Recent Patents on Anti-Infective Drug Discovery (v.6, #2)

Foreword by Leonard Amaral (76-76).
Resurgence of tuberculosis (TB) worldwide, coupled to increasing frequency of TB infections that are multi-drug (MDRTB) and extensive drug resistant (XDR TB), infections that are problematic to treat, extol high human and financial costs. Atthe present time there are no new drugs that are effective against these antibiotic resistant infections. Therefore, there is anurgent need for effective agents that are safe and within economic means of indigent countries where these infections arepredominant. This issue of PRI is dedicated to present reviews that support the use of the neuroleptic thioridazine (TZ) for therapy ofMDR and XDR TB either as a salvage drug or in combination with antibiotics to which the patient was initially nonresponsive.and this may also be extended to non-tuberculous mycobacterial disease. The reviews provide up-to the minuteperspective of the status of drug development for therapy of MDR and XDR TB infections (see SH Gillespie et al), current andfuture therapy of MDR and XDR TB infections (see M Boeree), the pharmacology of TZ (see HR Thanacoody), the use of TZas a “salvage drug” for improving the quality of life and possible extension of life of the MDR and XDR TB patient presentingwith a somber prognosis (see Z Udwadia), the successful therapy of 10 out of 12 XDR TB patients and protocols used (seeAmaral et al.), the targeting of the human macrophage for enhanced killing of intracellular strains of MDR and XDRMycobacterium tuberculosis by TZ and other agents that inhibit K+ and Ca++ efflux (see M Martins), the inhibition of effluxpumps that mediate MDR phenotypes of Mycobacterium tuberculosis by TZ and therefore reduce or reverse resistance toantibiotics to which the bacterium is initially resistant (see L Rodrigues et al.), the in vitro killing of mycobacteria (see-J vanIngen) and dormant mycobacteria by TZ-possibly suggestive of in vivo killing of latent mycobacteria of the infected patient(see C Sohaskey), the modulation of essential genes for survival of mycobacteria by TZ (see N Dutta et al.) and a revolutionarynew method for the alteration of anti-bacterial activity of an agent subsequent to exposure to specific high energy lasers (see MPascu et al.). All in all, the contents of this special issue provide the reader sufficient information regarding the role that TZ isto play in the therapy of MDR and XDR TB infections, for purposes of considering a variety of possibilities for patenting TZ asa “New Use ”. It is anticipated that as is usually the case, financial motivation will move TZ from its current status “potentialanti-MDR/XDR agent,” to one that will cure MDR and XDR TB. Therefore, consideration for patenting possibilities maycontribute significantly to the elimination of an infection that has plagued mankind since he left the cave and became a landedanimal (farmer).

New and active infections of tuberculosis continue to increase globally. Although antibiotic susceptible infections can be readily cured with isoniazide and rifampicin, infections resistant to these two antibiotics, named Multi- Drug Resistant TB (MDR TB), are problematic for therapy, extol high costs in terms of human suffering and finances, and when these MDR infections progress to Extensive Drug Resistant TB (XDR TB) status, they are not only difficult to treat, they produce high levels of mortality regardless of therapeutic modality employed. The neuroleptic thioridazine (TZ) has been shown to have wide spectrum in vitro and ex vivo activities against antibiotic susceptible, MDR and XDR strains, and has been successfully used for curing mice of active tuberculosis produced by antibiotic susceptible and MDR strains, and has cured 10 out of 12 XDR TB patients when used in combination with three antibiotics to which the XDR TB patients were non-responsive. Mycobacterium tuberculosis TZ has been recommended for “Compassionate Therapy” of MDR/XDR TB infections whose prognoses are significantly serious and anticipated to result in mortality. This review of TZ activity and its potential to cure MDR/XDR TB supports the contention that this neuroleptic offers patenting opportunities for “New Use”. The motivation for patents therefore is expected to rapidly bring TZ to the forefront for therapy of MDR/XDR TB and therefore, the striving for new patents is expected to contribute to the prevention of new infections of antibiotic resistant tuberculosis.

Safety and Efficacy of Thioridazine as Salvage Therapy in Indian Patients with XDR-TB by Zarir F. Udwadia, Tiyas Sen, Lancelot M. Pinto (88-91).
New drugs are desperately needed to combat XDR-TB as effective treatment involves at least four drugs towhich the patient is sensitive or has never received in the past. Most Indian patients have received almost all second linedrugs and have amplified resistance to most of the available drugs. Thioridazine has proven anti tuberculous effectsin vitro and in vivo mouse models and we used this drug as salvage therapy in 4 Indian patients with XDR (near total drugresistance) with advanced disease. We found this drug to be well tolerated, even in this malnourished and ill patientpopulation. It also resulted in clinical improvement in 3 of the 4 patients. Larger studies are being planned with this drugbeing added on to standardized or individualized XDR-TB regimens at an earlier stage. Because thioridazine has beenused successfully for therapy of XDR-TB when in combination with antibiotics to which the patients were nonresponsive,the suggestion has been made that Thioridazine is eligible for patent as “New Use”.

Thioridazine: The Good and the Bad by Ruben H.K. Thanacoody (92-98).
Thioridazine is a phenothiazine drug which has previously been extensively used for its antipsychotic properties as it is associated with a low risk of extra-pyramidal side-effects. There is good evidence to suggest that, in common with other phenothiazine drugs, thioridazine has important anti-microbial activity and is a potential candidate for development as an anti-microbial drug against multi-resistant organisms, including drug-resistant strains of Mycobacterium tuberculosis. The clinical pharmacology and toxicity profile of thioridazine are reviewed in this article and the implications for future drug development along with the patent are discussed.

Current evidence shows that thioridazine (THZ) is ready for global clinical evaluation, while some of its derivatives and other efflux pump inhibitors reach the end stage of preclinical evaluation. In this paper, a clinical trial plan is described that investigates the antituberculosis potency, the safety profile and the role of THZ and/or its derivatives in the treatment of TB in humans, both in patients infected with drug sensitive strains as in patients infected with multi or extensive drug resistant strains of Mycobacterium tuberculosis and some of the patents related to thioridazine are also discussed.

The phenothiazines are neuroleptic drugs that have long been known to have antimycobacterial activity, in vitro. Of the various commercially available phenothiazines, thioridazine, chlorpromazine and trifluoperazine are most active against mycobacteria, in vitro. Their MICs for Mycobacterium tuberculosis are in the 8-16 μg/ml range and MICs for Mycobacterium avium in the 10-32 μg/ml range, depending on methods and media. These concentrations cannot be safely attained in humans, where maximum serum concentrations are 0.5 μg/ml (thioridazine) to 1 μg/ml (chlorpromazine) or 4 μg/ml (trifluoperazine). Phenothiazines still have potential as antimycobacterial drugs because they accumulate in macrophages; concentrations inside macrophages are at least 10 fold higher than in serum. When applied to mycobacteria inside macrophages, concentrations as low as 0.1 μg/ml (thioridazine) or 0.1-3.6 μg/ml (chlorpromazine) kill phagocytized M. tuberculosis and M. avium in 3-7 days. Owing to their multiple and novel drug targets, phenothiazine resistance has not been observed. The drug targets and less toxic phenothiazine derivatives (patents include WO2005105145A and WO2010122012A) provide excellent patentable potentials. Thioridazine itself may be patented as “new use”. New drugs for treatment of mycobacterial disease, most notable multidrug- and extensively drug-resistant tuberculosis, are urgently needed; phenothiazines and their targets should be exploited for this use.

The emergence of resistance in Tuberculosis has become a serious problem for the control of this disease. For that reason, new therapeutic strategies that can be implemented in the clinical setting are urgently needed. The design of new compounds active against mycobacteria must take into account that Tuberculosis is mainly an intracellular infection of the alveolar macrophage and therefore must maintain activity within the host cells. An alternative therapeutic approach will be described in this review, focusing on the activation of the phagocytic cell and the subsequent killing of the internalized bacteria. This approach explores the combined use of antibiotics and phenothiazines, or Ca2+ and K+ flux inhibitors, in the infected macrophage. Targeting the infected macrophage and not the internalized bacteria could overcome the problem of bacterial multi-drug resistance. This will potentially eliminate the appearance of new multi-drug resistant tuberculosis (MDR-TB) cases and subsequently prevent the emergence of extensively-drug resistant tuberculosis (XDR-TB). Patents resulting from this novel and innovative approach could be extremely valuable if they can be implemented in the clinical setting. Other patents will also be discussed such as the treatment of TB using immunomodulator compounds (for example: betaglycans).

Inhibition of Drug Efflux in Mycobacteria with Phenothiazines and Other Putative Efflux Inhibitors by Liliana Rodrigues, Jose A. Ainsa, Leonard Amaral, Miguel Viveiros (118-127).
Mycobacteria are responsible for some of the oldest diseases known to man, usually associated with high morbility and mortality rates. An example is tuberculosis (TB), a major public health problem that accounts for eight million new cases each year. Furthermore, the increase of multidrug and extremely-drug resistance seriously threatens the success of the TB control programmes. Resistance to anti-mycobacterial drugs is often due to spontaneous mutations in target genes, followed by selection of resistant mutants during treatment. However, this does not explain all cases of drug resistance and other mechanism(s) may be involved, namely efflux pumps that extrude the drug to the exterior of the cell. Efflux pumps are becoming attractive drug targets for the development of new anti-mycobacterial compounds and several efflux inhibitors have been developed and published in patent applications (i.e. WO2004062674, US2004204378, US2003118541, WO2008141012, WO2009110002, WO2010054102). However, none of these inhibitors is used in clinical practice. This review will focus on the potential use of efflux inhibitors as adjuvants of the anti-mycobacterial therapy, an approach that may restore the activity of antibiotics that are subject to efflux and render the mycobacteria more susceptible to drugs transported by these pumps.

New Patentable Use of an Old Neuroleptic Compound Thioridazine to Combat Tuberculosis: A Gene Regulation Perspective by Noton K. Dutta, Kaushiki Mazumdar, Sujata G. Dastidar, Petros C. Karakousis, Leonard Amaral (128-138).
Use of the old antipsychotic phenothiazine thioridazine (THZ) for therapy of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) infection is now being seriously considered. It is reported that THZ primarily acts on enzymes involved in fatty acid metabolism and membrane proteins, particularly efflux pumps, as well as oxidoreductases and proteins involved in aerobic respiration that overlap with a number of conventional antituberculous drugs. It targets the products of the Rv3160c-Rv3161c operon, which are perhaps required for the detoxification of THZ, members of the sigma factor SigB regulon that play a crucial role in protecting the pathogen against cell envelope damage, and Rv2745c, a transcription factor that regulates ATP-dependent proteolysis. Some of these genes have been shown to be essential for the survival or persistence of Mycobacterium tuberculosis in the infected host. Since THZ targets multiple pathways, including those involved in cell wall processes and respiratory chain components, it may serve as a model for multi-target drug development, as well as constitute a highly potent addition to a combination of antituberculous drug regimens. The discussion of some of the patents relevant to thioridazine to combat tuberculosis is also included in the present manuscript.

Approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis. In the vast majority of cases this results in latent not active disease. Latent disease is defined as a positive reaction to tuberculin antigens but without any further clinical symptoms. Models have been developed to study latent tuberculosis with the two most prominent being the in vivo Murine model and the in vitro Wayne model. In both cases M. tuberculosis undergoes a change in its respiratory profile as it shifts down to a nonreplicating state. However in both the mouse and the Wayne model, dormant M. tuberculosis is sensitive to the phenothiazine thioridazine. This antibiotic has several targets, and the main one is respiration. There is a growing burden of multidrug resistant and extensively drug resistant tuberculosis. Treatment of these cases is expensive with high mortality. We propose that thioridazine alone, or with other antibiotics, be used to treat drug resistant latent tuberculosis. The advantages are that thioridazine is inexpensive, effective against drug resistant tuberculosis, well characterized and unlikely to induce drug resistance. The disadvantages include possible side effects, although these should be rare at the doses and length of time of treatment. Recent patents involving analogs of thioridazine suggest this class of drugs may hold great promise for the future treatment of the most drug resistant strains.

Direct Modification of Bioactive Phenothiazines by Exposure to Laser Radiation by Mihail-Lucian Pascu, Viorel Nastasa, Adriana Smarandache, Andra Militaru, Ana Martins, Miguel Viveiros, Mihai Boni, Ionut R. Andrei, Alexandru Pascu, Angela Staicu, Joseph Molnar, Seamus Fanning, Leonard Amaral (147-157).
Whereas exposure of combinations of a phenothiazine and bacterium to incoherent UV increases the activity of the phenothiazine, exposure of the phenothiazine alone does not yield an increase of its activity. Because the laser beam energy is greater than that produced by the incoherent UV sources, exposure of phenothiazines to specific lasers may yield molecules with altered activities over that of the unexposed parent. Chlorpromazine, thioridazine and promethazine active against bacteria were exposed to two distinct lasers for varying periods of time. Absorption and fluorescence spectra were conducted prior to and post-exposure and the products of laser exposure evaluated for activity against a Staphylococcus aureus ATCC strain via a disk susceptibility assay. Exposure to lasers alters the absorption/fluorescence spectra of the phenothiazines; reduces the activity of thioridazine against the test bacterium; produces a highly active chlorpromazine compound against the test organism. Exposure of phenothiazines to lasers alters their structure that results in altered activity against a bacterium. This is the first report that lasers can alter the physico-chemico characteristics to the extent that altered bioactivity results. Exposure to lasers is expected to yield compounds that are difficult to make via chemical manipulation methods. A survey of selected patents of interest, even co-lateral for the subject of this article is shortly made.

Targeting Hepatitis B Virus and Human Papillomavirus Induced Carcinogenesis: Novel Patented Therapeutics by Rupinder K. Kanwar, Neha Singh, Sneha Gurudevan, Jagat R. Kanwar (158-174).
Viral infections leading to carcinogenesis tops the risk factors list for the development of human cancer. The decades of research has provided ample scientific evidence that directly links 10-15% of the worldwide incidence of human cancers to the infections with seven human viruses. Moreover, the insights gained into the molecular pathogenetic and immune mechanisms of hepatitis B virus (HBV) and human papillomavirus (HPV) viral transmission to tumour progression, and the identification of their viral surface antigens as well as oncoproteins have provided the scientific community with opportunities to target these virus infections through the development of prophylactic vaccines and antiviral therapeutics. The preventive vaccination programmes targeting HBV and high risk HPV infections, linked to hepatocellular carcinoma (HCC) and cervical cancer respectively have been recently reported to alter age-old cancer patterns on an international scale. In this review, with an emphasis on HBV and HPV mediated carcinogenesis because of the similarities and differences in their global incidence patterns, viral transmission, mortality, molecular pathogenesis and prevention, we focus on the development of recently identified HBV and HPV targeting innovative strategies resulting in several patents and patent applications.

Commercial Development and Application of Type A Lantibiotics by Shawanda Wilson-Stanford, Leif Smith (175-185).
Lantibiotics are an interesting group of antimicrobial peptides. There are a number of reviews that describe the potential application of lantibiotics for controlling foodborne illnesses and their potential to treat Gram positive infections caused by organisms like Staphylococcus aureus. In this review, commercial potential for the producing organism for promoting health and their potential for protein chemistry applications are discussed. Lantibiotics are ribozomally synthesized as a prepropeptide, comprised of a leader sequence that directs the propeptide to the enzymes that perform a number of post-translational modifications. This system affords several possibilities for the synthesis of a library of peptides that may have therapeutic use. We also take a look at a couple of lantibiotic producing organisms that have potential applications in the field of probiotics, given their ability to displace pathogenic microorganisms. The use of their application along with the discussion of recent patents are discussed in this review article.

Patent Selections by Bentham Science Publishers (186-188).
The patents annotated in this section have been selected from various patent databases. These recent patents are relevant to the articles published in this journal issue, categorized by therapeutic areas/targets and therapeutic agents related to antiinfective drug discovery

Traumatic injuries present a major public health concern worldwide. A role of gut and/or its derived factors hasbeen suggested in the pathogenesis associated with injury. Injury causes the activation of various signaling pathwaysalong with the release and activation of pro-inflammatory cytokines, chemokines and adhesion molecules in the intestine.This mediates infiltration of neutrophils into the intestine. Primed and subsequently activated neutrophils release variousproteolytic enzymes and superoxide anion which ultimately result in intestinal inflammation and/or injury. This reviewwill discuss the various factors contributing to intestinal inflammation following injury. Some of the patents relevant tointestinal inflammation have also been discussed in the present manuscript.