Identification of a new antitubercular drug candidate, SQ109, from a combinatorial library of 1,2-ethylenediamines

Marina Protopopova*, Colleen Hanrahan, Boris Nikonenko, Rowena Samala, Ping Chen, Jackie Gearhart, Leo Einck and Carol A. Nacy

Sequella, Inc., 9610 Medical Center Drive, Suite 200, Rockville, MD 20850, USA


* Corresponding author. Tel: +1-301-217-3832; Fax: +1-301-762-7778; E-mail: marinaprotopopova{at}sequella.com

Received 25 March 2005; returned 14 May 2005; revised 14 July 2005; accepted 7 August 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The aim of this study was to identify a candidate drug for clinical development from a previously synthesized combinatorial library based on the 1,2-ethylenediamine structure of ethambutol.

Methods: Sixty-nine of the most potent hits against Mycobacterium tuberculosis from the original studies were subjected to a sequential set of tests in vitro and in vivo—determination of MIC for M. tuberculosis H37Rv, cytotoxicity, intracellular antimycobacterial activity, permeability evaluation and in vivo efficacy testing.

Results: Twenty-seven compounds with MICs of ≤15.6 µM were tested on Vero cells to determine in vitro cytotoxicity (IC50) and to establish a selectivity index (SI) (SI = IC50/MIC). Ten compounds with acceptable SI were tested for activity against intracellular bacteria—all were equivalent (within 1%) or superior to ethambutol and several demonstrated cidal activity. Five of the most potent compounds were tested for in vivo efficacy in a murine model of chronic tuberculosis infection.

Conclusion: Compound SQ109 with an MIC of 0.7–1.56 µM (H37Rv, Erdman and drug-resistant strains of M. tuberculosis), an SI of 16.7 and 99% inhibition activity against intracellular bacteria, demonstrated potency in vivo and limited toxicity in vitro and in vivo, and was selected for further development.

Keywords: tuberculosis , drugs , in vivo efficacy , cytotoxicity


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Despite a 5000 year history, tuberculosis (TB) remains the leading single-agent infectious disease killer in the world. The need to develop new and more effective drugs to shorten the standard 6 months multidrug regimen and to treat emerging drug-resistant infection has been well recognized, but neglected for decades. Only within the last few years have several promising drug candidates emerged.13 In our attempts to develop a second-generation agent from a first-line drug ethambutol,4,5 in collaboration with Clif Barry (NIH/NIAID), a library of 63 238 compounds based on a 1,2-ethylenediamine pharmacophore of ethambutol was synthesized and tested for activity against Mycobacterium tuberculosis. Several hundred hits (mostly lipophilic non-symmetric diamines) were discovered and 26 demonstrated in vitro activity equal to or greater (up to 14-fold) than ethambutol. Here, we present the results of in vitro and in vivo studies that led to the identification of a specific compound for pre-clinical development.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Compounds

All experimental compounds were synthesized as described previously;4 isoniazid and ethambutol were purchased from Sigma.

MIC determination

Experimental compounds and control drugs isoniazid and ethambutol were serially diluted from 500 to 0.244 µM and screened against M. tuberculosis H37Rv in triplicate using the broth microdilution method as described previously,4 except the results were read after 10 days of incubation. Testing of SQ109 against ethambutol-resistant strains of M. tuberculosis (ATCC 35837) was carried out in the BACTEC system. Independently, the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF, http://www.taacf.org) tested SQ109 in the Microplate Alamar Blue assay against H37Rv (ATCC 27294), Erdman (ATCC 35801) and single drug-resistant (SDR) strains of M. tuberculosis, including those resistant to ethambutol (ATCC 35837), isoniazid (ATCC 35822) and rifampicin (ATCC 35838).

Cytotoxicity

Cytotoxicity of the compounds was determined with the Vero cell line ATCC CCL-81 using an MTS assay.6

Intracellular antimycobacterial activity

Activity of selected compounds against intracellular M. tuberculosis was determined at their MIC using the RAW 264.7 (ATCC TIB-71) murine macrophage cell line infected with M. tuberculosis luciferase reporter strain pSMT1 (hsp60 promoter driven luciferase).7 Drug treatment continued for 6 days with timepoints at 1, 2, 3 and 6 days. Ethambutol at 12.5 µM and isoniazid at 1 µM were used as positive controls.

Log P determination

The octanol/water partition coefficient log P of the diamines was calculated using online services of Advanced Chemistry Development, Inc. (ACD) (http://www.acdlabs.com).

TC7 cell permeability of SQ109

Permeability of SQ109 in TC7 cells was determined at 50 µM in the apical to the basolateral (A–B) direction by Cerep (http://www.cerep.com) using established in-house protocols; experimental value of <2.5 x 10–6 cm/s of D-[14C]mannitol (50 mCi/mmol) was used to assess the cell monolayer integrity.

In vivo studies

All animal experiments were performed by Sequella personnel at the facilities of BIOQUAL, Inc. (Rockville, MD, USA) via the Institutional Agreement, and were carried out according to the guidelines of the US Government Policy on care and use of laboratory animals in biomedical research (assurance no. A2796.02). An aliquot of a frozen stock of M. tuberculosis H37Rv Pasteur was prepared and stored as described previously.8 Female C57BL/6 mice were infected intravenously with 105 cfu of M. tuberculosis H37Rv in 0.2 mL of PBS. Chemotherapy was initiated 20 days following infection and continued for up to 45 days with timepoints at 15, 30 and 45 days. Drugs (SQ58, 59, 73, 109, 111, isoniazid and ethambutol) were administered by gavage as solutions in a 5:95 mixture of ethanol and water, 200 µL per mouse, 5 days/week; one group of six mice was used for every compound and each dose. The cfu in organs were determined as described previously.8

Maximum tolerated dose

C57BL/6 female mice were administered a single dose of SQ109 at 100, 300, 400, 600, 700, 800 and 1000 mg/kg orally by gavage, three mice per dose. Mice were observed post-administration at 4 and 6 h, and then twice daily for the duration of the study (1 week). Surviving mice were sacrificed 7 days after drug administration and lungs, spleen, liver and kidneys were observed for evidence of drug toxicity.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The most potent hit compounds from the previous study4 were subjected to the following sequential set of tests: MIC, cytotoxicity screen, activity in infected macrophages, permeability evaluation and in vivo efficacy testing.

Sixty-nine 1,2-ethylenediamines4 were re-tested for in vitro activity against M. tuberculosis H37Rv in a microbroth dilution assay. The best MIC of 1.56 µM was determined for compound SQ109. The data (Table 1) were reproducible with minimal variation for four separate experiments, but varied significantly from previously published MIC values4 that could be attributed, among other possibilities, to the differences in protocols for MIC determination (reading at 10 days in this study versus 7 days). Despite these variations, the same active compounds were identified in both studies; diamine SQ73 was the only new addition to the list of potential leads. SQ109 was also tested against Erdman and SDR strains of M. tuberculosis and demonstrated significant activity (MIC, µM): Erdman (0.7), ethambutol-resistant (1.4 in Alamar Blue, 0.99 in the BACTEC), isoniazid-resistant (1.4), rifampicin-resistant (≤0.7). The fact that SQ109 works against an ethambutol-resistant strain suggests different specific target, mechanism of action and/or different activation pathways for SQ109 and ethambutol.


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Table 1. Structures, in vitro activity against M. tuberculosis H37Rv (MIC), cytotoxicity on Vero cells (IC50), log P values and intracellular activity against M. tuberculosis (% inhibition) of the top 27 1,2-ethylenediamines

 
Active compounds were then assessed for cytotoxicity using an in vitro assay with monkey kidney Vero cells (Table 1). Compounds with a 2-adamantyl moiety had the lowest toxicity (SQ58, 73 and 78), except for SQ109 which was in an intermediate category with an in vitro cytotoxicity (IC50) of 26 µM. Diamines with diphenylalkyl and myrtanyl fragments SQ7, 40, 41, 43, 117 and 151 were found to be the most toxic. These findings were somewhat disappointing, since those structural elements were among the most frequently occurring in the hit molecules,4 although they did substantially increase the log P values. The IC50 and MIC data were used to calculate the selectivity index (SI) of each compound as an estimate of a therapeutic window and a mechanism to identify candidates for efficacy studies in mice (Table 1). Top compounds were SQ58, 73, 78, 109 and 111 with SIs ranging from 5.8 to 16.7. The highest value of 16.7 was determined for SQ109.

Since M. tuberculosis is an intracellular pathogen of macrophages in mammalian hosts, and overall efficacy of any anti-TB drug will be improved by its ability to traffic into the macrophage phagosome containing replicating bacteria, we evaluated the effect of representative compounds on the viability of intracellular M. tuberculosis in target mammalian cells. Compounds SQ58, 73, 78, 109 and 111 along with other diamines with lower SIs (SQ34 and 37) and their close structural analogues (SQ41, 59 and 151) were tested at their MIC for activity against RAW 264.7 mouse macrophages infected with an M. tuberculosis luciferase reporter construct.7 Drug treatment continued for 6 days, and ethambutol and isoniazid served as controls. Mycobacterial growth was estimated based on light production and compared with infected not-treated controls. All experimental compounds exhibited intracellular activity equivalent to (within 1%) or better than ethambutol (83.8% at the MIC); SQ37, 59 and 109 demonstrated 99% inhibition.

Estimation of intestinal absorption was done based upon a predictive log P factor and using an in vitro absorption model (for SQ109 only). In general, compounds in the series demonstrated high log P values. However, SQ58, 73, 78, 109 and 111, which scored highest in vitro, had log P values in the high end of the acceptable range from –0.4 to 5.6.1 Permeability coefficient Paap of SQ109 in the A–B direction across TC7 cell monolayers was found to be 13.14 x 10–6 cm/s (with 27% recovery), which classifies SQ109 as a compound of medium permeability compared with the controls propranolol (26.42 x 10–6 cm/s), ranitidine (0.87 x 10–6 cm/s) and vinblastine (0.68 x 10–6 cm/s).

Thus, based on the results of in vitro studies, compounds SQ58, 73, 78, 109 and 111 (all with good potency in vitro and suitable log P values) as well as SQ59 (with an SI of 2.3, but high activity in macrophages) were advanced into in vivo efficacy testing.

In vivo efficacy of SQ58, 59, 73, 109 and 111 were studied in mice using a low-dose infection model of chronic TB (Figure 1). 9 SQ78 was not tested in this model, since it was inactive in a preliminary rapid (3 week) in vivo screen8 (data not shown; use of this model in the discovery of new anti-TB agents will be presented in a separate paper). In the studies, SQ73 and 109 were evaluated at doses of 1, 10 and 25 mg/kg; SQ59 at 1 and 10 mg/kg; SQ111 at 10 and 25 mg/kg; and SQ58 at 25 mg/kg. Mice treated with isoniazid (25 mg/kg) and ethambutol (100 mg/kg) were used as positive controls. All experimental compounds substantially decreased the concentration of viable bacteria in lungs and spleen, but were less effective than isoniazid. In lungs, SQ73 and 109 at 1 mg/kg performed as well as ethambutol. At 10 mg/kg, SQ59 and 73 showed good potency in both organs, and SQ109 during the 6 week therapy was more effective in lungs than ethambutol. Treatment with SQ109 at 25 mg/kg for 30 days showed a slight improvement over 10 mg/kg dose in spleen. Compounds SQ58 and 111, despite good MIC and SI, were the least effective in vivo. In contrast, SQ59 with a moderate MIC and low SI of 2.3 was able to reduce infection in spleen by two log even at a dose of 1 mg/kg, pointing to the fact that selection of compounds for in vivo efficacy studies strictly on SI can be misleading.



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Figure 1. In vivo efficacy studies of diamines SQ58, 59, 73, 109 and 111 in a mouse model of chronic TB infection. C57BL/6 female mice were inoculated intravenously with 105 cfu M. tuberculosis H37Rv. Chemotherapy was initiated 3 weeks following infection, drugs were administered by gavage 5 days/week. Infected untreated control (filled diamonds), isoniazid at 25 mg/kg (filled triangles), ethambutol at 100 mg/kg (filled squares), SQ58 at 25 mg/kg, SQ59 at 1 and 10 mg/kg (oblique crosses), SQ73 at 1, 10 and 25 mg/kg (stars), SQ109 at 1, 10 and 25 mg/kg (filled circles) and SQ111 at 10 and 25 mg/kg (crosses).

 
To enable further studies, compound SQ109 was also tested for acute toxicity in mice. Maximum tolerated dose for SQ109 was 600 mg/kg, with no visible changes observed in critical organs of the sacrificed animals.

Within the series, compound SQ109 was identified as the most potent. It had an MIC of 0.7–1.56 µM, an SI of 16.7 and demonstrated activity against drug-resistant strains of M. tuberculosis. Furthermore, SQ109 was able to reduce intracellular M. tuberculosis burden by 99% at its MIC (1.56 µM). In vivo, SQ109 demonstrated the highest activity, mainly in lungs, and was as effective in curing TB infection in mice at 1 mg/kg as ethambutol at 100 mg/kg; toxicity to mice was at suitable values. Ongoing collaborations with the NIH demonstrated good pharmacokinetic properties of SQ109, and, importantly, a continuous high concentration in the respiratory tract5 significantly above the MIC. This drug may provide sufficient therapeutic activity with a comfortable safety margin during antitubercular therapy. At the present time, SQ109 is nearing completion of formal pre-clinical safety pharmacology and toxicology studies in preparation for Phase I clinical trials. Originally developed as a second-generation antibiotic, SQ109 (like other hits in the series) shares the same 1,2-ethylenediamine pharmacophore with ethambutol. However, other structural dissimilarities between SQ109 and ethambutol, significant activity of SQ109 against drug-resistant strains of M. tuberculosis, including those that are ethambutol-resistant, different pharmacological profiles and also recently reported differences in its intracellular target(s)10 suggest that SQ109 is a new anti-TB drug, not an ethambutol analogue.


    Acknowledgements
 
We would like to thank Dr Douglas Young of Imperial College London (UK) for the generous gift of luciferase reporter construct pSMT1 and Dr Richard Lee of the University of Tennessee for his contribution into the development of a scalable synthesis of the diamine compounds. We also would like to thank TAACF for determination of the MICs. These studies were supported by the Challenge Grant (1UC1 AI-049514-01) of the National Institute of Allergic and Infectious Diseases (NIAID) of the NIH.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1. Barry CE III, Slayden RA, Sampson AE et al. Use of genomics and combinatorial chemistry in the development of new antimycobacterial drugs. Biochem Pharmacol 2000; 59: 221–31.[CrossRef][ISI][Medline]

2. Andries K, Verhasselt P, Guillemont J et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005; 307: 223–7.[Abstract/Free Full Text]

3. Stover KC, Warrener P, Vandevanter DR et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 2000; 405: 962.[CrossRef][ISI][Medline]

4. Lee RE, Protopopova M, Crooks E et al. Combinatorial lead optimization of [1,2]-diamines based on ethambutol as potential antituberculosis preclinical candidates. J Comb Chem 2003; 5: 172–87.[CrossRef][ISI][Medline]

5. Jia L, Tomaszewski JE, Hanrahan C et al. Pharmacodynamics and pharmacokinetics of SQ109, a new diamine-based antitubercular drug. Br J Pharmacol 2005; 144: 80–7.[CrossRef][ISI][Medline]

6. Cory AH, Owen TC, Barltrop JA et al. Use of an aqueous tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 1991; 3: 207–12.[ISI][Medline]

7. Snewin VA, Gares MP, Gaora PÓ et al. Assessment of immunity to mycobacterial infection with luciferase reporter constructs. Infect Immun 1999; 67: 4586–93.[Abstract/Free Full Text]

8. Nikonenko BV, Samala R, Einck L et al. Rapid, simple in vivo screen for new drugs active against Mycobacterium tuberculosis. Antimicrob Agents Chemother 2004; 48: 4550–5.[Abstract/Free Full Text]

9. Kelly BP, Furney SK, Jessen MT et al. Low-dose aerosol infection model for testing drugs for efficacy against Mycobacterium tuberculosis. Antimicrob Agents Chemother 1996; 40: 2809–12.[Abstract]

10. Boshoff HI, Myers TG, Copp BR et al. The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. J Biol Chem 2004; 279: 40174–84.[Abstract/Free Full Text]





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