NLRP3 inflammasome activation drives bystander cone photoreceptor cell death in a P23H rhodopsin model of retinal degeneration.

The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1ß and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

Novel oral amphotericin B formulation (iCo-010) remains highly effective against murine systemic candidiasis following exposure to tropical temperature.

PURPOSE: To evaluate the antifungal activity of amphotericin B (AmB) in a mouse model of systemic candidiasis following administration of a novel oral AmB formulation (iCo-010) that has been pre-exposed to tropical temperatures. METHODS: Amphotericin B (AmB) was prepared as a 5 mg/mL dispersion in a mixture of Peceol, Gelucire 44/14 and VitE-TPGS 2,3 (iCo-010). The formulation was protected from light and incubated in a sealed container at 43 °C for 60 days. Mice infected with Candida albicans were treated with either iCo-010 formulation pre-incubated at 43 °C for 60 days or freshly prepared iCo-010 formulation at doses of 5, 10 and 20 mg/kg once daily for five consecutive days. Single intravenous 5 mg/kg dose of AmBisome® was used as a positive control group. Seven days following the last dose, the kidney, liver, spleen, lung, heart and brain were removed and the number of colony forming units (CFUs) was determined as a measure of tissue fungal load. In addition, the concentration of AmB within each tissue was determined using high performance liquid chromatography (HPLC). RESULTS: There were no significant differences in the reduction of CFUs and the concentration of AmB recovered in all organs at all iCo-010 doses tested between the freshly prepared iCo-010 formulation compared to the formulation that was incubated at 43 °C for 60 days. CONCLUSIONS: A novel oral AmB formulation, iCo-010, incubated at 43 °C for 60 days to simulate the exposure of the formulation to tropical temperatures remained highly effective against murine systemic candidiasis.

A simple and sensitive method for determination of vitamins D3 and K1 in rat plasma: application for an in vivo pharmacokinetic study.

PURPOSE: To develop and to validate a simple but sensitive method for determination of vitamins D3 and K1 in rat plasma. METHODS: The sample treatment included protein precipitation by cold acetonitrile, evaporation, reconstitution with methanol and filtration. The chromatography conditions included Xterra RP18 3.5 µm 4.6 × 100 mm column at ambient temperature and mobile phase consisting of methanol/water (93/7, v/v) at 0.5 mL/min flow rate. Vitamin D3 and probucol were detected at 265 nm and vitamin K1 at 239 nm. Rats were administered intravenously by 0.1 mg/kg of vitamin D3 or K1 and the blood samples were withdrawn pre-administration and at pre-determined time points post-administration. The pharmacokinetic analysis was performed using a non-compartmental approach. RESULTS: The calibration curves in rat plasma were linear up to 5000 ng/mL for both vitamins. The limit of quantification (LOQ) was 20 ng/mL for vitamin D3 and 40 ng/mL for K1. Inter- and intra-day precision and accuracy were below 15%. The pharmacokinetic parameters of vitamin D3 following intravenous administration were: AUC0-∞ = 11323 ± 1081 h × ng/mL, Vd = 218 ± 80 mL/kg, CL = 8.9 ± 0.8 mL/h/kg, t1/2 = 16.8 ± 5 h; and of vitamin K1: AUC0-∞ = 2495 ± 297 h × ng/mL, Vd = 60 ±24 mL/kg, CL = 40.5 ± 5.1 mL/h/kg, t1/2 = 1.1 ±0.5 h. CONCLUSION: The developed HPLC-UV assay is a simple and sensitive method for the determination of vitamins D3 and K1 in rat plasma. A higher dose of vitamin K1 should be used in future studies for accurate estimation of pharmacokinetic parameters. The data show the suitability of the assay for pharmacokinetic studies in rats.

Efficacy of an oral and tropically stable lipid-based formulation of Amphotericin B (iCo-010) in an experimental mouse model of systemic candidiasis.

OBJECTIVE: An oral lipid based formulation that exhibits tropical stability (iCo-010) was developed to enhance the absorption of orally administered amphotericin B (AmB). iCo-010 has previously shown high efficacy in an acute model of systemic candidiasis in rats, directing the focus of this study to be its efficacy in a chronic model of systemic candidiasis in mice. METHODS: Mice were infected with 0.6 to 1×108 CFUs of Candida albicans ATCC 18804 strain by tail vein injection and were left for three days to develop the infection after which time treatment was initiated. The infected animals were assigned to the following treatment groups: no treatment (control) or iCo-010 at 5, 10 and 20 mg/kg administered by oral gavage once daily (QD) for 5 consecutive days. The animals were sacrificed 7 days after the last dose and the concentration of AmB and the fungal burden were assessed within the liver, kidneys, heart, lungs, spleen and brain. RESULTS: Although the infection was relatively low (~ 60-100 CFUs/ 1 ml tissue homogenate) in the liver, lungs and heart, the infection level was very high (70 000 CFUs / 1 ml tissue homogenate) in the kidney tissues for the control group. The highest concentrations of AmB were recovered in the kidneys and the spleen. The fungal burden in the tissues was lowered by 69-96% in the treatment groups when compared to the control group. CONCLUSION: Oral iCo-010 is an effective treatment of systemic candidiasis in the mouse model.

Pharmacokinetics and tissue distribution of amphotericin B following oral administration of three lipid-based formulations to rats.

The objective of this study was to assess the pharmacokinetics and tissue distribution of amphotericin B (AmB) in rats following oral administration of three lipid-based formulations (iCo-009, iCo-010 and iCo-011). The lipid-based formulations were administered to rats at a dose of 10 mg/kg and blood samples were withdrawn at predose, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 72 h, after which the animals were sacrificed and the body organs were collected for AmB quantification using a validated HPLC method. Plasma pharmacokinetics parameters were determined using non-compartmental analysis. The disappearance of AmB from plasma was the slowest following the administration of iCo-010 with MRT of 63 h followed by iCo-009 then iCo-011 (36 and 27 h). The AUC(0-24h) of iCo-009 and iCo-010 was 1.5-2-fold higher than that of iCo-011. The kidney exposure was comparable between iCo-009 and iCo-010 and was higher than that of iCo-011. The lung exposure was the highest following iCo-010 administration as compared to that of iCo-009. The distribution of AmB from plasma to tissues resulted in a high accumulation of AmB overtime with slow back-distribution to plasma. The pharmacokinetics profiles varied among the three formulations, despite the similarity in lipid composition between iCo-010 and iCo-011 and the presence of Peceol® as a common component in the formulations. The administration of oral iCo-010 could lead to higher steady state concentrations in the tissues after multiple dosing, which could lead to enhanced eradication of tissue borne fungal and parasitic infections.

ASCL1 is activated downstream of the ROR2/CREB signaling pathway to support lineage plasticity in prostate cancer.

Lineage plasticity is a form of therapy-induced drug resistance. In prostate cancer, androgen receptor (AR) pathway inhibitors potentially lead to the accretion of tumor relapse with loss of AR signaling and a shift from a luminal state to an alternate program. However, the molecular and signaling mechanisms orchestrating the development of lineage plasticity under the pressure of AR-targeted therapies are not fully understood. Here, a survey of receptor tyrosine kinases (RTKs) identifies ROR2 as the top upregulated RTK following AR pathway inhibition, which feeds into lineage plasticity by promoting stem-cell-like and neuronal networks. Mechanistically, ROR2 activates the ERK/CREB signaling pathway to modulate the expression of the lineage commitment transcription factor ASCL1. Collectively, our findings nominate ROR2 as a potential therapeutic target to reverse the ENZ-induced plastic phenotype and potentially re-sensitize tumors to AR pathway inhibitors.

ASCL1 activates neuronal stem cell-like lineage programming through remodeling of the chromatin landscape in prostate cancer.

Treatment with androgen receptor pathway inhibitors (ARPIs) in prostate cancer leads to the emergence of resistant tumors characterized by lineage plasticity and differentiation toward neuroendocrine lineage. Here, we find that ARPIs induce a rapid epigenetic alteration mediated by large-scale chromatin remodeling to support activation of stem/neuronal transcriptional programs. We identify the proneuronal transcription factor ASCL1 motif to be enriched in hyper-accessible regions. ASCL1 acts as a driver of the lineage plastic, neuronal transcriptional program to support treatment resistance and neuroendocrine phenotype. Targeting ASCL1 switches the neuroendocrine lineage back to the luminal epithelial state. This effect is modulated by disruption of the polycomb repressive complex-2 through UHRF1/AMPK axis and change the chromatin architecture in favor of luminal phenotype. Our study provides insights into the epigenetic alterations induced by ARPIs, governed by ASCL1, provides a proof of principle of targeting ASCL1 to reverse neuroendocrine phenotype, support luminal conversion and re-addiction to ARPIs.

Simultaneous determination of a novel antitrypanosomal compound (OSU-36) and its ester derivative (OSU-40) in plasma by HPLC: application to first pharmacokinetic study in rats.

PURPOSE: To develop an HPLC-UV method for determination of a novel antitrypanosomal compound (OSU-36) and its ester prodrug (OSU-40) in rat plasma and to apply the method for pharmacokinetic evaluation of both compounds in rats. METHODS: Since an attempt to assay for OSU-36 and OSU-40 in non-stabilized plasma resulted in highly non-linear calibration curves and poor sensitivity due to instability of the compounds, the plasma was stabilized using paraoxon and ascorbic acid. The sample treatment included protein precipitation by acetonitrile; evaporation; reconstitution with acetonitrile and filtration. The chromatography conditions included Xterra RP18 3.5 µm 4.6X100 mm column and gradient mobile phase system of acetonitrile-water. RESULTS: The limits of quantification (LOQ) were 50 ng/mL and 40 ng/mL for OSU-36 and OSU-40, respectively. The intra- and interday precision and accuracies were below 13% for low, medium and high quality control samples for both compounds. While OSU-40 has been stable in all tested handling conditions, OSU-36 was unstable in plasma after 20 days storage in -80 °C or 4h 28 °C storage. The developed method has been applied for a pharmacokinetic study in rats which revealed that an ester prodrug OSU-40 is rapidly converted to OSU-36 within the plasma compartment by plasma esterases. OSU-36, in turn, relatively quickly undergoes oxidative metabolism, including within the plasma compartment. CONCLUSIONS: A supplementation of rat plasma with an esterase inhibitor to prevent degradation of ester prodrug (OSU-40), and with antioxidant to prevent oxidation of OSU-36, is necessary for reliable determination of both compounds. Due to limited stability of OSU-36 in stabilized rat plasma, long-term storage of samples or prolonged handling in room temperature conditions is not recommended.

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

BACKGROUND: The 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). METHODS: BALB/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. RESULTS: The 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. CONCLUSIONS: Given 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.

An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer.

Cancers adapt to increasingly potent targeted therapies by reprogramming their phenotype. Here we investigated such a phenomenon in prostate cancer, in which tumours can escape epithelial lineage confinement and transition to a high-plasticity state as an adaptive response to potent androgen receptor (AR) antagonism. We found that AR activity can be maintained as tumours adopt alternative lineage identities, with changes in chromatin architecture guiding AR transcriptional rerouting. The epigenetic regulator enhancer of zeste homologue 2 (EZH2) co-occupies the reprogrammed AR cistrome to transcriptionally modulate stem cell and neuronal gene networks-granting privileges associated with both fates. This function of EZH2 was associated with T350 phosphorylation and establishment of a non-canonical polycomb subcomplex. Our study provides mechanistic insights into the plasticity of the lineage-infidelity state governed by AR reprogramming that enabled us to redirect cell fate by modulating EZH2 and AR, highlighting the clinical potential of reversing resistance phenotypes.

Biodistribution and tissue toxicity of amphotericin B in mice following multiple dose administration of a novel oral lipid-based formulation (iCo-009).

OBJECTIVES: The purpose of this study was to assess the biodistribution and toxicity of amphotericin B (AMB) following multiple dose administration of an oral lipid-based formulation (iCo-009). METHODS: BALB/c female mice were used. ICo-009 was administered twice daily for 5 days at doses of 2.5-20 mg/kg. Untreated animals, oral vehicle or intravenous Fungizone® (1 or 2 mg/kg) served as control groups. The animals were sacrificed 12 h following the last administration of AMB, and blood and multiple tissues were harvested for drug analysis and histopathological evaluation. Plasma or tissue samples were analysed for concentrations of AMB or creatinine by means of HPLC-UV. RESULTS: A dose-dependent accumulation of AMB in liver, spleen, kidney and lung tissues was found. The concentration of the drug in all these organs exceeded the corresponding concentrations in plasma at the same dose. The concentrations of AMB in heart and brain were similar to the corresponding concentrations in plasma. Creatinine concentrations were elevated above normal concentrations in the 2 mg/kg Fungizone® group only. Histopathological analysis of kidney and liver tissues revealed a normal pattern in all treated groups, except the 2 mg/kg Fungizone® group. No gastrointestinal toxicity was found in this study. CONCLUSIONS: A multiple dose treatment regimen with iCo-009 in mice results in a gradual accumulation of AMB in tissues. Despite significant concentrations of AMB, no kidney or liver toxicity of orally administered AMB was detected in this study. Furthermore, multiple oral administration of iCo-009 or of vehicle control did not induce gastrointestinal toxicity.

Visceral leishmaniasis affects liver and spleen concentrations of amphotericin B following administration to mice.

OBJECTIVES: To assess the impact of visceral leishmaniasis (VL) on the concentration of amphotericin B (AmB) recovered in the liver and spleen following either intravenous (AmBisome) or oral (iCo-009) AmB administration to mice. METHODS: Livers and spleens previously obtained from VL-infected BALB/c mice (following intravenous AmBisome or oral AmB treatments) were analysed for AmB concentrations. Then, non-infected BALB/c mice were divided into three treatment groups: a single dose of intravenous AmBisome (2 mg/kg, n = 5); and oral AmB every 12 h for 5 days (10 mg/kg, n = 6 and 20 mg/kg, n = 6). The animals were sacrificed 7 days after the initiation of the treatment and the livers and spleens were harvested for drug analysis by HPLC. RESULTS: The single intravenous injection of AmBisome resulted in a 77-fold lower concentration of AmB in infected compared with non-infected liver tissue, while the difference in AmB concentration in the spleen was only 5-fold. The multiple dose oral administration of AmB resulted in a 3-fold lower concentration of AmB in infected compared with non-infected livers for both oral doses, while the differences in AmB concentrations in the spleen were not statistically different for the oral treatment groups. CONCLUSIONS: VL significantly lowered the concentration of AmB in the liver and the spleen when compared with uninfected animals. This effect seems to correlate with the degree of infection of the tissue. In the case of the intravenous liposomal formulation (AmBisome), the differences between the infected and non-infected tissues are of a higher magnitude than in the case of orally administered AmB (iCo-009).

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.

Protonated nanostructured aluminosilicate (NSAS) reduces plasma cholesterol concentrations and atherosclerotic lesions in Apolipoprotein E deficient mice fed a high cholesterol and high fat diet.

The aim of this work was to assess the effect of chronic administration of protonated nanostructured aluminosilicate (NSAS) on the plasma cholesterol levels and development of atherosclerotic lesions in Apolipoprotein (ApoE) deficient mice fed a high cholesterol and high fat diet. Apolipoprotein E (ApoE) deficient mice were divided into the following treatment groups: protonated NSAS 1.4% (w/w), untreated control and 2% (w/w) stigmastanol mixed with high-cholesterol/high-fat diet. Animals were treated for 12 weeks, blood samples were withdrawn every 4 weeks for determination of plasma cholesterol and triglyceride levels. At the end of the study the aortic roots were harvested for assessment of atherosclerotic lesions. NSAS at 1.4% (w/w) and stigmastanol at 2% (w/w) treatment groups showed significant decreases in plasma cholesterol concentrations at all time points relative to the control animals. The lesion sum area in 1.4% (w/w) NSAS and 2% (w/w) stigmastanol groups were significantly less from the control animals. In conclusion, in this study, the effectiveness of chronic administration of protonated NSAS material in the reduction of plasma cholesterol levels and decrease in development of atherosclerotic lesions was demonstrated in Apo-E deficient mice model.

Assessing the antifungal activity, pharmacokinetics, and tissue distribution of amphotericin B following the administration of Abelcet and AmBisome in combination with caspofungin to rats infected with Aspergillus fumigatus.

The purpose of this study was to assess the antifungal activity, pharmacokinetics, and tissue distribution of amphotericin B (AmpB) following the administration of Abelcet and AmBisome alone and in combination with Caspofungin to rats infected with Aspergillus fumigatus. Aspergillus fumigatus inoculum (2.1-2.5 x 10(7) colony forming units [CFU]) was injected via the jugular vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single intravenous (i.v.) dose of Abelcet (5 mg AmpB/kg; n = 6), AmBisome (5 mg AmpB/kg; n = 6), Caspofungin (3 mg/kg; n = 5), Abelcet (5 mg AmpB/kg) plus Caspofungin (3 mg/kg) (n = 6), AmBisome (5 mg AmpB/kg) plus Caspofungin (3 mg/kg) (n = 7), or physiologic saline (non-treated controls; n = 6) once daily for 4 days. Antifungal activity was assessed by organ CFU concentrations and plasma galactomannan levels. Plasma and tissue samples were taken from each animal for AmpB pharmacokinetic analysis and tissue distribution determinations. Abelcet treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 73% compared to non-treated controls. Ambisome treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 69% compared to non-treated controls. Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 80% compared to non-treated controls. Abelcet plus Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 81% compared to non-treated controls. Ambisome plus Caspofungin treatment significantly decreased total fungal CFU concentrations recovered in all the organs added together by 98% compared to non-treated controls. Abelcet treatment significantly decreased plasma galactomannan levels by 50 and 75% 96 h following the initiation of treatment in the absence and presence of Caspofungin co-therapy, respectively. AmBisome treatment significantly decreased plasma galactomannan levels by 73 and 78% 96 h following the initiation of treatment in the absence and presence of Caspofungin co-therapy, respectively. Co-administration of Caspofungin with Abelcet and AmBisome did not significantly alter the plasma concentration-time profile, pharmacokinetic parameters, and tissue distribution of AmpB. Taken together, our findings suggest that an alternative mechanism, possibly at the cellular level rather than altered AmpB disposition, may be an explanation for the differences in organ CFU concentrations following Abelcet plus Caspofungin versus AmBisome plus Caspofungin administration.

Efficacy of Postnatal In Vivo Nonsense Suppression Therapy in a Pax6 Mouse Model of Aniridia.

Nonsense mutations leading to premature stop codons are common occurring in approximately 12% of all human genetic diseases. Thus, pharmacological nonsense mutation suppression strategies would be beneficial to a large number of patients if the drugs could be targeted to the affected tissues at the appropriate time. Here, we used nonsense suppression to manipulate Pax6 dosage at different developmental times in the eye of the small eye (Pax6Sey/+; G194X) mouse model of aniridia. Efficacy was assessed by functional assays for visual capacity, including electroretinography and optokinetic tracking (OKT), in addition to histological and biochemical studies. Malformation defects in the Pax6Sey/+ postnatal eye responded to topically delivered nonsense suppression in a dose- and time-dependent manner. Elevated levels of Mmp9, a direct downstream target of Pax6 in the cornea, were observed with the different treatment regimens. The lens capsule was particularly sensitive to Pax6 dosage, revealing a potential new role for Pax6 in lens capsule maintenance and development. The remarkable capacity of malformed ocular tissue to respond postnatally to Pax6 dosage in vivo demonstrates that the use of nonsense suppression could be a valuable therapeutic approach for blinding diseases caused by nonsense mutations.

The Effect of Digestion and Drug Load on Halofantrine Absorption from Self-nanoemulsifying Drug Delivery System (SNEDDS).

A super-saturated self-nanoemulsifying drug delivery system (super-SNEDDS), containing the poorly water-soluble drug halofantrine (Hf) at 150% of equilibrium solubility (S eq), was compared in vitro and in vivo with a conventional SNEDDS (75% of S eq) with respect to bioavailability and digestibility. Further, the effect of digestion on oral absorption of Hf from SNEDDS and super-SNEDDS was assessed by incorporation of the lipase inhibitor tetrahydrolipstatin (orlistat) into the SNEDDS. The SNEDDS contained soybean oil/Maisine 34-I (1:1), Kolliphor RH40, and ethanol at a ratio of 55:35:10, w/w percent. For the dynamic in vitro lipolysis, the precipitation of Hf at 60 min was significantly larger for the super-SNEDDS (66.8 ± 16.4%) than for the SNEDDS (18.5 ± 9.2%). The inhibition of the in vitro digestion by orlistat (1% (w/w)) lowered drug precipitation significantly for both the super-SNEDDS (36.8 ± 1.7%) and the SNEDDS (3.9 ± 0.7%). In the in vivo studies, the super-SNEDDS concept proved valid in a rat model with a significantly larger C max for the super-SNEDDS (964 ± 167 ng/mL) than for the SNEDDS (506 ± 112 ng/mL). The bioavailability of Hf dosed in super-SNEDDS (32.9 ± 3.6%) and SNEDDS (22.5 ± 6.3%) did not change significantly with co-administration of orlistat (45.5 ± 7.3% and 21.9 ± 6.5%, respectively). However, the pharmacokinetic parameters changed; the t max of the super-SNEDDS (1.3 ± 0.1 h) and SNEDDS (2.8 ± 1.2 h) were significantly lower when dosed with orlistat (6.0 ± 1.3 and 6.3 ± 1.2 h, respectively). These findings suggest that the role of lipid digestion for the absorption of drugs from SNEDDS may be less important than previously thought.

Dietary fats and pharmaceutical lipid excipients increase systemic exposure to orally administered cannabis and cannabis-based medicines.

There has been an escalating interest in the medicinal use of Cannabis sativa in recent years. Cannabis is often administered orally with fat-containing foods, or in lipid-based pharmaceutical preparations. However, the impact of lipids on the exposure of patients to cannabis components has not been explored. Therefore, the aim of this study is to elucidate the effect of oral co-administration of lipids on the exposure to two main active cannabinoids, Δ(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD). In this study, oral co-administration of lipids enhanced the systemic exposure of rats to THC and CBD by 2.5-fold and 3-fold, respectively, compared to lipid-free formulations. In vitro lipolysis was conducted to explore the effect of lipids on the intestinal solubilisation of cannabinoids. More than 30% of THC and CBD were distributed into micellar fraction following lipolysis, suggesting that at least one-third of the administered dose will be available for absorption following co-administration with lipids. Both cannabinoids showed very high affinity for artificial CM-like particles, as well as for rat and human CM, suggesting high potential for intestinal lymphatic transport. Moreover, comparable affinity of cannabinoids for rat and human CM suggests that similar increased exposure effects may be expected in humans. In conclusion, co-administration of dietary lipids or pharmaceutical lipid excipients has the potential to substantially increase the exposure to orally administered cannabis and cannabis-based medicines. The increase in patient exposure to cannabinoids is of high clinical importance as it could affect the therapeutic effect, but also toxicity, of orally administered cannabis or cannabis-based medicines.

Assessing the antifungal activity and toxicity profile of amphotericin B lipid complex (ABLC; Abelcet) in combination with caspofungin in experimental systemic aspergillosis.

The purpose of this study was to assess the antifungal activity and renal and hepatic toxicity of amphotericin B lipid complex (ABLC; Abelcet) following co-administration of Caspofungin to rats infected with Aspergillus fumigatus. Aspergillus fumigatus inoculum (1.3-2.3 x 10(7) colony forming units [CFU]) was injected via the jugular vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single intravenous (i.v.) dose of Fungizone(R) (1 mg AmpB/kg), ABLC (1 or 5 mg AmpB/kg), or an equivalent volume of normal saline (NS) (vehicle control) once daily for 4 days. Rats were further randomized into groups to receive 3 mg/kg Caspofungin or physiologic saline i.v. once daily for 4 days. To assess antifungal activity, brain, lung, heart, liver, spleen, and kidney sections were homogenized with NS (2 mL; 1 g of each tissue/mL) and a 0.1-mL aliquot was spread plated onto a Sabouraud dextrose agar plate. The plates were incubated for 48 h at 37 degrees C, at which time the numbers of CFU were determined and corrected for tissue weight. To assess renal and hepatic toxicity, serum creatinine and aspartate aminotransferase levels were determined. Fungizone and ABLC at a dosing regimen of 1 mg/kg i.v. once daily for four consecutive days and Caspofungin at a dosing regimen of 3 mg/kg i.v. once daily for four consecutive days had similar effectiveness in decreasing the total number of Aspergillus fumigatus CFUs found in all organs analyzed compared to non-treated controls. A combination of ABLC (1 mg/kg i.v. x 4 days) and Caspofungin (3 mg/kg i.v. x 4 days) significantly decreased the total number of Aspergillus fumigatus CFUs found in all organs analyzed compared to Caspofungin alone and non-treated controls. ABLC at a dosing regiment of 5 mg/kg i.v. once daily for four consecutive days was more effective in decreasing the total number of Aspergillus fumigatus CFUs found in all organs analyzed compared to Fungizone or ABLC alone at 1 mg/kg and Caspofungin alone at 3 mg/kg. However, a combination of ABLC (5 mg/kg i.v. x 4 days) and Caspofungin (3 mg/kg i.v. x 4 days) was not more effective than ABLC at 5 mg/kg or the combination of ABLC at 1 mg/kg and Caspofungin 3 mg/kg in reducing the total number of Aspergillus fumigatus CFUs compared to controls. Except for non-treated infected control rats, none of the treatment groups tested displayed a greater than 50% increase in serum creatinine concentrations from baseline. In addition, only ABLC at a dosing regimen of 1 mg/kg i.v. once daily for four consecutive days displayed a greater than 50% increase in AST concentration from baseline. Taken together, these findings suggest that ABLC at 5 mg/kg once daily x 4 days appears to be the best therapeutic choice in this animal model.

Plasma protein binding, lipoprotein distribution and uptake of free and lipid-associated BCL-2 antisense oligodeoxynucleotides (G3139) in human melanoma cells.

The objectives of this study were to determine the protein binding and lipoprotein distribution of G3139 and G3139 lipoplexes following incubation in human plasma, assess complement activation of, and the effect of pre-incubation of G3139 and G3139 lipoplexes in human plasma on in vitro cellular uptake of G3139. Effect of concentration and time on incorporation of free and lipid associated (lipoplexes) [3H]Bcl-2 AO (25-600 ng/ml) into normolipidemic human plasma lipoproteins was determined by density gradient ultracentrifugation after incubation at 37 degrees C for 5, 30, 60 and 120 min. Protein binding in the lipoprotein deficient fractions (LPDP) was determined by equilibrium dialysis. Complement interaction was determined by ELISA after exposure of human plasma to AO+/- liposomes prepared in serial dilution. In vitro uptake of G3139 and G3139 lipoplexes into human melanoma cells was assessed qualitatively by fluorescence microscopy after 4-h exposure to G3139 (free or as lipoplexes) with or without pre-incubation of G3139 in normal human plasma. Analysis of Bcl-2 AO-lipoprotein interaction over time and concentration indicated no significant movement of the compound within the different lipoprotein and LPDP fractions. Majority of drug was recovered within LPDP fraction, and more than 85% of drug recovered within LPDP fraction was protein bound. No significant activation of complement was noted for either free AO or lipoplexes. Pre-incubation of free AO or AO-lipoplexes in human plasma resulted in a greater cellular uptake of AO-lipoplexes compared with plasma free controls. These findings suggest that the majority of [3H]Bcl-2 AO is plasma protein bound with little lipoprotein association and no significant movement between different lipoprotein and LPDP fractions. Plasma protein binding other than lipoprotein binding may be responsible for the difference in cellular uptake of free AO vs. cationic lipoplexes.