Broad Dose Range Research Articles

The long-term efficacy of cannabidiol in the treatment of refractory epilepsy.

Cannabidiol (CBD) has been shown to reduce seizures among patients with refractory epilepsies of various etiologies in recent clinical trials and an expanded access program (EAP). Most studies report efficacy over short time periods (<1year), with little published on longer term efficacy. Here, we investigate the efficacy of CBD for a treatment period of up to 60months (median = 45.5months). We conducted a retrospective review of patient-reported seizure logs and medical records for 54 subjects with refractory epilepsy who enrolled in the Massachusetts General Hospital's open-label EAP for CBD as a new treatment for epilepsy. We analyzed the effect of CBD on seizure frequencies and concomitant antiepileptic drug (AED) use at 1year after starting treatment and the most recent study visit. Our results indicate that CBD maintains its efficacy for controlling seizures from Year 1 to the most recent study visit. The percentage of seizure responders remained similar at these time points (41.7%-42.6%), and the seizure response rate was also maintained (p=.12). Efficacy was also seen over a broad dose range, and up to 50mg/kg/day. CBD was particularly effective for controlling seizures in the setting of tuberous sclerosis complex and for reducing epileptic spasms and absence seizures. Although CBD use did not lead to an overall decrease in concomitant AEDs, most subjects reduced the dose of at least one concomitant AED compared to baseline. CBD was generally well tolerated, with drowsiness and diarrhea as the primary adverse reactions. This study demonstrates CBD does not lose its efficacy in controlling seizures over a treatment period of up to 60months. Taken alongside other results on the efficacy and tolerability of CBD in the treatment of refractory epilepsies, our results provide evidence that CBD is an effective, safe, and well-tolerated AED for long-term use.

Interplay Between Endocrine Disruptors and Immunity: Implications for Diseases of Autoreactive Etiology.

The sex-bias of disease susceptibility has remained a puzzling aspect of several autoimmune conditions, including post-infection viral autoimmunity. In the last half of the twentieth century, the incidence rate of female-biased autoimmunity has steadily increased independent of medical advances. This has suggested a role for environmental factors, such as endocrine disrupting chemicals, which have been described to interfere with endocrine signaling. Endocrine involvement in the proper function of innate and adaptive immunity has also been defined, however, these two areas have rarely been reviewed in correlation. In addition, studies addressing the effects of endocrine disruptors have reported findings resulting from a broad range of exposure doses, schedules and models. This experimental heterogeneity adds confusion and may mislead the translation of findings to human health. Our work will normalize results across experiments and provide a necessary summary relevant to human exposure. Through a novel approach, we describe how different categories of ubiquitously used environmental endocrine disruptors interfere with immune relevant endocrine signaling and contribute to autoimmunity. We hope this review will guide identification of mechanisms and concentration-dependent EDC effects important not only for the sex-bias of autoimmunity, but also for other conditions of immune dysfunction, including post-infection autoreactivity such as may arise following severe acute respiratory syndrome coronavirus 2, Epstein-Barr virus, Herpes Simplex virus.

Impacts of ionization radiation on the cuticular hydrocarbon profile and mating success of male house crickets (Acheta domesticus)

Purpose Ionizing radiation is well known to have drastic impacts on major life history features including survivorship, growth, fertility, and longevity. What is much less appreciated is how radiation stress can cause changes to more subtle traits, such as those associated with sexual signaling, an underappreciated but vital aspect of insect reproduction. In the House Cricket (Acheta domesticus) cuticular hydrocarbons are vital for sex and species recognition, as well as a possible indicator of stress, making them crucial for successful mating and reproduction. Materials and methods Here, we analyze the impacts of ionizing radiation on the cuticular hydrocarbons of male crickets and its subsequent impacts on mating success. We exposed juvenile (14-day, 4th instar) male crickets to a broad range of radiation doses (2 Gy − 2 Gy). Results We detected significant changes in individual cuticular hydrocarbons across a broad range of doses in mature male crickets using gas-liquid chromatography. Specifically, dose was identified as a significant contributing factor to hydrocarbon increases p < .0001. Mating success was significantly reduced in 12 Gy (p < .0001), 10 Gy (0.0001), and 7 Gy (0.0060) groups compared to non-irradiated controls. Conclusion Insect chemical communication can be species specific, and functionally specialized. Here, we show that radiation can alter the chemical signals used to attract mates in a large bodied insect and this may be a contributing factor to the described reduction in male mating success. Further research should be conducted to further analyze the various modes of communication employed by male crickets to attract mates i.e. acoustic signaling, and how this may also contribute to the reduction in mating success seen in irradiated males.

Investigational CD47-Blocker TTI-622 Shows Single-Agent Activity in Patients with Advanced Relapsed or Refractory Lymphoma: Update from the Ongoing First-in-Human Dose Escalation Study

Investigational CD47-Blocker TTI-622 Shows Single-Agent Activity in Patients with Advanced Relapsed or Refractory Lymphoma: Update from the Ongoing First-in-Human Dose Escalation Study

Dose- and Schedule-Dependent Immunomodulatory Effects of the Novel Celmod Agent CC-92480 in Patients with Relapsed/Refractory Multiple Myeloma

Dose- and Schedule-Dependent Immunomodulatory Effects of the Novel Celmod Agent CC-92480 in Patients with Relapsed/Refractory Multiple Myeloma

Magnesium Calcium Phosphate Cement Incorporating Citrate for Vascularized Bone Regeneration.

The development of bioactive bone cement is still a challenge for vascularized bone regeneration. Citrate participated in multiple biological processes, such as energy metabolism, osteogenesis, and angiogenesis. However, it is difficult to obtain a thorough and comprehensive understanding on osteogenic effects of exogenous citrate from different experimental conditions and treatment methods. In this study, by using a magnesium calcium phosphate cement (MCPC) matrix, we investigated the dual effect of exogenous citrate on osteogenesis and angiogenesis. Our studies show that citrate elevates the osteogenic function of osteoblasts under low doses and the angiogenic function of vascular endothelial cells under a broader dose range. These findings furnish a new strategy for regulating angiogenesis and osteogenic differentiation by administration of citrate in MCPC, driving the development of bioactive bone repair materials.

Induction and repression effects on CYP and transporter protein abundance by azole mixture uptake in rat liver.

Detection of mixture effects is a major challenge in current experimental and regulatory toxicology. Robust markers are needed that are easy to quantify and responsive to chemical stressors in a broad dose range. Several hepatic enzymes and proteins related to drug metabolism like cytochrome-P-450 (CYP) enzymes and transporters have been shown to be responsive to pesticide active substances in a broad dose range and are therefore good candidates to be used as markers for mixture toxicity. Even though they can be well quantified at the mRNA level, quantification on the protein level is challenging because most of these proteins are membrane bound. Here we report the development of mass spectrometry-based assays using triple-x-proteomics (TXP) antibodies in combination with targeted selected ion monitoring (tSIM) to quantify changes of protein levels due to exposure to mixtures of pesticide active substances. Our results indicate that changes on the protein level of CYP1A1, ABCB2, ABCC3 are in line with observations on the mRNA and enzyme activity level and are indicative of mixture effects. Therefore, the tests are promising to reveal effects by chemical mixture effects in toxicological studies in rats.

Low doses of Bisphenol S affect post-translational modifications of sperm proteins in male mice

BackgroundBisphenol S (BPS) is increasingly used as a replacement for bisphenol A in the manufacture of products containing polycarbonates and epoxy resins. However, further studies of BPS exposure are needed for the assessment of health risks to humans. In this study we assessed the potential harmfulness of low-dose BPS on reproduction in male mice.MethodsTo simulate human exposure under experimental conditions, 8-week-old outbred ICR male mice received 8 weeks of drinking water containing a broad range of BPS doses [0.001, 1.0, or 100 μg/kg body weight (bw)/day, BPS1–3] or vehicle control. Mice were sacrificed and testicular tissue taken for histological analysis and protein identification by nano-liquid chromatography/mass spectrometry (MS) and sperm collected for immunodetection of acetylated lysine and phosphorylated tyrosine followed by protein characterisation using matrix-assisted laser desorption ionisation time-of-flight MS (MALDI-TOF MS).ResultsThe results indicate that compared to vehicle, 100 μg/kg/day exposure (BPS3) leads to 1) significant histopathology in testicular tissue; and, 2) higher levels of the histone protein γH2AX, a reliable marker of DNA damage. There were fewer mature spermatozoa in the germ layer in the experimental group treated with 1 μg/kg bw (BPS2). Finally, western blot and MALDI-TOF MS studies showed significant alterations in the sperm acetylome and phosphorylome in mice treated with the lowest exposure (0.001 μg/kg/day; BPS1), although the dose is several times lower than what has been published so far.ConclusionsIn summary, this range of qualitative and quantitative findings in young male mice raise the possibility that very low doses of BPS may impair mammalian reproduction through epigenetic modifications of sperm proteins.

Experimental Single-Platform Approach to Enhance the Functionalization of Magnetically Targetable Cells.

Magnetic guidance shows promise as a strategy for improving the delivery and performance of cell therapeutics. However, clinical translation of magnetically guided cell therapy requires cell functionalization protocols that provide adequate magnetic properties in balance with unaltered cell viability and biological function. Existing methodologies for characterizing cells functionalized with magnetic nanoparticles (MNP) produce aggregate results, both distorted and unable to reflect variability in either magnetic or biological properties within a preparation. In the present study, we developed an inverted-plate assay allowing determination of these characteristics using a single-platform approach, and applied this method for a comparative analysis of two loading protocols providing highly uniform vs. uneven MNP distribution across cells. MNP uptake patterns remarkably different between the two protocols were first shown by fluorimetry carried out in a well-scan mode on endothelial cells (EC) loaded with BODIPY558/568-labeled MNP. Using the inverted-plate assay we next demonstrated that, in stark contrast to unevenly loaded cells, more than 50% of uniformly functionalized EC were captured within 5 min over a broad range of MNP doses. Furthermore, magnetically captured cells exhibited unaltered viability, substrate attachment, and proliferation rates. Conducted in parallel, magnetophoretic mobility studies corroborated the markedly superior guidance capacity of uniformly functionalized cells, confirming substantially faster cell capture kinetics on a clinically relevant time scale. Taken together, these results emphasize the importance of optimizing cell preparation protocols with regard to loading uniformity as key to efficient site-specific delivery, engraftment, and expansion of the functionalized cells, essential for both improving performance and facilitating translation of targeted cell therapeutics.

Multiple Biological Effects of Citrate in Magnesium Phosphate - Based Cement During Bone Regeneration

The development of multiple biofunction materials is still a challenge in bone tissue engineering biomaterials. Exogenous citrate is involved in multiple biological processes such as metabolism, osteogenesis and angiogenesis. However, it is difficult to obtain a thorough and comprehensive understanding on osteogenic effects of exogenous citrate from different experimental conditions and treatment methods. In this study, by using a magnesium phosphate - based cement (MPBC) matrix, we not only investigated the dual effect of exogenous citrate to osteogenesis and angiogenesis in vivo and vitro, but also explored the regulation of citrate on bone apatite crystal structure in an osteoblast differentiation model. Our studies show that citrate elevates the osteogenic function of osteoblasts under low doses and angiogenic function of vascular endothelial cells under a broader dose range. Furthermore, citrate, as an ionic chelator and mineralization inhibitor, improves the cell survival in high calcium and phosphorus environment and regulates the phase structure of osteogenic minerals. These findings provide a new strategy to regulate angiogenesis and osteogenic differentiation by adjusting citrate content and release in MPBC, guiding the development of bioactive materials for bone tissue regeneration. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (No. 51672206), the National Key Research and Development Program of China (2016YFC1101605 and 2018YFB1105500), the Major Special Projects of Technological Innovation of Hubei Province (No. 2019ACA130) and the Application Foundation and Front Research Program of Wuhan (No. 2018010401011273). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: All animal experiments were conducted using protocol approved by the China National Institutional Animal Care and Use Committee.

Toxic Responses Induced at High Doses May Affect Benchmark Doses.

To derive reference points (RPs) for health-based guidance values, the benchmark dose (BMD) approach increasingly replaces the no-observed-adverse-effect level approach. In the BMD approach, the RP corresponds to the benchmark dose lower confidence bounds (BMDLs) of a mathematical dose–response model derived from responses of animals over the entire dose range applied. The use of the entire dose range is seen as an important advantage of the BMD approach. This assumes that responses over the entire dose range are relevant for modeling low-dose responses, the basis for the RP. However, if part of the high-dose response was unnoticed triggered by a mechanism of action (MOA) that does not work at low doses, the high-dose response distorts the modeling of low-dose responses. Hence, we investigated the effect of high-dose specific responses on BMDLs by assuming a low- and a high-dose MOA. The BMDLs resulting from modeling fictitious quantal data were scattered over a broad dose range overlapping with the toxic range. Hence, BMDLs are sensitive to high-dose responses even though they might be irrelevant to low-dose response modeling. When applying the BMD approach, care should be taken that high-dose specific responses do not unduly affect the BMDL that derives from low doses.

The Dual Amylin and Calcitonin Receptor Agonist, KBP-066, Induces an Equally Potent Weight Loss Across a Broad Dose Range While Higher Doses May Further Improve Insulin Action.

Pharmacological treatment with dual amylin and calcitonin receptor agonists (DACRAs) cause significant weight loss and improvement of glucose homeostasis. In this study, the maximally efficacious dose of the novel DACRA, KeyBiosciencePeptide (KBP)-066, was investigated. Two different rat models were used: high-fat diet (HFD)-fed male Sprague-Dawley rats and male Zucker diabetic fatty (ZDF, fa/fa) rats to determine the maximum weight loss and glucose homeostatic effect, respectively. One acute study and one chronic study was performed in HFD rats. Two chronic studies were performed in ZDF rats: a preventive and an interventive. All studies covered a dose range of 5, 50, and 500 µg/kg KBP-066 delivered by subcutaneous injection. Treatment with KBP-066 resulted in a significant weight reduction of 13%-16% and improved glucose tolerance in HFD rats, which was independent of dose concentration. Dosing with 50 and 500 µg/kg led to a transient but significant increase in blood glucose, both in the acute and the chronic study in HFD rats. All doses of KBP-066 significantly improved glucose homeostasis in ZDF rats, both in the preventive and interventive study. Moreover, dosing with 50 and 500 µg/kg preserved insulin secretion to a greater extent than 5 µg/kg when compared with ZDF vehicle rats. Taken together, these results show that maximum weight loss is achieved with 5 µg/kg, which is within the range of previously reported DACRA dosing, whereas increasing dosing concentration to 50 and 500 µg/kg may further improve preservation of insulin secretion compared with 5 µg/kg in diabetic ZDF rats. SIGNIFICANCE STATEMENT: Here we show that KeyBiosciencePeptide (KBP)-066 induces an equally potent body weight loss across a broad dose range in obese rats. However, higher dosing of KBP-066 may improve insulin action in diabetic rats both as preventive and interventive treatment.

The Combination of the BCL-2 Antagonist Venetoclax with the CD70-Targeting Antibody Cusatuzumab Synergistically Eliminates Primary Human Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a very heterogeneous hematological malignancy characterized by the accumulation of myeloid blasts. Treatment options for unfit AML patients greater than 65 year of age are still limited and outcomes are dismal. The current standard of care for older AML patients are hypomethylating agents (HMA) or low dose Ara-C. However, even though inducing hematological remissions in up to 30 percent of the patients, responses are not durable and survival of these patients is only marginally prolonged. The poor durability is due to insufficient action on leukemia stem cells (LSC) which drive and maintain the disease and are resistance to therapy (Craddock et al, 2013 and DiNardo et al, 2019). We described how aberrant CD70/CD27 signaling drives stemness of AML LSCs and identified CD70 as potential new target for the treatment of AML patients (Riether et al, 2017). In a recent Phase 1 clinical trial, treatment of older and unfit AML patients with the ADCC-enhanced humanized monoclonal anti CD70 antibody (mAb) cusatuzumab in combination with HMA demonstrated promising clinical activity and a favorable tolerability profile. The BCL-2 antagonist, venetoclax, targets and eliminates LSCs by suppression of oxidative phosphorylation and demonstrated very promising activity in older AML patients in clinical phase I and II studies in combination with standard of care (Pollyea et al, 2018). However, even with novel agents such as venetoclax, there are still patients that become refractory or relapse. We hypothesized that combining venetoclax and cusatuzumab with distinct but complementary mechanisms of action could successfully eliminate LSCs. Experimental design To test this hypothesis, we performed a drug-combination study according to the Chou-Talalay method (Chou 2010) in CD70-expressing AML cell lines such as MOLM-13, MV4-11, and NOMO-1 cells in vitro. In addition, we tested the effect of the cusatuzumab/venetoclax and the cusatuzumab/venetoclax/HMA combination on colony formation and re-plating capacity of primary CD34+CD38- LSCs from newly diagnosed AML patients. Results We first treated MOLM-13, MV4-11, and NOMO-1 AML cells with vehicle, cusatuzumab alone or in combination with venetoclax or decitabine in a constant ratio in the presence of CFSE-labeled NK cells (ratio 1:1). AML cell numbers were assessed 72 hours later. Cusatuzumab in combination with venetoclax or decitabine and NK cells synergistically eliminated CD70-expressing AML cells in a broad dose range (Figure 1). To assess the effect of the cusatuzumab/venetoclax combination on primary human AML LSCs, we treated CD34+ CD38- LSCs with cusatuzumab or venetoclax monotherapy or in combination in the presence of NK cells and assessed colony formation. Cusatuzumab/venetoclax co-treatment was more efficacious than each monotherapy alone and strongly reduced LSCs and leukemia progenitors (Figure 2). To analyze the effect of the cusatuzumab/venetoclax treatment on LSC function in a more stringent way, we performed serial re-plating experiments in vitro. The impaired colony formation after combination treatment observed after the first plating was maintained during subsequent the re-plating, even though cusatuzumab and venetoclax were not present in the re-plating, indicating an effective reduction of LSCs. Mechanistically, we could show that treatment with venetoclax results in up-regulation of CD70 on LSCs, suggesting that venetoclax renders LSCs more susceptible to cytolytic killing with cusatuzumab. In older AML patients, cusatuzumab and venetoclax each have demonstrated promising clinical activity in combination with HMA. We determined whether addition of HMA to the cusatuzumab/venetoclax co-treatment could more effectively eliminate LSCs. The triplet combination cusatuzumab/venetoclax/decitabine did not further reduce growth of AML cell lines in vitro (Figure 1) nor colony and re-plating capacity of human LSCs compared to the cusatuzumab/venetoclax co-treatment. Conclusions Overall, these results indicate that a combination of cusatuzumab with venetoclax eliminates LSCs synergistically and more efficiently than as a monotherapy. Tolerability and efficacy will be tested in mouse models using both primary AML and cell line xenografts. The results suggest that targeting LSCs by combining venetoclax and cusatuzumab treatments is a promising novel treatment strategy in AML. Disclosures Johnson: Janssen R&amp;D: Employment. Drenberg:Janssen R&amp;D: Employment. Syed:Janssen R&amp;D: Employment. Moshir:Argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Leupin:Argenx: Employment, Equity Ownership, Patents &amp; Royalties. De Haard:Argenx: Employment, Equity Ownership, Patents &amp; Royalties.

Distinct Pathogenic Patterns of Burkholderia pseudomallei Isolates Selected from Caenorhabditis elegans and Dictyostelium discoideum Models.

Burkholderia pseudomallei is a selective agent that causes septic melioidosis and exhibits a broad range of lethal doses in animals. Host cellular virulence and phagocytic resistance are pathologic keys of B. pseudomallei. We first proposed Caenorhabditis elegans as the host cellular virulence model to mimic bacterial virulence against mammals and second established the resistance of B. pseudomallei to predation by Dictyostelium discoideum as the phagocytosis model. The saprophytic sepsis-causing Burkholderia sp. (B. pseudomallei, Burkholderia thailandensis, Burkholderia cenocepacia, and Burkholderia multivorans) exhibited different virulence patterns in both simple models, but B. pseudomallei was the most toxic. Using both models, attenuated isolates of B. pseudomallei were selected from a transposon-mutant library and a panel of environmental isolates and reconfirmed by in vitro mouse peritoneal exudate cell association and invasion assays. The distinct pathological patterns of melioidosis were inducted by different selected B. pseudomallei isolates. Fatal melioidosis was induced by the isolates with high virulence in both simple models within 4-5 day, whereas the low-virulence isolates resulted in prolonged survival greater than 30 day. Infection with the isolates having high resistance to D. discoideum predation but a low C. elegans killing effect led to 83% of mice with neurologic melioidosis. By contrast, infection with the isolates having low resistance to D. discoideum predation but high C. elegans killing effect led to 20% cases with inflammation in the salivary glands. Our results indicated that individual B. pseudomallei isolates selected from simple biological models contribute differently to disease progression and/or tissue tropism.

Caenorhabditis elegans As a Promising Alternative Model for Environmental Chemical Mixture Effect Assessment-A Comparative Study.

A key challenge of mixture toxicity testing is that a multitude of substances with even more combinations need to be tested in a broad dose range. Consequently testing in rodent bioassays, the current gold standard of toxicity testing, is hardly feasible. High-throughput compatible cell culture systems, however, suffer from limitations with respect to toxicokinetics, tissue interactions, and compensatory mechanisms. Therefore, simple organisms like the nematode Caenorhabditis elegans, combining relevant advantages of complex in vivo and fast in vitro assays might prove highly valuable within a testing strategy for mixtures. To investigate the comparability between results obtained with C. elegans and traditional rodent assays, we used five azole fungicides as well investigated model substances. Our findings suggest that azoles act additively in C. elegans which is in line with previous results in rats. Additionally, we show that toxicokinetics are one important factor for the differences in the relative toxicity of the azoles in both species. Importantly, we also demonstrate that in contrast to most rodent in vivo studies, C. elegans assays provide well-defined concentration-response relationships which are a very good basis for the prediction of mixture effects. We conclude that C. elegans may be an appropriate model for mixture toxicity testing at least within a first step to identify and prioritize relevant mixtures for further testing.

Caffeine modulates voluntary alcohol intake in mice depending on the access conditions: Involvement of adenosine receptors and the role of individual differences

Caffeine is the most consumed psychoactive stimulant and the main active ingredient of energy drinks. Epidemiology studies have shown a positive correlation between the consumption of energy drinks and that of ethanol. The popular belief is that caffeine antagonizes the intoxicating effects of alcohol. Both drugs act on the adenosine system but have opposite effects. Caffeine is a methylxanthine that acts as a nonselective adenosine receptor antagonist, binding to A1 and A2A receptor subtypes. In contrast, ethanol increases extracellular adenosinergic tone. The purpose of this study was to examine the impact of a broad range of doses of caffeine and of selective adenosine A1 and A2A receptor antagonists on voluntary ethanol intake under different ethanol access conditions. C57BL/6 J male mice had access to ethanol (10% w/v) under different conditions: restricted (2 h in the dark), unrestricted (24 h access), or after 4 days of alcohol removal following several periods of unrestricted access. Mice reduced ethanol intake in the restricted access condition after receiving caffeine (20.0 mg/kg), or theophylline (20.0 mg/kg), another methylxanthine. Selective A1 and A2A adenosine receptor antagonists, or their combination, did not have any effect. However, under unrestricted access conditions caffeine and the adenosine A2A receptor antagonist increased ethanol intake. After splitting animals into high, moderate and low ethanol consumers, caffeine (2.5–20.0 mg/kg) significantly increased ethanol consumption in moderate consumers with no effect on low or high consumers. In addition, after reintroducing ethanol access, caffeine (5.0 mg/kg) decreased ethanol consumption among moderate consumers. Thus, caffeine produced different effects on ethanol intake depending on the access condition and the baseline consumption of ethanol.

Impact of capacity‐limited binding on recombinant factor VIII and von Willebrand factor pharmacokinetics in hemophilia A rats

Unlabelled Box BackgroundUnderstanding of the pharmacokinetics (PK) interplay between factor VIII (FVIII) and von Willebrand factor (VWF) following high‐dose FVIII treatment is lacking. ObjectivesTo characterize the PK of recombinant FVIII (rFVIII), VWF, and the rFVIII:VWF complex in hemophilia A rats following intravenous administration of rFVIII using PK modeling. A second aim was to investigate the effect of high daily dosing and constant expression of rFVIII on VWF exposure using PK simulations. MethodsWe developed a population PK model based on the principles of target‐mediated drug disposition modeling, using data on total rFVIII and VWF plasma concentrations, and the rFVIII:VWF complex luminescent oxygen channeling immunoassay signal in hemophilia A rats following intravenous administration of rFVIII (17.5, 100, 1000, and 5000 IU kg−1). Additionally, we evaluated the influence of high‐dose rFVIII treatment on the exposure of VWF using PK simulations. ResultsThe plasma concentration‐time profiles of total rFVIII and VWF, and the luminescent oxygen channeling immunoassay signal‐time profiles of the rFVIII:VWF complex were adequately described using a two‐compartment quasi‐steady‐state target‐mediated drug disposition model (Kss = 0.14 nmol L−1). The elimination half‐life of the rFVIII:VWF complex was dependent on the unbound plasma concentration of rFVIII. Additionally, we showed that high‐dose rFVIII treatment may significantly reduce the endogenous VWF levels. ConclusionsWe developed a population‐based PK model describing the time‐course of total rFVIII, total VWF, and the rFVIII:VWF complex over a broad range of rFVIII doses in hemophilia A rats.

Formulation and processability screening for the rational design of ethylene-vinyl acetate based intra-vaginal rings

The application of ethylene-vinyl acetate (EVA) copolymers in reservoir-type intra-vaginal rings (IVRs) offers advantages over silicones including i) versatile properties, ii) absence of curing chemistry, and iii) continuous and flexible processing via co-extrusion. Thus, we investigated the capability of EVA based IVRs to deliver broad ranges of estradiol (E2) thereby, fulfilling the requirements of local and systemic hormone replacement therapy (HRT) and contraception. To circumvent the high material needs associated with co-extrusion, we implemented a small-scale screening procedure that accurately predicts the E2 release from IVRs comprising E2 below its solubility concentration in the core. Rational formulation design yielded the target release for local HRT (<10 µg/day), systemic HRT (50–100 µg/day) and contraception (>150 µg/day, combined with a progestin). Low E2 release was achieved by the combination of low E2 loadings, low VA content of the membrane polymer (also known as coat polymer or outer shell), and increased membrane thickness. Medium E2 release was provided by medium E2 loading, low VA content of the membrane polymer, and low membrane thickness. Combining high E2 loadings, high VA content of the membrane polymer, and low membrane thickness yielded high E2 release. This makes EVA based IVRs a versatile platform that can be used to deliver a broad range of E2 doses.

OR33-4 A Single Subcutaneous Injection Of The Kisspeptin Analogue, MVT-602, Induces A More Prolonged LH Surge Compared With Kisspeptin-54 In Healthy Women

Background Kisspeptin is a hypothalamic neuropeptide that is requisite for normal reproductive health. A subcutaneous bolus of the native form of kisspeptin (kisspeptin-54; KP54) induces an LH-surge lasting 12-14hrs, to safely mature oocytes during in vitro fertilisation (IVF) treatment. The kisspeptin analogue, MVT-602, has a longer half-life (t1/2 1.5-2.2h) than native KP54 (t1/2 0.5h), which could therefore confer significant advantages as an oocyte maturation trigger in the treatment of infertility. Whilst MVT-602 has been shown to stimulate LH release in men, its effects in women have not previously been investigated. To address this, we studied the effect of MVT-602 on gonadotrophin release in healthy women for the first time. Methods A two-phase dose-finding study was undertaken during the early follicular phase (Day 1-4) of 12 healthy women aged 18-35yrs with regular menstrual cycles. During phase 1, a broad dose-range for MVT-602 was determined: three women received a single subcutaneous bolus of one of five doses of MVT-602 (0.003, 0.03, 0.1, 0.3, or 1.0nmol/kg) in successive menstrual cycles. Reproductive hormones were measured every 30min for 14hrs post-MVT-602, and again at 24hrs and 48hrs post-injection. During phase 2, an additional 9 women received either KP54 (9.6nmol/kg), or MVT-602 at doses of 0.01 or 0.03nmol/kg during the follicular phase of successive menstrual cycles, following which reproductive hormone levels were monitored every 30mins for 24hrs post-injection. Intervention groups were compared by one-way ANOVA with post hoc Tukey’s test. ResultsPhase 1: Peak serum LH levels occurred between 14-24hrs and no further increases occurred with MVT-602 at doses greater than 0.03 nmol/kg. Phase 2: Peak amplitude of serum LH was found to be similar following KP54 and MVT-602 (mean±SD of peak LH KP54 12.3 ±5.3iU/L; MVT-602 0.01nmol/kg 11.4 ±4.2iU/L; MVT-602 0.03nmol/kg 11.1 ±3.0iU/L, p>0.05). However, time to peak LH was later following MVT-602 (22hrs) when compared with KP54 (4.9hrs; P<0.0001). Thus, the area under the curve (AUC) for LH exposure was significantly increased following MVT-602 when compared with KP54 (mean±SD of AUC LH KP54 9,095 ±5,632iU.min/L; MVT-602 0.01nmol/kg 18,129 ±6,471iU.min/L, P=0.02 vs KP54; MVT-602 0.03nmol/kg 20,086 ±6,227iU.min/L, P=0.004 vs KP54). Conclusion A single subcutaneous bolus of MVT-602 in healthy women resulted in an LH surge of similar amplitude, but of longer duration than by KP54. During studies evaluating the utility of KP54 as a trigger of oocyte maturation in IVF treatment, extending the duration of LH surge with a second bolus of KP54 safely optimised oocyte maturation. Thus, the prolonged gonadotrophin surge induced by MVT-602 could be of significant utility not only to safely optimise oocyte maturation in IVF treatment, but also to treat other reproductive disorders affecting women’s health.

Development of a PLS approach for the determination of the conversion in UV-cured white-pigmented coatings by NIR chemical imaging and its transfer to other substrates

Near-infrared (NIR) chemical imaging was used for in-line monitoring of the conversion in thick UV-cured white-pigmented acrylate coatings applied to various substrates such as glass, stainless steel, PVC and glass fiber reinforced plastic (GRP) boards. Quantitative results were obtained by means of chemometric calibration models based on the partial least square (PLS) algorithm. Two spectroscopic techniques were tested for their potential for the characterization of the conversion after UV irradiation with a broad range of UV doses in order to provide reliably precise reference data for the calibration. NIR reflection spectroscopy using band integration of the acrylate band at 1620 nm was selected as method of choice. Using glass as substrate for the coatings at the beginning, a PLS model was established and evaluated for its performance to predict the conversion in independent samples. The acrylate conversion was predicted with an error of 2%. Generally, any calibration model is specific to a well-defined sample system (e.g. material and thickness of substrate and coating). In order to reduce the effort required for the development of specific calibration models for each substrate used in this study, methods for calibration transfer were developed. It was found that the requirements for such transfer depend on the optical properties of the substrate. In case of stainless steel and PVC boards, simple preprocessing of the spectra by baseline correction and normalization (similar to samples on glass) led to satisfactory results, which is confirmed by prediction of the conversion with similar error margins as for coatings on glass. In case of glass fiber boards, the spectrum of the pristine GRP board had to be subtracted from the spectrum of the coated sample before the transfer of the PLS model. The resulting prediction error (RMSEP) was found to be 3.6%. The comparison between this transferred and a specific PLSGRP model that was built up for evaluation only proved the high performance of the transferred PLS model. These results clearly demonstrate the high efficiency of the transfer of specific PLS models to different (but similar) sample systems such as coatings applied to other substrates. In addition to the quantitative determination of the conversion, the developed calibration models were also used for the evaluation of its spatial distribution across the surface of UV-cured coatings.