And Alpha 1-acid Glycoprotein Research Articles

Quantitative determination of lurbinectedin, its unbound fraction and its metabolites in human plasma utilizing ultra-performance LC-MS/MS.

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods to quantify total lurbinectedin, its plasma protein binding to derive the unbound fraction and its main metabolites 1',3'-dihydroxy-lurbinectedin (M4) and N-desmethyl-lurbinectedin (M6) in human plasma, were developed and validated. For lurbinectedin, sample extraction was performed using supported liquid extraction. For metabolites, liquid-liquid extraction with stable isotope-labeled analogue internal standards was used. Plasma protein binding was evaluated using rapid equilibrium dialysis. In vitro investigations at different plasma protein concentrations were carried out to estimate dissociation rate constants to albumin and alpha-1-acid glycoprotein (AAG). Calibration curves displayed good linearity over 0.1 to 50 ng/mL for lurbinectedin and 0.5 to 20 ng/mL for the metabolites. Methods were validated in accordance with established guidance. The inter-day precision and accuracy ranged from 5.1% to 10.7%, and from -5% to 6% (lurbinectedin in plasma); from 3.1% to 6.6%, and from 4% to 6% (lurbinectedin in plasma:PBS); from 4.5% to 12.9%, and from 4% to 9% (M4); and from 7.5% to 10.5%, and from 6% to 12% (M6). All methods displayed good linearity (r2 >0.99). Recovery was evaluated for lurbinectedin in plasma:PBS (66.4% to 86.6%), M4 (7.82% to 13.4%) and M6 (22.2% to 34.3%). The method for lurbinectedin in plasma has been applied in most clinical studies, while the plasma:PBS and metabolites methods were used to evaluate the impact of special conditions on lurbinectedin PK. Lurbinectedin plasma protein binding was 99.6% and highly affected by AAG concentration. These UPLC-MS/MS methods enable the rapid and sensitive quantification of lurbinectedin and its main metabolites in clinical samples.

Species differences in plasma protein binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitor nirmatrelvir

Plasma protein binding (PPB) studies on the SARS-CoV-2 main protease inhibitor nirmatrelvir revealed considerable species differences primarily in dog and rabbit, which prompted further investigations into the biochemical basis for these differences. The unbound fraction (fu) of nirmatrelvir in dog and rabbit plasma was concentration (2–200 µM)-dependent (dog fu,p 0.024–0.69, rabbit fu,p 0.010–0.82). Concentration (0.1–100 µM)-dependent binding in serum albumin (SA) (fu,SA 0.040–0.82) and alpha-1-acid glycoprotein (AAG) (fu,AAG 0.050–0.64) was observed in dogs. Nirmatrelvir showed minimal binding to rabbit SA (1–100 µM: fu,SA 0.70–0.79), while binding to rabbit AAG was concentration-dependent (0.1–100 µM: fu,AAG 0.024–0.66). In contrast, nirmatrelvir (2 µM) revealed minimal binding (fu,AAG 0.79–0.88) to AAG from rat and monkeys. Nirmatrelvir showed minimal-to-moderate binding to SA (1–100 µM; fu,SA 0.70–1.0) and AAG (0.1–100 µM; fu,AAG 0.48–0.58) from humans across tested concentrations. Nirmatrelvir molecular docking studies using published crystal structures and homology models of human and preclinical species SA and AAG were used to rationalise the species differences to plasma proteins. This suggested that species differences in PPB are primarily driven by molecular differences in albumin and AAG resulting in differences in binding affinity.

Preclinical pharmacokinetics, CYP phenotyping, and tissue distribution study of novel anti-breast cancer candidate S-011-1559

S-011-1559 is a tyrosine-derived novel benzoxazine CDRI molecule targeted to the oestrogen-related receptor (ER-α/β) modulator in breast cancer. To explore the pharmacokinetics of S-011-1559, a selective and sensitive bioanalytical method using LC-MS/MS was established and validated in different biological matrices of female rats. Blood-to-plasma ratio and plasma protein binding (PPB) of S-011-1559 were found to be <1 and >97% in both rats and humans, respectively. The human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) binding was found in the range of > 68 to 45% and >14% respectively. Half-life and intrinsic clearance by microsomal stability study were found to be 28.83 min and 0.05 mL/min/mg in rats, 78.35 min and 0.036 mL/min/mg in humans, respectively. The IC50 value of S-011-1559 against CYP isoforms was revealed to moderately inhibit CYP2D6 by a reversible non-competitive mechanism. Tissue distribution of S-011-1559 on single intravenous injection at 2 mg/kg was found in the order of C lungs > C mammary gland > C spleen > C heart > C kidney > C liver > C brain. The data from the present study provides crucial information about S-011-1559 for further development as a novel potential drug candidate in modulating ER-α/β receptors of lung and breast neoplasia.

Neglecting Plasma Protein Binding in COVID-19 Patients Leads to a Wrong Interpretation of Lopinavir Overexposure.

Boffito et al. recalled the critical importance to correctly interpret protein binding. Changes of lopinavir pharmacokinetics in coronavirus disease 2019 (COVID‐19) are a perfect illustration. Indeed, several studies described that total lopinavir plasma concentrations were considerably higher in patients with severe COVID‐19 than those reported in patients with HIV. These findings have led to a reduction of the dose of lopinavir in some patients, hypothesizing an inhibitory effect of inflammation on lopinavir metabolism. Unfortunately, changes in plasma protein binding were never investigated. We performed a retrospective cohort study. Data were collected from the medical records of patients hospitalized for COVID‐19 treated with lopinavir/ritonavir in intensive care units or infectious disease departments of Toulouse University Hospital (France). Total and unbound concentrations of lopinavir, C reactive protein, albumin, and alpha‐1‐acid glycoprotein (AAG) levels were measured during routine care on the same samples. In patients with COVID‐19, increased total lopinavir concentration is the result of an increased AAG‐bound lopinavir concentration, whereas the unbound concentration remains constant, and insufficient to reduce the severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) viral load. Although international guidelines have recently recommended against using lopinavir/ritonavir to treat severe COVID‐19, the description of lopinavir pharmacokinetics changes in COVID‐19 is a textbook case of the high risk of misinterpretation of a total drug exposure when changes in protein binding are not taken into consideration.

Mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha-1-acid glycoprotein and permeability-limited hepatic uptake

1. The plasma clearance of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was shown to be markedly lower in human compared to the preclinical species, mouse, rat and dog.2. This led to vertical allometry where various interspecies scaling methods were applied to the data, with fold-errors between 4 and 13. We had previously reported the elimination and metabolic pathways of EPZ-5676 were similar across species. Therefore, the aim of this work was to explore the mechanistic basis for the species difference in clearance for EPZ-5676, focusing on other aspects of disposition.3. The protein binding of EPZ-5676 in human plasma demonstrated a non-linear relationship suggesting saturable binding at physiologically relevant concentrations. Saturation of protein binding was not observed in plasma from preclinical species. Kinetic determinations using purified serum albumin and alpha-1-acid glycoprotein (AAG) confirmed that EPZ-5676 is a high affinity ligand for AAG with a dissociation constant (Kd) of 0.24 μM.4. Permeability limited uptake was also considered since hepatocyte CLint was much lower in human relative to preclinical species. Passive unbound CLint for EPZ-5676 was estimated using a correlation analysis of logD and data previously reported on seven drugs in sandwich cultured human hepatocytes.5. Incorporation of AAG binding and permeability limited hepatic uptake into the well-stirred liver model gave rise to a predicted clearance for EPZ-5676 within 2-fold of the observed value of 1.4 mL min−1 kg−1. This analysis suggests that the marked species difference in EPZ-5676 clearance is driven by high affinity binding to human AAG as well as species-specific hepatic uptake invoking the role of transporters.

Serum and Urinary Biomarkers Endothelin-1, Beta-2 Microglobulin, Cystatin C, Galectin-3 and Alpha-1-acid Glycoprotein; Can they Surrogate Clinical and Histological Staging in Lupus Nephritis Patients?

Background: Lupus nephritis (LN) affects up to 50% of patients with Systemic lupus erythematosus (SLE) and is a major cause of morbidity. It is thus essential to identify biomarkers as indices with substantial predictive power to reduce the serious sequelae. However, criteria for disease remission have not been clearly established for these indices, except for the SLE Disease Activity Index (SLEDAI). Objectives: To investigate the relationship of non-invasively renal protein biomarkers and established measures of renal function to histologic findings in LN, and to test whether certain combinations of the above mentioned laboratory measures are diagnostic for specific histologic features of LN and to evaluate their relations to SLEDAI and chronicity. Methods: The study was conducted on 40 SLE female patients, recruited from renal unit of Internal Medicine department and Rheumatology and Rehabilitation department, and Neurology department, Assuit and Aswan University Hospitals, Egypt from May 2011 to January 2014, Renal biopsies were evaluated using the International Society of Nephrology/Renal Pathology Society classification (ISN/RPS), and scored for Activity Index and Chronicity Index; Clinical responders (CR) were required to have ≥50% reduction in proteinuria, normal or improved renal function, and inactive urinary sediment. Histopathological responders (HR) were required to have ≥50% improvement in Activity Index In addition, 40 age and sex matched healthy persons as a control group were enrolled in the study. The GFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Novel serum biomarkers; Endothelin-1 (ET-1), cystatin C, beta-2 microglobulin (B2M), galectin-3 (Gal-3) and alpha-1-acid glycoprotein (AAG) were collected. Urine samples from patients were collected within 2 months of kidney biopsy and assayed for the urinary biomarkers Endothelin-1 (ET-1), α1-acid glycoprotein (AAG), Cystatin C (CysC) and beta-2 microglobulin (B2M). Renal disease activity was estimated using the British Isles Lupus Assessment Group (BILAG) index. Results: The urinary and plasma biomarkers; ET-1, AAG, Cys C and B2M and GAL-3 were statistically significantly higher in patients with LN compared to controls that is reflective of specific histologic features seen in active LN. The combination of ET-1, AAG and CRP levels plus protein: creatinine ratio was excellent in predicting LN activity. The urinary B2M together with ET-1 and AAG plus creatinine clearance was an excellent diagnostic test for LN chronicity. However, plasma and urinary Cys C showed insignificant correlation with chronicity indices with lowest sensitivity and specificity. The statistically significantly high levels of serum and urinary ET-1 and AAG were related to specific histologic findings in LN with significant positive correlations with SLEDAI and chronicity indices in renal biopsy and highest sensitivity and specificity. Notably, these plasma biomarkers were increased linearly as renal function declined whereas urinary ET-1 and AAG rose exponentially. Thus, urinary ET-1 and AAG may be considered as a useful measure of renal inflammatory disease activity while measured renal function is still normal. Nevertheless, urinary and serum B2M exhibit a statistically insignificantly positive correlations and serum GAL-3 show insignificantly statistically negative correlations with SLEDAI and chronicity indices with lowest specificity and sensitivity reflecting the difficulty of being these biomarkers were useful markers for assessing activity and detection of early disease flares in patients with LN. Conventional clinical parameters such as creatinine clearance, proteinuria, urine sediments, anti-dsDNA, and complement levels are not sensitive or specific enough for detecting ongoing disease activity in the LNs and early relapse of nephritis. Conclusions: In this study biomarkers namely; Endothelin -1(ET-1) and α_1-acid glycoprotein (AAG) found to be associated with specific tissue changes observed in conjunction with LN activity and chronicity. The preliminary results suggest that these biomarkers may be part of a panel that in combination may eventually be able to predict histology without the need of an invasive biopsy. Currently, however we try to discover if these promising biomarkers actually alter patient outcomes and improve the lives of the patients with this life-threatening disease complication of SLE

Comprehensive Analysis Of The In Vitro Potency Of Ponatinib, and All Other Approved BCR-ABL Tyrosine Kinase Inhibitors (TKIs), Against a Panel Of Single and Compound BCR-ABL Mutants

BackgroundSecondary mutations in BCR-ABL are the most common cause of resistance to TKIs in patients (pts) with chronic myeloid leukemia (CML). Ponatinib is a potent pan-BCR-ABL TKI that has been shown to suppress the emergence of any single mutation in vitro, including T315I, at clinically achievable concentrations (40 nM), though higher concentrations were required to suppress emergence of certain compound mutations (2 mutations in the same BCR-ABL allele). Ponatinib has demonstrated significant clinical activity in pts in the phase 2 PACE trial, 60% of whom received 3 or more prior TKIs. Responses were observed for each of the 15 mutations present in >1 chronic phase CML pt at baseline, and no single mutation conferring resistance to ponatinib has emerged to date, though in some cases development of compound mutations has been observed. To gain a more precise understanding of the effects of specific mutations on the clinical efficacy of ponatinib, IC50s for ponatinib, and all other approved TKIs, against 31 single or compound BCR-ABL mutants were determined. To explore the relationship between in vitro potency and clinical efficacy, IC50s were related to “effective” TKI levels achieved in patients. MethodsTKI potency was assessed in engineered Ba/F3 cells by measuring cell viability at 72 hours. The effective plasma concentration for each TKI was calculated from published average steady-state concentration values for the recommended dose, and adjusted for the functional effects of protein binding. These effects were assessed by determining the degree to which TKI potency was reduced by the presence of physiological concentrations of human serum albumin (HSA) and alpha 1-acid glycoprotein (AAG). ResultsThe activity of ponatinib, and 5 other TKIs, against 21 single BCR-ABL mutants is shown in Figure A. Ponatinib potently inhibited viability of native BCR-ABL and all mutants, including T315I (IC50s (nM): 3-16). The IC50s for the other TKIs, excluding T315I (>4000 for all) ranged from: 201-10,000 (imatinib), 12-784 (nilotinib), 2-104 (dasatinib), 40-1,280 (bosutinib) and 18-5,216 (radotinib). IC50 values were compared to the effective plasma concentration for each TKI (Figure A). Mutants that have previously been associated with clinical resistance to a particular TKI tended to have IC50s that approached or substantially exceeded the effective concentration for that TKI, including most mutants for imatinib, E255K/V, Y253H, L248R, T315I for nilotinib, and V299L, F317C/I/V, T315A/I for dasatinib. The most problematic mutants for bosutinib predicted by this analysis were F317V, L248R, V299L, and T315I. Notably, all mutant IC50s fell below the effective concentration for ponatinib. The activity of all TKIs against 10 clinically-observed BCR-ABL compound mutants was also assessed. Four compound mutants had IC50s near or above the effective concentration for ponatinib (T315I+M351T, E255V+F317I, T315I+E255K, T315I+E255V) (Figure B). All 4, plus others, are also predicted to be problematic for the other TKIs. [Display omitted] ConclusionsRelating in vitro TKI potency to “effective” steady-state plasma concentrations in patients identified mutations known to confer clinical resistance to imatinib, nilotinib and dasatinib. This method of analysis suggests that ponatinib may be able to inhibit all single BCR-ABL mutants, but not all compound mutants, a prediction that is thus far consistent with results observed in patients. Compound mutations that are predicted to confer resistance to ponatinib are also predicted to confer resistance to all other approved TKIs. Early introduction of ponatinib may prevent the emergence of single mutations, and thus the sequential development of compound mutations. Disclosures:Gozgit:ARIAD: Employment, Equity Ownership. Schrock:ARIAD: Employment, Equity Ownership. Chen:ARIAD: Employment, Equity Ownership. Clackson:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Rivera:ARIAD: Employment.

Characterization of Binding of Raltegravir to Plasma Proteins

The objective of this study was to characterize raltegravir (RAL) binding to albumin and alpha-1-acid glycoprotein (AAG). Unbound and bound RAL were separated by ultrafiltration. The association constant (Ka) was estimated by a graphical method. In HIV-infected patients, the average plasma protein binding is 76%. RAL did not bind to AAG but bound to nonsaturable, low-affinity albumin sites with an n (number of sites) · Ka product of 9.8 × 10(2) liters/mol. A pH increase of 0.2 U led to a 2% increase in the bound fraction.

In Vitro–In Vivo Extrapolation of Clearance: Modeling Hepatic Metabolic Clearance of Highly Bound Drugs and Comparative Assessment with Existing Calculation Methods

In vitro-in vivo extrapolation (IVIVE) is an important method for estimating the hepatic metabolic clearance (CL) of drugs. This study highlights a problematic area observed when using microsomal data to predict in vivo CL of drugs that are highly bound to plasma proteins, and further explores mechanisms for human CL predictions by associating additional processes to IVIVE disconnect. Therefore, this study attempts to develop a novel IVIVE calculation method, which consists of adjusting the binding terms in a well-stirred liver model. A comparative assessment between the IVIVE method proposed here and previously published methods of Obach (1999. Drug Metab Dispos 27:1350-1359) and Berezhkovskiy (2010. J Pharm Sci 100:1167-1783) was also performed. The assessment was confined by the availability of measured in vitro and in vivo data in humans for 25 drugs highly bound to plasma proteins, for which it can be assumed that metabolism is the major route of elimination. Here, we argue that a difference in drug ionization and binding proteins such as albumin (AL) and alpha-1-acid glycoprotein (AAG) in plasma and liver also needs to be considered in IVIVE based on mechanistic studies. Therefore, converting unbound fraction in plasma to liver essentially increased the predicted CL values, which resulted in much more accurate estimates of in vivo CL as compared with the other IVIVE methods tested. The impact on CL estimate was more apparent for drugs binding to AL than to AAG. This is a mechanistic rational for explaining a considerable proportion of the divergence between previously estimated and observed CL values. Human CL was predicted within 1.5-fold, twofold, and threefold of the observed CL for 84%, 96%, and 100% of the compounds, respectively. Overall, this study demonstrates a significant improvement in the mechanism-based prediction of metabolic CL for these 25 highly bound drugs from in vitro data determined with microsomes, which should facilitate the application of physiologically based pharmacokinetic (PBPK) models in drug discovery and development.

Seroprevalence and serum profile of cytomegalovirus infection among patients with hematologic disorders in Bahia State, Brazil

Human cytomegalovirus (CMV) is a ubiquitous herpesvirus with from 30% to 100% of the general population exhibiting prior exposure by serology. This cross-sectional study evaluated the serological profile of anti-CMV antibodies and two acute-phase reaction proteins in Haematologic Disorder Patients (HDPs) from Bahia State, Brazil. Immuno-chemiluminescence assays were performed to detect anti-CMV IgM and IgG antibodies. Serological levels of High Sensitivity C-Reactive Protein (CRPH) and Alpha-1-Acid Glycoprotein (AAG) were measured using immunonephelometry. A total of 470 HDPs were enrolled, 238 (50.6%) males and 232 (49.4%) females. The overall seroprevalence of CMV was 89.4%, directly proportional to age and to the amount of blood units transfused. There was no difference between seroprevalence rates according to gender (P = 0.12). Four HDPs (0.9%) were seropositives for anti-CMV IgM, only one could be characterized as recent acute infection. The most CMV seropositive HDPs had anti-CMV IgG in low titers. There was a tendency for females to have higher anti-CMV IgG titers than men (P < 0.05). CRPH levels were different among HDPs CMV negative and positive groups (P < 0.001). There was no difference in the AAG levels between groups (P = 0.15). The high CMV seroprevalence found underscores the importance of using strategies to provide "CMV safe" blood to HDPs who are at high risk of developing severe CMV infection. CRPH can be used as a biomarker associated with CMV seropositivity; however, more efforts are needed to better characterize the clinical profile of active CMV infection in this group of patients.

Microquantitative Estimation of Serum C-Reactive Protein and Alpha 1-Acid Glycoprotein in Patients with Ovarian Cancer

Both serum C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) were measured in 22 patients with benign ovarian tumors, 58 patients with ovarian cancer and 31 normal controls, using a single radial immunodiffusion technique. In most normal women as well as patients with benign ovarian tumors, serum CRP was lower than 2 μg/ml and AAG lower than 80 mg/dl. In 15 of 25 untreated patients with ovarian cancer and in 14 of 16 patients with recurrence, both serum CRP and AAG were above 2 μg/ml and 80 mg/dl respectively. Serum CRP and AAG levels were measured sequentially in 9 patients with ovarian cancer after initial treatment. Serum CRP and AAG levels in 5 patients without recurrence had returned to and remained at normal levels after successful treatment. In contrast, 4 patients with recurrence exhibited elevations in serum CRP and AAG levels above the normal range. It is worth notions that these proteins increased before clinical evidence of recurrence. These studies suggest that serial measurement of CRP and AAG may be of great use in the surveillance of patients with ovarian cancer following initial treatment.

Serum Acute Phase Protein Concentrations in Dogs with Autoimmune Hemolytic Anemia

Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported. Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and alpha1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations. Twenty-seven dogs with AIHA and 11 control dogs. Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365. Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1-7; P < .002) and normalized with disease stabilization (days 9-365; P > .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions (P= .153-.940). PCV correlated with APP concentrations over time (CRP r=-.600, AAG r=-.665, albumin r= .533; P < .0001) as did WBC count (CRP r= .253, AAG r= .486, albumin r=-.246; P < .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 microg/mL) compared with dogs that did not (191.2 microg/mL; P= .02). An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.

Biomarkers of Inflammation and Malnutrition Associated with Early Death in Healthy Elderly People

To determine whether malnutrition and biomarkers of inflammation predict all-cause, cancer, and cardiovascular mortality in healthy elderly subjects. Population-based study, prospective cohort. Sète, on the French Mediterranean coast. Five hundred and fifty-three men and 888 women aged 60 and older from the Pathologies Oculaires Liées à l'Age cohort free of known comorbidities. Plasma levels of cholesterol, albumin, transthyretin (TTR), C-reactive protein (CRP), and alpha 1-acid glycoprotein (AAG) were measured at baseline. To investigate the risks of 5-year (early) and 5- to 9-year (late) mortality, hazard ratios (HR) were evaluated using Cox models. In men, early death was associated with high CRP and AAG and low albumin and TTR. In women, early death was associated with high AAG, low TTR and low cholesterol. For late death, the only significant association was with CRP in men. A synergistic effect was observed between biomarkers of inflammation and malnutrition. In men, the adjusted HR of early death was 4.98 (95% confidence interval (CI)=2.25-11.01) for both CRP in the highest quartile and albumin in the lowest. In men, this risk was greatest for both AAG in the highest quartile and TTR in the lowest (HR=6.86, 95% CI=3.20-14.71). In women, this risk was greatest for both AAG in the highest quartile and TTR in the lowest (HR=4.64, 95% CI=1.79-12.05). Cancer mortality was greater for high CRP and AAG and for low albumin and TTR in men but not in women. Biomarkers of inflammation and malnutrition together predict early mortality. In healthy elderly subjects, TTR and AAG could be helpful in identifying elderly subjects at higher risk of death.

Binding of gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase, to plasma proteins and blood cells: in vitro and in cancer patients

Gefitinib exhibits wide inter-subject pharmacokinetic variability which may contribute to differences in treatment outcome. Unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug. Thus it is desirable to determine gefitinib binding in plasma and factors affecting this process. An equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (fu) gefitinib in human plasma. Gefitinib binding in plasma from four different species and isolated protein solutions as well as drug partitioning in human blood cells were investigated. Unbound gefitinib plasma concentrations were measured in 21 cancer patients receiving daily oral gefitinib 250 mg or 500 mg. It was found that gefitinib was extensively bound in human rat mouse and dog plasma with mean fu values of 3.4%, 3.8%, 5.1% and 6.0% respectively. In isolated protein solutions approximately 90% and 78% of gefitinib was bound to human serum albumin (HSA) (40 mg/dL) and alpha1-acid glycoprotein (AAG) (1.4 mg/dL) with binding constants of 1.85 x 10(4) M(-1) and 1.13 x 10(5) M(-1) respectively. In whole blood 2.8% of gefitinib existed as the free drug while 79.4% and 17.8% was bound to plasma proteins and blood cells respectively. In plasma from cancer patients fu at pre-treatment varied 2.4-fold (mean 3.4 +/- 0.6%; range 2.2-5.4%) and fu was constant over the 28-days of treatment (P > 0.05). Pre-treatment AAG concentration was negatively correlated with pre-treatment fu (R2 = 0.28, P = 0.01). In conclusion gefitinib is highly protein bound (approximately 97%) in human plasma. Variable AAG concentrations observed in cancer patients may affect gefitinib fu with implications for inter-subject variation in drug toxicity and response.

High-density lipoproteins contribute to the alpha-1-globulin zone in capillary electrophoresis of human serum proteins.

Alpha-1-antitrypsin (AAT) and alpha-1-acid glycoprotein (AAG) are reported to be the main proteins contributing to the alpha-1-globulin capillary zone electrophoresis (CZE) zone, but the sum (AAT + AAG) showed lower than the alpha-1-globulin. We investigated the role of high-density lipoprotein (HDL), an additional protein migrating in the alpha-1-globulin zone, as a possible cause for such a gap. In a set of 98 sera we measured the alpha-1-globulin with a dedicated clinical capillary electrophoresis system, and AAT, AAG and apolipoprotein A-1 (ApoA) by immunonephelometry. The alpha-1-globulin were consistently higher than the sum (AAT + AAG), by (mean value +/- standard deviation) 1.70 +/- 0.88 g/L in 49 sera with low ApoA, and by 3.59 +/- 0.75 g/L in 49 sera with high ApoA. Corresponding figures in the comparison alpha-1-globulin/(AAT + AAG + ApoA) were reduced to 1.08 +/- 0.77 g/L and 1.67 +/- 0.70 g/L. It is concluded that HDL significantly contribute to the CZE alpha-1-globulin zone, but do not completely explain the differences between the electrophoretic and the immunochemical measurements. However, CZE alpha-1-globulin measurements give information about increases of the two major acute phase proteins comparable to specific protein measurements.

Contributions of CYP3A4, P-glycoprotein, and serum protein binding to the intestinal first-pass extraction of saquinavir.

Using CYP3A4-expressing Caco-2 cell monolayers, we assessed the roles of CYP3A4-mediated metabolism, P-glycoprotein (P-gp)-mediated efflux, and serum protein binding in determining the extent of the intestinal first-pass extraction (E(i)) of saquinavir. Saquinavir (5-40 microM) was added to the apical compartment of culture inserts. After 3 h, apical and basolateral media and cell scrapings were analyzed for saquinavir and a major CYP3A4-mediated metabolite (M7). The intracellular concentration of saquinavir was estimated from the degree of inhibition of CYP3A4 catalytic activity (midazolam 1'-hydroxylation). Compared with vehicle, the P-gp inhibitor LY335979 (zosuquidar trihydrochloride) (0.5 microM, apical) increased saquinavir cell content and M7 formation rate, but decreased the E(i) by approximately 50% due to a >90% increase in the amount of saquinavir recovered in the basolateral compartment. Compared with LY335779, physiological concentrations of basolateral serum proteins [human serum albumin and alpha1-acid glycoprotein (AAG)] increased saquinavir permeability by a similar degree but decreased the E(i) by approximately 50% due to a marked reduction in M7 formation. Increasing AAG concentration (1.0-2.5 g/l) had no additional effect on permeability or E(i). An estimate of the range of the E(i) of saquinavir (7-60%) was less than has been predicted based on in vitro data (>99%) but was consistent with a clinical study involving grapefruit juice. The incidental finding of greater M7 formation after basolateral compared with apical dosing could not be explained by differences in saquinavir cell content. We conclude that variable intestinal first-pass extraction of saquinavir in human immunodeficiency virus-infected patients could reflect variation in P-gp-mediated efflux and/or CYP3A4-catalyzed metabolism, but not in blood AAG levels.

Investigating the ultrastructure of fibrous long spacing collagen by parallel atomic force and transmission electron microscopy

The ultrastructure of fibrous long spacing (FLS) collagen fibrils has been investigated by performing both atomic force microscopy (AFM) and transmission electron microscopy (TEM) on exactly the same area of FLS collagen fibril samples. These FLS collagen fibrils were formed in vitro from type I collagen and alpha1-acid glycoprotein (AAG) solutions. On the basis of the correlated AFM and TEM images obtained before and after negative staining, the periodic dark bands observed in TEM images along the longitudinal axis of the FLS collagen fibril correspond directly to periodic protrusions seen by AFM. This observation is in agreement with the original surmise made by Gross, Highberger, and Schmitt (Gross J, Highberger JH, Schmitt FO, Proc Natl Acad Sci USA 1954;40:679-688) that the major repeating dark bands of FLS collagen fibrils observed under TEM are thick relative to the interband region. Although these results do not refute the idea of negative stain penetration into gap regions proposed by Hodge and Petruska (Petruska JA, Hodge AJ. Aspects of protein structure. Ramachandran GN, editor. New York: Academic Press; 1963. p. 289-300), there is no need to invoke the presence of gap regions to explain the periodic dark bands observed in TEM images of FLS collagen fibrils.

Assessment of nutritional status and prognosis in advanced cancer: interleukin-6, C-reactive protein, and the prognostic and inflammatory nutritional index

The Prognostic Inflammatory Nutritional Index (PINI) is a simple scoring system that has been used to evaluate nutritional status and prognosis in critically ill patients. The PINI has never been evaluated in advanced cancer. Fifty consecutive patients with advanced cancer, weight loss, and anorexia were studied. C-reactive protein (CRP), albumin, pre-albumin, interleukin-6 (IL-6), and alpha 1-acid glycoprotein (AAG) were evaluated. The individual values for AAG, CRP, and IL-6 were markedly elevated. In contrast to albumin and prealbumin, CRP levels were very high. The PINI was significantly elevated, and higher than reported in critically ill intensive care patients. Elevated IL-6 levels correlated with high PINI and CRP values. CRP, IL-6, and PINI should be considered in future research on nutritional status and prediction of prognosis in advanced cancer.

Pharmacokinetics and pharmacodynamics of vecuronium in children receiving phenytoin or carbamazepine for chronic anticonvulsant therapy

The pharmacokinetics and time course of action of vecuronium in normal children and children receiving anticonvulsant drugs for prolonged periods were characterized. A bolus dose of vecuronium 0.15 mg kg(-1) was administered i.v. to 10 non-epileptic children and to 10 children on phenytoin and 10 children on carbamazepine, who were matched for age and weight. Plasma concentrations of vecuronium, 3-OH desacetylvecuronium (the primary metabolite of vecuronium) and alpha1-acid glycoprotein (AAG) were determined. Pharmacokinetic variables were derived from plasma samples collected before and after administration of vecuronium. Neuromuscular transmission was monitored by evoked compound electromyography. Recovery of the first twitch of the train-of-four (T1/T0) and the recovery index (RI), the time for 25-75% recovery of T1/T0, were determined. The elimination half-life of vecuronium was significantly reduced in both anticonvulsant groups compared with control [control 48.2 (SD 40.3), phenytoin 23.5 (13.1), carbamazepine 18.4 (16.6) min, P<0.05]. Vecuronium clearance was increased in both anticonvulsant groups [control 9.0 (3.6), phenytoin 15.1 (8.9), carbamazepine 18.8 (13.1) ml kg(-1) min(-1), 0.05<P<0.1]. Children on chronic anticonvulsant therapy had a significantly shorter RI than control [control 21.8 (11), phenytoin 12.5 (8.3), carbamazepine 10.6 (5.9) min, P<0.05]. Concentrations of vecuronium at different degrees of recovery of T1, volumes of distribution and AAG concentrations were not different between groups. Our data confirm anticonvulsant-induced resistance to vecuronium in children and support a pharmacokinetic component contributing to the resistance.

Factors influencing the protein binding of a new phosphodiesterase V inhibitor, DA‐8159, using an equilibrium dialysis technique

Various factors influencing the protein binding of DA-8159 to 4% human serum albumin (HSA) were evaluated using an equilibrium dialysis technique at an initial DA-8159 concentration of 5 microg/mL. It took approximately 8 h incubation to reach an equilibrium between 4% HSA and an isotonic phosphate buffer of pH 7.4 containing 3% of dextran ('the buffer') using a Spectra/Por 2 membrane (mol. wt. cut-off: 12,000--14,000) in a water bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. The extent of binding was dependent on DA-8159 concentrations, HSA concentrations, incubation temperature, buffer pH, and alpha-1-acid glycoprotein (AAG) concentrations. The binding of DA-8159 in heparinized human plasma (93.9%) was significantly higher than in rats (81.4%), rabbits (80.4%), and dogs (82.2%), and this could be due to differences in AAG concentrations in plasma.