Identification of critical formulation parameters affecting the in vitro release, permeation, and rheological properties of the acyclovir topical cream.

To understand effects of formulation variables on the critical quality attributes (CQA) of acyclovir topical cream, this study investigated effects of propylene glycol (PG), poloxamer, and sodium lauryl sulfate (SLS) concentrations, acyclovir particle size, and formulation pH of the acyclovir cream. Fifteen formulations were prepared and characterized for rheological properties, particle size distribution, drug release and in vitro skin permeation. Drug distribution between various phases of the cream was determined. The concentration of soluble acyclovir in the aqueous phase was determined as a surrogate of the equilibrium with other acyclovir species in the cream. The interaction among effects of the formulation variables on the amount of acyclovir retained by skin was also evaluated. The results showed that PG significantly (p < 0.05) increased the yield stress, viscosity, drug concentration in the aqueous phase, and drug release. The PG and SLS significantly (p < 0.05) increased acyclovir retention by skin samples. Particle size of acyclovir inversely affected the drug release. This study revealed that the employed concentrations of PG and SLS and particle size of the dispersed acyclovir are critical formulation variables that should be carefully controlled when developing acyclovir topical creams with desired performance characteristics.

Synthetic methylated CpG ODNs are potent in vivo adjuvants when delivered in liposomal nanoparticles.

Although it is well documented that the immunological activity of cytosine-guanine (CpG) motifs is abrogated by 5' methylation of the cytosine residue, encapsulation within stabilized lipid nanoparticles endows these methylated cytosine-guanine- (mCpG-) containing oligonucleotides (ODNs) with potent immunostimulatory activity in murine animal models. Surprisingly, not only do liposomal nanoparticulate (LN) mCpG ODN possess immunostimulatory activity, their potency is found to be equivalent and often greater than the equivalent unmethylated form, as judged by a number of ex vivo innate and adaptive immune parameters and anti-tumor efficacy in murine models. Preliminary data indicate that both methylated and unmethylated CpG ODN act through a common receptor signaling pathway, specifically via toll-like receptor (TLR) 9, based on observations of up-regulated TLR9 expression, induction of nitric oxide and dependence on endosomal maturation. This is confirmed in TLR9 knockout animals which show no immunostimulatory activity following treatment with LN-mCpG ODN. These data, therefore, indicate that the mCpG DNA is fully competent to interact with TLR9 to initiate potent immune responses. Furthermore, this work implicates an as yet unidentified mechanism upstream of TLR9 which regulates the relative activities of free methylated versus unmethylated CpG ODN that is effectively bypassed by particulate delivery of CpG ODN.

The effect of circulation lifetime and drug-to-lipid ratio of intravenously administered lipid nanoparticles on the biodistribution and immunostimulatory activity of encapsulated CpG-ODN.

The encapsulation of conventional drugs in lipid nanoparticles (LNs) has been extensively utilized to enhance therapeutic activity by altering their pharmacokinetic (PK) and biodistribution (BD) properties. We have previously shown that the immunostimulatory activity of unmethylated cytidine-guanosine (CpG)-containing immunostimulatory oligodeoxynucleotides (ODN) is greatly enhanced when encapsulated in an LN (LN CpG-ODN). Here, we investigate the effect of circulation lifetime (determined by lipid composition) and drug-to-lipid (D/L) ratio of intravenously (i.v.) administered LN CpG-ODN on PK, BD, and cellular uptake and correlate these parameters with the immunostimulatory activity. Results from these studies show that despite significant differences in the circulation lifetime and the D/L ratio, the immune response is similar with respect to immune cell activation and cytolytic activity in the spleen and the blood compartments. Our findings indicate that the benefits of liposomal nanoparticles for the delivery of immunomodulatory drugs such as CpG-ODN are defined by a different paradigm than that for conventional drugs.

Effects of intravenous and subcutaneous administration on the pharmacokinetics, biodistribution, cellular uptake and immunostimulatory activity of CpG ODN encapsulated in liposomal nanoparticles.

We have previously demonstrated that the immune response to an unmethylated cytidine-guanosine (CpG)-containing oligonucleotide (ODN) is greatly enhanced when encapsulated in a lipid nanoparticle (LN-CpG ODN). In this study, the pharmacokinetics, biodistribution and cellular uptake of LN-CpG ODN following intravenous (i.v.) and subcutaneous (s.c.) administration was characterized and correlated with immunostimulatory activity. It is shown that, despite dramatic differences in tissue distribution profiles and considerable differences in uptake by CD11c-positive, CD11b-positive, Mac-3-positive and CD45R/B220-positive cells following i.v. and s.c. administration, the resultant immune response is very similar with respect to levels of cellular activation (DX5, Mac-3, CD11b, CD45/B220, CD4, CD8 and CD11c) and cytolytic activity of immune cells [natural killer (NK) cells and monocytes/macrophages] in the spleen and blood compartments. Some differences in response kinetics and antibody-dependent cellular cytotoxicity (ADCC) activity were noted in the peripheral blood NK cell population. Analyses of particle biodistribution and cell types involved in uptake leads to the conclusion that the inherent ability of antigen-presenting cells (APCs) to sequester LN-CpG ODN results in efficient uptake of the particle, even when present at very low concentrations, leading to similar responses following i.v. and s.c. administration. These results contrast with the behavior of free CpG ODN, for which distinctly different immune responses are observed following i.v. or s.c. administration.

The effect of bilayer and hexagonal H(II) phase lipid films on transepidermal water loss.

The common membrane phospholipids tend to adopt either the familiar bilayer phase or the less familiar hexagonal H(II) phase when isolated and hydrated in excess water. The objective of this study was to compare the effects of these very different macroscopic lipid structures on transepidermal water loss (TEWL) when they are applied to the surface of pig skin mounted in Franz diffusion cells. First, a novel in vitro method for monitoring TEWL was developed and characterized in which the flux of water from the subphase through skin was measured through the absorption of (3H)-water by lyophilized polyethylene glycol (PEG) mounted above the skin surface. TEWL was varied by disrupting the skin barrier to different degrees by tape stripping or solvent extraction. Bilayer-forming egg phosphatidylcholine (EPC) or hexagonal H(II)-forming dioleoyl phosphatidylethanolamine (DOPE) were applied topically as solutions in ethanol and subsequently dried to films. The molecular configuration adopted by each lipid at the skin surface was confirmed by phosphorus NMR. TEWL for normal skin was approximately 2 g H2O/h/m2, increasing to a maximum of 80 g H2O/h/m2 after the stratum corneum was completely removed by tape stripping. On tape-stripped skin, films of lipid doses as low as 10 mg/cm2 significantly reduced TEWL, and DOPE (hexagonal H(II)) was approximately twofold more effective than EPC (bilayer). Furthermore, the effects of EPC and Vaseline on reducing TEWL from damaged skin were readily reversed by a simple aqueous wash, whereas the DOPE effect was unaltered even by vigorous washing. Similar results were obtained with lipid films applied to solvent-extracted skin. The data are consistent with the formation of extensive hydrophobic interactions between the skin and the outwardly facing acyl chains of the inverted, hexagonal H(II) phase adopted by DOPE. This results in the formation of a durable surface barrier capable of significantly reducing TEWL from damaged skin.

Methods for the preparation of protein-oligonucleotide-lipid constructs.

A mixture of ionizable cationic lipids, steric barrier lipids, and colipids is used to encapsulate oligonucleotide DNA in lipidic particles called SALP. This material is under development as an adjuvant for vaccines. Previously we have shown that coupling the antigen directly to the surface of SALP can lead to enhanced immunological responses in vivo. Two different methods for preparing ovalbumin-SALP were assessed in this work. Originally the conjugates were prepared by treating SALP containing a maleimide-derivatized lipid with thiolated ovalbumin, a method we refer to as active coupling. This reaction was found to be difficult to control and generally resulted in low coupling efficiencies. The issues relating to this approach were characterized. We have recently developed alternative techniques based on first coupling ovalbumin to a micelle and then incubating the resultant product with SALP, methods we refer to as passive coupling. We have shown that this method allows accurate control of the levels of protein associated SALP and does not suffer from surface saturation effects seen with the active coupling method that places maximum limits on the amount of protein that can be coupled to the SALP surface. The products from the passive coupling protocol are shown to have activity comparable to those derived from the active coupling protocol in investigations of in vivo immune responses.