Patent Application
20240101564
The present invention relates to salts of a PI3Kdelta inhibitor (referred to as “Compound A” hereinafter), preferably fumarate, and the crystalline forms thereof. The present invention also relates to the process of preparation and uses of the salts and crystalline forms of Compound A.
Phosphatidylinositol-4,5-bisphosphate 3-kinase δ (PI3Kδ) is frequently active in B-cell malignancies and is central to multiple signaling pathways that drive proliferation, survival, homing, and retention of malignant B-cells in lymphoid tissue and bone marrow. In B-cell malignancies, PI3K pathway activity is significantly elevated, which is driven by altered B-cell receptor (BCR) signaling together with other co-stimulatory signals present in lymphoid tissues such as chemokines and cytokines (Puri and Gold 2012, Okkenhaug and Vanhaesebroeck 2003). PI3Kδ functions to integrate and transduce these signals from the microenvironment, thus promoting malignant B-cell proliferation, growth, survival, adhesion, and homing, making it an attractive drug target for B-cell malignancies (Yang et al 2015).
PI3Kδ is also important for the homeostasis and function of T-regulatory (Treg) cells (Lim and Okkenhaug 2019). The inactivation of PI3Kδ in mice can stimulate immune responses against solid tumors via the inhibition of Treg cells (Ali et al 2014). With PI3Kδ expression at low or undetectable levels in most organs, inhibitors against PI3Kδ should be selective for the immune system and less toxic (Okkenhaug and Fruman 2010).
Because of the specific and critical functions of PI3Kδ in adaptive immune responses, inhibitors of PI3Kδ are being developed for the treatment of autoimmune and inflammatory disorders, hematologic and solid tumors, and activated PI3Kδ syndrome (Lucas et al 2016; Okkenhaug and Burger 2016). PI3Kδ inhibitors are also being developed for the treatment of solid tumors because PI3Kδ is essential for the homeostasis and function of Foxp3+ Treg cells (Patton et al 2006). Loss of PI3Kδ activity, especially by specific deletion in Treg cells, can restrict the growth of transplanted tumors in mice (Ali et al 2014), providing a rationale for the evaluation of PI3Kδ inhibitors in solid tumors.
WO2019/047915A1 disclose a series of PI3Kδ inhibitors, in particular (S)-3-(1-(8-amino-1-methylimidazo[1,5-a]pyrazin-3-yl)ethyl)-5-chloro-6-fluoro-2-isopropoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide
Compound A is a potent and selective inhibitor of PI3Kδ in biochemical and cellular assays, it inhibits cellular growth of several cancer cell lines in vitro and induces dose-dependent antitumor effects against tumor xenografts engrated either subcutaneously or systemically in mice.
Compound A was confirmed to be amorphous (as shown in
In order to be manufactured into pharmaceutical products, it is strictly required that the active ingredient must have high purity and stability. Particularly, in order to maintain high stability in a longer shelf period, the active ingredient must have low hygroscopicity so that the influence on the quality by moisture can be avoided. Thus, the free base of Compound A needs to be converted into other forms such as salt to pursue improved properties.
For orally administered solid formulations comprising the desired active ingredient, the active ingredient needs to have the desired bioavailability so that the active ingredient could be absorbed into the blood circulation of the body as much as possible. However, the relationship between the bioavailability and the specific salt is unknown in the art, and a new salt of Compound A with higher bioavailability is highly desired.
Therefore, it remains the need for the discovery of new solid forms of Compound A or the salts thereof to meet the above pharmaceutical formulation requirements.
The present application discloses an invention to address the foregoing challenges and needs by providing stable salts of Compound A, and especially fumarate of Compound A, which shows the desired crystallinity and improved bioavailability suitable for pharmaceutical formulation.
In addition, the inventors have found that among different salts of Compound A, fumarate salt of Compound A shows unpredictable high bioavailability, which makes the fumarate salt of Compound A suitable for pharmaceutical formulation.
Surprisingly, salts of Compound A, preferably fumarate salt of Compound A, even more preferably the crystalline of fumarate is a solid with very low viscosity. The salts of Compound A, preferably fumarate salt of Compound A, even more preferably the crystalline of fumarate can be used in the large-scale production of formulation process without the viscous problem.
Even more surprisingly, the fumarate salt type D showed an excellent long-term stability during the 3-month experiment. From the current data, we also could expect that fumarate salt type D should have a very good long-term stability, such as 6-month long-term stability, 12-month long-term stability, 24-month long-term stability and 36-month long-term stability.
Before the filing date of the instant application, the inventors of the instant application have unexpectedly found that only fumaric acid can form crystalline forms with the desired crystallinity, high stability, low hygroscopicity and low viscosity. with Compound A.
Although a freebase may theoretically form pharmaceutically acceptable salts with many acids, Compound A as a specific freebase disclosed herein has been found cannot form a salt with many acids or cannot form a crystalline salt with the desired crystallinity. Among the many conventional acids or salt-forming agents including hydrochloric acid, sulfuric acid, phosphoric acid, L-tartaric acid, L-aspartic acid, maleic acid, fumaric acid, succinic acid, adipic acid, L-malic acid, citric acid, hippuric acid, L-ascorbic acid, acetic acid, glycolic acid, lauric acid, stearic acid, glutamic acid, D-gluconic acid, DL-lactic acid, benzenesulfonic acid, methanesulfonic acid, gentistic acid, oxalic acid, nicotinic acid. Among the acids (salt-forming agents), the inventors of the instant invention have found that fumaric acid is the only one that could form a crystalline with sharp peaks and a smooth baseline in the XRPD pattern. Inventors suprisingly found that fumarate of Compound A has a good crystallinity, safety and production compatibility.
In one aspect, provided herein is the crystalline form of Compound A fumarate Type A. As shown in
More specifically, the XRPD pattern of Compound A fumarate Type A has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern thereof typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern of Compound A fumarate Type E typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern thereof typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern thereof typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern of Compound A fumarate Type H typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern thereof typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern of Compound A fumarate Type J typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern of Compound A fumarate Type K typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern of Compound A fumarate Type L typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
More specifically, the XRPD pattern of Compound A fumarate Type M typically has the following peak diffraction angles (where “spacing” is shown as the “d-value” in
In one aspect, provided herein is the crystalline form of Compound A fumarate Type F. As shown in
The crystalline forms described above are rather stable crystalline forms.
For crystalline forms described above, only the main peaks (i.e., the most characteristic, significant, unique and/or reproducible peaks) are summarized; additional peaks may be obtained from the diffraction spectra by conventional methods. The main peaks described above can be reproduced within the margin of error (+ or −2 at the last given decimal place, or + or −0.2 at the stated value).
The method for preparing the free base of Compound A is disclosed in WO2019/047915A1. For the above-mentioned crystalline forms, the crystallization step can be conducted in an appropriate solvent system containing at least one solvent by evaporation of solvent, cooling and/or by addition of anti-solvents (solvents that are less able to solubilize the Compound A or its salts, including but not limited to those described herein) to achieve super-saturation in the solvent system.
Crystallization may be done with or without seed crystals, which is described in the present invention.
In an embodiment in this aspect, provided herewith is the fumarate of Compound A, preferably in the above-mentioned crystalline forms, more preferably in the crystalline forms of Types B, C, D and F, even more preferably in the crystalline forms of Types D and F, most preferably in the crystalline form of Type D.
The individual crystalline forms provided by the present invention develop under specific conditions dependent on the particular thermodynamic and equilibrium properties of the crystallization process. Therefore, a person skilled in the art will know that the crystals formed are a consequence of the kinetic and thermodynamic properties of the crystallization process. Under certain conditions (such as in a specific solvent), a particular crystalline form may have better properties than another crystalline form (or in fact have better properties than any other crystalline forms).
In another aspect, provided herein is a pharmaceutical composition each containing an effective amount of fumarate of Compound A, preferably in any of the above-described crystalline forms. The active compound can be 1-99% (by weight), preferably 1-70% (by weight), or more preferably 1-50% (by weight), or most preferably, 5-40% (by weight), of the composition.
In another aspect, provided herein is the use of the above-described salt or crystalline forms of Compound A in the manufacture of medicaments for the treatment of a cancer associated with PI3K delta inhibition.
In another aspect, provided herein is a pharmaceutical composition each containing an effective amount of fumarate salt of Compound A, preferably in any of the above-described crystalline forms, more preferably fumarate salt type D. The active compound can be 1-99% (by weight), preferably 1-70% (by weight), or more preferably 1-50% (by weight), or most preferably, 5-40% (by weight), of the composition.
The term “about” as used herein, unless indicated otherwise, denotes that a number (e.g., temperature, pH, volume, etc.) can vary within ±10%, preferably within ±5%.
A solvate herein is defined as a compound formed by solvation, for example as a combination of solvent molecules with molecules or ions of a solute. The known solvent molecules include water, alcohols and other polar organic solvents. Alcohols include methanol, ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol and t-butanol. The preferred solvent is typically water. The solvate compounds formed by solvation with water are sometimes termed as hydrates.
In some embodiments, the crystalline form has a crystalline purity at least about 80%, preferably at least about 90%, preferably at least about 95% crystalline purity, preferably about 97% crystalline purity, more preferably about 99% or more crystalline purity, and most preferably about 100% crystalline purity.
The term “crystalline purity,” as used herein, means the percentage of a particular crystalline form of a compound in a sample, which may contain the amorphous form of the compound, one or more other crystalline forms of the compound (other than the particular crystalline form of the compound), or a mixture thereof. Crystalline purity is determined by X-ray powder diffraction (XRPD), Infrared Raman spectroscopy and other solid state methods.
The following synthetic methods, specific examples, and efficacy tests further describe certain aspects of the present invention. They shall not limit or restrict the scope of the present invention in any way.
To a solution of (S)-3-(1-(8-amino-1-methylimidazo[1,5-a]pyrazin-3-yl)ethyl)-5-chloro-6-fluoro-2-isopropoxybenzoic acid (20 g, 49.2 mmol) in dichloromethane (100 mL) was added SOCl2 (29 g, 244 mmol) dropwise. The mixture was stirred at room temperature overnight. The mixture was concentrated under vacuum. The residue was dissolved in dichloromethane (200 mL). To the solution was added N-ethyl-N-isopropylpropan-2-amine (19 g, 147 mmol) at 0° C., and then a solution of 2-(4-methylpiperazin-1-yl)ethan-1-amine HCl salt (10.5 g, 70.3 mmol) in DCM (20 mL) was added dropwise. The mixture was stirred at 0° C. for 2 hours. The mixture was diluted with water (200 mL), extracted with dichloromethane (3×200 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography (eluent with dichloromethane:MeOH:ammonia water=100:10:0.5) to give the title compound (7.2 g, 27%). LC-MS (M+H)=531.9.
1H NMR (400 MHz, dmso) δ 8.63 (t, J=5.7 Hz, 1H), 7.38 (d, J=8.6 Hz, 1H), 7.25 (d, J=5.0 Hz, 1H), 6.85 (d, J=5.0 Hz, 1H), 6.43 (brs, 2H), 4.77 (q, J=6.9 Hz, 1H), 4.52-4.45 (m, 1H), 3.36-3.29 (m, 2H), 2.56 (s, 3H), 2.46-2.26 (m, 10H), 2.16 (s, 3H), 1.58 (d, J=7.1 Hz, 3H), 1.19 (d, J=6.0 Hz, 3H), 1.09 (d, J=6.0 Hz, 3H).
Salt formations were performed using each of 25 acids (HCl, H2SO4, H3PO4, L-tartaric acid, L-aspartic acid, Maleic acid, Fumaric acid, Succinic acid, Adipic acid, L-malic acid, Citric acid, Hippuric acid, L-ascorbic acid, Acetic acid, Glycolic acid, Laurie acid, Stearic acid, Glutamic acid, D-gluconic acid, DL-Lactic acid, Benzenesulfonic acid, Methanesulfonic acid, Gentistic acid, Oxalic acid, Nicotinic acid) as well as blank as the control in four solvent systems (Solvent: A was IPA/n-heptane (1:4, v/v); B was acetone/n-heptane (1:4, v/v); C was IPAc/n-heptane (4:1, v/v); D was 1,4-dioxane) via solvent-assisted reaction crystallization. In detail, about 15 mg of amorphous freebase (Compound A) and corresponding acid were mixed into each HPLC vial with the desired molar ratio of 1:1. 0.3 mL of the corresponding solvent was then added to form a suspension, which was magnetically stirred (˜800 rpm) at RT for about three days. Solids were isolated for XRPD analysis. The results are summarized in Table 1.
As summarized in Table 1, a total of seven potential crystalline salts (sulfate Type A, fumarate Type A, laurate Type A, stearate Type A, gentisate Type A, nicotinate Type A and nicotinate Type B) and two freebases (freebase Type A and B) were observed based on the XRPD comparison, wherein the two freebases (freebase Type A and B) were obtained as either in an amorphous form or in a gel. Another two crystalline salts (fumarate Type B and fumarate Type C) were obtained in the re-preparation process. The other experiments gave either amorphous salts or acids (indicating that no salt has been formed).
15.01 mg the free base of Compound A and 3.28 mg of fumaric acid were mixed into a vial. 0.3 mL acetone/n-heptane (1:4. V/V) was added to form a suspension. The suspension is stirred at room temperature at 800 rpm for 2 days and transferred to slurry at 5° C. at 800 rpm for another 2 days. The fumarate product was isolated by centrifugation and vacuum dried at room temperature for 3 days to obtain fumarate of Compound A.
Two batches of fumarate Type α were obtained via slurry of equimolar amorphous freebase and fumaric acid in acetone/n-heptane (1:4, v/v) at RT and then vacuum drying at RT. XRPD patterns were displayed in
To a solution of (S)-3-(1-(8-amino-1-methylimidazo[1,5-a]pyrazin-3-yl)ethyl)-5-chloro-6-fluoro-2-isopropoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide (1.0 g, freebase of Compound A) in EtOH (2 mL) was added a solution of fumaric acid (220 mg) in EtOH (4 mL). The mixture was stirred for 10 minutes. Then to the mixture was added n-butanol (6 mL). The resulting mixture was stirred at room temperature for 72 hours, then the product was obtained. 1H NMR spectra were was collected on Bruker 400M NMR Spectrometer using DMSO-d6. 1H NMR spectrum showed the molar ratio of acid/free base was 1.5:1 (
To a solution of (S)-3-(1-(8-amino-1-methylimidazo[1,5-a]pyrazin-3-yl)ethyl)-5-chloro-6-fluoro-2-isopropoxy-N-(2-(4-methylpiperazin-1-yl)ethyl)benzamide (5.0 g, the free base of Compound A) in EtOH (30 mL) was added a solution of fumaric acid (970 mg) in EtOH (50 mL). The mixture was stirred for 30 minutes. Then to the mixture was concentrated until about 24 g residue in the bottom. The resulting mixture was stirred at room temperature overnight, then the product was obtained. 1H NMR spectrum showed the molar ratio of acid/free base was 1:1 (
300 mg the free base of Compound A and 93 mg of D-tartaric acid was mixed into a vial with EtOH (10 mL), which was magnetically stirred at room temperature for about 30 min, then the product was obtained. 1H NMR (400 MHz, DMSO) δ 8.65 (t, J=5.3 Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.26 (d, J=5.0 Hz, 1H), 6.86 (d, J=4.9 Hz, 1H), 6.50 (brs, 2H), 4.77 (q, J=6.7 Hz, 1H), 4.51-4.41 (m, 1H), 4.18 (s, 3H), 3.70-2.90 (m, 11H), 2.75-2.55 (m, 7H), 2.47-2.40 (m, 6H), 1.58 (d, J=7.0 Hz, 3H), 1.19 (d, J=5.9 Hz, 3H), 1.09 (d, J=5.9 Hz, 3H). 1H NMR spectrum showed the molar ratio of acid/freebase was 1.5:1 (
Sulfate of Compound A was obtained via slurry of the equimolar free base of Compound A and sulfuric acid in isopropyl alcohol/n-heptane (1:4, v/v) at room temperature and then vacuum drying at room temperature. 1H NMR was shown in
Laurate of Compound A was obtained via slurry of the equimolar free base of Compound A and lauric acid in isopropyl acetate/n-heptane (4:1, v/v) at room temperature and then vacuum drying at room temperature. 1H NMR in
Stearate of Compound A was obtained via slurry of the equimolar free base of Compound A and stearic acid in isopropyl alcohol/n-heptane (1:4, v/v) at room temperature and then vacuum drying at room temperature. 1H NMR in
Gentisate of Compound A was obtained via slurry of the equimolar free base of Compound A and gentisic acid in 1,4-dioxane at room temperature and then vacuum drying at room temperature. 1H NMR in
Nicotinate of Compound A was obtained via slurry of the equimolar free base of Compound A and nicotinic acid in isopropyl alcohol/n-heptane (1:4, v/v) at room temperature and then vacuum drying at room temperature. 1H NMR in
Nicotinate of Compound A was obtained via slurry of the equimolar free base of Compound A and nicotinic acid in acetone/n-heptane (1:4, v/v) at room temperature and then vacuum drying at room temperature. 1H NMR in
Freebase type A was obtained via slurry of amorphous freebase (Compound A) in 1,4-dioxane at RT. XRPD pattern was shown in
Freebase type B was obtained via slurry of equimolar amorphous freebase (Compound A) and hippuric acid in 1,4-dioxane at RT XRPD pattern was shown in
Upon our findings that fumarate is the only salt that could potentially form a crystalline, the development of further crystalline is performed using different crystallization or solid transition methods, including anti-solvent addition, liquid vapor diffusion, solid vapor diffusion, slow evaporation, slurry conversion at RT, slurry conversion at 50° C., temperature cycling, polymer induced crystallization, and etc. In the above methods, DMSO, NMP, MeOH, EtOH, water, toluene, THF, 2-MeTHF, MEK, MIBK, MTBE, EtOAc, DCM, anisole, IPA, IPAc, n-heptane, ACN, acetone, butyl acetate, CHCl3, 1,4-dioxane and the mixture thereof are used as the solvent and/or anti-solvent. Types A, D, E, F, G, H, I, J, K, L and M are prepared in the processes specified below.
Experiments were performed using compound A fumarate (1:1) as the starting material. A total of 11 crystal forms were obtained and characterized by X-ray powder diffraction (XRPD), thermo gravimetric analysis (TGA), differential scanning calorimetry (DSC), and solution proton nuclear magnetic resonance (1H NMR). Further form identification study confirmed that among the 11 crystal forms, there are three hydrates (fumarate Type A, Type F and Type G), one anhydrate (fumarate Type D), three metastable anhydrates (fumarate Type K, Type L and Type M), two solvates (fumarate Type H and Type J) and two to be identified forms (fumarate Type E and Type I) that were challenging to re-prepare. Characterization summary for all the crystal forms was presented in Table 2.
Crystalline form of fumarate Type A was obtained via the following procedure: fumarate (20.7 mg) was dissolved in a mixture of EtOAc/MeOH (2:1, v/v, 0.6 mL). The clear solution was stayed in a quiet place and slow evaporated for 7 days to give fumarate type A.
Type K was obtained via heated Type A to 140° C. under nitrogen atmosphere, and cooled to 30° C.
XRPD pattern of fumarate Type A was displayed in
DVS testing on fumarate Type A was performed starting from 25° C./70% RH. As the result in
For further identification, VT-XRPD was performed on fumarate Type A. As VT-XRPD result showed in
XRPD pattern of fumarate Type K was displayed in
Crystalline form of fumarate Type D was obtained via the following procedure: Fumarate (11.8 g) was dissolved in EtOH (500 mL) at r. t. The solution was concentrated under vacuum at 50° C. to remove most of EtOH until the resulting material was 22 g left. The resulting material was stayed in a quiet place overnight to give a crystalline solid. The solid was rinsed with EtOH twice and dried under vacuum at 50° C. for 4 h to give fumarate type D.
Type L was obtained via heated Type D to 140° C. under nitrogen atmosphere.
XRPD pattern of fumarate Type D was displayed in
DVS test on Type D was started at 25° C./60% RH to avoid any unnecessary form change for the starting form. As shown in
To further identify fumarate Type D, VT-XRPD was performed. As VT-XRPD result showed in
XRPD pattern of fumarate Type L was displayed in
Crystalline form of fumarate Type F can be obtained via the following procedures: Fumarate (20.8 mg) was dissolved in EtOH (0.3 mL). To the mixture was added n-heptane (0.6 mL) dropwise. The mixture was stirred at r. t. overnight. The solid was separated by centrifugal separation.
Type M was obtained via heated Type F to 140° C. under nitrogen atmosphere, and cooled to 30° C.
XRPD pattern of fumarate Type F was displayed in
DVS testing on fumarate Type F was started at 25° C./80% RH to avoid any unnecessary form change for the starting form. DVS result in
To further identify fumarate Type F, VT-XRPD was performed. As VT-XRPD result showed in
XRPD pattern of fumarate Type M was displayed in
Crystalline form of fumarate Type G can be obtained via solid vapor diffusion in H2O for 8 days, followed by air-drying at RT overnight. A 3 mL of bottle contented Fumarate (19.5 mg) was placed in a 20 mL of bottle contented water (4 mL) for 8 days. The solid was collected.
XRPD pattern of fumarate Type G was displayed in
Crystalline form of fumarate Type H was obtained via the following procedures: Fumarate (59.5 mg) was dissolved in a mixture of 1,4-dioxane and water (9/1, v/v, 0.5 mL). The mixture was stirred at r. t. for 2 days and at −4° C. for 8 days. The solid was collected by filtration.
XRPD pattern of fumarate Type H was displayed in
Crystalline form of fumarate Type J can be obtained via recrystallization of fumarate Type D in EtOH. Fumarate (500.5 mg) was dissolved in EtOH (3.17 mL) at 70° C. The resulting clear solution was stirred at r. t. for 2 days. The solid was collected by Centrifugal separation. XRPD pattern of fumarate Type J was displayed in
Crystalline form of fumarate Type E was obtained via the following procedure: fumarate (20.7 mg) was dissolved in NMP (0.2 mL). To the clear solution was added EtOAc (1.8 mL) dropwise. The resulting mixture was stirred at room temperature over night.
As displayed by XRPD pattern in
Crystalline form of fumarate Type I can be obtained via solid vapor diffusion in EtOH for 8 days, followed by air-drying at RT overnight. A 3 mL of bottle contented Fumarate (20 mg) was placed in a 20 mL of bottle contented EtOH (4 mL) for 8 days. The solid was collected.
As displayed by XRPD pattern in
Fumarate Type D and Type F were further evaluated by solid-state stability tests under 25° C./60% RH and 40° C./75% RH for one week.
In the experiments, about 15 mg of solids was added into an HPLC vial, which was then sealed with parafilm and pricked with 10 holes. Place the vial under corresponding condition and test the solids by HPLC and XRPD after one week. The results were summarized in Table 24 below.
For fumarate Type D: XRPD results in
For fumarate Type F: XRPD results in
The oral dosing solution was prepared as follows: 5.0 mg of a test compound was weighed and dispersed in 10 mL of 0.5% methyl cellulose (MC). The final concentration of the test compound is 1 mg·mL−1 (Calculated by free freebase).
Male Sprague-Dawley rats (also summarized in Table 27) were housed in solid bottom polypropylene cages with sterilized bedding and receive sterilized diet and sterilized water. The room was controlled and monitored for humidity (targeted mean range 40% to 70%) and temperature (targeted mean range 18° C. to 26° C.) with 10 to 20 air changes/hour. The light cycle was maintained at 12-h light and 12-h dark. Only animals that appeared to be healthy were selected for this study based on overall health, body weight, or other relevant information. The animals were treated in accordance with a certain treatment schedule as summarized in Table 28.
All procedures performed on animals were in accordance with established guidelines and reviewed and approved by an independent institutional review board.
The male Sprague-Dawley rats were fasted overnight with free access to drinking water prior to treatment. On day 1, the animals were weighed and actual dose volume for each animal was calculated using the formula below:
Dose Volume (mL)=[Nominal Dose (mg·kg−1)/Dose Concentration (mg·mL−1)]×Animal Body Weight (kg)
Three rats for each group were given a single oral dose of 10 mg·kg−1. The dosing solutions were freshly prepared prior to dose administration. The actual body weights and actual volume injected were recorded accordingly. Four hours after dosing, the rats were allowed to intake food.
Blood samples (˜150 μL) were collected at different times from the jugular vein catheter into EDTA-K2 coated tubes. Whole blood was processed by centrifugation at 3000 g for 10 min. Plasma samples were collected and kept at −80° C. freezer prior to analysis. The blood sampling time was recorded accordingly.
The dose samples of PO were diluted with MeOH:H2O (4:1, v/v) to achieve the concentration of 2 μg·mL−1, respectively. Then, 2.5 μL of the diluted samples were added with 47.5 μL blank plasma, and then were handled as the plasma sample procedure. An aliquot of 10 μL of the mixture was injected into the LC-MS/MS system. The pharmacokinetic (PK) data of the test compounds were generated as shown in Table 29.
The foregoing examples and description of certain embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All references cited are incorporated herein by reference in their entireties.
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in any country.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/096509 | May 2021 | WO | international |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/095129 | May 2022 | US |
| Child | 18517894 | US |
