Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity.

Amphotericin B (AmB) is an amphiphilic drug commonly formulated in liposomes and administered intravenously to treat systemic fungal infections. Recent studies on the liposomal drug product have shed light on the AmB aggregation status in the bilayer, which heat treatment (curing) modifies. Although toxicity was found related to aggregation status - loose aggregates significantly more toxic than tight aggregates - the precise mechanism linking aggregation and toxicity was not well understood. This study directly measured drug release rate from various AmB liposomal preparations made with modified curing protocols to evaluate correlations among drug aggregation state, drug release, and in vitro toxicity. UV-Vis spectroscopy of these products detected unique curing-induced changes in the UV spectral features: a ∼25 nm blue-shift of the main absorption peak (λmax) in aqueous buffer and a decrease in the OD346/OD322 ratio upon thermal curing, reflecting tighter aggregation. In vitro release testing (IVRT) data showed, by applying and fitting first-order release kinetic models for one or two pools, that curing impacts two significant changes: a 3-5-fold drop in the overall drug release rate and a ten-fold decrease in the ratio between the loosely aggregated and the tightly aggregated, more thermodynamically stable drug pool. The kinetic data thus corroborated the trend independently deduced from the UV-Vis spectral data. The in vitro toxicity assay indicated a decreased toxicity with curing, as shown by the significantly increased concentration, causing half-maximal potassium release (TC50). The data suggest that the release of AmB requires dissociation of the tight complexes within the bilayer and that the reduced toxicity relates to this slower rate of dissociation. This study demonstrates the relationship between AmB aggregation status within the lipid bilayer and drug release (directly measured rate constants), providing a mechanistic link between aggregation status and in vitro toxicity in the liposomal formulations.

Optimization of the manufacturing process of a complex amphotericin B liposomal formulation using quality by design approach.

In this work, the manufacturing process of a complex liposomal amphotericin B (AmB) product was optimized using quality by design (QbD) approach. A comprehensive QbD-based process understanding and design space (DS) to the critical process parameters (CPPs) is essential to the drug development and consistent quality control. The process was based on the acid-aided formation of drug-lipid complexes in a methanol-chloroform mixture (step I) followed by spray drying (step II), hydration and liposome formation by microfluidization (step III), and lyophilization (step IV). Firstly, the risk assessment was conducted to identify the critical process parameters among the four key steps. Nine CPPs and five CQAs (API Monomer identity (absorbance main peak at 321 nm), API Aggregation identity (absorbance peak ratio, OD 415 nm/321 nm), particle size, in-vitro toxicity, and the cake quality) were determined based on their severity and occurrences with their contribution to the quality target product profile (QTPP). Based on the risk assessment results, the final screening design of experiments (DoE) was developed using fractional factorial design. Secondly, the empirical equation was developed for each CQA based on experimental data. The impact of CPPs on the CQAs was analyzed using the coefficient plot and contour plot. In addition to the effect of individual formulation parameters and process parameters, the effects of the four key separate steps were also evaluated and compared. In general, the curing temperature during microfluidization has been identified as the most significant CPP. Finally, design space exploration was carried out to demonstrate how the critical process parameters can be varied to consistently produce a drug product with desired characteristics. The design space size increased at the higher value of the curing temperature, the API to phospholipid ratio (API:PL), and the lower value of the DSPG to phospholipid ratio (PG:PL) and aspirator rate.

Critical process parameters in manufacturing of liposomal formulations of amphotericin B.

Identifying the critical process parameters (CPPs) of a complex drug product manufacture and the associated impact on critical quality attributes (CQAs) is essential to the development and quality control of both new and generic drugs. AmBisome, a liposomal amphotericin B (AMB) macrolide antibiotic widely adopted as an important antifungal drug product, was used as a model complex drug product in the current study. This study investigated how multi-step production approaches and related manufacturing conditions may affect essential physico-chemical and toxicological properties of the final drug product. A key challenge in the manufacture and analysis of liposomal AMB was the drug substance's propensity to aggregate, with associated poor solubility in water and organic solvents. This study identified three key CPPs in a four step manufacturing process: (i) proper acidification during formation of the drug-lipid complexes (Step 1), (ii) liposome heat curing following liposomal particle sizing (Step 3), and (iii) flash-freezing at the initial stages of the lyophilization cycle (Step 4). Over-acidification led to rapid degradation of the drug, whereas under-acidification hampered full solubilization and formation of the soluble drug-lipid complexes. Extended heat treatment of the formed liposomes at 65 °C, just above the lipid phase transition temperature, brought dramatic changes in the aggregated state and/or packing of the drug in the liposomal bilayer, as followed by the complex changes in the UV/Vis spectra. Such thermal conditioning resulted in a five- to ten-fold reduction in the in-vitro toxicity of the drug product, bringing it close to the values for AmBisome used as control and measured by the RBC assay. Finally, flash-freezing conditions during lyophilization was critical to prevent aggregation and maintaining the 80-120 nm liposome size when reconstituted. Our research found that changes in the amphotericin's UV/Vis spectra were a sensitive CQA measure and provided a set of quantitative parameters for a facile non-destructive process monitoring in-situ, as well as for comparison of the quality of final formulations.

Evaluating a novel panel of sperm function tests for utility in predicting intracytoplasmic sperm injection (ICSI) outcome.

PURPOSE: The objective of this study was to evaluate a panel of three sperm function tests; tests known to assess different aspects of sperm functionality and genomic integrity, the: 1) Sperm DNA Accelerated Decondensation (SDAD(TM)) Test, 2) Sperm DNA Decondensation (SDD(TM)) Test, and 3) Sperm Penetration Assay (SPA), determining if positive and negative test scores correlated with failed and successful ICSI outcomes, respectfully. METHODS: A prospective, double blinded, cohort study was performed. One study sample (ejaculated semen) was collected by each of the 60 male partners of the 60 couples enrolled in the study; males whose female partners were found to have no major female factor issues. The sperm from each male was analyzed in the SPA, and SDAD and SDD Tests, and used for ICSI (1 ICSI cycle per couple). RESULTS: The ICSI cycle pregnancy rate for this study was 50 %, with a delivery rate=40 % (n=60 ICSI cycles). The SPA and SDD Test scores did not significantly predict ICSI outcome when used as stand-alone tests (p>>0.05). However, when the SPA and SDD Test scores were used together, ICSI outcomes for a subgroup of 10 (16.7 %) males, were significantly predicted (p=0.03), with 1 live birth, and 9 negatives where the transferred embryos did not implant. In total, 38.4 % of the couples in this study were found to have a very poor chance for a successful ICSI cycle. CONCLUSION: SDAD Test scores alone, and SPA and SDD Test scores used together, significantly predicted failed ICSI outcomes. This indicates that the scores obtained when analyzing patients' sperm using a panel of sperm function tests; specifically, the SPA, and SDAD and SDD Tests, can be used to identify infertile couples who should not be directed to ICSI.

PreImplantation Factor (PIF) orchestrates systemic antiinflammatory response by immune cells: effect on peripheral blood mononuclear cells.

OBJECTIVE: Embryo-derived PreImplantation Factor (PIF) is essential for pregnancy immune modulation and synthetic PIF (sPIF), reverses neuroinflammation, and prevents diabetes mellitus through its immune modulatory properties. Herein, we explore sPIF's systemic effects on peripheral blood mononuclear cells (PBMCs). STUDY DESIGN: sPIF's effects on PBMCs and subset populations from nonpregnant patients (n = 7) and male patients were evaluated by the assessment of binding characteristics, mixed lymphocyte reaction, proliferation, cytokine secretion, and associated gene expression. Data analysis was by analysis of variance (P < .05). RESULTS: Fluorescein isothiocyanate-sPIF bound all myelomonocytic cells; binding was 30-fold up-regulated in mitogen-activated T and B cells (P < .05). sPIF decreased mixed lymphocyte reaction by 70% and blocked anti-CD3 antibody stimulated-PBMC proliferation by approximately 80% (P < .05). In naïve PBMCs, sPIF reduced interleukin (IL)-10 and -2; in activated PBMCs, sPIF increased IL-4, -5, -10, and -2, tumor necrosis factor-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor (P < .05). CONCLUSION: Physiologic concentrations of PIF exert potent systemic antiinflammatory effects on nonpregnant activated immune cells.

Serum inhibin A concentration in women with polycystic ovarian syndrome and the correlation to ethnicity, androgens and insulin resistance.

A prospective case-series in an academic hospital clinic was performed to determine whether there is a relationship between polycystic ovarian syndrome (PCOS) and ethnicity. Also, serum inhibin A concentrations were compared between PCOS and normal-ovulatory women. The possibility of a correlation between inhibin A, androgens and insulin resistance in PCOS women was evaluated. Serum inhibin A concentrations were measured in anovulatory PCOS patients (n=32) and in control women of reproductive age (n=16). Statistical analysis was performed using the Mann-Whitney U-test. Serum concentrations of inhibin A, follicle-stimulating hormone, LH, prolactin, thyroid-stimulating hormone, fasting glucose, insulin, testosterone, 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone sulphate (DHEAS) were measured. Inhibin A concentrations were significantly lower (4.5+/-4.8 pg/ml) when compared with the control group (13.2+/-14.4 pg/ml; P=0.003) and were not significantly different between Hispanic and Caucasian women diagnosed with PCOS. There was no correlation between inhibin A concentrations and insulin, testosterone, free testosterone, 17-OHP, or DHEAS concentrations. In PCOS women, inhibin A concentrations are similar between Hispanic and Caucasian women; however, women with PCOS, regardless of ethnicity, have a lower inhibin A concentration compared with normal-ovulatory women. No correlation was observed between inhibin A androgens and insulin resistance in women diagnosed with PCOS.

Inhibin A: marker for diagnosis of ectopic and early abnormal pregnancies.

A prospective case-control study was performed to determine whether inhibin A concentration is a clinically useful marker of ectopic pregnancy (EP). Inhibin A concentration in patients diagnosed with EP by laparoscopic and pathological findings (n = 17) was compared with that in missed miscarriage (n = 35), incomplete miscarriage (n = 14), spontaneous miscarriage (n = 5), threatened miscarriage (n = 6), normal pregnancy (n = 24) and non-pregnant controls (n = 20). The data were analysed using the Mann-Whitney U-test. EP yielded significantly lower inhibin A concentrations compared with normal pregnancy, 12.7 +/- 11.7 versus 237.3 +/- 125.9 pg/ml (P < 0.0002), and similar concentrations to non-pregnant controls (13.3 +/- 14.3 pg/ml). Inhibin A concentrations in abnormal pregnancies were significantly lower than in the normal pregnancy group: missed miscarriage 42.4 +/- 54.9 pg/ml (P < 0.0002); spontaneous miscarriage 47.5 +/- 55.6 pg/ml (P < 0.0002); and incomplete miscarriage 12.2 +/- 10.5 pg/ml (P < 0.0002). Threatened miscarriage was not statistically different to normal pregnancy (183.1 +/- 119.4 pg/ml). Human chorionic gonadotrophin concentrations in EP were not statistically significantly different compared with missed miscarriage and incomplete miscarriage. In conclusion, serum inhibin A concentration may be a reliable marker of EP.

Improved development of very-poor-quality human preembryos by coculture with human fallopian ampullary cells.

PURPOSE: To determine whether a confluent culture of fallopian ampullary epithelial cells, taken from women at the end of their reproductive life, is capable of rescuing very-poor-quality preembryos from cleavage arrest and/or degeneration. METHODS: Human preembryos. rejected for transfer or freezing because of very poor quality, and arrested within 24 h of cleavage, were cultured for 5 days in medium alone or over a confluent culture of fallopian ampullary epithelia] cells. Morphological criteria were utilized to assess preembryo degeneration and stage of development. RESULTS: The described coculture rescued preembryos from degeneration, enhancing development to the blastocyst stage 2.2-fold, compared with cultures in medium alone. Furthermore, fully expanded and hatching blastocysts were observed only under coculture conditions. CONCLUSIONS: Very-poor-quality human preembryos may be rescued from degeneration, and their growth and development dramatically improved, when cocultured with a confluent culture of fallopian ampullary epithelial cells.