Analysis of N15-rat growth hormone after incubation with rat subcutaneous tissue and immune cells using ultra-pressure chromatography-mass spectrometry.

Therapeutic proteins (TPs) are exposed to various immune cells like macrophages and neutrophils, especially after subcutaneous (SC) administration. It is well known that the immune cells can generate reactive oxygen species (ROS) and this may lead to oxidation of TPs. The oxidation can occur in the SC tissue after SC administration, during distribution to the immune organs like lymph nodes and spleen, and even in the blood circulation. The oxidation can lead to alteration of their pharmacokinetics and efficacy. Therefore, it is important to study the oxidation of TPs in the biological matrices using ultra-pressure chromatography-mass spectrometry. Rat growth hormone (rGH) was selected as a test protein due to its similarity with human growth hormone (hGH), which is widely used for treatment of growth hormone deficiency. In this manuscript, we have summarized sample processing strategy and ultra-pressure chromatography-mass spectrometry methodology to identify rGH and its degradation products after ex-vivo incubation with rat SC tissue, and in vitro incubation with rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as a model foreign host species. We did not observe oxidation of rGH in these biological matrices. This could be due to very minor yields of oxidation products, lack of sensitivity of the mass spectrometry method, loss of protein during sample processing, rapid turnover of oxidized protein or a combination of all factors.

Further exploration of the structure-activity relationship of imidazoquinolines; identification of potent C7-substituted imidazoquinolines.

Small molecule agonists of TLR7/8, such as imidazoquinolines, are validated agonists for the treatment of cancer and for use in vaccine adjuvants. Imidazoquinolines have been extensively modified to understand the structure-activity relationship (SAR) at the N1- and C2-positions resulting in the clinical drug imiquimod, resiquimod, and several other highly potent analogues. However, the SAR of the aryl ring has not been fully elucidated in the literature. This initial study examines the SAR of C7-substituted imidazoquinolines. These compounds not only demonstrated that TLR7/8 tolerate changes at the C7-position but can increase potency and change their cytokine profiles. The most notable TLR7/8 agonists developed from this study 5, 8, and 14 which are up to 4-fold and 2-fold more active than resiquimod for TLR8 and/or TLR7, respectively, and up to 100-fold more active than the FDA approved imiquimod for TLR7.

Formulation and preclinical evaluation of a toll-like receptor 7/8 agonist as an anti-tumoral immunomodulator.

The toll-like receptor 7 and 8 (TLR7/8) agonist Resiquimod (R848) has been recognized as a promising immunostimulator for the treatment of cutaneous cancers in multiple clinical trials. However, systemic administration of R848 often results in strong immune-related toxicities while having limited therapeutic effects to the tumor. In the present study, a prodrug-based nanocarrier delivery system was developed that exhibited high therapeutic efficiency. R848 was conjugated to α-tocopherol to constitute an R848-Toco prodrug, followed by formulating with a tocopherol-modified hyaluronic acid (HA-Toco) as a polymeric nano-suspension. In vitro evaluation showed that the delivery system prolonged the release kinetics while maintaining TLR agonist activities. When administered subcutaneously, the nano-suspension formed a depot at the injection site, inducing localized immune responses without systemic expansion. This formulation also suppressed tumor growth and recruited immune cells to the tumor in a murine model of head and neck cancer. In a preclinical canine study of spontaneous mast cell tumors, the treatment led to a 67% response rate (three partial remissions and one complete remission).

Cabozantinib Loaded DSPE-PEG2000 Micelles as Delivery System: Formulation, Characterization and Cytotoxicity Evaluation.

Cabozantinib, a potent pan-tyrosine kinase inhibitor, has been reported to provide enhanced antitumor efficacy by simultaneously inhibiting both MET and VEGF pathways, which are critical to tumor angiogenesis, survival and migration. It's very poor water solubility prevents its administration by the intravenous route, which may be important in patients unable to take the drug orally. In this study, we developed an efficient PEG-lipid-based polymeric micelle formulation with enhanced drug solubility and stability for cabozantinib delivery. DSPE-PEG2000 micelles encapsulating cabozantinib were prepared by a thin-film rehydration method followed by a lyophilization process to generate the dry dosage form. The average hydrodynamic diameter of freshly prepared micelles was 11 nm with a narrow size distribution, and the dry micelle cake could be fully reconstituted by rehydration. Approximately 75% of the drug was encapsulated into the lyophilized cake, and a sustained drug release profile was observed in simulated normal physiological release medium. Compared with the free cabozantinib solution, the drug-loaded micelles displayed significantly enhanced intracellular accumulation and cytotoxicity in human glioblastoma cancer cells and non-small lung cancer cells. These results suggest that the micellar formulation of cabozantinib may serve as a promising nanocarrier in anticancer treatments.