Pharmacodynamic Measures Research Articles

Pain management in chronic kidney disease: The pharmacokinetics and pharmacodynamics of hydromorphone and hydromorphone-3-glucuronide in hemodialysis patients

To describe the pharmacokinetics of hydromorphone (HM) and its primary metabolite hydromorphone-3-glucuronide (H3G) both on and off dialysis in relation to the pharmacodynamic measurements of pain. Prospective, open-label, observational study. Canadian, university-based renal program. Twelve anuric hemodialysis patients with chronic pain, established on immediate-release HM. HM and H3G plasma concentrations were measured during and between hemodialysis treatments using a reverse-phase high-performance liquid chromatography assay with liquid chromatography/mass spectrometer/mass spectrometer detection. The McGill Pain Questionnaire (MPQ) and a Visual Analogue Scale (VAS) were used to measure pain. Noncompartmental analyses were conducted. Adverse effects were recorded. HM did not substantially accumulate (accumulation factor R = 2.7 (1.6)), most likely due to the rapid conversion to H3G. Conversely, H3G accumulated between dialysis treatments (R = 12.5 (12.1)) but appeared to be effectively removed during hemodialysis (1.8 (0.7), p = 0.03). HM resulted in > 65 percent reduction in pain over dosing intervals. Mean MPQ pain scores decreased from 39.8 (18.2) to 12.3 (16.2) on dialysis and from 35.0 (18.5) to 15.5 (13.6) between dialysis treatments. Mean VAS pain scores decreased from 7.5 (2.5) to 3.0 (1.5) on dialysis and from 5.9 (3.2) to 4.4 (1.6) between dialysis treatments. No clinically significant opioid toxicity was observed. The accumulation of H3G between hemodialysis treatments was associated with greater sensory-type pain (r = 0.76, p < 0.0001) and reduced duration of analgesia. HM may be a safe and effective opioid for use in selected hemodialysis patients.

Efficacy and Safety Profiles of Oral Atorvastatin-Loaded Nanoparticles: Effect of Size Modulation on Biodistribution.

Atorvastatin calcium (AC)-loaded nanoparticles (NPs) of mean particle diameter <100 nm and narrow distribution were prepared and characterized. Their in vivo PK as well as PD measures following oral administration in different dosage regimens in hyperlipidemic rats were evaluated. The results revealed that the oral bioavailability of two selected AC-NPs formulations was 235% and 169% relative to Lipitor. However, the treatment regimens were not superior in reducing serum total cholesterol (TC), low-density lipoproteins (LDL), and triglycerides (TG) levels compared to Lipitor. Moreover, the AC-NPs treatments were associated with significant adverse effects observed biochemically and histologically. These results were contradictory with those obtained from a previous study in which similarly formulated AC-NPs of mean particle diameter >200 nm were found to be more safe and effective in reducing TC, LDL, and TG levels when administered to hyperlipiemic rats at reduced dosing frequency compared to daily dose of Lipitor despite their lower oral bioavailability. The discrepant correlation between pharmacokinetics (PK) and pharmacodynamics (PD) results was suggested to pertain to the different biodistribution profiles of AC-NPs depending on their sizes. Hereby, we provide a simple approach of particle size modulation to enhance the efficacy and safety of atorvastatin.

TP-0184 Inhibits ALK2/ACVR1, Decreases Hepcidin Levels, and Demonstrates Activity in Preclinical Mouse Models of Functional Iron Deficiency

BackgroundHepcidin is a liver peptide hormone that functions as a master regulator of bioavailable iron. Hepcidin levels become constitutively high in chronic inflammatory conditions where functional iron deficiency (FID) becomes a challenge. Approaches to target hepcidin expression or function have emerged as attractive strategies to reverse the complications of functional iron deficiency. Targeting the activation of the ALK2/ACVR1 receptor has been validated as a promising approach to target hepcidin. Activation of ALK2/ACVR1 in the liver induces the SMAD-driven transcription of hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Current therapeutic approaches for anemia of chronic disease (ACD) rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which adequately address the underlying pathological deficit of functionally low iron levels. TP-0184 has been developed as a potent and selective inhibitor of ALK2/ACVR1, and it was hypothesized that targeting of ALK2/ACVR1 with TP-0184 would reverse the effects of FID in preclinical models of anemia. TP-0184 is a small-molecule inhibitor of ALK2 kinase activity, exhibiting an IC50 of 5 nM in biochemical assays.AimsThe current study seeks to establish proof-of-concept that TP-0184 targets ALK2/ACVR1 and reverses the effects of FID. Utilizing the unique experience in a single patient IND, the clinical effects of TP-0184 on serum hepcidin levels was evaluated.MethodsMultiple preclinical models of anemia were used to evaluate the efficacy of TP-0184. These models included: a short-term chemical induced anemia by turpentine oil (TO), a model of anemia of cancer using the TC-1 syngenic lung cancer cell line, and an infectious disease model using heat-inactivated Brucella abortus (HKBA).ResultsTP-0184 has demonstrated consistent preclinical activity in three mouse efficacy models of FID. In the first model, TP-0184 reversed hepcidin induction more than 6.8-fold in mice induced with TO. The simplicity of the TO model has allowed the exploration of numerous dose levels and treatment schedules of TP-0184 and the ability to correlate these with pharmacokinetic plasma levels of the compound. In the second model, TP-0184 reversed elevated hepcidin levels and increased iron levels induced in TC-1 tumor bearing mice. Hepcidin was reduced nearly 3-fold with doses as low as 25 mg/kg. In the third model, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection of HKBA. In addition to these preclinical models, a single cancer patient received oral administration of TP-0184 in an emergency setting. Although the primary objectives of the single patient study did not include anemia, pharmacokinetic and pharmacodynamic measurements related to anemia were measured in the patient. The patient was administered 2 cycles of TP-0184, where each cycle evaluated a different formulation. Before the first cycle, the patient had a baseline serum hepcidin level of 559 pg/mL. These levels dropped to below the detectable limit within 24 hours of the first dose with TP-0184. Hepcidin levels remained undetectable for the duration of TP-0184 treatment. After a break in treatment, the patient was administered a second cycle of TP-0184 in an alternative formulation. During this cycle, the patient's hepcidin levels decreased from 182 pg/mL to 69 pg/mL after 24 hours of the first dose (cycle 2). With daily dosing, the patient's serum hepcidin levels continued to decrease to undetectable levels by at least 9 days. Importantly, serum iron levels mirrored serum hepcidin levels well. Plasma pharmacokinetics were also obtained in this patient.ConclusionTargeting ALK2/ACVR1 is an attractive strategy to downregulate hepcidin levels and reverse the effects of FID. TP-0184, a potent ALK2/ACVR1 inhibitor, has demonstrated consistent activity in multiple preclinical models and now has preliminary proof-of-concept evidence that it can modulate hepcidin levels in man. The development of TP-0184 is currently progressing through preclinical development and a Phase I first-in-human study focusing on FID is currently being planned. DisclosuresPeterson:Tolero Pharmaceuticals, Inc.: Employment. K:Tolero Pharmaceuticals, Inc.: Employment. Whatcott:Tolero Pharmaceuticals, Inc.: Employment. Siddiqui-Jain:Tolero Pharmaceuticals, Inc.: Employment. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Bearss:Tolero Pharmaceuticals, Inc.: Employment. Warner:Tolero Pharmaceuticals, Inc.: Employment.

A double-blind, placebo-controlled, single ascending-dose study of remyelinating antibody rHIgM22 in people with multiple sclerosis.

ObjectiveThe objective of this paper is to assess, in individuals with clinically stable multiple sclerosis (MS), the safety, tolerability, pharmacokinetics (PK) and exploratory pharmacodynamics of the monoclonal recombinant human antibody IgM22 (rHIgM22).MethodsSeventy-two adults with stable MS were enrolled in a double-blind, randomized, placebo-controlled, single ascending-dose, Phase 1 trial examining rHIgM22 from 0.025 to 2.0 mg/kg. Assessments included MRI, MR spectroscopy, plasma PK, and changes in clinical status, laboratory values and adverse events for three months. The final cohort had additional clinical, ophthalmologic, CSF collection and exploratory biomarker evaluations. Participants were monitored for six months.ResultsrHIgM22 was well tolerated with no clinically significant safety signals. Noncompartmental PK modeling demonstrated linear dose-proportionality both of Cmax and AUC0–Last. The steady-state apparent volume of distribution of approximately 58 ml/kg suggested primarily vascular compartmentalization. CSF:plasma rHIgM22 concentration increased from 0.003% on Day 2 for both 1.0 and 2.0 mg/kg to 0.056% and 0.586% for 1.0 and 2.0 mg/kg, respectively, on Day 29. No statistically significant treatment-related changes were observed in exploratory pharmacodynamic outcome measures included for the 21 participants of the extension cohort.ConclusionsSingle doses of rHIgM22 were well tolerated and exhibited linear PK, and antibody was detected in the CSF.

Reversal of apixaban anticoagulation by four‐factor prothrombin complex concentrates in healthy subjects: a randomized three‐period crossover study

Background Currently, there is no approved reversal agent for direct activated factor Xa (FXa) inhibitors; however, several agents are under investigation, including prothrombin complex concentrates (PCCs). Objective This open-label, randomized, placebo-controlled, three-period crossover study assessed the effect of two four-factor PCCs on apixaban pharmacodynamics and pharmacokinetics in 15 healthy subjects. Methods Subjects received apixaban 10 mg twice daily for 3 days. On day 4, 3 h after apixaban, subjects received a 30-min infusion of 50 IU kg-1 Cofact, Beriplex P/N (Beriplex), or saline. Change in endogenous thrombin potential (ETP), measured with a thrombin generation assay (TGA), was the primary endpoint. Secondary endpoints included changes in other TGA parameters, prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time, anti-FXa activity, apixaban pharmacokinetics, and safety. Results Apixaban-related changes in ETP and several other pharmacodynamic measures occurred following apixaban administration. Both PCCs reversed apixaban's effect on ETP; the differences in adjusted mean change from pre-PCC baseline to end of infusion were 425 nm min (95% confidence interval [CI] 219.8-630.7 nm min; P < 0.001) for Cofact, and 91 nm min (95% CI - 31.3 to 212.4 nm min; P > 0.05) for Beriplex. Both PCCs returned ETP to pre-apixaban baseline levels 4 h after PCC infusion, versus 45 h for placebo. For both PCCs, mean ETP peaked 21 h after PCC initiation, and then slowly decreased over the following 48 h. Both PCCs reversed apixaban's effect on TGA peak height, PT, and INR. Apixaban pharmacokinetic and anti-FXa profiles were consistent across treatments. Conclusions Cofact and Beriplex reversed apixaban's steady-state effects on several coagulation assessments.

Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides

BackgroundEpidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins.MethodsWe evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins.ResultsThe treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non–high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events.ConclusionsOligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850.)

Abstract CT120: Assessment of myeloblast CD33 receptor occupancy (RO) by vadastuximab talirine in patients with acute myeloid leukemia (AML) receiving monotherapy treatment

Abstract Background CD33 is expressed on myeloblasts in the majority of patients with AML. Vadastuximab talirine (33A) is a potent, highly stable antibody-drug conjugate composed of a humanized CD33-directed antibody conjugated at engineered cysteine residues to 2 molecules of pyrrolobenzodiazepine (PBD) via protease cleavable linkers. Upon binding to CD33, 33A is internalized and PBD is released leading to DNA cross-linking and cell death. RO on targeted cells following treatment administration is a pharmacodynamic measure of myeloblast exposure to 33A. Methods A phase 1 study (NCT01902329) assessed the safety and antileukemic activity of 33A as monotherapy. Best response was evaluated as CRc (CR + CR with incomplete count recovery), morphologic leukemia-free state, or resistant disease. Peripheral blood and bone marrow (BM) aspirates were collected for pharmacokinetic (PK) and biomarker analysis, including assessment of minimal residual disease negative (MRDneg) remission by 10-color flow cytometry. Calculated 33A PK parameters included the observed maximum concentration (Cmax) and the area under the concentration-time curve to the last quantifiable value (AUClast). Myeloblast CD33 levels were measured by multi-color flow cytometry using 2fluorophore conjugated anti-CD33 reagents (1 competitive with 33A). To assess RO, the relative fluorescence values for each reagent were compared in samples drawn before and 2 hours after IV 33A administration. RO was obtained from 71 evaluable patients; 57 (80%) had relapsed/refractory disease (median age 72); 14 patients were treatment naïve (median age 76). Results Cmax and AUClast increased with increasing dose. Dose-normalized (DN) AUClast declined with increasing baseline BM cellularity and myeloblast percentage. Greater RO was associated with greater Cmax and AUClast. In a univariate analysis, variables associated with odds of CRc included treatment naivety, DN Cmax, and AUClast. Variables associated with odds of MRDneg included RO, DN Cmax, Cmax and AUClast. RO ranged from &amp;lt;10% to &amp;gt;80%; across RO tertiles (32%, 55%), the odds of MRDneg remission increased from 4% (1/23) to 25% (6/24) to 50% (12/24). After accounting for dose, baseline BM myeloblasts, treatment naivety, and NPM1 mutation, the odds of CRc associated with DN Cmax and cytogenetic risk by MRC, while the odds of MRDneg associated with DN Cmax, Cmax, RO, and DN AUClast. Conclusions Dose normalized serum exposure to 33A declined with increased disease burden, as assessed by BM cellularity and myeloblasts. Exposure-related RO on peripheral blood myeloblasts confirmed CD33 targeting by 33A in patients. Together, these results support target-mediated disposition of 33A. An association of the odds of achieving MRDneg with RO, Cmax, and AUClast was observed with 33A monotherapy treatment. High RO was not required for patients to achieve CRc or MRDneg remission. Further efforts are underway to better understand the pharmacologic and molecular drivers of the deep, MRDneg remissions mediated by 33A. Citation Format: Charles Biddle-Snead, Jenna L. Voellinger, Phoenix Ho. Assessment of myeloblast CD33 receptor occupancy (RO) by vadastuximab talirine in patients with acute myeloid leukemia (AML) receiving monotherapy treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT120. doi:10.1158/1538-7445.AM2017-CT120

A phase I trial of ALKS 4230, an engineered cytokine activator of NK and effector T cells, in patients with advanced solid tumors.

TPS3111 Background: ALKS 4230 is an engineered fusion protein comprised of a circularly permuted interleukin-2 (IL-2) and IL-2 Receptor (IL-2R) α designed to selectively activate the intermediate-affinity (ia) IL-2R, comprised of IL-2Rβ and γc. The iaIL-2R is expressed predominantly on effector lymphocytes, which play an important role in driving antitumor immune responses. In contrast, unmodified IL-2 activates high-affinity (ha) IL-2R, driving the expansion of haIL-2R-expressing cell types including immunosuppressive CD4+ regulatory T (Treg) cells at concentrations below those at which iaIL-2R bearing effector cells are activated. Also, the haIL-2R is expressed on endothelial cells and may contribute to IL-2 mediated toxicity via capillary leak syndrome. Thus, selective activation of the iaIL-2R by ALKS 4230 has the potential to provide enhanced tumor killing as well as improved tolerability. Methods: ALKS 4230 is being studied in a phase 1 first-in-human trial in patients with advanced solid tumors. Key study objectives are to determine a recommended phase 2 dose and characterize the safety profile, pharmacokinetics (PK), pharmacodynamics (PD) and evidence of antitumor activity. A dose-escalation phase in patients with refractory solid tumors (Part A) will be followed by expansion cohorts in defined populations (Part B). ALKS 4230 is administered as a 30-minute intravenous infusion once daily for five days each cycle. Eligibility requires age 18, ECOG PS 0-1 and adequate bone marrow, liver and kidney function. The dose will be escalated until reaching MTD or an Optimal Biologic Dose. The first two dose cohorts will use a 3+3 design. Subsequent cohorts in Part A will enroll a minimum of 6 subjects. In Part B up to 21 patients will be enrolled into each of four tumor-specific cohorts. Peripheral blood samples will be collected for PK, immunogenicity and PD assessments. The primary PD endpoint is change-from-baseline in CD8+ T, NK and Treg cell counts. Other PD measures include serum concentrations of multiple proinflammatory cytokines and immunohistochemical assessment of markers of immune activation in tumor tissue from selected patients. Recruitment for Part A is ongoing. Clinical trial information: NCT02799095.

Platelet reactivity during mild therapeutic hypothermia in patients with acute myocardial infarction treated with ticagrelor: study protocol of a single-centre study

In summary, the available data on the antiplatelet efficacy of P2Y12 receptor inhibitors suggest their less potent and/or delayed effect in patients undergoing mild therapeutic hypothermia (MTH). However, previous studies do not explain the mechanisms of the impact of MTH on platelet function. Moreover, there is a lack of evidence of any relationship between the increased prevalence of thrombotic complications in MTH patients and the anti-platelet effect of P2Y12 receptor inhibitors. We hypothesise that MTH may interfere with the absorption of ticagrelor and consequently results in significant changes in the pharmacokinetic and pharmacodynamic profile of this P2Y12 receptor inhibitor. It cannot be ruled out that the high initial level of platelet activation in MTH patients may additionally modify the pharmacodynamics of ticagrelor. Malabsorption may slow down the desired antiplatelet effect, while high levels of platelet activation may reduce the antiplatelet effect of ticagrelor. The aim of this study is to verify whether the use of MTH after resuscitation in out-of-hospital cardiac arrest (OHCA) patients with STEMI (ST-segment elevation myocardial infarction) treated with primary percutaneous coronary intervention (pPCI), affects the anti-platelet effect of ticagrelor. Moreover, we attempt to elucidate the mechanisms of impaired effect of ticagrelor in MTH patients based on pharmacodynamic and pharmacokinetic measurements. To achieve the aim of the study, we planned the following: 1) comparison of the pharmacokinetic and pharmacodynamic results obtained from the study population (MTH + pPCI + ticagrelor) with results obtained from a demographically and clinically comparable population of patients with STEMI treated with primary PCI and receiving ticagrelor (pPCI + ticagrelor); and 2) analysis of the pharmacodynamic results in relation to the pharmacokinetic measurements in the target population. Understanding the mechanisms standing behind the impact of MTH on the efficacy of platelet inhibition with P2Y12 inhibitors is pivotal in reducing the risk of thrombotic complications. The study is expected to provide information leading to improvement of the safety of MTH in STEMI patients with OHCA treated with pPCI and receiving ticagrelor.

Evaluation of Brain Pharmacokinetic and Neuropharmacodynamic Attributes of an Antiepileptic Drug, Lacosamide, in Hepatic and Renal Impairment: Preclinical Evidence.

The knowledge of pharmacokinetic and pharmacodynamic properties of antiepileptic drugs is helpful in optimizing drug therapy for epilepsy. This study was designed to evaluate the pharmacokinetic and pharmacodynamic properties of lacosamide in experimentally induced hepatic and renal impairment in seizure animals. Hepatic or renal impairment was induced by injection of carbon tetrachloride or diclofenac sodium, respectively. After induction, the animals were administered a single dose of lacosamide. At different time points, maximal electroshock (MES) seizure recordings were made followed by isolation of plasma and brain samples for drug quantification and pharmacodynamic measurements. Our results showed a significant increase in the area under the curve of lacosamide in hepatic and renal impairment groups. Reduced clearance of lacosamide was observed in animals with renal impairment. Along with pharmacokinetic alterations, the changes in pharmacodynamic effects of lacosamide were also observed in all the groups. Lacosamide showed a significant protection against MES-induced seizures, oxidative stress, and neuroinflammatory cytokines. These findings revealed that experimentally induced hepatic or renal impairment could alter the pharmacokinetic as well as pharmacodynamic properties of lacosamide. Hence, these conditions may affect the safety and efficacy of lacosamide.

(238) The relationship between the pharmacokinetics of morphine abuse-deterrent, extended-release, injection-molded tablets and pharmacodynamic outcomes in oral and intranasal human abuse potential studies

It is generally accepted that rapid drug absorption and high plasma concentrations contribute to opioid abuse potential; however, few studies have evaluated this relationship. The US FDA has stated that the correlation between pharmacokinetics (PK) results and pharmacodynamics (PD) outcomes in abuse-deterrent (AD) opioid studies needs additional research. Herein, we examined the PK/PD relationships of morphine AD, extended-release (ER), injection-molded tablets (morphine-ADER-IMT; Egalet Corporation, Wayne, PA) and other treatments in two single-center, double-blind crossover human abuse potential studies in volunteers who were nondependent, recreational opioid users. In the oral study, subjects received 60 mg of intact morphine-ADER-IMT, manipulated morphine-ADER-IMT, crushed morphine ER, and placebo. In the intranasal study, subjects received 60 mg of manipulated intranasal high-volume morphine-ADER-IMT, manipulated intranasal low-volume morphine-ADER-IMT, crushed intranasal low-volume morphine ER, oral intact morphine-ADER-IMT, and placebo. The relationships between the PK parameters of peak plasma concentration (Cmax), time to Cmax (tmax), and abuse quotient (AQ; Cmax/tmax) and the PD abuse potential outcomes of at-the-moment Drug Liking peak effect (Emax), and Overall Drug Liking and Take Drug Again at 12 hours Visual Analog Scale scores were evaluated. The 10-unit slope, the predicted change in a PD outcome for a 10-unit change in the PK parameter, was determined for each PK/PD association. All PK parameters were significantly associated with all PD measures (P≤0.02) except between tmax and Take Drug Again in the intranasal study (P=0.12). Abuse potential measures were positively associated with Cmax and AQ and negatively associated with tmax. PK parameters were most effective in predicting Drug Liking Emax, followed by Overall Drug Liking and Take Drug Again. Cmax was numerically a slightly better predictor than AQ. The model may have potential predictive value for other AD products in development and advance the understanding of this important relationship for AD opioid development. Funded by Egalet Corporation.

Low-cost, rapidly-developed, 3D printed in vitro corpus callosum model for mucopolysaccharidosis type I.

The rising prevalence of high throughput screening and the general inability of (1) two dimensional (2D) cell culture and (2) in vitro release studies to predict in vivo neurobiological and pharmacokinetic responses in humans has led to greater interest in more realistic three dimensional (3D) benchtop platforms. Advantages of 3D human cell culture over its 2D analogue, or even animal models, include taking the effects of microgeometry and long-range topological features into consideration. In the era of personalized medicine, it has become increasingly valuable to screen candidate molecules and synergistic therapeutics at a patient-specific level, in particular for diseases that manifest in highly variable ways. The lack of established standards and the relatively arbitrary choice of probing conditions has limited in vitro drug release to a largely qualitative assessment as opposed to a predictive, quantitative measure of pharmacokinetics and pharmacodynamics in tissue. Here we report the methods used in the rapid, low-cost development of a 3D model of a mucopolysaccharidosis type I patient's corpus callosum, which may be used for cell culture and drug release. The CAD model is developed from in vivo brain MRI tracing of the corpus callosum using open-source software, printed with poly (lactic-acid) on a Makerbot Replicator 5X, UV-sterilized, and coated with poly (lysine) for cellular adhesion. Adaptations of material and 3D printer for expanded applications are also discussed.

Low-cost, rapidly-developed, 3D printed in vitro corpus callosum model for mucopolysaccharidosis type I

The rising prevalence of high throughput screening and the general inability of (1) two dimensional (2D) cell culture and (2) in vitro release studies to predict in vivo neurobiological and pharmacokinetic responses in humans has led to greater interest in more realistic three dimensional (3D) benchtop platforms. Advantages of 3D human cell culture over its 2D analogue, or even animal models, include taking the effects of microgeometry and long-range topological features into consideration. In the era of personalized medicine, it has become increasingly valuable to screen candidate molecules and synergistic therapeutics at a patient-specific level, in particular for diseases that manifest in highly variable ways. The lack of established standards and the relatively arbitrary choice of probing conditions has limited in vitro drug release to a largely qualitative assessment as opposed to a predictive, quantitative measure of pharmacokinetics and pharmacodynamics in tissue. Here we report the methods used in the rapid, low-cost development of a 3D model of a mucopolysaccharidosis type I patient’s corpus callosum, which may be used for cell culture and drug release. The CAD model is developed from in vivo brain MRI tracing of the corpus callosum using open-source software, printed with poly (lactic-acid) on a Makerbot Replicator 5X, UV-sterilized, and coated with poly (lysine) for cellular adhesion. Adaptations of material and 3D printer for expanded applications are also discussed.

A Highly Durable RNAi Therapeutic Inhibitor of PCSK9

BackgroundInclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin–kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol.MethodsIn this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated.ResultsThe most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headache, back pain, and diarrhea. All the adverse events were mild or moderate in severity. There were no serious adverse events or discontinuations due to adverse events. There was one grade 3 elevation in the γ-glutamyltransferase level, which was considered by the investigator to be related to statin therapy. In the single-dose phase, inclisiran doses of 300 mg or more reduced the PCSK9 level (up to a least-squares mean reduction of 74.5% from baseline to day 84), and doses of 100 mg or more reduced the LDL cholesterol level (up to a least-squares mean reduction of 50.6% from baseline). Reductions in the levels of PCSK9 and LDL cholesterol were maintained at day 180 for doses of 300 mg or more. All multiple-dose regimens reduced the levels of PCSK9 (up to a least-squares mean reduction of 83.8% from baseline to day 84) and LDL cholesterol (up to a least-squares mean reduction of 59.7% from baseline to day 84).ConclusionsIn this phase 1 trial, no serious adverse events were observed with inclisiran. Doses of 300 mg or more (in single or multiple doses) significantly reduced levels of PCSK9 and LDL cholesterol for at least 6 months. (Funded by Alnylam Pharmaceuticals and the Medicines Company; ClinicalTrials.gov number, NCT02314442.)

Knemometry is more sensitive to systemic effects of inhaled corticosteroids in children with asthma than 24-hour urine cortisol excretion

Pharmacodynamic assessment of the systemic effect of inhaled corticosteroids (ICSs) is often done by measuring 24-hour urine free cortisol (UFC) excretion. Knemometry assessing short-term lower-leg growth rate (LLGR) is a more rarely used alternative. The primary aim of this study was to compare the sensitivity of LLGR and 24-hour UFC excretion for evaluating systemic exposure to ICSs in prepubertal children with asthma. The secondary aim was to evaluate factors influencing the precision of LLGR calculated by the traditional 1 leg nonparametric method versus a new 2 leg parametric method. The study evaluated 60 children with mild asthma aged 5 to 12years participating in a randomized controlled trial of ICSs with longitudinal concomitant assessments of LLGR and 24-hour UFC excretion. The sensitivity of the safety assessments was analyzed by comparing LLGR and 24-hour UFC in the placebo run-in period with values in the ICS treatment period by using paired t tests. Factors with a potential influence on LLGR were analyzed by means of ANOVA and the Levene test of homogeneity. The mean LLGR was significantly reduced during the ICS versus placebo run-in periods: 0.18mm/wk (SD, 0.55mm/wk) versus 0.45mm/wk (SD, 0.39mm/wk), with a mean difference of 0.27mm/wk (95% CI, 0.05-0.48mm/wk; P=.02). In contrast, there was no difference in 24-hour UFC excretion: 6.91nmol/mmol (SD, 4.67nmol/mmol) versus 7.58nmol/mmol (SD, 6.17nmol/mmol), with a mean difference of 0.67nmol/mmol (95% CI, -1.13 to 2.48nmol/mmol; P=.46). We observed no significant difference in parametric determined LLGR caused by the child's age or sex, investigator, or season of measurement, whereas some differences were observed for the nonparametric LLGR. These findings suggest that knemometry is a more sensitive pharmacodynamic measure of systemic effects of ICSs than 24-hour UFC excretion and that a parametric determination of LLGR increases the sensitivity of the method. These findings should be considered by legislative authorities in the future.

Low-cost, rapidly-developed, 3D printed in vitro corpus callosum model for mucopolysaccharidosis type I

The rising prevalence of high throughput screening and the general inability of (1) two dimensional (2D) cell culture and (2)in vitrorelease studies to predictin vivoneurobiological and pharmacokinetic responses in humans has led to greater interest in more realistic three dimensional (3D) benchtop platforms. Advantages of 3D human cell culture over its 2D analogue, or even animal models, include taking the effects of microgeometry and long-range topological features into consideration. In the era of personalized medicine, it has become increasingly valuable to screen candidate molecules and synergistic therapeutics at a patient-specific level, in particular for diseases that manifest in highly variable ways. The lack of established standards and the relatively arbitrary choice of probing conditions has limitedin vitrodrug release to a largely qualitative assessment as opposed to a predictive, quantitative measure of pharmacokinetics and pharmacodynamics in tissue. Here we report the methods used in the rapid, low-cost development of a 3D model of a mucopolysaccharidosis type I patient’s corpus callosum, which may be used for cell culture and drug release. The CAD model is developed fromin vivobrain MRI tracing of the corpus callosum using open-source software, printed with poly (lactic-acid) on a Makerbot Replicator 5X, UV-sterilized, and coated with poly (lysine) for cellular adhesion. Adaptations of material and 3D printer for expanded applications are also discussed.

The ‘Photosensitivity Model’ is (also) a model for focal (partial) seizures

The ‘Photosensitivity Model’ uses a standardized stimulation protocol of repeated intermittent photic stimulation (IPS) over a three-day period, with administration of a single dose of an investigational antiepileptic drug (AED) after a baseline IPS day in photosensitive patients, followed by a third IPS day to determine duration of effect. This ‘Photosensitivity Model’ has shown its value in the development of new AEDs. Levetiracetam (LEV), currently a first-line AED in new-onset focal epilepsies, was not effective in classical animal models, but showed dose-dependent efficacy in the human ‘Photosensitivity Model’. Nevertheless, concerns have been expressed that AEDs selectively suppressing focal seizures might not suppress generalized photoparoxysmal EEG responses (PPR), the pharmacodynamic outcome measure in the Model. Herein, the following questions have been addressed: I. Can patients with generalized epileptiform discharges, evoked by IPS, so-called PPR, have focal epilepsy (focal seizures)? II. Are the photosensitive patients with focal epilepsy, who have participated in the photosensitivity trials, non-responsive to a new AED under investigation, as compared to those with generalized epilepsies? III. Are “focal epilepsy” AEDs effective both in the ‘Photosensitivity Model’ and in real life in photosensitive patients? We performed a systematic literature review of PPR in focal seizures and focal epilepsy and we analyzed data (published and unpublished) from 20 different potential AEDs studied prospectively in the ‘Photosensitivity Model’. Finally, the PPR effects of Na+ channel-blocking AEDs (considered as the most typical AEDs for focal epilepsy) are discussed with unequivocal examples given of the focal nature of a patient’s PPR. Based on the entire data evidence, we conclude that: 1. PPRs certainly exist in focal epilepsy (17% on average); 2. Clinical signs and symptoms of PPRs can be focal and 3. PPRs can definitely be used to identify or to prove efficacy of new AEDs for patients with focal epilepsy.

Thioguanine Combined with Decitabine Can Overcome Resistance to Hypomethylating Agents: Final Results of a Phase I Trial of a Pharmacodynamically-Conceived Thioguanine/Decitabine Combination in Patients with Advanced Myeloid Malignancies

Background: Outcomes are poor for older patients with acute myeloid leukemia (AML), high-risk myelodysplastic syndromes (MDS), or relapsed/refractory disease, and new therapies are needed. Using a chemosensitivity screening assay, we previously demonstrated that combination treatment with thioguanine and decitabine can restore therapeutic efficacy in primary leukemia cells isolated from patients with relapsed/refractory AML. To test the safety and synergistic efficacy of this combination in patients with advanced myeloid malignancies, we performed a Phase I dose-escalation trial of thioguanine given with decitabine.Patients and Methods: Patients with untreated AML ≥60 years of age and ineligible for standard induction, relapsed/refractory AML, and high-risk or relapsed MDS were eligible. Two thioguanine dose levels were evaluated: 80 and 120 mg/m2/day, given on Days 1-12 of induction and Days 1-7 of maintenance. Decitabine at 20mg/m2 was administered on Days 3-12 during induction and on Days 3-7 during maintenance. The primary objective was to determine the maximum tolerated dose (MTD) of thioguanine when given with decitabine. Key secondary objectives were to evaluate the overall response rate (ORR) and progression-free survival (PFS). Patient-specific pharmacodynamic measures to assess the biologic activity of thioguanine-decitabine were also performed. These included an in vitro chemosensitivity assay, BH3 profiling to measure the degree to which the leukemic blasts were primed for apoptosis, and genome-wide analysis of DNA methylation changes.Results: Twelve patients (median age 67; range 56-83) with de novo AML (n=1), secondary AML (n=6), relapsed/refractory AML (n=4), and chronic myelomonocytic leukemia (CMML) (n=1) were treated. Three patients experienced dose-limiting toxicity (DLT), which were acute renal failure requiring hemodialysis (80 mg/m2), persistent grade 4 leukopenia and thrombocytopenia (120 mg/m2), and grade 4 sepsis preventing continued treatment (120 mg/m2). Thioguanine at 80 mg/m2 was determined to be the MTD. Eleven of the 12 patients completed the first induction cycle, and 6 patients completed a second, identical induction cycle. The median number of cycles administered was 3 (range 1-8). One patient experienced a DLT prior to the first response assessment and was removed from study. The ORR in this intent-to-treat study was 67% (8/12). Six patients achieved a CR or CRi, one obtained a morphologic leukemia-free state, and one patient had a PR. Responses were observed in all disease types. Five of the 8 responses, including 4 CR/CRi, were achieved with thioguanine at 80 mg/m2, suggesting no loss of efficacy at the MTD compared with the higher dose level. All 11 evaluable patients had ≥50% reduction in bone marrow blast percentages after induction therapy. Six patients had previously received single-agent hypomethylating therapy, and 5 (83%) of these patients responded, demonstrating that thioguanine-decitabine can rescue prior hypomethylating agent failure. Out of the 8 responders, four (50%) proceeded to allogeneic stem cell transplantation (SCT), two relapsed after CR or CRi, one had a CNS-only relapse after achieving a CR, and one patient experienced DLT and was removed from the study. Of the four patients who proceeded to allogeneic SCT, two patients died in CR from transplant-related toxicity, one relapsed, and one patient remains alive and in remission greater than 2 years. Median PFS in responding patients was 42 weeks (range, 10-not reached, weeks). In vitro pharmacodynamic studies currently have been completed on samples from the first 6 patients treated on this trial. The chemosensitivity assay results on pre-treatment mononuclear cells directly correlated with initial response. In addition, significant apoptotic priming of the blasts, as suggested from BH3 profiling, also corresponded to initial clinical response.Conclusions: Thioguanine-decitabine can be administered safely and induce remission, even among patients who had previously been treated with hypomethylating agents. Intriguingly, preliminary results from the chemosensitivity screening assay and BH3 profiling correlated well with clinical responses. Additional correlative studies including DNA methylation analysis are ongoing to better understand the mechanism of synergy between thioguanine and decitabine. A multi-center Phase II trial is planned. DisclosuresJurcic:Astellas: Research Funding. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding.

Impact of timing from blood sampling to pharmacodynamic assessment on measures of platelet reactivity in patients treated with P2Y12 receptor inhibitors

Several platelet function tests (PFT) are available to assess the pharmacodynamic (PD) effects of P2Y12 inhibitors. However, there are technical variances between PFT, and P2Y12 inhibitors differ in pharmacological properties. Manufactures of PFT recommend a time-frame within which assessments needs to be executed. However, if the timing from blood sampling to processing affects PD results is unknown. We conducted a prospective study assessing the impact of timing from blood sampling to processing on PD measures using three different PFT. We studied 60 aspirin-treated patients with coronary artery disease (CAD) on maintenance P2Y12 inhibiting therapy [clopidogrel 75 mg/day (n=20), prasugrel 10 mg/day (n=20) and ticagrelor 90 mg bid (n=20)]. PD assessments (trough levels) were performed by VerifyNow P2Y12 (VN), light transmittance aggregometry (LTA) and vasodilator-stimulated phosphoprotein (VASP) at 30 minutes, 2 and 4 hours post-sampling; VASP was also performed at 24 hours. P2Y12 reaction units (PRU) by VN significantly decreased over time with all P2Y12 inhibitors (clopidogrel p<0.001; prasugrel p=0.016; ticagrelor p<0.001). PRU at 30 minutes and 2 hours were similar, but decreased at 4 hours. LTA showed consistent findings with VN. Conversely, PD measures as assessed by VASP were stable over time (p>0.1 for all P2Y12 inhibitors). In conclusion, in CAD patients on maintenance therapy with P2Y12 inhibitors, timing from blood sampling to processing significantly influences PD measures as assessed by VN and LTA, but not by VASP.

No evidence of potentiation of buprenorphine by milnacipran in healthy subjects using a nociceptive test battery.

Serotonin-norepinephrine reuptake inhibitors inhibit the reuptake of serotonin and noradrenalin and are used in the treatment of neuropathic pain. Animal studies suggest that milnacipran co-administered with opioids may potentiate the analgesic effect of μ-opioid receptor agonists. This study hypothesized that co-administration of milnacipran and buprenorphine would have a synergistic effect in evoked pain models in healthy subjects. This was a randomized double-blinded, placebo-controlled, four-way cross-over, multiple dose clinical trial to investigate the analgesic effects of buprenorphine (placebo, 0.5, 1 and 3μg/kg) in combination with milnacipran (placebo, 25 and 50mg) in healthy subjects. 11 healthy men were enrolled in the study. Buprenorphine alone showed a dose-response relationship indicative of anti-nociception in the pain tests. Following milnacipran administration, no changes were seen in the pharmacodynamic measurements for pain, psychomotor function, body stability or eye movements. For the electrical tests, cold pressor test and pressure pain test, buprenorphine alone was superior when compared with buprenorphine plus milnacipran. No differences in pharmacodynamic variables, besides an increase in pupil/iris ratio, were observed after repeated administration of milnacipran 50mg. Single and multiple doses of 25 or 50mg milnacipran did not further potentiate the anti-nociceptive effects of buprenorphine. Buprenorphine showed dose-dependent effects consistent with its pharmacological profile. Milnacipran alone did not affect any of the pain variables. The combination of both buprenorphine and milnacipran did not potentiate or show a synergistic effect on the pain models used in this study. Buprenorphine is known to be a potent opioid agonist. Animal studies suggest that milnacipran co-administered with opioids may potentiate the analgesic effect of μ-opioid receptor agonists. Here, we found that buprenorphine showed a dose-dependent analgesic effect, but that no potentiation or synergy on a battery of evoked pain tasks could be observed after co-administration of both milnacipran and buprenorphine.