Anders Andersen

Pharmaceutical-grade albumin: impaired drug-binding capacity in vitro

BackgroundAlbumin is the most abundant protein in blood plasma, and due to its ligand binding properties, serves as a circulating depot for endogenous and exogenous (e.g. drugs) compounds. Hence, the unbound drug is the pharmacologically active drug. Commercial human albumin preparations are frequently used during surgery and in critically ill patients. Recent studies have indicated that the use of pharmaceutical-grade albumin is controversial in critically ill patients. In this in vitro study we investigated the drug binding properties of pharmaceutical-grade albumins (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn), native human serum, and commercially available human serum albumin from Sigma Chemical Company.MethodsThe binding properties of the various albumin solutions were tested in vitro by means of ultrafiltration. Naproxen, warfarin, and digitoxin were used as ligands. HPLC was used to quantitate the total and free drug concentrations. The data were fitted to a model of two classes of binding sites for naproxen and warfarin and one class for digitoxin, using Microsoft Excel and Graphpad Prism.ResultsThe drugs were highly bound to albumin (95–99.5%). The highest affinity (lowest K1) was found with naproxen. Pharmaceutical-grade albumin solutions displayed significantly lower drug-binding capacity compared to native human serum and Sigma albumin. Thus, the free fraction was considerably higher, approximately 40 times for naproxen and 5 and 2 times for warfarin and digitoxin, respectively. The stabilisers caprylic acid and N-acetyl-DL-tryptophan used in the manufacturing procedure seem to be of importance. Adding the stabilisers to human serum and Sigma albumin reduced the binding affinity whereas charcoal treatment of the pharmaceutical-grade albumin from Octapharma almost restored the specific binding capacity.ConclusionThis in vitro study demonstrates that the specific binding for warfarin and digitoxin is significantly reduced and for naproxen no longer detectable in pharmaceutical-grade albumin. It further shows that the addition of stabilisers may be of major importance for this effect.

High dose methotrexate chemotherapy: pharmacokinetics, folate and toxicity in osteosarcoma patients

AIMSnTo investigate the relationships between pretreatment folate concentrations, MTX pharmacokinetics and acute toxicities following high dose methotrexate (HD MTX) therapy.nnnMETHODSnMTX and its major extracellular metabolite 7-OH-MTX were measured in eight serum samples per HD MTX cycle in 65 consecutive osteosarcoma patients (288 cycles) and AUC (area under the blood concentration-time curve) was calculated. Pretreatment concentrations of folate in serum (S) and erythrocytes (ER) were determined. Hepatic, renal and haematological toxicities, assessed by routine laboratory parameters, as well as mucositis were graded according to National Cancer Institute Common Terminology Criteria for adverse events (CTCAE v.3.0). Dermatitis and pleuritis were reported as occurred or not.nnnRESULTSnS- and ER-folate pretreatment concentrations increased significantly with increasing number of HD MTX cycles (P < 0.001). ER-folate pretreatment concentrations were higher among males (median 610u2003nmolu2003l⁻¹, 95% CI 550, 680) compared with females (median 465u2003nmolu2003l⁻¹, 95% CI 430, 520, P < 0.001), but showed no correlation with MTX or 7-OH-MTX pharmacokinetics. We found correlations between alanine aminotransferase peak concentration (ALAT(max) ) and clearance of MTX (P < 0.001), gender (P < 0.001), age (P < 0.001) and 7-OH-MTX concentrations (P < 0.001), the latter being the main factor influencing ALAT(max) .nnnCONCLUSIONnOur results suggest that 7-OH-MTX is involved in the development of HD MTX hepatic toxicity and that young female patients are most affected.

High dose methotrexate chemotherapy

AIMSnTo investigate the relationships between pretreatment folate concentrations, MTX pharmacokinetics and acute toxicities following high dose methotrexate (HD MTX) therapy.nnnMETHODSnMTX and its major extracellular metabolite 7-OH-MTX were measured in eight serum samples per HD MTX cycle in 65 consecutive osteosarcoma patients (288 cycles) and AUC (area under the blood concentration-time curve) was calculated. Pretreatment concentrations of folate in serum (S) and erythrocytes (ER) were determined. Hepatic, renal and haematological toxicities, assessed by routine laboratory parameters, as well as mucositis were graded according to National Cancer Institute Common Terminology Criteria for adverse events (CTCAE v.3.0). Dermatitis and pleuritis were reported as occurred or not.nnnRESULTSnS- and ER-folate pretreatment concentrations increased significantly with increasing number of HD MTX cycles (P < 0.001). ER-folate pretreatment concentrations were higher among males (median 610u2003nmolu2003l⁻¹, 95% CI 550, 680) compared with females (median 465u2003nmolu2003l⁻¹, 95% CI 430, 520, P < 0.001), but showed no correlation with MTX or 7-OH-MTX pharmacokinetics. We found correlations between alanine aminotransferase peak concentration (ALAT(max) ) and clearance of MTX (P < 0.001), gender (P < 0.001), age (P < 0.001) and 7-OH-MTX concentrations (P < 0.001), the latter being the main factor influencing ALAT(max) .nnnCONCLUSIONnOur results suggest that 7-OH-MTX is involved in the development of HD MTX hepatic toxicity and that young female patients are most affected.

Disease Mechanisms and Clonidine Treatment in Adolescent Chronic Fatigue Syndrome: A Combined Cross-sectional and Randomized Clinical Trial

IMPORTANCEnChronic fatigue syndrome (CFS) is a disabling condition with unknown disease mechanisms and few treatment options.nnnOBJECTIVEnTo explore the pathophysiology of CFS and assess clonidine hydrochloride pharmacotherapy in adolescents with CFS by using a hypothesis that patients with CFS have enhanced sympathetic activity and that sympatho-inhibition by clonidine would improve symptoms and function.nnnDESIGN, SETTING, AND PARTICIPANTSnParticipants were enrolled from a single referral center recruiting nationwide in Norway. A referred sample of 176 adolescents with CFS was assessed for eligibility; 120 were included (34 males and 86 females; mean age, 15.4 years). A volunteer sample of 68 healthy adolescents serving as controls was included (22 males and 46 females; mean age, 15.1 years). The CSF patients and healthy controls were assessed cross-sectionally at baseline. Thereafter, patients with CFS were randomized 1:1 to treatment with low-dose clonidine or placebo for 9 weeks and monitored for 30 weeks; double-blinding was provided. Data were collected from March 2010 until October 2012 as part of the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial.nnnINTERVENTIONSnClonidine hydrochloride capsules (25 µg or 50 µg twice daily for body weight <35 kg or >35 kg, respectively) vs placebo capsules for 9 weeks.nnnMAIN OUTCOMES AND MEASURESnNumber of steps per day.nnnRESULTSnAt baseline, patients with CFS had a lower number of steps per day (Pu2009<u2009.001), digit span backward score (Pu2009=u2009.002), and urinary cortisol to creatinine ratio (Pu2009=u2009.001), and a higher fatigue score (Pu2009<u2009.001), heart rate responsiveness (Pu2009=u2009.02), plasma norepinephrine level (Pu2009<u2009.001), and serum C-reactive protein concentration (Pu2009=u2009.04) compared with healthy controls. There were no significant differences regarding blood microbiology evaluation. During intervention, the clonidine group had a lower number of steps per day (mean difference, -637 steps; Pu2009=u2009.07), lower plasma norepinephrine level (mean difference, -42 pg/mL; Pu2009=u2009.01), and lower serum C-reactive protein concentration (mean ratio, 0.69; Pu2009=u2009.02) compared with the CFS placebo group.nnnCONCLUSIONS AND RELEVANCEnAdolescent CFS is associated with enhanced sympathetic nervous activity, low-grade systemic inflammation, attenuated hypothalamus-pituitary-adrenal axis function, cognitive impairment, and large activity reduction, but not with common microorganisms. Low-dose clonidine attenuates sympathetic outflow and systemic inflammation in CFS but has a concomitant negative effect on physical activity; thus, sympathetic and inflammatory enhancement may be compensatory mechanisms. Low-dose clonidine is not clinically useful in CFS.nnnTRIAL REGISTRATIONnclinicaltrials.gov Identifier: NCT01040429.

Experience of unpleasant sensations in the mouth after injection of saline from prefilled syringes

BackgroundNurses at The Norwegian Radium Hospital have reported that some patients notice an unpleasant smell or taste in accordance with flushing of intravenous lines with commercially available prefilled syringes. We have conducted a study in healthy volunteers to investigate the occurrence, consistency and intensity of this phenomenon.MethodsA randomised, blinded, crossover study comparing commercial available prefilled saline 9 mg/ml syringes to saline 9 mg/ml for injection in polyethylene package was performed in 10 healthy volunteers. The volunteers were given intravenous injections of varying volume and speed. Data were analysed using descriptive statistics, and also Wilcoxon Signed Rank Test to compare groups.ResultsAfter intravenous injection, 2 of 15 recordings demonstrated any sensation of smell or taste after injection of saline from polyethylene package, while 14 of 15 recordings noted a sensation after injection of saline from prefilled syringes. The intensity of the unpleasant sensation was rated significantly higher after injection of saline from prefilled syringes compared to saline from polyethylene (p = 0.001).ConclusionsInjection of saline from prefilled syringes in healthy volunteers resulted in an experience of bad taste or smell. It is important that nurses and health workers are aware of the phenomenon as described in this article in order to choose the preferred product for a given patient.

Pharmacokinetic analysis of two different docetaxel dose levels in patients with non-small cell lung cancer treated with docetaxel as monotherapy or with concurrent radiotherapy

BackgroundPrevious pharmacokinetic studies with docetaxel have mostly used 3-weekly (75 mg/m2 and 100 mg/m2) or weekly regimens (35–40 mg/m2). The pharmacokinetics and radiosensitizing efficacy of weekly 20 mg/m2 docetaxel, has however not been well characterized. We examined the pharmacokinetics of weekly docetaxel when administered with concurrent radiotherapy and compared the results with a 3-weekly 100 mg/m2 regimen.MethodsThirty-four patients with non small cell lung cancer (NSCLC) were included in this study, 19 receiving 100 mg/m2 docetaxel 3-weekly as single therapy, and 15 receiving 20 mg/m2 docetaxel weekly with concurrent radiotherapy. A newly developed HPLC method was used for measuring docetaxel levels, capable of quantifying docetaxel in plasma down to the nanomolar level.ResultsThe HPLC method showed detectable concentrations of docetaxel in plasma even after 72 hours. In the present study we have demonstrated that median docetaxel plasma levels of 3 nM can be obtained 72 hours after a dose of 20 mg/m2.ConclusionThe pharmacokinetics of docetaxel is characterized by great inter-individual variability and at some time points plasma concentrations for 20 mg/m2 and 100 mg/m2 docetaxel were overlapping. Extrapolation of these results indicates that radio sensitizing docetaxel concentrations may be present for as long as 1 week, thus supporting the use of 20 mg/m2 weekly docetaxel.

Evaluation of methotrexate tissue exposure by in situ microdialysis in a rat model.

The feasibility of using a microdialysis technique to obtain pharmacokinetic data on tissue exposure to methotrexate (MTX) was investigated. Microdialysis probes were implanted in the jugular vein, femoral muscle, and liver ofanesthetized male Wistar rats. MTX (100 mg/kg) was given as a bolus injection through an indwelling venous catheter, and blood samples were obtained through a second venous access and by microdialysis for a total of 6 h. Heparinized plasma, ultrafiltered plasma, and microdialysis effluent from tissue and venous probes were analyzed by high-performance liquid chromatography. Centrifugal ultrafiltration of rat plasma spiked in vitro with MTX (1–100 μM) revealed a mean binding to plasma proteins of 21%. In vitro microdialysis of this spiked plasma resulted in 23% relative recovery of the unbound fraction. In rats receiving MTX, plasma protein binding was 23% and the relative drug recovery as assessed with venous microdialysis probes was 18%. Plotting of unbound (i.e., ultrafiltrate) MTX concentrations in the blood against venous microdialysis perfusate values in the blood gave a good linear correlation with a coefficient of correlation (r2) of 0.98. There was also a linear correlation between the total MTX concentrations in venous blood and the drug levels in microdialysis samples from muscle and liver (r2=0.93 and 0.74, respectively). Area under the curve estimations were consistent with an MTX exposure of 30% and 46% for the muscle and liver as compared with the circulation. The present study demonstrates that the microdialysis technique can provide reproducible data on tissue exposure to MTX in an animal model and indicates that the methodology is adaptable to clinical settings.

Mycophenolate pharmacokinetics and inosine monophosphate dehydrogenase activity in liver transplant recipients with an emphasis on therapeutic drug monitoring

Abstract Background. The pharmacokinetics of the immunosuppressant mycophenolic acid (MPA) demonstrates high inter- and intra-patient variability. Variation in the binding of MPA to albumin has been postulated to be an important factor in this variability, and monitoring of free MPA has been suggested to improve therapeutic drug monitoring (TDM) of MPA. Inosine monophosphate dehydrogenase (IMPDH) is the target enzyme for MPA, therefore the IMPDH activity in lymphocytes can serve as a marker of the MPA-specific response. This study aimed to explore how the albumin concentration influences the free concentration of MPA in liver transplant recipients and to assess whether alteration in the free MPA influences IMPDH activity in CD4 + cells. Methods. Blood samples were taken from 20 liver transplant recipients on two separate occasions (days 3–5 and 16–21). Total and free concentrations of MPA, and IMPDH activity were measured during the first 4 h of each dose interval. Results. Albumin levels correlated with the free fraction of MPA. However, the total MPA and free MPA were equal predictors of the immunosuppressive response as defined by IMPDH activity. Conclusion. Total and free MPA are equally good predictors of the immunosuppressive effect exerted by MPA as defined by IMPDH activity. IMPDH activity measurements represent a promising approach to TDM in patients treated with MPA.

A sensitive and simple high-performance liquid chromatographic method for the determination of mitoxantrone in plasma.

A sensitive high-performance liquid chromatographic (HPLC) method for measuring the anthracene derivative, mitoxantrone, in plasma is described. After protein precipitation with 5-sulfosalicylic acid, samples are resolved by isocratic elution from a C18 reverse phase support and quantified by ultraviolet (UV) detection. Recovery of mitoxantrone after deproteinization was >70%. Within-run and between-day coefficients of variation (CVs) were 5.1 and 13.7%, respectively, at mitoxantrone concentrations of 20 nM (n = 6). The limit of detection was 2.5 nM and the standard curve linear up to 1 μM. Stability studies have shown that mitoxantrone is stable in spiked whole blood for 3–6 h, provided the samples are kept on ice. The drug is stable in plasma and deproteinized plasma samples for at least 24 h. The method requires few manipulations and is readily adaptable to automation.

SNPs in genes coding for ROS metabolism and signalling in association with docetaxel clearance

The dose of docetaxel is currently calculated based on body surface area and does not reflect the pharmacokinetic, metabolic potential or genetic background of the patients. The influence of genetic variation on the clearance of docetaxel was analysed in a two-stage analysis. In step one, 583 single-nucleotide polymorphisms (SNPs) in 203 genes were genotyped on samples from 24 patients with locally advanced non-small cell lung cancer. We found that many of the genes harbour several SNPs associated with clearance of docetaxel. Most notably these were four SNPs in EGF, three SNPs in PRDX4 and XPC, and two SNPs in GSTA4, TGFBR2, TNFAIP2, BCL2, DPYD and EGFR. The multiple SNPs per gene suggested the existence of common haplotypes associated with clearance. These were confirmed with detailed haplotype analysis. On the basis of analysis of variance (ANOVA), quantitative mutual information score (QMIS) and Kruskal–Wallis (KW) analysis SNPs significantly associated with clearance of docetaxel were confirmed for GSTA4, PRDX4, TGFBR2 and XPC and additional putative markers were found in CYP2C8, EPHX1, IGF2, IL1R2, MAPK7, NDUFB4, TGFBR3, TPMT (2 SNPs), (P<0.05 or borderline significant for all three methods, 14 SNPs in total). In step two, these 14 SNPs were genotyped in additional 9 samples and the results combined with the genotyping results from the first step. For 7 of the 14 SNPs, the results are still significant/borderline significant by all three methods: ANOVA, QMIS and KW analysis strengthening our hypothesis that they are associated with the clearance of docetaxel.