Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N-Acetylgalactosamine-Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.

Nonclinical Pharmacokinetics and Absorption, Distribution, Metabolism, and Excretion of Givosiran, the First Approved N-Acetylgalactosamine-Conjugated RNA Interference Therapeutic.

Givosiran is an N-acetylgalactosamine-conjugated RNA interference therapeutic that targets 5'-aminolevulinate synthase 1 mRNA in the liver and is currently marketed for the treatment of acute hepatic porphyria. Herein, nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion properties of givosiran were characterized. Givosiran was completely absorbed after subcutaneous administration with relatively short plasma elimination half-life (t1/2; less than 4 hours). Plasma exposure increased approximately dose proportionally with no accumulation after repeat doses. Plasma protein binding was concentration dependent across all species tested and was around 90% at clinically relevant concentration in human. Givosiran predominantly distributed to the liver by asialoglycoprotein receptor-mediated uptake, and the t1/2 in the liver was significantly longer (∼1 week). Givosiran was metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species with no human unique metabolites. Givosiran metabolized to form one primary active metabolite with the loss of one nucleotide from the 3' end of antisense strand, AS(N-1)3' givosiran, which was equipotent to givosiran. Renal and fecal excretion were minor routes of elimination of givosiran as approximately 10% and 16% of the dose was recovered intact in excreta of rats and monkeys, respectively. Givosiran is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, givosiran has a low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion (ADME) properties of givosiran were characterized. Givosiran shows similar pharmacokinetics and ADME properties across rats and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in adequate exposure of givosiran to the target organ (liver). These studies support the interpretation of toxicology studies, help characterize the disposition of givosiran in humans, and support the clinical use of givosiran for the treatment of acute hepatic porphyria.

Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

In Vitro Drug-Drug Interaction Evaluation of GalNAc Conjugated siRNAs Against CYP450 Enzymes and Transporters.

Small interfering RNAs (siRNAs) represent a new class of medicines that are smaller (∼16,000 Da) than biologic therapeutics (>150,000 Da) but much larger than small molecules (<900 Da). Current regulatory guidance on drug-drug interactions (DDIs) from the European Medicines Agency, Food and Drug Administration, and Pharmaceutical and Medical Devices Agency provides no recommendations for oligonucleotide therapeutics including siRNAs; therefore, small molecule guidance documents have historically been applied. Over ∼10 years, in vitro DDI investigations with siRNAs conjugated to a triantennary N-acetylgalactosamine [(GalNAc)-siRNA] ligand have been conducted during nonclinical drug development to elucidate the potential clinical DDI liability. GalNAc siRNAs were evaluated as substrates, inhibitors, or inducers of major cytochrome P450s (P450s) and as substrates and inhibitors of transporters. Aggregate analysis of these data demonstrates a low potential for DDI against P450s. Zero of five, 10, and seven are inducers, time-dependent inhibitors, or substrates, respectively, and nine of 12 do not inhibit any P450 isoform evaluated. Three GalNAc siRNAs inhibited CYP2C8 at supratherapeutic concentrations, and one mildly inhibited CYP2B6. The lowest K i value of 28 µM is >3000-fold above the therapeutic clinical C max at steady state, and importantly no clinical inhibition was projected. Of four GalNAc siRNAs tested none were substrates for transporters and one caused inhibition of P-glycoprotein, calculated not to be clinically relevant. The pharmacological basis for DDIs, including consideration of the target and/or off-target profiles for GalNAc siRNAs, should be made as part of the overall DDI risk assessment. If modulation of the target protein does not interfere with P450s or transporters, then in vitro or clinical investigations into the DDI potential of the GalNAc siRNAs are not warranted. SIGNIFICANCE STATEMENT: Recommendations for evaluating DDI potential of small molecule drugs are well established; however, guidance for novel modalities, particularly oligonucleotide-based therapeutics are lacking. Given the paucity of published data in this field, in vitro DDI investigations are often conducted. The aggregate analysis of GalNAc-siRNA data reviewed herein demonstrates that, like new biological entities, these oligonucleotide-based therapeutic drugs are unlikely to result in DDIs; therefore, it is recommended that the need for in vitro or clinical investigations similarly be determined on a case-by-case basis. Given the mechanism of siRNA action, special consideration should be made in cases where there may be a pharmacological basis for DDIs.

The Diagnosis of Metastatic Axillary Lymph Nodes of Breast Cancer By Diffusion Weighted Imaging: a meta-analysis and systematic review.

BACKGROUND: The purpose of this meta-analysis was to evaluate the clinical significance of diffusion-weighted imaging in assessing the status of axillary lymph nodes in patients with breast cancer. METHODS: We searched the PubMed, Cochrane, and EMBASE databases, selected studies by inclusion and exclusion criteria, and assessed the quality of selected studies. We explored the source of heterogeneity; calculated sensitivity, specificity, positive and negative likelihood ratios, and pretest probability. A summary receiver operating characteristic curve was performed. Student's t test was used to compare the different mean apparent diffusion coefficient values of different status lymph nodes. RESULTS: In selected 10 studies, a total of 801 patients and 2305 lymph nodes were included following inclusion criteria. All scores of the quality assessment of the included studies were greater than or equal to 10 points. The sensitivity was 0.89 (95 % CI 0.79-0.95), the specificity was 0.83 (95 % CI 0.71-0.91), the positive and negative likelihood ratios were 3.86 (95 % CI 2.75-5.41) and 0.17 (95 % CI 0.09-0.32), the pretest probabilities were 53 and 54 %, the area under the curve were 0.93 (95 % CI 0.90-0.95), respectively. The mean apparent diffusion coefficient value of metastatic lymph nodes was significantly lower than that of nonmetastatic axillary lymph nodes. CONCLUSIONS: Diffusion-weighted imaging is a promising tool to discriminate between metastatic and nonmetastatic axillary lymph nodes. Combined with the mean apparent diffusion coefficient value, it can quantitatively diagnose lymph node metastases. Conducting large-scale, high-quality researches can improve the clinical significance of diffusion-weighted imaging to distinguish metastatic and nonmetastatic axillary lymph nodes in patients with breast cancer and provide the evidence to assess the status of axillary lymph nodes.

Radiological findings of malignant peripheral nerve sheath tumor: reports of six cases and review of literature.

BACKGROUND: Malignant peripheral nerve sheath tumor (MPNST) is a kind of rare neurogenic tumor. If associated with neurofibromatosis type 1, MPNST usually has a higher mortality. The aim of the article is to assess the imaging characteristics of MPNST and compare them with those of benign peripheral nerve sheath tumor (BPNST) to characterize this tumor. METHODS: Clinical and imaging data of six cases with MPNST and 28 cases with BPNST in our institution since 2011 were retrospectively reviewed. Thirty-three patients have available MR imaging data, and two patients of MPNST also accepted CT scan. One patient accepted CT scan only. Location, size, shape, signal or density, boundary, bone destruction, relation to adjacent nerve, contrast-enhanced features as well as some other signs were assessed and compared with statistical software. Student's t test was used for comparison of continuous variables. Fisher's exact test was used for analysis of nominal variable. A P value ≤0.05 was considered to be statistically significant. RESULTS: Differences existed between two groups in tumor size ((7.2 ± 3.3)cm in MPNST vs. (3.8 ± 1.4)cm in BPNST), unclear margin (4/6 in MPNST vs. 1/28 in BPNST), eccentricity to the nerve (1/6 in MPNST vs. 21/28 in BPNST), intratumoral lobulation (4/6 in MPNST vs. 2/28 in BPNST), peritumoral edema (3/6 in MPNST vs. 0 in BPNST), and peripheral enhancement (4/6 in MPNST (three of five MR, one CT) vs. 4/28 in BPNST). Bone destruction was observed in one MPNST. CONCLUSIONS: MR imaging is a valuable, non-invasive modality for the diagnosis of MPNST. Peripheral enhancement with non-cystic appearance or remarkable heterogeneous enhancement may be useful for differential diagnosis. Other imaging features such as large size (over 5 cm in diameter), ill-defined margin, intratumoral lobulation, peritumoral edema, and adjacent bone destruction are also supportive of MPNST.

Phase 1 dose-escalation, pharmacokinetic, and cerebrospinal fluid distribution study of TAK-285, an investigational inhibitor of EGFR and HER2.

INTRODUCTION: This phase 1 study assessed safety, maximum tolerated dose (MTD), pharmacokinetics, cerebrospinal fluid (CSF) distribution, and preliminary clinical activity of the receptor tyrosine kinase inhibitor TAK-285. METHODS: Patients with advanced, histologically confirmed solid tumors and Eastern Cooperative Oncology Group performance status ≤2 received daily oral TAK-285; daily dose was escalated within defined cohorts until MTD and recommended phase 2 dose (RP2D) were determined. Eleven patients were enrolled into an RP2D cohort. Blood samples were collected from all cohorts; CSF was collected at pharmacokinetic steady-state from RP2D patients. Tumor responses were assessed every 8 weeks per Response Evaluation Criteria in Solid Tumors. RESULTS: Fifty-four patients were enrolled (median age 60; range, 35-76 years). The most common diagnoses were cancers of the colon (28 %), breast (17 %), and pancreas (9 %). Escalation cohorts evaluated doses from 50 mg daily to 500 mg twice daily; the MTD/RP2D was 400 mg twice daily. Dose-limiting toxicities included diarrhea, hypokalemia, and fatigue. Drug absorption was fast (median time of maximum concentration was 2-3 h), and mean half-life was 9 h. Steady-state average unbound CSF concentration (geometric mean 1.54 [range, 0.51-4.27] ng/mL; n = 5) at the RP2D was below the 50 % inhibitory concentration (9.3 ng/mL) for inhibition of tyrosine kinase activity in cells expressing recombinant HER2. Best response was stable disease (12 weeks of nonprogression) in 13 patients. CONCLUSIONS: TAK-285 was generally well tolerated at the RP2D. Distribution in human CSF was confirmed, but the free concentration of the drug was below that associated with biologically relevant target inhibition.

Imaging findings of solitary fibrous tumor in the abdomen and pelvis.

PURPOSE: The purpose of this study was to investigate the imaging characteristics of solitary fibrous tumor (SFT) in the abdomen and pelvis. METHODS: Nine cases of SFT confirmed by surgery and pathology were retrospectively analyzed in terms of computed tomography (CT, eight cases) and magnetic resonance imaging (MRI, one case). RESULTS: SFT were located in the retroperitoneum (4/9), abdominal cavity (1/9), pelvis (4/9). Eight cases were single (8/9) and one case (1/9) with three tumors. The average tumor size of 11 lesions was 9.7 cm (4.7-20 cm). Nine tumors were round or ovoid, and two lesions were irregular. The CT value of the plain scans ranged from 33 to 43 Hounsfield units (HU, mean 37.6 HU) in five cases. Arterial-phase CT found solid parts demonstrate avid enhancement (eight cases) and five of them presented with multiple circuitous vessels along the periphery with a CT value of 68-89 HU (mean 76.6 HU). In the venous and delayed phases, enhancement was strengthened progressively. The CT values at venous (eight cases) and delayed phases (five cases) were 108-115 and 112-123 HU respectively, with averages of 109.8 and 114.8 HU. Patch or nodular no-enhanced areas were observed in eight cases during the enhanced phases. One case showed isointensity on T1-weighted images and high signal intensity on T2-weighted images accompanied by linear or curvilinear hypointense lines. Intense enhancements along with linear no-enhancement areas are seen in the arterial and venous phases. CONCLUSION: The possibility of SFT should be considered when a single or multiple masses with sharp border, inhomogeneous density or signal are detected, especially, with inhomogeneous intense enhancement in the arterial phase being maintained in the venous and delayed phases.

CT features of retroperitoneal solitary fibrous tumor: report of three cases and review of the literature.

CT findings in three cases with solitary fibrous tumors (SFTs) confirmed by histopathology and immunohistochemistry were reviewed retrospectively, and compared with pathological results. The three tumors were large, well-defined, and smooth contour masses and SFT consisted of solid components of two different densities. On enhanced CT scans, tumors were strongly enhancing, the multiple vascular shadows were seen within the tumor in the arterial phase. There is progressive enhancement from the arterial to the venous phase, and the tumor capsule can be observed. Histologically, the tumors are composed of spindle cells within a background of collagen stroma, and showed a wide range of growth patterns, alternating hypercellular (tumor cell-rich) and hypocellular (collagen-rich) areas. The diagnosis is confirmed by characteristic positive immunohistochemical staining for CD34.

The multislice CT findings of renal carcinoma associated with XP11.2 translocation/TFE gene fusion and collecting duct carcinoma.

BACKGROUND: Renal cell carcinoma associated with Xp11.2 translocation and TFE gene fusion (Xp11.2/TFE RCC), and collecting duct carcinoma (CDC) are uncommon subtypes of renal cell carcinomas. PURPOSE: To investigate the multislice CT (MSCT) characteristics of these two tumor types. MATERIAL AND METHODS: Nine patients with Xp11.2/TFE RCC and 10 patients with CDC were studied retrospectively. MSCT was undertaken to investigate differences in tumor characteristics and enhancement patterns. RESULTS: All patients had single tumors centered in the renal medulla. Two patients with each tumor type had lymph node involvement and there was a single case of hepatic metastasis (Xp11.2/TFE RCC). The mean tumor diameter of Xp11.2/TFE RCC tumors was significantly larger than for CDC tumors. Two patients with Xp11.2/TFE RCC had cystic components as did eight patients with CDC (P < 0.05). Calcifications were present in six patients, each with CDC. Clear tumor boundaries were visible in two patients with CDC and in nine with Xp11.2/TFE RCC (P < 0.05). The density of Xp11.2/TFE RCC tumors was greater than that of CDC tumors, normal renal cortex, or medulla on unenhanced CT. Enhancement was higher with Xp11.2/TFE RCC than with CDC tumors during all phases. Xp11.2/TFE RCC enhancement was higher than in the renal medulla during cortical and medullary phase but lower than in normal renal medulla during the delayed phase. CDC tumor enhancement was lower than that for normal renal medulla during all enhanced phases. CONCLUSION: Both tumor types originated from the renal medulla. Distinguishing features included density on unenhanced CT, enhancement patterns, and capsule signs. Identifying these differences may aid diagnosis.

Primary choriocarcinoma of the colon: a case report and review of the literature.

Choriocarcinoma usually arises in the uterus and gonads. Primary choriocarcinoma (PCC) in an extragenital organ is rare. When it occurs in the gastrointestinal tract, the stomach is the most common site. Only 12 cases of PCC of the colon have been reported in the world literature. Most cases were associated with adenocarcinoma. We report the case of a 36-year-old man with PCC of the colon and review the clinical characteristics of previously documented cases.

[Study of resting state functional connectivity of the red nucleus and substantia nigra were in normal adult].

OBJECTIVE: To detect the functional networks of the red nucleus and substantia nigra during the resting state in normal subjects with functional magnetic resonance imaging (fMRI). METHODS: Sixteen normal subjects were performed resting state fMRI scanning and susceptibility weighted imaging. The function connectivity networks base on seed regions of the red nucleus and substantia nigra were extracted from low frequency fluctuation signal in fMRI data by using a temporal correlation method. Individual functional maps were entered two-tailed one-sample t test to determine brain regions with significant positive correlation to the seeds. The statistic threshold was set at P < 0.001, cluster size>42 (336 mm(3)), cluster connectivity criterion 5 min with Alphasim correction. RESULTS: Brain regions involved in the functional connectivity network of the red nucleus include: dorsal anterior cingutate, supramarginal gyrus, the ventrolateral and the ventromedial nucleus of the thalamus, globus pallidus, dorsal thalamus, hippocampus, substantia nigra, red nucleus, pons, dentate nucleus, vermis; Brain regions involved in the functional connectivity network of the substantia nigra include: anterior cingutate, supramarginal gyrus, globus pallidus, dorsal thalamus, hippocampus, lobus insularis, substantia nigra, red nucleus, pons, dentate nucleus. The distribution of the networks of the red nucleus and substantia nigra presented symmetrical. Although the functional networks of the red nucleus and substantia nigra over lapped largely with each other, the rubral network was slightly different with the nigral network, witch showed strong correlations with more wide-spread striatum and thalamus areas. CONCLUSION: The functional networks of the red nucleus and substantia nigra reflected strong interplay within the extrapyramidal subcortical system, as well as correlations between some limited cerebral cortices; Functional magnetic resonance imaging is a potential powerful tool to explore the extrapyramidal system.

[Preliminary study of breast cancer magnetic resonance targeting diagnosis basing on magnetic nanoparticles in vitro].

OBJECTIVE: To develop a magnetic nanoparticles based magnetic resonance (MR) probe targeting CD40 mutant in the imaging of breast cancer cells in vitro. METHODS: For preparing an immunologically competent probe, monoclonal antibody was conjugated with ultrasmall superparamagnetic iron oxide (USPIO) particles basing on chemical cross-linking method.Its bioactivity was analyzed with flow cytometry, confocal microscopy and Prussian blue staining. The probe's cell MR imaging in vitro was conducted on breast cancer cells (M231) high expressing CD40 mutant. The signal data from different groups were collected and analyzed with one-way variance and least significant difference-t test. RESULTS: The molecular probe carrying nanoparticles and CD40 mutant antibody was constructed and separated successfully. The probe had similar magnetic property compared with original USPIO particles.It could recognize CD40 mutant on breast cancer cells (M231) with high specificity. MR cell imaging in vitro shows that T2 and T2(*) obviously shortened after probe binding with M231 cells and T2 weighted imaging become darker than control groups, the time of T2 is 5H6-USPIO (51.66 ± 5.31) , 5C11-USPIO (92.89 ± 4.72), USPIO (64.56 ± 3.85) ms. The T2 and T2(*) relaxation time of experiment group was shorter than control groups with statistical significance (P < 0.01) . CONCLUSION: MR molecular probe targeting CD40 mutant may bind with breast cancer cells (M231) to provide further in vivo animal MR imaging. And CD40 mutant is expected to provide a new target for MR molecular imaging of breast cancer.

Quantification of oxalate by novel LC-MS/MS: assay development, validation and application in lumasiran clinical trials.

Background: Measurement of plasma oxalate (POx) is challenging, but critical, for management of patients with primary hyperoxaluria type 1. A novel LC-MS/MS assay was developed, validated and used to quantify POx in patients with primary hyperoxaluria type 1. Methods: Samples (100 µl of plasma in K2EDTA) were spiked with internal standard (13C2-labeled oxalic acid), acidified and cleaned by protein precipitation before analysis using anion HPLC-ESI-MS/MS. The assay was validated with a quantitation range of 0.500-50.0 µg/ml (5.55-555 µmol/l). All parameters successfully met acceptance criteria, including 15% (20% at lower limit of quantification) for accuracy and precision. Conclusion: This assay has advantages over previously published POx quantitation methods, was validated in accordance with regulatory guidelines and accurately determined POx levels in humans.

A novel assay to measure plasma oxalate was developed and validated successfully in accordance with regulatory guidelines. The required sample volume was only 100 µl of plasma, which is especially favorable in the pediatric population, and there is no need to acidify blood at the collection site before processing. The assay accurately determines plasma oxalate levels, which were used as a measure of efficacy in the lumasiran clinical trials.

OSWG Recommended Approaches to the Nonclinical Pharmacokinetic (ADME) Characterization of Therapeutic Oligonucleotides.

This white paper summarizes the recommendations of the absorption, distribution, metabolism, and excretion (ADME) Subcommittee of the Oligonucleotide Safety Working Group for the characterization of absorption, distribution, metabolism, and excretion of oligonucleotide (ON) therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across ON classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional ONs of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.

Cinnamaldehyde Alleviates Bone Loss by Targeting Oxidative Stress and Mitochondrial Damage via the Nrf2/HO-1 Pathway in BMSCs and Ovariectomized Mice.

Osteoporosis (OP) is typically brought on by disruption of bone homeostasis. Excessive oxidative stress and mitochondrial dysfunction are believed to be the primary mechanisms underlying this disorder. Therefore, in order to restore bone homeostasis effectively, targeted treatment of oxidative stress and mitochondrial dysfunction is necessary. Cinnamaldehyde (CIN), a small molecule that acts as an agonist for the nuclear factor erythroid 2-related factor (Nrf2), has been found to possess antiapoptotic, anti-inflammatory, and antioxidant properties. We found that CIN, while rescuing apoptosis, can also reduce the accumulation of reactive oxygen species (ROS) to improve mitochondrial dysfunction and thus restore the osteogenic differentiation potential of BMSCs disrupted by hydrogen peroxide (H2O2) exposure. The role of CIN was preliminarily considered to be a consequence of Nrf2/HO-1 axis activation. The ovariectomized mice model further demonstrated that CIN treatment ameliorated oxidative stress in vivo, partially reversing OVX-induced bone loss. This improvement was seen in the trabecular microarchitecture and bone biochemical indices. However, when ML385 was concurrently injected with CIN, the positive effects of CIN were largely blocked. In conclusion, this study sheds light on the intrinsic mechanisms by which CIN regulates BMSCs and highlights the potential therapeutic applications of these findings in the treatment of osteoporosis.

The effects of febuxostat on the pharmacokinetic parameters of rosiglitazone, a CYP2C8 substrate.

AIMS: To determine the effect of febuxostat on cytochrome P450 2C8 (CYP2C8) activity using rosiglitazone as a CYP2C8 substrate. METHODS: Healthy subjects received febuxostat 120 mg daily (regimen A) or matching placebo (regimen B) for 9 days along with a single oral dose of rosiglitazone 4 mg on day 5 in a double-blind, randomized, cross-over fashion (≥7 day washout between periods). Plasma samples for analysis of the impact of febuxostat on the pharmacokinetics (PK) of rosiglitazone and its metabolite, N-desmethylrosiglitazone, were collected for 120 h after co-administration. RESULTS: Of the 39 subjects enrolled, 36 completed the study and were included in the PK analyses. Rosiglitazone PK parameters were comparable between regimens A and B. Median time to maximal plasma concentration, mean maximal plasma concentration (C(max)), area under the concentration-time curve (AUC) from time zero to the last quantifiable concentration (AUC(0-tlqc)), AUC from time zero to infinity (AUC(0-∞)), and terminal elimination half-life for regimen A were 0.50 h, 308.6 ng ml⁻¹, 1594.9 ng h ml⁻¹, 1616.0 ng h ml⁻¹ and 4.1 h, respectively, and for regimen B they were 0.50 h, 327.6 ng ml⁻¹, 1564.5 ng h ml⁻¹, 1584.2 ng h ml⁻¹ and 4.0 h, respectively. Point estimates for the ratio of regimen A to regimen B (90% confidence intervals) for rosiglitazone C(max) , AUC(0-tlqc) and AUC(0-∞) central values were 0.94 (0.89-1.00), 1.02 (1.00-1.04) and 1.02 (1.00-1.04), respectively. CONCLUSIONS: Co-administration of febuxostat had no effect on rosiglitazone or N-desmethylrosiglitazone PK parameters, suggesting that febuxostat can be given safely with drugs metabolized through CYP2C8.

The effects of xanthine oxidase inhibition by febuxostat on the pharmacokinetics of theophylline.

OBJECTIVE: Febuxostat, a non-purine selective xanthine oxidase (XO) inhibitor, may affect the metabolism of theophylline as XO hydroxylates 1-methylxanthine to 1-methyluric acid. The objective of this study was to examine the effects of febuxostat on the pharmacokinetics of theophylline and its metabolites. METHODS: 24 healthy subjects received febuxostat 80 mg (Regimen A) or matching placebo (Regimen B) daily for 7 days along with a single oral dose of theophylline 400 mg on Day 5 in a double-blind, randomized, cross-over fashion (≥ 7 day washout between periods) followed by collection of plasma and urine samples for 72 h. RESULTS: For Regimens A and B, mean theophylline Cmax values were 4.4 and 4.1 µg/ml, respectively, and mean theophylline AUC0-tlqc was 122.3 and 115.2 µg x h/ml, respectively. The ratios of theophylline Cmax and AUC0-tlqc central values following coadministration with febuxostat or placebo were 1.03 (90% confidence intervals (CIs), 0.917 - 1.149) and 1.04 (90% CI, 0.927 - 1.156). Both 90% CIs fell within the no-effect range of 0.8 and 1.25. Mean excreted amounts in urine for 1-methylxanthine levels were higher in Regimen A vs. B (40.1 vs. 0.1 mg), while 1-methyluric acid levels were lower (3.1 vs. 56.2 mg). Mean excreted amounts of theophylline and other metabolites were comparable between Regimen A and B. CONCLUSIONS: No dose adjustment for theophylline is necessary when coadministered with febuxostat 80 mg, as coadministration does not affect the plasma pharmacokinetics of theophylline and neither 1-methylxanthine nor 1-methyluric have any pharmacological effect.

[Multi-slice computed tomography diagnosis of collecting duct carcinoma].

OBJECTIVE: To explore the diagnostic features of collecting duct carcinoma (CDC). METHODS: A total of 7 CDC patients were retrospectively examined by multi-slice computed tomography (MSCT). The relevant diagnostic parameters were assessed. RESULTS: All lesions were located in renal medulla. Among them, infiltrations extended to renal calyx (n = 3) and cortex (n = 5). There were indistinct boundaries (capsule sign) on enhanced phase (n = 6) and pre-capsule (n = 1). On non-enhanced CT, CDC attenuation was greater than normal renal cortex or medulla (43.8 ± 5.3 vs 37.6 ± 5.1 or 32.6 ± 4.1, P < 0.05). The degree of enhancement was less than normal renal cortex and medulla during all enhanced phases (P < 0.05 or 0.01). Excellent consistency existed between CT appearances of CDC and pathological characteristics. CONCLUSION: Dynamic contrast enhanced-CT can show distinct imaging features of CDC correlated with pathological characteristics so as to allow a better differential diagnosis.