In vivo rAAV-mediated human TGF-ß overexpression reduces perifocal osteoarthritis and improves osteochondral repair in a large animal model at one year.

OBJECTIVE: Osteoarthritis (OA) is a serious consequence of focal osteochondral defects. Gene transfer of human transforming growth factor beta (hTGF-ß) with recombinant adeno-associated virus (rAAV) vectors offers a strategy to improve osteochondral repair. However, the long-term in vivo effects of such rAAV-mediated TGF-ß overexpression including its potential benefits on OA development remain unknown. METHOD: Focal osteochondral defects in minipig knees received rAAV-lacZ (control) or rAAV-hTGF-ß in vivo. After one year, osteochondral repair and perifocal OA were visualized using validated macroscopic scoring, ultra-high-field MRI at 9.4 T, and micro-CT. A quantitative estimation of the cellular densities and a validated semi-quantitative scoring of histological and immunohistological parameters completed the analysis of microarchitectural parameters. RESULTS: Direct rAAV-hTGF-ß application induced and maintained significantly improved defect filling and safranin O staining intensity and overall cartilage repair at one year in vivo. In addition, rAAV-hTGF-ß led to significantly higher chondrocyte densities within the cartilaginous repair tissue without affecting chondrocyte hypertrophy and minimized subarticular trabecular separation. Of note, rAAV-hTGF-ß significantly improved the adjacent cartilage structure and chondrocyte density and reduced overall perifocal OA development after one year in vivo. CONCLUSIONS: rAAV-hTGF-ß treatment improves long-term osteochondral repair and delays the progression of perifocal OA in a translational model. These findings have considerable potential for targeted molecular approaches to treat focal osteochondral defects.

Alteration of sarcoplasmic reticulum Ca2+ ATPase expression in lower limb ischemia caused by atherosclerosis obliterans.

Atherosclerosis is a disease caused by a build-up of fatty plaques and cholesterol in the arteries. The lumen of the vessels is obliterated resulting in restricted blood supply to tissues. In ischemic conditions, the cytosolic Ca2+ level of skeletal muscle may increase, indicating the alteration of Ca2+ removal mechanisms. Ca2+ is transported from cytosol into the sarcoplasmic reticulum by Ca2+ ATPase (SERCA), with its 1a isoform expressed in adult, while its 1b isoform in neonatal and regenerating fast-twitch skeletal muscle. To investigate the role of these isoforms in ischemic skeletal muscle, biopsies from musculus biceps femoris of patients who underwent amputation due to atherosclerosis were examined. Samples were removed from the visibly healthy and hypoxia-affected tissue. Significantly increased SERCA1a expression was detected under the ischemic conditions (246 ± 69%; p < 0.05) compared with the healthy tissue. Furthermore, the ratio of SERCA1a-positive fibers was slightly increased (46 ± 4% in healthy tissue and 60 ± 5% in ischemic tissue; p > 0.05), whereas SERCA2a did not change. In addition, in primary cultures derived from hypoxia-affected tissue, the diameter and fusion index of myotubes were significantly increased (30 ± 1.6 µm vs. 41 ± 2.4 µm and 31 ± 4% vs. 45 ± 3%; p < 0.05). We propose that the increased SERCA1a expression indicates the existence and location of compensating mechanisms in ischemic muscle.

The protein composition of reconstituted 30S ribosomal subunits: the effects of single protein omission.

Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.

In vitro and in vivo evaluation of dihydropyrimidinone C-5 amides as potent and selective alpha(1A) receptor antagonists for the treatment of benign prostatic hyperplasia.

alpha(1) Adrenergic receptors mediate both vascular and lower urinary tract tone, and alpha(1) receptor antagonists such as terazosin (1b) are used to treat both hypertension and benign prostatic hyperplasia (BPH). Recently, three different subtypes of this receptor have been identified, with the alpha(1A) receptor being most prevalent in lower urinary tract tissue. This paper explores 4-aryldihydropyrimidinones attached to an aminopropyl-4-arylpiperidine via a C-5 amide as selective alpha(1A) receptor subtype antagonists. In receptor binding assays, these types of compounds generally display K(i) values for the alpha(1a) receptor subtype <1 nM while being greater than 100-fold selective versus the alpha(1b) and alpha(1d) receptor subtypes. Many of these compounds were also evaluated in vivo and found to be more potent than terazosin in both a rat model of prostate tone and a dog model of intra-urethral pressure without significantly affecting blood pressure. While many of the compounds tested displayed poor pharmacokinetics, compound 48 was found to have adequate bioavailability (>20%) and half-life (>6 h) in both rats and dogs. Due to its selectivity for the alpha(1a) over the alpha(1b) and alpha(1d) receptors as well as its favorable pharmacokinetic profile, 48 has the potential to relieve the symptoms of BPH without eliciting effects on the cardiovascular system.

Mechanistic investigation of ionization suppression in electrospray ionization.

We show results from experiments designed to determine the relative importance of gas phase processes and solution phase processes into ionization suppression observed in biological sample extracts. The data indicate that gas phase reactions leading to the loss of net charge on the analyte is not likely to be the most important process involved in ionization suppression. The results point to changes in the droplet solution properties caused by the presence of nonvolatile solutes as the main cause of ionization suppression in electrospray ionization of biological extracts.

Synthesis of conformationally constrained 5,6,7, 8-Tetrahydroimidazo[1,5-a]pyridine inhibitors of farnesyltransferase.

[reaction: see text] Synthesis of the 8-amino-5,6,7,8-tetrahydroimidazo[1,5-a]pyridine ring system was accomplished by intramolecular cyclization of an iminium ion, derived from condensation of an amine and a substituted gamma-(1-imidazolyl)butyraldehyde. The reaction was used to produce conformationally restricted farnesyltransferase inhibitor analogues which exhibit improved in vivo metabolic stability.

Rizatriptan, a novel 5-HT1B/1D agonist for migraine: single- and multiple-dose tolerability and pharmacokinetics in healthy subjects.

Rizatriptan is a novel 5-HT1D/1B agonist for relief of migraine headache. The pharmacokinetics, metabolite profiles, and tolerability of rizatriptan were examined in a multiple-dose study in healthy subjects. Rizatriptan (N = 24) (or placebo, N = 12) was administered as a single 10 mg dose, followed 48 hours later by administration of one 10 mg dose every 2 hours for three doses on 4 consecutive days, corresponding to the maximum daily dose for a migraine attack. The AUC of rizatriptan and its active N-monodesmethyl metabolite after three 10 mg doses was approximately threefold greater than the plasma concentrations following a single 10 mg dose. Metabolite profiles were similar after single and multiple doses. Adverse events during rizatriptan were mild and transient; similar events occurred during placebo, with a somewhat reduced incidence. Diastolic blood pressure tended to increase compared with placebo (approximately 5 mmHg), particularly on the first multiple-dose day (p < .01 vs. placebo). In conclusion, rizatriptan is well tolerated by healthy subjects during multiple-dose administration, with no unexpected accumulation of drug in plasma.

Emergency esophagogastrectomy for treatment of hydrochloric acid injury.

Eight patients with severe hydrochloric acid injury of the esophagus and stomach had early esophagogastrectomy. Four patients survived and 6 to 8 weeks later underwent successful substernal colon replacement. The value of early diagnosis and subsequent aggressive surgical management of patients who have ingested hydrochloric acid is stressed.

The inhibitory effects of allopurinol on the production and cytotoxicity of tumor necrosis factor.

Allopurinol, a xanthine oxidase inhibitor, impaired the cytotoxic effect of human recombinant tumor necrosis factor (TNF) against WEHI cells. Actinomycin D abolished the inhibition of cytotoxicity by allopurinol. Allopurinol also exerted an inhibitory effect on the production of TNF by human mononuclear cells stimulated by either heat-killed Staphylococcus aureus or E. coli lipopolysaccharide. It is suggested that allopurinol inhibits TNF cytotoxicity by decreasing the level of oxygen free radicals generated (among other mechanisms) by the action of xanthine oxidase. Whatever the mechanism, the fact that allopurinol counteracts the toxicity of TNF can help towards an understanding of the complex nature of TNF toxicity.

Quantitation of the 5HT1D agonists MK-462 and sumatriptan in plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

The 5HT1D agonist sumatriptan is efficacious in the treatment of migraines. MK-462 is a drug of the same class which is under development in our laboratories. Bioanalytical methods of high efficiency, specificity and sensitivity were required to support the preclinical and clinical programs. These assays were based on HPLC with tandem MS-MS detection. MK-462 and sumatriptan were extracted using an automated solid-phase extraction technique on a C2 Varian Bond-Elut cartridge. The n-diethyl analogues of MK-462 and sumatriptan were used as internal standards. The analytes were chromatographed using reversed-phase (nitrile) columns coupled via a heated nebulizer interface to an atmospheric pressure chemical ionization source. The chromatographic run times were less than 7 min. Both methods were precise, accurate and selective down to plasma concentrations of 0.5 ng/ml. The assay for MK-462 was adapted to separately monitor the unlabeled and 14C-labeled species of the drug following intravenous administration of radiolabeled material to man.

Determination of the beta-adrenergic blocker timolol in plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A method based on LC-MS-MS has been developed for the determination of timolol in plasma using the (CD3)3-labelled species as the internal standard. Timolol is isolated from plasma by a simple solid-phase extraction and converted to its oxazolidin-2-one prior to analysis on a 50 x 4.6 mm reversed-phase high-performance liquid chromatography column packed with SynChropak, C18, 5 microns. The column eluate is passed by means of a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source where the analyte and its internal standard are detected using multiple reaction monitoring (MRM). The very high specificity of this technique permits chromatographic run times of less than 2 min. The method has a lower quantifiable limit of 0.5 ng ml-1, with intra- and inter-day relative standard deviations less than 10%, and enables the determination of timolol in plasma after ocular administration to volunteers.

A direct technique for the simultaneous determination of 10 drug candidates in plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry interfaced to a Prospekt solid-phase extraction system.

New drug candidates are being synthesized at an ever increasing rate and, until recently, the pharmacokinetics of only a few of these could be evaluated. Our laboratory is taking a novel approach to rapid multiple pharmacokinetic screening of potential drug candidates in which mixtures of new substances are co-administered to animals and analyzed simultaneously in plasma using liquid chromatography with tandem MS/MS detection in conjunction with a Prospekt automated on-line solid-phase extraction system. Plasma is sampled via an autosampler and extracted by the Prospekt with the eluent being introduced directly via a reverse phase HPLC column and a heated nebulizer interface to the mass spectrometer. Generic extraction and chromatographic conditions generally give good recoveries. The chromatographic run-times are less than 8 min. The accuracy and precision of these assays are carefully controlled with recoveries generally in the range 80-120% and coefficients of variation less than 20%. Lower quantifiable limits range from 2.5 to 5 ng ml-1. This approach considerably reduces the number of animals needed to screen drug candidates and its power is illustrated by determination of the pharmacokinetics of 10 substances after their simultaneous administration to dogs.

Determination of MK-383, a non-peptide fibrinogen receptor antagonist, in human plasma and urine by radioimmunoassay.

MK-383 is a novel, non-peptide fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via the N-hydroxysuccinimide ester from which the radioligand was also prepared by reaction with [I125]iodotyrosine. The method was specific and no immunoreactive material other than the parent drug was detectable in plasma and urine from dosed volunteers. This direct assay, using 5 microliters of plasma or 0.5 microliter of urine, is sensitive to 1 and 10 ng ml-1, respectively, without matrix interference and has sufficient sensitivity, specificity, accuracy, and precision for the analysis of clinical samples.

A rapid and specific assay, based on liquid chromatography-atmospheric pressure chemical ionization mass spectrometry, for the determination of MK-434 (a 5 alpha-reductase inhibitor) and its metabolites in plasma.

MK-434 is a new 5 alpha-reductase inhibitor. A sensitive and specific assay based on combined liquid chromatography-mass spectrometry (LC-MS) has been developed for the determination of this compound in plasma. The analyte was isolated from plasma by solid-phase extraction on a C18 cartridge. A related substance, L-654,066, was used as the internal standard. Extracts were separated on a 5-cm C18-reversed-phase high performance liquid chromatography column interfaced via the heated nebulizer probe to a corona discharge chemical ionization source. The mass spectrometer was operated in the positive ion MS-MS mode. The method had sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples containing MK-434 and its two principal metabolites at concentrations in the range 0.5-50 ng ml-1. The chromatographic run time was < 5 min.

Determination of L-691,121, a new class III antiarrhythmic, and its principal metabolite in plasma by differential radioimmunoassay.

A sensitive and specific method based on radioimmunoassay (RIA) has been developed for the analysis of L-691,121, a new antiarrhythmic agent, and its major metabolite, L-692,199, in plasma. Two RIAs using immunogens and radioligands prepared from different derivatives of L-691,121 were used in conjunction to determine both parent compound and metabolite concentrations by solving simultaneous equations, since neither assay alone was adequately specific. Variable cross-reactivity factors were incorporated into the calculations to correct for non-parallel drug and metabolite displacement curves. The direct assay using 30 microliters of plasma is sensitive to 0.1 ng ml-1 and has sufficient precision, accuracy and specificity for the analysis of clinical samples.

Determination of MK-852, a new fibrinogen receptor antagonist, in plasma and urine by radioimmunoassay.

MK-852 is a novel fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via a dinitrophenylene bridge and the radioligand by reaction of the drug with the 125I-labelled Bolton-Hunter reagent. The method was specific and no immunoreactive material other than parent drug was detectable in plasma from dosed volunteers. The direct assay using 0.05 ml of plasma is sensitive to 0.2 ng ml-1 without matrix interference and has sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples. The lower quantifiable limit in (diluted) urine is 50 ng ml-1.

The development and cross-validation of methods based on radioimmunoassay and LC/MS-MS for the quantification of the class III antiarrhythmic agent, MK-0499, in human plasma and urine.

An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired student's t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.

The use of stable isotope labeling and liquid chromatography-tandem mass spectrometry techniques to simultaneously determine the oral and ophthalmic bioavailability of timolol in dogs.

Assays have been established for the quantitation of timolol and its 13C3- and 2H9-stable-isotope-labeled analogs in plasma and urine using liquid chromatography with atmospheric-pressure chemical-ionization tandem mass spectrometry. For the analysis of urine, underivatized timolol and its labeled analogs are monitored while timolol in plasma is assayed down to concentrations of 0.2 ng/mL after derivatization with phosgene. The great power of this technique is illustrated by simultaneously assaying three different species of timolol in plasma and urine obtained from dogs receiving simultaneous ophthalmic, oral, and intravenous doses of unlabeled and [2H9]- and [13C3]-labeled timolol. Thus, the ophthalmic and oral bioavailabilities of timolol are measured in a single experiment rather than as a three-phase crossover experiment. This approach yields accurate and precise analytical data, obviates intrasubject variability, and saves both analytical and animal resources.

[Emergency treatment of corrosive gastrointestinal injuries]. / Korrozív gastrointestinalis sérülések akut ellátása.

Authors discuss their experience of management for caustic gastrointestinal injury. Importance of urgent endoscopy in establishing the extend of injury is emphasized. In eight out of 12 surgical intervention an esophagogastrectomy were performed. According to their experience in case of serious caustic burn the only hope to save the patients life is the early radical operation.

[Management of injuries of the artificial esophagus]. / Arteficiális nyelöcsö sérülések ellátása.

The authors analyse their experiences in connection with 20 cases of artificial esophageal injuries. Attention is called to the intraoperative lesions of the esophageal wall and to the damages caused by drugs. They underline the importance of the time factor and good results were achieved by using so called "conservative surgical treatment".