Development of an Accelerated Rotator-based Drug Release Method for the Evaluation of Bupivacaine Multivesicular Liposomes.

PURPOSE: A multivesicular liposome (MVL) is a liposomal vehicle designed to achieve sustained release characteristics for drugs with short half-lives. For example, a commercial MVL formulation of bupivacaine has been approved by the U.S. Food and Drug Administration for local and regional analgesia. For complex formulations like those containing MVLs, challenges in developing an in vitro release testing (IVRT) method may hinder generic development and regulatory approval. In this study, we developed an accelerated rotator-based IVRT method with the ability to discriminate bupivacaine MVLs with different quality attributes. METHODS: Three IVRT experimental setups including mesh tube, horizontal shaker, and vertical rotator were screened to ensure that at least 50% of bupivacaine can release from MVLs in 24 h. Sample dilution factors, incubation temperature, and the release media pH were optimized for the IVRT. The reproducibility of the developed IVRT method was validated with commercial bupivacaine MVLs. The discriminative capacity was assessed via comparing commercial and compromised bupivacaine MVL formulations. RESULTS: The rotator-based release setup was chosen due to the capability to obtain 70% of drug release within 24 h. The optimized testing conditions were chosen with a 50-fold dilution factor, a temperature of 37ºC, and a media pH of 7.4. CONCLUSIONS: An accelerated rotator-based IVRT method for bupivacaine MVLs was developed in this study, with the discriminatory ability to distinguish between formulations of different qualities. The developed IVRT method was a robust tool for generic development of MVL based formulations.

Novel ProTide prodrugs of 5-fluoro-2'-deoxyuridine for the treatment of liver cancer.

ProTide prodrug technology has emerged as a promising way for the development of anti-viral and anti-tumor drugs, whereas, there are fewer applications for the treatment of liver cancer. Herein, a series of distinct 3'-ester ProTide prodrugs of 5-fluoro-2'-deoxyuridine (FdUR) were synthesized and evaluated for their anti-liver cancer activity. The most efficient prodrug 11b reached a sub-micromolar activity (IC50 = 0.42 ± 0.13 µM) against HepG2 and over 100-fold and 200-fold improvements compared to 5-FU, respectively. 11b also demonstrated favorable selectivity towards normal liver cells L-02 (IC50 > 100 µM). In vitro metabolic stability studies revealed that 11b is stable in the plasma and could be activated rapidly in the liver, which supported that 11b is liver-targeted. Importantly, to more accurately evaluate the anti-HCC activity of 11b, the liver orthotopic model was built and 11b significantly suppressed tumor growth (TGI = 75.5%) at a dose of 60 mg/kg/2d in vivo without obvious toxicity. Overall, these promising results indicated that 11b could serve as a safe and effective prodrug of 5-FU nucleoside for liver cancer therapy.

Translocation of IGF-1R in endoplasmic reticulum enhances SERCA2 activity to trigger Ca2+ER perturbation in hepatocellular carcinoma.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rß is translocated into the ER by ß-arrestin2 (ß-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rß. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rß levels. ER IGF-1Rß phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rß with SERCA2, and therefore ER IGF-1Rß failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rß and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.

Activation of insulin-like growth factor-1 receptor (IGF-1R) promotes growth of colorectal cancer through triggering the MEX3A-mediated degradation of RIG-I.

Insulin-like growth factor-1 receptor (IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers. However, targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings. Here, we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I. The active ß-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A. In response to ligands, IGF-1Rß activated the basal ßarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250, opening the middle loop (Leu130‒Cys141) to the RING domain of MEX3A through the conformational changes of ßarr2. The models of ßarr2/IGF-1Rß and ßarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I. The assay of the mutants ßarr2Y64A and ßarr2Y250A further confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rß and the RING domain of MEX3A. The truncated-ßarr2 and the peptide ATQAIRIF, which mimicked the RING domain of MEX3A could prevent the formation of ßarr2/IGF-1Rß and ßarr2/MEX3A complexes, thus blocking the IGF-1R-triggered RIG-I degradation. Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway. Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I. In summary, we revealed a disobliging IGF-1R signaling by which IGF-1Rß promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.

Characterization of exparel bupivacaine multivesicular liposomes.

Exparel is a bupivacaine multivesicular liposomes (MVLs) formulation developed based on the DepoFoam technology. The complex composition and the unique structure of MVLs pose challenges to the development and assessment of generic versions. In the present work, we developed a panel of analytical methods to characterize Exparel with respect to particle size, drug and lipid content, residual solvents, and pH. In addition, an accelerated in vitro drug release assay was developed using a rotator-facilitated, sample-and-separate experimental setup. The proposed method could achieve over 80% of bupivacaine release within 24 h, which could potentially be used for formulation comparison and quality control purposes. The batch-to-batch variability of Exparel was examined by the established analytical methods. Four different batches of Exparel showed good batch-to-batch consistency in drug content, particle size, pH, and in vitro drug release kinetics. However, slight variation in lipid contents were observed.

Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo.

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.

Comparative Pharmacokinetics and Tissue Distribution of M10 and Its Metabolite Myricetin in Normal and Dextran-Sodium-Sulfate-Induced Colitis Mice.

M10, a novel myricetin derivative, is an anti-inflammatory agent designed for treatment of colitis. Here, we aim to investigate its pharmacokinetic behavior and tissue distribution in a mouse model with colitis. Pharmacokinetics and tissue distribution of M10 and its metabolite myricetin were compared in normal mice and in dextran-sodium-sulfate (DSS)-induced colitis mice. The role of fecal microbiota was also analyzed during metabolism of M10 in vitro. After oral administration, M10 was very low in the plasma of both normal and diseased mice. However, both M10 and myricetin were mainly distributed in the gastrointestinal tract, including the stomach, colon and small intestine, in physiological and pathological conditions. Significantly, M10 and myricetin were found in higher levels in gastrointestinal tracts with inflamed tissues than in normal tissues of mice. An in vitro assay revealed that 80% of M10 was metabolized to myricetin via fecal microbiota. After oral administration, M10 was not absorbed into circulation but mainly distributed in the inflamed submucosal tissues of colitic mice, where it was metabolized into myricetin to prevent colitis development.

Comparative Transcriptome Analysis of Slow-Twitch and Fast-Twitch Muscles in Dezhou Donkeys.

The skeletal muscle fiber profile is closely related to livestock meat quality. However, the molecular mechanisms determining muscle fiber types in donkeys are not completely understood. In this study, we selected the psoas major muscle (PM; mainly composed of oxidative-type muscle fibers) and biceps femoris muscle (BF; mainly composed of glycolytic-type muscle fibers) and systematically compared their mRNA and microRNA transcriptomes via RNA-seq. We identified a total of 2881 differentially expressed genes (DEGs) and 21 known differentially expressed miRNAs (DEmiRs). Furthermore, functional enrichment analysis showed that the DEGs were mainly involved in energy metabolism and actin cytoskeleton regulation. The glycolysis/gluconeogenesis pathway (including up-regulated genes such as PKM, LDHA, PGK1 and ALDOA) was more highly enriched in BF, whereas the oxidative phosphorylation pathway and cardiac muscle contraction (including down-regulated genes such as LDHB, ATP2A2, myosin-7 (MYH7), TNNC1, TPM3 and TNNI1) was more enriched in PM. Additionally, we identified several candidate miRNA-mRNA pairs that might regulate muscle fiber types using the integrated miRNA-mRNA analysis. Combined with the results of protein-protein interaction (PPI) analysis, some interesting DEGs (including ACTN3, TNNT3, TPM2, TNNC2, PKM, TNNC1 and TNNI1) might be potential candidate target genes involved in the miRNA-mediated regulation of the myofibril composition. This study is the first to indicate that DEmiRs, especially eca-miR-193a-5p and eca-miR-370, and potential candidate target genes that are mainly involved in actin binding (e.g., ACTN3, TNNT3 and TNNC1) and the glycolysis/gluconeogenesis pathways (e.g., PKM) might coregulate the myofibril composition in donkeys. This study may provide useful information for improving meat quality traits in Dezhou donkeys.

Vaccine nanodiscs plus polyICLC elicit robust CD8+ T cell responses in mice and non-human primates.

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications.

Myricetin and M10, a myricetin-3-O-ß-d-lactose sodium salt, modify composition of gut microbiota in mice with ulcerative colitis.

Our previous studies found that M10, a myricetin-3-O-ß-d-lactose sodium salt, possessed higher effects of ameliorating ulcerative colitis (UC) than Myricetin in mice. Here, we aim to investigate whether the inhibition of UC is the consequence of the effects of M10 that leads to the changed microbiota. Mice model of UC was induced by dextran sulfate sodium (DSS) treatment. M10 and Myricetin were orally administrated for 12 weeks. We performed 16S rDNA sequencing assay to analyze the composition of gut microbiota isolated from ileocecum. Both M10 and Myricetin normalized the composition of Firmicutes and Actinobacteria as healthy mice had. At genus level, the effects of M10 and Myricetin on colitis were associated to the increase of probiotics, such as Akkermansia, and the inhibition of pathogenic microorganisms, such as Ruminococcus and Parabacteroides. M10 had stronger activity than Myricetin in the improvement of biosynthesis and degradation activities, resulting to increasing metabolism of sulfur, pyruvate, steroid biosynthesis and unsaturated fatty acid biosynthesis in gut. Furthermore, M10 normalized the proportion of Firmicutes and Actinobacteria in gut microbiota. It suggests that the improvements in UC are the consequence of the effect of M10 that leads to the changed intestinal microbiota. Conclusion: M10 contributed the pharmacological effects on UC by modification of the intestinal microbiota.

Synthetic high-density lipoproteins delivering liver X receptor agonist prevent atherogenesis by enhancing reverse cholesterol transport.

Liver X nuclear receptor (LXR) agonists are promising anti-atherosclerotic agents that increase the expression of cholesterol transporters on atheroma macrophages leading to increased efflux of cholesterol to endogenous high-density lipoprotein (HDL) acceptors. HDL subsequently delivers effluxed cholesterol to the liver by the process of reverse cholesterol transport, resulting in reduction of atherosclerotic plaques. However, LXR agonists administration triggers undesirable liver steatosis and hypertriglyceridemia due to increased fatty acid and sterol synthesis. LXR-induced liver toxicity, poor drug aqueous solubility and low levels of endogenous HDL acceptors in target patient populations limit the clinical translation of LXR agonists. Here, we propose a dual-antiatherogenic strategy for administration of the LXR agonist, T0901317 (T1317), by encapsulating in synthetic HDL (sHDL) nanoparticles. sHDL had been clinically proven to serve as cholesterol acceptors, resulting in plaque reduction in atherosclerosis patients. In addition, the hydrophobic core and endogenous atheroma-targeting ability of sHDL allow for encapsulation of water-insoluble drugs and their subsequent delivery to atheroma. Several compositions of sHDL were tested to optimize both T1317 encapsulation efficiency and ability of T1317-sHDL to efflux cholesterol. Optimized T1317-sHDL exhibited more efficient cholesterol efflux from macrophages and enhanced atheroma-targeting relative to free drug. Most importantly, in an apolipoprotein E deficient (ApoE-/-) atherosclerosis progression murine model, T1317-sHDL showed superior inhibition of atherogenesis and reduced hypertriglyceridemia side effects in comparison to the free drug and blank sHDL. The T1317-sHDL pharmacological efficacy was observed at doses lower than those previously described for LXR agents, which may have additional safety benefits. In addition, the established clinical manufacturing, safety and efficacy of blank sHDL nanoparticles used in this study could facilitate future clinical translation of LXR-loaded sHDLs.

Analytical method development and comparability study for AmBisome® and generic Amphotericin B liposomal products.

Liposomal Amphotericin B, known as AmBisome®, is a life-saving antifungal product that sold $407 million in 2019. AmBisome® has a rather complex physical structure in that Amphotericin B (AmpB) forms a stable ionic complex with the lipid bilayer to maintain AmBisome®'s low toxicity and high stability in systemic circulation. Failed attempts to reproduce AmBisome®'s precise structure has resulted in faster drug release and higher toxicity both in vitro and in vivo. In this study, we established several analytical methodologies to quantify liposomal AmpB components, characterize thermal properties of the liposome, and determine particle size distribution, AmpB aggregation state, and drug release kinetics. We applied these methodologies together with in vitro hemolytic potential and antifungal activity tests to characterize multiple lots of AmBisome® and two generic products approved in India, Phosome® and Amphonex®. We also used Fungizone®, a micellar AmpB formulation, and "leaky" AmpB liposomes as negative controls. Our results showed that Phosome® and Amphonex® were both similar to AmBisome®, while Fungizone® and 'leaky" liposomes exhibited differences in both thermal properties and AmpB aggregation state, leading to faster drug release and higher toxicity. Due to the increased interest of the pharmaceutical industry in making generic AmBisome® and the lack of standard analytical methods to characterize liposomal AmpB products, the methodologies described here are valuable for the development of generic liposomal AmpB products.

Structural analysis of lecithin:cholesterol acyltransferase bound to high density lipoprotein particles.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

Synergetic Effect of rHDL and LXR Agonist on Reduction of Atherosclerosis in Mice.

High-density lipoproteins (HDLs) are unique in that they play an important role in the reverse cholesterol transport process. However, reconstituted HDL (rHDL) infusions have demonstrated limited beneficial effect in clinical practice. This is perhaps a consequence of the limited cholesterol efflux abilities of atheroma macrophages due to decreased expression of cholesterol transporters in advanced atheromas and following rHDL infusion treatment. Thus, we propose that a combination therapy of rHDL and a liver X receptor (LXR) agonist could maximize the therapeutic benefit of rHDL by upregulating ATP-binding cassette transporters A-1 (ABCA1) and ATP-binding cassette transporter G-1 (ABCG1), and enhancing cholesterol efflux to rHDL. In macrophages, rHDL downregulated the expression of ABCA1/G1 in a dose- and rHDL composition-dependent manner. Although LXR agonist, T0901317 (T1317), upregulated the expression of ABCA1 and ABCG1, the drug itself did not have any effect on cholesterol efflux (6.6 ± 0.5%) while the combination of rHDL and T1317 exhibited enhanced cholesterol efflux from [3H]-cholesterol loaded J774A.1 macrophages (23.3 ± 1.3%). Treatment with rHDL + T1317 significantly reduced the area of aortic plaque in ApoE-/- mice compared to PBS treated control animals (24.16 ± 1.42% vs. 31.59 ± 1.93%, p < 0.001), while neither rHDL nor T1317 treatment alone had a significant effect. Together, we show that rHDL paired with an LXR agonist can induce a synergetic effect in reducing atheroma burden. This synergy could lead to lower overall effective dose for both drugs, potentially overcoming the existing barriers in clinical development and renewing pharmaceutical interest in these two drug classes.

Robust anti-tumor T cell response with efficient intratumoral infiltration by nanodisc cancer immunotherapy.

Potent anti-tumor T cell response and efficient intratumoral T cell infiltration are the major challenges for therapeutic cancer vaccines. To address these issues, a nano-vaccine system has been designed to promote anti-tumor T cell responses, and intratumoral infiltration was examined in various murine tumor models. Subcutaneous vaccination with nanodiscs carrying human papillomavirus (HPV)-16 E7 antigen elicits as high as ~32% E7-specific CD8 α + T cell responses in circulation, representing a 29-fold improvement over the soluble peptide vaccination. Importantly, nanodisc vaccination also promotes robust intratumoral T cell infiltration and eliminates HPV16 E6/E7-expressing TC-1 tumors at mucosal sites, including lungs, inner lip, and intravaginal tissues. In a benchmark study with a live Listeria vaccine combined with anti-PD-1 IgG, nanodiscs plus anti-PD-1 immune checkpoint blockade elicits comparable levels of T cell responses with anti-tumor efficacy. Furthermore, compared with Complete Freund's Adjuvant combined with tetanus toxoid, nanodisc vaccination in HLA-A02 mice generates >200-fold stronger IFN-γ+ T cell responses against a neoantigen from an HLA-A02 melanoma patient. Overall, these results show that the nanodisc system is a promising cancer vaccine platform for inducing anti-tumor T cell responses.

Predicting drug release kinetics from nanocarriers inside dialysis bags.

Dialysis methods are frequently used to determine the in vitro drug release kinetics of nanoparticle drug delivery systems. However, the need for the released drug to diffuse through the dialysis membrane delays its appearance in the sampling compartment. Thus, the apparent drug release data outside the dialysis bag typically does not match the desired release kinetics inside the bag adjacent to the nanocarriers. To address this issue, here we describe a simple approach to determine the actual drug release kinetics from nano drug carriers inside the dialysis bag from the experimental data measured from the sampling compartment. First, a calibration experiment is carried out to determine the diffusion barrier properties of the dialysis membranes. The apparent drug release profile of the nanocarrier is then determined using the dialysis method, and a mathematical model is applied to determine the actual drug release kinetics from the experimental data. The model was tested on DOXIL® (doxorubicin liposomes), and an excellent agreement was found between the predicted and measured drug concentration inside the dialysis membranes. By taking the barrier effects of dialysis membranes into consideration, our model independent of drug carrier not only enables the proper interpretation of the data from dialysis studies but also helps to evaluate the dialysis methodology applied to in vitro drug release assays.

Characterization of apolipoprotein A-I peptide phospholipid interaction and its effect on HDL nanodisc assembly.

Background: Synthetic HDLs (sHDLs), small nanodiscs of apolipoprotein mimetic peptides surrounding lipid bilayers, were developed clinically for atheroma regression in cardiovascular patients. Formation of HDL involves interaction of apolipoprotein A-I (ApoA-I) with phospholipid bilayers and assembly into lipid-protein nanodiscs. Purpose: The objective of this study is to improve understanding of physico-chemical aspects of HDL biogenesis such as the thermodynamics of ApoA-I-peptide membrane insertion, lipid binding, and HDL self-assembly to improve our ability to form homogeneous sHDL nanodiscs that are suitable for clinical administration. Methods: The ApoA-I-mimetic peptide, 22A, was combined with either egg sphingomyelin (eSM) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid vesicles to form sHDL. The sHDL assembly process was investigated through lipid vehicle solubilization assays and characterization of purity, size, and morphology of resulting nanoparticles via gel permeation chromatography (GPC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Peptide-lipid interactions involved were further probed by sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). The pharmacokinetics of eSM-sHDL and POPC-sHDL nanodiscs were investigated in Sprague Dawley rats. Results: sHDL formation was temperature-dependent, with spontaneous formation of sHDL nanoparticles occurring only at temperatures exceeding lipid transition temperatures as evidenced by DLS, GPC, and TEM characterization. SFG and ATR-FTIR spectroscopy findings support a change in peptide-lipid bilayer interactions at temperatures above the lipid transition temperature. Lipid-22A interactions were stronger with eSM than with POPC, which resulted in the formation of more homogeneous sHDL nanoparticles with longer in vivo circulation time as evidenced the PK study. Conclusion: Physico-chemical characteristics of sHDL are in part determined by phospholipid composition. Optimization of phospholipid composition may be utilized to improve the stability and homogeneity of sHDL.

Development of a flow-through USP 4 apparatus drug release assay for the evaluation of amphotericin B liposome.

AmBisome® is a liposomal formulation of amphotericin B (Amp B), a complex parenteral antifungal product with no US FDA approved generic version available to date. For generic Amp B liposomal product development, examination of the drug release profile is important for product quality control and analytical comparability evaluation with the reference listed drug. Yet, there is no standardized in vitro drug release (IVR) assay currently available for Amp B liposomes. In this study, we describe the development of a USP-4 apparatus-based IVR assay capable of discriminating liposomal Amp B formulations based on the drug release profile. The goal of the IVR assay development was to identify release media compositions and assay temperatures capable of facilitating 70-100% of drug release from AmBisome® in 24 h without Amp B precipitation or disruption of liposome structure. We found that an addition of 5% w/v of γ-cyclodextrin to the release media of 5% sucrose, 10 mM HEPES, and 0.01% NaN3 (pH = 7.4) prevented Amp B precipitation and facilitated drug release. Increased IVR assay temperature led to increased drug release rate, and 55 °C was selected as the highest temperature that induced drug release close to our target without causing product precipitation. The developed IVR assay was used to discriminate between drug release rates from AmBisome® and micellar Amp B products like Fungizone® and Fungcosome. The IVR assay was also capable of discriminating between Amp B liposomes with the same composition as AmBisome® but prepared by either extrusion or homogenization processes, both of which resulted in measurable liposomal particle size heterogeneity and Amp B concentration differences. Finally, the USP-4 IVR assay was used to compare Amp B release profiles between AmBisome® and two generic products approved in India, Amphonex® (Bharat Serums and Vaccines Ltd.) (f2 = 66.3) and Phosome® (Cipla Ltd.) (f2 = 55.4). Taken together, the developed USP-4 IVR assay can be a useful tool for drug release profile characterization in generic liposomal Amp B formulation development.

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol.

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.

Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2.

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.