Degradation of poly-L-lactide. Part 1: in vitro and in vivo physiological temperature degradation

Poly-L-Lactide is a bioresorbable polymer which degrades through hydrolysis of its ester linkage influenced by initial molecular weight and degree of crystallinity. Polymers belonging to the aliphatic polyester family currently represent the most attractive group of polymers that meet the medical and physical demands for safe clinical applications. Compression moulded PLLA pellets were produced as rods, sterilized and degraded both in vitro and in vivo (sub-dermal implantation model). The material molecular weight, crystallinity, mechanical strength and thermal properties were evaluated. In both in vitro and in vivo environments, degradation proceeded at the same rate and followed the general sequence of aliphatic polyester degradation, ruling out enzymes accelerating the degradation rate in vivo. By 44 weeks duration of implantation the PLLA rods were still biocompatible, before any mass loss was observed.

Roles of norepinephrine and ATP in sympathetically evoked vasoconstriction in rat tail and hindlimb in vivo.

In anesthetized rats, we characterized the contributions of norepinephrine (NE) and ATP to changes in tail and hindlimb (femoral) vascular resistances (TVR and FVR, respectively) evoked by three patterns of sympathetic stimulation: 1) couplets (2 impulses at 20 Hz), 2) short trains (20 impulses at 20 Hz), and 3) a natural irregular pattern previously recorded from a sympathetic fiber innervating the rat tail artery. All stimuli evoked greater changes in TVR than FVR. Judging from the effects of the -adrenoceptor antagonist phentolamine, the purinergic receptor antagonist suramin, or ,-methylene ATP (which desensitizes P2X receptors), we propose that NE has a major role in the constriction evoked by the couplet, as well as by the short train and by the low- and high-frequency components of the natural pattern, but that considerable synergy occurred between the actions of ATP and NE. This contrasts with previous in vitro studies that indicated that ATP dominates vascular responses evoked by sympathetic stimulation with a few impulses at low frequency and that NE dominates responses to longer trains or at high frequencies.

Comparative effects of GLP-1 and GIP on cAMP production, insulin secretion, and in vivo antidiabetic actions following substitution of Ala8/Ala2 with 2-aminobutyric acid

The two major incretin hormones, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP), are currently being considered as prospective drug candidates for treatment of type 2 diabetes. Interest in these gut hormones was initially spurred by their potent insulinotropic activities, but a number of other antihyperglycaemic actions are now established. One of the foremost barriers in progressing GLP-1 and GIP to the clinic concerns their rapid degradation and inactivation by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV). Here, we compare the DPP IV resistance and biological properties of Abu(8)/ Abu(2) (2-aminobutyric acid) substituted analogues of GLP-1 and GIP engineered to impart DPP IV resistance. Whereas (Abu(8))GLP-1 was completely stable to human plasma (half-life > 12h), GLP-1, GIP, and (Abu(2))GIP were rapidly degraded (half-lives: 6.2, 6.0, and 7.1 h, respectively). Native GIP, GLP-1, and particularly (Abu(8))GLP-1 elicited significant adenylate cyclase and insulinotropic activity, while (Abu(2))GIP was less effective. Similarly, in obese diabetic (ob/ob) mice, GIP, GLP-1, and (Abu(8))GLP-1 displayed substantial glucose-lowering and insulin -releasing activities, whereas (Abu(2))GIP was only weakly active. These studies illustrate divergent effects of penultimate amino acid Ala(8)/Ala(2) substitution with Abu on the biological properties of GLP-1 and GIP, suggesting that (Abu(8))GLP-1 represents a potential candidate for future therapeutic development. (C) 2004 Elsevier Inc. All rights reserved.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Dynamics and Function of Langerhans Cells In Vivo: Dermal Dendritic Cells Colonize Lymph Node Areas Distinct from Slower Migrating Langerhans Cells

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.