Immunotherapy of advanced malignancy by direct gene transfer of an interleukin-2 DNA/DMRIE/DOPE lipid complex: phase I/II experience.

PURPOSE: We have completed a phase I study, followed by three phase I/II studies, in patients with metastatic melanoma, renal cell carcinoma (RCC), and sarcoma in order to evaluate the safety, toxicity, and antitumor activity of Leuvectin (Vical Inc, San Diego, CA), a gene transfer product containing a plasmid encoding human interleukin (IL)-2 formulated with the cationic lipid 1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/dioleyl-phosphatidyl-ethanolamine (DMRIE/DOPE) and administered intratumorally. PATIENTS AND METHODS: Twenty-four patients were treated in the phase I study. Leuvectin doses were 10 microg, 30 microg, or 300 microg weekly for 6 weeks. In three subsequent phase I/II studies, a total of 52 patients (18 with melanoma, 17 with RCC, and 17 with sarcoma) were treated with further escalating doses of Leuvectin: 300 microg twice a week for 3 weeks, 750 microg weekly for 6 weeks, and 1,500 microg weekly for 6 weeks. RESULTS: There were no drug-related grade 4 toxicities and only one grade 3 toxicity, but the majority of patients experienced mild constitutional symptoms after treatment. In the phase I/II studies, 45 patients were assessable for response (14 with RCC, 16 with melanoma, and 15 with sarcoma). Two patients with RCC and one with melanoma have achieved partial responses lasting from 16 to 19 months and continuing. In addition, two RCC, three melanoma, and six sarcoma patients had stable disease lasting from 3 to 18 months and continuing. The plasmid was detected by polymerase chain reaction assay in the posttreatment samples of 29 of 46 evaluated patients. Immunohistochemistry studies on serial biopsy specimens showed increased IL-2 expression and CD8(+) infiltration after treatment in the tumor samples of several patients (12 and 16, respectively). CONCLUSION: Direct intratumoral injection of Leuvectin is a safe and possibly effective immunotherapeutic approach in the treatment of certain tumor types.

In vitro studies of a new thrombus-specific ultrasound contrast agent.

Ultrasound is used as a primary diagnostic technique for the detection of deep venous thrombosis. The purpose of this study is to describe the development of a new thrombus-specific ultrasound contrast agent: The linear hexapeptide (lysine-glutamine-alanine-glycine-aspartate-valine) was synthesized and coupled to a lipid moiety. The targeted lipid was then incorporated into the lipid blend for the contrast agent Aerosomes (ImaRx, Tucson, AZ, USA). The lipid blend was used to entrap perfluorobutane microbubbles. The microbubbles were sized and studied in vitro for acoustic stability, binding to blood clot, and ultrasound enhancement in vitro of blood clot. The results showed the mean size of the specific ultrasound contrast agent (MRX-408) was about 2.0 microm. The microbubbles appeared as smooth spherical structures. Microscopy showed that the targeted bubbles bound to blood clot whereas control, nontargeted bubbles did not bind to blood clot. In vitro acoustic study showed similar stability of the microbubbles compared with control microbubbles. The targeted microbubbles enhanced blood clot in vitro whereas nontargeted microbubbles did not enhance clot. Thus this promising new thrombus-specific ultrasound contrast agent could potentially improve detection of thrombosis by ultrasound and might be useful for distinguishing between new and old thrombosis. In vivo studies are in progress.

Ultrasound enhances gene expression of liposomal transfection.

RATIONALE AND OBJECTIVES: Cationic liposomes are under development as delivery agents for gene therapy. The authors studied the effect of ultrasound on gene expression in cell cultures during liposomal transfection experiments. METHODS: Cationic liposomes of dipalmitoylethylphosphocholine and dioleoylphosphatidylethanolamine were used to transfect cultured HeLa, NIH/3T3, and C127I cells with the chloramphenicol acetyl transferase (CAT) gene. A cell viability assay was performed on cultured HeLa cells that were exposed to varying durations (5 seconds or 30 seconds) and intensities of 1 MHz continuous-wave therapeutic ultrasound after transfection, and gene expression was measured 48 hours later. RESULTS: Cells survived 30 seconds or less at a power level of 0.5 watts/cm2 but died when exposed for 60 seconds or longer. Exposures of 5 seconds and 30 seconds of ultrasound resulted in significant increases in gene expression in all three cell types tested in this experiment. CONCLUSIONS: Relatively low levels of ultrasound energy can be used to enhance gene expression from liposomal transfection. Additional experiments are needed to optimize this process and clarify the mechanisms involved.

Phase I clinical trials of MRX-115. A new ultrasound contrast agent.

RATIONALE AND OBJECTIVES: Stabilized microbubbles are under development as contrast agents for medical ultrasound. The authors report the results of Phase I clinical trials of a new ultrasound contrast agent based on lipidencapsulated perfluorocarbon gas microbubbles. METHODS: Lipids encapsulating perfluoropropane gas (Aerosomes MRX-115, ImaRx Pharmaceutical Corp., Tucson, AZ) were evaluated in Phase I clinical trials. Two separate studies were performed. The first was a single escalating-dose study (n = 30 subjects), and the second was a multiple-dose study (n = 18 subjects) with rechallenge in several subjects (n = 4) after 21 days. Echocardiographic examinations were performed before and after contrast agent for each test drug administration for both studies, with the exception of the rechallenge group. Doses tested in the single-dose study ranged from 0.005 mL/kg to 0.100 mL/kg body weight. In the multiple-dose study, five doses of 0.005 mL/ kg to 0.030 mL/kg (0.025-0.150 mL/kg total dose) were evaluated. Studies were single-masked, placebo-controlled, and safety assessment and adverse events were monitored. RESULTS: All doses in both studies were well tolerated with no treatment-related changes in safety measures for either study. Left ventricular cavity and myocardial enhancement were seen with all doses of MRX-115. CONCLUSIONS: MRX-115 is a promising new intravascular ultrasound contrast agent that was safe and well tolerated at the doses evaluated in these studies.

Acoustically active lipospheres containing paclitaxel: a new therapeutic ultrasound contrast agent.

RATIONALE AND OBJECTIVES: Paclitaxel-carrying lipospheres (MRX-552) were developed and evaluated as a new ultrasound contrast agent for chemotherapeutic drug delivery. METHODS: Paclitaxel was suspended in soybean oil and added to an aqueous suspension of phospholipids in vials. The headspace of the vials was replaced with perfluorobutane gas; the vials were sealed, and they were agitated at 4200 rpm on a shaking device. The resulting lipospheres containing paclitaxel were studied for concentration, size, acute toxicity in mice, and acoustic activity and drug release with ultrasound. Lipospheres containing sudan black dye were produced to demonstrate the acoustically active liposphere (AAL)-ultrasound release concept. RESULTS: Acoustically active lipospheres containing paclitaxel had a mean particle count of approximately 1 x 10(9) particles per mL and a mean size of 2.9 microns. Acute toxicity studies in mice showed a 10-fold reduction in toxicity for paclitaxel in AALs compared with free paclitaxel. The AALs reflected ultrasound as a contrast agent. Increasing amounts of ultrasound energy selectively ruptured the AALs and released the paclitaxel. CONCLUSIONS: Acoustically active lipospheres represent a new class of acoustically active drug delivery vehicles. Future studies will assess efficacy of AALs for ultrasound-mediated drug delivery.

Magnetic resonance imaging using gadolinium labeled monoclonal antibody.

Gadolinium was attached to antibodies and tested in vitro and in vivo for its effect on proton relaxation enhancement. Using the cyclic anhydride method, diethylenetriaminepentaacetic acid (DTPA) was attached to albumin, IgG and anti-CEA monoclonal antibody. Gadolinium (Gd) was then chelated to the protein complexes forming protein-DTPA-Gd complex. With this technique approximately 9 atoms of Gd could be attached to each albumin molecule, 4 to each IgG molecule and 1.5 to each monoclonal antibody molecule. The minimal in vitro concentration of Gd in the form of IgG-DTPA-Gd necessary to produce proton relaxation enhancement at 0.35 tesla was 10(-1) mM. An in vivo experiment using anticarcinoembryonic antigen (CEA) monoclonal antibody-DTPA-Gd in hamsters implanted with human colon carcinoma resulted in a tumor concentration of Gd of less than 10(-4) mM. No enhancement of the tumors was detected at that concentration. For monoclonal antibodies to function as selective MR contrast agents, substantial advances in technology must occur.

Binding and lysing of blood clots using MRX-408.

RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.

Sensitivity of muscle proton spin-spin relaxation time as an index of muscle activation.

The purpose of this study was to determine the minimum number of contractions that are needed to detect an increase in the muscle proton spin-spin relaxation time (T2) at a given exercise intensity. Five healthy human subjects performed five sets of an exercise that included concentric and eccentric contractions of the elbow-flexor muscles with loads that were 25 or 80% of maximum. With the 80% load, the five sets involved 1, 2, 5, 10, or 20 repetitions of the exercise; with the 25% load the five sets were 2, 5, 10, 20, or 40 repetitions. The upper arm of each subject was imaged before and immediately after each set of the exercise. Spin-echo images (repetition time/echo time = 2,000 ms/30, 60, 90, and 120 ms) were collected using an extremity coil, and T2 values were calculated. The signal intensity was measured from the elbow-flexor and -extensor muscles and from the bone marrow of the humerus. With the 80% load, T2 increased in the short head of the biceps brachii after two repetitions of the elbow exercise and after five repetitions in the brachialis and the long head of the biceps brachii. With the 25% load, T2 became longer after five repetitions of the exercise for the short head of the biceps brachii and after 10 repetitions for the brachialis and the long head of the biceps brachii. T2 varied linearly with the number of contraction repetitions for each of the elbow-flexor muscles at either load (r2 > or = 0.97, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced visualization of intravascular and left atrial appendage thrombus with the use of a thrombus-targeting ultrasonographic contrast agent (MRX-408A1): In vivo experimental echocardiographic studies.

Echocardiographic evaluation for the recognition of intravascular and left atrial appendage thrombus remains a difficult problem. A thrombus-specific ultrasonographic contrast agent has the potential for an alternative approach for their delineation. The aim of this study was to investigate the usefulness of thrombus-specific contrast agent MRX-408A1 for the detection of acute experimentally created intravascular and intracardiac thrombus. In the first study, we created inferior vena cava thrombus in 9 dogs. With the use of fundamental 2-dimensional echocardiography imaging, we recorded images of the inferior vena cava thrombus at baseline (n = 9), with the thrombus-specific contrast agent MRX-408A1 (n = 9), and with nonspecific contrast agent MRX-113 (n = 6). In the second study, we created a left atrial appendage thrombus in 8 dogs. We imaged left atrial appendage thrombus at baseline and during MRX-113 and MRX-408A1 infusion. Thrombus was successfully created in all dogs in study 1 and in 6 of 8 dogs in study 2. MRX-408A1 produced a visually apparent increase in ultrasonographic contrast enhancement of the thrombus in all cases in which thrombus was found on autopsy. In both studies, MRX-408A1 increased the videointensity of the thrombus significantly compared with baseline images and images obtained during MRX-113 infusion. The size of the visually detectable thrombus on the image was also significantly larger during MRX-408A1 infusion than at baseline and during MRX-113 infusion. These data provide in vivo demonstration of the efficacy of a thrombus-specific contrast agent, MRX-408A1, in the detection of acute intravascular and intracardiac thrombus. It has the potential to improve the diagnostic accuracy of ultrasonography for the detection of acute thrombi at various cardiovascular sites in the clinical setting.

Liposomal Gd-DTPA: effect of encapsulation on enhancement of hepatoma model by MRI.

Liposomes entrapping gadolinium-DTPA (Gd-DTPA) were synthesized from 60 mole percent egg phosphatidylcholine (EPC) and 40 mole percent cholesterol or EPC alone entrapping Gd-DTPA in diameters of 100 and 200 nm. Rats bearing Morris hepatoma in their flanks were imaged by MR pre- and post-contrast with free Gd-DTPA and liposomal Gd-DTPA for up to four hours after IV contrast. Comparison of images after free and liposomal Gd-DTPA showed dramatic differences in tumor and organ enhancement. Liposomal Gd-DTPA enhancement of tumor corresponded more closely to histologically proven vascularized portions of tumor than free Gd-DTPA. Hepatic enhancement was greater with liposomal than free Gd-DTPA and time course of liver, kidney and tumor enhancement was prolonged. The 100-nm EPC Gd-DTPA liposomes caused the greatest enhancement. Gd-DTPA liposomes may be useful as liver and blood pool contrast agents. By varying lipid composition and vesicle size, patterns of enhancement may be selectively modified.

Pulse sequence optimization for T2-weighted MR imaging of the brain.

The authors implemented bipolar velocity compensated pulse techniques for T2-weighted MR imaging of the brain. Signal-to-noise (S/N) and image quality was compared for pulse sequences with standard and optimized RF pulses, low and regular bandwidth versions and cardiac triggering. Images from bipolar velocity compensated sequences allowed better visualization of vessels and basilar cisterns and improved image quality relative to standard sequences without velocity compensation. The implementation of optimized RF pulses with bipolar sequences resulted in further improvement in image quality. Single echo sequences consistently had improved image quality and signal-to-noise relative to the second echo of a double echo sequence. Low bandwidth bipolar sequences with extended sampling period had 30% higher S/N, but at the cost of slight loss in edge definition. The highest image quality was obtained with the bipolar, optimized RF, single echo sequence. Using this technique contiguous high quality image slices could be obtained with velocity compensation. The addition of cardiac triggering to bipolar sequences resulted in slight improvement in image quality, but this difference was marginal and probably rarely necessary for MR imaging of the brain.

Gradient-echo imaging of hemorrhage at 1.5 Tesla.

We report in vitro and in vivo MR studies of hemorrhage using the gradient-echo pulse sequence, FISP (steady state free precession) and FLASH (spoiling of transverse magnetization) at 1.5 Tesla. Phantoms containing methemoglobin, ferromagnetic particles, human serum and blood clot were scanned using both spin-echo and gradient-echo techniques. FLASH signal intensities were more sensitive to methemoglobin concentration than high T1-weighted spin-echo images. FISP showed little change in signal intensity with varying concentrations of methemoglobin and a contrast relationship similar to T2-weighted spin-echo techniques. FISP and FLASH showed intensity changes at lower concentrations of ferromagnetic material than T2-weighted spin-echo sequences. In vitro blood clot was less intense when observed by FISP and FLASH sequences than on the T2-weighted spin-echo sequences. Maximum contrast between clot and other blood components occurred at a flip angle of 45 degrees for FLASH and 60 degrees for FISP. FISP and FLASH scans of patients with hemorrhage demonstrated a marked decrease in signal intensity in the region of blood clot. This decrease was more pronounced with the gradient-echo sequences than with T2-weighted spin-echo images. We conclude that FLASH is useful for detecting methemoglobin and that both FISP and FLASH are useful for evaluating hemorrhage because of their sensitivity to methemoglobin.

Magnetic resonance imaging in human lymphedema: comparison with lymphangioscintigraphy.

Magnetic resonance (MR) imaging and isotope lymphography (lymphangioscintigraphy, LAS) was done in 32 patients with peripheral lymphedema (19 primary and 13 secondary). MRI characteristically showed diffuse dermal and subcutaneous edema, a nonedematous, occasionally hypertrophied skeletal muscle compartment, variability in regional lymph node size and appearance depending on the underlying clinical disorder, serpiginous "channels" or "lakes" consistent with dermal collateral lymphangiectasis and sequestered lymph, and increased subcutaneous fat. In contrast, LAS showed dermal diffusion ("backflow"), cross-over with retrograde tracer backflow (reflux), delayed tracer transport, and depending on the cause of lymphedema (i.e., primary or secondary), discrete or poorly defined lymph trunks (tracer "bands") and delayed or nonvisualization of regional lymph nodes. Although not a first-line clinical test, MR particularly in conjunction with LAS noninvasively provides accurate anatomical definition of the peripheral lymphatic system. In contradistinction to LAS, MR can visualize lymph trunks, nodes, and soft tissues proximal to sites of lymphatic obstruction. Together these imaging modalities may substitute for conventional oil contrast lymphography in the evaluation of the pathogenesis and evolution of most lymphologic disorders.

A new nonionic macrocyclic gadolinium(III) chelate as a potential magnetic-resonance-imaging contrast agent.

A new GdIII complex of a 17-membered macrocycle with three pendant carboxymethyl groups has been synthesized; the ligand has been obtained in a single step from diethylenetriaminepentaacetic dianhydride (pentetic dianhydride) and 1,4-butanediamine (putrescine). An X-ray crystal analysis has shown that the complex is a nonionic metal chelate with a coordinated water molecule. The near-zero electrical conductivity, 1 omega-1 cm2 mol-1, of a 1 mM solution indicated that the metal chelate is essentially undissociated. The NMR T1 relaxivity is 2.5 s-1 mM-1 at a resonance field of 64 MHz, and 3.4 s-1 mM-1 at 250 MHz. This new GdIII complex is a potentially important magnetic resonance imaging contrast agent.

Bone marrow imaging using STIR at 0.5 and 1.5 T.

We retrospectively examined MR images in 82 patients to evaluate the usefulness of short inversion time inversion recovery (STIR) in bone marrow imaging at 0.5 and 1.5 T. The study included 56 patients at 1.5 T and 26 patients at 0.5 T with a variety of pathologic bone marrow lesions (principally oncological), and compared the contrast and image quality of STIR imaging with spin-echo short repetition time/echo time (TR/TE), long TR/TE, and gradient-echo sequences. The pulse sequences were adjusted for optimal image quality, contrast, and fat nulling. STIR appears especially useful for the evaluation of red marrow (e.g., spine), where contrast between normal and infiltrated marrow is greater than with either gradient-echo or T1-weighted images. STIR is also extremely sensitive for evaluation of osteomyelitis, including soft tissue extent. In more peripheral (yellow) marrow, T1-weighted images are usually as sensitive as STIR. Limitations of STIR include artifacts, in particular motion artifact that at high field strength necessitates motion compensation. At 0.5 T, however, motion compensation is usually not necessary. Also, because of extreme sensitivity to water content, STIR may overstate the margins of a marrow lesion. With these limitations in mind, STIR is a very effective pulse sequence at both 0.5 and 1.5 T for evaluation of marrow abnormalities.

Magnetic resonance guidance of percutaneous ethanol injection in liver.

RATIONALE AND OBJECTIVES: Percutaneous ethanol injection (PEI) is used as a form of treatment for cancer, particularly malignant hepatic tumors. Little is known about the intratumoral distributions of ethanol following PEI. We assessed, using magnetic resonance (MR) imaging, the distribution of ethanol in liver and the concentration of ethanol needed to kill tumor cells in vivo. METHODS: MR imaging studies were performed using phantoms of alcohol, ex vivo bovine liver, and healthy human volunteers. A variety of pulse sequences were tested for their ability to maximize the signal intensity from alcohol while minimizing the signal from liver tissues as well as the regions of necrosis following ethanol injection. A cell culture model of in vitro cytotoxicity was developed to predict the target concentration of alcohol necessary for killing tumor cells. RESULTS: At 1.5 T, we found that an inversion-recovery spin-echo sequence using an inversion time of 250 msec and an echo time of 150 msec in combination with water saturation pulses effectively suppressed the tissue water signal from human liver while obtaining a clear signal from the ethanol. The cytotoxicity experiments suggested that a concentration of 20% or more ethanol is sufficient to completely kill all the tumor cells. CONCLUSION: A critical concentration of ethanol (e.g., 10%) is necessary for full tumoricidal effect. MR imaging should be able to determine the volume of distribution and the intratumoral concentrations of ethanol, thus potentially allowing researchers to achieve the requisite concentrations for maximal tumoricidal effects.

Accuracy of CT biopsy: laser guidance versus conventional freehand techniques.

RATIONALE AND OBJECTIVES: The purpose of this study was to determine whether laser-guided computed tomographic (CT) biopsy is more accurate than CT-guided biopsy with conventional freehand techniques. MATERIALS AND METHODS: Two independent operators performed an equal number of freehand and laser-guided needle passes at varying single and double angles (0 degree, 30 degrees, 60 degrees, 25 degrees/30 degrees, and 25 degrees/60 degrees) on targets within six pork and beef phantoms. A total of 180 biopsy passes were performed, and error distances of needle tip to target were tabulated. Data were analyzed by means of repeated measures analysis of variance (ANOVA) to compare the accuracy of laser guidance with freehand passes. ANOVA and correlation analysis were also used to confirm the relative equivalency of phantom targets and biopsy parameters. RESULTS: Overall, laser-guided passes were statistically significantly more accurate than freehand passes. Mean error with laser guidance was 5.01 mm (standard error [SE] = 0.41 mm), whereas mean error with freehand techniques was 10.58 mm (SE = 0.82 mm) (F = 52.0, df = 1.17, P = .0001). Ninety-three percent of laser-guided passes and 56% of freehand passes were within 1 cm of the intended target. Error increased for both laser-guided and freehand techniques with larger angles or double-angle biopsies, but the increases were greater with freehand technique. No statistically significant differences existed between the targets themselves or biopsy parameters for the two operators. CONCLUSION: Laser-guided CT biopsies were more accurate than freehand CT biopsies. Practical advantages of laser guidance over freehand CT biopsy methods may include decreased procedure times and reduced patient morbidity.

Estimation of volumes of distribution and intratumoral ethanol concentrations by computed tomography scanning after percutaneous ethanol injection.

RATIONALE AND OBJECTIVES: We developed a technique for estimating the volumes of distribution and intratumoral ethanol concentrations using computed tomography (CT) scanning in patients undergoing percutaneous ethanol injection (PEI) treatment of malignant hepatic tumors. METHODS: A phantom containing anhydrous ethanol diluted with deionized distilled water to concentrations of 0-100% ethanol was scanned by CT. Thirty-seven treatment sessions were performed on eight patients with malignant hepatic tumors undergoing PEI under CT guidance. The patients were scanned pre- and post-PEI, and a region of interest containing the treated hepatic tissue was selected for pixels between -250 and 15 Hounsfield units (H). The mean density of the pixels in this range was computed and the concentration of ethanol estimated. Volumes of distribution of ethanol and intratumoral concentration were then correlated with volume of ethanol injected during PEI. The ratios of volumes of distribution of ethanol to ethanol injected (adjusted in-range [IR]/volume injected) were compared for responders (n = 4) and nonresponders (n = 4). RESULTS: CT numbers in the phantom scaled linearly with ethanol concentration; 100% ethanol measured -234 H. On CT scans after PEI, the volume of distribution of ethanol correlated positively with the volume injected. Calculated intratumoral ethanol concentrations ranged from 4% to 31%. The adjusted IR/volume injected was significantly higher for responders than nonresponders (p < .5). CONCLUSION: CT density data can be used to estimate volume of ethanol distribution in tissue; a larger relative intratumoral distribution of alcohol appears to correlate with a favorable response to PEI. However, CT measurement of intratumoral ethanol concentrations may require more complex computational techniques.