Predicting In Vivo Target Occupancy (TO) Profiles via Physiologically Based Pharmacokinetic-TO Modeling of Warfarin Pharmacokinetics in Blood: Importance of Low Dose Data and Prediction of Stereoselective Target Interactions.

Warfarin is well recognized for its high-affinity and capacity-limited binding to the pharmacological target and undergoes target-mediated drug disposition. Here, we developed a physiologically based pharmacokinetic (PBPK) model that incorporated saturable target binding and other reported hepatic disposition components of warfarin. The PBPK model parameters were optimized by fitting to the reported blood pharmacokinetic (PK) profiles of warfarin with no stereoisomeric separation after oral dosing of racemic warfarin (0.1, 2, 5, or 10 mg) using the Cluster Gauss-Newton method (CGNM). The CGNM-based analysis yielded multiple "accepted" sets for six optimized parameters, which were then used to simulate the warfarin blood PK and in vivo target occupancy (TO) profiles. When further analyses examined the impact of dose selection on uncertainty in parameter estimation by the PBPK modeling, the PK data from 0.1 mg dose (well below target saturation) was important in practically identifying the target binding-related parameters in vivo. When stereoselective differences were incorporated for both hepatic disposition and target interactions, our PBPK modeling predicted that R-warfarin (of slower clearance and lower target affinity than S-warfarin) contributes to TO prolongation after oral dosing of racemic warfarin. Our results extend the validity of the approach by which the PBPK-TO modeling of blood PK profiles can yield TO prediction in vivo (applicable to the drugs with targets of high affinity and abundance and limited distribution volume via nontarget interactions). Our findings support that model-informed dose selection and PBPK-TO modeling may aid in TO and efficacy assessment in preclinical and clinical phase 1 studies. SIGNIFICANCE STATEMENT: The current physiologically based pharmacokinetic modeling incorporated the reported hepatic disposition components and target binding of warfarin and analyzed the blood pharmacokinetic (PK) profiles from varying warfarin doses, practically identifying target binding-related parameters in vivo. By implementing the stereoselective differences between R- and S-warfarin, our analysis predicted the role of R-warfarin in prolonging overall target occupancy. Our results extend the validity of analyzing blood PK profiles to predict target occupancy in vivo, which may guide efficacy assessment.

Experimental and Modeling Evidence Supporting the Trans-Inhibition Mechanism for Preincubation Time-Dependent, Long-Lasting Inhibition of Organic Anion Transporting Polypeptide 1B1 by Cyclosporine A.

Cyclosporine A (CsA) and rifampin are potent inhibitors of organic anion transporting polypeptide (OATP) 1B1 and are widely used to assess the risk for drug-drug interactions. CsA displays preincubation time-dependent, long-lasting inhibition of OATP1B1 in vitro and in rats in vivo, and a proposed mechanism is the trans-inhibition by which CsA inhibits OATP1B1 from the inside of cells. The current study aimed to experimentally validate the proposed mechanism using human embryonic kidney 293 cells stably expressing OATP1B1. The uptake of CsA reached a plateau following an approximate 60-minute incubation, with the cell-to-buffer concentration ratio of 3930, reflective of the high-affinity, high-capacity intracellular binding of CsA. The time course of CsA uptake was analyzed to estimate the kinetic parameters for permeability clearance and intracellular binding. When the OATP1B1-mediated uptake of [3H]estradiol-17ß-glucuronide was measured following preincubation with CsA for 5 to 120 minutes, apparent Ki values became lower with longer preincubation. Our kinetic modeling incorporated the two reversible inhibition constants [Ki,trans and Ki,cis for the inhibition from inside (trans-inhibition) and outside (cis-inhibition) of cells, respectively] and estimated Ki,trans value of CsA was smaller by 48-fold than the estimated Ki,cis value. Rifampin also displayed preincubation time-dependent inhibition of OATP1B1, albeit the extent of enhancement was only twofold. The current study provides experimental evidence for the preincubation time-dependent shift of apparent Ki values and a mechanistic basis for physiologically based pharmacokinetic modeling that incorporates permeability clearance, extensive intracellular binding, and asymmetry of Ki values between the inside and outside of cells. SIGNIFICANCE STATEMENT: In vitro data and kinetic modeling support that preincubation time-dependent, long-lasting inhibition of OATP1B1 by CsA can be explained by the extensive intracellular binding and reversible OATP1B1 inhibition intracellularly (trans-inhibition) as well as extracellularly (cis-inhibition). For inhibitors to display time-dependency, the following factors were found important: time to reach a steady-state cellular concentration, trans-inhibition potency relative to cis-inhibition, and the degree of cellular inhibitor accumulation. This study would aid in the accurate prediction of drug-drug interactions mediated by OATP1B1 inhibition.

Successful Treatment of Thrombotic Thrombocytopenic Purpura in a Patient with Mixed Connective Tissue Disease.

BACKGROUND: Although the survival rate of thrombocytopenic purpura (TTP) has increased significantly due to the introduction of therapeutic plasma exchange (TPE). TTP in patients with mixed connective tissue disease (MCTD) has a very high mortality rate and a very small number of reported cases. In TTP, daily TPE is administered until a treatment response is achieved; however, in practice, TPE is often not performed for such long durations. METHODS: We report a case of TTP with MCTD in a female patient. She had developed thrombocytopenia and hemolytic anemia 9 months after delivery. She had status epilepticus and lapsed into a coma. RESULTS: The patient was successfully treated with extended sessions of TPE with corticosteroids and rituximab. CONCLUSIONS: Although the TTP regimen has not yet been established and remains controversial, this report demonstrates the importance of continuing daily TPE until achieving a treatment response.

Developing a Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Seven Respiratory Viruses including SARS-CoV-2.

Background and Objectives: The coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a pandemic even in 2022. As the initial symptoms of COVID-19 overlap with those of infections from other respiratory viruses, an accurate and rapid diagnosis of COVID-19 is essential for administering appropriate treatment to patients. Currently, the most widely used method for detecting respiratory viruses is based on real-time polymerase chain reaction (PCR) and includes reverse-transcription real-time quantitative PCR (RT-qPCR). However, RT-qPCR assays require sophisticated facilities and are time-consuming. This study aimed to develop a real-time quantitative loop-mediated isothermal amplification (RT-qLAMP) assay and compare its analytical performance with RT-qPCR. Materials and Methods: A total of 315 nasopharyngeal swabs from patients with symptoms of respiratory infections were included in this study. A primary screening of the specimens was performed using RT-qPCR. RNA/DNA from standard strains for respiratory viruses and heat-inactivated preparations of standard strains for SARS-CoV-2 were used to evaluate the accuracy and target specificity of the RT-qLAMP assay. Results: We successfully developed an RT-qLAMP assay for seven respiratory viruses: respiratory syncytial virus (RSV) A, RSV B, adenovirus, influenza (Flu) A (H1N1 and H3N2), Flu B, and SARS-CoV-2. RT-qLAMP was performed in a final reaction volume of 9.6 µL. No cross-reactivity was observed. Compared with the RT-PCR results, the sensitivity and specificity of the RT-qLAMP assay were 95.1% and 100%, respectively. The agreement between the two methods was 97.1%. The median amplification time to RT-qLAMP positivity was 22:34 min (range: 6:80-47:98 min). Conclusions: The RT-qLAMP assay requires a small number of reagents and samples and is performed with an isothermal reaction. This study established a fast, simple, and sensitive test that can be applied to point-of-care testing devices to facilitate the detection of respiratory viruses, including SARS-CoV-2.

Post-translational regulation of the major drug transporters in the families of organic anion transporters and organic anion-transporting polypeptides.

The organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs) belong to the solute carrier (SLC) transporter superfamily and play important roles in handling various endogenous and exogenous compounds of anionic charge. The OATs and OATPs are often implicated in drug therapy by impacting the pharmacokinetics of clinically important drugs and, thereby, drug exposure in the target organs or cells. Various mechanisms (e.g. genetic, environmental, and disease-related factors, drug-drug interactions, and food-drug interactions) can lead to variations in the expression and activity of the anion drug-transporting proteins of OATs and OATPs, possibly impacting the therapeutic outcomes. Previous investigations mainly focused on the regulation at the transcriptional level and drug-drug interactions as competing substrates or inhibitors. Recently, evidence has accumulated that cellular trafficking, post-translational modification, and degradation mechanisms serve as another important layer for the mechanisms underlying the variations in the OATs and OATPs. This review will provide a brief overview of the major OATs and OATPs implicated in drug therapy and summarize recent progress in our understanding of the post-translational modifications, in particular ubiquitination and degradation pathways of the individual OATs and OATPs implicated in drug therapy.

Revisiting Nonlinear Bosentan Pharmacokinetics by Physiologically Based Pharmacokinetic Modeling: Target Binding, Albeit Not a Major Contributor to Nonlinearity, Can Offer Prediction of Target Occupancy.

Bosentan is a high-affinity antagonist of endothelin receptors and one of the earliest examples for target-mediated drug disposition [a type of nonlinear pharmacokinetics (PKs) caused by saturable target binding]. The previous physiologically based PK (PBPK) modeling indicated that the nonlinear PKs of bosentan was explainable by considering saturable hepatic uptake. However, it remained unexamined to what extent the saturable target binding contributes to the nonlinear PKs of bosentan. Here, we developed a PBPK model incorporating saturable target binding and hepatic uptake and analyzed the clinical bosentan PK data using the cluster Gauss-Newton method (CGNM). The PBPK model without target binding fell short in capturing the bosentan concentrations below 100 nM, based on the PK profiles and the goodness-of-fit plot. Both global and local identifiability analyses (using the CGNM and Fisher information matrix, respectively) informed that the target binding parameters were identifiable only if the observations from the lowest dose (10 mg) were included. By analyzing blood PK profiles alone, the PBPK model with target binding yielded practically identifiable target binding parameters and predicted the maximum target occupancies of 0.6-0.8 at clinical bosentan doses. Our results indicate that target binding, albeit not a major contributor to the nonlinear bosentan PKs, may offer a prediction of target occupancy from blood PK profiles alone and potential guidance on achieving optimal efficacy outcomes, under the condition when the high-affinity drug target is responsible for the efficacy of interest and when the dose ranges cover varying degrees of target binding. SIGNIFICANCE STATEMENT: By incorporating saturable target binding, our physiologically based pharmacokinetic (PBPK) model predicted in vivo target occupancy of bosentan based only on the blood concentration-time profiles obtained from a wide range of doses. Our analysis highlights the potential utility of PBPK models that incorporate target binding in predicting target occupancy in vivo.

Sustained Delivery of Carfilzomib by Tannic Acid-Based Nanocapsules Helps Develop Antitumor Immunity.

A group of chemotherapeutic drugs has gained increasing interest in cancer immunotherapy due to the potential to induce immunogenic cell death (ICD). A critical challenge in using the ICD inducers in cancer immunotherapy is the immunotoxicity accompanying their antiproliferative effects. To alleviate this, a nanocapsule formulation of carfilzomib (CFZ), an ICD-inducing proteasome inhibitor, was developed using interfacial supramolecular assembly of tannic acid (TA) and iron, supplemented with albumin coating. The albumin-coated CFZ nanocapsules (CFZ-pTA-alb) attenuated CFZ release, reducing toxicity to immune cells. Moreover, due to the adhesive nature of the TA assembly, CFZ-pTA-alb served as a reservoir of damage-associated molecular patterns released from dying tumor cells to activate dendritic cells. Upon intratumoral administration, CFZ-pTA-alb prolonged tumor retention of CFZ and showed consistently greater antitumor effects than cyclodextrin-solubilized CFZ (CFZ-CD) in B16F10 and CT26 tumor models. Unlike CFZ-CD, the locally injected CFZ-pTA-alb protected or enhanced CD8+ T cell population in tumors, helped develop splenocytes with tumor-specific interferon-γ response, and delayed tumor development on the contralateral side in immunocompetent mice (but not in athymic nude mice), supporting that CFZ-pTA-alb contributed to activating antitumor immunity. This study demonstrates that sustained delivery of ICD inducers by TA-based nanocapsules is an effective way of translating local ICD induction to systemic antitumor immunity.

Suppression of Canine ATP Binding Cassette ABCB1 in Madin-Darby Canine Kidney Type II Cells Unmasks Human ABCG2-Mediated Efflux of Olaparib.

Endogenous canine ATP binding cassette B1 (cABCB1) is expressed abundantly in Madin-Darby canine kidney type II (MDCKII) cells, and its presence often complicates phenotyping of the transport process. Errors in estimating the corrected efflux ratio (cER), as a result of the variable expression of cABCB1, were examined for the dual substrates of ABCB1 and ABCG2 in MDCKII cells expressing human ABCG2 (hABCG2). cABCB1 mRNA and protein expression was 60% and 55% lower, respectively, in MDCKII cells expressing hABCG2 compared with the wild type, suggesting that the expression of endogenous cABCB1 became variable after the expression of hABCG2. To minimize the contribution of endogenous efflux, cABCB1 was suppressed kinetically (using verapamil as a selective inhibitor) or biochemically (transfecting short-hairpin RNA against cABCB1). Under these suppression conditions, cER values for irinotecan and topotecan (dual substrates of ABCB1 and ABCG2) were elevated by more than 4-fold and 2-fold, respectively, compared with cER values without the suppression. The cER of olaparib was similarly increased to 3- and 5-fold in MDCKII cells under the kinetic and biochemical suppression of cABCB1, respectively, suggesting that hABCG2-mediated efflux cannot be ruled out for olaparib. Since the substrate selectivity for ABCB1 and ABCG2 overlapped considerably, the possibility of an inaccurate estimation of cER must be considered for dual substrates in the case of the variable expression of cABCB1 in MDCKII cells.

A Comparative In Vivo Study of Albumin-Coated Paclitaxel Nanocrystals and Abraxane.

Nanoparticulate drug carriers exploit the enhanced permeability of tumor vasculature to achieve selective delivery of chemotherapeutic drugs. For this purpose, nanoparticles (NPs) need to circulate with a long half-life, enter tumors via the permeable vasculature and stay in tumors via favorable interactions with tumor cells. To fulfill these requirements, albumin-coated nanocrystal formulation of paclitaxel (PTX), Cim-F-alb, featuring high drug loading content, physical stability in serum, and surface-bound albumin in its native conformation is prepared. The pharmacokinetic and biodistribution (PK/BD) profiles of Cim-F-alb in a mouse model of B16F10 melanoma show that Cim-F-alb exhibits a longer plasma half-life and a greater PTX deposition in tumors than Abraxane by ≈1.5 and ≈4.6 fold, respectively. Biolayer interferometry analysis indicates that Cim-F-alb has less interaction with serum proteins than nanocrystals lacking albumin coating, indicating the protective effect of the surface-bound albumin against opsonization in the initial deposition phase. With the advantageous PK/BD profiles, Cim-F-alb shows greater and longer-lasting anticancer efficacy than Abraxane at the equivalent dose. This study demonstrates the significance of controlling circulation stability and surface property of NPs in efficient drug delivery to tumors and enhanced anticancer efficacy.

Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins.

Current nanoparticle (NP) drug carriers mostly depend on the enhanced permeability and retention (EPR) effect for selective drug delivery to solid tumors. However, in the absence of a persistent EPR effect, the peritumoral endothelium can function as an access barrier to tumors and negatively affect the effectiveness of NPs. In recognition of the peritumoral endothelium as a potential barrier in drug delivery to tumors, poly(lactic-co-glycolic acid) (PLGA) NPs are modified with a quinic acid (QA) derivative, synthetic mimic of selectin ligands. QA-decorated NPs (QA-NP) interact with human umbilical vein endothelial cells expressing E-/P-selectins and induce transient increase in endothelial permeability to translocate across the layer. QA-NP reach selectin-upregulated tumors, achieving greater tumor accumulation and paclitaxel (PTX) delivery than polyethylene glycol-decorated NPs (PEG-NP). PTX-loaded QA-NP show greater anticancer efficacy than Taxol or PTX-loaded PEG-NP at the equivalent PTX dose in different animal models and dosing regimens. Repeated dosing of PTX-loaded QA-NP for two weeks results in complete tumor remission in 40-60% of MDA-MB-231 tumor-bearing mice, while those receiving control treatments succumb to death. QA-NP can exploit the interaction with selectin-expressing peritumoral endothelium and deliver anticancer drugs to tumors to a greater extent than the level currently possible with the EPR effect.

Physiologically Based Pharmacokinetic Modeling of Bosentan Identifies the Saturable Hepatic Uptake As a Major Contributor to Its Nonlinear Pharmacokinetics.

Bosentan is a substrate of hepatic uptake transporter organic anion-transporting polypeptides (OATPs), and undergoes extensive hepatic metabolism by cytochrome P450 (P450), namely, CYP3A4 and CYP2C9. Several clinical investigations have reported a nonlinear relationship between bosentan doses and its systemic exposure, which likely involves the saturation of OATP-mediated uptake, P450-mediated metabolism, or both in the liver. Yet, the underlying causes for the nonlinear bosentan pharmacokinetics are not fully delineated. To address this, we performed physiologically based pharmacokinetic (PBPK) modeling analyses for bosentan after its intravenous administration at different doses. As a bottom-up approach, PBPK modeling analyses were performed using in vitro kinetic parameters, other relevant parameters, and scaling factors. As top-down approaches, three different types of PBPK models that incorporate the saturation of hepatic uptake, metabolism, or both were compared. The prediction from the bottom-up approach (models 1 and 2) yielded blood bosentan concentration-time profiles and their systemic clearance values that were not in good agreement with the clinically observed data. From top-down approaches (models 3, 4, 5-1, and 5-2), the prediction accuracy was best only with the incorporation of the saturable hepatic uptake for bosentan. Taken together, the PBPK models for bosentan were successfully established, and the comparison of different PBPK models identified the saturation of the hepatic uptake process as a major contributing factor for the nonlinear pharmacokinetics of bosentan.

Enhancing Docetaxel Delivery to Multidrug-Resistant Cancer Cells with Albumin-Coated Nanocrystals.

Intravenous delivery of poorly water-soluble anticancer drugs such as docetaxel (DTX) is challenging due to the low bioavailability and the toxicity related to solubilizing excipients. Colloidal nanoparticles are used as alternative carriers, but low drug loading capacity and circulation instability limit their clinical translation. To address these challenges, DTX nanocrystals (NCs) were prepared using Pluronic F127 as an intermediate stabilizer and albumin as a functional surface modifier, which were previously found to be effective in producing small and stable NCs. We hypothesize that the albumin-coated DTX NCs (DTX-F-alb) will remain stable in serum-containing medium so as to effectively leverage the enhanced permeability and retention effect. In addition, the surface-bound albumin, in its native form, may contribute to cellular transport of NCs through interactions with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC). DTX-F-alb NCs showed sheet-like structure with an average length, width, and thickness of 284 ± 96, 173 ± 56, and 40 ± 8 nm and remained stable in 50% serum solution at a concentration greater than 10 µg/mL. Cytotoxicity and cellular uptake of DTX-F-alb and unformulated (free) DTX were compared on three cell lines with different levels of SPARC expression and DTX sensitivity. While the uptake of free DTX was highly dependent on DTX sensitivity, DTX-F-alb treatment resulted in relatively consistent cellular levels of DTX. Free DTX was more efficient in entering drug-sensitive B16F10 and SKOV-3 cells than DTX-F-alb, with consistent cytotoxic effects. In contrast, multidrug-resistant NCI/ADR-RES cells took up DTX-F-alb more than free DTX with time and responded better to the former. This difference was reduced by SPARC knockdown. The high SPARC expression level of NCI/ADR-RES cells, the known affinity of albumin for SPARC, and the opposing effect of SPARC knockdown support that DTX-F-alb have exploited the surface-bound albumin-SPARC interaction in entering NCI/ADR-RES cells. Albumin-coated NC system is a promising formulation for the delivery of hydrophobic anticancer drugs to multidrug-resistant tumors.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test.

BACKGROUND: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). METHODS: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0-1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). RESULTS: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). CONCLUSIONS: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

Limitations of Preoperative PFA-200 as a Predictor of Postoperative Blood Loss in Total Knee Arthroplasty: According to Use of Tranexamic Acid.

BACKGROUND: Recently, anti-fibrinolytic agents (tranexamic acid) have been used to reduce blood loss or and the need for transfusion. We evaluated the clinical utility of PFA-200 as a predictor of postoperative blood loss in a clinical setting, where hemostasis can be affected by variables such as existing comorbidities and medication use. METHODS: We analyzed retrospectively 243 TKA patients having PFA-200 results between March 2014 and May 2016. We collected the medication history of anti-thrombotic agents, closure time measured by PFA-200, postoperative drainage volume, and perioperative change of hemoglobin values. Parameters associated with postoperative blood loss were compared to the prolongation of closure time and medication use. The influence of variables on postoperative blood loss was analyzed. RESULTS: The closure times of PFA-200 were prolonged in 68 patients (295.8 ± 10.7) and were not prolonged in 175 patients (125.4 ± 35.8). The prolonged closure time did not affect any parameters associated with postoperative blood loss. A history of preoperative anti-platelet agents used was a factor that statistically significantly increased the amount of postoperative drainage and reduction of perioperative hemoglobin values. In addition, postoperative tranexamic acid treatment was the factor that decreased the postoperative hemoglobin levels (OR = 0.297 with p = 0.010) most. CONCLUSIONS: The effectiveness of preoperative PFA-200 in assessments of postoperative bleeding risk is reduced with postoperative use of tranexamic acid.

Modified Complete Blood Count Indices as Predicting Markers of Preeclampsia from Gestational Hypertension: Neutrophil to Lymphocyte Ratio, Platelet to Lymphocyte Ratio, and Platelet to Neutrophil Ratio.

BACKGROUND: To evaluate modified complete blood count (CBC) indices as a predicting marker of preeclampsia (PE) from gestational hypertension (GH), we analyzed the neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and platelet to neutrophil ratio (PNR). PNR was a newly designed index in this study based on results of PE patients having a tendency toward higher neutrophil count and lower platelet count compared to normal pregnant women in previous studies. METHODS: We recruited 86 normal pregnant women, 33 patients with GH and 68 patients with PE. Subjects with any history of membrane rupture, infection, or multiple pregnancies were excluded. PNR, NLR, and PLR values including other CBC indices were statistically analyzed. RESULTS: NLR value of PE patients was significantly higher than GH patients (p = 0.011). PNR value was the most statistically significant index separating patients with PE and GH (p < 0.001). PLR value was lower in patients with PE compared to GH; however, statistical significance was low. CONCLUSIONS: NLR as well as PNR is a useful index to help predicting progression from GH to PE. Further studies are required to evaluate the full extent of utility of PNR as a predictive index in PE patients.

Pure Red Cell Aplasia Associated with Monoclonal Gammopathy of Undetermined Significance and Literature Review.

BACKGROUND: Pure red cell aplasia (PRCA) is an uncommon disease which involves an almost complete absence of the erythroid lineage in bone marrow (BM) and causes severe anemia. Cases due to monoclonal gammopathy occurring in plasma cell disorder have been infrequently reported. Here we report a case of PRCA associated plasma cell disorder, especially monoclonal gammopathy of undetermined significance (MGUS). METHODS: A 55-year-old male visited the ER due to general weakness. At his initial visit he exhibited severe anemia. Mild intravascular hemolysis was suspected. For anemia evaluation, BM examination was performed. In BM aspiration, almost no erythroid precursor cells were observed. Also, plasma cells were relatively elevated, at 7.2%. Serum electrophoresis and immunofixation revealed paraproteinemia of 5.1 g/L (IgG and lambda). No hypercalcemia, renal insufficiency or lytic bone lesions were found. This unusual case showed MGUS accompanied by PCRA. We were also able to assume the erythroid cell-specific restriction due to paraprotein, because we ruled out possible causes of PRCA. RESULTS: We discovered several reported cases associated with plasma cell dyscrasia. However, most of these cases involved plasma cell myeloma, characterized by high immunoglobulin burden. Our case demonstrates that PRCA is also observed in cases with MGUS, where immunoglobulin burden is low. CONCLUSIONS: It is not yet accurately known, what parts of erythroid precursors are targeted by M-protein nor what the mechanism is. Therefore, additional research into this matter is necessary.

High-Resolution Snapshots of Proteasome Inhibitors in Action Revise Inhibition Paradigms and Inspire Next-Generation Inhibitor Design.

New high-resolution crystal structures reported by Schrader and colleagues refine our understanding of how peptide epoxyketone anticancer drugs inactivate their target: the human proteasome. These findings provide important clues for the design of next-generation proteasome inhibitor drugs.

Polymer micelle formulations of proteasome inhibitor carfilzomib for improved metabolic stability and anticancer efficacy in human multiple myeloma and lung cancer cell lines.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor drug approved for the treatment of multiple myeloma. Contrary to its excellent antimyeloma activity, CFZ has shown only limited efficacy in patients with solid malignancies. This lack of efficacy has been attributed in part to rapid degradation of CFZ in the body, possibly hindering the ability of CFZ to access the proteasome target in solid tumors. We hypothesized that polymer micelles, a currently Food and Drug Administration-approved nanoparticle drug delivery formulation, may protect CFZ from metabolic degradation and thus expand the clinical utility of the drug as an anticancer agent. To test our hypothesis, we prepared CFZ-entrapped polymer micelle particles with various compositions and drug release profiles and examined the extent of the CFZ metabolism in vitro using mouse liver homogenates. We also assessed the cytotoxic activities of the CFZ-entrapped micelle formulations in human cancer cell lines derived from B lymphocytes (RPMI-8226) and the lung (H460). Our data indicated that polymer micelle-based formulations can improve metabolic stability and cytotoxic effects of CFZ compared with free CFZ in human cancer cell lines tested. Taken together, these results suggest that polymer micelles may have potential as a delivery system for CFZ with an extended therapeutic utility for nonhematologic malignancies in the future.

Elucidating the catalytic subunit composition of distinct proteasome subtypes: a crosslinking approach employing bifunctional activity-based probes.

In addition to two well-recognized proteasome subtypes-constitutive proteasomes and immunoproteasomes-mounting evidence also suggests the existence of intermediate proteasome subtypes containing unconventional mixtures of catalytic subunits. Although they appear to play unique biological roles, the lack of practical methods for detecting distinct proteasome subtypes has limited functional investigations. Here, we report the development of activity-based probes that crosslink two catalytic subunits within intact proteasome complexes. Identification of the crosslinked subunit pairs provides direct evidence of the catalytic subunit composition of proteasomes. Using these probes, we found that U266 multiple myeloma cells contain intermediate proteasomes comprising both ß1i and ß2, but not ß1 and ß2i, consistent with previous findings with other cell types. Our bifunctional probes can be utilized in functional investigations of distinct proteasome subtypes in various biological settings.

Gender differences in the hepatic elimination and pharmacokinetics of lobeglitazone in rats.

The pharmacokinetics of lobeglitazone (LB) was studied after intravenous administration at a dose of 1 mg/kg and oral administration at doses of 0.1, 1 and 10 mg/kg in male and female rats. The area under the plasma concentration-time curve from time zero to infinity (AUCinf ) after intravenous administration was approximately 7.1 times higher in female rats than in male rats. In addition, the AUCinf in the case of oral administration was at least 4.4 times higher in female rats and appeared to increase in proportion to the dose in both genders. The in vitro half-lives were 18.8 ± 4.45 min and 60.7 ± 11.2 min, as evidenced by incubating liver microsomes obtained from male and female rats, respectively. As a result, the estimated CLint for LB for male rat liver microsomes (0.0779 ± 0.0233 ml/min/mg protein) was much higher than that for female rat liver microsomes (0.0233 ± 0.0039 ml/min/mg protein, p < 0.05). These observations suggest that there are gender differences in the pharmacokinetics and hepatic metabolism for LB in rats. Copyright © 2015 John Wiley & Sons, Ltd.