A phase I/II dose-escalation multi-center study to evaluate the safety of infusion of natural killer cells or memory T cells as adoptive therapy in coronavirus pneumonia and/or lymphopenia: RELEASE study protocol.

BACKGROUND: Moderate/severe cases of COVID-19 present a dysregulated immune system with T cell lymphopenia and a hyper-inflammatory state. This is a study protocol of an open-label, multi-center, double-arm, randomized, dose-finding phase I/II clinical trial to evaluate the safety, tolerability, alloreactivity, and efficacy of the administration of allogeneic memory T cells and natural killer (NK) cells in COVID-19 patients with lymphopenia and/or pneumonia. The aim of the study is to determine the safety and the efficacy of the recommended phase 2 dose (RP2D) of this treatment for patients with moderate/severe COVID-19. METHODS: In the phase I trial, 18 patients with COVID-19-related pneumonia and/or lymphopenia with no oxygen requirement or with an oxygen need of ≤ 2.5 liters per minute (lpm) in nasal cannula will be assigned to two arms, based on the biology of the donor and the patient. Treatment of arm A consists of the administration of escalating doses of memory T cells, plus standard of care (SoC). Treatment of arm B consists of the administration of escalating doses of NK cells, plus SoC. In the phase II trial, a total of 182 patients with COVID-19-related pneumonia and/or lymphopenia requiring or not oxygen supplementation but without mechanical ventilation will be allocated to arm A or B, considering HLA typing. Within each arm, they will be randomized in a 1:1 ratio. In arm A, patients will receive SoC or RP2D for memory T cells plus the SoC. In arm B, patients will receive SoC or RP2D for NK cells plus the SoC. DISCUSSION: We hypothesized that SARS-CoV-2-specific memory T-lymphocytes obtained from convalescent donors recovered from COVID-19 can be used as a passive cell immunotherapy to treat pneumonia and lymphopenia in moderate/severe patients. The lymphopenia induced by COVID-19 constitutes a therapeutic window that may facilitate donor engraftment and viral protection until recovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT04578210 . First Posted : October 8, 2020.

Phase I dose-escalation single centre clinical trial to evaluate the safety of infusion of memory T cells as adoptive therapy in COVID-19 (RELEASE).

BACKGROUND: Effective treatments are still needed to reduce the severity of symptoms, time of hospitalization, and mortality of COVID-19. SARS-CoV-2 specific memory T-lymphocytes obtained from convalescent donors recovered can be used as passive cell immunotherapy. METHODS: Between September and November 2020 a phase 1, dose-escalation, single centre clinical trial was conducted to evaluate the safety and feasibility of the infusion of CD45RA- memory T cells containing SARS-CoV-2 specific T cells as adoptive cell therapy against moderate/severe cases of COVID-19. Nine participants with pneumonia and/or lymphopenia and with at least one human leukocyte antigen (HLA) match with the donor were infused. The first three subjects received the lowest dose (1 × 105 cells/kg), the next three received the intermediate dose (5 × 105 cells/kg) and the last three received the highest dose (1 × 106 cells/kg) of CD45RA- memory T cells. Clinicaltrials.gov registration: NCT04578210. FINDINGS: All participants' clinical status measured by National Early Warning Score (NEWS) and 7-category point ordinal scales showed improvement six days after infusion. No serious adverse events were reported. Inflammatory parameters were stabilised post-infusion and the participants showed lymphocyte recovery two weeks after the procedure. Donor microchimerism was observed at least for three weeks after infusion in all patients. INTERPRETATION: This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent CD45RA- memory T cells is feasible and safe. FUNDING: Clinical Trial supported by Spanish Clinical Research Network PT17/0017/0013. Co-funded by European Regional Development Fund/European Social Fund. CRIS CANCER Foundation Grant to AP-M and Agencia Valenciana de Innovación Grant AVI-GVA COVID-19-68 to BS.

Cross-cutting view of current challenges in paediatric solid organ and haematopoietic stem cell transplantation in Europe: the European Reference Network TransplantChild.

The low prevalence of European paediatric transplanted patients and scarcity of resources and expertise led to the need for a multidisciplinary network able to improve the quality of life of paediatric patients and families requiring a solid organ or haematopoietic stem cell transplantation. The European Reference Network (ERN) TransplantChild is one of the 24 ERNs established in a European legal framework to improve the care of patients with rare diseases. ERN TransplantChild is the only ERN focused on both solid organ and haematopoietic stem cell paediatric transplantation, based on the understanding of paediatric transplantation as a complex and highly specialised process where specific complications appear regardless the organ involved, thus linking the skills and knowledge of different organ disciplines. Gathering European centres of expertise in paediatric transplantation will give access to a correct and timely diagnosis, share expertise and knowledge and collect a critical mass of patients and data that increases the speed and value of clinical research outcomes. Therefore, the ERN TransplantChild aims for a paediatric Pan-European, Pan-transplant approach.

A preliminary model to avoid the overestimation of sample size in bioequivalence studies.

Often the only available data in literature for sample size estimations in bioequivalence studies is intersubject variability, which tends to result in overestimation of sample size. In this paper, we proposed a preliminary model of intrasubject variability based on intersubject variability for Cmax and AUC data from randomized, crossovers, bioequivalence (BE) studies. From 93 Cmax and 121 AUC data from test-reference comparisons that fulfilled BE criteria, we calculated intersubject variability for the reference formulation and intrasubject variability from ANOVA. Lineal and exponential models (y=a(1-e-bx)) were fitted weighted by the inverse of the variance, to predict the intrasubject variability based on intersubject variability. To validate the model we calculated the coefficient of cross-validation of data from 30 new BE studies. The models fit very well (R2=0.997 and 0.990 for Cmax and AUC respectively) and the cross-validation correlation were 0.847 for Cmax and 0.572 for AUC. A preliminary model analyses allow us to estimate the intrasubject variability based on intersubject variability for sample size calculation purposes in BE studies. This approximation provides an opportunity for sample size reduction avoiding unnecessary exposure of healthy volunteers. Further modelling studies are desirable to confirm these results especially suggestions of the higher intersubject variability range.

A multiplex assay to detect variations in the CYP2C9, VKORC1, CYP4F2 and APOE genes involved in acenocoumarol metabolism.

OBJECTIVES: We have developed a genotyping system to determine the alleles of genes related to interindividual variability in acenocoumarol dosage requirements. This genotyping system is intended for routine clinical use and therefore it is essential that it be simple, fast and inexpensive. DESIGN AND METHODS: We developed a PCR multiplex SNaPshot reaction that targets 6 SNPs (single nucleotide polymorphisms) in CYP2C9, CYP4F2, VKORC1 and APOE genes, which are associated with acenocoumarol dose requirements. RESULTS: We tested the multiplex in 152 samples and found it to be 100% concordant with the results of other methods. CONCLUSIONS: We successfully produced a reliable multiplex system for simultaneously typing 6 SNPs. This system may be used as a model for accurate, simple and inexpensive genotyping of SNPs related to dose requirements. This information allows the prediction of drug efficiency in patients prior to treatment with acenocoumarol and the prevention of adverse drug reactions.

Use of antifungal agents in pediatric and adult high-risk areas.

The purpose of this investigation was to describe the characteristics of the use of systemic antifungal agents (AFAs) and to evaluate their appropriateness of use. A prospective drug-utilisation study was conducted in intensive-care areas: haematology-oncology services and transplant units. Data were collected in three periods over 9 months. The required sample size was determined to be 113 patients (margin of error ±7%, 95% confidence interval [CI]), assuming a variability of 50%. Two different investigator groups evaluated the appropriateness of use separately; Cohen's Kappa index was used to calculate the degree of agreement between groups. A total of 114 patients we included, of which 62 (54.4%) were children. A total of 150 prescriptions were administered; fluconazole was the most frequently prescribed (38%), followed by liposomal amphotericin B (22.7%) and caspofungin (18.7%). The indications were: (1) pre-emptive treatment of Candida in non-neutropaenic critically ill patients (35.1%), (2) treatment of systemic fungal infection (24.6%), (3) prophylaxis for systemic fungal infection (SFI) in immunocompromised patients (16.7%), (4) prophylaxis of SFI in transplant recipients (12.3%), (5) prophylaxis of SFI in preterm infants (5.3%), (6) treatment of SFI in neonates (6.1%). The Kappa index showed a substantial agreement (Kappa = 0.73). The indications were considered to be inappropriate in 71 (47.3%) episodes. The indications or dosages were inappropriate in 79 cases (52.7%). The indications, dosages or duration of treatment were inappropriate in 83 cases (55.3%). We conclude that AFAs are prescribed for a significant number of inappropriate indications.

A pharmacovigilance program from laboratory signals for the detection and reporting of serious adverse drug reactions in hospitalized patients.

The detection and reporting of serious adverse drug reactions (SADRs) have become important components of monitoring and evaluation activities performed in hospitals. We present the implementation of a prospective pharmacovigilance program based on automatic laboratory signals (ALSs) at a hospital. We also report the general findings after the first year of operation of the program, which involved ALSs that indicate various SADRs: agranulocytosis, aplastic anemia, liver injury, thrombocytopenia, hyponatremia, and rhabdomyolysis. The number of hospitalizations during the year was 54,525, and 1,732 patients experienced at least one ALS. The review of electronic medical records (EMRs) showed that no alternative cause (i.e., no non-SADR explanation) for the ALS was identified in 520 (30%) of the patients. After the individual ALS-patient evaluation, a total of 110 SADRs (6.35% of those identified after reviewing EMRs and 21.15% of those requiring individual patient evaluations) were identified. In other words, in order to identify a single SADR, we had to review the electronic records of approximately 16 patients and personally visit 5 patients.

The importance of sample size, log-mean ratios, and intrasubject variability in the acceptance criteria of 108 bioequivalence studies.

PURPOSE: Fulfilling bioequivalence criteria with highly variable drugs is difficult. The aim of this study was to compare the importance of sample size, intrasubject variability, and the point estimate of test and reference formulations with regard to meeting bioequivalence (BE) criteria [maximum observed plasma concentration (C(max)) and area under the concentration-time curve (AUC)]. METHODS: We compared 137 pairs of data from BE studies with a conventional number of subjects, approximately 31-32 volunteers, developed in the last 10 years. RESULTS: The third part of the studies failed to demonstrate BE, in part due to an unacceptable difference between the mean ratios (T/R) (18) but also due to high variability with small differences between formulations (17). Increasing the number of subjects is hard to justify, and expanding the confidence interval (CI) was insufficient for the most highly variable drugs. CONCLUSIONS: Therefore, for low-variable drugs, the difference between formulations was the cornerstone of the fulfillment of BE criteria, but for highly variable drugs, the intrasubject coefficient of variability (ICV) was decisive. Our proposal is that for highly variable drugs that fall outside BE 90% CI limits could result in BE in the absence of formulation effect and maximal differences between formulations below 20%.

Gender differences in the disposition of metronidazole.

OBJECTIVE: Gender is usually considered to be one of the factors influencing disposition of drugs, but the evidence available is sometimes conflicting and information for a large number of frequently used drugs is lacking. An evaluation of sex differences in the disposition ofmetronidazole was carried out during a bioequivalence study. SUBJECTS AND METHODS: Twenty-four volunteers (12 males and 12 females) were included in an open, single-dose, two-sequence, crossover randomized trial with a one-week washout interval. All volunteers received in each period, a single 250 mg dose of one of the two study formulations of metronidazole. Venous blood samples were collected immediately before and at 15 time points in an 48-hour interval after drug administration; metronidazole concentrations were determined by HPLC. Non-compartmental pharmacokinetic analysis was performed and log-transformed AUC(0-infinity) and Cmax were tested for bioequivalence. Sex differences were evaluated by means of a 4-factor (sex, sequence, treatment and period) ANOVA. RESULTS: The studied formulations were found bioequivalent according to international standards: average 90% confidence interval for AUC(0-infinity) was 98 to 104 and for Cmax 93 to 115. After correction for the administered dose/kg, AUC was about 12% lower in females than in males (p = 0.0388) and, therefore, a higher calculated oral Cl/kg was found in females. Apparent distribution volume, after correction for weight, was significantly higher in males (p = 0.0019). Metronidazole half-life and MRT were shorter in females than in males (p - 0.0014 and p = 0.0002, respectively). CONCLUSIONS: Data obtained in this study suggest that metronidazole clearance in females is about 12% higher than in males although these differences are probably of no clinical relevance.

Cefminox versus metronidazole plus gentamicin intra-abdominal infections: a prospective randomized controlled clinical trial.

BACKGROUND: The aim of this prospective study was to compare the safety and efficacy of a new cephamycin, cefminox 2 g/12 h, to those of the usual regimen combining metronidazole 500 mg/8 h and gentamicin 80 mg/8 h (M+G). PATIENTS AND METHODS: 160 patients with clinically proven intra-abdominal infection were prospectively included in an open parallel randomized comparative multicenter trial. Antibiotics were started preoperatively and discontinued after clinical and laboratory evidence of resolution of the infection. Serum and peritoneal fluid levels and serum bactericidal activities were also studied. RESULTS: 150 patients were clinically evaluable. There was one failure in the cefminox group and three in the M+G group (not significant, RR: 1.07, 95% CI: 1-1.15). No differences were found in the number of wound infections, length of stay or duration of antibiotic therapy. Adverse effects were reported in 11 cases, all of them mild to moderate. Escherichia coli and Bacteroides fragilis were the most frequently found microorganisms. CONCLUSION: Cefminox is as effective and as safe as M+G in the treatment of intra-abdominal infections.

Digoxin level and clinical manifestations as determinants in the diagnosis of digoxin toxicity.

The aim of this study was to determine the relative importance of different risk factors in the diagnosis of digitalis toxicity. The authors recruited inpatients for whom serum digoxin level was requested and prospectively followed them for a week to ascertain if they showed digitalis toxicity. The predictive value of different factors for the assessment of digoxin toxicity was analyzed by multiple logistic regression. Forty-one toxic and 58 nontoxic patients were included. In the univariant analysis, intoxicated patients were older, most were women, and they had worse renal function and higher digoxin level; but there were no differences in serum electrolytes or other risk factors. In the multivariant analysis, digoxin level was the only independent factor related to digitalis toxicity. A different risk of toxicity for each clinical manifestation was found for a certain digoxin level. Patients with signs of automaticity in the electrocardiogram had a higher likelihood of being intoxicated than patients with gastrointestinal symptoms, atrioventricular block, or bradycardia. Therefore, in the population evaluated in this study, digoxin level is the key independent factor in digoxin intoxication, although the probability of being intoxicated is also a function of the type of clinical manifestations. A graphic approximation of this probability based on these two factors is presented.

Tobramycin penetration into epithelial lining fluid of patients with pneumonia.

OBJECTIVE: To study the penetration of tobramycin in lung tissue evaluated as the concentration in epithelial lining fluid and to characterize the time course of the drug in the treatment of patients with pneumonia. METHODS: The subjects were 16 patients with pneumonia and taking tobramycin who had clinical indications for bronchoscopy. Bronchoscopy with bronchoalveolar lavage of the pneumonic area was performed once on each patient 1/2%, 2, 4, or 8 hours after the previous tobramycin dose. Urea was used as an endogenous marker for quantification of epithelial lining fluid obtained at bronchoalveolar lavage. Tobramycin concentrations in serum were measured for all patients at the aforementioned 4 time points. Tobramycin concentration was determined by means of fluorescent polarization immunoassay modified for bronchoalveolar samples. RESULTS: Levels of tobramycin in the fluid of the epithelial lining were 2.33+/-0.5 at 1/2 hour, 1.67+/-0.6 at 2 hours, 1.62+/-1.19 at 4 hours, and 0.77+/-0.38 microg/mL at 8 hours. The ratio of epithelial lining fluid to serum concentration of tobramycin was 0.30+/-0.03 at 1/2 hour, 0.42+/-0.16 at 2 hours, 0.64+/-0.37 at 4 hours, and 1.53+/-0.76 at 8 hours. The ratio at peak serum time was similar to that reported for tobramycin and netilmicin. CONCLUSIONS: High peak serum concentrations of tobramycin are necessary to obtain microbiologically active concentrations at the alveolar level. The fluid of the epithelial lining constitutes a deep compartment for aminoglycosides. The disappearance of tobramycin was slower than at the serum level.

Bioequivalence evaluation of 2 different oral formulations of ciprofloxacin in healthy volunteers.

A bioequivalence study of 2 oral formulations of 500 mg tablets of ciprofloxacin was carried out in 24 healthy volunteers according to a single dose, two-sequence, crossover randomized design. Blood samples were taken prior to each administration and at 13 points within 32 hours after the dose, and plasma concentrations of ciprofloxacin were determined by HPLC. The pharmacokinetic parameters Cmax and tmax were obtained directly from plasma data, ke was estimated by log-linear regression, and AUC was calculated by the trapezoidal rule. The pharmacokinetic parameters AUC and Cmax were tested for bioequivalence after log-transformation of data, differences of tmax were evaluated nonparametrically. The 90% standard confidence intervals of the mean values for the test/reference ratios were 0.87-0.97 for AUC and 0.91-1.05 for Cmax, within the bioequivalence acceptable range of 0.80-1.25 limits. So, we conclude that both formulations were found bioequivalent and, therefore, interchangeable.

Pharmacokinetic study of ebrotidine administered in multiple doses to healthy volunteers for 4 days.

The safety of ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4- thiazolyl]methyl]thio]ethyl]amino] methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542), a new H2-receptor antagonist with gastroprotective activity, was assessed and its main pharmacokinetic parameters were determined in order to establish the dose linearity after the repeated administration of three different dose levels. The study was carried out in a group of 8 healthy volunteers of either sex, aged between 20 to 29 years. Oral doses of ebrotidine were administered in a randomized, single-blind design. Volunteers remained in the Unit for two days at each of the three study phases with washout intervals of 2 weeks and received seven doses of ebrotidine (150, 300 and 500 mg b.i.d). Pharmacological evaluation included vital signs, laboratory tests, adverse events and blood and urine samplings for pharmacokinetic analysis. Ebrotidine was determined by high performance liquid chromatography (HPLC) with UV detection. The results showed a good tolerability of ebrotidine after the administration of seven doses for 4 days, with no changes in the vital signs or laboratory parameters. No clinically significant dose-related adverse events were reported during the study. The absorption of ebrotidine was relatively rapid (tmax approximately 2 h) and linear within the dose range from 150 to 500 mg. Drug biotransformation was linear with doses tested, and no metabolic saturation occurred. The terminal elimination half-life of ebrotidine was between 7 and 11 h or even longer. There was no accumulation of ebrotidine and the steady state was reached, regardless of the dose administered, within the first 24-48 h.

Cefminox versus Cefoxitin in Hysterectomy Prophylaxis : Clinical Efficacy and Serum and Tissue Concentrations.

This phase III, prospective, randomised, open, controlled clinical trial compared the efficacy of single-dose cefminox (2g) versus triple-dose cefoxitin (2g every 4 hours) as antibiotic prophylaxis in 112 women undergoing gynaecological surgery (vaginal or abdominal hysterectomy). Peak, intraoperative and trough serum concentrations were determined for both antibiotics, as well as their concentrations in myometrial tissue in a subset of patients from the study (22 patients from the cefminox group and 18 from the cefoxitin group). Clinical response was satisfactory in all women treated with cefminox (59 of 59) and in 52 of 53 patients treated with cefoxitin. Fever-related morbidity, hospital stay and adverse reactions were similar in both groups. Peak serum concentrations were 132.3 mg/L for cefminox and 82.2 mg/L for cefoxitin. 12-hour concentrations were 2.82 mg/L for cefminox and 2.17 mg/L for cefoxitin, and were higher than the respective minimum inhibitory concentrations (MICs) for pathogens commonly associated with this pathology. Uterine tissue concentrations were 24.5 and 41.6 mg/L for cefminox and cefoxitin, respectively, and also clearly exceeded MIC. It was shown that the use of a single preoperative dose of cefminox was similar in efficacy to 3 doses of cefoxitin administered every 4 hours, and that the serum and tissue concentrations attained provide adequate antibiotic coverage. In view of the general trend towards the use of a single dose for prophylaxis, cefminox offers a new alternative for antibiotic prophylaxis in gynaecological surgery.

Evaluation of sex differences in the pharmacokinetics of ranitidine in humans.

A bioequivalence study of two oral formulations of 300 mg ranitidine was carried out in 16 healthy volunteers (8 men and 8 women), and the pharmacokinetics in both sexes were compared. There was bioequivalence of both formulations. The terminal half-life of ranitidine was 7% shorter and the oral apparent clearance 10.5% higher in women (1.44 L/h/kg) than in men (1.29 L/h/kg), although this difference did not reach statistical significance. No differences were observed in maximum concentration (Cmax) or the time of its occurrence (tmax). Sex, age, and weight did not correlate significantly with oral clearance. These results suggest that there are no sex differences in the pharmacokinetics of ranitidine, or that any differences would not be of clinical relevance. It also should be emphasized that bioequivalence trials also can be used to study other pharmacokinetic or pharmacodynamic characteristics of drugs without damaging the main endpoint of the study.

Nimodipine transfer into human breast milk and cerebrospinal fluid.

OBJECTIVE: To report nimodipine concentrations in breast milk and cerebrospinal fluid (CSF) of a lactating woman who was given the drug to prevent a vascular spasm secondary to angiographic examination. METHODS: A 36-year-old woman received a total dose of nimodipine 46 mg iv over 24 hours. She extracted milk when she noted mammary tightness, and blood samples were taken simultaneously by venipuncture in the arm contralateral to that of the nimodipine infusion. A CSF sample also was taken in a diagnostic lumbar puncture. RESULTS: Nimodipine concentration in milk was much lower than that in serum, with a milk/serum ratio of 0.06-0.15. The CSF/serum ratio was 0.01. We estimate that the infant would have received between 0.008% and 0.092% of the weight-adjusted dose that was administered to the mother if the baby had been nursed. CONCLUSIONS: Nimodipine is transferred to human milk in a lower proportion than are other calcium-channel blockers. These results suggest that treating the mother with nimodipine would entail no risk to the nursing infant.