Safety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson's disease.

BACKGROUND: Neuroinflammation plays a pathogenic role in Parkinson's disease (PD). Immunotherapies that restore brain homeostasis can mitigate neurodegeneration by transforming T cell phenotypes. Sargramostim has gained considerable attention as an immune transformer through laboratory bench to bedside clinical studies. However, its therapeutic use has been offset by dose-dependent adverse events. Therefore, we performed a reduced drug dose regimen to evaluate safety and to uncover novel disease-linked biomarkers during 5 days/week sargramostim treatments for one year. METHODS: Five PD subjects were enrolled in a Phase 1b, unblinded, open-label study to assess safety and tolerability of 3 µg/kg/day sargramostim. Complete blood counts and chemistry profiles, physical examinations, adverse events (AEs), immune profiling, Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) scores, T cell phenotypes/function, DNA methylation, and gene and protein patterns were evaluated. FINDINGS: Sargramostim administered at 3 µg/kg/day significantly reduced numbers and severity of AEs/subject/month compared to 6 µg/kg/day treatment. While MDS-UPDRS Part III score reductions were recorded, peripheral blood immunoregulatory phenotypes and function were elevated. Hypomethylation of upstream FOXP3 DNA elements was also increased. INTERPRETATION: Long-term sargramostim treatment at 3 µg/kg/day is well-tolerated and effective in restoring immune homeostasis. There were decreased numbers and severity of AEs and restored peripheral immune function coordinate with increased numbers and function of Treg. MDS-UPDRS Part III scores did not worsen. Larger patient numbers need be evaluated to assess conclusive drug efficacy (ClinicalTrials.gov NCT03790670). FUNDING: The research was supported by community funds to the University of Nebraska Foundation and federal research support from 5 R01NS034239-25.

Facile Preparation of Peptides for Mass Spectrometry Analysis in Bottom-Up Proteomics Workflows.

Mass spectrometry (MS) is routinely used to identify, characterize, and quantify biological molecules. For protein analysis, MS-based workflows can be broadly categorized as top-down or bottom-up, depending on whether the proteins are analyzed as intact molecules or first digested into peptides. This article outlines steps for preparing peptide samples for MS as part of a bottom-up proteomics workflow, providing versatile methods suitable for discovery and targeted analyses in qualitative and quantitative workflows. Resulting samples contain peptides of suitable size for analysis by MS instrumentation generally available to modern research laboratories, including MS coupled to either liquid chromatography (LC) or matrix-assisted laser desorption/ionization (MALDI) interfaces. This article incorporates recent developments in methodologies and consumables to facilitate sample preparation. The protocols are well-suited to users without prior experience in proteomics and include methods for universally applicable suspension trap processing and for alternate in-solution processing to accommodate a range of sample types. Cleanup, quantification, and fractionation procedures are also described. © 2021 The Authors. Basic Protocol: Preparation of high-complexity peptide samples for mass spectrometry analysis using S-Trap™ processing Alternate Protocol 1: Preparation of low- to moderate-complexity peptide samples for mass spectrometry analysis using in-solution processing Alternate Protocol 2: Detergent, polymer, and salt removal from peptide samples before mass spectrometry analysis using SP2 processing Support Protocol 1: Protein quantification using Pierce 660 nm assay Support Protocol 2: Peptide quantification using Pierce quantitative fluorometric peptide assay Support Protocol 3: High-pH fractionation of complex peptide samples.

Synthesis and Characterization of Long-Acting Darunavir Prodrugs.

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.

A long acting nanoformulated lamivudine ProTide.

A long acting (LA) hydrophobic and lipophilic lamivudine (3TC) was created as a phosphoramidate pronucleotide (designated M23TC). M23TC improved intracellular delivery of active triphosphate metabolites and enhanced antiretroviral and pharmacokinetic (PK) profiles over the native drug. A single treatment of human monocyte derived macrophages (MDM) with nanoformulated M23TC (NM23TC) improved drug uptake, retention, intracellular 3TC triphosphates and antiretroviral activities in MDM and CD4+ T cells. PK tests of NM23TC administered to Sprague Dawley rats demonstrated sustained prodrug and drug triphosphate levels in blood and tissues for 30 days. The development of NM23TC remains a substantive step forward in producing LA slow effective release antiretrovirals for future clinical translation.

Synthesis and characterization of a long-acting emtricitabine prodrug nanoformulation.

PURPOSE: A palmitoylated prodrug of emtricitabine (FTC) was synthesized to extend the drug's half-life, antiretroviral activities and biodistribution. METHODS: A modified FTC prodrug (MFTC) was synthesized by palmitoyl chloride esterification. MFTC's chemical structure was evaluated by nuclear magnetic resonance. The created hydrophobic prodrug nanocrystals were encased into a poloxamer surfactant and the pharmacokinetics (PK), biodistribution and antiretroviral activities of the nanoformulation (NMFTC) were assessed. The conversion of MFTC to FTC triphosphates was evaluated. RESULTS: MFTC coated with poloxamer formed stable nanocrystals (NMFTC). NMFTC demonstrated an average particle size, polydispersity index and zeta potential of 350 nm, 0.24 and -20 mV, respectively. Drug encapsulation efficiency was 90%. NMFTC was readily taken up by human monocyte-derived macrophages yielding readily detected intracellular FTC triphosphates and an extended PK profile. CONCLUSION: NMFTC shows improved antiretroviral activities over native FTC. This is coordinate with its extended apparent half-life. The work represents an incremental advance in the development of a long-acting FTC formulation.

Creation of a long-acting rilpivirine prodrug nanoformulation.

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug half-life and improve tissue biodistribution. These results warrant further development for human use.

Synthesis of a long acting nanoformulated emtricitabine ProTide.

While antiretroviral therapy (ART) has revolutionized treatment and prevention of human immunodeficiency virus type one (HIV-1) infection, regimen adherence, viral mutations, drug toxicities and access stigma and fatigue are treatment limitations. These have led to new opportunities for the development of long acting (LA) ART including implantable devices and chemical drug modifications. Herein, medicinal and formulation chemistry were used to develop LA prodrug nanoformulations of emtricitabine (FTC). A potent lipophilic FTC phosphoramidate prodrug (M2FTC) was synthesized then encapsulated into a poloxamer surfactant (NM2FTC). These modifications extended the biology, apparent drug half-life and antiretroviral activities of the formulations. NM2FTC demonstrated a >30-fold increase in macrophage and CD4+ T cell drug uptake with efficient conversion to triphosphates (FTC-TP). Intracellular FTC-TP protected macrophages against an HIV-1 challenge for 30 days. A single intramuscular injection of NM2FTC, at 45 mg/kg native drug equivalents, into Sprague Dawley rats resulted in sustained prodrug levels in blood, liver, spleen and lymph nodes and FTC-TP in lymph node and spleen cells at one month. In contrast, native FTC-TPs was present for one day. These results are an advance in the transformation of FTC into a LA agent.

Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost.

Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained.