CN113559103B - Docosahexaenoic acid coupling prodrug of mTOR inhibitor PP242, preparation and application thereof - Google Patents
Docosahexaenoic acid coupling prodrug of mTOR inhibitor PP242, preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a docosahexaenoic acid coupling prodrug of an mTOR inhibitor PP242 and application thereof. The PP242-DHA coupling prodrug is applied to the anti-transplant rejection medicament, so that the residence time of the medicament in vivo and the accumulation of peripheral immune organs are improved, the immune rejection reaction can be effectively inhibited, the survival time of a transplant recipient graft is prolonged, and the medication safety is high.
Description
Technical Field
The invention belongs to the technical field of anti-organ transplant rejection medicines, and particularly relates to preparation of a docosahexaenoic acid (Docosahexaenoic Acid, DHA) coupled prodrug of an mTOR inhibitor PP242 and application of the coupled prodrug in anti-heart transplant rejection.
Background
Rapamycin is the only clinically applied mTOR (mammalian target protein of rapamycin) inhibitor in the current organ transplantation field, and is widely applied to solid organ transplantation such as kidney, liver, heart and the like. At present, rapamycin has the problems of poor water solubility and quick in-vivo metabolism, is difficult to be administrated by intravenous drip, and has the problems of low bioavailability, insufficient local drug accumulation in transplanted organs, large toxic and side effects and easy 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 having the structure:
PP242 is capable of simultaneously inhibiting both mTORC1 and mTORC2 activity. Studies have shown that PP242 can inhibit proliferation of various tumor cells and promote apoptosis. At present, no report that PP242 can be used for preparing anti-transplant rejection medicines exists.
Disclosure of Invention
The inventor screens out mTOR inhibitor with obvious proliferation inhibition effect on T cells through an in vitro CFSE marked T cell proliferation test system based on mTOR inhibitor small molecule compound library, and discovers that mTOR inhibitor PP242 inhibits CD4 + /CD8 + T cell proliferation effects are most pronounced. The mouse heart transplant model demonstrates an anti-transplant rejection effect. In order to further improve the in vivo bioavailability and the curative effect of anti-transplant rejection, the esterification reaction is coupled with long-chain unsaturated alkane molecule DHA with strong hydrophobicity, so that the self-assembled nano immunosuppressant is constructed, the anti-rejection curative effect of the nano immunosuppressant in a mouse transplanting model is obviously improved compared with that of the original drug, no obvious toxic or side effect is generated, and a novel technical strategy is provided for the development of a novel clinical transplant rejection immunosuppressant and the prevention and treatment of transplant rejection.
The invention provides preparation and application of a docosahexaenoic acid coupling prodrug of an mTOR inhibitor PP 242; the prodrug has simple synthesis method, no special auxiliary materials and easy subsequent industrial batch synthesis production. In vivo heart transplantation animal experiments show that the PP242-DHA nanometer preparation can effectively treat heart transplant rejection reaction after short-term administration, and prolong the survival time of transplanted heart.
The invention provides application of PP242 or a docosahexaenoic acid coupling prodrug of PP242 in resisting transplant rejection. Wherein the structural formula of the docosahexaenoic acid coupling prodrug of PP242 (namely the PP242-DHA coupling prodrug) is as follows:
the docosahexaenoic acid coupling prodrug of the PP242 can realize self-assembly nanocrystallization.
We earlier applied in vitro T cell proliferation assay screening to obtain CD4 + 、CD8 + The invention constructs a PP242-DHA coupled prodrug self-assembled nano preparation and a polyethylene glycol PP242-DHA coupled prodrug nano preparation by utilizing a strategy of modifying a prodrug by docosahexaenoic acid, thereby better improving the pharmacokinetic characteristics of the PP242, prolonging the internal circulation time of the drug, realizing better inhibition of immune rejection after transplantation and achieving the purpose of prolonging the survival time of the graft.
Preferably, the anti-transplant rejection drug is an anti-transplant rejection targeting drug.
Preferably, the anti-transplant rejection drug is a drug having a function of suppressing proliferation of T cells and reducing transplant immune rejection.
Our earlier experiments show that PP242 has significant effect of inhibiting T cell proliferation, but PP242 has poor water solubility and low in vivo bioavailability, resulting in less significant anti-rejection effect in the transplantation model. According to the invention, the PP242-DHA nano preparation is constructed, so that the water solubility of the PP242-DHA nano preparation is greatly increased, the internal circulation time of the PP242 is prolonged, and the effective blood concentration is maintained; meanwhile, the nano preparation can target to spleen and lymph node of peripheral immune organs, thereby realizing better anti-transplant rejection effect.
As a further preferred aspect, the anti-transplant rejection drug is a drug for treating transplant rejection.
The preparation method of the PP242-DHA coupling prodrug comprises the following steps: and under the action of a condensing agent and a catalyst, performing esterification reaction on the PP242 and docosahexaenoic acid (DHA) to obtain the PP242-DHA coupling prodrug as 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-coupled prodrug of PP242 is in the form of a nano-preparation.
As a further preferred aspect, the DHA-coupled prodrug of PP242 employs self-assembled nanoformulations or pegylated nanoformulations (i.e., pegylated PP 242-DHA-coupled prodrug nanoformulations).
As a specific preference, the preparation method of the self-assembled nano-preparation of the PP242-DHA coupling prodrug comprises the steps of injecting an organic solvent dissolved with the PP242-DHA coupling prodrug into an aqueous phase under ultrasonic conditions, and dialyzing to obtain uniformly dispersed nano-particles, namely the self-assembled nano-preparation of the PP242-DHA coupling prodrug;
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 preferred aspect, the volume ratio of dimethyl sulfoxide to aqueous phase is 1:9.
as a further preferred option, the pegylated nanoformulation is prepared from 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] (DSPE-PEG) coupled to the PP242-DHA prodrug.
As a still further preference, the PP242-DHA is combined with 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) based on the mass of PP242]The mass ratio of (2) is 8-12:1; as a further preference the PP242-DHA is combined with 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol)]Is 10 mass ratio: 1. preferably, the 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol)]Is DSPE-PEG 2k (or DSPE-PEG 2000).
As a specific preference, the preparation method of the polyethylene glycol PP242-DHA coupled prodrug nano-preparation comprises the steps of dissolving the PP242-DHA coupled prodrug and containing DSPE-PEG 2k The organic solvent of (2) is injected into the aqueous phase under the ultrasonic condition, and the uniformly dispersed nano particles are obtained through dialysis.
Prepared byIn the course, PP242-DHA coupled prodrug and DSPE-PEG can be used 2k Respectively dissolving in organic solvent, fully and uniformly mixing the organic solvent, finally injecting the uniformly mixed organic solvent into water phase under ultrasonic condition, and dialyzing to obtain uniformly dispersed nano particles.
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 self-assembled nano-preparation based on the PP242-DHA coupled prodrug, and the PEGylated PP242-DHA coupled prodrug nano-preparation. The nanometer preparation in the form obviously improves the water solubility of PP242, and DHA 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 PP242-DHA nanometer preparation obviously improves the pharmacokinetics characteristic of the medicine, prolongs the internal circulation time of the medicine, can effectively deliver the medicine to peripheral immune organs, plays a role in more effectively maintaining the immune suppression of the peripheral immune microenvironment, and further reduces rejection reaction after organ transplantation.
Compared with the prior art, the invention has the advantages that:
1) The mTOR inhibitor PP242 with an immunosuppression 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 PP242 has good safety and biocompatibility, and is convenient for clinical transformation.
2) According to the invention, the unsaturated fatty acid DHA is modified for the PP242, so that the water solubility of the medicine is greatly improved, the use of auxiliary materials such as solubilizer, surfactant and the like is avoided, and the safety is improved.
3) The PP242-DHA coupling prodrug constructed by the invention has the targeting property of peripheral immune organs, increases the accumulation effect of spleen and lymph nodes, thereby achieving better peripheral immune tolerance of organ transplantation and reducing rejection reaction of grafts.
In conclusion, the PP242-DHA coupled prodrug is applied to the anti-transplant rejection medicament, so that the residence time of the medicament in vivo and the accumulation of peripheral immune organs are improved, the immune rejection reaction can be effectively inhibited, the survival time of a transplant recipient graft is prolonged, and the medication safety is high.
Drawings
FIG. 1 is a synthetic scheme for PP242-DHA coupled prodrugs of example 1;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the PP242-DHA coupling prodrug prepared in example 1;
FIG. 3 is a graph of electron microscope and particle size of the nano-preparation of example 2 (1);
FIG. 4 is a graph showing the in vivo organ profile of (2) a nanofabricated device of example 2;
FIG. 5 is a graph showing the comparison of the anti-transplant rejection effect of the nano-agent of example 3 (1);
FIG. 6 is a graph of in situ heart section HE of the (2) nanofabricated graft recipient mouse in example 3;
FIG. 7 is a HE view of liver sections of (2) nanofabricated graft recipient mice in example 3;
FIG. 8 is a graph of (2) a lung section HE of a recipient mouse transplanted with a nanofabricated preparation in example 3;
FIG. 9 is a HE chart of kidney sections of the (2) nanofabricated graft recipient mice in example 3.
Detailed Description
Example 1
Synthesis of PP242-DHA coupled prodrug, as shown in FIG. 1:
EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide, 25.6mg,0.165 mmol) and DMAP (4-dimethylaminopyridine, 20.2mg,0.165 mmol) were added dropwise to a reaction flask containing PP242 (46.2 mg,0.15 mmol) and DHA (cis-4, 7,10,13,16, 19-docosahexaenoic acid, 52.5mg,0.16 mmol) dissolved in 2mL anhydrous DMF. Stirring at 43deg.C for 6 hr, removing solvent, diluting the reaction mixture with dichloromethane, and washing with 0.1M hydrochloric acid solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution, respectively; the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and the solvent was removed under reduced pressure; the product DHA-PP242 coupled prodrug (42.3 mg, 45.8% yield) was obtained after purification by column chromatography (DCM: meOH=20:1) (DHA-PP 242).
1H NMR nuclear magnetic data of the product PP242-DHA coupled prodrug are as follows, and nuclear magnetic spectrum is shown in FIG. 2:
1 H 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) Preparation of PEGylated PP242-DHA nanometer preparation:
the DHA-PP242 coupling prodrug prepared in the example 1 is dissolved in a dimethyl sulfoxide solvent containing DSPE-PEG2000 (the mass ratio of the DHA-PP242 coupling prodrug to the DSPE-PEG2000 is 10:1, and the dimethyl sulfoxide is the dissolving amount), so as to obtain a mixed solution. Adding water, wherein the volume ratio of dimethyl sulfoxide to water phase is 1:9, removing the organic solvent by dialysis (MW 3500) to obtain uniformly dispersed PEGylated PP242-DHA nanoformulations (DPNP or PEGylated NPs or PEGylated DPNPs). The particle size distribution and transmission electron microscope are shown in FIG. 3.
In FIG. 3, the preparation method of PP242-DHA Self-assembled nanoparticles (Self-assembled DHA-PP242nanoparticles, or bare DPNP) is similar to that of the PEGylated PP242-DHA nanoparticles, except that DSPE-PEG2000 is not added.
In fig. 3 a and B: "-" means a nanofabricated without DSPE-PEG 2000; "+" indicates the addition of a nano-formulation of DSPE-PEG 2000; in panel a, "PP242" represents a nanoparticle system with or without DSPE-PEG 2000) PP242 stock directly dissolved in water. As can be seen from the left graph in the A, compared with the experimental group without DSPE-PEG2000, the PP242 technical material nano-particle system of the added DSPE-PEG2000 has obvious precipitation phenomenon after 24 hours; from the right graph in the A, the polyethylene glycol PP242-DHA nanometer preparation still presents a good soluble nanometer suspension state in 24 hours. From panel B, it can be seen that the PEGylated PP242-DHA nano-preparation has a remarkable tyndall effect. From panels C and D, it can be seen that PEGylated PP242-DHA nanoparticles (PEGylated NPs) are significantly smaller than the diameter of PP242-DHA self-assembled nanoparticles without DSPE-PEG2000 added. Panel D shows the average particle diameter (D H ) Relation to time, from D-graphThe DHA-PP242 self-assembled nano-preparation particles and the polyethylene glycol DHA-PP242 nano-preparation have relatively stable average particle size.
2) Drug in vivo organ distribution experiment:
DHA-PP242 was synthesized using PP242 and DHA according to the procedure presented in FIG. 4A, using the procedure of example 1. DHA-Cy5.5 was synthesized in the same manner.
DHA-PP242 was then dissolved in DMSO and mixed with a DMSO solution of DSPE-PEG2000 and DHA-Cy5.5 according to a similar method as in step 1) above in this example, the mass ratio of DHA-PP242, DSPE-PEG2000 and DHA-Cy5.5 (calculated as the amount of Cy5.5 therein) being in turn 100:10:1, preparing a fluorescent probe Cy5.5 marked PEGylated PP242-DHA nanometer preparation (Cy5.5 marked DPNP) after dialysis, and injecting the nanometer preparation into a mouse in an intraperitoneal mode.
In fig. 4B, mice were sacrificed at 6, 12, 24, 48 and 72 hours after injection, spleens of the mice were removed, and fluorescence intensity of cy5.5 was detected by a small animal imaging system, with radiation efficiency as a criterion. The abscissa in C is time and the ordinate is the average radiation efficiency.
In fig. 4, D is the fluorescence intensity of cy5.5 detected by the small animal imaging system by killing mice at 6, 12, 24, 48 and 72 hours after injection, taking out the abdominal aortic side lymph nodes of the mice, and using the radiation efficiency as a judgment index. E is time on the abscissa and average radiation efficiency on the ordinate.
As can be seen from FIG. 4, the fluorescent probe Cy5.5-labeled DHA-PP242 has good peripheral immune organ targeting, and is mainly accumulated in spleen and drainage lymph nodes after 12-48 hours of administration, wherein the spleen and the lymph nodes are the main sites for immune activation of organ transplantation T cells, and the fluorescent probe control group has no obvious accumulation and trend effects in the spleen and the lymph nodes.
Example 3
1) Allogeneic ectopic heart transplantation model and immunosuppressive treatment experiment
The stable donor BALB/C to receptor C57BL/6 mice skin transplantation and the mice ectopic heart transplantation model are constructed and obtained by the method of anastomosis of the heart aorta and the receptor abdominal aorta and the heart and lung arteries and the receptor inferior vena cava, and the allograft model rejection effect is strong and stable and is easy to observe and evaluate. The administration was carried out 1 time a day after the heart transplantation operation, and the administration was carried out 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 administration, and the survival time of the ectopic transplanted cores of the mice is used as the evaluation basis of the therapeutic effect of the medicaments.
In FIG. 5, A is heart transplantation of Balb/C mice into the C57BL/6 mice abdominal cavity, followed by continuous administration of PP242 or PEGylated DHA-PP242nanoparticles (DPNP) for 10 days after surgery, and normal saline as a control. B is the survival time of transplanted hearts of three groups of mice. C is the change in body weight of three groups of mice over the dosing time. D is 4 days after heart transplantation (POD 4), taking transplanted hearts for HE staining, and finding that immune cell infiltration appears at the edges of the transplanted hearts of the normal saline group and the PP242 group on the fourth day, but DPNP group has no obvious immune cell infiltration; at 8 days after heart transplantation (POD 8), the transplanted hearts were HE stained, and immune cell infiltration occurred in the muscle layers of the transplanted hearts of the saline group and PP242 group at the eighth day, but only a small amount of immune cell infiltration occurred in the DPNP group. E. Eight days post-operative transplanted hearts were ground to extract RNA, and 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 were detected by quantitative PCR techniques.
As shown in FIG. 5, compared with the physiological Saline group (Saline), the PP242 original drug and the nano drug group (DPNP, prepared in example 2) effectively prolong the median survival time of the transplanted heart, and the weights of the three groups have no obvious difference. Tissue sections have been shown to effectively reduce rejection of transplanted hearts and reduce the expression of inflammatory cytokines such as IL-2. Compared with the original drug form, the nano-drug group has more remarkable treatment effect.
2) Effect of drugs on toxicity of major organs in allogeneic ectopic heart transplant model recipient mice
In-situ heart (non-ectopic transplanted heart), kidney, liver and lung of the transplanted recipient mice were taken 4 days and 8 days after transplantation, and after formalin fixation, HE observation of the slices was performed, which revealed that 40 mg/kg/day, administration was continued for 10 days, and no obvious organ toxic or side effects were observed in both the original drug and the nano-preparation group. As shown in fig. 6-9. FIG. 6 shows HE staining of hearts in the chest of three groups of C57BL/6 mice receiving heart transplants on postoperative days 4 and 8. FIG. 7 is a graph showing HE staining of the livers of three groups of C57BL/6 mice receiving heart transplants on postoperative days 4 and 8. FIG. 8 shows HE staining of lungs from three groups of C57BL/6 mice receiving heart transplants on postoperative days 4 and 8. FIG. 9 shows HE staining of kidneys of three groups of C57BL/6 mice receiving heart transplants on postoperative days 4 and 8.
In conclusion, the nano preparation in the form of the invention obviously improves the water solubility of the PP242, and avoids the use of auxiliary materials such as solubilizers and the like; meanwhile, the unsaturated fatty acid used for modification is a substance required by a human body, has good biocompatibility, is convenient for clinical transformation, and has better application prospect. More importantly, the self-assembled nano preparation of the polyethylene glycol unsaturated fatty acid coupling prodrug remarkably improves the pharmacokinetic property of the drug, prolongs the in vivo circulation time of the drug, can deliver PP242 to a target part, exerts the drug effect, has better anti-transplant rejection effect compared with the original drug, and has no obvious toxic or side effect.
Claims (7)
1. A docosahexaenoic acid coupled prodrug nano-formulation of an mTOR inhibitor PP242, comprising a coupled prodrug of the structure:
2. the nano-formulation of docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242 of claim 1, wherein the nano-formulation is self-assembled from the coupled prodrug of the structure of claim 1; or from a coupled prodrug of the structure of claim 1 by pegylation.
3. The nano-formulation of docosahexaenoic acid coupled prodrug of mTOR inhibitor PP242 of claim 2, wherein the nano-formulation is obtained by reacting 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] with a coupled prodrug of the structure shown in claim 1.
4. A method of preparing a docosahexaenoic acid coupled prodrug nano-formulation of an mTOR inhibitor PP242 of claim 2, comprising:
injecting an organic solvent dissolved with the PP242-DHA coupling prodrug into an aqueous phase under ultrasonic conditions, and dialyzing to obtain uniformly dispersed nano particles, namely the PP242-DHA coupling prodrug self-assembled nano preparation;
or, injecting the PP242-DHA coupling prodrug dissolved in the nanometer emulsion and an organic solvent containing 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ methoxy (polyethylene glycol) ] into an aqueous phase under ultrasonic conditions, and dialyzing to obtain uniformly dispersed nanometer particles, namely the polyethylene glycol PP242-DHA coupling prodrug nanometer preparation.
5. Use of a docosahexaenoic acid-coupled prodrug nano-formulation of an mTOR inhibitor PP242 as in claim 1 for the preparation of a medicament for treating transplant rejection.
6. The use according to claim 5, wherein the anti-transplant rejection medicament is a medicament having a function of inhibiting a pathological reaction of transplant immune rejection.
7. The use according to claim 5, wherein the medicament is an anti-transplant rejection targeting medicament.
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Citations (3)
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| WO2013053919A2 (en) * | 2011-10-14 | 2013-04-18 | Inserm | Biomarkers of renal disorders |
| CN109306339A (en) * | 2017-07-28 | 2019-02-05 | 上海市血液中心 | A kind of preparation method and applications keeping stable tolerogenic dendritic cells under inflammatory environment |
| EP3494208A1 (en) * | 2016-08-05 | 2019-06-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Ex vivo generation of gamma delta foxp3+ regulatory t cells and therapeutic uses thereof |
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| WO2010006072A2 (en) * | 2008-07-08 | 2010-01-14 | The Regents Of The University Of California | Mtor modulators and uses thereof |
| CA2859986A1 (en) * | 2011-12-22 | 2013-06-27 | Tokai Pharmaceuticals, Inc. | Methods and compositions for combination therapy using pi3k/mtor inhibitors |
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| WO2013053919A2 (en) * | 2011-10-14 | 2013-04-18 | Inserm | Biomarkers of renal disorders |
| EP3494208A1 (en) * | 2016-08-05 | 2019-06-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Ex vivo generation of gamma delta foxp3+ regulatory t cells and therapeutic uses thereof |
| CN109306339A (en) * | 2017-07-28 | 2019-02-05 | 上海市血液中心 | A kind of preparation method and applications keeping stable tolerogenic dendritic cells under inflammatory environment |
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| Comparative metabolomics and lipidomics study to evaluate the metabolic differences between first- and second-generation mammalian or mechanistic target of rapamycin inhibitors;Md Mamunur Rashid 等;《Biomed Chromatogr》;第35卷(第11期) * |
| Evolving perspectives of mTOR complexes in immunity and transplantation;D. Fantus等;《American Journal of Transplantation》;第15卷(第4期);891-902 * |
| New perspectives on mTOR inhibitors (rapamycin, rapalogs and TORKinibs) intransplantation;Matthias Waldner等;《Br J Clin Pharmacol》;第82卷(第5期);1158-1170 * |
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