Anti-melanoma Activity Research Articles

In silico-guided synthesis of a new, highly soluble, and anti-melanoma flavone glucoside: Skullcapflavone II-6'-O-β-glucoside.

Guided by in silico analysis tools and biotransformation technology, new derivatives of natural compounds with heightened bioactivities can be explored and synthesized efficiently. In this study, in silico data mining and molecular docking analysis predicted that glucosides of skullcapflavone II (SKII) were new flavonoid compounds and had higher binding potential to oncogenic proteins than SKII. These benefits guided us to perform glycosylation of SKII by utilizing four glycoside hydrolases and five glycosyltransferases (GTs). Findings unveiled that exclusive glycosylation of SKII was achieved solely through the action of GTs, with Bacillus subtilis BsUGT489 exhibiting the highest catalytic glycosylation efficacy. Structure analysis determined the glycosylated product as a novel compound, skullcapflavone II-6'-O-β-glucoside (SKII-G). Significantly, the aqueous solubility of SKII-G exceeded its precursor, SKII, by 272-fold. Furthermore, SKII-G demonstrated noteworthy anti-melanoma activity against human A2058 cells, exhibiting an IC50 value surpassing that of SKII by 1.4-fold. Intriguingly, no substantial cytotoxic effects were observed in a murine macrophage cell line, RAW 264.7. This promising anti-melanoma activity without adverse effects on macrophages suggests that SKII-G could be a potential candidate for further preclinical and clinical studies. The in silico tool-guided synthesis of a new, highly soluble, and potent anti-melanoma glucoside, SKII-G, provides a rational design to facilitate the future discovery of new and bioactive compounds.

Enhanced Anti-Melanoma Activity of Nutlin-3a Delivered via Ethosomes: Targeting p53-Mediated Apoptosis in HT144 Cells.

This study evaluated ethosomes as a novel nanodelivery system for nutlin-3a, a known MDM2 inhibitor and activator of the p53 pathway, to improve nutlin-3a's poor solubility, limiting its bio-distribution and therapeutic efficacy. The potential of nutlin-3a-loaded ethosomes was investigated on two in vitro models of melanoma: the HT144 cell line p53wild-type and the SK-MEL-28 cell line p53mutated. Nutlin-3a-loaded ethosomes were characterized for their physicochemical properties and used to treat melanoma cells at different concentrations, considering nutlin-3a solution and empty ethosomes as controls. The biological effects on cells were evaluated 24 and 48 h after treatment by analyzing the cell morphology and viability, cell cycle, and apoptosis rate using flow cytometry and the p53 pathway's activation via Western blotting. The results indicate that ethosomes are delivery systems able to maintain nutlin-3a's functionality and specific biological action, as evidenced by the molecular activation of the p53 pathway and the biological events leading to cell cycle block and apoptosis in p53wild-type cells. Nutlin-3a-loaded ethosomes induced morphological changes in the HT144 cell line, with evident apoptotic cells and a reduction in the number of viable cells of over 80%. Furthermore, nutlin-3a-loaded ethosomes successfully modulated two p53-regulated proteins involved in survival/apoptosis, with up to a 2.5-fold increase in membrane TRAIL-R2 and up to an 8.2-fold decrease in Notch-1 (Notch intracellular domain, NICD) protein expression. The expression of these molecules is known to be altered or dysfunctional in a large percentage of melanoma tumors. Notably, ethosomes, regardless of their nutlin-3a loading, exhibited the ability to reduce HT144 melanoma cellular migration, as assessed in real time using xCELLigence, likely due to the modification of lipid rafts, suggesting their potential antimetastatic properties. Overall, nutlin-3a delivery using ethosomes appears to be a significantly effective means for upregulating the p53 pathway and downregulating active Notch-1, while also taking advantage of their unexpected ability to reduce cellular migration. The findings of this study could pave the way for the development of specific nutlin-3a-loaded ethosome-based medicinal products for cutaneous use.

Predictive Production of a New Highly Soluble Glucoside, Corylin-7-O-β-Glucoside with Potent Anti-inflammatory and Anti-melanoma Activities.

Computational tools can now facilitate screening precursors and selecting suitable biotransformation enzymes for producing new bioactive compounds. This study applied the data-mining approach to screen for candidate precursors of glycosyltransferases to produce new glucosides from 412 commercial natural compounds. Among five candidates, experimental results showed that only corylin could be glycosylated by the bacterial glycosyltransferase, BsUGT489. Analysis of interaction potential between candidates and glycosyltransferase by molecular docking tools also found that corylin was the only compatible substrate. The new glucoside was purified and confirmed to be corylin-7-O-β-glucoside. The aqueous solubility of corylin-7-O-β-glucoside was 14.2 times more than its precursor aglycone, corylin. Corylin-7-O-β-glucoside retained anti-inflammatory activity in lipopolysaccharide-induced nitric oxide production of murine macrophage RAW 264.7 cells, with an IC50 value of 121.1 ± 9.5µM. Further, corylin-7-O-β-glucoside exhibited more potent anti-melanoma activity against murine B16 and human A2058 melanoma cells than corylin. Together, predictive studies facilitate the production of a new glucoside, corylin-7-O-β-glucoside, which is highly soluble and possesses anti-inflammatory and anti-melanoma activities and therefore has promising future applications in pharmacology.

Benzoxaborinine, New Chemotype for Carbonic Anhydrase Inhibition: Ex Novo Synthesis, Crystallography, In Silico Studies, and Anti-Melanoma Cell Line Activity.

The benzoxaborinine scaffold, a homologue of benzoxaborole with an additional carbon atom in the boracycle, shows significant potential in developing new therapeutic agents. This study reports the synthesis, inhibition assays against four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, and anti-melanoma evaluation of 7-aryl(thio)ureido-substituted benzoxaborinines. Some derivatives, particularly compound 11, exhibited potent inhibitory activity (below 65 nM) against hCA IX and XII and stronger antiproliferative effects than SLC-0111 on human melanoma cells under hypoxia. Crystallographic studies of benzoxaborinine 3 adducts with hCA I and II demonstrated the binding mode of this chemotype, revealing that although both benzoxaborinine 3 and benzoxaborole 10 share a similar zinc-binding mode, the expanded ring in benzoxaborinine led to a different orientation within the active site. These findings suggest that benzoxaborinines hold promise for designing novel carbonic anhydrase inhibitors.

Repurposing of Antidiarrheal Loperamide for Treating Melanoma by Inducing Cell Apoptosis and Cell Metastasis Suppression In vitro and In vivo.

Melanoma is the most common skin tumor worldwide and still lacks effective therapeutic agents in clinical practice. Repurposing of existing drugs for clinical tumor treatment is an attractive and effective strategy. Loperamide is a commonly used anti-diarrheal drug with excellent safety profiles. However, the affection and mechanism of loperamide in melanoma remain unknown. Herein, the potential anti-melanoma effects and mechanism of loperamide were investigated in vitro and in vivo. In the present study, we demonstrated that loperamide possessed a strong inhibition in cell viability and proliferation in melanoma using MTT, colony formation and EUD incorporation assays. Meanwhile, xenograft tumor models were established to investigate the anti-melanoma activity of loperamide in vivo. Moreover, the effects of loperamide on apoptosis in melanoma cells and potential mechanisms were explored by Annexin V-FITC apoptosis detection, cell cycle, mitochondrial membrane potential assay, reactive oxygen species level detection, and apoptosis-correlation proteins analysis. Furthermore, loperamide-suppressed melanoma metastasis was studied by migration and invasion assays. What's more, immunohistochemical and immunofluorescence staining assays were applied to demonstrate the mechanism of loperamide against melanoma in vivo. Finally, we performed the analysis of routine blood and blood biochemical, as well as hematoxylin- eosin (H&E) staining, in order to investigate the safety properties of loperamide. Loperamide could observably inhibit melanoma cell proliferation in vitro and in vivo. Meanwhile, loperamide induced melanoma cell apoptosis by accumulation of the sub-G1 cells population, enhancement of reactive oxygen species level, depletion of mitochondrial membrane potential, and apoptosis-related protein activation in vitro. Of note, apoptosis-inducing effects were also observed in vivo. Subsequently, loperamide markedly restrained melanoma cell migration and invasion in vitro and in vivo. Ultimately, loperamide was witnessed to have an amicable safety profile. These findings suggested that repurposing of loperamide might have great potential as a novel and safe alternative strategy to cure melanoma via inhibiting proliferation, inducing apoptosis and cell cycle arrest, and suppressing migration and invasion.

Nitric Oxide-Releasing Topical Treatments for Cutaneous Melanoma.

Melanoma is an aggressive skin cancer notorious for high levels of drug resistance. Additionally, current treatments such as immunotherapies are often associated with numerous adverse side effects. The use of nitric oxide (NO) may represent an attractive treatment for melanoma due to NO's various anticancer properties, unlikeliness to foster resistance, and limited toxicity toward healthy tissues. The anticancer effects of chemical NO donors have been explored previously but with limited understanding of the needed characteristics for exerting optimal antimelanoma activity. Herein, the in vitro therapeutic efficacy of three macromolecular NO donor systems (i.e., cyclodextrin, mesoporous silica nanoparticles, and hyaluronic acid) with tunable NO-release kinetics was explored by evaluating skin permeation along with toxicity against melanoma and healthy skin cells. Cytotoxicity against melanoma cells was dependent on NO payload and not donor identity or NO-release kinetics. In contrast, cytotoxicity against healthy cells was primarily influenced by the macromolecular NO donor, with cyclodextrin- and hyaluronic acid-based NO donors having the highest therapeutic indices. In vitro skin permeation was influenced by both the size and charge of the NO donor, with smaller, more neutral donors resulting in greater permeation. A Pluronic F127 organogel was optimized for the delivery of a cyclodextrin-based NO donor. Delivery of the NO donor in this manner resulted in increased in vitro skin permeation and reduced tumor growth in an in vivo model.

Anti-melanoma cancer activity and chemical profile of the essential oil of Seseli yunnanense Franch

Abstract Seseli yunnanense appertains to the Apiaceae family. S. yunnanense is known as an herbal remedy to treat several illnesses in Chinese traditional medicine. In this research, the essential oil (EO) of the aerial part of S. yunnanense (SYEO) was isolated by hydro-distillation apparatus. The chemical composition of the volatile oil was recognized using Gas chromatography (GC)/flame ionization detector and GC-Mass spectrometry methods. 1,1-Diphenyl-2-picrylhydrazyl and MTT assays were carried out to investigate the antioxidant and anti-melanoma cancer activity of the oil. The chromatography results showed SYEO was dominated by sesquiterpenes of α-bisabolol 40.19%, β-eudesmol 13.12%, and the phenolic compound eugenol 12.06%. SYEO scavenged the free radical of DPPH by IC50 of 454.66 ± 7.25 μg/mL. In the MTT assay, SYEO showed a dose depending on the viability of the melanoma cell lines of HMV-II, SK-ML-2, G-361, and HT144. The highest activity was obtained against the HMV-II cell line with IC50 of 116.82 ± 2.34 μg/mL. The cytotoxicity of SYEO was examined against HUVEC cell lines. The results exhibited the safety of EO for the human normal cells. The results showed the EO of S. yunnanense is a potent agent for treating melanoma cancer.

Development of Quercetin Solid Dispersion-Loaded Dissolving Microneedles and In Vitro Investigation of Their Anti-Melanoma Activities.

Background: Melanoma is a skin cancer that requires early treatment to prevent metastasis. In particular, the superficial spreading melanoma, excisional surgery with local administration of anti-cancer drugs via microneedles is currently considered a potential combination therapy. Quercetin is a natural flavonoid having activities against melanoma cells. Unfortunately, the therapeutic effect is limited by its poor water solubility. Objectives: This study aimed to develop formulations of solid dispersion-loaded dissolving microneedles (SD-DMNs) of quercetin and to investigate their in vitro activities against melanoma cells. Methods: Quercetin solid dispersions (Q-SDs) were prepared using polyvinylpyrrolidone K30 (PVP) via a solvent technique. The optimized Q-SD was selected for preparing Q-SD-loaded dissolving microneedles (Q-SD-DMNs) using a mold casting method. Results: Q-SDs had higher water solubility than that of quercetin by 5-10 times depending on the ratio of quercetin-to-PVP. The presence of quercetin in the Q-SD and Q-SD-DMN were in an amorphous form. The obtained Q-SD-DMNs had pyramid-shaped microneedles. Their strength depended on the compositions, i.e., ratios of hyaluronic acid-to-sodium carboxymethylcellulose and the content of Q-SD. An optimized Q-SD-DMN increased the in vitro skin permeation of quercetin compared to that of microneedles containing quercetin (without being processed). From the molecular investigations, the optimized Q-SD-DMN reduced the viability of the A375 cells (melanoma cells) through the induction of cell apoptosis. It suppressed Bcl-2 gene expression and led to a lower content of Bcl-2 in the cells. Conclusions: The optimized Q-SD-DMN has a potential for use in further in vivo studies as a synergistic method of melanoma treatment.

Protein Structure Inspired Discovery of a Novel Inducer of Anoikis in Human Melanoma.

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

Ethyl Acetate Fraction from Eleutherococcus divaricatus Root Extract as a Promising Source of Compounds with Anti-Hyaluronidase, Anti-Tyrosinase, and Antioxidant Activity but Not Anti-Melanoma Activity.

Eleutherococcus divaricatus (Siebold and Zucc.) S. Y. Hu. has been used in Traditional Chinese Medicine (TCM) due to its anticancer, immunostimulant, and anti-inflammatory activities. However, its mechanism of action and chemical composition are still insufficiently understood and require more advanced research, especially for cases in which anti-inflammatory properties are beneficial. The aim of this study was to evaluate the impact of E. divaricatus root extracts and fractions on proinflammatory serum hyaluronidase and tyrosinase in children diagnosed with acute lymphoblastic leukemia. Antioxidant and anti-melanoma activities were also examined and correlated with metabolomic data. For the first time, we discovered that the ethyl acetate fraction significantly inhibits hyaluronidase activity, with mean group values of 55.82% and 63.8% for aescin used as a control. However, interestingly, the fraction showed no activity against human tyrosinase, and in A375 melanoma cells treated with a doxorubicin fraction, doxorubicin activity decreased. This fraction exhibited the most potent antioxidant activity, which can be attributed to high contents of polyphenols, especially caffeic acid (24 mg/g). The findings suggest an important role of the ethyl acetate fraction in hyaluronidase inhibition, which may additionally indicate its anti-inflammatory property. The results suggest that this fraction can be used in inflammatory-related diseases, although with precautions in cases of patients undergoing chemotherapy.

Modulation of the Antimelanoma Activity Imparted to Artemisinin Hybrids by the Monoterpene Counterpart.

Molecular hybridization is a widely used strategy in drug discovery and development processes that consists of the combination of two bioactive compounds toward a novel entity. In the current study, two libraries of hybrid derivatives coming from the linkage of sesquiterpene counterparts dihydroartemisinin and artesunic acid, with a series of monoterpenes, were synthesized and evaluated by cell viability assay on primary and metastatic melanoma cell lines. Almost all the obtained compounds showed micromolar antimelanoma activity and selectivity toward the metastatic form of this cancer. Four hybrid derivatives containing perillyl alcohol, citronellol, and nerol as monoterpene counterpart emerged as the best compounds of the series, with nerol being active in combination with both sesquiterpenes, dihydroartemisinin and artesunic acid. Preliminary studies on the mechanism of action have shown the dependence of the pharmacological activity of newly synthesized hybrids on the formation of carbon- and oxygen-centered radical species. This study demonstrated the positive modulation of the pharmacodynamic effect of artemisinin semisynthetic derivatives dihydroartemisinin and artesunic acid due to the hybridization with monoterpene counterparts.

Polyfunctionalized organoselenides: New synthetic approach from selenium-containing cyanohydrins and anti-melanoma activity

A series of seleno-containing polyfunctionalized compounds was synthesized exploring cyanohydrin chemistry, including α-hydroxy esters, α-hydroxy acids, 1,2-diols, and 1,2-diacetates, with yields ranging from 26 up to 99 %. The cytotoxicity of all synthesized compounds was then evaluated using a non-tumor cell line (BALB/3T3 murine fibroblasts), and those deemed non-cytotoxic had their anti-melanoma activity evaluated using B16-F10 murine melanoma cells. These assays identified two compounds with selective cytotoxic activity against the tested melanoma cell line, showing a potential anti-melanoma application.

New insights into the anti-inflammatory and anti-melanoma mechanisms of action of azelaic acid and other Fusarium solani metabolites via in vitro and in silico studies

Metabolites exploration of the ethyl acetate extract of Fusarium solani culture broth that was isolated from Euphorbia tirucalli root afforded five compounds; 4-hydroxybenzaldehyde (1), 4-hydroxybenzoic acid (2), tyrosol (3), azelaic acid (4), malic acid (5), and fusaric acid (6). Fungal extract as well as its metabolites were evaluated for their anti-inflammatory and anti-hyperpigmentation potential via in vitro cyclooxygenases and tyrosinase inhibition assays, respectively. Azelaic acid (4) exhibited powerful and selective COX-2 inhibition followed by fusaric acid (6) with IC50 values (2.21 ± 0.06 and 4.81 ± 0.14 μM, respectively). As well, azelaic acid (4) had the most impressive tyrosinase inhibitory effect with IC50 value of 8.75 ± 0.18 μM compared to kojic acid (IC50 = 9.27 ± 0.19 μM). Exclusive computational studies of azelaic acid and fusaric acid with COX-2 were in good accord with the in vitro results. Interestingly, this is the first time to investigate and report the potential of compounds 3–6 to inhibit cyclooxygenase enzymes. One of the most invasive forms of skin cancer is melanoma, a molecular docking study using a set of enzymes related to melanoma suggested pirin to be therapeutic target for azelaic acid and fusaric acid as a plausible mechanism for their anti-melanoma activity.

Antimelanoma Effects of Alchemilla vulgaris: A Comprehensive In Vitro and In Vivo Study.

Due to the rich ethnobotanical and growing evidence-based medicine records, the Alchemillae herba, i.e., the upper parts of the Lady's mantle (Alchemilla vulgaris L.), was used for the assessment of antimelanoma activity. The ethanolic extract of A. vulgaris strongly suppressed the viability of B16F1, B16F10, 518A2, and Fem-X cell lines. In contrast to the in vitro study, where the B16F1 cells were more sensitive to the treatment than the more aggressive counterpart B16F10, the results obtained in vivo using the corresponding syngeneic murine model were quite the opposite. The higher sensitivity of B16F10 tumors in vivo may be attributed to a more complex response to the extract compared to one triggered in vitro. In addition, the strong immunosuppressive microenvironment in the B16F1 model is impaired by the treatment, as evidenced by enhanced antigen-presenting potential of dendritic cells, influx and activity of CD4+ T and CD8+ T lymphocytes, decreased presence of T regulatory lymphocytes, and attenuation of anti-inflammatory cytokine production. All these effects are supported by the absence of systemic toxicity. A. vulgaris extract treatment results in a sustained and enhanced ability to reduce melanoma growth, followed by the restoration of innate and adopted antitumor immunity without affecting the overall physiology of the host.

Nanoemulsified Essential Oil of Melaleuca leucadendron Leaves for Topical Application: In Vitro Photoprotective, Antioxidant and Anti-Melanoma Activities.

Melanoma, primarily caused by solar ultraviolet (UV) radiation, can be prevented by the use of sunscreens. However, the use of synthetic sunscreens raises environmental concerns. Natural compounds with antioxidant photoprotective properties and cytotoxic effects against cancer cells can be promising for the prevention and treatment of melanoma with less environmental effect. This study focuses on Melaleuca leucadendron essential oil (EO) for photoprotection and antitumor applications. EO was hydrodistilled from M. leucadendron leaves with a 0.59% yield. Gas chromatography-mass spectrometry detected monoterpenes and sesquiterpenes. Nanoemulsions were prepared with (NE-EO) and without EO (NE-B) using the phase inversion method, showing good stability, spherical or oval morphology, and a pseudoplastic profile. Photoprotective activity assessed spectrophotometrically showed that the NE-EO was more effective than NE-B and free EO. Antioxidant activity evaluated by DPPH and ABTS methods indicated that pure and nanoemulsified EO mainly inhibited the ABTS radical, showing IC50 40.72 and 5.30 µg/mL, respectively. Cytotoxicity tests on L-929 mouse fibroblasts, NGM human melanocyte, B16-F10 melanoma, and MeWo human melanoma revealed that EO and NE-EO were more cytotoxic to melanoma cells than to non-tumor cells. The stable NE-EO demonstrates potential for melanoma prevention and treatment. Further research is required to gain a better understanding of these activities.

Regorafenib in patients with pretreated advanced melanoma: a single-center case series.

Melanoma patients failing all approved treatment options have a poor prognosis. The antimelanoma activity of regorafenib (REGO), a multitargeted kinase inhibitor, has not been investigated in this patient population. The objective response rate and safety of REGO treatment in advanced melanoma patients was investigated retrospectively. Twenty-seven patients received REGO treatment. All patients had progressed on anti-programmed cell death protein 1 (PD-1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint inhibition and BRAF/MEK inhibitors (in case of a BRAF V600 mutation). REGO was administered in continuous dosing and combined (upfront or sequentially) with nivolumab ( n = 5), trametinib ( n = 8), binimetinib ( n = 2), encorafenib ( n = 1), dabrafenib/trametinib ( n = 9), or encorafenib/binimetinib ( n = 7). The best overall response was partial response (PR) in five patients (18.5%) and stable disease in three patients (11.1%). Three of seven (42.8%) BRAF V600mut patients treated with REGO in combination with BRAF/MEK inhibitors obtained a PR (including regression of brain metastases in all three patients). In addition, PR was documented in a BRAF V600mut patient treated with REGO plus anti-PD-1, and a NRASQ61mut patient treated with REGO plus a MEK inhibitor. Common grade 3-4 treatment-related adverse events included arterial hypertension ( n = 7), elevated transaminase levels ( n = 5), abdominal pain ( n = 3), colitis ( n = 2), anorexia ( n = 1), diarrhea ( n = 1), fever ( n = 1), duodenal perforation ( n = 1), and colonic bleeding ( n = 1). Median progression-free survival was 11.0 weeks (95% confidence interval, 7.1-14.9); median overall survival was 23.1 weeks (95% confidence interval, 13.0-33.3). REGO has a manageable safety profile in advanced melanoma patients, in monotherapy as well as combined with BRAF/MEK inhibitors or PD-1 blocking monoclonal antibodies. The triplet combination of REGO with BRAF/MEK inhibitors appears most active, particularly in the BRAF V600mut patients.

Design, synthesis, and evaluation of novel quindoline derivatives with fork-shaped side chains as RNA G-quadruplex stabilizers for repressing oncogene NRAS translation

NRAS mutation is the second most common oncogenic factor in cutaneous melanoma. Inhibiting NRAS translation by stabilizing the G-quadruplex (G4) structure with small molecules seems to be a potential strategy for cancer therapy due to the NRAS protein's lack of a druggable pocket. To enhance the effects of previously reported G4 stabilizers quindoline derivatives, we designed and synthesized a novel series of quindoline derivatives with fork-shaped side chains by introducing (alkylamino)alkoxy side chains. Panels of experimental results showed that introducing a fork-shaped (alkylamino)alkoxy side chain could enhance the stabilizing abilities of the ligands against NRAS RNA G-quadruplexes and their anti-melanoma activities. One of them, 10b, exhibited good antitumor activity in the NRAS-mutant melanoma xenograft mouse model, showing the therapeutic potential of this kind of compounds.

Abstract 5938: Development of HH044, an orally bioavailable nNOS inhibitor, for the treatment of cutaneous melanoma

Abstract Background: Human cutaneous melanoma is one of the few cancers in which the incidence rate continues to increase, and despite recent advances in immune and targeted therapies, the 5-year survival rate of metastatic disease remains low. Neuronal nitric oxide synthase (nNOS) has been identified to contribute to melanoma development, stimulating disease progression, and facilitating escape of melanoma cells from the immune response. We developed a novel small molecule, HH044, which specifically inhibits nNOS activity and reduced nitric oxide production in melanoma cells. We aim to conduct a proof-of-concept study using our nNOS inhibitor administered orally for anti-melanoma treatment. Methods: Human melanoma cell lines (A375, Sk-Mel-28, wm3211) were employed to analyze the anti-cancer activities of HH044 in vitro. An immune-competent syngeneic melanoma mouse model was used to determine the anti-tumor activity of HH044 when administered orally (50 mg/kg/day) or intraperitoneally (10 mg/kg/day) for 21 days. A pharmacokinetic study of drug levels in serum and tumor samples was conducted after a single dose given intraperitoneally or orally using LC-MS/MS analysis at different time points. Results: HH044 has high selectivity of nNOS over iNOS (340-fold) and eNOS (540-fold). The IC50 of HH044 in melanoma cell lines was 5.27 ± 3.3 μM, while iNOS inhibitor 1200W did not exhibit any cytotoxicity up to 100 μM concentration. Of note, nNOS blockade by HH044 did not result in reactivation of iNOS, and expression levels of iNOS were not changed after HH044 treatment. In vivo studies demonstrated that HH044 treatment given intraperitoneally exhibited potent anti-tumor activity and reduced tumor weight to 45% of control. When administered orally, HH044 at 50 mg/kg significantly reduced tumor growth to 61% of control and extended mice median survival from 32 days to 53 days. Pharmacokinetic analysis showed that oral administration achieved sufficient drug levels in xenograft tumors, which were well above the Ki value (0.005 μM) despite a wide range of bioavailability. The drug level peaked at 2 hours after a single dose (2.27 ± 0.666 μM in the tumor and 23.68 ± 17.43 μM in the serum). Intraperitoneal injection achieved much higher levels in serum and tumor samples, which also declined slower compared to oral gavage administration. By 24 hours, the drug levels in serum and xenograft tumors remained at 1.55 ± 0.12 μM and 1.77 ± 0.24 μM concentration, respectively. No significant systemic toxicities were observed with HH044 treatments either given via i.p. injection or orally. Further in vivo studies combining nNOS inhibitors with immune checkpoint blockade are currently underway. Conclusions: HH044, as the first-in-class nNOS inhibitor, exhibits potent anti-melanoma activity by oral administration. Developing selective nNOS inhibitors has shown to be a novel therapeutic strategy to improve melanoma treatment. Citation Format: Anika Patel, Shirley Tong, Richard B. Silverman, Sun Yang. Development of HH044, an orally bioavailable nNOS inhibitor, for the treatment of cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5938.

Discovery of novel peptide–dehydroepiandrosterone hybrids inducing endoplasmic reticulum stress with effective in vitro and in vivo anti-melanoma activities

Steroid hybrids have emerged as a type of advantageous compound as they could offer improved pharmacological and pharmaceutical properties. Here, we report a series of novel peptide–dehydroepiandrosterone hybrids, which would effectively induce endoplasmic reticulum stress (ERS) and lead to apoptosis with outstanding in vitro and in vivo anti-melanoma effects. The lead compound IId among various steroids conjugated with peptides and pyridines showed effective in vivo activity in B16 xenograft mice: in medium- and high-dose treatment groups (60 and 80 mg/kg), compound IId would significantly inhibit the growth of tumours by 98%–99% compared to the control group, with the highest survival rate as well. Further mechanism studies showed that compound IId would damage the endoplasmic reticulum and upregulate the ERS markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), which could further regulate caspase and Bcl-2 family proteins and lead to cell apoptosis. The compound IId was also proven to be effective in inhibiting B16 cell migration and invasion.

Development of nanoemulgel of 5-Fluorouracil for skin melanoma using glycyrrhizin as a penetration enhancer

The purpose of this study was to enhance the topical delivery of 5-Fluorouracil (5-FU), a cancer treatment, by developing a nanoemulgel formulation. Glycyrrhizin (GLY), a natural penetration enhancer has been investigated to exhibit synergistic effects with 5-FU in inhibiting melanoma cell proliferation and inducing apoptosis, Hence, GLY, along with suitable lipids was utilized to create an optimized nanoemulsion (NE) based gel. Solubility studies and ternary phase diagram revealed isopropyl myristate (IPM), Span 80, Tween 80 as Smix and Transcutol P as co-surfactant. IPM demonstrates excellent solubilizing properties facilitates higher drug loading, ensuring efficient delivery to the target site.,The optimized formulation consisting of 40 % IPM, 30 % of mixture of Tween80: Span80 (Smix) and 15 % Transcutol P provides with a nanometric size of 64.1 ± 5.13 nm and drug loading of 97.3 ± 5.83 %. The optimized formulation observed with no creaming and breakeing of NE and found thermodynamically stable during different stress conditions (temperatures of 4.0 °C and 45.0 °C) and physical thawing (−21.0 ± 0.50 °C to 20.0 ± 0.50 °C). The NE was then transformed into a nanoemulgel (NEG) using 1.5 % w/w Carbopol base and 0.1 % w/w glycyrrhizin. The ex vivo permeability studies showed significant enhancements in drug permeability with the GLY-based 5-FU-NEG formulation compared to pure 5-FU gel in excised pig skin upto1440 min in PBS 7.4 as receptor media. The IC50 values for Plain 5-FU gel, 5-FU-NEG, and GLY-based 5-FU-NEG were found to be 20 µg/mL, 1.1 µg/mL, and 0.1 µg/mL, respectively in B16F10 cell lines. The percentage intracellular uptake of GLY-5-FU-NEG and 5-FU-NEG was found to be 44.3 % and 53.6 %, respectively. GLY-based 5-FU-NEG formulation showed alterations in cell cycle distribution, in compared to 5-FU-NE gel. The overall findings suggest that the GLY-based 5-FU-NEG holds promise for improving anti-melanoma activity.