Human Serum Protein Binding Research Articles

Chemical Functionalization of Bioceramics To Enhance Endothelial Cells Adhesion for Tissue Engineering

To control the selective adhesion of human endothelial cells and human serum proteins to bioceramics of different compositions, a multifunctional ligand containing a cyclic arginine-glycine-aspartate (RGD) peptide, a tetraethylene glycol spacer, and a gallate moiety was designed, synthesized, and characterized. The binding of this ligand to alumina-based, hydroxyapatite-based, and calcium phosphate-based bioceramics was demonstrated. The conjugation of this ligand to the bioceramics induced a decrease in the nonselective and integrin-selective binding of human serum proteins, whereas the binding and adhesion of human endothelial cells was enhanced, dependent on the particular bioceramics.

Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface

The human commensal bacterium Staphylococcus aureus is renowned as a causative agent of severe invasive diseases. Upon entering the bloodstream, S. aureus can infect almost every tissue and organ system in the human body. To withstand insults from the immune system upon invasion, several immune-evasive mechanisms have evolved in S. aureus, such as complement inhibition by secreted proteins and IgG-binding by surface-exposed protein A. While it is generally accepted that S. aureus cells bind a range of host factors for various purposes, no global analyses to profile staphylococcal host factor binding have so far been performed. Therefore, we explored the possibility to profile the binding of human serum proteins to S. aureus cells by "surface shaving" with trypsin and subsequent MS analysis of liberated peptides. This resulted in the identification of several components of the complement system, the platelet factor 4 and the isoform 1 of the inter-α-trypsin inhibitor heavy chain H4 on the staphylococcal cell surface. We conclude that surface shaving is a versatile tool to profile global interactions between human serum proteins and the S. aureus cell surface.

Binding of Human Serum Proteins to Titanium Dioxide Particles In Vitro

To determine the capacity of human serum proteins to bind to titanium dioxide (TiO(2)) particles of different polymorphs and sizes. TiO(2) particles were mixed with diluted human serum, purified human serum albumin (HSA) or purified human serum gamma-globulin (HGG) solutions. After incubation at 37°C for 1 h, the particles were sedimented by centrifugation, and proteins in the supernatant, as well as those bound to the particles, were analyzed. The total protein concentration in the supernatant was lowered by TiO(2), whereas the albumin/globulin ratio was elevated by the particles. Incubation with TiO(2) also lowered the immunoglobulin, pre-albumin, beta2-microglobulin, ceruloplasmin and retinol-binding protein levels, but not ferritin levels, in the supernatant. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins in the supernatant, especially HGG, were observed to decrease, while those released from the particles (after adding 1% SDS and heating) increased, depending on the dose of TiO(2). Purified HGG and HSA were also bound to TiO(2), although the former appeared to have a higher affinity. All the proteins tested showed the highest binding potency to the amorphous particles (<50 nm) and the lowest to the rutile particles (<5,000 nm), while binding to anatase particles was intermediate. The affinity to the larger anatase was higher than that to smaller anatase particles in most cases. Human serum proteins, including the two major components, HSA and HGG, are bound by TiO(2) particles. The polymorph of the particles seems to be important for determining the binding capacity of the particles and it may affect distribution of the particles in the body.

In Vitro Characterization of GS-8374, a Novel Phosphonate-Containing Inhibitor of HIV-1 Protease with a Favorable Resistance Profile

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4(+) T cells (50% effective concentration [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC(50)s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

(1S)-1,5-Anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-d-glucitol (TS-071) is a Potent, Selective Sodium-Dependent Glucose Cotransporter 2 (SGLT2) Inhibitor for Type 2 Diabetes Treatment

Derivatives of a novel scaffold, C-phenyl 1-thio-D-glucitol, were prepared and evaluated for sodium-dependent glucose cotransporter (SGLT) 2 and SGLT1 inhibition activities. Optimization of substituents on the aromatic rings afforded five compounds with potent and selective SGLT2 inhibition activities. The compounds were evaluated for in vitro human metabolic stability, human serum protein binding (SPB), and Caco-2 permeability. Of them, (1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol (3p) exhibited potent SGLT2 inhibition activity (IC(50) = 2.26 nM), with 1650-fold selectivity over SGLT1. Compound 3p showed good metabolic stability toward cryo-preserved human hepatic clearance, lower SPB, and moderate Caco-2 permeability. Since 3p should have acceptable human pharmacokinetics (PK) properties, it could be a clinical candidate for treating type 2 diabetes. We observed that compound 3p exhibits a blood glucose lowering effect, excellent urinary glucose excretion properties, and promising PK profiles in animals. Phase II clinical trials of 3p (TS-071) are currently ongoing.

Immunocompatibility properties of lipid–polymer hybrid nanoparticles with heterogeneous surface functional groups

Here we report the immunological characterization of lipid–polymer hybrid nanoparticles (NPs) and propose a method to control the levels of complement activation induced by these NPs. This method consists of the highly specific modification of the NP surface with methoxyl, carboxyl, and amine groups. Hybrid NPs with methoxyl surface groups induced the lowest complement activation, whereas the NPs with amine surface groups induced the highest activation. All possible combinations among carboxyl, amine, and methoxyl groups also activated the complement system to a certain extent. All types of NPs activated the complement system primarily via the alternative pathway rather than the lectin pathway. The classical pathway was activated to a very small extent by the NPs with carboxyl and amine surface groups. Human serum and plasma protein binding studies showed that these NPs had different protein binding patterns. Studies of both complement activation and coagulation activation suggested that NPs with methoxyl surface groups might be an ideal candidate for drug delivery applications, since they are not likely to cause any immunological adverse reaction in the human body.

2-(1H-Imidazol-4-yl)ethanamine and 2-(1H-pyrazol-1-yl)ethanamine side chain variants of the IGF-1R inhibitor BMS-536924

A series of IGF-1R inhibitors is disclosed, wherein the (m-chlorophenyl)ethanol side chain of BMS-536924 (1) is replaced with a series of 2-(1H-imidazol-4-yl)ethanamine and 2-(1H-pyrazol-1-yl)ethanamine side chains. Some analogs show improved IGF-1R potency and oral exposure. Analogs from both series, 16a and 17f, show in vivo activity comparable to 1 in our constitutively activated IGF-1R Sal tumor model. This may be the due to the improved protein binding in human and mouse serum for imidazole 16a and the excellent oral exposure of pyrazole 17f.

QSAR Modeling of Human Serum Protein Binding with Several Modeling Techniques Utilizing Structure−Information Representation

Four modeling techniques, using topological descriptors to represent molecular structure, were employed to produce models of human serum protein binding (% bound) on a data set of 1008 experimental values, carefully screened from publicly available sources. To our knowledge, this data is the largest set on human serum protein binding reported for QSAR modeling. The data was partitioned into a training set of 808 compounds and an external validation test set of 200 compounds. Partitioning was accomplished by clustering the compounds in a structure descriptor space so that random sampling of 20% of the whole data set produced an external test set that is a good representative of the training set with respect to both structure and protein binding values. The four modeling techniques include multiple linear regression (MLR), artificial neural networks (ANN), k-nearest neighbors (kNN), and support vector machines (SVM). With the exception of the MLR model, the ANN, kNN, and SVM QSARs were ensemble models. Training set correlation coefficients and mean absolute error ranged from r2=0.90 and MAE=7.6 for ANN to r2=0.61 and MAE=16.2 for MLR. Prediction results from the validation set yielded correlation coefficients and mean absolute errors which ranged from r2=0.70 and MAE=14.1 for ANN to a low of r2=0.59 and MAE=18.3 for the SVM model. Structure descriptors that contribute significantly to the models are discussed and compared with those found in other published models. For the ANN model, structure descriptor trends with respect to their affects on predicted protein binding can assist the chemist in structure modification during the drug design process.

Dasatinib (BMS-354825) Overcomes Multiple Mechanisms of Imatinib Resistance in Chronic Myeloid Leukemia (CML).

Resistance to imatinib is a growing concern in CML, particularly in advanced disease. The most common cause of resistance is mutations in BCR-ABL, but other mechanisms have also been identified, including over-expression of BCR-ABL, activation of SRC family kinases and the P-glycoprotein (PGP) efflux pump (via MDR1 over-expression). Dasatinib (BMS-354825) is a novel, oral, multi-targeted tyrosine kinase inhibitor that targets BCR-ABL and SRC kinases. Dasatinib has 325-fold greater potency versus imatinib in cell lines transduced with wild-type BCR-ABL and is active against 18 out of 19 BCR-ABL mutations tested that confer imatinib resistance (Shah et al, Science 305:399, 2004; O'Hare et al, Cancer Res 65:4500–5, 2005), and preliminary results from a Phase I study show that it is well tolerated and has significant activity in imatinib-resistant patients in all phases of CML (Sawyers et al, J Clin Oncol 23:565s, 2005; Talpaz et al, J Clin Oncol 23:564s, 2005). We assessed the ability of dasatinib to overcome a variety of mechanisms of imatinib resistance. First, the leukemic-cell killing activity of dasatinib was tested in vitro in three human imatinib-resistant CML cell lines (K562/IM, MEG-01/IM and SUP-B15/IM). Based on IC50 values, dasatinib had >1000-fold more potent leukemic-cell killing activity compared with imatinib versus all three cell lines. Furthermore, in mice bearing K562/IM xenografts, dasatinib was curative at doses >5 mg/kg, while imatinib had little or no impact at doses as high as 150 mg/kg, its maximum tolerated dose. We determined that the MEG-01/IM and SUP-B15/IM cell lines carried BCR-ABL mutations known to confer imatinib resistance to imatinib clinically (Q252H and F359V, respectively). In K562/IM cells, BCR-ABL mutations or BCR-ABL over-expression were not detected, but the SRC family member FYN was over-expressed. PP2, a known inhibitor of SRC family kinases but not BCR-ABL, could reverse the imatinib resistance in these cells. Together, these data suggest that activation of FYN may be a cause of imatinib resistance in K562/IM. Based on cell proliferation IC50, we found that the anti-leukemic activity of dasatinib in K562/IM cells was 29-fold more potent compared with AMN107 (a tyrosine kinase inhibitor that inhibits BCR-ABL but not SRC family kinases). Given that the human serum protein binding of dasatinib, imatinib and AMN107 were 93, 92 and >99% respectively, the difference in potency between dasatinib and AMN107 in vivo may be far greater than the simple fold-difference in the in vitro IC50 values. Finally, in K562 cells over-expressing PGP (K562/ADM), we found that dasatinib was only 6-fold less active than in parental K562 cells. Because of the extreme potency of dasatinib in K562 cells, this reduced potency still afforded an IC50 of 3 nM, which is readily achievable in vivo. Indeed, in mice bearing K562/ADM xenografts, dasatinib was curative at 30 mg/kg, with significant anti-leukemic activity at 15 mg/kg. In conclusion, the rational design of dasatinib as a multi-targeted kinase inhibitor allows this agent to overcome a variety of mechanisms of resistance to imatinib in CML, including mechanisms that are not overcome by agents with a narrower spectrum of inhibition, such as AMN107. Dasatinib is currently in Phase II evaluation in imatinib-resistant/-intolerant patients in the ‘START' program, and in Phase I evaluation in solid tumors.

N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent.

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 [N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, BMS-387032], has been identified as an ATP-competitive and CDK2-selective inhibitor which has been selected to enter Phase 1 human clinical trials as an antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC(50) = 48 nM and was 10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an A2780 cellular cytotoxicity assay in which 21 showed an IC(50) = 95 nM. Metabolism and pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor model and in a s.c./i.p. A2780 human ovarian carcinoma xenograft model.

Human serum opsonization of orthopedic biomaterial particles: protein-binding and monocyte/macrophage activation in vitro.

Wear particles generated after total joint arthroplasty activate monocyte/macrophages and incite formation of a granulomatous periprosthetic tissue associated with bone loss and implant loosening. This study tested the hypothesis that selective opsonization of orthopedic implant biomaterial wear particles by human serum proteins influences monocyte/macrophage activation. Serum protein binding to metallic, polymeric, and ceramic particles was determined by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Individual proteins bound to particles were subsequently identified using two-dimensional SDS-PAGE, microsequencing techniques, and SWISS-PROT analysis. Effects of selective protein opsonization on particle-induced monocyte/macrophage activation were assessed by quantification of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha release. Results from one-dimensional gel analyses revealed distinct serum protein-binding patterns specific for each material tested. Two-dimensional gel analysis together with amino acid sequencing of the prominent protein species confirmed the presence of albumin and alpha-1-antitrypsin bound to all particles tested. In contrast to the metallic particles, apolipoprotein was a major species associated with polymeric particles. Opsonization of PMMA particles with purified preparations of each of the identified proteins showed that albumin significantly enhanced particle-induced monocyte/macrophage activation. These data confirm orthopedic biomaterial specific binding of human serum proteins and demonstrate that albumin exacerbates particle-induced monocyte/macrophage activation. Alterations in the chemical and surface properties of orthopedic biomaterials to modulate protein interactions may improve implant longevity.

Serum protein binding of tolterodine and its major metabolites in humans and several animal species.

The aim of this study was to determine in vitro protein binding of tolterodine and its 5-hydroxymethyl (5-HM) and N-dealkylated metabolites in serum from humans and several animal species at concentrations similar to those obtained in clinical and preclinical studies. Binding of tolterodine and the two metabolites to human serum albumin and alpha1-acid glycoprotein (AAG) was also assessed, as was binding of tolterodine to red blood cells. Ex vivo protein binding of tolterodine and 5-HM was determined in serum samples from healthy volunteers treated with oral tolterodine 4 mg twice daily for 8 days. Tolterodine exhibited high protein binding in human serum; the unbound fraction (f(u)) was 3.7%. The unbound fraction of tolterodine in cat and dog serum (1.5 and 2.1%, respectively) was lower compared with human serum; f(u) was higher in the other species investigated (rat, 22%; mouse, 16-17%; rabbit, 39%). The unbound fraction of 5-HM was much higher in serum from humans (36%) and all animal species investigated (mouse, 72%; rabbit, 68%; cat, 32%; dog, 45%). Binding of N-dealkylated tolterodine to proteins in human serum was intermediate (f(u) 14%). AAG was the major binding protein for tolterodine and 5-HM, and the degree of binding increased with increasing concentration of the protein. The association constant of 5-HM for AAG was lower than that of tolterodine (1.3 x 10(5) M(-1) versus 2.1 x 10(6) M(-1)). The blood:plasma tolterodine concentration ratio was 0.6 in both humans and dog; thus, a minor fraction of tolterodine was present in red blood cells compared with plasma (0.18 and 0.36, respectively). In the mouse, tolterodine was equally present in blood and plasma. In ex vivo samples, f(u) values for tolterodine (pH adjusted) varied between 1.6 and 4.9% (mean 2.8%), which could be explained by differences in AAG concentrations. There was good correlation between observed f(u) values for tolterodine and those predicted on the basis of AAG levels. Similar findings were observed for 5-HM.

Serum protein binding of histamine H 1-antagonists. A comparative study on the serum protein binding of a sedating ([ 3H]mepyramine) and a non-sedating H 1-antagonist ([ 3H]loratadine)

Nowadays classical histamine H 1-receptor antagonists (antihistamines) are relatively rarely used in therapy against allergic disorders, mainly because they are able to penetrate the central nervous system (CNS) and induce subsequently unwanted side effects (most importantly sedation). In contrast, several modern H 1-antagonists have been claimed not to penetrate the CNS and lack the sedating properties. In this study, human serum protein binding, one of the properties which may influence brain penetration of H 1-antagonists, has been investigated by comparing the sedating H 1-antagonist [ 4H]mepyramine with the non-sedating H 1-antagonist [ 1H]loratadine. It is shown that human serum albumin (HSA) binding and total serum protein binding of [ 1H]mepyramine is low at therapeutic levels and at physiological pH (<30% bound) and strongly depends on the ionization state of this compound (p K d = 8.92). Our results demonstrate tha the unprotonated form of mepyramine in particular binds to serum proteins. This conclusion is supported by further results from plain HSA binding experiments, in this study, on six other unlabeled. ‘classical’ H 1-antagonists (i.e., diphenhydramine, chlorpheniramine, dimethindene, imipramine, azatadine, descarbocthoxyloratadine). On the other hand, the modern non-sedating H 1-antagonist loratadine (log P oct=4.40) binds to a high level to serum proteins at therapeutic levels (>95% bound). Our results further indicate that the main determinants of HSA binding for H 1-antagonists are lipophilicity and acid-base properties as expressed by log P and p K d, re (both included in the parameter log D oct). However, hydrogen bonding capacity ( Δlog P) is not correlated to serum protein bin series of H 1-antagonists investigated. We further propose that highly lipophilic non-sedating H 1-antagonists (i.e., loratadine, terfenadine, astemizole, temelastine) probably have a low extraction ratio in the brain due to the tight lipoid blood brain barrier and a relatively high extraction ratio in the porous peripheral tissues.

Identification of the bile acid binding proteins in human serum by photoaffinity labeling

The binding of conjugated and unconjugated bile acids to human serum lipoproteins was investigated by density gradient centrifugation and photoaffinity labeling studies. The binding of bile acids to high-density lipoprotein increased by substitution of the 3α-hydroxy group in cholate and taurocholate by a photolabile 3-azido or 3-azi-function. The affinity of bile acid derivatives to HDL showed the following ranking: 3β-azido-7α,12α-dihydroxy-,3,3-azo-7α,12α-dihydroxy- > 3α,7α,12α-trihydroxy-,11ζ-azido-3α,7α,12ζ-trihydroxy- > 11ζ;-azido-12-oxo-3α,7α-dihydroxy- > 7,7-azo-3α,12α-dihydroxy-,3α,7α-dihydroxy-,3α,12α-dihydroxy- > 3 a-hydroxy-cholan-24-oic acid. Based on the actual serum concentrations of albumin and HDL, a preference of hydrophilic bile acids to HDL is evident, the 3-azido- and 3-azi-derivatives showing a 5–23-fold higher binding to HDL compared to soluble serum proteins. For the identification of the bile acid binding proteins in human blood, photoaffinity labeling with a variety of photolabile conjugated and unconjugated bile acid derivatives was performed with subsequent analysis of radiolabeled serum proteins by one- and two-dimensional gel electrophoresis. In addition to albumin and the apolipoproteins A-I and A-II of high-density lipoproteins (Kramer et al. (1979) Eur. J. Biochem. 102, 1–9), three further proteins in the lipoprotein free serum fraction of M r 41000, 50 000 and 83 000 were specifically labeled. By two-dimensional electrophoresis and by immunoprecipitation these proteins were identified as α 1-acid glycoprotein ( M r 41 000), α 1-antitrypsin ( M r 50000) and transferrin ( M r 83 000). No binding of bile acids to haptoglobin, α 2-HS-glycoprotein, hemopexin or α 1-fetoprotein occurred. In conclusion, these studies show that bile acid derivatives bind to several serum proteins in addition to albumin and furthermore that the substituent in position 3 of the steroid nucleus greatly influences the affinity of bile acids to high density lipoproteins.

Circulating levels of IGFs and IGF binding proteins in human cord serum: relationships to intrauterine growth.

Cord sera were obtained from 44 term, human infants exhibiting various patterns of intrauterine growth and were assayed for IGF-1, IGF-2, and IGFBP-1, 2, and 3 by specific RIAs. Serum levels were correlated with birth weight (BW), ponderal index (PI), and placental weight (PW). Total IGF-1 levels correlated significantly with BW (r = 0.392), PW (r = 0.351), and PI (r = 0.481). By contrast, the correlation of IGF-2 with birth weight was not statistically significant (r = 0.264, P = 0.091). The association of IGF-2 with PI, however, was significant (r = 3.348, P = 0.024). IGFBP-3 exhibited significant correlations with BW, PI, and PW, similar to those seen with IGF-1. IGFBP-1 and IGFBP-2, however, were not significantly related to growth parameters. IGF-1 levels correlated strongly with IGFBP-3 levels (r = 0.646, P = 0.001). By contrast, IGF-1 correlated with the reciprocal of both IGFBP-1 and IGFBP-2. Based upon in vitro affinity constants, theoretical concentrations for each [IGF/IGFBP] complex, free IGFs, and free IGFBPs were calculated for each infant. Multiple regression analysis was performed including all 11 calculated variables and correlated with each growth parameter. This analysis revealed that an integrated expression of IGF activity exhibited stronger correlations with growth than each individual peptide species (BW, r = 0.681; PI, r = 0.660; PW, r = 0.658). These data further support roles for IGF related peptides (IGFRPs) in human fetal and placental growth and suggest regulatory/counterregulatory roles for the IGFBPs. It also supports the hypothesis that individual IGFRPs interact in a complex manner to define 'net IGF activity' in relation to fetal growth and/or metabolic status.

フェニトイン，バルプロ酸，サリチル酸とセフピラミドのｉｎ ｖｉｔｒｏ血清蛋白結合における相互作用

Cefpiramide (CPM) is a third-generation cephem antibiotic characterized by its higher protein binding percentage, higher therapeutic concentration and longer biological half-life. We studied drug interactions of human serum protein binding between CPM and phenytoin (PHT), valproic acid (VPA) and salicylic acid (SA) with higher protein binding percentages. When CPM and SA were added to serum concomitantly, the percentages of the unbound concentration (free percent) of both drugs increased significantly over that of CPM or SA alone. However, when CPM and PHT were added similarly and also when CPM and VPA added in the same way, the free percentages of both drugs did not change significantly. These results confirmed the necessity to pay close attention to the interaction of serum protein binding between CPM and SA because increases in the free percentages of both drugs may adversely influence the clinical effects and therapeutic safety.

Rates of dissociation of sex steroid hormones from human sex hormone-binding globulin: A reassessment

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure the rates of dissociation of dihydrotestosterone (DHT), testosterone (T), and estradiol (E 2) from their binding proteins in human serum at 37°C. Because measurements were begun after each hormone had fully (>99%) dissociated from albumin, the observed rates of dissociation correspond to the rates of dissociation of the sex hormone-binding globulin (SHBG)-hormone complexes. The dissociation rate constants of the hormone-SHBG complexes were determined to be 0.016±0.001, 0.056±0.002, and 0.083±0.003 s −1 for DHT, T, and E 2, respectively, corresponding to half-times of dissociation ( t 1 2 ) of 43, 12 and 8.4 s, respectively. The physiological significance of these findings can best be appreciated by comparing these t 1 2 s with the capillary and sinusoidal transit times of various tissues (<1 s to ∼ 10 s).

Insulin-Like Growth Factor Binding Proteins in Maternal Serum Throughout Gestation and in the Puerperium: Effects of a Pregnancy-Associated Serum Protease Activity*

The cDNAs encoding three major insulin-like growth factor-binding proteins (IGFBPs) have been cloned and sequenced. We have examined, by Western ligand blotting, the profiles of these binding proteins in human female serum in the normal menstrual cycle, throughout pregnancy, and during the postpartum period. There was no change in the serum profile of any of the binding proteins in early pregnancy compared to that in the secretory phase of the menstrual cycle. However, there was a marked decrease in circulating levels of the main serum IGFBP, IGFBP-3, after 6 weeks of gestation, continuing progressively to term and returning to nonpregnant levels by 5 days postpartum. IGFBP-2 decreased steadily throughout gestation. In contrast, IGFBP-1 levels were found to rise by the second trimester. Endoglycosidase-F digestion did not enhance detection of IGFBP-3 by ligand blotting. Immunoprecipitations with two separate antibodies against IGFBP-3 and IGFBP-2, followed by Western ligand blotting, confirmed the marked decrease in IGFBP-3 levels after 6 weeks of gestation and the more gradual decrease in IGFBP-2. In contrast, immunoprecipitations with IGFBP-1 monoclonal antibodies confirmed the increase in IGFBP-1 during gestation. Endogenous serum IGFs were separated from serum IGFBPs by acid chromatography, and an 80% decrease in total IGF-binding activity in the IGFBP fraction of chromatographed pregnancy vs. nonpregnancy serum was detected by charcoal absorption assay. Furthermore, immunoprecipitations of IGF affinity cross-linked IGFBP fractions with IGFBP-3-specific antiserum confirmed a marked diminution of IGFBP-3 in pregnancy compared to nonpregnancy serum, and revealed, only in pregnancy serum, the concomitant appearance of a band with a mol wt of 34K and three less intense bands with mol wt between 20-26K on sodium dodecyl sulfate gels. Incubation of nonpregnancy serum with 6-week pregnancy serum at 37 C for 5 h, followed by Western ligand blotting, showed only a slight reduction in the amount of IGFBP-3 in the mixture compared to that in controls. However, incubation of term pregnancy with nonpregnancy serum at 37 C for 5 h revealed a marked reduction of IGFBP-3 in the mixture. When iodinated recombinant IGFBP-3 was incubated with term pregnancy serum under the same conditions, the appearance of a 29K protein was identified by gel electrophoresis and autoradiography, along with three less intense bands with mol wt between 17-22K.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro activity of LY 264826 compared with that of vancomycin against 100 clinical isolates each of methicillin-resistant Staphylococcus aureus and Clostridium difficile

The in vitro activity of LY 264826, a new glycopeptide antibiotic, was compared with that of vancomycin against 100 strains each of methicillin-resistant Staphylococcus aureus and Clostridium difficile. LY 264826 was more active, by weight, than vancomycin against the isolates tested. The human serum protein binding of LY 264826 was 15.3% (range, 9.8 to 21.8%).

Characterization of Insulin-Like Growth Factor-Binding Proteins from Sheep Thyroid Cells*

The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins antigenically related to BP-53.