Single Intravenous Bolus Injection Research Articles

Prolonged efficacy of cefazolin in intraosseous regional prophylaxis for total knee arthroplasty: a rabbit model study

BackgroundA novel approach known as intraosseous regional administration (IORA) has emerged as a technique for delivering prophylactic antibiotics, and it results in higher tissue concentrations around the knee. It is hypothesized that IORA of cefazolin for antibiotic prophylaxis during total knee arthroplasty will result in sustained effective levels for a longer duration. The aim of the current study was to investigate temporal changes in peri-knee cefazolin blood concentrations after IORA of cefazolin.MethodsTwelve rabbits were randomly divided into two groups, with six rabbits in each group. In control group a single intravenous bolus injection of cefazolin (10 mL, 100 mg) was administered into the marginal ear vein. In experimental groupexperimental group the same dose of cefazolin was injected into the left tibial marrow cavity after tourniquet inflation at the base of the left thigh. Blood samples were collected periodically at different timepoints, and cefazolin concentrations were determined.ResultsThe intraosseous treatment resulted in significant differences in plasma cefazolin concentrations at all timepoints. Experimental group exhibited higher plasma cefazolin concentrations than control group.ConclusionsCefazolin in intraosseous regional prophylaxis exhibits effectiveness in intraoperative antibiotic prophylaxis by maintaining concentrations above the minimum inhibitory concentration for extended durations, rather than relying solely on high concentrations.

Effect on physiological parameters and anaesthetic dose requirement of isoflurane when tramadol given as a continuous rate infusion vs a single intravenous bolus injection during ovariohysterectomy in dogs.

Tramadol produces a significant reduction in both sevoflurane and isoflurane minimum alveolar concentrations in dogs under experimental conditions. This study aims to compare the effects of tramadol administered as a constant rate infusion (CRI) with those of tramadol administered as a single intravenous bolus on physiological parameters and isoflurane requirements in dogs undergoing ovariohysterectomy. In this study, forty female dogs undergoing ovariohysterectomy were enrolled. The bitches were anesthetized with 5 mg/kg of tiletamine/zolazepam combined with 0.05 mg/kg of acepromazine intravenously. Anesthesia was maintained with isoflurane delivered in 100% oxygen. The group A (n = 20) received tramadol 4 mg/kg in a single intravenous bolus, whereas the group B (n = 20) received tramadol 1.5 mg/kg in an intravenous bolus followed by tramadol 2.6 mg/kg/h as a CRI. The following parameters were recorded: heart rate, respiratory rate, non-invasive blood pressure, body temperature, EtCO2, SpO2 and inspired and expired concentrations of isoflurane. Parameter measurements were performed from pre-preedication (baseline) to skin suturing. The dogs were healthy subjects that demonstrated no abnormalities on laboratory investigations. Significant tachycardia was recorded after administration of tiletamine/zolazepam combined with acepromazine in both groups. Heart rate decreased after intubation but remained significantly higher compared to baseline values in both groups. Systolic blood pressure significantly decreased in both groups but the recorded values were within the physiological range. Mild reduction in body temperature was recorded in both groups. SpO2 and EtCO2 remained within the physiological range. Isoflurane requirement was significantly lower in the group B compared to the group A. Transient twitching was recorded in two dogs belonging to the group A after tramadol administration. Compared to tramadol given as a single intravenous bolus injection during ovariohysterectomy in dogs, tramadol administered as a CRI reduces isoflurane requirements in dogs anesthetized with tiletamine/zolazepam combined with acepromazine. Both tramadol given as a CRI and a single intravenous bolus injection, induce decrease in heart rate, respiratory rate and in body temperature but the values of these parameters remain within physiological range in dogs undergoing ovariohysterectomy.

Tumor-Associated Immune-Cell-Mediated Tumor-Targeting Mechanism with NIR-II Fluorescence Imaging.

The strategy of structure-inherent tumor targeting (SITT) with cyanine-based fluorophores is receiving more attention because no chemical conjugation of targeting moieties is required. However, the targeting mechanism behind SITT has not yet been well explained. Here, it is demonstrated that heptamethine-cyanine-based fluorophores possess not only targetability of tumor microenvironments without the need for additional targeting ligands but also second near-infrared spectral window (NIR-II) imaging capabilities, i.e., minimum scattering and ultralow autofluorescence. The new SITT mechanism suggests that bone-marrow-derived and/or tissue-resident/tumor-associated immune cells can be a principal target for cancer detection due to their abundance in tumoral tissues. Among the tested, SH1 provides ubiquitous tumor targetability and a high tumor-to-background ratio (TBR) ranging from 9.5 to 47 in pancreatic, breast, and lung cancer mouse models upon a single bolus intravenous injection. Furthermore, SH1 can be used to detect small cancerous tissues smaller than 2 mm in diameter in orthotopic lung cancer models. Thus, SH1 could be a promising cancer-targeting agent and have a bright future for intraoperative optical imaging and image-guided cancer surgery.

Microdosing as a Potential Tool to Enhance Clinical Development of Novel Antibiotics: A Tissue and Plasma PK Feasibility Study with Ciprofloxacin.

Background and ObjectiveIn microdose studies, drug pharmacokinetics is measured in humans after administration of subtherapeutic doses. While previous microdose studies focused primarily on plasma pharmacokinetics, we set out to evaluate the feasibility of microdosing for a pharmacokinetic assessment in subcutaneous tissue and epithelial lining fluid.MethodsHealthy subjects received a single intravenous bolus injection of a microdose of [14C]ciprofloxacin (1.1 µg, 7 kBq) with (cohort A, n = 9) or without (cohort B, n = 9) a prior intravenous infusion of a therapeutic dose of unlabeled ciprofloxacin (400 mg). Microdialysis and bronchoalveolar lavage were applied for determination of subcutaneous and intrapulmonary drug concentrations. Microdose [14C]ciprofloxacin was quantified by accelerator mass spectrometry and therapeutic-dose ciprofloxacin by liquid chromatography–tandem mass spectrometry.ResultsThe pharmacokinetics of therapeutic-dose ciprofloxacin (cohort A) in plasma, subcutaneous tissue, and epithelial lining fluid was in accordance with previous data. In plasma and subcutaneous tissue, the dose-adjusted area under the concentration–time curve of microdose ciprofloxacin was similar in cohorts A and B and within an 0.8-fold to 1.1-fold range of the area under the concentration–time curve of therapeutic-dose ciprofloxacin. Penetration of microdose ciprofloxacin into subcutaneous tissue was similar in cohorts A and B and comparable to that of therapeutic-dose ciprofloxacin with subcutaneous tissue-to-plasma area under the concentration–time curve ratios of 0.44, 0.44, and 0.38, respectively. Penetration of microdose ciprofloxacin into epithelial lining fluid was highly variable and failed to predict the epithelial lining fluid penetration of therapeutic-dose ciprofloxacin.ConclusionsOur study confirms the feasibility of microdosing for pharmacokinetic measurements in plasma and subcutaneous tissue. Microdosing combined with microdialysis is a potentially useful tool in clinical antimicrobial drug development, but its applicability for the assessment of pulmonary pharmacokinetics with bronchoalveolar lavage requires further studies.Clinical Trial RegistrationClinicalTrials.gov NCT03177720 (registered 6 June, 2017).Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-01091-1.

Pharmacokinetic Profile of Fentanyl in the Koala (Phascolarctos cinereus) after Intravenous Administration, and Absorption via a Transdermal Patch.

Simple SummaryKoalas can be injured by cars and bushfires, and be affected by painful infectious diseases. When koalas undergo surgery to repair broken bones, they require analgesia. Fentanyl is a potent opioid that can be administered during surgery to provide analgesia. This study describes the rate of elimination of fentanyl in koalas’ blood when administered as a single intravenous injection and consequently calculates the dose rate to administer a constant rate fentanyl infusion into the koalas’ veins to provide short-term pain control. Fentanyl can also be absorbed via the skin into the circulation when applied as a transdermal patch. Although the data for transdermal fentanyl patch absorption is from two koalas only, the results demonstrate that when a patch is applied, pain control is likely to occur 12 h after application to koalas’ skin. Fentanyl may provide effective pain control to koalas either as an intravenous infusion or as a transdermal patch.Fentanyl was administered as a single intravenous bolus injection at 5 µg/kg to five koalas and fentanyl plasma concentrations for a minimum of 2 h were quantified by an enzyme-linked immunosorbent assay (ELISA). The median (range) fentanyl elimination half-life and clearance were 0.53 (0.38–0.91) h, and 10.01 (7.03–11.69) L/kg/h, respectively. Assuming an analgesic therapeutic plasma concentration of 0.23 ng/mL (extrapolated from human studies), an intravenous constant infusion rate was estimated at approximately between 1.7 to 2.7 µg/kg/h (using the clearance 95% confidence intervals). A transdermal fentanyl patch was applied to the antebrachium of an additional two koalas for 72 h. Fentanyl plasma concentrations were determined during the patch application and after patch removal at 80 h. The fentanyl plasma concentration was greater than 0.23 ng/mL after 12 to 16 h. While the patch was applied, the maximum fentanyl concentration was approximately 0.7 ng/mL from 32 to 72 h. Fentanyl plasma concentrations increased to 0.89 ng/mL 1 h after the patch was removed, and then decreased to a mean of 0.47 ng/mL at 80 h. The transdermal fentanyl patch is likely to provide some level of analgesia but should be initially co-administered with another faster acting analgesic for the first 12 h.

Assessment of the safety, pharmacokinetics and pharmacodynamics of GSK3335065, an inhibitor of kynurenine monooxygenase, in a randomised placebo-controlled first-in-human study in healthy volunteers.

GSK3335065 is an inhibitor of kynurenine monooxygenase (KMO) being developed for the treatment of acute pancreatitis. Healthy male volunteers were administered ascending doses of GSK3335065 or matched placebo as a single intravenous bolus injection to assess safety, tolerability, pharmacokinetics and pharmacodynamics. GSK3335065 displayed an apparent volume of distribution between 20.6L and 44.6L, a clearance between 0.462 L/h and 0.805 L/hr and a terminal half-life between 31.3 and 34.5hr. In the single subject who received 1.3mg GSK3335065, changes in tryptophan pathway metabolites were observed consistent with the changes seen in preclinical species suggesting that KMO enzyme activity was partially inhibited. However, a broad complex ventricular tachycardia was observed in this subject, which was judged to be a Serious Adverse Event (SAE) and resulted in early termination of the study. While development of GSK3335065 was subsequently discontinued, significant confounding factors hinder a clear interpretation that the tachycardia was directly related to administration of the compound.

Stereoselective methadone disposition after administration of racemic methadone to anesthetized Shetland ponies assessed by capillary electrophoresis.

The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l‐methadone and d‐methadone and their major metabolites, l‐ and d‐2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated γ‐cyclodextrin as chiral selector and electrokinetic analyte injection from liquid/liquid extracts prepared at alkaline pH. In both trials, the d‐methadone concentrations were lower than those of l‐methadone and the d‐EDDP levels were lower than those of L‐EDDP. For the case of a single intravenous bolus injection, the plasma concentration versus time profile of methadone enantiomers was analyzed with a two‐compartment pharmacokinetic model. l‐methadone showed a slower elimination rate constant, a lower body clearance, and a smaller steady‐state volume of distribution than d‐methadone. d‐methadone and d‐EDDP were eliminated faster than their respective l‐enantiomers. This is the first study that outlines that the disposition of racemic methadone administered to anesthetized equines is enantioselective.

Pharmacokinetic Model Analysis of Supralingual, Oral and Intravenous Deliveries of Mycophenolic Acid.

Mycophenolic acid (MPA) is commonly used for organ rejection prophylaxis via oral administration in the clinic. Recent studies have shown that MPA also has anticancer activities. To explore new therapeutic options for oral precancerous/cancerous lesions, MPA was designed to release topically on the dorsal tongue surface via a mucoadhesive patch. The objective of this study was to establish the pharmacokinetic (PK) and tongue tissue distribution of mucoadhesive MPA patch formulation after supralingual administration in rats and also compare the PK differences between oral, intravenous, and supralingual administration of MPA. Blood samples were collected from Sprague Dawley rats before and after a single intravenous bolus injection, a single oral dose, or a mucoadhesive patch administration on the dorsal tongue surface for 4 h, all with a dose of 0.5 mg/kg of MPA. Plots of MPA plasma concentration versus time were obtained. As multiple peaks were found in all three curves, the enterohepatic recycling (EHR) model in the Phoenix software was adapted to describe their PK parameters with an individual PK analysis method. The mean half-lives of intravenous and oral administrations were 10.5 h and 7.4 h, respectively. The estimated bioavailability after oral and supralingual administration was 72.4% and 7.6%, respectively. There was a 0.5 h lag-time presented after supralingual administration. The results suggest that the systemic plasma MPA concentrations were much lower in rats receiving supralingual administration compared to those receiving doses from the other two routes, and the amount of MPA accumulated in the tongue after patch application showed a sustained drug release pattern. Studies on the dynamic of drug retention in the tongue after supralingual administration showed that ~3.8% of the dose was accumulated inside of tongue right after the patch removal, ~0.11% of the dose remained after 20 h, and ~20.6% of MPA was not released from the patches 4 h after application. The data demonstrate that supralingual application of an MPA patch can deliver a high amount of drug at the site of administration with little systemic circulation exposure, hence lowering the potential gastrointestinal side effects associated with oral administration. Thus, supralingual administration is a potential alternative route for treating oral lesions.

Abstract PS1-21: Molecular fluorescence-guided surgery using Beva800 for the assessment of tumor margins during breast conserving surgery of patients with primary breast cancer (MARGIN-II)

Abstract Introduction:The goal of breast conserving surgery (BCS) for early breast cancer (EBC) is to remove the tumor and a surrounding rim of normal tissue, while preserving as much of the normal breast tissue as possible. Incomplete resections are associated with higher rates of surgical re-excision. Repeat surgeries are not only a burden to patients physically but also psychologically and can delay recommended adjuvant therapies. Accurate determination of tumor margins during surgery is therefore critical for successful outcome. Breast cancer tissue produces significantly higher amounts of VEGF-A than healthy tissue. VEGF-A stimulates tumor angiogenesis and is therefore an excellent target for molecular imaging techniques. The fluorescence imaging agent bevacizumab-IRDye800CW (Beva800) is a conjugate of bevacizumab (a humanized antibody targeting human VEGF) and IRDye800CW (a near-infrared fluorescence dye) which binds specifically to VEGF-A. Beva800 provides a potentially highly efficacious approach to imaging specimen and cavity margins during BCS. Herein we present a phase II study that combined Beva800 with the SurgVision Explorer Air camera for intraoperative margin assessment during BCS for EBC.Methods:MARGIN II is a multicenter open-label single arm prospective clinical trial aimed at evaluating Beva800 for assessment of tumor margins in women with EBC scheduled for BCS. The study was a within-patient comparison of positive tumor margin rates using BCS standard of care compared to intraoperative assessment with 4.5 mg Beva800 and fluorescence imaging with the SurgVision Explorer Air camera. Patients undergoing neoadjuvant chemotherapy were excluded. All patients received a single intra-venous bolus injection of 4.5 mg of Beva800 three days before surgery. The fluorescent signal was visualized during surgery using NIR fluorescence imaging (700-1000 nm). This wavelength window typically has very low tissue auto-fluorescence (filtering out background noise) and greater tissue penetration depth due to reduced haemoglobin absorption. Standard of care assessment was defined as visual inspection, palpation and, in cases of pre-operative wire marking, specimen sonography or mammography. Beva800 efficacy was determined as the number of patients in which a pathology-confirmed positive margin was identified by fluorescence guided surgery using Beva800 but not by standard of care BCS. The results per patient were divided into two clusters: results after standard of care BCS and results after fluorescence guided surgery, according to their margin status at pathology. The need for re-operation because of involved margins within 30 days after the first BCS and the safety of 4.5 mg Beva800 was assessed.Results:The recruitment goal of 40 patients in 5 centers has almost been reached and results of the final analysis will be presented at the meeting.Conclusion:Molecular fluorescence-guided surgery using Beva800 has the potential to change the practice of breast conserving surgery by avoiding unnecessary re-operations. This would lead to fewer interventions, a reduced burden on patients through repeat surgery and reduced delay of adjuvant therapies. Citation Format: Hans-Christian Kolberg, Carmen Röhm, Angrit Stachs, Florian Schütz, Jens-Uwe Blohmer, Sarah Wetzig, Steffi Hartmann, Jörg Heil, Markus Hahn. Molecular fluorescence-guided surgery using Beva800 for the assessment of tumor margins during breast conserving surgery of patients with primary breast cancer (MARGIN-II) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS1-21.

Use of Intravenous Infusion Study Design to Simultaneously Determine Brain Penetration and Systemic Pharmacokinetic Parameters in Rats.

In drug discovery, the extent of brain penetration as measured by free brain/plasma concentration ratio (Kp,uu) is normally determined from one experiment after constant intravenous infusion, and pharmacokinetics (PK) parameters, including clearance (CL), volume of distribution at steady state (Vss), and effective half-life (t 1/2 ,eff) are determined from another experiment after a single intravenous bolus injection. The objective of the present study was to develop and verify a method to simultaneously determine Kp,uu and PK parameters from a single intravenous infusion experiment. In this study, nine compounds (atenolol, loperamide, minoxidil, N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine, sulpiride, and four proprietary compounds) were intravenously infused for 4 hours at 1 mg/kg or 24 hours at 1 or 6 mg/kg or bolus injected at 1 mg/kg. Plasma samples were serially collected, and brain and cerebrospinal fluid samples were collected at the end of infusion. The PK parameters were obtained using noncompartmental analysis (NCA) and compartmental analysis. The Kp,uu,brain values of those compounds increased up to 2.86-fold from 4 to 24 hours. The CL calculated from infusion rate over steady-state concentration from the 24-hour infusion studies was more consistent with the CL from the intravenous bolus studies than that from 4-hour infusion studies (CL avg. fold of difference 1.19-1.44 vs. 2.10). The compartmental analysis using one- and two-compartment models demonstrated better performance than NCA regardless of study design. In addition, volume of distribution at steady state and t 1/2,eff could be accurately obtained by one-compartment analysis within 2-fold difference. In conclusion, both unbound brain-to-plasma ratio and PK parameters can be successfully estimated from a 24-hour intravenous infusion study design. SIGNIFICANCE STATEMENT: We demonstrated that the extent of brain penetration and pharmacokinetic parameters (such as clearance, Vss, and effective t 1/2) can be determined from a single constant intravenous infusion study in rats.

Development of Pharmacodynamic Assays to Assess Ex Vivo Masp-2 Inhibition and Their Use to Characterize the Pharmacodynamics of Narsoplimab (OMS721) in Humans and Monkeys

Introduction Narsoplimab (OMS721) is a fully human monoclonal antibody that binds to and inhibits mannan-binding lectin-associated serine protease 2 (MASP-2). MASP-2 is the key enzyme responsible for activation of the lectin pathway of the complement system. The lectin pathway is activated when plasma is exposed to molecular patterns present on microbes and injured host cell surfaces, initiating a proteolytic cascade that results in the activation of complement components C4, C2, C3 and C5 to yield multiple biologically active products. Inhibiting MASP-2 activity prevents lectin-mediated generation of complement activation products, thereby reducing inflammation and tissue injury. Narsoplimab is the first lectin pathway-specific complement inhibitor in clinical development. In published clinical studies and case reports, improvement has been observed in narsoplimab-treated patients with hematopoietic stem cell transplant-associated thrombotic microangiopathy, immunoglobulin A (IgA) nephropathy and atypical hemolytic uremic syndrome. Here we describe the development of pharmacodynamic (PD) assays to quantify MASP-2 inhibition ex vivo and their use to study duration of action and to establish the pharmacokinetic (PK)/PD relationship of narsoplimab in monkeys and humans. Methods PD assays to measure lectin pathway activity, a measure of MASP-2 activity, in minimally diluted (90%) serum from human and monkey were developed using immobilized mannan as a lectin pathway-specific complement activator with C4b as the activation endpoint. ELISA and flow cytometry assay platforms were employed to assess the PD response of monkeys or normal healthy human volunteers administered narsoplimab, respectively. The PD response was used to characterize the time-course and dose-response of lectin pathway inhibition by narsoplimab. Drug concentration data obtained from matched serum samples were used to estimate the ex vivo EC50 of lectin pathway inhibition in monkeys and humans. Lectin pathway-specific complement activation assays applicable to minimally diluted serum samples were developed by evaluating the time-course of lectin-induced complement activation response under varying reaction temperatures and selecting optimized test conditions within the dynamic range of the assay platform. The test methods were used to characterize monkey and human lectin pathway inhibition profiles of narsoplimab in vitro, and the flow cytometric method was validated for analysis of human serum samples. Results The PD assays were used to characterize the PD response of monkeys administered a single intravenous (IV) bolus injection of narsoplimab at 0.05, 0.15, 0.5, 1.5 or 5 mg/kg, and of normal human healthy volunteers administered a single IV infusion of the drug at 0.25, 0.625 or 2 mg/kg, or 6 weekly IV infusions at 2 or 4 mg/kg. A clear, dose-dependent initial PD response was observed in monkeys in the dose range of 0.5 mg/kg to 5 mg/kg, which subsequently declined over time. In humans, a minimal PD response was observed at 0.025 mg/kg. A near maximal initial PD response was seen at 0.675 mg/kg or higher, followed by a decline over time. In the 6-week repeat-dose study, once-weekly dosing of narsoplimab at 2 mg/kg resulted in a mean trough PD response of ~55% lectin pathway inhibition, which increased to an ~80% mean trough response with once-weekly dosing of 4 mg/kg. PK sample analysis revealed a clear PK/PD relationship in monkeys and humans. Modeling of the PK/PD relationship in monkeys indicated an EC50 value of ~7 µg/mL (~ 46 nM), which is comparable to the IC50 value of lectin pathway inhibition in vitro (33 nM). In humans, modeling of the PK/PD relationship data indicated an EC50 value of ~500 ng/mL (3.3 nM), again very similar to the IC50 value of lectin pathway inhibition in vitro (3.4 nM). Conclusion We successfully developed PD assays to monitor blockade of lectin pathway activation in monkeys and humans administered narsoplimab. Using a validated PD assay, we demonstrate that a high level of lectin pathway blockade can be maintained in normal human healthy volunteers by once-weekly administration of narsoplimab at 4 mg/kg IV. We also demonstrate a consistent PK/PD relationship across the study population, indicating that narsoplimab concentration ranges can be used to target the desired level of lectin pathway inhibition. Figure 1 Disclosures Freeman: Omeros Corporation: Current Employment. Cummings:Omeros Corporation: Current Employment. Chuidian:Omeros Corporation: Current Employment. Dudler:Omeros Corporation: Current Employment.

Pharmacology and Pharmacokinetics of NXT007; Emicizumab-Based Engineered Fixa/Fx Bispecific Antibody with Improved Properties

Background Emicizumab (HEMLIBRA®) is a factor (F) VIII function-mimetic therapeutic bispecific antibody (BsAb) to FIXa and FX able to prevent bleeding in persons with hemophilia A (PwHA) when injected subcutaneously once every 1, 2 or 4 weeks. To develop a next generation version, we sought an agent able to keep hemostatic potential in non-hemophilic range with more convenient dosing regimen (dosing frequency/volume). We successfully created the emicizumab-based engineered four-chain BsAbs, NXT series. Among these, we selected NXT007 as a clinical candidate. Objectives The aim of this study is to clarify the in vitro and in vivo properties of NXT007 and predict its therapeutic potency non-clinically. Methods We evaluated the pharmacological activities of NXT007 in vitro using a thrombin generation assay (TGA) with FVIII-deficient patient plasma, and in vivo by inducing bleeding in FVIII-neutralizing antibody-treated acquired hemophilia A cynomolgus monkey (cyno) model. To clarify the FVIII-cofactor activity of NXT007, we performed an enzymatic kinetics analysis of FIXa-catalyzed FX activation with and without NXT007, as well as surface plasmon resonance analysis to determine the dissociation constant (KD) of NXT007 to FIX, FIXa, FX and FXa. We obtained its pharmacokinetic (PK) profile in non-human primates in a single dose SC/IV study. Results In vitro addition of NXT007 at 30 μg/mL increased the peak height of TGA in FVIII-deficient plasma to the same levels achieved by recombinant human FVIII at 40-100 IU/dL (FXIa-triggering) or 100-150 IU/dL (tissue factor-triggering). A single bolus intravenous injection of NXT007 (0.075 mg/kg) ameliorated bleeding symptoms in the cyno model to similar as a twice daily intravenous injection of recombinant porcine FVIII (20 U/kg). The in vitro and in vivo results were roughly concordant. NXT007 increased the turnover rate (kcat) of FIXa-catalyzed FX activation by approximately 4,000-folds compared to the condition without cofactor. The impact of NXT007 on the kcat was similar to that of emicizumab. As for binding affinities, the KD values of NXT007 to FIX, FIXa, FX and FXa were 1.08, 0.728, 0.0538 and 0.0231 μM, respectively in buffer solution. Compared to emicizumab, NXT007 bound more strongly to FX/FXa and with similar affinity to FIX/FIXa. This means that NXT007 would have an ability to form more FIX-BsAb-FX ternary complex than emicizumab. Calculated using the above KD values, at 30 μg/mL of BsAb the estimated concentration of FIX-NXT007-FX ternary complex in plasma is approximately 10-fold higher than that of the FIX-emicizumab-FX ternary complex which is roughly concordant with the difference in their FVIII equivalent thrombin generation activity. Prothrombin time (PT) was not clearly prolonged suggesting minimal impact on FX function by in vitro addition of NXT007 at up to 30 μg/mL, which was enough to induce sufficient thrombin burst in FVIII-deficient plasma as described above. A half-life of NXT007 was 19.6 to 24.4 days (0.02-2 mg/kg, SC) and SC bioavailability was 84.4% (2 mg/kg) in the in vivo cyno PK study, in which no obvious change in plasma FIX or FX levels was observed after 0.02-2 mg/kg single SC administration. Conclusions Based on the nonclinical results, NXT007, delivered in every-4-week SC injections, will keep a non-hemophilic range of equivalent FVIII thrombin generation in PwHA, Compared with emicizumab, NXT007's improved cofactor activity may be attributed to its more efficient ternary complex formation while keeping turnover rate with minimal impact on FX function suggested by PT value and antigen accumulation. A phase 1/2 clinical study of NXT007 is now on-going (NXTAGE; JapicCTI-194919). Disclosures Yamaguchi: Chugai Pharmaceutical Co., Ltd: Current Employment. Soeda:Chugai Pharmaceutical Co., Ltd.: Current Employment. Sato:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shibahara:Chugai Pharmaceutical Co., Ltd.: Current Employment. Koga:Chugai Pharmaceutical Co., Ltd.: Current Employment. Ichiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Joyashiki:Chugai Pharmaceutical Co., Ltd.: Current Employment. Teranishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Nishimura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Shiraiwa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitamura:Chugai Pharmaceutical Co., Ltd.: Current Employment. Igawa:Chugai Pharmaceutical Co., Ltd.: Current Employment. Konishi:Chugai Pharmaceutical Co., Ltd.: Current Employment. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Current Employment.

Simultaneous multipurpose fluorescence imaging with IRDye® 800BK during laparoscopic surgery.

IRDye® 800BK is a fluorophore, currently undergoing clinical translation, which has both biliary and renal clearance. To date, there is no description of a fluorophore, which can be simultaneously used for non-invasive, near-infrared fluorescence-based (NIRF) visualization of different structures and perfusion evaluation. The purpose of this study was to evaluate IRDye® 800BK for the simultaneous assessment of bowel perfusion, lymphography, ureter and bile duct delineation. Six pigs received a 0.15mg/kg dye as a single bolus intravenous injection (IV). With the FLER (fluorescence-based enhanced reality) software, fluorescence intensity (FI) of 5 regions of interest (ROI) in an ischemic bowel loop was measured along with the time to reach the FI peak, and capillary lactate was measured from the same ROI, followed by the assessment of the ureters and bile ducts for a maximal duration of 180min after dye administration. In 3 animals, the procedure was initiated via gastroscopic injection of a 0.6mg (1mg/mL) dye in the gastric submucosa followed by lymphography in a NIRF setting. Excellent visualization of the ureters and bowel perfusion was obtained under NIRF imaging. Additionally, the bile duct and gastric lymph ducts and nodes were visualized. A positive correlation was found between the time to peak FI in the ischemic bowel loop and the corresponding capillary lactate levels (rho 0.59, p < 0.001). In this study, we successfully demonstrated the simultaneous multipurpose IRDye® 800BK applicability during laparoscopic surgery. This fluorophore has the potential to become a powerful and versatile image-guided surgery tool.

Population analysis to assess the influence of age and body weight on pharmacokinetics and pharmacodynamics of dexmedetomidine in New Zealand White rabbits.

The purpose of this work was i) to develop a population pharmacokinetic (PK) and pharmacodynamic (PD) model of dexmedetomidine (DEX) in New Zealand White rabbits, ii) to investigate the influence of the age and weight of the animals on the model parameters, and iii) to assess the linearity of DEX PKs in the examined dose range. This was a prospective, crossover study, using a total of 18 New Zealand White rabbits. DEX was administered as a single intravenous bolus injection in the doses from 25 to 300 μg kg-1 . Each New Zealand White rabbit was given the same dose of drug in its three developmental stages. To determine the DEX PK, seven blood samples were taken from each animal. The pedal withdrawal reflex was the PD response used to assess the degree of sedation. Nonlinear mixed effects modelling was used for the population PK/PD analysis. The typical value of elimination clearance was 0.061 L min-1 and was 35% higher in younger New Zealand White rabbits compared with older animals. The PK of DEX was linear in the examined concentration range. Age-related changes in sensitivity to DEX were not detected. The results suggest that due to the pharmacokinetics, younger animals will have lower DEX concentrations and a shorter duration of sedation than older animals given the same doses of DEX per kg of body weight.

OR23-05 Human Absorption, Metabolism, Excretion, and Absolute Oral Bioavailability of 14C-CRN00808, an Orally Bioavailable, Nonpeptide, Selective, Somatostatin Receptor 2 (sST2) Biased Agonist for the Treatment of Acromegaly

Injected depot formulations of somatostatin peptide analogs are routinely used to treat acromegaly and neuroendocrine tumors (NETs). CRN00808, a small molecule nonpeptide selective somatostatin receptor 2 (sst2) agonist, is being evaluated for efficacy and safety in patients with acromegaly. The current Phase 1 study was conducted in two Parts: In Part A, the absorption, metabolism, excretion, and mass balance of a single oral dose of 20 mg [14C]-CRN00808 (3.0 MBq) oral solution was characterized in six healthy male subjects. Plasma, blood, urine, and feces were collected for up to 432 hours, and were analyzed for total radioactivity and CRN00808 concentrations (plasma only). Metabolite profiling was conducted on the plasma, urine, and feces samples. In Part B, the absolute bioavailability of CRN00808 was determined by administering a single oral dose of 20 mg CRN00808 compared with a single micro-tracer intravenous (IV) bolus injection of 50 µg [14C]-CRN00808 (0.0185 MBq) in five healthy male subjects. The IV dose was administered approximately 90 minutes after the oral dose. Plasma samples were collected for up to 144 hours and were analyzed for total radioactivity and CRN00808 concentrations (plasma only).Key data from Part A and Part B will be presented. Available data from Part A of the study show that >90% of radioactivity was recovered within 7 days of dosing. The primary route of excretion was the feces (>90%) with minimal excretion in the urine (<10%). Absorption of total [14C]-CRN00808-derived radioactivity in plasma was rapid (median Tmax=1 hour), and the mean Cmax, AUC0-∞, and t1/2 were determined to be 194 ng-equivalents/mL, 3340 ng-equivalents.hr/mL, and 31 hours, respectively. The pharmacokinetic parameters of unchanged CRN00808 in plasma were similar, suggesting that majority of the circulating drug-derived radioactivity is accounted for by unchanged CRN00808 and there are no abundant circulating metabolites. Treatment emergent adverse events associated with CRN00808 were generally mild and transient, and consistent with those reported with other somatostatin agonists. In conclusion, results from this clinical trial in healthy volunteers confirm that CRN00808 has excellent drug-like properties for chronic once-daily oral treatment of patients with acromegaly.

Intraoperative tranexamic acid to decrease blood loss during myomectomy: a randomized, double-blind, placebo-controlled trial

Myomectomy is associated with a significant risk of hemorrhage. Tranexamic acid is a synthetic lysine derivative with antifibrinolytic activity used in other surgical disciplines to reduce blood loss during surgery. However, its utility in gynecologic surgery is not well understood. This study aimed to determine the effect of early administration of intravenous tranexamic acid on perioperative bleeding and blood transfusion requirements in women undergoing myomectomy. This study was a double-blinded, randomized, placebo-controlled trial conducted in an academic teaching hospital. Women with symptomatic fibroids thought to be at risk for large intraoperative blood loss who met the following criteria were included in the study: (1) at least 1 fibroid ≥10 cm, (2) any intramural or broad ligament fibroid ≥6 cm, and/or (3) at least 5 total fibroids based on preoperative imaging. Patients were randomized to receive a single intravenous bolus injection of tranexamic acid 15 mg/kg (intervention group) versus an intravenous bolus injection of saline of equivalent volume (placebo group) 20 minutes before the initial surgical incision. Perioperative bleeding was defined by measuring intraoperative estimated blood loss, change between pre- and postoperative hemoglobin, and frequency of blood transfusions. Estimated blood loss was calculated by combining the blood volume collected within the suction canister and the weight of used sponges. The 2 groups were compared for age; body mass index; perioperative hemoglobin and hematocrit; perioperative blood loss; duration of surgery; blood transfusion requirements; and the number, total weight, and volume of myomas removed. A total of 60 patients (30 per arm) were enrolled into the study between March 1, 2015, and January 29, 2018. Age, body mass index, baseline hemoglobin and/or hematocrit, number and total weight of myomas removed, and size of myomas did not differ between arms. Of 60 patients, 32 (53%) had laparoscopic myomectomy, 24 (40%) had robotic myomectomy, and 4 (7%) had laparotomy. Median estimated blood loss was 200 mL for the tranexamic acid group and 240 mL for the placebo group (P=.88). There was no difference in median duration of surgery (165 vs 164 minutes; P=.64) or change in perioperative hemoglobin (1.00 vs 1.1 g/dL; P=.64). Patients in the tranexamic acid group did not require blood transfusions; however, 4 patients (13.3%) in the placebo group (P=.11) required blood transfusions. Intravenous administration of tranexamic acid in patients undergoing laparoscopic or robotic myomectomies was not associated with decreased blood loss.

Evaluation of the Human Factor IX Gene Therapy Vector TAK-748 in Hemophilia: Results from Non-Clinical Studies in Factor IX Knockout Mice and Rhesus Monkeys

Introduction: Adeno-associated virus (AAV)-based factor IX (FIX) gene therapy has the potential to provide clinical benefit in patients with hemophilia B. TAK-748 is a novel next-generation AAV vector for FIX gene therapy. The vector design includes the insertion of 3 hepatocyte-specific cis-regulatory elements to increase the strength of the liver-specific transthyretin promoter driving expression of a human FIX transgene. Aims: The objectives of this study were to investigate the TAK-748 dose-response relationship for FIX activity, and evaluate its efficacy, in FIX knockout (KO) mice and rhesus monkeys. Methods: Male FIX KO mice (N=12/group) received single intravenous doses of TAK-748 (7.4×1010, 1.5×1011, 7.4×1011, or 1.5×1012 vector genomes [vg]/kg) or buffer. Blood samples were taken on days 7, 14, 28, 42, and 56 for the analysis of plasma FIX activity. At the end of the observation period, the bleeding phenotype was assessed by a tail-tip bleeding assay. The viral transduction efficiency of TAK-748 in liver tissue was analyzed by quantitative real-time polymerase chain reaction. Safety assessments included monitoring for clinical signs, and histopathological analysis of selected organs (liver, spleen, kidney, and heart). Male rhesus monkeys (N=3/group) were administered single TAK-748 intravenous bolus injections (3.8×1011, 9.5×1011, or 1.9×1012 vg/kg). Blood samples were collected before dosing and weekly after dosing up to week 18. Plasma FIX activity, human (hu)FIX antigen, and anti-hu-Padua FIX neutralizing antibodies were analyzed. Results: No clinical signs or deaths were recorded in animals treated with TAK-748, and there were no TAK-748-related histopathological findings in the tissues collected from the mice. FIX activity levels in plasma from FIX KO mice treated with buffer were below the lower limit of quantification. Administration of TAK-748 resulted in a dose-dependent increase in mean plasma FIX activity, and supraphysiologic mean FIX levels up to 41.0 IU/mL (1.5×1012 vg/kg). In the tail-tip bleeding assay, blood loss was significantly reduced in the TAK-748 groups at dose levels above 7.4×1010 vg/kg (P&lt;0.05 vs. buffer control). Administration of TAK-748 to rhesus monkeys resulted in a dose-dependent increase in mean plasma FIX activity and antigen, and peak levels of huFIX expression were detected 2-4 weeks after treatment. Mean huFIX activity was 0.3, 0.6, and 1.9 IU/mL after treatment with 3.8×1011 vg/kg, 9.5×1011 vg/kg, and 1.9×1012 vg/kg TAK-748, respectively. A significant reduction in FIX activity and huFIX protein was observed in most of the animals starting about 4 weeks after dosing with TAK-748. In most animals, anti-huFIX Padua neutralizing antibody titers were detected at about week 6 of the study and correlated with the preceding reductions in huFIX expression. Conclusions: Treatment with TAK-748 resulted in dose-dependent increases in plasma FIX activity and was efficacious in FIX KO mice and rhesus monkeys. There were no treatment-related safety findings. Disclosures Weiller: Baxalta Innovations GmbH, a Takeda company: Employment. Wang:Shire US Inc., a Takeda company: Employment, Equity Ownership. Coulibaly:Baxalta Innovations GmbH, a Takeda company: Employment. Schuster:Baxalta Innovations GmbH, a Takeda company: Employment. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Sun:Shire US Inc., a Takeda company: Employment. Chuah:Shire, a Takeda company: Consultancy, Research Funding; Catalyst Bio: Consultancy, Research Funding; Pfizer: Research Funding. Vandendriessche:Shire, a Takeda company: Consultancy, Honoraria, Research Funding; Catalyst Bio: Consultancy, Research Funding; Pfizer: Research Funding; Biotest: Honoraria. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Höllriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.

Pharmacokinetics of oral and intravenous cannabidiol and its antidepressant-like effects in chronic mild stress mouse model

Cannabidiol (CBD) exhibits significant efficacy in mental and inflammatory diseases. Several studies have recently reported on the rapid antidepressant-like effects of CBD, suggesting that CBD is a potential anti-depressant or anti-stress drug. However, CBD is mainly administered orally or by inhalation with poor bioavailability, resulting in high costs. We aim to explore the efficacy of long-term periodic administration of CBD in chronic mild stress (CMS) via two routes and its pharmacokinetics. We treated ICR mice with CBD administered orally and intravenously and then determined the kinetic constants. A single bolus intravenous injection of CBD resulted in a half-life of 3.9 h, mean residence time of 3.3 h, and oral bioavailability of about 8.6%. The antidepressant-like effects of periodically administered CBD on the chronic mild stress mouse model are evaluated. Results demonstrated that such treatment at a high dose of 100 mg/kg CBD (p.o.) or a low dose of 10 mg/kg CBD (i.v.), elicited significant antidepressant-like behavioral effects in forced swim test, following increased mRNA expression of brain-derived neurotrophic factor (BDNF) and synaptophysin in the prefrontal cortex and the hippocampus. Our findings are expected to provide a reference for the development of intravenous antidepressant formulations of CBD.

Pharmacokinetics and Safety of a Diclofenac Sodium 75mg/1mL Solution (Akis®/Dicloin®) Administered as a Single Intravenous Bolus Injection in Healthy Men and Women.

BackgroundA 1-mL aqueous solution for parenteral injection containing diclofenac sodium and hydroxypropyl-β-cyclodextrin, presently on the market for intramuscular and subcutaneous administration (Akis®/Dicloin®), was further developed for intravenous (i.v.) bolus administration.ObjectivesThe study objective was to compare the tolerability and diclofenac pharmacokinetics after a single i.v. bolus of the investigational solution to those of other parenteral diclofenac products.MethodsThe study comprised three parts: (i) Part 1: an exploratory dose-escalation study to evaluate the tolerability of 25 mg/1 mL, 50 mg/1 mL and 75 mg/1 mL diclofenac sodium formulations administered as a single 5-s i.v. bolus; (ii) Part 2: an exploratory, randomised, crossover study to evaluate the pharmacokinetics of diclofenac following 5-, 15-, and 30-s i.v. bolus injections of diclofenac sodium 75 mg/1 mL; (iii) Part 3: a randomised crossover study to compare the pharmacokinetics of diclofenac following a 5-s i.v. bolus of the 75 mg/1 mL solution to the pharmacokinetics of diclofenac following a 30-min i.v. infusion or intramuscular administration of a 75 mg/3 mL reference formulation.ResultsThe extent of exposure to diclofenac sodium afforded by the 5-s i.v. bolus of 75 mg/1 mL was equivalent to that provided by the 30-min i.v. infusion of 75 mg/3 mL, since the 90% confidence interval of the geometric mean ratio (GMR) of the area under the curve (AUC) from time 0 to the last plasma concentration time t (AUC0-t) was within the limits 80.00–125.00%, as was the 90% confidence interval of the GMR of the AUC from time 0 extrapolated to infinity (AUC0-∞). The maximum observed plasma concentration (Cmax) was approximately 2.7-fold higher and was achieved earlier (0.05 vs. 0.50 h) with the 1 mL than with the 3 mL formulation, and was similar to data published for a 75 mg/2 mL formulation given as a 15-s i.v. bolus.ConclusionsDiclofenac sodium 75 mg/1 mL solution administered as a 5-s i.v. bolus was well tolerated. The pharmacokinetic profile, which showed a faster onset and a higher concentration peak than seen for other products and administration routes, suggests a superior analgesic effect.

Abstract 3029: FAP-mediated tumor accumulation of a T-cell agonistic FAP/4-1BB DARPin drug candidate analyzed by SPECT/CT and quantitative biodistribution

Abstract Immunomodulating agents have revolutionized anti-cancer therapy. However, monotherapy is often not sufficient, and development of combination treatments is hampered by cumulative toxicity. In an attempt to overcome this challenge, a tumor-restricted agonistic 4-1BB/FAP DARPin drug candidate, which induces T-cell co-stimulation only when clustered by binding to fibroblast activation protein alpha (FAP) expressing cells, has been developed. FAP is a type II membrane-bound glycoprotein abundantly expressed in the stroma of many solid tumors by cancer-associated fibroblasts. As shown previously using in vitro and in vivo models (HT-29), co-stimulation induced by a FAP-targeted 4-1BB agonistic DARPin molecule leads to enhanced activation and expansion of CD8+ T-cells. To support clinical development of the drug candidate, tumor localization and accumulation were studied by whole-body SPECT/CT imaging and quantitative biodistribution using Indium-111 labeled DARPin molecules in a human colorectal adenocarcinoma (HT-29) xenograft model in CD1 nude mice. Immunohistochemical staining of tumor stroma confirmed local expression of FAP. Labeled 4-1BB/FAP DARPin molecules specifically accumulated in FAP-expressing tumor in vivo. SPECT/CT imaging and biodistribution revealed a maximum tumor accumulation of around 15% of the injected dose per gram of tissue around 72 h post injection. High tumor/blood ratios were observed one week post injection because the activity in the blood decreased according to the expected serum half-life of 26 h, determined in separate pharmacokinetic studies in BALB/c mice following single dose intravenous bolus injections. Based on the decrease of radioactivity in the tumor, a tumor residence half-life of approximately 4 days was calculated, indicating an extended tumor retention potentially due to FAP binding. No accumulation was observed in the muscle tissue that was choosen as a rather weakly-perfused control tissue. Taken together, FAP-targeting of a 4-1BB agonist DARPin molecule resulted in expected high tumor accumulation and retention compared to an untargeted version of the molecule, both relevant observations for further preclinical and clinical studies. These findings suggest that tumor-targeting via FAP has the potential to induce T-cell activation restricted to the tumor site, and thereby reducing toxicities caused by systemic 4-1BB activation. In conclusion, immunostimulatory drugs with tumor-targeted activity may have the potential to circumvent current limitations of immunotherapy and allow safe and effective use, in particular in combination therapy. Citation Format: Christian Reichen, Ralph Bessey, Christine DePasquale, Stefan Imobersteg, Martin Behe, Alain Blanc, Roger Schibli, Alexander Link, Laurent Juglair, Joanna Taylor, Patricia Schildknecht, Julia Hepp, Elmar vom Baur, Hong Ji, Christof Zitt, Victor Levitsky, Keith M. Dawson, Michael T. Stumpp, Dan Snell. FAP-mediated tumor accumulation of a T-cell agonistic FAP/4-1BB DARPin drug candidate analyzed by SPECT/CT and quantitative biodistribution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3029.