Pharmacokinetics of oseltamivir phosphate and oseltamivir carboxylate in non-pregnant and pregnant rhesus monkeys.

Oseltamivir is an antiviral drug approved to treat influenza in humans. Although the dosing regimen of this drug is well established for non-pregnant patients, it is not clear if the significant physiological alterations associated with pregnancy affect the pharmacokinetics of oseltamivir and, thus, warrant different dosing regimens to assure efficacy. In this study, we investigated the suitability of rhesus macaques as an animal model for studying oseltamivir pharmacokinetics during all trimesters of pregnancy in comparison to pre-pregnant conditions. Specifically, we compared the pharmacokinetics of oseltamivir and its pharmacologically active metabolite oseltamivir carboxylate in rhesus monkeys after intravenous and nasogastric administration of 2.5 mg oseltamivir phosphate/kg body weight given prior to and during the first, second, and third trimesters of pregnancy. Pregnancy had only a modest effect upon the pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate. Monkeys treated intravenously in the third trimester had a reduction in Vd and CL, compared to non-pregnant monkeys. These changes did not occur in the other two trimesters. Pregnant monkeys treated intravenously had 20-25% decrease in AUC0-∞ of oseltamivir carboxylate and a corresponding increase in Vd and CL. Pregnant monkeys treated nasogastrically with oseltamivir phosphate demonstrated a pattern that recapitulated intravenous dosing. Taken together these data indicate that rhesus monkeys are an acceptable model for studying drug-pregnancy interactions.

Tick-Borne Pathogens Associated with Medically Important Ticks in Alabama: A Four-Year Survey.

Background: Tick-borne diseases (TBDs) represent a significant threat to human health in the United States. Based on reported cases of notifiable TBDs to the Centers for Disease Control and Prevention (CDC) the state of Alabama is no exception, yet previously there has been no active surveillance program in place to comprehensively assess the presence and prevalence of tick vectors and their associated TBD pathogens in Alabama. Here we summarize initial findings from a 4-year survey to address this unmet need. Materials and Methods: Beginning in 2018 and proceeding through 2021, ticks were collected throughout the state of Alabama and pooled before being screened for a panel of TBD pathogens known to circulate in the United States. Results: Consistent with previously reported cases, TBD pathogens associated with anaplasmosis, babesiosis, ehrlichiosis, and spotted fever rickettsiosis were detected in ticks of Alabama. Causative agents for tularemia and Lyme disease were not detected despite previously reported human disease cases. There was also no evidence of Heartland virus despite recent reports of the virus being detected in ticks in Northwestern counties. Conclusions: While these results serve to provide some insights into TBD pathogens associated with ticks in Alabama, they also raise many questions that highlight the need for additional studies and continued surveillance to fully understand the TBD threat to human health in Alabama.

A Trans-Agency Workshop on the Pathophysiology of Radiation-Induced Lung Injury.

Research and development of medical countermeasures (MCMs) for radiation-induced lung injury relies on the availability of animal models with well-characterized pathophysiology, allowing effective bridging to humans. To develop useful animal models, it is important to understand the clinical condition, advantages and limitations of individual models, and how to properly apply these models to demonstrate MCM efficacy. On March 20, 2019, a meeting sponsored by the Radiation and Nuclear Countermeasures Program (RNCP) within the National Institute of Allergy and Infectious Diseases (NIAID) brought together medical, scientific and regulatory communities, including academic and industry subject matter experts, and government stakeholders from the Food and Drug Administration (FDA) and the Biomedical Advanced Research and Development Authority (BARDA), to identify critical research gaps, discuss current clinical practices for various forms of pulmonary damage, and consider available animal models for radiation-induced lung injury.

Evaluation of DNA-Launched Virus-Like Particle Vaccines in an Immune Competent Mouse Model of Chikungunya Virus Infection.

Chikungunya virus (CHIKV) infection can result in chronic and debilitating arthralgia affecting humans in tropical and subtropical regions around the world, yet there are no licensed vaccines to prevent infection. DNA launched virus like particle (VLP) vaccines represent a potentially safer alternative to traditional live-attenuated vaccines; however, fully characterized immunocompetent mouse models which appropriately include both male and female animals for preclinical evaluation of these, and other, vaccine platforms are lacking. Utilizing virus stocks engineered to express mutations reported to enhance CHIKV virulence in mice, infection of male and female immunocompetent mice was evaluated, and the resulting model utilized to assess the efficacy of candidate DNA launched CHIKV VLP vaccines. Results demonstrate the potential utility of DNA launched VLP vaccines in comparison to a live attenuated CHIKV vaccine and identify gender differences in viral RNA loads that impact interpretation of vaccine efficacy and may have important implications for future CHIKV vaccine development.

Rickettsia prowazekii uses an sn-glycerol-3-phosphate dehydrogenase and a novel dihydroxyacetone phosphate transport system to supply triose phosphate for phospholipid biosynthesis.

Rickettsia prowazekii is an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion to sn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway is R. prowazekii open reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3P in vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of the R. prowazekii gpsA gene functioned to complement an Escherichia coli gpsA mutant. Furthermore, gpsA mRNA was detected in R. prowazekii purified from hen egg yolk sacs, and G3PDH activity was assayable in R. prowazekii lysed-cell extracts. Together, these data strongly suggested that R. prowazekii encodes and synthesizes a functional GpsA enzyme, yet R. prowazekii is unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned that R. prowazekii transports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purified R. prowazekii transported and incorporated DHAP into phospholipids, thus implicating a role for GpsA in vivo as part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.

AR12 (OSU-03012) suppresses GRP78 expression and inhibits SARS-CoV-2 replication.

AR12 is a derivative of celecoxib which no-longer acts against COX2 but instead inhibits the ATPase activity of multiple chaperone proteins, in particular GRP78. GRP78 acts as a sensor of endoplasmic reticulum stress and is an essential chaperone required for the life cycle of all mammalian viruses. We and others previously demonstrated in vitro and in vivo that AR12 increases autophagosome formation and autophagic flux, enhances virus protein degradation, preventing virus reproduction, and prolonging the survival of infected animals. In this report, we determined whether AR12 could act against SARS-CoV-2. In a dose-dependent fashion AR12 inhibited SARS-CoV-2 spike protein expression in transfected or infected cells. AR12 suppressed the production of infectious virions via autophagosome formation, which was also associated with degradation of GRP78. After AR12 exposure, the colocalization of GRP78 with spike protein was reduced. Knock down of eIF2α prevented AR12-induced spike degradation and knock down of Beclin1 or ATG5 caused the spike protein to localize in LAMP2+ vesicles without apparent degradation. HCT116 cells expressing ATG16L1 T300, found in the majority of persons of non-European descent, particularly from Africa, expressed greater amounts of GRP78 and SARS-CoV-2 receptor angiotensin converting enzyme 2 compared to ATG16L1 A300, predominantly found in Europeans, suggestive that ATG16L1 T300 expression may be associated with a greater ability to be infected and to reproduce SARS-CoV-2. In conclusion, our findings demonstrate that AR12 represents a clinically relevant anti-viral drug for the treatment of SARS-CoV-2.

Development of a validation algorithm for 'present on admission' flagging.

BACKGROUND: The use of routine hospital data for understanding patterns of adverse outcomes has been limited in the past by the fact that pre-existing and post-admission conditions have been indistinguishable. The use of a 'Present on Admission' (or POA) indicator to distinguish pre-existing or co-morbid conditions from those arising during the episode of care has been advocated in the US for many years as a tool to support quality assurance activities and improve the accuracy of risk adjustment methodologies. The USA, Australia and Canada now all assign a flag to indicate the timing of onset of diagnoses. For quality improvement purposes, it is the 'not-POA' diagnoses (that is, those acquired in hospital) that are of interest. METHODS: Our objective was to develop an algorithm for assessing the validity of assignment of 'not-POA' flags. We undertook expert review of the International Classification of Diseases, 10th Revision, Australian Modification (ICD-10-AM) to identify conditions that could not be plausibly hospital-acquired. The resulting computer algorithm was tested against all diagnoses flagged as complications in the Victorian (Australia) Admitted Episodes Dataset, 2005/06. Measures reported include rates of appropriate assignment of the new Australian 'Condition Onset' flag by ICD chapter, and patterns of invalid flagging. RESULTS: Of 18,418 diagnosis codes reviewed, 93.4% (n = 17,195) reflected agreement on status for flagging by at least 2 of 3 reviewers (including 64.4% unanimous agreement; Fleiss' Kappa: 0.61). In tests of the new algorithm, 96.14% of all hospital-acquired diagnosis codes flagged were found to be valid in the Victorian records analysed. A lower proportion of individual codes was judged to be acceptably flagged (76.2%), but this reflected a high proportion of codes used <5 times in the data set (789/1035 invalid codes). CONCLUSION: An indicator variable about the timing of occurrence of diagnoses can greatly expand the use of routinely coded data for hospital quality improvement programmes. The data-cleaning instrument developed and tested here can help guide coding practice in those health systems considering this change in hospital coding. The algorithm embodies principles for development of coding standards and coder education that would result in improved data validity for routine use of non-POA information.

Amoxicillin for postexposure inhalational anthrax in pediatrics: rationale for dosing recommendations.

We reviewed information about the safety and plasma pharmacokinetic data for amoxicillin, specifically related to its potential use for postexposure inhalational anthrax. Amoxicillin (45 mg/kg/d) given orally in 3 divided doses to pediatric patients <40 kg should yield an adequate time above the MIC for susceptible Bacillus anthracis (< or =0.5 microg/mL) over most of the dosing interval (75-100%). Doses <45 mg/kg/d and dosing intervals longer than 8 hours should not be used for postexposure inhalational anthrax.

FDA perspective on antivirals against biothreats: communicate early and often.

Development of antiviral products for certain highly pathogenic viruses with limited available treatments, such as viruses that may have biothreat potential, is critically important and challenging. The mission of the FDA is to protect the public health by assuring the safety, efficacy and quality of such products. Human clinical trials are critically important whenever relevant naturally occurring diseases can appropriately be studied. In selected situations when clinical studies are not ethical and field efficacy studies are not feasible, the Animal Rule (67 FR 37988, 2002) introduces the possibility of drug/biologic approval/licensure based on efficacy studies in animals, and appropriate human safety and pharmacokinetic information. This approach necessitates the development of well-delineated animal models predictive of human disease and treatment responses, and plans for adding human information if suitable circumstances arise. Efficient development of therapeutics against these agents requires collaborative efforts among industry, academia and federal agencies.

Immune response to recombinant Burkholderia pseudomallei FliC.

Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays with rFliC were performed ex vivo. TLR5-flagellin interactions trigger activation of transcription factor NF-κB in HEK-BlueTM-hTLR5 cells. Pro-inflammatory cytokine (IL-1ß, IL-6, and TNF-α) productions from whole blood in response to rFliC differed between fourteen healthy individuals. The levels of these cytokines changed in a dose and time-dependent manner. ELISA was used to determine rFliC-specific antibodies in serum samples from different groups of melioidosis patients and healthy subjects. IgG antibody to rFliC in melioidosis patients with diabetes were higher compared with non-diabetic patients. Our results show that B. pseudomallei flagellin is a potent immune stimulator and that the immune responses to rFliC are different among individuals. This may provide valuable insights toward the potential use of rFliC in vaccine development.

Child and adolescent psychopharmacology in the new millennium: a workshop for academia, industry, and government.

OBJECTIVE: To give academic researchers, government officials, and industry scientists an opportunity to assess the state of pediatric psychopharmacology and identify challenges facing professionals in the field. METHOD: Increased federal spending and the introduction of pediatric exclusivity led to large increases in pediatric psychopharmacology research in the 1990s. Despite the increase in research, concerns exist about methods and incentives for making new medications available for use in pediatric psychiatric disorders. In recognition of these concerns, the Duke Clinical Research Institute held a roundtable in September 2004. Participants from the National Institutes of Health, regulatory agencies, academia, and the pharmaceutical industry spoke about the effects of government regulations such as the U.S. Food and Drug Administration Modernization Act and the Pediatric Research Equity Act on pediatric research from academic, clinical, and industry perspectives, and bioethical considerations of such research. CONCLUSIONS: To ensure development of new drugs for treating psychiatric disorders in children and adolescents, we must address the challenges posed by the regulatory environment governing pediatric psychopharmacology research. Strategies were identified for improving the evidence base for psychopharmacologic interventions in youth before widespread use and for more effectively defining a research agenda for the future.

Peer-reviewed publication of clinical trials completed for pediatric exclusivity.

CONTEXT: Much of pediatric drug use is off-label because appropriate pediatric studies have not been conducted and the drugs have not been labeled by the US Food and Drug Administration (FDA) for use in children. In 1997, Congress authorized the FDA to grant extensions of marketing rights known as "pediatric exclusivity" if FDA-requested pediatric trials were conducted. As a result, there have been over 100 product labeling changes. The publication status of studies completed for pediatric exclusivity has not been evaluated. OBJECTIVE: To quantify the dissemination of results of studies conducted for pediatric exclusivity into the peer-review literature. DESIGN: Cohort study of all trials conducted for pediatric exclusivity between 1998 and 2004 as determined by MEDLINE and EMBASE searches through 2005, the subsequent labeling changes, and the publication of those studies in peer-reviewed journals. We categorized any labeling changes resulting from the studies as positive or negative for the drug under study. We then evaluated aspects of the studies and product label changes that were associated with subsequent publication in peer-reviewed medical journals. MAIN OUTCOME MEASURES: Publication of the trial data in peer-reviewed journals. RESULTS: Between 1998 and 2004, 253 studies were submitted to the FDA for pediatric exclusivity: 125 (50%) evaluated efficacy, 51 (20%) were multi-dose pharmacokinetic, 34 (13%) were single-dose pharmacokinetic, and 43 (17%) were safety studies. Labeling changes were positive for 127/253 (50%) of studies; only 113/253 (45%) were published. Efficacy studies and those with a positive labeling change were more likely to be published. CONCLUSIONS: The pediatric exclusivity program has been successful in encouraging drug studies in children. However, the dissemination of these results in the peer-reviewed literature is limited. Mechanisms to more widely disperse this information through publication warrant further evaluation.

Pediatric drug labeling: improving the safety and efficacy of pediatric therapies.

CONTEXT: Approximately 50% to 75% of drugs used in pediatric medicine have not been studied adequately to provide appropriate labeling information. In 1997, Congress passed the Food and Drug Administration Modernization Act (FDAMA), which encouraged pediatric drug development by providing an incentive in the form of additional marketing exclusivity. OBJECTIVE: To identify new drug labeling information from pediatric studies submitted to the FDA in response to written requests. DESIGN AND SETTING: Between July 1998 and April 1, 2002, the FDA requested studies on 242 drugs, and 53 drugs were granted exclusivity. As of January 2003, 49 drugs have new labels. Data from the studies of the first 33 drugs with new pediatric information on the label as of April 2002 are included. Significant labeling information was analyzed along with baseline data and types of studies requested. MAIN OUTCOME MEASURES: Safety data and pediatric information for labeled drugs. RESULTS: There were 53 studies for 33 drug products, 12 (23%) were evaluated for safety only; 23 (43%), safety and efficacy; and 18 (34%), pharmacokinetics and/or pharmacodynamics. Significant new dosing and/or safety information was identified for 12 (36%) drugs. New dosing information was determined for 7 of these drugs. Safety information was defined for gabapentin, propofol, sevoflurane, the combination of ribavirin and interferon alfa-2b, and various betamethasone-containing dermatologic preparations. There was a higher percentage of deaths reported with patients who received propofol compared with controls in the pediatric intensive care unit. Seizures were seen in patients administered sevoflurane. Patients receiving a combination of ribavirin and interferon alfa-2b experienced an increased incidence of suicidal ideation when compared with adults. An unexpectedly high percentage of those receiving betamethasone-containing dermatologic preparations had documented hypopituitary-adrenal axis suppression. CONCLUSION: The FDAMA has stimulated pediatric clinical studies resulting in improved understanding of the pharmacokinetics of drugs prescribed in pediatric medicine, important dose changes, and improved safety for children taking certain drugs.

Revised structures for the predominant O-polysaccharides expressed by Burkholderia pseudomallei and Burkholderia mallei.

O-Polysaccharides (OPS) were isolated from purified Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharides by mild-acid hydrolysis and gel-permeation chromatography. 1-D and 2-D (1)H and (13)C NMR spectroscopy experiments revealed that the OPS antigens were unbranched heteropolymers with the following structures: Collectively, our results demonstrate that the predominant OPS antigens expressed by B. pseudomallei and B. mallei isolates are structurally more complex than previously described and provide evidence that different capping residues are used by these closely related pathogens to terminate chain elongation. Additionally, they confirm that Burkholderia thailandensis and B. pseudomallei express OPS antigens that are essentially identical to one another.

Development of capsular polysaccharide-based glycoconjugates for immunization against melioidosis and glanders.

Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders.

A classification of hospital-acquired diagnoses for use with routine hospital data.

OBJECTIVE: To develop a tool to allow Australian hospitals to monitor the range of hospital-acquired diagnoses coded in routine data in support of quality improvement efforts. DESIGN AND SETTING: Secondary analysis of abstracted inpatient records for all episodes in acute care hospitals in Victoria for the financial year 2005-06 (n=2.032 million) to develop a classification system for hospital-acquired diagnoses; each record contains up to 40 diagnosis fields coded with the ICD-10-AM (International Classification of Diseases, 10th revision, Australian modification). MAIN OUTCOME MEASURE: The Classification of Hospital Acquired Diagnoses (CHADx) was developed by: analysing codes with a "complications" flag to identify high-volume code groups; assessing their salience through an iterative review by health information managers, patient safety researchers and clinicians; and developing principles to reduce double counting arising from coding standards. RESULTS: The dataset included 126,940 inpatient episodes with any hospital-acquired diagnosis (complication rate, 6.25%). Records had a mean of three flagged diagnoses; including unflagged obstetric and neonatal codes, 514,371 diagnoses were available for analysis. Of these, 2.9% (14,898) were removed as comorbidities rather than complications, and another 118,640 were removed as redundant codes, leaving 380,833 diagnoses for grouping into CHADx classes. We used 4345 unique codes to characterise hospital-acquired conditions; in the final CHADx these were grouped into 144 detailed subclasses and 17 "roll-up" groups. CONCLUSIONS: Monitoring quality improvement requires timely hospital-onset data, regardless of causation or "preventability" of each complication. The CHADx uses routinely abstracted hospital diagnosis and condition-onset information about in-hospital complications. Use of this classification will allow hospitals to track monthly performance for any of the CHADx indicators, or to evaluate specific quality improvement projects.

Improving pediatric dosing through pediatric initiatives: what we have learned.

OBJECTIVE: The goal was to review the impact of pediatric drug studies, as measured by the improvement in pediatric dosing and other pertinent information captured in the drug labeling. METHODS: We reviewed the pediatric studies for 108 products submitted (July 1998 through October 2005) in response to a Food and Drug Administration written request for pediatric studies, and the subsequent labeling changes. We analyzed the dosing modifications and focused on drug clearance as an important parameter influencing pediatric dosing. RESULTS: The first 108 drugs with new or revised pediatric labeling changes had dosing changes or pharmacokinetic information (n = 23), new safety information (n = 34), information concerning lack of efficacy (n = 19), new pediatric formulations (n = 12), and extended age limits (n = 77). A product might have had > or = 1 labeling change. We selected specific examples (n = 16) that illustrate significant differences in pediatric pharmacokinetics. CONCLUSIONS: Critical changes in drug labeling for pediatric patients illustrate that unique pediatric dosing often is necessary, reflecting growth and maturational stages of pediatric patients. These changes provide evidence that pediatric dosing should not be determined by simply applying weight-based calculations to the adult dose. Drug clearance is highly variable in the pediatric population and is not readily predictable on the basis of adult information.

"Black box" 101: How the Food and Drug Administration evaluates, communicates, and manages drug benefit/risk.

A prominently displayed boxed warning, the so-called "black box," is added to the labeling of drugs or drug products by the Food and Drug Administration when serious adverse reactions or special problems occur, particularly those that may lead to death or serious injury. Healthcare providers are often not knowledgeable about the origin, meaning, and implications of these "black box" warnings. In this review, our goal is to provide insight into how the Food and Drug Administration evaluates, communicates, and manages drug benefit/risk. We discuss drug labeling, the emphasis on safety throughout the drug approval process, legislative initiatives for safe use of drugs in children, and postmarketing safety surveillance. In addition, we encourage health care providers to report drug reactions to the Food and Drug Administration's MedWatch program. A discussion of new Food and Drug Administration initiatives to improve drug safety processes and methods to serve the public better are highlighted.