Immunomodulatory and toxic effects of free and liposome-encapsulated tumor necrosis factor alpha in rats.

Tumor necrosis factor alpha has potent immunomodulatory and antitumor activity, but its therapeutic applications may be limited by its significant host toxicity. We showed that liposome-encapsulated recombinant human tumor necrosis factor alpha (rHuTNF-alpha) retained full anticellular activity in vitro. We then assessed the immunomodulatory and toxic effects of two different doses of i.v. free or liposome-encapsulated rHuTNF-alpha in normal rats. Both free and liposome-encapsulated rHuTNF-alpha significantly enhanced alveolar macrophage- and blood monocyte-mediated interleukin 1 release and tumor cell lysis, as well as natural killer cell cytotoxicity, when compared to buffer-treated controls. However, administration of rHuTNF-alpha in liposomes substantially reduced tumor necrosis factor alpha-mediated toxicity. Animals receiving liposome-encapsulated rHuTNF-alpha showed significantly less tissue injury, gastric retention, and circulating leukocyte shifts than animals receiving free rHuTNF-alpha. In addition, liposome-based delivery significantly increased lung and liver uptake of rHuTNF-alpha. Therefore, liposome-encapsulated rHuTNF-alpha retains immunomodulatory activity, significantly reduces toxic inflammatory effects, and may allow targeting of tumor necrosis factor alpha to selected organs after i.v. administration.

Monocyte-mediated lysis of HIV-infected tumor cells.

Peripheral blood monocytes (PBM) can selectively lyse malignant or virus-infected cells. We investigated the effects of target cell infection with HIV-1 on PBM cytolytic function. Cytokine-activated PBM lysed uninfected, HSV-1-infected or vaccinia virus-infected tumor cells, but did not lyse the same cell lines when infected with the human immunodeficiency virus type 1 (HIV-1). HIV did not impair PBM viability, and actinomycin D (Act D) pretreatment of HIV-infected target cells restored their susceptibility to PBM-mediated lysis. Either antibody to CD4 (Leu3a) or a recombinant vaccinia virus that induces expression of the HIV envelope protein, also inhibited target cell lysis by PBM. These studies indicate that CD4 can function as a mediator of PBM cytolytic function, and that target cell expression of the HIV-1 envelope protein may inhibit monocyte-mediated antitumor responses.

Aerosol administration of antiviral agents to treat lung infection due to murine cytomegalovirus.

Cytomegalovirus (CMV) pneumonia causes significant morbidity and mortality in bone marrow transplant recipients and in patients with AIDS. 9-(1,3-Dihydroxy-2-propoxymethyl) guanine (ganciclovir) and phosphonoformic acid (PFA) demonstrate activity against CMV in human infections, although recurrent CMV and systemic drug toxicity frequently develop. We examined the efficacy of aerosol administration of antiviral agents against murine CMV (MCMV) infection. Animals were inoculated with MCMV intranasally and were treated with oral ganciclovir; with aerosolized ganciclovir, PFA, or ribavirin; or with buffer. MCMV in lung and salivary gland homogenates was quantified by plaque assay. Oral ganciclovir (200 mg/kg per day) reduced titers of MCMV in both tissues by greater than 95%. Aerosolized ganciclovir, 100 and 200 mg/kg per day, reduced lung titers of MCMV by 93% and 97%, respectively. Aerosolized PFA, 20 and 200 mg/kg per day, reduced lung titers of MCMV by 60% and 68%, respectively. Aerosolized ganciclovir and PFA inhibited replication of MCMV in salivary glands substantially less than did oral administration of either agent. Our results suggest that aerosol administration of antiviral agents can potently and selectively inhibit replication of MCMV in the lung.

Liposome-associated tumor necrosis factor retains bioactivity in the presence of neutralizing anti-tumor necrosis factor antibodies.

Cell-associated TNF-alpha, either bound to its receptor on monocyte membranes or expressed as an integral membrane protein, can exert potent tumor cytolytic activity. We assessed the interaction of TNF with the lipid bilayer membrane system, liposomes, and the effects of membrane association on TNF bioactivity. High levels of TNF can be encapsulated within liposomes. At neutral pH, TNF binds to the surface of preformed liposomes (liposome-associated TNF), but does not partition into the lipid bilayer. TNF appears to bind to negatively charged phospholipid head groups of the outer membrane leaflet. Free TNF, liposome-associated TNF, and liposome-encapsulated TNF display comparable abilities to activate human peripheral blood monocytes and to lyse tumor cells. However, liposome-encapsulated TNF, as well as TNF bound to the outer surface of preformed liposomes, retains bioactivity in the presence of anti-TNF antibodies that neutralize free TNF. The interaction of liposomal TNF with cell surface TNF receptors thus appears to be preserved in the presence of neutralizing antibodies.

Reduction in intensity of Pneumocystis carinii pneumonia in mice by aerosol administration of gamma interferon.

Pneumocystis carinii pneumonia in patients with AIDS may result from impaired local release of gamma interferon from lung lymphocytes and subsequent failure of macrophage activation. We tested this hypothesis with mice rendered immunodeficient by selective depletion of CD4+ lymphocytes and inoculated intratracheally with P. carinii. After aerosol administration of recombinant murine gamma interferon, the intensity of P. carinii infection in these mice was reduced in comparison with that in mice not treated with gamma interferon. Histologic scoring of lung tissue did not reveal additional pulmonary inflammation attributable to gamma interferon. Aerosol administration of gamma interferon may reduce the intensity of P. carinii infection by augmentation of host defense, despite persistent CD4+ lymphocyte depletion.

Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats.

The recombinant cytokines IFN-gamma and TNF-alpha stimulate several macrophage-mediated functions important in host defense. However, systemic administration of cytokines may be limited by significant host toxicity. We investigated whether aerosolized cytokines can stimulate alveolar macrophage and blood monocyte function, and whether they induce an inflammatory response in the lungs of normal rats. We found that aerosolized murine rIFN-gamma or recombinant human TNF-alpha increased IL-1 production by both alveolar macrophages and blood monocytes for at least 5 days after administration. Furthermore, murine rIFN-gamma increased the expression of Ia Ag on alveolar macrophages and human rTNF-alpha increased alveolar macrophage- and blood monocyte-mediated tumor lysis. Sequential aerosolization of IFN-gamma and TNF-alpha significantly increased both IL-1 release and Ia expression compared to either cytokine administered alone. Aerosolized human rTNF-alpha achieved lung levels comparable to those produced by an i.v. TNF-alpha dose reported to cause diffuse organ injury and death in rats. However, plasma TNF-alpha levels were several thousand-fold lower after aerosol administration. Aerosolized cytokines did not induce lung edema or an inflammatory cell infiltrate within the airways or alveoli. Aerosolized human rTNF-alpha alone, or murine rIFN-gamma and human rTNF-alpha, induced margination of leukocytes in pulmonary blood vessels 1 day after aerosolization, and a few small foci of pulmonary hemorrhage 5 days later. We conclude that aerosol administration of IFN-gamma or TNF-alpha enhances both pulmonary and systemic monocyte function, and that the combination of IFN-gamma and TNF-alpha produce additive or synergistic effects. Aerosolized cytokines induce only a minimal pulmonary inflammatory response. Aerosolized TNF-alpha produces high cytokine levels in the lung but very low uptake into the circulation.

Selective delivery of pentamidine to the lung by aerosol.

In 8 patients with diffuse infiltrates on chest radiograph undergoing fiberoptic bronchoscopy for suspected Pneumocystis carinii pneumonia, bronchoalveolar lavage sediment and supernatant concentrations of pentamidine were compared 18 to 24 h after administration of 4 mg/kg intravenous (n = 3) and aerosolized (n = 5) pentamidine isethionate. Aerosol was inhaled for 35 to 40 min with 300 mg of pentamidine isethionate in a jet nebulizer, baffled to decrease the particle size to 1.42 micron +/- 1.88 (mass median aerodynamic diameter +/- geometric standard deviation). Bronchoalveolar pentamidine concentrations were: In sediment, 9.34 +/- 1.74 postintravenous versus 705 +/- 242 ng/ml postaerosol (mean +/- SEM, p less than 0.05); supernatant, 2.64 +/- 0.73 postintravenous versus 23.2 +/- 7.75 ng/ml postaerosol (mean +/- SEM, p less than 0.05). Serum pentamidine levels were low or undetectable after aerosolization. Aerosol administration delivers significantly higher concentrations of pentamidine to the air spaces than does intravenous delivery in patients with diffuse alveolar infiltrates.

Selective enhancement of pentamidine uptake in the lung by aerosolization and delivery in liposomes.

We examined the tissue distribution of pentamidine, both as free drug and encapsulated in liposomes, after intravenous (iv) and aerosol administration in healthy rodents. After iv injection, drug levels in the lung were increased as much as 34-fold by administration in liposomes. Concurrently, peak liver uptake was increased 4-fold and renal deposition reduced 3-fold. Liposome-mediated lung delivery was highly dependent on liposome size. Decreasing liposome mean diameter from 2.5 to 0.4 micron reduced lung uptake of pentamidine 90-fold while affecting extrapulmonary organ deposition to a much lesser degree. Aerosol delivery of pentamidine produced high, sustained lung levels, with no evidence of drug clearance from the lung between 1 and 48 h after administration. Extrapulmonary drug levels produced by aerosol delivery were negligible throughout this period. There were no significant differences in the organ distribution of aerosolized free versus liposome-encapsulated drug, apparently because of sequestration of pentamidine within the lung. Comparison of drug levels in material recovered by bronchoalveolar lavage suggests that aerosol delivery of pentamidine produces substantially higher deposition in the alveolar space than does iv drug injection. Light and electron microscopic (EM) examination of lung, kidney, and liver after iv or aerosol administration of liposomes revealed no tissue abnormalities, although isolated platelet clumps were noted in pulmonary capillaries by EM examination.

Successful treatment with aerosolized pentamidine of Pneumocystis carinii pneumonia in rats.

We examined both the therapeutic efficacy and tissue distribution of aerosolized pentamidine in immunosuppressed rats with Pneumocystis carinii pneumonia. In rats immunosuppressed by 5 weeks of pretreatment with dexamethasone, a 2-week course of 5 mg of aerosolized pentamidine per kg per day, administered free or encapsulated in the drug carrier system (liposomes), eradicated P. carinii pneumonia in 75% of treated animals. At this dose, extrapulmonary drug uptake as measured by a sensitive high-pressure liquid chromatography assay was negligible. No significant differences in tissue distribution were noted between aerosolized free and liposome-encapsulated pentamidine. In rats receiving dexamethasone for 6 weeks prior to treatment with pentamidine, both lung uptake and therapeutic efficacy of aerosolized pentamidine (5 mg/kg per day) were substantially reduced. Aerosolized pentamidine appears to be an effective therapy for P. carinii pneumonia in rats and produces significantly lower extrapulmonary drug deposition than parenteral administration. The severity of P. carinii involvement at the time of treatment influences both the level of drug delivery to the lung and the response to aerosolized pentamidine therapy.

Aerosolised pentamidine as sole therapy for Pneumocystis carinii pneumonia in patients with acquired immunodeficiency syndrome.

15 patients with first episodes of Pneumocystis carinii pneumonia and the acquired immunodeficiency syndrome were treated with only aerosolised pentamidine, which they inhaled for 20 minutes every day for 21 days. 13 of the 15 responded to therapy. Mean PaO2 (mm Hg) and vital capacity (% predicted) were 67.9 and 50.8 before therapy and 80.1 and 67.9 after therapy in patients successfully treated. No systemic side-effects occurred and serum pentamidine concentrations were low in all patients. The only local adverse reaction was cough in 12 patients. Aerosolised pentamidine may be an effective non-toxic treatment for P carinii pneumonia.