The patient's view on rare disease trial design - a qualitative study.

BACKGROUND: Clinical trials in rare diseases are more challenging than trials in frequent diseases. Small numbers of eligible trial participants, often complicated by heterogeneity among rare disease patients, hamper the design and conduct of a 'classical' Randomized Controlled Trial. Therefore, novel designs are developed by statisticians. However, it is important to be aware of possible design aspects that may jeopardize the feasibility of trial conduct. If the burden of participation is considered out of proportion by patients or parents, recruitment may fail or participants may drop out before trial completion. In order to maximize the chance of success of trials in small populations, it is important to know which aspects of trial design are considered important by patients. RESULTS: We have interviewed all ten members of the Patient Think Tank (PTT) of the ASTERIX project, a European research consortium on methodology for clinical trials in small populations. The PTT members are rare disease patient representatives who have completed extensive training in clinical trial methodology. We have analyzed the interviews qualitatively according to Grounded Theory using a thematic analysis, and we structured the topics in four chronologically ordered themes: 1. Involvement in trial design; 2. Opinions on trial design; 3. Trial participation; 4. Phase after the trial. Our main findings are that the PTT-members recommend that patients are involved in trial design from an early stage on, and have influence on the outcomes and measurement instruments that are chosen in the trial, the length of the study, the choice of participants, and the information that is sent to potential participants. Also, according to the PTT-members, patient groups should consider setting up disease registries, placebo groups should be minimized, and more education on clinical trials is advised. CONCLUSIONS: Rare disease patient representatives who have been educated about clinical trial methodology think it is important to involve patient representatives in research at an early stage. They can be of advice in trial design in such a way that the ratio of potential benefit and burden of trial participation as well as the chosen outcome measures and in- and exclusion criteria are optimized.

The POWER-tool: Recommendations for involving patient representatives in choosing relevant outcome measures during rare disease clinical trial design.

In clinical trials, it is relevant to ask patients and/or their caregivers which aspects concerning their disease they consider important to measure when a new intervention is being investigated. Those aspects, useful as outcome measures in a trial, are of pivotal importance for the result of the trial and the subsequent decision-making. In rare diseases the choice of outcome measures may be even more important, due to the small numbers and heterogeneity of the patients that are included. We have developed a tool to involve patients in the determination of outcome measures and the choice of measurement instruments. This tool was developed together with a patient think tank, consisting of a group of rare disease patient representatives, and by interviewing end users. We have road-tested our tool in an ongoing trial, and evaluated it during a focus group meeting. The tool consists of three steps: 1) Preparation, 2) Consultation of patients, 3) Follow-up during which the consultation results are implemented in the trial design. The tool provides guidelines for researchers to include the patient's opinion in the choice of outcome measures in the trial design stage. We describe the development of the POWER-tool (Patient participation in Outcome measure WEighing for Rare diseases), and first experiences of the tool in an ongoing trial.

Development of a patient-reported outcome measure for upper limb function in Duchenne muscular dystrophy: DMD Upper Limb PROM.

AIM: To develop a patient-reported outcome measure (PROM) assessing upper limb function related to activities of daily living (ADL) that cannot be observed in a clinical setting, specifically for patients with Duchenne muscular dystrophy (DMD) across a wide age range, applicable in the different stages of the disease. METHOD: The developmental process was based on US Food and Drug Administration guidelines. This included item generation from a systematic review of existing tools and expert opinion on task difficulty and relevance, involving individuals with DMD. Cultural aspects affecting ADL were taken into consideration to make this tool applicable to the broad DMD community. Items were selected in relation to a conceptual framework reflecting disease progression covering the full range of upper limb function across different ADL domains. RESULTS: After pilot testing and iterative Rasch analyses, redundant or clinically irrelevant items were removed. The final questionnaire consists of 32 items covering four domains of ADL (food, self-care, household and environment, leisure and communication). Test-retest reliability was excellent. INTERPRETATION: A DMD-specific upper limb PROM was developed on the basis of clinical relevance and psychometric robustness. Its main purpose is to document the patient self-reported natural history of DMD and assess the efficacy of interventions.

Forty-Five Years of Duchenne Muscular Dystrophy in The Netherlands.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle disease. No curative therapy is currently available, but in recent decades standards of care have improved. These improvements include the use of corticosteroids and mechanical ventilation. OBJECTIVE: To present a detailed population based report of the DMD disease course in The Netherlands (1980-2006) and evaluate the effect of changes in care by comparing it with an historical Dutch DMD cohort (1961-1974). METHODS: Information about DMD patients was gathered through the Dutch Dystrophinopathy Database using a standardized questionnaire and information from treating physicians. RESULTS: The study population involved 336 DMD patients (70% of the estimated prevalence), of whom 285 were still alive. Mean age at disease milestones was: diagnosis 4.3 years, wheelchair dependence 9.7 years, scoliosis surgery 14 years, cardiomyopathy (fractional shortening <27%) 15 years, mechanical ventilation 17 years and death 19 years. Within our cohort, corticosteroid use was associated with an increased age of wheelchair dependence from 9.8 to 11.6 years (p < 0.001). When comparing the recent cohort to the historical cohort, mean survival improved from 17 to 27 years (p < 0.001). CONCLUSION: The current study gives detailed information about the disease course of DMD patients, provides evidence for the positive effect of steroid treatment and mechanical ventilation and supports the use of patient registries as a valuable resource for evaluating improvements in care.

Highly efficient synthesis of ampicillin in an "aqueous solution-precipitate" system: repetitive addition of substrates in a semicontinuous process.

The synthesis of ampicillin catalyzed by Escherichia coli penicillin acylase was optimized in an aqueous system with partially dissolved antibiotic nucleus 6-aminopenicillanic acid (6-APA). The yields of both 6-APA and acyl donor could be improved by repetitively adding substrates to the reaction, allowing the concentration of 6-APA to remain saturated throughout. In this reaction concept, with four subsequent additions of substrates, 97% conversion of 6-APA and 72% of D-(-)-phenylglycine methyl ester (D-PGM) to ampicillin was achieved. The synthetic potential of this concept was estimated using a mathematical model which showed that by increasing the amount of added substrates a nearly quantitative conversion of 6-APA and 85% conversion of acyl donor into ampicillin could be achieved.

Enzymatic synthesis of beta-lactam antibiotics using penicillin-G acylase in frozen media.

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.

Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors.

Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants. The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone. The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol. small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions. The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R. leguminosarum.

Site-specific cleavage of RNA by Fe(II).bleomycin.

Bleomycin is an antitumor agent whose activity has long been thought to derive from its ability to degrade DNA. Recent findings suggest that cellular RNA may be a therapeutically relevant locus. At micromolar concentrations, Fe(II)-bleomycin readily cleaved a Bacillus subtilis tRNAHis precursor in a highly selective fashion, but Escherichia coli tRNA(Tyr) precursor was largely unaffected even under more forcing conditions. Other substrates included an RNA transcript encoding a large segment of the reverse transcriptase from human immunodeficiency virus 1. RNA cleavage was oxidative, approximately 10-fold more selective than DNA cleavage, and largely unaffected by nonsubstrate RNAs. RNA sequence analysis suggested recognition of RNA tertiary structure, rather than recognition of specific sequences; subsets of nucleotides at the junction of single- and double-stranded regions were especially susceptible to cleavage. The ready accessibility of cellular RNAs to xenobiotic agents, the high selectivity of bleomycin action on RNAs, and the paucity of mechanisms for RNA repair suggest that RNA may be a therapeutically relevant target for bleomycin.

The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. Chemical synthesis and biological activity of cyclic nucleotide dimer, trimer, and phosphothioate derivatives.

An unusual compound, cyclic-bis(3'----5') diguanylic acid (c-di-GMP or cGpGp), is involved in the regulation of cellulose synthesis in the bacterium Acetobacter xylinum. This cyclic dinucleotide acts as an allosteric, positive effector of cellulose synthase activity in vitro (Ka = 0.31 microM) and is inactivated via degradation by a Ca2(+)-sensitive phosphodiesterase, PDE-A (Km = 0.25 microM). A series of 13 analogs cyclic dimer and trimer nucleotides were synthesized, employing a phosphotriester approach, and tested for the ability to mimick c-di-GMP as activators of cellulose synthase and as substrates for PDE-A. Seven of the synthetic compounds stimulate cellulose synthase activity and all of these activators undergo the Ca2(+)-inhibited degradation reaction. The order of affinities for synthase activators is cGpGp approximately cdGpGp approximately cGp(S)Gp (S-diastereomer) greater than cIpGp greater than cdGpdGp greater than cXpGp greater than cIpIp greater than cGp(S)Gp (R-diastereomer). Three cyclic dinucleotides of negligible affinity for either enzyme are cApAp, cUpUp, and cCpCp. This same order of affinities essentially pertains to the analogs as inhibitors of PDE-A activity, but at least one cyclic dinucleotide, cXpXp, which does not bind to cellulose synthase, is also a substrate for the degradation reaction, demonstrating that although the two enzymes share a similar, high degree of specificity for c-di-GMP, their cyclic dinucleotide binding sites are not identical. Phosphodiester bonds of activators in which an exocyclic oxygen is replaced with an atom of sulfur (cGp(S)Gp isomers) resist the action of PDE-A, and such derivatives may be prototypes for synthetic non-hydrolyzable c-di-GMP analogs.

Hairpin structures in DNA containing arabinofuranosylcytosine. A combination of nuclear magnetic resonance and molecular dynamics.

Nuclear magnetic resonance (NMR) and model-building studies were carried out on the hairpin form of the octamer d(CGaCTAGCG) (aC = arabinofuranosylcytosine), referred to as the TA compound. The nonexchangeable protons of the TA compound were assigned by means of nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY). From a detailed analysis of the coupling data and of the NOESY spectra the following conclusions are reached: (i) The hairpin consists of a stem of three Watson-Crick type base pairs, and the two remaining residues, T(4) and dA(5), participate in a loop. (ii) All sugar rings show conformational flexibility although a strong preference for the S-type (C2'-endo) conformer is observed. (iii) The thymine does not stack upon the 3' side of the stem as expected, but swings into the minor groove. (This folding principle of the loop involves an unusual alpha t conformer in residue T(4).) (iv) At the 5'-3' loop-stem junction a stacking discontinuity occurs as a consequence of a sharp turn in that part of the backbone, caused by the unusual beta + and gamma t torsion angles in residue dG(6). (v) The A base slides over the 5' side of the stem to stack upon the aC(3) residue at the 3' side of the stem in an antiparallel fashion. On the basis of J couplings and a set of approximate proton-proton distances from NOE cross peaks, a model for the hairpin was constructed. This model was then refined by using an iterative relaxation matrix approach (IRMA) in combination with restrained molecular dynamics calculations. The resulting final model satisfactorily explains all the distance constraints.

Conformational consequences of the incorporation of arabinofuranosylcytidine in DNA. An NMR study of the DNA fragments d(CGCTAGCG) and d(CGaCTAGCG) in solution.

The self-complementary octamers d(CGCTAGCG) and d(CGaCTAGCG) (aC, arabinofuranosylcytidine) were studied by means of NMR spectroscopy. It is shown that d(CGaCTAGCG), under suitable conditions of oligonucleotide concentration, ionic strength and temperature, exclusively adopts a hairpin structure. However, under the same experimental conditions (5 mM DNA, no added salt, 295 K) d(CGCTAGCG) mainly adopts a B-DNA-type duplex. At lower temperatures (less than or equal to 290 K) the hairpin form of d(CGaCTAGCG) occurs in slow exchange with an intact B-DNA-type duplex. When the DNA concentration of d(CGCTAGCG) is dramatically reduced (less than or equal to 0.5 mM) the hairpin form becomes highly favoured at the expense of the dimer. Moreover, proton-chemical-shift considerations indicate that the structural features of the hairpin structure of d(CGCTAGCG) mimic, in part, those of the modified octamer d(CGaCTAGCG), i.e. a loop comprising only the two central residues with the thymine located into the minor groove (Pieters, J. M. L., de Vroom, E., van der Marel, G. A., van Boom, J. H., Koning, T. M. G., Kaptein, R. and Altona, C. unpublished results). Thermodynamic analysis of d(CGCTAGCG) yields an average Tmd value of 342 K (1 M DNA) and a delta Hod value of -266 kJ/mol for the dimer/coil transition and an average Tmh value of 321 K and delta Hoh - 102 kJ/mol for the hairpin/coil equilibrium. For the duplex/coil equilibrium of d(CGaCTAGCG) an average Tmd value of 336 K (1 M DNA) and delta Hod value of -253 kJ/mol are deduced. The hairpin/coil transition of d(CGaCTAGCG) is characterized by a delta Hoh value of -104 kJ/mol and an average Tmh value of 331 K. It is concluded that incorporation of an arabinofuranosylcytidine in the octamer d(CGaCTAGCG) results in stabilization of the hairpin form, whereas the dimer is destablized by two aC.dG base pairs.

Synthesis of nucleic acid methylphosphonothioates.

The reagent obtained in situ by treating methylphosphonothioic dichloride with 1-hydroxy-6-trifluoromethylbenzotriazole could be used for the introduction of methylphosphonothioate linkages. The individual diastereomers of the protected dimer d-Tp(S,Me)A were applied in the synthesis of the chiral pure (R or S) hexamers d-[CpCpTp(S,Me)ApGpG]. The reagent showed also to be very effective for the preparation of the 3',5'-cyclic methylphosphonothioate of uridine.

Bulge-out structures in the single-stranded trimer AUA and in the duplex (CUGGUGCGG).(CCGCCCAG). A model-building and NMR study.

Model-building studies were carried out on the trimer AUA. Bulge-out structures which allow incorporation into a continuous RNA helix were generated and energy-minimized. All geometrical features obtained by previous NMR studies on purine-pyrimidine-purine sequences are accounted for in these models. One of the models was used to fit into a double helical fragment. Only minor changes were necessary to construct a central bulge-out in an otherwise intact duplex. NMR and model-building studies were performed on the duplex (CUGGUGCGG).(CCGCCCAG) which contains an unpaired uridine residue. NOE data, chemical-shift profiles and imino-proton resonances provided evidence that the extra U is bulged out of the duplex. The relatively small dispersion in 31P chemical shifts (approximately equal to 0.7 ppm) indicate the absence of t/g or g/t combinations for the phosphodiester angles zeta/alpha. An energy-minimized model of the duplex, which fits the present collection of data, is presented.

Synthesis of cyclic oligonucleotides by a modified phosphotriester approach.

Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGCG) and d(TAAAAA)] and a cyclic octadeoxyribonucleotide [d(CGTGCGTG)].

Influence of uracil on the conformational behaviour of RNA oligonucleotides in solution.

NMR studies were carried out on some alternating pyrimidine-purine sequences: the single-stranded tetramers CACA and UGUG and the self-complementary octamer CACAUGUG. Assignments, based upon COSY, homonuclear Hartmann-Hahn, and NOESY experiments, are given for the resonances of all base protons and of several sugar protons. Chemical shift vs temperature profiles were used to obtain thermodynamic parameters for the single-stranded stack in equilibrium with random coil and the duplex in equilibrium with random coil equilibria. The populations of N-type conformer of the ribose rings were estimated from the observed J1'2'. Comparisons with another alternating pyrimidine-purine sequence Um2(6)AUm2(6)A and with the deoxyribose counterparts d(CACA), d(TGTG) and d(CACATGTG) are given. Previous 1H-NMR investigations of Um2(6)AUm2(6)A revealed that the population of bulge-out structure diminishes compared to m2(6)AUm2(6)A due to the U(1)-m2(6)A(2) stacking interaction. In CACA a strong stacking proclivity (Tm = 310 K) together with a clear preference for N-type ribose is observed. However, the stacking interactions in UGUG are relatively less stable (Tm = 288 K) and a bias towards S-type sugar is present. Besides a small amount of stack, a significant contribution of bulge out structure is proposed for UGUG. We conclude that the nature of the pyrimidine base mainly determines the formation of bulge-out structures. The poor stacking properties of uracil now appear to be mainly responsible for this phenomenon. Comparison with the deoxyribose counterparts shows a reasonable agreement between the Tm values of CACA and d(CACA), whereas the Tm of UGUG (288 K) is much lower than the Tm of d(TGTG) (315 K). It is suggested that the absence of bulge-out structures in DNA purine-pyrimidine-purine sequences is related to the relatively strong stacking proclivity of dT residues compared to that of U residues. The Tm values (average 341 K) for the duplex in equilibrium with random coil transition obtained for each residue of CACAUGUG appear very similar. All ribose rings, except the G(8), adopt a pure N conformer in the duplex. This is taken to mean that the differences in conformational behaviour of the constituent tetramers disappear upon duplex formation.

Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.

Conformational analysis of the tetranucleotides m6(2)A-m6(2)A-U-m6(2)A(m6(2)A = N6-dimethyladenosine) and U-m6(2)A-U-m6(2)A and of the hybrid dA-r(U-A). A one- and two-dimensional NMR study.

A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.

Preparation of the individual diastereomers of adenylyl-(2'-5')-P-thioadenylyl-(2'-5')-adenosine and their 5'-phosphorylated derivatives.

The individual diastereomers of trimer A2'p5'A2'p(s)5'A, containing one phosphorothioate linkage, were prepared via a modified hydroxybenzotriazole phosphotriester approach. The 5'-phosphorylated derivatives of the latter compounds were obtained after phosphorylation with a 6-trifluoromethyl-1-benzotriazolyl activated phosphoromorpholidate.