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Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2 ) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2 ) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.
Assuntos
Carcinoma Ductal Pancreático , Citostáticos , Neoplasias Pancreáticas , Humanos , Gencitabina , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Citostáticos/uso terapêutico , Desoxicitidina/efeitos adversos , Cobicistat , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Paclitaxel/efeitos adversos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Ensaios Clínicos Fase I como AssuntoRESUMO
Neoadjuvant chemotherapy can improve the survival of individuals with borderline and unresectable pancreatic ductal adenocarcinoma; however, heterogeneous responses to chemotherapy remain a significant clinical challenge. Here, we performed RNA sequencing (n = 97) and multiplexed immunofluorescence (n = 122) on chemo-naive and postchemotherapy (post-CTX) resected patient samples (chemoradiotherapy excluded) to define the impact of neoadjuvant chemotherapy. Transcriptome analysis combined with high-resolution mapping of whole-tissue sections identified GATA6 (classical), KRT17 (basal-like) and cytochrome P450 3A (CYP3A) coexpressing cells that were preferentially enriched in post-CTX resected samples. The persistence of GATA6hi and KRT17hi cells post-CTX was significantly associated with poor survival after mFOLFIRINOX (mFFX), but not gemcitabine (GEM), treatment. Analysis of organoid models derived from chemo-naive and post-CTX samples demonstrated that CYP3A expression is a predictor of chemotherapy response and that CYP3A-expressing drug detoxification pathways can metabolize the prodrug irinotecan, a constituent of mFFX. These findings identify CYP3A-expressing drug-tolerant cell phenotypes in residual disease that may ultimately inform adjuvant treatment selection.
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Adenocarcinoma , Terapia Neoadjuvante , Humanos , Citocromo P-450 CYP3A , Adjuvantes Imunológicos , Queratina-17 , FenótipoRESUMO
BACKGROUND: Immunotherapy in microsatellite stable colorectal or pancreatic cancer has not shown promising results. It has been hypothesized that targeting immunosuppressive molecules like SDF1-alpha/CXCL12 could contribute to immunotherapy and animal models showed promising results on T cell activation and migration in combination with immune checkpoint inhibition. METHODS: Here, we describe the successful application of anti-CXCL12 (NOX-A12) in patients with advanced stage pretreated metastatic colorectal and pancreatic cancer (OPERA trial). The treatment consisted of 2 weeks of anti-CXCL12 monotherapy with NOX-A12 followed by combination therapy with pembrolizumab (n=20 patients) until progression or intolerable toxicity had occurred. RESULTS: The treatment was safe and well tolerated with 83.8% grade I/II, 15.5% grade III and 0.7% grade V adverse events. Of note, for a majority of patients, time on trial treatment was prolonged compared with their last standard treatment preceding trial participation. Systematic serial biopsies revealed distinct patterns of modulation. Tissue and clinical responses were associated with Th1-like tissue reactivity upon CXCL12 inhibition. A downregulation of a cytokine cassette of interleukin (IL)-2/IL-16/CXCL-10 was associated with tumor resistance and furthermore linked to a rare, CXCL12-associated CD14+CD15+promonocytic population. T cells showed aggregation and directed movement towards the tumor cells in responding tissues. Serum analyses detected homogeneous immunomodulatory patterns in all patients, regardless of tissue responses. CONCLUSIONS: We demonstrate that the combination of CXCL12 inhibition and checkpoint inhibition is safe and grants further exploration of synergistic combinatorial strategies.
Assuntos
Quimiocina CXCL12/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Neoplasias PancreáticasRESUMO
PURPOSE: To investigate the safety, clinical efficacy, virus pharmacokinetics, shedding, and immune response after administration of an oncolytic parvovirus (H-1PV, ParvOryx) to patients with metastatic pancreatic ductal adenocarcinoma (PDAC) refractory to first-line therapy. PATIENTS AND METHODS: This is a noncontrolled, single-arm, open-label, dose-escalating, single-center clinical trial. Seven patients with PDAC and at least one liver metastasis were included. ParvOryx was administered intravenously on 4 consecutive days and as an intralesional injection, 6 to 13 days thereafter. Altogether, three escalating dose levels were investigated. In addition, gemcitabine treatment was initiated on day 28. RESULTS: ParvOryx showed excellent tolerability with no dose-limiting toxicities. One patient had a confirmed partial response and one patient revealed an unconfirmed partial response according to RECIST criteria. Both patients showed remarkably long surivial of 326 and 555 days, respectively. Investigation of pharmacokinetics and virus shedding revealed dose dependency with no excretion of active virus particles in saliva or urine and very limited excretion in feces. H-1PV nucleic acids were detected in tumor samples of four patients. All patients showed T-cell responses to viral proteins. An interesting immunologic pattern developed in tumor tissues and in blood of both patients with partial response suggesting immune activation after administration of ParvOryx. CONCLUSIONS: The trial met all primary objectives, revealed no environmental risks, and indicated favorable immune modulation after administration of ParvOryx. It can be considered a good basis for further systematic clinical development alone or in combination with immunomodulatory compounds.
Assuntos
Adenocarcinoma/secundário , Adenocarcinoma/terapia , Carcinoma Ductal Pancreático/secundário , Carcinoma Ductal Pancreático/terapia , Parvovirus H-1 , Sistema Imunitário/imunologia , Terapia Viral Oncolítica , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Idoso , Humanos , Pessoa de Meia-Idade , Terapia Viral Oncolítica/efeitos adversosRESUMO
The inhibitor of anaplastic lymphoma kinase (ALK) crizotinib significantly increases survival in patients with ALK-positive non-small cell lung cancer (NSCLC). When evaluating crizotinib pharmacokinetics (PKs) in patients taking the standard flat oral dose of 250 mg b.i.d., interindividual PK variability is substantial and patient survival is lower in the quartile with the lowest steady-state trough plasma concentrations (Cmin,ss ), suggesting that concentrations should be monitored and doses individualized. We investigated whether the CYP3A inhibitor cobicistat increases Cmin,ss of the CYP3A substrate crizotinib in patients with low exposure. Patients with ALK-positive NSCLC of our outpatient clinic treated with crizotinib were enrolled in a phase I trial (EudraCT 2016-002187-14, DRKS00012360) if crizotinib Cmin,ss was below 310 ng/mL and treated with cobicistat for 14 days. Crizotinib plasma concentration profiles were established before and after a 14-day co-administration of cobicistat to construct the area under the plasma concentration-time curve in the dosing interval from zero to 12 hours (AUC0-12 ). Patients were also monitored for adverse events by physical examination, laboratory tests, and 12-lead echocardiogram. Enrolment was prematurely stopped because of the approval of alectinib, a next-generation ALK-inhibitor with superior efficacy. In the only patient enrolled, cobicistat increased Cmin,ss from 158 ng/mL (before cobicistat) to 308 ng/mL (day 8) and 417 ng/mL (day 14 on cobicistat), concurrently the AUC0-12 increased by 78% from 2,210 ng/mL*h to 3,925 ng/mL*h. Neither safety signals nor serious adverse events occurred. Pharmacoenhancement with cobicistat as an alternative for dose individualisation for patients with NSCLC with low crizotinib exposure appears to be safe and is cost-effective and feasible.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cobicistat/farmacocinética , Crizotinibe/farmacocinética , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/genética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/economia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Cobicistat/administração & dosagem , Cobicistat/economia , Análise Custo-Benefício , Crizotinibe/administração & dosagem , Crizotinibe/economia , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Inibidores do Citocromo P-450 CYP3A/economia , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Estudos de Viabilidade , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/economia , Inibidores de Proteínas Quinases/farmacocinéticaRESUMO
PIM447, a novel pan-PIM inhibitor, has shown preclinical activity in multiple myeloma (MM). In the multicenter, open-label, first-in-human study, patients with relapsed and/or refractory MM were enrolled to determine the maximum-tolerated dose (MTD) or recommended dose (RD), safety, pharmacokinetics, and preliminary anti-myeloma activity of PIM447. PIM447 was administered in escalating oral doses of 70-700 mg once daily (q.d.) for 28-day continuous cycles. Seventy-nine patients with a median of four prior therapies were enrolled. Seventy-seven patients (97.5%) had an adverse event (AE) suspected as treatment related, with treatment-related grade 3/4 AEs being mostly hematologic. Eleven dose-limiting toxicities occurred, and an MTD of 500 mg q.d. and an RD of 300 mg q.d. were established. The main reason for discontinuation was disease progression in 54 patients (68.4%). In the entire study population, a disease control rate of 72.2%, a clinical benefit rate of 25.3%, and an overall response rate of 8.9% were observed per modified International Myeloma Working Group criteria. Median progression-free survival at the RD was 10.9 months. PIM447 was well tolerated and demonstrated single-agent antitumor activity in relapsed/refractory MM patients, providing proof of principle for Pim (Proviral Insertions of Moloney Murine leukemia virus) kinase inhibition as a novel therapeutic approach in MM.
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Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Monitoramento de Medicamentos , Resistência a Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Recidiva , Resultado do TratamentoRESUMO
Posaconazole prophylaxis is recommended for patients with acute myeloid leukaemia during induction chemotherapy. Although a tablet formulation with better oral bioavailability is available, some patients have to rely on the oral suspension in clinical routine. Therefore, effectiveness of posaconazole oral suspension under real-life clinical conditions and impact of patient education about the correct intake on its plasma concentrations were assessed in this study. Altogether 96 patients receiving 160 cycles of induction chemotherapy were retrospectively (40 patients) and prospectively (56 patients) analysed. Patients were assigned into two groups for each chemotherapy cycle according to the application of antifungal prophylaxis (A: posaconazole oral suspension, 200 mg three times a day ≥7 days; B: intake <7 days, fluconazole or no prophylaxis). Antifungal prophylaxis and therapy were analysed for each cycle. Additionally, plasma concentrations were determined from prospectively included subjects of group A who were intensively educated to perform a correct drug intake. Systemic antifungal therapy was statistically started less often in group A (26% vs 53%; P = 0.002). Posaconazole prophylaxis was associated with a lower risk of proven invasive fungal infection (P = 0.003). Median plasma concentration apparently increased between the first and second time of determination effected by an initial intensive on-site patient education. The clinical effectiveness of posaconazole oral suspension was confirmed. A detailed patient education at the beginning of the treatment with posaconazole oral suspensions seems to be of primary importance for efficient plasma concentrations.
Assuntos
Antibioticoprofilaxia/métodos , Antifúngicos/uso terapêutico , Antineoplásicos/efeitos adversos , Infecções Fúngicas Invasivas/prevenção & controle , Leucemia Mieloide Aguda/tratamento farmacológico , Educação de Pacientes como Assunto , Triazóis/uso terapêutico , Administração Oral , Adulto , Idoso , Antifúngicos/sangue , Feminino , Humanos , Quimioterapia de Indução/efeitos adversos , Quimioterapia de Indução/métodos , Infecções Fúngicas Invasivas/epidemiologia , Infecções Fúngicas Invasivas/etiologia , Masculino , Pessoa de Meia-Idade , Avaliação de Programas e Projetos de Saúde , Estudos Prospectivos , Estudos Retrospectivos , Suspensões , Resultado do Tratamento , Triazóis/sangue , Adulto JovemRESUMO
VXM01 is a first-in-kind orally applied tumor vaccine based on live attenuated Salmonella typhi carrying an expression plasmid encoding VEGFR2, an antigen expressed on tumor vasculature and a stable and accessible target for anti-angiogenic intervention. A recent randomized, placebo-controlled, phase I dose-escalation trial in advanced pancreatic cancer patients demonstrated safety, immunogenicity and transient, T-cell response-related anti-angiogenic activity of four priming vaccinations applied within one week. We here evaluated whether monthly boost vaccinations are safe and can sustain increased frequencies of vaccine-specific T cells. Patients with advanced pancreatic cancer were randomly assigned at a ratio of 2:1 to priming with VXM01 followed by up to six monthly boost vaccinations, or placebo treatment. Vaccinations were applied orally at two alternative doses of either 106 colony-forming units (CFU) or 107 CFU, and concomitant treatment with standard-of-care gemcitabine during the priming phase, and any treatment thereafter, was allowed in the study. Immunomonitoring involved interferon-gamma (IFNγ) ELIspot analysis with long overlapping peptides spanning the entire VEGFR2 sequence. A total of 26 patients were treated. Treatment-related adverse events preferentially associated with VXM01 were decreases in lymphocyte numbers in the blood, increased frequencies of neutrophils and diarrhea. Eight out of 16 patients who received at least one boosting vaccination responded with pronounced, i.e. at least 3-fold, increase in VEGFR2-specific T cell response over baseline levels. In the VXM01 vaccination group, VEGFR2-specific T cells peaked preferentially during the boosting phase with an average 4-fold increase over baseline levels. In conclusion, prime/boost vaccination with VXM01 was safe and immunogenic and increased vaccine specific T cell responses compared with placebo treatment.
RESUMO
Adverse drug interactions due to grapefruit juice are well known prompting warnings even in drug labels. Similar issues have not been reported for clementines and available data is scarce, despite of genetic descent. We observed substantially increased tacrolimus trough concentrations in a renal transplant patient consuming high clementine amounts and, thus, scrutinised the effects of clementine juice on drug metabolism and drug transporters in vitro and compared it to the effects of mandarin and grapefruit juice. All citrus juices profoundly induced several drug transporters and drug metabolising enzymes, whereas the effects of grapefruit juice were most pronounced (e.g. 156-fold and 34-fold induction of cytochrome P450 (CYP) 3A4 mRNA by grapefruit juice and clementine juice, respectively). However, the juices also inhibited e.g. CYP3A4, raising the question which effect prevails in vivo. Using an enzymatic activity assay, we demonstrated that at least in vitro CYP3A4 inhibition prevails for both grapefruit and clementine juice, whereas for CYP1A2 induction appears to predominate. Thus, inhibition of CYP3A4 is presumably the underlying reason for the observed increase in the concentrations of the CYP3A4 substrate tacrolimus in the patient. Taken together, our data indicate that clementine juice as well as grapefruit juice and to a lesser extent also mandarin juice can induce several important drug metabolising enzymes and drug transporters, but also inhibit some of these proteins. Our data indicate that clementine juice similar to grapefruit juice bears the potential for profound interactions with drugs potentially leading to adverse drug effects e.g. through over-exposure to CYP3A4 substrates.
Assuntos
Citrus paradisi , Citrus sinensis , Citrus , Interações Alimento-Droga/fisiologia , Sucos de Frutas e Vegetais , Tacrolimo/metabolismo , Adulto , Animais , Antibióticos Antituberculose/efeitos adversos , Antibióticos Antituberculose/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Sucos de Frutas e Vegetais/efeitos adversos , Células HEK293 , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/metabolismo , Camundongos , Rifampina/efeitos adversos , Rifampina/metabolismo , Tacrolimo/efeitos adversosRESUMO
Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with anti-inflammatory, antiviral and anti-cancer properties. Beneficial cardiovascular effects such as increased nitric oxide (NO) production through enhancement of endothelial NO synthase (eNOS) activity and upregulation of eNOS expression have been demonstrated for this compound. In the present study, immortalized human EA.hy 926 endothelial cells were incubated for up to 1 h with 1-100 µM BA and with the phosphatidylinositol-3-kinase (PI3K) inhibitors LY294002 and wortmannin, or the estrogen receptor (ER) antagonist ICI 182,780. Phosphorylation status of eNOS and total eNOS protein were analyzed by Western blotting using a serine 1177 phosphosite-specific antibody. Bioactive NO production was assessed by determination of cGMP content in rat lung fibroblasts (RFL-6) reporter cells. Short-term incubation of EA.hy 926 cells with BA resulted in eNOS phosphorylation at the serine 1177 residue in a concentration- and time-dependent manner with a half-maximal effective concentration of 0.57 µM. This was associated with an enhanced production of NO. BA-induced eNOS phosphorylation and NO production was completely blocked by pretreatment with ICI 182,780, and was attenuated by pretreatment with the PI3K inhibitors wortmannin and LY294002. These results indicate that fast non-genomic effects of ER with downstream signaling through the PI3K/Akt pathway and consecutive eNOS phosphorylation at serine 1177 are involved in BA-induced eNOS activation.
Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/metabolismo , Triterpenos/farmacologia , Androstadienos/farmacologia , Animais , Linhagem Celular , Cromonas/farmacologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fulvestranto , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/citologia , Pulmão/metabolismo , Morfolinas/farmacologia , Triterpenos Pentacíclicos , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Wortmanina , Ácido BetulínicoRESUMO
VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points - pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.
Assuntos
Arsenicais/metabolismo , Citocromo P-450 CYP3A/sangue , Leucemia Promielocítica Aguda/sangue , Leucemia Promielocítica Aguda/tratamento farmacológico , Óxidos/metabolismo , Trióxido de Arsênio , Arsenicais/administração & dosagem , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Óxidos/administração & dosagemRESUMO
BACKGROUND: The investigational oral DNA vaccine VXM01 targets the vascular endothelial growth factor receptor 2 (VEGFR-2) and uses Salmonella typhi Ty21a as a vector. The immune reaction elicited by VXM01 is expected to disrupt the tumor neovasculature and, consequently, inhibit tumor growth. VXM01 potentially combines the advantages of anti-angiogenic therapy and active immunotherapy. METHODS/DESIGN: This phase I trial examines the safety, tolerability, and immunological and clinical responses to VXM01. The randomized, placebo-controlled, double blind dose-escalation study includes up to 45 patients with locally advanced and stage IV pancreatic cancer. The patients will receive four doses of VXM01 or placebo in addition to gemcitabine as standard of care. Doses from 106 cfu up to 1010 cfu of VXM01 will be evaluated in the study. An independent data safety monitoring board (DSMB) will be involved in the dose-escalation decisions. In addition to safety as primary endpoint, the VXM01-specific immune reaction, as well as clinical response parameters will be evaluated. DISCUSSION: The results of this study shall provide the first data regarding the safety and immunogenicity of the oral anti-VEGFR-2 vaccine VXM01 in cancer patients. They will also define the recommended dose for phase II and provide the basis for further clinical evaluation, which may also include additional cancer indications. TRIAL REGISTRATION: EudraCT No.: 2011-000222-29, NCT01486329, ISRCTN68809279.