Conducting Drug Treatment Trials in Children: Opportunities and Challenges.

Children were often referred to as "therapeutic orphans" in the past due to different reasons such as ethical, regulatory, economic, scientific, etc., ones. They were exposed to avoidable risks while missing out on therapeutic advances. Pediatric patients have suffered from a lack of scientific and regulatory standards (e.g., proper drug testing, authorization of medicines for their use, etc.), although the pharmaceutical legislative framework, which ensures the high standards of safety, quality, and efficacy of medicinal products for use in adults, was developed primarily in response to past "drug disasters," mainly involving children. The adoption of pediatric regulatory initiatives first in the USA and then in Europe and other countries and regions has significantly changed the worldwide frameworks and permanently changed pediatric drug research and development. This article tries to give various perspectives with historical context, a review of the different challenges and opportunities as well as important stakeholders in pediatric drug development. The pediatric trial networks are probably the most important stakeholder that enables efficient patient recruitment, access to better resource utilization, and global collaboration of different stakeholders necessary for performing quality and well-designed clinical trials.

Data from a microdosed recombinant human erythropoietin administration study applying the new biotinylated clone AE7A5 antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis protocol.

Erythropoietin (EPO) is a hormone, which stimulates the production of red blood cells. Due to its performance-enhancing effect, it is prohibited by the World Anti-Doping Agency (WADA). In order to reduce the detection window of EPO doping, athletes have been applying low doses of recombinant EPO (e.g., <10 IU/kg body weight, daily or every second day) instead of larger doses twice or more per week (e.g., 30 IU/kg). Microdoses of Retacrit (epoetin zeta), an EPO biosimilar, were administered intravenously and subcutaneously to human males and females. Urine and serum samples were collected and analysed applying the new biotinylated clone AE7A5 EPO antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE) protocol. With the improved protocol, microdosed Retacrit (7.5 IU/kg body weight [BW]) was detectable for at least 52 h after intravenous administration. Detection windows were approximately the same for serum and urine and doubled after subcutaneous administration (~104 h). Previous studies applying different electrophoretic techniques and the not further optimized SAR-PAGE protocol revealed considerably shorter detection windows for recombinant human erythropoietin (rhEPO) microdoses. Because the new biotinylated antibody performed significantly more sensitive than the nonbiotinylated version, the new protocol will improve the sensitivity and hence detectability of recombinant EPO in doping control.

Experience with comprehensive pharmacogenomic multi-gene panel in clinical practice: a retrospective single-center study.

AIM: To assess the prevalence of actionable pharmacogenetic interventions in patients who underwent pharmacogenetic testing with a multi-gene panel. METHODS: We retrospectively reviewed single-center electronic health records. A total of 319 patients were enrolled who underwent pharmacogenomic testing with the RightMed test panel using TaqMan quantitative real-time PCR method and copy number variation analysis to determine the SNPs in the 27 target genes. RESULTS: Actionable drug-gene pairs were found in 235 (73.7%) patients. Relevant guidelines on genotype-based prescribing were available for 133 (56.7%) patients at the time of testing. Based on the patients' genotype, 139 (43.6%) patients were using at least one drug with significant pharmacogenetic interactions. CONCLUSION: Two out of three patients had at least one drug-gene pair in their therapy. Further studies should assess the clinical effectiveness of integrating pharmacogenomic data into patients' electronic health records.

Association of bacterial load in drinking water and allergic diseases in childhood.

BACKGROUND: Treatment of drinking water may decrease microbial exposure. OBJECTIVE: To investigate whether bacterial load in drinking water is associated with altered risk of allergic diseases. METHODS: We recruited 1,110 schoolchildren aged 6-16 years between 2011 and 2013 in Pozega-Slavonia County in Croatia, where we capitalized on a natural experiment whereby individuals receive drinking water through public mains supply or individual wells. We obtained data on microbial content of drinking water for all participants; 585 children were randomly selected for more detailed assessments, including skin prick testing. Since water supply was highly correlated with rural residence, we compared clinical outcomes across four groups (Rural/Individual, Rural/Public, Urban/Individual and Urban/Public). For each child, we derived quantitative index of microbial exposure (bacterial load in the drinking water measured during the child's first year of life). RESULTS: Cumulative bacterial load in drinking water was higher (median [IQR]: 6390 [4190-9550] vs 0 [0-0]; P < .0001), and lifetime prevalence of allergic diseases was significantly lower among children with individual supply (5.5% vs 2.3%, P = .01; 14.4% vs 6.7%, P < .001; 25.2% vs 15.1%, P < .001; asthma, atopic dermatitis [AD] and rhinitis, respectively). Compared with the reference group (Urban/Public), there was a significant reduction in the risk of ever asthma, AD and rhinitis amongst rural children with individual supply: OR [95% CI]: 0.14 [0.03,0.67], P = .013; 0.20 [0.09,0.43], P < .001; 0.17 [0.10,0.32], P < .001. Protection was also observed in the Rural/Public group, but the effect was consistently highest among Rural/Individual children. In the quantitative analysis, the risk of allergic diseases decreased significantly with increasing bacterial load in drinking water in the first year of life (0.79 [0.70,0.88], P < .001; 0.90 [0.83,0.99], P = .025; 0.80 [0.74,0.86], P < .001; current wheeze, AD and rhinitis). CONCLUSIONS AND CLINICAL RELEVANCE: High commensal bacterial content in drinking water may protect against allergic diseases.

Expiratory variability index (EVI) is associated with the severity of acute bronchial obstruction in small children: A proof-of-concept study.

BACKGROUND: Lung function testing in small children is cumbersome. However, reduced variability of tidal breathing recorded using impedance pneumography (IP) during sleep was recently found to be a potential objective marker of wheeze in children aged 1-5 years. We aimed to investigate how an acute bronchial obstruction (BO) and its severity, and recovery thereof reflect in expiratory variability index (EVI). METHODS: EVI was measured using a wearable IP system (Ventica®) during sleep in 40 healthy controls (aged 1.5-5.9 years) and 30 patients hospitalized due to acute BO (aged 1.3-5.3 years). In healthy controls, EVI was measured for 1-3 nights at their homes. Patients were measured for several nights during hospitalization, as practically feasible, and at home 2 and 4 weeks post-discharge. RESULTS: We received 79 EVI results from 39 controls and 139 from 30 patients. 90% had previous BO episodes, 30% used asthma controller medication before and 100% after hospitalization. Compared to controls, EVI was significantly lower during hospitalization (P < .0001) having significant correlation with number of days to discharge (r = -.38, P = .004). At 2 or 4 weeks post-discharge, EVI was not significantly different from the controls (P = .14, P = .49, respectively). EVI was significantly associated with chest auscultation findings (P = .0001) being 17.5 (4.9) (median, IQR) with normal auscultation, 15.6 (7.4) in those with prolonged expiration and 11.4 (6.8) in those with wheeze and/or rales and crackles. CONCLUSIONS: EVI was found to be a sensitive, objective marker of acute BO, showing strong association with changes in clinical status in wheezy children aged 1-5 years.

Quantification of endogenous steroid sulfates and glucuronides in human urine after intramuscular administration of testosterone esters.

For an effective detection of doping with pseudo-endogenous anabolic steroids, the urinary steroid profile is of high value. In this work, the aim was to investigate steroid metabolism disruption after exogenous intramuscular administration of different testosterone esters. The investigation focused on both sulfo - and glucoro conjugated androgens. A single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido®) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanone®), was given to six healthy volunteers. Urine was collected throughout a testing period of 60 days. A LC-MS method was developed and validated for the analysis of eight conjugated steroids in their intact form. The results show that urinary changes in both sulfo - and glucuro conjugated steroid levels are prominent after the injection of testosterone esters. A promising potential marker for the intake of exogenous testosterone is the combined ratio of epitestosterone sulfate/epitestosterone glucuronide to testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) as a complementary biomarker for testosterone abuse. This represents a new piece of evidence to detect testosterone doping, representing a new approach and being independent from the metabolic connections of the markers in the steroid passport.

Pharmacogenomics at the center of precision medicine: challenges and perspective in an era of Big Data.

Pharmacogenomics (PGx) is one of the core elements of personalized medicine. PGx information reduces the likelihood of adverse drug reactions and optimizes therapeutic efficacy. St Catherine Specialty Hospital in Zagreb/Zabok, Croatia has implemented a personalized patient approach using the RightMed® Comprehensive PGx panel of 25 pharmacogenes plus Facor V Leiden, Factor II and MTHFR genes, which is interpreted by a special counseling team to offer the best quality of care. With the advent of significant technological advances comes another challenge: how can we harness the data to inform clinically actionable measures and how can we use it to develop better predictive risk models? We propose to apply the principles artificial intelligence to develop a medication optimization platform to prevent, manage and treat different diseases.

Challenges in anesthesia personalization: resolving the pharmacogenomic puzzle.

Clinicians are witnessing differences in the doses required for induction and maintenance of anesthesia, as well as prolonged recovery in some patients. Predictable factors like patient characteristics, factors related to the procedure, pharmacological characteristics of anesthetics and adjunctive drugs, might explain some of the observed differences. However, the role of various polymorphisms of genes encoding for drugs' molecular targets, transporters and metabolic enzymes can have a significant impact on anesthesia outcome, too. In the present paper, we critically discuss pharmacological characteristics of the most common drugs used in anesthesia, with a focus on the possible genetic background of unpredictable diversities in anesthesia outcomes.

Cystic fibrosis presentation in del. F508 and p. Tyr109Glyfs compound heterozygote CFTR state: a case report.

The diagnosis of cystic fibrosis (CF) is commonly confirmed by molecular genetics with the presence of specific mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report a case of cystic fibrosis (CF) in a 15-year-old female patient who is a compound heterozygote for CFTR gene, with delta F508 and Tyr109Glyfs mutations detected. This is the first detailed description of such a case in the medical literature. The primary CF presentation occurred at the age of 9 in the form of gastrointestinal symptoms including greasy, bulky, and foul-smelling stool. The patient exhibited delayed growth, with her height and weight being below the 5th centile for age according to the World Health Organization growth curves. Pancreatic enzyme supplement treatment was started immediately, alongside high-fat and high-calorie diet, resulting in patient's recovery and development. DNA analysis of CFTR gene demonstrated the presence of del. F508 mutation and a rare combining deletion and insertion mutation p. Tyr109Glyfs. The combination of the two mutations is very rare in CF patients and is therefore valuable to document this case in order to provide information on disease progression, therapy options, and outcomes. With standard treatment and early diagnosis, the patient is currently doing well and is not restricted by the disease in her daily and sports activities.

Diagnosis of perioperative anaphylaxis.

Every agent used during the perioperative period may be involved and have the potential to trigger both allergic, IgE and non-IgE reaction as well as non-specific (non-allergic) reactions. In many cases, an allergic mechanism cannot be ruled out and systematic investigations should be tested of all drugs and agents the patient was exposed to prior to the reaction. The complexity of agents used for anaesthesia and surgery present challenges when attempting to identify the culprit drugs and select proper testing to better recognition of trigger. The diagnosis of preoperative anaphylactic or anaphylactoid reaction is clinical and based upon the presence of characteristic symptom and signs that begin suddenly and developed rapidly in most cases. Elevations of mast cell mediators such as tryptase and histamine in the blood can help to distinguish anaphylaxis from other disorders that present with similar clinical picture. The secondary investigations of adverse perioperative drug reactions are highly specialised and include skin testing, in vitro testing and in some cases challenge tests. Any suspected reaction during anaesthesia must be extensively investigated and these diagnostic tests should be done in specialised centres. The cooperation between anaesthesiologists and allergists is necessary to provide the necessary diagnostic tests to identify the responsible drug, to carry out prevention and to provide recommendations for future anesthetic procedures.

Successful Treatment of Autoimmune Pulmonary Alveolar Proteinosis in a Pediatric Patient.

BACKGROUND Pulmonary alveolar proteinosis (PAP) is a rare condition characterized by the intra-alveolar accumulation of surfactant-derived material, which impairs gas exchange and results in respiratory insufficiency. Two major subtypes of PAP are autoimmune and non-autoimmune PAP. The diagnosis relies on clinical presentation, ground glass opacities on CT scan, bronchoscopy with PAS stain of BAL fluid (BALF), lung biopsy with PAS-positive material in the alveoli, and the presence of anti GM-CSF antibodies in serum or BALF for an autoimmune subtype. The therapeutic approach to pediatric cases varies according to age and the general clinical state of the child; however, whole lung lavage (WLL) and inhaled or subcutaneous GM-CSF are generally first-line therapy. CASE REPORT We report a unique case of an autoimmune type of PAP in a 12-year-old boy, who underwent successful bilateral lung transplantation after inefficacious treatment with GM-CSF, and who developed post-transplant lymphoproliferative disease (PTLD) and was successfully treated with a chemotherapeutic protocol. CONCLUSIONS Although lung transplantation is a rarely used therapeutic approach for patients with an autoimmune subtype of PAP, in cases of inefficacious treatment with other modalities, lung transplantation should be considered.

The effect of mouth breathing on exercise induced fall in lung function in children with allergic asthma and rhinitis.

OBJECTIVES: Exercise induced bronchospasm (EIB) represents a common feature of childhood asthma which is most commonly revealed during free running. On the other hand aerobic exercise shows significant beneficial effects in asthmatics especially on the reduction of the level of systemic inflammation and is recommended as part of its treatment. The aim of this study was to test how mandatory mouth breathing influences the exercise induced level of decrease in lung function according to the level of severity of allergic rhinitis (AR). METHODS: Free 6-minute running test preceded and followed by spirometry done with and without a nose clip a day apart was conducted in 55 children with moderate persistent asthma and AR. Children were divided into two groups according to the severity of nasal symptoms. RESULTS: There was a greater fall in forced expiratory volume in one second after exercise with a nose clip in children with less nasal symptoms than in children with more nasal symptoms (mean ± SD; -5.28 (7.91) vs. -0.08 (4.58), p = 0.0228) compared to testing without the nose clip (mean ± SD; LNS, -1.31 ± 3.89%, p = 0.2408; MNS, -1.47 ± 3.68%, p = 0.2883). CONCLUSION: Our results show that regular mouth breathing due to nasal congestion may lessen the degree of EIB in patients with persistent AR and allergic asthma.

Detection of testosterone esters in blood.

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma. Furthermore, the suitability of different types of blood collection devices was evaluated. Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested. A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®)) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon(®)). Blood was collected throughout a testing period of 60 days. The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All investigated testosterone esters could be detected in post-administration blood samples. The detection time depended on the type of ester administered. Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied. The testosterone ester detection window, however, was comparable.

Urates in exhaled breath condensate as a biomarker of control in childhood asthma.

OBJECTIVE: The aim of this study was to (1) investigate the possibility to use urates in exhaled breath condensate (EBC) as a biomarker of airway inflammation and control in childhood asthma and (2) explore their association with other biomarkers of airway inflammation and clinical indices of asthma control (Asthma Control Test [ACT], quality of life [PAQLQ], lung function, prn beta-agonist use, time from last exacerbation [TLE]. METHODS: This cross-sectional study comprised 103 consecutive patients (age 6-18 years) divided in groups of uncontrolled ([NC], n = 53) and controlled asthma ([C], n = 50). Measured lung function and biomarkers included: spirometry, eosinophilic cationic protein (ECP), high-sensitivity C-reactive protein (hs-CRP), exhaled NO (FENO), pH and urates in EBC and exhaled breath temperature (EBT). RESULTS: Statistically significant differences were found between groups for EBC urates, EBC pH and EBT (NC versus C: EBC urates, median [IQR], µmol/L; 10 [6] versus 45 [29], p < 0.001; EBC pH, mean [SD], 7.2 [0.17] versus 7.33 [0.16], p = 0.002; EBT mean [SD], °C; 34.26 [0.83], versus 33.90 [0.60], p = 0.014). EBC urates showed significant association with TLE and FENO (r = 0.518, p < 0.001; r = 0.369, p = 0.007, respectively) in NC, and EBC pH (r = 0.351, p < 0.001), FEV1 (r = 0.222, p = 0.024), ACT (r = 0.654, p < 0.001), PAQLQ (r = 0.686, p < 0.001) and prn salbutamol use (r = -0.527, p < 0.001) in all asthmatics. CONCLUSION: In our study, EBC urates were found to be the best single predictor of asthma control and underlying airway inflammation. Our results provide evidence supporting the potential utility to use EBC urates as an additional non-invasive biomarker of control in childhood asthma.

Lung diffusion capacity in children with respiratory symptoms and untreated GERD.

BACKGROUND: Gastroesophageal reflux disease (GERD) is associated with many respiratory disorders, among which, chronic cough, laryngitis, and asthma are among the most common. We investigated lung function, including gas diffusion capacity, in children with poor asthma control or chronic laryngitis with untreated GERD. MATERIAL AND METHODS: A total of 71 children, aged 6-17 years, with chronic respiratory and other symptoms suggestive for GERD, were enrolled and divided into 2 groups: chronic laryngitis and asthma. Participants underwent 24-hour pH monitoring and lung function assessment, measurement of single-breath diffusing capacity of the lung for carbon monoxide (DLCO), and fraction of exhaled nitric oxide (FENO) measurement. RESULTS: 24-hour pH monitoring was positive for GERD in 92.1% of preselected children with asthma and 90.1% of children with chronic recurrent laryngitis. All flows (PEF, MEF75, MEF50, and MEF25) were significantly lower in the asthma group, while FENO and DLCO were significantly lower in the laryngitis group. A significant inverse relationship was found between DLCO and all reflux indexes in the laryngitis group. Each unit change of Johnson-DeMeester score and Boix-Ochoa score increased the odds for significantly lower DLCO in laryngitis patients by 3.9% and 5.5%, respectively. CONCLUSIONS: In children with uncontrolled asthma and chronic laryngitis, the regurgitation of gastric contents due to GERD contributes to poor asthma control and aggravation of chronic laryngitis. Despite having normal lung function, the gas diffusion capacity should be controlled in patients with GERD and chronic laryngitis, and it might be the very first abnormality in distal airways.

Pulmonary arteriovenous malformations presenting as difficult-to-control asthma: a case report.

INTRODUCTION: Although pulmonary arteriovenous malformations are relatively rare disorders, they are an important part of the differential diagnosis of common pulmonary problems, such as hypoxemia, dyspnea on exertion and pulmonary nodules. CASE PRESENTATION: An 11-year-old Croatian boy of Mediterranean origin with a history of asthma since childhood was admitted to our hospital for evaluation of difficult-to-control asthma during the previous six months. A chest X-ray showed a homogeneous soft tissue mass in the lingual area. Computed tomography angiography of the thorax showed two pulmonary arteriovenous malformations, one on each side of the lungs. Diagnosis of hereditary hemorrhagic telangiectasia was made clinically by Curaçao criteria. Genetic analysis revealed a mutation in the endoglin gene. The patient was treated with embolotherapy with good clinical outcome. CONCLUSION: We present a case of pulmonary arteriovenous malformations masquerading as refractory asthma.

Inhaled corticosteroids used for the control of asthma in a "real-life" setting do not affect linear growth velocity in prepubertal children.

BACKGROUND: Recent guidelines recommend inhaled corticosteroids as the first-line treatment for persistent asthma. However, long-term corticosteroid treatment in children has raised concerns about potential growth rate deceleration. We aimed to assess the association of growth velocity with the use of inhaled corticosteroids in prepubertal children with asthma in a "real-life" setting. MATERIAL/METHODS: This study included 844 children aged 4-9.5 years coming to the hospital for regular check-ups between October 2006 and February 2009 for asthma with/without allergic rhinitis and no other known constraints of growth. Out of the 844 children, 790 had all data needed for analysis--245 children were not treated with ICS, 545 children received ICS (fluticasone, budesonide) with/without INCS (fluticasone, mometasone or budesonide). During the study period, 48 children with/without ICS received short SCS courses. RESULTS: Mean (SE) height at the first check-up was 123.1 (0.31) cm; range (100.0-147.8 cm). Mean (SE) linear growth velocity (LGV) of the included children was 0.185 (0.0035) mm/day between 2 check-ups. No significant difference was found in LGV between the group not treated with ICS (0.180 mm/day±0.0055) and the group treated with ICS (0.187±0.0044 mm/day). Also, there was no statistical difference between subgroups according to additional therapy with INCS and SCS. No significant correlation was found for LGV and daily dose of ICS (r=0.086, p>0.05). CONCLUSIONS: In our retrospective study using electronic hospital database, ICS and combined use of corticosteroids did not show any association with LGV in prepubertal asthmatic children in a "real-life" setting.

[Allergic reactions to vaccines]. / Alergijske reakcije na cjepiva.

Allergic reactions after vaccination are very rare. Even if the vaccine is not clearly the cause of possible side effects, including possible allergic reactions occurring after vaccine administration, they should be reported to the appropriate authorized body. Allergic reaction occuring to other vaccine components are more likely than to the active agent, for instance to gelatine, egg protein, chicken protein, dextran, thimerosal, antimicrobials, etc. All patients with suspected allergy to vaccine components should be evaluated by an allergist. Immediate-type allergy skin testing should be performed in patients who appear to have had an allergic reaction after vaccination. This testing should help confirm that the reaction was IgE mediated and identify the responsible vaccine component. If the skin test result is negative, it is extremely unlikely that the patient will develop an allergic reaction and the patient can be vaccinated. In patients with suggestive history and positive skin tests results to vaccine components, the clinician can consider administering the alternative vaccine or the same vaccine in graded doses while observing the patient. Mild local reactions, fever, and other constitutional symptoms after vaccinations are not contraindications to subsequent doses.

The effect of cyclic adenosine monophosphate (cAMP) on acute liver toxicity in mice induced by D-galactosamine and lipopolysaccharide.

The aim of this study was to examine the effect of cyclic adenosine monophosphate (cAMP) and its possible interference/synergism with calcium channel blocker in mice with acute liver injury induced with D-galactosamine (D-GalN) and lipopolysaccharide (LPS). C57Bl/6 mice were given i.p. simultaneously 300 mg/kg D-GalN and LPS 0.01 mg/kg. This treatment induced severe hepatitis, as evidenced by high mortality (80-90%) of control mice and large increase in concentration of aminotransferases in plasma (AST, ALT). Injection of stabile analogue of cAMP (dibutyryl-cAMP, db-cAMP) one hour before hepatotoxic agents increased survival of mice in dose-dependent manner and in medium dose significantly decreased plasma ALT level. Similar (protective) effect had also verapamil, calcium channel blocker, when given in non toxic doses and at the same time schedule as db-cAMP Combination of db-cAMP and verapamil had not synergistic effect in protection from D-GalN+LPS hepatotoxicity; the survival of mice was similar to that seen in protection caused by each agent alone.