Gwt1 enzyme

Fosmanogepix (APX001) has a minimum effective dose of 0.008-0.25 μg/ml to suppress the development of Aspergillus fumigatus, Candida albicans, Clostridium neoformans, and Clostridium gattii over a 40-72 hour period[1].Cryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited, especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. Here we evaluated the in vitro and in vivo activity of several compounds, including APX001A and its prodrug, APX001, currently in clinical development for the treatment of invasive fungal infections. These compounds target the conserved Gwt1 enzyme that is required for the localization of glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins in fungi. The Gwt1 inhibitors had low MIC values, ranging from 0.004 μg/ml to 0.5 μg/ml, against both C. neoformans and C. gattii. APX001A and APX2020 demonstrated in vitro synergy with fluconazole (fractional inhibitory concentration index, 0.37 for both). [1]

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APX001A inhibited the growth of A. fumigatus with a minimum effective concentration of 0.03 μg/ml. [2]

Fosmanogepix (APX001) (390 mg/kg, po, three times daily) lowers burden in the Swedish cryptococcal meningitis (CM) model [1]. Fosmanogepix (APX001) (100 mg/kg, mantra)

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In a CM model, APX001 and fluconazole each alone reduced the fungal burden in brain tissue (0.78 and 1.04 log10 CFU/g, respectively), whereas the combination resulted in a reduction of 3.52 log10 CFU/g brain tissue. Efficacy, as measured by a reduction in the brain and lung tissue fungal burden, was also observed for another Gwt1 inhibitor prodrug, APX2096, where dose-dependent reductions in the fungal burden ranged from 5.91 to 1.79 log10 CFU/g lung tissue and from 7.00 and 0.92 log10 CFU/g brain tissue, representing the nearly complete or complete sterilization of lung and brain tissue at the higher doses. These data support the further clinical evaluation of this new class of antifungal agents for the treatment of CM.[1]

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The use of 50 mg/kg 1-aminobenzotriazole (ABT), a suicide inhibitor of cytochrome P450 enzymes, enhanced APX001A exposures (area under the time-concentration curve [AUC]) 16- to 18-fold and enhanced serum half-life from ∼1 to 9 h, more closely mimicking human pharmacokinetics. We evaluated the efficacy of APX001 (with ABT) in treating murine IPA compared to posaconazole treatment. Treatment of mice with 78 mg/kg once daily (QD), 78 mg/kg twice daily, or 104 mg/kg QD APX001 significantly enhanced the median survival time and prolonged day 21 postinfection overall survival compared to the placebo. Furthermore, administration of APX001 resulted in a significant reduction in lung fungal burden (4.2 to 7.6 log10 conidial equivalents/g of tissue) versus the untreated control and resolved the infection, as judged by histopathological examination. The observed survival and tissue clearance were comparable to a clinically relevant posaconazole dose. These results warrant the continued development of APX001 as a broad-spectrum, first-in-class treatment of invasive fungal infections.[2]

Antifungal susceptibility testing. [1]
To establish the antimicrobial activity of the APX001A analogs, broth microdilution susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A3 for yeasts and M38-A2 for molds. APX001A and analogs were first diluted in DMSO to obtain intermediate dilutions. These were further diluted in microtiter plates to obtain a final concentration of 2 to 0.002 μg/ml. One microliter of DMSO was added to the no-drug control wells. The solutions were mixed on a plate shaker for 10 min, and the plates were incubated at 35°C for 40 to 48 h (C. albicans, A. fumigatus) and 72 h (C. neoformans). The minimum concentration that led to a 50% reduction in fungal growth compared to that for the control (determined with the aid of a reading mirror) was determined as the MIC for C. albicans and C. neoformans. The minimum concentration that led to the shortening of hyphae compared to the hyphal growth in DMSO control wells was determined as the minimum effective concentration (MEC) for A. fumigatus (as read for the echinocandins). The use of the MIC and MEC endpoints for APX001A (formerly E1210) against yeasts and molds, respectively, has been described previously. For the cryptococcal synergy studies, APX001A and APX2020 MIC values were read at 50% inhibition.

To establish the antimicrobial activity of APX001A analogs, broth microdilution susceptibility testing was performed according to CLSI guideline M38-A2 for molds. APX001A were first diluted in dimethyl sulfoxide (DMSO) to obtain intermediate dilutions. These were further diluted in microtiter plates to obtain a final concentration of 0.002 to 2 μg/ml. The, 1 μl of DMSO was added to “no drug” control wells. The solutions were mixed on a plate shaker for 10 min, and plates were incubated at 35°C for 40 to 48 h. The minimum concentration that led to shortening of hyphae compared to hyphal growth in DMSO control wells was determined as the MEC for A. fumigatus (as read for echinocandins). Similar methods were used to determine the effect of ABT on the growth of A. fumigatus, with the exception that DMSO was not used because ABT is a water-soluble molecule. The range of ABT concentrations was 0.016 to 16 μg/ml in one study and 0.25 to 250 μg/ml in a follow-up study. The use of the MIC and MEC endpoints for APX001A (formerly E1210) against yeasts and molds, respectively, has been described previously. Standard checkerboard assays were utilized to evaluate synergy between ABT and APX001A on A. fumigatus MYA3626 (APX001A concentrations ranged from 0.0005 to 0.125 μg/ml; ABT concentrations ranged from 0.016 to 16 μg/ml). Inhibition endpoints for the synergy assay were read using the MEC value, as read for assessment of the activity of APX001A against molds. [2]

Animal/Disease Models: CD-1 mice [1]
Doses: 100 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: The half-life of the active part APX001A was extended from 1.3 hrs (hrs (hours)) to 8.8 hrs (hrs (hours)), increasing the area under the curve (AUC) 9 times.

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Pharmacokinetic analysis. [1]
Single-dose PK experiments were performed in healthy male CD-1 mice following i.p. or oral dosing of 26 mg/kg of the prodrugs APX001, APX2096, APX2097, and APX2104. In half of the cohorts, mice received a single oral dose of 100-mg/kg ABT at 2 h prior to prodrug dosing. Plasma was collected at 0.083, 0.5, 2, 4, 8, and 24 h postdose (n = 3 per time point). The area under the curve (AUC) was calculated from time zero to the time of the last measurable concentration. The active metabolite concentrations in plasma (APX001A, APX2039, APX2020, and APX2041) were determined by liquid chromatography-tandem mass spectrometry. PK parameters were determined using Phoenix WinNonlin (v7.0) software and a noncompartmental model. Samples with concentrations that were below the limit of quantification (0.5 or 1 ng/ml) were not used in the calculation of averages.

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IPA model. [2]
The IPA model was performed as previously described. Briefly, immunosuppressed mice were challenged with A. fumigatus in an inhalation chamber by aerosolizing 12 ml of a 1 × 109 ml suspension of conidia with a small particle nebulizer driven by compressed air. A standard exposure time of 1 h was used for all experiments. Immediately after infection, a subset of the mice was sacrificed, and the lungs were removed for quantitative culture. Mice were rendered neutropenic using a regimen of 200 mg/kg cyclophosphamide and 500 mg/kg cortisone acetate 2 days before and on day 3 relative to infection. To prevent bacterial infection, mice were given Baytril (50 μg/ml of enrofloxacin; Bayer) added to the drinking water from day –3 to day 0. Ceftazidime (5 μg/dose/0.2 ml) replaced Baytril treatment on day 0 and was administered daily by subcutaneous injection from day 0 until day 8. We administered 50 mg/kg ABT orally 2 h before the administration of APX001 for 7 days. Posaconazole (20 mg/kg QD or 30 mg/kg BID) was administered orally for 7 days. Survival was monitored through day 21. Mice were given free access to water and standard laboratory diet. All drug treatments were initiated 16 h postinfection and continued for 8 consecutive days given by oral gavage.

The relationship between AUC values and changes in log10 CFU/g in tissue was analyzed. The MIC values of the three compounds evaluated in the efficacy model against the infecting strain (Cryptococcus neoformans H99) differed by 8 to 32 times: APX001A was 0.25 μg/ml; APX2020 was 0.031 μg/ml; and APX2039 was 0.008 μg/ml (Table 1). Data in Table 3 show that the AUC values after intraperitoneal injection (with ABT) ranged from 24.3 to 97.3 μg·h/ml, a difference of 4 times. To understand the impact of the differences in AUC and MIC, we assessed the magnitude of changes in log10 CFU/g in tissue during the three experiments. [1]
The AUC values of APX001 (with or without ABT) in the three studies ranged from 7.0 μg·h/ml (7.5 mg/kg APX001 QD in combination with ABT) to 196.3 μg·h/ml (390 mg/kg TID). A slight but significant reduction in pulmonary bacterial load (1.5 log10 CFU/g) was observed at an AUC of 196.3 μg·h/ml. Lower AUC values were ineffective. The AUC values of APX2097 ranged from 10.0 to 116.4 μg·h/ml, and those of APX2096 ranged from 27 to 224.3 μg·h/ml. We compared the efficacy of the three compounds at doses with an AUC of approximately 80 µg·h/ml. In the presence of ABT, doses of 20 mg/kg APX2096, 60 mg/kg APX2097, and 80 mg/kg APX001 produced very similar AUC values of 74.8, 82.1, and 79.4 μg·h/ml, respectively. However, in brain tissue, colony-forming units (CFU) decreased by 2.95, 1.45, and 0.85 log10 CFU/g, respectively; and in lung tissue, CFU decreased by 3.69, 1.55, and 0.9 log10 CFU/g, respectively. Therefore, although the AUC values of these three compounds were the same, lower MIC values (0.008 μg/ml, 0.031 μg/ml, and 0.25 μg/ml, respectively) were associated with better efficacy, suggesting that increased microbial activity was the primary reason for the improved efficacy. [1]

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The pharmacokinetics of APX001A were compared after oral administration of 26 mg/kg of the prodrug APX001 (equivalent to 20 mg/kg of the active portion APX001A, with a conversion factor of 1.3 to account for the methyl phosphate group), and the pharmacokinetics of APX001A were compared with those of ABT administered 2 hours before APX001 administration and those of ABT not administered. The doses of ABT were 25, 50, and 100 mg/kg once daily (QD) and 50 mg/kg twice daily (BID). Consistent with our previous findings (17), in male CD-1 mice, when the dose of the prodrug APX001 was 26 mg/kg, administration of 100 mg/kg ABT once daily (QD) increased the mean AUClast (area under the plasma concentration-time curve, from time zero to the last measurable concentration) of APX001A by 15-fold (Table 1). Interestingly, the increase in AUClast was maintained when ABT was administered once daily (QD) or twice daily (BID) (16.3-fold and 15-fold, respectively, compared with the control group that did not receive ABT, P > 0.62 for all ABT comparison regimens) (Table 1), suggesting that lower doses of ABT have the same efficacy as 100 mg/kg doses of ABT in increasing APX001A AUClast. In contrast, once daily (QD) administration of 25 mg/kg doses of ABT resulted in significantly lower AUC values for APX001A than once daily (QD) administration of 50 mg/kg doses of ABT (P = 0.02), although the AUC value increased by 12.8-fold compared with the control group that did not receive ABT (P = 0.0002) (Table 1) [2]. Since higher doses of APX001 may be used in efficacy models, it is crucial to understand the linear relationship of AUC values when ABT is used. Therefore, the pharmacokinetics of APX001A were evaluated after administration of 52 mg/kg APX001 prodrug (equivalent to 40 mg/kg active ingredient APX001A) in the presence of different doses of ABT. The data in Table 1 show that administration of ABT at 50 mg/kg BID and 50 mg/kg QD resulted in similar AUC values for APX001A (92.41 ± 7.70 and 94.29 ± 12.43, respectively), representing a 17.4 to 17.8-fold increase in AUC compared to the control group without ABT (5.30 ± 0.98) (P < 0.0003). In contrast, the APX001A AUC value was lower in the once-daily (QD) 25 mg/kg ABT group (52.00 ± 35.46), representing a 9.8-fold increase in AUC compared to the control group without ABT (Table 1). [1]
The AUC obtained after administration of 52 mg/kg APX001 in combination with 50 mg/kg ABT (once or twice daily) was approximately twice as high as that obtained after administration of 26 mg/kg APX001 (P > 0.14), which is consistent with the dose-linear relationship, at least within this dose range. In subsequent Aspergillus fumigatus mouse model experiments, we chose to use the lowest effective dose of ABT (50 mg/kg once daily) in combination with oral APX001. [2]

[1]. In Vitro and In Vivo Evaluation of APX001A/APX001 and Other Gwt1 Inhibitors against Cryptococcus. Antimicrob Agents Chemother. 2018 Jul 27;62(8). pii: e00523-18.

[2]. APX001 Is Effective in the Treatment of Murine Invasive Pulmonary Aspergillosis. Antimicrob Agents Chemother. 2019 Jan 29;63(2). pii: e01713-18.

[3]. In Vitro and In Vivo Evaluation of APX001A/APX001 and Other Gwt1 Inhibitors against Cryptococcus. Antimicrob Agents Chemother. 2018 Jul 27;62(8):e00523-18.

Fosmanogepix is currently undergoing the clinical trial NCT03604705 (Efficacy and safety study of APX001 in patients with non-neutropenic candidemia). Fosmanogepix is an orally administered small-molecule Gwt1 fungal enzyme inhibitor with potential antifungal activity. Fosmanogepix is an N-phosphonooxymethyl prodrug that is rapidly and completely metabolized by systemic alkaline phosphatases to its active moiety, APX001A (E1210), after administration. This active prodrug targets Gwt1, a highly conserved inositol acylate that catalyzes a key step in the glycosylphosphatidylinositol (GPI) anchoring biosynthesis pathway. Inhibition of Gwt1 prevents the localization of mannoproteins in the cell wall, thereby disrupting cell wall integrity and inhibiting biofilm formation, germ tube formation, and fungal growth.

C22H21N4O6P

468.3991

468.119

C, 56.41; H, 4.52; N, 11.96; O, 20.49; P, 6.61

2091769-17-2

Manogepix;936339-60-5

44123754

White to yellow solid powder

1.6

2

9

9

33

644

0

P(=O)([O-])(O[H])OC([H])([H])[N+]1=C([H])C([H])=C([H])C(=C1N([H])[H])C1=C([H])C(C([H])([H])C2C([H])=C([H])C(C([H])([H])OC3=C([H])C([H])=C([H])C([H])=N3)=C([H])C=2[H])=NO1

JQONJQKKVAHONF-UHFFFAOYSA-N

InChI=1S/C22H21N4O6P/c23-22-19(4-3-11-26(22)15-31-33(27,28)29)20-13-18(25-32-20)12-16-6-8-17(9-7-16)14-30-21-5-1-2-10-24-21/h1-11,13,23H,12,14-15H2,(H2,27,28,29)

[2-amino-3-[3-[[4-(pyridin-2-yloxymethyl)phenyl]methyl]-1,2-oxazol-5-yl]pyridin-1-ium-1-yl]methyl hydrogen phosphate

Fosmanogepix; 2091769-17-2; APX001; Fosmanogepix [INN]; Fosmanogepix [USAN]; APX-001; 1XQ871489P; E1211;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~5 mg/mL (~10.67 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.34 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.34 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)