CN117815239A - Small molecule pharmaceutical composition and application thereof - Google Patents

Abstract

The present invention provides a small molecule drug composition and its application. The drug composition comprises VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin, wherein each component is a simple, safe and low-cost small molecule compound. The drug composition can reprogram astrocytes of mice after cerebral hemorrhage into neurons through continuous administration in vitro and in vivo, and can provide potential therapeutic effects for the recovery of neurological function of cerebral hemorrhage in the future.

Description

Small molecule drug composition and its application

Technical Field

The present application relates to the field of medical technology, and in particular to a small molecule drug composition and its application.

Background technique

Cerebral hemorrhage is the second largest subtype of stroke, with high morbidity, mortality, and disability rates. The primary and secondary damage to brain tissue caused by cerebral hemorrhage leads to irreversible death of neurons, while the regeneration capacity of neurons is extremely limited. Therefore, patients with cerebral hemorrhage often have varying degrees of neurological deficits. Currently, the clinical treatment of cerebral hemorrhage is very limited, mainly conservative treatment and minimally invasive treatment, which have little effect on the recovery of lost neurons. Therefore, there is an urgent need for treatment methods that can effectively supplement dead neurons.

Stem cell transplantation technology can replenish lost neurons to a certain extent, but there are problems such as abnormal differentiation, tumorigenicity, and ethics, which limit its application in the field of regenerative medicine. The rise of the field of reprogramming in the past decade has promoted the development of regenerative medicine research. The application of reprogramming technology in the nervous system is mainly to reprogram non-neuronal cells into neurons. The current reprogramming methods mainly include virus-mediated transcription factor overexpression and small molecule compound combination. Virus-mediated transcription factor overexpression induces non-neuronal cells to reprogram into neurons, which has limitations, such as abnormal gene insertion into host cells, irreversible regulation, and high prices. Small molecule compounds have simple structures, low prices, good permeability, and can achieve reversible regulation and fine regulation. However, small molecule compounds often require the synergistic effect of several drugs to achieve reprogramming, and how to establish an effective formula is a key issue. Secondly, since small molecules are not integrated into the genome, continuous administration is required to achieve effective reprogramming. In order to avoid the side effects of drugs on other systems, local administration is a preferred option.

Therefore, it is necessary to provide a small molecule combination drug that can achieve the reprogramming of non-neuronal cells into neurons through local administration.

Summary of the invention

The present application provides a small molecule drug composition and its application to solve the technical problem that existing small molecule drug compositions require continuous administration.

In a first aspect, the present application provides a small molecule pharmaceutical composition comprising VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin.

Optionally, the molar concentration ratio of VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin in the composition is 500:3:1:10:5:7.5:10.

Optionally, the composition further comprises at least one of a stabilizer, a dispersant, a solubilizer, and a buffer.

In a second aspect, the present invention further provides, based on the same inventive concept, the use of the small molecule drug composition described in the first aspect in the reprogramming of astrocytes into neurons after cerebral hemorrhage.

Optionally, the small molecule drug composition is used to induce reprogramming of astrocytes into neurons after cerebral hemorrhage in vitro, and the small molecule drug composition includes 0.5mM VPA, 3μM CHIR99021, 1μM RepSox, 10μM RG108, 5μM DAPT, 7.5μM KC7f2 and 10μM forskolin.

Optionally, the method of using the small molecule drug composition to induce in vitro reprogramming of astrocytes into neurons after cerebral hemorrhage specifically comprises the following steps:

Obtain primary astrocytes in vitro, purify and culture them;

Using a small molecule drug composition to induce reprogramming of purified cultured astrocytes;

After induction of reprogramming, the culture medium is replaced and culture is continued until neuron-like cells appear.

Optionally, the inducing reprogramming of purified cultured astrocytes using the small molecule drug composition specifically includes: adding the small molecule drug composition to the purified cultured astrocytes, replacing the drug with a new one every two days for four days.

Optionally, a method for inducing reprogramming of astrocytes into neurons after cerebral hemorrhage in vivo using a small molecule drug composition, wherein the small molecule drug composition includes 1.5mM VPA, 9μM CHIR99021, 3μM RepSox, 30μM RG108, 15μM DAPT, 22.5μM KC7f2 and 30μM forskolin.

Optionally, the method of inducing astrocyte reprogramming into neurons after cerebral hemorrhage in vivo using the small molecule drug composition specifically comprises the following steps:

The small molecule drug composition solution is prepared by artificial cerebrospinal fluid, and the small molecule drug composition solution is injected into the skull through an Alzet pump to induce reprogramming of astrocytes into neurons after cerebral hemorrhage.

Optionally, the small molecule drug composition solution is infused into the brain via an Alzet pump at a frequency of 0.25 μL/h for 2 weeks.

The above technical solution provided by the present invention has the following advantages compared with the prior art:

The present invention provides a small molecule drug composition and its application. Each component of the drug composition is a simple, safe, low-cost small molecule compound. The drug composition can reprogram astrocytes of mice after cerebral hemorrhage into neurons through continuous targeted administration in vitro and in vivo, and can provide potential therapeutic effects for the recovery of neurological function of cerebral hemorrhage in the future.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

FIG1 is a schematic diagram of the chemical structure of the small molecule compound VPA provided in the present application;

FIG2 is a schematic diagram of the chemical structure of the small molecule compound CHIR99021 provided in the present application;

FIG3 is a schematic diagram of the chemical structure of the small molecule compound RepSox provided in the present application;

FIG4 is a schematic diagram of the chemical structure of the small molecule compound RG108 provided in the present application;

FIG5 is a schematic diagram of the chemical structure of the small molecule compound DAPT provided in the present application;

FIG6 is a schematic diagram of the chemical structure of the small molecule compound KC7f2 provided in the present application;

FIG7 is a schematic diagram of the chemical structure of the small molecule compound forskolin provided in the present application;

FIG8 is a schematic diagram of purified P3 astrocytes in Experimental Example 1;

FIG9 is a schematic diagram of cells in an intermediate state during the process of reprogramming astrocytes into neurons in Experimental Example 1;

FIG10 is a schematic diagram of astrocyte reprogramming into neuron-like cells;

FIG11 is the fluorescence identification result obtained after using NeuN, GFAP, DAPI, and Merge to label FIG10 respectively;

FIG12 is a schematic diagram of constructing a mouse cerebral hemorrhage model using the collagenase method in Experimental Example 2;

FIG13 is a schematic diagram showing the principle of the process of astrocyte reprogramming into neurons after induced cerebral hemorrhage in mice in Experimental Example 2;

FIG. 14 is a schematic diagram of the results of mouse brain sections and immunofluorescence staining after 12 weeks of drug administration.

Detailed ways

In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and/or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and/or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can both mean: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or plural, respectively.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

In a first aspect, the present application provides a small molecule pharmaceutical composition comprising VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin.

It should be noted that VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin were all purchased from MCE, and the corresponding chemical structures are shown in Figures 1 to 7, respectively.

In an optional embodiment, the molar concentration ratio of VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin in the composition is 500:3:1:10:5:7.5:10.

In an optional embodiment, the composition further comprises at least one of a stabilizer, a dispersant, a solubilizer, and a buffer.

In a second aspect, the present invention further provides, based on the same inventive concept, the use of the small molecule drug composition described in the first aspect in the reprogramming of astrocytes into neurons after cerebral hemorrhage.

In an optional embodiment, the small molecule pharmaceutical composition is used to induce reprogramming of astrocytes into neurons after cerebral hemorrhage in vitro.

In an alternative embodiment, the small molecule drug composition comprises 0.5 mM VPA, 3 μM CHIR99021, 1 μM RepSox, 10 μM RG108, 5 μM DAPT, 7.5 μM KC7f2 and 10 μM forskolin.

In an optional embodiment, the method of inducing astrocyte reprogramming into neurons after cerebral hemorrhage in vitro using the small molecule pharmaceutical composition specifically comprises the following steps:

Obtain primary astrocytes in vitro, purify and culture them;

Using a small molecule drug composition to induce reprogramming of purified cultured astrocytes;

After induction of reprogramming, the culture medium is replaced and culture is continued until neuron-like cells appear.

In an optional embodiment, primary astrocytes are obtained in vitro and purified and cultured, specifically comprising: obtaining primary astrocytes from mice in vitro, and purifying and culturing from P0 to P3.

In an optional embodiment, the inducing reprogramming of purified cultured astrocytes using a small molecule drug composition specifically includes: adding the small molecule drug composition to the purified cultured astrocytes, replacing the drug with a new one every two days for four days.

In an optional embodiment, the small molecule drug composition is used to induce reprogramming of astrocytes into neurons after cerebral hemorrhage in vivo, and the small molecule drug composition includes 1.5mM VPA, 9μM CHIR99021, 3μM RepSox, 30μM RG108, 15μM DAPT, 22.5μM KC7f2 and 30μM forskolin.

In an optional embodiment, the method of inducing astrocyte reprogramming into neurons after cerebral hemorrhage in vivo using the small molecule pharmaceutical composition specifically comprises the following steps:

The small molecule drug composition solution is prepared by artificial cerebrospinal fluid, and the small molecule drug composition solution is injected into the skull through an Alzet pump to induce reprogramming of astrocytes into neurons after cerebral hemorrhage.

In an optional embodiment, the small molecule drug composition solution includes 1.5 mM VPA, 9 μM CHIR99021, 3 μM RepSox, 30 μM RG108, 15 μM DAPT, 22.5 μM KC7f2 and 30 μM forskolin.

In an optional embodiment, the small molecule drug composition solution is infused into the brain via an Alzet pump at a frequency of 0.25 μL/h for 14 days.

In an optional embodiment, the small molecule drug composition solution is injected into the cranium via an Alzet pump, specifically comprising: loading the configured small molecule drug composition solution into the Alzet pump, then connecting a cannula to the front end of the Alzet pump, implanting the cannula around the intracranial cerebral hemorrhage lesion, and simultaneously burying the Alzet pump subcutaneously.

The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

Example 1

This embodiment provides a small molecule drug composition, in which the molar concentration ratio of VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin is 500:3:1:10:5:7.5:10.

The small molecule drug composition provided in Example 1 was used to induce reprogramming of astrocytes into neurons after cerebral hemorrhage in vitro and in vivo, respectively. The experimental process is as follows:

Experimental Example 1

This experimental example uses a small molecule drug composition to induce in vitro reprogramming of astrocytes into neurons after intracerebral hemorrhage in mice. Experimental method:

Primary cortical and striatal astrocytes from newborn mice were obtained for in vitro culture. The newborn mice used in this experiment were C57 red-skinned suckling mice within 24 hours of birth.

The obtained primary cortical and striatal astrocytes were subcultured until the astrocytes were purified from P0 to P3, and the cell morphology was shown in FIG8 .

Add a small molecule drug composition to the purified cultured astrocytes, replace the new drug every two days for four days, specifically including:

Wash the astrocytes collected previously with 10 ml DMEM/F12 culture medium once, centrifuge at 800 rpm for 4 min at room temperature, and resuspend in DMEM/F12 culture medium containing 10% FBS to adjust the cell density to 4×104 cells/ml. Pipette 100 μL of cell suspension and drop it in the center of the glass slide. After incubating in a cell culture incubator for 4 hours, add 500 μL of 10% FBS DMEM/F12 culture medium to each well, and place the 24-well plate in a cell culture incubator for standby use.

The small molecule drug composition solution was prepared using DMSO, and after being completely dissolved, it was added to the DMEM/F12 culture medium containing 10% FBS, so that the final concentrations were 0.5mM VPA, 3μM CHIR99021, 1μM RepSox, 10μM RG108, 5μMDAPT, 7.5μM KC7f2 and 10μM forskolin. The culture medium of the astrocytes that had been completely attached to the wall in the previous culture was replaced with this culture medium. The above culture medium was replaced every two days for four days, and the induced cells were shown in Figure 9.

On the fifth day of induction, the culture medium was replaced with a culture medium containing 20 ng/ml BDNF and 20 ng/ml GDNF. After 6 days of continuous culture, the obtained cell morphology is shown in FIG10 , from which neuron-like cells can be observed.

Experimental results:

The cells obtained were marked with markers NeuN and GFAP, respectively, and the immunofluorescence identification results were obtained after staining with dyes DAPI and Merge, as shown in Figure 11. It can be clearly seen from Figure 11 that most of the cells are neuron marker NeuN, while astrocyte marker GFAP is almost invisible, proving that the small molecule drug composition provided by the present invention successfully completes in vitro induction of astrocyte reprogramming into neurons.

Experimental Example 2

This experimental example uses a small molecule drug composition to induce astrocyte reprogramming into neurons after intracerebral hemorrhage in mice. Experimental method:

Eight-week-old Aldh1l1-cre/ERT2-Ai9 male mice were selected and intraperitoneally injected with tamoxifen for 5 days. After waiting for 7 days, a mouse cerebral hemorrhage model was established using the collagenase method, as shown in FIG12 .

After a 21-day recovery period, the mice were given topical medications, including:

First, DMSO was used to prepare a storage solution, and then artificial cerebrospinal fluid was used to prepare 100 μL of a small molecule drug composition solution, which included 1.5 mM VPA, 9 μM CHIR99021, 3 μM RepSox, 30 μM RG108, 15 μM DAPT, 22.5 μM KC7f2, and 30 μM forskolin; the prepared small molecule drug composition solution was loaded into an Alzet pump, and then a cannula was connected to the front end of the Alzet pump. The cannula was implanted around the intracranial cerebral hemorrhage lesion of the mouse, and the Alzet pump was buried subcutaneously in the mouse.

The small molecule drug composition solution was infused into the brain via an Alzet pump at a frequency of 0.25 μL/h. After continuous infusion for 14 days, administration was stopped.

After waiting for 12 weeks, the mice were killed and frozen sections and immunofluorescence staining were performed on the mouse skull. The section results are shown in Figure 14: ALDH1L1-Cre ert2-AI9 transgenic mice were used to trace astrocytes. After cerebral hemorrhage modeling and reprogramming, the sections were stained with NeuN, and double-labeled cells around the hematoma were seen, which can be considered as neurons reprogrammed from astrocytes.

In summary, the small molecule drug composition provided in the present invention can successfully reprogram astrocytes into neurons after cerebral hemorrhage both in vivo and in vitro.

The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims (10)

1. A small molecule drug composition, characterized in that it comprises: VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin. 2. The small molecule drug composition according to claim 1, characterized in that the molar concentration ratio of VPA, CHIR99021, RepSox, RG108, DAPT, KC7f2 and forskolin in the composition is 500:3:1:10:5:7.5:10. 3. The small molecule drug composition according to claim 1, characterized in that the composition further comprises at least one of a stabilizer, a dispersant, a solubilizer, and a buffer. 4. Use of the small molecule pharmaceutical composition according to any one of claims 1 to 3 in reprogramming astrocytes into neurons after cerebral hemorrhage. 5. The use according to claim 4, characterized in that astrocytes are reprogrammed into neurons after cerebral hemorrhage induced in vitro using a small molecule drug composition, wherein the small molecule drug composition comprises 0.5 mM VPA, 3 μM CHIR99021, 1 μM RepSox, 10 μM RG108, 5 μM DAPT, 7.5 μM KC7f2 and 10 μM forskolin. 6. The use according to claim 5, characterized in that the method of inducing astrocyte reprogramming into neurons after cerebral hemorrhage in vitro using the small molecule drug composition specifically comprises the following steps: Obtain primary astrocytes in vitro, purify and culture them; Using a small molecule drug composition to induce reprogramming of purified cultured astrocytes; After induction of reprogramming, the culture medium is replaced and culture is continued until neuron-like cells appear. 7. The use according to claim 6 is characterized in that the use of the small molecule drug composition to induce reprogramming of purified and cultured astrocytes specifically comprises: adding the small molecule drug composition to the purified and cultured astrocytes, replacing new drugs every two days for four days. 8. The use according to claim 4, characterized in that it is a method for inducing astrocyte reprogramming into neurons after cerebral hemorrhage in vivo using a small molecule drug composition, wherein the small molecule drug composition comprises 1.5 mM VPA, 9 μM CHIR99021, 3 μM RepSox, 30 μM RG108, 15 μM DAPT, 22.5 μM KC7f2 and 30 μM forskolin. 9. The use according to claim 8, characterized in that the method of inducing astrocyte reprogramming into neurons after cerebral hemorrhage in vivo using the small molecule drug composition specifically comprises the following steps: The small molecule drug composition solution is prepared by artificial cerebrospinal fluid, and the small molecule drug composition solution is injected into the skull through an Alzet pump to induce reprogramming of astrocytes into neurons after cerebral hemorrhage. 10 . The use according to claim 9 , characterized in that the small molecule drug composition solution is infused into the brain via an Alzet pump at a frequency of 0.25 μL/h for 2 weeks.