Philip Sassene

Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 1: Method Parameterization and Comparison of In Vitro Digestion Profiles Across a Range of Representative Formulations

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation.

Precipitation of a Poorly Soluble Model Drug during In Vitro Lipolysis: Characterization and Dissolution of the Precipitate

Precipitation of cinnarizine during in vitro lipolysis of a self-microemulsifying drug delivery system (SMEDDS) was characterized to gain a better understanding of the mechanisms behind the precipitation. During in vitro lipolysis of the SMEDDS with or without cinnarizine, samples were taken at several timepoints and ultracentrifuged. Cinnarizine content in the pellet increased from 4% to 59% during lipolysis. The precipitation of cinnarizine during in vitro lipolysis correlated well with the degree of lipid digestion, determined by sodium hydroxide addition. The pellet from the endpoint of lipolysis was isolated and subjected to dissolution in biorelevant media. Dissolution rate of cinnarizine from pellets containing precipitated cinnarizine was initially 10-fold higher than dissolution from blank pellet spiked with crystalline cinnarizine, reaching more than 50% drug dissolved in the first minute. Pellets were further characterized by X-ray powder diffraction (XRPD) and polarized light microscopy (PLM). Both methods indicated the presence of liquid crystalline phases of calcium fatty acid soaps, but no presence of crystalline cinnarizine in the pellet. Overall, dissolution studies along with XRPD and PLM analysis indicate that cinnarizine precipitating during in vitro lipolysis of this SMEDDS is not crystalline, suggesting an either amorphous form or a molecular dispersion.

In vitro lipolysis models as a tool for the characterization of oral lipid and surfactant based drug delivery systems

With the increasing interest in lipid and surfactant based drug delivery systems (LSBDDS) for oral delivery of poorly soluble drugs, the need for efficient development tools is emerging. In vitro lipolysis models, simulating the digestion in the small intestine, is a promising tool in this regard. Several different in vitro lipolysis models have been used for characterization of LSBDDS, all using porcine pancreatin as lipase source, and primarily differing in the addition scheme of calcium and the kind of bile acids employed. Both calcium and bile influence the lipolysis. Calcium have been used both as fixed addition at the beginning of the experiment and with a continuous addition during lipolysis. Both pure bile acids and crude porcine bile extract have been used. Lipolysis of LSBDDS will generate mixed micelles, as well as lamellar and hexagonal phases. These have been characterized by dynamic light scattering, cryogenic transmission electron microscopy and small angle X-ray scattering. The faith of drug during in vitro digestion of a LSBDDS is often studied by ultracentrifugation and quantification of drug in the different phases formed. Further, drug precipitated during in vitro lipolysis has been characterized by X-ray powder diffraction and polarized light microscopy.

Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 3: Understanding Supersaturation Versus Precipitation Potential During the In Vitro Digestion of Type I, II, IIIA, IIIB and IV Lipid-Based Formulations

PurposeRecent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation – precipitation balance for a range of LBFs.MethodsType I, II, IIIA/B LBFs containing medium-chain (MC) or long-chain (LC) lipids, and lipid-free Type IV LBF incorporating different doses of fenofibrate or tolfenamic acid were digested in vitro in a simulated intestinal medium. The degree of supersaturation was assessed through comparison of drug concentrations in aqueous digestion phases (APDIGEST) during LBF digestion and the equilibrium drug solubility in the same phases.ResultsIncreasing fenofibrate or tolfenamic acid drug loads (i.e., dose) had negligible effects on LC LBF performance during digestion, but promoted drug crystallization (confirmed by XRPD) from MC and Type IV LBF. Drug crystallization was only evident in instances when the calculated maximum supersaturation ratio (SRM) was >3. This threshold SRM value was remarkably consistent across all LBF and was also consistent with previous studies with danazol.ConclusionsThe maximum supersaturation ratio (SRM) provides an indication of the supersaturation ‘pressure’ exerted by formulation digestion and is strongly predictive of the likelihood of drug precipitation in vitro. This may also prove effective in discriminating the in vivo performance of LBFs.

Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 4: Proposing a New Lipid Formulation Performance Classification System

The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF performance, including the degree of supersaturation generated on dispersion/digestion. Stresses evaluated included decreasing LBF concentration (↓LBF), increasing bile salt, and decreasing pH. Their capacity to stress LBFs was dependent on LBF composition and drug type: ↓LBF was a stressor to medium-chain glyceride-rich LBFs, but not more hydrophilic surfactant-rich LBFs, whereas decreasing pH stressed tolfenamic acid LBFs, but not fenofibrate LBFs. Lastly, a new Performance Classification System, that is, LBF composition independent, is proposed to promote standardized LBF comparisons, encourage robust LBF development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance grading.

Elucidating the Molecular Interactions Occurring during Drug Precipitation of Weak Bases from Lipid-Based Formulations: A Case Study with Cinnarizine and a Long Chain Self-Nanoemulsifying Drug Delivery System

The aim of this study was to investigate if molecular interactions between the weak base cinnarizine and lipolysis products were affecting the morphology of precipitated drug formed during in vitro lipolysis. In vitro lipolysis studies of a self-nanoemulsifying drug delivery system with or without cinnarizine were conducted. The digestion phases (aqueous phase and pellet phase) were separated by ultracentrifugation, and the pellet was isolated and lyophilized. The lyophilized pellets were examined by X-ray powder diffraction, (13)C solid-state nuclear magnetic resonance ((13)C NMR), (1)H liquid-state NMR ((1)H NMR) spectroscopy and differential scanning calorimetry (DSC). The (13)C NMR data indicated that the carbonyl groups and aliphatic part of the lipids undergo structural changes when the pellet contains cinnarizine. The (1)H NMR data suggests interactions occurring around the nitrogens on cinnarizine and the carboxylic group of fatty acids. DSC thermograms showed cinnarizine to be homogeneously incorporated into the lipids of the pellet, and no free amorphous cinnarizine was present. The three techniques (13)C NMR, (1)H NMR, and DSC complement each other and suggest interactions to occur between fatty acids and cinnarizine, which in turn favors amorphous precipitation.

In vitro and in vivo evaluations of the performance of an indirubin derivative, formulated in four different self-emulsifying drug delivery systems

Anticancer indirubins are poorly soluble in water. Here, digestion of four self‐emulsifying drug delivery systems (SEDDS) containing E804 (indirubin‐3′‐oxime 2,3‐dihydroxypropyl ether) was compared by dynamic lipolysis and bioavailability studies. Used lipids were either medium‐chain or long‐chain glycerides.

Development of a high-throughput in vitro intestinal lipolysis model for rapid screening of lipid-based drug delivery systems.

PURPOSE To develop a high-throughput in vitro intestinal lipolysis (HTP) model, without any means of pH-stat-titration, to enable a fast evaluation of lipid-based drug delivery systems (LbDDS). MATERIAL AND METHOD The HTP model was compared to the traditionally used dynamic in vitro lipolysis (DIVL) model with regard to the extent of lipid digestion and drug distribution of two poorly soluble model drugs (cinnarizine and danazol), during digestion of three LbDDS (LbDDS I-III). RESULT The HTP model was able to maintain pH around 6.5 during digestion, without the addition of NaOH to neutralize the free fatty acids (FFAs), due to an increased buffer capacity. Cinnarizine was primarily located in the aqueous phase during digestion of all three LbDDS and did not differ significantly between the two models. The distribution of danazol varied from formulation to formulation, but no significant difference between the models was observed. The triacylglycerides (TAG) in LbDDS III were digested to the same extent in both models, whereas the TAG present in LbDDS II was digested slightly less in the HTP model. No TAG was present in LbDDS I and digestion was therefore not analyzed. CONCLUSION The HTP model is able to predict drug distribution during digestion of LbDDS containing poorly water soluble drugs in the same manner as the DIVL model. Thus the HTP model might prove applicable for high-throughput evaluation of LbDDS in e.g. 96 well plates or small scale dissolution equipment.

Rhamnogalacturonan-I based microcapsules for targeted drug release

Drug targeting to the colon via the oral administration route for local treatment of e.g. inflammatory bowel disease and colonic cancer has several advantages such as needle-free administration and low infection risk. A new source for delivery is plant-polysaccharide based delivery platforms such as Rhamnogalacturonan-I (RG-I). In the gastro-intestinal tract the RG-I is only degraded by the action of the colonic microflora. For assessment of potential drug delivery properties, RG-I based microcapsules (~1 μm in diameter) were prepared by an interfacial poly-addition reaction. The cross-linked capsules were loaded with a fluorescent dye (model drug). The capsules showed negligible and very little in vitro release when subjected to media simulating gastric and intestinal fluids, respectively. However, upon exposure to a cocktail of commercial RG-I cleaving enzymes, ~ 9 times higher release was observed, demonstrating that the capsules can be opened by enzymatic degradation. The combined results suggest a potential platform for targeted drug delivery in the terminal gastro-intestinal tract.

Comparison of lipases for in vitro models of gastric digestion: lipolysis using two infant formulas as model substrates

The aim of this study was to find a lipase suitable as a surrogate for Human Gastric Lipase (HGL), since the development of predictive gastrointestinal lipolysis models are hampered by the lack of a lipase with similar digestive properties as HGL. Three potential surrogates for HGL; Rhizopus Oryzae Lipase (ROL), Rabbit Gastric Lipase (RGL) and recombinant HGL (rHGL), were used to catalyze the in vitro digestion of two infant formulas (a medium-chain triacylglyceride enriched formula (MC-IF) and a predominantly long-chain triacylglyceride formula (LC-IF)). Digesta were withdrawn after 0, 5, 15, 30, 60 min of gastric digestion and after 90 or 180 min of intestinal digestion with or without the presence of pancreatic enzymes, respectively. The digesta were analyzed by scanning electron microscopy and gas chromatography to quantify the release of fatty acids (FAs). Digestions of both formulas, catalyzed by ROL, showed that the extent of gastric digestion was higher than expected from previously published in vivo data. ROL was furthermore insensitive to FA chain length and all FAs were released at the same pace. RGL and rHGL favoured the release of MC-FAs in both formulas, but rHGL did also release some LC-FAs during digestion of MC-IF, whereas RGL only released MC-FAs. Digestion of a MC-IF by HGL in vivo showed that MC-FAs are preferentially released, but some LC-FAs are also released. Thus of the tested lipase rHGL replicated the digestive properties of HGL the best and is a suitable surrogate for HGL for use in in vitro gastrointestinal lipolysis models.