Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.
We have examined the catabolism of the proteoglycans aggrecan, decorin and biglycan in fresh tendon samples and in explant cultures of tissue from the tensional and compressed regions of young and mature bovine tendons. A panel of well-characterized antibodies that recognize glycosaminoglycan or protein (linear or neoepitope) sequences was used to detect proteoglycans and proteoglycan degradation products that were both retained within the tissue and released into the culture medium. In addition, a reverse-transcriptase-mediated PCR analysis was used to examine the mRNA expression patterns of tendon proteoglycans and aggrecanases. The results of this study indicate a major role for aggrecanase(s) in the catabolism of aggrecan in bovine tendon. The study also provides a characterization of glycosaminoglycan epitopes associated with the proteoglycans of tendon, illustrating age-related changes in the isomers of chondroitin sulphate disaccharides that remain attached to the core protein glycosaminoglycan linkage region after digestion with chondroitinase ABC. Evidence for a rapid turnover of the small proteoglycans decorin and biglycan was also observed, indicating additional molecular pathways that might compromise the integrity of the collagen matrix and potentially contribute to tendon dysfunction after injury and during disease.
systematic review of the use of dosage form manipulation to obtain required doses to inform use of manipulation in paediatric practice.International Journal of Pharmaceutics http://dx.doi.org/10. 1016/j.ijpharm.2016.12.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. This study sought to determine whether there is an evidence base for drug manipulation to obtain 37 the required dose, a common feature of paediatric clinical practice. A systematic review of the 38 data sources, PubMed, EMBASE, CINAHL, IPA and the Cochrane database of systematic 39 reviews, was used. Studies that considered the dose accuracy of manipulated medicines of any 40 dosage form, evidence of safety or harm, bioavailability, patient experience, tolerability, 41 contamination and comparison of methods of manipulation were included. Case studies and 42 letters were excluded. Fifty studies were eligible for inclusion, 49 of which involved tablets 43 being cut, split, crushed or dispersed. The remaining one study involved the manipulation of 44 suppositories of one drug. No eligible studies concerning manipulation of oral capsules or 45 liquids, rectal enemas, nebuliser solutions, injections or transdermal patches were identified. 46Twenty four of the tablet studies considered dose accuracy using weight and/or drug content. In 47 studies that considered weight using adapted pharmacopoeial specifications, the percentage of 48 halved tablets meeting these specifications ranged from 30% to 100%. Eighteen studies 49 investigated bioavailability, pharmacokinetics or clinical outcomes following manipulations 50 which included nine delayed or modified release formulations. In each of these nine studies the 51 entirety of the dosage form was administered. Only one of the 18 studies was identified where 52 drugs were manipulated to obtain a proportion of the dosage form, and that proportion 53 administered. The five studies that considered patient perception found that having to manipulate 54 the tablets did not have a negative impact on adherence. Of the 49 studies only two studies 55 reported investigating children. This review yielded limited evidence to support manipulation of 56 medicines for children. The results cannot be extrapolated between dosage forms, methods of 57 manipulation or between different brands of the same drug. 58 59 4 INTRODUCTION 60Many medicines given to children are used "off-label" because the medicine has only been 61 researched and authorised for adults. Often the dosage form (e.g., tablets, capsules, 62 suppositories) is suitable for administration to adults but not to younger children (Waller, 2007). 63Age...
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