CN113559103A - Docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242, preparation and application thereof - Google Patents

Docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242, preparation and application thereof Download PDF

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CN113559103A
CN113559103A CN202110715908.0A CN202110715908A CN113559103A CN 113559103 A CN113559103 A CN 113559103A CN 202110715908 A CN202110715908 A CN 202110715908A CN 113559103 A CN113559103 A CN 113559103A
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mtor inhibitor
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docosahexaenoic acid
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CN113559103B (en
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谢海洋
周科
王杭祥
郑树森
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Zhejiang University ZJU
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Abstract

The invention discloses a docosahexaenoic acid coupled prodrug of an mTOR inhibitor PP242 and application thereof. The PP242-DHA coupled prodrug is applied to the anti-transplant rejection medicine, so that the retention time of the medicine in the body and the accumulation of peripheral immune organs are improved, the immune rejection reaction can be effectively inhibited, the survival time of transplant recipients is prolonged, and the medication safety is high.

Description

Docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242, preparation and application thereof
Technical Field
The invention belongs to the technical field of anti-organ transplant rejection medicines, and particularly relates to preparation of a Docosahexaenoic Acid (DHA) coupled prodrug of an mTOR inhibitor PP242 and application of the DHA coupled prodrug in anti-heart transplant rejection.
Background
Rapamycin is the only mTOR (mammalian target of rapamycin) inhibitor clinically used in the field of organ transplantation at present, and is widely used in solid organ transplantation such as kidney, liver, heart and the like. At present, rapamycin has the problems of poor water solubility and fast in vivo metabolism, and is difficult to administer by intravenous drip, while oral ingestion has the defects of low bioavailability, insufficient drug accumulation at local transplanted organs and large toxic and side effects, and is easy to cause metabolic complications after transplantation. Transplant rejection, particularly acute rejection, is primarily an inflammatory response mediated by T cell activation proliferation.
PP242 is a selective mTOR inhibitor, which has the following structure:
Figure BDA0003133232600000011
PP242 is able to inhibit the activity of both mTORC1 and mTORC 2. Studies have shown that PP242 can inhibit the proliferation of various tumor cells and promote apoptosis. At present, no report that PP242 can be used for preparing an anti-transplant rejection medicine exists.
Disclosure of Invention
The inventor screens out mTOR inhibitors with obvious proliferation inhibition effect on T cells through an in-vitro CFSE (circulating fluid bed assay) labeled T cell proliferation test system based on an mTOR inhibitor small molecule compound library, and finds that the mTOR inhibitor PP242 inhibits CD4+/CD8+The T cell proliferation effect was most pronounced. The mouse heart transplantation model proves that the mouse heart transplantation model has the effect of resisting transplant rejection. In order to further improve the in vivo bioavailability and the curative effect of graft rejection resistance, long-chain unsaturated alkane molecules DHA with strong hydrophobicity are coupled in esterification reaction, a self-assembled nano immunosuppressant is constructed, the rejection curative effect of the nano immunosuppressant in a mouse transplantation model is improved obviously compared with that of a raw pesticide, and no obvious toxic or side effect exists, so that a new technical strategy is provided for development of a novel clinical graft rejection immunosuppressant and prevention and treatment of graft rejection.
The invention provides a preparation method and application of a docosahexaenoic acid coupled prodrug of an mTOR inhibitor PP 242; the prodrug has simple synthesis method, no special auxiliary material and easy subsequent industrial batch synthesis production. In-vivo heart transplantation animal experiments show that the PP242-DHA nano preparation can effectively resist heart transplantation rejection reaction after short-term administration, and prolong the survival time of transplanted hearts.
The invention provides application of PP242 or a docosahexaenoic acid coupled prodrug of PP242 in resisting transplant rejection. Wherein the structure of the docosahexaenoic acid coupled prodrug (namely the PP242-DHA coupled prodrug) of the PP242 is as follows:
Figure BDA0003133232600000021
the docosahexaenoic acid coupled prodrug of the PP242 can realize self-assembly nano-crystallization.
In vitro T cell proliferation assay screening was previously applied to obtain CD4+、CD8+The invention constructs a PP242-DHA coupled prodrug self-assembly nano preparation and a PEGylated PP242-DHA coupled prodrug nano preparation by utilizing a strategy of modifying a prodrug by docosahexaenoic acid, better improves the pharmacokinetic characteristic of PP242, prolongs the internal circulation time of a medicament, can realize better inhibition of immunological rejection after transplantation by short-term administration, and achieves the purpose of prolonging the survival time of a transplant.
Preferably, the anti-transplant rejection drug is an anti-transplant rejection targeted drug.
Preferably, the anti-transplant rejection drug is a drug capable of inhibiting T cell proliferation and alleviating transplant immune rejection.
Our previous experiments show that PP242 has significant effect of inhibiting T cell proliferation, but PP242 has poor water solubility and low in vivo bioavailability, so that the in vivo anti-rejection effect of the transplantation model is not very significant. According to the invention, the water solubility of the PP242-DHA nano preparation is greatly increased by constructing the PP242-DHA nano preparation, the in-vivo circulation time of the PP242 is prolonged, and the effective blood concentration is maintained; meanwhile, the nano preparation can be targeted to spleen and lymph nodes of peripheral immune organs, so that a better anti-transplant rejection effect is realized.
As a further preferred, the anti-transplant rejection drug is a drug for treating transplant rejection.
The preparation method of the PP242-DHA coupled prodrug comprises the following steps: under the action of a condensing agent and a catalyst, the PP242 and docosahexaenoic acid (DHA) are subjected to esterification reaction to obtain the PP242-DHA coupled prodrug, which is shown in the formula.
The condensing agent is 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), and the catalyst is 4-Dimethylaminopyridine (DMAP). Preferably, the reaction solvent is N, N-dimethylformamide. Preferably, the reaction temperature is 40 to 50 ℃. The reaction time is 2-8 hours.
Preferably, the DHA conjugated prodrug of PP242 is in the form of a nanoformulation.
As a further preferred example, the DHA conjugated prodrug of PP242 is a self-assembled nano-formulation or a pegylated nano-formulation (i.e., pegylated PP242-DHA conjugated prodrug nano-formulation).
Specifically, the preparation method of the self-assembly nano preparation of the PP242-DHA conjugated prodrug comprises the steps of injecting an organic solvent dissolved with the PP242-DHA conjugated prodrug into an aqueous phase under the ultrasonic condition, and dialyzing to obtain uniformly dispersed nano particles, namely the self-assembly nano preparation of the PP242-DHA conjugated prodrug;
preferably, the organic solvent is a dimethyl sulfoxide solvent, and the volume ratio of the dimethyl sulfoxide to the water phase is 1: 8-15; as a further preference, the volume ratio of dimethyl sulfoxide to aqueous phase is 1: 9.
as a further preferred, the pegylated nanoformulation is prepared from 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] (DSPE-PEG) and the PP242-DHA conjugated prodrug.
As a further preference, said PP242-DHA is admixed with 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) by mass of PP242]The mass ratio of (A) to (B) is 8-12: 1; further preferred is the combination of said PP242-DHA and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol)]The mass ratio of (A) to (B) is 10: 1. preferably, the 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol)Alcohol)]Is DSPE-PEG2k(or DSPE-PEG 2000).
Specifically, the preparation method of the pegylated PP242-DHA conjugated prodrug nano-preparation comprises the steps of dissolving the PP242-DHA conjugated prodrug and the drug containing DSPE-PEG2kThe organic solvent is injected into the water phase under the ultrasonic condition, and the uniformly dispersed nano particles are obtained by dialysis.
In the preparation process, PP242-DHA conjugated prodrug and DSPE-PEG can be added2kRespectively dissolving in organic solvent, mixing the organic solvent, injecting the mixed organic solvent into water phase under ultrasonic condition, and dialyzing to obtain uniformly dispersed nanoparticles.
Preferably, the organic solvent is dimethyl sulfoxide solvent, and the volume ratio of dimethyl sulfoxide to water phase is 1: 8-15; as a further preference, the volume ratio is 1: 9.
the invention provides the PP242-DHA conjugated prodrug-based self-assembly nano preparation and the polyethylene glycol PP242-DHA conjugated prodrug nano preparation. The nanometer preparation in the form obviously improves the water solubility of the PP242, and meanwhile, the modified DHA is a substance required by human bodies, has good biocompatibility, is convenient for clinical transformation, and has better application prospect. More importantly, the PP242-DHA nano preparation obviously improves the pharmacokinetic property of the medicine, prolongs the in vivo circulation time of the medicine, can effectively deliver the medicine to peripheral immune organs, and plays a more powerful role in maintaining the immune suppression of peripheral immune microenvironment, thereby relieving rejection reaction after organ transplantation.
Compared with the prior art, the invention has the advantages that:
1) the mTOR inhibitor PP242 with the immunosuppressive effect is screened, and the human body essential fatty acid DHA is used as a modification material of the prodrug, so that the mTOR inhibitor has good safety and biocompatibility, and is convenient for clinical transformation.
2) According to the invention, the unsaturated fatty acid DHA is modified by the PP242, so that the water solubility of the medicine is greatly improved, auxiliary materials such as a solubilizer and a surfactant are avoided, and the safety is improved.
3) The PP242-DHA coupled prodrug constructed by the invention has the targeting property of peripheral immune organs, and increases the accumulation of spleen and lymph nodes, thereby achieving better peripheral immune tolerance of organ transplantation and reducing rejection reaction of transplants.
In conclusion, the PP242-DHA coupled prodrug is applied to the anti-transplant rejection medicine, so that the retention time of the medicine in the body and the accumulation of immune organs at the periphery are improved, the immune rejection reaction can be effectively inhibited, the survival time of transplant recipients is prolonged, and the medication safety is high.
Drawings
FIG. 1 is a scheme showing the synthesis scheme of a PP242-DHA conjugated prodrug of example 1;
FIG. 2 is a nuclear magnetic hydrogen spectrum of a PP242-DHA conjugated prodrug prepared in example 1;
FIG. 3 is an electron microscope and particle size chart of the nano-formulation in example 2 (1);
FIG. 4 is an in vivo organ distribution map of the nano-formulation of example 2 (2);
FIG. 5 is a graph showing the effect of (1) the nano-formulation on the anti-transplant rejection in example 3;
FIG. 6 is a HE diagram of an orthotopic heart slice of a mouse as a recipient of the (2) nanoformulation transplantation in example 3;
FIG. 7 is a HE map of liver slices of a recipient mouse transplanted with (2) the nano-formulation in example 3;
FIG. 8 is a HE map of lung sections of a recipient mouse transplanted with (2) the nano-formulation in example 3;
FIG. 9 is a HE map of kidney section of the mouse as a recipient of the (2) nano-formulation transplantation in example 3.
Detailed Description
Example 1
Synthesis of PP242-DHA conjugated prodrug, as shown in figure 1:
EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide, 25.6mg, 0.165mmol) and DMAP (4-dimethylaminopyridine, 20.2mg, 0.165mmol) were quickly added dropwise to a reaction flask containing PP242(46.2mg, 0.15mmol) and DHA (cis-4, 7,10,13,16, 19-docosahexaenoic acid, 52.5mg, 0.16mmol), and 2mL of anhydrous DMF was added to dissolve the mixture. Stirring at 43 deg.C for 6 hr, removing solvent, diluting the reaction mixture with dichloromethane, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate water solution, and saturated saline water solution; drying the organic phase with anhydrous sodium sulfate, filtering, collecting filtrate, and removing solvent under reduced pressure; purification by column chromatography (DCM: MeOH ═ 20: 1) gave the product, DHA-PP242, conjugated prodrug (42.3mg, 45.8% yield) (DHA-PP 242).
The 1H NMR nuclear magnetic data of the product PP242-DHA conjugated prodrug is as follows, and the nuclear magnetic spectrum is shown in fig. 2:
1H NMR(400MHz,Chloroform-d)δ8.95(s,1H),8.39(s,1H),7.43(d,J＝8.8Hz,1H),7.36(d,J＝2.2Hz,1H),6.98(dd,J＝8.6,2.3Hz,1H),6.86(d,J＝2.0Hz,1H),5.87(s,2H),5.45–5.31(m,12H),5.24–5.18(m,1H),2.90–2.80(m,10H),2.67(m,J＝7.2Hz,2H),2.59–2.53(m,2H),2.07(t,J＝7.4Hz,2H),1.25(s,6H),0.98(m,3H).
example 2
1) Preparing a PEGylated PP242-DHA nano preparation:
the DHA-PP242 conjugate prodrug prepared in example 1 was dissolved in a dimethyl sulfoxide solvent containing DSPE-PEG2000 (mass ratio of DHA-PP242 conjugate prodrug to DSPE-PEG2000 was 10: 1, and dimethyl sulfoxide was the amount of dissolved), to obtain a mixed solution. Adding water, wherein the volume ratio of the dimethyl sulfoxide to the water phase is 1: 9, removing the organic solvent by dialysis (MW 3500) to obtain uniformly dispersed PEGylated PP242-DHA nano-preparations (DPNP or PEGylated NPs or PEGylated DPNP). The particle size distribution and transmission electron microscopy are shown in FIG. 3.
In FIG. 3, Self-assembled nanoparticles of PP242-DHA (Self-assembled DHA-PP242nanoparticles, or bare DPNP) were prepared as distinct from PEGylated PP242-DHA nanoformulations without the addition of DSPE-PEG 2000.
In A and B of FIG. 3: "-" indicates the nanoformulation without DSPE-PEG 2000; "+" indicates a nanopreparation with DSPE-PEG2000 added; in panel A, "PP 242" represents a nanoparticle system in which bulk PP242 (with or without DSPE-PEG2000) is dissolved directly in water. The left graph in A shows that compared with the experimental group without DSPE-PEG2000, the PP242 original drug nanoparticle system with DSPE-PEG2000 added has obvious precipitation phenomenon after 24 hours; as shown in the right picture in A, the pegylation PP242-DHA sodiumThe rice formulation still exhibited a good state of soluble nanosuspension for 24 hours. As can be seen from the B picture, the PEGylated PP242-DHA nano preparation has an obvious Tyndall effect. As can be seen from the C and D graphs, the diameter of the PEGylated PP242-DHA nano-preparation (PEGylated NPs) particles is significantly smaller than that of the PP242-DHA self-assembled nano-particles without DSPE-PEG 2000. D is the average particle diameter (D)H) The graph D shows that the DHA-PP242 self-assembled nano preparation particles and the polyethylene glycol DHA-PP242nano preparation have relatively stable average particle size.
2) Drug organ distribution in vivo experiment:
DHA-PP242 was synthesized from PP242 and DHA using the method of example 1 according to the scheme A in FIG. 4. DHA-Cy5.5 was synthesized according to the same method.
Then, according to a method similar to that of the above step 1) of this example, DHA-PP242 is dissolved in DMSO and mixed with a DMSO solution of DSPE-PEG2000 and DHA-Cy5.5, and the mass ratio of DHA-PP242, DSPE-PEG2000 and DHA-Cy5.5 (calculated by the amount of Cy5.5 therein) is 100: 10: 1, preparing a fluorescent probe Cy5.5 marked polyethylene glycol PP242-DHA nano preparation (Cy5.5 labeled DPNP) after dialysis, and injecting the nano preparation into a mouse in an abdominal cavity.
In FIG. 4, B is a case where the mice were sacrificed at 6, 12, 24, 48 and 72 hours after the injection, spleens of the mice were removed, and the fluorescence intensity of Cy5.5 was measured by a small animal imaging system with the radiation efficiency as an index for evaluation. In C, the abscissa is time and the ordinate is the average radiation efficiency.
In FIG. 4, D is the result of the examination of mice at 6, 12, 24, 48 and 72 hours after the injection, and the ventral aortic lymph nodes were removed from the mice, and the fluorescence intensity of Cy5.5 was measured by a small animal imaging system, and the radiation efficiency was used as an index for evaluation. E is time on the abscissa and the average radiation efficiency on the ordinate.
As can be seen from FIG. 4, the DHA-PP242 labeled with the fluorescent probe Cy5.5 has good peripheral immune organ targeting property, and is mainly accumulated in spleen and draining lymph nodes 12-48 hours after administration, the spleen and the lymph nodes are main sites for immune activation of organ transplantation T cells, and the fluorescent probe control group has no obvious accumulation and trend effect in the spleen and the lymph nodes.
Example 3
1) Construction of allogeneic ectopic heart transplantation model and immunosuppressive treatment experiment
The stable donor BALB/C-acceptor C57BL/6 mouse skin transplantation and mouse ectopic heart transplantation model is constructed, the mouse ectopic heart transplantation model is constructed by a method that a donor aorta is matched with an acceptor abdominal aorta, and a donor pulmonary artery is matched with an acceptor inferior vena cava, and the allograft model has strong and stable rejection effect and is easy to observe and evaluate. The administration was performed 1 time per day after the heart transplantation operation by intraperitoneal injection for 10 days (40 mg/kg/day). The change of the body weight of the mice is recorded within 10 days after the administration, and the ectopic transplantation heart survival time of the mice is used as the evaluation basis of the treatment effect of the medicament.
In FIG. 5, A is the heart of Balb/C mice transplanted into the abdominal cavity of C57BL/6 mice and administered continuously for 10 days after surgery, and PP242 or PEG-DHA 242nanoparticles (DPNP) were used as a control. B is the survival time of the transplanted hearts of three groups of mice. C is the body weight change of the three groups of mice over the time of administration. D is 4 days after heart transplantation (POD 4), the transplanted heart is taken for HE staining, and immune cell infiltration appears at the edge of the transplanted heart of the normal saline group and the PP242 group at the fourth day, but no obvious immune cell infiltration exists in the DPNP group; at 8 days after heart transplantation (POD 8), the transplanted heart was HE stained, and at the eighth day, immune cell infiltration occurred in the transplanted cardiac muscle layer of the saline group and the PP242 group, but only a small amount of immune cell infiltration occurred in the DPNP group. E. Taking transplanted hearts eight days after surgery to grind and extract RNA, and detecting IL-2, IL-4, IL-6, IL-10, CX3CL, CXCL12, CXCL16, HMGB1, ICAM-1, GMCSF1, TGF-beta, TNF-alpha, IFN-gamma, NF-kappa B, CTLA4, PDL1, CD11B, CD11c, FOXP3, RAPTOR, Granzyme B and Perforin 1 by a quantitative PCR technology.
As shown in fig. 5, compared to the Saline group (salt), both the PP242 technical material and the nano-drug group (DPNP prepared in example 2) effectively prolonged the median survival time of the transplanted heart, and there was no significant difference in body weight among the three groups. The tissue section shows that the rejection reaction of transplanted heart is effectively reduced, and the expression of inflammatory cell factors such as IL-2 is reduced. Compared with the original medicine form, the nano medicine group has more obvious treatment effect.
2) Effect of drugs on toxicity of major organs of allogeneic ectopic Heart transplant model recipient mice
The heart in situ (non-ectopic transplantation heart), the kidney, the liver and the lung of a transplant recipient mouse are taken 4 days and 8 days after transplantation, slice HE observation is carried out after formalin fixation, 40 mg/kg/day and continuous 10 days of drug administration are found, and the original drug and the nano preparation have no obvious organ toxic or side effect. As shown in fig. 6-9. FIG. 6 shows HE staining of hearts in the thoracic cavities of three groups of C57BL/6 mice that received heart transplantation, on days 4 and 8 after surgery. FIG. 7 shows HE staining of liver from three groups of C57BL/6 mice that received heart transplantation on days 4 and 8 after surgery. FIG. 8 shows HE staining of lungs from three groups of C57BL/6 mice that received heart transplants on days 4 and 8 post-surgery. FIG. 9 shows HE staining of the kidneys of three groups of C57BL/6 mice that received heart transplantation on days 4 and 8 post-surgery.
In conclusion, the nano preparation in the form of the invention obviously improves the water solubility of PP242, and avoids the use of auxiliary materials such as solubilizer and the like; meanwhile, the unsaturated fatty acid used for modification is a substance required by human body, has good biocompatibility, is convenient for clinical transformation, and has better application prospect. More importantly, the self-assembly nanometer preparation of the polyethylene glycol unsaturated fatty acid coupling prodrug obviously improves the pharmacokinetic characteristic of the drug, prolongs the in vivo circulation time of the drug, can deliver PP242 to a target site to play the drug effect, has better anti-transplant rejection effect compared with the original drug, and has no obvious toxic or side effect.

Claims (10)

1. A docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242, having the structure:
Figure FDA0003133232590000011
2. a preparation method of a docosahexaenoic acid coupled prodrug of an mTOR inhibitor PP242 is characterized in that under the action of a condensing agent and a catalyst, the PP242 and docosahexaenoic acid are subjected to esterification reaction to obtain the coupled prodrug.
3. A nano-formulation of a docosahexaenoic acid conjugated prodrug of mTOR inhibitor PP242, comprising the conjugated prodrug of claim 1.
4. The nano-formulation of a docosahexaenoic acid conjugated prodrug of mTOR inhibitor PP242 according to claim 3, which is obtained by self-assembly of a conjugated prodrug of the structure shown in claim 1; or a conjugated prodrug of the structure of claim 1, which is pegylated.
5. The nano-formulation of a docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242 according to claim 4, wherein the nano-formulation is obtained by reacting 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] with a coupled prodrug of structure shown in claim 1.
6. A method for preparing the nano-formulation of docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242 of claim 4, comprising:
injecting an organic solvent dissolved with the PP242-DHA coupled prodrug into a water phase under an ultrasonic condition, and dialyzing to obtain uniformly dispersed nano particles, namely the PP242-DHA coupled prodrug self-assembled nano preparation;
or, the PP242-DHA conjugated prodrug and an organic solvent containing 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] are dissolved in the water phase under the ultrasonic condition, and the water phase is dialyzed to obtain uniformly dispersed nano particles, namely the pegylated PP242-DHA conjugated prodrug nano preparation.
7. Use of a PP242 mTOR inhibitor PP242 or a docosahexaenoic acid conjugated prodrug of the mTOR inhibitor PP242 of claim 1 for the manufacture of a medicament for the treatment of a disease benefiting from the inhibition of T-cell proliferation.
8. Use of the PP242 mTOR inhibitor PP242 or a docosahexaenoic acid conjugated prodrug of the mTOR inhibitor PP242 of claim 1 for the preparation of an anti-transplant rejection medicament.
9. The use according to claim 8, wherein said anti-transplant rejection drug is a drug having an effect of suppressing the pathological reaction of transplant immune rejection.
10. The use according to claim 8, wherein the medicament is an anti-transplant rejection targeted medicament.
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