Population Pharmacokinetics, Pharmacogenomics, and Adverse Events of Osimertinib and its Two Active Metabolites, AZ5104 and AZ7550, in Japanese Patients with Advanced Non-small Cell Lung Cancer: a Prospective Observational Study.

BACKGROUND: Potential novel strategies for adverse event (AE) management of osimertinib therapy, including therapeutic drug monitoring and the use of biomarkers, have not yet been fully investigated. This study aimed to evaluate (1) the relationship between exposure to osimertinib, especially its active metabolites (AZ5104 and AZ7550), and AEs, and (2) the relationship between germline polymorphisms and AEs. METHODS: We conducted a prospective, longitudinal observational study of 53 patients with advanced non-small cell lung cancer receiving osimertinib therapy from February 2019 to April 2022. A population pharmacokinetic model was developed to estimate the area under the serum concentration-time curve from 0 to 24 h (AUC0-24) of osimertinib and its metabolites. Germline polymorphisms were analyzed using TaqMan® SNP genotyping and CycleavePCR® assays. RESULTS: There was a significant association between the AUC0-24 of AZ7550 and grade ≥ 2 paronychia (p = 0.043) or anorexia (p = 0.011) and between that of osimertinib or AZ5104 and grade ≥ 2 diarrhea (p = 0.026 and p = 0.049, respectively). Furthermore, the AUC0-24 of AZ5104 was significantly associated with any grade ≥ 2 AEs (p = 0.046). EGFR rs2293348 and rs4947492 were associated with severe AEs (p = 0.019 and p = 0.050, respectively), and ABCG2 rs2231137 and ABCB1 rs1128503 were associated with grade ≥ 2 AEs (p = 0.008 and p = 0.038, respectively). CONCLUSION: Higher exposures to osimertinib, AZ5104, and AZ7550 and polymorphisms in EGFR, ABCG2, and ABCB1 were related to higher severity of AEs; therefore, monitoring these may be beneficial for osimertinib AE management.

Comparison of cultured cell attachment on a temperature-responsive polymer, poly-L-lysine, and collagen using modeling curves and a thermal-controlled quartz crystal microbalance.

The characteristics of cultured cell attachment onto poly-L-lysine (PLL), collagen, and the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAM) were studied using a quartz crystal microbalance (QCM). A QCM with microscope cameras enclosed in a Peltier chamber was developed to enable QCM measurements and microphotographic imaging to be conducted in a temperature-controlled CO2 incubator. Human hepatoma cell line HepG2 cells were cultured on the quartz crystals coated with PLL, collagen, and PNIPAM. Response curves of the resonant frequency of the quartz crystals during the cell attachment process were analyzed on the basis of the parameters of modeling curves fit to the experimentally obtained curves. Analysis of the fitting curves showed that the time constants of the first-lag response were 11 h for PLL, 16 h for collagen, and 38 h for PNIPAM and that the frequency change for the PNIPAM films was six times smaller than those for the PLL and collagen films. These findings were supported by photographic images showing wider cell spread on PLL and collagen than on PNIPAM. The response of cells on PNIPAM was measured during a thermal cycle from 37 to 20 °C to 37 °C. In the resonance frequency-resonance resistance (F-R) diagram, the slopes of ΔR/ΔF corresponding to the cell attachment process and those corresponding to the thermal cycling process differed; the positions in the F-R diagram also shifted to higher resonant frequencies after the thermal cycle. These results suggested that the mass effect decreased as a result of the weakening of the cell attachment strength by the thermal cycle because the molecular brushes of PNIPAM were disarranged.

Monitoring and Modeling of Living Cell Responses in the Attachment Process and Reaction to the Antitumor Reagent Cisplatin Studied by a Quartz Crystal Microbalance Combined with a Microscope.

The attachment process and response to an antitumor reagent for cultured cells were monitored with a quartz crystal microbalance (QCM) combined with a microscope. To fit the experimentally obtained curves of the resonant frequency, model equations of resonant frequency curves were built, and parameters of time constants and scale coefficients were determined. For the cell attachment process, a first-order lag response curve well fit the experimental curves. For the response to cisplatin, two response steps were observed in both QCM data and microscopic images, where the cells loosened in the first step and shrank in the second step. Resonant frequency responses for both processes were well fit by two logarithmic normal distribution functions. In addition, the dependence of the resonant frequency change on the cell number was also studied, and a cell-cell interaction model for attached cells was proposed to explain the saturation of the resonant frequency change in high density cell seeding.

Direct measurement of interaction forces between a platinum dichloride complex and DNA molecules.

The interaction forces between a platinum dichloride complex and DNA molecules have been studied using atomic force microscopy (AFM). The platinum dichloride complex, di-dimethylsulfoxide-dichloroplatinum (II) (Pt(DMSO)2Cl2), was immobilized on an AFM probe by coordinating the platinum to two amino groups to form a complex similar to Pt(en)Cl2, which is structurally similar to cisplatin. The retraction forces were measured between the platinum complex and DNA molecules immobilized on mica plates using force curve measurements. The histogram of the retraction force for λ-DNA showed several peaks; the unit retraction force was estimated to be 130 pN for a pulling rate of 60 nm/s. The retraction forces were also measured separately for four single-base DNA oligomers (adenine, guanine, thymine, and cytosine). Retraction forces were frequently observed in the force curves for the DNA oligomers of guanine and adenine. For the guanine DNA oligomer, the most frequent retraction force was slightly lower than but very similar to the retraction force for λ-DNA. A higher retraction force was obtained for the adenine DNA oligomer than for the guanine oligomer. This result is consistent with a higher retraction activation energy of adenine with the Pt complex being than that of guanine because the kinetic rate constant for retraction correlates to exp(FΔx - ΔE) where ΔE is an activation energy, F is an applied force, and Δx is a displacement of distance.

Fabrication of a polymer-metal combined atomic force microscopy probe for coarse food surface imaging.

We fabricated a polymer-metal combined atomic force microscopy (AFM) probe by two steps; a polymeric resin was used at first step, and a metal-ion was used at second step which needs more fabricating time than the resin. At first step, we fabricated a cylindrical base on to a commercial cantilever. At second step, we fabricated a conical probe on to the fabricated cylindrical base. To make the conical probe composed with silver, a 0.2 M aqueous solution of silver nitrate (AgNO3) was used. A 50 microm length polymeric-metallic hybrid tip has been fabricated to observe large bio and food samples. Generally, the AFM images of bio/food samples show cliff-like sharp patters in vertical. However, the AFM image by fabricated long tip shows clear structure of each brown rice flours. As most of commercial tips have three-angular pyramidal, the scanned results should be influenced by the lateral face of the three-angular pyramid, which results in cliff-like images. Because the sample size is large, the side area of the sample was adversely affected by the pyramidal structure during imaging. This problem may be resolved by designing conical structure tips. As the conical structure has no edge, the AFM image becomes clear. The fabricated tip has conical structure, and a clear AFM image was achieved.

Comparative evaluation of interaction force between cofilin protein and peptide probe to actin protein.

In current study, we report the direct interaction force comparison for a synthesized peptide probe to actin and cofilin protein to actin using atomic force microscopy. The peptide probe was synthesized following the actin binding module of cofilin protein. Thus, the functionality of the peptide probe was similar with that of the cofilin protein. The difference between the peptide probe and cofilin protein was the molecular size. The small peptide probe enables highly dense surface modification, thus produces different interaction force curves compared with that of original cofilin protein to actin. The results showed the peptide probe was possible to measure many numbers of the interaction force though the measurement of single molecular order interaction force was a weak point. These imply that the peptide probe has a merit when that was applied surface related applications such as protein sensor and protein-protein interaction separation.

Observation of interaction force in large area between actin modified surface and actin antibody modified microsphere probe.

In order to observe the force interaction in large areas, a novel force detection probe was fabricated by two-photon absorbed photopolymerization (TPAP) techniques. The probe was based on a commercial cantilever, and a docking structure for adopting a microsphere immobilized with actin antibody was fabricated by the TPAP techniques. The commercial AFM tip was also modified with the antibody for comparison. Using force curve measurement, the interaction force was compared between the modified probes and the sample surface which was immobilized with actin using a spotting system. The adhesive force of 1.3 nN was measured applying the commercial cantilever. The value was comparable to the measured interaction force of 130 nN applying the microsphere modified cantilever. The measured adhesive force of the novel probe was 100-fold larger than that obtained by the sharp AFM cantilever tip. This strong adhesive force of the microsphere modified cantilever to actin is explainable by the large contact area between the microsphere and the sample surface.

Increase the reliability and applicability of quartz crystal resonator coupled with other instruments.

The principle and applications of quartz-crystal resonator systems and the methods to increase their reliability and applicability coupled with other instruments were reported. The principle of quartz-crystal resonator system was documented with based on the three basic concepts for mass, viscosity, and viscoelastic changes. In the preliminary discussion, the realization of a resonant frequency-resonant resistance diagram was described in detail. As the examples of quartz-crystal resonator applications with introducing the resonant resistance concept and the resonant frequency, the fabrication of a carbon-coated quartz crystal sensor and monitoring changes in the viscoelastic properties of thin polymer films were carefully discussed. Examples for increasing their reliability and applicability of quartz crystal resonator systems combined with UV-visible spectroscopy, Atomic Force Microscope (AFM), Surface Plasmon Resonance (SPR), or Charge Coupled Device (CdCD) camera were described.

Analysis of HepG2 cell response to a wide concentration range of mitomycin C using a multichannel quartz crystal microbalance system with a microscope.

The morphological response of HepG2 cells to mitomycin C was analyzed using a multichannel quartz crystal microbalance system equipped with a home-built movable microscope that enables the simultaneous acquisition of cell images and measurements of eight-channel quartz crystal microbalance. After 24 h of cell seeding, mitomycin C was injected into the culture medium. During the attachment process, the resonant frequency decreased, and the curves fitted well with the first-order lag response. Analysis of the response to mitomycin C revealed that the resonant frequency response curves varied with mitomycin C concentration. When the mitomycin C concentration was <10 µmol L-1, the delay time was observed before the increase in resonant frequency. When the mitomycin C concentration was extremely low, an additional decrease in resonant frequency was observed in the middle of the delay time that fitted well with the cumulative log-normal distribution curve. The resonant frequency response curves after the delay time fitted well with the cumulative log-normal distribution curves. The delay time and mean cumulative log-normal distribution time for the increase in resonant frequency correlated with the mitomycin C concentration; however, the mean time for the additional decrease in the resonant frequency did not show a statistically significant difference as a function of mitomycin C concentration. For mitomycin C concentrations of >20 µmol L-1, the response to the change in resonant frequency was rapid, and the response curves fitted well with the first-order lag response. The first-order lag response indicates that the response occurred simultaneously for all cells. The results showed that the time constant was independent of the tested mitomycin C concentration between 20 and 100 µmol L-1. These results suggested that different cell death processes occurred by mitomycin C. The findings of this study suggest that the system can be used to investigate cell death in adherent cells.

Evaluation of interaction between histidine binding Cu2+ ion and histidine by atomic force microscopy.

This paper presents a direct interaction force measurement between histidine molecules using AFM force-distance curve measurement. AFM force-distance curves between the histidine-modified cantilever and substrate in the different conditions with or without intercalating Cu2+ ion were measured and interpreted via Gaussian curve fitting analyses. The adhesion force between histidine molecules was shown to be 110 pN under the presence of Cu2+. The result was compareable to the measured adhesion force about 0 pN, which was measured by the removal of Cu2+ ion with the addition of EDTA. The result indicated the direct histidine-histidie interaction was difficult without the role of the bridigible ionic component. From the results, the possibility of direct measurement on chemical affinities between biomolecules was suggested by using AFM force-distance curve analyses. Especially, the current approach showed the possible affinity measurement techniques that elucidate the role of bridge ions.

Development and validation of a new liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of afatinib, dacomitinib, osimertinib, and the active metabolites of osimertinib in human serum.

Reports on the therapeutic drug monitoring (TDM) of second- and third-generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer patients are limited and are required to improve the safety of EGFR-TKI therapy. Some EGFR-TKIs have active metabolites with similar or higher potency compared with the parent compounds; thus, monitoring the parent compound as well as its active metabolites is essential for truly effective TDM. In this study, we developed and validated a method that simultaneously quantifies second- and third-generation EGFR-TKIs (afatinib, dacomitinib, and osimertinib) and the active metabolites of osimertinib, AZ5104 and AZ7550, in the human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The clinical application of the method was also evaluated. The analytes were extracted from a 100 µL serum sample using a simple protein precipitation method and analyzed using LC-MS/MS. Excellent linearity of calibration curves was observed at ranges of 2.5-125.0 ng/mL for afatinib, 2.5-125.0 ng/mL for dacomitinib, 4.0-800.0 ng/mL for osimertinib, 1.0-125.0 ng/mL for AZ5104, and 2.5-125.0 ng/mL for AZ7550. The precision and accuracy were below 14.9% and within ± 14.9% of the nominal concentrations, respectively. The mean recovery was higher than 94.7% and the coefficient of variation (CV) was lower than 8.3%. The mean internal-standard normalized matrix factors ranged from 94.6 to 111.9%, and the CVs were lower than 9.7%. This analytical method met the acceptance criteria of the U.S. Food and Drug Administration guidelines. The method was also successfully applied to the analysis of 45 clinical samples; it supports the efficient and valuable analysis for TDM investigations of EGFR-TKIs.

Influence of diquat on growth and death of HepG2 cells using quartz crystal and micro CCD camera.

Diquat is widely used agent which produces toxicity in human and implicated as an environmental toxicity. HepG2 cell was cultured onto an indium tin oxide (ITO) surface of quartz crystal modified a collagen film. In this paper, we investigated the physical properties and the morphological change of the HepG2 cells cultured onto the ITO electrode of the quartz crystal sensor with micro CCD camera. The resonance responses of the quartz crystal and the morphological change were directly monitored. After seeding the cells and diquat injection into the chamber, the resonance frequency and the resonance resistance were obtained with real time morphologies. From the resonance characteristics and the series of morphologies, we could know the diquat to be death and weakening of the cells.

[A case of delayed colonic perforation after metallic stent placement for advanced descending colon cancer during bevacizumab-based chemotherapy].

A 73-year-old man with advanced descending colon cancer and peritoneal metastases underwent a self-expandable metallic stent placement under fluoroscopic guidance on October 2007. The stent placement was successful without early complication. After 6 courses of FOLFOX4 followed by 7 courses of FOLFIRI, he received Bevacizumab-based chemotherapy from August 2008. In April 2009, he was admitted to our hospital with severe abdominal pain due to perforation of descending colon. Although emergent surgery was performed, he developed DIC and died on the 21 postoperative days. This case suggests that metallic stent placement for colorectal cancer cases might increase the risk of bowel perforation during Bevacizumab-based chemotherapy.

Prospective feasibility study of indigo naturalis ointment for chemotherapy-induced oral mucositis.

OBJECTIVES: Indigo naturalis, a herbal medicine effective against ulcerative colitis, exhibits anti-inflammatory effects and induces interleukin-22-mediated antimicrobial peptide production. Anti-inflammatory activity and the prevention of secondary infection are essential for the management of chemotherapy-induced oral mucositis (CIOM); therefore, we developed an indigo naturalis ointment to be administered topically for CIOM and evaluated its feasibility. METHODS: We performed a single-centre, open-label, prospective feasibility study from March 2017 to December 2018. The key eligibility criteria for the subjects were as follows: (1) receiving chemotherapy for a malignant tumour; (2) grade 1 or 2 CIOM and (3) receiving continuous oral care. The treatment protocol comprised topical indigo naturalis ointment application three times a day for 7 days. The primary endpoint assessed was feasibility. The secondary endpoints assessed were the changes in oral findings, oral cavity pain and safety. RESULTS: Nineteen patients with CIOM were enrolled. The average feasibility (the proportion of prescribed applications that were carried out) observed in this study was 94.7%±8.9% (95% CI 90.5% to 99.0%), which was higher than the expected feasibility. The revised oral assessment guide scores of the mucous membrane domain and total scores were significantly improved. All patients reported a reduction in oral cavity pain, with a median pain resolution duration of 6 days. No serious adverse events were observed. CONCLUSIONS: The indigo naturalis ointment was feasible, and showed the potential for efficacy and safety. Larger randomised controlled trials are needed to further assess the efficacy and safety of indigo naturalis compared with a placebo. TRIAL REGISTRATION NUMBER: UMIN000024271.

Lorlatinib-induced visual and auditory hallucinations: A case report.

Lorlatinib can cause visual and auditory hallucinations. And, it is necessary to keep in mind that hallucinations can persist even after discontinuation in patients who develop hallucinations while receiving lorlatinib.

Construction of simultaneous SPR and QCM sensing platform.

To construct a novel simultaneous SPR and QCM sensing system, an AT-cut quartz crystal is fabricated by sputtering 250 nm of ITO on one side of the quartz plate over a 5-nm thick underlay of titanium, while a 50-nm thick layer of gold is sputter-deposited on the other side to induce a total light reflection of an incident laser beam on the thin gold layer. The signals of the sensing system are detected using a Handy-SPR and QCA922 when dropping 200 microL of Milli-Q water into the sensing cell. A decrease in the SPR reflected light intensity is clearly identified. In the same experiment, the changes in the resonant frequency and resistance are about 2 kHz and 200 Omega, respectively. Furthermore, the oscillation stabilities of the resonant frequency and resistance are about 50 Hz and 2 Omega, respectively, which are sufficient to detect a large mass change on the QCM/SPR chip.

Development of glucose sensor using two-photon adsorbed photopolymerization.

A novel glucose sensor was constructed, and its analytical potential examined. A chip-type three-electrode system for use in a flow-type electrochemical glucose sensor was fabricated using a UV lithography technique on a glass slide. An Ag/AgCl reference electrode was made by electroplating silver onto a Pt electrode and dipping in a saturated KCl solution for 30 min. In addition, a glucose-sensing electrode was fabricated using a two-photon adsorbed photopolymerization technique with a photo-reactive resin containing a glucose oxidase enzyme, ferrocene mediator, non-ionic surfactant, and carbon nanotubes. The cyclic voltammetry of the potassium ferrocyanide in the Pt sensor system showed a stable electrode condition. The response of the modified Pt sensor confirms the feasibility of using a two-photon adsorbed photopolymerization technique for the easy fabrication of functional biosensors.

A case of mosaic Klinefelter syndrome associated with isodicentric Yp.

We describe a case of mosaic Klinefelter syndrome demonstrating an isodicentric Y chromosome. A 70-year-old man visited our outpatient clinic complaining of dysuria resulting from atrophy of the penis. His height was 170 cm and his weight was 60 kg. A serum hormonal analysis revealed hypergonadotropic hypogonadism. A chromosomal analysis with fluorescence in situ hybridization revealed four cell lines in which the karyotypes were 47,XXY, 46,XY, 46,XX and 47,XX,idic(Y) (q11.2). To the best of our knowledge this is the first case of mosaic Klinefelter syndrome bearing an isodicentric Y chromosome. The origin of the isodicentric Y is discussed. (Reprod Med Biol 2008; 7: 177-180).

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance.

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.

Results of extracorporeal shock wave lithotripsy for the treatment of upper urinary tract stones.

The treatment results of 697 renal units in 687 patients treated for upper urinary tract stones using a Piezolith 2500 at the Ageo Central General Hospital during the 5-year period between August 1999 and July 2004 were analyzed. The stone-free rate and the success rate were calculated according to the stone size and location. Univariate and multivariate logistic regression analyses were performed to estimate the effect of the patient age, sex, affected side, stone location and stone size on the stone-free rate and the success rate. Overall, the stone-free rate and the success rate at 3 months after treatment were 82.6% and 91.9%, respectively. The stone-free rates in renal and ureteral stones were 60.5% and 88.7%, respectively. The success rates for renal and ureteral stones were 93.6% and 91.6%, respectively. In a univariate logistic regression analysis, patient age and stone size were significant negative factors for both the stone-free rate and the success rate and the stone location in ureter was a significant positive factor only for the stone-free rate. In a multivariate analysis, stone location in the ureter had a positive effect on the stone-free rate, although a negative effect on the success rate. Extracorporeal shock wave lithotripsy for upper urinary tract stones was an effective and safe treatment modality. However, if pre-treatment factors indicate unfavorable outcome, the other treatment options should be considered for the first line therapy.