Rational Drug Design of Targeted and Enzyme-Cleavable Vitamin E Analogs as a Neoadjuvant to Chemotherapy: In Vitro and In Vivo Evaluation on Reduction of the Cardiotoxicity Side Effect of Doxorubicin.

Traditional drug design focuses on specific biological targets where specific receptors or biomarkers are overexpressed by cancer cells. Cancer cells circumvent the interventions by activating survival pathways and/or downregulating cell death pathways for their survival. A priori activation of apoptosis pathways of tumor (AAAPT) is a novel tumor-sensitizing technology that sensitizes tumor cells that are not responding well to the current treatments by targeting specific survival pathways involved in the desensitization of tumor cells and tries to revive them selectively in cancer cells, sparing normal cells. Several vitamin E derivatives (AMP-001, AMP-002, AMP-003, and AMP-004) were synthesized, characterized, and studied for their anti-tumorigenic properties and their synergistic potential with the standard chemotherapy doxorubicin in various cancer cells including brain cancer stem cells in vitro. Preliminary studies revealed that AAAPT drugs (a) reduced the invasive potential of brain tumor stem cells, (b) synergized with Federal Drug Application-approved doxorubicin, and (c) enhanced the therapeutic index of doxorubicin in the triple-negative breast cancer tumor rat model, preserving the ventricular function compared to cardiotoxic doxorubicin alone at therapeutic dose. The AAAPT approach has the advantage of inhibiting survival pathways and activating cell death pathways selectively in cancer cells by using targeting, linkers cleavable by tumor-specific Cathepsin B, and PEGylation technology to enhance the bioavailability. We propose AAAPT drugs as a neoadjuvant to chemotherapy and not as stand-alone therapy, which is shown to be effective in expanding the therapeutic index of doxorubicin and making it work at lower doses.

Design of a Tumor Binding GMCSF as Intratumoral Immunotherapy of Solid Tumors.

Next-generation cancer immunotherapies may utilize immunostimulants to selectively activate the host immune system against tumor cells. Checkpoint inhibitors (CPIs) like anti-PD1/PDL-1 that inhibit immunosuppression have shown unprecedented success but are only effective in the 20-30% of patients that possess an already "hot" (immunogenic) tumor. In this regard, intratumoral (IT) injection of immunostimulants is a promising approach since they can work synergistically with CPIs to overcome the resistance to immunotherapies by inducing immune stimulation in the tumor. One such immunostimulant is granulocyte macrophage-colony-stimulating factor (GMCSF) that functions by recruiting and activating antigen-presenting cells (dendritic cells) in the tumor, thereby initiating anti-tumor immune responses. However, key problems with GMCSF are lack of efficacy and the risk of systemic toxicity caused by the leakage of GMCSF from the tumor tissue. We have designed tumor-retentive versions of GMCSF that are safe yet potent immunostimulants for the local treatment of solid tumors. The engineered GMCSFs (eGMCSF) were synthesized by recombinantly fusing tumor-ECM (extracellular matrix) binding peptides to GMCSF. The eGMCSFs exhibited enhanced tumor binding and potent immunological activity in vitro and in vivo. Upon IT administration, the tumor-retentive eGMCSFs persisted in the tumor, thereby alleviating systemic toxicity, and elicited localized immune activation to effectively turn an unresponsive immunologically "cold" tumor "hot".

Intratumoral Cancer Chemotherapy with a Carrier-Based Immunogenic Cell-Death Eliciting Platinum (IV) Agent.

A carrier-based, immunogenic cell death (ICD)-eliciting platinum(IV) chemotherapeutic agent was synthesized via complexation between an axially derivatized Pt(IV)-tocopherol and hyaluronan (HA)-tocopherol nanocarrier. The resultant HA-Pt(IV) complex demonstrated antiproliferative activity and induced calreticulin translocation, an indicator of ICD, in murine and human head and neck cancer (HNC) cells. The intratumorally administered HA-Pt(IV) treatments were tolerable and efficacious in both immunocompetent and immunodeficient mice with HNC, partially because of the direct cytotoxicity. Superior efficacy and survival were observed in the immunocompetent group, suggesting a possible Pt(IV)-induced immunological response, which would only manifest in animals with an intact immune system. Subsequent imaging of tumor tissues demonstrated increased macrophage infiltration in the HA-Pt(IV)-treated tumors compared to the nontreated controls and the cisplatin-treated tumors, suggesting favorable inflammatory activation. RNA sequencing of HA-Pt(IV)-treated tumors indicated that carbohydrate and vitamin metabolisms were the most important Kyoto Encyclopedia of Genes and Genomes pathways, and molecular function, biological process, and cellular component were highly enriched gene ontology categories.

Implantable hyaluronic acid-deferoxamine conjugate prevents nonunions through stimulation of neovascularization.

Approximately 6.3 million fractures occur in the U.S. annually, with 5-10% resulting in debilitating nonunions. A major limitation to achieving successful bony union is impaired neovascularization. To augment fracture healing, we designed an implantable drug delivery technology containing the angiogenic stimulant, deferoxamine (DFO). DFO activates new blood vessel formation through iron chelation and upregulation of the HIF-1α pathway. However, due to its short half-life and rapid clearance, maintaining DFO at the callus site during peak fracture angiogenesis has remained challenging. To overcome these limitations, we composed an implantable formulation of DFO conjugated to hyaluronic acid (HA). This compound immobilizes DFO within the fracture callus throughout the angiogenic window, making it a high-capacity iron sponge that amplifies blood vessel formation and prevents nonunions. We investigated implanted HA-DFO's capacity to facilitate fracture healing in the irradiated rat mandible, a model whereby nonunions routinely develop secondary to obliteration of vascularity. HA-DFO implantation significantly improved radiomorphometrics and metrics of biomechanical strength. In addition, HA-DFO treated mandibles exhibited a remarkable 91% bone union rate, representing a 3.5-fold improvement over non-treated/irradiated controls (20% bone union rate). Collectively, our work proposes a unique methodology for the targeted delivery of DFO to fracture sites in order to facilitate neovascularization. If these findings are successfully translated into clinical practice, millions of patients will benefit from the prevention of nonunions.

Alternol eliminates excessive ATP production by disturbing Krebs cycle in prostate cancer.

BACKGROUND: Alternol is a natural compound isolated from fermentation products of a mutant fungus. Our previous studies demonstrated that Alternol specifically kills cancer cells but spares benign cells. METHODS: To investigate the mechanism underlying alternol-induced cancer cell-specific killing effect, we took a comprehensive strategy to identify Alternol's protein targets in prostate cancer cells, including PC-3, C4-2, and 22RV1, plus benign BPH1 cell lines. Major experimental techniques included biotin-streptavidin pulldown assay coupled with mass-spectrometry, in vitro enzyme activity assay for Krebs cycle enzymes and gas chromatography-mass spectrometry (GC-MS) for metabolomic analysis. RESULTS: Among 14 verified protein targets, four were Krebs cycle enzymes, fumarate hydratase (FH), malate dehydrogenase-2 (MDH2), dihydrolipoamide acetyltransferase (DLAT) in pyruvate dehydrogenase complex (PDHC) and dihydrolipoamide S-succinyltransferase (DLST) in a-ketoglutarate dehydrogenase complex (KGDHC). Functional assays revealed that PDHC and KGDHC activities at the basal level were significantly higher in prostate cancer cells compared to benign prostate BPH1 cells, while alternol treatment reduced their activities in cancer cells close to the levels in BPH1 cells. Although FH and MDH2 activities were comparable among prostate cancer and benign cell lines at the basal level, Alternol treatment largely increased their activities in cancer cells. Metabolomic analysis revealed that Alternol treatment remarkably reduced the levels of malic acid, fumaric acid, and isocitric acid and mitochondrial respiration in prostate cancer cells. Alternol also drastically reduced mitochondrial respiration and ATP production in PC-3 cells in vitro or in xenograft tissues but not in BPH1 cells or host liver tissues. CONCLUSIONS: Alternol interacts with multiple Krebs cycle enzymes, resulting in reduced mitochondrial respiration and ATP production in prostate cancer cells and xenograft tissues, providing a novel therapeutic strategy for prostate cancer treatment.

Injectable Chemotherapy Downstaged Oral Squamous Cell Carcinoma from Nonresectable to Resectable in a Rescue Dog: Diagnosis, Treatment, and Outcome.

This case report documents the diagnosis, treatment, and outcome of a nonresectable oral squamous cell carcinoma in a dog with initial poor prognosis. An approximately 4-year-old female Staffordshire Bull Terrier presented with a large mass on the front of lower jaw which was diagnosed as oral papillary squamous cell carcinoma by histopathology. CT scans revealed invasion of the cancer to the frenulum of the tongue. The mass was inoperable due to location, expansiveness, and metastatic lymph nodes. The dog received 4 treatments of intralesional hyaluronan-platinum conjugates (HylaPlat™, HylaPharm LLC, Lawrence, Kansas) at 3-week intervals. Clinical chemistry and complete blood count were performed one week after each treatment and results were within normal limits. Complications included bleeding due to tumor tissue sloughing, as well as a single seizure due to unknown causes. Upon completion of chemotherapy, CT showed that the mass had regressed and was no longer invading the lingual frenulum, and multiple lymph nodes were free of metastasis. The mass thus became resectable and the dog successfully underwent rostral bilateral mandibulectomy. Over one year after chemotherapy and surgery, the cancer remains in complete remission.

DOX-Vit D, a Novel Doxorubicin Delivery Approach, Inhibits Human Osteosarcoma Cell Proliferation by Inducing Apoptosis While Inhibiting Akt and mTOR Signaling Pathways.

Doxorubicin (DOX) is a very potent and effective anticancer agent. However, the effectiveness of DOX in osteosarcoma is usually limited by the acquired drug resistance. Recently, Vitamin D (Vit-D) was shown to suppress the growth of many human cancer cells. Taken together, we synthesized DOX-Vit D by conjugating Vit-D to DOX in order to increase the delivery of DOX into cancer cells and mitigate the chemoresistance associated with DOX. For this purpose, MG63 cells were treated with 10 µM DOX or DOX-Vit D for 24 h. Thereafter, MTT, real-time PCR and western blot analysis were used to determine cell proliferation, genes and proteins expression, respectively. Our results showed that DOX-Vit D, but not DOX, significantly elicited an apoptotic signal in MG63 cells as evidenced by induction of death receptor, Caspase-3 and BCLxs genes. Mechanistically, the DOX-Vit D-induced apoptogens were credited to the activation of p-JNK and p-p38 signaling pathway and the inhibition of proliferative proteins, p-Akt and p-mTOR. Our findings propose that DOX-Vit D suppressed the growth of MG63 cells by inducing apoptosis while inhibiting cell survival and proliferative signaling pathways. DOX-Vit D may serve as a novel drug delivery approach to potentiate the delivery of DOX into cancer cells.

Pharmacokinetic Evaluation of a DSPE-PEG2000 Micellar Formulation of Ridaforolimus in Rat.

The rapamycin analog, ridaforolimus, has demonstrated potent anti-proliferative effects in cancer treatment, and it currently is being evaluated in a range of clinical cancer studies. Ridaforolimus is an extremely lipophilic compound with limited aqueous solubility, which may benefit from formulation with polymeric micelles. Herein, we report the encapsulation of ridaforolimus in 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000) (DSPE-PEG2000) via a solvent extraction technique. Micelle loading greatly improved the solubility of ridaforolimus by approximately 40 times from 200 µg/mL to 8.9 mg/mL. The diameters of the drug-loaded micelles were 33 ± 15 nm indicating they are of appropriate size to accumulate within the tumor site via the enhanced permeability and retention (EPR) effect. The DSPE-PEG2000 micelle formulation was dosed intravenously to rats at 10 mg/kg and compared to a control of ridaforolimus in ethanol/PEG 400. The micelle significantly increased the half-life of ridaforolimus by 170% and decreased the clearance by 58%, which is consistent with improved retention of the drug in the plasma by the micelle formulation.