The VideoToolbox software for visual psychophysics: transforming numbers into movies

BACKGROUND AND OBJECTIVE: To optimize photodynamic therapy, it is necessary to know the distribution of photosensitizer in normal tissue as well as tumors and to know how well animal models match human. This study measured the biodistribution of meta-Tetra(Hydroxyphenyl) Chlorin (mTHPC) in three species of animals and in humans. STUDY DESIGN/MATERIALS AND METHODS: mTHPC was injected intravenously into dogs, rabbits, and humans, and drug levels in various tissues were determined 6 days later. One dog was perfused with 3 L of saline to remove blood trapped within organs. RESULTS: Absolute and relative concentrations of drug in specific tissues varied between species and between individuals. There was a general pattern of distribution. Highly vascularized tissues had the highest levels of mTHPC, not simply due to trapping of blood. mTHPC did not localize in bone and did not cross the blood-brain barrier. Humans had much higher levels of drug in their plasma and tissues than did animals. CONCLUSIONS: First, drug retention varies from one tissue to another. Second, there is significant variability from one individual to another, whether animal or human. Third, current models cannot accurately predict from animal studies the optimum dose for humans. Measurement of photosensitizer level in plasma at time of treatment would allow optimal photodynamic dosing.     

Factors that facilitate compliance to lower fat intake

A construct of IL-2 and pseudomonas exotoxin (PE40) has been genetically engineered. An aliquot of 100 microliter of the chimeric protein, radiolabelled with I125, was administered to healthy rats by various routes. At different intervals, ocular and non ocular tissues were removed and the levels of the radiolabelled chimeric protein IL-2-PE40 measured. Systemic administration of IL2-PE40 either intravenously (IV) or intraperitoneally (IP) leads to high levels of the drug in the blood, liver and spleen. Little or no radioactivity is observed within the ocular tissues using this route. On the other hand, local administration of the drug either as subtenon injection or as eye drops resulted in a very high concentration of the drug within the conjunctiva, cornea and sclera, with little radioactivity detected systemically. Subtenon injection induced a significant drug level within the optic nerve. With the drops, the chimeric protein was also detected, in low levels, intraocularly.     

Altered tissue distribution and elimination of amikacin encapsulated in unilamellar, low-clearance liposomes (MiKasome)

PURPOSE: Amikacin in small unilamellar liposomes (MiKasome) has prolonged plasma residence (half-life > 24hr) and sustained efficacy in Gram-negative infection models. Since low-clearance liposomes may be subject to a lower rate of phagocytic uptake, we hypothesized this formulation may enhance amikacin distribution to tissues outside the mononuclear phagocyte system. METHODS: Rats received one intravenous dose (50 mg/kg) of conventional or liposomal amikacin. Amikacin was measured for ten days in plasma, twelve tissues, urine and bile. RESULTS: Liposomal amikacin increased and prolonged drug exposure in all tissues. Tissue half-lives (63-465 hr) exceeded the plasma half-life (24.5 hr). Peak levels occurred within 4 hours in some tissues, but were delayed 1-3 days in spleen, liver, lungs and duodenum, demonstrating the importance of characterizing the entire tissue concentration vs. time profile for liposomal drugs. Predicted steady-state tissue concentrations for twice weekly dosing were >100 microg/g. Less than half the liposomal amikacin was recovered in tissues and excreta, suggesting metabolism occurred. Amikacin was not detected in plasma ultrafiltrates. Tissue-plasma partition coefficients (0.2-0.8 in most tissues) estimated from tissue-plasma ratios at Tmax were similar to those estimated from tissue AUCs. CONCLUSIONS: Low-clearance liposomal amikacin increased and prolonged drug residence in all tissues compared to conventional amikacin. The long tissue half-lives suggest liposomal amikacin is sequestered within tissues, and that an extended dosing interval is appropriate for chronic or prophylactic therapy with this formulation.     

The behavioral syndrome induced by adreno corticotropic hormone in rats is prevented by Ca++ channel blockade

Cellular levels of O6-methylguanine-DNA methyltransferase (MGMT) correlate strongly with cellular resistance to carcinogenic and chemotherapeutic agents that produce adducts at the O6-position of guanine in DNA. Although biochemical and molecular assays can indicate the average MGMT content of tissues or tumors, they cannot distinguish mixed populations of cells, such as those that exist in tumor biopsy samples. We have determined MGMT at the cellular level in a panel of pediatric rhabdomyosarcoma xenografts by in situ immunostaining with a human MGMT-specific antibody employing a very sensitive procedure that involves biotin-avidin coupled horseradish peroxidase with silver-enhanced diaminobenzidine-nickel staining. Two xenograft tumor lines known to be MGMT-deficient were not stained, whereas the nuclei in three MGMT-expressing lines were clearly stained. This is the first demonstration of an in situ procedure that discriminates drug-sensitive MGMT-deficient tumors from drug-resistant MGMT expressing tumors. This procedure should prove useful, therefore, for predicting the susceptibility of tissues and tumors to O6-guanine alkylating agents.     

Theoretical models for drug delivery to solid tumors

The effectiveness of anti-cancer drug therapies is often limited by the difficulty of achieving drug delivery throughout solid tumors. Mathematical models permit an analysis of the factors leading to inadequate drug delivery to tumors and can suggest strategies for improving delivery. An overview is given of key factors that influence drug delivery and the extent to which they have been incorporated into existing theoretical models. These factors include spatial gradients of drug concentration and other variables within tumors and other parts of the body, and the relative magnitudes of the time scales involved in drug transport, tumor cell kinetics, and host toxicity. Models for both systemic and regional delivery methods are considered, including intravenous, intraarterial, intraperitoneal, intrathecal, and intratumoral delivery. Strategies for improving delivery are discussed, including use of two-step therapies, hyperthermia, liposome encapsulation, and magnetic targeting. Until now, modeling has mainly developed in separate subfields of tumor growth and cell kill kinetics, compartmental modeling of the body, spatially distributed models for single tissues, radiation dose calculations, tumor oxygenation, tumor blood flow, and cellular pharmacokinetics. In the future, models that integrate these subfields should be developed.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 +/- 1.2 pmol/g ww (mean +/- SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 +/- 1.8 fmol/10(5) cells/24h, mean +/- SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10(5) cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

Dental disease and current treatment needs in a group of physically handicapped children

The enzyme glutathione reductase (GR) (GSSG+NADPH+H+-->2 GSH+NADP+) plays a key role in the cellular defense against oxidative stress. High levels of GR activity are often associated with tumor growth and/or resistance mechanisms against drug and radiation therapy. In order to investigate the molecular basis of elevated glutathione reductase activities we studied the enzyme at the DNA, mRNA and protein levels in murine experimental tumor cell lines and in human lung tumors. A modified ultracentrifugation procedure was developed which allowed the simultaneous isolation of DNA and total cellular RNA. Out of 11 human bronchial carcinomas obtained from patients without prior chemotherapy, five tumors showed a GR activity which was 2.4 to 3.8 times higher than in the respective control tissues. In each case the elevated enzyme activity was accompanied by an elevated GRmRNA levels. For none of the tumors, GR gene rearrangement or amplification was observed by Southern blot analyses. The mouse tumor cell lines ASB XIV, Lewis lung carcinoma and EAT cells, also showed high levels of GRmRNA whereas this mRNA was hardly detectable in normal mouse lung tissue.     

Expression of the multidrug resistance-associated protein (MRP) gene in human cancers

We determined the expression of a newly recognized drug resistance gene, the multidrug resistance-associated protein (MRP) gene, [Cole et al., Science (Washington DC), 258: 1650-1654, 1992], in normal human tissues and in >370 human tumor biopsies using a quantitative RNase protection assay and immunohistochemistry. MRP mRNA appeared to be ubiquitously expressed at low levels in all normal tissues, including peripheral blood, the endocrine glands (adrenal and thyroid), striated muscle, the lymphoreticular system (spleen and tonsil), the digestive tract (salivary gland, esophagus, liver, gall bladder, pancreas, and colon), the respiratory tract (lung), and the urogenital tract (kidney, bladder, testis, and ovary). The human cancers analyzed could be divided into three groups with regard to MRP expression. Group 1 consists of tumors that often exhibit high to very high MRP mRNA levels (e.g., chronic lymphocytic leukemia). Group 2 comprises the tumors that often exhibit low, but occasionally exhibit high MRP mRNA expression (e.g., esophagus squamous cell carcinoma, non-small cell lung cancer, and acute myelocytic leukemia). Group 3 comprises the tumors with predominantly low levels of MRP mRNA, comparable to the levels found in normal tissues (e.g., other hematological malignancies, soft tissue sarcomas, melanoma, and cancers of the prostate, breast, kidney, bladder, testis, ovary, and colon). Using the MRP-specific mAbs MRPr1 and MRPm6, we confirmed the elevated MRP mRNA levels in tumor tissues by immunohistochemistry. We conclude that hyperexpression of MRP is observed in several human cancers, and that additional studies are needed to assess the clinical relevance of MRP.     

Antitumor activity of nigericin and 5-(N-ethyl-N-isopropyl)amiloride: an approach to therapy based on cellular acidification and the inhibition of regulation of intracellular pH

The extracellular pH (pHe) in solid tumors is frequently lower than the pHe in normal tissues, but the intracellular pH (pHi) is regulated to physiological levels. Cell killing can be achieved in an acidic environment in tissue culture by nigericin, which acidifies cells by transporting H+ from the extracellular space into the cytoplasm; this cell killing can be enhanced when used with 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a potent inhibitor of membrane-based Na+/H+ exchange, which plays a major role in the regulation of pHi (R. P. Maidorn; E. J. Cragoe; I. F. Tannock, Br. J. Cancer 67:297-303; 1993). We have therefore assessed the ability of nigericin and EIPA to kill cells in two murine solid tumors (the KHT fibrosarcoma and the EMT-6 sarcoma). Hydralazine, which reduces tumor blood flow, or glucose, which stimulates glycolysis leading to accumulation of lactate, were also administered to mice to lower pHe in the tumors. We observed only a small decrease in the surviving fractions of cells in the tumors when tolerated doses of nigericin and EIPA were given IP to tumor-bearing mice. When nigericin and EIPA were combined with administration of hydralazine, the surviving fraction of cells in both tumors was reduced by a factor of 0.01, but there were minimal effects on growth delay. Administration of glucose with nigericin and EIPA led to a smaller reduction in surviving fraction of the KHT tumor (by approximately 0.1), although glucose was more effective than hydralazine in lowering the mean tumor pHe. When KHT tumors were treated with 15 Gy X-rays followed immediately by nigericin, EIPA, and hydralazine, a reduced surviving fraction as well as an increase in tumor growth delay was observed compared to radiation alone; however, there was little evidence to suggest that these agents were selectively toxic to the cells that survived radiation. Nigericin and EIPA, with or without hydralazine, had minimal effects on normal tissues, as assessed by changes in body weight, number of leukocytes, and serum creatinine levels. We conclude that pharmacological effects to acidify cells and to prevent regulation of pHi under the acidic conditions that exist in solid tumors can lead to moderate levels of cell killing, if additional strategies are used to lower tumor pHe.     

Ocular pharmacokinetics of orally administered azithromycin in rabbits

Azithromycin was orally administered to Dutch-belted rabbits following extracapsular lens extraction in one eye. At various times the animals were sacrificed, and serum and ocular tissues were obtained for drug level determination by HPLC-EC. Following a single dose, peak levels of drug in ocular tissues were measured within 8 hours (cornea > 0.5 micrograms/g [15mg/kg]; > 1.5 micrograms/g [3Omg/kg]). Highest levels were obtained in iris and ciliary body ( > 15 micrograms). Measurable tissue levels persisted for at least 120 hours. Trough levels increased proportionately during drug multiple dose administration. Five days following five daily 15mg/kg doses, corneal levels exceeded 0.5 micrograms/g, and iris and ciliary levels were higher than 15 micrograms/g. Aqueous humor and serum levels were equivalent. Vitreous humor levels, though higher than aqueous humor, were consistently < 1 microgram/ml. Extracapsular cataract extraction did not significantly affect drug uptake.     

Soft tissue osteosarcoma with telangiectatic features: MR imaging findings in two cases

Extraskeletal osteosarcomas are rare tumors, and the telangiectatic variety is the least common histological variety in this group. This report describes the clinical and MR imaging findings in two cases arising in the pretibial soft tissues. Both tumors demonstrated marked inhomogeneity with T2-weighted spin echo and STIR sequences. One of the tumors revealed numerous fluid levels within the lesion. A review of the MRI features of these tumors is provided. Osteosarcoma with telangiectatic features should be considered in the differential diagnosis of a soft tissue mass with fluid-fluid levels in patients 40 years of age or older.     

Relevance of circadian cell kinetics in the timing of chemotherapy in animal tumors

Circadian rhythmicity in cell division has been proved in the actively proliferating healthy tissues of rodents (cornea, G.I.-tract, bone marrow, gonads, epidermis) as well as in most spontaneous or transplanted tumors growing in solid or ascites fluid phases. This circadian division accounts in part for the circadian varying sensitivity of healthy target tissues to oncolytic agents (chronotoxicity/chronotolerance). Similarly, antitumor activity as gauged from analysis of impact on cell kinetics and/or tumor shrinkage or cure was also shown to depend upon the dose and doing time of anticancer drug administration. Variations in the timing of internal cell kinetics after initial drug administration were also shown to depend on the type of drug (alkylating agent, antimitotic, or antimetabolite agent). Interestingly, internal desynchronization may occur in both healthy and tumor tissues, it may allow the selection of given circadian stages for the administration of a second drug at the point of highest tumor sensitivity and healthy tissue resistance (time of least sensitivity). These observations have been utilized to test strategies in sequential drug scheduling aimed at improving the overall therapeutic index. Observations indicate that in some instances these approaches could be translated to human beings.     

Intratumoral conversion of 5-fluorocytosine to 5-fluorouracil by monoclonal antibody-cytosine deaminase conjugates: noninvasive detection of prodrug activation by magnetic resonance spectroscopy and spectroscopic imaging

The monitoring of antibody-directed enzyme-prodrug therapies requires evaluation of drug activation within the tissues of interest. We have demonstrated the feasibility of noninvasive magnetic resonance spectroscopy and spectroscopic imaging (chemical shift imaging) to detect activation of the prodrug 5-fluorocytosine (5-FCyt) to the cytotoxic species 5-fluorouracil (5-FU) by monoclonal antibody-cytosine deaminase (CD) conjugates. In vitro, L6-CD but not 1F5-CD selectively metabolized 5-FCyt to 5-FU on H2981 human lung adenocarcinoma cells because of the presence and absence of cell surface L6 and CD20 antigens, respectively. After pretreatment of H2981 tumor-bearing mice with L6-CD, in vivo metabolism of 5-FCyt to 5-FU within the tumors was detected by 19F magnetic resonance spectroscopy; the chemical shift separation between 5-FCyt and 5-FU resonances was approximately 1.2 ppm. 5-FU levels were 50-100% of 5-FCyt levels in tumors 10-60 min after 5-FCyt administration. Whole body 19F chemical shift imaging (6 x 6 mm in-plane resolution) of tumor-bearing mice demonstrated the highest signal intensity of 5-FU within the tumor region. This study supports further development of noninvasive magnetic resonance methods for preclinical and clinical monitoring of CD enzyme-prodrug therapies.     

Osteopontin expression in mammary gland development and tumorigenesis

Osteopontin (OPN) is a secreted phosphoprotein that binds to cells via an Arg-Gly-Asp sequence and to mineralized surfaces. The protein can mediate cell adhesion and is strongly implicated in transformation and tumorigenesis. We have examined the expression pattern of OPN in mouse mammary glands at different stages of postnatal development. Whereas OPN is expressed at low-to-moderate levels in mammary glands from virgin and pregnant mice, the levels of OPN mRNA are extremely high in the lactating gland, consistent with the presence of the protein in milk. Expression is highest at 2 days of lactation and declines thereafter, but it remains high through involution. OPN expression is restricted to small nests or groups of cells at 9 days of involution. These results suggest that OPN may play a specific role in the process of involution that may be distinct from its role during lactation. In mammary tumors arising spontaneously in transgenic mice expressing the oncogenes c-myc and/or v-Ha-ras under the control of the mouse mammary tumor virus promoter, the level of OPN expression is increased dramatically over that in the normal gland in these same animals. Numerous cells expressing OPN mRNA are widespread throughout the tumors. OPN protein is detectable by Western blotting in extracts from the mammary gland at 2 days of lactation and from the tumors, but not in mammary glands at other stages of development. We hypothesize that OPN is exported from most tissues and that the protein is only detectable in tissues elaborating fluids, such as the lactating mammary gland, or in pathological situations when expression of OPN is abnormally high, such as in tumors.     

Comparative kinetics of oxytetracycline and oxyglucocycline cotent in tissues of the ENT organs

The kinetics of oxytetracycline and oxyglucocycline levels (calculated for oxytetracycline) was studied on 80 patients with chronic inflammatory diseases of the upper respiratory organs, the antibiotics being administered intramuscularly. After a single injection of oxytetrachcline hydrochloride in a dose of 1500 Units/kg body weight its levels in the blood serum and certain tissues of the ENT organs were rather low. When the dose was 3000 Units/kg the maximum antibiotic level in the blood level reached 1 Unit/ml. The drug was detected in the bacteriostatic concentrations in the blood serum and tissues within 12 hours. When the dose of oxyglucocycline was 1500 Units/kg the rate of its absorption was 4 times higher than that of oxytetracycline absorption. However, the character of its distribution in the blood and tissues of the ENT organs did not differ from distribution of oxytetracycline as dependent on the duration of the injection period. The both pharmaceutical forms had a tendency to some retention of the drugs in the tissues of the palatine tonsils. Because of better absorption and retention in the tonsils tissue oxyglucocycline may be recommended for therapy of inflammatory processes in the lymphadenoid apparatus of the pharynx.     

Transcriptional activation of NF-kappa B activity by Epstein-Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases

Epstein-Barr virus (EBV) has been known to be associated with many malignant tumors, including nasopharyngeal carcinoma (NPC). Previous studies have indicated that an EBV-encoded oncoprotein, latent membrane protein 1 (LMP1), is expressed in many NPC tissues. LMP1 has been shown to stimulate HIV LTR through the two NF-kappa B binding sites within this promoter. In this study, we examined the feasibility of using this property of LMP1 as a therapeutic strategy for the treatment of NPC. This therapy consists of the preferential killing of the LMP1-expressing cells by gene transfer using the NF-kappa B-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system. The 800-bp HIV-LTR, which contains two NF-kappa B binding sites, was used to drive the HSVtk gene. Stable C33A cell clones expressing the LMP1 and the HSVtk genes were subjected to the GCV sensitivity test. Results showed that cells expressing both the LMP1 and the HSVtk genes were highly sensitive to GCV treatment. These cells were introduced into nude mice subcutaneously and tumors became palpable within 2 weeks. GCV was then introduced intraperitoneally to these mice and the sizes of the tumors were measured daily. Results showed that the tumors regressed in the group of mice carrying cells that stably expressed both the LMP1 and the HSVtk genes, but not in mice carrying cells containing LMP1 or HSVtk alone. Our data indicate that the HSVtk gene expressed from a NF-kappa B-binding motif-containing promoter that is regulated by LMP1 may be used as an in vivo gene therapy strategy of EBV LMP1-expressing cancers such as NPC.     

Effect of ifosfamide treatment on glutathione and glutathione conjugation activity in patients with advanced cancers

Several studies have suggested that the glutathione/glutathione S-transferase (GSH/GST) system is involved in resistance of tumors toward ifosfamide and other cytostatic agents. Besides, ifosfamide metabolites (in vitro) as well as ifosfamide treatment (in vivo) have been shown to decrease cellular GSH availability. In the present study, the in vivo effects of three different ifosfamide treatment schedules on the GSH/GST system were studied in patients with advanced cancers (n = 24): continuous i.v. infusions of 1300 mg/m2 daily for 10 days and 5000 mg/m2/day for 24 h, as well as a 4-h infusion of 3000 mg/m2 daily for 3 days. The GSH/GST system was characterized by administering bromisoval, a probe drug to assess GSH conjugation activity in vivo, as well as by daily monitoring of GSH concentrations in blood cells and plasma. Bromisoval pharmacokinetics was assessed before and at the end of the ifosfamide treatment. Blood cell GSH levels decreased significantly (P < 0.05) during the 3- and 10-day ifosfamide treatment schedules; the 24-h treatment had no effect. The ifosfamide treatment schedules had only minimal effects on bromisoval pharmacokinetics. Assuming that the kinetics of the probe drug provide an accurate reflection of enzyme activity, this suggests that GST activity remains unchanged. Because GSH conjugation of bromisoval enantiomers requires both GST activity and GSH availability, these results also indicate that, despite the 35% decrease in GSH in blood cells of two patient groups, the GSH availability of the cancer patients was not rate-limiting for GSH conjugation of bromisoval enantiomers. If GSH levels in blood cells reflect those in tumors/other tissues, the present results indicate that ifosfamide may be used clinically to decrease GSH levels. However, whether a 35% decrease is sufficient to increase tumor sensitivity toward (other) cytostatics remains uncertain.     

Oxygenation of head and neck tumors

BACKGROUND: Tumor hypoxia could play a role in the response to radiation therapy. Few data are available on oxygen tension (pO2) measurements in head and neck tumors. METHODS: The KIMOC-6650 Histograph (Eppendorf, Hamburg, Germany) was used to measure the oxygenation status of normal tissues and head and neck tumors in 20 patients. RESULTS: The median pO2 for normal tissues was 43 mmHg with very low pO2 values (2.0 mmHg or less) recorded in two patients. Low median pO2 levels (10 mmHg or less) were recorded in 2 of 5 primary tumors and in 11 of 15 metastatic lymphadenopathies, with very low values in 11 nodes. The median pO2 in tumors was lower than that of normal tissues in 12 of 15 patients with comparative measurements. Oxygen tension was recorded in three nodes after an evaluation of tissue density (by computed tomographic scanner); in two nodes, the mean and median pO2 values were lower in the hypodense areas than in isodense areas. The data for N2 and N3 nodes showed significantly more values below 2.0 mmHg as nodal size increased (P < 10(-4), by chi-square test). No systematic decrease in pO2 was recorded from the periphery to the center of the tumors. CONCLUSIONS: Very low pO2 values, corresponding to radiobiologic hypoxia, were found in most of these tumors. The prognostic value of these pO2 measurements in regard to treatment response remains to be demonstrated.