Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

Vitamin E is a generic term that refers to a family of compounds that is further divided into two subgroups called tocopherols and tocotrienols. Although all natural forms of vitamin E display potent antioxidant activity, tocotrienols are significantly more potent than tocopherols in inhibiting tumor cell growth and viability, and anticancer activity of tocotrienols is mediated independently of their antioxidant activity. In addition, the anticancer effects of tocotrienols are observed using treatment doses that have little or no effect on normal cell function or viability. This review will summarize experimental studies that have identified the intracellular mechanism mediating the anticancer effects of tocotrienols. Evidence is also provided showing that combined treatment of tocotrienol with other cancer chemotherapies can result in a synergistic inhibition in cancer cell growth and viability. Taken together, these findings strongly indicate that tocotrienols may provide significant health benefits in the prevention and/or treatment of cancer when used either alone as monotherapy or in combination with other anticancer agents.

Breast cancer is a multi-stage process which leads to the accumulation of abnormal cells arising from excessive proliferation, lack of apoptosis or a combination of both. Natural compounds such as g-tocotrienol have been shown to selectively inhibit cancer cell growth without harming normal cell viability with little or no adverse side effects. The antiproliferative and apoptotic effects of the tocotrienol isoform, γ-tocotrienol, have been firmly established in various cancer types. However, in vivo studies have provided mixed results, attributed to γ-tocotrienol rapid clearance and low bioavailability. In order to improve anticancer potency and bioavailability in vivo, γ- and Δ-tocotrienol were chemically modified with electrophilic substitution reactions on their chromane ring using Mannich and Lederer-Manasse reactions, resulting in the synthesis of various oxazine derivatives. Several of these oxazine derivatives (compounds 26, 31, 39, 40 and 44) were found to display potent anticancer activity as compared to their parent compounds when tested on +SA mammary cancer cells grown in culture. These in vitro studies were followed up with in vivo studies to determine the anticancer effects of oxazine derivatives on the growth of mammary tumors in mice. Female syngeneic BALB/c mice, 4-6 week old were inoculated with 1×106 +SA mammary tumor cells in the left mammary pad. Once tumor size reached 5mm in diameter, animals were divided into different treatment groups and received an intra-tumoral injection injected of 0-120μg/20μl tocotrienol or its derivative every other day for 11 days. Afterwards, mice were sacrificed, tumors removed and placed in -80°C until further analysis. Results from these studies showed that tumor growth rate was significantly reduced in the oxazine derivative treated animals as compared to the vehicle-treated controls. Western blot analysis of tumor samples showed that the growth inhibitory effects of tocotrienol derivatives was also associated with a significant reduction in phosphorylated (activated) Akt and reductions in cell cycle regulatory proteins cyclin D1 and cyclin dependent kinases (CDK2, CDK4 and CDK6). In addition, oxazine derivative treatment was also associated with a large increase in CDK inhibitors p21 and p27, as compared to tumors obtained from the vehicle-treated control mice. Western blot analysis also showed that tumor from oxazine-derivative treated mice displayed a large reduction in NFκB levels and its downstream gene product COX-2. In summary, oxazine derivatives of tocotrienols display more potent anticancer activity both in vitro and in vivo, as compared to their parent compounds and suggest that these tocotrienol derivatives may provide some benefit as novel anticancer therapeutic agents. This work was supported, in part, by First Tec International Ltd. (Hong Kong), Malaysian Palm oil Council and the Louisiana Cancer Foundation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-11.

Resident mesenchymal stem cells (MSCs) promote cancer progression. However, pathways and mechanisms involved in recruiting MSCs into breast tumors remain largely undefined. Here we show that geminin-dependent acetylation releases HMGB1 from the chromatin to the cytoplasm and extracellular space. Extracellular acetylated HMGB1 (Ac-HMGB1) promotes geminin overexpressing (GemOE) cells survival by binding to RAGE and activating NF-κB signaling. Extracellular Ac-HMGB1 also triggers expression and activation of RAGE in the non-expressing MSCs. RAGE activation induces expression of CXCR4 in MSCs and directional migration towards SDF1 (aka CXCL12)-expressing GemOE cells in vitro and in vivo. These effects augmented by the necrotic and hypoxic environment in GemOE tumors, especially within their cores. Reciprocal interactions between newly recruited MSCs and GemOE tumor cells elevate tumor-initiating (TIC), basal and epithelial-to-mesenchymal transition (EMT) traits and enhance aggressiveness in vitro and in vivo in GemOE tumor cells. Indeed, faster, larger and more aggressive tumors develop when GemOE cells are co-injected with MSCs in orthotopic breast tumor model. Concurrently, inhibiting c-Abl (and thus geminin function), RAGE or CXCR4 prevented MSCs recruitment to GemOE cells in vitro and in vivo, and decreased the TIC, basal and EMT phenotypes in these tumor cells. Accordingly, we propose that GemOE tumor cells present within tumor cores represent metastatic precursors, and suppressing the GemOE→HMGB1/RAGE→SDF1/CXCR4 signaling circuit could be a valid target for therapies to inhibit GemOE tumors and their metastases.

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Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a β-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

In response to low oxygen supply, cancer cells elevate production of HIF-1α, a hypoxia-inducible transcription factor that subsequently acts to stimulate blood vessel formation and promote survival. Studies were conducted to determine the role of δ-tocotrienol and a semisynthetic δ-tocotrienol oxazine derivative, compound 44, on +SA mammary tumor cell hypoxic response. Treatment with 150 µM CoCl2 induced a hypoxic response in +SA mammary tumor cells as evidenced by a large increase in HIF-1α levels, and combined treatment with compound 44 attenuated this response. CoCl2-induced hypoxia was also associated with a large increase in Akt/mTOR signaling, activation of downstream targets p70S6K and eIF-4E1, and a significant increase in VEGF production, and combined treatment with compound 44 blocked this response. Additional in vivo studies showed that intralesional treatment with compound 44 in BALB/c mice bearing +SA mammary tumors significantly decreased the levels of HIF-1α, and this effect was associated with a corresponding decrease in Akt/mTOR signaling and activation of downstream targets p70S6kinase and eIF-4E1. These findings demonstrate that treatment with the δ-tocotrienol oxazine derivative, compound 44, significantly attenuates +SA mammary tumor cell compensatory responses to hypoxia and suggests that this compound may provide benefit in the treatment of rapidly growing solid breast tumors.

Biodegradable polymer-based injectable in situ forming depot (ISD) systems that solidify in the body to form a solid or semisolid reservoir are becoming increasingly attractive as an injectable dosage form for sustained (months to years) parenteral drug delivery. Evaluation of long-term drug release from the ISD systems during the formulation development is laborious and costly. An accelerated release method that can effectively correlate the months to years of long-term release in a short time such as days or weeks is economically needed. However, no such accelerated ISD system release method has been reported in the literature to date. The objective of the current study was to develop a short-term accelerated in vitro release method for contraceptive levonorgestrel (LNG)-containing ISD systems to screen formulations for more than 3-month contraception after a single subcutaneous injection. The LNG-containing ISD formulations were prepared by using biodegradable poly(lactide-co-glycolide) and polylactic acid polymer and solvent mixtures containing N-methyl-2-pyrrolidone and benzyl benzoate or triethyl citrate. Drug release studies were performed under real-time (long-term) conditions (PBS, pH 7.4, 37 °C) and four accelerated (short-term) conditions: (A) PBS, pH 7.4, 50 °C; (B) 25% ethanol in PBS, pH 7.4, 50 °C; (C) 25% ethanol in PBS, 2% Tween 20, pH 7.4, 50 °C; and (D) 25% ethanol in PBS, 2% Tween 20, pH 9, 50 °C. The LNG release profile, including the release mechanism under the accelerated condition D within two weeks, correlated (r2 ≥ 0.98) well with that under real-time conditions at four months.