Standard dosing of infusional 5-FU results in subtherapeutic drug exposure for up to half of all patients. We evaluated plasma uracil concentration and DPYD rs4294451 genotype as candidate predictors of individual-level 5-FU exposure. We conducted a prospective study of drug exposure in patients with gastrointestinal cancer receiving infusional 5-FU. Participants were evaluated by measurement of fasting pretreatment plasma uracil concentration, DPYD gene sequence (including genotyping of rs4294451) and 5-FU area under the curve (5-FU AUC). We used a linear mixed-effects model to evaluate the association of 5-FU AUC with uracil concentration and rs4294451 genotype, adjusting for cycle number, sex, serum creatinine, and 5-FU dose. There were 29 evaluable participants with a median age of 64 years (range 41–81); nine (31

751 Background: Plasma uracil concentration >16 ng/mL is used in some countries as a screening test for DPD deficiency. We conducted this exploratory study to evaluate the association of uracil concentration with 5-FU exposure, toxicity, and DPYD genotype in patients without known DPD deficiency. The main hypothesis was that lower uracil concentration would be predictive of subtherapeutic 5-FU drug exposure (5-FU AUC < 20 mg*h/L) in patients treated with infusional 5-FU. Methods: The study prospectively enrolled patients with GI cancer who were receiving initial chemotherapy with a regimen containing a 46-hour 5-FU infusion initially dosed at 2400 mg/m2. Study candidates were screened for the DPYD *2A, *13 and c.2846A>T gene variants; variant carriers were excluded. Participants gave pretreatment fasting morning blood samples for determination of plasma uracil concentration and DPYD whole-gene sequencing. Plasma samples were collected at hour 45 of 5-FU infusion in chemotherapy cycles 1 and 2 for determination of 5-FU exposure, measured as the 5-FU AUC. We estimated a linear mixed-effects model to test the association of uracil concentration with 5-FU AUC, adjusting for cycle, sex, creatinine, and 5-FU infusion dose (in mg/m2). Results: There were 29 evaluable participants with a median age of 64 years (range 41-81); nine (31%) were female. The most common cancer sites were colorectal (n = 15) and pancreatic (10). The median (IQR) plasma uracil concentration was 10.4 ng/mL (7.2, 12.6). Data for 5-FU AUC were available for 19 subjects in cycle 1 (median = 22.0 mg*h/L) and 27 subjects in cycle 2 (median = 19.3 mg*h/L). In the mixed-effects model, male sex was significantly associated with 5-FU AUC (beta = -12.0 [95% CI -17.2, -6.7], p <0.001) but plasma uracil concentration was not (beta = 0.35 [-0.16, 0.86], p=0.16). Holding other covariates constant, male sex was associated with a 29% decrease in 5-FU AUC (vs female) and plasma uracil concentration in the lowest quartile was associated with a 12% decrease in 5-FU AUC (vs highest quartile). Three of 29 participants (10%, all male) had uracil concentrations of >16 ng/mL, suggestive of DPD deficiency. The subject with the highest uracil concentration (23.1 ng/mL) had severe toxicity in cycle 1 (neutropenia, mucositis) requiring 5-FU dose reduction; whole gene sequencing identified a rare DPYD missense variant in this subject (c.2185G>A [p.A729T]). The other two subjects with uracil concentration >16 ng/mL did not have culprit DPYD gene variants and tolerated infusional 5-FU at 2400 mg/m2 without early dose-limiting toxicity. Conclusions: Our findings suggest that male sex is more important than low pretreatment plasma uracil concentration as a predictor of risk for subtherapeutic 5-FU drug exposure. This result is consistent with prior evidence that men experience less 5-FU associated toxicity than women.

Cholesterol homeostasis is pivotal for cellular function. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also abbreviated as SOAT1, is an enzyme responsible for catalyzing the storage of excess cholesterol to cholesteryl esters. ACAT1 is an emerging target to treat diverse diseases including atherosclerosis, cancer, and neurodegenerative diseases. F12511 is a high-affinity ACAT1 inhibitor. Previously, we developed a stealth liposome-based nanoparticle to encapsulate F12511 to enhance its delivery to the brain and showed its efficacy in treating a mouse model for Alzheimer’s disease (AD). In this study, we introduce F26, a close derivative of F12511 metabolite in rats. F26 was encapsulated in the same DSPE-PEG2000/phosphatidylcholine (PC) liposome-based nanoparticle system. We employed various in vitro and in vivo methodologies to assess F26’s efficacy and toxicity compared to F12511. The results demonstrate that F26 is more effective and durable than F12511 in inhibiting ACAT1, in both mouse embryonic fibroblasts (MEFs), and in multiple mouse tissues including the brain tissues, without exhibiting any overt systemic or neurotoxic effects. This study demonstrates the superior pharmacokinetic and safety profile of F26 in wild-type mice, and suggests its therapeutic potential against various neurodegenerative diseases including AD.

Background Autoimmune Hemolytic Anemia (AIHA) is a disorder in which red blood cells are destroyed by autoantibodies produced by B-cells and plasma cells. The goal of treatment is to maintain a Hemoglobin (Hgb) concentration ≥ 10 g/dL which involves high-dose steroids with a taper; however, steroids are frequently not curative as they do not eliminate the antibody-producing clone. The use of rituximab, an anti-CD20 monoclonal antibody (mAb), improved outcomes for AIHA, including response rates and cure rates. It is approved in the first-line setting and for relapsed/refractory AIHA. However, for patients who do not respond or relapse after rituximab, options are generally limited. One putative mechanism of resistance to rituximab is that the B-cell clone matures into a non-CD-20-expressing plasma cell. Using that hypothesis, we designed this study to test the safety and efficacy of daratumumab, an anti-CD38 mAb approved in several combinations for treatment of multiple myeloma as it eliminates CD38 expressing plasma cells. Study Design Main inclusion criteria were age ≥ 18, AIHA based on a Hgb < 10 g/dL and a positive direct antiglobulin test, and clinical signs of acute hemolysis including an elevated lactate dehydrogenase (LDH) and reticulocyte count (RC) or low haptoglobin. All patients had to have relapsed after prior treatment with steroids at ≥ 1mg/kg of prednisone (or equivalent) and rituximab ≥100mg. Patients also had to have an Eastern Cooperative Oncology Group function 0-2. Main exclusion criteria were uncontrolled medical disorders, lymphoid malignancies other than low grade lymphoproliferative disorders not requiring treatment, pregnancy, breastfeeding, COPD with FEV1 < 50%, renal or liver dysfunction, or prior anti-CD38 therapy. A baseline evaluation included a complete blood count (CBC), complete metabolic panel (CMP), LDH, DAT, haptoglobin, and RC. They received 6 weekly doses of subcutaneous (SC) daratumumab 1800mg and hyaluronidase PH20 2,000 U/ml. A CBC, CMP, LDH, and RC were checked weekly. Follow up with labs occurred every 8 weeks for 1 year. The primary objective was safety monitoring. Unacceptable toxicity necessitating withdrawal was defined as any grade ≥3 event based on CTCAE version 5. The secondary objective was treatment efficacy. A complete response (CR) was defined as a normalization in Hgb without evidence of hemolysis. A partial response was defined as an increase in Hgb > 10g/dL while allowing hemolysis with an elevated RC, bilirubin, and/or LDH. PR could include use of prednisone <10mg daily (or equivalent) to maintain Hgb > 10g/dL. Trough serum concentrations of daratumumab were measured prior to each treatment and post last dose using an ELISA assay. Results This study enrolled 2 patients. For our primary endpoint, there were no unexpected toxicities attributed to daratumumab. Patient 1 was a 57-year-old Caucasian female. Baseline Hgb and absolute RC were 9.3 g/dL and 0.28g/dL on the day of dose 1, respectively. At dose 2, her Hgb was 10.1 g/dL and her RC 0.24g/dL. By week 5, her Hgb and RCnormalized at 12.5 g/dL and 0.11, respectively and have remained in normal range at 9 months. Her bilirubin and LDH remained in normal range throughout the study. She remains in complete remission 10 months from first daratumumab dose. Patient 2 was a 45-year-old Caucasian female. Baseline Hgb and RC were 9.2 g/dL and 0.23g/dL on day 1, respectively. She displayed normalization of her RC by week 4 measured at 0.08. Her Hgb improved to 10.5 g/dL at week 3 and normalized at 11.8 g/dL at week 8. Total bilirubin and LDH remained in normal limits in this timeframe. At week 14, she displayed evidence of relapsed disease with a Hgb 6.4 g/dL, a RC 0.45, and LDH 785 units/L. At that time, she was diagnosed with a COVID infection and was removed from the study. Daratumumab trough concentrations increased steadily after the first dose in both patients, peaking after the sixth dose at 268.1 mcg/mL and 366.8 mcg/mL. Conclusions Due to the rarity of the disease, we were only able to recruit 2 patients to this single-institution trial, making it difficult to draw definitive conclusions. However, we found it promising that treatment was well-tolerated without unexpected toxicities, and that both patients achieved CRs. We suggest further investigation into daratumumab for relapsed or refractory AIHA where other treatment options are limited.

Cholesterol is essential for cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is catalyzed by the enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 being the primary isoenzyme in most cells in humans. In Alzheimer's Disease, CEs accumulate in vulnerable brain regions. Therefore, ACATs may be promising targets for treating AD. F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for antiatherosclerosis. Previously, we developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG2000 with phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) in wild-type mice and performed an HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not overaccumulate in the brain or other tissues after repeated IVs. A histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that a 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and nonphosphorylated tau, and reduced neuroinflammation. This work lays the foundation for nanoparticle F to be used as a possible therapy for AD and other neurodegenerative diseases.

IntroductionSonidegib and vismodegib are currently the only US Food and Drug Administration and European Medicines Agency-approved small-molecule Hedgehog pathway inhibitors (HHIs)for treating adults with advanced or refractory basal cell carcinoma (BCC) that is not amenable to conventional surgery or radiotherapy. At this time, there are no head-to-head clinical trials comparing these two HHIs for efficacy and safety to assist clinicians with determining which HHI may be best suited for their patients.Areas coveredThis review briefly describes the pathogenesis of BCC, provides a detailed overview of the key pharmacokinetic profile differences between sonidegib and vismodegib, explains their pharmacodynamics, and highlights the therapeutic considerations when either HHI is used to treat special patient populations.Expert opinionAlthough both HHIs act at the same molecular target in the Hedgehog pathway, there are significant differences in their pharmacokinetic profiles that may play a potential role in their efficacy and safety. Evidence-based recommendations serve to inform clinicians until direct comparative clinical trials of sonidegib versus vismodegib are conducted to determine the clinical relevance of the reported differences in their pharmacokinetic properties.

Background: Sunitinib is a multi-target tyrosine kinase inhibitor (TKI) that inhibits VEGF receptor 1, 2, 3 (VEGFRs), platelet-derived growth factor receptor (PDGFR), colony-stimulating factor receptor (CSFR), and the stem cell factor receptor c-KIT. Temsirolimus inhibits mammalian target of rapamycin (mTOR) through binding to intracellular protein FKBP-12. Both agents are approved for the treatment of metastatic renal cell carcinoma (mRCC), have different anticancer mechanisms, and non-overlapping toxicities. These attributes form the scientific rationale for sequential combination of these agents. The primary objective of the study was to investigate the efficacy of alternating sunitinib and temsirolimus therapy on progression-free survival (PFS) in mRCC.Methods: We undertook a phase II, multi-center, single cohort, open-label study in patients with mRCC. Patients were treated with alternating dosing of 4 weeks of sunitinib 50 mg PO daily, followed by 2 weeks rest, then 4 weeks of temsirolimus 25 mg IV weekly, followed by 2 weeks rest (12 weeks total per cycle). The primary endpoint was PFS. Secondary endpoints included clinical response rate and characterization of the toxicity profile of this combination therapy.Results: Nineteen patients were enrolled into the study. The median observed PFS (n = 13 evaluable for PFS) was 8.8 months (95% CI 6.8-25.2 months). Best responses achieved were five partial response, nine stable disease, and three disease progression according to RECIST 1.1 guidelines (two non-evaluable). The most commonly observed toxicities were fatigue, platelet count decrease, creatinine increased, diarrhea, oral mucositis, edema, anemia, rash, hypophosphatemia, dysgeusia, and palmar-plantar erythrodysesthesia syndrome.Conclusion: Alternating sunitinib and temsirolimus did not improve the PFS in patients with mRCC.

AIM:Metformin is used for the management of type 2 diabetes mellitus (T2DM) and is being tested clinically as an anticancer agent. Metformin concentrations safely achievable in human solid tissues including tumours are unknown. This study was designed to determine metformin concentration in tissue compartments as a function of dose to inform rational dosing in preclinical models and interpretation of clinical results." METHODS:Subjects with solid tumours to be treated by resection and either (A) willingness to take metformin for 7-10 days before surgery or (B) taking metformin for T2DM were eligible. Whole blood, plasma, tumour, tumour-adjacent uninvolved tissue and subcutaneous adipose tissue were obtained for liquid chromatography with tandem mass spectrometry to measure metformin concentrations. RESULTS:All subjects had primary lung tumours. Metformin dose was significantly correlated with drug concentrations in all tissues analysed. Intersubject metformin concentrations varied by over two orders of magnitude. Metformin concentrations were significantly higher in tumour tissues and lower in adipose tissues compared to other tissues. Concentrations in blood and plasma were significantly correlated with concentrations in solid tissues. CONCLUSION:Metformin accumulates in cellular compartments. Concentrations observed in plasma, blood, lung and tumour tissues in subjects treated with US Food and Drug Administration-approved doses for T2DM are lower than those typically used in tissue culture studies. However, such tissue concentrations are in line with those found within cultured cells treated with supra-pharmacological doses of metformin. Given the large intersubject variability in metformin concentrations, it is imperative to determine whether there is an association between tissue metformin concentration and anticancer activity in humans.

Chronic lymphocytic leukemia (CLL) was projected to cause 4320 deaths in 2021 in the US [1]. Chemo-immunotherapy regimens are commonly associated with unfavorable adverse events (AEs) [2, 3]. Targeted therapies such as the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib are better tolerated and have become standard of care for CLL. Ibrutinib causes egress of CLL cells from their stromal niche leading to peripheral lymphocytosis [4]. While initially successful in treatment of naïve, relapsed/refractory, and high risk CLL (deletion 17p), most high risk patients experienced disease progression, indicating a need to improve BTK-targeted therapy regimens [5-8]. Microtubule inhibitors kill CLL cells ex vivo with dependence on c-Jun N-terminal kinase (JNK) activation and induction of NOXA [9]. Vincristine activated JNK but not NOXA in circulating CLL cells in vivo [10]. BNC105, a colchicine-site microtubule inhibitor, was more potent at activating the JNK-NOXA apoptotic cascade [11]. BNC105P, the prodrug of BNC105, had a monotherapy maximum tolerated dose (MTD) of 16 mg/m2 in solid tumor patients with on-target polymerized tubulin reduction activity starting at 12.6 mg/m2 [12]. The study rationale was to target CLL cell egress from the stromal niche with ibrutinib and eliminate CLL cells in circulation with BNC105P to potentially obtain deep, long-lasting responses [13]. We therefore conducted an open-label, Phase Ib, dose escalation study of BNC105P and ibrutinib in patients with relapsed/refractory CLL (NCT03454165). Patients were enrolled at the Dartmouth Cancer Center using a Dartmouth College and Dartmouth-Hitchcock Health Institutional Review Board approved protocol and written informed consent. Study patients provided written informed consent prior to the performance of any study procedures and given a copy of the signed written informed consent. BNC105P (Bionomics Limited, Thebarton, Australia) was administered via IV infusion on days 1 and 8 (cycle 1) and days 8 and 15 (cycles 2–6) of 21-day cycles. Ibrutinib 420 mg was self-administered orally on days 1–21 (cycles 2–6). In drug combination cycles, ibrutinib was taken ≥30 min prior to BNC105P infusion. A standard 3 + 3 Phase I dose escalation design was used with planned BNC105P dose cohorts of 8 mg/m2 (cohort 1), 12 mg/m2 (cohort 2), and 16 mg/m2 (cohort 3). The primary study objective was to determine the MTD of BNC105P in combination with ibrutinib in CLL patients. All patient inclusion and exclusion criteria for study NCT03454165 are detailed at https://clinicaltrials.gov/ct2/show/NCT03454165. Prior ibrutinib therapy was allowed but not required. Enrolled patient demographics are listed in Table 1. Clinical responses were assessed via computerized tomography scans. Modified IWCLL guidelines were used to categorize objective responses [14]. Dose limiting toxicities (DLTs) were defined as: all nonhematological toxicities of ≥ grade 3 except: reversible grade 3 nausea, vomiting or diarrhea; reversible asymptomatic grade 3–4 laboratory abnormalities; hematological toxicities: grade ≥ 3 febrile neutropenia; infection of grade 4, or accompanied by fever or fails to resolve within 7 days; failure of adequate recovery of neutrophils/platelets or neutrophil count to > 1000/platelets 50,000/mm3 (whichever is lower) within 14 days of the scheduled start day for cycle 2 or cycle 3. Grading of nonhematological toxicities utilized NCI CTCAE v4.03, while hematological toxicities were graded according to 1996 modified NCI-WG criteria for CLL/SLL, updated in 2008 [14]. The MTD was defined as the dose at which no more than 1/6 of the subjects (16.7%) experience a DLT. Three patients were treated in the 8 mg/m2 BNC105P plus 420 mg ibrutinib cohort (Cohort 1) without developing any DLTs. Subsequently three patients were treated with 12 mg/m2 BNC105P plus 420 mg ibrutinib (Cohort 2). One of the cohort 2 patients who initially received 12 mg/m2 of BNC105P in cycle 1 and 420 mg ibrutinib C2D1-7 was subsequently dose reduced (as per protocol because he developed Gr 1→Gr2 thrombocytopenia, but this was not a defined DLT) to 8 mg/m2 BNC105P and 280 mg ibrutinib, which was tolerated for a further six cycles. Patient outcomes, serious AEs (SAEs), and AEs are summarized in Table 1. One DLT of protracted grade 2 thrombocytopenia in cohort 2 was attributed to ibrutinib. Two SAEs occurred: one sudden death (unknown cause) occurred 10 days after completing study combination treatment while receiving ibrutinib monotherapy; one patient in cohort 1 developed cryptococcal pneumonia on C3D7 following heavy exposure to bat droppings (guano) at a timepoint beyond the defined DLT time window. Treatment-related AEs included (n of six patients): fatigue (3), anemia (3), constipation (2), bruising (2), hypertension (2), dizziness (1), neutropenia (1), thrombocytopenia (2), elevated ALT (2), lymphocytosis (1), hyponatremia (1), headaches (1), elevated AST (1), ankle edema (1), arthralgia (1), dysgeusia (1), and grade 2 protracted thrombocytopenia resulting in a DLT (1). All AEs were grade 1 or 2 except neutropenia (grade 4, baseline grade 3) and lymphocytosis (grade 3). Of the six patients treated, one had a partial remission, three initially had stable disease (one sustained), and two were not evaluable for response. Absolute lymphocyte counts (ALCs) were measured (Figure 1A) with a marked increase (21%–433%) a week after ibrutinib therapy (first administered on C2D1). ALCs on C2D8 decreased following the BNC105P dosing by 6%–34% in five patients. Other peripheral blood mononuclear cells (PBMC) biomarkers were assessed by collecting whole blood pre-dose, 0.5, 3, and 6 h post-BNC105P infusion on C1D1 and C2D8, and preibrutinib dose on C2D1 as described previously [10, 11]. JNK was phosphorylated/activated (n = 6/6) and NOXA induced (n = 5/6) after C1D1 BNC105P treatment (Figure 1B,C). Apoptosis marker, cleaved PARP, was negligible (Figure 1D). Tubulin was destabilized and found in the soluble fraction of PBMCs after BNC105P treatment (n = 5/6) (Figure 1E). This study tested the novel combination of BNC105P with ibrutinib in patients with relapsed/refractory CLL. Unfortunately, the primary objective of this study (MTD determination) was not achieved due to study termination mandated by institutional policy, which defines that the rate of six patients enrolled over 3.5 years was excessively slow. This inadequate rate of enrollment was contributed to by the proven efficacy of the venetoclax plus rituximab combination [15] and Federal Drug Administration (FDA) approval of acalabrutinib. The study drug combination at a dose of 8 mg/m2 IV BNC105P plus 420 mg PO ibrutinib was safe and well tolerated (no DLTs). Three patients were treated at the 12 mg/m2 IV BNC105P and 420 mg PO ibrutinib dose level. One patient was dose reduced as per protocol because of Gr 2 thrombocytopenia and was treated for 6 cycles with partial remission but having completed study treatment and while taking ibrutinib monotherapy suddenly died (unknown cause; no autopsy). A second patient had a DLT of protracted thrombocytopenia (Gr 2) and was unable to continue therapy beyond cycle 2. The third patient tolerated the Cohort 2 study doses for six cycles with initial stable disease. The common major toxicities (>30%) were grades 1–2 anemia, fatigue, bruising, constipation, hypertension, and thrombocytopenia and are known adverse effects of ibrutinib therapy. Our study supports the utility of tubulin depolymerization as a biomarker for BNC105P on-target activity. However, larger studies are required to establish the MTD, full safety profile, and apoptotic biomarker utility of this drug combination in CLL patients. Concept and trial design: DBP, AVD, TDT, AE, and LDL. Clinical support: BML, LDL, and FL. Data collection and analysis: DBP, DBN, BML, LDL, and FL. Manuscript original draft: DBP. Data interpretation and final manuscript review: Darcy B. Pooler, Dylan B. Ness, Alexey V. Danilov, Bridget M. Labrie, Tor D. Tosteson, Alan Eastman, Lionel D. Lewis, and Frederick Lansigan. We thank all of our patients and their families for their generous participation in this study and Bionomics Limited for supplying the BNC105P. The authors acknowledge the services of the Clinical Pharmacology Shared Resource and the Biostatistical Resource at the Dartmouth Cancer Center with funding from NCI Cancer Center Support Grant 5P30 CA023108. DBP, DBN, BML, TDT, AE, FL have no competing interests to report in relation to this study. AVD is a paid consultant to Abbvie, AstraZeneca, BeiGene, Gamida, Genentech, Janssen, Morphosys, Pharmacyclics, and TG Therapeutics and has ongoing research funding from AstraZeneca, Bayer Oncology, Bristol Meyers Squibb, MEI Pharma, SecuraBio, TG Therapeutics, and Takeda Oncology. LDL is a paid consultant to G1 Therapeutics and 7 Hills Pharma LLC and receives support for clinical trials from Bristol Myers Squibb, AbbVie, and AstraZeneca. Data may be requested from the corresponding author. All data provided are anonymized to respect the privacy of patients who have participated in the trial in line with applicable laws and regulations. The Hopeman Foundation; Bench to Bedside Funding from The Dartmouth Cancer Center. Data may be requested from the corresponding author. All data provided are anonymized to respect the privacy of patients who have participated in the trial in line with applicable laws and regulations.

Estimating statistical significance of the difference between two spectra or series is a fundamental statistical problem. Multivariate significance tests exist but the limitations preclude their use in many common cases; e.g. one-sided testing, unequal variance and when few repetitions are acquired all of which are required in magnetic spectroscopy of nanoparticle Brownian motion (MSB). We introduce a test, termed the T-S test, that is powerful and exact (exact type I error). It is flexible enough to be one- or two-sided and the one-sided version can specify arbitrary regions where each spectrum should be larger. The T-S test takes the-one or two-sided p -value at each frequency and combines them using Stouffer’s method. We evaluated it using simulated spectra and measured MSB spectra. For the single-sided version, mean of the spectrum, A-T, was used as a reference; the T-S test is as powerful when the variance at each frequency is uniform and outperforms when the noise power is not uniform. For the two-sided version, the Hotelling T2 two-sided multivariate test was used as a reference; the two-sided T-S test is only slightly less powerful for large numbers of repetitions and outperforms rather dramatically for small numbers of repetitions. The T-S test was used to estimate the sensitivity of our current MSB spectrometer showing 1 ng sensitivity. Using eight repetitions the T-S test allowed 15 pM concentrations of mouse IL-6 to be identified while the mean of the spectra only identified 76 pM.

OBJECTIVE:The aryl hydrocarbon receptor (AHR) plays a key role in obesity. In vitro studies revealed that the tryptophan metabolite kynurenine (Kyn) activates AHR signaling in cultured hepatocytes. The objective of this study was to determine whether Kyn activated the AHR in mice to induce obesity. METHODS:Mice were fed a low-fat diet and the same diet supplemented with Kyn. Body mass, liver status, and the expression of identified relevant genes were determined. RESULTS:Kyn caused mice to gain significant body mass, develop fatty liver and hyperglycemia, and increase expression levels of cytochrome P450 1B1 and stearoyl-CoA desaturase 1. The hyperglycemia was accompanied with decreased insulin levels, which may have been due to the repression of genes involved in insulin secretion. Kyn plasma concentrations and BMI were measured in female patients, and a significant association was observed between Kyn and age in patients with obesity but not in patients who were lean. CONCLUSIONS:Results show that (1) Kyn or a metabolite thereof is a ligand responsible for inducing AHR-based obesity, fatty liver, and hyperglycemia in mice; (2) plasma Kyn levels increase with age in women with obesity but not in lean women; and (3) an activated AHR is necessary but not sufficient to attain obesity, a status that also requires fat in the diet.

AIMS:We evaluated the potential effect of sonidegib at an oral dose of 800 mg once daily (QD) on the pharmacokinetics (PK) of the probe drugs warfarin (CYP2C9) and bupropion (CYP2B6).METHODS:This was a multicentre, open-label study to evaluate the effect of sonidegib on the PK of the probe drugs warfarin and bupropion in patients with advanced solid tumours. Cohort 1 patients received a single warfarin 15-mg dose on Day 1 of the run-in period and on Cycle 2 Day 22 (C2D22) of sonidegib administration. Cohort 2 patients received a single bupropion 75-mg dose on Day 1 of run-in period and on C2D22 of sonidegib administration. Sonidegib 800 mg QD oral dosing began on Cycle 1 Day 1 of a 28-day cycle after the run-in period in both cohorts.RESULTS:The geometric means ratios [90% confidence interval] for (S)-warfarin with and without sonidegib were: area under the concentration-time curve from time 0 to infinity (AUCinf ) 1.15 [1.07, 1.24] and maximum plasma concentration (Cmax ) 0.88 [0.81, 0.97]; and for (R)-warfarin were: AUCinf 1.10 [0.98, 1.24] and Cmax 0.93 [0.87, 1.0]. The geometric means ratios [90% confidence interval] of bupropion with and without sonidegib were: AUCinf 1.10 [0.99, 1.23] and Cmax 1.16 [0.95, 1.42]. Sonidegib 800 mg had a safety profile that was similar to that of lower dose sonidegib 200 mg and was unaffected by single doses of the probe drugs.CONCLUSIONS:Sonidegib dosed orally at 800 mg QD (higher than the Food and Drug Administration-approved dose) did not impact the PK or pharmacodynamics of warfarin (CYP2C9 probe substrate) or the PK of bupropion (CYP2B6 probe substrate).

We are developing magnetic nanoparticle (NP) methods to characterize inflammation and infectionin vivo. Peritoneal infection in C57BL/6 mice was used as a biological model. An intraperitoneal NP injection was followed by measurement of magnetic nanoparticle spectroscopy of Brownian rotation (MSB) spectra taken over time. MSB measures the magnetization of NPs in a low frequency alternating magnetic field. Two groups of three mice were studied; each group had two infected mice and one control with no infection. The raw MSB signal was compared with two derived metrics: the NP relaxation time and number of NPs present in the sensitive volume of the receive coil. A four compartment dynamic model was used to relate those physical properties to the relevant biological processes including phagocytic activity and migration. The relaxation time increased over time for all of the mice as the NPs were absorbed. The NP number decreased over time as the NPs were cleared from the sensitive volume of the receive coil. The composite p-values for all three rate constants were significant: raw signal, 0.0002, relaxation, <10(-16)and local NP clearance, <10(-16). However, not all the individual mice had significant changes: Only half the infected mice had significantly different rate constants for raw signal reduction. All infected mice had significantly smaller relaxation time constants. All but one of the infected mice had significantly lower rate constants for local clearance. Relaxation is affected by both phagocytic activity, edema and temperature changes and it should be possible to better isolate those effects to more completely characterize inflammation using more advanced MSB methods. The MSB NP signal can be used to identify inflammationin vivobecause it has the unique ability to monitor phagocytic absorption through relaxation measurements.

Under physiological conditions biomarker concentrations tend to rise and fall over time e.g. for inflammation. Ex vivo measurements provide a snapshot in time of biomarker concentrations, which is useful, but limited. Approaching real time monitoring of biomarker concentration(s) using a wearable, implantable or injectable in vivo sensor is therefore an appealing target. As an early step towards developing an in vivo biomarker sensor, antibody (AB) tagged magnetic nanoparticles (NPs) are used here to demonstrate the in vitro measurement of similar to 5 distinct biomarkers with high specificity and sensitivity. In previous work, aptamers were used to target a given biomarker in vitro and generate magnetic clusters that exhibit a characteristic rotational signature quite different from free NPs. Here the method is expanded to detect a much wider range of biomarkers using polyclonal ABs attached to the surface of the NPs. Commercial ABs exist for a wide range of targets allowing accurate and specific concentration measurements for most significant biomarkers. We show sufficient detection sensitivity, using an in-house spectrometer to measure the rotational signatures of the NPs, to assess physiological concentrations of hormones, cytokines and other signaling molecules. Detection limits for biomarkers drawn mainly from pain and inflammation targets were: 10 pM for mouse Granzyme B (mGZM-B), 40 pM for mouse interferon-gamma (mIFN-gamma), 7 pM for mouse interleukin-6 (mIL-6), 40 pM for rat interleukin-6 (rIL-6), 40 pM for mouse vascular endothelial growth factor (mVEGF) and 250 pM for rat calcitonin gene related peptide (rCGRP). Much lower detection limits are certainly possible using improved spectrometers and nanoparticles.

We are developing magnetic nanoparticle (NP) methods to characterize inflammation and infection in vivo. Peritoneal infection in C57BL/6 mice was used as a biological model. An intraperitoneal NP injection was followed by measurement of magnetic nanoparticle spectroscopy of Brownian rotation (MSB) spectra taken over time. MSB measures the magnetization of NPs in a low frequency alternating magnetic field. Two groups of three mice were studied; each group had two infected mice and one control with no infection. The raw MSB signal was compared with two derived metrics: the NP relaxation time and number of NPs present in the sensitive volume of the receive coil. A four compartment dynamic model was used to relate those physical properties to the relevant biological processes including phagocytic activity and migration. The relaxation time increased over time for all of the mice as the NPs were absorbed. The NP number decreased over time as the NPs were cleared from the sensitive volume of the receive coil. The composite p-values for all three rate constants were significant: raw signal, 0.0002, relaxation, <10-16 and local NP clearance, <10-16. However, not all the individual mice had significant changes: Only half the infected mice had significantly different rate constants for raw signal reduction. All infected mice had significantly smaller relaxation time constants. All but one of the infected mice had significantly lower rate constants for local clearance. Relaxation is affected by both phagocytic activity, edema and temperature changes and it should be possible to better isolate those effects to more completely characterize inflammation using more advanced MSB methods. The MSB NP signal can be used to identify inflammation in vivo because it has the unique ability to monitor phagocytic absorption through relaxation measurements.

Deep vein thrombosis, the development of blood clots in the peripheral veins, is a very serious, life threatening condition that is prevalent in the elderly. To deliver proper treatment that enhances the survival rate, it is very important to detect thrombi early and at the point of care. We explored the ability of magnetic particle spectroscopy (MSB) to detect thrombus via specific binding of aptamer functionalized magnetic nanoparticles with the blood clot. MSB uses the harmonics produced by nanoparticles in an alternating magnetic field to measure the rotational freedom and, therefore, the bound state of the nanoparticles. The nanoparticles’ relaxation time for Brownian rotation increases when bound [A.M. Rauwerdink and J. B. Weaver, Appl. Phys. Lett. 96, 1 (2010)]. The relaxation time can therefore be used to characterize the nanoparticle binding to thrombin in the blood clot. For longer relaxation times, the approach to saturation is more gradual reducing the higher harmonics and the harmonic ratio. The harmonic ratios of nanoparticles conjugated with anti-thrombin aptamers (ATP) decrease significantly over time with blood clot present in the sample medium, compared with nanoparticles without ATP. Moreover, the blood clot removed from the sample medium produced a significant MSB signal, indicating the nanoparticles are immobilized on the clot. Our results show that MSB could be a very useful non-invasive, quick tool to detect blood clots at the point of care so proper treatment can be used to reduce the risks inherent in deep vein thrombosis.

A series of techniques have been developed to use magnetic nanoparticles as biosensors to characterize their local microenvironment. Two approaches have been used to obtain quantitative information: model based approaches and scaling based approaches. We have favored scaling based approaches, because approximations made in models can lead to limitations in the accuracy. Currently all the scaling approaches use harmonic ratios to retrieve physical parameters like temperature, viscosity and relaxation time. In this work, we showed that the phase angle of the signal at a single harmonic frequency is an alternative to the ratio. The phase angle is nanoparticle density-independent, and can be used to improve sensitivity, enabling us to measure smaller biomedical effects. With the phase angle as an example, we showed that scaling methods are general and do not depend on specific approximations. We showed that the same scaling techniques can be used with both the phase angle and harmonic ratio because they both depend on the same combinations of physical parameters. Using the phase angle improves the precision and using the combination of phase angles and harmonic ratio provides the best precision.

The extremely high sensitivity that has been suggested for magnetic particle imaging has its roots in the unique signal produced by the nanoparticles at the frequencies of the harmonics of the drive field. That sensitivity should be translatable to other methods that utilize magnetic nanoparticle probes, specifically towards magnetic nanoparticle spectroscopy that is used to measure molecular biomarker concentrations for an “in vivo ELISA” assay approach. In this paper, we translate the predicted sensitivity of magnetic particle imaging into a projected sensitivity limit for in vivo ELISA. The simplifying assumptions adopted are: 1) the limiting noise in the detection system is equivalent to the minimum detectable mass of nanoparticles; 2) the nanoparticle’s signal arising from Brownian relaxation is completely eliminated by the molecular binding event, which can be accomplished by binding the nanoparticle to something so massive that it can no longer physically rotate and is large enough that Neel relaxation is minimal. Given these assumptions, the equation for the minimum concentration of molecular biomarker we should be able to detect is obtained and the in vivo sensitivity is estimated to be in the attomolar to zeptomolar range. Spectrometer design and nonspecific binding are the technical limitations that need to be overcome to achieve the theoretical limit presented.