Liposome for detecting drug release in plasma and application thereof in predicting ABC phenomenon of PEG drug

Abstract

The invention provides lipidosome for detecting drug release in plasma and application of the lipidosome in predicting the ABC phenomenon of a PEG drug. Researches find that the plasma drug release amount measured by the PEGylated liposome of the hydrophilic camptothecin derivative prodrug provided by the invention is related to the ABC phenomenon of a PEG drug, the method can be used for predicting the ABC phenomenon of the PEG drug, the method does not need to measure the concentration of an anti-PEG antibody in plasma, and the effect of predicting the ABC phenomenon of the PEG liposome is superior to that of the anti-PEG antibody. Compared with other liposome drug release determination methods, the method does not need additional steps to separate liposome encapsulated drugs and release drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis and relates to the prediction of the accelerated blood clearance (ABC) phenomenon of PEG drugs, and particularly relates to a liposome capable of detecting drug release in plasma and its application in predicting the ABC phenomenon of PEG drugs. Background Art

[0002] Accelerated blood clearance (ABC) refers to the phenomenon of rapid clearance of polyethylene glycol (PEG) liposomes from the blood circulation after repeated administration. The process is as follows: PEGylated liposomes activate reactive B cells in the marginal zone of the spleen to produce anti-PEG IgM, which forms immune complexes with the re-injected PEGylated liposomes, activates the complement system, and accelerates the uptake of PEGylated liposomes by macrophages through complement receptors, resulting in rapid clearance of PEGylated liposomes. Anti-PEG antibodies are widely found in healthy people who have not been exposed to PEG drug treatment. In one study, 43.1% of 2404 healthy people tested positive for anti-PEG antibodies by ELISA (see Chen, BM et al., Acs Nano, 2021.15(9): p.14022-14048). This may be due to the widespread use of daily products and vaccines containing PEG, such as soaps, cosmetics, sunscreens, and foods. It is reported that pre-existing anti-PEG antibodies in mouse models can cause a strong ABC phenomenon in PEGylated liposomal doxorubicin, thereby reducing the tumor targeting and anti-tumor activity of liposome drugs. In addition, anti-PEG antibodies can also lead to the rapid clearance of PEG protein drugs, thereby reducing the therapeutic effect of PEG protein drugs (see Nishio, A et al. Science Translational Medicine, 2021.13(587)). Therefore, pre-existing anti-PEG antibodies will reduce the efficacy of existing PEG drugs, which has caused widespread concern about the effectiveness of PEG drugs.

[0003] Predicting the ABC phenomenon is a viable approach to preventing clinical failure of PEGylated liposome drugs. Anti-PEG antibody testing is a conventional method for predicting ABC. ELISA, with its high sensitivity, reliability, and quantitative detection capabilities, is widely used to measure anti-PEG antibody levels. However, because the affinity of standard antibodies differs from that of anti-PEG antibodies in vivo, and the affinity of standard antibodies varies between laboratories, ELISA testing can only provide relative values ​​of antibody content, not absolute concentrations. Furthermore, the antibody-PEG binding mechanism and affinity in ELISA differ from the binding of antibodies to PEGylated liposomes in plasma. Therefore, conventional ELISA methods have difficulty establishing a correlation between antibody concentration and ABC phenomenon and using it to predict ABC. New methods for predicting ABC in PEGylated drugs are urgently needed.

[0004] In summary, there is currently no method that can effectively predict the ABC phenomenon of PEG drugs. Therefore, developing new ABC phenomenon prediction methods to overcome the bottleneck of the current lack of unified quantitative standards for anti-PEG antibody detection will effectively promote the rational application of PEG drugs in clinical practice. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention provides a hydrophilic camptothecin derivative prodrug and a PEGylated liposome encapsulating the hydrophilic camptothecin derivative prodrug. The liposome is used to quickly detect the amount of liposome drug released in plasma, which can effectively predict the ABC phenomenon of PEG drugs. Figure 1 As shown: After PEGylated liposomes encapsulating a hydrophilic camptothecin derivative prodrug are mixed with plasma, anti-PEG antibodies in the plasma bind to PEG and activate complement, forming membrane attack complex pores on the liposome membrane. As a result, the hydrophilic camptothecin derivative is released from the liposome into the plasma through the pores and rapidly degraded into the camptothecin prototype drug. By measuring the content of the camptothecin prototype drug, the drug release amount from the liposome can be calculated. Since the drug release amount is proportional to the ABC phenomenon of PEG drugs, it can be used to predict the ABC phenomenon of PEG drugs.

[0006] Unless otherwise specified, all parts described in the present invention are parts by weight and all percentages described are mass percentages.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] A hydrophilic camptothecin derivative prodrug having the following structural formula:

[0009]

[0010] R is H or CH2CH3, n=1-10.

[0011] Furthermore, the hydrophilic camptothecin derivative prodrug has the following structure:

[0012]

[0013] R is H or CH2CH3.

[0014] Preferably, the hydrophilic camptothecin derivative prodrug has the following structure:

[0015]

[0016] The present invention also provides PEGylated liposomes encapsulating hydrophilic camptothecin derivative prodrugs.

[0017] The PEGylated liposome containing a hydrophilic camptothecin derivative prodrug comprises the above-mentioned hydrophilic camptothecin derivative prodrug and PEGylated phospholipid.

[0018] The PEGylated phospholipid is a conjugate of PEG and DSPE connected by an amide bond, preferably DSPE-mPEG 2000 or DSPE-mPEG 5000 .

[0019] The above-mentioned liposomes also include phospholipids, which are natural, semi-synthetic and fully synthetic phospholipids such as egg yolk phosphatidylcholine (EPC), soybean lecithin, sphingomyelin, hydrogenated soybean lecithin (HSPC), distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), etc., preferably hydrogenated soybean lecithin HSPC or DSPC.

[0020] The above-mentioned liposomes also include cholesterol.

[0021] The present invention also provides the application of the hydrophilic camptothecin derivative prodrug in predicting the ABC phenomenon of PEG drugs.

[0022] The present invention also provides the application of PEGylated liposomes encapsulating hydrophilic camptothecin derivative prodrugs in predicting the ABC phenomenon of PEG drugs.

[0023] The application method of the hydrophilic camptothecin prodrug-encapsulated PEG liposomes described in all of the above items to predict the ABC phenomenon of PEG drugs is as follows: human plasma or human serum is added to an EP tube, and hydrophilic camptothecin derivative prodrug PEG liposomes diluted with physiological saline are added. After mixing evenly, the mixture is incubated at 38°C for 10-20 minutes (min). Methanol containing 1% acetic acid (or 0.1% trifluoroacetic acid) is added, and the mixture is shaken for 10 minutes. After centrifugation at 10,000 rpm for 10 minutes, the supernatant is collected for high-performance liquid chromatography analysis. By measuring the ratio of the camptothecin derivative prototype and the camptothecin derivative prodrug, the drug release ratio of the hydrophilic camptothecin derivative prodrug PEG liposomes is determined, and then the ABC phenomenon of the PEG drug is predicted.

[0024] Beneficial effects:

[0025] The plasma drug release measured from PEGylated liposomes of hydrophilic camptothecin derivative prodrugs provided by the present invention correlates with the ABC phenomenon of PEG drugs and can be used to predict the ABC phenomenon of PEG drugs. This method does not require measuring the concentration of anti-PEG antibodies in plasma and is more effective in predicting the ABC phenomenon of PEG liposomes than anti-PEG antibodies. Compared to other liposome drug release assays, this method does not require the additional step of separating the liposome-encapsulated drug from the released drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of SN38-GSH liposomes for measuring plasma drug release, where MAC is the membrane attack complex mediated by anti-PEG antibodies;

[0027] Figure 2 This is the high-resolution mass spectrum of SN38-GSH;

[0028] Figure 3 This is the liquid phase analysis spectrum of SN38-GSH generated by the reaction of SN38-MAL and GSH liposomes;

[0029] Figure 4 is the particle size distribution and electron microscopy image of SN38-GSH liposomes;

[0030] Figure 5 is the drug release curve of SN38-GSH liposomes and free SN38-GSH in healthy rat plasma;

[0031] Figure 6 The drug release curves (A) and HPLC analysis spectra (B) of SN38-GSH liposomes in rat immune plasma and healthy rat plasma;

[0032] Figure 7Figure 1 shows the effects of plasma treatment (2 mg / ml PEG (A), heating at 56°C for 30 min (B), and 10 mM EDTA (C)) on plasma ADR in rats immunized with SN38-GSH liposomes.

[0033] Figure 8 This is the result of the precision determination of SN38-GSH liposome in the ADR detection of immunized rat plasma;

[0034] Figure 9 This is the result graph showing the effect of SN38-GSH liposome concentration on the accelerated release of plasma liposome drugs;

[0035] Figure 10 This is a graph showing the changes in ADR in the plasma of immunized rats at different time points;

[0036] Figure 11 This is a graph showing the correlation between ADR and ABC phenomena at different times after rat immunization;

[0037] Figure 12 This is a graph showing the correlation between ADR and ABC phenomenon in rat plasma after multiple PEG immunizations;

[0038] Figure 13 This is a graph showing the correlation between the ABC phenomenon and ADR in rats immunized with different doses of PEG liposomes;

[0039] Figure 14 The graph shows the correlation between the ABC phenomenon of SN38-GSH liposomes and ADR (A), anti-PEG IgM (B) and IgG (C) in rats immunized with different PEG excipients.

[0040] Figure 15 The graph shows the correlation between the ABC phenomenon of DOXIL liposomes and the accelerated drug release phenomenon (A), anti-PEG IgM (B) and IgG (C);

[0041] Figure 16 Statistical chart of ADR (A) and its incidence (B) in human plasma. DETAILED DESCRIPTION

[0042] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention is further described below by specific embodiments. It should be understood that these embodiments are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention. Unless otherwise specified, the parts described in the present invention are all by weight and the percentages are all by mass.

[0043] Example 1

[0044] The present invention is only described by way of example in the following embodiments. However, after reading this patent application, those skilled in the art may make various modifications to the present invention without departing from the spirit and scope of the present invention.

[0045] Related nouns:

[0046] Accelerated drug release (ADR) refers to the drug release rate of hydrophilic camptothecin derivative prodrug PEGylated liposomes induced by unit volume of plasma.

[0047] The area under the plasma drug concentration-time (0-24h) curve (AUC 0-24h ), refers to the integrated area under the plasma SN38-GSH concentration versus time curve from 0 to 24 h after intravenous injection of SN38-GSH liposomes.

[0048] Example 1. Synthesis of SN38-GSH

[0049] 1 g of 3-maleimidopropionic acid was dissolved in 40 ml of dichloromethane (DCM), and dicyclohexylcarbodiimide (DCC, 1.2 eq.) and 2 ml of ethylene glycol were added. After stirring for 5 min, 50 mg of DMAP was added and the reaction was stirred at room temperature for 2 h. The reaction solution was spin-dried, ethyl acetate was added, and the filtrate was filtered and spin-dried. The product was then separated using a silica gel column to obtain the conjugate of MAL and ethylene glycol (MAL-2C-OH). SN38 was then weighed into a reaction flask, vacuumed for 5 min, dissolved in anhydrous DCM, and filled with nitrogen. Under stirring, accurately weighed triphosgene (BTC, 0.35 eq.) and DMAP (2.0 eq.) were dissolved in anhydrous DCM and added to the reaction flask sequentially via syringe. The reaction was stirred for 5 min. Accurately weighed MAL-2C-OH was added to the reaction flask. The reaction was stirred for 30 min. After the reaction was confirmed to be complete by thin-layer chromatography, the reaction solution was poured into a separatory funnel, washed once with saturated citric acid and saturated sodium chloride solution, and the organic phase was collected. The aqueous phase was washed three times with DCM and then discarded. The organic phase was dehydrated with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and applied to a large silica gel plate to separate SN38-MAL. GSH was dissolved in 100mM pH 5.0 phosphate buffer (5mg / ml), and SN38-MAL was dissolved in a mixed solvent of acetonitrile and dimethyl sulfoxide (9:1, v:v) (10mg / ml). After mixing equal volumes of GSH solution and SN38-MAL solution, the mixture was placed in a 60°C water bath and incubated for 10min to obtain hydrophilic SN38-GSH. The product was identified by high-resolution mass spectrometry ( Figure 2 ), confirming the successful synthesis.

[0050]

[0051] Example 2: Preparation of PEGylated liposomes encapsulating hydrophilic camptothecin derivative prodrugs (taking SN38-GSH liposomes as an example)

[0052] 42mg HSPC, 20mg cholesterol and 13.5mg mPEG2000-DSPE were weighed and dissolved in 0.5ml ethanol; the above phospholipid solution was then added dropwise to 5ml 400mM glutathione (GSH) aqueous solution to obtain crude liposomes. The liposomes were repeatedly extruded through a polycarbonate membrane with a pore size of 80nm, and the resulting liposomes were repeatedly dialyzed 7 times in a 150mM NaCl solution to finally obtain GSH liposomes. SN38-MAL was then dissolved in a mixed solvent of acetonitrile and dimethyl sulfoxide (9:1, v:v) at a concentration of 10mg / ml, 100μl of SN38-MAL solution was added dropwise to 1ml of GSH liposomes, and incubated at 60°C for 5min under stirring to obtain SN38-GSH liposomes. The particle size of the SN38-GSH liposomes was determined by laser scattering, and the particle size of the liposomes was observed by dialysis electron microscopy. As shown in FIG. Figure 3 As shown, SN38-MAL reacts with GSH in liposomes to generate SN38-GSH; Figure 4 As shown, the average particle size of the obtained SN38-GSH liposomes was 113.6 nm, and SN38-GSH drug crystals were visible in the liposomes, indicating that SN38-GSH was successfully encapsulated in the liposomes.

[0053] Example 3: Drug release study of SN38-GSH liposomes and free SN38-GSH in plasma

[0054] 25 μl of healthy rat plasma was collected and placed in a 0.5 ml EP tube. SN38-GSH liposomes and free SN38-GSH were added to the tubes. At 1, 3, 5, and 12 hours, 2.5 μl of plasma was added to 200 μl of methanol containing 1% acetic acid. The mixture was ultrasonicated in a water bath for 1 minute and centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected and the concentrations of SN38-GSH and SN38 were determined by HPLC. Figure 5 As shown in the figure, free SN38-GSH is rapidly degraded in plasma, while SN38-GSH in liposomes is not degraded, indicating that SN38-GSH can be rapidly degraded into the prototype drug after being released from liposomes. Therefore, the release rate of SN38 can be calculated by the formula SN38 / (SN38-GSH+SN38).

[0055] Example 4: Drug release study of SN38-GSH liposomes in PEG-immunized rat plasma and normal plasma

[0056] Rats were immunized with SN38-GSH liposomes (0.3 mg / kg, phospholipid concentration) by subcutaneous injection. Seven days later, 500 μl of rat plasma was collected. 500 μl of plasma from healthy rats (not PEG-immunized) was used as control plasma. 200 μl of plasma from PEG-immunized rats and 200 μl of normal plasma were added to SN38-GSH liposomes and the SN38 concentration was 1 μg / ml. The cells were incubated in a 38°C water bath for different times. Then, 2.5 μl of plasma was added to 200 μl of methanol containing 1% acetic acid, sonicated in a water bath for 1 minute, and centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected and the concentrations of SN38-GSH and SN38 were determined by HPLC. The release rate of SN38 was calculated using the formula SN38 / (SN38-GSH+SN38). HPLC analysis used a C18 column with a mobile phase of 15% 0.1% TFA aqueous solution and 85% methanol at a flow rate of 1.0 ml / min. Detection was performed using a fluorescence detector with an excitation wavelength of 353 nm and an emission wavelength of 535 nm. The results showed that in the plasma of PEG-immunized rats, SN38-GSH liposomes released SN38 rapidly; whereas in the plasma of healthy rats, SN38-GSH liposomes released SN38 slowly. Figure 6 Heating the plasma at 56°C for 30 min completely inhibited ADR, and adding EDTA also completely inhibited ADR, indicating that ADR was caused by complement activation; adding free PEG could now inhibit ADR in the plasma of PEG-immunized rats, indicating that ADR was induced by anti-PEG antibodies combined with PEG liposomes ( Figure 7 ); In addition, the RSD of ADR detection was less than 5% ( Figure 8 ); The above results indicate that anti-PEG antibodies can bind to PEG liposomes and activate complement to induce ADR, while SN38-GSH liposomes can effectively detect ADR.

[0057] Example 6: Study on the effect of SN38-GSH liposome concentration on its plasma drug release

[0058] Rats were immunized with subcutaneous injection of SN38-GSH liposomes (0.3 mg / kg). Seven days later, 500 μl of rat plasma was collected to determine the ADR of SN38-GSH liposomes in the plasma. The determination method is as follows: 25 μl of the above plasma was placed in a 0.5 ml EP tube, and SN38-GSH liposomes were added to make the concentrations 1 μg / ml, 5 μg / ml and 25 μg / ml respectively; after mixing evenly, incubate at 38°C for 1, 2, 3, 5 and 10 minutes respectively, take 2.5 μl and add 200 μl of 200 μl of methanol containing 1% acetic acid. The sample was sonicated in a water bath for 1 minute and centrifuged at 10,000 rpm for 5 minutes, and then the supernatant was collected. The concentrations of SN38-GSH and SN38 were determined by HPLC, and the release ratio of SN38 was calculated by the formula SN38 / (SN38-GSH+SN38). As Figure 9 As shown in the figure, as the liposome concentration increases, the liposome drug release ratio gradually decreases, indicating that the quantitative determination of drug release requires a fixed liposome concentration, indicating that the liposome plasma drug release amount can be quantitatively determined by fixing the liposome concentration.

[0059] Example 7: Development of the phenomenon of accelerated drug release from SN38-GSH liposomes

[0060] Rats were immunized subcutaneously with SN38-GSH liposomes (0.3 mg / kg). Plasma was collected 1-6 days and 1-6 weeks after immunization. The ADR of SN38-GSH liposomes in the plasma was determined as follows: 25 μl of the plasma was added to a 0.5 ml EP tube, followed by SN38-GSH liposomes at a concentration of 1 (or 5 and 25) μg / ml. After mixing, the tube was incubated at 38°C for 20 minutes, followed by the addition of 200 μl of methanol containing 1% acetic acid. The sample was sonicated in a water bath for 1 minute and centrifuged at 10,000 rpm for 5 minutes. The supernatant was then collected and the concentrations of SN38-GSH and SN38 were determined by HPLC. The release rate of SN38 was calculated using the formula SN38 / (SN38-GSH+SN38). If the release rate of SN38 was greater than 70%, the concentration of SN38-GSH liposomes was increased to 5 μg / ml, and the drug release ratio was determined to determine the maximum ADR of the plasma sample. Figure 10 As shown in the figure, the ADR phenomenon of rat immune plasma appeared on the 4th day, reached the peak on the 7th day, and then gradually decreased in the following weeks.

[0061] Example 8: Correlation between SN38-GSH liposome ADR and ABC phenomenon at different times after immunization in rats

[0062] Rats were immunized with SN38-GSH liposomes (0.3 mg / kg) by subcutaneous injection, and the pharmacokinetic experiments of SN38-GSH liposomes were conducted on days 7, 28, and 56, respectively. The specific method was as follows: SN38-GSH liposomes were injected intravenously at a dose of 3 mg / kg, and 100 μl of rat blood was collected at 5 minutes, 0.5, 1, 2, 4, 8, and 24 hours, respectively. The rat blood was centrifuged for 5 minutes (5000 rpm) and the rat plasma was collected; 10 μl of rat plasma was taken, 490 μl of methanol containing 1% acetic acid was added, the sample was ultrasonicated in a water bath for 1 minute, centrifuged at 10000 rpm for 5 minutes, and then the supernatant was collected. SN38-GSH was determined by HPLC, and the blood drug concentration-time area under 0-24 hours (AUC 0-24h ) was used as an indicator to evaluate the ABC phenomenon in rats. Before the pharmacokinetic experiment, blank plasma was collected, the plasma drug ADR was determined, and the correlation between ADR and ABC phenomenon was analyzed. Figure 11 As shown, the plasma ADR at different times after rat immunization was proportional to the ABC phenomenon of PEG liposomes, indicating that the ADR measured by SN38-GSH liposomes can predict the ABC phenomenon.

[0063] Example 9: Correlation between SN38-GSH liposome ADR and ABC phenomenon in rats immunized with multiple PEG

[0064] Rats were first immunized with subcutaneous injection of SN38-GSH liposomes (0.3 mg / kg). The rats were immunized twice and three times 2 and 4 months later, respectively. 7 days after each immunization, the pharmacokinetic experiment of SN38-GSH liposomes was conducted. The dose of SN38-GSH liposomes was 3 mg / kg. The sampling time points were 5 min, 0.5, 1, 2, 4, 8 and 24 h. At each time point, 10 μl of rat plasma was collected and 490 μl of methanol containing 1% acetic acid was added. The sample was ultrasonicated in a water bath for 1 min and centrifuged at 10,000 rpm for 5 min. The supernatant was then collected and the SN38-GSH was determined by HPLC. The blood drug concentration-time area (AUC 0-24 h) was used as the plot. 0-24h ) was used as an indicator to evaluate the ABC phenomenon in rats. Before each pharmacokinetic experiment, blank plasma was collected, the ADR of plasma was measured, and the correlation between ADR and ABC phenomenon was analyzed. Figure 12 As shown, the plasma ADR of rats immunized with multiple PEG liposomes was proportional to the ABC phenomenon of PEG liposomes, indicating that the ADR of SN38-GSH liposomes can predict the ABC phenomenon.

[0065] Example 10: Correlation between the ADR of SN38-GSH liposomes and the ABC phenomenon in rats immunized with different doses of liposomes

[0066] Rats were immunized with PEG by intravenous injection of SN38-GSH liposomes at doses of 0.01, 0.1, 0.3, and 3 mg / kg, respectively. Seven days later, a pharmacokinetic study of SN38-GSH liposomes was conducted. The dose of SN38-GSH liposomes was 3 mg / kg, and the sampling time points were 5 min, 0.5, 1, 2, 4, 8, and 24 h. At each time point, 10 μl of rat plasma was collected, 490 μl of methanol containing 1% acetic acid was added, the sample was sonicated in a water bath for 1 min, centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. SN38-GSH was determined by HPLC, and the blood drug concentration-time area under the time (AUC 0-24 h) was used. 0-24h ) was used as an indicator to evaluate the ABC phenomenon in rats. Before each pharmacokinetic experiment, blank plasma was collected, the ADR of plasma was measured, and the correlation between ADR and ABC phenomenon was analyzed. Figure 13 As shown, the plasma ADR of rats immunized with different doses was proportional to the ABC phenomenon of PEG liposomes, indicating that the ADR of SN38-GSH liposomes can predict the ABC phenomenon.

[0067] Example 11: The role of SN38-GSH liposome ADR in predicting the ABC phenomenon in rats immunized with PEG-containing pharmaceutical excipients

[0068] Rats were immunized with PEG by intravenous injection of 100 mg / kg of polyoxyethylene castor oil (CrE), Tween 80 (Tween80), and poloxamer F68, respectively. Seven days later, a pharmacokinetic study of SN38-GSH liposomes was conducted. The dose of SN38-GSH liposomes was 0.3 mg / kg, and the sampling time points were 5 min, 0.5, 1, 2, 4, 8, and 24 h. At each time point, 10 μl of rat plasma was collected, 490 μl of methanol containing 1% acetic acid was added, the sample was sonicated in a water bath for 1 min, centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. SN38-GSH was determined by HPLC, and the blood drug concentration-time area under the time (AUC 0-24 h) was used. 0-24h ) as an indicator to evaluate the ABC phenomenon in rats. Before each pharmacokinetic experiment, blank plasma was collected, the plasma ADR was determined, and the correlation between the accelerated drug release and the ABC phenomenon was analyzed. At the same time, the levels of anti-PEG IgM and IgG in plasma were determined, and their correlation with the ABC phenomenon was analyzed. Figure 14 As shown in the data, the plasma ADR of rats immunized with different PEG pharmaceutical excipients was proportional to the ABC phenomenon of PEG liposomes, and the correlation was better than the correlation between PEG antibodies and the ABC phenomenon, indicating that the accelerated release ratio of SN38-GSH liposome drugs can predict the ABC phenomenon, and the prediction effect is better than that of PEG antibodies.

[0069] Example 12: The role of SN38-GSH liposome ADR in predicting the clinical ABC phenomenon of doxorubicin liposomes (DOXIL)

[0070] Rats were immunized with PEG by intravenous injection of SN38-GSH liposomes at doses of 0.1 and 0.3 mg / kg, respectively. Seven days later, a pharmacokinetic experiment of DOXIL was conducted. The dose of doxorubicin was 3 mg / kg, and the sampling time points were 5 min, 0.5, 1, 2, 4, 8 and 24 h. 50 μl of rat plasma was collected at each time point, 200 μl of methanol was added, the sample was ultrasonicated in a water bath for 1 min, centrifuged at 10,000 rpm for 5 min, and then the supernatant was collected. The concentration of doxorubicin was determined by HPLC. HPLC analysis used a C18 column with a mobile phase of 30% 0.1% trifluoroacetic acid and 70% methanol at a flow rate of 1.0 ml / min. A fluorescence detector was used for detection with an excitation wavelength of 470 nm and an emission wavelength of 555 nm. The area under the blood drug concentration-time curve (AUC 0-24 h) was used. 0-24h ) was used as an indicator to evaluate the ABC phenomenon in rats. Before each pharmacokinetic experiment, blank plasma was collected, the ADR of the plasma was determined, and the correlation between ADR and ABC phenomenon was analyzed. At the same time, the levels of anti-PEG IgM and IgG in the plasma were determined, and their correlation with ABC phenomenon was analyzed. Figure 15 As shown in the results, ADR is proportional to the DOXILABC phenomenon, and its correlation is better than the correlation between PEG antibody and ABC phenomenon, indicating that the ADR measured by SN38-GSH liposomes can predict the ABC phenomenon of clinical DOXIL, and the prediction effect is better than that of PEG antibody.

[0071] Example 13: Study on the incidence of ADR of SN38-GSH liposomes in human plasma

[0072] Plasma was collected from the physical examination population, and the ADR of SN38-GSH liposomes in the plasma was determined. The determination method was as follows: 25 μl of plasma was placed in a 0.5 ml EP tube, and SN38-GSH liposomes were added to a concentration of 1, 5 or 25 μg / ml; after mixing evenly, the mixture was incubated at 38°C for 20 minutes, and then 200 μl of methanol containing 1% acetic acid was added. The sample was ultrasonicated in a water bath for 1 minute, centrifuged at 10,000 rpm for 5 minutes, and then the supernatant was collected. The concentrations of SN38-GSH and SN38 were determined by HPLC, and the release rate of SN38 was calculated by the formula SN38 / (SN38-GSH+SN38), and the ADR was calculated. Figure 16As shown in the data, about 5% of the plasma in the population had a weak liposome ADR phenomenon (20-200%), and about 0.28% of the plasma had a strong liposome ADR phenomenon (greater than 200%), indicating that PEG liposomes may experience ABC phenomenon during clinical use, thereby reducing their efficacy.

Claims

1. A hydrophilic camptothecin derivative prodrug having the following structural formula: R is H or CH2CH3, n=1-10.

2. The hydrophilic camptothecin derivative prodrug according to claim 1, wherein Has the following structure: R is H or CH2CH3.

3. The hydrophilic camptothecin derivative prodrug according to claim 1, wherein Has the following structure:

4. PEGylated liposomes encapsulating a hydrophilic camptothecin derivative prodrug, characterized in that: The invention comprises the hydrophilic camptothecin derivative prodrug according to any one of claims 1 to 3 and PEGylated phospholipid.

5. The PEGylated liposome according to claim 4, wherein The PEGylated phospholipid is a conjugate of PEG and DSPE connected via an amide bond.

6. The PEGylated liposome of claim 4, wherein The PEGylated phospholipid is DSPE-mPEG 2000 or DSPE-mPEG 5000 .

7. The PEGylated liposome of claim 4, wherein The liposomes further comprise phospholipids, which are natural phospholipids, semi-synthetic phospholipids or fully synthetic phospholipids; preferably egg yolk phosphatidylcholine (EPC), soybean lecithin, sphingomyelin, hydrogenated soybean phospholipids (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC); more preferably hydrogenated soybean phospholipids HSPC and DSPC.

8. The PEGylated liposome of claim 4, wherein The liposomes also include cholesterol.

9. Use of the hydrophilic camptothecin derivative prodrug according to any one of claims 1 to 3 in predicting the ABC phenomenon of PEG drugs.

10. Use of the PEGylated liposomes encapsulating a hydrophilic camptothecin derivative prodrug as claimed in any one of claims 4 to 8 in predicting the ABC phenomenon of PEG drugs.

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