Rescimod prodrug as well as preparation method and application thereof

Abstract

The invention provides a racimod prodrug as well as a preparation method and application thereof. The lecimod prodrug comprises a carrier and a riboflavin derivative loaded on the carrier. Wherein the carrier is obtained by bonding of R848-TK and mPEG-b-PHEA (mPEG-b-PHEA). According to the invention, R848 NPs is taken as a carrier to physically support a fat-soluble riboflavin derivative, and the R848 NPs is used for constructing a lecimod prodrug capable of being ultrasonically activated. Wherein the riboflavin derivative, as a fat-soluble substance, can be activated under an ultrasonic condition so as to generate < 1 > O2 for cutting a TK bond, so that the active drug R848 is released. According to the invention, by preparing the ultrasonic activated racimod prodrug with stronger specificity, the tumor selectivity of the drug can be effectively enhanced, and time-space controllable precise treatment is realized.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a resiquimod prodrug and a preparation method and application thereof. Background Art

[0002] Prodrugs refer to bioreversible derivatives that are inactive in themselves but can effectively release active drugs after biotransformation in the body. The term prodrug was first proposed by Adrian Albert in 1958. It was originally used to describe a concept that can produce a compound with pharmacological effects after biotransformation. Later, Rautio et al. further improved the definition of prodrugs. In recent years, prodrugs have been widely used due to their advantages over original drugs, such as solubility, chemical stability, slow metabolism, and lower toxic side effects. They have become an important strategy to improve the pharmacokinetic and pharmacodynamic properties of active drugs and reduce toxic side effects.

[0003] Prodrugs can be divided into endogenous stimulus-responsive prodrugs and exogenous stimulus-responsive prodrugs according to the driving strategy. Endogenous stimulus response is the most commonly approved prodrug activation strategy, which usually uses enzymes or microenvironmental differences between diseased tissues and normal tissues to activate prodrugs. For example, the thioketal (TK) bond is a commonly used ROS-responsive bond that can be cleaved under the action of ROS and has been widely used in the construction of endogenous ROS-stimulated responsive prodrugs. However, most studies have shown that the limited differences between diseased tissues and normal tissues often lead to insufficient specificity and selectivity of drugs, which in turn leads to poor therapeutic effects and is accompanied by serious side effects. Therefore, based on the TK bond, designing a prodrug with higher sensitivity, stronger selectivity, and the ability to release active drugs on demand in a spatiotemporally controllable manner is still a major problem that needs to be solved.

[0004] Exogenous stimulus-responsive prodrugs provide a more promising prodrug strategy. Ultrasound, as a non-invasive physical radiation source, has been used as an exogenous stimulus to activate ultrasound-responsive prodrugs in recent years. Compared with endogenous stimulation, ultrasound activation has many advantages. First, ultrasound has extremely high tissue penetration, usually >10 cm. More importantly, ultrasound can achieve spatiotemporally controllable point activation, is highly specific, and is not affected by individual differences. In the past decade, ultrasound-mediated bond cleavage has been successfully applied in prodrug development, providing a more promising prodrug development strategy for the preparation of highly tumor-selective, on-demand release of active drugs, and spatiotemporally controllable precision therapy.

[0005] As a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, resiquimod (R848) has been shown to have good anti-tumor activity in multiple clinical trials and is very effective in stimulating immune responses and inhibiting tumor growth. However, due to the lack of tumor selectivity, R848 will cause severe systemic toxicity after administration, resulting in its failure to achieve further clinical application. Therefore, it is urgent to develop a prodrug with high specificity and selectivity that releases active R848 on demand at the lesion site, while reducing drug toxicity and achieving selective activation of the drug at the tumor site. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a resiquimod prodrug and a preparation method and application thereof. The resiquimod prodrug can achieve selective activation of the drug at the tumor site while reducing drug toxicity.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] The present invention provides a resiquimod prodrug, which comprises a carrier and a riboflavin derivative loaded on the carrier;

[0009] The carrier is obtained by bonding R848-TK and mPEG-b-PHEA.

[0010] Preferably, the riboflavin derivative is selected from any one or more of riboflavin tetrabutyrate, riboflavin tetraacetate, riboflavin tetrapropionate, riboflavin 2',3',4',5'-tetra(decanoate), riboflavin 5'-butyrate, riboflavin 5'-acetate, riboflavin adamantanoate, riboflavin 5'-(hydrogen malonate) or riboflavin butyrate.

[0011] Preferably, the mass ratio of R848 to the riboflavin derivative in the carrier is 1:(1-10).

[0012] Preferably, the resiquimod prodrug has ultrasound activation properties.

[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned resiquimod prodrug, comprising the following steps:

[0014] After mixing the carrier and the riboflavin derivative in the presence of an organic solvent, the organic solvent is removed, and the obtained product is self-assembled in water to form micelles to obtain a resiquimod prodrug;

[0015] The carrier is obtained by bonding R848-TK and mPEG-b-PHEA.

[0016] Preferably, the organic solvent is selected from any one or more of chloroform, dichloromethane or methanol.

[0017] In a third aspect, the present invention also provides a use of a resiquimod prodrug in the preparation of an anti-tumor drug.

[0018] In a fourth aspect, the present invention further provides an anti-tumor drug, which comprises the above-mentioned resiquimod prodrug and a pharmaceutically acceptable adjuvant.

[0019] Preferably, the anti-tumor drug releases R848 in the resiquimod prodrug under ultrasonic conditions.

[0020] Preferably, the frequency of the ultrasound is 0.5-3 MHz; the power is 0-3 W / cm 2 .

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a resiquimod prodrug, which includes a carrier and a riboflavin derivative loaded on the carrier. The carrier is obtained by bonding R848-TK and mPEG-b-PHEA. The present invention uses R848 NPs as a carrier to physically load a fat-soluble riboflavin derivative to construct an ultrasound-activated resiquimod prodrug. The riboflavin derivative, as a fat-soluble substance, can be activated under ultrasound conditions to produce 1 O2 is used to cut the TK bond, thereby releasing the active drug R848. The present invention can effectively enhance the tumor selectivity of the drug and achieve time-space controllable precision treatment by preparing a more specific ultrasound-activated resiquimod prodrug. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The comparison chart of the breakage rate of TK-PrOH under different sonosensitizers;

[0024] Figure 2 Schematic diagram of the synthesis process of R848 / TBR NPs in Example 3;

[0025] Figure 3 The color comparison diagram of R848 NPs aqueous solution and R848 / TBR NPs solution;

[0026] Figure 4 is the UV-visible absorption spectrum of R848 / TBR NPs in Example 3;

[0027] Figure 5 is the hydrated particle size diagram of R848 / TBR NPs in Example 3;

[0028] Figure 6 The UV-visible absorption spectra of ICG and R848 / TBR NPs with different concentrations after ultrasonic mixing;

[0029] Figure 7 This is a schematic diagram of drug administration to mice in Application Example 1;

[0030] Figure 8 The tumor inhibition curves of different groups are shown;

[0031] Fig. 9 Figure 2 shows the tumor weights of different groups;

[0032] Fig.10 Figure 3 shows the body weight of mice in different groups. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Since single R848 lacks tumor selectivity, it will cause serious systemic toxicity after administration, resulting in its failure to achieve further clinical application. Therefore, this application is dedicated to preparing a prodrug that releases active R848 on demand at the lesion site, while reducing drug toxicity and achieving selective activation of the drug at the tumor site.

[0035] Based on this, the present invention provides a resiquimod prodrug, which includes a carrier and a sonosensitizer loaded on the carrier.

[0036] In the present invention, the carrier is R848 NPs, which is obtained by bonding R848 and mPEG-b-PHEA through an amide condensation reaction and an esterification reaction of a linker containing a ROS-responsive bond thioketal (TK) bond, respectively. The specific preparation method can be referred to patent 202311219082.4. To avoid complexity, this application will not be repeated.

[0037] In the preliminary experiment, the present invention studied the ultrasonic mixing of the above-mentioned types of sonosensitizers (including: hematoporphyrin methyl ether (HMME), Bengal rose red (RB), doxorubicin (DOX), IR780, riboflavin (Rf) and indocyanine green (ICG)) with the substrate TK-PrOH, and analyzed the cleavage rate of the substrate. The results show that after ultrasonic mixing with different types of sonosensitizers, the substrate shows different cleavage rates, which indicates that different types of sonosensitizers do have an effect on the cleavage efficiency of the TK bond, and among the above six sonosensitizers, Rf shows the highest substrate cleavage rate after ultrasound, so the present invention selects Rf for subsequent experiments. However, Rf itself is a water-soluble substance, which makes it difficult to achieve a higher loading efficiency. Therefore, in order to achieve a higher loading efficiency, the present invention preferably uses riboflavin derivatives, i.e., riboflavin esters, to construct ultrasound-activated resiquimod prodrugs by utilizing their fat solubility, and accurately releases the active drug R848 on the basis of achieving a higher loading rate.

[0038] In some embodiments of the present invention, the riboflavin derivative is selected from any one or more of riboflavin tetrabutyrate, riboflavin tetraacetate, riboflavin tetrapropionate, riboflavin 2',3',4',5'-tetra(decanoate), riboflavin 5'-butyrate, riboflavin 5'-acetate, riboflavin adamantanoate, riboflavin 5'-(hydrogen malonate) or riboflavin butyrate, preferably riboflavin tetrabutyrate, also known as riboflavin tetrabutyrate, abbreviated as: TBR.

[0039] It should be noted that too low an amount of riboflavin derivative may result in insufficient TK bond cleavage, thereby resulting in insufficient release of R848 and reduced drug efficacy, while too high an amount may require further optimization of the preparation method to ensure a higher loading efficiency. Therefore, the present invention preferably has a mass ratio of R848 to riboflavin derivative in the carrier of 1:(1-10), preferably 1:(3-5).

[0040] The present invention uses R848 NPs as a carrier to physically load fat-soluble riboflavin derivatives to construct ultrasound-activated resiquimod prodrugs. Among them, riboflavin derivatives, as a fat-soluble substance, can be activated under ultrasound conditions to produce 1 O2 is used to cut the TK bond, thereby releasing the active drug R848. The present invention can effectively enhance the tumor selectivity of the drug and achieve time-space controllable precision treatment by preparing a more specific ultrasound-activated resiquimod prodrug.

[0041] The present invention also provides a method for preparing the above-mentioned resiquimod prodrug, which comprises the following steps:

[0042] After the carrier and the riboflavin derivative are mixed in the presence of an organic solvent, the organic solvent is removed, and the obtained product is self-assembled in water to form micelles to obtain the resiquimod prodrug.

[0043] The organic solvent is selected from any one or more of chloroform, dichloromethane or methanol, preferably chloroform.

[0044] In some embodiments of the present invention, it is preferred that after the carrier and the riboflavin derivative are mixed in the presence of an organic solvent, the organic solvent is removed under vacuum conditions, and after the solvent is removed, a uniform thin film is formed on the wall of the container.

[0045] Then, according to the present invention, water is added to hydrate the film, and micelles, i.e., resiquimod prodrugs, are formed by self-assembly. The water is preferably deionized water, and the present invention has no particular limitation on the amount of water used, as long as the film is hydrated.

[0046] The present invention also provides a use of the above-mentioned resiquimod prodrug in preparing an anti-tumor drug.

[0047] The present invention also provides an anti-tumor drug, which comprises the above-mentioned resiquimod prodrug and a pharmaceutically acceptable adjuvant. The adjuvant is a substance well known to those skilled in the art.

[0048] The present invention also provides a method for using an anti-tumor drug, which comprises the following steps:

[0049] After the anti-tumor drug is injected into the body, it is treated with ultrasound.

[0050] In some specific embodiments of the present invention, 6 hours after the in vivo injection of the above-mentioned drug, the tumor site is subjected to ultrasound treatment.

[0051] The frequency of the ultrasonic treatment is 0.5-3 MHz, preferably 1 MHz; the power is 0-3 W / cm 2 , preferably 1~2W / cm 2 The duty cycle of the ultrasonic treatment is preferably 50%.

[0052] In the actual application process of the present invention, one course of treatment is preferably two days, and a total of three courses of treatment are continuous. According to the test, the anti-tumor drug shows a significant tumor inhibition rate under ultrasound treatment, and the tumor inhibition rate can reach 98.1%.

[0053] In order to further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention are all common commercially available products.

[0054] Example 1

[0055] This example investigates the cleavage efficiency of the substrate TK-PrOH containing a TK bond in vitro at different ultrasonic frequencies, ultrasonic powers and ultrasonic times in the absence of a sonosensitizer. The specific method is as follows:

[0056] The substrate TK-PrOH was fully dissolved in a mixed solution of methanol and water, and then the solution was ultrasonically treated under different ultrasonic conditions. The specific conditions are shown in Table 1. 1 The integration of the -CH3 peak at 1.48 ppm in the H NMR spectrum was used to determine the fragmentation rate of the substrate TK-PrOH.

[0057] The results are shown in Table 1 (the BLANK group is the NMR integral data of TK-PrOH in aqueous solution; the H2O2 group and groups 1 to 12 are all NMR integral data in 100 μM hydrogen peroxide solution.):

[0058] Table 1

[0059]

[0060] The results showed that when the ultrasonic frequency and ultrasonic power were fixed, the degree of substrate cleavage was positively correlated with the ultrasonic time. When the ultrasonic frequency and ultrasonic time were fixed, the degree of substrate cleavage was positively correlated with the ultrasonic power, while the fixed ultrasonic power was negatively correlated with the ultrasonic time. In short, long-term, high-power low-frequency ultrasound is more conducive to the cleavage of TK bonds (Table 1). However, in the absence of a sonosensitizer, the substrates all showed lower cleavage efficiency.

[0061] Therefore, the present invention further introduces a sonosensitizer, as shown in Example 2.

[0062] Example 2

[0063] This example explores the effects of different types of sonosensitizers (including hematoporphyrin methyl ether (HMME), rose Bengal (RB), doxorubicin (DOX), IR780, riboflavin (Rf) and indocyanine green (ICG)) on the cleavage efficiency of the TK bond. The specific method is as follows:

[0064] The above-mentioned types of sonosensitizers were mixed with substrate TK-PrOH and subjected to ultrasound. The concentrations of the above-mentioned types of sonosensitizers and substrate TK-PrOH were all 200 μM. The ultrasound conditions used were: 1 MHz, 2 W / cm 2 , 10 min, 50% duty cycle, and the fragmentation rate of the substrate was analyzed.

[0065] The test results are as follows Figure 1 As shown in Table 2:

[0066] Table 2

[0067]

[0068] As shown in Table 2, after being mixed with different types of sonosensitizers and subjected to ultrasound, the substrates showed different cleavage rates, which indicates that different types of sonosensitizers do have an effect on the cleavage efficiency of the TK bond. Among the six sonosensitizers mentioned above, Rf showed the highest substrate cleavage rate after ultrasound ( Figure 1 ), so the present invention uses Rf for subsequent experiments.

[0069] Example 3

[0070] This embodiment provides a resiquimod prodrug (abbreviated as: R848 / TBR NPs), and the schematic diagram of its synthesis is as follows Figure 2 As shown, the specific method is as follows:

[0071] First, according to patent 202311219082.4, R848 nanoparticles, referred to as R848 NPs, are prepared. The specific structure of the R848 NPs is shown in Formula 2:

[0072]

[0073] Formula 2;

[0074] Then, R848 NPs (100 mg) and riboflavin tetrabutyrate (TBR, 30 mg) were fully dissolved in CHCl3 (10 mL), and then the solvent was removed under vacuum conditions. A uniform film was formed on the surface of the solid product, and then deionized water was added to hydrate the film to form micelles through self-assembly. The micelles were filtered through a 0.22 μm aqueous phase filter membrane to remove insoluble matter, and then freeze-dried for 48 h to obtain ultrasound-activated resiquimod prodrug (R848 / TBR NPs).

[0075] In the present invention, the aqueous solution of the R848 NPs is colorless and transparent. After the introduction of TBR, the obtained R848 / TBR NPs has a light yellow solution ( Figure 3 ).

[0076] The successful synthesis of R848 / TBR NPs was confirmed by UV-visible absorption spectroscopy. The characteristic peaks of R848 appeared at 313 nm and 327 nm, and the characteristic peaks of TBR appeared at 375 nm and 444 nm, respectively. The molar ratio of R848 to TBR was calculated to be 1:1.5 ( Figure 4 ).

[0077] Furthermore, the hydrated particle size of R848 NPs was measured by DLS to be approximately 170 nm ( Figure 5 ).

[0078] The present invention also uses ICG as 1 O2 probe was used to detect the R848 / TBR NPs in vitro 1 O2 production capacity, the UV results showed that after being mixed with different concentrations of R848 / TBR NPs and ultrasonically, the UV absorption value of ICG decreased significantly, proving that R848 / TBR NPs can effectively produce 1 O2( Figure 6 ).

[0079] Application Example 1

[0080] In this application example, the prepared ultrasound-activated resiquimod prodrug (R848 / TBR NPs) was combined with ultrasound to study the anti-tumor effect on the CT26 tumor model.

[0081] In order to construct a CT26 subcutaneous tumor model, the present invention inoculated CT26 tumor cells into the abdomen of BALB / C mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). When the tumor volume reached about 150 mm 3 At the same time, the mice were randomly divided into 9 groups: PBS, US, TBR, TBR+US, R848 NPs, R848 / TBR NPs, R848 NPs+US, R848 NPs+TBR+US, and R848 / TBR NPs+US, with 5 mice in each group. Each mouse in each group was injected with R848 NPs (eq. to R848 1.5 mg / kg) or R848 / TBR NPs (eq. to R848 1.5 mg / kg) or TBR (4.8 mg / kg) through the tail vein once every other day (0, 2, 4 days). Specifically, the mice in the PBS group were injected with PBS (200 μL each), the mice in the TBR group and the TBR+US group were injected with TBR, the mice in the R848 NPs and R848 NPs+US group were injected with R848 NPs, the mice in the R848 NPs+TBR+US group were injected with a mixed solution of R848 NPs and TBR, the mice in the R848 / TBR NPs and R848 / TBR NPs+US groups were injected with R848 / TBRNPs, and 6 hours after the tail vein injection of the above drugs, the tumor site was treated with ultrasound (1 MHz, 2 W / cm 2 , 50% duty cycle, 2 min), one course of treatment for two days, a total of 3 courses of treatment ( Figure 7 ).

[0082] Tumor volume was recorded every two days, and the results showed that the PBS group showed the fastest tumor growth rate, with the tumor volume reaching approximately 1500 mm on day 12.3 , R848 / TBR NPs+US showed a significant tumor inhibition rate of up to 98.1% ( Figure 8 On day 12, the mice were euthanized and the tumors were collected and weighed. It was observed that the tumor weight of mice treated with R848 / TBRNPs+US was significantly reduced ( Fig. 9 ), and the mice did not show significant weight loss during the treatment ( Fig.10 ).

[0083] The above results show that the R848 / TBR NPs+US group can significantly inhibit the growth of CT26 tumors. The main reason is that TBR can be activated under ultrasound conditions to produce a large amount of 1 O2 induces tumor cell apoptosis and simultaneously cuts the TK bond, releasing R848, effectively inhibiting tumor growth.

[0084] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A resiquimod prodrug, characterized in that It comprises a carrier and a riboflavin derivative loaded on the carrier; The carrier is composed of R848-TK and mPEG- b -PHEA bonding is obtained.

2. The resiquimod prodrug according to claim 1, characterized in that The riboflavin derivative is selected from any one or more of riboflavin tetrabutyrate, riboflavin tetraacetate, riboflavin tetrapropionate, riboflavin 2',3',4',5'-tetra(decanoate), riboflavin 5'-butyrate, riboflavin 5'-acetate, riboflavin adamantanoate, riboflavin 5'-(hydrogen malonate) or riboflavin butyrate.

3. The resiquimod prodrug according to claim 1 or 2, characterized in that The mass ratio of R848 to the riboflavin derivative in the carrier is 1:(1-10).

4. The resiquimod prodrug according to any one of claims 1 to 3, characterized in that The resiquimod prodrug has ultrasound activation properties.

5. A method for preparing a resiquimod prodrug as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: After mixing the carrier and the riboflavin derivative in the presence of an organic solvent, the organic solvent is removed, and the obtained product is self-assembled in water to form micelles to obtain a resiquimod prodrug; The carrier is composed of R848-TK and mPEG- b -PHEA bonding is obtained.

6. The preparation method according to claim 5, characterized in that: The organic solvent is selected from any one or more of chloroform, dichloromethane or methanol.

7. Use of the resiquimod prodrug according to any one of claims 1 to 4 or the resiquimod prodrug prepared by the preparation method according to claim 5 or 6 in the preparation of antitumor drugs.

8. An anti-tumor drug, characterized in that: The invention comprises the resiquimod prodrug according to any one of claims 1 to 4 or the resiquimod prodrug prepared by the preparation method according to claim 5 or 6, and a pharmaceutically acceptable adjuvant.

9. The anti-tumor drug according to claim 8, characterized in that: The anti-tumor drug releases R848 in the resiquimod prodrug under ultrasonic conditions.

10. The anti-tumor drug according to claim 9, characterized in that: The frequency of the ultrasound is 0.5-3 MHz; the power is 0-3 W / cm 2 .

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