Drug Dilution Research Articles

Antibacterial activity of selected siddha herbo-mineral preparations against clinically isolated enterococcus species

Objective: In siddha medicinal practice the use of plant extracts as well as inorganic natural preparations has a vital role as medicines. The present research in the field of phytomedicine is to evaluate the antibacterial properties of three herbo- mineral siddha drugs (Palakarai parpam, Padikara parpam, Uppu chenduram) on clinically isolated Enterococcus strains. Materials and Methods: Fifteen bacterial strains isolated from clinical samples were identified as Enterococcus faecalis(7), E. faecieum(8).The susceptibility of the microorganisms to the siddha drugs (Palakarai parpam, Padikara parpam, Uppu chendooram) were screened by disc diffusion method and the effective drug’s MIC value was calculated by agar dilution method. Results: Padikara parpam was considered sensitive against the tested Enterococcus faecalis and Enterococcus faecium .Agar dilution was performed with 1%, 0.5%, 0.25% and 0.12% of the drug dilution. MIC was found to be 0.5%. Conclusion: The drug Padikara parpam contains essential elements which are considered to be good anti-microbial activity against En-terococcus spp. To conclude, the claim in the siddha system of medicine is scientifically validated.

BEA-001 Building Up a Regional and Interdisciplinary Network For Better Use of Medicines in Intensive Care Units

BackgroundClinical pharmacy in intensive care units (ICUs) showed beneficial effects on safety and economics. The establishment of a regional network including pharmacists, physicians and nurses of all ICUs seemed useful...

A study on the effects of a calcium drug on the bone mineral density (BMD) by using dual-energy X-ray Absorptiometry (DXA)

Measurements of osteoporosis might contain errors caused by the calcium drug used in the prevention and the treatment of osteoporosis. This study conducted a lumbar spine phantom experiment to examine whether a calcium drug can influence the measured values of the bone mineral density (BMD) because of the drug taken by a real patient remaining undigested in the stomach. Dual-energy X-ray Absorptiometry (DXA) was used to measure the BMD for a calcium-drug in an equipment-dedicated lumbar spine phantom and 10 patients selected for the BMD measurement. Three types of drugs that are prescribed in actual clinical practice calcium drugs were used for the phantom experiment, and the drugs were divided into a fixed dose, 1/2 of the fixed dose, 1/4 of the fixed dose and 1/8 of the fixed dose. Without the drugs included, the phantom was scanned 60 times continuously to calculate the baseline BMD. The BMD was measured as the calcium drug coated with paraffin was placed in the lumbar vertebra 2 and the soft tissue region of the phantom. To determine when the drug was invisible to the naked eye are measured, the BMD at different drug dilutions. The measurements were conducted three times to calculate the mean. In the patient experiment, patients were selected who visited hospital after taking the drug before measuring the BMD. After a certain time had passed, the BMD was measured again to examine the difference in images and the change in BMD values due to the calcium-drug intake. The BMD measurements of lumbar 1–4 in the phantom were higher, with statistical significant, than the least significant change (LSC) in the bone region for all three drugs (Ca carbonate, Ca citrate and Ca cholecalciferol), showing a significant increase. On the other hand, there was no significant change in the soft tissue. When Ca Cholecalciferol was used in a fixed dose, the BMD of L2 increased by 11.6%, showing the largest increase among the drugs examined, but only a 2.8% increase in the BMD of L1–L4 was observed. The dependence of the results on the degree of dilution of the calcium drug showed that the three drugs had values that exceeded the LSC significantly. The measured the BMD was higher in 7 out of 10 patients when the patients took the calcium drug than when they did not. In addition, one of the patients showed a 3.6% increase in BMD. In addition, the calcium drug appeared in the image in two of the patients who showed an increase in the BMD. This confirmed that the drug remained undigested, with a certain amount of it remaining in the body system. Overall, the measured BMD is affected when the calcium drug taken by a real patient remains undigested in the stomach.

Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilum to nine antimicrobials

A multi-laboratory broth microdilution method trial was performed to standardize the specialized test conditions required for the fish pathogens Flavobacterium columnare and F. psychrophilum. Nine laboratories tested the quality control (QC) strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 against 10 antimicrobials (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole) in diluted (4 g l-1) cation-adjusted Mueller-Hinton broth incubated at 28 and 18°C for 44-48 and 92-96 h, respectively. QC ranges were set for 9 of the 10 antimicrobials. Most of the minimal inhibitory concentration (MIC) distributions (16 of 18, 9 drugs at both temperatures) for A. salmonicida ATCC 33658 were centered on a single median MIC ± 1 two-fold drug dilution resulting in a QC range that spanned 3 dilutions. More of the E. coli ATCC 25922 MIC distributions (7 of 16) were centered between 2 MIC dilutions requiring a QC range that spanned 4 dilutions. A QC range could not be determined for E. coli ATCC 25922 against 2 antimicrobials at the low temperature. These data and their associated QC ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI), and will be included in the next edition of the CLSI M49-A Guideline. This method represents the first standardized reference method for testing fish pathogenic Flavobacterium spp.

Daratumumab, a CD38 Monoclonal Antibody in Patients with Multiple Myeloma - Data From a Dose-Escalation Phase I/II Study

AbstractAbstract 73 BackgroundDaratumumab (HuMax™-CD38) is a human CD38 monoclonal antibody with broad-spectrum killing activity and effectively mediates killing of CD38-expressing tumor cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis. In this present ongoing first-in-human (FIH) dose-escalation study of daratumumab in pts with multiple myeloma (MM) (ClinicalTrials.gov CT00574288), the safety profile has been acceptable and preliminary efficacy data have already been published1,2. Here we present data from the dose escalation part of the study. ObjectivesThe primary objective was to establish the safety profile. The secondary objectives were to establish the maximum tolerated dose (MTD), assessment of efficacy, pharmacokinetics (PK) and immunogenicity – Anti-Drug-Antibodies (ADA). MethodsPts ≥18 years and diagnosed with MM requiring systemic therapy and considered relapsed or refractory to at least two different prior lines of therapy and ineligible for ASCT were enrolled. The study was based on a 3+3 dose-escalation design. Daratumumab was administered over a 9-week period consisting of 2 pre- and 7 full doses. The doses ranged from 0.005 mg/kg to 24 mg/kg. Prednisolone/methylprednisolone was administered to prevent infusion related events (IREs) in a maximum dose equivalent to 27mg dexamethasone per week. Daratumumab plasma concentrations were measured by ELISA. Evaluation of efficacy data was according to IMGW guidelines3. A bridging ElectroChemiLuminesence (ELC) method on the MesoScale Discovery platform was used to detect ADA responses in pts to daratumumab.The results presented are based on data analyzed before database lock. ResultsData from 32 pts including 2 pts in the ongoing 24mg/kg cohort were collected until now. Median age was 61 years (42–76). The median number of prior treatment lines was: 6.3 (2–12).PK analysis showed plasma peak levels as expected, but relatively rapid clearance at low dose levels. The clearance rate decreased with increasing dose suggesting an effect of target binding on the PK. At doses ≥ 4 mg/kg, daratumumab trough levels were consistent ≥ 10 μg/ml and observed PK values approximately estimated PK values (Figure 1).Preliminary efficacy evaluation in this abstract was based on best response to paraprotein as reflected by change in serum or urine M-component or free light chains (FLC) according to IMGW guidelines3. For groups ≤ 2 mg/kg, 4/20 pts achieved a reduction in paraprotein (12%, 14%, 19%, 55%); in the 4 mg/kg group, 3/3 pts had a reduction in paraprotein of 49%, 100%, and 64%, respectively. In the 8 mg/kg group, 2/3 pts had a reduction in paraprotein of 39%, and 100%, respectively whereas in the 16mg/kg cohort, 2/3 pts had a reduction in paraprotein of 50%, and 33%, respectively. A reduction of 80%-100% in the bone marrow plasma cells was seen in the 4 mg/kg group and onwards. No detectable ADA responses were found in the pts.No DLTs were reported in the 2, 4, 8 and 16mg/kg cohorts. The most common adverse events reported were infusion related events. The observed IREs occurred predominantly during the initial infusions, 10% of pts reported IREs during the pre-dose, 30% during the first full infusion with a gradual decrease in frequency after the 2nd full infusion. No dose relationship was observed. Most IREs had onset within 3–4 hours of infusion, two of the IREs were grade 3 and the remaining grade 1–2. Four IREs were SAEs; however, since implementation of steroids before all infusions and dilution of trial drug, no serious IREs were reported in the 4, 8 and 16mg/kg cohorts. ConclusionIn pts with relapsed or refractory MM treated with daratumumab, a marked reduction in paraprotein and bone marrow plasma cells was observed in the higher dose cohorts. This has not previously been demonstrated with a single-agent monoclonal antibody in MM. No unexpected buildup of daratumumab was seen and in pts treated with 4mg/kg and upwards the observed plasma concentrations were close to those predicted. No ADA responses were detected. The MTD was not yet established and the toxicity was manageable. All data available from part 1 will be presented at the meeting. [Display omitted] Disclosures:Plesner:Genmab: Consultancy; Janssen Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees. Lokhorst:Genmab: Consultancy. Lisby:Genmab: Employment. Richardson:Millenium Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees.

Identification of Small Molecules Selectively Targeting Leukemic Stem Cells within Their Stromal Niche

Abstract 413Current therapies for acute myeloid leukemia (AML) are highly toxic, yet the relapse rate remains high. New therapies are needed to improve cure rates while decreasing toxicity. Because therapies may be affected by the tumor niche, we aimed to test new compounds on leukemic stem cells (LSCs) within their stromal microenvironment. A niche-based high throughput screen identified candidate small molecules potentially toxic to MLL-AF9 murine leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell et al, Blood 118, Abs 760, 2011.) Three such compounds, including a selective serotonin receptor antagonist highly specific for the 5-HT1B receptor, SB-216641, and two antihelminthics, parbendazole and methiazole, were found to be effective and selected for studies on human leukemias.We first examined SB-216641, studying the effects of this compound on 7 human primary AML samples. We began by assessing the compound’s effect on LSCs using the week 5 cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. CD34+ cells were isolated with immunomagnetic beads. The leukemic cells were pulse treated for 18 hours and washed prior to placement on MS-5 murine stroma. We performed serial drug dilutions using the CAFC assay with the human primary samples as well as with HSCs derived from cord blood. All human leukemic samples formed cobblestone areas in the control setting (46-200 CAFCs/106 cells plated). IC50 for the human primary leukemia CAFCs was 630 nm, and at 10 μM all LSCs were killed while normal human HSCs had 100% survival. A combination of the AML cell line HL60 transduced with GFP-luciferase and normal cord blood CD34+ cells (1:200) were then pre-incubated overnight with SB-216641 at 5 and 10 μM and injected into Nod Scid IL2R-gamma null (NSG) mice. The control mice had leukemic engraftment by luciferase imaging and flow cytometry and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. Primary patient AML samples were also pre-incubated overnight with SB-216641 at 10 μM and injected into NSG mice. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had no engraftment following exposure to SB-216641. Finally, an in vivo study was completed on NSG mice injected intraperitoneally with 20 mg/kg/day beginning on day 1 or day 8 after inoculation with HL60 (500 cells). The mice were imaged at 2 and 3 week time points and both treatment groups had significantly less leukemia on imaging than the control group with minimal toxicity noted. Another specific 5-HT1B receptor antagonist, SB-224289, was found to have similar activity to SB-216641 against leukemic cells and to spare HSCs in preliminary studies.Similar CAFC studies with serial dilutions on primary AML samples were performed on the two anti-helminthic agents. IC50 for parbendazole was 1.25 μM and for methiazole 5 μM. As shown by luciferase imaging and flow cytometry, when injected with combined HL60 and cord blood pre-incubated overnight at 5 and 10 μM with each compound as described above, the control mice engrafted with leukemia and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. NSG mice were then injected with primary AML pretreated overnight with parbendazole at 10 μM. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had significantly lower engraftment following exposure to parbendazole (p = 0.01).Two new avenues of leukemia therapy were discovered warranting further investigation. SB-216641, an agent with a completely novel receptor target in leukemia therapy, has shown both in vitro success in human leukemia as well as preliminary success in vivo with minimal toxicity. We aim to move forward with this agent while also testing parbendazole in vivo, as this compound is already known to have good pharmacokinetics and minimal toxicity in animals. The high toxicity to LSCs and sparing of normal HSCs give both these agents an attractive profile for future clinical trials. Disclosures:Ebert:Genoptix: Consultancy; Celgene: Consultancy.

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority. Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9'A mice (1) and α6 L9'S mice (2), allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices. In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

Visualization of subcutaneous insulin injections by x-ray computed tomography

We report how the three-dimensional structure of subcutaneous injections of soluble insulin can be visualized by x-ray computed tomography using an iodine based contrast agent. The injections investigated are performed ex vivo in porcine adipose tissue. Full tomography scans carried out at a laboratory x-ray source with a total acquisition time of about 1 min yield CT-images with an effective pixel size of 109 × 109 μm2. The depots are segmented using a modified Chan–Vese algorithm and we are able to observe differences in the shape of the injection depot and the position of the depot in the skin among equally performed injections. To overcome the beam hardening artefacts, which affect the quantitative prediction of the volume injected, we additionally present results concerning the visualization of two injections using synchrotron radiation. The spatial concentration distribution of iodine is calculated to show the dilution of the insulin drug inside the depot. Characterisation of the shape of the depot and the spatial concentration profile of the injected fluid is important knowledge when improving the clinical formulation of an insulin drug, the performance of injection devices and when predicting the effect of the drug through biomedical simulations.

Estudio Multicéntrico español para la Prevención de Errores de Medicación. Resultados de cuatro años (2007-2011)

ObjectiveThe primary objective of the Spanish Multicentre Study for the Prevention of Medication Errors 2007-2011 was to increase patient safety by improving drug practices in 26 participating Spanish hospitals. The secondary objective was to ascertain the medication error rate. MethodWe used a modified Barker and McConnell observation method. ResultsDuring 2007-2008, 21 009 observations were recorded In 23 hospitals; during 2008-2009, 11 320 observations were recorded in 10 hospitals; during 2009-2010, 6819 observations were recorded in 8 hospitals, and during 2010-2011, 5876 observations were recorded in six hospitals. Error rates, including medication time errors and failure to inform patients came to 21.7%, 33.3%, 35.6% and 25.7% in each of the years respectively. Excluding time errors, the rates were 18.2%, 32.2%, 33.4% and 23.5%; excluding failure to inform patients as well, rates dropped to 12.6%, 14.8%, 12.8% and 8.6%. Strategies for improvement: a normalised drug administration timetable, normalised IV drug dilutions and rates, better drug administration coordination with mealtimes, electronic prescription, improved patient identification measures, adjusting doses for kidney function, drug reconciliation programmes and patient and drug bar code scanning. Conclusions1. The applied measures improved patient safety through better drug practices in participating EMOPEM hospitals. 2. Drug error rates obtained for this sample of Spanish hospitals are some of the highest published. They are significantly lower, however, when we exclude time errors and failure to inform the patient.

A New Photometric Assay with Bromocresol Purple for Testing in vitro Antitrichomonal Activity in Aerobic Environment

A new colorimetric assay relying on the acidic metabolism of Trichomonas vaginalis was developed for in vitro screening of various compounds against axenically grown trichomonads. Parasites from continuous culture were exposed to series of drug dilutions in a microtiter plate. After an incubation period of 48 h at 37 degrees C, the pH indicator of the medium had changed its colour in non-inhibited cultures due to the production of lactate and acetate. Inhibited cultures showed no colour changes. The use of bromocresol purple, a pH indicator, was suitable for several reasons: it is not toxic at the concentration used in the assay; the absorbance of bromocresol purple at 405 nm showed a linear correlation with both the pH of the medium and the viability of the trichomonads observed microscopically; plates could easily be read by eye or using an enzyme-linked immunosorbant assay (ELISA) reader. By comparison of the decreases in absorbance in test cultures with those in control cultures, IC50 values could be determined. Thus, IC50 of metronidazole was calculated at 25.5 +/- 2.3 mumol/l (n = 5) with the bromocresol purple assay and about 25 mumol/l after microscope observation. Minimal lethal concentrations (MLCs) could be read by eye. This test is now routinely used for anti-trichomonas evaluation of various chemical compounds and natural products.

A Phase I/II, Dose-Escalation Study of Daratumumab, A CD38 Mab in Patients with Multiple Myeloma – Preliminary Safety Data

Abstract 1873 Background:Patients with multiple myeloma (MM) relapsed or refractory to current treatment options and ineligible for ASCT have a poor prognosis. Therefore new treatment options are highly needed for this patient population. Malignant MM cells exhibit very strong CD38 surface expression. Daratumumab is a human CD38 antibody with broad spectrum killing activity. Daratumumab mediates MM cell death via antibody dependent cellular cytotoxicity, complement dependent cytotoxicity and apoptosis. Daratumumab has been shown to inhibit growth of CD38-expression tumor cells in SCID mouse xenografts. We report the preliminary safety results from the ongoing first in man dose-escalation study (clin.trial.gov. NCT00574288). Methods:Patients (age > 18 yrs) ineligible for ASCT and relapsed or refractory to at least two different prior therapies receive 7 full and 2 pre-dose infusions of daratumumab (1st pre-dose day 1, 1st full dose day 2, 2nd pre-dose day 21, 2nd full-dose day 22 and thereafter weekly full-dose daratumumab). The study design encompasses a classic 3 + 3 dose escalation with the possibility to limit exposure to 1 patient in the first 2 cohorts pending safety data. The dose range is from 0.005 mg/kg to 24 mg/kg. The primary objective is to establish the safety profile and secondary objectives are to establish MTD and evaluate the efficacy. An independent Data Monitoring Committee evaluates the safety data for each cohort before dose-escalation. Results:Until May 31st, 2011 safety data for 20 patients, including dose range from 0.005 mg/kg up to 2 mg/kg cohort, were collected. Median age 62 yrs (42–76), F/M: 7/13, stage of MM (ISS): 1: 5 patients; 2: 9 patients; 3: 5 patients and 1 unknown. The most common AEs reported across all cohorts are pyrexia (35%), free hemoglobin (25%), anemia (25%), proteinuria (25%), cough (15%), dizziness (10%), flushing (10%), influenza-like-illness (10%), nausea (10%) hypo/hypertension (10%/10%), thrombocytopenia (10%), hemolysis (15%), lymphopenia (10%), arthralgia (10%), abdominal pain (10%) and chest pain (10%). Five SAE were assessed as related to daratumumab. One patient had anemia grade 3 and thrombocytopenia grade 4, one patient experienced cytokine release syndrome grade 2 and based on these DLT events 3 more patients were enrolled in the 0.1 mg/kg and 1.0mg/kg cohorts, one patient experienced a grade 3 bronchospasm 40 hours after end of trial drug infusion and one patient had AST elevation. All patients recovered after relevant treatment. Since implementation of relevant pre-medication and dilution of trial drug no severe infusion related AEs have been reported.Overall summary of AEs – safety population≤0.1 mg/kg0.5 mg/kg1 mg/kg2 mg/kgTotalPatients836320Related AE grade > 330003Related SAEs30115All data are number of patientsThe MTD has not been reached at the cut-off time point for this evaluation.Efficacy evaluation is ongoing and preliminary data will be presented at the meeting. Conclusion:Daratumumab has a favorable safety profile as monotherapy in patients with multiple myeloma in doses up to and including 2 mg/kg. The dose-escalation is ongoing and updated safety data will be presented at the meeting. When the MTD is established, an extension study with daratumumab as monotherapy will be conducted. Disclosures:Plesner:Genmab A/S: Consultancy. Lokhorst:Genmab: Consultancy. Valentin:Genmab A/S: Employment. Lisby:Genmab A/S: Employment. Richardson:Genmab: Consultancy.

A Synthetic HIV-1 Subtype C Backbone Generates Comparable PR and RT Resistance Profiles to a Subtype B Backbone in a Recombinant Virus Assay

In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C) viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT) fragments from 8 HIV-1 subtype C-infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT) using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample) were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE), p24 and Viral Load (VL) were monitored. The resulting HIV-1-C recombinant virus stocks (RVS) were added to MT4-eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI) to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i) functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii) their rate of infection was slower than viruses generated in the subtype B backbone; iii) they did not produce clear CPE in MT4 cells; and iv) drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT.

Developing a passive sensitization procedure for basophils to diagnose IgE-mediated drug hypersensitivity (151.19)

Abstract Introduction: The basophil activation test is quite well established for protein allergens. It is normally done with the patients’ basophils and requires fresh cells and rapid processing. It would be a particularly interesting test for drug allergy, as only very few drug specific in vitro IgE assays exist and it seems to be a highly relevant read out system. Objective: To set up a passive sensitization protocol to test IgE-mediated drug hypersensitivity. Methods: PMBCs of basophil donors are incubated with lactic acid (pH=3.9) to remove surface bound IgE. Afterwards, re-sensitization with patient serum and subsequent stimulation with serial dilutions of drugs. Degranulation can be measured by flow cytometry (anti-CCR3-APC/anti-CD63-PE). Results: We were able to establish a well functional procedure for protein allergens and for some drug allergens (cephalosporines, NMBAs). Passive sensitization assays using serum of patients with well-documented IgE-mediated amoxicillin allergy were reproducibly false negative. Conclusion: Using defined donor basophils for passive sensitizations with serum would have several advantages over direct assays, as it would facilitate diagnosis, as patient’s sera is easy to store and as it would provide a direct proof of an IgE-mediated hypersensitivity reaction. However, a better understanding of how small drugs are actually able to cross-link cell surface bound IgE seems to be a prerequisite to improve this assay and to widen its application.

Effects of Flow Rate Modifications on Reported Analgesia and Quality of Life in Chronic Pain Patients Treated with Continuous Intrathecal Drug Therapy

We compared the analgesia and the quality of life of a constant daily dose of intrathecal drug administered at different flow rates in patients treated for chronic pain. We postulate that the quality of the analgesia, at the same daily dose, will show an infusion rate dependent pattern with decreased pain at higher flow rates. Twenty consecutive patients on stable intrathecal treatment were included in a double-blind three-period crossover study where the same daily dose was administered at single, double, and quadruple flow rates in a randomized sequence. The mean daily pain score and the quality of life (EuroQol measure of health outcome [EQ-5D]) were measured following each flow rate change, after 1 week of stabilization. Results. Visual analog scale (VAS) scores remained unchanged with all flow rates. Compared with the lowest flow rate, the EQ-5D index decreased with double and even more with quadruple flow rate, suggesting a clinically relevant worsening of the health state with higher flow rates. Adverse events were equally distributed in all groups. Pain VAS did not significantly change with flow rate. This is consistent with preclinical data showing very limited increase in drug distribution in the cerebrospinal fluid with much larger flow rate augmentation. However, the quality of life decreased consistently as the flow rate increased. This was entirely due to a worsening of the pain and anxiety dimension of the EQ-5D questionnaire, caused presumably by a slight increase in pain rather than adverse events. We suggest that at higher flow rates increased drug dilution results in a decreased effect at the receptor site.

Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs

Tumor tissues are often hypoxic because of defective vasculature. We previously showed that tumor tissues are also often deprived of glucose. The efficacy of anticancer drugs is affected by the tumor microenvironment, partly because of the drug delivery and cellular drug resistance; however, the precise mechanisms remain to be clarified. In the present study, we attempted to clarify whether hypoglycemic/hypoxic condition, which mimics the tumor microenvironment, might induce drug resistance, and if it did, to elucidate the underlying mechanisms. Pancreatic cancer-derived PANC-1 cells were treated with serial dilutions of anticancer drugs and incubated in either normoglycemic (1.0 g/L glucose) or hypoglycemic (0 g/L glucose) and normoxic (21% O(2)) or hypoxic (1% O(2) ) conditions. The 50% inhibitory concentration of gemcitabine was 1000 times higher for PANC-1 cells incubated under the hypoglycemic/hypoxic condition than for those incubated under the normoglycemic/normoxic condition. Conventional anticancer drugs target rapidly growing cells, so that non-proliferating or slowly proliferating cells usually show resistance to drugs. Though the cell cycle was delayed, sufficient cellular uptake and DNA incorporation of gemcitabine occurred under the hypoglycemic/hypoxic condition to cause DNA lesions and S-phase arrest. To overcome hypoglycemic/hypoxia-induced drug resistance, we examined kinase inhibitors targeting Chk1 or cell-survival signaling pathways. Among the compounds examined, the combination of UCN-01 and LY294002 partially sensitized the cells to gemcitabine under the hypoglycemic/hypoxic condition. These findings suggested that the adoption of suitable strategies may enhance the cytotoxicities of clinically used anticancer drugs against cancer cells.

Paclitaxel Gelatin Nanoparticles for Intravesical Bladder Cancer Therapy

We have noted that inadequate drug delivery to tumor cells is a major cause of failed intravesical therapy for nonmuscle invading bladder cancer, partly due to the dilution of drug concentration by urine production during treatment. To address this problem we developed gelatin nanoparticles of paclitaxel designed to yield constant drug concentrations. The hypothesis that a constant, therapeutic concentration in urine, bladder tissue and tumors can be attained was evaluated in dogs. We studied drug release from paclitaxel gelatin nanoparticles in culture medium in vitro. In vivo studies were performed in tumor-free dogs and in pet dogs with naturally occurring transitional cell carcinoma, in which the pharmacokinetics of paclitaxel gelatin nanoparticles were determined in plasma, urine and tumors. Paclitaxel release from paclitaxel gelatin nanoparticles in vitro and in vivo was rate limited by the drug solubility in aqueous medium. This property yielded constant drug concentrations independent of changes in urine volume during the 2-hour treatment. Intravesical paclitaxel gelatin nanoparticles showed low systemic absorption, and favorable bladder tissue/tumor targeting and retention properties with pharmacologically active concentrations retained in tumors for at least 1 week. Constant drug release from paclitaxel gelatin nanoparticles may overcome the problem of drug dilution by newly produced urine and the sustained drug levels in tumors may decrease treatment frequency.

Screening of Antimicrobial Activity of Alcoholic & Aqueous Extract of Some Indigenous Plants

Plants known to possess several antimicrobial compounds are used in all traditional medicine. Our study was to screen the leaf extracts of Aloe vera, Datura stromonium, pongamia pinnata, Lantona camara , Calotropis procera. These plants were collected and aqueous and alcoholic extracts were prepared by decoction and hot percolation process. Microbes for study are staphylococcus, E.coli and Aspergillus species. They were isolated from different sources and identified by morphological and chemical tests. Different dilutions of the test drugs were prepared with saline and by using turbidity method MIC was found and anti fungal activity by spore germination method. Alcoholic extracts displayed higher antibacterial and anti fungal activity than aqueous extract. Results concluded that Aloevera had the highest and strong activity against S.aureus and E.coli. Lanata camara does not show anti bacterial and anti fungal activity. Pongamia pinnata aqueous extract showed better anti E.coli activity than its alcoholic extract. Datura stramonium showed better activity against staphylococcus aureus & showed little anti Aspergillus activity. Calotropis procera showed antibacterial activity against Staphyloccus aureus & E.coli and does not showed anti Aspergillus activity. © 2011 IGJPS. All rights reserved

Abstract P6-15-13: Biologic Activity of a Novel Heat Shock Protein 90 Inhibitor, PF-4928473, in Human Breast Cancer Cell Lines

Abstract Background: Heat shock protein 90 (HSP90) is a key molecular chaperone involved with the post-translational folding and maturation of various cellular proteins, including Raf, Src, ER, EGFR, HER2/ErbB2, IGFR, and VEGFR. Therefore, targeting HSP90 represents an attractive strategy for modulating aberrant oncogenic signal transduction in human malignancies. In the present study, we investigate the anti-cancer activity of PF-4928473, a novel small molecule inhibitor of HSP90, in a diverse panel of human breast cancer cell lines in vitro. Methods: 49 breast cancer cell lines (representing 10 luminal, 18 HER2- amplified, 18 non-luminal, 3 immortalized subtypes), including two HER2- amplified lines with conditioned trastuzumab and lapatinib resistance (SKBR3 parental), were assessed. Cell proliferation assays with two-fold drug dilutions over 9 concentrations were performed over 6 days to determine growth-adjusted IC50 and IC100 values, the latter as a surrogate of in vitro drug lethality. Flow-cytometry was performed in a selected subset of 21 cell lines to assess effects on cell cycle and apoptosis after 48 hours and 5 days of drug exposure, respectively. Western blot analysis evaluated the biochemical effects of HSP90 drug inhibition. Results: In our panel, IC50s ranged from 8 (±1) nM to 525 (±2) nM, with 45 cell lines exhibiting IC50s ≥50 nM. Differential drug sensitivity was observed with HER2-amplified lines, with 15 of 18 demonstrating IC50s ≥28 nM. For SKBR3-trastuzumab and -lapatinib-resistant lines, IC50s were 13 (±1) nM and 21 (±2) nM, respectively. IC100s ranged from 9 (±3) nM to > 1000 nM. 17 of 18 HER2-amplified lines had IC100s between 9 (±3) nM and 69 (±1) nM, including SKBR3-trastuzumab and -lapatinib-resistant lines at 31 (±2) nM and 66 (±2) nM, respectively. Flow cytometry with 50 nM of PF-4928473 revealed moderate early apoptotic effects in cell lines with IC50s < 30 nM, encompassing the majority of HER2-amplified lines. Mild effects on cell cycle, primarily G0/G1 arrest, were observed in 8 lines at 50 and/or 100 nM. Western blots of representative cell lines at 50 and 100 nM of PF-4928473 identified downregulation of p-AKT and p-ERK in the HER2-amplified SKBR3 line (IC50: 19 ±4 nM), vs. relative lack of effect for the non-luminal MDA-MB-468 (IC50: 24 ±5 nM) and luminal KPL-1 (IC50: 68 ±2 nM) lines. Upregulation of HSP70, a known biomarker associated with HSP90 inhibition, was observed at 12 hours post-drug exposure regardless of cell sensitivity. Further biochemical studies are ongoing. Conclusions: PF-4928473 demonstrated low nanomolar in vitro anti-proliferative activity, lethality, and apoptosis in a wide panel of human breast cancer cell lines, including trastuzumab and lapatinib-resistant lines. Biological effects were most pronounced in HER2-amplified lines, in keeping with HER2 as a known protein client of HSP90. Based on these results, further translational evaluation of HSP90 inhibitors in this population merits consideration. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-13.

Blood Pressure, but Not Cerebrospinal Fluid Fentanyl Concentration, Predicts Duration of Labor Analgesia From Spinal Fentanyl

Obstetric Anesthesia Digest: September 2010 - Volume 30 - Issue 3 - p 164-165 doi: 10.1097/01.aoa.0000386830.43127.d6

Maximum Non-Reactive Concentration of Midazolam and Ketamine for Skin Testing Study in Non-Allergic Healthy Volunteers

The objective of our study was to determine the maximal non-reactive concentrations for midazolam and ketamine in healthy volunteers using both prick and intradermal skin tests. Twenty-one healthy Caucasian volunteers were tested for midazolam and ketamine using more clustered concentrations (identical for both prick and intradermal tests) than those resulting from decimal dilutions. The criteria for positivity were based on dilutions of drugs that cause wheal and flare reactions in subjects without history of allergy. For the prick method, the concentrations that did not produce wheal and flare were 1 mg/ml for midazolam and 10 mg/ml for ketamine. For intradermal tests, using serial dilutions, we found that the highest concentration for which the subjects did not pass the positivity criteria was 0.25 mg/ml for both drugs.