Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.

Amorphous solid dispersions (ASDs) are an attractive option to improve the bioavailability of poorly water-soluble compounds. However, the material attributes of ASDs can present formulation and processability challenges, which are often mitigated by the addition of excipients albeit at the expense of tablet size. In this work, an ASD manufacturing train combining co-precipitation and thin film evaporation (TFE) was used to generate high bulk-density co-precipitated amorphous dispersion (cPAD). The cPAD/TFE material was directly compressed into tablets at amorphous solid dispersion loadings up to 89 wt%, representing a greater than 60% reduction in tablet size relative to formulated tablets containing spray dried intermediate (SDI). This high ASD loading was possible due to densification of the amorphous dispersion during drying by TFE. Pharmacokinetic performance of the TFE-isolated, co-precipitated dispersion was shown to be equivalent to an SDI formulation. These data highlight the downstream advantages of this novel ASD manufacturing pathway to facilitate reduced tablet size via high ASD loading in directly compressed tablets.

Electrospun polymeric nanofibers have garnered substantial interest for biomedical applications in recent years due to their ability to enhance solubility, dissolution rate, and provide targeted and sustained release for a range of active pharmaceutical ingredients (APIs). A design of experiments (DOE) was applied to select formulation polymer properties and incorporation of an API ciprofloxacin hydrochloride to study electrospinning process parameters and nanofiber characteristics. Polymer solutions were characterized by viscosity and surface tension, and the resultant nanofibers were studied by scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, and in vitro drug release. Incorporation of ciprofloxacin HCl improved nanofiber uniformity and reduced the nanofiber breakage and cracking that was evident in poly(vinyl acetate) based nanofibers that were fabricated without the ciprofloxacin HCl. In vitro, extended drug release was observed with poly(vinyl acetate) nanofibers (up to 6 days), while a rapid drug release (less than 2 h) was shown by poly(vinyl alcohol) nanofibers. This work shows the utility of DOEs for studying API-containing electrospinning formulations to determine unique trends as compared with the electrospinning formulations without a drug such as ciprofloxacin HCl, and DOEs should be considered as a rapid approach to bring electrospinning forward as a process method for biomedical materials.

Long-acting parenteral (LAP) implant has garnered the attraction as a drug delivery technique in recent years. Understanding the drug release process is critical for the study of underlying release mechanism. In this paper, we present a novel application of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MADLI-MSI) for the direct visualization of the drug release process from non-conductive polymeric based LAP implants at molecular level. Custom-made sample holders were designed for LAP sample introduction in place of traditional conductive glass slides. The main technical obstacles of applying MALDI-MSI to study non-conductive materials are surface conductivity which can lead to charge build-up. In order to obtain homogeneous imaging of non-conductive sample surfaces, we developed a new sample surface treatment procedure, which is a critical control step to ensure the data reliability and accuracy in understanding kinetics of drug release process of LAP. Overall, this is the first comprehensive report of a sample preparation methodology tailored for imaging LAP at molecular level, allowing for the direct chemical identification and 2D mapping of an active pharmaceutical ingredient (API) distribution during LAP release process. Furthermore, this work has established the foundation to apply MALDI-MSI to the understanding of LAP implant formulation homogeneity, chemical composition, and degradation. More importantly, this work enabled the extension of MALDI-MSI technique to study a wide range of non-conductive materials.

Dissolution of amorphous solid dispersions (ASD) can lead to the formation of amorphous drug-rich nano species (nanodroplets) via liquid-liquid phase separation or glass-liquid phase separation when the drug concentration exceeds the amorphous solubility. These nanodroplets have been shown to be beneficial for ASD performance both in vitro and in vivo. Thus, understanding the generation and stability of nanodroplets from ASD formulations is important. In this study, the impacts of polymer selection and active pharmaceutical ingredient (API) physicochemical properties (wet glass transition temperature (Tg) and log P) on nanodroplet release were studied. Six APIs with different physicochemical properties were formulated as ASDs with two polymers, polyvinylpyrrolidone/vinyl acetate (PVPVA) and hydroxypropyl methylcellulose acetate succinate (HPMCAS). Their release performance was evaluated using both powder and surface normalized dissolution of compacts. In general, HPMCAS-based dispersions resulted in higher drug release compared to PVPVA-based dispersions. The two polymers also exhibited different trends in nanodroplet formation as a function of drug loading (DL). PVPVA ASDs exhibited a "falling-off-the-cliff" effect, with a dramatic decline in release performance with a small increase in drug loading, while HPMCAS ASDs exhibited a negative "slope" in the release rate as a function of drug loading. For both polymers, low Tg compounds achieved higher levels of nanodroplet formation compared to high Tg compounds. The nanodroplets generated from ASD dissolution were also monitored with dynamic light scattering, and HPMCAS was found to be more effective at stabilizing nanodroplets against size increase. Insights from this study may be used to guide formulation design and selection of excipients based on API physicochemical properties.

Implants offer the opportunity to improve patient adherence and real-world outcomes. However, most polymers used today are hydrophobic and limit drug properties suitable for development. Thermoplastic poly(urethanes) (TPUs) form pores upon hydration and may facilitate the development of implants containing drugs exhibiting broadly different properties. We sought to investigate the effect of drug physicochemical properties on permeability through membranes of varying TPU mixture composition; leverage imaging to visualize microstructural changes to the membrane across the TPU mixture composition range; and quantitatively characterize the membrane microstructure using equivalent pore analysis. We observed a correlation between drug hydrophobicity and its permeability through hydrophobic-rich TPU membranes. Conversely, all compounds diffused through hydrophilic-rich TPU membranes at similar rates, regardless of drug properties. Imaging revealed significant microstructure differences between hydrophobic-rich and hydrophilic-rich TPU membranes, supporting hypotheses proposed in our previous study. The hydrated hydrophilic TPU membrane pore area was determined to be 0.583% and its equivalent pore radius was found to be 128 nm, suggesting that hydrophilic TPU membranes may be used to modify the release of small molecular weight drugs and macromolecules. These findings highlight the benefits of hydrophilic TPUs as rate-controlling membranes to modulate the release rate of drugs with varying physicochemical properties.

Amorphous Solid Dispersion (ASD) based formulations have been frequently used to improve the bioavailability of poorly water soluble drugs, however, common processes to produce ASDs are not feasible for Absorption, Distribution, Metabolism and Excretion (ADME) studies with radio-labeled Active Pharmaceutical Ingredients (API) due to the complications associated with radioactive material handling. Liquid formulations are routinely used to support the ADME studies, though bridging the bioperformance between a liquid formulation to the amorphous dosage form for poorly soluble compounds has not been well studied, and can be challenging due to the potentially rapid in vitro and in vivo recrystallization and precipitation. Here we report the development of a fit for purpose liquid formulation that could accommodate the radioactive API and provide comparable bioavailability relative to the amorphous formulation without the need for dose adjustment. A number of formulation approaches were explored and the prototype formulations were evaluated by dissolution and preclinical pharmacokinetic studies. A PolyEthylene Glycol 400 (PEG 400) based solution formulation impregnated with a polymer, HydroxyPropyl MethylCellulose Acetate Succinate-L (HPMCAS-L), was identified as the lead formulation. It was found that the bioavailability of the formulation can be compromised by the presence of undissolved crystalline seeds, and the inclusion of HPMCAS-L can mitigate this effect, as well as potentially facilitate the nanoparticle formation. During the study, it is also noted that although dissolution test is instrumental in the formulation development, the in vitro study over predicted the extent of in vivo precipitation for PEG 400 formulation containing no crystalline seeds.

A novel laser induced breakdown spectroscopy (LIBS) 2D imaging method is used to directly visualize the drug release process of long-acting injectable (LAI) implants for the first time.

Laser induced breakdown spectroscopy (LIBS) has emerged as an innovative tool for quantitative and qualitative elemental analysis in pharmaceutical research. Herein, the potential use of LIBS for rapid characterization of tablet coatings is illustrated, including the investigation of coating thickness, coating uniformity and localized coating contamination. The laser shot number required for penetrating the coating correlates well with coating thickness determined from traditional scanning electron microscopy measurements. Each laser shot represents a 2.58 μm coating thickness. The inter-tablet coating uniformity was directly visualized using LIBS-based 3D chemical imaging, and the intra-tablet coating uniformity was quantitatively investigated. To our knowledge, this is the first report of 3D LIBS-based chemical imaging being utilized for quantitative analysis of pharmaceutical tablet coatings. In addition to elemental information, the accurate location of contaminants on the tablet coating was rapidly identified using 2D imaging. These results pave the way for LIBS to be a valuable technique for the analysis of pharmaceutical tablet coatings.

PURPOSE:Miscibility between the drug and the polymer in an amorphous solid dispersion (ASD) is considered to be one of the most important factors impacting the solid state stability and dissolution performance of the active pharmaceutical ingredient (API). The research described herein utilizes emerging fluorescence-based methodologies to probe (im)miscibility of itraconazole (ITZ)-hydroxypropyl methylcellulose (HPMC) ASDs. METHODS:The ASDs were prepared by solvent evaporation with varying evaporation rates and were characterized by steady-state fluorescence spectroscopy, confocal imaging, differential scanning calorimetry (DSC), and solid state nuclear magnetic resonance (ssNMR) spectroscopy. RESULTS:The size of the phase separated domains for the ITZ-HPMC ASDs was affected by the solvent evaporation rate. Smaller domains (<10 nm) were observed in spray-dried ASDs, whereas larger domains (>30 nm) were found in ASDs prepared using slower evaporation rates. Confocal imaging provided visual confirmation of phase separation along with chemical specificity, achieved by selectively staining drug-rich and polymer-rich phases. ssNMR confirmed the results of fluorescence-based techniques and provided information on the size of phase separated domains. CONCLUSIONS:The fluorescence-based methodologies proved to be sensitive and rapid in detecting phase separation, even at the nanoscale, in the ITZ-HPMC ASDs. Fluorescence-based methods thus show promise for miscibility evaluation of spray-dried ASDs.

A variety of formulation strategies have been developed to mitigate the inadequate aqueous solubility of certain therapeutic agents. Among these, achieving supersaturation in vivo is a promising approach to improve the extent of oral absorption. Because of the thermodynamic instability of supersaturated solutions, inhibitors are needed to kinetically hinder crystallization. In addition to commonly used polymeric additives, bile salts, naturally present in the gastrointestinal tract, have been shown to exhibit crystallization inhibition properties. However, the impact of bile salts on solution thermodynamics is not well understood, although this knowledge is essential in order to explore the mechanism of crystallization inhibition. To better describe solution thermodynamics in the presence of bile salts, a side-by-side diffusion cell was used to evaluate solute flux for solutions of telaprevir in the absence and presence of the six most abundant bile salts in human intestinal fluid at various solute concentrations; flux measurements provide information about the solute thermodynamic activity and hence can provide an improved measurement of supersaturation in complex solutions. Trihydroxy bile salts had minimal impact on solution phase boundaries as well as solute flux, while micellar dihydroxy bile salts solubilized telaprevir leading to reduced solute flux across the membrane. An inconsistency between the concentration-based supersaturation ratio and that based on solute thermodynamic activity (the fundamental driving force for crystallization) was noted, suggesting that the activity-based supersaturation should be determined to better interpret any modification in crystallization kinetics in the presence of these additives. These findings indicate that bile salts are not interchangeable from a thermodynamic perspective and provide a foundation for further studies evaluating the mechanism of crystallization inhibition.

Bile salts impact crystal nucleation and growth of supersaturated solutions of poorly water soluble drugs.

Currently, it is of interest to improve the oral absorption of poorly water-soluble therapeutic agents using supersaturating formulations. Understanding crystallization kinetics of supersaturated drug solutions is central to the design and evaluation of such formulations. Bile salts have drawn increasing attention in this context as they serve important roles in biorelevant dissolution media, in vivo, and have been shown to slow down the crystallization of active pharmaceutical ingredients. The goal of this study was to evaluate the impact of bile salt monomers and micelles on the crystallization of telaprevir, a poorly water-soluble drug, from aqueous solution. To better describe the crystallization driving force in the presence of the bile salts, a side-by-side diffusion cell was used to evaluate telaprevit mass flow rate, and hence solute activity, in the absence and presence of different bile salts. The effectiveness of monomeric and miceller bile salts as crystallization inhibitors was then evaluated by performing crystallization induction time experiments at constant, activity-based supersaturation. The six most abundant biologically relevant bile salts were investigated (sodium taurocholate, sodium taurodeoxycholate, sodium taurochenodeoxycholate, sodium glycocholate, sodium glycodeoxycholate, and sodium glycochenodeoxycholate). All six bile salts exhibited nucleation inhibition properties in both homogeneous supersaturated telaprevir solutions and highly supersaturated telaprevir solutions containing a second phase. The ability to retard telaprevir nucleation, however, varied among the bile salts and also depended on the aggregation state. Monomeric bile salts were found to be effective crystallization inhibitors. At higher bile salt concentrations, trihydroxy bile salts showed better inhibition compared to dihydroxy bile salts. These results highlight the importance of considering the composition of the test medium used to evaluate product performance, in particular in the context of evaluating crystallization kinetics.

Supersaturating formulations have become a popular approach to address the issue of inadequate aqueous solubility for small molecules. Given that prevention of nucleation may not be always possible, it is of particular interest to control crystal growth from supersaturated solutions in order to maximize the extent of oral absorption of therapeutic agents. Bile salts are endogenous surfactants that are present in biorelevant dissolution media and can have direct impact on the in vivo performance of therapeutic agents due to their presence in the gastrointestinal tract. In this study, an in situ common history seeding method was implemented to provide seeds that better mimic crystals formed from aqueous supersaturated solutions. By forming seeds in situ and then regenerating supersaturation, we were able to probe the influence of biorelevant bile salts on telaprevir crystal growth rates and secondary nucleation. The extent of growth inhibition was found to vary with bile salt chemistry and aggregation level. It was also noted that sodium taurocholate and sodium taurochenodeoxycholate inhibited secondary nucleation of telaprevir from highly supersaturated solutions. The impact of bile salts on crystal growth and secondary nucleation is of relevance for improved understanding of precipitation kinetics of supersaturating dosage forms.

Bile salts are natural surfactants present in the human gastrointestinal tract. Therefore, it is essential to consider their effect on the dissolution and crystallization tendency of oral drug formulations. Although a recent study showed that sodium taurocholate delayed nucleation for 11 structurally diverse compounds, there is limited information about the crystallization inhibition properties of other bile salts and whether they are interchangeable in this context. In this study, we evaluated the ability of 13 bile salts to maintain supersaturated aqueous solutions of three compounds: celecoxib, nevirapine, and fibanserin. Most bile salts extended nucleation induction times. However, their inhibitory effects varied depending on the structure and concentration of the bile salt and the drug. The R5 group and hydrophobicity of the bile salt appeared to be essential. Molecular dynamics simulations indicated that van der Waals and hydrogen bonding interactions occurred between nevirapine and bile salts, with variations in different systems. These results are important to better understand the crystallization tendency of orally delivered poorly water-soluble compounds in vivo.

The renal outer medullary potassium (ROMK) channel, located at the apical surface of epithelial cells in the thick ascending loop of Henle and cortical collecting duct, contributes to salt reabsorption and potassium secretion, and represents a target for the development of new mechanism of action diuretics. This idea is supported by the phenotype of antenatal Bartter's syndrome type II associated with loss-of-function mutations in the human ROMK channel, as well as, by cardiovascular studies of heterozygous carriers of channel mutations associated with type II Bartter's syndrome. Although the pharmacology of ROMK channels is still being developed, channel inhibitors have been identified and shown to cause natriuresis and diuresis, in the absence of any significant kaliuresis, on acute oral dosing to rats or dogs. Improvements in potency and selectivity have led to the discovery of MK-7145 [5,5 '-((1R,1 ' R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)], a potential clinical development candidate. In spontaneously hypertensive rats, oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the entire treatment period, and that displays additive/synergistic effects when administered in combination with hydrochlorothiazide or candesartan, respectively. Acute or chronic oral administration of MK-7145 to normotensive dogs led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. Elevations in bicarbonate and aldosterone were found after 6 days of dosing. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure. In addition, Bartter's syndrome type II features are manifested on exposure to ROMK inhibitors.

Amorphous solid dispersions (ASDs) formulated with acid-insoluble (enteric) polymers form suspensions in acidic media where the polymer is largely insoluble. However, a small amount of drug can dissolve and a supersaturated solution may be generated. The goal of this study was to gain insight into the leaching mechanisms of both drug and polymer from the suspended particles, studying the impact of solution additives such as surfactants.