Nonlinear Protein Binding: Not What You Think

Nonlinear protein binding is traditionally thought of as an increasing fraction unbound with increasing total drug concentration. In the past several years, research into the protein binding of several tetracyclines has shown that an unexpected and counterintuitive phenomenon has been observed, specifically that of decreasing unbound drug fraction with increasing total concentrations of drug over certain concentration ranges. Although several studies of tigecycline have shown the importance calcium and its chelation may play in the protein-drug interaction, the potential clinical implications and relevance have not been explored. Here, we define typical and atypical nonlinear protein binding, overview protein binding theory, and discuss theoretical implications on pharmacokinetics. Using tigecycline as an example, in silico simulations and calculations show how when atypical nonlinear protein binding is not accounted for free drug exposure, and drug tissue penetration may be overestimated. It is important to revisit the impacts of nonlinearity in protein binding on clinical pharmacokinetics and pharmacodynamics, and ultimately, clinical efficacy. Although this phenomenon could potentially warrant clinical dose adjustment for certain compounds, it also presents a potential opportunity to exploit underlying mechanisms to develop new therapies and better understand molecular interactions of xenobiotics within the physiological system.     

Enhanced in vitro activity of tigecycline in the presence of chelating agents

The lack of availability of novel antibiotic agents and the rise of resistance to existing therapies has led clinicians to utilise combination therapy to adequately treat bacterial infections. Here we examined how chelators may impact the in vitro activity of tigecycline (TIG) against Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Minimum inhibitory concentrations (MICs) were determined by broth dilution with and without various combinations of chelators (EDTA and other tetracyclines) and metal ions (i.e. calcium, magnesium). Trimethoprim (TMP) was used as a non-chelating control. Addition of metal ions led to increases in MICs, whilst addition of EDTA led to decreases in MICs. The chelating effects of EDTA were reversed by addition of magnesium and most profoundly calcium. Similar effects of EDTA and calcium were observed for tetracycline (TET) and TMP. When other tetracyclines (TET, oxytetracycline (OXY) and chlortetracycline (CHL)) were used as chelators at concentrations below their MICs, TIG MICs decreased for P. aeruginosa but not for E. coli. Some decreases in TIG MICs were observed for K. pneumoniae when TET and CHL were added. A dose-dependent decrease in TIG MIC was observed for TET and was reversed by the addition of calcium. The presence of effects of EDTA and calcium on TMP MICs indicates that mechanisms outside of TIG chelation likely play a role in enhanced activity. Full characterisation of an unexpected interaction such as TIG–TET with different microorganisms could provide valuable insights into the underlying mechanisms and design of physiologically viable chelators as candidates for future combinations regimens.     

Survival and Functional Outcomes in Boys with Cerebral Adrenoleukodystrophy with and without Hematopoietic Stem Cell Transplantation

Cerebral adrenoleukodystrophy (CALD) is a rapidly progressing, often fatal neurodegenerative disease caused by mutations in the ABCD1 gene, resulting in deficiency of ALD protein. Clinical benefit has been reported following allogeneic hematopoietic stem cell transplantation (HSCT). We conducted a large multicenter retrospective chart review to characterize the natural history of CALD, to describe outcomes after HSCT, and to identify predictors of treatment outcomes. Major functional disabilities (MFDs) were identified as having the most significant impact on patients’ abilities to function independently and were used to assess HSCT outcome. Neurologic function score (NFS) and Loes magnetic resonance imaging score were assessed. Data were collected on 72 patients with CALD who did not undergo HSCT (untreated cohort) and on 65 patients who underwent transplantation (HSCT cohort) at 5 clinical sites. Kaplan-Meier (KM) estimates of 5-year overall survival (OS) from the time of CALD diagnosis were 55% (95% confidence interval [CI], 42.2% to 65.7%) for the untreated cohort and 78% (95% CI, 64% to 86.6%) for the HSCT cohort overall (P?=?.01). KM estimates of 2-year MFD-free survival for patients with gadolinium-enhanced lesions (GdE⁺) were 29% (95% CI, 11.7% to 48.2%) for untreated patients (n?=?21). For patients who underwent HSCT with GdE⁺ at baseline, with an NFS ≤1 and Loes score of 0.5 to ≤9 (n?=?27), the 2-year MFD-free survival was 84% (95% CI, 62.3% to 93.6%). Mortality rates post-HSCT were 8% (5 of 65) at 100days and 18% (12 of 65) at 1 year, with disease progression (44%; 7 of 16) and infection (31%; 5 of 16) listed as the most common causes of death. Adverse events post-HSCT included infection (29%; 19 of 65), acute grade II-IV graft-versus-host disease (GVHD) (31%; 18 of 58), and chronic GVHD (7%; 4 of 58). Eighteen percent of the patients (12 of 65) experienced engraftment failure after their first HSCT. Positive predictors of OS in the HSCT cohort may include donor-recipient HLA matching and lack of GVHD, and early disease treatment was predictive of MFD-free survival. GdE⁺ status is a strong predictor of disease progression in untreated patients.? This study confirms HSCT as an effective treatment for CALD when performed early. We propose survival without MFDs as a relevant treatment goal, rather than solely assessing OS as an indicator of treatment success.     

Phenotypic and molecular characterization of Escherichia albertii: Further surrogates to avoid potential laboratory misidentification

Escherichia albertii is an emerging gastrointestinal pathogen, related to Escherichia coli, which can be misidentified as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), due to the presence of the eae gene in E. albertii. The aim of this study was to verify our hypothesis that E. coli cytolethal distending toxin-II (Eccdt-II) gene-positive E. coli is E. albertii and to accumulate the data regarding the bacteriological characteristics of E. albertii. For these purposes, we attempted to detect E. albertii in eae gene-positive bacteria previously identified as E. coli and to examine if re-identified E. albertii contained Eccdt-II-homologous gene and remaining eae gene-positive E. coli did not. A total of 373 eae gene-positive E. coli strains were analyzed by biochemical tests, multilocus sequence analysis and an E. albertii-specific PCR. The strains re-identified as E. albertii were also examined for the presence of cdt genes by using ³²P-labled DNA probes, followed by their toxin-typing. Of the 373 strains, 17 were re-identified as E. albertii by three above-mentioned methods. Furthermore, all the 17 re-identified E. albertii possessed cdt genes highly homologous to Eccdt-II and Eacdt genes. Moreover, Eccdt-I or both Eccdt-I and stx2f genes were detected in two re-identified E. albertii strains. However, the remaining 356 strains did not carry such cdt genes. These data indicate that all re-identified E. albertii isolates specifically carried cdt genes homologous to Eccdt-II and Eacdt genes. We suggest that Eccdt-II gene-positive E. coli may be identical to E. albertii.     

Reduction in Mortality after Umbilical Cord Blood Transplantation in Children Over a 20-Year Period (1995-2014)

Infections and graft-versus-host disease (GVHD) have historically resulted in high mortality among children undergoing umbilical cord blood transplantation (UCBT). However, recent advances in clinical practice have likely improved outcomes of these patients. We conducted a retrospective cohort study of children (<18years of age) undergoing UCBT at Duke University between January 1, 1995 and December 31, 2014. We compared 2-year all-cause and cause-specific mortality during 3 time periods based on year of transplantation (1995 to 2001, 2002 to 2007, and 2008 to 2014). We used multivariable Cox regression to identify demographic and UCBT characteristics that were associated with all-cause mortality, transplantation-related mortality, and death from invasive aspergillosis after adjustment for time period. During the 20-year study period 824 children underwent UCBT. Two-year all-cause mortality declined from 48% in 1995 to 2001 to 30% in 2008 to 2014 (P?=?.0002). White race and nonmalignant UCBT indications were associated with lower mortality. Black children tended to have a higher risk of death for which GVHD (18% versus 11%; P?=?.06) or graft failure (9% versus 3%; P?=?.01) were contributory than white children. Comparing 2008 to 2014 with 1995 to 2001, more than half (59%) of the reduced mortality was attributable to a reduction in infectious mortality, with 45% specifically related to reduced mortality from invasive aspergillosis. Antifungal prophylaxis with voriconazole was associated with lower mortality from invasive aspergillosis than low-dose amphotericin B lipid complex (hazard ratio, .09; 95% confidence interval, .01 to .76). With the decline in mortality from invasive aspergillosis, adenovirus and cytomegalovirus have become the most frequentinfectious causes of death in children after UCBT. Advances in clinical practice over the past 20years improved survival of children after UCBT. Reduced mortality from infections, particularly invasive aspergillosis, accounted for the largest improvement in survival and was associated with use of voriconazole for antifungal prophylaxis.     

Qualitative and quantitative expression of bovine bone mineral in experimental bone defects. Part 1: Description of a dog model and histological observations

BACKGROUND: The purpose of this study was to evaluate histologically the contribution of inorganic bovine bone biomaterial in a new experimental bone defect in dogs at different healing periods and to examine newly formed bone around the grafted mineral particles and their relationship in membrane-protected (test) and non-protected intrabony (control) defects. METHODS: Four round intrabony defects, 5 x 4 mm were made bilaterally (at different times) on the lateral bony mandibular angle in eight dogs. Two defects were filled with bovine bone mineral (BBM) particles and two remained non-grafted but were blood clotted. A collagen membrane covered each defect type (n = 4). This procedure was repeated on the contralateral side at a different given time to obtain two different healing periods in each dog. Thus, four specimens were obtained at 3, 6, 12, and 24 months postoperatively for each healing period. The non-decalcification method (Donath technique) with Stevenel's blue and van Gieson's picro fuchsin staining was used for histological examination. RESULTS: Newly formed bone was observed at all examined defect types. The BBM particles were clearly evident regardless of the healing period. At 3 and 6 months, newly formed bone, woven in nature, was incorporated with the grafted particles. High cellular bone with occasional osteoclasts was noted towards the surface of the mineral particles. No substantial difference was observed between the protected and the non-protected defects except for higher ossified centers around the membrane-protected defects. At the non-grafted sites, the membrane-protected defect showed newly formed bone near the bony walls, and particularly under the membrane, establishing a bony bridge over the defect at the healing periods. The non-grafted unprotected defect (control) showed bone formation only at the base and close to the bony walls leaving a healed concave configuration. At 1 and 2 years, the grafted sites showed full bone healing configuration. However, the grafted particles still dominated the previous defect area and were completely surrounded by the newly formed bone. Osteons and lamellar bone arrangement were established but the bone was still highly cellular and osteoclasts could still be identified. The non-grafted membrane-protected sites showed excellent bone healing although areas of non-mineralized soft tissue were often seen. The control sites healed but still presented with a concave surface configuration. CONCLUSIONS: BBM biomaterial is a highly osteoconductive material. In a 4-wall bony defect, newly formed bone was well evident in establishing excellent bone healing configuration with or without a regenerative biological barrier. The grafted material dominated the experimental sites with no substantial resorption at any healing period up to 2 years observation.