A method for producing peptide maps of picomolar quantities of protein labelled with the Bolton-Hunter reagent in polyacrylamide gel

A method of radioiodination to high specific activity by Bolton Hunter reagent of fixed and stained histones in polyacrylamide gel is described. The comparative peptide maps of the histones H1 and H3, labelled in solution and the gel are presented.     

Route-dependent stereoselective pharmacokinetics of tramadol and its active O-demethylated metabolite in rats

The effects of route of administration on the stereoselective pharmacokinetics of tramadol (T) and its active metabolite (M1) were studied in rats. A single 20 mg/kg dose of racemic T was administered through intravenous, intraperitoneal, or oral route to different groups of rats, and blood and urine samples were collected. Samples were analyzed using chiral chromatography, and pharmacokinetic parameters (mean +/- SD) were estimated by noncompartmental methods. Following intravenous injection, there was no stereoselectivity in the pharmacokinetics of T. Both enantiomers showed clearance values (62.5 +/- 27.2 and 64.4 +/- 39.0 ml/min/kg for (+)- and (-)-T, respectively) that were equal or higher than the reported liver blood flow in rats. Similar to T, the area under the plasma concentration-time curves (AUCs) of M1 did not exhibit stereoselectivity after intravenous administration of the parent drug. However, the systemic availability of (+)-T was significantly (P < 0.05) higher than that of its antipode following intraperitoneal (0.527 +/- 0.240 vs. 0.373 +/- 0.189) and oral (0.307 +/- 0.136 vs. 0.159 +/- 0.115) administrations. The AUC of the M1 enantiomers, on the other hand, remained mostly nonstereoselective regardless of the route of administration. Pharmacokinetic analysis indicated that the stereoselectivity in the pharmacokinetics of oral T is due to stereoselective first pass metabolism in the liver and, possibly, in the gastrointestinal tract. The direction and extent of stereoselectivity in the pharmacokinetics of T and M1 in rats were in agreement with those previously reported in humans, suggesting that the rat may be a suitable model for enantioselective studies of T pharmacokinetics.     

Iodotyrosylation of peptides using tertiary-butyloxycarbonyl-l-[125I]iodotyrosine N-hydroxysuccinimide ester

A rapid method for the iodotyrosylation of peptides using tertiary-butyloxycarbonyl-l-[¹²⁵I]-iodotyrosine N-hydroxysuccinimide ester is described. The method, like that of Bolton and Hunter (1973, Biochem. J.133, 529–539), uses nonoxidizing conditions, is applicable to tyrosine-free peptides, and results in an easily isolated product with decreased cationic charge. The present method has the additional advantage that the derivatized peptide is readily deblocked to form a radiolabeled product with the same net charge as the starting material.     

IgG‐single chain Fv fusion protein therapeutic for alzheimer's disease: Expression in CHO cells and pharmacokinetics and brain delivery in the rhesus monkey

Monoclonal antibodies (MAb) directed against the Abeta amyloid peptide of Alzheimer's disease (AD) are potential new therapies for AD, since these antibodies disaggregate brain amyloid plaque. However, the MAb is not transported across the blood–brain barrier (BBB). To enable BBB transport, a single chain Fv (ScFv) antibody against the Abeta peptide of AD was re‐engineered as a fusion protein with the MAb against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the ScFv therapeutic antibody across the BBB. Chinese hamster ovary (CHO) cells were stably transfected with a tandem vector encoding the heavy and light chains of the HIRMAb–ScFv fusion protein. A high secreting line was isolated following methotrexate amplification and dilutional cloning. The HIRMAb–ScFv fusion protein in conditioned serum‐free medium was purified by protein A affinity chromatography. The fusion protein was stable as a liquid formulation, and retained high‐affinity binding of both the HIR and the Abeta amyloid peptide. The HIRMAb–ScFv fusion protein was radiolabeled with the ¹²⁵I‐Bolton–Hunter reagent, followed by measurement of the pharmacokinetics of plasma clearance and brain uptake in the adult Rhesus monkey. The HIRMAb–ScFv fusion protein was rapidly cleared from plasma and was transported across the primate BBB in vivo. In conclusion, the HIRMAb–ScFv fusion protein is a new class of antibody‐based therapeutic for AD that has been specifically engineered to cross the human BBB. Biotechnol. Bioeng. 2010; 105: 627–635. © 2009 Wiley Periodicals, Inc.     

Rat plasma kallikrein: purification, NH2-terminal sequencing and development of a specific radioimmunoassay

Rat plasma kallikrein (rPK) was purified to homogeneity form plasma using affinity and high-performance liquid chromatography techniques, and subjected to NH2-terminal sequencing. The data showed that the sequenced segments of the regulatory (heavy) and catalytic (light) chains of the proteinase, respectively, display 73 and 91% sequence similarity with their counterpart in human plasma kallikrein. This sequence homology in conjunction with the determined molecular structure and inhibitor sensitivity support the identity of the isolated enzyme as plasma kallikrein. A polyclonal antiserum against rPK was obtained after immunization of rabbits with the purified enzyme, and a specific radioimmunoassay was developed. Since Tyr-iodinated rPK was not recognized by the antiserum, two alternative approaches were found to be successful. These included the use of a tracer consisting of rPK modified with either the affinity reagent 125I-labeled DTyr-Glu-Phe-Lys-Arg chloromethyl ketone or with the Bolton Hunter reagent. The usable range of the assay is between 15-150 fmol per tube. The antibody was shown to bind both monomeric and dimeric forms of rPK. Denaturation of the enzyme in sodium dodecyl sulfate does not abolish immune recognition only as long as the regulatory subunit is attached to the catalytic chain. Oxidation or reduction of rPK results in complete loss of immunoreactivity. This observation suggests that perhaps the disulfide linkage of the catalytic and regulatory polypeptides somehow helps to protect the antigenic epitope from denaturation. Alternatively, the epitope(s) recognized by the antibody spans a domain which includes both Tyr and Cys residues necessary for immune recognition.     

A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan.

A major portion of the humoral immune response to peptidoglycans is directed against the non-cross-linked pentapeptide side chains of these ubiquitous bacterial antigens. At present, no specific and sensitive assay for pentapeptide antibody determination is available. Therefore, a radioimmunoassay has been developed which employs the synthetic pentapeptide hapten L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, labeled by the active ester method of Bolton and Hunter to high specific activities (6.74 to 18.18 muCi/mug) with 125I, and used as a reagent for measuring pentapeptide antibody. A-variant streptococcal antisera, known to contain pentapeptide antibodies as shown by quantitative precipitation, would bind more than 95% of the radiolabeled hapten in contrast to 2 to 3% by preimmune rabbit sera. Specificity of the binding reaction was demonstrated by inhibition experiments imploying various synthetic oligopeptides related or unrelated to the pentapeptide in the radioimmunoassay. Binding curves established with serial dilutions of peptidoglycan antiserum were linear from 15 to 500 mug/ml of antibody permitting pentapeptide antibody measurement within this range. Comparative data on pentapeptide antibody determinations by quantitative precipitation and radioimmunoassay are given and the time course of the production of this antibody in 14 rabbits hyperimmunized with A-variant streptococcal vaccine is reported.     

Pharmacokinetics of methyl parathion: a comparison following single intravenous,oral or dermal administration

Assessment of the risks posed by the residential use of methyl parathion requires an understanding of its pharmacokinetics after different routes of exposure. Thus, studies were performed using adult female rats to define the pharmacokinetic parameters for methyl parathion after intravenous injection and to apply the described model to an examination of its pharmacokinetics after single oral or dermal exposure. The pharmacokinetics of methyl parathion after intravenous administration (1.5 mg/kg) were best described by a three-compartment model; the apparent volume of the central compartment was 1.45 liters/kg, clearance was 1.85 liters/h/kg and the terminal half-life was 6.6 h with an elimination constant of 0.50 h(-1). The apparent oral absorption coefficient for methyl parathion (1.5 mg/kg) was 1.24 h(-1), and its oral bioavailability was approximately 20%. The latter likely includes a significant first pass effect. Concentrations of methyl parathion increased during the initial 10-60 min and then declined during the next 15-36 h. After dermal administration (6.25-25 mg/kg), methyl parathion concentrations peaked within 12-26 h and then declined dose dependently. The apparent dermal absorption coefficient was approximately 0.41 h(-1), and only two pharmacokinetic compartments could be distinguished. In conclusion, the pharmacokinetics of methyl parathion are complex and route dependent. Also, dermal exposure, because of sustained methyl parathion concentrations, may pose the greatest risk.     

Synthesis and biological activities of substance P iodinated derivatives

The synthesis of four substance P (SP) derivatives obtained by coupling the monoiodo and diiodo Bolton and Hunter reagents with synthetic SP is described. These compounds were proved to be good ligands for SP antibodies. As seen for SP, they exhibit a high biological activity in the guinea pig ileum bioassay.     

Determination of the renin inhibitor Ro 42-5892 in human plasma by automated pre-column derivatization, reversed-phase high-performance liquid chromatographic separation and electrochemical detection after post-column irradiation

The renin inhibitor Ro 42-5892 has been found to be very potent, thereby necessitating a sensitive assay method for the evaluation of its pharmacokinetics in man. We report here the development of a very sensitive and selective HPLC assay for the analysis of this compound in human plasma. Ro 42-5892 was extracted from plasma with dichloromethane, derivatized with 2,4-dinitrofluorobenzene and then chromatographed on a Novapak C₁₈ column (150 × 3.9 mm I.D.) with acetic acid buffer (pH 7)-acetonitrile (100:85). Detection was performed by irradiation at 254 nm, followed by electrochemical oxidation at 550 mV. The extraction recovery of Ro 92-5 from human plasma (mean 102%) was quantitative. With this method a limit of quantitation of 0.3 ng/ml was achieved. The assay was linear up to 5 ng/ml, had acceptable inter-assay precision (12.2%) and accuracy (9.3%) and was successfully tested for selectivity. This assay was successfully applied to over 250 samples from a pharmacokinetic study in hypertensive patients.     

Receptor-mediated biliary transport of immunoglobulin A and asialoglycoprotein: sorting and missorting of ligands revealed by two radiolabeling methods

In the rat, all receptor-bindable immunoglobulin A (IgA), and 1-4% of injected asialoglycoprotein (ASG), are transported from blood to bile intact. The major fraction of the ASG is degraded in hepatic lysosomes. The study described here was designed to elucidate the sorting that occurs in hepatocytes subsequent to receptor binding of ligands not sharing the same fate. We show that conjugation of protein with the Bolton and Hunter reagent can be used as a probe for the lysosomal pathway, since 50% of the reagent is released into bile after lysosomal degradation of internalized protein. Radiolabeling by iodine monochloride was alternatively used to follow the direct pathways that deliver intact IgA and ASG to bile. After intravenous injection of labeled proteins, first intact ASG and IgA, and then radioactive catabolites from degraded protein, were released into bile. No proteolytic intermediates were detected, and the transport of IgA or ASG directly to bile was not affected by the lysosomal protease inhibitor leupeptin. These observations indicate that divergence of the direct biliary transport pathways from the degradation pathway occurs at a stage preceding delivery to lysosomes, possibly at the cell surface. Competition studies showed that all three pathways (including the biliary transport of intact ASG) are receptor mediated, but even at supersaturating doses the uptake and processing of IgA and ASG occur independently. We propose that IgA and ASG receptors are not frequently in juxtaposition on the plasma membrane, but that ASG, after binding to its receptor, is occasionally missorted into the biliary transport pool.     

Plasma concentrations of 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in Rhesus monkeys after administration by various routes

A method is described for the estimation of 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ by double antibody radioimmunoassay. Plasma samples obtained from animals treated with 9-methylene-16,16-dimethyl-PGE₂,1-adamantanamine salt were extracted with diethyl ether to recover the prostaglandin. The validation of sample preparation and assay procedure are presented. Rhesus females were treated by several routes of administration and the samples assayed for drug content. Maximum blood levels were probably reached 30 minutes following subcutaneous injection and within 30 seconds of an intravenous injection. Results of the acute intravenous injection indicate an initial half-life of approximately one minute in peripheral circulation. Continuous intravenous infusion at 3 increasing doses of this compound resulted in a stepwise increase in plasma drug concentrations. Vaginal administration of 9-methylene-16,16-dimethyl-PGE₂,1-adamantanamine salt in suppositories produced a dose dependent increase in plasma drug concentration. Higher plasma drug concentrations were produced when the prostaglandin was delivered in H-15 base suppositories than in E-76 base suppositories.     

Tissue distribution and plasma clearance of heparin-binding EGF-like growth factor (HB-EGF) in adult and newborn rats

Heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells from various forms of injury in vitro and attenuate intestinal ischemia/reperfusion damage in vivo. With the goal of eventual clinical use of HB-EGF to protect the intestines from injury in neonates, children, and adults, the pharmacokinetics and biodistribution of 125I-labeled HB-EGF were investigated. After intravenous bolus, HB-EGF had a distribution half-life of 0.8 min and an elimination half-life of 26.67 min. After gastric administration, the bioavailability was 7.8%, with a 2.38 h half-life in the absorption phase and an 11.13 h half-life in the elimination phase. After intravenous dosing, most radioactivity was found in the plasma, liver, kidneys, bile, and urine, whereas it was mainly distributed in the gastrointestinal tract after intragastric administration. The degradation of 125I-HB-EGF in plasma from newborn rats was lower than that in adult rats after gastric administration. This supports the feasibility of enteral administration of HB-EGF in the treatment of gastrointestinal diseases, including newborns afflicted with necrotizing enterocolitis.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

Stability-indicating high-performance liquid chromatographic assay of busulfan in aqueous and plasma samples

A sensitive, specific and stability-indicating high-performance liquid chromatographic (HPLC) assay, involving pre-column derivatization and solid-phase extraction (SPE), was developed and validated for the quantitation of busulfan (BU) in aqueous and plasma samples. The linearity of the assay was in the concentration ranges of 0.15–10 μg/ml and 0.15–3 μg/ml for aqueous and plasma samples, respectively. The within-day and between-day variations were 2.90 and 3.31%, respectively, for the aqueous samples, and 9.24 and 14.56%, respectively, for the plasma samples. The overall recovery, derivatization yield and SPE efficiency of BU from plasma samples were 82.03, 108.01 and 86.69%, respectively. Forced degraded samples, either in highly acidic, neutral or basic medium, produced no interfering peaks in the chromatogram. The reported assay requires only 0.2 ml of plasma for the analysis, and its sensitivity is 150 ng/ml by monitoring samples at a wavelength of 254 nm, sufficient to study the plasma pharmacokinetics of BU in rats after a clinically relevant oral dose. Moreover, the sensitivity of the assay can be significantly increased to 30 ng/ml by monitoring samples at a wavelength of 278 nm. The applications of the assay were demonstrated with BU solubility measurements in two aqueous systems and with plasma samples from a Sprague–Dawley rat for an in vivo pharmacokinetic study. In addition, the assay has been employed in the development of a patented intravenous formulation, and in evaluations of stability, preclinical pharmacokinetics in rats and dogs, and clinical phase I trial of the formulation. The assay is readily adaptable to clinical therapeutic drug monitoring.     

Radioimmunoassay for a novel lignan-related hypocholesterolemic agent, S-8921, in human plasma after high-performance liquid chromatography purification and in human urine after immunoaffinity extraction

The novel compound methyl-1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) has hypocholesterolemic activity in animals and is expected to exhibit a similar activity in human. Reversed-phase high-performance liquid chromatography (HPLC) separation followed by radioimmunoassay (RIA) for human plasma samples (HPLC–RIA) and immunoaffinity extraction (IAE) followed by RIA for human urine samples (IAE–RIA) were developed for investigation of S-8921 behavior in clinical studies. For the RIA, antisera from rabbit and a radioiodine-labelled S-8921 were prepared by immunizing a conjugate of S-8921 with bovine serum albumin and by the Bolton and Hunter method, respectively. HPLC–RIA using a semi-micro column was very sensitive, that is a 0.05 ng/ml limit of quantitation in human plasma, and specific for unchanged form of S-8921. IAE–RIA using a centrifugal filtration tube completely eliminated the matrix effect of human urine, and was very feasible. The limit of quantitation was 0.10 ng/ml. RIA detection following HPLC or IAE proved to be very useful for the pharmaceutical analysis of extremely low drug concentrations in body fluids.     

Adaptation of the plasma renin radioimmunoassay for use with HIV-1 protease

We have demonstrated the use of a radioimmunoassay to quantitate the peptidolytic activity of human immunodeficiency virus, type 1 (HIV-1) protease using a tetradecapeptide substrate of porcine renin, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser. HIV-1 protease catalyzes cleavage of this substrate at the same Leu-Leu bond as does porcine renin, resulting in the formation of authentic angiotensin-I. The angiotensin-I product is then detected by use of a commercially available renin plasma assay kit, which constitutes the basis of the RIA. The radioimmunoassay provides detection of the protease-catalyzed formation of angiotensin-I at picomolar concentrations in vitro. We demonstrate the use of this assay in determining IC50 values for two HIV-1 protease inhibitors present in cell culture media and in standard assay buffer. An example of the potential development of this assay for the quantitation of these inhibitors present in ex vivo plasma samples is also presented.     

Determination of amiprilose in human plasma by high-performance liquid chromatography with fluorimetric detection

A reversed-phase HPLC method to quantify amiprilose in human plasma is described. The method involves liquid–liquid extraction of amiprilose and the internal standard from plasma. The extracted compounds are derivatized with 1,8-naphthalic dicarboxylic acid using 2-chloro-1-methylpyridinium iodide as a coupling reagent. The derivatized products are separated on a reversed-phase column and monitored fluorimetrically using 280 nm and 340 nm as excitation and emission wavelengths, respectively. The derivatized products which exhibit two peaks on chromatogram, are shown to be the interconvertible isomers. This assay has been used in pharmacokinetic studies of amiprilose in humans.     

Comparison of the effects of perfusion in determining brain penetration (brain-to-plasma ratios) of small molecules in rats

In the process of drug discovery, brain and plasma measurements of new chemical entities in rodents are of interest, particularly when the target receptors are in the brain. Brain-to-plasma ratios (B/P) obtained from a rodent pharmacokinetic assay are useful in helping determine which compounds are brain penetrant. The study reported here was performed to determine whether whole-body saline perfusion for complete blood removal was required to accurately measure brain tissue compound concentrations. Diazepam was used as a positive control since it is highly brain penetrant. Compound A was used as a negative control since it had known poor brain penetration. After intravenous dosing with either diazepam or compound A, rats were anesthetized and blood was collected, then the brain was removed following no perfusion or whole-body perfusion with saline. The analytes described (compound A, diazepam, and the internal standard) were recovered from plasma or brain homogenate by use of protein precipitation, and were subsequently analyzed by use of liquid chromatography/tandem mass spectrometry (LC/MS/MS). The B/P values determined by use of LC-MS were not significantly different in perfused vs. non-perfused rats (P > or = 0.05). This approach (whole brain collected from non-perfused male rats) is an attractive alternative over brain penetration studies of perfused rats, since it has markedly reduced the technical time and potential for pain and distress required for generating B/P data due to elimination of the requirement for anesthesia and surgical preparation of animals.     

Progenitor cells as remote "bioreactors": neuroprotection via modulation of the systemic inflammatory response

Acute central nervous system(CNS)injuries such as spinal cord injury,traumatic brain injury,autoimmune encephalomyelitis,and ischemic stroke are associ- ated with significant morbidity,mortality,and health care costs worldwide.Preliminary research has shown potential neuroprotection associated with adult tissue derived stem/progenitor cell based therapies.While initial research indicated that engraftment and transdif- ferentiation into neural cells could explain the observed benefit,the exact mechanism remains controversial.A second hypothesis details localized stem/progenitor cell engraftment with alteration of the loco-regional milieu;however,the limited rate of cell engraftment makes this theory less likely.There is a growing amount of pre-clinical data supporting the idea that,after intravenous injection,stem/progenitor cells interact with immuno- logic cells located in organ systems distant to the CNS,thereby altering the systemic immunologic/inflammatory response.Such distant cell"bioreactors"could modulate the observed post-injury pro-inflammatory environment and lead to neuroprotection.In this review,we discuss the current literature detailing the above mechanisms of action for adult stem/progenitor cell based therapies in the CNS.