Standardising clinical outcomes measures for adult clinical trials in Fabry disease: A global Delphi consensus.

BACKGROUND: Recent years have witnessed a considerable increase in clinical trials of new investigational agents for Fabry disease (FD). Several trials investigating different agents are currently in progress; however, lack of standardisation results in challenges to interpretation and comparison. To facilitate the standardisation of investigational programs, we have developed a common framework for future clinical trials in FD. METHODS AND FINDINGS: A broad consensus regarding clinical outcomes and ways to measure them was obtained via the Delphi methodology. 35 FD clinical experts from 4 continents, representing 3389 FD patients, participated in 3 rounds of Delphi procedure. The aim was to reach a consensus regarding clinical trial design, best treatment comparator, clinical outcomes, measurement of those clinical outcomes and inclusion and exclusion criteria. Consensus results of this initiative included: the selection of the adaptative clinical trial as the ideal study design and agalsidase beta as ideal comparator treatment due to its longstanding use in FD. Renal and cardiac outcomes, such as glomerular filtration rate, proteinuria and left ventricular mass index, were prioritised, whereas neurological outcomes including cerebrovascular and white matter lesions were dismissed as a primary or secondary outcome measure. Besides, there was a consensus regarding the importance of patient-related outcomes such as general quality of life, pain, and gastrointestinal symptoms. Also, unity about lysoGb3 and Gb3 tissue deposits as useful surrogate markers of the disease was obtained. The group recognised that cardiac T1 mapping still has potential but requires further development before its widespread introduction in clinical trials. Finally, patients with end-stage renal disease or renal transplant should be excluded unless a particular group for them is created inside the clinical trial. CONCLUSION: This consensus will help to shape the future of clinical trials in FD. We note that the FDA has, coincidentally, recently published draft guidelines on clinical trials in FD and welcome this contribution.

Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance.

BACKGROUND: Screening in subjects with left ventricular hypertrophy (LVH) reveals a high prevalence of Fabry disease (FD). Often, a diagnosis is uncertain because characteristic clinical features are absent and genetic variants of unknown significance (GVUS) in the α-galactosidase A (GLA) gene are identified. This carries a risk of misdiagnosis, inappropriate counselling and extremely expensive treatment. We developed a diagnostic algorithm for adults with LVH (maximal wall thickness (MWT) of >12 mm), GLA GVUS and an uncertain diagnosis of FD. METHODS: A Delphi method was used to reach a consensus between FD experts. We performed a systematic review selecting criteria on electrocardiogram, MRI and echocardiography to confirm or exclude FD. Criteria for a definite or uncertain diagnosis and a gold standard were defined. RESULTS: A definite diagnosis of FD was defined as follows: a GLA mutation with ≤ 5% GLA activity (leucocytes, mean of reference value, males only) with ≥ 1 characteristic FD symptom or sign (neuropathic pain, cornea verticillata, angiokeratoma) or increased plasma (lyso)Gb3 (classical male range) or family members with definite FD. Subjects with LVH failing these criteria have a GVUS and an uncertain diagnosis. The gold standard was defined as characteristic storage in an endomyocardial biopsy on electron microscopy. Abnormally low voltages on ECG and severe LVH (MWT>15 mm) <20 years exclude FD. Other criteria were rejected due to insufficient evidence. CONCLUSIONS: In adults with unexplained LVH and a GLA GVUS, severe LVH at young age and low voltages on ECG exclude FD. If absent, an endomyocardial biopsy with electron microscopy should be performed.

Outcomes of patients treated through the Canadian Fabry disease initiative.

BACKGROUND: The Canadian Fabry disease initiative (CFDI) tracks outcomes of subjects with Fabry disease treated enzyme replacement therapy (ERT) given to subjects who meet evidence-based treatment guidelines and cardiovascular risk factor modification. METHODS: We report 5 year follow-up data on 362 subjects for a composite endpoint (death, neurologic or cardiovascular events, development of end-stage renal disease or sustained increase in serum creatinine of 50% from baseline). RESULTS: At enrollment, 86 subjects had previously received ERT (Cohort 1a) and 67 subjects were newly started (Cohort 1b) and randomized to agalsidase alfa or agalsidase beta. 209 subjects did not initially meet ERT criteria (Cohort 1c), 25 of whom met ERT criteria in follow-up and were moved to Cohort 1b (total N=178 ERT treated subjects). Use of supportive therapies such as aspirin (78%), renin-angiotensin blockade (59%), and statins (55%) was common in ERT treated subjects. In Cohort 1a, 32 subjects met the composite endpoint with 8 deaths. In Cohort 1b, 16 subjects met the composite endpoint with 1 death. Cohort 1b had fewer clinical events than Cohort 1a (p=0.039) suggesting that the treatment protocol was effective in targeting subjects at an earlier stage. 19.4% of Cohort 1b subjects on agalsidase alfa and 13.3% on agalsidase beta had a clinical event (p=0.57). 10 Cohort 1c subjects had clinical events, none of which would have been prevented by earlier use of ERT. CONCLUSIONS: Cardiovascular risk factor modification and targeted use of ERT reduce the risk of adverse outcomes related to Fabry disease.

L-selectin is dispensable for T regulatory cell function postallogeneic bone marrow transplantation.

In murine models, the adoptive transfer of CD4(+) /CD25(+) regulatory T cells (T(regs) ) inhibited graft-versus-host disease (GvHD). Previous work has indicated a critical role for the adhesion molecule L-selectin (CD62L) in the function of T(regs) in preventing GvHD. Here we examined the capacity of naive wild-type (WT), CD62L(-/-) and ex vivo expanded CD62L(Lo) T(regs) to inhibit acute GvHD. Surprisingly, we found that CD62L(-/-) T(regs) were potent suppressors of GvHD, whereas CD62L(Lo) T(regs) were unable to inhibit disease despite being functionally competent to suppress allo T cell responses in vitro. Concomitant with improved outcomes, WT and CD62L(-/-) T(regs) significantly reduced liver pathology and systemic pro-inflammatory cytokine production, although CD62L(-/-) T(regs) were less effective in reducing lung pathology. While accumulation of CD62L(-/-) T(regs) in GvHD target organs was equivalent to WT T(regs) , CD62L(-/-) T(regs) did not migrate as well as WT T(regs) to peripheral lymph nodes (PLNs) over the first 2 weeks posttransplantation. This work demonstrated that CD62L was dispensable for T(reg) -mediated protection from GvHD.

Baseline characteristics of patients enrolled in the Canadian Fabry Disease Initiative.

The Canadian Fabry Disease Initiative [CFDI] is a longitudinal study evaluating all Canadians diagnosed with Fabry disease [FD]. The study has 3 cohorts: Cohort 1A which includes 81 subjects who were on enzyme replacement therapy [ERT] prior to October 2006, Cohort 1B which has ongoing enrolment of subjects newly started on ERT who are randomized to agalsidase alfa or agalsidase beta, and Cohort 1C where subjects who do not meet nationally accepted Canadian criteria for ERT are followed to assess the natural history of disease complications. The study currently enrols 244 patients [95 males and 149 females] with a mean age of 41.9+/-14.5years. There is a high prevalence of the c.427G>C mutation. Cohort 1A contains 82 patients [59 males, 23 females] of whom 42% are known to have cardiac complications of FD and 38% renal complications. Cohort 1B at the time of writing contained 37 patients [15 males, 22 females] of whom the indications for ERT were cardiac in 55% and renal in 60%. Cohort 1C at the time of writing contained 125 patients [22 males, 103 females]. Enrolment is ongoing in both Cohorts 1B and 1C. When compared to subjects in the Fabry Outcome Survey and the Fabry Registry, subjects in the CFDI are less likely to be male reflecting less ascertainment bias. The CFDI is a robust national data set that will contribute to available data on the natural history of FD and on the comparative efficacy of the two commercially available ERT products.

Neural substrates of facial emotion processing using fMRI.

We identified human brain regions involved in the perception of sad, frightened, happy, angry, and neutral facial expressions using functional magnetic resonance imaging (fMRI). Twenty-one healthy right-handed adult volunteers (11 men, 10 women; aged 18-45; mean age 21.6 years) participated in four separate runs, one for each of the four emotions. Participants viewed blocks of emotionally expressive faces alternating with blocks of neutral faces and scrambled images. In comparison with scrambled images, neutral faces activated the fusiform gyri, the right lateral occipital gyrus, the right superior temporal sulcus, the inferior frontal gyri, and the amygdala/entorhinal cortex. In comparisons of emotional and neutral faces, we found that (1) emotional faces elicit increased activation in a subset of cortical regions involved in neutral face processing and in areas not activated by neutral faces; (2) differences in activation as a function of emotion category were most evident in the frontal lobes; (3) men showed a differential neural response depending upon the emotion expressed but women did not.

Relationship between attachment-felt security and history of suicidal behaviours in clinical adolescents.

OBJECTIVE: This study was designed to test the hypothesis that adolescents who perceive their attachment figures as unavailable (low felt security) would be overrepresented in the case group of adolescents with a history of suicidal behaviours. METHOD: One hundred and eighty-seven adolescents in psychiatric treatment participated in this retrospective case-comparison study of attachment-felt security and history of suicidal behaviours. All participants completed the following measures: Adolescent Attachment Questionnaire, Perceived Social Support From Friends Scale, Rosenberg Self-Esteem Scale, Beck Hopelessness Scale, the depression syndrome scale of the Youth Self Report, and Adam's Suicidal Ideation and Behavior protocol. RESULTS: The comparison group comprised 101 adolescents who had never experienced suicidal ideation or behaviour; the case group included 86 adolescents with a history of suicidal behaviour. We found that perceived unavailability and high levels of depressive symptomatology were predictive of suicidal behaviours. We also found a strong association between being older and having high levels of angry distress in adolescents with a history of suicidal behaviours. CONCLUSION: The advantage of including an assessment of parent-adolescent attachment with clinical adolescents is noted.

Anti-tumour activity in vitro and in vivo of selective differentiating agents containing hydroxamate.

A series of hydroxamates, which are not metalloprotease inhibitors, have been found to be selectively toxic to a range of transformed and human tumour cells without killing normal cells (fibroblasts, melanocytes) at the same concentrations. Within 24 h of treatment, drug action is characterized by morphological reversion of tumour cells to a more normal phenotype (dendritic morphology), and rapid and reversible acetylation of histone H4 in both tumour and normal cells. Two hydroxamates inhibited growth of xenografts of human melanoma cells in nude mice; resistance did not develop in vivo or in vitro. A third hydroxamate, trichostatin A, was active in vitro but became inactivated and had no anti-tumour activity in vivo. Development of dendritic morphology was found to be dependent upon phosphatase activity, RNA and protein synthesis. Proliferating hybrid clones of sensitive and resistant cells remained sensitive to ABHA, indicating a dominant-negative mechanism of sensitivity. Histone H4 hyperacetylation suggests that these agents act at the chromatin level. This work may lead to new drugs that are potent, and selective anti-tumour agents with low toxicity to normal cells.

Towards protein surface mimetics.

Proteins are generally poor drug candidates due to bioavailability problems that stem from conformational instability, susceptibility to proteolytic degradation, poor membrane penetration, and unfavourable pharmacokinetics. Since many proteins exert their biological activity through relatively small regions of their folded surfaces, their actions could in principle be reproduced by much smaller designers molecules that retain these localised bioactive surfaces but have potentially improved pharmacokinetic/dynamic properties. Unlike proteins, smaller peptides generally lack well defined three dimensional structure in aqueous solution and tend to be conformationally mobile. Considerable progress has been made in recent years towards the use of molecular constraints to stabilise bioactive conformations. By affixing or incorporating templates that fix secondary and tertiary structures of small peptides, synthetic molecules (protein surface mimetics) can be devised to mimic the localised elements of protein structure that constitute bioactive surfaces. This is a promising growth area of medicinal chemistry that could impact significantly on biology and medicine. In this perspective review we summarise and prescribe methods for mimicking individual elements of secondary structure (helices, turns, strands, sheets) and for assembling their combinations into tertiary structures (helix bundles, multiple loops, helix-loop-helix motifs). A detailed understanding of the features that stabilise secondary and tertiary structures is the key to developing appropriate templates to support and correctly position residues in smaller folded surfaces. The goal is to direct critical amino acids (or surrogates) into the same conformational space and orientation as in bioactive surfaces of a native protein, yet retain sufficient flexibility to bind cooperatively, and with complementarity, to a given receptor. The requirements of size, shape, and directionality for templates to control peptide assembly and folding are discussed in relation to selected mimetics of secondary and tertiary structures. Particularly striking is the general tendency for protease inhibitors and MHC-binding peptides to adopt strand conformations; agonists and antagonists for G protein-coupled receptors to predominate in turn structures; transcription factors, cytokines and DNA/RNA-binding motifs to be helical; and antigen-recognition segments of antibodies to involve multiple loops.

Tumor selectivity and transcriptional activation by azelaic bishydroxamic acid in human melanocytic cells.

Azelaic bishydroxamic acid (ABHA), a potent differentiating agent for lymphoid cells, was selectively toxic for 5 human tumor cell lines and transformed human melanocytes and keratinocytes (dose for 37% survival, D37, 30-100 microg/mL) compared with normal cells (melanocytes, fibroblasts; D37 > 300 microg/mL). Dendritic morphology was the only indicator found for increased differentiation, markers for the pigmentation pathway being unchanged or inhibited by ABHA. In contrast to hexamethylene bisacetamide and azelaic acid, ABHA significantly increased the HIV LTR, SV40 and c-fos promoter activities during a 24 hr treatment. Metallothionein promoter activity was enhanced by 5 hr treatment with ABHA in a sensitive melanoma cell line (MM96L) but was inhibited in a more resistant line (HeLa); c-fos promoter activity was inhibited in HeLa during this time. Transcription from a p53 binding response element was inhibited in MM96L by a 24 hr ABHA treatment but enhanced in HeLa. ABHA may represent a structural prototype for designing more potent and selective anti-melanoma agents.

Reflective capacity and its significance to the attachment concept of the self.

Attachment writing about the self systematically regards the working model of the self as a social product. At the same time, reflective self-capacity is regarded as a particularly salient aspect of the individual's current state of mind with respect to attachment. Additionally, evidence has accumulated that some individuals have been able to create a coherent working model of the self despite a history of negative attachment experiences. This paper proposes that in these circumstances it is necessary to conceive of a private self through which the history of attachment experiences may be creatively transformed.

Biphasic response of the metallothionein promoter to ultraviolet radiation in human melanoma cells.

Because metallothionein (MT) is elevated and may be protective in UV-irradiated skin, we have studied the effects of UV and other agents on MT transcription using the sheep MT 1A promoter, linked to the beta-galactosidase gene and stably transfected into human cell lines. beta-galactosidase reporter activity was inducible by adding Zn2+ ions to the medium (100 microM for 2-4 h). Two differentiating agents, butyric acid and azelaic bishydroxamic acid (ABHA), significantly increased the response to Zn2+ in a melanoma cell line (MM96L-gal). UVB (280-315 nm) had two distinct, time-dependent effects. During the first 4 h after irradiation, high doses of UVB inhibited induction by Zn2+, an effect that was made more acute by simultaneous exposure to the differentiating agents. These changes in reporter activity were not due to alterations in Zn2+ transport into the cell. The UVB-depressed MT response subsequently recovered and by 24 h was double the control, yet remained sensitive to ABHA. Reporter activity in transfected HeLa cells differed from that in MM96L, being depressed 4 and 24 h after UVB and insensitive to ABHA at both times. Galactosidase reporter activity driven by non-MT promoters was not affected by these treatments. Dependence of MT transcriptional activity on UV-related DNA damage could be inferred because equitoxic UVC (254 nm) affected the response to Zn2+ in a similar fashion, whereas UVA, cisplatin and a methylating agent had no effect. The MT response was partly dependent on the PKC signal transduction pathway because it was inhibited by phorbol ester in HeLa, and by bisindolyl maleimide in HeLa and MM96L. The biphasic MT transcriptional response may model a signal transduction pathway that gives an early, depressed response to acute UV damage, with exacerbation by concurrent differentiation stimuli, but switches to a positive, cell-specific and potentially protective response at later times.

Tolypodiol, an antiinflammatory diterpenoid from the cyanobacterium Tolypothrix nodosa.

A new diterpenoid, tolypodiol (1), has been isolated from the terrestrial cyanobacterium Tolypothrix nodosa (HT-58-2) and identified by NMR and mass spectral analysis. The monoacetate derivative 2 was prepared. Tolypodiol (1) and its monoacetate derivative 2 show potent antiinflammatory activity in the mouse ear edema assay.

Possible role of the intestinal P-450 enzyme system in a cyclosporine-clarithromycin interaction.

Clarithromycin is a macrolide antibiotic similar in structure to erythromycin, but suggested to have fewer drug interactions. Although a pharmacokinetic interaction between clarithromycin and cyclosporine was recently reported, its magnitude and mechanism have not been explored. A 43-year-old renal transplant recipient receiving cyclosporine was treated with clarithromycin because of pneumonia. A cyclosporine pharmacokinetic study was performed 8 days after the initiation of the clarithromycin and 14 days after stopping the drug. Clarithromycin coadministration caused an approximately 2-fold increase in the area under the whole blood concentration versus time curve of cyclosporine. The oral clearance of cyclosporine was halved by clarithromycin, but the terminal elimination rate constant decreased only 15% and mean residence time 20%. These observations suggest that clarithromycin inhibits not only the hepatic metabolism but also the intestinal metabolism of cyclosporine. Caution is advised when administering the two drugs concurrently, and additional studies are necessary to elucidate the mechanism of this interaction.

Binding kinetics and bioassay of RRE mRNA fragments to a peptide containing the recognition domain of HIV-1 Rev.

Surface plasmon resonance techniques have been used to examine the kinetics of binding for two RNA fragments to an RNA binding domain of HIV-1 REv. RBE3 RNA elicited an apparent dissociation constant (KD) of 121 nM while RREIIB41-79 RNA exhibited an apparent dissociation constant (KD) of 2.5 nM. The dissociation rates for both RNA fragments were comparable. However, the shorter sequence, RBE3, exhibited considerably slower association kinetics. A series of known inhibitors were assayed against these RNA' and the derived K1's were consistent with those reported in the literature, validating the method for routine inhibitor assays.

Affinity purification of a correctly folded fragment of synthetic HIV-1 mRNA using a HIV-1 Rev peptide-ligand.

Formation of a macromolecular complex between the RNA binding protein HIV-1 Rev and HIV-1 mRNA is an essential prerequisite for nuclear export and subsequent expression of HIV-1 mRNA. The arginine rich peptide TRQARRNRRRRWRARQR, corresponding to residues 34-50 of HIV-1 Rev, contains the mRNA binding motif. We prepared a thioether linked Rev34-50-cellulose conjugate to affinity purify a fragment of synthetic mRNA corresponding to the high affinity binding site for Rev. The correctly folded fraction of mRNA (27.5%) was isolated from a crude synthetic mixture.

Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations.

The carboxyl-terminal domain (CTD) of the RNA polymerase II largest subunit plays an essential but poorly understood role in transcription. The CTD is highly phosphorylated in vivo and this modification may be important in the transition from transcription initiation to elongation. We report here the development of a strategy for creating novel yeast CTDs. We have used this approach to show that the minimum viable CTD in yeast contains eight consensus (Tyr1Ser2Pro3Thr4Ser5Pro6Ser7) heptapeptide repeats. Substitution of alanine or glutamate for serines in positions two or five is lethal. In addition, changing tyrosine in position one to phenylalanine is lethal. The effects of mutations that alter potential phosphorylation sites are consistent with a requirement for CTD phosphorylation in vivo.

NMR solution structure of the RNA-binding peptide from human immunodeficiency virus (type 1) Rev.

NMR spectroscopy has been used to solve the three-dimensional solution structure of a minimal RNA-binding domain of the Rev protein from the human immunodeficiency virus (type 1), an essential regulatory protein for viral replication. The presence of 10 arginine residues in the 17-residue peptide Rev34-50 caused significant problems in assignment of the NMR spectra. To improve spectral resolution, the peptide was synthesized with an alanine replacing a nonessential arginine and with selectively 15N-labeled residues. Contrary to Chou-Fasman modeling predictions an alpha-helix was detected in both water and 20% trifluoroethanol (TFE) and was found to span residues that constitute the RNA-binding and nuclear-localizing domains of Rev. The sequence-specific information provided by the NMR data gives a full description of the solution conformation of Rev34-50 which serves as a template for investigating binding of the peptide to RNA from the Rev response element (RRE). Preliminary modeling suggests that the helix can fit neatly into the expanded major groove of the RRE where interactions between the peptide side chains and the RNA can be identified. These data may aid the construction of a suitable pharmacophore model for the rational design of molecules that block Rev-RNA binding and inhibit HIV replication.

Targeting HIV-1 protease: a test of drug-design methodologies.

The proteinase of the human immunodeficiency virus (HIV-1 protease) is an obvious example of a receptor for which drug design methodologies have been successfully applied. In this article, Michael West and David Fairlie outline the specific progress made to date towards the rational design of protease inhibitors as anti-HIV drugs, and compare their pharmacological profiles. The rationale employed in designing protease inhibitors illustrates evolving trends in drug design, problems in comparing assay data, and obstacles to developing enzyme inhibitors into drugs.

Experience with not offering dialysis to patients with a poor prognosis.

Despite ongoing discussion of dialysis rationing in the nephrology community, there are little available data describing current practice in treatment selection for very ill renal patients with a poor prognosis. We report a prospective survey of end-stage renal patients referred to our Canadian regional dialysis center who were not accepted to the dialysis program on the grounds of poor prognosis and low quality of life. One quarter of patients referred during 1992 were not accepted to the program, with a mean age of 74 +/- 11 years. Patients were predominantly female and most suffered from a combination of renovascular and cardiovascular disease, with very poor functional capacity as determined by the Karnofsky scale. Nonacceptance to the dialysis program did not create legal difficulties or requests for second opinions. Based on our experience, we propose guidelines for nonacceptance of patients to dialysis programs.