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.

Analysis of AUC values versus the change in the number of log10 CFU per gram of tissue. The three compounds evaluated in the efficacy model had MIC values for the infecting strain (C. neoformans H99) that differed by 8- to 32-fold: for APX001A, 0.25 μg/ml; for APX2020, 0.031 μg/ml; and for APX2039, 0.008 μg/ml (Table 1). The data in Table 3 show that AUC values after i.p. dosing (plus ABT) ranged from 24.3 to 97.3 μg · h/ml, representing a 4-fold difference. To understand the influence of AUC-versus-MIC differences, we assessed the magnitude of changes in the number of log10 CFU/g of tissue across the three experiments.[1]
AUC values across the three experiments for APX001 (with or without ABT) ranged from 7.0 μg · h/ml (7.5-mg/kg APX001 QD plus ABT) to 196.3 μg · h/ml (390 mg/kg TID). At an AUC of 196.3 μg · h/ml, a modest but significant reduction in the lung burden was observed (1.5 log10 CFU/g). Lower AUC values were not efficacious. AUC values ranged from 10.0 to 116.4 μg · h/ml for APX2097 and from 27 to 224.3 μg · h/ml for APX2096. We compared the efficacy of the three compounds at a dose that gave rise to AUC values 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 resulted in very similar AUC values of 74.8, 82.1, and 79.4 μg · h/ml, respectively. However, the reductions were 2.95, 1.45, and 0.85 log10 CFU/g, respectively, in brain and 3.69, 1.55, and 0.9 log10 CFU/g, respectively, in lung. Thus, despite the same AUC values for the 3 compounds, better efficacy was associated with lower MIC values (0.008 μg/ml, 0.031 μg/ml, and 0.25 μg/ml, respectively), suggesting that improved microbiological activity largely accounts for improved efficacy.[1]

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The PK of APX001A after oral administration of 26 mg/kg of the prodrug APX001 (equivalent to 20 mg/kg of the active moiety APX001A using a conversion factor of 1.3 to account for the methyl phosphate group) were compared with and without the administration of ABT given 2 h prior to APX001 dosing. ABT doses were tested at 25, 50, and 100 mg/kg once daily (QD) and at 50 mg/kg twice daily (BID). Consistent with our previous findings (17), administration of ABT at 100 mg/kg QD resulted in a 15-fold increase in the average APX001A AUClast (area under the plasma concentration-time curve from time zero to time of last measurable concentration) in male CD-1 mice when the prodrug APX001 was dosed at 26 mg/kg (Table 1). Interestingly, this increase in AUClast was maintained when ABT was dosed at 50 mg/kg QD or BID (16.3- or 15-fold versus the no-ABT control, P > 0.62 for all ABT comparison regimens) (Table 1), suggesting that this lower dose of ABT is as efficient as the 100-mg/kg ABT dose in enhancing APX001A AUClast. In contrast, the 25-mg/kg QD dose of ABT resulted in a lower APX001A AUC value that was statistically significant from the 50-mg/kg QD dose (P = 0.02), although a 12.8-fold increase in the AUC value versus the no-ABT control was observed (P = 0.0002) (Table 1).[2]

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Since higher APX001 doses could potentially be utilized in efficacy models, it was important to understand the linearity of AUC values while utilizing ABT. Thus, the PK of APX001A after the administration of 52 mg/kg APX001 prodrug (equivalent to 40 mg/kg of the active moiety APX001A) was evaluated in the presence of different doses of ABT. The data in Table 1 show that the administration of ABT at 50 mg/kg BID and 50 mg/kg QD resulted in similar APX001A AUC values (92.41 ± 7.70 and 94.29 ± 12.43, respectively), which translated into a 17.4- to 17.8-fold increase in AUC versus the no-ABT control (5.30 ± 0.98) (P < 0.0003). In contrast, the 25-mg/kg QD ABT dose resulted in a lower APX001A AUC value (52.00 ± 35.46), representing a 9.8-fold increase versus the no-ABT control (Table 1).[1]
The AUC values obtained after dosing 52 mg/kg APX001 plus 50 mg/kg ABT (QD or BID) were ∼2-fold higher than the parallel values obtained when 26 mg/kg APX001 was dosed (P > 0.14), consistent with dose linearity, at least within that dosing range. We chose to use the lowest, optimal dose of ABT at a 50-mg/kg QD dose in conjunction with the oral administration of APX001 in the subsequent A. fumigatus mouse model experiments.[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 under investigation in clinical trial NCT03604705 (An Efficacy and Safety Study of APX001 in Non-Neutropenic Patients With Candidemia).
Fosmanogepix is an orally available small molecule inhibitor of the Gwt1 fungal enzyme with potential antifungal activity. Upon administration, fosmanogepix, a N-phosphonooxymethyl prodrug, is rapidly and completely metabolized by systemic alkaline phosphatases to its active moiety, APX001A (E1210). The active prodrug targets Gwt1, a highly conserved inositol acylase which catalyzes an essential step in the glycosylphosphatidylinositol (GPI)-anchor biosynthesis pathway. Inhibition of Gwt1 prevents localization of cell wall mannoproteins, which compromises cell wall integrity, 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.)