PLL-g-PEG Polymer Inhibits Antibody-Drug Conjugate Uptake into Human Corneal Epithelial Cells In Vitro.

Purpose: Antibody-drug conjugates (ADCs) are a relatively recent advance in the delivery of chemotherapeutics that improve targeting of cytotoxic agents. However, despite their antitumor activity, severe ocular adverse effects, including vision loss, have been reported for several ADCs. The nonspecific uptake of ADCs into human corneal epithelial cells (HCECs) and their precursors via macropinocytosis has been proposed to be the primary mechanism of ocular toxicity. In this study, we evaluated the ability of a novel polymer, poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), to decrease the ADC rituximab-mc monomethylauristatin F (MMAF) (RIX) uptake into human corneal epithelial (HCE-T) cells. Methods: HCE-T cells were exposed to increasing concentrations of RIX to determine inhibition of cell proliferation. HCE-T cells were treated with PLL-g-PEG, the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA), or vehicle. After 30 min of incubation, RIX was added. ADC was detected by fluorescent anti-human immunoglobulin G and fluorescently conjugated dextran as viewed by microscopy. Results: RIX caused dose-dependent inhibition of HCE-T cell proliferation. EIPA significantly reduced RIX uptake and decreased macropinocytosis as assessed by direct quantification of RIX using a fluorescently conjugated anti-human antibody as well as quantification of macropinocytosis using fluorescently conjugated dextran. PLL-g-PEG resulted in a dose-dependent inhibition of RIX uptake with half-maximal inhibitory concentrations of 0.022%-0.023% PLL-g-PEG. Conclusion: The data show PLL-g-PEG to be a potent inhibitor of RIX uptake by corneal epithelial cells and support its use as a novel therapeutic approach for the prevention of ocular adverse events associated with ADC therapy.

Setting the record straight-Correcting uterine cancer incidence and mortality in the Nordic countries by reallocation of unspecified cases.

INTRODUCTION: The incidence of and mortality from cancers of the cervix uteri and corpus uteri are underestimated if the presence of uterine cancers, where the exact topography (site of origin) is not specified, is omitted. In this paper we present the corrected figures on mortality from and incidence of cervix and corpus uteri cancers in the Nordic countries by reallocating unspecified uterine cancer deaths and cases to originate either from the corpus uteri or cervix uteri. To further validate the accuracy of reallocation, we also analyzed how well the reallocation captures the changes occurring as the result of a transition in cause of death coding in Norway that took place in 2005. MATERIAL AND METHODS: This study uses data available in the NORDCAN database, which contains aggregated cancer data from all the Nordic countries for the years 1960-2016. The unspecified uterine cancer cases and deaths were reallocated to either cervix uteri or corpus uteri based on the estimated probability that follows the distribution of cases and deaths with verified topography. The estimated proportions of cases and deaths for both cancers were calculated for each combination of age group, year, and country as a proportion of cases (and deaths, respectively) with known topography. Annual age-standardized rates were calculated by direct age-adjustment. RESULTS: The proportions of unspecified uterine cancers were higher in the mortality data than in incidence data, with mean values for 1960-2016 ranging between 5.1% and 26.6% and between 0.2% and 6.8% by country, respectively. In the Nordic countries combined, the reallocation increased the number of cases by 4% and deaths by approximately 20% for both cancers. Finland was the only Nordic country where the mortality rate did not increase substantially after reallocation. CONCLUSIONS: The reallocation procedure had a significant impact on mortality from cancers of the cervix and corpus uteri for countries where the proportion of cancer deaths coded as uterus, not otherwise specified, is substantial. More effort to validate cause of death data with incidence data from cancer registries is warranted to avoid erroneous conclusions of temporal trends based on uncorrected cancer burden.

Decreasing trend and changing indications of hysterectomy in Finland.

INTRODUCTION: Hysterectomy has been one of the most common surgical procedures in women in Finland. We studied the population-based trends of hysterectomy and its indications from 1986 to 2017. MATERIAL AND METHODS: A retrospective population-based cohort was created from the Care Register for Health Care by identifying women who had a hysterectomy from 1986 to 2017 and calculating the number of women from the Digital and Population Data Services Agency. We estimated the number and incidence of hysterectomy by period and age as well as by indication. We considered the primary diagnosis at the time of surgery as the indication of hysterectomy. RESULTS: The number of hysterectomies increased from 7492 procedures in 1986 to 12 404 procedures in 1998, and reduced substantially after that to 5971 procedures in 2017, the turning point being in 1999. The incidence rate of hysterectomy has decreased on average by 2.5% annually from 432.6 per 100 000 women in 1998-2001 to 224.5 per 100 000 women in 2014-2017. The median age at the time of hysterectomy has increased from 51 years in 1998-2001 to 55 years in 2014-2017. The cumulative burden of hysterectomy by age of 60 years has nearly halved from the first 4-year period (23%) to the last (12%). After 2010, the most common indication has been genital prolapse and incontinence, whereas earlier it was uterine fibroids. CONCLUSIONS: The number and incidence of hysterectomies have fluctuated during the observation period 1986-2017 and decreased considerably during the past 17 years in Finland. This is probably a result of the availability of hormonal and other conservative treatment options for bleeding disorders and uterine fibroids. As hysterectomy practically removes the risk for endometrial cancer, the change in hysterectomy incidence over time emphasizes the importance of correcting endometrial cancer incidence according to hysterectomy incidence.

Efficacy of Trabodenoson in a Mouse Keratoconjunctivitis Sicca (KCS) Model for Dry-Eye Syndrome.

Purpose: To determine the efficacy of trabodenoson, an adenosine mimetic with highly selective adenosine A1 receptor binding properties, in a preclinical mouse model for dry-eye disease. Methods: Dry-eye disease was induced in adult male C57BL/6 mice using a combination of desiccating environment and transdermal administration of scopolamine. Mice were treated concurrently and twice daily with either vehicle, 6% trabodenoson, or 0.05% cyclosporine (Restasis). Efficacy (P < 0.05 versus vehicle) was determined by clinical assessment of dry-eye symptoms using corneal fluorescein staining and tear volumes and histopathologically by quantifying lacrimal gland pathology and conjunctival goblet cells. Results: Twice-daily topical (ocular) administration of trabodenoson increased tear levels and reduced corneal fluorescein staining (P < 0.05) as compared with vehicle-treated eyes in a mouse model of dry-eye disease. Furthermore, significant infiltration of immune cells in the lacrimal gland and reduced number of mucin-producing conjunctival goblet cells were noted in both untreated and vehicle-treated eyes. Comparatively, trabodenoson treatment significantly reduced lacrimal gland infiltration and increased the number of goblet cells (P < 0.05 for both versus vehicle). These trabodenoson-related effects on lacrimal gland pathology and goblet cells were similar to or better than the effects observed with cyclosporine treatment. Conclusions: Topical ocular delivery of trabodenoson significantly improves the clinical and histopathological signs associated with dry-eye disease in mice. This improvement appears to be related to anti-inflammatory effects from targeting adenosine signaling and represents a novel therapeutic approach to develop for the management of dry-eye disease.

Brain pharmacokinetics of ganciclovir in rats with orthotopic BT4C glioma.

Ganciclovir (GCV) is an essential part of the Herpes simplex virus thymidine kinase (HSV-tk) gene therapy of malignant gliomas. The purpose of this study was to investigate the brain pharmacokinetics and tumor uptake of GCV in the BT4C rat glioma model. GCV's brain and tumor uptakes were investigated by in vivo microdialysis in rats with orthotopic BT4C glioma. In addition, the ability of GCV to cross the blood-brain barrier and tumor vasculature was assessed with in situ rat brain perfusion. Finally, the extent to which GCV could permeate across the BT4C glioma cell membrane was assessed in vitro. The areas under the concentration curve of unbound GCV in blood, brain extracellular fluid (ECF), and tumor ECF were 6157, 1658, and 4834 µM⋅min, respectively. The apparent maximum unbound concentrations achieved within 60 minutes were 46.9, 11.8, and 25.8 µM in blood, brain, and tumor, respectively. The unbound GCV concentrations in brain and tumor after in situ rat brain perfusion were 0.41 and 1.39 nmol/g, respectively. The highly polar GCV likely crosses the fenestrated tumor vasculature by paracellular diffusion. Thus, GCV is able to reach the extracellular space around the tumor at higher concentrations than that in healthy brain. However, GCV uptake into BT4C cells at 100 µM was only 2.1 pmol/mg of protein, and no active transporter-mediated disposition of GCV could be detected in vitro. In conclusion, the limited efficacy of HSV-tk/GCV gene therapy may be due to the poor cellular uptake and rapid elimination of GCV.

Similar molecular descriptors determine the in vitro drug permeability in endothelial and epithelial cells.

Endothelial and epithelial cells are commonly used for assessing blood-brain barrier (BBB) permeability of the drug candidates. However, the additional value provided by the endothelial cells as an in vitro BBB drug permeability model is not clear. The aim of the study was to identify the molecular descriptors that impact on drug permeability through the primary bovine brain microvessel endothelial cell model (BBMEC) and compare descriptors with those determined for epithelial cell models. In addition, we intended to clarify the reasons for previously reported similar in vitro-in vivo correlations between endothelial and epithelial cell models and to evaluate whether BBMEC can provide additional value. The permeability of model drugs through the BBMEC was determined. Principal component analysis (PCA) model was created with twenty-two model drugs and this model was used to interpret the molecular descriptors. The present study demonstrates that hydrophobic interactions, the balance between the hydrophilic and lipophilic moieties in the drug, hydrophilic interactions and hydrogen bonding interactions are the key descriptors depicting drug permeability through the BBMEC. There were no clear differences between the molecular descriptors determining the in vitro permeability of drugs in the endothelial and epithelial cells. In conclusion, the predominance of passive permeability in in vitro setups may explain the similar in vitro-in vivo correlations previously obtained between endothelial and epithelial cell models. Therefore, the present results further support previous findings that epithelial cell models can be used instead of laborious primary endothelial cells as an in vitro BBB permeability model when passive transport is mainly being evaluated.

Human and bovine milk oligosaccharides inhibit Neisseria meningitidis pili attachment in vitro.

Milk oligosaccharides have been shown to interfere with adhesion of many pathogens to host mucosal surfaces. Characterization of the adhesion mechanisms of the bacteria to host cell surface is needed to develop novel functional food, infant formulas, and anti-infective drugs. Adhesion of Neisseria meningitidis, a human specific pathogen causing meningitis and septicemia, is not completely understood but is mediated by type IV pili. Here, we developed a microtiter well pili binding assay to investigate the binding activities of N. meningitidis isolated type IV pili to different glycoproteins. Pili binding activities to bovine thyroglobulin and human salivary agglutinin but not to chicken ovalbumin were present. Inhibition of these binding activities was demonstrated by fractionated human or bovine milk oligosaccharides. The binding of neisserial pili to bovine thyroglobulin was most effective and was clearly inhibited by human milk neutral or bovine milk acidic oligosaccharides.