Once-Weekly Subcutaneous Delivery of Polymer-Linked Rotigotine (SER-214) Provides Continuous Plasma Levels in Parkinson's Disease Patients.

BACKGROUND: Extensive scientific and clinical evidence indicates that continuous delivery of a dopaminergic agent is associated with significant reduction in motor complications compared with intermittent oral dosing with the same agent. There has been an intensive effort to develop a method of providing continuous plasma levels of a dopaminergic agent that avoids the need for surgical therapy or an infusion system. Studies in MPTP-treated monkeys demonstrate that once-weekly injections of polymer-linked rotigotine provide continuous plasma levels and antiparkinsonian benefits. METHODS: We performed a multicenter open-label, multiple-ascending-dose-ranging cohort study to evaluate the safety, tolerability, and pharmacokinetics of polymer-linked rotigotine in PD patients. RESULTS: A total of 19 patients were evaluated in 4 cohorts in doses of 20 50, 100, and 200 mg of polymer-linked rotigotine, administered subcutaneously once weekly. The study demonstrated remarkably stable dose-related plasma levels of total and free rotigotine with no accumulation or dumping. Treatment was generally safe and well tolerated. One subject in the 50-mg group discontinued because of hives, which cleared rapidly with antihistamine treatment. CONCLUSIONS: This study demonstrates that once-a-week subcutaneous administration of polymer-linked rotigotine provides relatively constant plasma levels of rotigotine and is safe and well tolerated. These findings suggest that this convenient method of delivery of rotigotine has the potential to treat or prevent motor complications in PD patients without the need for a surgical procedure or an infusion system. This approach may also prove applicable to other agents such as apomorphine that can be linked to this polymer © 2020 International Parkinson and Movement Disorder Society.

Higher cannabidiol plasma levels are associated with better seizure response following treatment with a pharmaceutical grade cannabidiol.

OBJECTIVE: The objective of this study was to determine the relationship between cannabidiol (CBD) dose, CBD plasma level, and seizure control in a large open-label single-center study. METHODS: All participants with treatment-refractory epilepsy participating in our expanded access program (EAP) were approached for participation. Highly purified grade CBD (Epidiolex®) dosing was weight-based and could be increased every 2 weeks by 5 mg/kg/day up to a maximum dosage of 50 mg/kg/day depending on tolerance and seizure control. Seizure counts were obtained at each visit with frequency calculated per 2-week periods. Cross-sectional plasma peak levels of CBD were obtained ~4 h after dosing in consecutively presenting patients. RESULTS: We evaluated 56 adults and 44 children (100 total; 54 female) at two time points - one before initiating CBD and one at the time of CBD plasma level testing. There was a positive linear correlation between CBD dosage (range from 5 to 50 mg/kg/day) and level (range from 7.1-1200 ng/mL) in all participants (r = 0.640; p < 0.001). The quantile regression model supported the notion of increased CBD levels being associated with improvement in seizure frequency after adjusting for age - specifically, a 100 ng/mL increase in CBD level was associated with approximately two counts reduction in seizure frequency per time period (1.87 96% confidence interval [CI] 0.34-3.39; p = 0.018). In participants with the same CBD level, differences in seizure improvement did not depend on age (p = 0.318). CONCLUSIONS: In this open-label study, we found evidence of a linear correlation between CBD dosage and plasma levels, and that higher dose/levels are associated with a higher response rate for seizure improvement. Children and adults responded to CBD similarly. However, seizure control response rates suggest children may respond to lower dosages/plasma levels than adults. Findings reported in this study are specific to Epidiolex® and should not be extrapolated to other CBD products.

Rotigotine polyoxazoline conjugate SER-214 provides robust and sustained antiparkinsonian benefit.

Currently available dopaminergic drugs such as levodopa and dopamine (DA) receptor agonists impart considerable improvement in Parkinson's disease (PD) motor symptoms but often lead to significant motor complications including "wearing-off" and dyskinesia. Such complications are believed to stem from the pulsatile nature of dopaminergic stimulation with these agents. Continuous dopaminergic drug delivery using polyoxazoline (POZ) polymer conjugation may improve motor symptoms, while avoiding development of side effects. The purposes of the current study are to characterize the in vitro and in vivo pharmacokinetics of POZ conjugation of a U.S. Food and Drug Administration (FDA)-approved DA agonist, rotigotine, and to evaluate their effects in an established rat model of PD. After determination of release profiles of several POZ-conjugated constructs ("fast": SER-212; "moderate": SER-213; and "slow": SER-214) using in vitro hydrolysis, normal male Sprague-Dawley rats were used for determination of the pharmacokinetic profile of both acute and chronic exposure. Finally, a separate group of rats was rendered hemiparkinsonian using intracranial 6-hydroxydopamine (6-OHDA) infusions, treated acutely with POZ-rotigotine, and assessed for rotational behavior and antiparkinsonian benefit using the cylinder test. POZ-rotigotine formulations SER-213 and SER-214 led to substantial pharmacokinetic improvement compared to unconjugated rotigotine. In addition, SER-214 led to antiparkinsonian effects in DA-lesioned rats that persisted up to 5 days posttreatment. Repeated weekly dose administration of SER-214 to normal rats for up to 12 weeks demonstrated highly reproducible pharmacokinetic profiles. The continuous dopaminergic stimulation profile afforded by SER-214 could represent a significant advance in the treatment of PD, with potential to be a viable, once-per-week therapy for PD patients.

Effects of Ad5FGF-4 in patients with angina: an analysis of pooled data from the AGENT-3 and AGENT-4 trials.

OBJECTIVES: The goal of this study was to explore the effects of angiogenic gene therapy. BACKGROUND: Preclinical studies with intracoronary administration of Ad5FGF-4 (alferminogene tadenovec, Generx, Berlex Biosciences, Richmond, California) suggested it could induce angiogenesis and provide a new clinical approach to the treatment of chronic angina pectoris. Two preliminary clinical trials provided evidence that it could improve exercise treadmill test (ETT) time and myocardial perfusion. The AGENT (Angiogenic GENe Therapy)-3 and -4 trials of a low and high dose of Ad5FGF-4 for chronic angina were initiated in the U.S. and other countries and enrolled 532 patients in a randomized, double-blind, placebo-controlled fashion. Both studies were halted when an interim analysis of the AGENT-3 trial indicated that the primary end point change from baseline in total ETT time at 12 weeks would not reach significance. METHODS: We performed a pooled data analysis from the 2 nearly identical trials to investigate possible treatment effects on primary and secondary end points in prespecified subgroups. RESULTS: The effect of placebo was large and not different than active treatment in men, but the placebo effect in women was negligible and the treatment effect was significantly greater than placebo. We found a significant, gender-specific beneficial effect of Ad5FGF-4 on total ETT time, time to 1 mm ST-segment depression, time to angina, and Canadian Cardiovascular Society class in women. This is the first clinical report of a gender difference in response to cardiac angiogenic therapy. CONCLUSIONS: The potential importance of the observed gender-specific angiogenic response on the clinical treatment of refractory angina is substantial and deserves further investigation. (Efficacy and Safety of Intracoronary Ad5FGF-4 in Patients With Stable Angina; http://www.clinicaltrials.gov/ct/show/NCT00346437; NCT00346437) (Safety and Efficacy of Intracoronary Ad5FGF-4 in Patients With Stable Angina [AGENT-4]; http://www.clinicaltrials.gov/ct/show/NCT00185263; NCT00185263) (AWARE; http://www.clinicaltrials.gov/ct/show/NCT000438867; NCT000438867).

Clinical development of PEGylated recombinant staphylokinase (PEG-Sak) for bolus thrombolytic treatment of patients with acute myocardial infarction.

The development of bolus thrombolytic agents, in conjunction with bolus anti-thrombotics (e.g. low molecular weight heparins), remains an ambitious but achievable goal of therapy for acute myocardial infarction-a disease which takes the lives of millions each year. This chapter summarizes the data accumulated over nearly a decade of investigation of recombinant staphylokinase (Sak) as a safe, cost-effective thrombolytic agent. The results of extensive animal investigations suggested this agent exhibited a uniquely fibrin-selective mechanism of action. Administration of various recombinant versions of the molecule to over 1000 patients on a global scale suggest this agent may be as effective as tissue-type plasminogen activator (rt-PA) in achieving prompt arterial recanalization of acutely occluded coronary arteries. The development of this protein as a single bolus agent is described in detail, and the results of recently completed international trials comparing this bolus agent to front-loaded rt-PA are summarized.

Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography.

Staphylokinase variant SY161 is a recombinant mutant of the Staphylococcus aureus polypeptide staphylokinase (Sak), and is currently in human clinical trials as a thrombolytic agent. The 15 kDa single chain SY161 protein is expressed as a soluble cytoplasmic product in E. coli with a single cysteine inserted near the N-terminus. The protein as extracted from E. coli is a mixture of both monomeric and intermolecularly disulfide crosslinked species. To improve protein purification yields SY161 is sulfitolyzed during the early stages of production, preventing disulfide formation. The protein is later modified during manufacturing to incorporate a single 5 kDa polyethylene glycol group on the single sulfhydryl sidechain. We have developed and qualified a reverse-phase chromatographic method to quantitate SY161 during product manufacturing. We discuss the use of the assay during manufacturing development to monitor fermentation yields, the SY161 PEGylation reaction, and as an in-process manufacturing control assay. The assay has been applied as a product purity and identity release assay and is suitable for use in assessing product structural integrity during stability testing. The assay has a linear range of quantitation for SY161 from at least 0.15 to 16 micro g, and is-in addition capable of detecting and quantitating protein de-PEGylation events and host cell-derived protein contaminants.

Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains.

Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene-targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (129/SvEv, 129/SvJ, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola, C3H/HeN, BALB/c, and FVB/N). Germline transmission was established by blastocyst injection of established ES cell lines after 10 or more passages from all of seven strains tested (129/SvJ, C57BL/6N, C57BL/6JOla, DBA/2N, DBA/1Ola, BALB/c, and FVB/N), by diploid aggregation of ES cell lines from all of four strains tested (129/SvEv, C57BL/6N, CBA/ CaOla, and FVB/N), or by tetraploid aggregation of ES cell lines from all of three strains tested (129/SvEv, C57BL/6N, and CBA/CaOla). Thus, these inbred ES cell lines may constitute useful tools to derive gene-targeted mice and isogenic controls in selected genetic backgrounds.

An enzyme-linked immunosorbent assay for host cell protein contaminants in recombinant PEGylated staphylokinase mutant SY161.

Staphylokinase, a bacterially-derived protein which functions as a plasminogen activator, has potential utility as a human therapeutic for thrombotic disorders. A recombinant version of this protein, SY161, contains 13 amino acid substitutions designed to decrease immunogenicity, and has been covalently modified by crosslinking a 5 kDa polyethyleneglycol (PEG) group to the N-terminal region to prolong the drug circulating half-life. The recombinant PEG-modified SY161 staphylokinase is currently in phase II clinical trials as a treatment for acute myocardial infarction. We have developed a sensitive product specific host cell protein (HCP) assay in the ELISA format to monitor in process host-derived contaminant clearance and final drug product purity. The assay is based upon use of goat polyclonal antibodies raised against E. coli host strain cell proteins from a null cell line, extracted by the same manufacturing process used to produce SY161. The identification and clearance of HCP contaminants was confirmed during drug product production using SDS-PAGE and Western blotting utilizing the same polyclonal HCP antibodies. The assay is specific for E. coli host cell strain proteins with a useful detection range from 1 to 100 ng/ml, and is not affected by product level. The level of residual HCPs in the clinical product produced by our manufacturing process was determined to be less than 1 ng/ml at a product concentration of 1 mg/ml.

Cardiac dysfunction in mice lacking cytochrome-c oxidase subunit VIaH.

Cytochrome-c oxidase subunit VIaH (COXVIaH) has been implicated in the modulation of COX activity. A gene-targeting strategy was undertaken to generate mice that lacked COXVIaH to determine its role in regulation of oxidative energy production and mechanical performance in cardiac muscle. Total COX activity was decreased in hearts from mutant mice, which appears to be a consequence of altered assembly of the holoenzyme COX. However, total myocardial ATP was not significantly different in wild-type and mutant mice. Myocardial performance was examined using the isolated working heart preparation. As left atrial filling pressure increased, hearts from mutant mice were unable to generate equivalent stroke work compared with hearts from wild-type mice. Direct measurement of left ventricular end-diastolic volume using magnetic resonance imaging revealed that cardiac dysfunction was a consequence of impaired ventricular filling or diastolic dysfunction. These findings suggest that a genetic deficiency of COXVIaH has a measurable impact on myocardial diastolic performance despite the presence of normal cellular ATP levels.

Preformulation development of recombinant pegylated staphylokinase SY161 using statistical design.

The goal of this study was to perform preformulation development of SY161 by using statistical design methods to understand the effects of buffer strength, NaCl concentration, and pH on conformation and stability of the protein. It was also important to elucidate interactions between these factors. A central composite design using a 2-level full-factorial study was performed. Secondary structure was evaluated using circular dichroism. Stability toward unfolding was investigated using high-sensitivity differential scanning calorimetry. Depegylation, aggregation, and protein loss were evaluated using SEC-HPLC with on-line light scattering, at time zero and after a 2-week stability study. Response surface plots clearly show optimal pH, NaCl, and buffer conditions. Interactions between pH and NaCl as well as pH and buffer concentration are observed. Tm is seen to be predictive of SY161 stability. Secondary structure changes were minimal and did not influence stability. Statistical design was very effective in providing an understanding of the effects of the formulation components on SY161 stability.