Diana S.-L. Chow

Acute safety and pharmacokinetics of intravenous busulfan when used with oral busulfan and cyclophosphamide as pretransplantation conditioning therapy: A phase I study

The unpredictable intestinal absorption and erratic bioavailability of oral busulfan (Bu) has limited the drugs use in high-dose pretransplantation conditioning therapy. To standardize drug delivery, we solubilized Bu for parenteral use. This new intravenous (i.v.) Bu formulation was combined with oral Bu and cyclophosphamide (Cy) to evaluate (1) the human acute toxicity of i.v. Bu and its solvent system and (2) the pharmacokinetics of Bu in patients undergoing hematopoietic progenitor cell transplantation (HPCT). One dose of i.v. Bu (escalating from 0.08 to 0.8 mg/kg) was given over 2 hours by pump; 6 hours later, an oral Bu regimen was begun, consisting of 1 mg/kg every 6 hours for 15 doses, followed by Cy, 60 mg/kg daily for 2 days. After 1 day of rest, HPCT was performed. The i.v. Bu dose was well tolerated and did not produce any acute toxicity reaction that could be attributed to the solvent system of dimethylacetamide and polyethylene glycol (PEG)-400. All observed treatment-related toxicity was as would be expected after high-dose oral Bu plus Cy. When the i.v. Bu was used as reference solution, the pharmacokinetic analysis indicated an average bioavailability of oral high-dose Bu of 69%, ranging from <10% to virtually 100%. Further, the 2-hour infusion of i.v. Bu gave a time to maximum plasma concentration following drug administration similar to that of oral Bu (2 hours and 1.8 hours, respectively), and i.v. Bu had a clearance similar to that of oral Bu. Based on the data in this study, we suggest that the optimal (starting) dose of i.v. Bu (in combination with Cy) in our forthcoming phase 2 trial should be on the order of 0.8 mg/kg to target an area under the curve (AUC) of 1100 to 1200 micromol/L per minute. This would secure myeloablation and engraftment but save the vast majority of patients from the increased risk of serious hepatic veno-occlusive disease that has been reported when the AUC level exceeds 1500 micromol/L per minute. Bu administration via the i.v. route will assure complete bioavailability and reliable systemic drug exposure with more predictable blood levels and, therefore, possibly lower the risks for serious/life-threatening toxicity, graft rejection, and recurrent leukemia.

A prospective, multicenter, phase I matched-comparison group trial of safety, pharmacokinetics, and preliminary efficacy of riluzole in patients with traumatic spinal cord injury.

A prospective, multicenter phase I trial was undertaken by the North American Clinical Trials Network (NACTN) to investigate the pharmacokinetics and safety of, as well as obtain pilot data on, the effects of riluzole on neurological outcome in acute spinal cord injury (SCI). Thirty-six patients, with ASIA impairment grades A-C (28 cervical and 8 thoracic) were enrolled at 6 NACTN sites between April 2010 and June 2011. Patients received 50 mg of riluzole PO/NG twice-daily, within 12 h of SCI, for 14 days. Peak and trough plasma concentrations were quantified on days 3 and 14. Peak plasma concentration (Cmax) and systemic exposure to riluzole varied significantly between patients. On the same dose basis, Cmax did not reach levels comparable to those in patients with amyotrophic lateral sclerosis. Riluzole plasma levels were significantly higher on day 3 than on day 14, resulting from a lower clearance and a smaller volume of distribution on day 3. Rates of medical complications, adverse events, and progression of neurological status were evaluated by comparison with matched patients in the NACTN SCI Registry. Medical complications in riluzole-treated patients occurred with incidences similar to those in patients in the comparison group. Mild-to-moderate increase in liver enzyme and bilirubin levels were found in 14-70% of patients for different enzymes. Three patients had borderline severe elevations of enzymes. No patient had elevated bilirubin on day 14 of administration of riluzole. There were no serious adverse events related to riluzole and no deaths. The mean motor score of 24 cervical injury riluzole-treated patients gained 31.2 points from admission to 90 days, compared to 15.7 points for 26 registry patients, a 15.5-point difference (p=0.021). Patients with cervical injuries treated with riluzole had more-robust conversions of impairment grades to higher grades than the comparison group.

Delayed Post-Injury Administration of Riluzole Is Neuroprotective in a Preclinical Rodent Model of Cervical Spinal Cord Injury

Riluzole, a sodium/glutamate antagonist has shown promise as a neuroprotective agent. It is licensed for amyotrophic lateral sclerosis and is in clinical trial development for spinal cord injury (SCI). This study investigated the therapeutic time-window and pharmacokinetics of riluzole in a rodent model of cervical SCI. Rats were treated with riluzole (8 mg/kg) at 1 hour (P1) and 3 hours (P3) after injury or with vehicle. Afterward, P1 and P3 groups received riluzole (6 (mg/kg) every 12 hours for 7 days. Both P1 and P3 animals had significant improvements in locomotor recovery as measured by open field locomotion (BBB score, BBB subscore). Von Frey stimuli did not reveal an increase in at level or below level mechanical allodynia. Sensory-evoked potential recordings and quantification of axonal cytoskeleton demonstrated a riluzole-mediated improvement in axonal integrity and function. Histopathological and retrograde tracing studies demonstrated that delayed administration leads to tissue preservation and reduces apoptosis and inflammation. High performance liquid chromatography (HPLC) was undertaken to examine the pharmacokinetics of riluzole. Riluzole penetrates the spinal cord in 15 min, and SCI slowed elimination of riluzole from the spinal cord, resulting in a longer half-life and higher drug concentration in spinal cord and plasma. Initiation of riluzole treatment 1 and 3 hours post-SCI led to functional, histological, and molecular benefits. While extrapolation of post-injury time windows from rat to man is challenging, evidence from SCI-related biomarker studies would suggest that the post-injury time window is likely to be at least 12 hours in man.

Rationale, design and critical end points for the Riluzole in Acute Spinal Cord Injury Study (RISCIS): a randomized, double-blinded, placebo-controlled parallel multi-center trial

Background:Riluzole is a sodium channel-blocking agent used in treating amyotrophic lateral sclerosis. It has been approved by the U.S. Food and Drug Administration, Canadian and Australian authorities, and in many other countries. A phase I trial of riluzole for acute spinal cord injury (SCI) provided safety and pharmacokinetic data and suggested neuroprotective benefits. A phase IIB/III double-blinded randomized controlled trial (RCT) started in January 2014 (https://clinicaltrials.gov, NCT01597518). This article describes the pathophysiological rationale, preclinical experience and design of the phase IIB/III RCT of Riluzole in Acute Spinal Cord Injury Study (RISCIS).Objectives:The primary objective of the trial is to evaluate the superiority of riluzole, at a dose of 100 mg BID in the first 24 h followed by 50 mg BID for the following 13 days post injury, compared with placebo in improving neurological motor outcomes in patients with C4–C8 level, International Standards for Neurological Classification of Spinal Cord Injury Examination (ISNCSCI) grade A, B or C acute (within 12 h post injury) SCI.Setting:Acute trauma centers worldwideMethods:A double-blind, multi-center, placebo-controlled RCT will enroll 351 participants randomized 1:1 to riluzole and placebo. The primary end point is the change between 180 days and baseline in ISNCSCI Motor Score. This study has 90% power to detect a change of nine points in ISNCSCI Motor Score at one-sided α=0.025.Results:Currently enrolling in 11 centers.Conclusion:This study will provide class I evidence regarding the safety and neuroprotective efficacy of riluzole in patients with acute cervical SCI.

Stability-indicating high-performance liquid chromatographic assay of busulfan in aqueous and plasma samples

A sensitive, specific and stability-indicating high-performance liquid chromatographic (HPLC) assay, involving pre-column derivatization and solid-phase extraction (SPE), was developed and validated for the quantitation of busulfan (BU) in aqueous and plasma samples. The linearity of the assay was in the concentration ranges of 0.15-10 microg/ml and 0.15-3 microg/ml for aqueous and plasma samples, respectively. The within-day and between-day variations were 2.90 and 3.31%, respectively, for the aqueous samples, and 9.24 and 14.56%, respectively, for the plasma samples. The overall recovery, derivatization yield and SPE efficiency of BU from plasma samples were 82.03, 108.01 and 86.69%, respectively. Forced degraded samples, either in highly acidic, neutral or basic medium, produced no interfering peaks in the chromatogram. The reported assay requires only 0.2 ml of plasma for the analysis, and its sensitivity is 150 ng/ml by monitoring samples at a wavelength of 254 nm, sufficient to study the plasma pharmacokinetics of BU in rats after a clinically relevant oral dose. Moreover, the sensitivity of the assay can be significantly increased to 30 ng/ml by monitoring samples at a wavelength of 278 nm. The applications of the assay were demonstrated with BU solubility measurements in two aqueous systems and with plasma samples from a Sprague-Dawley rat for an in vivo pharmacokinetic study. In addition, the assay has been employed in the development of a patented intravenous formulation, and in evaluations of stability, preclinical pharmacokinetics in rats and dogs, and clinical phase I trial of the formulation. The assay is readily adaptable to clinical therapeutic drug monitoring.

Pharmacology of riluzole in acute spinal cord injury

OBJECT The aim of this paper was to characterize individual and population pharmacokinetics of enterally administered riluzole in a Phase 1 clinical trial of riluzole as a neuroprotective agent in adults 18-70 years old with acute spinal cord injury (SCI). METHODS Thirty-five individuals with acute SCI, American Spinal Injury Association Impairment Scale Grades A-C, neurological levels from C-4 to T-12, who were enrolled in the Phase 1 clinical trial sponsored by the North American Clinical Trials Network for Treatment of Spinal Cord Injury, received 50 mg riluzole twice daily for 28 doses. The first dose was administered at a mean of 8.7 ± 2.2 hours postinjury. Trough plasma samples were collected within 1 hour predose, and peak plasma samples were collected 2 hours postdose on Days 3 and 14 of treatment. Riluzole concentrations were quantified by high-performance liquid chromatography assay. The data were analyzed for individual and population pharmacokinetics using basic structural and covariate models. The pharmacokinetic measures studied were the peak concentration (C(max)), trough concentration (C(min)), systemic exposure (AUC(0-12)), clearance (CL/F), and volume of distribution (V_F) normalized by the bioavailability (F). RESULTS The C(max) and AUC(0-12) achieved in SCI patients were lower than those in ALS patients on the same dose basis, due to a higher CL and larger V. The pharmacokinetics of riluzole (C(max), C(min), AUC(0-12), CL, and V) changed during the acute and subacute phases of SCI during the 14 days of therapy. It was consistently observed in patients at all clinical sites that C(max), C(min), and AUC(0-12) (128.9 ng/ml, 45.6 ng/ml, and 982.0 ng × hr/ml, respectively) were significantly higher on Day 3 than on Day 14 (76.5 ng/ml, 19.1 ng/ml, and 521.0 ng × hr/ml, respectively). These changes resulted from lower CL (49.5 vs 106.2 L/hour) and smaller V (557.1 vs 1297.9/L) on Day 3. No fluid imbalance or cytochrome P 1A2 induction due to concomitant medications was identified during the treatment course to account for such increases in V and CL, respectively. Possible mechanisms underlying these changes are discussed. CONCLUSIONS This is the first report of clinical pharmacokinetics of riluzole in patients with SCI. The C(max) and AUC(0-12) achieved in SCI patients were lower than those in ALS patients on the same dose basis, due to a higher clearance and larger volume of distribution in SCI patients. The finding in SCI patients of an increase in the clearance and distribution of riluzole between the 3rd and 14th days after SCI, with a lower plasma concentration of riluzole on the 14th day, stresses the importance of monitoring changes in drug metabolism after SCI in interpreting the safety and efficacy of therapeutic drugs that are used in clinical trials in SCI. Clinical trial registration no.: NCT00876889.

A validated ultra-performance liquid chromatography–tandem mass spectrometry method for the quantification of polymyxin B in mouse serum and epithelial lining fluid: application to pharmacokinetic studies

OBJECTIVES A rapid, sensitive and robust ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of four major polymyxin B components (polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-polymyxin B1) in serum and epithelial lining fluid (ELF) samples. METHODS A Waters Acquity UPLC HSS C18 column was used with 0.1% formic acid in water/acetonitrile as mobile phases. Analysis was performed in a positive ionization mode with multiple-reactions monitoring scan type. Five percent trichloroacetic acid was used to precipitate proteins in biological samples and to increase the sensitivity of detection. RESULTS Our results showed a linear concentration range of 0.0065-3.2 mg/L for all the major polymyxin B components in both serum and ELF, respectively; the interday variation was <10% and the accuracy was 88%-115%. The validated method was used to characterize the pharmacokinetics (serum and ELF) of polymyxin B in mice. CONCLUSIONS This is the first report, to date, examining the individual pharmacokinetics of various polymyxin B components in mice. Our results revealed no considerable differences in clearances among the components. The limited exposure of polymyxin B in ELF observed was consistent with the less favourable efficacy of polymyxin B reported for the treatment of pulmonary infections. This method can be used to further examine the pharmacokinetics of polymyxin B in a variety of clinical and experimental settings.

Pharmacokinetics and efficacy of liposomal polymyxin B in a murine pneumonia model.

Polymyxin B (PB) is increasingly used as the last treatment for multidrug-resistant (MDR) Gram-negative bacterial infections. In this study, serum and epithelial lining fluid (ELF) pharmacokinetics and the efficacy of a PB liposomal formulation were investigated. Two groups of 24 Swiss Webster mice were intravenously administered PB liposomes or PB aqueous solution at ca. 3 mg/kg. Serum and ELF samples were collected for up to 6 h to quantify major PB components. Three groups of neutropenic mice (n = 6/group) were infected with a clinical MDR Pseudomonas aeruginosa strain followed by intravenous administration of PB liposomes or PB aqueous solution at 3 mg/kg every 6 h or sham (drug-free) liposomes every 6 h. Bacterial burden in animal lung tissues was quantified after 24 h of therapy and was compared using one-way ANOVA. Survival of infected animals over time (n = 10/group) was evaluated by Kaplan-Meier analysis and log-rank test. In the pharmacokinetic study, the AUC ratio in ELF between liposome and aqueous solution groups ranged from 4.6 to 11.1 for various major PB components. In the efficacy study, for strain PA 9019 a significantly lower bacterial burden was seen in the liposomal group (3.8 ± 0.7 vs. 7.9 ± 0.8 log(10)CFU/g in the aqueous solution group), which subsequently prolonged survival of infected animals. In this study, treatment with a PB liposomal formulation yielded higher drug penetration into pulmonary ELF, which resulted in superior efficacy. However, further investigations on the clinical utility of the PB liposomal formulation are warranted.

Development and validation of a highly sensitive LC–MS/MS assay for the quantification of arginine vasopressin in human plasma and urine: Application in preterm neonates and child

Arginine vasopressin is an endogenous neuropeptide secreted in response to situations such as hyperosmolality, hypotension and hypovolemia. The purpose of this study was to develop a reliable assay using small volumes of plasma and urine samples to quantify vasopressin levels in preterm infants. Weak cation solid-phase extraction was used to extract vasopressin from 200μl human plasma and urine samples. Separation was achieved on a Waters Acquity UPLC BEH C18 column by gradient elution at 0.55ml/min, with a mobile phase composed of methanol and 0.02% aqueous acetic acid solution. Analysis was performed under a hybrid triple quadrupole linear ion trap mass spectrometer, operated in multiple reaction monitoring mode using positive ionization. The linear response range was 1.0-40pg/ml for vasopressin, with the lower limit of quantification (LLOQ) of 1.0pg/ml in human plasma and urine. Recoveries at concentrations of 3, 10 and 32pg/ml were all greater than 70%, and matrix effects were within 15%. The method was validated with intra-day and inter-day precision of less than 8% for human plasma and urine. The intra-day and inter-day accuracy for human plasma were 91.9-100.6% and 92.3-104.8%, respectively. The intra-day and inter-day accuracy for human urine were 89.2-95.9% and 89.3-91.3%, respectively. The validated method was successfully applied to analyze two preterm neonate plasma samples and one child urine sample. In conclusion, the developed and validated method was sensitive and reliable, and was successfully used to quantify endogenous vasopressin levels in neonate plasma and child urine.

Evaluation of the sodium-glutamate blocker riluzole in a preclinical model of ervical spinal cord injury

Study type: Basic research Introduction: Because sodium and glutamate play integrated roles in the pathology of spinal cord injury (SCI), there is intense interest in the potential role of the sodium-glutamate blocker riluzole as a neuroprotective agent for spinal cord injury. A phase I safety clinical trial of riluzole is about to commence in the USA and Canada. Objective: The key challenges in translating riluzole to the clinic relate to uncertainty regarding the time window for post-injury administration of riluzole and the optimal dose. Hence, the current study was undertaken to study these issues in a preclinical model of cervical SCI in rodents. Methods: Adult female rats received moderate cervical spinal cord clip compression injury (35 g × 1 min at C7–T1) and were randomly assigned to one of the following three groups (n = 12/group): a) 8 mg/kg of riluzole intraperitoneally at 1 hour (P1 group) ; b) 3 hours (P3 group) after injury or c) a control group (vehicle). The P1 and P3 groups then received 6 mg/kg of riluzole intraperitoneally every 12 hours for 7 days. A smaller group of rats (n = 10) received a higher dose (8 mg/kg IP Q12 h for 7 days) of riluzole following the initial dosing. Functional recovery was tested by open field locomotion (BBB score, BBB subscore) and grid walk. Sensory function (tactile allodynia) was assessed by quantitative von Frey filament testing. Somatosensory evoked potentials (SSEP) were performed to quantitatively assess central axonal conduction. Western blotting for neurofilament 200 (NF200) was used to assess axonal integrity. Quantitative image analysis of lesion histology (HE/LFB stained sections) was undertaken to assess tissue sparing at the injury site. High performance liquid chromatography (HPLC) was used to test pharmacokinetics of riluzole. ANOVA followed by the Tukeys post-hoc testing was used to compare the results. Results: A total of 92.3% of injured rats that received repeated dose of 6 mg/kg riluzole survived during the term of 6 weeks, showing tolerance to this dosage. In contrast, higher doses of riluzole (8mg/kg IP q12h post-injury) were associated with significant respiratory depression and high mortality (7/10 rats). Riluzole treatment (P1 group) decreased the functional deficit significantly (P < 0.05) at 6 weeks after injury by showing 11.4 in BBB, 3.8 in BBB subscore and 2.8 in grid walk test, compared with 10.25 in BBB, 1.1 in BBB subscore, and 8.0 in grid walk test of control group (Figure 1). Quantitative analysis of SSEPs confirmed the neuroprotective effects of riluzole (P1 group) by showing significantly larger amplitude (control = 20.67 µV, P1 = 42.67 µV and P3 = 37.00 µV, Figure 2) and reduced response latency (control = 4.05 ms and P1 = 3.9 ms). Western blotting of NF200 indicated greater axonal preservation with riluzole treatment (P1 group) at 1 week after injury. The P3 group showed trends to improved neurobehavioral recovery and axonal preservation that did not attain significance. HPLC showed riluzole penetrated into the spinal cord as fast as 15 minutes, accumulated in the spinal cord at a concentration six times higher than in plasma, and two times higher than in brain. The drug stayed in spinal cord with a high concentration before the next injection.Figure 1 Riluzole treatment improved functional recovery. The most robust effects were seen when the drug was injected at one hour after SCIFigure 2 Riluzole treatment increased amplitude of somatosensory evoked potentials Conclusions: The sodium-glutamate blocker riluzole confers neurobehavioral and anatomical neuroprotection when administered one hour after moderately severe cervical contusive/compressive injury.