Regulatory utility of physiologically-based pharmacokinetic modeling to support alternative bioequivalence approaches and risk assessment: A workshop summary report.

This report summarizes the proceedings for day 2 sessions 1 and 3 of the 2-day public workshop entitled "Regulatory Utility of Mechanistic Modeling to Support Alternative Bioequivalence Approaches," a jointly sponsored workshop by the US Food and Drug Administration (FDA) and the Center for Research on Complex Generics (CRCG). The aims of this workshop were: (1) to discuss how mechanistic modeling, including physiologically-based pharmacokinetic (PBPK) modeling and simulation, can support product development, and regulatory submissions; (2) to share the current state of mechanistic modeling for bioequivalence (BE) assessment through case studies; (3) to establish a consensus on best practices for using PBPK modeling for BE assessment to help drive further investment by the generic drug industry into mechanistic modeling and simulation; and (4) to introduce the concept of a Model Master File to improve model-sharing. The theme of day 2 covered PBPK absorption model for oral products as an alternative BE approach and a tool for supporting risk assessment and biowaiver (session 1), oral PBPK for evaluating the impact of food on BE (session 2), successful cases, and challenges for oral PBPK (session 3). This report summarizes the topics of the presentations of day 2 sessions 1 and session 3 from FDA, academia, and pharmaceutical industry, including the current status of oral PBPK, case examples as well as the challenges and opportunities in this area. In addition, panel discussions on the utility of oral PBPK in both new drugs and generic drugs from regulatory and industry perspective are also summarized.

Principles and Experimental Considerations for In Vitro Transporter Interaction Assays.

Drug transporters are universally acknowledged as important determinants of the absorption, distribution, metabolism, and excretion of both endogenous and exogenous compounds. Altered transporter function, whether due to genetic polymorphism, DDIs, disease, or environmental factors such as dietary constituents, can result in changes in drug efficacy and/or toxicity due to changes in circulating or tissue levels of either drugs or endogenous substrates.Prediction of whether and to what extent the biological fate of a drug is influenced by drug transporters, therefore, requires in vitro test systems that can accurately predict the risk and magnitude of clinical DDIs. While these in vitro assessments appear simple in theory, practitioners recognize that there are multiple factors that can influence experimental outcomes. A better understanding of these variables, including test compound characteristics, test systems, assay formats, and experimental design, will enable clear, actionable steps and translatable outcomes that may avoid unnecessary downstream clinical engagement. This chapter will delineate the role of these variables in improving in vitro assay outcomes.

Case Study 7: Transporters Case Studies-In Vitro Solutions for Translatable Outcomes.

Assessing the interactions of a new drug candidate with transporters, either as a substrate, inhibitor, or inducer, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to 11 for the EMA as of 2020. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the most relevant tools for the study and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter are unpublished at the time of the compilation of this book. It has been included in this chapter to provide a sense of the complexities in transporter kinetics to the reader.

Psychosocial health effects of Covid-19 infection on persons in treatment centers in Lagos, Nigeria.

INTRODUCTION: Prior research has highlighted the psychosocial impact of infectious diseases on individuals and the community at large. However, little is known about the psychosocial implications of COVID-19. This study set out to determine the rate as well as correlates of anxiety and depressive symptoms among persons managed as in-patients for COVID-19 in Lagos, Nigeria. MATERIALS AND METHODS: We conducted an online survey between April to June ending 2020 using a consecutive sampling technique of persons positive for COVID-19 and who were managed as in-patients across five (5) treatment centres in Lagos, Nigeria. The survey collected information on demographic as well as clinical data including suicidality. Anxiety and depressive symptoms were assessed using the Hospital Anxiety and Depression Scale (HADS). RESULTS: There were one hundred and sixty participants in total. The mean age of respondents was 36.4 (±9.7) years with a higher proportion (56.9%) being males. With regards to diagnosis, 28.1% and 27.5% of the respondents were categorised as probable cases of depression and anxiety respectively, while 3.8% respondents reported suicidal ideation. Majority of the respondents (61.9%) reported the fear of infecting their loved ones. The variables that showed association with psychiatric morbidity were a past history of an emotional concern, employment status, guilt about infecting others and boredom. CONCLUSION: This study revealed a high burden of psychological/psychiatric morbidity among persons treated for COVID-19, particularly persons who have had prior emotional concerns. The findings from this study reiterate the need to pay attention to the mental health of people during disease outbreaks and to incorporate psychosocial interventions as part of the management package.

Is the plastibell of any haemostatic value after 24 h?

BACKGROUND: The Plastibell is the most popular circumcision method among mothers in our city. Haemorrhage is its major problem. At our centre, we have recorded many circumcision problems resulting from prolonged retention of the Plastibell ring and this study, therefore, sought to explore the ways of reducing complications resulting from prolonged retention of the ring. PATIENTS AND METHODS: This was a prospective study, in which a total of sixty consecutive male neonates were recruited with all undergoing circumcision using the Plastibell device. Thirty patients were assigned to the subject group, in whom the Plastibell ring was removed by the investigator at 24 h while the other thirty constituted the control group whose Plastibell rings were allowed to fall off on their own. The patients selected were aged between 7 and 28 days. RESULTS: Overall, 4 (6.6%) of the sixty neonatal circumcisions in this study were complicated by haemorrhage. There was minor bleeding in 3 (10%) of the thirty subjects and 1 (3.3%) of the thirty controls. There was no statistically significant difference between the groups (P = 0.3006). One patient each from the subject and control groups bled following slipped ligature a few hours after Plastibell circumcision. The other two patients in the subject group bled following the removal of the Plastibell ring at 24 h. All the bleeding episodes were effectively controlled within 5 min by firm digital pressure only administered through a piece of dry, sterile gauze. CONCLUSION: Post-circumcision haemorrhage was not significantly different between circumcised babies whose Plastibell rings were removed at 24 h and those in whom it was left to fall off on its own.

The Effect of Excipients on the Permeability of BCS Class III Compounds and Implications for Biowaivers.

PURPOSE: Currently, the FDA allows biowaivers for Class I (high solubility and high permeability) and Class III (high solubility and low permeability) compounds of the Biopharmaceutics Classification System (BCS). Scientific evidence should be provided to support biowaivers for BCS Class I and Class III (high solubility and low permeability) compounds. METHODS: Data on the effects of excipients on drug permeability are needed to demonstrate that commonly used excipients do not affect the permeability of BCS Class III compounds, which would support the application of biowaivers to Class III compounds. This study was designed to generate such data by assessing the permeability of four BCS Class III compounds and one Class I compound in the presence and absence of five commonly used excipients. RESULTS: The permeability of each of the compounds was assessed, at three to five concentrations, with each excipient in two different models: Caco-2 cell monolayers, and in situ rat intestinal perfusion. No substantial increases in the permeability of any of the compounds were observed in the presence of any of the tested excipients in either of the models, with the exception of disruption of Caco-2 cell monolayer integrity by sodium lauryl sulfate at 0.1 mg/ml and higher. CONCLUSION: The results suggest that the absorption of these four BCS Class III compounds would not be greatly affected by the tested excipients. This may have implications in supporting biowaivers for BCS Class III compounds in general.

Principles and experimental considerations for in vitro transporter interaction assays.

Drug transporters are now universally acknowledged as important determinants of the absorption, distribution, metabolism and excretion of both endogenous and exogenous compounds. Altered transporter function, whether due to genetic polymorphism, DDIs, disease, or environmental factors such as dietary constituents, can result in changes in drug efficacy and/or toxicity due to changes in circulating or tissue levels of either drugs or endogenous substrates.Prediction of whether and to what extent the biological fate of a drug is influenced by drug transporters, therefore, requires in vitro test systems that can accurately predict the risk and magnitude of clinical DDIs. While these in vitro assessments appear simple in theory, practitioners recognize that there are multiple factors that can influence experimental outcomes. A better understanding of these variables, including test compound characteristics, test systems, assay formats, and experimental design will enable clear, actionable steps and translatable outcomes that may avoid unnecessary downstream clinical engagement. This chapter will delineate the role of these variables in improving in vitro assay outcomes.

Case study 6. Transporter case studies: in vitro solutions for translatable outcomes.

Assessing the interactions of a new drug candidate with transporters, either as a substrate or as an inhibitor, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to eleven for the EMA as of 2013. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to really appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the best tools for the job and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in transporter kinetics to the reader.

Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.

Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes.

Non-trauma related paediatric abdominal surgical emergencies in Lagos, Nigeria: Epidemiology and indicators of survival.

BACKGROUND: Pediatric surgical emergencies are associated with higher morbidity and mortality. The aim of this study is to describe the epidemiology of non-trauma related pediatric abdominal surgical emergencies in our centre and determine the indicators for survival in a cohort of patients. PATIENTS AND METHODS: A retrospective study of children aged 1 day to 15 years who presented with non-trauma related abdominal emergencies at the Lagos University Teaching Hospital (LUTH). RESULTS: There were 129 children. The median age at presentation was 5 months (range: 1 day-15 years). There were 104 males and 25 females. Sixty-four (49.6%) patients presented within 48 hours of the onset of the symptoms while 65 (50.4%) presented after 48 hours. Intestinal obstruction is the commonest indication for pediatric emergency surgery in our centre accounting for 76 patients (58.9%). Appendicitis is the second most common indication for emergency surgery with 13 patients (10.1%). Thirteen patients (10.1%) had postoperative complications. There were 13 deaths in all (10.1% mortality rate). Eleven out of 43 (25.6%) neonates died compared with 2 (2.3%) out of 86 patients in the other age groups (P=0.002). Seven out of 107 (6.5%) patients that had surgery within 72 hours died while 5/22 (22.7%) patients died who had surgery after 72 hours (P=0.003). There were 4 mortalities (28.6%) among patients with postoperative complications compared with 9 (7.8%) mortalities among 116 patients without any postoperative complications (Pp=0.001). CONCLUSION: Intestinal obstruction is the commonest pediatric surgical emergency seen in LUTH. Neonatal age, admission to surgery intervention time >72 hours, and severe postoperative complications are associated with high mortality.

Pediatric trauma in sub-Saharan Africa: Challenges in overcoming the scourge.

All over the world, pediatric trauma has emerged as an important public health problem. It accounts for the highest mortality in children and young adults in developed countries. Reports from Africa on trauma in the pediatric age group are few and most have been single center experience. In many low-and middle-income countries, the death rates from trauma in the pediatric age group exceed those found in developed countries. Much of this mortality is preventable by developing suitable preventive measures, implementing an effective trauma system and adapting interventions that have been implemented in developed countries that have led to significant reduction in both morbidity and mortality. This review of literature on the subject by pediatric and orthopedic surgeons from different centers in Africa aims to highlight the challenges faced in the care of these patients and proffer solutions to the scourge.

Surgically correctable morbidity from male circumcision: indications for specialist surgical care in lagos.

AIM/OBJECTIVE: To determine the pattern of morbidity and outcome among patients referred to the Pediatric Surgery Unit of the Lagos University Teaching Hospital (LUTH) following circumcision. MATERIALS AND METHODS: Retrospective descriptive study of all patients with complications of circumcision who were managed in LUTH between 2008 and 2010. RESULTS: There were 36 patients. The age range was between 2 days and 9 years (median-3 months). Fifteen cases (42.9%) were due to urethro-cutaneous fistula while there were six cases (16.7%) of postcircumcision bleeding. There were four cases (11.1%) each of partial penile amputation and buried penis. There were also cases of meatal stenosis, penile implantation cyst and glanulo-preputial skin bridge. With respect to the treatment offered, eleven (30.6%) patients had urethroplasty for the urethro-cutaneous fistulae while seven (19.4%) patients had penile refashioning for the buried penis and penile amputation. Appropriate surgical treatments were performed for the other complications. CONCLUSION: Urethrocutaneous fistula and penile amputation are the commonest complications of circumcision for which referral is made to LUTH. Treatment outcome was satisfactory. Health education and legislation to ensure procedure is performed by qualified medical and paramedical staff may reduce the morbidity.

Early outcome of open primary pull through versus staged pull through in Hirschsprung's disease: a single centre experience from Nigeria.

BACKGROUND: The treatment of Hirschsprung's disease has followed a trend from 2 or 3-staged pull-through (SPT) procedures to a single stage primary pull-through (PPT) procedure and from open surgery to laparoscopy-assisted, and trans-anal pull through procedures. The (PPT) procedure has the advantage of avoiding a stoma and its complications. OBJECTIVE: This study compares the outcomes in open PPT and SPT in our centre. METHODS: Retrospective observational study at a single centre from Nigeria over a 4year period. RESULTS: Of 46 patients with Hirschsprung's disease, 29 patients had pull-through procedures during the study period; 19 had SPT and 10 had PPT. There were 21 boys and 8 girls (M:F = 2.6:1). Five (17.1%) were diagnosed in the neonatal period and median age at surgery was 30 months (1 month - 31 yrs). The mean length of hospital stay was 30 days (+/- 7) in the SPT group while it was 16 days (+/- 3) in the PPT group, p < 0.05. Colostomy morbidity such as prolapse and skin excoriation showed statistical significant difference compared to other complications associated with pull-through (p < 0.05); however morbidity from pull-through procedures alone were similar. There was no statistically significant difference in post operative bowel habit irrespective of type of pull through operation performed (p > 0.05). Patients were followed up for between 3 months and 36 months. Two patients died giving a mortality rate of 6.7% overall. CONCLUSION: PPT significantly reduced both hospital stay and colostomy complications compared to SPT.

Survey of teaching, research and conference experiences of paediatric surgical trainees in Nigeria.

AIM: To determine the teaching methods used by residents in paediatric surgery in Nigeria and their exposure to research and conferences. MATERIALS AND METHODS: A structured questionnaire was administered to trainees in paediatric surgery in Nigeria seeking information regarding different teaching methods used, frequency of use, involvement in research and participation in conferences. RESULTS: There were 11 respondents (91.6%) of 12 questionnaires that were distributed. All of them were training in accredited teaching hospitals in Nigeria. All of them had been involved in teaching medical students. Ten residents were involved in teaching in wards/bedside two times or more in a week and all were involved in teaching at the clinics. Only one resident used audiovisual aid at least once a week to teach students. Eight trainees used tutorial or seminar group discussion as a teaching tool once a week. Four trainees had not used written essay as a way of teaching students while five had never given students lectures in a classroom before. All the respondents had participated in retrospective research while nine had been involved in prospective research. Nine residents had attended conferences nationally while two had attended international conferences. Six trainees presented a paper or more at national conferences while one presented at an international conference. CONCLUSION: Trainees in paediatric surgery in Nigeria are significantly involved in the teaching of undergraduate medical students and clinical research. This should be encouraged and further enhanced by motivating the trainees to attend international conferences.

Use of transporter knockdown Caco-2 cells to investigate the in vitro efflux of statin drugs.

The objective of the present study was to determine the efflux transporters responsible for acid and lactone statin drug efflux using transporter knockdown Caco-2 cells. The bidirectional transport was determined in Caco-2 cell monolayers in which the expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), or multidrug resistance associated protein 2 (MRP2) was knocked down by transduction with lentivirus containing human transporter-targeted small hairpin RNAs (shRNAs). Cells transduced with lentivirus containing nontargeted shRNA served as the vector control. Atorvastatin, lovastatin, and rosuvastatin displayed extremely low apical-to-basolateral (A-to-B) transport, which made the P(app,A-B) values too unreliable to calculate the efflux ratio. Thus, transport comparisons were performed using the B-to-A permeability (P(app,B-A)) values. Presented in the order of vector control, P-gp, BCRP, and MRP2 knockdown Caco-2 cells, the P(app,B-A) values (×10(-6), cm/s) were 28.1 ± 1.3, 8.6 ± 2.9, 20.3 ± 1.8, and 21.5 ± 1.6 for atorvastatin; 96.1 ± 7.1, 25.3 ± 3.5, 57.3 ± 9.8, and 48.2 ± 2.3 for fluvastatin; and 14.1 ± 1.9, 4.6 ± 1.7, 5.8 ± 0.7, and 6.6 ± 1.8 for rosuvastatin, respectively. Lovastatin and simvastatin showed no efflux in the vector control or knockdown cell monolayers in either lactone or acid forms. Results indicate that atorvastatin, fluvastatin, and rosuvastatin were transported by P-gp, BCRP, and MRP2. On the other hand, neither the lactone nor the resulting acid of lovastatin and simvastatin was transported by P-gp, BCRP, or MRP2. The current study demonstrated that the transporter knockdown Caco-2 cells are useful tools for studying drug-transporter interactions and should help eliminate some of the ambiguity associated with the identification of drug-transporter interactions based on chemical inhibitors alone.

The nasty surprise of a complex drug-drug interaction.

In vitro investigation of pharmacokinetic drug-drug interactions (DDIs) has officially been part of the regulatory pathway for new drugs in the USA since the publication of an FDA guidance on the subject in 1997. The field has continued to evolve, driven by preclinical and clinical experience, improved understanding of the molecular basis of DDIs, technological advances, and a continuous dialogue between the FDA and pharmaceutical industry scientists. Some striking DDIs involve multiple molecular species and targets; their mechanisms and magnitude would have been difficult or impossible to predict with available in vitro tools. This article focuses on one such example.

A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells.

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).