Final Drug Concentration Research Articles

P041 Preliminary evaluation of gradient concentration strips for detection of terbinafine resistance in Trichophyton spp.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesDermatophytosis is the most common superficial fungal infection. Trichophyton rubrum and T. mentagrophytes are the most frequently isolated species, but their incidence varies according to geographical regions. Terbinafine is the main molecule used to treat this type of infection. In recent years, a high incidence of chronic infections, reinfections, and treatment failures due to a newly described specie, T. indotineae, have been reported in India and recently described in Europe. It is currently a public health problem for the management of these infections in this country.Until now, the monitoring of dermatophyte susceptibility to antifungals was rarely performed due to the lack of standardized in vitro tests. Since then, an in vitro technique has been standardized by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) to test terbinafine and other antifungals. Recently, a gradient concentration strip method has been marketed.The aim of this study was to compare terbinafine susceptibility testing by the gradient concentration strip (GCS) method and the EUCAST standardized method.MethodsA panel of 47 molecularly identified isolates of T. interdigitale, T. mentagrophytes, and T. indotineae was used. The panel included 39 terbinafine- susceptible isolates and 8 terbinafine resistant isolates for which the squalene epoxidase gene was sequenced.Minimum inhibitory concentration (MIC) of terbinafine was determined using EUCAST microdilution broth method for dermatophytes. Inoculum was supplemented with cycloheximide and chloramphenicol. Final drug concentrations ranged from 0.008 to 8 μg/ml and microtiter plates were incubated at 25°C for 5 days. The MIC was determined spectrophotometrically with a 90% growth inhibition endpoint.MIC of terbinafine was also determined using GCS (Terbinafine Ezy MIC™ Strip, HiMedia, India) on RPMI agar. The plates were incubated for 5 days at 25°C. After incubation, MIC was read by using a complete inhibition endpoint. Isolates were considered wild-type when MIC was ≤ 0.125 μg/ml.ResultsEUCAST MIC values ranged from 0.008 to 0.0625 μg/mL and from 0.25 to 16 μg/ml for susceptible and resistant isolates, respectively.GCS MIC values ranged from 0.002 to 0.03 μg/ml and 0.125 to >32 for susceptible and resistant isolates, respectively.The categorical agreement (percentage of strains found in the same category) by the two techniques was 98%.ConclusionThese preliminary results show that GCS can detect resistance to terbinafine and could be used as a screening method. These results must be confirmed on a larger panel of isolates.

Shape-Tunable UV-Printed Solid Drugs for Personalized Medicine.

Several recent advances have emerged in biotherapy and the development of personal drugs. However, studies exploring effective manufacturing methods of personal drugs remain limited. In this study, solid drugs based on poly(ethylene glycol)diacrylate (PEGDA) hydrogel and doxorubicin were fabricated, and their final geometry was varied through UV-light patterning. The results suggested that the final drug concentration was affected by the geometrical volume as well as the UV-light exposure time. The analysis of PEGDA showed no effect on the surrounding cells, indicating its high biocompatibility. However, with the addition of doxorubicin, it showed an excellent therapeutic effect, indicating that drugs inside the PEGDA structure could be successfully released. This approach enables personal drugs to be fabricated in a simple, fast, and uniform manner, with perfectly tuned geometry.

Anti-tumor Synergistic Effect of a Dual Cancer-Specific Recombinant Adenovirus and Paclitaxel on Breast Cancer.

This study aimed at investigating the anticancer potential of the recombinant adenovirus Ad-apoptin-hTERTp-E1a (Ad-VT) and its synergistic combination with paclitaxel (PTX) in breast cancer treatment. First, we used the Calcusyn software to analyze the synergy between the Ad-VT and paclitaxel, and to determine the final drug concentration. Second, we used crystal violet staining and WST-1 assays to analyze the inhibitory effect of Ad-VT and paclitaxel combination treatment on MCF-7, MDA-MB-231, and MCF-10A cells. Subsequently, we used Hoechst, Annexin V, JC-1 staining to analyze the inhibition pathway of drugs on breast cancer cells. We also used Transwell assays to analyze the cell migration and invasion of MCF-7 and MDA-MB-231 cells. The pGL4.51 plasmid was used to transfect and to generate MDA-MB-231 cells, that stably express luciferase (MDA-MB-231-LUC). The in vivo tumor inhibition effect of Ad-VT and paclitaxel combination treatment was subsequently confirmed using a tumor-bearing nude mouse model. This combination treatment can increase the inhibition of breast cancer cells and reduce paclitaxel toxicity. Ad-VT had a strong apoptosis-inducing effect on MCF-7 and MDA-MB-231 cells, that was mainly mediated through the mitochondrial apoptotic pathway. The combination of Ad-VT and paclitaxel could significantly increase the inhibition of breast cancer cell migration and invasion. Combination of Ad-VT and paclitaxel can inhibit tumor growth and reduce toxicity in vivo. Ad-VT can also inhibit the growth of breast cancer cells and promote their apoptosis. Meanwhile, when it is combined with paclitaxel, Ad-VT could play a significant role in a synergistic tumor inhibition.

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp.

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL-1) and IVM at 1.1 μm (1 μg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Reply.

Arshinoff and Modabber5 call into question two assumptions of our mathematical model describing the residence times of moxifloxacin in the anterior segment with two different intracameral injection strategies.1 The authors of the letter agree with us on the underlying principle of our publication but disagree on the volume of the anterior chamber (AC) and the half-life of antibiotics. We selected two representative mean pseudophakic AC volumes (0.19 mL and 0.33 mL), substantiated by the literature,2,3 to demonstrate the principle that injecting the same dose of antibiotics (eg, 500 μg) into ACs with varying volumes produces varying concentrations of antibiotics within the total aqueous humor volume. The larger volume of 0.33 mL of Matsuura was derived from aqueous humor sampling immediately after intracameral injection in humans, before any possible fibrosis of the capsule. Libre and Mathews4 also agree with this volume. Arshinoff and Modabber's5 estimation of 0.5 mL is based on a mean phakic AC volume in the human eye of 0.25 mL; however, measurements of the AC volume in the elderly phakic eye average only 0.15 mL.6,7 With this experimentally derived volume, Arshinoff's estimation of the mean pseudophakic aqueous volume would be 0.4 mL, close to Matsuura and Libre's reported value of 0.33 mL. The AC size is known to vary from patient to patient, which cataract surgeons recognize when operating on smaller eyes and myopic eyes with longer axial lengths and larger chambers. Therefore, attempting to replace the aqueous contents with an antibiotic solution of known concentration will achieve the more consistent final concentration of drug, irrespective of the patient's pseudophakic AC volume. This same principle would have been demonstrated had we chosen two different AC volumes. There is a wide variation in the reported half-life elimination of antibiotics in the AC. Generally speaking, interdrug variation in elimination times, especially in the eye, may be due to factors such as differences in the drug molecular size, tissue binding, active pump mechanisms, and obstructions or reductions to outflow and drug elimination. Asena obtained antibiotic half-lives between 1.2 hours and 13 hours in rabbits, depending on which time points were used8; Lipnitzki et al.9 found between 0.64 hours and 1.8 hours in rabbits. Matsuura et al.3,10 demonstrated a half-life in rabbits and humans that averaged 1.2 hours over most time points. We chose 1.2 hours as it agrees with the 1% per minute aqueous turnover rate noted by Goel et al.11 The varied measures of the intradrug half-life elimination time of moxifloxacin and other antibiotics in the AC may be due to imprecision in injection and sampling techniques, pharmacokinetics that are not single compartment, and variations in individual subject anatomy and physiology. We would caution against the speculation of postantibiotic effects in regard to half-life because efficacy should be related to the target microorganism.12 Furthermore, principles that apply to the multidose administration of antibiotics in the treatment of systemic infections should not be presumptively applied to the scenario of single-dose intraocular injection without empirical evidence. Our study used two different and substantiated aqueous volumes and demonstrated that injecting small volumes of an agent can result in unequal final drug concentrations in the AC. Given the patient-to-patient variations, the “flushing” or large-volume injection technique should offer some degree of standardization and assurance of a more uniform final drug concentration in the pseudophakic AC as compared with smaller-volume–injected aliquots, thereby minimizing the interpatient variable of AC volume differences during cataract surgery.

Systematic Review of Variations in Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for Peritoneal Metastasis from Colorectal Cancer

Background: Cytoreductive surgery (CRS), followed by hyperthermic intraperitoneal chemotherapy (HIPEC), combines radical surgery with abdominal heated chemotherapy, constituting a multimodal treatment approach. Since clear standards for HIPEC conduct in colorectal carcinoma (CRC) are lacking, we aimed to provide a comprehensive structured survey. Data sources and study eligibility criteria: A systematic literature search was performed in PubMed, with keywords “HIPEC” and “colorectal cancer”, according to established guidelines. Articles were systematically screened, selecting 87 publications complemented by 48 publications identified through extended search for subsequent synthesis and evaluation, extracting inter alia details on used drugs, dosage, temperature, exposure times, and carrier solutions. Results: Compiled publications contained 171 reports on HIPEC conduct foremost with mitomycin C and oxaliplatin, but also other drugs and drug combinations, comprising at least 60 different procedures. We hence provide an overview of interconnections between HIPEC protocols, used drugs and carrier solutions as well as their volumes. In addition, HIPEC temperatures and dosing benchmarks, as well as an estimate of in vivo resulting drug concentrations are demonstrated. Conclusions and implications: Owing to recent developments, HIPEC conduct and practices need to be reassessed. Unfortunately, imprecise and lacking reporting is frequent, which is why minimal information requirements should be established for HIPEC and the introduction of final drug concentrations for comparability reasons seems sensible.

Mechanistic Modeling of Antibody-Drug Conjugate Internalization at the Cellular Level Reveals Inefficient Processing Steps.

Antibody-drug conjugates (ADC) offer an avenue for specific drug delivery to target cells. Here, parameters with important roles in the cellular processing of ADCs were quantitatively measured for Ab033, an antibody against EGFR. In EGFR-overexpressing cancer cell lines, Ab033 internalized at rates of 0.047/min and 0.15/min for A431 and H441 cells, respectively. Once internalized, Ab033 either trafficked to the lysosome or was recycled; up to 45% of internalized Ab033 returned to the cell surface. Despite such recycling, intracellular accumulation of Ab033 continually increased over 24 hours. Ab033 was conjugated to form a dual toxin ADC containing both cleavable and non-cleavable linker-drug payloads for release rate comparisons. Intracellular concentrations of freed drug from cleavable linker were greater than from non-cleavable linker and exceeded 5 × 106 drug molecules per A431 cell after 24 hours. Compared with intracellular antibody accumulation, formation of released drug was delayed, likely due to the time needed for endo-lysosomal trafficking and subsequent linker/antibody proteolysis. Informed by the quantitative data, a cellular ADC model was constructed and used to summarize processing inefficiencies. Modeling simulations were conducted to determine parameter sensitivity on intracellular drug concentrations, with rates of EGFR internalization and recycling as well as ADC trafficking found to be the most sensitive toward final intracellular drug concentrations. Overall, this study shows Ab033 ADCs to be a viable strategy for delivery of cytotoxic drugs into tumor cells with subsequent modeling efforts able to highlight key processing steps to be improved for increased drug delivery. Mol Cancer Ther; 17(6); 1341-51. ©2018 AACR.

Cathinone stability in authentic urine specimens

PurposeSynthetic cathinones are encountered in a variety of antemortem and postmortem forensic toxicology investigations. Earlier experimental studies using fortified urine have evaluated analyte, temperature and pH-dependent variables associated with their stability. The purpose of this study was to compare experimental findings with those obtained using authentic urine from cathinone users. MethodsIn this report we compare cathinone concentrations in 180 authentic unpreserved urine specimens, following known periods of refrigerated storage. These findings are compared with previously published experimental data using fortified drug-free urine. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) was used to target 22 cathinones. Quantitative results were compared in urine specimens (pH 4.5–10) following 5–17 months of storage. ResultsThe 180 specimens resulted in 164 quantitative findings involving α-PVP, ethylone, methylone, MDPV and pentylone. Initial drug concentrations ranged from 25ng/mL to over 100,000ng/mL. Upon reanalysis, the percentage of drug remaining (0–119%) was correlated with storage time and specimen pH. The ability to reconfirm original results was not correlated with storage time. Instead, specimen pH was far more predictive. The relationship between initial and final drug concentration was highly pH-dependent, yielding significant correlations for α-PVP, ethylone and methylone, particularly under acidic conditions. ConclusionsThese results are in good agreement with experimental findings and highlight the critical importance of specimen pH, rather than conventional time dependent variables, when considering cathinone stability in biological samples. The potential for pre-analytical changes in cathinone concentrations must be carefully considered when interpreting their results.

Editor's Highlight: PlacentalDisposition and Effects of Crizotinib: An Ex Vivo Study in the Isolated Dual-Side Perfused Human Cotyledon.

Tyrosine kinase inhibitors (TKIs) play an important role in cancer pharmacotherapy, yet there is limited data on their use during pregnancy. We studied placental disposition and placental toxicity of crizotinib, a TKI used to treat nonsmall cell lung cancer. Term placentas were perfused for 3 h with crizotinib (1 µM) using the ex vivo dual-side cotyledon perfusion technique. Interference of TKIs with trophoblast viability was studied using BeWo cells. Expression of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) in placental tissue was assessed by immunohistochemistry and inhibition of these transporters was determined in vitro by transport studies with membrane vesicles overexpressing human P-gp or BCRP. We found that crizotinib rapidly and strongly accumulates in cotyledon perfusion experiments, reaching a concentration of 3.1 ± 0.4 µM in placental tissue. Final drug concentrations in the maternal and foetal reservoirs were 0.2 ± 0.05 and 0.08 ± 0.01 µM, respectively. Furthermore, crizotinib inhibited BeWo cell viability (IC50: 234 nM, 95% CI: 167-328 nM) 10 times more potently than other TKIs tested. In vitro transport studies revealed that crizotinib is a potent inhibitor of the transport activities of BCRP (IC50: 5.7 µM, 95% CI: 2.7-11.8 µM) and P-gp (IC50: 7.8 µM, 95% CI: 3.4-18.0 µM). In conclusion, crizotinib strongly accumulated in placental tissue at clinically relevant concentrations. IC50 values for transporter inhibition and trophoblast cell viability were similar to the tissue concentrations reached, suggesting that crizotinib can inhibit placental BCRP and P-gp function and possibly affect trophoblast viability.

Nanoemulsion loaded gel for topical co-delivery of clobitasol propionate and calcipotriol in psoriasis

Current work reports the development and optimization of clobitasol propionate (CP) and calcipotriol (CT) loaded nanoemulsion based gel for topical treatment of psoriasis. Components of nanoemulsion viz., oil and surfactant/co-surfactant were selected depending upon solubility and emulsification potential respectively. The optimized ratio of 5:3:2 of Capmul MCM C8 EP, Cremophor RH 40 and Labrafil 1944 CS was selected. Carbopol 980 was used as gelling agent to achieve final drug concentration of 0.05% w/w and 0.005% w/w respectively for CP and CT. HaCaT cell lines showed higher uptake of drug from nanoemulsion in correlation with the enhancement in penetration of both drugs in stratum corneum (SC) and viable layer from nanoemulsion and gel as compared to free drugs. Imiquimod induced psoriatic BALB/c mice revealed significantly higher anti-psoriatic activity of nanoemulsion gel as compared to free drugs and marketed formulation. The developed formulation showed negligible skin irritation despite increased penetration into the skin.

Anticancer and Cytotoxic Potential of Turmeric (Curcuma longa), Neem (Azadirachta indica), Tulasi (Ocimum sanctum) and Ginger (Zingiber officinale) Extracts on HeLa cell line

Anticancer and Cytotoxic Potential of Turmeric (Curcuma longa), Neem (Azadirachta indica), Tulasi (Ocimum sanctum) and Ginger (Zingiber officinale) Extracts on HeLa cell line

W2: Practical considerations when using oral fluid as toxicological matrix

Introduction Over the last decades, the possibilities for monitoring the presence of drugs of abuse in oral fluid (OF) has increased enormously. Especially in the domain of testing drivers under influence of drugs (DUID), OF has gained popularity due to its non-invasive collected, directly on-site without hampering privacy. Several on-site immunosorbent tests (OIT) were developed over the years. For confirmatory analysis, the use of OF has been hampered by the influence of the salivary composition on the final drug concentrations. The OF collection protocol, the degree of stimulation of salivary flow, the physicochemical properties of drugs of abuse, and the type of drug administration are some of the parameters that influence the OF drug concentrations. Moreover, the final analytical result will be influenced by the OF collection. As a result, toxicological analysis of OF samples and the final interpretation is not always straightforward. Methods Laboratory and roadside studies, as well as double-blind placebo-controlled studies are used to evaluate the problems encountered using OF as confirmation matrix. The focus is put on Δ 9 -tetrahydrocannabinol (THC) analysis. The placebo-controlled study involves 10 test subjects smoking two subsequent doses of THC; 300 μg/kg and 150 μg/kg with a pause of 75 minutes using a Volcano vaporizer. Using the above mentioned studies, OIT (Drug-Test ® , Drager and Drugwipe ® , Securetec) and several OF collection devices (StatSure ® , Diagnostic Systems Inc; Quantisal ® , Immunalysis and Certus ® , Concateno) will be evaluated via UPLC-MS/MS analysis. Results Sensitivity of OIT seem to increase. The DrugTest 5000 and Drugwipe demonstrated respectively a sensitivity of 93% and 72% for Δ 9 -tetrahydrocannabinol during roadside studies. The Drugwipe is very sensitive just after smoking, but it rapidly decreases within 1.5 hours. The type of OF collection has to be specified when using OF as confirmation matrix, as THC recovery ranged from 50 to 70% and is dependent on the storage conditions. While matrix effects due to the collector buffers are observed, stability is often guaranteed via the stabilizing buffers. In a roadside study, we observed that after 5 minutes collection time, about 0.11–1.15 mL of OF was collected. Therefore, the final drug concentrations in neat OF have to be calculated via the following formula: C = [C UPLC/MS · (V1 + (W1–W2))] / [V2 x (W1–W2)] with C being the drug concentration in neat OF, C UPLC/MS the concentration obtained via the calibration curve, V1 the buffer volume, V2 the theoretical total collected volume and W1 and W2 respectively the weight of the collection device after and before OF collection. Neat OF THC concentrations in the placebo controlled study ranged from 12,361 ng/g 5 minutes after smoking down to 34 ng/g 80 minutes after 2 smoking sessions. Under placebo conditions, a median of 8 ng/g THC in OF was observed. The impact of all these observations on DUID legislations will be discussed. Conclusion OF has its limitations as a toxicological matrix. However, in situations where ‘recent’ drug use in combination with analytical cut-offs for interpretations are used, OF combines an easy collection of samples with a quick analysis of large batches.

Dosage uniformity problems which occur due to technological errors in extemporaneously prepared suppositories in hospitals and pharmacies

The availability of suppositories in Hungary, especially in clinical pharmacy practice, is usually provided by extemporaneous preparations. Due to the known advantages of rectal drug administration, its benefits are frequently utilized in pediatrics. However, errors during the extemporaneous manufacturing process can lead to non-homogenous drug distribution within the dosage units. To determine the root cause of these errors and provide corrective actions, we studied suppository samples prepared with exactly known errors using both cerimetric titration and HPLC technique. Our results show that the most frequent technological error occurs when the pharmacist fails to use the correct displacement factor in the calculations which could lead to a 4.6% increase/decrease in the assay in individual dosage units. The second most important source of error can occur when the molding excess is calculated solely for the suppository base. This can further dilute the final suppository drug concentration causing the assay to be as low as 80%. As a conclusion we emphasize that the application of predetermined displacement factors in calculations for the formulation of suppositories is highly important, which enables the pharmacist to produce a final product containing exactly the determined dose of an active substance despite the different densities of the components.

Influence of drug content, type of semi-solid vehicle and rheological properties on the skin penetration of the model drug fludrocortisone acetate

Throughout Europe, topical creams containing corticosteroids are diluted with various neutral cream bases to meet the specific needs of patients. Even though this practice has been common for years, its effect has not been thoroughly investigated and so the effectiveness of the diluted topical steroidal creams is difficult to predict. In the present study, the model drug fludrocortisone acetate was incorporated into three cream bases of different hydrophilicity that are commonly used in Austria. Different final drug concentrations were chosen for comparative studies. Additionally, a semi-solid preparation developed by our group was investigated for comparison. These formulations were tested in diffusion and tape stripping experiments. Diffusion cell studies showed that changes in drug concentration do not necessarily change the skin permeation behaviour in vitro. The tape stripping protocol was successfully optimised for investigation of semi-solid preparations to provide reproducible and accurate results despite the challenges of investigating semi-solid formulations. The results showed that tape stripping experiments are more suitable to elucidate subtle differences between formulations. The composition of the cream bases exhibited stronger effects on the skin penetration of the steroidal drug irrespective of its concentration than the rheological properties. No correlation between formulation viscosity and skin penetration was found.

Effect of Sedation on Pain Perception

Sedation or anesthesia is used to facilitate many cases of an estimated 45 million diagnostic and therapeutic medical procedures in the United States. Preclinical studies have called attention to the possibility that sedative-hypnotic drugs can increase pain perception, but whether this observation holds true in humans and whether pain-modulating effects are agent-specific or characteristic of IV sedation in general remain unclear. To study this important clinical question, the authors recruited 86 healthy volunteers and randomly assigned them to receive one of three sedative drugs: midazolam, propofol, or dexmedetomidine. The authors asked participants to rate their pain in response to four experimental pain tasks (i.e., cold, heat, ischemic, or electrical pain) before and during moderate sedation. Midazolam increased cold, heat, and electrical pain perception significantly (10-point pain rating scale change, 0.82 ± 0.29, mean ± SEM). Propofol reduced ischemic pain and dexmedetomidine reduced both cold and ischemic pain significantly (-1.58 ± 0.28, mean ± SEM). The authors observed a gender-by-race interaction for dexmedetomidine. In addition to these drug-specific effects, the authors observed gender effects on pain perception; female subjects rated identical experimental pain stimuli higher than male subjects. The authors also noted race-drug interaction effects for dexmedetomidine, with higher doses of drug needed to sedate Caucasians compared with African Americans. The results of the authors' study call attention to the fact that IV sedatives may increase pain perception. The effect of sedation on pain perception is agent- and pain type-specific. Knowledge of these effects provides a rational basis for analgesia and sedation to facilitate medical procedures.

Selection in culture of HIV resistance to dolutegravir by mutations at integrase positions R263K and H51Y that diminish viral replication fitness

Purpose of the studyWe selected for resistance in tissue culture against dolutegravir (DTG), a second‐generation HIV integrase strand transfer inhibitor, which is now in phase 2/3 clinical trials, in order to try to characterize the resistance profile of this compound.MethodsHIV‐1 of different subtypes was grown in both MT‐2 cells and peripheral blood mononuclear cells over protracted periods, with the concentration of DTG being incrementally increased from an initial concentration of 0.05 nM, i.e. 4 times less than the EC50. After a total of 6 months of growth, a final drug concentration of 50–100 nM was achieved, beyond which virus could no longer be grown. Viral DNA was then sequenced to reveal the presence of mutations that might confer resistance to DTG. The biological relevance of these mutations was confirmed through site‐directed mutagenesis experiments in which individual mutations or combinations of mutations were studied in comparison with wild‐type (wt) virus in tissue culture and with recombinant HIV integrase enzyme in biochemical assays.Summary of resultsThe most common integrase resistance mutation to arise in subtype B and recombinant A/G viruses was R263K followed by H51Y. In the case of subtype C viruses, the most common mutation was G118R followed by H51Y. The presence of R263K alone conferred an approximate 3–6‐fold level of resistance to DTG in culture, a 30% drop in levels of recombinant integrase strand transfer activity, as well as an approximate 20–30% loss in viral replicative capacity. Biochemical experiments indicated that the t½ residency times of DTG for subtype B and C viruses for wt integrase were 26 h and 38 h, respectively, and for R263K, 16h and 22h, respectively. In contrast, H51Y by itself did not significantly affect either strand transfer activity or resistance to DTG. However, the combination of R263K together with H51Y led to an increase in levels of DTG resistance to about 15‐fold accompanied by an ~50% loss in both viral replication capacity and integrase strand transfer activity.ConclusionsR263K and H51Y can combine to augment levels of resistance to DTG yet result in a more severe attenuation of viral replication capacity and integrase strand transfer activity than R263K alone. These data suggest that viruses containing both mutations may be at a severe replicative disadvantage and help to explain why primary resistance to DTG is so rare to arise in clinical studies performed to date.

Determination of antituberculosis drug concentration in human plasma by MALDI‐TOF/TOF

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix-assisted laser desorption/ionization source equipped with tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti-TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis-infected patients is reported. The volume of the plasma sample was 200 microL. Plasma samples were cleaned-up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 microL of 50% methanol-0.03% formic acid solution (v/v), spiked with known amounts of anti-TB drugs, mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI-TOF/TOF sample target plate. The anti-TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti-TB drug concentration range explored (the final anti-TB drug concentration ranged from 0.20 to 200 pmol/microL). The absolute recovery of the anti-TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI-TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/microL, respectively.

Reduction in Variation of Intravenous Drug Administration in Seventeen San Diego Hospitals with Standardized Drug Concentrations and Dosage Units

Purpose To standardize intravenous (IV) medication concentrations and dosage units across adult hospitals in San Diego County. Methods The San Diego Patient Safety Consortium Task Force comprised representatives from 17 adult hospitals. The first step was a survey of concentration standards and dosage units for the most common and highest-risk IV medications in use at all sites. Survey results were compared with manufacturers' recommendations and reference materials. A Delphi decision-making process was used to determine final drug concentration and dosage units for each drug. Results Survey results showed more than 85 concentrations used for 34 drugs. Nine of these were high-risk medications, for which 27 different concentrations were identified. With the exception of magnesium sulfate for nonobstetric use, a single concentration was developed for each medication, resulting in a greater than 94% reduction in variation from concentration standards. A single concentration standard was developed for each high-risk medication, resulting in a 77% reduction in variation. A single dosage unit standard was developed for all medications reviewed, resulting in a 100% reduction in variation. Of the 17 hospitals, 15 were assessed for adoption of the standards. Of these 15 hospitals, adoption of concentrations standards ranged from 72.2% to 100% and adoption of dosage units ranged from 84.8% to 100%. Conclusion County-wide standardization of high-risk IV drug concentrations and dosage units reduces variability in IV therapy, which can help promote safer and more consistent practices in IV medication administration. It is expected that standardization will decrease the potential for medication errors within hospitals and on patient transfer to other health care facilities.

Fosaprepitant: a neurokinin-1 receptor antagonist for the prevention of chemotherapy-induced nausea and vomiting

Chemotherapy-induced nausea and vomiting (CINV) is a distressing and common adverse event associated with cancer treatment. Updated antiemetic guidelines were published in 2008 by the National Comprehensive Cancer Network and, in 2006, by the American Society of Clinical Oncology, which have included the use of the new and more effective antiemetic agents 5-hydroxytryptamine-3 (5-HT3) receptor antagonist and neurokinin (NK)-1 receptor antagonist. Aprepitant is a selective NK-1 receptor antagonist approved as part of combination therapy with a corticosteroid and a 5-HT3 receptor antagonist for the prevention of acute and delayed CINV in patients receiving moderately and highly emetogenic chemotherapy. Fosaprepitant (also known as MK-0517 and L-758,298) is a water-soluble phosphoryl prodrug for aprepitant, which, when administered intravenously, is converted to aprepitant within 30 min of intravenous administration via the action of ubiquitous phosphatases. Owing to the rapid conversion of fosaprepitant to the active form (aprepitant), fosaprepitant 115 mg provided the same aprepitant exposure in terms of AUC as aprepitant 12 mg orally, and fosaprepitant is expected to provide a correspondingly similar antiemetic effect as aprepitant. Clinical studies have suggested that fosaprepitant could be appropriate as an intravenous alternative to the aprepitant oral capsule. In a study in healthy subjects, fosaprepitant 115 mg was generally well tolerated at a final drug concentration of 1 mg/ml, and fosaprepitant 115 mg was AUC bioequivalent to aprepitant 125 mg. Fosaprepitant in the dose of 115 mg has been approved by the US FDA, the EU and the Australian authorities on day 1 of a 3-day oral aprepitant regimen, with oral aprepitant administered on days 2 and 3. Fosaprepitant may be a useful parenteral alternative to oral aprepitant. Further study is needed to clarify the utility of fosaprepitant in the prevention of CINV and to clarify optimal dosing regimens that may be appropriate substitutes for oral aprepitant.

Rapid Throughput Screening of Apparent KSP values for Weakly Basic Drugs Using 96-Well Format

A rapid-throughput screening assay was developed to estimate the salt solubility parameter, KSP, with a minimal quantity of drug. This assay allows for early evaluation of salt limited solubility with a large number of counter-ions and biologically promising drug leads. Drugs dissolved (typically 10 mM) in DMSO are robotically distributed to a 96-well plate. DMSO is evaporated, and drugs are equilibrated with various acids at different concentrations (typically <1 M) to yield final total drug concentrations around 2.5 mM. The plate is checked for precipitation. Filtrates from only those precipitated wells were subjected to rapid gradient HPLC analysis. An iterative procedure is employed to calculate all species concentrations based on mass and charge balance equations. The apparent KSP values assuming 1:1 stoichiometry are determined from counter-ion and ionized drug activities. A correlation coefficient >0.975 for eight drugs totaling 16 salts is reported. Intra-day and inter-day reproducibility was <10%. Conventional apparent KSP measurements were translated to 96-well format for increased throughput and minimal drug consumption (typically 10 mg) to evaluate at least eight different counter-ions. Although the current protocol estimates KSP from 10−3 to 10−7 M, the dynamic range of the assay could be expanded by adjusting drug and counter-ion concentrations.