Patent Application
20230365492
The application claims priority to Chinese patent application No. 202011424798.4, filed on Dec. 9, 2020, the entire contents of which are incorporated herein by reference.
The present invention belongs to the field of medicine, and specifically relates to a novel lithium organic acid-amino acid salt, and a crystal form, a composition, a preparation method and an application thereof in the field of neuropsychiatric diseases.
Lithium salts can be used in the field of mental diseases, have a good curative effect and a preventive effect on recurrent episodes of mania and depression in bipolar disorder, and also has a unique prevention effect on suicide risk. Recent studies have also found that the lithium salts can play a neuroprotective role by acting on GSK-3, WNT, AKT and neurotransmitters. The lithium salts have selective activity for GSK-3β, through two main mechanisms: (1) directly inhibiting the activity of the GSK-3β as a competitive inhibitor of Mg2+; and (2) indirectly inhibiting the activity of the GSK-3β by increasing the expression of phosphorylated AKT, phosphorylated GSK-3β (ser9) and MCL-1. Such protective role has the potential to the prevention and treatment of neurodegenerative diseases, including Alzheimer's disease. At present, lithium salts which are commonly used clinically include lithium carbonate, lithium citrate, lithium acetate and long-acting lithium salts, among which the lithium carbonate is most commonly used.
At present, inorganic lithium salts commonly used in clinical applications have a narrow safety range, and long-term medication is usually required in the treatment of mental diseases. Potential kidney damage and thyroid dysfunction caused by the long-term medication greatly limited their clinical applications. Meanwhile, after long-term exposure of those currently common-used lithium inorganic acid salts (such as lithium chloride and lithium carbonate), disorder of the blood pH value will also be caused, leading to metabolic acidosis and aggravated burden of the kidney. In recent years, it has been found that a variety of small molecule organic acids, including butyric acid, isobutyric acid, valproic acid and folic acid, have an important influence on the central nervous system, which can effectively relieve anxiety, depression and other emotional abnormalities and delay degenerative diseases of the central nervous system. In addition, organic acid salts usually have great differences from inorganic acid salts in absorption, distribution and metabolism characteristics, so that the in vivo distribution defects of existing inorganic lithium salts are expected to be changed. Therefore, organic lithium salts have a higher new drug development value and a better market prospect.
Technical problems to be solved: A purpose of the present invention is to provide a novel lithium organic acid-amino acid salt, so as to solve the problems of insufficient efficacy and high toxicity and side effects of existing lithium preparations.
Meanwhile, the present invention provides a crystal form of a lithium organic acid-L-proline salt and a composition.
The present invention also provides a preparation method of the lithium organic acid-amino acid salt, the crystal form or the composition.
The present invention further provides an application of the lithium organic acid-amino acid salt, the crystal form or the composition in preparation of drugs for treatment of neurological diseases or mental diseases.
A lithium organic acid-amino acid salt is provided, where a lithium organic acid is one or more of lithium isobutyrate, lithium n-butyrate, lithium lactate, lithium citrate or lithium cholesterol and other lithium organic acids; an amino acid is one of L-proline, valine, lysine or other natural amino acids or artificially synthetic amino acids; and the lithium organic acid-amino acid salt is a salt formed by the lithium organic acid and the amino acid.
As a preference, the lithium organic acid is lithium isobutyrate.
As a preference, the amino acid is L-proline.
As a preference, the lithium organic acid-amino acid salt is a lithium isobutyrate-L-proline salt.
As a preference, the lithium isobutyrate and the amino acid in the lithium organic acid-amino acid salt are combined at a molar ratio of (0.8-1.2):(0.8-1.2). As a further preference, the lithium organic acid-amino acid salt is a lithium isobutyrate-L-proline salt. The lithium isobutyrate and the L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of (0.8-1.2):(0.8-1.2).
As a preference, the lithium isobutyrate and the L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1. As a further preference, the lithium organic acid-amino acid salt is a lithium isobutyrate-L-proline salt. The lithium isobutyrate and the L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1.
A crystal form of a lithium organic acid-amino acid salt is provided, where lithium isobutyrate and L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1, and the crystal form has characteristic peaks at a diffraction angle (2θ) of 8.201°±0.2°, 11.735°±0.2°, 20.395°±0.2°, 23.669°±0.2° and 24.930°±0.2° in an X-ray powder diffraction spectrum.
A crystal form of a lithium organic acid-amino acid salt is provided, where lithium isobutyrate and L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1, and the crystal form has characteristic peaks at a diffraction angle (2θ) of 7.9090±0.2°, 8.201°±0.2°, 11.735°±0.2°, 16.525°±0.2°, 20.395°±0.2°, 23.669°±0.2°, 24.930°±0.2°, 30.232°±0.2°, 31.427°±0.2° and 33.451°±0.2° in an X-ray powder diffraction spectrum.
A crystal form of a lithium organic acid-amino acid salt is provided, where lithium isobutyrate and L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1, and the crystal form has characteristic peaks at a diffraction angle (2θ) of 8.255°±0.2°, 11.782°±0.2°, 16.584°±0.2°, 16.811°±0.2°, 18.711°±0.2°, 18.800°±0.2°, 20.451°±0.2°, 23.715°±0.2°, 25.017°±0.2°, 25.040°±0.2°, 30.281°±0.2°, 31.490°±0.2°, 33.537°±0.2° and 35.886°±0.2° in an X-ray powder diffraction spectrum.
Meanwhile, the present invention provides a preparation method of the lithium organic acid-amino acid salt (preferably lithium isobutyrate-L-proline salt) described in any one of the technical solutions or the crystal form described in any one of the technical solutions. The preparation method includes: preparation by a single solvent method or a mixed solvent method.
The preparation method of the lithium organic acid-amino acid salt (preferably lithium isobutyrate-L-proline salt) described in any one of the technical solutions, or the preparation method of the crystal form described in any one of the technical solutions includes:
As a preference, in the single solvent method, the solvent is n-butanol; and in the mixed solvent crystallization method, the good solvent is ethanol, and the poor solvent is tetrahydrofuran.
In the single solvent method, the added amount of the solvent is determined based on the situation that proper dissolution is realized under the heating reflux; and in the mixed solvent crystallization method, the good solvent is added first to realize proper dissolution, then the poor solvent is added to realize proper precipitation, the good solvent is supplemented to realize dissolution by heating, and the reflux is conducted for 1-5 hours, followed by natural cooling and crystallization.
A crystal form of a lithium organic acid-amino acid salt is provided, where lithium isobutyrate and L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1, and the crystal form has characteristic peaks at a diffraction angle (2θ) of 7.909°, 8.201°, 11.735°, 16.525°, 20.395°, 23.669°, 24.930°, 30.232°, 31.427° and 33.451° in an X-ray powder diffraction spectrum.
A preparation method of the crystal form includes: adding an appropriate amount of a good solvent (n-butanol) to equal chemical equivalents of the lithium isobutyrate and the L-proline, conducting heating reflux to realize proper dissolution, and recording the use amount of the solvent; conducting stirring for about 3 hours, and conducting heat filtration, followed by natural cooling and crystallization; and filtering a solid, and conducting vacuum drying until a constant weight to obtain the crystal form of a lithium organic acid-amino acid salt.
A crystal form of a lithium organic acid-amino acid salt is provided, where lithium isobutyrate and L-proline in the lithium organic acid-amino acid salt are combined at a molar ratio of 1:1, and the crystal form has characteristic peaks at a diffraction angle (2θ) of 8.255°, 11.782°, 16.584°, 16.811°, 18.711°, 18.800°, 20.451°, 23.715°, 25.017°, 25.040°, 30.281°, 31.490°, 33.537° and 35.886° in an X-ray powder diffraction spectrum.
A preparation method of the crystal form includes: adding an appropriate amount of a good solvent to equal chemical equivalents of the lithium isobutyrate and the L-proline, conducting heating reflux to realize proper dissolution, and then adding an appropriate amount of a poor solvent until proper precipitation of a solid; supplementing the good solvent to realize re-dissolution of the solid, and conducting reflux for 3 hours, followed by natural cooling and crystallization; and filtering a solid, and conducting vacuum drying until a constant weight to obtain a white solid of the crystal form.
A pharmaceutical composition is provided, where the pharmaceutical composition includes the lithium organic acid-amino acid salt described in any one of the technical solutions or the crystal form described in any one of the technical solutions.
The present invention provides an application of the lithium organic acid-amino acid salt described in any one of the technical solutions, the crystal form described in any one of the technical solutions, or the pharmaceutical composition in preparation of a medicine for treatment of a disease that benefits from the inhibition of the GSK-3β activity, the increase of the β-catenine protein expression, or the inhibition of the tau protein expression.
As a preference, the disease includes one or more of a neurodegenerative disease and a mental disease.
As a preference, the neurodegenerative disease is one or more of Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, amyotrophic lateral sclerosis and spinocerebellar ataxia.
As a preference, the mental disease is one or more of mild, moderate, or severe depression and bipolar disorder.
The novel lithium isobutyrate-L-proline salt is prepared by the applicant based on the single solvent method and the mixed solvent method with the lithium isobutyrate and the L-proline as raw materials. In the single solvent method, an appropriate amount of a good solvent is added to different chemical equivalents of the lithium isobutyrate and the L-proline, heating reflux is conducted for 1-5 hours, and heat filtration is conducted, followed by natural cooling and crystallization. In the mixed solvent crystallization method, an appropriate amount of a good solvent is added to different chemical equivalents of the lithium isobutyrate and the L-proline, heating reflux is conducted for dissolution, then an appropriate amount of a poor solvent is added for precipitation of a solid, the good solvent is supplemented for redissolution of the solid, and reflux is conducted for 1-5 hours, followed by natural cooling and crystallization. According to XRD and hydrogen spectrum verification, a novel salt is formed by the lithium isobutyrate and the L-proline. On this basis, the potential application value and safety of the salt for the treatment of Alzheimer's disease are preliminarily evaluated.
According to the present invention, the nuclear magnetic hydrogen spectrum of the obtained lithium isobutyrate-L-proline salt, lithium isobutyrate and L-proline is separately detected under same conditions, and then superimposed. According to a superimposed spectrum, it can be seen that the hydrogen spectrum of the solid precipitated under the condition of a single solvent or the solid obtained after mixed crystallization is different from that of the single compound lithium isobutyrate, that of the single compound L-proline or the superimposed spectrum of the two single compounds. Therefore, it can be determined that the solid precipitated under the condition of n-butanol is a salt different from the two raw materials.
According to the present invention, the lengths of C—O bonds of the L-proline in a single crystal lattice are measured by Mercury software, which are 1.233 Å and 1.268 Å respectively, and the ratio is 1.028. The lengths of the C—O bonds of the L-proline are basically the same, indicating that due to obvious proton transfer of the L-proline in a complex, the lengths of the C—O bonds of a carboxylate group tend to be the same, and it is determined that a prepared product is a lithium isobutyrate-L-proline salt.
Experimental results show that the lithium isobutyrate-L-proline salt significantly inhibits the GSK-3β activity, as evidenced by the significantly decreased phosphorylated GSK-3β (Ser9) expression in a dose-dependent manner, and the effect is significantly better than that of an existing and clinically common-used lithium preparation, lithium carbonate. Meanwhile, the lithium isobutyrate-L-proline salt significantly increases the β-catenine protein expression, indicating that the salt has the effect of promoting proliferation and survival of cells, and the effect is significantly better than that of the lithium carbonate. An SH-SY5Y cell experiment shows that the lithium isobutyrate-L-proline salt can significantly down-regulate the neurodegeneration related protein (tau) expression, indicating that the salt has the effect of inhibiting degeneration of nerve cells, and the effect is significantly better than that of the lithium carbonate. Moreover, compared with the lithium carbonate, the cytotoxicity of the lithium isobutyrate-L-proline salt is significantly reduced, and the Notch expression is not affected while the GSK-3β is inhibited. The lithium isobutyrate-L-proline salt has no significant influence on the survival of HEK293 cells. Finally, experimental results further prove that compared with the lithium carbonate, the lithium isobutyrate-L-proline salt has a higher blood lithium level, while having no obvious difference in the level of lithium in the brain.
The present invention has the following beneficial effects. The present invention has a positive curative effect and a preventive effect on recurrent episodes of mania and depression in bipolar disorder, and also has a unique prevention effect on suicide risk. Meanwhile, the salt can effectively relieve anxiety, depression and other emotional abnormalities and delay degenerative changes of the central nervous system. Moreover, organic acid salts usually have great differences from inorganic acid salts in absorption, distribution and metabolism characteristics, so that in vivo distribution defects of existing inorganic lithium salts are changed. In addition, the novel small molecule organic lithium salt designed and developed by the present invention has better safety while the efficacy for diseases of the central nervous system is improved, so that the clinical dosage of the lithium salt is reduced, and the occurrence of peripheral adverse reactions is avoided.
The following embodiments are described to make the present invention fully understood by persons skilled in the art, rather than to limit the present invention in any manner. Lithium isobutyrate in the present invention can be replaced with one or more of lithium butyrate, lithium lactate, lithium cholesterol and other organic lithium salts, and proline can be replaced with valine, lysine and other natural essential amino acids or artificially synthetic amino acids.
An appropriate amount of a good solvent (n-butanol) was added to equal chemical equivalents of lithium isobutyrate (500 mg) and L-proline (612 mg), heating reflux was conducted to realize proper dissolution, and the use amount of the solvent was recorded (the added volume of the n-butanol was 55 ml). Stirring was conducted for about 3 hours, and heat filtration was conducted, followed by natural cooling and crystallization. A solid was filtered, and vacuum drying was conducted until a constant weight to obtain 589.3 mg of a white solid (a lithium isobutyrate-L-proline salt obtained by a single solvent crystallization method) with a mass yield of 53.06%. XRD and hydrogen spectrum detection were conducted (with CD3OD as a deuterated reagent).
The hydrogen spectrum of a single compound lithium isobutyrate is (as shown in
The hydrogen spectrum of a single compound L-proline is (as shown in
The hydrogen spectrum of a physical mixture of equal chemical equivalents of lithium isobutyrate and L-proline is (as shown in
The hydrogen spectrum of a lithium isobutyrate-L-proline salt is (as shown in
From
From the superimposed spectrum, it can be seen that the hydrogen spectrum of the solid precipitated under the condition of n-butanol in Example 1 is different from that of the single compound lithium isobutyrate, that of the single compound L-proline or the superimposed spectrum of the two single compounds. Therefore, it can be determined that the solid precipitated under the condition of n-butanol is different from the single lithium isobutyrate or the L-proline. Combining with the nuclear magnetic spectrum, it is further proved that the solid obtained in Example 1 of the present invention is a lithium isobutyrate-L-proline salt at a ratio of 1:1.
An appropriate amount (50 ml) of a good solvent (ethanol) was added to equal chemical equivalents of lithium isobutyrate (500 mg) and L-proline (612 mg), heating reflux was conducted to realize proper dissolution, and then an appropriate amount (30 ml) of a poor solvent (tetrahydrofuran) was added until proper precipitation of a solid. The good solvent (7 ml) was supplemented to realize redissolution of the solid, and reflux was conducted for 3 hours, followed by natural cooling and crystallization. A solid was filtered, and vacuum drying was conducted until a constant weight to obtain 374.8 mg of a white solid (a lithium isobutyrate-L-proline salt obtained by a mixed solvent crystallization method) with a mass yield of 33.75%. XRD and hydrogen spectrum detection were conducted separately (with CD3OD as a deuterated reagent).
Combing
From the superimposed spectrum, it can be seen that the hydrogen spectrum of the solid precipitated in the mixed solvent of ethanol and tetrahydrofuran in Example 2 is different from that of the single compound lithium isobutyrate, that of the single compound L-proline or the superimposed spectrum of the two single compounds. Therefore, it can be determined that the solid precipitated in the mixed solvent of ethanol and tetrahydrofuran is a salt different from the two raw materials. From the XRD spectrum of the solid precipitated in the mixed solvent of ethanol and tetrahydrofuran and the XRD spectrum of the solid precipitated in the single solvent (n-butanol), it can be seen that the two solids have the 20 characteristic peaks within a deviation range of 0.2 and same peak intensity characteristics. Therefore, it can be determined that the two solids are a same crystal form of a same salt.
According to the XRD and hydrogen spectrum results, an n-propanol system and an n-butanol system were selected for single crystal culture, and structures of formed solid products were determined. Finally, the mass of a crystal precipitated under the condition of n-butanol meets test requirements, and a single crystal structure is as shown in
The lengths of C—O bonds of the L-proline in a single crystal lattice were measured by Mercury software, which were 1.233 Å and 1.268 Å respectively, and the ratio was 1.028. The lengths of the C—O bonds of the L-proline are basically the same, indicating that due to obvious proton transfer of the L-proline in the solid product, the lengths of the C—O bonds of a carboxylate group tend to be the same, and it is determined that the prepared product is a lithium isobutyrate-L-proline salt.
Under conventional culture conditions, CHO cells were treated with lithium isobutyrate or a lithium isobutyrate-L-proline salt with a concentration of 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 mM (mmol/L) for 2 hours (or treated with lithium carbonate or a lithium isobutyrate-L-proline salt with a concentration of 0, 5, 10, 20, 40, 60 and 90 mM for 2 hours), and then washed with PBS for three times for 5 minutes each time. The cells were continuously subjected to lysis by an RIPA buffer containing a protease inhibitor, followed by detection by Western blot with phosphorylated GSK-3β (Ser9) or a total GSK-3β antibody as an antibody.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference protein. Experimental results are as shown in
The lithium isobutyrate-L-proline salt has a better effect of promoting survival of cells.
Under conventional culture conditions, CHO cells were treated with lithium carbonate or a lithium isobutyrate-L-proline salt with a concentration of 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 mM for 2 hours, and then washed with PBS for three times for 5 minutes each time. The cells were continuously subjected to lysis by an RIPA buffer containing a protease inhibitor, followed by detection by Western blot with a β-catenine antibody as an antibody. GAPDH was used as an internal reference protein. Experimental results are as shown in
Under conventional culture conditions, SH-SY5Y cells were treated with lithium carbonate or a lithium isobutyrate-L-proline salt with a concentration of 0, 3.125, 6.25, 12.5, 25 and 50 mM for 3 hours, and then washed with PBS for three times for 5 minutes each time. The cells were continuously subjected to lysis by an RIPA buffer containing a protease inhibitor, followed by detection by Western blot with a total tau protein antibody as an antibody. GAPDH was used as an internal reference protein. Experimental results are as shown in
Under conventional culture conditions, SH-SY5Y cells were treated with lithium carbonate or a lithium isobutyrate-L-proline salt with a concentration of 0, 3.125, 6.25, 12.5, 25 and 50 mM for 3 hours, and then washed with PBS for three times for 5 minutes each time. The cells were continuously subjected to lysis by an RIPA buffer containing a protease inhibitor, followed by detection by Western blot with a total Notch protein antibody as an antibody. GAPDH was used as an internal reference protein. A Notch protein is an important signaling molecule at the downstream of GSK-3, and plays an important positive role in regulating the proliferation and survival of cells. Experimental results are as shown in
The lithium isobutyrate-L-proline salt has lower cytotoxicity than lithium carbonate.
Under conventional culture conditions, HEK293 cells were treated with lithium carbonate (represented by a black dotted line) or a lithium isobutyrate-L-proline salt (represented by a black solid line) with a concentration of 0, 0.7, 1.56, 3.12, 6.25, 12.5, 25 and 50 mM for 12 and 24 hours, and then detected by a CCK-8 test to obtain the cytotoxicity. The absorbance was read by a microplate reader at 450 nm (results are expressed as mean±standard error). Experimental results are as shown in
Under conventional culture conditions, HEK293 cells were treated with lithium isobutyrate (represented by a black dotted line) or a lithium isobutyrate-L-proline salt (represented by a black solid line) with a concentration of 0, 0.7, 1.56, 3.12, 6.25, 12.5, 25 and 50 mM for 12 and 24 hours, and then detected by a CCK-8 test to obtain the cytotoxicity. The absorbance was read by a microplate reader at 450 nm (results are expressed as mean±standard error). Experimental results are as shown in
HEK293 cells were separately treated with a lithium isobutyrate-L-proline salt and lithium carbonate with a concentration of 5 mM for 4 hours, then 5 μg/mL of wheat germ agglutinin and an Alexa Fluor 488 conjugate cell membrane dye were added, and the cells were incubated at 37° C. for 10 minutes, washed with PBS for two times and observed under a confocal microscope. Experimental results (as shown in
Compared with the lithium carbonate, the lithium isobutyrate-L-proline salt has better in vivo pharmacokinetics, inhibitory activity for GSK-3 in brain tissues and safety.
38 male SD rats were administrated with lithium carbonate or a lithium isobutyrate-L proline salt by gavage (at 0.08 mmol lithium/rat, dissolved in 400 μL of double distilled water). After the administration, the rats were sacrificed (1 rat was sacrificed at the time point of 0, and 3 rats were sacrificed at other time points) at different time points (including 0, 0.5, 1, 4, 8, 12 and 24 hours), blood and brain tissues were quickly collected, plasma was separated, and a homogenate of the brain tissues was prepared. The content of lithium in the plasma and the brain tissues was detected by an ICP-MS method (results are expressed as mean±standard error). Experimental results are as shown in
APP/PS1 transgenic AD model mice were orally administrated with a lithium isobutyrate-L proline salt or lithium carbonate (by gavage at 0.02, 0.08 and 0.16 mmol/mouse, with 3 mice in each group), and then sacrificed 4 hours later. Brain tissues were extracted, and a homogenate of the brain tissues was prepared. The phosphorylated GSK-3β (Ser9) expression in the brain tissues was detected by western blot with a β-actin protein as an internal reference. Experimental results are as shown in
According to preliminary experimental results, the oral acute toxicity of a lithium isobutyrate-L-proline salt and lithium carbonate was detected by a limited test method and a Horn's method, respectively. According to the limited test, 20 Kunming mice (with the weight of 19.6-22 g, including half male mice and half female mice) were orally administrated with 5,000 mg/kg of a lithium isobutyrate-L-proline salt at one time. According to the Horn's method, 40 Kunming mice (with the weight of 19.8-22 g, including half male mice and half female mice) were divided into four groups at an equal ratio of 2.15, including a 464 mg/kg dose group, a 1,000 mg/kg dose group, a 2,150 mg/kg dose group and a 4,640 mg/kg dose group (each group was orally administrated at one time and includes 10 mice with half male mice and half female mice). After the administration, the mice were observed at 0.5, 2 and 4 hours. 24 hours later, poisoning symptoms, occurrence time and death time of the experimental animals were observed every day for an observation period of 14 days. Dead animals and other animals sacrificed within 14 days were subjected to gross anatomy. When gross pathological changes were observed by naked eyes, pathological examination was performed. Experimental results are as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011424798.4 | Dec 2020 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/131789 | Nov 2021 | US |
| Child | 18328801 | US |
