Gene R. DiResta

midazolam Changes Cerebral Blood Flow in Discrete Brain Regions : an H2-15o Positron Emission Tomography Study

Background: Changes in regional cerebral blood flow (rCBF) determined with H215 O positron emission tomographic imaging can identify neural circuits affected by centrally acting drugs. Methods: Fourteen volunteers received one of two midazolam infusions adjusted according to electroencephalographic response. Low or high midazolam effects were identified using post‐hoc spectral analysis of the electroencephalographic response obtained during positron emission tomographic imaging based on the absence or presence of 14‐Hz spindle activity. The absolute change in global CBF was calculated, and relative changes in rCBF were determined using statistical parametric mapping with localization to standard stereotactic coordinates. Results: The low‐effect group received 7.5 +/‐ 1.7 mg midazolam (serum concentrations, 74 +/‐ 24 ng/ml), and the high‐effect group received 9.7 +/‐ 1.3 mg midazolam (serum concentrations, 129 +/‐ 48 ng/ml). Midazolam decreased global CBF by 12% from 39.2 +/‐ 4.1 to 34.4 +/‐ 6.1 ml [center dot] 100 g sup ‐1 [center dot] min sup ‐1 (P < 0.02 at a partial pressure of carbon dioxide of 40 mmHg). The rCBF changes in the low‐effect group were a subset of the high‐effect group. Decreased rCBF (P < 0.001) occurred in the insula, the cingulate gyrus, multiple areas in the prefrontal cortex, the thalamus, and parietal and temporal association areas. Asymmetric changes occurred, particularly in the low‐effect group, and were more significant in the left frontal cortex and thalamus and the right insula. Relative rCBF was increased in the occipital areas. Conclusion: Midazolam causes dose‐related changes in rCBF in brain regions associated with the normal functioning of arousal, attention, and memory.

Elevated Physiologic Tumor Pressure Promotes Proliferation and Chemosensitivity in Human Osteosarcoma

Purpose: This study investigates the effect of constitutively raised interstitial fluid pressure on osteosarcoma physiology and chemosensitivity. Experimental Design: We did pressure and blood flow assessments at the time of open biopsy in patients with the diagnosis of high-grade osteosarcoma and correlated this to survival and chemotherapy-associated tumor necrosis. Osteosarcoma cell lines were then evaluated for proliferative and therapeutic indices in a replicated high-pressure environment. Results: Sixteen osteosarcomas in vivo were assessed and exhibited elevated interstitial fluid pressures (mean 35.2 ± SD, 18.6 mmHg). This was not associated with significantly impeded blood flow as measured by a Doppler probe at a single site (P < 0.12). Nonetheless, greater chemotherapy-associated necrosis and associated longer survival were seen in tumors with higher interstitial fluid pressures (P < 0.05). In vitro, cells undergo significant physiologic changes under pressure. Osteosarcoma cell lines grown in a novel hydrostatically pressurized system had variable cell line–specific growth proportional to the level of pressure. They were more proliferative as indicated by cell cycle analysis with more cells in S phase after 48 hours of pressurization (P < 0.01). There was a significant elevation in the cell cycle–related transcription factors E2F-1 (P < 0.03) and E2F-4 (P < 0.002). These changes were associated with increased chemosensitivity. Cells tested under pressure showed an increased sensitivity to cisplatin (P < 0.00006) and doxorubicin (P < 0.03) reminiscent of the increased chemotherapy-associated necrosis seen in tumors with higher interstitial fluid pressure in the clinical study. Conclusions: The results of this study suggest that cells in the in vivo pressurized environment are at a higher state of regenerative activity than is demonstrable in conventional cell culture systems. Variations in tumor interstitial fluid pressure have the potential to alter chemotherapeutic effects.

Development of a method to measure kinetics of radiolabelled monoclonal antibody in human tumour with applications to microdosimetry: positron emission tomography studies of iodine-124 labelled 3F8 monoclonal antibody in glioma

We present a method to assess quantitatively the immunological characteristics of tumours using radiolabelled monoclonal antibody and positron emission tomography (PET) to improve dosimetry for radioimunotherapy. This method is illustrated with a glioma patient who was injected with 96.2 MBq of iodine-124 labelled 3F8, a murine antibody (IgG3) specific against the ganglioside GD2. Serial PET scans and plasma samples were taken over 11 days. A three-compartment model was used to estimate the plasma to tumour transfer constant (K1), the tumour to plasma transfer constant k2, the association and dissociation constants (k3, k4) of antibody binding, and the binding potential. Tumour radioactivity peaked at 18 h at 0.0045% ID/g. The kinetic parameters were estimated to be: K1 = 0.048 ml h−1 g−1, k2 = 0.16 h−1, k3 = 0.03 h−1, k4 = 0.015 h−1 and BP = 2.25. Based on these kinetic parameters, the amount of tumour-bound radiolabelled monoclonal antibody was calculated. This method permits estimates of both macrodosimetry and microdosimetry at the cellular level based on in vivo non-invasive measurement.

PMMA to stabilize bone and deliver antineoplastic and antiresorptive agents.

Antineoplastic and antiresorptive drugs added to polymethylmethacrylate cement may prevent local cancer progression and failure of reconstructive devices used to treat patients with pathologic fractures. We tested the mechanical properties of cement containing various amounts of the drugs and found that as much as 2 g of either doxorubicin or pamidronate can be added to Simplex ® cement and the cement retains 87% of its compressive and tensile strength after 6 months of wet storage. Approximately 1 mg pamidronate elutes from experimental pellets. One half of the drug elution occurs within the first day in experiments that combined doxorubicin and pamidronate, and within 3 days when pamidronate was the only additive. Cement containing these drugs seems to be strong enough, but its fatigue strength should be tested before using it clinically. Sufficient amounts of the tested drugs elute to have potential biologic activity.

Tumor interstitial fluid pressure may regulate angiogenic factors in osteosarcoma

We have previously shown that osteosarcomas (OS) have states of increased interstitial fluid pressure (IFP), which correlate with increased proliferation and chemosensitivity. In this study, we hypothesized that constitutively raised IFP in OS regulates angiogenesis. Sixteen patients with the clinical diagnosis of OS underwent blood flow and IFP readings by the wick‐in‐needle method at the time and location of open biopsy. Vascularity was determined by capillary density in the biopsy specimens. We performed digital image analysis of immunohistochemical staining for CD31, VEGF‐A, VEGF‐C, and TPA on paraffin‐embedded tissue blocks of the biopsy samples. Clinical results were validated in a pressurized cell culture system. Interstitial fluid pressures in the tumors (mean 33.5 ± SD 17.2 mmHg) were significantly higher (p = 0.00001) than that in normal tissue (2.9 ± 5.7 mmHg). Pressure readings were significantly higher in low vascularity tumors compared to high vascularity tumors (p < 0.001). In the OS cell lines, growth in a pressurized environment was associated with VEGF‐A downregulation, VEGF‐C upregulation, and TPA upregulation. The reverse was seen in the OB cell line. Growth in the HUVEC cell line was not significantly inhibited in a pressurized environment. Immunohistochemical assessment for VEGF‐A (p = 0.01), VEGF‐C (p = 0.008), and TPA (p = 0.0001) translation were consistent with the findings on PCR. Our data suggests that some molecules in angiogenesis are regulated by changes in IFP.

Cell proliferation of cultured human cancer cells are affected by the elevated tumor pressures that exist in vivo.

Elevated interstitial fluid pressure (IFP) is observed in most solid tumors. However, the study of the cellular processes of tumors and the development of chemotherapy are routinely studied using in vitro culture systems at atmospheric pressure. Using a new pressurized cell culture system, we investigated the influence of hydrostatic pressure on population dynamics of three primary osteosarcoma (HOS, U2OS, SaOS2) and two metastatic tumor cell lines (MCF7 breast, H1299 lung) that invade bone. Values of IFP in normal human bone and muscle, and in osteosarcoma tumors obtained during their surgical biopsy established the hydrostatic pressure range for the in vitro cell studies. The IFP values were obtained from a retrospective review of patient records. IFP from confirmed osteosarcoma was 35.9± 16.2 mmHg. Tumor IFP was significantly higher than muscle IFP (p < 0.001) and bone IFP (p < 0.003).The in vitro study measured the cell-line proliferation using hydrostatic pressures of 0, 20, 50 and 100 mmHg. The findings suggest that hydrostatic pressure either increases or decreases tumor proliferation rates depending on cell type. Furthermore, cell death was not associated with apoptosis.

Biomechanical analysis of anterior poly-methyl-methacrylate reconstruction following total spondylectomy for metastatic disease.

Study Design. Three reconstruction options were evaluated biomechanically following total spondylectomy using human cadaveric spine specimens. Objectives. To evaluate and compare the stability of combined anterior and posterior fixation incorporating poly-methyl-methacrylate with alternative accepted reconstruction techniques. Summary of Background Data. Total spondylectomy represents the most radical option for decompression in metastatic spinal cord compression. Poly-methyl-methacrylate is considered a useful adjunct in spinal column stabilization and arthrodesis; however, there is little published biomechanical data to support its use in this setting. Methods. Ten fresh-frozen human cadaveric spines (T9-L3) were used. After intact analysis, a total spondylectomy was performed at T12. Three potential reconstruction techniques were tested for their ability to restore stiffness to the specimen: 1) multilevel posterior pedicle screw instrumentation from T10-L2; 2) anterior instrumentation (ATL Z plate II) and rib graft at T11-L1 with multilevel posterior pedicle screw instrumentation from T10-L2; and 3) anterior cement (Simplex P) and pins construct (T12) with multilevel posterior pedicle screw instrumentation from T10-L2. Each of the three potential reconstruction techniques was tested on each specimen in random order using nondestructive testing under load control. Results. Only combined stabilization techniques (e.g., anterior instrumentation and rib graft with multilevel posterior pedicle screw instrumentation and anterior cement-and-pins construct with multilevel posterior pedicle screw instrumentation) restored stiffness to a level equivalent to or higher than that of the intact spine in all loading modes (P < 0.05). Anterior cement-and-pins construct with multilevel posterior pedicle screw instrumentation provided more stability to the specimen than anterior instrumentation and rib graft with multilevel posterior pedicle screw instrumentation in compression and flexion testing (P < 0.05). Posterior instrumentation alone did not restore stiffness to the intact level in compression and flexion testing (P < 0.005). Conclusions. Combined anterior and posterior reconstruction using a cement construct provides equal to or more stability than the intact spine in all testing modes. Posterior stabilization alone is an inferior method of reconstruction following total spondylectomy. Poly-methyl-methacrylate has the advantage over traditional anterior reconstruction techniques in that it can be inserted using a posterior approach.

Measurement of brain tissue specific gravity using pycnometry

In this paper we introduce and characterize pycnometry, a method used to measure fluid density, for determining a tissues specific gravity. It uses a 2-ml glass pycnometer filled with distilled water to determine a tissue samples displacement volume. The tissues density is determined when its weight is divided by this volume and specific gravity is computed by dividing the tissue density by the density of water. Pycnometry was validated using pre-calibrated glass, specific gravity standards over the range 1.03-1.26, and compared to the density gradient method using rat brain tissue. We observed that the specific gravity values obtained using pycnometry were highly correlated with the specific gravity standards (slope = 1.0107, r = 0.996) and with the density gradient column when tissue volumes larger than 0.120 ml were used with the pycnometer (slope = 1.0707, r = 0.9826). Good correlation was also observed between percent water content values computed using the Nelson equation with pycnometry or density gradient specific gravity values versus the measured percent water content values obtained with the wet weight/dry weight method. Pycnometry is an accurate, reproducible technique to measure tissue specific gravity and brain edema and is best suited for use in a laboratory that engages sporadically in brain edema measurement.

“Artificial Lymphatic System”: A New Approach to Reduce Interstitial Hypertension and Increase Blood Flow, pH and pO2 in Solid Tumors

AbstractA mechanical drainage system, the “artificial lymphatic system” (ALS), consisting of a vacuum source and drain, is evaluated for its ability to aspirate the interstitial fluids responsible for the elevated interstitial fluid pressure (IFP) observed in solid tumors. IFP, pH, and pO2 radial profiles were measured before and after aspiration using wick-in-needle (WIN) probes, needle pH and oxygen electrodes, respectively. Laser Doppler flowmetry measured temporal changes in blood flow rate (BFR) at the tumor surface during aspiration. The WIN probe and IFP profile data were analyzed using numerical simulation and distributed mathematical models, respectively. The model parameter, pE reflecting central tumor IFP, was reduced from 15.3 to 5.7 mm Hg in neuroblastoma and from 13.3 to 12.1 mm Hg in Walker 256, respectively, following aspiration. The simulation demonstrated that spatial averaging inherent in WIN measurements reduced the calculated magnitude of the model parameter changes. IFP was significantly lower (p < 0.05), especially in regions surrounding the drain, and BFR was significantly higher (p < 0.05) following 25 and 45 min of aspiration, respectively; pH and pO2 profiles increased following aspiration. The experimental and mathematical findings suggest that ALS aspiration may be a viable way of reducing IFP and increasing BFR, pO2 and pH and should enhance solid tumor chemo and radiation therapy.

Pharmacokinetics and acute toxicology of intraventricular 131I-monoclonal antibody targeting disialoganglioside in non-human primates

Tumors metastatic to the leptomeninges are often incurable despitecurrent aggressive treatment modalities. Regional therapy by intrathecaladministration of monoclonal antibodies (MoAbs) can maximize theirconcentration to tumor sites while reducing systemic toxicities.Anti-GD2 antibody 3F8 has successfully targeted humanneuroectoderm derived tumors. Disialoganglioside GD2expression in the central nervous system is identical between humans andcynomolgus monkeys. We studied the pharmacokinetics and the acute andsubacute toxicities of intraventricular 131I-3F8 in 8cynomolgus monkeys. Four animals were purposely immunized with intravenous3F8 administered 2–4 weeks prior to injections. All animals remainedclinically stable. Toxicities included weight loss, fever and CSFleukocytosis. One animal developed a left-sided hemiparesis following hisseventh injection, presumably due to a local drug accumulation in thesetting of an intermittently patent catheter. The estimated radiation doseto the CSF was 19–48 Gy in the immunized monkeys and 19–82 Gy inthe nonimmunized monkeys, and to blood was 0.11–0.98 Gy and0.29–2.03 Gy, respectively. Histopathology revealed chronic reactivechanges adjacent to the region of catheter placement and a focal vasculitisin 2 animals. Peripheral blood counts and bone marrow examinations remainednormal. Because of the blood-brain barrier, CSF monkey-anti-mouse antibodytiters were less than 10 per cent of those in the serum. In contrast to theCSF radioactivity clearance which was similar in all animals, bloodclearance was substantially accelerated in 3F8-immunized animals versuscontrols. Correspondingly, the CSF to blood dose ratio was improved 1.3 to6.6 fold (mean 3.5). We conclude that intraventricular administration of131I-3F8 in primates is tolerable. It can deliver very highdoses of radiation to the CSF space with minimal toxicity to blood and bonemarrow. Serum anti-mouse antibody accelerates the clearance of131I-3F8 in blood and may improve the therapeutic index.