Plasma

Example 66

The activity of SYN-PKU-2002 was assessed in vivo. To prepare the cells for the study, SYN-PKU901 and SYN-PKU-2002 overnight cultures were each used to inoculate 4 2 L flasks containing 500 mL of LB with DAP100 ug/mL. These cultures were grown for 1 hr and 45 min and then moved to the anaerobic chamber supplying 90% N₂, 5% CO₂, and 5% H₂ for 4 hours. Cells were then spun down at 4600×G for 12 min and resuspended in 10 mL of formulation buffer (Glycerol: 15% (v/v), Sucrose: 10% (w/v) (100 g/L), MOPS: 10 mM (2.1 g/L), NaCl: 25 mM (1.46 g/L)). Several 40 ul aliquots were removed to be used for cell counting and activity determination. The viability as determined by cellometer count (in quadruplicate) 6.94e10 cfu/ml (+/−5.78e9).

Activity was determined using a plate based assay. Briefly, 1×10⁸ cfu as determined by cellometer were added to 1 ml of prewarmed assay buffer (1× M9 minimal media containing 0.5% glucose, 50 mM MOPS, and 50 mM phenylalanine) in a microfuge tube, vortexed briefly, and immediately placed in a heat block or water bath at 37 degrees Celsius for static incubation (t=0). Supernatant samples from cells re-suspended in assay buffer were analyzed for the abundance of TCA over several time points using spectrophotometer at an absorbance of 290 nm. The accurate OD290 window for TCA detection occurs in a relatively narrow concentration range. For this reason, supernatant samples were diluted to ensure that the absorbance measurement fell into the linear range for detection. Measurements were compared to a TCA standard curve. Activity was determined to be 2.72 umol/hr/le9 cfu (+/−0.15 umol/hr/le9 cfu).

Beginning 4 days prior to the study (i.e., Days −4-1), Pah ENU2/2 mice (˜11-15 weeks of age) were maintained on phenylalanine-free chow and water that was supplemented with 0.5 grams/L phenylalanine. On the day of the study, mice were randomized into treatment groups according to weight as follows: Group 1: SYN-PKU901 (n=9); Group 2: Group 2: SYN-PKU-2002 (n=9). Blood samples were collected by sub-mandibular skin puncture to determine baseline phenylalanine levels. Mice were then administered single dose of phenylalanine by subcutaneous injection at 0.1 mg per gram body weight, according to the average group weight. At 1, 2 and 3 h post Phe challenge, the bacteria (or water) were administered to mice by oral gavage (3×250 ul). Whole blood was collected via submandibular bleed at each time point. Urine collection in metabolic caging commenced immediately after the 1^st bacterial dose and continued to be collected for the duration of the study (4 hours).

Blood samples were kept on ice until processing for plasma in a centrifuge (2000 g for 10 min at 4 C) within 20 min of collection. Plasma was then transferred into a 96-well plate for MS analysis. Urine was collected in 5 mL tubes and volumes were recorded before transferring samples to MS for analysis. Results are shown in FIG. 17A and FIG. 17B and show that SYN-PKU-2002 causes decreased changes in phenylalanine post-Phe injection and produces hippurate, in a similar manner as SYN-PKU-710.

Example 8

Characterization of Absorption, Distribution, Metabolism, and Excretion of Oral [¹⁴C]Vorasidenib with Concomitant Intravenous Microdose Administration of [¹³C₃¹⁵N₃]Vorasidenib in Humans

Metabolite profiling and identification of vorasidenib (AG-881) was performed in plasma, urine, and fecal samples collected from five healthy subjects after a single 50-mg (100 μCi) oral dose of [14C]AG-881 and concomitant intravenous microdose of [¹³C3 ¹⁵N3]AG-881.

Plasma samples collected at selected time points from 0 through 336 hour postdose were pooled across subjects to generate 0—to 72 and 96-336-hour area under the concentration-time curve (AUC)-representative samples. Urine and feces samples were pooled by subject to generate individual urine and fecal pools. Plasma, urine, and feces samples were extracted, as appropriate, the extracts were profiled using high performance liquid chromatography (HPLC), and metabolites were identified by liquid chromatography-mass spectrometry (LC-MS and/or LC-MS/MS) analysis and by comparison of retention time with reference standards, when available.

Due to low radioactivity in samples, plasma metabolite profiling was performed by using accelerator mass spectrometry (AMS). In plasma, AG-881 was accounted for 66.24 and 29.47% of the total radioactivity in the pooled AUC_{0-72 h} and AUC_{96-336 h} plasma, respectively. The most abundant radioactive peak (P7; M458) represented 0.10 and 43.92% of total radioactivity for pooled AUC_0-72 and AUC_{96-336 h} plasma, respectively. All other radioactive peaks accounted for less than 6% of the total plasma radioactivity and were not identified.

The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the dose), while no AG-881 was detected in urine. In comparison, metabolites in excreta accounted for approximately 18% of dose in feces and for approximately 4% of dose in urine. M515, M460-1, M499, M516/M460-2, and M472/M476 were the most abundant metabolites in feces, and each accounted for approximately 2 to 5% of the radioactive dose, while M266 was the most abundant metabolite identified in urine and accounted for a mean of 2.54% of the dose. The remaining radioactive components in urine and feces each accounted for <1% of the dose.

Overall, the data presented indicate [¹⁴C]AG-881 underwent moderate metabolism after a single oral dose of 50-mg (100 μCi) and was eliminated in humans via a combination of metabolism and excretion of unchanged parent. AG-881 metabolism involved the oxidation and conjugation with glutathione (GSH) by displacement of the chlorine at the chloropyridine moiety. Subsequent biotransformation of GSH intermediates resulted in elimination of both glutamic acid and glycine to form the cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further converted by a series of biotransformation reactions such as oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation and N-dealkylation resulting in the formation multiple metabolites.

A summary of the metabolites observed is included in Table 2

Example 1

95 g of manganese (purity: 99.95%; purchased from Taewon Scientific Co., Ltd.) and 5 g of high-purity graphite (purity: 99.5%; purchased from Taewon Scientific Co., Ltd.) were placed in a water-cooled copper crucible of an argon plasma arc melting apparatus (manufactured by Labold AG, Germany, Model: vacuum arc melting furnace Model LK6/45), and melted at 2,000 K under an argon atmosphere. The melt was cooled to room temperature at a cooling rate of 10⁴ K/min to obtain an alloy ingot. The alloy ingot was crushed to a particle size of 1 mm or less by hand grinding. Thereafter, the obtained powders were magnetically separated using a Nd-based magnet to remove impurities repeatedly, and the Mn₄C magnetic powders were collected. The collected Mn₄C magnetic powders were subjected to X-ray diffraction (XRD) analysis (measurement system: D/MAX-2500 V/PO, Rigaku; measurement condition: Cu—K_α ray) and energy-dispersive X-ray spectroscopy (EDS) using FE-SEM (Field Emission Scanning Electron Microscope, MIRA3 LM).

FIGS. 2(a) and 2 (b) show an X-ray diffraction pattern and an energy-dispersive X-ray spectroscopy graph of the Mn₄C magnetic material produced according to Example 1 of the present disclosure, respectively.

As can be seen in FIG. 2(a), the Mn₄C magnetic material showed diffraction peaks of (111), (200), (220), (311) and (222) crystal planes at 2θ values of 40°, 48°, 69°, 82° and 88°, respectively, in the XRD analysis. Thus, it can be seen that the XRD patterns of the Mn₄C magnetic material produced according to Example 1 are well consistent with the patterns of the cubic perovskite Mn₄C. In addition, the Mn₄C magnetic material shows several very weak diffraction peaks that can correspond to Mn₂₃C₆ and Mn. That is, the diffraction peak intensity at 2θ values of 43° and 44°, which correspond to Mn and Mn₂₃C₆ impurities, is as very low as about 2.5% of the diffraction intensity of the peak corresponding to the (111) plane. Through this, it can be seen that the powders obtained in Example 1 have high-purity Mn₄C phase. The lattice parameter of the Mn₄C is estimated to be about 3.8682 Å.

FIG. 2(b) shows the results of analyzing the atomic ratio of Mn:C in the powder by EDS. The atomic ratio of Mn:C is 80.62:19.38, which is very close to 4:1 within the experimental uncertainties. Thus, it can be seen that the powder is also confirmed to be Mn₄C.

The M-T curve of the field aligned Mn₄C powder obtained in Example 1 was measured under an applied field of 4 T and at a temperature ranging from 50 K to 400 K. Meanwhile, the M-T curve of the randomly oriented Mn₄C powder was measured under an applied field of 1 T. The Curie temperature of Mn₄C was measured under 10 mT while decreasing temperature from 930 K at a rate of 20 K/min.

FIGS. 3(a) to 3(c) show the M-T curves of the Mn₄C magnetic material, produced according to Example 1 of the present disclosure, under magnetic fields of 4 T, 1 T, and 10 mT, respectively.

FIG. 3 shows magnetization-temperature (M-T) curves indicating the results of measuring the temperature-dependent magnetization intensity of the Mn₄C magnetic material, produced in Example 1, using the vibrating sample magnetometer (VSM) mode of Physical Property Measurement System (PPMS®) (Quantum Design Inc.).

According to the Néel theory, the ferrimagnets that contain nonequivalent substructures of magnetic ions may have a number of unusual forms of M-T curves below the Curie temperature, depending on the distribution of magnetic ions between the substructures and on the relative value of the molecular field coefficients. The anomalous M-T curves of Mn₄C, as shown in FIG. 3(a), can be explained to some extent by the Néel's P-type ferrimagnetism, which appears when the sublattice with smaller moment is thermally disturbed more easily. For Mn₄C with two sublattices of Mn_I and Mn_II, as shown in FIG. 1, the Mn_I sublattice might have smaller moment.

FIG. 3(a) shows the temperature dependence of magnetization of the Mn₄C magnetic material produced in Example 1. The magnetization of Mn₄C measured at 4.2K is 6.22 Am²/kg (4 T), corresponding to 0.258μ_B per unit cell. The magnetization of the Mn₄C magnetic material varies little at temperatures below 50 K, and is quite different from that of most magnetic materials, which undergo a magnetization deterioration with increasing temperature due to thermal agitation. Furthermore, the magnetization of the Mn₄C magnetic material increases linearly with increasing temperature at temperatures above 50 K. The linear fitting of the magnetization of Mn₄C at 4 T within the temperature range of 100 K to 400 K can be written as M=0.0072T+5.6788, where M and T are expressed in Am²/kg and K, respectively. Thus, the temperature coefficient of magnetization of Mn₄C is estimated to be about ˜2.99*10⁻⁴μ_B/K per unit cell. The mechanisms of the anomalous thermomagnetic behaviors of Mn₄C may be related to the magnetization competition of the two ferromagnetic sublattices (Mn_I and Mn_II) as shown in FIG. 1.

FIG. 3(b) shows the M-T curves of the Mn₄C powders at temperatures within the range of 300 K to 930 K under 1 T. The linear magnetization increment stops at 590 K, above which the magnetization of Mn₄C starts to decrease slowly first and then sharply at a temperature of about 860 K. The slow magnetization decrement at temperatures above 590 K is ascribed to the decomposition of Mn₄C, which is proved by further heat-treatment of Mn₄C as described below.

According to one embodiment of the present disclosure, the saturation magnetization of Mn₄C increases linearly with increasing temperature within the range of 50 K to 590 K and remains stable at temperatures below 50 K. The increases in anomalous magnetization of Mn₄C with increasing temperature can be considered in terms of the Néel's P-type ferrimagnetism. At temperatures above 590 K, the Mn₄C decomposes into Mn₂₃C₆ and Mn, which are partially oxidized into the manganosite when exposed to air. The remanent magnetization of Mn₄C varies little with temperature. The Curie temperature of Mn₄C is about 870 K. The positive temperature coefficient (about 0.0072 Am²/kgK) of magnetization in Mn₄C is potentially important in controlling the thermodynamics of magnetization in magnetic materials.

The Curie temperature T_e of Mn₄C is measured to be about 870 K, as shown in FIG. 3(c). Therefore, the sharp magnetization decrement of Mn₄C at temperatures above 860 K is ascribed to both the decomposition of Mn₄C and the temperature near the Tc of Mn₄C.

FIG. 4 is a graph showing the magnetic hysteresis loops of the Mn₄C magnetic material, produced according to Example 1 of the present disclosure, at 4.2 K, 200 K and 400 K. The magnetic hysteresis loops were measured by using the PPMS system (Quantum Design) under a magnetic field of 7 T while the temperature was changed from 4 K to 400 K.

As shown in FIG. 4, the positive temperature coefficient of magnetization was further proved by the magnetic hysteresis loops of Mn₄C as shown in FIG. 4. The Mn₄C shows a much higher magnetization at 400 K than that at 4.2 K. Moreover, the remanent magnetization of Mn₄C varies little with temperature and is Δ3.5 Am²/kg within the temperature range of 4.2 K to 400 K. The constant remanent magnetization of Mn₄C within a wide temperature range indicates the high stability of magnetization against thermal agitation. The coercivities of Mn₄C at 4.2 K, 200 K, and 400 K were 75 mT, 43 mT, and 33 mT, respectively.

The magnetic properties of Mn₄C measured are different from the previous theoretical results. A corner Mn_I moment of 3.85μ_B antiparallel to three face-centered Mn_II moments of 1.23μ_B in Mn₄C was expected at 77 K. The net moment per unit cell was estimated to be 0.16μ_B. In the above experiment, the net moment in pure Mn₄C at 77 K is 0.26μ_B/unit cell, which is much larger than that expected by Takei et al. It was reported that the total magnetic moment of Mn₄C was calculated to be about 1μ_B, which is almost four times larger than the 0.258μ_B per unit cell measured at 4.2 K, as shown in FIG. 4.

FIG. 5 is an enlarged view of the temperature-dependent XRD patterns of the Mn₄C magnetic material produced according to Example 1 of the present disclosure.

The thermomagnetic behaviors of Mn₄C are related to the variation in the lattice parameters of Mn₄C with temperature. It is known that the distance of near-neighbor manganese atoms plays an important role in the antiferro- or ferro-magnetic configurations of Mn atoms. Ferromagnetic coupling of Mn atoms is possible only when the Mn—Mn distance is large enough. FIG. 5 shows the diffraction peaks of the (111) and (200) planes of Mn₄C at temperatures from 16 K to 300 K. With increasing temperature, both (111) and (200) peaks of Mn₄C shifted to a lower degree at temperatures between 50 K and 300 K, indicating an enlarged distance of Mn—Mn atoms in Mn₄C. No peak shift is obviously observed for Mn₄C at temperatures below 50 K. The distance of nearest-neighbor manganese atoms plays an important role in the antiferro- or ferro-magnetic configurations of Mn atoms and thus has a large effect on the magnetic properties of the compounds.

Thus, it can be seen that the abnormal increase in magnetization of Mn₄C with increasing temperature occurs due to the variation in the lattice parameters of Mn₄C with temperature.

The powder produced in Example 1 was annealed in vacuum for 1 hour at each of 700 K and 923 K, and then subjected to X-ray spectroscopy, and the results thereof are shown in FIG. 6.

The magnetization reduction of Mn₄C at temperatures above 590 K is ascribed to the decomposition of Mn₄C, which is proved by the XRD patterns of the powders after annealing Mn₄C at elevated temperatures. FIG. 6 shows the structural evolution of Mn₄C at elevated temperatures. When Mn₄C is annealed at 700 K, a small fraction of Mn₄C decomposes into a small amount of Mn₂₃C₆ and Mn. The presence of manganosite is ascribed to the spontaneous oxidation of the Mn precipitated from Mn₄C when exposed to air after annealing. The fraction of Mn₂₃C₆ was enhanced significantly for Mn₄C annealed at 923 K, as shown in FIG. 6.

These results prove that the metastable Mn₄C decomposes into stable Mn₂₃C₆ at temperatures above 590 K. The presence of Mn₄C in the powder annealed at 923 K indicates a limited decomposition rate of Mn₄C, from which the Tc of Mn₄C can be measured. Both Mn₂₃C₆ and Mn are weak paramagnets at ambient temperature and elevated temperatures. Therefore, the magnetic transition of the Mn₄C magnetic material at 870 K is ascribed to the Curie point of the ferrimagnetic Mn₄C.

The Mn₄C shows a constant magnetization of 0.258μ_B per unit cell below 50 K and a linear increment of magnetization with increasing temperature within the range of 50 K to 590 K, above which Mn₂₃C₆ precipitates from Mn₄C. The anomalous M-T curves of Mn₄C can be considered in terms of the Néel's P-type ferrimagnetism.