Poly (γ-glutamic acid) based combination of water-insoluble paclitaxel and TLR7 agonist for chemo-immunotherapy
Introduction
Cancer continues to be one of the leading cause of death, accounting for more than 1.5 million new cases and 0.5 million deaths [1]. Tumor is a complex and heterogeneous structure involving co-evolution of vasculature, tumor-resistant immune cells, extracellular matrix including fibroblasts which assist tumor cells escape therapeutic intervention [2], [3]. While chemotherapy has been used as a prominent anti-cancer modality, anti-cancer drugs are known to have high incidence of side-effects and disease recurrence [4]. Paclitaxel is a potent chemotherapeutic agent used for treatment of various cancers such as breast, non-small cell lung cancers, ovarian cancer, malignant brain tumors, and a variety of other solid tumors [5]. The clinical efficacy is jeopardized because of the systemic side-effects, insolubility in water and low bioavailability of paclitaxel [6], [7]. While various new approaches for effective and targeted delivery of paclitaxel are in progress [8], [9], it has become a desirable candidate drug for combination with various other modalities, but has gained limited success in clinical trials [10], [11], [12], [13].
Immune system is indispensable aspect of tumor microenvironment because tumor has the ability to evade the immune system by various mechanisms such as suppression of tumor reactive T cells by transforming growth factor-β (TGF-β) secretion or through regulatory T cells or by direct upregulation of death ligands such as Fas-L [14]. Consequently, immunotherapeutic approaches for cancer such as dendritic cells and T cells based vaccine, cytokines, toll-like receptor (TLR) agonists, viral vaccine, peptide based vaccine, DNA based vaccines have gained importance [15], [16]. Lately, the role of TLR stimulation has been emphasized for cancer treatment [17], [18], [19], [20] and combination of TLR agonists with other treatment modalities such as T cell modulation, anti-CTLA4 therapy or CD40L plasmid DNA for cancer treatment has been reported [21], [22], [23]. While TLR agonists are being explored for anti-cancer treatment, on a contrary, cancer cells are known to express TLRs and TLR agonists are also found to facilitate tumor proliferation, metastasis and inhibit apoptosis [24]. Thus, selection of suitable TLR agonist for cancer therapy is a critical step. TLR-7 agonist imiquimod also known as R837 has been approved by FDA for topical administration in cancer therapy and diseases such as genital warts, actinic keratoses, superficial basal cell carcinoma and lentigo maligna [25], [26]. Imiquimod is capable of imparting cytotoxic T cells with enhanced anti-tumor properties [27]. Imiquimod is also known to induce systemic immunity in cryosurgery patients [28]. In clinical trials, although it was well-tolerated with minimum side-effects, effective therapeutic response was not observed [29]. Because of the dual nature of TLR agonists in cancer, monotherapy using standalone TLR agonists is leading to inadequate treatment and has great scope for improvisation [30], [31].
Taking into consideration the limitations of chemotherapy and immunotherapy, chemo-immunotherapy has emerged as a new branch of cancer research with highly promising results [32]. The efficacy of chemotherapeutic agent can be increased if the host immunity is also taken into consideration. It has been found in phase III trial using combination of chemotherapeutic agent 5-fluorouracil (5-FU) and adriamycin with TLR3 agonist polyadenylic-polyuridylic acid (poly A:U) leads to significantly prolonged patient survival as compared to chemotherapy alone [33]. In a study, combination of paclitaxel and TLR4 agonist illustrated 40% reduction in tumor in mice as compared to paclitaxel alone [34]. In another report, it was demonstrated that pre-conditioning with chemotherapeutic agent complemented by adoptive T cell transfer, viral vaccine and immunostimulatory TLR agonist is capable of getting rid of melanoma tumor completely [35]. These results suggest that if the tumor surveillance is broken, then even low concentrations of anti-cancer drug is expected to be effective with reduced side-effects. However, the challenge is to determine an appropriate combination of anti-cancer drug and TLR agonist, which can completely eliminate the tumor without any reappearance of the disease. Another consideration is that anti-cancer drug should have minimum adverse effect on the immune cells while it should be able to kill tumor cells at the same concentrations of treatment. Also, the TLR agonist selected should not only be able to counter the immuno-suppressive environment within the tumor but should also be capable of triggering release of cytokines by immune cells which is sufficient for development of anti-tumor milieu.
In this research, poly (γ-glutamic acid) (γ-PGA) based combination of low dose of anti-cancer drug (paclitaxel) with immune-stimulatory agent (imiquimod) was tested for synergetic effect against solid tumor. Paclitaxel treatment was speculated to cause tumor cell death which should lead to production of tumor specific antigens as well as danger signals also known as damage associated molecular patterns (DAMPs). Imiquimod was used as an adjuvant and was expected to induce activation and maturation of immune cells for eliciting anti-tumor immune response. Since, both paclitaxel and imiquimod are water-insoluble drugs, γ-PGA which is a water soluble bioderived anionic polymer was used to form a stable aqueous micro-dispersion of the two drugs. The memory response against tumor was also assessed in order to test the longevity and clinical applicability of the formulation.
Section snippets
Preparation of polymer-drug micro-dispersion
For preparation of γ-PGA-paclitaxel micro-dispersion (γ-PGA/Ptx), 10 mg of paclitaxel (ChemieTek, Indianapolis, USA) in 500 μl DMSO was added to 2 ml of 2.5% (w/v) of 50 kDa poly (γ-glutamic acid) (BioLeaders Corporation, Daejeon, South Korea) while sonicating using a probe sonicator for 2 min. For preparation of γ-PGA-imiquimod micro-dispersion (γ-PGA/Imq), 10 mg of imiquimod (TCI, Tokyo, Japan) was dissolved in 2 ml DMSO by heating at 60 °C. This solution was added to 2 ml of 2.5% of 50 kDa
Characterization of γ-PGA-drug complex
The electron micrographs of the dried micro-dispersions showed presence of elongated paclitaxel and cubic imiquimod crystals embedded in polymeric matrix of γ-PGA (Fig. 1A). From the TEM images, well dispersed crystals of individual drugs were clearly demonstrated. The aqueous dispersion of lyophilized micro-dispersion was analyzed for size and size distribution. Distinct monodisperse peaks for γ-PGA/Imq and γ-PGA/Ptx/Imq was observed, indicating uniform size distribution (Table 1). In case of
Discussion
Effective delivery of water-insoluble therapeutic agent is one of the major hurdle in their clinical application. Various approaches such as solid dispersions, micro-suspensions and nano-suspensions by wet milling process, melt extrusion or using stabilizers are being explored [41], [42], [43], [44]. The selected anti-cancer drug paclitaxel and immuno-stimulating agent imiquimod are water-insoluble in nature. Therefore, the primary aim of this work was effective administration of the two drugs,
Conclusion
Standalone chemotherapy or immunotherapy has limitations such as systemic side-effects, insufficient immuno-stimulation, tumor directed tolerance of immune cells, lack of systemic and tumor specific immune response and tumor recurrence. This report emphasizes the concept of combination of chemotherapy with immunotherapy for eradication of solid tumors using water insoluble therapeutic agents. While most of these agents are water-insoluble in nature, using poly (γ-glutamic acid) for formulation
Acknowledgments
This work was supported by financial support from the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. 2009-0083540, No. 2012M3A9C6050070), the Korea Health Technology R&D Project, Ministry of Health & Welfare (no. A111918), the National Agenda Project grant from Korea Research Council of Fundamental Science and Technology, and KRIBB Research Initiative Program (KGM1211332).
References (52)
- et al.
Tumors as organs: complex tissues that interface with the entire organism
Dev Cell
(2010) - et al.
Paclitaxel and its formulations
Int J Pharm
(2002) - et al.
OncoGel (ReGel/paclitaxel) – Clinical applications for a novel paclitaxel delivery system
Adv Drug Deliv Rev
(2009) - et al.
Combination chemotherapy of the taxanes and antimetabolites: its use and limitations
Eur J Cancer
(2001) - et al.
TLR based therapeutics
Curr Opin Pharmacol
(2011) - et al.
Anti-tumor immune responses of tumor-associated macrophages via toll-like receptor 4 triggered by cationic polymers
Biomaterials
(2013) - et al.
T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy
Blood
(2009) - et al.
Dual character of toll-like receptor signaling: pro-tumorigenic effects and anti-tumor functions
Biochim Biophys Acta -Rev Cancer
(2013) - et al.
Imiquimod. a toll-like receptor-7 agonist, induces perforin in cytotoxic T lymphocytes in vitro
Mol Immunol
(2004) - et al.
Imiquimod enhances the systemic immunity attained by local cryosurgery destruction of melanoma lesions
J Invest Dermatol
(2007)
Phase III trial of adjuvant 5-fluorouracil and adriamycin versus 5-fluorouracil, adriamycin, and polyadenylic–polyuridylic acid (poly A: U) for locally advanced gastric cancer after curative surgery: final results of 15-year follow-up
Ann Oncol
Nanoparticle mediated co-delivery of paclitaxel and a TLR-4 agonist results in tumor regression and enhanced immune response at the tumor microenvironment in a mouse model
Int J Pharm
Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice
Cell Immunol
Solid dispersion of poorly water-soluble drugs: early promises, subsequent problems, and recent breakthroughs
J Pharm Sci
Formation of nano/micro-dispersions with improved dissolution properties upon dispersion of ritonavir melt extrudate in aqueous media
Eur J Pharm Sci
Cancer statistics, 2013
CA: A Cancer J Clin
Influence of tumour micro-environment heterogeneity on therapeutic response
Nature
Side effects and emotional distress during cancer chemotherapy
Cancer
Paclitaxel (Taxol)
New Engl J Med
Paclitaxel: a review of adverse toxicities and novel delivery strategies
Expert Opin Drug Saf
Paclitaxel in cancer treatment: perspectives and prospects of its delivery challenges
Crit Rev Ther Drug Carrier Syst
Combination neratinib (HKI-272) and paclitaxel therapy in patients with HER2-positive metastatic breast cancer
Br J Cancer
Enhanced antitumor activity of paclitaxel in combination with the anticarcinoma immunoconjugate BR96-doxorubicin
Clin Cancer Res
Paclitaxel combination therapy in the treatment of metastatic breast cancer: a review
Semin Oncol
Tolerance and cancer: mechanisms of tumor evasion and strategies for breaking tolerance
J Clin Oncol
Cancer immunotherapy: moving beyond current vaccines
Nat Med
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Two authors equally contributed to this work.