TWI453034B - Pharmaceutical compositions for treating tumors and use thereof - Google Patents

Description

Medicinal composition for treating tumors and use thereof

The present invention relates to a pharmaceutical composition for treating a tumor using a polymeric micelle that encapsulates an antitumor drug. In one embodiment, the polymeric micelle comprises a block copolymer comprising at least one hydrophilic block, at least one hydrophobic block, and at least one amphoteric Ions (zwitterions). The anti-tumor drug is, for example, hydrophobic. The invention also relates to methods of enhancing the solubility of anti-tumor drugs, methods of increasing the blood circulation time of anti-tumor drugs, and methods of delivering anti-tumor drugs to one or more solid tumors.

Many anti-tumor drugs are hydrophobic and therefore have limited solubility in aqueous media. For example, camptothecin (CPT), an inhibitor of DNA topoisomerase I, has been shown to be a therapeutic candidate for the treatment of tumors. CPT has a terminal ring that is converted between a lactone form in an acidic medium (pH < 5) and a ring-opened carboxylate form in an alkaline medium (pH > 8), but only internally The ester form CPT is pharmaceutically active. However, this active form is hydrophobic and therefore has difficulty in delivery in a physiological environment.

There is another problem in delivering CPT or its analogs. For example, since the lactone form CPT and the carboxylate form CPT are interconvertible in a pH dependent equilibrium, the lactone form CPT can be rapidly converted to the carboxylate form CPT in a physiological environment. Furthermore, because the carboxylate form CPT can bind very efficiently to human serum albumin (HSA), more lactone form CPT will be converted to the carboxylate form CPT in the presence of HSA to achieve equilibrium.

Like CPT, its biological analogues, such as 7-ethyl-10-hydroxycamptothecin (SN38, 7-ethyl-10-[4-(1-piperidino)-1- Piperidono] a metabolite of carbonyloxycamptothecin (CPT11) and some other anti-tumor drugs also have poor solubility and similar active form-inactive form conversion problems in physiological environments. Because these drugs may be highly toxic and rapidly metabolized, it is desirable to introduce and deliver therapeutic levels of the drugs to solid tumors while reducing their toxicity.

Several methods have been developed for these purposes, including the use of micelles as vectors, since well-designed micelles, such as biodegradable and biocompatible micelles, are capable of solubilizing hydrophobic resistance in physiological environments. A tumor drug that increases the blood circulation time of the drug, and thereby delivers the desired therapeutic level of the drug to a solid tumor. However, there is still a need for a better alternative.

The inventors of the present invention have unexpectedly discovered that certain polymeric micelles can provide better properties when delivering anti-tumor drugs. In one embodiment, the present invention provides a pharmaceutical composition for treating a tumor using a polymer microcapsule coated with an antitumor drug, wherein the polymer microcell comprises a block copolymer, and the block copolymer contains One or more hydrophilic blocks, one or more hydrophobic blocks, and one or more zwitterions.

The hydrophobic block may comprise at least one selected from the group consisting of, for example, polycaprolactone (PCL), polyvalerolactone (PVL), poly(lactide co-glycolide) (poly(lactide-co-) Glycolide), PLGA), polylactic acid (PLA), polybutyrolactone (PBL), polyglycolide and polypropiolactone (PPL). The hydrophilic block may comprise at least one selected from the group consisting of, for example, polyethylene glycol (PEG), hyaluronic acid (HA), and poly-gamma-glutamine acid (γ). -PGA). And the zwitterion may comprise at least one molecule selected from the group consisting of, for example, phosphorylcholine (PC), sulfobetaine (NS) and amino acids. The antitumor drug coated in the polymer micelle may be a single drug or a combination of different drugs.

The invention also relates to a method of enhancing the solubility of an anti-tumor drug, a method of increasing the blood circulation time of the drug, and a method of delivering the drug to one or more solid tumors. These methods use the polymeric microcapsules described above to coat at least one anti-tumor drug to increase the solubility of the drug, blood circulation time, and/or deliver the drug to one or more solid tumors.

It is to be understood that the foregoing general description and claims

The accompanying drawings, which are incorporated in the claims

Exemplary embodiments of the present invention will now be described in detail, examples of which are illustrated in the accompanying drawings.

The present invention relates to a pharmaceutical composition for treating tumors using a polymer microcapsule coated with an antitumor drug. The polymeric micelles comprise a block copolymer comprising at least one hydrophilic block, at least one hydrophobic block, and at least one zwitterion. The block copolymer can be, for example, amphiphilic. In one embodiment, the hydrophobic block has a molecular weight of, for example, from about 500 to about 30,000 Daltons. The hydrophobic block may comprise, for example, at least one selected from the group consisting of polycaprolactone (PCL), polyvalerolactone (PVL), poly(lactide co-glycolide) (PLGA), polylactic acid (PLA), Polybutyrolactone (PBL), polyglycolide and polypropiolactone (PPL). The hydrophilic block has a molecular weight of, for example, from about 500 to about 30,000 Daltons. The hydrophilic block may comprise, for example, at least one selected from the group consisting of polyethylene glycol (PEG), hyaluronic acid (HA), and poly-gamma-glutamic acid (γ-PGA). And the zwitterion may comprise, for example, at least one member selected from the group consisting of choline (PC), sulfobetaine (NS), and an amino acid.

An exemplary block copolymer, PEG-PCL-PC, has the following structure:

Wherein R is a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a fluorenyl group, and the C _1-6 alkyl group, benzyl group or fluorenyl group may be unsubstituted or substituted with a functional group, the hydrogen atom, C _{The 1-6} alkyl group, the benzyl group or the fluorenyl group may be protected; m and n may be the same or different and each is an integer; preferably, m and n are each an integer of from 1 to 200, more preferably, m And n are each an integer of from 10 to 100, more preferably, m is an integer of from 30 to 85, and n is an integer of from 10 to 80. The block copolymers disclosed herein can be prepared by the process disclosed in U.S. Patent Application Publication No. 2007/0104654.

The block copolymer disclosed in the present invention is capable of forming a polymer microcell in an aqueous medium when a critical micelle concentration (CMC) is exceeded, wherein the hydrophobic portion is embedded in the core. The polymeric micelles may, for example, have a diameter of from about 20 to 1,000 nm. Due to the chain flexibility of the hydrophilic block and the presence of zwitterions, the polymeric micelles are substantially non-immunogenic. The hydrophobic block can be decomposed by enzymatic or hydrolysis. The polymeric micelles are biodegradable and/or biocompatible. Therefore, after the hydrophobic block is decomposed, the remaining harmless substances such as hydrophilic blocks and zwitterions are soluble in the blood and then removed from the renal system.

The antitumor drug coated in the polymer micelle may be a single drug or a combination of different drugs.

The polymeric microcells disclosed herein can be used as an effective pharmaceutical carrier and are capable of absorbing at least one hydrophobic drug into its hydrophobic core to form a pharmaceutical composition. Accordingly, the present invention is also directed to methods of enhancing the solubility of an anti-tumor drug, methods of increasing the blood circulation time of the drug, and methods of delivering the drug to one or more solid tumors. These methods use the polymeric microvesicles disclosed herein to coat at least one anti-tumor drug to increase the solubility, effectiveness or potency of the drug, and to deliver the drug to one or more solid tumors. In one embodiment, the invention relates to a method of delivering an anti-tumor drug to a solid tumor, the method comprising coating the anti-tumor drug in a polymeric microcell disclosed herein to form a coating complex (encapsulation complex) The coated complex is then delivered to the human body by known methods of drug delivery, such as by oral administration, transdermal administration, injection or inhalation.

The polymer micelle coated with at least one antitumor drug disclosed in the present invention can be produced, for example, by the following method. A certain amount of the antitumor drug and the block copolymer were stirred and dissolved in 1 ml of dimethyl hydrazine (DMS0). After removing DMSO by freeze drying, 1 ml of a 10% sucrose solution was added, and then the freeze-dried solid was dissolved to form a suspension. Ten minutes after undergoing ultra-sonication, the suspension is further filtered through a 0.45 μm screening procedure to remove uncoated drug crystals, and then a polymer microparticle coated with at least one antitumor drug can be obtained. Cell. The drug encapsulation efficiency (E.E.) is calculated using the following formula:

E. E (%) = (total mass of drug in microvesicle / total mass of drug added) x 100.

Table 1 shows the selection of various antitumor drugs (CPT or SN38), block copolymers and their amounts in each formulation. PEG, PCL, PVL, and PC represent polyethylene glycol, polycaprolactone, polyvalerolactone, and choline, respectively, and the attached numbers represent approximate molecular weights of PEG, PCL, and PVL. For example, PEG ₅₀₀₀ PCL ₁₉₀₀ PC means that the block copolymer comprises PEG having a molecular weight of about 5000 Daltons, which is attached to a PCL having a molecular weight of about 1900 Daltons, which PCL is further attached to the PC.

The composition code is given arbitrarily to indicate different compositions. The CC201, CC301, CC701, CV201 and SC201 compositions do not contain any zwitterions and are therefore used for comparison purposes.

The particle size distribution can be obtained, for example, by a laser particle size analyzer (Coulter N4 plus), and the amount of CPT or SN38 coated in each formulation can be determined by HPLC. P.S., P.I., and E.E. in Table 1 represent the particle size, polydispersity index, and encapsulation ratio, respectively. These parameters can be measured and/or calculated according to techniques known in the art.

The invention is explained in more detail on the basis of the following examples, which are not to be construed as limiting the scope of the invention.

[Example 1]: Release test using a dialysis bag

A 50 μL solution of the pharmaceutical composition prepared according to the method described above was added to a dialysis bag having a molecular weight cutoff of about 3,500 Daltons, followed by 50 ml of phosphate buffered saline (PBS) at 37 ° C (pH). 7.4) Perform dialysis. After 0.5, 1, 1.5, 2, 4, and 8 hours of dialysis, 250 μL of out-of-bag buffer was taken out, and then each was mixed with 750 μL of methanol (solution in 0.6 N HCl). The amount of each drug released from the polymer micelles and then dialyzed into the extra-buffer buffer was determined by HPLC. 50 μL of CPT-containing DMSO solution (CPT-DMSO) was used as a control.

Figure 1 shows the release profile of CPT (or SN38) with incubation time for various compositions using a dialysis bag. Table 2 shows the raw data.

As shown in Figure 1 and Table 2, after 8 hours of dialysis, more than 90% of the CPT contained in CPT-DMSO was dialyzed into the extra-buffer buffer, but only 30% or less of the drug contained in the pharmaceutical composition. Dialysis into the extra-buffer buffer. Figures 1 and 2 also show that, in general, zwitterionic polymeric micelles such as CCP201 and SCP201 compositions are more effective in keeping the drug coated than the zwitterionic polymer micelles.

[Example 2]: Direct dilution test

150 μL of the pharmaceutical composition of the present invention prepared according to the method described above was mixed with 1350 μL of PBS (pH 7.4), followed by culturing at 37 °C. After 0.5, 1, 2, 4, and 8 hours of culture, 10 μL of the culture solution was separately taken out, and then each was mixed with 990 μL of methanol. The amount of lactone form CPT or SN38 in the mixture was determined by HPLC. 150 μL of a CPT-containing DMSO solution (CPT-DMSO) was used as a control.

Figure 2 shows the ratio of the lactone form CPT (or SN38) remaining with the incubation time for each composition using the direct dilution method. Table 3 shows the raw data.

As shown in Figure 2 and Table 3, only about 20% of the CPT in CPT-DMSO remained in the lactone form after 8 hours of culture, but more than 50% of the CPT and SN38 initially contained in the pharmaceutical composition remained in the lactone form. . Figure 2 and Table 3 also show that, in general, zwitterionic polymer micelles such as CCP201 and SCP201 are more effective in maintaining CPT and SN38 compared to zwitterionic polymer micelles. Ester form.

[Example 3]: In vivo kinetic test of CPT

CPT at a dose of 1 mg/kg in DMSO, CC201, CCP201 and CV201 was introduced into SD mice by intravenous injection, respectively. The concentration of the lactone form CPT in the blood over time was then determined by HPLC.

Figure 3 shows the quantitative distribution of lactone form CPT in plasma after injection in an in vivo kinetic test. Table 4 shows the raw data.

^b ND means undetectable.

Where ng means 10 ^-9 grams.

Table 5 shows the kinetic data. T _1/2 (hr), AUC _INF (hr*ng/ml), CL (mL/hr/kg), and Vss (mL/kg) in Table 5 represent the half-life, up to the area under the infinite curve (area under The curve to infinity), the clearance rate and the steady state distribution volume and their units. n represents the number of samples. These parameters can be measured and/or calculated according to techniques known in the art.

According to Table 5, after 4 hours, in the case of CPT-DMSO, only trace amounts of lactone form CPT were detected in the blood, but conversely, significant significance was detected with the CC201, CV201 and CCP201 compositions. Higher plasma lactone form CPT concentration. Table 5 shows that CCP201 provides the best protection against lactone form CPT in the blood because the T _1/2 (hr) and AUC _INF (hr*ng/ml) values of the plasma lactone form CPT for CCP201 The T _1/2 (hr) and AUC _INF (hr*ng/ml) values of the plasma lactone form CPT are about 4 times and 9.5 times higher than for CPT-DMSO, and with the zwitterionic polymer micelles. In contrast, polymeric mice containing zwitterionics such as CCP201 are more effective at maintaining CPT in the form of lactone in the blood. Furthermore, Table 5 demonstrates that exemplary embodiments of the present invention are capable of substantially improving the stability of the lactone form of CPT in the presence of HSA and reducing the amount of CPT convertible to the carboxylate form in the presence of HSA.

[Example 4]: In vivo kinetics test of SN38

SN38 at a dose of 4 mg/kg in DMSO, SC201 and SCP201 was introduced into SD mice by intravenous injection, respectively. The concentration of the lactone form SN38 in the blood over time was then determined by HPLC.

Figure 4 shows the quantitative distribution of lactone form SN38 in plasma after injection in an in vivo kinetic assay. Table 6 shows the raw data.

Table 7 shows the kinetic data. T _1/2 (hr), AUC _INF (hr*ng/ml), CL (mL/hr/kg), and Vss (mL/kg) in Table 7 represent half-life, area under the infinite curve, and clearance rate, respectively. And steady-state volume of distribution and their units. n represents the number of samples. These parameters can be measured and/or calculated according to techniques known in the art.

According to Table 7, after 4 hours, with SN38-DMS0, only a limited amount of SN38 was detected in the blood, but conversely, a significantly higher plasma SN38 concentration was detected with SC201 and SCP201. Table 7 shows that SCP201 provides the best protection against SN38 in the blood because the T _1/2 (hr) and AUC _INF (hr*ng/ml) values of plasma SN38 are comparable to those of SN38-DMSO for SCP201, respectively. The T _1/2 (hr) and AUC _INF (hr*ng/ml) values of plasma SN38 were approximately 2-fold and 50-fold higher.

F Example 5]: Comparison of in vivo drug efficacy of CCP201 and free CPT11

Human colon cancer HT29 cells were subcutaneously implanted into the dorsal muscle of immunodeficient mice. After the tumor size reached about 300-500 mm ³ , the mice were randomly divided into 4 groups, and then saline, CPT11 and CCP201 were introduced into the mice by intravenous injection, respectively. The frequency of administration was twice a week for a total of five doses. Tumor size and weight were monitored for each mouse. Tumor size was measured and calculated according to the formula V = 1/2 ab ² , where V is the tumor volume, a is the longest diameter of the tumor, and b is the shortest diameter of the tumor.

Figure 5 shows the size of HT29 tumors after treatment with CCP201 and free CPT11. Table 8 shows the raw data and Table 9 shows the general information. The results indicate that, in general, CCP201 is able to provide higher drug efficacy or potency than free CPT11. Specifically, the tumor inhibition rate of the 18 mg/kg CCP201 dose exceeded 60%, which was significantly higher than the tumor inhibition rate of free CPT11.

[Example 6]: Comparison of in vivo drug efficacy of SN38 and free CPT11

Human colon cancer HT29 cells were subcutaneously implanted into the dorsal muscle of immunodeficient mice. After the tumor size reached approximately 100-200 mm ³ , the mice were randomly divided into 5 groups, and then saline, CPT11 and SCP201 were introduced into the mice by intravenous injection, respectively. The frequency of administration was twice a week for a total of five doses. Tumor size and weight were monitored for each mouse. Tumor size was measured and calculated according to the formula V = 1/2 ab ² , where V is the tumor volume, a is the longest diameter of the tumor, and b is the shortest diameter of the tumor. Human colon cancer Colo205 cells were also tested by the same method as described above except that the mice were divided into 6 groups.

Figure 6 shows the size of HT29 tumors after treatment with SCP201 and free CPT11. Table 10 shows the raw data and Table 11 shows the general information.

Figure 7 shows the size of Colo205 tumors after treatment with SCP201 and free CPT11. Table 12 shows the raw data and Table 13 shows the general information.

^b The maximum body weight measured after the first treatment.

Figures 7, 12 and 13 show that, in general, SCP201 is able to provide higher drug efficacy than free CPT11. Specifically, Tables 11 and 13 both show that when 20 mg/kg of SCP201 is used, the tumor inhibition rate for HT29 and Colo205 can be higher than 90%.

[Example 7]: In vivo drug efficacy test of SCP201 using MTT assay

Various human cancer cell lines such as A549, AS2 and H460 were implanted into the multiwell plate, and then Dulbecco's Modified Eagle Media (high glucose) containing 10% fetal bovine serum and 1% P/S was added. Go to each hole. After 24 hours of incubation at 37 ° C and CO ₂ , various amounts of CPT11, SN38 and SCP201 were added to each well, respectively, and the mixture was further cultured at 37 ° C and CO ₂ for 72 hours. Then 20 μL of 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well to start the reaction. . After 2 hours, the suspension in each well was removed and DMSO was then added to the wells to dissolve the formazan formed during the reaction. Live cell concentrations were then obtained by analyzing the OD570 and OD600 data for each well.

Table 14 shows the selection of cancer cell lines and CPT11, SN38, respectively, and SCP201 half maximal inhibitory concentration of each tumor cell line (IC _50). The differences in drug activity between CPT11 and SN38 are consistent with the information disclosed in the publication. Table 14 shows that there is no reduction in the in vitro pharmaceutical activity of the pharmaceutical composition comprising SN38 disclosed herein.

The human lung cancer cell lines (A549 and AS2) used in the present invention are provided by Prof. Wu-Chou Su (National Cheng Kung University Hospital College of Medicine, Taiwan). Human colorectal cancer (Colo 205 and HT29) was provided by Dr. Ming-Jium Shieh (National Taiwan University College of Medicine and College of Engineering, Taiwan). The human liver cancer cell line SK-HEP-1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). All remaining cell lines tested in the present invention were obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan).

Other embodiments of the invention will be apparent to those skilled in the < The specification and examples are to be considered as illustrative only,

Figure 1 shows the release profile of CPT (or SN38) with incubation time for various compositions using a dialysis bag.

Figure 2 shows the ratio of lactone form CPT (or SN38) remaining with the time of incubation for each composition using direct dilution.

Figure 3 shows the quantitative distribution of lactone form CPT in plasma after injection in an in vivo kinetic assay.

Figure 4 shows the quantitative distribution of lactone form SN38 in plasma after injection in an in vivo kinetic assay.

Figure 5 shows the size of HT29 tumors after treatment with CCP201 and free CPT11.

Figure 6 shows the size of HT29 tumors after treatment with SCP201 and free CPT11.

Figure 7 shows the size of Colo205 tumors after treatment with SCP201 and free CPT11.

Claims (10)

A pharmaceutical composition for treating a tumor, the composition comprising: at least one polymeric microcell; and at least one antitumor drug coated in the polymeric microcell; wherein the polymeric microcell comprises a block copolymer of the formula, (wherein R is a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a fluorenyl group; m and n may be the same or different and each are an integer of from 1 to 200); and the antitumor drug and the block copolymer are The weight ratio is 2:10~3:10. The pharmaceutical composition for treating a tumor according to claim 1, wherein the polymer micelle has a diameter ranging from about 20 nm to about 1,000 nm. The pharmaceutical composition for treating a tumor according to claim 1, wherein the polymeric microcell has a hydrophobic inner and a hydrophilic surface. The pharmaceutical composition for treating a tumor according to claim 1, wherein the at least one antitumor drug is hydrophobic. The pharmaceutical composition for treating a tumor according to claim 1, wherein the tumor is a solid tumor. The pharmaceutical composition for treating a tumor according to claim 1, wherein the at least one antitumor drug is selected from the group consisting of 7-ethyl-10-hydroxycamptothecin, camptothecin, and derivatives thereof. Medicine for treating tumors as described in claim 1 A composition wherein the block copolymer is amphoteric. The pharmaceutical composition for treating a tumor according to claim 1, wherein the block copolymer is biodegradable. A pharmaceutical composition for treating a tumor according to claim 1, wherein the block copolymer is biocompatible. A method for enhancing water solubility of an antitumor drug, the method comprising: forming a polymer microcell; and coating the antitumor drug in the polymer microcell; wherein the polymer microcell comprises the following formula Block copolymer (wherein R is a hydrogen atom, a C _1-6 alkyl group, a benzyl group or a fluorenyl group; m and n may be the same or different and each are an integer of from 1 to 200); and the antitumor drug and the block copolymer are The weight ratio is 2:10~3:10.