Functionality of bone marrow mesenchymal stromal cells derived from head and neck cancer patients - A FDA-IND enabling study regarding MSC-based treatments for radiation-induced xerostomia.

PURPOSE: Salivary dysfunction is a significant side effect of radiation therapy for head and neck cancer (HNC). Preliminary data suggests that mesenchymal stromal cells (MSCs) can improve salivary function. Whether MSCs from HNC patients who have completed chemoradiation are functionally similar to those from healthy patients is unknown. We performed a pilot clinical study to determine whether bone marrow-derived MSCs [MSC(M)] from HNC patients could be used for the treatment of RT-induced salivary dysfunction. METHODS: An IRB-approved pilot clinical study was undertaken on HNC patients with xerostomia who had completed treatment two or more years prior. Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated and cultured. Culture-expanded MSC(M) were stimulated with IFNγ and cryopreserved prior to reanimation and profiling for functional markers by flow cytometry and ELISA. MSC(M) were additionally injected into mice with radiation-induced xerostomia and the changes in salivary gland histology and salivary production were examined. RESULTS: A total of six subjects were enrolled. MSC(M) from all subjects were culture expanded to > 20 million cells in a median of 15.5 days (range 8-20 days). Flow cytometry confirmed that cultured cells from HNC patients were MSC(M). Functional flow cytometry demonstrated that these IFNγ-stimulated MSC(M) acquired an immunosuppressive phenotype. IFNγ-stimulated MSC(M) from HNC patients were found to express GDNF, WNT1, and R-spondin 1 as well as pro-angiogenesis and immunomodulatory cytokines. In mice, IFNγ-stimulated MSC(M) injection after radiation decreased the loss of acinar cells, decreased the formation of fibrosis, and increased salivary production. CONCLUSIONS: MSC (M) from previously treated HNC patients can be expanded for auto-transplantation and are functionally active. Furthermore IFNγ-stimulated MSC(M) express proteins implicated in salivary gland regeneration. This study provides preliminary data supporting the feasibility of using autologous MSC(M) from HNC patients to treat RT-induced salivary dysfunction.

Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction (MARSH): Study protocol for a phase 1 dose-escalation trial of patients with xerostomia after radiation therapy for head and neck cancer: MARSH: Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction.

BACKGROUND: Xerostomia, or dry mouth, is a common side effect of head and neck radiation. Current treatment options for radiation-induced xerostomia are generally supportive in nature. Adult stem cells are the ultimate source for replenishment of salivary gland tissue. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a viable cell-based therapy for xerostomia. We have undertaken studies enabling U.S. Food and Drug Administration Investigational New Drug status, demonstrating the normal phenotype, intact functionality, and pro-growth secretome of interferon-γ (IFNγ)-stimulated BM-MSCs taken from patients with head and neck cancer who have undergone radiation ± chemotherapy. Here we present the protocol of MARSH, a first-in-human clinical trial of bone marrow-derived, IFNγ-activated BM-MSCs for the treatment of radiation-induced xerostomia. METHODS: This single-center phase 1 dose-escalation with expansion cohort, non-placebo-controlled study will assess the safety and tolerability of BM-MSCs for the treatment of radiation-induced xerostomia in patients who had head and neck cancer. The phase 1 dose-escalation study will be a 3 + 3 design with staggered enrollment. A total of 21 to 30 subjects (9 to 18 in phase 1 study, 12 in expansion cohort) will be enrolled. The primary endpoint is determining the recommended phase 2 dose (RP2D) of IFNγ-stimulated BM-MSCs to enable further studies on the efficacy of BM-MSCs. Patients' bone marrow will be aspirated, and BM-MSCs will be expanded, stimulated with IFNγ, and injected into the submandibular gland. The RP2D will be determined by dose-limiting toxicities occurring within 1 month of BM-MSC injection. Secondary outcomes of saliva amounts and composition, ultrasound of salivary glands, and quality of life surveys will be taken at 3-, 6-, 12-, and 24-month visits. DISCUSSION: Autotransplantation of IFNγ-stimulated BM-MSCs in salivary glands after radiation therapy or chemoradiation therapy may provide an innovative remedy to treat xerostomia and restore quality of life. This is the first therapy for radiation-induced xerostomia that may be curative. TRIAL REGISTRATION: World Health Organization International Clinical Trials Registry Platform: NCT04489732.

Role of Virus-Specific T Cell Therapy for Cytomegalovirus and BK Infections in Kidney Transplant Recipients.

Cytomegalovirus (CMV) and BK virus (BKV) are common viral infections after kidney transplant. Their negative effects on patient and graft outcomes have been well described. However, despite improvement in screening and prophylaxis strategies, CMV and BKV continue to negatively affect both short- and long-term graft survival. Adequate cell-mediated immunity is essential for the control and prevention of opportunistic viral infections, such as CMV and BKV. Therefore, immune reconstitution, in particular T cell recovery, is a key factor in antiviral control after kidney transplantation. Cell-based immunotherapy offers an attractive alternative approach to traditional interventions. Adoptive T cell transfer, via infusions of allogeneic virus-specific T lymphocytes is capable of restoring virus-specific T cell immunity, and are safe and effective in the treatment of viral infections after hematopoietic stem cell transplantation. In this article, we review the emerging role of virus-specific T cell therapy in the management of CMV and BKV after kidney transplantation. On the basis of the available data, virus-specific T cell therapy may be a promising addition to the antiviral treatment armamentarium after kidney transplantation. Future studies are needed to more clearly define the efficacy and risks of virus-specific T cell therapy in the kidney transplant population.

Synthesis, characterization, and anti-melanoma activity of tetra-O-substituted analogs of nordihydroguaiaretic acid.

Synthesis of seven semi-synthetic analogs of NDGA is described. An approach to NDGA derivatization is described in which the ortho-phenolic groups are tethered together by one atom, forming a 5-membered heterocyclic ring. The analogs were evaluated for cytotoxicity in four cancer cell lines and compared to NDGA and tetra-O-methyl-NDGA (M4N) (1a). NDGA bis-cyclic sulfate (2a), NDGA bis-cyclic carbonate (2b), and methylenedioxyphenyl-NDGA (2d) and NDGA tetra acetate (1b) showed anti-cancer activity in vitro. Two compounds, (1b) and (2b), were evaluated for anticancer activity in a mouse xenograft model of human melanoma and showed dose-dependent activity.

Cytotoxic activity of Apomine is due to a novel membrane-mediated cytolytic mechanism independent of apoptosis in the A375 human melanoma cell line.

Apomine, a novel bisphosphonate ester, has demonstrated anticancer activity in a variety of cancer cell lines; however, its mechanism of cytotoxicity is not well understood. Previous work has demonstrated that Apomine induces cell death by activation of caspase-3 in several cancer cell types. However, we have demonstrated that Apomine induces cell death in the A375 human melanoma cell line through a novel membrane-mediated mechanism that is independent of caspase-3 activation. This mechanism of membrane lysis may apply to other bisphosphonates and may be an important mechanism for overcoming resistance to apoptosis. Interestingly, Apomine-mediated cell death in the A375 and UACC 3093 human melanoma cell lines is also independent of N-Ras farnesylation, which was a previously described mechanism of action for Apomine in other cancer cell types. These data suggest that Apomine induces cell death through a novel plasma membrane-mediated cytolytic pathway, independent of caspase-3 activation and N-Ras farnesylation.

Preclinical antitumor activity, pharmacokinetics and pharmacodynamics of imexon in mice.

Imexon, a novel pro-oxidant, thiol-binding agent, is currently in phase I/II clinical trials in patients with advanced solid tumors. The aim of this study was to characterize the preclinical pharmacology of imexon in vivo. We investigated the anticancer activity of imexon in several cancer cell lines grown as xenografts in severe combined immunodeficient mice. Imexon was active against both hematologic and solid tumor types. The maximally tolerated dose, at the selected dosing schedule, was 150 mg/kg. Using the maximally tolerated dose of imexon, we sought to identify a potential pharmacodynamic biomarker to monitor the mechanistic effect systemically. As imexon binds cellular thiols in vitro, thiol depletion by imexon in vivo was evaluated as a potential biomarker. Following a single 150 mg/kg dose of imexon by intraperitoneal injection, glutathione levels decreased by 40% at 3 h in mouse erythrocytes. In mouse plasma, imexon treatment led to a significant decrease in cystine levels 2-4 h after drug administration. Notably, by this time, free imexon plasma levels were nondetectable. By investigating the pharmacokinetics of imexon, we also found that imexon undergoes rapid clearance from plasma in a dose-independent fashion with a half-life of 12-15 min. In summary, imexon is active against several cancer types in vivo. Imexon also decreases circulating thiols and exhibits dose-independent pharmacokinetics in mice. Plasma cystine levels may represent a biomarker of imexon activity in vivo.

Cytotoxic lignans from Larrea tridentata.

Six lignans, including the cyclolignan 3,4'-dihydroxy-3',4'-dimethoxy-6,7'-cyclolignan, were isolated from the flowering tops of Larrea tridentata. Additionally the flavanone, (S)-4',5-dihydroxy-7-methoxyflavanone, was isolated for the first time from L. tridentata or any member of the family Zygophyllaceae. All of the compounds were assessed for their growth inhibitory activity against human breast cancer, human colon cancer and human melanoma cell lines. The lignans had IC50 values of 5-60 microM with the linear butane-type lignans being the most potent, and it was found that colon cancer cells were the least sensitive cell type tested. The relative potency of linear butane type lignans against human breast cancer appears to correlate positively with the number of O-methyl groups present on the molecule.

Nordihydroguaiaretic acid: hepatotoxicity and detoxification in the mouse.

Larrea tridentata (Moc & Sess) Cov. (Zygophyllaceae) is an ethnobotanically important plant found in the American Southwest and northern Mexico. Although numerous beneficial effects have been attributed to this plant, several case reports have demonstrated high doses of Larrea-containing herbals induce hepatotoxicity and nephrotoxicity in humans. Nordihydriguaiaretic acid (NDGA) is a lignan found in high amounts (up to 10% by dry weight) in the leaves and twigs of L. tridentata. Previously, NDGA has been shown to induce cystic nephropathy in the rat, however, no reports have been made concerning this compound's hepatotoxic potential. Here, we report that intraperitoneal administration of NDGA is lethal in the mouse (LD(50)=75 mg/kg). Administration is associated with a time and dose-dependent increase in serum alanine aminotransferase levels, which suggest liver damage. Indeed, freshly isolated mouse hepatocytes are more sensitive to NDGA than human melanoma cells. Furthermore, we have identified glucuronidation as a potential detoxification mechanism for NDGA. Both mono and diglucuronide conjugates of NDGA are formed after intravenous dosing. The monoglucuronide is also formed after incubation of NDGA with human hepatic microsomes; suggesting that glucuronide conjugation is important in the metabolism of NDGA by humans. In summary, this report indicates that NDGA may contribute to the hepatotoxicity of L. tridentata and provides preliminary information on NDGA metabolism.