Optimization of Benzoxazinorifamycins to Minimize hPXR Activation for the Treatment of Tuberculosis and HIV Coinfection.

Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (ß-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.

Optimization of Benzoxazinorifamycins to Improve Mycobacterium tuberculosis RNA Polymerase Inhibition and Treatment of Tuberculosis.

Rifampin (RMP), a very potent inhibitor of the Mycobacterium tuberculosis (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIFR) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIFR) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue (27a) that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (∼30-fold > RMP) activity against RIFR MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, 27a has a 7 day MBC that is ∼10-fold more potent than RMP. These results suggest that 27a may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of ∼2 g of 27a at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies.

Cyst fluid metabolites distinguish malignant from benign pancreatic cysts.

OBJECTIVES: Current standard of care imaging, cytology, or cystic fluid analysis cannot reliably differentiate malignant from benign pancreatic cystic neoplasms. This study sought to determine if the metabolic profile of cystic fluid could distinguish benign and malignant lesions, as well as mucinous and non-mucinous lesions. METHODS: Metabolic profiling by untargeted mass spectrometry and quantitative nuclear magnetic resonance was performed in 24 pancreatic cyst fluid from surgically resected samples with pathological diagnoses and clinicopathological correlation. RESULTS: (Iso)-butyrylcarnitine distinguished malignant from benign pancreatic cysts, with a diagnostic accuracy of 89%. (Iso)-butyrylcarnitine was 28-fold more abundant in malignant cyst fluid compared with benign cyst fluid (P=.048). Furthermore, 5-oxoproline (P=.01) differentiated mucinous from non-mucinous cysts with a diagnostic accuracy of 90%, better than glucose (82% accuracy), a previously described metabolite that distinguishes mucinous from non-mucinous cysts. Combined analysis of glucose and 5-oxoproline did not improve the diagnostic accuracy. In comparison, standard of care cyst fluid carcinoembryonic antigen (CEA) and cytology had a diagnostic accuracy of 40% and 60% respectively for mucinous cysts. (Iso)-butyrylcarnitine and 5-oxoproline correlated with cyst fluid CEA levels (P<.0001 and P<.05 respectively). For diagnosing malignant pancreatic cysts, the diagnostic accuracies of cyst size > 3 cm, ≥ 1 high-risk features, cyst fluid CEA, and cytology are 38%, 75%, 80%, and 75%, respectively. CONCLUSIONS: (Iso)-butyrylcarnitine has potential clinical application for accurately distinguishing malignant from benign pancreatic cysts, and 5-oxoproline for distinguishing mucinous from non-mucinous cysts.

Respiratory Colonization and Short-Term Temporal Changes in the Urinary Metabolome of Children.

The human metabolome may vary based on age, over time, and in the presence of viral carriage and bacterial colonization-a common scenario in children. We used nuclear magnetic resonance spectroscopy to identify and quantify urinary metabolites of children without signs or symptoms of respiratory illness. A urine sample and two nasopharyngeal swabs were collected to test for respiratory viral pathogens and colonization by Streptococcus pneumoniae (Sp). Urine samples were collected at the initial visit, 24 h post-enrollment, and 10-14 days post-enrollment. Of the 122 children enrolled, 24% had a virus detected and 19.7% had Sp detected. Intraclass correlation coefficients demonstrated greater within-subject versus between-subject variability for all metabolites detected. In linear mixed models adjusted for age, time, history of asthma, Sp, and viruses, 1-methylnicotinamide was increased by 50% in children with Sp and decreased by 35% in children with rhinovirus/enterovirus. Children with Sp had 83% higher levels of trimethylamine-N-oxide compared with those without Sp. However, when adjusting for multiple comparisons, the association was no longer statistically significant. In conclusion, there appear to be short-term changes within the urinary metabolome of healthy children, but levels of metabolites did not statistically differ in children with viral carriage or Sp detected.

Genetic and Metabolite Variability in One-Carbon Metabolism Applied to an Insulin Resistance Model in Patients With Schizophrenia Receiving Atypical Antipsychotics.

Background: Patients with schizophrenia are at high risk of pre-mature mortality due to cardiovascular disease (CVD). Our group has completed studies in pharmacogenomics and metabolomics that have independently identified perturbations in one-carbon metabolism as associated with risk factors for CVD in this patient population. Therefore, this study aimed to use genetic and metabolomic data to determine the relationship between folate pharmacogenomics, one-carbon metabolites, and insulin resistance as measured using the homeostatic model assessment for insulin resistance (HOMA-IR) as a marker of CVD. Methods: Participants in this pilot analysis were on a stable atypical antipsychotic regimen for at least 6 months, with no diabetes diagnosis or use of antidiabetic medications. Participant samples were genotyped for MTHFR variants rs1801131 (MTHFR A1298C) and rs1801133 (MTHFR C677T). Serum metabolite concentrations were obtained with NMR. A least squares regression model was used to predict log(HOMA-IR) values based on the following independent variables: serum glutamate, glycine, betaine, serine, and threonine concentrations, and carrier status of the variant alleles for the selected genotypes. Results: A total of 67 participants were included, with a median age of 47 years old (IQR 42-52), 39% were female, and the median BMI was 30.3 (IQR 26.3-37.1). Overall, the model demonstrated an ability to predict log(HOMA-IR) values with an adjusted R 2 of 0.44 and a p-value of < 0.001. Glutamate, threonine, and carrier status of the MTHFR 1298 C or MTHFR 677 T allele were positively correlated with log(HOMA-IR), whereas glycine, serine, and betaine concentrations trended inversely with log(HOMA-IR). All factors included in this final model were considered as having a possible effect on predicting log(HOMA-IR) as measured with a p-value < 0.1. Conclusions: Presence of pharmacogenomic variants that decrease the functional capacity of the MTHFR enzyme are associated with increased risk for cardiovascular disease, as measured in this instance by log(HOMA-IR). Furthermore, serine, glycine, and betaine concentrations trended inversely with HOMA-IR, suggesting that increased presence of methyl-donating groups is associated with lower measures of insulin resistance. Ultimately, these results will need to be replicated in a significantly larger population.

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidines as atypical protein kinase C inhibitors to control retinal vascular permeability and cytokine-induced edema.

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1-2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and Cmax), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.

Disruption of histidine and energy homeostasis in chronic obstructive pulmonary disease.

Background: Chronic obstructive pulmonary disease (COPD) is a systemic condition that is too complex to be assessed by lung function alone. Metabolomics has the potential to help understand the mechanistic underpinnings that contribute to COPD pathogenesis. Since blood metabolomics may be affected by sex and body mass index (BMI), the aim of this study was to determine the metabolomic variability in male smokers with and without COPD who have a narrow BMI range. Methods: We compared the quantitative proton nuclear magnetic resonance acquired serum metabolomics of a male Chinese Han population of non-smokers without COPD, and smokers with and without COPD. We also assessed the impact of smoking status on metabolite concentrations and the associations between metabolite concentrations and inflammatory markers such as serum interleukin-6 and histamine, and blood cell differential (%). Metabolomics data were log-transformed and auto-scaled for parametric statistical analysis. Mean normalized metabolite concentration values and continuous demographic variables were compared by Student's t-test with Welch correction or ANOVA with post-hoc Tukey's test, as applicable; t-test p-values for metabolomics data were corrected for false discovery rate (FDR). A Pearson association matrix was built to evaluate the relationship between metabolite concentrations, clinical parameters and markers of inflammation. Results: Twenty-eight metabolites were identified and quantified. Creatine, glycine, histidine, and threonine concentrations were reduced in COPD patients compared to non-COPD smokers (FDR ≤15%). Concentrations of these metabolites were inversely correlated with interleukin-6 levels. COPD patients had overall dampening of metabolite concentrations including energy-related metabolic pathways such as creatine metabolism. They also had higher histamine levels and percent basophils compared to smokers without COPD. Conclusion: COPD is associated with alterations in the serum metabolome, including a disruption in the histidine-histamine and creatine metabolic pathways. These findings support the use of metabolomics to understand the pathogenic mechanisms involved in COPD.Trial registration www.clinicaltrials.gov, NCT03310177.

Serum amino acid concentrations and clinical outcomes in smokers: SPIROMICS metabolomics study.

Metabolomics is an emerging science that can inform pathogenic mechanisms behind clinical phenotypes in COPD. We aimed to understand disturbances in the serum metabolome associated with respiratory outcomes in ever-smokers from the SPIROMICS cohort. We measured 27 serum metabolites, mostly amino acids, by 1H-nuclear magnetic resonance spectroscopy in 157 white ever-smokers with and without COPD. We tested the association between log-transformed metabolite concentrations and one-year incidence of respiratory exacerbations after adjusting for age, sex, current smoking, body mass index, diabetes, inhaled or oral corticosteroid use, study site and clinical predictors of exacerbations, including FEV1% predicted and history of exacerbations. The mean age of participants was 53.7 years and 58% had COPD. Lower concentrations of serum amino acids were independently associated with 1-year incidence of respiratory exacerbations, including tryptophan (ß = -4.1, 95% CI [-7.0; -1.1], p = 0.007) and the branched-chain amino acids (leucine: ß = -6.0, 95% CI [-9.5; -2.4], p = 0.001; isoleucine: ß = -5.2, 95% CI [-8.6; -1.8], p = 0.003; valine: ß = -4.1, 95% CI [-6.9; -1.4], p = 0.003). Tryptophan concentration was inversely associated with the blood neutrophil-to-lymphocyte ratio (p = 0.03) and the BODE index (p = 0.03). Reduced serum amino acid concentrations in ever-smokers with and without COPD are associated with an increased incidence of respiratory exacerbations.

Pharmacometabolomics reveals a role for histidine, phenylalanine, and threonine in the development of paclitaxel-induced peripheral neuropathy.

PURPOSE: Approximately 25% of breast cancer patients experience treatment delays or discontinuation due to paclitaxel-induced peripheral neuropathy (PN). Currently, there are no predictive biomarkers of PN. Pharmacometabolomics is an informative tool for biomarker discovery of drug toxicity. We conducted a secondary whole blood pharmacometabolomics analysis to assess the association between pretreatment metabolome, early treatment-induced metabolic changes, and the development of PN. METHODS: Whole blood samples were collected pre-treatment (BL), just before the end of the first paclitaxel infusion (EOI), and 24 h after the first infusion (24H) from sixty patients with breast cancer receiving (80 mg/m2) weekly treatment. Neuropathy was assessed at BL and prior to each infusion using the sensory subscale (CIPN8) of the EORTC CIPN20 questionnaire. Blood metabolites were quantified from 1-D-1H-nuclear magnetic resonance spectra using Chenomx® software. Metabolite concentrations were normalized in preparation for Pearson correlation and one-way repeated measures ANOVA with multiple comparisons corrected by false discovery rate (FDR). RESULTS: Pretreatment histidine, phenylalanine, and threonine concentrations were inversely associated with maximum change in CIPN8 (ΔCIPN8) (p < 0.02; FDR ≤ 25%). Paclitaxel caused a significant change in concentrations of 2-hydroxybutyrate, 3-hydroxybutyrate, pyruvate, o-acetylcarnitine, and several amino acids from BL to EOI and/or 24H (p < 0.05; FDR ≤ 25%), although these changes were not associated with ΔCIPN8. CONCLUSIONS: Whole blood metabolomics is a feasible approach to identify potential biomarker candidates of paclitaxel-induced PN. The findings suggest that pretreatment concentrations of histidine, phenylalanine, and threonine may be predictive of the severity of future PN and paclitaxel-induced metabolic changes may be related to disruption of energy homeostasis.

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 1 H-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.

Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism.

Prolonged (8 weeks) oral administration of clofazimine results in a profound pharmacodynamic response-bioaccumulation in macrophages (including Kupffer cells) as intracellular crystal-like drug inclusions (CLDIs) with an associated increase in interleukin-1 receptor antagonist production. Notably, CLDI formation in Kupffer cells concomitantly occurs with the formation of macrophage-centric granulomas. Accordingly, we sought to understand the impact of these events on host metabolism using 1H-nuclear magnetic resonance metabolomics. Mice received a clofazimine or vehicle-enriched (sham) diet for at least 8 weeks. At 2 weeks, the antimicrobial activity of clofazimine was evident by changes in urine metabolites. From 2 to 8 weeks, there was a striking change in metabolite levels indicative of a reorientation of host energy metabolism paralleling the onset of CLDI and granuloma formation. This was evidenced by a progressive reduction in urine levels of metabolites involved in one-carbon metabolism with corresponding increases in whole blood, and changes in metabolites associated with lipid, nucleotide and amino acid metabolism, and glycolysis. Although clofazimine-fed mice ate more, they gained less weight than control mice. Together, these results indicate that macrophage sequestration of clofazimine as CLDIs and granuloma formation is accompanied by a profound metabolic disruption in energy homeostasis and one-carbon metabolism.

1D-¹H-nuclear magnetic resonance metabolomics reveals age-related changes in metabolites associated with experimental venous thrombosis.

OBJECTIVE: Age is a significant risk factor for the development of venous thrombosis (VT), but the mechanism(s) that underlie this risk remain(s) undefined and poorly understood. Aging is known to adversely influence inflammation and affect metabolism. Untargeted metabolomics permits an agnostic assessment of the physiological landscape and lends insight into the mechanistic underpinnings of clinical phenotypes. The objective of this exploratory study was to test the feasibility of a metabolomics approach for identifying potential metabolic mechanisms of age-related VT. METHODS: We subjected whole blood samples collected from young and old nonthrombosed controls and VT mice 2 days after thrombus induction using the electrolytic inferior vena cava, to a methanol:chloroform extraction and assayed the resulting aqueous fractions using 1D-(1)H- nuclear magnetic resonance. Normalized mouse metabolite data were compared across groups using analysis of variance (ANOVA) with Holm-Sidak post-testing. In addition, associations between metabolite concentrations and parameters of thrombosis such as thrombus and vein wall weights, and markers of inflammation, vein wall P- and E-selectin levels, were assessed using linear regression. The relatedness of the found significant metabolites was visually assessed using a bioinformatics tool, Metscape, which generates compound-reaction-enzyme-gene networks to aid in the interpretation of metabolomics data. RESULTS: Old mice with VT had a greater mean vein wall weight compared with young mice with VT (P < .05). Clot weight differences between old and young mice followed the same trend as vein wall weight (0.011 ± 0.04 g vs 0.008 ± 0.003 g; P = not significant). Glutamine (ANOVA, P < .01), proline (ANOVA, P < .01), and phenylalanine (ANOVA, P < .05) levels were increased in old VT mice compared with age-matched controls and young VT mice. Betaine and/or trimethylamine N-oxide levels were increased in aged mice compared with young animals. Vein wall weight was strongly associated with glutamine (P < .05), and phenylalanine (P < .01) concentrations and there was a trend toward an association with proline (P = .09) concentration. Vein wall P-selectin, but not E-selectin levels, were increased in old VT mice and were associated with the three found metabolites of age-related VT. Collectively, with the addition of glutamate, these metabolites form a single compound-reaction-enzyme-gene network that was generated by Metscape. CONCLUSIONS: We used 1D-(1)H-nuclear magnetic resonance-metabolite profiling to identify, for the first time, in an experimental model, three potential metabolites, glutamine, phenylalanine, and proline, associated with age-related VT. These metabolites are metabolically related and their levels are associated with vein wall weight and P-selectin concentrations. In aggregate, these findings provide a "roadmap" of pathways that could be interrogated in future studies, which could include provocation of the glutamine, phenylalanine, and proline pathways in the vein wall. This study introduces metabolomics as a new approach to furthering knowledge about the mechanisms of age-related VT.

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.

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

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 H-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.

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 ß-glucosylserine residue, Ser(ß-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.

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 Phe(4) 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.

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.

Phosphorylation of enkephalins: NMR and CD studies in aqueous and membrane-mimicking environments.

Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed µ/δ-agonist, and one related to DAMGO, a highly selective µ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its ß-d-glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human µ, δ and κ-opiate receptors. Antinociception was measured in mice using the 55 °C tail-flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high-field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations.

Glycosylation improves the central effects of DAMGO.

A series of mu-agonist DAMGO analogs were synthesized and pharmacologically characterized to test the 'biousian' hypothesis of membrane hopping. DAMGO was altered by incorporating moieties of increasing water solubility into the C-terminus via carboxamide and simple glycoside additions. The hydrophilic C-terminal moieties were varied from glycinol in DAMGO (1) to l-serine amide (2), l-serine amide beta-d-xyloside (3), l-serine amide beta-d-glucoside (4), and finally to l-serine amide beta-lactoside (5). Opioid binding and mouse tail-flick studies were performed. Antinociceptive potency (intravenous) increased, passing through a maximum (A(50) approximately 0.2 micromol/kg) for 2 and 3 as membrane affinity versus water solubility became optimal, and dropped off (A(50) approximately 1.0 micromol/kg) for 4 and 5 as water solubility dominated molecular behavior. Intravenous A(50) values were plotted versus hydrodynamic values (glucose units, g.u.) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A(50) versus e(-Awater/Alipid)), and provided either a V-shaped or a U-shaped curve, as predicted by the 'biousian' hypothesis. The mu-selective receptor profile was maintained (K(i)'s = 0.66-1.3 nm) upon modifications at the C-terminus. The optimal 'degree of glycosylation' for the DAMGO peptide message appears to be between 1.25 and 1.75 g.u. (hydrodynamic g.u.), or 0.75 and 0.90 in terms of the surface-derived amphipathicity values.