Larisa Yeomans

Development of a Bioavailable μ Opioid Receptor (MOPr) Agonist, δ Opioid Receptor (DOPr) Antagonist Peptide That Evokes Antinociception without Development of Acute Tolerance

We have previously described a cyclic tetrapeptide, 1, that displays μ opioid receptor (MOPr) agonist and δ opioid receptor (DOPr) antagonist activity, a profile associated with a reduced incidence of opioid tolerance and dependence. Like many peptides, 1 has poor bioavailability. We describe here an analogue of 1 with an added C-terminal β-glucosylserine residue, Ser(β-Glc)NH2, a modification that has previously been shown to improve bioavailability of opioid peptides. The resulting peptide, 4, exhibits full antinociceptive efficacy in the mouse warm water tail withdrawal assay after intraperitoneal administration with potency similar to that of morphine. Further, 4 does not give rise to acute tolerance and thus represents a promising lead for the development of opioid analgesics with reduced side effects.

Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy μ Opioid Receptor (MOR) Agonist/δ Opioid Receptor (DOR) Antagonist Ligands

We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a μ opioid receptor (MOR) agonist, δ opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood-brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.

Whole Blood Reveals More Metabolic Detail of the Human Metabolome than Serum as Measured by 1H-NMR Spectroscopy: Implications for Sepsis Metabolomics.

ABSTRACT Serum is a common sample of convenience for metabolomics studies. Its processing time can be lengthy and may result in the loss of metabolites including those of red blood cells (RBCs). Unlike serum, whole blood (WB) is quickly processed, minimizing the influence of variable hemolysis while including RBC metabolites. To determine differences between serum and WB metabolomes, both sample types, collected from healthy volunteers, were assayed by 1H-NMR (proton nuclear magnetic resonance) spectroscopy. A total of 34 and 50 aqueous metabolites were quantified from serum and WB, respectively. Free hemoglobin (Hgb) levels in serum were measured, and the correlation between Hgb and metabolite concentrations was determined. Most metabolites detected in serum were at higher concentrations in WB with the exception of acetoacetate and propylene glycol. The 18 unique metabolites of WB included adenosine, AMP, ADP, and ATP, which are associated with RBC metabolism. The use of serum results in the underrepresentation of a number of metabolic pathways including branched-chain amino acid degradation and glycolysis and gluconeogenesis. The range of free Hgb in serum was 0.03 to 0.01 g/dL, and eight metabolites were associated (P ⩽ 0.05) with free Hgb. The range of free Hgb in serum samples from 18 sepsis patients was 0.02 to 0.46 g/dL. Whole blood and serum have unique aqueous metabolite profiles, but the use of serum may introduce potential pathway bias. Use of WB for metabolomics may be particularly important for studies in diseases such as sepsis in which RBC metabolism is altered, and mechanical and sepsis-induced hemolysis contributes to variance in the metabolome.

Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition

In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman, and powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples and a 5-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4-5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl(-)/CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing the massive bioaccumulation of a weakly basic drug.

Further Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists: Balancing MOR and DOR Affinities.

In a previously described peptidomimetic series, we reported the development of bifunctional μ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: (1) probing bioavailability and improving metabolic stability, (2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the κ-opioid receptor (KOR), and (3) improving in vivo efficacy. Here, we establish that, through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h.

Emerging Biomarkers of Illness Severity: Urinary Metabolites Associated with Sepsis and Necrotizing Methicillin-Resistant Staphylococcus aureus Pneumonia

Our objective was to illustrate the potential of metabolomics to identify novel biomarkers of illness severity in a child with fatal necrotizing pneumonia caused by methicillin‐resistant Staphylococcus aureus (MRSA). We present a case report with two control groups and a metabolomics analysis: an infant with fatal MRSA pneumonia, four children with influenza pneumonia (pneumonia control group), and seven healthy children with no known infections (healthy control group). Urine samples were collected from all children. Metabolites were identified and quantified using 1H‐nuclear magnetic resonance spectrometry. Normalized metabolite concentration data from children with influenza pneumonia and healthy controls were compared by using an unpaired Student t test. To identify differentiating metabolites of MRSA pneumonia, the fold change of each metabolite was calculated by dividing each urine metabolite concentration of the patient with fatal MRSA pneumonia by the median urine concentration values of the same metabolite of the patients with influenza pneumonia and healthy controls, respectively. MetScape (http://metscape.ncibi.org/), a bioinformatics tool, was used for data visualization and interpretation. Urine metabolite concentrations previously identified as associated with sepsis in children (e.g., 3‐hydroxybutyrate, carnitine, and creatinine) were higher in the patient with fatal MRSA pneumonia compared with those of patients with influenza pneumonia and healthy controls. The concentrations of additional metabolites—acetone, acetoacetate, choline, fumarate, glucose, and 3‐aminoisobutyrate—were more than 25‐fold higher in the patient with MRSA pneumonia than those of patients with influenza pneumonia and healthy controls. These metabolic changes in the urine preceded the clinical severe sepsis phenotype, suggesting that detection of the extent of metabolic disruption can aid in the early identification of a sepsis phenotype in advance of the clinical diagnosis. These data also support the utility of metabolomics for the development of clinical assays to identify patients with pediatric pneumonia at high risk for deterioration.

Translation of structure‐activity relationships from cyclic mixed efficacy opioid peptides to linear analogues

Most opioid analgesics used in the treatment of pain are mu opioid receptor (MOR) agonists. While effective, there are significant drawbacks to opioid use, including the development of tolerance and dependence. However, the coadministration of a MOR agonist with a delta opioid receptor (DOR) antagonist slows the development of MOR‐related side effects, while maintaining analgesia. We have previously reported a series of cyclic mixed efficacy MOR agonist/DOR antagonist ligands. Here we describe the transfer of key features from these cyclic analogs to linear sequences. Using the linear MOR/DOR agonist, Tyr‐DThr‐Gly‐Phe‐Leu‐Ser‐NH2 (DTLES), as a lead scaffold, we replaced Phe4 with bulkier and/or constrained aromatic residues shown to confer DOR antagonism in our cyclic ligands. These replacements failed to confer DOR antagonism in the DTLES analogs, presumably because the more flexible linear ligands can adopt binding poses that will fit in the narrow binding pocket of the active conformations of both MOR and DOR. Nonetheless, the pharmacological profile observed in this series, high affinity and efficacy for MOR and DOR with selectivity relative to KOR, has also been shown to reduce the development of unwanted side effects. We further modified our lead MOR/DOR agonist with a C‐terminal glucoserine to improve bioavailability. The resulting ligand displayed high efficacy and potency at both MOR and DOR and no efficacy at KOR.