Intraocular penetration of oral levofloxacin in rabbits

The intraocular penetration of levofloxacin was studied in albino and pigmented rabbits after oral administration of 14C-levofloxacin at a dose of 20 mg/kg. The radioactivity of the eyeball of albino rabbits in the autoradiogram was much higher at 1 hr than at 2 or 6 hr after administration. On the other hand, the distribution of radioactivity in pigmented rabbits was similar at 1, 2, and 6 hr after administration. The radioactivity levels in melanin-containing tissues such as the iris-ciliary body and the choroid-retina were significantly higher in pigmented rabbits than in albino rabbits 2 hr after oral administration. The radioactivity levels in the ocular tissues without melanin except for the cornea were similar in albino and pigmented rabbits. These results indicate that the intraocular pharmacokinetics of levofloxacin are markedly affected by its affinity for melanin.     

Human glioma cells overexpress receptors for interleukin 13 and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin

Recently, we have demonstrated that a spectrum of human adenocarcinoma cell lines express binding sites for interleukin 13 (IL-13). These cells are killed by a chimeric protein composed of human (h) IL-13 and a derivative of Pseudomonas exotoxin, PE38QQR (Debinski et al., J. Biol. Chem., 270: 16775-16780, 1995). The cell killing was hIL-13- and hIL-4-specific, indicating that a common binding site for the two cytokines is present in several solid tumor cell lines. Herein, we report that an array of established glioma cell lines is killed by very low concentrations of hIL-13-PE38QQR, often reaching <1 ng/ml (<20 pM). Glioma cells express up to 30,000 molecules of IL-13 receptor/cell which has intermediate affinity toward hIL-13. hIL-13-PE38QQR is more active (up to 3 logs difference in cytotoxic activities) than are the corresponding chimeric toxins containing hIL-4 or hIL-6. The cytotoxic action of hIL-13-PE38QQR is blocked by an excess of hIL-13 on all cell lines studied, and it is not neutralized by hIL-4 on some of these cells. Our results show that human brain cancers richly express receptors for IL-13. Furthermore, the interaction detected previously between receptors for IL-13 and IL-4 on solid tumors cell lines is of a qualitatively different character in U-251 MG and U-373 MG glioma cells. The receptor for IL-13 may represent a new marker of brain cancers and an attractive target for anticancer therapies.     

In vivo and in vitro biotransformation of the lithium salt of gamma-linolenic acid by three human carcinomas

Lipid metabolism has been considered recently as a novel target for cancer therapy. In this field, lithium gamma-linolenate (LiGLA) is a promising experimental compound for use in the treatment of human tumours. In vivo and in vitro studies allowed us to assess the metabolism of radiolabelled LiGLA by tumour tissue and different organs of the host. In vitro studies demonstrated that human pancreatic (AsPC-1), prostatic (PC-3) and mammary carcinoma (ZR-75-1) cells were capable of elongating GLA from LiGLA to dihomo-gamma-linolenic acid (DGLA) and further desaturating it to arachidonic acid (AA). AsPC-1 cells showed the lowest delta5-desaturase activity on DGLA. In the in vivo studies, nude mice bearing the human carcinomas were given Li[1-(14)C]GLA (2.5 mg kg(-1)) by intravenous injection for 30 min. Mice were either sacrificed after infusion or left for up to 96 h recovery before sacrifice. In general, the organs showed a maximum uptake of radioactivity 30 min after the infusion started (t = 0). Thereafter, in major organs the percentage of injected radioactivity per g of tissue declined below 1% 96 h after infusion. In kidney, brain, testes/ovaries and all three tumour tissues, labelling remained constant throughout the experiment. The ratio of radioactivity in liver to tumour tissues ranged between 16- and 24-fold at t = 0 and between 3.1- and 3.7-fold at 96 h. All tissues showed a progressive increase in the proportion of radioactivity associated with AA with a concomitant decrease in radiolabelled GLA as the time after infusion increased. DGLA declined rapidly in liver and plasma, but at a much slower rate in brain and malignant tissue. Seventy-two hours after the infusion, GLA was only detected in plasma and tumour tissue. The sum of GLA + DGLA varied among tumour tissues, but it remained 2-4 times higher than in liver and plasma. In brain, DGLA is the major contributor to the sum of these fatty acids. Data showed that cytotoxic GLA and DGLA, the latter provided either by the host or by endogenous synthesis, remained in human tumours for at least 4 days.     

Expression of interleukin-6 receptors by pediatric acute lymphoblastic leukemia cells with the t(4;11) translocation: a possible target for therapy with recombinant IL6-Pseudomonas exotoxin

We have detected expression of interleukin-6 receptors (IL-6R) by primary leukemic cells from three of six patients with t(4;11)+ ALL. Scatchard analysis revealed from 960 to 2100 high-affinity IL-6R/cell on these cells (median, 1560; mean, 1540). All three IL-6R+ cases also expressed CD33, which was not expressed on IL-6R-negative cases. To determine if these receptors could serve as a target for a recombinant ligand-toxin, we examined the sensitivity of primary IL-6R+ ALL cells to a recombinant IL6-Pseudomonas exotoxin (IL6-PE4E) fusion protein, in which the toxicity and specificity of the chimeric toxin was enhanced by substitution of four glutamine residues for naturally occurring amino acids in PE domain I. Primary cells from IL-6R+ cases were sensitive to IL6-PE4E in a 48-h cytotoxicity assay, with ID50 values (concentrations causing 50% decrease in viability) ranging from 23 ng/ml to 92 ng/ml (median, 61; mean, 58). Furthermore, incubation of these cells with 10(3) ng/ml IL6-toxin for 24 h prevented their subsequent engraftment in SCID mice. Thus, IL6-PE4E may be useful for ex vivo purging of IL-6R+ leukemic cells in an autologous bone marrow transplantation setting and possibly for therapy of residual, chemotherapy-resistant disease.     

Biodistribution and kinetics of holmium-166-chitosan complex (DW-166HC) in rats and mice

The fate of 166Ho-chitosan complex, a radiopharmaceutical drug for cancer therapy, was determined by studying its absorption, distribution and excretion in rats and mice. METHODS: Holmium-166-chitosan complex [0.75 mg of Ho(NO3)3 x 5H2O and 1 mg chitosan/ head] was administered intrahepatically to male rats. Radioactive concentrations in blood, urinary and fecal excretion and radioactive distribution in tissues were examined. To determine the effects of chitosan in 166Ho-chitosan complex, 166Ho alone [0.75 mg of Ho(NO3)3 x 5H2O/head] was intrahepatically administered to male rats, and radioactive concentrations in blood, urinary and fecal excretion and radioactive distribution were examined. In B16 melanoma-transplanted nude mice, radioactive distribution after intratumoral administration of 166Ho-chitosan complex [0.075 mg of Ho(NO3)3 x 5H2O and 0.10 mg chitosan/head] was investigated also. RESULTS: After administration of 166Ho-chitosan complex, the radioactive concentrations in blood were low, and cumulative urinary and fecal excretions over a period of 0-72 hr were 0.53% and 0.54%, respectively. The radioactive concentrations in tissues and the whole-body autoradiography images showed that most of the administered radioactivity was localized at the administration site, and only slight radioactivity was detected from the liver, spleen, lungs and bones. On the other hand, results of intrahepatic administration of 166Ho alone showed high radioactive concentrations in the blood, and the whole-body autoradiographs showed that the administered radioactivity was distributed in many organs and tissues. These results strongly suggest that 166Ho is retained at the administration site only when it forms a chelate complex with chitosan. Autoradiographs after intratumoral administration of 166Ho-chitosan complex showed that radioactivity was localized at the site of administration without distribution to the other organs and tissues. CONCLUSION: Administered 166Ho-chitosan complex is retained at the administration site after either intrahepatic or intratumoral administration to rats or tumor-transplanted nude mice.     

Complete regression of established human glioblastoma tumor xenograft by interleukin-4 toxin therapy

No curative therapy is available for malignant gliomas. We have discovered that human glioblastoma cells express high affinity interleukin-4 receptor (IL-4R), which is an attractive target for receptor-directed IL-4 toxin therapy. The IL-4 toxin, IL-4(38-37)-PE38KDEL, is a fusion protein containing translocation and enzymatic domains of Pseudomonas exotoxin and a circularly permuted human IL-4. The IL-4 toxin binds specifically to the IL-4R and is highly cytotoxic to glioblastoma cells, as determined by clonogenic and protein synthesis inhibition assays. Intratumoral administration of the IL-4 toxin given on alternate days for 3-4 doses into U251 glioblastoma flank tumors in nude mice, showed a complete remission of small (approximately 13 mm3) and large (approximately 60 mm3) tumors in all animals, without any evidence of toxicity. A significant antitumor activity was also observed when the IL-4 toxin was administered via i.p. and i.v. routes. These results demonstrate that the IL-4 toxin may be a new therapeutic drug for the treatment of human glioblastoma. Therefore, we have begun a Phase I clinical trial with IL-4(38-37)-PE38KDEL for treatment of human glioblastoma.     

Interleukin-2 receptor beta and CD57 expression in orbital tissues from patients with chronic, stable thyroid-associated ophthalmopathy

In order to determine whether components of the interleukin-2 receptor (IL-2R) and lymphoid cells are present in extra ocular and periocular tissues from patients with chronic, stable thyroid-associated ophthalmopathy (TAO) we studied 16 specimens of extra ocular muscle and periorbital connective tissue from 14 patients with chronic, stable, TAO using an immunohistochemical assay and a panel of murine monoclonal antibodies reactive with IL-2R alpha and beta components and lymphoid cell surface markers. As controls we studied orbital tissues from 11 patients undergoing surgery for unrelated orbital disorders. All extra ocular muscle specimens from patients with TAO exhibited IL-2R beta expression primarily on the perimysium and endomysium surrounding the ocular muscle fasciculi and fibers of which nine specimens stained intensely. The Natural Killer (NK) cell marker CD57 was the most common cell surface antigen detected, in seven of nine specimens, whose localization often corresponded to that of IL-2R beta distribution. No IL-2R alpha expression was detected in any specimen. Seven of the 11 control specimens were positive for IL-2R beta but in a less intense fashion than in TAO specimens while no CD57 staining was detected. T cell, B cell, and cells of granulocyte and monocyte lineage were only occasionally found in both TAO and control specimens. The aberrant expression of IL-2R beta and CD57 which may be representative of NK cell presence in extra ocular muscle tissues from patients with stable, chronic TAO may play a role in the pathogenesis of the ophthalmopathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone and IL-6 requirements for human C-reactive protein gene expression in transgenic mice

In vitro, IL-6 is the main inducer of the human C-reactive protein (CRP) gene, and IL-1 and steroids can enhance this effect. However, in mice, IL-6 is necessary but not sufficient for induction of the human CRP transgene, and testosterone is required for its constitutive expression by males. To examine the relative contributions of testosterone and IL-6 in the regulation of CRP gene expression, we produced CRP-transgenic (CRPtg), IL-6-deficient (IL-6-/-) mice. Male CRPtg/IL-6-/- mice expressed CRP constitutively, but CRP levels were not increased after injection of LPS. However, acute-phase CRP levels were attained after injection of IL-6. In contrast, female CRPtg/IL-6-/- mice did not express CRP constitutively or after administration of LPS, IL-6, IL-1, or IL-6 plus IL-1. Like males, testosterone-treated CRPtg/IL-6-/- females expressed CRP constitutively, and their transgene responded to injection of IL-6. The endogenous acute-phase protein serum amyloid P (SAP) was expressed constitutively equally by male and female IL-6-/- mice, responded minimally to LPS, and did not respond to either IL-6 or IL-1 alone. Acute-phase levels of SAP were induced in IL-6-/- mice by injection of IL-6 together with IL-1 or LPS. We conclude that in vivo, both constitutive and IL-6-dependent acute-phase expression of the CRP transgene require testosterone. In contrast, testosterone is not required for expression of the SAP gene, which requires IL-1 plus IL-6 for acute-phase induction.     

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.     

Absorption, distribution, and excretion of [14C]patulin by rats

Adult rats of both sexes were given a single oral dose of [14C] patulin and were sacrificed at various time intervals from 4 hr to 7 days following administration of the mycotoxin. Two groups of rats were employed; the treated group had been exposed to daily oral doses of unlabeled patulin (dissolved in pH 5.0 citrate buffer) in utero and for 41-66 wk after weaning, while the controls were given the buffer only throughout gestation and for 38-81 wk after weaning. Approximately 49% of the administered 14C radioactivity was recovered from feces and 36% from urine within 7 days after dosing. Most of the excretion of labeled material occurred within the first 24 hr. All of the 14C activity detected in the urine samples was either metabolites and/or conjugates of the original [14C]patulin. About 1-2% of the total radioactivity was recovered as 14CO2 from expired air. Carbon-14 radioactivity in various tissues and organs was determined throughout the 7 day period; the most significant retention site was the red blood cells.     

Role of prolactin in the modulation of NK and LAK cell activity after short- or long-term morphine administration in neoplastic patients

In a previous work we demonstrated that chronic in vivo antalgic therapy of cancer patients with morphine reduced the endogenous cytotoxic activity of natural killer (NK) cells, while increasing the development of lymphokine activated killer (LAK) cell cytotoxicity. In order to investigate the mechanisms by which morphine affects NK and LAK cell function further, we evaluated the modulation exerted by short- or long-term morphine administration on either NK/LAK cell cytotoxicities or plasma levels of prolactin (PRL) and other immunomodulating neurohormones. An intravenous morphine injection (10 mg) significantly increased the plasma levels of PRL, reduced the cytotoxic activity of NK cells, and increased the development of LAK cell activity 30 min after drug injection in neoplastic patients. The administration of bromocriptine before the injection of morphine prevented both PRL augmentation and the increase in LAK cell activation, although it did not prevent the inhibition of NK cytotoxicity. The chronic oral administration of morphine (90 +/- 30 mg/day for 1 month) also resulted in higher PRL levels; the NK and LAK cell activities were, respectively, lower than or higher than those found in neoplastic patients untreated with morphine. The plasma levels of thyrotropin (TSH), adrenocorticotropic hormone (ACTH) and cortisol were not significantly modified in either short- or long-term experiments. The absolute number and the percentages of lymphocyte populations, as well as the percentage of IL-2 receptors, were not modified after short-term morphine administration whereas little changes of T lymphocyte populations and NK cell number were observed after oral treatment with morphine. In vitro morphine did not affect the development of LAK cell activity. In conclusion, our findings indicate that morphine reduces NK cytotoxicity and increases the development of LAK cell cytotoxicity after short- and long-term administration. The effect of morphine on LAK cell activation but not on NK cell reduction is related to the modulation of PRL levels determined by the opioid drug.     

Fate of etiproston, a synthetic analogue of PGF2 alpha, in cows

The pharmacokinetics and metabolic fate of labelled compounds were investigated after intramuscular administration of 3H-radiolabelled etiproston to nine cows. Elimination was rapid (t 1/2 beta = 2.8 h). Forty-eight h after administration 92.6% of the radioactivity had been eliminated, mainly via the urinary (66% at 48 h) and faecal routes (26% at 48 h). In comparison, little elimination in milk occurred (less than 0.034% dose/l by 24 h). Radioactivity at the injection site 48 h after administration was seen in one cow (< 4.68 x 10(-5%) dose/g). No radioactivity was detected in the tissues. Urinary metabolites were purified and isolated using XAD-2 extraction and preparative HPLC in reverse and normal phases. The main urinary metabolite, identified by mass spectrometry, was the tetranor acid derivative in equilibrium with its lactone form.     

Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors

We have studied the ability of adenoviral (Ad) vectors expressing the cytokines IL-2 or IL-12 to mediate regression of established tumors in a mouse model of mammary adenocarcinoma. Previous results indicated that intratumoral injection of vectors expressing IL-2 (AdCAIL-2), or IL-12 (AdmIL-12.1) induced complete tumor regression in approximately 30-40% of treated animals. In the current studies, we investigated the mechanism of tumor killing in responding animals and the efficacy of AdIL-2 and AdIL-12 vector administration in combination compared with the use of either vector alone. Animals bearing subcutaneous mammary tumors were injected intratumorally with Ad vectors expressing IL-2 or IL-12 or were coinjected with both vectors. Animals receiving the combination treatment responded substantially better than animals which had received either vector alone, with 65% of animals treated with both vectors undergoing complete tumor regression. In all three treatment regimens, tumor regression was associated with the presence of specific antitumor antigen cytotoxic T-lymphocytes (CTLs), which secreted elevated levels of IFN-gamma. Consistent with circulating CTLs being involved in regression, when animals bearing bilateral tumors were inoculated in a single tumor with IL-2 or IL-12 expressing vectors, both tumors regressed in many cases. Again, treatment with both AdCAIL-2 and AdmIL-12.1 was most effective, with 63% of animals undergoing complete regression of both treated and untreated tumors, compared to 18 or 22% of animals injected with either AdCAIL-2 or AdmIL-12.1 alone. These data indicate that the combination of IL-2 and IL-12 is a more effective inducer of antitumor immune responses than either one alone, and that the resulting antitumor responses are effective in mediating the regression of distal untreated tumors, a property which may aid in the treatment of metastatic disease.     

Studies on the toxicology of hexachlorobenzene. II. Identification and determination of metabolites

Female rats were dosed intraperitoneally with 14C-hexaxhlorobenzene. The drug was administered on 2 or 3 occasions. The total doses amounted to 260 and 390 mg/kg 14C-hexachlorobenzene, respectively. Urine and feces from the animals were collected over a period of 4 weeks after the first injection. Both excreta and some tissues of the animals were examined for their content of radioactivity and for hexachlorobenzene and its metabolites. Gas chromatography, isotope dilution analysis, and combined gas chromatography-mass spectrometry were used to identify the metabolites of hexachlorobenzene. In urine pentachlorophenol, tetrachlorohydroquinone, and pentachlorothiophenol were present as major metabolites. One of the isomers of tetrachlorothiophenol was present as a minor metabolite. In the feces pentachlorophenol and pentachlorothiophenol only were identified. At the end of the experiment, carbon-14 excreted with urine and feces amounted to 7% and 27%, respectively, of the radioactivity administered. More than 90% of carbon-14 excreted in urine was contained in the major metabolites. In the feces about 30% of the excreted radioactivity was bound to metabolites and about 70% was contained in the unchanged drug, while in the tissues of the animals only pentachlorophenol was detected in measurable amounts, accounting for 10% of label in blood and less than 0.1% of carbon-14 determined in body fat. Total radioactivity contained in the metabolites detected in the animal body and in the excreta at the end of the experiment accounted for about 16% of the administered radioactivity.     

Metabolic fate of ethylenethiourea in pregnant rats

Metabolic fate of ETU was investigated in the rats administered orally 100 mg/kg of C14-ETU on the twelfth day of gestation. ETU was absorbed readily from the gastrointestinal tract and passed away from the whole body tissues including the fetus rapidly. Only the exception was the thyroid gland and the radio-activity was accumulated in the gland. Most of the administered activity (80.2%,4,5-C14-ETU) was eliminated into the urine in 24 hr and the tissues (including the fetus) levels of radioactivity from 2-C14-ETU reached maximal within 2 hr and fell down to negligible levels by 24 hr. Radiocarbon(s) of 4,5-C14-ETU was expired as radioactive carbon dioxide and was incorporated into the serum and fetal cell constituents (crude protein fraction), but that of 2-C14-ETU was neither expired or incorporated into the cell constituents. From the fetus extract ETU and several radioactive metabolites were detected.     

The kinetics of 1,2-3H-cortisol after intra-amniotic administration

Foetuses of random-bred H mice (Velax) received on day 13 of foetal development 1,2-3H-cortisol by the intra-amniotic administration, and 10, 20, 30, 60 and 120 min. after drug injection the radioactivity in the amniotic fluid, foetuses and placentae was determined by methanol extraction. The mean value of total radioactivity in the foetuses and placentae attained 2.2% and 2.7%, respectively, of the injected dose during the first 10 min. and did no longer change substantially. Total radioactivity in the amniotic fluid, after an initial acute fall, corresponded to 16.4% of injected dose from 20 min. to 2 hours after drug administration.     

Nitric oxide modulation of retinal, choroidal, and anterior uveal blood flow in newborn piglets

The purpose of the present study was to investigate the role of nitric oxide (NO) in modulating the resting vascular tone of the choroidal and anterior uveal circulations and the autoregulatory gain of the retina. Blood flow (ml/min/100 gm dry weight) to tissues was determined in 23 anesthetized piglets (3-4 kg) using radiolabelled microspheres. Ocular Perfusion Pressure (OPP) was defined as mean arterial pressure minus intraocular pressure (IOP) which was manipulated hydrostatically by cannulation of the anterior eye chamber. The OPP was decreased during intravenous infusion (30 mg/kg/hr) of either the NO-synthase inhibitor L-NAME or the inactive enantiomer D-NAME. Blood flows were determined at OPP of 60, 50, 40, 30, and 20 mmHg following initial ocular blood flow measurements. Mean initial choroidal and anterior uveal blood flows with L-NAME showed a 47+/-12% and a 43+/-6% reduction (p <.001), respectively. Mean choroidal blood flows were significantly reduced (p<.01) in the L-NAME treated animals at an OPP of 60 and 50 when compared to D-NAME. Uveal blood flows were linearly correlated with OPP in the L-NAME and D-NAME treated groups. Uveal blood flow was greater following exogenous administration of L-arginine (180 mg/kg). Mean initial retinal blood flow did not differ significantly in either group. Retinal blood flow with L-NAME was reduced at OPP of 60 mmHg and below compared to D-NAME (p<.05). The degree of compensation in the autoregulatory gain of the retinal vasculature was reduced in the presence of L-NAME at an OPP of 50 mmHg and below compared to D-NAME. These data support the hypothesis that NO may be a primary mediator in maintaining resting vascular tone to the choroid and anterior uvea in vivo and that NO blockade reduces the degree of compensation in the autoregulatory gain of the retinal vasculature within a specific range of ocular perfusion pressures.