ABSTRACT
BACKGROUND AND PURPOSE: Disodium 2,4-disulphophenyl-N-tert-butylnitrone (NXY-059) was neuroprotective in experimental stroke models but ineffective in a large clinical trial. This first-ever individual animal meta-analysis was used to assess the preclinical studies. EXPERIMENTAL APPROACH: Studies were obtained from AstraZeneca and PubMed searches. Data for each animal were obtained from the lead author of each study and/or AstraZeneca. Published summary data were used if individual data were not available. Infarct volume and motor impairment were standardized to reflect different species and scales. Standardized mean difference (SMD), coefficients from multilevel models and 95% confidence intervals (95% CI) are presented. KEY RESULTS: Fifteen studies (26 conditions, 12 laboratories) involving rats (544), mice (9) and marmosets (32) were identified (NXY-059: 332, control: 253) with individual data for 442 animals. Four studies were unpublished. Studies variably used randomization (40%), blinding of surgeon (53%) and outcome assessor (67%). NXY-059 reduced total (SMD -1.17, 95% CI -1.50 to -0.84), cortical (SMD -2.17, 95% CI -2.99 to -1.34) and subcortical (-1.43, 95% CI -2.20 to -0.86) lesion volume; efficacy was seen in transient, permanent and thrombotic ischaemia, up to 180 min post occlusion. NXY-059 reduced motor impairment (SMD -1.66, 95% CI -2.18 to -1.14) and neglect. Evidence for performance, attrition and publication bias was present. CONCLUSIONS AND IMPLICATIONS: NXY-059 was neuroprotective in experimental stroke although bias may have resulted in efficacy being overestimated. Efficacy in young, healthy, male animals is a poor predictor of clinical outcome. We suggest the use of preclinical meta-analysis before initiation of future clinical trials.
Subject(s)
Benzenesulfonates/therapeutic use , Neuroprotective Agents/therapeutic use , Stroke/drug therapy , Animals , Benzenesulfonates/blood , Brain/pathology , Brain/physiopathology , Callithrix , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Male , Mice , Neuroprotective Agents/blood , Random Allocation , Rats , Stroke/pathology , Stroke/physiopathologyABSTRACT
Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/metabolism , DNA/metabolism , Magnesium/pharmacology , Manganese/pharmacologyABSTRACT
Type II restriction endonucleases usually recognize 4-6-base pair (bp) sites on DNA and cleave each site in a separate reaction. A few type II endonucleases have 8-bp recognition sites, but these seem unsuited for restriction, since their sites are rare on most DNA. Moreover, only one endonuclease that recognizes a target containing 8 bp has been examined to date, and this enzyme, SfiI, needs two copies of this site for its DNA cleavage reaction. In this study, several endonucleases with 8-bp sites were tested on plasmids that have either one or two copies of the relevant sequence to determine if they also need two sites. SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, and SdaI all acted through equal and independent reactions at each site. AscI cleaved the DNA with one site at the same rate as that with two sites but acted processively on the latter. In contrast, SgrAI showed a marked preference for the plasmid with two sites and cleaved both sites on this DNA in a concerted manner, like SfiI. Endonucleases that require two copies of an 8-bp sequence may be widespread in nature, where, despite this seemingly inappropriate requirement, they may function in DNA restriction.
