Direct transport of cocaine from the nasal cavity to the brain following intranasal cocaine administration in rats.

Individuals who consume cocaine illegally have long since adopted or explored the nasal route of administration. This study was designed to determine in an animal model whether nasally applied cocaine could be transported directly from the nasal cavity to the central nervous system. Male Sprague-Dawley rats were used in the study. The nasal cavity was isolated to prevent drainage of nasally applied dosing solution to nonnasal regions. Cocaine was then administered, either by intranasal (in) administration or by intravenous (iv) injection. At different times post dose, blood and tissues from different regions of the brain were collected. Cocaine concentrations in plasma and tissue samples were analyzed by HPLC. After iv administration, similar cocaine contents in different brain regions were observed. Following in administration, cocaine content in samples collected within 60 min post dose were found to differ considerably in different brain regions. The highest content was observed in the olfactory bulb, followed by the olfactory tract and then the remaining part of the brain. To allow comparison of brain cocaine content after iv and in administration, brain cocaine contents were normalized by plasma cocaine concentrations. The ratios of the area under the cocaine concentration-time curve (AUC) between the olfactory bulb and plasma at early times following in administration were significantly higher than those obtained after the iv dose (13.4 +/- 5.56 vs 6.16 +/- 0.94, p < 0.05, for AUC ratio up to 2 min post dose; 9.39 +/- 1.47 vs 7.34 +/- 0.59, p < 0.05, for AUC ratio up to 4 min post dose). At 1 min post dose, the olfactory bulb-to-plasma cocaine concentration ratios following in administration was three times those obtained after iv administration. After 1 min, the olfactory bulb-to-plasma concentration ratios following in administration were found to be similar to or smaller than those obtained after iv administration. The tissue-to-plasma concentration ratios in other brain regions following in administration were found to be smaller than those obtained following iv dosing. We conclude that nasally administered cocaine was transported directly from the nasal cavity to the brain but that only a very small fraction of the dose was transported via the direct pathway.

Trolox attenuates cortical neuronal injury induced by iron, ultraviolet light, glucose deprivation, or AMPA.

The vitamin E analog, trolox, protected cultured cortical neurons against damage induced by exposure to either iron ions or ultraviolet (UV) light, consistent with an ability to inhibit free radical-mediated cytotoxicity. Trolox also reduced neuronal death induced by 24 h exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), but not that induced by N-methyl-D-aspartate (NMDA). When combined with the NMDA receptor antagonist dextrorphan, trolox also reduced the neuronal injury induced by glucose deprivation.

Induction of peroxisomal enzymes in cultured porcine endothelial cells by the hypolipidemic drug ciprofibrate.

The purpose of this study was to determine if the hypolipidemic peroxisome proliferator ciprofibrate, which induces peroxisomes in the liver, can induce peroxisomes in cultured porcine pulmonary endothelial cells. Ciprofibrate was added at three concentrations to cell cultures for a 6-day period. The induction of peroxisomes in the cells was detected by determining total peroxisomal beta-oxidation and peroxisomal catalase activity. The addition of ciprofibrate was found to increase peroxisomal enzyme activities in a dose-dependent manner, with the highest activity being reached at 1000 microM ciprofibrate. Ciprofibrate also caused an increased transfer of albumin across endothelial cells cultured on micropore filters. This study shows that peroxisomal enzyme activities can be induced by ciprofibrate in endothelial cells, which may have implications in diseases mediated by vascular injury.

Formation of macrophage (M) colony-stimulating factor by murine leukemia x fibroblast hybrid cells.

Clonogenic assays for M, GM, and G precursors of rat or mouse marrow cells were performed in the presence of medium conditioned by the growth of ASL-1 leukemia X LM fibroblast hybrid cells. Both GM- and M-colony-forming units (CFUs) were present in marrow cultures maintained in conditioned medium (CM) from hybrid cells (up to 162 +/- 10 total colonies per 10(5) cells) and from LM cells (65 +/- 5). Conditioned medium from ASL-1 cells did not lead to the formation of CFUs. The hybrid cell-derived CM supported the development of M and GM-CFUs from the marrows of DBA, CAF1, BDF1, C3D2F1, and C57B1/6 mice as well as Lewis, Brown Norway, and Wistar Furth rats. G-CFU were not detected in any of the preparations. Hybrid cell-CM supported the long-term growth and proliferation of macrophage-like cells from mouse spleen, consistent with the presence of M-colony-stimulating factor (CSF). Evidence that M-CSF formed by the hybrid cells and M-CSF formed by L cells shared structural features was provided by antibody neutralization studies. The CFU-promoting activity of hybrid cell-derived M-CSF was neutralized by an antiserum raised in goats against M-CSF purified from L cells. Independently prepared ASL-1 X LM hybrid cells, like the original, led to the formation of GM and M-CFUs. Attempts to detect each of several other previously defined growth factors in medium conditioned by the hybrid cells were unsuccessful. Interleukins 1, 2, and 3; B cell growth factors interferons alpha, beta, and gamma; erythropoietin; and burst promoting factor were not detected.

Biotransformation of primaquine in vitro with human K562 and bone marrow cells.

Although the antimalarial activity, hemolytic and methemoglobinemic side effects, and detoxification of primaquine are all thought to depend on various biotransformation products of the drug, their site and mechanism of formation and degradation are unknown and their specific biologic effects remain very poorly understood, particularly in humans. We have therefore explored the feasibility of studying primaquine metabolism in cultured human cells. We found that the biotransformation of primaquine can be investigated in vitro in serum-supplemented liquid cultures of partially synchronized and exponentially growing human erythroleukemic K562 cells. Further, these cells can be replaced by cells present in normal bone marrow. Primaquine is rapidly and predominantly converted in vitro into carboxyprimaquine (CPQ) in a quantitative manner and without further modification. In addition to CPQ, a compound Xc that is not 6-methoxy-8-aminoquinoline, and is not derived from CPQ, appears in minor amounts in a delayed fashion. With the K562 as well as with the bone marrow cells the formation of CPQ from primaquine can be totally blocked by large concentrations of the nitrosourea, 1,3-bis-(2-chloroethyl)-nitrosourea (BCNU). With bone marrow, increasing blockade of CPQ formation by BCNU leads invariably to a progressive and striking accumulation of Xc. The availability of reproducible, quantitative, and practical new tools for the study of primaquine metabolism in vitro raises a number of challenging questions and may improve understanding of the mode of action, toxicology, and pharmacogenetics of 8-aminoquinolines.

Regeneration of T lymphocytes from human bone marrow cells after depletion with methylprednisolone.

This study evaluates the propagation of T lymphocytes in bone marrow cells after in vitro methylprednisolone treatment. Previous studies from this laboratory showed that immunocompetent T lymphocytes could be eliminated from the bone marrow cells by incubation with 15 mg/ml (0.04 M) of methylprednisolone for 1 hr. The effectiveness of the chemoseparation was assessed by E-rosette forming cell assay, mitogen-induced lymphoblastogenic responses, and lymphocyte surface markers. In this study, bone marrow cells treated with different concentrations of methylprednisolone were cultured in the presence of PHA and Interleukin-2. The mitogen-induced lymphoblastogenesis was restored in the 15 mg/ml MP-treated bone marrow cells by the 8th day of culture in the presence of Interleukin-2. The cells in the culture were analyzed by E-rosette forming cell assay. It was shown that the predominant cells in the cultures were E-rosette forming cells. This study demonstrates that immunocompetent T lymphocytes can be regenerated in the presence of mitogen and Interleukin-2 from bone marrow cells treated with 15 mg/ml of methylprednisolone.

Autologous bone marrow transplantation (ABMT) in the treatment of cancer.

Clinical trials in autologous BMT to date have indicated that significant salvage and potential cures can be obtained in patients with high grade non-Hodgkin's lymphoma (NHL) who have failed primary therapy and are treated with high dose chemoradiotherapy and autologous marrow rescue. The major need in NHL is to better define those patients who might benefit by autologous BMT and to reduce the relapse rate by improved pre- or post-transplant therapy. Similar results to those in NHL could be obtained in acute leukemia if occult tumor cells could be eliminated from autologous marrow. Animal model experiments have shown that it is feasible to eliminate low level contamination with tumor cells by in vitro immunologic or pharmacologic treatment. While it is too early to accurately assess the efficacy of ongoing clinical trials using those marrow purging techniques, a few patients have exhibited encouraging durations of CCR. Should these approaches prove to be effective in only a fraction of cases, combination in vitro treatment or the use of more efficient effector mechanisms for cell killing (e.g., ricin conjugated antibody) may very well clear occult tumor from the marrow of most patients. The encouraging results with autologous BMT in leukemia and lymphoma stand in sharp contrast to the disappointing results so far achieved with the non-hematologic solid tumors. It is, however, possible that those cancers have not been subjected to the most rigorous test for successful autologous BMT and that the search for newer agents which can produce operationally irreversible aplasia may provide a fairer test of this approach. It is to be hoped that future research will settle this point.

Detection and significance of granulocyte alloimmunization in leukocyte transfusion therapy on neutropenic dogs.

Studies were carried out in dogs to detect alloimmunization to granulocytes by indirect immunofluorescence (GIIF) and to determine the significance of cross-matching in selecting donors for immunized recipients. All recipients were rendered neutropenic (less than 500 per microliter) by chemotherapy. Three groups of transfusions were evaluated. Group I consisted of 14 transfusions administered to sensitized dogs that were GIIF-negative with donor cells; Group II, 12 transfusions to the same dogs with GIIF-positive donor cells; Group III, 16 transfusions to nonimmunized control dogs. At 1 hour following transfusion, circulating granulocytes demonstrated increments of 956 +/- 137 per microliter (mean +/- SEM) in Group I dogs compared to 236 +/- 57 per microliter for Group II (p less than 0.001). Group III transfusions yielded increments of 888 +/- 116 per microliter, not significantly different from Group I. GIIF correlated better with transfusion results than granulocyte cytotoxicity or lymphocyte cytotoxicity tests or leukoagglutination cross-matching. Nineteen sera with alloantibodies to granulocytes were produced by random or intrafamilial immunizations with granulocytes. Partial characterization of specificities showed that granulocyte specificities were independent of DLA, and consistent with a genetic system of dominant alleles. It was concluded that a) alloimmunization to granulocytes is detected by the GIIF, b) positive tests predict transfusion effects, and c) a genetic system independent of DLA could be recognized on dog granulocytes.

An analysis of quantitative relationships of granulocyte transfusion therapy in canines.

Granulocyte transfusions were administered to dogs rendered neutropenic by prior cyclophosphamide treatment. Linear regression analysis established significant direct relationships between numbers of granulocytes infused per kg body weight and blood increment achieved. Migration of granulocytes to cerebrospinal fluid during induced Candida albicans meningitis related to concurrent increments in blood following transfusion. Levels of C. albicans systemic infection were inversely related to numbers of granulocytes administered during a four-day course of therapy. These studies demonstrate the quantitative relationships of granulocyte transfusion therapy to potential therapeutic effects at the tissue level.