Vaccination of seronegative volunteers with a human immunodeficiency virus type 1 env/rev DNA vaccine induces antigen-specific proliferation and lymphocyte production of beta-chemokines.

There is a pressing need to test novel vaccine concepts in an effort to develop an effective vaccine for human immunodeficiency virus (HIV) type 1. A phase I clinical study was done to test the immunogenicity of an HIV env/rev DNA vaccine, which was administered intramuscularly to HIV-1-seronegative persons. Subjects received 3 doses of vaccine at a single concentration (100 or 300 microgram) at 0, 4, 8, and 24 weeks. In at least 1 of multiple assays, the 6 subjects who received the 300-microgram dose had DNA vaccine-induced antigen-specific lymphocyte proliferative responses and antigen-specific production of both interferon-gamma and beta-chemokine. Furthermore, 4 of 5 subjects in the 300 microgram-dose group responded to both the rev and env components of the vaccine. The responses did not persist within inoculated individuals and scored in different individuals at different times in the trial. This study supports that HIV-1 DNA vaccine antigens can stimulate multiple immune responses in vaccine-naive individuals, and it warrants additional studies designed to enhance DNA vaccine immunogenicity.

HIV-1 DNA vaccines and chemokines.

DNA vaccines have a demonstrated ability to induce humoral and cellular immune responses in animal models and humans. The technology, although it dates back to the 1950's, has had an insurgence of interest within the past few years following concurrent research papers. The basic technology is being applied broadly to viral, bacterial and parasitic infections. It has also been demonstrated that genes delivered via plasmid expression vectors result in expression of functional proteins in the inoculated host. Further, injection of plasmids encoding cytokine, chemokine or co-stimulatory molecules, also referred to as immunomodulatory plasmids can lead to the further expansion of this technology to include directed immunology. We have been developing DNA technology specifically with a focus as a vaccine against HIV-1 infection. We report that such vaccines can stimulate immune responses in a variety of relevant animal systems including humoral and cellular responses as well as the production of beta-chemokines. We describe that the beta-chemokines can both modulate the immune response induced by DNA vaccines and be modulated by the DNA vaccines in the murine and chimpanzee models as well as in humans.

Enhancement of cellular immune response in HIV-1 seropositive individuals: A DNA-based trial.

A DNA-based vaccine containing HIV-1 Env and Rev genes was tested for safety and host immune response in 15 HIV-infected asymptomatic patients with CD4-positive lymphocyte counts >/=500/microl of blood and receiving no antiviral therapy. Successive groups of patients received three doses of vaccine at 30, 100, or 300 microg at 10-week intervals in a dose-escalation trial. Some changes were noted in cytotoxic T-lymphocyte activity against gp160-bearing targets. Importantly, enhanced specific lymphocyte proliferative activity against HIV-1 envelope was observed in multiple patients. Three of three patients in the 300-microg dose group also developed increased MIP-1alpha levels which were detectable in their serum. Interestingly patients in the lowest dose group showed no overall changes in the immune parameters measured. The majority of patients who exhibited increases in any immune parameters were contained within the 300 microg, which was the highest dose group. These studies support further investigation of this technology for the production of antigen-specific immune responses in humans.

First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response.

A DNA-based vaccine containing human immunodeficiency virus type 1 (HIV-1) env and rev genes was tested for safety and host immune response in 15 asymptomatic HIV-infected patients who were not using antiviral drugs and who had CD4+ lymphocyte counts of > or = 500 per microliter of blood. Successive groups received three doses of vaccine (30, 100, or 300 microg) at 10-week intervals in a dose-escalation trial. Vaccine administration induced no local or systemic reactions, and no laboratory abnormalities were detected. Specifically, no patient developed anti-DNA antibody or muscle enzyme elevations. No consistent change occurred in CD4 or CD8 lymphocyte counts or in plasma HIV concentration. Antibody against gp120 increased in individual patients in the 100- and 300-/microg groups. Some increases were noted in cytotoxic T lymphocyte activity against gp160-bearing targets and in lymphocyte proliferative activity. The safety and potential immunogenicity of an HIV-directed DNA-based vaccine was demonstrated, a finding that should encourage further studies.

Liposome-encapsulated-gentamicin therapy of Mycobacterium avium complex infection in beige mice.

The efficacy of liposome-encapsulated gentamicin and free gentamicin was evaluated with the beige (C57BL/6J-bgj/bgj) mouse model of disseminated Mycobacterium avium complex infection. Approximately 10(7) viable M. avium complex cells were given intravenously. Seven days later, treatment with either encapsulated or free gentamicin at 20 mg/kg of body weight was started. Treatment was given daily for 5 consecutive days or twice weekly for 3 weeks. The mice were sacrificed 5 days after the last dose. Spleens, livers, and lungs were homogenized, and viable cell counts were determined. An analysis of variance and subsequent Tukey honestly significant difference tests indicated that both encapsulated and free gentamicin reduced viable cell counts in each of the organs compared with no treatment. Encapsulated gentamicin significantly reduced viable cell counts in the spleen and liver compared with the free gentamicin. A dose-response experiment was performed with a daily dose of 0.2, 2, or 20 mg/kg. Dose-related reductions in viable cell counts were observed for spleens and livers, although none of the regimens resulted in sterilization of these organs. Liposome-encapsulated gentamicin should be considered for further evaluation in the treatment of M. avium complex infection in humans.

Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients.

Studies from this laboratory (Mayer et al. (1986) Biochim. Biophys. Acta 857, 123-126) have shown that doxorubicin can be accumulated into liposomal systems in response to transmembrane pH gradients (inside acidic). Here, detailed characterizations of the drug uptake and retention properties of these systems are performed. It is shown that for egg phosphatidylcholine (EPC) vesicles (mean diameter of 170 nm) exhibiting transmembrane pH gradients (inside acidic) doxorubicin can be sequestered into the interior aqueous compartment to achieve drug trapping efficiencies in excess of 98% and drug-to-lipid ratios of 0.36:1 (mol/mol). Drug-to-lipid ratios as high as 1.7:1 (mol/mol) can be obtained under appropriate conditions. Lower drug-to-lipid ratios are required to achieve trapping efficiencies in excess of 98% for smaller (less than or equal to 100 nm) systems. Doxorubicin trapping efficiencies and uptake capacities are related ito maintenance of the transmembrane pH gradient during encapsulation as well as the interaction between doxorubicin and entrapped citrate. This citrate-doxorubicin interaction increases drug uptake levels above those predicted by the Henderson-Hasselbach relationship. Increased drug-to-lipid ratios and trapping efficiencies are observed for higher interior buffering capacities. Retention of a large transmembrane pH gradient (greater than 2 units) after entrapment reduces the rate of drug leakage from the liposomes. For example, EPC/cholesterol (55:45, mol/mol) liposomal doxorubicin systems can be achieved which released less than 5% of encapsulated doxorubicin (drug-to-lipid molar ratio = 0.33:1) over 24 h at 37 degrees C. This pH gradient-dependent encapsulation technique is extremely versatile, and well characterized liposomal doxorubicin preparations can be generated to exhibit a wide range of properties such as vesicle size, lipid composition, drug-to-lipid ratio and drug release kinetics. This entrapment procedure therefore appears well suited for use in therapeutic applications. Finally, a rapid colorimetric test for determining the amount of unencapsulated doxorubicin in liposomal systems is described.

Pharmacokinetics and in vivo activity of liposome-encapsulated gentamicin.

Gentamicin sulfate was encapsulated in liposomes composed solely of egg phosphatidylcholine and administered via intravenous injection to rats and mice. The total gentamicin activity (regardless of whether it was free or liposome associated) in serum and selected tissues was determined for 24 h (serum) or up to 15 weeks (tissues) by using a microbiological assay. The mean half-lives in serum of a single 20-mg/kg dose of free (nonencapsulated) gentamicin in mice and rats were estimated to be 1.0 and 0.6 h, respectively, whereas a similar dose of encapsulated drug had apparent mean half-lives of 3.8 h in mice and 4.0 h in rats. In both species, the apparent half-life in serum of the liposomal formulation increased as the dose increased. Liposome encapsulation resulted in higher and more prolonged activity in organs rich in reticuloendothelial cells (especially spleen and liver). In acute septicemia infections in mice, the liposomal formulation showed enhanced prophylactic activity (as determined by calculation of the 50% protective dose). In a model of murine salmonellosis, liposomal gentamicin greatly enhanced survival when given as a single dose (10 mg/kg) at 1 or 2 days after infection as well as up to 7 days before infection.

Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice.

The effects of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice have been investigated using a versatile procedure for encapsulating doxorubicin inside liposomes. In this procedure, vesicles exhibiting transmembrane pH gradients (acidic inside) were employed to achieve drug trapping efficiencies in excess of 98%. Drug-to-lipid ratios as high as 0.3:1 (wt:wt) could be obtained in a manner that is relatively independent of lipid composition and vesicle size. Egg phosphatidylcholine (EPC)/cholesterol (55:45; mol/mol) vesicles sized through filters with a 200-nm pore size and loaded employing transmembrane pH gradients to achieve a doxorubicin-to-lipid ratio of 0.3:1 (wt/wt) increased the LD50 of free drug by approximately twofold. Removing cholesterol or decreasing the drug-to-lipid ratio in EPC/cholesterol preparations led to significant decreases in the LD50 of liposomal doxorubicin whereas, the LD50 increased 4- to 6-fold when distearoylphosphatidylcholine was substituted for EPC. The results suggest that the stability of liposomally entrapped doxorubicin in the circulation is an important factor in the toxicity of this drug in liposomal form. In contrast, the antitumor activity of liposomal doxorubicin is not influenced dramatically by alterations in lipid composition. Liposomal doxorubicin preparations of EPC, EPC/cholesterol (55:45; mol:mol), EPC/egg phosphatidylglycerol (EPG)/cholesterol (27.5:27.5:45; mol:mol), and distearoylphosphatidylcholine/cholesterol (55:45; mol:mol) all demonstrated similar efficacy to that of free drug when given at doses of 20 mg/kg and below. Higher dose levels of the less toxic formulations could be administered, leading to enhanced increases in life span (ILS) values. Variations in vesicle size, however, strongly influenced the antitumor activity of liposomal doxorubicin. At a dose of 20 mg/kg, large EPC/cholesterol systems are significantly less effective than free drug (with ILS values of 65% and 145%, respectively). In contrast, small systems sized through filters with a 100-nm pore size are more effective than free drug, resulting in an ILS of 375% and a 30% long term (greater than 60 days) survival rate when administered at a dose of 20 mg/kg. Similar size-dependent effects are observed for distearoylphosphatidylcholine/cholesterol systems.

Liposome-encapsulated-amikacin therapy of Mycobacterium avium complex infection in beige mice.

Efficacy of liposome-encapsulated amikacin and free amikacin against Mycobacterium avium complex was evaluated in the beige mouse (C57BL/6J-bgJ/bgJ) acute infection model. Approximately 10(7) viable M. avium complex serotype 1 cells for which the MIC of amikacin was 8 micrograms/ml were given intravenously. Treatment was started with encapsulated or free amikacin at approximately 110 or 40 mg/kg of body weight 7 or 14 days later. In the former experiment, treatment was given two or three times per week. In the latter experiment, treatment was given daily for 5 days. The animals were sacrificed 5 days after the last dose. Liver, spleen, and lung were homogenized, and viable cell counts were determined on 7H10 agar. An analysis of variance and subsequent Tukey HSD (honestly significant difference) tests indicated that both encapsulated and free amikacin significantly reduced viable cell counts in each of the organs compared with counts in the control group. Compared with free amikacin, encapsulated amikacin significantly reduced viable cell counts in the liver and spleen. Liposome encapsulation of an active agent appears to be a promising therapeutic approach to M. avium complex infection.

Analysis of the effect of liposome encapsulation on the vesicant properties, acute and cardiac toxicities, and antitumor efficacy of doxorubicin.

Numerous studies have demonstrated that liposomal encapsulation decreases the life-threatening chronic and acute toxicities of doxorubicin in the face of unaltered or improved antitumor activity. Minimal attention has been paid to the encapsulation effect on the lesser toxicities of the drug, specifically the vesicant properties. In this report we assess the effect of the encapsulation of doxorubicin in an egg-yolk phosphatidylcholine (EPC) cholesterol liposome on the drug's topical toxicity. In addition, to ensure acceptable activity and reduction in toxicity comparable with those of previously assessed formulations, the cardiac and acute toxicities and antitumor activity of the liposomal doxorubicin complex were also investigated. Antitumor efficacy was assessed using the metastatic murine P815 mastocytoma model. Equivalent doses of free and encapsulated doxorubicin possessed the same antitumor activity in the prolongation of animal survival in 14-day survival studies conducted to assess the effect of liposomal encapsulation on the acute toxicity of this drug. The LD50 of liposomal doxorubicin was found to be 40 mg/kg, 53% higher than that of free doxorubicin (26 mg/kg). Histologic examination of cardiac sections taken from DBA/2J mice 7 days after a single i.v. injection of free or liposomal doxorubicin (25 mg/kg) revealed that the liposomal preparation was much less cardiotoxic. In animals receiving the free drug, edema, monocytic infiltration, and cell necrosis were evident. In contrast, those receiving the liposomal preparation demonstrated slight cellular edema but showed no evidence of cellular necrosis. To assess vesicant properties, DBA/2J mice were given a single s.c. injection (0.2 ml) of free or liposomal doxorubicin (2 mg/ml). Those receiving the free drug immediately developed erythema and edema at the injection site, which progressed to ulceration. Those receiving the liposomal complex developed slight erythema and edema but did not ulcerate at any time. All signs of irritation in this group had subsided 3 weeks postinjection. In summary, the liposomal complex used eliminated the vesicant properties of doxorubicin as well as significantly decreasing its cardiac and acute toxicities in the face of unaltered antitumor activity.

Encapsulation of indomethacin in liposomes provides protection against both gastric and intestinal ulceration when orally administered to rats.

Encapsulation of indomethacin into egg phosphatidylcholine (EPC) monophasic vesicles (MPV) or into stable plurilamellar vesicles (SPLV) before oral administration to rats substantially reduced or eliminated the gastric and intestinal ulceration normally associated with ingestion of this drug. Ulcers were assessed by the 4-hour single-dose gastric ulceration model and the 4- or 14-day repeated-dose intestinal ulceration model, using microscopic/planimetric quantitation. Oral dosages of up to 10 mg/kg of indomethacin in polyethylene glycol-400 resulted in substantial gastric ulceration, but not when given in methylcellulose suspension or as EPCMPV. Severe intestinal ulcers resulted following oral administration of indomethacin in either vehicle at daily 3-4-mg/kg doses, but did not result from EPCMPV formulations, whether dosed for 4 days or 14 days. Oral administration of pH-sensitive indomethacin liposomes constructed from cholesterol hemisuccinate resulted in loss of the protective action. Indomethacin-MPV showed both comparable bioactivity and comparable blood levels of the drug when contrasted with free drug in vehicles. Biodistribution studies demonstrated that when delivered from liposomes, drug and phospholipid are rapidly cleared through the stomach but then are differentially absorbed. Empty EPCMPV given by mouth also offered some protection against ulcers induced by systemic (subcutaneous) introduction of indomethacin, although better protective action was noted when the drug was first liposome-encapsulated and then given orally. The application of liposomes to the development of nonsteroidal antiinflammatory drugs that have minimal gastrointestinal side effects is discussed.

Preparation and use of liposomes in the treatment of microbial infections.

The potential application of liposomes to drug delivery has been apparent since 1965, when these phospholipid vesicles were first described by Bangham. Since then, experiments on animals have shown that liposome encapsulation can dramatically alter the distribution of drugs in the body and their rate of clearance. These pharmacokinetic differences, as well as other less well-understood effects, can result in reduced toxicity and enhanced efficacy of the encapsulated drug. The vast majority of studies on the therapeutic use of liposomes have involved the delivery of drugs used in cancer chemotherapy and metabolic storage diseases, but there is now more literature on the use of liposomes for the delivery of antimicrobial drugs and immunomodulating agents. This review briefly discusses the general properties of liposomes and the rationale for their use in antimicrobial drug delivery and immunomodulation, as well as the encapsulation of specific agents and the effect of encapsulation on the treatment of infectious diseases.