Targeted modulation of acute inflammation.

Localized inflammation of the lungs was induced with the neutrophil chemoattractant, N-formylmethionylleucylphenylalanine (FMLP) in combination with magnetically responsive albumin microspheres, a drug carrier that provides efficient, extremely rapid localization in tissue. Intravenous targeting to rat lungs was accomplished by means of an external thoracic magnet. This caused progressive local accumulation of neutrophils, extravascular cell migration, and acute tissue injury. Microscopic findings favored chemotaxis as the principal mechanism of cell accumulation. This system provides a new experimental model for acute alveolar damage, a rapid in vivo assay for drugs that modulate neutrophil chemotaxis, and a new therapeutic approach to focusing inflammation in patients with chemotactic defects.

5-Azacytidine-induced uncoupling of differentiation and tumorigenicity in a murine cell line.

Highly tumorigenic, myogenically defective T984-15 murine cells were treated with the hypomethylating agent 5-azacytidine (5-azaC). In response to drug treatment, T984-15 cells formed colonies that were myosin-positive and contained fused myotubes. When cloned, these differentiated colonies gave rise to cells that maintained their myogenic potential even after prolonged growth in tissue culture. The myogenic differentiation observed in response to 5-azaC treatment was not the result of selection of a preexisting myogenic subpopulation, inasmuch as treatment of a subcloned population of nondifferentiating cells with 5-azaC also resulted in the induction of myogenesis. In addition to inducing myogenic potential, 5-azaC generally suppressed the tumorigenic potential of the treated cells. Whereas 19 of 19 untreated T984-15 clones when injected into BALB/c nude mice produced tumors, 8 of 9 of the 5-azaC-treated clones injected displayed suppressed tumorigenicity under identical conditions. Tumorigenic suppression, however, was independent of the induction of differentiation: 1 myogenic clone remained tumorigenic and 5 clones whose tumorigenic potential was suppressed were nonmyogenic. Thus treatment with the hypomethylating agent 5-azaC not only affected the expression of differentiated and tumorigenic phenotypes but also dissociated their usually coordinate regulation.

In vitro release of biologically active adriamycin by magnetically responsive albumin microspheres.

The kinetic release of therapeutically active Adriamycin from two different heat-stabilized preparations of magnetically responsive albumin microspheres (1 micron) has been evaluated using a rapid in vitro bioassay-harvesting system. Release products are added to freshly plated monolayers of a malignant Fisher 344 rat fibrosarcoma cell line, and the inhibition of [3H]uridine incorporation into trichloroacetic acid-precipitable material (RNA) and whole cells is determined in a 6-hr microtiter assay. The latter harvesting technique utilizes semiautomated cell collection using a multiple sample harvester. Standard fluorometric drug analyses are used to quantitate the chemical release rates of Adriamycin and related degradation products (aglycones). By altering the temperature of albumin matrix stabilization from 22 to 135 degrees, the half-time for the release of therapeutically active drug has been varied from 15 min to 9 hr. The biological activity of drug products released by the highest temperature (135 degrees) preparation is 78% of that for the native free drug. These in vitro antitumor assays are used to predict the maximal rates of release that could occur at the tissue level under optimal conditions of in vivo targeting.

Biomimetic transport and rational drug delivery.

Medicine and pharmaceutics are encountering critical needs and opportunities for transvascular drug delivery that improves site targeting and tissue permeation by mimicking natural tissue addressing and transport mechanisms. This is driven by the accelerated development of genomic agents requiring targeted controlled release. Although rationally designed for in vitro activity, such agents are not highly effective in vivo, due to opsonization and degradation by plasma constituents, and failure to transport across the local vascular endothelium and tissue matrix. A growing knowledge of the addresses of the body can be applied to engineer "Bio-Logically" staged delivery systems with sequential bioaddressins complementary to the discontinuous compartments encountered--termed discontinuum pharmaceutics. Effective tissue targeting is accomplished by leukocytes, bacteria, and viruses. We are increasingly able to mimic their bioaddressins by genomic means. Approaches described in this commentary include: (a) endothelial-directed adhesion mediated by oligosaccharides and carbohydrates (e.g. dermatan sulfate as a mimic of sulfated CD44) and peptidomimetics interacting with adhesins, selectins, integrins, hyaluronans, and locally induced growth factors (e.g. vascular endothelial growth factor, VEGF) and coagulation factors (e.g. factor VIII antigen); (b) improved tissue permeation conferred by hydrophilically "cloaked" carrier systems; (c) "uncloaking" by matrix dilution or selective triggering near the target cells; and (d) target binding-internalization by terminally exposed hydrophobic moieties, cationic polymers, and receptor-binding lectins, peptides, or carbohydrates. This commentary also describes intermediate technology solutions (e.g. "hybrid drugs"), and highlights the high-resolution, dynamic magnetic resonance imaging and radiopharmaceutical imaging technologies plus the groups and organizations capable of accelerating these important initiatives.

A novel heparin-coated hydrophilic preparation of amphotericin B hydrosomes.

Amphotericin B Hydrosomes (AH; Access Pharmaceuticals Inc) are a novel formulation of hydrophilic, heparin-surfaced nanoparticles (mean diameter 105 nm) containing amphotericin B (AmB) designed to target infected sites by local adhesion. AH are cleared in part by a hepatobiliary mechanism, which results in a reduction of AmB concentration in major organs by about 50% in 24 h. In mice with pulmonary blastomycosis, unlike Fungizone (Bristol-Myers Squibb Inc), a deoxycholate micellar formulation of AmB, AH accumulates 3-fold more in infected lungs than normal lungs, between 3 and 24 h post-injection. Histologically, AH accumulates at the sites of lesions. AH is approximately 7-fold less toxic than Fungizone based on acute lethality and histopathological assessment of renal damage. In vitro, AH and Fungizone were equally active against Blastomyces dermatitidis and in vivo they were equivalent in prolonging mouse survival, when compared with equal dosing of AmB. In reducing infectious burdens in vivo, Fungizone was 3-fold more effective than AH on a mg/kg basis of administered AmB. However, AH at 4.8 mg/kg cured 50 to 60% of mice, whereas Fungizone at a near lethal dose of 1.2 mg/kg cured none. The AH formulation of AmB has an improved therapeutic index, relative renal-site avoidance and selective accumulation in infected tissues, which combine to merit additional studies in appropriate fungal models.

Single donor, HL-A matched platelet transfusions for thrombocytopenic patients undergoing surgery.

Thrombocytopenic patients, who displayed hemostatic disorders and had been previously sensitized by repeated blood transfusions and/or pregnancies, were supported for surgical procedures by platelet transfusions obtained from a single ABO and HL-A matched donor by the use of continuous collection centrifugation. Because of the low incidence of HL-A identical donors, compatibility was assessed by known serological cross-reactivity of the HL-A determinants. In three cases repeated platelet transfusions had excellent in vivo survival, and sensitization could not be detected by a battery of immunological assays. In one case there was immune sensitization and refractoriness to repeated platelet transfusion, as documented by accelerated in vivo destruction of donor and third-party platelets bearing the disparate factor HL-A8. Although serologic tests for lymphocytotoxic and leukoagglutinating antibodies were negative, the patient displayed cellular immunity in leukocyte aggregation and cell-mediated plateletolysis tests. The single donor, continuous collection technique appears to have the technical advantage of rapid, efficient collection and the immunological benefit of a restricted spectrum of allosensitization.

Simplified toxicologic monitoring of adriamycin, its major metabolites and nogalamycin by reverse-phase high pressure liquid chromatography, Part I: Analytical techniques for isolated human plasma.

A simplified method is presented for routine toxicologic analysis of the antineoplastic drug, adriamycin, and its major metabolites in human plasma. This method employs a single solvent extraction of plasma using an acetonitrile-aqueous NaOH mixture, followed by reverse-phase isocratic high pressure liquid chromatographic (HPLC) analysis. Recoveries of greater than 95% are obtained for adriamycin and its two major derivatives over the range of concentration: 0.2 to 8.0 nmol/mL of plasma. This method of drug and metabolite isolation and HPLC analysis from human plasma provides an efficient and accurate approach to the individualized toxicologic monitoring of adriamycin pharmacokinetics and metabolism.

Suppression of stimulating cell activity by microtubule-disrupting alkaloids.

Microtubule-disrupting alkaloids and protein fixatives were used to investigate the nature of an active process that must occur within stimulator cells in order for them to initiate a unidirectional mixed lymphocyte response (MLR). Brief treatment of the stimulator cells (SC) with glutaraldehyde (0.15%), formalin (0.6%), or lanthanum chloride (10-3 M) abolished their capacity to activate responder cells (RC). Pretreatment of SC with microtubule-disrupting alkaloids, colchicine (c) (10-4 to 10-6 M) or colchicine + vincristine (c+v) (10-4 to 10-6 M) also abrogated their stimulating capacity. This capacity was not restored by the addition of supernates from untreated cultures, thereby excluding the possibility that the alkaloids acted by decreasing the release of soluble stimulatory factors from SC. The introduction of alkaloid-inactivated, mitomycin-treated RC as drug carriers did not affect the mitogenic response of untreated RC to concanavalin A. This excluded a significant leakage of alkaloids from the treated SC and uptake by RC during culture. Lumicolchicine produced no decrease in the stimulating capacity of SC. This suggested that the suppression induced by low concentrations of colchicine resulted from its specific disruption of microtubules. None of the above treatments quantitatively reduced the antigenicity of SC, as evaluated by humoral and cell-mediated lysis of the treated cells. Also, these treatments produced no significant changes in the specific binding of concanavalin A by SC. These results indicate there is a functional interaction of microtubular structures with cell surface antigens that appears to regulate either the capacity of SC to associate with RC, or the ability of SC to form and stabilize stimulatory antigenic configurations on the cell surface.

A microfluorometric mithramycin assay for quantitating the effects of immunotoxicants on lymphocyte activation.

A semiautomated, microfluorometric assay has been developed for the detection of toxicant-induced changes in lymphocyte DNA content at standard intervals after mitogen activation. DNA is quantitated by solubilizing the cells and determining the fluorescence enhancement that results from formation of the highly specific mithramycin:DNA adduct. The limit of detection is 0.21 microgram (30,000 resting cell equivalents) per microtiter well. Correlation with the less sensitive, nonautomatable, diphenylamine DNA assay gives a correlation coefficient r = 0.91. Prototype substances representative of true immunotoxicants (prostaglandin E2) and common interfering substances (thymidine at less than 5 X 10(-4) M)m have been tested. The latter substance produces false positive results in the standard [3H]thymidine assay. The mithramycin assay does not inappropriately detect this interfering substance. It has the characteristics of a highly specific, accurate technique for screening and quantitating immunotoxic drugs, agents, and mediators in patient sera and other complex biological fluids.

Semiautomated microfluorometric instrument and reagent system for monitoring disease-related immunosuppression.

Several common metabolites and drugs in the serum in of patients with inflammatory, infectious, autoimmune, immunodeficient, neoplastic, and toxicant-induced diseases can produce artifactual suppression of the [methyl-3H]-thymidine assay, which is widely used to evaluate lymphocyte responsiveness. We have developed a sensitive, semiautomated, fluorescence-enhancement assay in which true immunosuppressors are measured in the presence of absence of such interfering substances. Peripheral blood lymphocytes are activated with mitogens in standard microtiter culture trays. Changes in lymphocyte DNA content are quantified with a reagent formulation containing mithramycin, the fluorescence of which is enhanced on binding to DNA in the presence of MgCl2. We solubilize cells within the intact microtiter tray by using an automated, inverted "Array Sonicator," and measure fluorescence with an automated, photon-counting fluorometer. With this system, immune response modulation can be accurately assessed in the presence of patients' sera and other complex test substances (e.g., supernates from hybridomas, fermentation vats, viral preparations, and macrophage cultures.

Differences in the age-dependent release of a low molecular weight suppressor (LMWS) and stimulators by normal and nzb/w lymphoid organs.

The age-dependent release of soluble suppressors and stimulators of DNA synthesis by cultured thymocytes and spleen cells from C57BL/6 and BALB/c mice was compared with their release by NZB/W lymphoid organs. Spleen cells from the normal strains released high levels of suppressor early in life and gradually decresing quantities with age, NZB/W spleen cells exhibited an early deficiency followed by a later excess in the production of suppressor. These differences were quantitated by dose-response studies. Thymocytes from the normal strains released stimulatory factors throughout life. In contrast, NZB/W thymocytes stopped releasing stimulatory activity and began to produce suppressor after 2 1/2 to 4 months of life. This abnormal elaboration of suppressor by thymocytes occurred 2 months before its reappearance in the autologous spleen cell supernatant. Both the early and late-appearing (less than 1000). This activity was designed as low molecular weight suppressor (LMWS). Its aberrant production by their reported functional immunologic abnormalities. The following items were discussed: the production of LMWS by adherent spleen cells, its relationship to previously described regulators, its partial purification and initial chemical characterization, and exclusion of the naturally occurring inhibitors of lymphocyte activation, cortisol, corticosterone, cold thymidine, and cyclic AMP as the active molecule.