Options of Therapeutics and Novel Delivery Systems of Drugs for the Treatment of Melanoma.

Melanoma is one of the most severe cancerous diseases. The cells employ multiple signaling pathways, such as ERK, HGF/c-MET, WNT, and COX-2 to cause the cell proliferation, survival, and metastasis. Treatment of melanoma, including surgery, chemotherapy, immunotherapy, radiation, and targeted therapy, is based on 4 major or 11 substages of the disease. Fourteen drugs, including dacarbazine, interferon α-2b, interleukin-12, ipilimumab, peginterferon α-2b, vemurafenib, trametinib, talimogene laherparepvec, cobimetinib, pembrolizumab, dabrafenib, binimetinib, encorafenib, and nivolumab, have been approved by the FDA for the treatment of melanoma. All of them are in conventional dosage forms of injection solutions, suspensions, oral tablets, or capsules. Major drawbacks of the treatment are side effects of the drugs and patients' incompliance to them. These are consequences of high doses and long-term treatments for the diseases. Currently more than 350 NCI-registered clinical trials are being carried out to treat advanced and/or metastatic melanoma using novel treatment methods, such as immune cell therapy, cancer vaccines, and new therapeutic targets. In addition, novel delivery systems using biomaterials of the approved drugs have been developed attempting to increase the drug delivery, targeting, stability, bioavailability, thus potentially reducing the toxicity and increasing the treatment effectiveness. Nanoparticles and liposomes have been emerging as advanced delivery systems which can improve drug stability and systemic circulation time. In this review, the most recent findings in the options for treatment and development of novel drug delivery systems for the treatment of melanoma are comprehensively discussed.

Identification of (R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator.

A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.

Localized irradiation of tumors prior to synthetic dsRNA therapy enhanced the resultant anti-tumor activity.

BACKGROUND AND PURPOSE: Despite the potent tumoricidal activity of the synthetic dsRNA in culture, its in vivo anti-tumor activity has proven to be limited. We sought to devise and validate a new strategy to improve the in vivo anti-tumor activity by integrating localized irradiation into dsRNA therapy. MATERIALS AND METHODS: Using a mouse lung cancer model and a mouse melanoma model in immuno-competent mice or athymic nude mice, we evaluated the combined anti-tumor activity using a synthetic dsRNA, polyinosine-cytosine (poly(I:C)). RESULTS: Localized irradiation of tumors prior to the poly(I:C) therapy significantly delayed the tumor growth as compared to monotherapies using the radiation or poly(I:C) alone. The poly(I:C) enhanced the tumor response to radiation with a dose modification factor as large as 20. The combined effect was synergistic only in immuno-competent mice with highly immunogenic tumors. The anti-tumor activity of the combination therapy was significantly impaired when the type I interferons in the mice were neutralized. CONCLUSIONS: This combination modality may represent a promising approach to exploit synthetic dsRNA in cancer therapy and to enhance tumor response to radiation. T cell-mediated immunity was likely responsible for the combined synergistic effect. Type I interferons contributed significantly to the combined anti-tumor activity.

Nasal immunization with the mixture of PA63, LF, and a PGA conjugate induced strong antibody responses against all three antigens.

A new generation anthrax vaccine is expected to target not only the anthrax protective antigen (PA) protein, but also other virulent factors of Bacillus anthracis. It is also expected to be amenable for rapid mass immunization of a large number of people. This study aimed to address these needs by designing a prototypic triantigen nasal anthrax vaccine candidate that contained a truncated PA (rPA63), the anthrax lethal factor (LF), and the capsular poly-gamma-D-glutamic acid (gammaDPGA) as the antigens and a synthetic double-stranded RNA (dsRNA), polyriboinosinic-polyribocytodylic acid (poly(I:C)) as the adjuvant. This study identified the optimal dose of nasal poly(I:C) in mice, demonstrated that nasal immunization of mice with the LF was capable of inducing functional anti-LF antibodies (Abs), and showed that nasal immunization of mice with the prototypic triantigen vaccine candidate induced strong immune responses against all three antigens. The immune responses protected macrophages against an anthrax lethal toxin challenge in vitro and enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge in vivo. The anti-PGA Abs were shown to have complement-mediated bacteriolytic activity. After further optimization, this triantigen nasal vaccine candidate is expected to become one of the newer generation anthrax vaccines.

Tumor chemo-immunotherapy using gemcitabine and a synthetic dsRNA.

Both gemcitabine and synthetic double-stranded RNA (dsRNA) are known to be proapoptotic and immuno-stimulatory (-modulatory). We sought to evaluate the extent to which a combination therapy using gemcitabine and a synthetic dsRNA, polyinosine-cytosine (poly(I:C)), would improve the resultant anti-tumor activity. Using model lung and breast cancers in mice, we demonstrated that combination treatment of tumor-bearing mice with the poly(I:C) and gemcitabine synergistically delayed the tumor growth and prolonged the survival of the mice. The combination treatment also synergistically inhibited tumor cell growth in vitro and promoted more tumor cells to undergo apoptosis in vivo. Finally, the combination therapy generated a strong and durable specific anti-tumor immune response, although the immune response alone was unable to control the tumor growth after the termination of the therapy. This approach represents a promising therapy to improve the clinical outcomes for tumors sensitive to both dsRNA and gemcitabine.

Immunization by application of DNA vaccine onto a skin area wherein the hair follicles have been induced into anagen-onset stage.

An attractive approach to immunization is to apply DNA vaccine topically onto the skin. However, it is important to ensure that a strong immune response is induced without disrupting the skin stratum corneum. The hair follicles have been shown to be the major portal of entry for DNA applied onto the skin, and it has been reported that the transfection of hair follicle cells occurs mainly at the onset of a new growing stage of the hair cycle. Using an anthrax protective antigen (PA) protein-encoding plasmid in mice, we demonstrated that the anti-PA immune responses were significantly stronger when the hair follicles in the application area were induced into anagen-onset stage than when in telogen stage. The anti-PA antibodies enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge. The enhanced immune responses can be partially attributed to the enhanced antigen gene expression and plasmid DNA uptake in the skin area wherein the hair follicles were induced into anagen-onset stage. Moreover, the moderate dermal inflammation associated with the anagen induction may also have contributed to the enhancement of the resultant immune response. This represents a novel approach to enhancing the immune response induced by a topically applied DNA vaccine.

Learning from viruses: the necrotic bodies of tumor cells with intracellular synthetic dsRNA induced strong anti-tumor immune responses.

PURPOSE: Coaxing dead tumor cells to induce specific immune responses is an attractive tumor therapy. However, there continues to be a need for adjuvants that can promote the cross-presentation of the dead tumor cells to induce specific anti-tumor response. Viral dsRNA has multiple mechanisms to promote the cross-presentation of viral antigens in virus-infected cells. We propose to learn from viruses by generating dead tumor cells having synthetic dsRNA delivered inside them to allow the dsRNA to promote the cross-presentation of dead tumor cells. MATERIALS AND METHODS: Using synthetic dsRNA, poly(I:C), and the TC-1 cervical cancer model, we evaluated the extent to which the poly(I:C) can promote the necrotic bodies of TC-1 cells to induce specific anti-tumor immune response. The poly(I:C) was either simply mixed with the dead TC-1 cells or pre-loaded inside them. RESULTS: Immunization of tumor-bearing mice with the necrotic bodies of tumor cells admixed with poly(I:C) significantly inhibited the tumor growth. More importantly, immunization with the necrotic bodies having poly(I:C) pre-loaded inside led to a significantly stronger anti-tumor response than when the necrotic bodies were simply admixed with the poly(I:C), apparently through a CD8(+) CTL response-mediated mechanism. CONCLUSIONS: These findings are expected to be clinically relevant for devising improved whole cell-based tumor vaccines.

Biodistribution and tumor-accumulation of gadolinium (Gd) encapsulated in long-circulating liposomes in tumor-bearing mice for potential neutron capture therapy.

To deliver and maintain a sufficient amount of Gd into tumors is required for a successful Gd neutron capture therapy (Gd-NCT), but it has been proven to be rather challenging to achieve. Previously, we have reported a Gd-encapsulated liposome formulation that has the potential to overcome this challenge. In the present study, we sought to systemically evaluate the biodistribution and the tumor-accumulation of the Gd in model tumor-bearing mice. The Gd-encapsulated liposomes were injected into mice pre-grafted with two different model tumors. The Gd content in the tumors and other organs were determined at various time after the injection. A sufficient amount of Gd was readily delivered into those two different model tumors. Increasing the dose of Gd by injecting the Gd-encapsulated liposomes multiple times tended to increase the uptake of the Gd by the tumors. Finally, the uptake of Gd by tumors was inversely correlated with the size of the tumors. The Gd-encapsulated liposomes hold great potentials as a Gd delivery system for NCT of small- and medium-size tumors. Alternative strategies may have to be adopted in order to use NCT to treat large, advanced solid tumors, although for which, Gd-NCT might be advantageous over boron-NCT.

Long-circulating gadolinium-encapsulated liposomes for potential application in tumor neutron capture therapy.

Gadolinium neutron capture therapy (Gd-NCT) is a promising cancer therapy modality. One of the key factors for a successful Gd-NCT is to deliver and maintain a sufficient amount of Gd in tumor tissues during neutron irradiation. We proposed to prepare a Gd delivery system by complexing a Gd-containing compound, diethylenetriaminepentaacetic acid (Gd-DTPA), with a polycationic peptide, poly-L-lysine (pLL), and then encapsulate the complexed Gd-DTPA into PEGylated liposomes. Complexation of Gd-DTPA with pLL not only enhanced the encapsulation efficiency of Gd-DTPA in liposomes, but also significantly limited the release of Gd-DTPA from the liposomes. A Gd-DTPA-encapsulated liposome formulation that contained 6.8+/-0.3 mg/mL of pure encapsulated Gd was prepared. The blood half-life of the Gd encapsulated into the liposome formulation was estimated to be about 24 h in healthy tumor-free mice. About 12 h after the Gd-encapsulated liposomes were intravenously injected into mice with pre-established model tumors, the Gd content in the tumors reached an average of 159 microg/g of wet tumor tissue. This Gd-DTPA encapsulated liposome may be used to deliver Gd into solid tumors for NCT and tumor imaging.