Ellen K. Wasan

Biological barriers to cellular delivery of lipid-based DNA carriers.

Although lipid-based DNA delivery systems are being assessed in gene therapy clinical trials, many investigators in this field are concerned about the inefficiency of lipid-based gene transfer technology, a criticism directed at all formulations used to enhance transfer of plasmid expression vectors. It is important to recognize that many approaches have been taken to improve transfection efficiency, however because of the complex nature of the formulation technology being developed, it has been extremely difficult to define specific carrier attributes that enhance transfection. We believe that these optimization processes are flawed for two reasons. First, a very defined change in formulation components affects the physical and chemical characteristics of the carrier in many ways. As a consequence, it has not been possible to define structure/activity relationships. Second, the primary endpoint used to assess plasmid delivery has been transgene expression, an activity that is under the control of cellular processes that have nothing to do with delivery. Gene expression following administration of a plasmid expression vector involves a number of critical steps: (i) DNA protection, (ii) binding to a specific cell population, (iii) DNA transfer across the cell membrane, (iv) release of DNA into the cytoplasm, (v) transport through the cell and across the nuclear membrane as well as (vi) transcription and translation of the gene. The objective of this review is to describe lipid-based DNA carrier systems and the attributes believed to be important in regulating the transfection activity of these formulations. Although membrane destabilization activity of the lipid-based carriers plays an important role, we suggest here that a critical element required for efficient transfection is dissociation of lipids bound to the plasmid expression vector following internalization.

Development and characterization of oral lipid-based amphotericin B formulations with enhanced drug solubility, stability and antifungal activity in rats infected with Aspergillus fumigatus or Candida albicans.

OBJECTIVE To develop an oral formulation of Amphotericin B (AmpB) with: (A) medium chain triglycerides, fatty acids and nonionic surfactants as a self-emulsifying drug delivery system (SEDDS); or (B) glyceryl mono-oleate (Peceol) with poly(ethylene glycol) (PEG)-phospholipids. METHODS SEDDS formulations were prepared by simple mixing at 40 degrees C. Peceol/DSPE-PEG-lipid formulations were prepared by solvent evaporation. Parameters evaluated included: miscibility, solubility and emulsion droplet size after incubation in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) via dynamic light scattering. The stability of AmpB in Peceol/DSPE-PEG was evaluated in SGF and SIF. Phase stability of AmpB in Peceol+/-DSPE-PEG following thermal cycling was evaluated by atomic force microscopy (AFM). Aspergillus fumigatus (2.9-3.45 x 10(7) colony forming units per mL [CFU]) or Candida albicans (3-3.65 x 10(6) CFU per mL) were injected via the jugular vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single oral gavage of a Peceol-DSPE/PEG2000-based AmpB (10 mg AmpB/kg and 5 mg AmpB/kg for the Candida albicans study only) twice daily for 2 consecutive days, a single intravenous (i.v.) dose of Abelcet (5mg AmpB/kg), or physiologic saline (non-treated controls; n=9) once daily for 2 consecutive days. Antifungal activity was assessed by organ CFU concentrations and plasma galactomannan levels in the case of A. fumigatus and organ CFU concentrations in the case of Candida albicans. Plasma samples were taken from each animal prior to infection, 48 h after initiation of infection but prior to drug treatment and at the end of the study for plasma creatinine determinations as a measure of renal toxicity. RESULTS Mean diameter of SEDDS after 30 min in 150 mM NaCl at 37 degrees C was 200-400 nm. However, the Peceol/DSPE-PEG, where PEG MW was 350, 550, 750 or 2000, showed a greater solubilization of AmpB (5 mg/mL) compared to SEDDS formulations (100-500 microg/mL). Upon dispersion in SIF, Peceol/DSPE-PEG formulations generated submicron emulsion particle sizes varying slightly with PEG MW. Stability of the AmpB in Peceol/DSPE-PEG formulations in SGF or SIF was >80% after 2 h, and best for formulations containing DSPE-PEG 750 or 2000 compared to 350, 550 or Peceol only. Monoglyceride-Peceol-DSPE/PEG2000-based oral AmpB treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by >80% compared to non-treated controls without significant changes in plasma creatinine levels in the A. fumigatus infected rats. In addition, this formulation significantly decreased kidney fungal CFU concentrations by >75% at the 5 mg/kg dose and by >95% at the 10 mg/kg dose compared to non-treated controls without significant changes in the plasma creatinine levels in the Candida albicans-infected rats. CONCLUSIONS Novel lipid-based AmpB oral formulations were prepared that provide excellent drug solubilization, drug stability in simulated gastric and intestinal fluids and antifungal activity without renal toxicity in rats infected with A. fumigatus and C. albicans.

The functional roles of poly(ethylene glycol)-lipid and lysolipid in the drug retention and release from lysolipid-containing thermosensitive liposomes in vitro and in vivo.

Triggered release of liposomal contents following tumor accumulation and mild local heating is pursued as a means of improving the therapeutic index of chemotherapeutic drugs. Lysolipid-containing thermosensitive liposomes (LTSLs) are composed of dipalmitoylphosphatidylcholine (DPPC), the lysolipid monostearoylphosphatidylcholine (MSPC), and poly(ethylene glycol)-conjugated distearoylphosphatidylethanolamine (DSPE-PEG(2000)). We investigated the roles of DSPE-PEG(2000) and lysolipid in the functional performance of the LTSL-doxorubicin formulation. Varying PEG-lipid concentration (0-5 mol%) or bilayer orientation did not affect the release; however, lysolipid (0-10 mol%) had a concentration-dependent effect on drug release at 42 degrees C in vitro. Pharmacokinetics of various LTSL formulations were compared in mice with body temperature controlled at 37 degrees C. As expected, incorporation of the PEG-lipid increased doxorubicin plasma half-life; however, PEG-lipid orientation (bilayer vs. external leaflet) did not significantly improve circulation lifetime or drug retention in LTSL. Approximately 70% of lysolipid was lost within 1 h postinjection of LTSL, which could be due to interactions with the large membrane pool of the biological milieu. Considering that the present LTSL-doxorubicin formulation exhibits significant therapeutic activity when used in conjunction with mild heating, our current study provided critical insights into how the physicochemical properties of LTSL can be tailored to achieve better therapeutic activity.

Highly Effective Oral Amphotericin B Formulation against Murine Visceral Leishmaniasis

Visceral leishmaniasis is a deadly parasitic disease caused by obligate intramacrophage protozoans of the Leishmania genus. The World Health Organization estimates the annual death toll to be 50,000, with 500,000 new cases each year. Without treatment, visceral leishmaniasis is inevitably fatal. For the last 70 years, the first line of defense has been pentavalent antimonials; however, increased resistance has brought amphotericin B to the forefront of treatment options. Unfortunately, the difficult route of drug administration, toxicity issues, and cost prevent amphotericin B from reaching the infected population, and mortality continues to rise. Our reformulation of amphotericin B for oral administration has resulted in a highly efficacious antileishmanial treatment that significantly reduces or eradicates liver parasitemia in a murine model of visceral leishmaniasis. This formulation has overcome amphotericin Bs significant physicochemical barriers to absorption and holds promise for the development of a self-administered oral therapy for the treatment of visceral leishmaniasis.

Pharmacokinetics and biodistribution of amphotericin B in rats following oral administration in a novel lipid-based formulation

OBJECTIVES To assess the pharmacokinetics and biodistribution of amphotericin B (AmB) following oral administration in a novel mono/diglyceride-phospholipid formulation and to compare with intravenous (iv) administrations using commercial formulations. METHODS Rats were allocated into the following treatment groups: oral gavage of AmB dispersed in mono/diglyceride-phospholipid formulation at doses of 4.5 and 10 mg/kg; iv bolus administration of 0.8 mg/kg Fungizone; iv bolus of 5 mg/kg Abelcet and iv bolus of 5 mg/kg AmBisome. Blood was sampled from jugular vein cannula at certain time points. The animals were sacrificed 72 h following administration of AmB and multiple tissues were harvested. The concentration of AmB in plasma and tissues was determined by means of HPLC. The plasma creatinine concentrations were determined using an enzymatic kit. RESULTS The pharmacokinetics and tissue distribution of AmB following iv administrations of the commercial formulations were found to be highly formulation dependent. The terminal half-life and biodistribution of orally administered AmB in a mono/diglyceride-phospholipid formulation resembled those of Fungizone. The larger volume of the co-administered lipid-based formulation in the case of the higher dose of orally administered AmB resulted in flip-flop kinetics and in preferential distribution into the kidneys. No nephrotoxicity was detected for any formulation and route of administration. CONCLUSIONS Oral administration of AmB in a mono/diglyceride-phospholipid formulation to rats resulted in significant intestinal absorption into the systemic circulation with pharmacokinetic and biodistribution properties similar to a micellar iv preparation.

A Novel Tropically Stable Oral Amphotericin B Formulation (iCo-010) Exhibits Efficacy against Visceral Leishmaniasis in a Murine Model

Purpose To develop an oral formulation of amphotericin B (AmB) that is stable at the temperatures of WHO Climatic Zones 3 and 4 (30–43°C) and to evaluate its efficacy in a murine model of visceral leishmaniasis (VL). Methods The stability testing of four novel oral lipid AmB formulations composed of mono- and di-glycerides and pegylated esters (iCo-010 to iCo-013) was performed over 60 d and analyzed by HPLC-UV. In addition, the four formulations were incubated 4 h in fasted-state simulated intestinal fluid. AmB concentration was measured spectrophotometrically and emulsion droplet diameter was assessed by dynamic light scattering. Antileishmanial activity of iCo-010 was evaluated at increasing oral doses (2.5 to 10 mg/kg) in a murine model of VL. Results AmB stability in the lipid formulation (iCo-010) was >75% over 60 days. After 4 h in fasted-state simulated intestinal fluid, AmB concentration was >95%. iCo-010 demonstrated significant efficacy when orally administered to VL-infected mice bid for five days (inhibition of 99%, 98%, and 83% at 10, 5 and 2.5 mg/kg compared to the vehicle control). In addition, the qd dose of 20 mg/kg provided 96% inhibition compared to the vehicle control. Conclusions The oral AmB formulation iCo-010 is stable at the temperatures of WHO Climatic Zones 3 and 4 (30–43°C). iCo-010 showed excellent antileishmanial activity at both 10 mg/kg po bid for 5 days (<99% reduction in parasitic infection) and 20 mg/kg po qd for 5 days (95% inhibition when compared to control).

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.

Lipid/DNA complexes as an intermediate in the preparation of particles for gene transfer: an alternative to cationic liposome/DNA aggregates

Abstract When cationic liposomes are mixed with plasmid DNA an aggregation reaction occurs. The heterogeneous membrane structures which arise following this reaction are dependent on liposomal lipid composition, liposome/DNA ratio as well as the presence of added salts or proteins. The resulting structures are also unstable, resulting in time dependent changes in the physical attributes of the aggregates. Pharmaceutical development of the liposome/DNA aggregates will be challenging because of these factors. For these reasons we have pursued development of alternative lipid-based systems as a vehicle for gene transfer. The pivotal step that led to development of these novel systems was the identification and isolation of a hydrophobic cationic lipid/DNA complex. This complex can be used as an intermediate in the preparation of well-defined particles. The hydrophobic lipid/DNA complex and the liposome/DNA aggregates are both formed as a consequence of multivalent electrostatic interactions. In contrast to the liposome/DNA aggregates, however, we believe that the particles formed when using lipid/DNA complex intermediates are a consequence of hydrophobic interactions.

Tropically stable novel oral lipid formulation of amphotericin B (iCo-010): biodistribution and toxicity in a mouse model

BackgroundThe purpose of this study was to evaluate the biodistribution and toxicity of amphotericin B (AmB) following multiple oral administrations of a novel tropically stable lipid-based formulation (iCo-010).MethodsBALB/c mice were allocated into six groups: oral iCo-010 twice daily for 5 days in the dose of 20, 10, 5 and 2.5 mg/kg; vehicle control; and intravenous boluses of Fungizone® 2 mg/kg once daily for 5 days. The animals were sacrificed 12 h following the last administration and blood and tissues were collected.ResultsThe plasma concentrations of AmB were similar to previously reported after administration of iCo-009. Somewhat lower concentrations of AmB were detected in reticulo-endothelial system in the case of iCo-010 when compared with iCo-009. The concentration in kidney was higher with iCo-010 than with iCo-009. The creatinine levels in all oral treatment groups were in a normal range as in the case of iCo-009. Administration of Fungizone® resulted in elevated plasma creatinine levels. Histopathology analysis detected no GI, liver or kidney toxicity following multiple dose oral administration of iCo-010. Fungizone® treatment induced necrotic changes in hepatic and kidney tissues.ConclusionsGiven the tropical stability of iCo-010, near identical activity against visceral leishmaniasis and significant concentrations in target organs this formulation has a potential to become a treatment of choice in tropical developing countries.

Assessment of novel oral lipid-based formulations of amphotericin B using an in vitro lipolysis model

The purpose of this study was to investigate the intraluminal processing of novel oral lipid-based formulations of amphotericin B using an in vitro lipolysis model. Amphotericin B (AmB) was formulated in three lipid-based formulations consisting of different lipid components: iCo-009, iCo-010 and iCo-011. Various lipid loads (0.25, 0.5, 1 and 2 g) were digested using the lipolysis model to assess AmB distribution among the lipolysis phases. The duration of lipolysis was comparable among the three formulations except for 2 g load of iCo-009 which had a significantly longer lipolysis than iCo-010 and iCo-011. The lipid components of iCo-009 experienced lower extent of lipolysis as compared to other formulations. Amphotericin B concentration in the aqueous phases was the highest with iCo-010 which also had the lowest sediment recovery. Amphotericin B levels in the undigested lipid layers were comparable between iCo-009 and iCo-010 and were higher than with iCo-011. Given the observation that iCo-010 had the highest aqueous micellar solubilization and the lowest sediment recovery of AmB among the tested formulations, these results could potentially be used to interpret and predict the in vivo performance of AmB- SEDDS formulations in future studies.