TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1−/− mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-α, IL-6, IL-12, MCP-1, IFN-γ, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1−/− macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Infection of C57BL/6J mice with Mycoplasma pulmonis (MP) enhanced NK cell function 3-7 days later, as detected by in vitro and in vivo assays. Moreover, spleen and lung cells of acutely infected C57BL/6J mice inhibited MP growth in vitro. The effectors were eliminated by treatment with anti-NK antibody in vivo and anti-asialo GM1 serum or anti-3A4 antibody plus complement in vitro. Clearance of viable and radiolabeled MP from the lungs was also enhanced in acutely infected mice. Acutely infected mice with severe combined immunodeficiency (SCID) eliminated viable MP faster than did uninfected mice. Antibodies to interferon-gamma (IFN-gamma) impaired clearance of MP from the lungs of SCID mice and decreased their survival times. Activated NK cells can function in resistance to early stages of infection with MP. NK cells directly inhibit MP with secrete IFN-gamma, which may activate macrophages or inhibit the growth of MP or both.

siRNA has the potential to be a selective targeted cancer therapy but currently lacks an adequate delivery mechanism. Ensysce has been exploring the use of single-walled carbon nanotubes (SWCNT) as delivery vehicles for siRNA and has previously reported on the safety, in vitro and in vivo efficacy of this platform. We have determined that siRNA/SWCNT complexes are well tolerated, and can be administered in a multi-weekly intravenous (i.v.) dosing schedule over extended periods of time. The pharmacokinetics and distribution of the SWCNT in animals have been examined. The goal of the current study was to determine an optimal formulation of siRNA/SWCNT that provides maximal tumor accumulation and antitumor efficacy. siRNA/SWCNT complexes were formulated in aqueous solutions with PEG-lipid analogues and the circulation and tissue distribution were evaluated. All solutions prepared were examined by atomic force microscopy (AFM), dynamic light scattering (DLS) and for z-potential. siRNA payloads were quantified and siRNA/SWCNT complexes were examined for stability in serum and systemic efficacy. Solutions of siRNA/SWCNT formulated complexes were administered i.v. to C57Bl6 mice or to nu/nu mice bearing MiaPaCa human pancreatic or A431 human epidermoid carcinoma tumors. Mice were sacrificed at various times after i.v. administration. Blood and tissues were collected and SWCNT concentrations were determined by measuring near infrared fluorescence using NS2 Nanospectralizer. The presence and persistence of SWCNT in the tissues was determined. Animals bearing A431 xenografts were treated with SWCNT/siEGFR/PEG-lipid analogue complexes and tumor growth control was compared to that produced by erlotinib. Data to be presented will include details of the analytical characterization of the SWCNT, circulating half-life (t1/2) and tissue concentrations versus time of administration. Tumor and tissue SWCNT content were found to depend on the t1/2 of the formulation of preparations that were delivered. Microscopic examination of tumors indicated that SWCNT presence in tumor increased with the increasing t1/2. The siRNA target knockdown in tumor xenografts of up to 90% was observed with complexes comprised of a number of siRNA payloads including siKRAS, siEGFR and siTRX. Antitumor activity has also been demonstrated with complexes targeting one or two growth driving proteins. Formulations providing t1/2 of 13 hr for the circulating SWCNT were evaluated with siEGFR against A431 EGFR overexpressing cells, with the optimal formulation showing tumor growth delay of 16 to 20 days versus the free siRNA treated control. In summary, SWCNT offer a novel approach to deliver oligonucleotides safely and effectively and formulation of the complexes can offer a means to control the siRNA biodistribution. Citation Format: D. Lynn Kirkpatrick, Olga Gliko, Michelle Weiss, Iris Owusu, Anton Naumov. Single-walled carbon nanotubes for delivery of siRNA: Antitumor efficacy comparing formulated complexes in EGFR overexpressing A431 xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5621. doi:10.1158/1538-7445.AM2013-5621