Drug Permeability - Best Practices for Biopharmaceutics Classification System (BCS)-Based Biowaivers: A workshop Summary Report.

The workshop "Drug Permeability - Best Practices for Biopharmaceutics Classification System (BCS) Based Biowaivers" was held virtually on December 6, 2021, organized by the University of Maryland Center of Excellence in Regulatory Science and Innovation (M-CERSI), and the Food and Drug Administration (FDA). The workshop focused on the industrial, academic, and regulatory experiences in generating and evaluating permeability data, with the aim to further facilitate implementation of the BCS and efficient development of high-quality drug products globally. As the first international permeability workshop since the BCS based biowaivers was finalized as the ICH M9 guideline, the workshop included lectures, panel discussions, and breakout sessions. Lecture and panel discussion topics covered case studies at IND, NDA, and ANDA stages, typical deficiencies relating to permeability assessment supporting BCS biowaiver, types of evidence that are available to demonstrate high permeability, method suitability of a permeability assay, impact of excipients, importance of global acceptance of permeability methods, opportunities to expand the use of biowaivers (e.g. non-Caco-2 cell lines, totality-of-evidence approach to demonstrate high permeability) and future of permeability testing. Breakout sessions focused on 1) in vitro and in silico intestinal permeability methods; 2) potential excipient effects on permeability and; 3) use of label and literature data to designate permeability class.

Lymphoma incidence, survival and prevalence 2004-2014: sub-type analyses from the UK's Haematological Malignancy Research Network.

BACKGROUND: Population-based information about cancer occurrence and survival are required to inform clinical practice and research; but for most lymphomas data are lacking. METHODS: Set within a socio-demographically representative UK population of nearly 4 million, lymphoma data (N=5796) are from an established patient cohort. RESULTS: Incidence, survival (overall and relative) and prevalence estimates for >20 subtypes are presented. With few exceptions, males tended to be diagnosed at younger ages and have significantly (P<0.05) higher incidence rates. Differences were greatest at younger ages: the <15 year male/female rate ratio for all subtypes combined being 2.2 (95% CI 1.3-3.4). These gender differences impacted on prevalence; most subtype estimates being significantly (P<0.05) higher in males than females. Outcome varied widely by subtype; survival of patients with nodular lymphocyte predominant Hodgkin lymphoma approached that of the general population, whereas less than a third of those with other B-cell (e.g., mantle cell) or T-cell (e.g., peripheral-T) lymphomas survived for ≥5 years. No males/female survival differences were detected. CONCLUSIONS: Major strengths of our study include completeness of ascertainment, world-class diagnostics and generalisability. The marked variations demonstrated confirm the requirement for 'real-world' data to inform aetiological hypotheses, health-care planning and the future monitoring of therapeutic changes.

Determinants of survival in patients with chronic myeloid leukaemia treated in the new era of oral therapy: findings from a UK population-based patient cohort.

OBJECTIVES: To examine contemporary survival patterns in the general population of patients diagnosed with chronic myeloid leukaemia (CML), and to identify patient groups with less than optimal outcomes. DESIGN: Prospective population-based cohort. SETTING: The UK's Haematological Malignancy Research Network (catchment population 3.6 million, with >2000 new haematological malignancies diagnosed annually). PARTICIPANTS: All patients newly diagnosed with CML, from September 2004 to August 2011 and followed up to 31 March 2013. MAIN OUTCOME MEASURE: Incidence and survival. RESULTS: With a median diagnostic age of 59 years, the CML age standardised (European) incidence was 0.9/100 000 (95% CIs 0.8 to 0.9), 5-year overall survival was 78.9% (72.3 to 84.0) and 5-year relative survival 88.6% (81.0 to 93.3). The efficacy of treatment across all ages was clearly demonstrated; the relative survival curves for those under 60 and over 60 years being closely aligned. Survival findings were similar for men and women, but varied with deprivation; the age and sex adjusted HR being 3.43 (1.89 to 6.22) for deprivation categories 4-5 (less affluent) versus 1-3 (more affluent). None of these differences were attributable to the biological features of the disease. CONCLUSIONS: When therapy is freely provided, population-based survival for CML is similar to that reported in clinical trials, and age loses its prognostic significance. However, although most of the patients with CML now experience close to normal lifespans, those living in more deprived areas tend to have poorer outcomes, despite receiving the same clinical care. A significant improvement in overall population outcomes could be achieved if these socioeconomic differences, which may reflect the treatment compliance, could be eliminated.

Cortical processing of visceral and somatic stimulation: differentiating pain intensity from unpleasantness.

Visceral and somatic pain perception differs in several aspects: poor localization of visceral pain and the ability of visceral pain to be referred to somatic structures. The perception of pain intensity and affect in visceral and somatic pain syndromes is often different, with visceral pain reported as more unpleasant. To determine whether these behavioral differences are due to differences in the central processing of visceral and somatic pain, non-invasive imaging tools are required to examine the neural correlates of visceral and somatic events when the behavior has been isolated and matched for either unpleasantness or pain intensity. In this study we matched the unpleasantness of somatic and visceral sensations and imaged the neural representation of this perception using functional magnetic resonance imaging in 10 healthy right-handed subjects. Each subject received noxious thermal stimuli to the left foot and midline lower back and balloon distension of the rectum while being scanned. Stimuli were matched to the same unpleasantness rating, producing mild-moderate pain intensity for somatic stimuli but an intensity below the pain threshold for the visceral stimuli. Visceral stimuli induced deactivation of the perigenual cingulate bilaterally with a relatively greater activation of the right anterior insula-i.e. regions encoding affect. Somatic pain induced left dorso-lateral pre-frontal cortex and bilateral inferior parietal cortex activation i.e. regions encoding spatial orientation and assessing perceptual valence of the stimulus. We believe that the observed patterns of activation represent the differences in cortical process of interoceptive (visceral) and exteroceptive (somatic) stimuli when matched for unpleasantness.

Genotyping demonstrates that the strains of Proteus mirabilis from bladder stones and catheter encrustations of patients undergoing long-term bladder catheterization are identical.

PURPOSE: We established the incidence of bladder stones in patients who experienced recurrent encrustation and blockage of indwelling bladder catheters and examined the relationship between isolates of Proteus mirabilis from the stones and from the crystalline biofilms on the catheters. MATERIALS AND METHODS: The first 100 patients attending a clinic for patients experiencing problems with the management of long-term bladder catheters were studied. Flexible cystoscopy was used to detect bladder stones. Catheter encrustation was assessed visually and by electron microscopy. Bacteriological analysis was performed on the stones and catheter biofilms. P. mirabilis isolates were genotyped by pulsed field gel electrophoresis of restriction enzyme digests of bacterial DNA. RESULTS: Most patients (85%) had been referred because of catheter blockage and in 61 (72%) the catheters were encrusted. P. mirabilis was recovered from 37 of 47 encrusted catheters (79%) that were examined but not from any nonencrusted catheters. Of the 61 patients with encrusted catheters 38 (62%) had bladder stones. Pairs of isolates of P. mirabilis from the stones and the catheter biofilms from 6 patients were genotyped. The DNA profiles of each pair of isolates were identical. CONCLUSIONS: The majority of patients (62%) with recurrent catheter encrustation had bladder stones. The stones harbored the strains of P. mirabilis that rapidly colonize replacement catheters with crystalline biofilm. Flexible cystoscopy to detect and remove stones might help resolve the problem of recurrent catheter encrustation.

Ecological monitoring for assessing the state of the nearshore and open waters of the Great Lakes.

The Great Lakes Water Quality Agreement stipulates that the Governments of Canada and the United States are responsible for restoring and maintaining the chemical, physical and biological integrity of the waters of the Great Lakes Basin Ecosystem. Due to varying mandates and areas of expertise, monitoring to assess progress towards this objective is conducted by a multitude of Canadian and U.S. federal and provincial/state agencies, in cooperation with academia and regional authorities. This paper highlights selected long-term monitoring programs and discusses a number of documented ecological changes that indicate the present state of the open and nearshore waters of the Great Lakes.

Placental tissue interleukin-10 receptor distribution in pre-eclampsia.

PROBLEM: Reduced placental (trophoblast) cytokine interleukin-10 (IL-10) occurs in human pre-eclampsia. Along with an increase in inflammatory cytokines this may play an important role in the development of hypertension in pregnancy. It is not clear whether the changes in placental IL-10 are due to a change in the placental cell production of IL-10 or a result of changes in cytokine receptor status in adjacent tissues. This study is aimed at qualifying the presence and distribution of IL-10 receptors in women with a pre-eclamptic outcome compared to normal pregnancy and gestational hypertension. METHOD OF STUDY: Patients at the KGV Hospital, Sydney was selected for the study. Placentas were collected fresh and paraffin serial sections made. Sections were stained with IL-10 receptor antibody (10 microgram/mL) using avidin-biotin immunohistochemistry. Tissues of patients with pre-eclampsia (n=11) were compared with normal pregnancy (n=12). Pre-eclampsia was defined as a blood pressure >140/90 mmHg on two occasions and de nova proteinuria >300 mg per day which resolved post-partum. The fetal weights, gestational ages and maternal ages at delivery were compared (ANOVA) and the differences in staining of decidual and villous tissues were graded according to density. Statistical comparisons were made using the Kruskal-Wallis test. RESULTS: The groups were similar for maternal gestational age but delivered at earlier gestation and with lower fetal weight. There was significantly less villous cytotrophoblast staining for IL-10 receptor in all groups (P=0.012) compared to decidual trophoblast cells. There was equal intensity and density of extravillous straining observed in normal pregnancy (45 +/- 12%) positive cells compared to pre-eclampsia (27 +/- 12%). CONCLUSION: IL-10 receptors are present in greater concentration in the extravillous (decidual) trophoblast compared to villi. The decrease in IL-10 produced by trophoblast cells in pre-eclampsia is not explained by a difference in the IL-10 receptor distribution compared to normal pregnancy.

Placental endothelial nitric oxide synthase localization and expression in normal human pregnancy and pre-eclampsia.

1. The aim of the present study was to investigate whether pre-eclampisa, a state of placental hypoxia, is associated with placental abnormalities in the amount, distribution and expression of endothelial nitric oxide synthase (eNOS). 2. Localization and intensity of eNOS was determined by immunohistochemistry using an antibody specific for eNOS. The amount of eNOS mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the densitometry of gel bands was expressed as a ratio of the band density of the housekeeping gene beta2-microglobulin. 3. Endothelial NOS staining was localized to syncytiotrophoblast cells within the villi and decidual trophoblast cells. It was not present in the endothelium of terminal villous vessels. There was no significant difference in eNOS villous or decidual staining intensity between normal pregnancy (NP; n = 12), pre-eclampsia (n = 14), or gestational hypertension (GH; n = 4). Staining for eNOS was not significantly different in the decidua compared with the villi in NP, GH or pre-eclampsia. Within the decidua, the depth of eNOS staining was similar in NP, pre-eclampisa and GH. 4. There was no significant difference in eNOS mRNA expression between NP (0.70 +/- 0.11), pre-eclampsia (0.5 +/- 0.07) or GH (0.69 +/- 0.26). 5. These findings suggest that the amount of eNOS in the placenta is not deficient in pre-eclampsia, excluding a possible pathogenic role for eNOS in this disease. Furthermore, placental hypoxia, which is associated with pre-eclampsia, did not induce an upregulation of eNOS

p53 and DCC immunohistochemistry in curative rectal cancer surgery.

BACKGROUND AND AIMS: In rectal cancer altered expression of p53 or DCC may be indicative of poor patient prognosis. This study determined by immunohistochemistry the tumour status of p53 protein and DCC protein in patients with rectal cancer who had a curative resection and were followed-up prospectively and examined the correlation to clinical and pathology variables. PATIENTS AND METHODS: The study included 171 who had a curative resection for rectal cancer: 88 at Concord Hospital (CH) followed up for a mean of 11 years and 83 at Royal Prince Alfred Hospital (RPAH) followed up prospectively for a mean of 4.3 years. Specimens were assessed by immunohistochemical assay of p53 expression ( n=170) and of DCC expression ( n=168). RESULTS: p53 over-expression was demonstrated in 58% of CH tumours and 59% of RPAH tumours. Absence of normal DCC expression was demonstrated in 66% of CH tumours and 52% of RPAH tumours. On both separate and combined analysis of these groups there were no significant associations by univariate analysis between p53 expression or DCC expression or combinations of p53 and DCC expression and the pathology variables: extent of penetration through bowel wall, lymph node involvement, presence of venous invasion, and tumour differentiation. CONCLUSION: The immunohistochemical p53 and DCC status of rectal tumours was not associated with other clinical or pathology variables, nor predictive of outcome.

p53, DCC and thymidylate synthase as predictors of survival after resection of hepatic metastases from colorectal cancer.

BACKGROUND: Hepatic metastasis from colorectal cancer is a common problem. Hepatic resection offers the only chance of cure. Prognosis of patients following hepatic resection is currently based on clinicopathological factors (of both the primary cancer and the hepatic metastasis), which do not accurately predict the subsequent behaviour of the tumour. The aim of this study was to evaluate three molecular genetic markers - p53, DCC (deleted in colonic cancer) and thymidylate synthase - in both the primary colorectal tumour and the resected hepatic metastases, and to determine their correlation, if any, with survival in patients with resected hepatic metastases from colorectal cancer. METHODS: Sixty-three patients with hepatic metastases and 40 corresponding colorectal primary tumours were studied using immunohistochemical staining for p53, DCC and thymidylate synthase, as well as p53 gene mutations using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) analysis. The results were correlated with survival. RESULTS: There was no correlation between p53, DCC or thymidylate synthase immunohistochemical staining, or between p53 PCR-SSCP analysis, and survival for either hepatic metastases or the colorectal primary tumour. CONCLUSION: Prediction of prognosis in patients having resection of hepatic metastases from colorectal cancer continues to be problematic. Other genetic markers or combination of markers need to be evaluated.

Transforming growth factor-beta 1 does not relate to hypertension in pre-eclampsia.

1. Pre-eclampsia is a human disease of pregnancy characterized by high blood pressure, proteinuria and end-organ damage, if severe. Pre-eclampsia is thought to be related to changes in early placental development, with the formation of a shallower than normal placental bed. 2. Transforming growth factor (TGF)-beta1 is a multifunctional fibrogenic growth factor involved in immune regulation that is elevated in some populations with a high risk of hypertensive end-organ disease related to increases in endothelin release. Transforming growth factor-beta1 is also an important factor in placental implantation. Alterations in TGF-beta1 may be related to abnormal placental development in early pregnancy and, thus, are a candidate for the development of hypertension in pre-eclampsia. 3. The aim of the present study was to examine the placental distribution and serum concentration of TGF-beta1 in patients with pre-eclampsia compared with normal pregnancy. 4. Patients with pre-eclampsia (n = 12) were compared with patients with normal pregnancy (n = 14). Transforming growth factor-beta1 was determined by TGF-beta1 Max ELISA (Promega, Madsion, WI, USA) after serum dilution (1/150) and acid activation. Placental distribution was determined by immunostaining with TGF-beta1 (Santa Cruz, Santa Cruz, CA, USA; 20 ng/mL) and the villi and decidual trophoblast were scored for intensity and extent of staining. 5. Patients with pre-eclampsia had a mean gestational age of 36 weeks, whereas those with a normal pregnancy had a mean gestational age of 39.0 +/- 0.4 weeks. There was no difference in TGF-beta1 concentration between the two groups (mean (+/-SEM) 27.1 +/- 1.0 vs 26.4 +/- 0.7 pg/mL for normal pregnancy and pre-eclampsia, respectively; P = 0.73, Mann-Whitney U-test). There was no correlation between systolic or diastolic blood pressure and TGF-beta1 concentration (regression analysis P = 0.4 and 0.2). Immunostaining was absent in the villous trophoblast cells and endovascular and extravillous trophoblast of term placentas. 6. Although TGF-beta1 is present in trophoblast cells in early pregnancy during placental development, TGF-beta1 concentrations were not increased in the placenta at term in pre-eclampsia and there was no correlation between blood pressure and serum TGF-beta1, suggesting that TGF-beta1 does not play a role in the development of late gestation pre-eclampsia and hypertension.

Histological tumour response to pre-operative combined modality therapy in locally advanced rectal cancer.

BACKGROUND: Pre-operative combined modality therapy (CMT) is used in locally advanced rectal cancer. Its use affects the clinicopathological staging based on the resected specimen. Assessment of the tumour response in the resected specimen may provide prognostic information. This study was undertaken to determine the histological response to pre-operative chemoradiation and to assess the interobserver reliability of a newly developed tumour response grading system for rectal cancer. METHODS: Pre-operative biopsy specimens and the resected specimens of 21 patients with low rectal cancer were assessed. The patients underwent pre-operative CMT consisting of radiotherapy (45 Gy) with 5-FU either as a continuous infusion or as a bolus intravenous infusion with leucovorin. After four to six weeks tumour response was assessed by comparing pre-operative transrectal ultrasound (TRUS) findings (uT1-4, uN0-1) with postoperative histopathological assessment (pT1-4, pN0-1) using UICC TNM characteristics. Tumour response was defined as a decrease in T status. The histological response to CMT was based on the tumour regression grade (TRG) and ranged from fibrosis extending through the rectal wall with no residual cancer (TRG 1), to no evidence of tumour response (TRG 5). Inter-observer reliability was assessed using weighted and unweighted kappa statistics. RESULTS: Local downstaging was demonstrated in 11/21 (52%) of patients. Three of 21 patients had a TRG 1 response. Thirteen of 21 (62%) patients had TRG 1-3 responses to CMT. There was no significant correlation between local downstaging and TRG. The interobserver correlation coefficient for assessment of TRG was 0.88 (unweighted kappa). CONCLUSIONS: Local downstaging by pre-operative CMT can be demonstrated if pre-operative TRUS staging is compared to standard pathology staging in patients with rectal cancer. Local downstaging is not directly related to histologic response as assessed by TRG. Inter-observer reporting of tumour regression grade (TRG) is reliable.

A giant multiacinar macroregenerative nodule in an explanted liver.

Macroregenerative nodules (MRN) have been detected with increased incidence in explanted livers since orthotopic liver transplantation (OLTx) has become a routine treatment for end-stage liver disease. Autopsy series suggest that MRN may be more common than once thought, and several studies point to the malignant potential of these lesions. With increasing waiting times for OLTx, the propensity for these premalignant lesions to arise in cirrhotic livers has important implications for the supervision of patients on OLTx waiting lists. We present here a striking example of a MRN and review a topic that is generating considerable interest.

In vivo image-guided (1)H-magnetic resonance spectroscopy of the serial development of hepatocarcinogenesis in an experimental animal model.

Histology on a core or open biopsy is considered the gold standard for the diagnosis of tumours. While the non-invasive technique of magnetic resonance imaging can direct some of the decision diagnostic making, it has limitations and disadvantages, that can be partly overcome with the use of in vivo magnetic resonance spectroscopy (MRS). In vivo MRS is able to provide a specific biochemical profile on tumour tissue, compared with normal tissue. The capability of this technique is demonstrated here by the long-term development of hepatocellular carcinoma in an animal model. It allows the observation of the biochemical changes that occur in tumour tissue during its progression from preneoplastic nodules to hepatocellular carcinoma. Specifically the changes in the lipid profiles of tumour tissue at various stages of development are observed with proton ((1)H) MRS. Significant increases occurred in the lipid acyl chain methylene and methyl hydrogens during the early developmental stages of hepatocarcinogenesis, whereas during later stages associated with tumour development there was a significant increase in the levels of olefinic acyl chain hydrogens from unsaturated lipids. It is anticipated that this model will precede the application of the same technology to the non-invasive diagnosis and grading of human hepatocellular carcinoma.

New concepts in the treatment of ureteral calculi.

The management of patients with ureteral stones remains under debate in several areas. The ability to predict spontaneous passage has improved but remains imprecise, whilst the range of therapeutic options continues to widen. Excellent results can be obtained by both shockwave lithotripsy and ureteroscopic methods, with relatively minimal complications. Routine ureteral stenting is not warranted whichever treatment is chosen. In future, directly comparative studies should be designed to incorporate quality-of-life parameters rather than just stone-free status, to improve our understanding of the effect of treatment decisions on patients.

Suprapubic track pressure and force--deformation measurements in a (live) human subject and in animal models post-mortem.

Tests have been performed on animal models shortly post-mortem and on a healthy human subject in order to obtain estimates of the forces which act on suprapubic urinary catheters and similar devices and to develop an abdominal wall simulator. Such data and test methods are required for the systematic design of suprapubic devices because of the dual need to maintain the functionality of devices and to avoid excessive pressure on soft body tissue which could lead to ischaemia and in turn necrosis. In the post-mortem animal models, electrical excitation was applied to the abdominal wall in order to stimulate muscle activity. Two types of transducers were used: a soft membrane transducer (SMT) for pressure measurement and novel instrumented 'tongs' to determine indentation stiffness characteristics in the suprapubic track or artificial pathway created for a device. The SMT has been extensively used in the urethras and bladders of human subjects while the tongs were built specifically for these tests. Only the well-established SMT was used with the human subject; a peak pressure of 22 kPa was obtained. In the animal models the pressure profile given by the SMT had a peak whose position corresponded well with the estimated location of the rectus muscle measured on the fixed tissue section. The peak value was 5.5 kPa, comparable with values likely to cause necrosis if maintained for more than 1 day. Remarkably consistent indentation stiffness values were obtained with the instrumented tongs; all values were close to 0.45 N/mm (33 kPa/mm).

Posttransplant administration of donor leukocytes induces long-term acceptance of kidney or liver transplants by an activation-associated immune mechanism.

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-gamma at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-gamma mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.