PAK1 inhibition reduces tumor size and extends the lifespan of mice in a genetically engineered mouse model of Neurofibromatosis Type 2 (NF2).

Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.

Advances in Targeted Therapy for Neurofibromatosis Type 2 (NF2)-Associated Vestibular Schwannomas.

PURPOSE OF REVIEW: Neurofibromatosis 2 (NF2) is an autosomal-dominant genetic disorder characterized by bilateral vestibular schwannomas (VS), meningiomas, ependymomas, spinal and peripheral schwannomas, optic gliomas, and juvenile cataracts. Ongoing studies provide new insight into the role of the NF2 gene and merlin in VS tumorigenesis. RECENT FINDINGS: As NF2 tumor biology becomes increasingly understood, therapeutics targeting specific molecular pathways have been developed and evaluated in preclinical and clinical studies. NF2-associated VS are a source of significant morbidity with current treatments including surgery, radiation, and observation. Currently, there are no FDA-approved medical therapies for VS, and the development of selective therapeutics is a high priority. This manuscript reviews NF2 tumor biology and current therapeutics undergoing investigation for treatment of patients with VS.

Peri-Insular Hemispherotomy: A Systematic Review and Institutional Experience.

INTRODUCTION: Peri-insular hemispherotomy (PIH) is a hemispheric separation technique under the broader hemispherotomy group, a surgical treatment for patients with intractable epilepsy. Hemispherotomy techniques such as the PIH, vertical parasagittal hemispherotomy (VPH), and modified-lateral hemispherotomy are commonly assessed together, despite significant differences in anatomical approach and patient selection. We aim to describe patient selection, outcomes, and complications of PIH in its own right. METHODS: A systematic review of the literature, in accordance with the Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was conducted, with searches of the PubMed and Embase databases. A local series including patients receiving PIH and followed up at the Queensland Children's Hospital between 2014 and 2020 was included. RESULTS: Systematic review of the literature identified 393 patients from 13 eligible studies. Engel class 1 outcomes occurred in 82.4% of patients, while 8.6% developed post-operative hydrocephalus. Hydrocephalus was most common in the youngest patient cohorts. Developmental pathology was present in 114 (40.8%) patients, who had fewer Engel 1 outcomes compared to those with acquired pathology (69.1% vs. 83.7%, p = 0.0167). The local series included 13 patients, 11/13 (84.6%) had Engel class 1 seizure outcomes. Post-operative hydrocephalus occurred in 2 patients (15.4%), and 10/13 (76.9%) patients had worsened neurological deficit. CONCLUSION: PIH delivers Engel 1 outcomes for over 4 in 5 patients selected for this procedure, greater than described in combined hemispherectomy analyses. It is an effective technique in patients with developmental and acquired pathologies, despite general preference of VPH in this patient group. Finally, very young patients may have significant seizure and cognitive benefits from PIH; however, hydrocephalus is most common in this group warranting careful risk-benefit assessment. This review delivers a dedicated PIH outcomes analysis to inform clinical and patient decision-making.

In Vitro Metabolism and CYP-Modulating Activity of Lavender Oil and Its Major Constituents.

The application of essential oils has historically been limited to topical (massage therapy) and inhalational (aromatherapy) routes of administration. More recently, however, evaluation of the therapeutic effects of essential oils has expanded to include the oral route of administration, which increases the herb-drug interaction potential. The purpose of this study was to evaluate the herb-drug interaction potential of lavender essential oil and two of its primary phytoactive constituents, namely linalool and linalyl acetate. The metabolic stability of linalool and linalyl acetate was determined in human liver microsomes (HLM) and S9 fractions by quantitative analysis using UPLC-MS/MS system. Linalool was metabolically unstable in HLM and S9 fractions with an intrinsic clearance of 31.28 mL·min-1·kg-1, and 7.64 mL·min-1·kg-1, respectively. Interestingly, it was observed that linalyl acetate converted to linalool both in HLM and S9 fractions. Lavender oil showed weak inhibitory effect on the catalytic activity of CYP3A4 and CYP1A2 enzymes (IC50 12.0 and 21.5 µg/mL). Linalyl acetate inhibited CYP3A4 (IC50 4.75 µg/mL) while linalool did not show any inhibitory effect on any of the enzymes. The lavender oil and its constituents did not activate PXR to a considerable extent, and no activation of AhR was observed, suggesting a lack of potential to modify the pharmacokinetic and pharmacodynamic properties of conventional medications if used concurrently.

Development of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for Characterizing Linalool Oral Pharmacokinetics in Humans.

Lavender (Lavandula angustifolia Miller or Lavandula officinalis Chaix) is an ethnopharmacological plant commonly known as English lavender. Linalool and linalyl acetate are putative phytoactives in lavender essential oil (LEO) derived from the flower heads. LEO has been used in aroma or massage therapy to reduce sleep disturbance and to mitigate anxiety. Recently, an oral LEO formulation was administered in human clinical trials designed to ascertain its anxiolytic effect. However, human pharmacokinetics and an LC-MS/MS method for the measurement of linalool are lacking. To address this deficiency, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of linalool in human serum. Prior to the analysis, a simple sample preparation protocol including protein precipitation and liquid-liquid extraction of serum samples was created. The prepared samples were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to quantitatively determine linalool and IS according to transitions of m/z 137.1→95.1 (tR 0.79 min) and 205.2→149.1 (tR 1.56 min), respectively. The method was validated for precision, accuracy, selectivity, linearity, sensitivity, matrix effects, and stability, and it was successfully applied to characterize the oral pharmacokinetics of linalool in humans. The newly developed LC-MS/MS-based method and its application in clinical trial serum samples are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

Vagus nerve stimulation: a 20-year Australian experience.

BACKGROUND: Vagus nerve stimulation (VNS) therapy was first approved in the mid-1990s in the USA, Europe and Australia, with demonstrable efficacy in paediatric populations. Benefit in seizure frequency reduction can be observed up to 2 years post-intervention; however, few studies assess outcomes beyond this period. Furthermore, paediatric cohort sizes are small, limiting generalisability of outcome assessments. We evaluate VNS insertion outcomes and complications or side-effects in a large paediatric cohort, over a 20-year period from Queensland's first VNS insertion. METHODS: A retrospective review was conducted of all paediatric VNS insertions at the Queensland Children's Hospital (QCH) and the Mater Children's Hospital/Mater Children's Private Hospital (MCH/MCPH) Brisbane. A minimum of 1-year follow-up from 1999 to 2020 was required for inclusion. Patients were assessed on demographics, epilepsy details, seizure outcomes and complications or side-effects. RESULTS: In this extended follow-up cohort (76 patients, 7.2 ± 5.3 years), 51.3% of patients had ≥ 50% seizure frequency reduction, while 73.7% experienced an Engel III outcome (worthwhile benefit) or better. Eleven patients (14.9%) were seizure-free at follow-up, and 81.6% retained long-term therapy. Stimulation-related side-effects are common (17.1%) but rarely result in stimulation cessation (3.9%). Cessation occurred in 14 patients (18.4%) and most commonly related to minimal benefit (13.2%). Demographics, aetiology, seizure nature and surgical factors did not influence outcomes. CONCLUSION: Over extended treatment periods, a large proportion of patients will benefit significantly from VNS therapy. Approximately 4 of 5 patients will retain VNS therapy, and in cases of cessation, this is most commonly related to minimal benefit. Underlying demographics, aetiology or seizure nature do not influence outcomes. This 20-year Queensland assessment of VNS therapy outcomes informs long-term expectation of VNS therapy.

Development of a GC/Q-ToF-MS Method Coupled with Headspace Solid-Phase Microextraction to Evaluate the In Vitro Metabolism of ß-Caryophyllene.

Sample preparation remains both a challenging and time-consuming process in the field of bioanalytical chemistry. Many traditional techniques often require multi-step processes, which can introduce additional errors to the analytical method. Given the complexity of many biological matrices, thorough analyte extraction presents a major challenge to researchers. In the present study, a headspace solid-phase microextraction (HS-SPME) coupled with a GC/Q-ToF-MS method, was developed to quantify in vitro metabolism of ß-caryophyllene by both human liver microsome (HLM) and S9 liver fractions. Validation of the method was demonstrated both in terms of linearity (R2 = 0.9948) and sensitivity with a limit of detection of 3 ng/mL and a limit of quantitation of 10 ng/mL. In addition, the method also demonstrated both inter- and intra-day precision with the relative standard deviation (RSD) being less than 10% with four concentrations ranging from 50-500 ng/mL. Since this method requires no solvents and minimal sample preparation, it provides a rapid and economical alternative to traditional extraction techniques. The method also eliminates the need to remove salts or buffers, which are commonly present in biological matrices. Although this method was developed to quantify in vitro metabolism of one analyte, it could easily be adapted to detect or quantify numerous volatiles and/or semi-volatiles found in biological matrices.

A proteasome-resistant fragment of NIK mediates oncogenic NF-κB signaling in schwannomas.

Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.

Metabolomic Analysis Identified Reduced Levels of Xenobiotics, Oxidative Stress, and Improved Vitamin Metabolism in Smokers Switched to Vuse Electronic Nicotine Delivery System.

INTRODUCTION: Switching to noncombustible tobacco products presents an opportunity for cigarette smokers to potentially reduce the health risks associated with smoking. Electronic Nicotine Delivery Systems (ENDS) are one such product because the vapor produced from ENDS contains far fewer toxicants than cigarette smoke. To investigate the biochemical effects of switching from smoking to an ENDS, we assessed global metabolomic profiles of smokers in a 7-day confinement clinical study. METHODS: In the first 2 days of this clinical study, the subjects used their usual brand of cigarettes and then switched to exclusive ENDS ad libitum use for 5 days. Urine and plasma samples were collected at baseline and 5 days after switching. The samples were analyzed using a mass spectrometry-based metabolomic platform. RESULTS: Random forest analyses of urine and plasma metabolomic data revealed excellent predictive accuracy (>97%) of a 30-metabolite signature that can differentiate smokers from 5-day ENDS switchers. In these signatures, most biomarkers are nicotine-derived metabolites or xenobiotics. They were significantly reduced in urine and plasma, suggesting a decreased xenobiotic load on subjects. Our results also show significantly decreased levels of plasma glutathione metabolites after switching, which suggests reduced levels of oxidative stress. In addition, increased urinary and plasma levels of vitamins and antioxidants were identified, suggesting enhanced bioavailability due to discontinuation of cigarette smoking and switching to Vuse ENDS use. CONCLUSIONS: Our results suggest reduced toxicant exposure, reduced oxidative stress, and potential beneficial changes in vitamin metabolism within 5 days in smokers switching to Vuse ENDS. IMPLICATIONS: Switching from smoking to exclusive ENDS use in clinical confinement settings results in significant reduction of nicotine metabolites and other cigarette-related xenobiotics in urine and plasma of subjects. Significantly decreased oxidative stress-related metabolites and increased urinary and plasma levels of vitamin metabolites and antioxidants in 5-day short-term ENDS switchers suggest less toxic physiological environment for consumers of ENDS products and potential health benefits if such changes persist.

Chiari 1 malformation: age-based outcomes in a paediatric surgical cohort.

PURPOSE: Substantial evidence exists describing differences between paediatric and adult Chiari 1 malformation (CM1) patients. Differences in clinical presentation between very young (0-6 years old) and older (7-18 years old) paediatric patients is similarly well-established. However, progression on these findings with regard to surgical outcomes is limited. We aimed to establish whether inter-paediatric age group modifies surgical outcome for CM1 decompression. METHODS: Retrospective chart review was conducted for 65 patients receiving posterior fossa decompression between 2006 and 2018. Presenting features, surgical management, and outcome were evaluated and stratified into very young patients (0-6 years) or older patients (7-18 years). Outcomes were assessed using the Chicago Chiari Outcome Scale (CCOS), a validated 16-point framework for comparison. RESULTS: Very young patients (21 patients) scored significantly lower in surgical outcome overall compared with older patients (44 patients) (12.1 ± 3.2/16 vs 14.2 ± 1.6/16, p = 0.011), and across 3/4 CCOS subscores: non-pain symptoms, functionality, and complications. Very young patients also returned to theatre more commonly (47.6% vs 13.6%, p = 0.003), primarily for re-do decompression (7/10 patients, 70%). Finally, the presentation of very young patients differed to older patients with significantly more oropharyngeal (38.1% vs 9.0%, p = 0.014) and motor symptoms (47.6% vs 22.7%, p = 0.042). DISCUSSION: Very young patients (0-6 years) do not appear to respond as well to standard posterior fossa decompression, as their older (7-18 years) paediatric counterparts, in the absence of several baseline cohort characteristic differences. We hypothesise underlying anatomical differences may contribute to this finding.

Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor.

RATIONALE: Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches. OBJECTIVES: To identify genetic susceptibility targets for ARDS. METHODS: A genome-wide association study was performed on 232 African American patients with ARDS and 162 at-risk control subjects. The Identify Candidate Causal SNPs and Pathways platform was used to infer the association of known gene sets with the top prioritized intragenic SNPs. Preclinical validation of SELPLG (selectin P ligand gene) was performed using mouse models of LPS- and ventilator-induced lung injury. Exonic variation within SELPLG distinguishing patients with ARDS from sepsis control subjects was confirmed in an independent cohort. MEASUREMENTS AND MAIN RESULTS: Pathway prioritization analysis identified a nonsynonymous coding SNP (rs2228315) within SELPLG, encoding P-selectin glycoprotein ligand 1, to be associated with increased susceptibility. In an independent cohort, two exonic SELPLG SNPs were significantly associated with ARDS susceptibility. Additional support for SELPLG as an ARDS candidate gene was derived from preclinical ARDS models where SELPLG gene expression in lung tissues was significantly increased in both ventilator-induced (twofold increase) and LPS-induced (5.7-fold increase) murine lung injury models compared with controls. Furthermore, Selplg-/- mice exhibited significantly reduced LPS-induced inflammatory lung injury compared with wild-type C57/B6 mice. Finally, an antibody that neutralizes P-selectin glycoprotein ligand 1 significantly attenuated LPS-induced lung inflammation. CONCLUSIONS: These findings identify SELPLG as a novel ARDS susceptibility gene among individuals of European and African descent.

Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin Signaling Complex to Inhibit VEGF-Induced Retinal Angiogenesis.

Targeting vascular endothelial growth factor (VEGF) is a common treatment strategy for neovascular eye disease, a major cause of vision loss in diabetic retinopathy and age-related macular degeneration. However, the decline in clinical efficacy over time in many patients suggests that monotherapy of anti-VEGF protein therapeutics may benefit from adjunctive treatments. Our previous work has shown that through decreased activation of the cytoskeletal protein paxillin, growth factor-induced ischemic retinopathy in the murine oxygen-induced retinopathy model could be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome is required for human retinal endothelial cell migration and proliferation. Specifically, the disruption of focal adhesion kinase (FAK) and paxillin interactions using the small molecule JP-153 inhibited Src-dependent phosphorylation of paxillin (Y118) and downstream activation of Akt (S473), resulting in reduced migration and proliferation of retinal endothelial cells stimulated with VEGF. However, this effect did not prevent the initial activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded microemulsion affected the hallmark features of pathologic retinal angiogenesis, reducing neovascular tuft formation and increased avascular area, in a dose-dependent manner. In conclusion, our results suggest that using small molecules to modulate the focal adhesion protein paxillin is an effective strategy for treating pathologic retinal neovascularization. To our knowledge, this is the first paradigm validating modulation of paxillin to inhibit angiogenesis. As such, we have identified and developed a novel class of small molecules aimed at targeting focal adhesion protein interactions that are essential for pathologic neovascularization in the eye.

A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation.

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.

Mitigation of radiation injury by selective stimulation of the LPA(2) receptor.

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA(2) receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA(2) receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24h after radiation exposure. Our findings suggest that by specifically activating LPA(2) receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Quinic acid derivative KZ-41 exhibits radiomitigating activity in preclinical models of radiation injury.

Acute radiation syndrome is induced when a significant portion of the body receives high-dose, as well as high-dose rate, radiation. We have previously identified a quinic acid-based derivative, KZ-41, that protects from radiation injury. Further preclinical efficacy studies were conducted to determine the radiomitigating activity of KZ-41. C57BL/6 mice received total body irradiation (TBI-LD80/30, ¹³7Cs; ∼2 min) followed by either normal saline or KZ-41 (100 mg/kg sc ∼26 h post-TBI). KZ-41 increased 30-day survival by approximately 45% compared with vehicle controls (P < 0.05). To further investigate the potential radiomodulating mechanisms of KZ-41, we developed a combined radiation and vascular injury model. C57BL/6 mice surgically fixed with dorsal windows for dermal vasculature imaging received either sham or TBI (¹³7Cs; 6 Gray). Postcapillary venule injury was induced (24, 48, 72, and 96 h post-TBI) followed by imaging at 5 min and 24 h to assess clot formation and blood flow. Impairment in flow (P < 0.05) and clot formation (P < 0.05) were observed as early as 48 and 72 h, respectively. Thus, vascular injury 72 h post-TBI was used to evaluate intervention (KZ-41; 100 mg/kg i.p. at 12, 36, and 60 h post-TBI) on radiation-induced changes in both flow and clot formation. KZ-41, although not improving flow, increased clot formation (P < 0.05). Platelet counts were lower in both irradiated groups compared with sham controls (P < 0.05). In summary, KZ-41 exerts radiomitigating activity in lethally irradiated mice. Imaging results suggest KZ-41 exerts radiomitigating activity through mechanisms involving promotion of initial clot formation and vascular flow restoration. The imaging model described herein is useful for further examination of radiation-induced vascular injury repair mechanisms.

Synthesis and characterization of an anti-apoptotic immunosuppressive compound for improving the outcome of islet transplantation.

Mycophenolic acid (MPA) is a commonly used immunosuppressive drug for human islet transplantation. However, it is toxic to transplanted islets, causing primary nonfunction. We recently synthesized a quinic acid derivative, 1,3,4,5-tetrahydroxy-N-propylcyclohexanecarboxamide (KZ41), which has anti-inflammatory and anti-apoptotic effects. We hypothesized that the conjugate (E)-2,3,5-trihydroxy-5-(propylcarbamoyl) cyclohexyl 6-(4-ethoxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (JP-3-110), which is composed of KZ41 and MPA through esterification, can suppress the immune rejection while inducing less toxicity. Early characterization showed that the solubility of JP-3-110 was significantly higher than that of MPA, though JP-3-110 was still poorly water-soluble. The ester bond connecting KZ41 and MPA is stable for a limited duration (<4 weeks). Pharmacological studies demonstrated that JP-3-110 induced significantly less activated caspase 3 and apoptotic cell death of human islets than MPA, while maintaining an equally potent immunosuppressive effect. A similar immunosuppressive effect of JP-3-110 and MPA in humanized NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NOD scid gamma, NSG) mice with adoptively transferred human immunity was observed. Taken together, our results demonstrated that JP-3-110 can be a safer immunosuppressive agent for human islet transplantation.

LC-MS/MS identification of the one-carbon cycle metabolites in human plasma.

The one-carbon cycle is composed of four major biologically important molecules: methionine (L-Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy). In addition to these key metabolites, there are multiple enzymes, vitamins, and cofactors that play essential roles in the cascade of the biochemical reactions that convert one metabolite into another in the cycle. Simultaneous quantitative measurement of four major metabolites can be used to detect possible aberrations in this vital cycle. Abnormalities in the one-carbon cycle might lead to hyper- or hypomethylation, homocystinemia, liver dysfunction, and accumulation of white-matter hyperintensities in the human brain. Previously published methods describe evaluation of several components of the one-carbon cycle, but none to our knowledge demonstrated simultaneous measurement of all four key molecules (L-Met, SAM, SAH, and Hcy). We describe a novel analytical method suitable for simultaneous identification and quantification of L-Met, SAM, SAH, and Hcy with LC-MS/MS. Moreover, we tested this method to identify these metabolites in human plasma collected from patients with multiple sclerosis and healthy individuals. In a pilot feasibility study, our results indicate that patients with multiple sclerosis showed abnormalities in the one-carbon cycle.

In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1.

We define previously unrecognized in vivo pathways of vitamin D(3) (D3) metabolism generating novel D3-hydroxyderivatives different from 25-hydroxyvitamin D(3) [25(OH)D3] and 1,25(OH)(2)D3. Their novel products include 20-hydroxyvitamin D(3) [20(OH)D3], 22(OH)D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, 1,20(OH)(2)D3, 1,20,23(OH)(3)D3, and 17,20,23(OH)(3)D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ∼20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α-hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)(2)D3 → 17,20,23(OH)(3)D3 with further 1α-hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)(2)D3 and D3 → 22(OH)D3 → 20,22(OH)(2)D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)(2)D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)(2)D3, 20,22(OH)(2)D3, and 17,20,23(OH)(3)D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)(2)D3.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing pomegranate extract pharmacokinetics in humans.

Pomegranate extracts standardized to punicalagins are a rich source of ellagitannins including ellagic acid (EA). Recent evidence suggests that gut microbiota-derived urolithin (Uro) metabolites of ellagitannins are pharmacologically active. Studies have evaluated the pharmacokinetics of EA, however, little is known about the disposition of urolithin metabolites (urolithin A (UA) and B (UB)). To address this gap, we developed and applied a novel ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the characterization of EA and Uro oral pharmacokinetics in humans. Subjects (10/cohort) received a single oral dose (250 or 1000 mg) of pomegranate extract (Pomella® extract) standardized to contain not less than 30 % punicalagins, < 5 % EA, and not less than 50 % polyphenols. Plasma samples, collected over 48 h, were treated with ß-glucuronidase and sulfatase to permit comparison between unconjugated and conjugated forms of EA, UA and UB. EA and urolithins were separated by gradient elution (acetonitrile/water, 0.1 % formic acid) using a C18 column connected to a triple quadrupole mass spectrometer operating in the negative mode. Conjugated EA exposure was ∼5-8-fold higher than unconjugated EA for both dose groups. Conjugated UA was readily detectable beginning ∼8 h post-dosing, however, unconjugated UA was detectable in only a few subjects. Neither form of UB was detected. Together these data indicate EA is rapidly absorbed and conjugated following oral administration of Pomella® extract. Moreover, UA's delayed appearance in the blood, primarily in the conjugated form, is consistent with gut microbiota-mediated metabolism of EA to UA, which is then rapidly converted to its conjugated form.

Extraordinary Speech and Language Outcomes After Auditory Brainstem Implantation: Guidance From a Case Study.

PURPOSE: Large individual differences and poor speech recognition outcomes are routinely observed in most patients who have received auditory brainstem implants (ABIs). A case report of an ABI recipient with exceptionally good speech recognition outcomes presents an opportunity to better understand the core information processing mechanisms that underlie variability and individual differences in outcomes. METHOD: A case study is reported of an adult ABI recipient (ID-006) with postlingually acquired, Neurofibromatosis Type 2 (NF2)-related hearing loss who displayed exceptional postoperative speech recognition scores. A novel battery of assessment measures was used to evaluate ID-006's auditory, cognitive, and linguistic information processing skills. RESULTS: Seventeen years following ABI activation, ID-006 scored 77.6% correct on the AzBio Sentences in quiet. On auditory processing tasks, ID-006 scored higher on tasks with meaningful sentences and much lower on tasks that relied exclusively on audibility. ID-006 also demonstrated exceptionally strong abilities on several cognitive and linguistic information processing tasks. CONCLUSIONS: Results from a novel battery of information processing tests suggest that ID-006 relies extensively on top-down predictive processing and cognitive control strategies to efficiently encode and process auditory information provided by his ABI. Results suggest that current measures of outcomes and benefits should be expanded beyond conventional speech recognition measures to include more sensitive and robust measures of speech recognition as well as neurocognitive measures such as executive function, working memory, and lexical access.