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Medullary thyroid carcinoma is a rare endocrine tumor, which shows overexpression of somatostatin receptor subtype 2. There is no systemic therapy for medullary thyroid carcinoma. Previously we reported that octreotide-PEG liposomes loaded with irinotecan, which target somatostatin receptor subtype 2, showed high therapeutic efficacy for medullary thyroid carcinoma xenografts compared with free irinotecan or non-targeted non-PEGylated liposomal irinotecan. In this study, we evaluated octreotide-PEG liposomes loaded with irinotecan in terms of the biodistribution of irinotecan and its active metabolite, and its therapeutic efficacy, compared with PEGylated liposomes. Furthermore, to elucidate the effect of octreotide ligand after cellular association, we assessed the cytotoxicity in tumor cells and the inhibition of protein phosphorylation in the tumor cells and xenografts using empty octreotide-PEG liposomes, which were loaded with no drug. In a therapeutic study, octreotide-PEG liposomes loaded with irinotecan significantly improved median survival compared with PEGylated liposomes. In tumor tissue at 6 h after injection, octreotide-PEG liposome-treated mice showed significantly higher concentrations of irinotecan and 7-ethyl-10-hydrocamptothecin compared with PEGylated liposome-treated mice, indicating that octreotide-PEG liposomes accumulated rapidly and to a high level in the tumor. Furthermore, empty octreotide-PEG liposome inhibited the phosphorylation of p70S6K in vitro and in vivo. These findings indicated that octreotide-PEG liposomal irinotecan has dual functions with targeted tumor delivery and assistance of cellular cytotoxicity, which led to higher therapeutic efficacy than PEGylated liposomes for medullary thyroid carcinoma xenografts. (Cancer Sci 2012; 103: 310–316)
Medullary thyroid carcinoma (MTC) originates in C cells of the thyroid gland. It is known to overexpress somatostatin receptor (SSTR) subtypes 1, 2, and 5.(1) Somatostatin receptor 2 expression was the most frequently detected subtype in human MTC and was significantly higher than the other SSTR subtypes in an established human MTC cell line, TT.(1–3) A therapeutic approach for MTC using irinotecan (CPT-11) was reported, but the results were inconclusive.(4,5) Overall, there is no systemic therapy for MTC.
CPT-11 is a water-soluble prodrug and is converted to 7-ethyl-10-hydrocamptothecin (SN-38), an active metabolite of CPT-11, by carboxylesterase (CE).(6,7) CPT-11 inhibits the resealing of single-strand DNA breaks mediated by topoisomerase I by stabilizing cleavable complexes and is a cell cycle-specific drug.(8–11) From this, a long period of exposure to CPT-11 induces cytotoxicity of tumor cells. The CPT-11 therapy, however, has failed owing to its short half-life and low tumor distribution. Therefore, selective delivery of CPT-11 to tumor sites could lead to successful CPT-11 therapy for MTC. For this purpose, we developed Oct-CL, namely, octreotide-PEG liposomes loaded with CPT-11, in which octreotide (Oct) has high binding affinity for SSTR2.(12) This approach proved promising because Oct-CL led to higher therapeutic efficacy for MTC xenografts than free CPT-11 or non-targeted non-PEGylated liposomal CPT-11.(13)
PEGylated liposomes (SL) can passively accumulate into tumor tissue due to the enhanced permeability and retention effect.(14) Therefore, we compared the therapeutic efficacy of antitumor activity of Oct-CL with SL to clarify whether the active-targeting ability of Oct-CL was superior to the passive-targeting ability of SL. Generally, because active-targeting liposome biodistribution is different from that of passive-targeting liposomes, we examined the biodistribution of CPT-11 and SN-38 after i.v. injection of Oct-CL and SL into MTC xenograft mice.
Moreover, we found that non-loaded Oct-CL, or empty Oct-CL, showed higher cytotoxicity in TT cells compared with empty SL.(13) To gain more insight into tumor suppression, we examined the mechanism of cytotoxicity of the Oct ligand. It was reported that Oct alone can produce an antiproliferative action to inhibit the phosphorylation of p70S6K in the Akt/mTOR/p70S6K pathway in insulinoma cells.(15) From this information, we tried to clarify the effects of Oct ligand on the phosphorylation of p70S6K in vitro and in vivo. We provide an answer to the mechanism of cytotoxicity of empty Oct-CL in this study.
Here we present a more extensive investigation of Oct-CL. We evaluated the function of Oct using Oct-CL from viewpoints of therapeutic efficacy and the biodistribution of CPT-11 and SN-38 in MTC tumor xenografts after injection of Oct-CL or SL. Furthermore, we assessed the cytotoxicity mechanism of Oct ligand using empty Oct-CL in vitro and in vivo.
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In this study, we showed that Oct-CL loaded with CPT-11 showed the early and higher accumulation of CPT-11 in tumor, enhanced the antitumor effect, and significantly prolonged median survival compared with SL. Moreover, the mechanism of action of Oct associated with liposomes was investigated by measuring the biodistribution of Oct-CL loaded with CPT-11 and the phosphorylation of proteins after empty Oct-CL treatment. Recently, we have reported that Oct-CL loaded with CPT-11 showed higher therapeutic efficacy compared with free CPT-11 or non-targeted non-PEGylated liposome in TT tumor xenografts.(13) However, the effect of Oct and its mechanism still remained after association with liposomes.
In our study, Oct-CL showed a significantly higher distribution to TT tumor tissue compared with SL at least until 6 h after injection. This result suggested that Oct-CL accumulated rapidly in the tumor after injection as a result of Oct-targeting, whereas SL accumulated slowly in the tumor by the enhanced permeability and retention effect for 24 h. Twenty-four hours after injection, there was no significant difference between Oct-CL and SL. The CPT-11 level of Oct-CL in the tumor was sustained for 24 h after injection.
Why was the SN-38 concentration of tumors in Oct-CL-treated mice significantly higher than that of SL-treated mice at 24 h even though the CPT-11 concentration was similar? Generally, it is reported that CPT-11 conversion to SN-38 mainly occurs through the action of liver CE.(26) Accordingly, it can be regarded that SN-38 transformed in the liver accumulated in the tumor. When the accumulation of CPT-11 in the liver was similar between Oct-CL- and SL-treated mice, converted SN-38 in the liver should be present in similar amounts, and, therefore, the amount of SN-38 accumulating in tumor should be similar. We previously reported that there were no significant differences between Oct-CL- and SL-treated mice in terms of drug release over 48 h at 37°C in PBS.(13) Furthermore, an in vitro conversion study showed that the conversion activity of CPT-11 to SN-38 in TT tumor tissue was 0.8-fold that of the liver. From these findings, higher SN-38 concentration of Oct-CL at 24 h might reflect early direct distribution of Oct-CL to tumor tissue by SSTR targeting. To the best of our knowledge, this is the first report regarding CPT-11 conversion activity in TT cells and TT tumor tissue.
When we examined other tissues, there were significant differences between Oct-CL- and SL-treated mice in terms of kidney and liver distribution. The CPT-11 concentration in the kidneys of Oct-CL-treated mice was significantly lower compared with that of SL-treated mice. Because the kidney is known to express SSTR2,(27) Oct-CL might be selectively and rapidly distributed to the kidneys, as well as to the tumor, then excreted more rapidly than in SL-treated mice. With regard to the liver, Oct-CL accumulated to a higher level in the liver than SL, because SL was modified with Oct ligand and it may be taken up by the reticuloendothelial system.
Empty Oct-CL exerted cell growth inhibition at low concentrations (0.42 and 4.2 μM), whereas free Oct did not exert cell growth inhibition below 100 μM (Fig. 4a,c). Empty Oct-CL showed effective cell growth inhibition compared with free Oct, suggesting that the affinity of empty Oct-CL to SSTR may be higher than that of free Oct. Phosphorylation of p70S6K is reported to activate cell growth and proliferation.(24) p70S6K is the downstream protein of the PI3K/Akt/mTOR pathway and phosphorylation of p70S6K is generally used as a marker of the inhibition of the PI3K/Akt/mTOR pathway.(28) Treatment with empty Oct-CL caused the suppression of phosphorylation of p70S6K in vitro and in vivo (Fig. 5a,b) but empty SL did not. Therefore, this result suggested that empty Oct-CL inhibited cell growth and proliferation by suppressing the phosphorylation of p70S6K in TT cells and TT tumor tissue. In other words, the targeting ligand Oct had not only tumor targeting activity but also assisted with antitumor effects. Therefore, the Oct ligand has dual functionality.
However, treatment with empty Oct-CL did not show any antitumor effect under this condition (Fig. 6). For MTC, it was reported that clinically s.c. injection of free Oct at 500 μg (=458 nmol)/day for 90 days, and 150 μg (=137 nmol)/day for 6 months showed no effect on therapeutic efficacy.(29,30) In this therapeutic study, Oct originated from Oct-CL was injected twice as 867 nmol Oct/kg/day. From these different schedules, it is difficult to judge the therapeutic efficacy by Oct alone in our study.
Figure 7 illustrates the proposed antitumor effects of Oct-CL loaded with CPT-11 for MTC. Oct-CL was selectively associated with TT cells. CPT-11 was released from Oct-CL, then CPT-11 was converted to SN-38 by CE in TT tumor. SN-38 produced in the tumors showed cytotoxicity. Oct-CL suppressed the phosphorylation of p70S6K. These suppressions led to inhibition of cell growth and proliferation, which assisted in antitumor effects for MTC using Oct-CL loaded with CPT-11.
Figure 7. Proposed antitumor effects of octreotide-PEG liposomes loaded with CPT-11 (Oct-CL) for human medullary thyroid carcinoma.
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In conclusion, the present study showed that Oct-CL loaded with CPT-11 showed enhanced therapeutic efficacy, which correlated with a strong antitumor effect and the significant improvement of median survival, which was superior to SL in TT tumor xenografts. The improvement in therapeutic efficacy was due to the dual functions of Oct; the rapid and highly selective distribution of Oct-CL to tumor by SSTR targeting and the assistance of cell growth suppression by Oct.