds is approximately equally moderate in sensitive and resistant strains. MIC values were 16 to 64. mu.g/ml (data not shown).

Example 5 synergistic Effect of phenothiazine derivatives

The synergistic effect of ciprofloxacin, an anti-infective agent, against ciprofloxacin-resistant clinical bacterial isolates of enterococcus faecalis was investigated.

Synergy was studied by a checkerboard combination test by contacting cells with 0-8 μ g/ml of an anti-infective agent in the presence or absence of chlorpromazine, promazine, or perphenazine. Each experiment was repeated 2 times. MIC values represent the average of two separate experiments.

The log phase cultures of bacteria were diluted with fresh pre-warmed medium and adjusted to the specified OD at 600nm to give a final concentration of 1X 10 per well^4-5Bacteria/ml medium. Addition of each chemosensitising compound to bacterial cultures in wells at two-fold dilution to final concentrationThe degree is 0 to 16. mu.g/ml. Anti-infective agents were added to bacterial cultures in wells at a double dilution to give a final concentration of 0 to 16 μ g/ml. The plates were incubated at 37 ℃ for 16 hours. The growth of the pores was visually evaluated. The Fractional Inhibitory Concentration (FIC) of the anti-infective agent alone and in combination with chlorpromazine, promazine or perphenazine was calculated. The FIC index is calculated using the formula:

FIC＝FIC_{chemosensitizing compounds}+FIC_{Anti-infective agents}Wherein:

Synergy was defined as FIC index < 0.5. The calculated FIC index is shown in table 6 below:

the FIC index of chlorpromazine, promazine, or perphenazine indicates that these compounds may synergistically enhance the bacteriostatic effects of anti-infective agents in drug resistant cells. All FIC indices of the chemosensitising compounds determined on resistant cells were < 0.5. The ability of chlorpromazine, promazine, or perphenazine to enhance ciprofloxacin action is equivalent. The minimum effective concentration of the enhancing compound is 1 to 2. mu.g/ml. Thus, clinical use of, for example, chlorpromazine, promazine, or perphenazine, in combination with an anti-infective agent will likely cause the MIC of the anti-infective agent for DR cells to become significantly lower than clinically achievable concentrations, showing effective concentrations < 2 μ g/ml.

Table 6.^*: ciprofloxacin (ciprofloxacin)

Example 6 development of insensitivity to chemosensitising Compounds

One possible limitation for the combination of an anti-infective agent and an inhibitor of the resistance mechanism is the possibility of the microorganism developing mutations that render it insensitive to the inhibitor. This has been observed in, for example, bacteria, viruses, fungi and yeasts.

The effect of the inhibitors on the rate of appearance of single-step resistance to ciprofloxacin selected in vitro in a clinical isolate of staphylococcus aureus O11 was determined.

Staphylococcus aureus cells were plated on LB agar plates containing ciprofloxacin at a concentration of 1. mu.g/ml (twice the MIC) in the presence or absence of 1. mu.g/ml of trans-clopenthixol, and spontaneous mutants were obtained after 24 hours. The frequency of mutant selection was determined to be 3X 10 by comparing the number of colonies grown on plates containing anti-infective agent with the number of colonies obtained by suitable dilution of plates in the absence of anti-infective agent^-8。

When evaluating the use of inhibitors in the clinic, perhaps the most important aspect is the effect of these inhibitors on the emergence of resistant mutants. Importantly, as shown in table 7, the tested inhibitors reduced the frequency of spontaneous emergence of resistance to ciprofloxacin by a factor of 100 or more. This significant effect cannot be attributed to the toxic effect of the inhibitor, since the same concentration of inhibitor (at least 10-fold lower than its MIC for staphylococcus aureus) does not affect neither the colony forming ability nor the colony size of staphylococcus aureus cells plated in the absence of ciprofloxacin. In conclusion, trans-clopenthixol inhibits the emergence of resistance to ciprofloxacin in staphylococcus aureus.

TABLE 7 frequency of appearance of selected in vitro ciprofloxacin variants resistant to 1. mu.g/ml (twice the MIC for Staphylococcus aureus strains) in the presence or absence of inhibitor.

Example 7 enhancement of Trans-clopenthixol in a mouse model of peritonitis

Bacteria: a clinical isolate of enterococcus faecalis BG-029 from human urine was used. The strain is resistant to ciprofloxacin (MIC, 4 mu g/ml). The MIC of trans-clopenthixol was 6. mu.g/ml.

MIC: MIC was determined by microdilution assay according to NCCLS guidelines.

Animals: female NMRI mice (about 6 to 8 weeks old; weight, 30 ± 2g) were used in a mouse model of peritonitis pneumonia (described below).

Antibiotics: ciprofloxacin was obtained as a 2mg/ml infusion from Bayer A/S (Lyngby, Denmark). Trans-clopenthixol was obtained as a powder reference substance from the british pharmacopoeia committee laboratory (Middlesex, uk).

Mouse pharmacokinetic study of trans-clopenthixol:

pharmacokinetic studies were performed using NMRI mice. Serum concentrations were determined after a single dose of 0.3mg was administered to each mouse. The drug was administered by subcutaneous injection in the cervical region at 0.2 ml/dose. Blood samples were taken from mice at 1, 2, 4, 6 and 24 hours post-injection, with 3 per group. After collection, the blood was centrifuged and the serum was stored at-80 ℃ until it was analyzed by high pressure liquid chromatography.

Mouse peritonitis model:

bacterial suspensions were prepared from fresh overnight cultures (prepared from frozen stock cultures) on 5% blood agar plates as described above. An inoculum for a mouse peritonitis model was prepared just prior to use and adjusted to a density of about 10 at 540nm⁷CFU/ml. The size of the inoculum was determined by viability counts on 5% blood agar.

Neutropenia was induced by pre-treatment of mice with cyclophosphamide (6 mg daily for 3 days). Intraperitoneal injection of mice with 0.5ml of enterococcus suspension resulted in bacteremia within 1 hour of inoculation. Antibiotic treatment was started 1 hour after inoculation. Ciprofloxacin and trans-clopenthixol were administered subcutaneously in the cervical region at 0.1 ml/dose. Each treatment group included five mice. All experiments included vaccinated untreated control mice. (see: Erlandsdottir et al; Antimicrob AgentsChemother.2001, 4 months; 45 (4): 1078-85)

TABLE 8 treatment regimen for infected mice

Group of	Treatment of
		1. Control	Is free of
2. Ciprofloxacin alone	12.5mg ciprofloxacin/mouse
		3. Trans-clopenthixol alone	5mg/kg mice
4. Trans-clopenthixol and ciprofloxacin	5mg of Trans-clopenthixol/kg mouse, followed immediately by 12.5mg of ciprofloxacin/mouse

The effect of various treatment regimens was determined by evaluating bacterial counts in peritoneal fluid over a 6 hour treatment period. After sacrifice, peritoneal washes were performed as follows: peritoneal fluid was collected by intraperitoneal injection of 2ml of sterile saline, followed by massaging of the abdomen, followed by opening of the peritoneum. Immediately, the peritoneal fluid was diluted 10-fold with saline, from which 20. mu.l were spotted on 5% blood agar plates, followed by overnight incubation at 35 ℃ and colony counting. The lowest detected levels of bacterial counts in blood and peritoneal fluid were 50 and 250CFU/ml, respectively.

The bactericidal efficacy of the treatment regimen in the mouse model was calculated by subtracting the results of each treated mouse from the average results of the control mice at the end of the treatment (6 hours). P was considered significant with < 0.05. All statistical comparisons are two-tailed.

As a result:

strong potentiating activity of trans-clopenthixol in mouse peritoneum

The bactericidal activity of ciprofloxacin and trans-clopenthixol, alone or in combination, in the mouse peritoneum is shown in figure 3. It can be seen that a sub-therapeutic amount of ciprofloxacin alone had no effect on the infection and that the drug-resistant bacteria were not eliminated from the mouse peritoneum. However, when mice were treated with ciprofloxacin and trans-clopenthixol in combination, the bacteria were eliminated (p < 0.05). Trans-clopenthixol (TC) alone does not affect the bacteria at the sub-therapeutic doses given. (TC serum values for mice were below 300 ng/ml-this value was well below the MIC value of 6. mu. gTC/ml, as shown in FIG. 4).

Example 8 synergistic Effect of thioxanthene derivatives on fungi

The synergistic effect of trans-clopenthixol was studied by a checkerboard combination test by contacting cells with 0-256 μ g/ml of an anti-infective agent in the presence or absence of trans-clopenthixol (concentrations of 0 to 8 μ g/ml at two-fold dilution). Each experiment was repeated 3 times. MIC values represent the average of two separate experiments.

Fungus strain: clinical isolates of fluconazole-resistant candida albicans from patients with candidemia.

Antifungal agents: fluconazole (Pfizer, Ballerup, Denmark)

Before susceptibility testing, isolates were subcultured on Sabouraud glucose agar for 24 hours.

Broth microdilution tests were performed according to NCCLS document M27-A (reference: national Committee for clinical laboratory standards (1997). reference to the Broth dilution antifungal susceptibility test of Alcoholic cultures: approved standards M27-A. NCCLS, Wayne, Pa.).

After mixing the wells by pipetting into the resuspended yeast pellet, the microtiter plate is read at 530nm by spectrophotometer. The MIC of fluconazole was defined as the lowest drug concentration that inhibited 80% growth. The following experimental cut points were applied: sensitive to fluconazole (S), and the MIC is more than or equal to 8 mu g/ml; sensitive dose-dependence (SDD), 8 μ g/ml < MIC < 64 μ g/ml; and drug resistance (R), MIC is less than or equal to 64 mu g/ml.

The Fractional Inhibitory Concentration (FIC) of the anti-infective agent alone and in combination with trans-clopenthixol was calculated. The FIC index is calculated using the formula:

FIC＝FIC_{chemosensitizing compounds}+FIC_{Anti-infective agents}Wherein:

Synergy is defined as FIC index < 0.5. The calculated FIC index is shown in table 9 below:

TABLE 9

Fungal strains	MIC mu g/ml Fluconazole (FL)	MIC ug/ml trans-clopenthixol (TC)	MICμg/ml FL+TC (3μg/ml)	FIC index
					Candida albicans	128	32	4	0.16

The FIC index of trans-clopenthixol indicates that the compound can strongly and synergistically enhance the antifungal effect of an antifungal agent on drug-resistant cells. It can be seen that the FIC index of the chemosensitising compound measured in resistant cells is < 0.5. Thus, clinical use of, for example, trans-clopenthixol in combination with an antifungal agent will likely result in the MIC of the antifungal agent for DR cells becoming significantly lower than clinically attainable, exhibiting an effective concentration of ≦ 3 μ g/ml.

Example 9 potentiation of antiviral Compounds by thioxanthene derivatives

The potentiating effect of trans-clopenthixol on antiviral agents was investigated by a checkerboard combination test by contacting HIV infected cells with 0-3 μ M antiviral agent in the presence or absence of trans-clopenthixol at concentrations of 0 to 6 μ M. Each experiment was repeated 3 times. MIC values represent the average of two separate experiments.

The method comprises the following steps:

viruses and cells

HIV-1 strain HTLV-IIIB at 37 ℃ and 5% CO₂Propagation was performed in H9 cells using RPMI1640 (growth medium) containing 10% heat-inactivated Fetal Calf Serum (FCS) and antibiotics. The culture supernatant was filtered (0.45nm), an aliquot was taken and stored at-80 ℃ until use. HIV-1 strain was obtained from NIH AIDS Research and Reference Program.

Compound (I)

Antiviral drugs: AZT, (3 '-azido-3' -deoxythymidine), Glaxo Wellcome.

Enhancing compound (b): trans-clopenthixol was obtained as a powder reference substance from the british pharmacopoeia committee laboratory (Middlesex, uk).

Inhibition of HIV-1 replication

The possible antiviral activity of the compounds against HIV-1 strain IIIB was determined using MT4 cells as target cells. MT4 cells were incubated with virus (0.005MOI) and growth medium containing dilutions of test compound for 6 days, and virus-infected and non-virus-infected control cultures were incubated in parallel. The MTT assay as described previously was used to indirectly quantify HIV expression in culture. Compounds that mediate less than a 30% reduction in HIV expression are considered to be biologically inactive. The cytotoxic effect of the compounds in uninfected MT4 cultures containing dilutions of the test compounds as described above was determined in parallel. Cultures for the antiviral activity and cytotoxicity assays were prepared in triplicate, 200ml per culture in microtiter plates.

Inhibition of cell growth by 30% relative to control cultures was considered significant.

The 50% inhibitory concentration was determined by interpolation from a plot of percent inhibition versus compound concentration.

EC50 is defined as the effective concentration to inhibit 50% of viral production, 50% of viral infectivity or 50% of viral-induced cytopathic effects.

CC50 is defined as the inhibitory concentration that reduces cell growth or viability of uninfected cells by 50%.

Results

As can be seen from table 10, the combination of trans-clopenthixol and AZT enhances the antiviral effect of AZT by a factor of 10, and thus it may be sufficient to inhibit drug-resistant viral strains. Trans-clopenthixol alone has no antiviral or cytotoxic effect at the concentrations used.

Table 10: potentiation of the antiviral compound AZT (A) by trans-clopenthixol (TC). The concentration unit is μ M. (see below)

EC50 A	CC50 A	EC50 TC	CC50 TC	EC50 A+TC(1μM)	CC50 A+TC(1μM)
						0.05	＞3	＞3	＞3	0.01	＞3

CC50 is defined as the inhibitory concentration that reduces cell growth or viability of uninfected cells by 50%. Viral assay methods references: petersen L, Jrgensen PT，Nielsen C，Hansen TH，Nielsen J，Pedersen EB.Synthesis and Evaluation of Double-Prodrugsagainst HIV.Conjugation of D4T with 6-Benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442，Emivirine)Type Reverse TranscriptaseInhibitors via the SATE Prodrug Approach.J.Med.Chem.2005，48，1211-1220。

Claims

1. Use of a clinically relevant amount of a compound or salt of formula (I) that yields a steady state serum concentration of less than 8.0mg/l in the manufacture of a medicament for use in combination with an anti-infective agent in the treatment or prevention of an infectious disease,

wherein

V is selected from S, SO₂And SO;

w is C＝CH-(CHX)_m-N(R₁₀)(R₁₁)；

m is an integer of 1 to 5;

R₂selected from F, Cl, Br, I, CH₂Y、CHY₂And CY₃Wherein Y is a halogen atom;

R₁、R₃、R₄、R₆、R₇、R₈and R₉Each independently selected from hydrogen, optionally substituted C_1-6Alkyl and optionally substituted C_1-6An alkoxy group;

R₁₀and R₁₁Together with the nitrogen atom to which they are attached form an optionally substituted nitrogen-containing heteroaryl or an optionally substituted heterocyclyl;

wherein the infectious disease is caused by an infectious agent selected from the group consisting of a virus, a bacterium, and a fungus, said infectious agent being resistant to a drug; and wherein said infectious agent is not selected from the group consisting of Staphylococcus aureus strain SA-1199, SA-1199B, SA-K1712, SA-K1748, SA 8325-4, and SA-K2068.

2. Use according to claim 1, wherein R₂Selected from F, Cl, CF₃And CCl₃。

3. Use according to claim 2, wherein R₂Is Cl or CF₃。

4. Use according to any one of claims 1 to 3, wherein R₁、R₃、R₄、R₆、R₇、R₈And R₉Is hydrogen.

5. Use according to any one of claims 1 to 3, wherein V is S or SO.

6. Use according to claim 5, wherein V is S.

7. Use according to any one of claims 1 to 3, wherein m is 2 or 3.

8. Use according to claim 7, wherein m is 2.

9. Use according to claim 8, wherein W is C ═ CH- (CH)₂)₂-N(R₁₀)(R₁₁) And R is₁₀And R₁₁As defined in claim 1.

10. Use according to any one of claims 1 to 3, wherein R₁₀And R₁₁Together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic group.

11. The use according to claim 10, wherein the optionally substituted heterocyclic group is selected from the group consisting of an optionally substituted 2-pyrrolinyl group, an optionally substituted 3-pyrrolinyl group, an optionally substituted pyrrolidinyl group, an optionally substituted 2-imidazolinyl group, an optionally substituted imidazolidinyl group, an optionally substituted 2-pyrazolinyl group, an optionally substituted 3-pyrazolinyl group, an optionally substituted pyrazolidinyl group, an optionally substituted piperidyl group, an optionally substituted morpholinyl group, an optionally substituted thiomorpholinyl group and an optionally substituted piperazinyl group.

12. The use according to claim 11, wherein said optionally substituted heterocyclyl is an optionally substituted piperidinyl or an optionally substituted piperazinyl.

13. The use according to claim 12, wherein said optionally substituted heterocyclic group is an optionally substituted piperazinyl group.

14. Use according to claim 13, wherein the piperazinyl is substituted in the para position.

15. Use according to claim 13 or 14, wherein said piperazinyl is optionally substituted C_1-6Alkyl substitution.

16. The use according to claim 15, wherein said optionally substituted C_1-6Alkyl is selected from-CH₃、-CH₂OH、-CH₂-CH₃and-CH₂-CH₂OH。

17. The use according to claim 16, wherein said optionally substituted C_1-6Alkyl is-CH₃or-CH₂-CH₂OH。

18. The use according to claim 17, wherein said optionally substituted C_1-6Alkyl is-CH₂-CH₂OH。

19. The use according to claim 15, wherein said optionally substituted C_1-6The alkyl group is in the para position.

20. The use according to claim 13, wherein said piperazinyl is unsubstituted.

21. Use according to any one of claims 1 to 3, wherein C ═ CH- (CHX)_m-N(R₁₀)(R₁₁) The group is in the trans configuration.

22. The use according to claim 19, wherein said compound is selected from the group consisting of trans-flupentixol, cis-flupentixol, trans-clopenthixol and cis-clopenthixol.

23. Use according to claim 22, wherein said compound is trans-flupenthixol or trans-clopenthixol.

24. Use according to claim 23, wherein said compound is trans-clopenthixol.

25. Use according to claim 1, wherein said steady state serum concentration is from 0.01 μ g/l to less than 8.0 mg/l.

26. Use according to any one of claims 1 to 3, wherein said infectious agent is multidrug resistant.

27. A pharmaceutical composition comprising a clinically relevant amount of a compound as defined in any one of claims 1 to 24 which produces a steady state serum concentration of less than 8.0mg/l and an anti-infective agent capable of killing, inhibiting or retarding the growth of an infectious agent selected from the group consisting of bacteria, viruses and fungi, together with at least one pharmaceutically acceptable carrier or excipient; wherein the composition is in the form selected from the group consisting of tablets, capsules, pills, powders, granules, emulsions, gels including hydrogels, pastes, ointments, plasters, suppositories, enemas, injections, implants, sprays and aerosols.

28. The pharmaceutical composition according to claim 27, wherein said composition is in unit dosage form.

29. The pharmaceutical composition according to claim 28, wherein said composition is in the form of a tablet.

30. The pharmaceutical composition according to claim 27, wherein the compound as defined in any one of claims 1 to 24 is selected from the group consisting of trans-clopenthixol and trans-flupentixol.

31. The pharmaceutical composition according to claim 30, wherein the compound as defined in any one of claims 1 to 24 is trans-flupentixol.

32. A kit comprising a first dosage unit containing a clinically relevant amount of a compound as defined in any one of claims 1 to 24 that produces a steady state serum concentration of less than 8.0mg/l and another dosage unit containing an antimicrobial agent.

33. A composition or kit according to any one of claims 27 to 32 for use as a medicament.