A novel treatment planning methodology for high dose 166Ho-DOTMP therapy in patients with multiple myeloma

Bone marrow ablation, i.e., the complete sterilization of the active bone marrow, followed by bone marrow transplantation (BMT) is a comment treatment of hematological malignancies. The use of targeted bone-seeking radiopharmaceuticals to selectively deliver radiation to the adjacent bone marrow cavities while sparing normal tissues is a promising technique. Current radiopharmaceutical treatment planning methods do not properly compensate for the patient-specific variable distribution of radioactive material within the skeleton. To improve the current method of internal dosimetry, novel methods for measuring the radiopharmaceutical distribution within the skeleton were developed. 99mTc-MDP was proven as an adequate surrogate for measuring 166Ho-DOTMP skeletal uptake and biodistribution, allowing these measures to be obtained faster, safer, and with higher spatial resolution. This translates directly into better measurements of the radiation dose distribution within the bone marrow. The resulting bone marrow dose-volume histograms allow prediction of the patient disease response where conventional organ scale dosimetry failed. They indicate that complete remission is only achieved when greater than 90% of the bone marrow receives at least 30 Gy. ^ Comprehensive treatment planning requires combining target and non-target organ dosimetry. Organs in the urinary tract were of special concern. The kidney dose is primarily dependent upon the mean transit time of 166 Ho-DOTMP through the kidney. Deconvolution analysis of renograms predicted a mean transit time of 2.6 minutes for 166Ho-DOTMP. The radiation dose to the urinary bladder wall is dependent upon numerous factors including patient hydration and void schedule. For beta-emitting isotopes such as 166Ho, reduction of the bladder wall dose is best accomplished through good patient hydration and ensuring a partially full bladder at the time of injection. Encouraging the patient to void frequently, or catheterizing the patient without irrigation, will not significantly reduce the bladder wall dose. ^ The results from this work will produce the most advanced treatment planning methodology for bone marrow ablation therapy using radioisotopes currently available. Treatments can be tailored specifically for each patient, including the addition of concomitant total body irradiation for patients with unfavorable dose distributions, to deliver a desired patient disease response, while minimizing the dose or toxicity to non-target organs. ^

DEVELOPMENT AND IMPLEMENTATION OF A REMOTE AUDIT TOOL FOR HIGH DOSE RATE (HDR) 192IR BRACHYTHERAPY USING OPTICALLY STIMULATED LUMINESCENCE DOSIMETRY

This work aimed to create a mailable and OSLD-based phantom with accuracy suitable for RPC audits of HDR brachytherapy sources at institutions participating in NCI-funded cooperative clinical trials. An 8 × 8 × 10 cm3 prototype with two slots capable of holding nanoDot Al2O3:C OSL dosimeters (Landauer, Glenwood, IL) was designed and built. The phantom has a single channel capable of accepting all 192Ir HDR brachytherapy sources in current clinical use in the United States. Irradiations were performed with an 192Ir HDR source to determine correction factors for linearity with dose, dose rate, and the combined effect of irradiation energy and phantom construction. The uncertainties introduced by source positioning in the phantom and timer resolution limitations were also investigated. It was found that the linearity correction factor was where dose is in cGy, which differed from that determined by the RPC for the same batch of dosimeters under 60Co irradiation. There was no significant dose rate effect. Separate energy+block correction factors were determined for both models of 192Ir sources currently in clinical use and these vendor-specific correction factors differed by almost 2.6%. For Nucletron sources, this correction factor was 1.026±0.004 (99% Confidence Interval) and for Varian sources it was 1.000±0.007 (99% CI). Reasonable deviations in source positioning within the phantom and the limited resolution of the source timer had insignificant effects on the ability to measure dose. Overall measurement uncertainty of the system was estimated to be ±2.5% for both Nucletron and Varian source audits (95% CI). This uncertainty was sufficient to establish a ±5% acceptance criterion for source strength audits under a formal RPC audit program. Trial audits of eight participating institutions resulted in an average RPC-to-institution dose ratio of 1.000 with a standard deviation of 0.011.

Pencil -beam redefinition algorithm dose calculations for electron therapy treatment planning

The electron pencil-beam redefinition algorithm (PBRA) of Shiu and Hogstrom has been developed for use in radiotherapy treatment planning (RTP). Earlier studies of Boyd and Hogstrom showed that the PBRA lacked an adequate incident beam model, that PBRA might require improved electron physics, and that no data existed which allowed adequate assessment of the PBRA-calculated dose accuracy in a heterogeneous medium such as one presented by patient anatomy. The hypothesis of this research was that by addressing the above issues the PBRA-calculated dose would be accurate to within 4% or 2 mm in regions of high dose gradients. A secondary electron source was added to the PBRA to account for collimation-scattered electrons in the incident beam. Parameters of the dual-source model were determined from a minimal data set to allow ease of beam commissioning. Comparisons with measured data showed 3% or better dose accuracy in water within the field for cases where 4% accuracy was not previously achievable. A measured data set was developed that allowed an evaluation of PBRA in regions distal to localized heterogeneities. Geometries in the data set included irregular surfaces and high- and low-density internal heterogeneities. The data was estimated to have 1% precision and 2% agreement with accurate, benchmarked Monte Carlo (MC) code. PBRA electron transport was enhanced by modeling local pencil beam divergence. This required fundamental changes to the mathematics of electron transport (divPBRA). Evaluation of divPBRA with the measured data set showed marginal improvement in dose accuracy when compared to PBRA; however, 4% or 2mm accuracy was not achieved by either PBRA version for all data points. Finally, PBRA was evaluated clinically by comparing PBRA- and MC-calculated dose distributions using site-specific patient RTP data. Results show PBRA did not agree with MC to within 4% or 2mm in a small fraction (<3%) of the irradiated volume. Although the hypothesis of the research was shown to be false, the minor dose inaccuracies should have little or no impact on RTP decisions or patient outcome. Therefore, given ease of beam commissioning, documentation of accuracy, and calculational speed, the PBRA should be considered a practical tool for clinical use. ^

IL-2 REGULATION OF IL-24 EXPRESSION LEADS TO GROWTH SUPPRESSION OF MELANOMA CELLS

Melanoma is known to be highly resistant to chemotherapy. Treatment with high dose IL-2 has shown significant clinical benefit in a minority of metastatic melanoma patients and has lead to long term survival in a few cases. However, this treatment is associated with excessive multiorgan toxicities, which severely limits its use. We hypothesize that one mechanism of effective IL-2 therapy is through the direct upregulation of IL-24 production in melanoma tumors and subsequent IL-24 mediated tumor growth suppression. Five melanoma cell lines were treated with high dose recombinant hIL-2 at 1000U/ml. Three of the cell lines (A375, WM1341, WM793) showed statistically significant increases in their levels of IL-24 protein when measured by Western blotting, while the remaining two lines (WM35, MeWo) remained negative for IL-24 message and protein. This increase in IL-24 was abolished by either preincubating with an anti-IL-2 antibody or by blocking the IL-2 receptor directly with antibodies against the receptor chains. We also demonstrated by ELISA that these three cell lines secrete IL-24 protein in higher amounts when stimulated with IL-2 than do untreated cells. These cells were found to contain IL-2R beta and gamma message by RT-PCR and also expressed higher levels of IL-24 when treated with IL-15, which shares the IL-2R beta chain. Thus we propose that IL-2 is signaling through IL-2R beta on some melanoma cells to upregulate IL-24 protein expression. To address the biological function of IL-2 in melanoma cells, five cell lines were treated with IL-2 and cell viability determined. Cell growth was found to be significantly decreased by day 4 in the IL-24 positive cell lines while no effect on growth was seen in WM35 or MeWo. Incubating the cells with anti-IL-24 antibody or transfecting with IL-24 siRNA effectively negated the growth suppression seen with IL-2. These data support our hypothesis that in addition to its immunotherapeutic effects, IL-2 also acts directly on some melanoma tumors and that the IL-24 and IL-2R beta status of a tumor may be useful in predicting patient response to high dose IL-2.

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Effects of Combined Bevacizumab and Paclitaxel on Tumor Interstitial Fluid Pressure in a Preclinical Breast Cancer Model

Effects of Combined Bevacizumab and Paclitaxel on Tumor Interstitial Fluid Pressure in a Preclinical Breast Cancer Model by Ricardo H. Alvarez Several mechanisms of cell resistance are often accountable for unsuccessful chemotherapy against cancer. Another reason, which has received increased attention, is the inefficient transport of anticancer drugs into tumor tissue. These impaired transports of chemotherapy into the tumor have been attributed to abnormal microvasculature and to pathologically increased tumor hypertension also called: interstitial fluid pressure (IFP). The pathophysiological processes leading to elevated tumor IFP are poorly understood. Here, in a preclinical breast cancer model, it is argued that a condition of raised IFP is a major factor in preventing optimal access of systemically administered chemotherapy agents. In our experimental model, we used a GILM2 human breast cancer in xenografts; mice were treated with different doses of paclitaxel –a widely used antimicrotubular agent, and bevacizumab –monoclonal antibody against vascular endothelial growth factor (VEGF). The proposed research project is designed to test the hypothesis that paclitaxel in combination with bevacizumab decreases the tumor IPF by restoring tumor permeability and increasing chemotherapy delivery. We demonstrated that the combination of paclitaxel and bevacizumab produced greater tumor control than either agent given alone and this combination reduced the IFP, producing an increment of 75% of apoptosis compared with the control arm. In addition, the intra-tumor paclitaxel quantification by liquid chromatography/Mass Spectrometry (LC/MS) demonstrated that lower dose of both agents showed a synergistic effect compared with high dose of treatment, where there is no significantly increase of paclitaxel into the tumor. These preclinical results are likely to have broad implications for the utility of anti-angiogenic therapies alone and in combination with chemotherapeutic agents.

Patient Characteristics and Outcomes of Gastrointestinal Stromal Tumor Patients Undergoing Imatinib Plasma Level Testing

Although gastrointestinal stromal tumor (GIST) is effectively treated with imatinib, there are a number of clinical challenges in the optimal treatment of these patients. The plasma steady-state trough level of imatinib has been proposed to correlate with clinical outcome. Plasma imatinib level may be affected by a number of patient characteristics. Additionally, the ideal plasma trough concentration of imatinib is likely to vary based on the KIT genotype (genotype determines imatinib binding affinity) of the individual patient. Patients’ genotype or plasma imatinib level may influence the type and duration of response that is appreciable by clinical evaluation. The objectives of this study were to determine effects of genotype on the type of response appreciable by current imaging criteria, to determine the distribution of plasma imatinib levels in patients with GIST, to determine factors that correlate with plasma imatinib level, to determine the incremental effects of imatinib dose escalation; and to explore the median plasma levels and outcomes of patients with various KIT mutations. We therefore obtained KIT mutation information and analyzed CT response for size and density measurement of GISTs at baseline and within the first four moths of imatinib treatment. In 126 patients with metastatic/unresectable disease, the KIT genotype of patients’ tumor was significantly associated with unique response characteristics measurable by CT. Furthermore, hepatic and peritoneal metastases differed in their response characteristics. A subgroup of patients with KIT exon 9 mutation, who received higher doses of imatinib and experienced higher trough imatinib levels, experienced improved progression-free survival similar to that of KIT exon 11 patients. Therefore, we have found that imatinib plasma levels were higher in patients with elevated Aspartate amino transferase, were women, were older, or were being treated concomitantly with CYP450 substrate drugs. As expected, CYP450 inducers correlated with a lower plasma imatinib levels in GIST patients. Renal metabolism of imatinib accounts for <10%, so it was not included in the analysis but may affect covariates. Interestingly, there was a trend for low imatinib levels and inferior progression-free survival in patients who had undergone complete gastrectomy. Patients with KIT exon 9 mutation in our cohort received higher imatinib doses, experienced higher trough imatinib levels, and experienced a PFS similar to that of KIT exon 11 patients. In conclusion, imatinib plasma levels are influenced by a number of patient characteristics. The optimal imatinib plasma level for individual patients is not known but is an area of intense investigation. Our study confirms patients with KIT exon 9 mutations benefit from high-dose imatinib and higher trough imatinib levels.

DUAL ENERGY COMPUTED TOMOGRAPHY FOR PROTON THERAPY TREATMENT PLANNING

Proton radiation therapy is gaining popularity because of the unique characteristics of its dose distribution, e.g., high dose-gradient at the distal end of the percentage-depth-dose curve (known as the Bragg peak). The high dose-gradient offers the possibility of delivering high dose to the target while still sparing critical organs distal to the target. However, the high dose-gradient is a double-edged sword: a small shift of the highly conformal high-dose area can cause the target to be substantially under-dosed or the critical organs to be substantially over-dosed. Because of that, large margins are required in treatment planning to ensure adequate dose coverage of the target, which prevents us from realizing the full potential of proton beams. Therefore, it is critical to reduce uncertainties in the proton radiation therapy. One major uncertainty in a proton treatment is the range uncertainty related to the estimation of proton stopping power ratio (SPR) distribution inside a patient. The SPR distribution inside a patient is required to account for tissue heterogeneities when calculating dose distribution inside the patient. In current clinical practice, the SPR distribution inside a patient is estimated from the patient’s treatment planning computed tomography (CT) images based on the CT number-to-SPR calibration curve. The SPR derived from a single CT number carries large uncertainties in the presence of human tissue composition variations, which is the major drawback of the current SPR estimation method. We propose to solve this problem by using dual energy CT (DECT) and hypothesize that the range uncertainty can be reduced by a factor of two from currently used value of 3.5%. A MATLAB program was developed to calculate the electron density ratio (EDR) and effective atomic number (EAN) from two CT measurements of the same object. An empirical relationship was discovered between mean excitation energies and EANs existing in human body tissues. With the MATLAB program and the empirical relationship, a DECT-based method was successfully developed to derive SPRs for human body tissues (the DECT method). The DECT method is more robust against the uncertainties in human tissues compositions than the current single-CT-based method, because the DECT method incorporated both density and elemental composition information in the SPR estimation. Furthermore, we studied practical limitations of the DECT method. We found that the accuracy of the DECT method using conventional kV-kV x-ray pair is susceptible to CT number variations, which compromises the theoretical advantage of the DECT method. Our solution to this problem is to use a different x-ray pair for the DECT. The accuracy of the DECT method using different combinations of x-ray energies, i.e., the kV-kV, kV-MV and MV-MV pair, was compared using the measured imaging uncertainties for each case. The kV-MV DECT was found to be the most robust against CT number variations. In addition, we studied how uncertainties propagate through the DECT calculation, and found general principles of selecting x-ray pairs for the DECT method to minimize its sensitivity to CT number variations. The uncertainties in SPRs estimated using the kV-MV DECT were analyzed further and compared to those using the stoichiometric method. The uncertainties in SPR estimation can be divided into five categories according to their origins: the inherent uncertainty, the DECT modeling uncertainty, the CT imaging uncertainty, the uncertainty in the mean excitation energy, and SPR variation with proton energy. Additionally, human body tissues were divided into three tissue groups – low density (lung) tissues, soft tissues and bone tissues. The uncertainties were estimated separately because their uncertainties were different under each condition. An estimate of the composite range uncertainty (2s) was determined for three tumor sites – prostate, lung, and head-and-neck, by combining the uncertainty estimates of all three tissue groups, weighted by their proportions along typical beam path for each treatment site. In conclusion, the DECT method holds theoretical advantages in estimating SPRs for human tissues over the current single-CT-based method. Using existing imaging techniques, the kV-MV DECT approach was capable of reducing the range uncertainty from the currently used value of 3.5% to 1.9%-2.3%, but it is short to reach our original goal of reducing the range uncertainty by a factor of two. The dominant source of uncertainties in the kV-MV DECT was the uncertainties in CT imaging, especially in MV CT imaging. Further reduction in beam hardening effect, the impact of scatter, out-of-field object etc. would reduce the Hounsfeld Unit variations in CT imaging. The kV-MV DECT still has the potential to reduce the range uncertainty further.

Uptake And Metabolism Of 5’-AMP In The Erythrocyte Play Key Roles In The 5’-AMP Induced Model Of Deep Hypometabolism

UPTAKE AND METABOLISM OF 5’-AMP IN THE ERYTHROCYTE PLAY KEY ROLES IN THE 5’-AMP INDUCED MODEL OF DEEP HYPOMETABOLISM Publication No. ________ Isadora Susan Daniels, B.A. Supervisory Professor: Cheng Chi Lee, Ph.D. Mechanisms that initiate and control the natural hypometabolic states of mammals are poorly understood. The laboratory developed a model of deep hypometabolism (DH) initiated by uptake of 5’-adenosine monophosphate (5’-AMP) into erythrocytes. Mice enter DH when given a high dose of 5’-AMP and the body cools readily. Influx of 5’-AMP appears to inhibit thermoregulatory control. In a 15°C environment, mice injected with 5’-AMP (0.5 mg/gw) enter a Phase I response in which oxygen consumption (VO2) drops rapidly to 1/3rd of euthermic levels. The Phase I response appears independent of body temperature (Tb). This is followed by gradual body temperature decline that correlates with VO2 decline, called Phase II response. Within 90 minutes, mouse Tb approaches 15°C, and VO2 is 1/10th of normal. Mice can remain several hours in this state, before gradually and safely recovering. The DH state translates to other mammalian species. Our studies show uptake and metabolism of 5’-AMP in erythrocytes causes biochemical changes that initiate DH. Increased AMP shifts the adenylate equilibrium toward ADP formation, consequently decreasing intracellular ATP. In turn, glycolysis slows, indicated by increased glucose and decreased lactate. 2,3-bisphosphoglycerate levels rise, allosterically reducing oxygen affinity for hemoglobin, and deoxyhemoglobin rises. Less oxygen transport to tissues likely triggers the DH model. The major intracellular pathway for AMP catabolism is catalyzed by AMP deaminase (AMPD). Multiple AMPD isozymes are expressed in various tissues, but erythrocytes only have AMPD3. Mice lacking AMPD3 were created to study control of the DH model, specifically in erythrocytes. Telemetric measurements demonstrate lower Tb and difficulty maintaining Tb under moderate metabolic stress. A more dramatic response to lower dose of 5’-AMP suggests AMPD activity in the erythrocyte plays an important role in control of the DH model. Analysis of adenylates in erythrocyte lysate shows 3-fold higher levels of ATP and ADP but similar AMP levels to wild-type. Taken together, results indicate alterations in energy status of erythrocytes can induce a hypometabolic state. AMPD3 control of AMP catabolism is important in controlling the DH model. Genetically reducing AMP catabolism in erythrocytes causes a phenotype of lower Tb and compromised ability to maintain temperature homeostasis.

A dosimetric study of radionuclide therapy for bone marrow ablation

In a phase I clinical trial, six multiple myeloma patients, who were non-responsive to conventional therapy and were scheduled for bone marrow transplantation, received Holmium-166 ($\sp{166}$Ho) labeled to a bone seeking agent, DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene-phosphonic acid), for the purpose of bone marrow ablation. The specific aims of my research within this protocol were to evaluate the toxicity and efficacy of $\sp{166}$Ho DOTMP by quantifying the in vivo pharmacokinetics and radiation dosimetry, and by correlating these results to the biologic response observed. The reproducibility of pharmacokinetics from multiple injections of $\sp{166}$Ho DOTMP administered to these myeloma patients was demonstrated from both blood and whole body retention. The skeletal concentration of $\sp{166}$Ho DOTMP was heterogenous in all six patients: high in the ribs, pelvis, and lumbar vertebrae regions, and relatively low in the femurs, arms, and head.^ A novel technique was developed to calculate the radiation dose to the bone marrow in each skeletal ROI, and was applied to all six $\sp{166}$Ho DOTMP patients. Radiation dose estimates for the bone marrow calculated using the standard MIRD "S" factors were compared with the average values derived from the heterogenous distribution of activity in the skeleton (i.e., the regional technique). The results from the two techniques were significantly different; the average of the dose estimates from the regional technique were typically 30% greater. Furthermore, the regional technique provided a range of radiation doses for the entire marrow volume, while the MIRD "S" factors only provided a single value. Dose volume histogram analysis of data from the regional technique indicated a range of dose estimates that varied by a factor of 10 between the high dose and low dose regions. Finally, the observed clinical response of cells and abnormal proteins measured in bone marrow aspirates and peripheral blood samples were compared with radiation dose estimates for the bone marrow calculated from the standard and regional technique. The results showed the regional technique values correlated more closely to several clinical response parameters. (Abstract shortened by UMI.) ^

Impact of COP9 signalosome subunit 6 (CSN6) on MDM2-p53 axis in DNA damage-mediated apoptosis and tumorigenesis

Mammalian COP9 signalosome, which connects signaling with the ubiquitin-mediated proteasome degradation pathway, is implicated in cell cycle regulation and DNA damage response. However, whether COP9 is dysregulated in cancers has not been well established. Here, we showed that COP9 subunit 6 (CSN6) was upregulated in malignant breast and thyroid tumors and positively correlated with MDM2 expression. Investigation of the underlying mechanism suggested that CSN6 stabilized MDM2, thereby accelerating the degradation of p53. We generated mice carrying a targeted disruption of the Csn6 gene, and found that the mice with both alleles disrupted (Csn6-/- ) died in early embryogenesis (E7.5). Csn6+/- mice were sensitized to undergo γ-radiation-induced p53-dependent apoptosis in both thymus and developing central nervous system. Consequently. Csn6 +/- mice were more susceptible to the lethal effects of high-dose γ-radiation than wild-type mice. Notably, Csn6+/- mice were less susceptible to γ-radiation-induced tumorigenesis and had better long-term survival after low-dose γ-radiation exposure compared with wild-type animals, indicating that loss of CSN6 enhanced p53-mediated tumor suppression in vivo. In summary, the regulation of MDM2-p53 signaling by CSN6 plays a significant role in DNA damage-mediated apoptosis and tumorigenesis, which suggests that CSN6 may potentially be a valuable diagnostic marker for cancers with a dysregulated MDM2-p53 axis. ^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^

Fate and effects of drilling wastes in a shallow estuarine mesocosm

To address concerns expressed about the possible effect of drilling mud discharges on shallow, low-energy estuarine ecosystems, a 12 month study was designed to detect alterations in water quality and sediment geochemistry. Each drilling mud used in the study and sediments from the study site were analyzed in the laboratory for chemical and physical characteristics. Potential water quality impacts were simulated by the EPA-COE elutriation test procedure. Mud toxicity was measured by acute and chronic bioassays with Mysidopsis bahia, Mercenaria mercenaria, and Nereis virens.^ For the field study, a relatively pristine, shallow (1.2 m) estuary (Christmas Bay, TX) without any drilling activity for the last 30 years was chosen for the study site. After a three month baseline study, three stations were selected. Station 1 was an external control. At each treatment station (2, 3), mesocosms were constructed to enclose a 3.5 m$\sp3$ water column. Each treatment station included an internal control site also. Each in situ mesocosm, except the controls, was successively dosed at a mesocosm-specific dose (1:100; 1:1,000; or 1:10,000 v/v) with 4 field collected drilling muds (spud, nondispersed, lightly-treated, and heavily-treated lignosulfonate) in sequential order over 1.5 months. Twenty-four hours after each dose, water exchange was allowed until the next treatment. Station 3 was destroyed by a winter storm. After the last treatment, the enclosures were removed and the remaining sites monitored for 6 months. One additional site was similarly dosed (1:100 v/v) with clean dredged sediment from Christmas Bay for comparison between dredged sediments and drilling muds.^ Results of the analysis of the water samples and field measurements showed that water quality was impacted during the discharges, primarily at the highest dose (1:100 v/v), but that elevated levels of C, Cr (T,F), Cr$\sp{+3}$ (T, F), N, Pb, and Zn returned to ambient levels before the end of the 24 hour exposure period or immediately after water exchange was allowed (Al, Ba(T), Chlorophyll ABC, SS, %T). Barium, from the barite, was used as a geochemical tracer in the sediments to confirm estimated doses by mass balance calculations. Barium reached a maximum of 166x background levels at the high dose mesocosm. Barium levels returned to ambient or only slightly elevated levels at the end of the 6 month monitoring period due to sediment deposition, resuspension, and bioturbation. QA/QC results using blind samples consisting of lab standards and spiked samples for both water and sediment matrices were within acceptable coefficients of variation.^ In order to avoid impacts on water quality and sediment geochemistry in a shallow estuarine ecosystem, this study concluded that a minimal dilution of 1:1,000 (v/v) would be required in addition to existing regulatory constraints. ^

Why does low dose morphine augment fentanyl analgesia?

Opioids dominate the field of pain management because of their ability to provide analgesia in many medical circumstances. However, side effects including respiratory depression, constipation, tolerance, physical dependence, and the risk of addiction limit their clinical utility. Fear of these side effects results in the under-treatment of acute pain. For many years, research has focused on ways to improve the therapeutic index (the ratio of desirable analgesic effects to undesirable side effects) of opioids. One strategy, combining opioid agonists that bind to different opioid receptor types, may prove successful.^ We discovered that subcutaneous co-administration of a moderately analgesic dose of the mu-opioid receptor (MOR) selective agonist fentanyl (20μg/kg) with subanalgesic doses of the less MOR-specific agonist morphine (100ng/kg-100μg/kg), augmented acute fentanyl analgesia in rats. Parallel [35S]GTPγS binding studies using naïve rat substantia gelatinosa membrane treated with fentanyl (4μM) and morphine (1nM-1pM) demonstrated a 2-fold increase in total G-protein activation. This correlation between morphine-induced augmentation of fentanyl analgesia and G-protein activation led to our proposal that interactions between MORs and DORs underlie opioid-induced augmentation. We discovered that morphine-induced augmentation of fentanyl analgesia and G-protein activity was mediated by DORs. Adding the DOR-selective antagonist naltrindole (200ng/kg, 40nM) at doses that did not alter the analgesic or G-protein activation of fentanyl, blocked increases in analgesia and G-protein activation induced by fentanyl/morphine combinations. Equivalent doses of the MOR-selective antagonist cyprodime (20ng/kg, 4nM) did not block augmentation. Substitution of the DOR-selective agonist SNC80 for morphine yielded similar results, further supporting our conclusion that interactions between MORs and DORs are responsible for morphine-induced augmentation of fentanyl analgesia and G-protein activation. Confocal microscopy of rat substantia gelatinosa showed that changes in the rate of opioid receptor internalization did not account for these effects.^ In conclusion, fentanyl analgesia augmentation by subanalgesic morphine is mediated by increased G-protein activation resulting from functional interactions between MORs and DORs, not changes in MOR internalization. Additional animal and clinical studies are needed to determine whether side effect incidence changes following opioid co-administration. If side effect incidence decreases or remains unchanged, these findings could have important implications for clinical pain treatment. ^