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Cisplatin (CDDP) has been a key drug for chemotherapy in patients with head and neck squamous cell carcinoma. Nephrotoxicity is one of its adverse reactions that are dose limiting. To increase its antitumor effects and reduce such toxicity problems, polymeric micelles carrying CDDP (NC-6004) have been developed. The present study was designed to evaluate the efficacy and safety of NC-6004 for oral squamous cell carcinoma. In vitro antitumor activity was assayed in four oral squamous cell carcinoma cell lines. To investigate the antitumor and nephrotoxic effects of NC-6004, nude mice bearing OSC-19 were administered NC-6004 or CDDP. The in vitro growth-inhibitory effect of NC-6004 was significantly less than that of CDDP. However, both NC-6004 and CDDP showed equivalent antitumor effects in vivo. Mice with CDDP developed renal cell apoptosis; however, those injected with NC-6004 were almost free of renal cell injury. Moreover, in an orthotopic tongue cancer model using OSC-19, NC-6004 reduced the rate of sentinel lymph node metastasis to lower than that with CDDP. In conclusion, considering the potential advantages in terms of noticeable antitumor activity, lymphatic drug delivery and reduced nephrotoxicity, NC-6004 represents a significant structural improvement in the development of a platinum complex.
Head and neck cancer remains a significant public health problem and ranks in the six leading cancers by incidence worldwide, with an estimated 600 000 new cases every year. Cisplatin (cis-dichlorodiammineplatinum; CDDP) has been demonstrated to be one of the most effective cytotoxic agents  and the CDDP-based chemotherapy regimen has gained widespread use in patients with head and neck squamous cell carcinoma (HNSCC). However, its administration has been hindered by its adverse reactions, for example, nephrotoxicity, neurotoxicity, gastrointestinal toxicity, hematological toxicity and ototoxicity. Among these, the significant risk of nephrotoxicity frequently hinders the use of high doses to maximize its antineoplastic effects, which might be the cause of treatment failure.
To overcome these problems and improve the therapeutic effect of CDDP, we have been applying superselective supradose intra-arterial CDDP infusion for advanced HNSCC. However, since this technique is more complicated than that of i.v. infusion of antitumor drugs, it is not prevalent in the chemotherapy scene. Recently, several kinds of nanoparticle therapeutic platforms, including liposomes, nanoparticles and polymeric micelles, have been developed based on the idea that the drug delivery system (DDS) can accumulate in the tumor selectively, with reduced distribution in normal tissues and minimized undesirable side-effects.[5-7] NC-6004, which is a CDDP-incorporating polymeric micellar nanoparticle, enhanced antitumor activity and reduced the nephrotoxicity and neurotoxicity of CDDP in gastric cancer.[8-10] Poly(ethylene glycol)-poly(glutamic acid) block copolymers (PEG-P[Gu]) confer a stealth property to the formulation, which allows the micellar drug preparation to be less avidly taken up by the reticuloendothelial system and retained in the circulation for a longer period of time. This has been recognized as the enhanced permeability and retention (EPR) effect where extravasation of high-molecular-weight micelles through leaky tumor capillary fenestrations, which result from abnormalities in angiogenesis at the tumor site, lead to accumulation and retention for a long time. A prolonged circulation time and the ability of EPR lead to the accumulation of CDDP in tumor tissues. NC-6004 has been previously shown to possess significant antitumor activity for human gastric cancer cells in a mouse model. However, the efficacy of NC-6004 in head and neck cancer has never been studied.
In the present study, we analyzed the cytotoxicity of NC-6004 using the MTS assay and apoptosis assay on oral squamous cell carcinoma cell lines. Antitumor and nephrotoxic effects of NC-6004 were investigated and compared with those of CDDP in in vivo experiments. As frequent involvement of regional lymph nodes, especially the sentinel lymph node (SLN), is one of the outstanding features of oral SCC, we further evaluated the lymphatic distribution of NC-6004 in order to develop a target therapy for cervical lymph node metastasis, which also has never been evaluated.
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Cancer nanotechnology is a new field of interdisciplinary research aiming to enhance the methods of cancer diagnosis and treatment. From the standpoint of DDS targeting solid carcinoma, a variety of polymeric micelle-based anticancer drugs have been developed to achieve high and selective accumulation at the tumor site.[16-18] CDDP is a key drug in chemotherapy for malignancies such as lung, gastrointestinal, genitourinary and head and neck cancer. Studies have shown that NC-6004 caused better selective accumulation of CDDP in tumors while lessening its distribution in normal tissue. NC-6004, of which the drug-loaded core is covered by a hydrophilic poly(ethylene glycol) shell layer, is not detected by macrophages, can be retained in the circulation for long periods, is redistributed in tissue and can extravagate preferentially to infiltrate solid tumors. In the present study, we have investigated the antitumor properties and nephrotoxic effects of NC-6004 on human oral cancer cell lines. Moreover, a submucosal injection of NC-6004 exhibited substantial therapeutic efficacy in inhibiting cervical lymphatic metastasis in an orthotopic tongue cancer model.
To characterize the cellular basis of NC-6004 cytotoxicity relative to CDDP, we tested the two drugs in four different oral squamous carcinoma cell lines. The success of CDDP lies in its ability to arrest DNA synthesis, induce oxidative stress and activate apoptotic pathways in tumor cells. The growth-inhibitory effect of NC-6004 was significantly less than that of CDDP. The different effects between these two drugs lies in the slow release of CDDP in the presence of abundant chloride ions as NC-6004 contains coordination bonds between the atoms of Pt in CDDP and the carboxylic group in the side chain, which is in line with previous studies.[9, 10] The caspase cascade is activated when a CDDP stimulus induces the release of cytochrome c from mitochondria. This activation leads to an irreversible commitment to apoptotic cell death. We found that activation of caspase-3 and caspase-7 was induced in both CDDP- and NC-6004-treated OSC-19 cells.
Nephrotoxicity is one of the most significant adverse effects of CDDP. CDDP accumulates in cells from all nephron segments, but is preferentially taken up by the highly susceptible proximal tubular epithelial cells, which bear the brunt of the damage. This nephrotoxicity limits the dose that can be administered and prevents the potential efficacy of CDDP. The size of NC-6004 is approximately 30 nm in diameter, which is large enough to avoid renal secretion. The Cmax value for Pt concentrations in the kidney after NC-6004 administration is much lower than that of free CDDP administration. In the present study, the number of apoptotic renal cells in NC-6004-treated mice was significantly lower than that in CDDP-treated mice by approximately 66% (CDDP, 14.2%; NC-6004, 4.2%). Reductions in nephrotoxicity of NC-6004 might allow patients to undergo therapy without hospitalization for hydration and the treatment of CDDP-related toxicities.
We evaluated the in vivo antitumor activity of NC-6004 in mice. Reductions in tumor size after administration of NC-6004 or CDDP were approximately 14–20%, respectively, and were not significantly different from each other. The data obtained could be interpreted as these micelles having efficacy against oral squamous cell carcinoma cell lines similar to that of CDDP, but with much less renal toxicity toward the host.
The presence of cervical lymph node metastasis is an indicator of poor prognosis in patients with HNSCC.[24-27] Recently, the SLN has been highlighted as the lymph node that first receives lymphatic drainage from the primary site of a tumor. To control lymph node metastasis, especially SLN micrometastasis in the early stages, drugs need to be delivered in tumoricidal concentrations from the site of application. The drug is highly selective depending on the size of molecule to access the lymphatic system, which was reported as the “blood–lymph barrier”. The blood–lymph barrier makes conventional anticancer agents fail to effectively enter the lymphatic system. The present study showed that sublingual injection of NC-6004 in an orthotopic tongue cancer mouse model significantly reduced lymphatic tumor metastasis. Moreover, a high concentration of Pt in cervical lymph nodes was maintained for at least 24 h after administration of NC-6004, whereas CDDP was not delivered to lymph nodes, which is attributable to the lymphatic drug delivery of NC-6004 from the primary tumor. These results suggest that this new drug-delivery system and the accumulation of micelles in lymph nodes are feasible for local chemotherapy targeting SLN in patients with occult lymphatic metastasis or micrometastasis.
We did not examine Pt concentrations in cervical lymph nodes with intravenous infusion of NC-6004 and CDDP. However, Pt concentrations in the SNL were higher in the NC-6004-injected group than CDDP in topically infused groups, indicating that NC-6004 is more suitable for a lymphatic delivery system. These results suggest that such polymeric micelled-drugs, which accumulate in higher concentrations around the primary tumor than NC-6004, might be more suitable for the control of lymphatic disease in HNSCC.
The present study demonstrated the superior safety and antitumor efficacy of NC-6004 against head and neck cancer cells over that of CDDP. Considering the potential advantages in terms of noticeable antitumor activity, lymphatic drug delivery and reduced nephrotoxicity, NC-6004 represents a significant structural improvement in the development of a platinum complex. NC-6004 has progressed to a phase I clinical trial in the UK, and phase I/II trials are currently underway in East Asia.