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SQ109 (NSC-722041) is an antituberculosis drug which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, it is a novel, potent and selective inhibitor of the trypomastigote form of the parasite with IC50 for cell killing of 50±8 nM. SQ109 showed activity against both drug susceptible and Multi-drug-resistant tuberculosis bacteria, including Extensively drug-resistant tuberculosis strains. In preclinical studies SQ109 enhanced the activity of anti-tubercular drugs isoniazid and rifampin and reduced by >30% the time required to cure mice of experimental TB. SQ109 may also have potential as a drug lead against Chagas disease.
Physicochemical Properties
| Molecular Formula | C22H38N2 |
| Molecular Weight | 330.55 |
| Exact Mass | 330.303 |
| Elemental Analysis | C, 79.94; H, 11.59; N, 8.47 |
| CAS # | 502487-67-4 |
| PubChem CID | 5274428 |
| Appearance | Light yellow to yellow oily liquid |
| Density | 0.97±0.1 g/cm3 |
| LogP | 5.464 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 2 |
| Rotatable Bond Count | 9 |
| Heavy Atom Count | 24 |
| Complexity | 431 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | C/C(C)=C/CC/C(C)=C/CNCCNC1[C@H]2C[C@H]3C[C@@H]1C[C@H](C3)C2 |
| InChi Key | JFIBVDBTCDTBRH-WUROFCERSA-N |
| InChi Code | InChI=1S/C22H38N2/c1-16(2)5-4-6-17(3)7-8-23-9-10-24-22-20-12-18-11-19(14-20)15-21(22)13-18/h5,7,18-24H,4,6,8-15H2,1-3H3/b17-7+/t18-,19+,20-,21?,22? |
| Chemical Name | N1-((1r,5R,7S)-adamantan-2-yl)-N2-((E)-3,7-dimethylocta-2,6-dien-1-yl)ethane-1,2-diamine |
| Synonyms | SQ109; SQ-109; SQ 109; NSC 722041; 502487-67-4; N-Geranyl-N'-(2-adamantyl)ethane-1,2-diamine; N'-(2-adamantyl)-N-[(2E)-3,7-dimethylocta-2,6-dienyl]ethane-1,2-diamine; 9AU7XUV31A; CHEMBL561057; NSC-722041; NSC722041. |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Trypanosoma; MmpL3 |
| ln Vitro |
With a selectivity index of approximately 10 to 20, SQ109 also inhibits clinically relevant intracellular amastigotes (IC50, ~0.5 to 1 μM) and extracellular epimastigotes (IC50, 4.6±1 μM). SQ109 performs poorly in an assay measuring hemolysis of red blood cells (EC50, ~80 μM). Furthermore, as shown by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and light microscopy (LMC), SQ109 significantly alters the ultrastructural characteristics of all three life cycle forms[1].
Researchers tested the antituberculosis drug SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, for its in vitro activity against the trypanosomatid parasite Trypanosoma cruzi, the causative agent of Chagas disease. SQ109 was found to be a potent inhibitor of the trypomastigote form of the parasite, with a 50% inhibitory concentration (IC50) for cell killing of 50 ± 8 nM, but it had little effect (50% effective concentration [EC50], ∼80 μM) in a red blood cell hemolysis assay. It also inhibited extracellular epimastigotes (IC50, 4.6 ± 1 μM) and the clinically relevant intracellular amastigotes (IC50, ∼0.5 to 1 μM), with a selectivity index of ∼10 to 20. SQ109 caused major ultrastructural changes in all three life cycle forms, as observed by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). It rapidly collapsed the inner mitochondrial membrane potential (Δψm) in succinate-energized mitochondria, acting in the same manner as the uncoupler FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone], and it caused the alkalinization of internal acidic compartments, effects that are likely to make major contributions to its mechanism of action. The compound also had activity against squalene synthase, binding to its active site; it inhibited sterol side-chain reduction and, in the amastigote assay, acted synergistically with the antifungal drug posaconazole, with a fractional inhibitory concentration index (FICI) of 0.48, but these effects are unlikely to account for the rapid effects seen on cell morphology and cell killing. SQ109 thus most likely acts, at least in part, by collapsing Δψ/ΔpH, one of the major mechanisms demonstrated previously for its action against Mycobacterium tuberculosis. Overall, the results suggest that SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, may also have potential as a drug lead against Chagas disease [1]. |
| ln Vivo |
For 28 days, mice given SQ109 orally (0.1–25 mg/kg daily) showed dose-dependent reductions in lung and spleen mycobacterial loads, which were similar to those of EMB given daily at 100 mg/kg, though less effective than isoniazid (INH) given daily at 25 mg/kg. After a single administration, the pharmacokinetic profiles of SQ109 in mice revealed a Cmax of 1038 for intravenous (i.v.) and 135 ng/mL for oral administration, along with an oral Tmax of 0.31 hours.Regarding SQ109, the elimination t1/2 is 3.5 (i.v.) and 5.2 h (p.o.). Oral bioavailability amounts to 4% [2]. Dogs have a significantly larger volume of distribution for SQ109 than rats do (7-8 h, mean 7.4 h), indicating that dogs have a longer terminal half-life (t1/2) of SQ109. It has been found that SQ109 has an oral bioavailability of 12% in rats and 5% in dogs[3].
SQ109 is a novel [1,2]-diamine-based ethambutol (EMB) analog developed from high-throughput combinatorial screening. The present study aimed at characterizing its pharmacodynamics and pharmacokinetics. The antimicrobial activity of SQ109 was confirmed in vitro (Mycobacterium tuberculosis-infected murine macrophages) and in vivo (M. tuberculosis-infected C57BL/6 mice) and compared to isoniazid (INH) and EMB. SQ109 showed potency and efficacy in inhibiting intracellular M. tuberculosis that was similar to INH, but superior to EMB. In vivo oral administration of SQ109 (0.1-25 mg kg(-1) day(-1)) to the mice for 28 days resulted in dose-dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg(-1) day(-1), but was less potent than INH at 25 mg kg(-1) day(-1). Monitoring of SQ109 levels in mouse tissues on days 1, 14 and 28 following 28-day oral administration (10 mg kg(-1) day(-1)) revealed that lungs and spleen contained the highest concentration of SQ109, at least 10 times above its MIC. Pharmacokinetic profiles of SQ109 in mice following a single administration showed its C(max) as 1038 (intravenous (i.v.)) and 135 ng ml(-1) (p.o.), with an oral T(max) of 0.31 h. The elimination t(1/2) of SQ109 was 3.5 (i.v.) and 5.2 h (p.o.). The oral bioavailability was 4%. However, SQ109 displayed a large volume of distribution into various tissues. The highest concentration of SQ109 was present in lung (>MIC), which was at least 120-fold (p.o.) and 180-fold (i.v.) higher than that in plasma. The next ranked tissues were spleen and kidney. SQ109 levels in most tissues after a single administration were significantly higher than that in blood. High tissue concentrations of SQ109 persisted for the observation period (10 h). This study demonstrated that SQ109 displays promising in vitro and in vivo antitubercular activity with favorable targeted tissue distribution properties. [2] This study aimed at characterizing the interspecies absorption, distribution, metabolism and elimination (ADME) profile of N-geranyl-N'-(2-adamantyl)ethane-1,2-diamine (SQ109), a new diamine-based antitubercular drug. Single doses of SQ109 were administered (intravenously (i.v.) and per os (p.o.)) to rodents and dogs and blood samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Based on i.v. equivalent body surface area dose, the terminal half-life (t1/2) of SQ109 in dogs was longer than that in rodents, reflected by a larger volume of distribution (Vss) and a higher clearance rate of SQ109 in dogs, compared to that in rodents. The oral bioavailability of SQ109 in dogs, rats and mice were 2.4-5, 12 and 3.8%, respectively. After oral administration of [14C]SQ109 to rats, the highest level of radioactivity was in the liver, followed by the lung, spleen and kidney. Tissue-to-blood ratios of [14C]SQ109 were greater than 1. Fecal elimination of [14C]SQ109 accounted for 22.2% of the total dose of [14C]SQ109, while urinary excretion accounted for only 5.6%. The binding of [14C]SQ109 (0.1-2.5 microg ml-1) to plasma proteins varied from 6 to 23% depending on the species (human, mouse, rat and dog). SQ109 was metabolized by rat, mouse, dog and human liver microsomes, resulting in 22.8, 48.4, 50.8 or 58.3%, respectively, of SQ109 remaining after a 10-min incubation at 37 degrees C. The predominant metabolites in the human liver microsomes gave intense ion signals at 195, 347 and 363m/z, suggesting the oxidation, epoxidation and N-dealkylation of SQ109. P450 reaction phenotyping using recombinant cDNA-expressed human CYPs in conjunction with specific CYP inhibitors indicated that CYP2D6 and CYP2C19 were the predominant CYPs involved in SQ109 metabolism[3]. |
| Enzyme Assay |
Microsomal metabolism of SQ109 [3] The microsomal assay was similar to that described previously (Jia et al., 2003). Briefly, SQ109 (10 μM) was incubated with mouse, rat, dog and human liver microsomes, respectively, in an NADPH-generating system containing 1.3 mM NADP, 3.3 mM glucose-6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase and 3.3 mM MgCl2 in 100 mM potassium phosphate buffer (pH 7.4). Reaction mixtures were prepared in duplicate and were preincubated for 5 min at 37°C. The reactions were then initiated by the addition of microsomes (30 μl of a 20 mg ml−1 solution in 250 mM sucrose, yielding a final protein concentration of 0.5 mg ml−1). The final volume of each reaction mixture was 1.2 ml. Negative control reactions were prepared by incubating mixtures that excluded either microsomes or SQ109 from the mixture. For negative control incubation where microsomes were excluded, they were added back to the reaction mixture after quenching with acetonitrile. Samples were removed at 0, 10, 20, 40 or 80 min and vortex-mixed with cold acetonitrile to stop the reaction. After centrifugation, a portion of each resulting supernatant was analyzed by mass spectrometry for unchanged SQ109. Metabolism of SQ109 by cDNA-expressed recombinant human CYPs [3] SQ109 metabolism was also evaluated in microsomes prepared from insect cells transfected with cDNAs encoding for human CYP1A2, CYP2A6, CYP3A4, CYP2B6, CYP2C8, CYP2C9, CYP2C19 or CYP2D6. The recombinant enzymes and microsomes from untransfected insect cells were used in parallel as a control. SQ109 (10 μM) was preincubated in duplicate with the above-mentioned NADPH-generating system for 5 min at 37°C. The reactions were then initiated by the addition of the individual CYPs (final 100 pmol CYP ml−1) or corresponding untransfected cells. Samples were mixed by inversion, removed at 0 and 30 min and mixed with ice-cold acetonitrile to stop the reactions. After centrifugation (14,000 × g for 20 min at 4°C), each extract was analyzed by using the mass spectrometry to monitor metabolite formation or SQ109 depletion. Electrospray ionization (ESI) full mass scans were performed to obtain the ion chromatograms of the expected metabolites according to predicted mass gains and losses as compared with the molecular mass of SQ109. The ESI as a gentle ionization technique is preferred in metabolite analysis, since ESI usually does not dissociate compounds extensively. The metabolite profiling was based on the detection of protonated, deprotonated or adduct ions, but not on the fragment ions (Kostiainen et al., 2003). Samples were assayed in both the negative and positive ion to ensure detection of all potential metabolite(s), and define the structure(s). Metabolite quantitation was based on percentages of peak areas of each metabolite as a function of incubation time compared to the total area of all chromatographic peaks. Radioactivity determination [3] The radioactivity of all samples was measured in the Tri-Carb 2100TR liquid scintillation analyzer. All counts were converted to absolute radioactivity (d.p.m.) by automatic chemiluminescence and quench correction. Samples having radioactivity (d.p.m.) less than or equal to twice background d.p.m. were considered to be below the limit of quantitation, and therefore the reading was considered zero d.p.m. for calculation purposes. SQ109 equivalents in biological samples were determined by dividing the sample d.p.m. by the specific activity of [14C]SQ109 in d.p.m. per microgram, and expressed in microgram per gram of tissue. [14C]SQ109 equivalents were also expressed as a percentage of [14C]SQ109 amount in organs or tissues over the administered total [14C]SQ109 amount per animal. The radioactivity in rat urine and feces was expressed as a percentage of the administered dose for each time interval and as a cumulative percentage. Chemical inhibition studies [3] The following inhibitors, at the concentrations shown, were incubated with the corresponding CYP isoforms. These concentrations were based on literature information (Parkinson, 1996; Tucker et al., 2001; Bjornsson et al., 2003): furafylline (CYP1A2; 0.1, 1 and 10 μM), quinidine (CYP2D6; 0.5, 1 and 10 μM), ticlopidine (CYP2C19; 5, 20 and 100 μM) (Donahue et al., 1997; Tateishi et al., 1999; Ha-Duong et al., 2001) and troleandomycin (CYP3A; 0.5, 1 and 10 μM). All inhibitors were dissolved in methanol prior to addition to the incubation mixtures. Reaction mixtures containing human cDNA expressed CYP2D6 or CYP2C19 (100 pmol P450 ml−1 reaction mixture), the NADPH-generating system, selective CYP inhibitors and 100 mM potassium phosphate buffer (pH 7.4) were preincubated for 15 min at 37°C. Each reaction was then started by the addition of SQ109 (10 μM) with subsequent mixing of each sample by inversion. The samples were immediately removed and mixed with cold acetonitrile to stop reaction at 0 and 30 min of incubation. SQ109 and its metabolites were identified by the LC/MS/MS method. Peak areas formed were used for quantitative analyses. Control incubation mixtures included mixtures without inhibitors, and mixtures with untransfected insect cell microsomes used as microsomal control, and mixtures that contained methanol instead of inhibitor (methanol control). Quantitative analyses were performed by comparing the peak areas of the inhibition reactions to their respective methanol controls. The total organic solvent content of the in vitro reaction mixtures was less than 2%. |
| Cell Assay |
SQ109 (2.5–20 μM) is applied to the LLC-MK2 cells, and they are then incubated for 96 hours at 37°C. To the untreated samples, fresh RPMI 1640 medium containing 10% FBS is added as a control. The MTS/PMS test is used to assess toxicity. Based on its activities against the trypomastigote and intracellular amastigote forms of T.cruzi, SQ109's selectivity index is calculated as the ratio of the parasite's 50% lysing concentration (LC50) or IC50 to the 50% cytotoxic concentration (CC50) of mammalian cells. Every experiment is run in duplicate. Three or more experiments yield the means[1].
In vitro infection model [2] The RAW 264.7 (ATCC TIB-71) murine macrophage cell line was seeded overnight at 5 × 105 cells per well in 24-well plates at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) with essential amino acids and glutamine supplemented with 10% heat-inactivated fetal bovine serum (FBS). Log-phase cultures of recombinant M. tuberculosis H37Rv containing the luciferase reporter construct pSMT1 (hsp60 promoter-driven luciferase) (Snewin et al., 1999) were grown in Middlebrook 7H9 supplemented with bovine serum albumin (BSA), dextrose, catalase and 50 μg ml−1 of hygromycin B at 37°C with 5% CO2 from frozen stocks. Mycobacteria were harvested by centrifugation at 2500 × g for 10 min and washed twice with serum-free DMEM before re-suspension in DMEM supplemented with 5% heat-inactivated FBS at 5 × 106 cells ml−1. Macrophages were infected by incubation with the bacteria at a ratio of 10 : 1 (M. tuberculosis: cell) overnight at 37°C; they were then washed three times in Dulbecco's phosphate-buffered saline (PBS, pH 7.4). The infected cells were treated in triplicate with INH, EMB and SQ109 dissolved in DMEM containing 5% FBS at various MIC for 7 days. Macrophage cells were lysed by the addition of 1 ml per well sterile distilled H2O containing 0.1% Triton X-100 with stirring for 2 min. In all, 100 μl lysate was sampled from each well and an equal amount of 1% N-decyl aldehyde in ethanol was added. Luminescence was immediately quantified using a Luminiskan luminometer (Packard) with a dwell-time of 10 s per well to test the activities of each drug on the infected RAW 264.7 cells. The bioluminescence-based assay employs a reporter strain of M. tuberculosis, which endogenously expresses firefly luciferase that catalyzes the substrate to produce luminescence. Therefore, mycobacterial growth inside the infected cells can be estimated based on luminous intensity (Snewin et al., 1999). Plasma protein binding [3] The percent binding of [14C]SQ109 to mouse, rat, dog and human plasma proteins was determined by using ultracentrifugation (Barre et al., 1985; Boulton et al., 1998). Briefly, spiking solutions of [14C]SQ109 were prepared by diluting the stock [14C]SQ109 (1 mCi of 5.9 mg−1 ml−1) with absolute ethanol to yield spiking solutions containing 5, 25 or 125 μg ml−1 of [14C]SQ109. For each species, a 10.8 ml aliquot of plasma was mixed with 0.22 ml of the appropriate spiking solution of [14C]SQ109 to yield final SQ109 concentrations of 0.1, 0.5 or 2.5 μg ml−1. The plasma mixtures were placed into individual polycarbonate ultracentrifuge tubes and centrifuged at 100,000 × g for 24 h at 4°C. [3] At the end of the centrifugation period, the upper chylomicron layer, middle aqueous layer and lower protein pellet were separated and the volume of each layer was determined. The protein pellets were dissolved in Soluene 350 tissue solubilizer. The radioactivity of duplicate portions of the chylomicron and aqueous layers as well as the solubilized protein layer was determined, and the total amount of radioactivity in each layer was calculated. The percentage of radioactivity in each layer was determined by comparing the amount of radioactivity in each layer with the sum of the total amount of radioactivity in all three postcentrifugation plasma samples. The percent of the total radioactivity in the aqueous layer was considered to represent the unbound fraction of SQ109, while the sum of the radioactivity in the chylomicron and lower (pellet) protein layers was considered to represent the bound fraction of the drug. |
| Animal Protocol |
Mice [2] Eight-week-old female C57BL/6 mice are utilized. Twenty days after inoculation, mice are given oral doses of INH (25 mg/kg), ethambutol (EMB) (100 mg/kg), and SQ109 (0.1, 10 and 25 mg/kg). Mice infected but untreated control groups are euthanized either at the start of therapy (early controls) or at the conclusion of the treatment period. Every group consists of six mice. Four weeks after the start of treatment, the mice receive chemotherapy five days a week until they die. Weighing and aseptic removal of the lungs and spleen are performed.Two milliliters of sterile PBS containing 0.05% Tween-80 are used to homogenize the organs. After being diluted ten times in PBS, homogenate samples from distinct tissues are plated on 7H10 agar plates. Before calculating CFU, inoculated dishes are incubated for three weeks at 37°C in room temperature. A logarithmic scale is used to convert viable counts, and readings are adjusted to reflect totals for all organs. The survival rate and the mean number of CFU in mouse organs are used to evaluate the degree of infection and the efficacy of the treatments. Pharmacokinetic studies [2] Male CD2F1 mice (23–27 g) were administered SQ109 at 3 mg kg−1 (intravenous (i.v.)), or 25 mg kg−1 (p.o.). Four or five mice were anesthetized with isoflurane at the following times after administration of SQ109 to collect blood from the brachial region of each animal: 2, 5, 10, 15 and 30 min and 1, 3, 6, 10 and 24 h after a single i.v. dosing; 5, 15 and 30 min and 1, 2, 4, 6, 10 and 24 h after a single oral dosing. Each blood sample was collected into a tube containing EDTA and centrifuged (2000 × g, 10 min) to separate plasma and red blood cells. To each 200 μl of plasma sample, 10 μl of internal standard solution (10 μg ml−1) was added. SQ109 was then separated and analyzed according to the previously described procedures. Peak area ratios of SQ109 to the internal standard were plotted against theoretical concentrations. Drug concentrations in samples were calculated from the standard calibration curves. Pharmacokinetic parameters were calculated using the computer program WinNonlin (Pharsight Co., Mountain View, CA, U.S.A.), and bioavailability was calculated as (AUCp.o. AUCi.v.−1) × (dosei.v. × dosep.o.−1) × 100%. SQ109 levels in vital tissues following multiple dosing for 28 days [2] In order to determine whether tissue levels of SQ109 correlate with its efficacy in the H37Rv-infected mouse model, and whether SQ109 accumulates in the targeted tissues over a long period of multiple dose administration, SQ109 levels in the lung, spleen, liver, kidney and heart were monitored during the period of multiple dose administration. Briefly, C57BL/6 mice were orally given SQ109 by gavage at 10 mg kg−1 day−1 for 28 days. Groups of five mice each were anesthetized at 1 h after oral administration on days 1, 14 and 28 with CO2/O2. Blood and the five vital tissues were collected. Plasma and tissue homogenates were prepared for analysis using the below-mentioned procedures. Standard curves for SQ109 in different tissue matrices were plotted for quantitative purposes. Tissue distribution and elimination of SQ109 after a single administration [2] Male CD2F1 mice (25–27 g) were housed in suspended wire metabolism cages in order to collect their urine and feces to determine SQ109 excretion. The mice were fasted overnight before dosing. Water was provided throughout the study. Mice were dosed with SQ109 at either 3 mg kg−1 (i.v.) or 25 mg kg−1 (p.o.). Groups of four mice each were anesthetized with isoflurane at 1, 4 and 10 h after dosing, and blood was collected from the brachial region of each mouse into a tube containing EDTA. Tissues and organs were immediately removed, individually weighed, washed with cold saline and stored at −20°C prior to analysis for levels of SQ109. On the day of analysis, tissues and organs were minced with scissors and homogenized in ice-cold 5 mM ammonium acetate buffer (1 : 5, w : w). Aliquots of the homogenate (200 μl) were extracted as described previously. [2] For elimination studies with SQ109, groups of four mice resided in metabolism cages, where urine and feces were separated by a cone-shaped device. Pooled urine and feces were cumulatively collected prior to drug administration, and at 4, 8, 24 and 32 h after a single dose (3 mg kg−1, i.v.; 25 mg kg−1, p.o.). Feces were homogenized in 12 volumes by fecal weight of 5 mM ammonium acetate buffer, and 200 μl aliquots were extracted after centrifugation as described previously. Urine samples were diluted 1 : 10 with 5 mM ammonium acetate without further preparation. Rat and dog pharmacokinetic studies with SQ109 [3] Rats with an indwelling jugular vein catheter were used for the pharmacokinetic studies. Rats were given either a single intravenous (i.v.) bolus dose of 1.5 mg kg−1 (9 mg m−2) or an oral dose of 13 mg kg−1 (78 mg m−2) of SQ109 (n=8 per dose group); rats were divided into subgroups consisting of four rats per subgroup. Rat blood (0.7 ml) was withdrawn from the jugular vein catheter at alternating time points from individual rats in each subgroup. Blood samples were collected at 2, 5, 10, 15 and 30 min and 1, 3, 6, 10 and 24 h after a single i.v. administration, or 5, 15 and 30 min and 1, 2, 4, 6, 10 and 24 h after a single oral administration. Each blood sample was centrifuged to separate plasma, which was then stored at −70°C until analysis. [3] Beagle dogs were dosed by gavage at either 3.75 or 15 mg kg−1 (75 or 300 mg m−2), or intravenously at either 0.45 or 4.5 mg kg−1 (9 and 90 mg m−2). Dog blood (0.7 ml) was withdrawn from the jugular vein at 2, 5, 10, 20 and 30 min and 1, 2, 4, 8, 12, 18 and 24 h after a single i.v. administration, or 10, 20 and 30 min and 1, 2, 4, 8, 12, 18 and 24 h after a single oral administration. [3] Each blood sample was collected into a tube containing EDTA and centrifuged (2000 × g, 10 min) to separate plasma and red blood cells. To each 200 μl of plasma sample, 10 μl of internal standard solution (10 μg ml−1) was added. SQ109 was then separated and analyzed by the LC/MS/MS method according to the previously described procedures (Jia et al., 2005b). Peak area ratios of SQ109 to the internal standard were plotted against theoretical concentrations. Drug concentrations in the plasma samples were calculated from the standard calibration curves. Pharmacokinetic parameters were calculated using the computer program WinNonlin, and bioavailability was calculated as (AUCp.o. AUCi.v.−1) × (dosei.v. dosep.o.−1) × 100%. Tissue distribution and elimination of [14C]SQ109 in rats [3] [14C]SQ109 (5.8 mg ml−1) was diluted 4.4-fold with 0.9% sterile saline to yield a formulation containing 1.3 mg ml−1 SQ109 (225 μCi ml−1). Male Fischer rats (271–289 g) were individually housed in metabolism cages from which urine and feces were cumulatively collected to determine [14C]SQ109 excretion rate. The rats were orally dosed by gavage with 13 mg kg−1 of [14C]SQ109. Rats were killed at 0.5, 5, 10 and 24 h after dosing (n=3 per time point) in order to collect blood, tissues and organs for quantitative analysis. Tissues, intestinal tract contents and feces were homogenized in 10 volumes of water. Duplicate aliquots of whole blood, homogenates of tissues, intestinal contents and feces were digested with tissue solubilizer Soluene 350, and decolorized with 30% hydrogen peroxide to eliminate chemiluminescence. The samples were radioassayed after mixing with glacial acetic acid and the scintillation cocktail. Duplicate aliquots of urine and cage rinses were radioassayed after mixing with scintillation cocktail. [3] Pieces of carcasses were digested with 650 ml of 10 N sodium hydroxide, maintained at 37°C for 3 days, and then at room temperature until complete dissolution of the carcasses (∼2 weeks). Quadruplicate aliquots of each dissolved carcass were diluted with water (1 : 20, v v−1); portions of each diluted sample were radioassayed after the addition of an appropriate scintillator. After correction for volume by dilution and volume assayed, the radioactivity expressed as disintegrations per min (d.p.m.) of [14C]SQ109 in each sample was determined. |
| ADME/Pharmacokinetics |
Once it entered systemic circulation of mice after a single i.v. or p.o. administration, SQ109 rapidly reached peak concentrations in the lung and spleen far above its MIC level for M. tuberculosis, without noticeable side effects such as injection site irritation, inability to move, ruffled fur, ataxia, tremors, convulsions, emesis, diarrhea, labored breathing and acute death. SQ109 concentrations in the respiratory tract remained above the MIC for more than 10 h after oral dosing (Figure 5). This rapid transfer of compound from circulation to vascularized tissue of the lung may be related to the fact that SQ109 has an adamantane tricyclic fragment in the chemical structure (Figure 1). Adamantane-containing antiviral drugs on the market (Rimantadine and Amantadine) are used to combat viral lung pathogens like Influenza A by inhibiting initiation of infection and virus assembly (Hay et al., 1985) and by blocking the ion channels in lipid membranes of virus (Griffin et al., 2003). These drugs distribute specifically to lung and have large Vss (Hoffman et al., 1988). It is very likely that the adamantane structure is also integral for both in vivo and in vitro antitubercular activity: four EMB analogues out of seven of the most potent compounds in vitro and in vivo have an adamantyl moiety in the molecules (unpublished observation).
[2] SQ109 possesses a large Vss, as do the other EMB analogs. Its large Vss might also be attributed to hydrophobic moieties of the compound and diamine structure that results in rapid penetration into extravascular compartments with favorable tissue kinetics, especially in the lung and spleen (Figures 3 and 5). The lung and spleen concentrations of SQ109 oscillated above the MIC over the course of daily administration. This finding suggests that orally delivered SQ109 in animal models and patients may beneficially concentrate in these organs, where replicating Mycobacteria happen during disease. That SQ109 was easily detected in organs of mice administered a 25 mg kg−1 oral dose clearly demonstrated that much of the dose was absorbed into tissues. [2] However, pharmacokinetic studies indicate that SQ109 has a poorer oral bioavailability than its in vivo efficacy in mycobacterial disease and tissue distribution would indicate (Table 2). The reasons for poor oral bioavailability of any compound include: [2] Poor oral absorption: The Cmax level of oral SQ109 was about 80-fold less than that of i.v. SQ109 when compared on the basis of same dose, suggesting issues with intestinal absorption. Many candidate drugs fail due to problems with intestinal absorption, since molecules must be able to permeate cell membranes composed of phospholipid bilayers to traverse the intestine and enter circulation. In addition, two findings in the present study argue against profound intestinal permeability issues with SQ109: (i) the drug is able to pass through several cell membranes and achieve effective concentrations in the phagosome of infected macrophages in vitro (Figure 2), and (ii) sufficient drug traversed the intestine and distributed to the lung and spleen to achieve levels far above the in vitro MIC and create an effective antimicrobial effect, even at the lowest dose tested in vivo, 0.1 mg kg−1 (Table 1). [2] First-pass effect: After i.v. administration, the concentration of SQ109 in liver, the main organ of drug metabolism, was lower than the concentration observed in most tissues, including the brain (Figure 5). Relative to the concentration of SQ109 in other tissues, however, the concentration of SQ109 in liver was higher after oral administration than after i.v. administration to mice (Figures 3 and 5). These data suggest that SQ109 may undergo a first-pass metabolic step in the liver after oral administration. So far we have identified four metabolites of SQ109 after incubation of the compound with mouse and human liver microsomes and recombinant CYP450 isoforms. [2] High dissociation constants of SQ109 from blood proteins, causing quick disappearance from the systemic circulation by tissue redistribution. The time course of tissue concentrations of SQ109 after oral administration is compatible with an initial hepatic sequestration of this material, with subsequent redistribution to the lung, spleen and kidney (Figures 3 and 5). [2] There are a number of drugs with apparent bioavailability issues that have good, even excellent efficacy against their target pathogen. For example, Halofuginone is a drug used to prevent coccidiosis in poultry, treat cryptosporidiosis and theileriosis in cattle, and treat various diseases in humans. This drug has no oral bioavailability (Stecklair et al., 2001). Like SQ109, Halofuginone shows concentrations in the targeted tissues 50–2000 times higher than in plasma after oral administration. Most antibiotic drugs exert their effects not within the plasma compartment but in defined targets into which these drugs must be distributed from the central compartment (Muller et al., 2004). Recent studies have further indicated that antibiotic drug levels at the target site may substantially differ from corresponding plasma drug levels, and the concentration profile at the target site is more helpful than concentration in plasma in designing clinical trials, and is an important determinant of clinical outcome (Ryan, 1993; Presant et al., 1994; Joukhadar et al., 2001). In this respect, SQ109 shows favorable targeted tissue distribution properties in relation to its antitubercular activity. [2] Interestingly, SQ109 appears to preferentially accumulate in the lungs and spleen over a 28-day period of repeated administration. There was statistically significant accumulation of SQ109 in these two tissues and slight increase of SQ109 in other tissues when we compare concentrations of the drug at steady state on days 14 and 28 with that achieved on day 1 after a single administration (Figure 3). When the drug disposition kinetics are in first order, tissue concentrations following multiple doses should be higher at steady state. This suggests that enzymatic involvement in the up- or downregulation of SQ109 in the tissues is not an issue. These results also explain the complex relationship between concentration–time profiles of SQ109 and its observed antitubercular effect (Figures 4 and 5). [2] In conclusion, SQ109 exhibited both in vitro antimicrobial activity against M. tuberculosis strain H37Rv grown inside the host murine macrophage cells and in vivo antimicrobial activity on the mouse model inoculated with the H37Rv. Oral administration of SQ109 to the mice for 28 consecutive days resulted in significant reductions in mycobacterial burden in both spleen and lungs of the mice. Monitoring SQ109 levels in mouse vital tissues in the course of 28-day oral administration showed that the potential sites of action (e.g., lungs and spleen) contained SQ109 at least 10 times more than its MIC. SQ109 displayed a large volume of distribution to various tissues. Despite its low oral bioavailability, the targeted tissue concentrations of SQ109 were at least 120-fold higher than that in plasma. This study provides important insights into the integration of in vitro efficacy parameters, into the in vivo pharmacokinetic and pharmacodynamic evaluation of SQ109, and should facilitate future clinical trials of the compound.[2] SQ109 is lipophilic and has low aqueous solubility. Its absorption time in the tested animal species should be comparable due to the similar nature of the biomembrane of intestinal epithelial cells across species and the absorptive process (simple diffusion) that is basically an interaction between a specific drug and the biomembrane (Lin, 1995; Martinez et al., 2002). This is supported by the results of the present studies, which show that the Tmax of SQ109 (∼0.5 h) was similar among mice, rats and dogs (Tables 1 and 2). [3] SQ109 possesses a large Vss as do the other ethambutol analogs in mice (Jia et al., 2005b, 2005c). The same observation was made in rats and dogs in the present studies. Although methods for the prediction of Vss based on experimentally determined physicochemical parameters can predict the Vss close to the actual value, it must be kept in mind that drug- or plasma-based models refer to the process of penetration into hypothetical homogeneous compartments as ‘tissue penetration'. This concept is misleading, as it does not take into account the uniqueness of separate heterogeneous organ systems (Muller et al., 2004). Therefore, it rarely corresponds to a real volume, such as plasma volume, extracellular water or total body water. Drug distribution may be to any one or a combination of the tissues and fluids of the body. Furthermore, binding to tissue components may be so great that the Vss is many times the total body size (Rowland & Tozer, 1995). Most importantly, it must be considered that the actual target space for anti-infective agents is the interstitial space fluid (Ryan, 1993). [3] Many drugs are subject to a ‘first-pass effect' when absorbed from the gastrointestinal tract into the systemic circulation. The organs responsible for this effect (i.e., intestine, liver and lung) are serially arranged and can potentially reduce the extent of bioavailability. The contribution of each organ has been assessed indirectly by comparison of AUC values obtained by different routes of administration (Pang & Gillette, 1978). Based on SQ109 tissue concentration–time data (Figure 3), one may conclude that SQ109 readily permeates the biomembrane of intestinal epithelial cells because of the drug's relatively fast Tmax (∼0.5 h). The tissue concentration–time data also suggest that the liver plays a significant role in eliminating and metabolizing SQ109. This finding is based on the data that show that (1) liver contained the highest concentration of [14C]SQ109 (excluding the gastrointestinal concentrations of [14C]SQ109); (2) [14C]SQ109 in the liver was at least 200-fold higher than that in whole blood; and (3) direct measurement of SQ109 per se by using the LC/MS/MS method showed that SQ109 concentration in the liver was significantly lower than those in the lung, spleen, kidney and heart (Jia et al., 2005b). However, radioactive measurement indicated that the liver contains the highest amount of radioactivity. This discrepancy can be explained on the basis that the LC/MS/MS method was developed to be highly specific for the parent molecule of the drug only and did not detect any of the compound's metabolites. The radioactivity measurement detects all 14C-containing components, which would include the parent compound and any corresponding metabolites of SQ109. These data suggest that SQ109 does undergo a first-pass metabolic step in the liver before it reaches the systemic circulation. [3] Several factors may affect the CL of a certain drug, including plasma protein binding, metabolism and hepatic blood flow. For a drug with high CL, hepatic blood flow limits the drug's CL (Boxenbaum, 1980). This rule may explain the species difference in CL of SQ109. Although there were slight interspecies differences in the metabolism of SQ109 by liver microsomes (Figure 4), the CL of SQ109 in dogs was four times higher than in mice and rats, probably because dogs have a high hepatic blood flow (Davies & Morris, 1993). Kinetically, drug distribution is often defined as the ratio of amount of drug in the body to the plasma or blood concentration. SQ109 has relatively low binding to plasma proteins (Table 5), but high binding to tissue proteins (Jia et al., 2005b) and high permeability across biomembranes. Therefore, its volume of distribution across species is extremely high (Table 3). A drug's half-life is proportional to its volume of distribution, but inversely proportional to its clearance. The theory is supported by the results of the present studies that indicate a longer t1/2 of SQ109 in dogs than in mice and rats (Table 3). [3] In the present studies, we used the ultracentrifugation technique to determine plasma protein binding, which avoids the nonspecific membrane binding in contrast to other plasma protein binding techniques (Barre et al., 1985). To determine the binding capacity and affinity of plasma protein to a drug, it is suggested that at least three concentrations of the drug (e.g., 0.1, 0.5 and 2.5 μg ml−1) be applied and tested (Kariv et al., 2001; Jia et al., 2003) to clarify concentration-related changes in the extent of binding. As shown in Table 5, increases in total [14C]SQ109 by five- and 25-fold resulted in only a slight increase in bound fraction of [14C]SQ109. This result implies that the binding seems to be of low affinity and high capacity because the fraction of bound SQ109 is relatively constant over a 25-fold concentration range and independent of drug concentration. [3] Because of the first-pass effect, the oral bioavailability of SQ109 across animal species tested was relatively low (4–12%; Table 3), and was likely to be related to a high hepatic clearance of SQ109. Using liver microsomes from various species and CYP reaction phenotyping, we found that SQ109 was rapidly metabolized when incubated with liver microsomes (Figure 4), although the metabolism rate varied from species to species. The liver microsome-induced metabolism of SQ109 was observed again when SQ109 was incubated in individual CYP2D6 or CYP2C19 isozymes. Furthermore, selective inhibitors of CYP2D6 and CYP2C19 prevented the metabolism in a dose-dependent manner (Figures 6 and 7). Therefore, both CYP2D6 and CYP2C19 can be singled out as primary CYPs that catalyze SQ109 metabolism. CYP2D6 and CYP2C are involved in biotransformation of about 25 and 15% of all drugs (Parkinson, 1996; Schlichting et al., 2000; Tucker et al., 2001), respectively. Although low oral bioavailability of many drugs can be attributed to the extensive metabolism by CYP3A4, the present studies demonstrated little effect of CYP3A4 on SQ109 metabolism. [3] Based on the P450 reaction phenotyping study, we proposed the in vitro metabolic pathway of SQ109 shown in Figure 5. N-nitroso-SQ109 (M1) could be formed via CYP2D6- and CYP2C19-catalyzed metabolism. Out of two amino groups in the molecule, the most vulnerable site of SQ109 known to be prone to electrophilic attack from −NO may be the second, the geranyl-substituted, amino group in the molecule. Biological nitrosation remains an issue that engenders controversy about biological reactivity and functions of nitrosation (Williams, 1988; Jia et al., 1996). Possible metabolism of SQ109 may also include addition of a single oxygen to SQ109, resulting in the formation of metabolite M2 with m/z 347. One of the metabolites (M2-1, m/z 347) is likely to be produced by oxidation of the adamantane ring, similar to the metabolism of a marketed adamantane-containing antiviral drug rimantadine. Another single oxygenated SQ109 metabolite (M2-2, m/z 347) may form through epoxidation of the N-allyl double bond in the geranyl moiety, leading to the corresponding epoxide that may further undergo thermal rearrangement resulting in the ketodiamine SQ109 (M2-2). N-(2-adamantyl)ethylene-1,2-diamine (M3, m/z 195) may be formed by fragmentation of the parent compound via hydroxylation of an activated carbon atom in the geranyl fragment followed by N-dealkylation of the unstable intermediate. The formation of M3 was demonstrated by a single mass chromatographic peak (not shown). There are several pathways to explain formation of M4 that may appear as different structures with the same m/z 363 because of the locations of the two oxygen atoms added to SQ109. It is highly likely that consequent oxidation reactions take place, such as ring oxidation and N-hydroxylation (M4-1, m/z 363) and/or ring oxidation and epoxidation (M4-2, m/z 363) processes that lead to the formation of corresponding hydroxy metabolites. [3] In the metabolic events occurring to SQ109 such as C-oxidation and N-hydroxylation (Figure 5), oxygen from the ferric state (FeO)3+ of CYP450 could be incorporated into SQ109, which otherwise remains intact. In the case of N-dealkylation, oxygenation of SQ109 may be followed by a thermal rearrangement, leading to cleavage of an N-amantadine structure (M3) from the remaining aliphatic structure with incorporation of oxygen from the (FeO)3+ complex into the aliphatic structure. Nonetheless, the reality of the metabolic pathway and significance of the in vivo effects of the produced end metabolites need to be further investigated and validated using authentic metabolites. [3] In conclusion, the present study systematically showed interspecies similarities and differences in ADME profile of SQ109, and its large Vss and relatively low oral bioavailability. Oral [14C]SQ109 produced the highest level of radioactivity in liver. This may represent a significant first-pass effect on SQ109, as evidenced by rapid liver microsomal metabolism of SQ109, primarily catalyzed by CYP2D6 and CYP2C19. Fecal and urinary elimination accounted for 22.2 and 5.6% of the total dose of [14C]SQ109, respectively. None of these and our previous findings guarantee that SQ109 will be a successful antibiotic. Like any drug, it could turn out to be toxic during long-term use. Nevertheless, this is clearly the most promising new antituberculosis agent that has passed through many preclinical tests (Lee et al., 2003; Jia et al., 2005a, 2005b, 2005c). |
| References |
[1]. SQ109, a new drug lead for Chagas disease. Antimicrob Agents Chemother. 2015 Apr;59(4):1950-61. [2]. Pharmacodynamics and pharmacokinetics of SQ109, a new diamine-based antitubercular drug. Br J Pharmacol. 2005 Jan;144(1):80-7 [3]. Interspecies pharmacokinetics and in vitro metabolism of SQ109. Br J Pharmacol. 2006 Mar;147(5):476-85. |
| Additional Infomation |
SQ-109 is an orally active, small molecule antibiotic for treatment of pulmonary TB. Currently in Phase I clinical trials, SQ-109 could replace one or more drugs in the current first-line TB drug regimen, simplify therapy, and shorten the TB treatment regimen. Antibiotic SQ109 is an orally available, acid-stable diamine antibiotic, with potential antimicrobial activity against a variety of bacteria including Helicobacter pylori (H. pylori) and Mycobacterium tuberculosis (M. Tuberculosis). As an ethambutol analogue with asymmetric structure, SQ109 does not act on the same target as ethambutol. However, this agent interferes with cell wall synthesis, thereby causing weakening of the cell wall and ultimately cell lysis. Drug Indication Investigated for use/treatment in bacterial infection, infectious and parasitic disease (unspecified), and tuberculosis. Mechanism of Action With a mechanism of action distinct from other antibiotics used in TB therapy, SQ109 inhibits cell wall synthesis and acts on multiple cellular pathways in a select group of microorganisms. The intensity of drug pharmacologic effects of an antimicrobial is a function of the amount of drug in the body and, more specifically, the achievement of effective drug concentration at the site(s) of bacterial infection. In the present study, we evaluated the change in SQ109 concentration over time as assessed by pharmacokinetics and compared these results to the static relationship between the concentration at the infection site and the intensity of observed activity as quantified by pharmacodynamic analysis and efficacy against multiplying M. tuberculosis. The efficacy of SQ109 against M. tuberculosis was demonstrated through in vitro macrophage model followed by further testing in an in vivo animal model. Both models were infected with the same M. tuberculosis strain H37Rv. SQ109 showed its ability to penetrate into macrophage phagosome, where M. tuberculosis replicates, and result in inhibition of the bacteria that usually behave as an intracellular parasite of macrophages in mammalian hosts. The activity of SQ109 in this regard was comparable to that of INH in the macrophage test system, but superior over that of EMB (Figure 2). With demonstrated in vitro activity, SQ109 also exerted in vivo antitubercular activity in a dose-dependent manner by reducing bacterial load of 1–1.9 log units in liver and spleen homogenates over a period of 28 days by conventional monitoring of CFU levels. INH (25 mg kg−1 day−1) was demonstrated to be more potent than SQ109 (0.1, 10 and 25 mg kg−1 day−1) and EMB (100 mg kg−1 day−1) in reducing mycobacterial burden in both spleen and lung (Table 1). The discrepancies in in vitro and in vivo activities among SQ109, INH and EMB are presently not clear, although it is likely that they reflect the differences of their molecular targets and pharmacokinetic properties. [2] |
Solubility Data
| Solubility (In Vitro) | DMSO : ≥ ~25 mg/mL (~75.63 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0253 mL | 15.1263 mL | 30.2526 mL | |
| 5 mM | 0.6051 mL | 3.0253 mL | 6.0505 mL | |
| 10 mM | 0.3025 mL | 1.5126 mL | 3.0253 mL |