Patent Application
20200232961
The disclosure relates to the characterization of chemical compounds using an in vitro mimic of the vertebrate intestinal tract and, more particularly, to the dissolution and absorption characteristics of chemical compounds and materials used in such characterizations of chemical compounds.
The United States Pharmacopeia (USP) established some of the most ubiquitous dissolution methodologies, equipment, and standards used today around the world. In 1950, the USP's only official test for tablet and capsules was the disintegration test, but this test was known to be indirectly related to drug bioavailability and product performance. In 1962, dissolution testing was beginning to gain support as a more discriminating test for drug bioavailability and drug product performance. In 1968, the USP1 basket-stirred flask apparatus was introduced and by 1975, through a collaborative effort of industry and government, there was convincing evidence that the USP1 could provide an in vivo-in vitro correlation. These findings provided momentum for a movement away from an in vitro-in vivo USP bioavailability standard. The USP1 was complemented by the USP paddle apparatus (USP2) in 1978 and, at the same time, the USP adopted the sink condition (3 times volume required to saturate a solution (about 500 mL-1000 mL; typically, 900 mL), in simple aqueous media to eliminate the use of enzymes in simulated intestinal fluid) and 50 rpm paddle speed to maximize product discrimination. Between 1985-1990, after the advent of more complex dosage forms (e.g., enteric coated, extend/delayed release), the USP implemented an official system for accounting in vivo-in vitro correlations (levels A, B, C, D).52 This development provided a framework to compare how well a drug product performed relative to a pharmacokinetic clinical study. The trend of using dissolution testing as a method to discriminate between formulations, which is the primary use in industry to date, is contrary to the original intentions of the implementation of dissolution testing. This development was well intentioned and rational, due to the need to quantify formulation consistency and to ensure product safety and efficacy for the general public. However, this disclosure resulted from experiments that questioned the utility of the USP dissolution test, with one focus being to make the dissolution test more physiologically meaningful. Typical efforts along these lines attempt to make the content of the dissolution medium biomimetic, while few efforts focus on the apparatus or in vivo mechanical/hydrodynamic forces.53-62
For orally administered drugs, in vivo relevant (ivR) dissolution methodology examines the drug product in an in vitro experimental system that strives to accurately simulate the critical parameters of the in vivo environment and processes of the vertebrate, e.g., human, gastrointestinal tract. The information is important in focusing pharmaceutical development pipelines on therapeutics, e.g., drugs such as biologics and small-molecule chemicals, that exhibit physico-chemical properties compatible with delivery to, dissolution in, and absorption by the vertebrate GI tract at efficacious yet safe dosages.
Various in vitro systems have been used in the past to attempt to integrate absorption kinetics into dissolution methods, such as the octanol-water system, Caco-2 cell membrane permeability assay, and parallel artificial membrane permeability assay (PAMPA). These systems fall short, however, in providing realistic human GI absorption kinetics in a robust and facile in vitro setting.
Biphasic systems or organic-solvent-based absorption systems (OSAS) have been utilized in the pharmaceutical industry to incorporate a passive absorption compartment into ivR in vitro dissolution systems.7-11 Systems such as octanol-water, however, can be challenging to use. The boundary that occurs between the aqueous and organic phases is more dynamic than a physical barrier.12 Large agitations can create mixing between the organic and aqueous layers, which can result in a poorly defined interface. A significant challenge in OSAS systems is adjusting the absorptive surface area to dissolution volume ratio to modulate the interfacial mass transfer rate to accurately simulate human oral absorption.
Other popular systems for simultaneous dissolution and absorption studies are the cell-based membrane systems. Yamashita et al. have done extensive work with Caco-2 and MDCK II monolayer cell membranes in side-by-side diffusion cells in an effort to include absorption kinetics in dissolution tests for the purpose of in vitro-in vivo correlation and evaluation of drug candidates during the early discovery phase of drug development.13-16 They also have examined formulation effects (micelle transport and food effect) in vitro on Caco-2 membrane transport.17-19 The donor and receiver volumes (on the order of 1-10 mL) make these assays attractive to industry and academia for higher throughput permeation studies (used in well-plate assays), in addition to containing biological drug transporters in some systems that allow for active transport to occur in an in vitro environment. However, Caco-2 cell membrane assays to date have not attempted to achieve the correct membrane surface area to dissolution volume ratio to replicate human absorption rates. On balance, there are significant challenges in using Caco-2 or similar systems. Caco-2 cells are known for their significant inter-laboratory variability, which requires an external system of validation to verify any results generated using Caco-2 permeability assays. In the recent past, a clone of Caco-2 (TC7) was developed to increase the consistency of Caco-2 assays. Cell based systems require prolonged periods of time for cells to culture prior to use, unlike a synthetic membrane like PDMS or PAMPA. Additionally, drug molecules tend to be retained in the Caco-2 membrane when the drug exhibits poor solubility, and the hydrodynamic boundary layer severely affects the permeation of drug molecules through the membrane.20-21
PAMPA is another tool that was developed in the late 1990s to improve and expedite the evaluation of new chemical entities in terms of permeability and estimated oral absorption rates. The development of PAMPA focused on high-throughput screening, which gives PAMPA a distinct advantage over Caco-2 or other cell-based assays. This advantage is particularly valuable in the pharmaceutical industry, where the rapid pace of early discovery-phase pharmaceutical development demands robust, repeatable, and fast/real-time analytical techniques in data analysis.22 There are a range of structural configurations for different routes of drug administrations, i.e., oral, brain, skin, and different combinations of polymer/organic/phospholipid phases) for PAMPA systems.23-26 PAMPA has even been used in parallel studies with Caco-2 assays to determine effects of reflux and efflux transporters on permeability and oral absorption.27 These characteristics make PAMPA a powerful tool for permeability screening and mechanism determination, but a poor method for incorporating realistic vertebrate, e.g., mammalian such as human, GI absorption kinetics into dissolution methods. PAMPA suffers the same limitations as cell-based assays where interfacial area is determined artificially by the micro well-plates used in the assay, and the available volumes for dissolution are not scaled to match the human GI situation. The hydrodynamic boundary layer can also significantly affect the permeation of lipophilic drugs.28
Computer-aided design (CAD) and additive manufacturing have been used since the 1980s to reduce the cost, effort, and production time of prototype models for engineered parts. CAD models of a part are made in a variety of commercially available software packages and exported into a “.stl” file (Standard Tessellation Language or STereoLithographic file). The stl file provides coordinates for triangular planes which represent small portions of surfaces of the CAD model. Higher tessellation translates to more resolution the surface has, which ultimately leads to smoother or more detailed printed parts. One of the most popular additive manufacturing techniques is stereolithography (SL), which is one method within the colloquial term “3D printing”. SL uses photosensitive monomer resins that polymerize when exposed to ultraviolet light. The penetration of ultraviolet light is shallow in the liquid monomer resin, thus the polymerization reaction occurs near the liquid surface. Once the photopolymerization is complete, the build substrate is lowered into the reservoir of liquid monomer resin and then the photopolymerization process is repeated over the slice of the two-dimensional cross section until the part is completed. Another common additive manufacturing technique is fused deposited modeling (FDM). FDM relies on hot melt extrusion, specifically, heating the material 0.5° C. above the melting temperature and then depositing it through a movable head. The material rapidly cools after the extrusion and cold welds in place to the adjoining layers. The resolution of the additive manufacturing device is described in terms of voxels, or three-dimensional pixels.66-68
CAD, FDM, and SL have recently begun to enter the field of pharmaceutical science in the form of drug delivery systems, such as modified and immediate release tablets, caplets, and disks.69-77 While there appears to be interest in using additive manufacturing to develop new ways to control dose weight and dissolution properties, there is no information about using additive manufacturing to improve the science of dissolution itself.
In view of the disadvantages and limitations of existing technologies for characterizing the dissolution and absorption properties of orally administrable compounds, e.g., therapeutics, a need continues to exist in the art for assessing the dissolution and absorption properties of such compounds in a manner that reflects their in vivo behavior upon oral administration.
The disclosure provides investigations establishing the use of a PDMS membrane, such as found in the context of an ultra-thin, large-area poly(dimethylsiloxane) membrane diffusion cell or UTLAM PDMS, to overcome the experimental challenges encountered in other in vitro permeation assays/methods. A properly selected material can yield a membrane that mitigates or eliminates the experimental challenges found in other in vitro absorption systems. The selection of an optimal polymer membrane is more than a question of lipophilicity, however. There are in vitro aspects that must be considered as well, such as erosion, degradation, and physical stability. Water intrusion/swellability, creation of aqueous pores, diffusion of drug, diffusion of polymer degradation products, micro-environmental pH changes, diffusion through pores, hydrogen/hydroxide ion transport into the membrane causing micro-environment pH changes, osmotic effects, convection, adsorption/desorption, and partitioning behavior may all affect mass transport.29
An in vivo relevant (ivR) in vitro model of drug absorption uses physiologically relevant fluids (e.g., pH, volume, temperature, buffer, buffer capacity, surfactants), hydrodynamic conditions (e.g., shear, advection), and mass transfer rates (e.g., diffusion, permeation to simulate the absorption process) in an in vitro system can better simulate in vivo performance than current and common compendial dissolution methodologies.1-4, 92 Disclosed herein, in relevant part, is the design, fabrication, and evaluation of a new in vitro device that incorporates new knowledge of the human gastrointestinal (GI) tract from a unique clinical study performed in humans and computation fluid dynamics simulations.50, 51
An ultra-thin, large area poly(dimethylsiloxane) membrane diffusion cell (UTLAM) (PDMS) simulates the passive diffusion mechanism of the human oral absorption pathway (duodenum and jejunum).92 It is a single compartment vessel that incorporates a hydrofoil impeller to reduce bulk fluid shear rates while maintaining axial mixing that promotes particle re-suspension. It is contemplated that various embodiments of the UTLAM diffusion cell will emulate different human GI tract compartmental segments using buffer and pumping parameters similar to those used in artificial stomach and duodenum (ASD) and gastrointestinal simulator (GIS) devices.
Since there is no significant convective passive drug transport through the human GI tract, polymers that were known to have convective transport behaviors such as sieving or size exclusion were eliminated from consideration for the biomimetic in vitro membrane. For robust and simple in vitro mass transport analysis, the membrane needs to be stable across the pH spectrum and be unaffected by the solution conditions present in the donor and receiver compartments confining donor and receiver fluids, respectively. The donor fluid or compartment contains the initial concentration of the compound of interest while the receiver fluid or compartment is separated, at least in part, therefrom by the polymer and the receiver fluid or compartment initially lacks the compound of interest. Selecting the polymer to not include highly reactive functional groups ensures physical and chemical stability. Polymers containing carboxylic anhydrides, ketal-, and ortho-ester functional groups were excluded because they are known to be some of the most reactive functional groups. The type of drug (acid or base) and the drug's interaction with the membrane can contribute to degradation or hydrolysis of the membrane. Acidic and basic drugs can autocatalyze degradation by changing the micro-environment pH in the polymer.29 These reasons resulted in the exclusion of hydrogels and other common control-released polymers. While employed in other pharmaceutical applications, the use of these types of polymers in in vivo Relevant (ivR) absorption applications would be problematic due to the diffusion coefficient becoming dependent on the aqueous content in the membrane, and because such polymers exhibit a time-dependent interfacial surface area. Silicone-based polymers, including poly (dimethyl siloxanes), poly-dimethyl silicones, and poly-siloxanes, are identified as suitable polymers for use in the ivR methodologies disclosed herein. An exemplary silicone-based polymer, poly(dimethyl siloxane) (PDMS), is characterized herein because it exhibits the desirable characteristics of this class of polymer, i.e., it is non-swelling, has non-interconnected porosity, is lipophilic/organophilic, and is pH stable. In addition, PDMS is easy to fabricate, is inexpensive, and is widely available.
PDMS as an in vitro biomimetic analog of the passive drug absorption process in the human gastrointestinal (GI) tract was assessed. PDMS is biomimetic because of similarities to the GI tract in small molecule transport, such as mechanism, ionization selectivity, and lipophilicity.
The disclosure provides improved in vitro methods for measuring the absorption characteristics of orally administrable compounds such as therapeutics, including biologics and small-molecule compounds. As noted above, the methods rely on the identification of a material that closely mimics the in vivo behavior of the vertebrate gastrointestinal tract based on significant similarities of the structural and functional properties of the material to the structural and functional properties of the vertebrate GI tract. That material is a silicone-based polymer, e.g., poly (dimethyl siloxane), which is shown herein to possess the structural characteristics of a stable polymer exhibiting unconnected pores establishing gut-like porosity without deteriorating in the presence of aqueous fluids. The disclosure also provides methods of producing such materials, e.g., in the form of polymeric membranes of various sizes and thicknesses, useful in assessing the absorption characteristics of compounds in a simple and cost-effective manner.
Using physiologically relevant fluids (e.g., pH, volumes, temperature, buffer, buffer capacity, surfactants), hydrodynamic conditions (e.g., shear, advection), and mass transfer rates (e.g., diffusion, permeation to simulate the absorption process) in an in vitro system can better simulate in vivo performance than current and common compendial dissolution methodologies. The disclosure provides a synthetic polymer that closely approximates the passive absorption kinetics of the human intestinal tract. More particularly, disclosed is a silicone-based polymer that meets the requirements of a robust, semipermeable, and in vivo-relevant (i.e., ivR), in vitro membrane. By measuring the drug permeability in the disclosed membrane system, its capacity to act as an ivR membrane was demonstrated for a variety of drugs that span the lipophilicity spectrum.
The disclosure is drawn to an in vitro method of measuring absorption of an orally administrable compound as a method of assessing the absorption of the compound in the vertebrate gastrointestinal tract, the method comprising: (a) contacting a silicone-based polymer with an orally administrable compound in vitro; and (b) measuring the absorption rate of the compound. In some embodiments, the polymer is a poly (dimethyl siloxane), a poly di-methyl silicone or a poly siloxane polymer. In some embodiments, the polymer is a poly (di-methyl siloxane) (PDMS) polymer. In some embodiments, the absorption measure comprises: (a) determining the aqueous initial concentration of compound before exposure to the polymer (e.g., poly (dimethyl siloxane) (PDMS)); (b) measuring the rate of appearance of compound after exposure to the polymer in a receiver compartment; and (c) using a scaled surface area of the polymer and scaled volume available for diffusion to assess the absorption of the compound in the vertebrate gastrointestinal tract. Typically, assessment of the absorption of the compound will reveal that the absorption of the compound by the polymer simulates the absorption of the compound by the vertebrate intestinal tract. Embodiments of this aspect of the disclosure are contemplated wherein the polymer (e.g., poly (dimethyl siloxane)) comprises pores having an average pore diameter of 0.4 to 0.9 nanometers, such as a pore diameter that is 0.8 to 0.9 nanometers. The pore dimensions are found over a broad temperature range (see
Other features and advantages of the disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosed subject matter, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
Through a combination of intubation studies (pressure wave motility of segments of the GI tract, gastric contents and pH), real-time magnetic resonance imaging (MRI) manometry (real-time free water flow, motility patterns), and computational fluid-dynamic simulations (CFDS) of peristaltic fluid and mass flow, the first scientifically derived criteria for orally administered dissolution testing has been achieved. With the ability to understand the distinct segmental nature of the gastrointestinal tract, it is possible to capture the most critical parameters (pH, fluid volume, shear rates, secretion rates, intercompartmental transfer rates, antero-retrograde flow rates, and the like) in each segment and in between segments and use these parameters to develop an apparatus that accurately simulates the current understanding of gastrointestinal dissolution processes. See Table 1. These measured and predicted values governed the design, simulation, evaluation, and implementation of the UTLAM devices disclosed herein. It is expected that UTLAM devices will be connected in series to form an artificial organ or organ system, such as an artificial stomach and duodenum or to form a gastrointestinal stimulator, where the design of concatenated UTLAM devices will be influenced by the average residence time of compounds in, e.g., the duodenum and/or the jejunum.
Disclosed herein are in vitro methods for measuring the absorption of a compound by a type of polymeric material that may be in the form of a membrane of various thicknesses, wherein the in vitro absorption measurements are in close agreement with the absorption characteristics of the compound in the vertebrate gastrointestinal tract, thereby providing an in vitro assessment of the in vivo absorption characteristics of a given compound in the vertebrate intestinal tract. The material is a poly-silicone polymer such as poly (dimethyl siloxane), poly (dimethyl silicone) or poly siloxane that provides a material of stable structure comprising unconnected pores establishing a porosity closely mimicking the porosity of the vertebrate GI tract, such as the human gastrointestinal tract. The measurement and assessment methods disclosed herein will accelerate efforts to identify compounds such as therapeutics (e.g., small molecule compounds and biologics such as peptides and proteins) that exhibit desirable absorption characteristics in the vertebrate gastrointestinal tract. In addition, the methods will facilitate efforts to characterize known therapeutics and allocate such compounds to administration regimens where their absorption characteristics would be useful. With respect to the devices disclosed herein, construction is aided by the use of various forms of 3D printing. The resolution of the three-dimensional printers used in the experiments disclosed herein range from 16 μm to 85 μm for the SL resin printers (Projet 3500 HD Max/Stratasys J750) and 178 μm-500 μm for the FDM ABS printer (Stratasys Dimension Elite). The ability to rationally design dissolution methodologies and equipment that are relevant to physiologic dissolution are now possible because of rapid prototyping simulated parts (vessels and impellers) from computational fluid dynamics simulations, and informed by clinical studies.
One of the main kinetic processes involved in gastrointestinal dissolution is absorption, but this process is rarely captured in dissolution testing. Poly(dimethyl siloxane) (PDMS) membranes have been demonstrated to be adequate biomimetic analogue for the passive oral absorption pathway in human beings.92 The ability to implement a biomimetic polymer membrane is highly desirable for the experimental advantages over similar organic solvent based systems. To achieve absorption rates for BCS class I, II, and III compounds that are within the expected physiological norm, PDMS membranes must be thin and have a large surface area. One method to produce highly homogenous, uniformly thick, ultra thin, large surface area membranes is to use a spin coater. Spin coaters are known for the ability to produce high quality, homogenous films at scales as low as single nanometers.47, 78, 79 PDMS membranes have been produced that were larger than 5 cm2 and approximately 100 nm thick.47 A similar method was used to generate PDMS membranes that simulate passive oral absorption in humans using a sacrificial polyvinyl alcohol (PVA) film for release instead of gelatin. PVA's solvent (deionized water) is orthogonally soluble to the PDMS solvent (hexane), which makes it a convenient choice for a water-soluble sacrificial release layer. PVA was also found to dissolve in about two hours at room temperature (about 60 nm×25 πcm2 PVA). Upon release from the silicon wafer, the PDMS can become wrinkled if a weight is used to submerge the wafer. These wrinkles relax in deionized water at room temperature over several hours uninfluenced or manually relaxed with the aid of a tweezers in seconds. To measure the thicknesses of the PVA, ellipsometric measurements sufficed, but because of the target thicknesses of the biomimetic PDMS UTLAM, ellipsometry of the bilayer film was not possible. Scanning electron microscopy, after a liquid nitrogen fracture, was used to analyze the thickness of the PDMS film that had been spun out of hexane onto a prepared PVA-coated silicon wafer. When the PDMS UTLAM was utilized for diffusion experiments, it was released from the silicon wafer in a long, wide, and shallow tray containing deionized water, which allows the PDMS to float to the surface of the water, completely unsupported by any structure other than itself. The PDMS UTLAM is mechanically strong enough to be handled, but because the UTLAMs are semi-self-adherent, it is difficult to flatten a membrane should it come into contact with itself. Therefore, PDMS UTLAMs are transferred from the release/transfer vessel into the diffusion cell using a part of the diffusion cell disclosed herein.
An exemplary polymer is poly(dimethyl siloxane) (PDMS), which was commercialized in 1943 by the Dow Corning company and was obtained for these studies as the Sylgard 184 elastomer kit.30 This kit contains two components, the polymer base and the polymer curing agent. The polymer base contains 60% dimethylsiloxane, which is dimethylvinyl terminated, 30%-40% dimethylvinylated and trimethylated silica, and 1-5% tetra(trimethylsiloxy)silane. The base material is viscous, with a ηbase=5000 cS. The curing agent contains dimethyl methylhydrogen siloxane that, through a platinum catalyst, initiates a step-wise polymerization using a hydrosilation reaction at the vinyl groups in the base material.31-32 At room temperature (RT; 20° C.) PDMS forms a transparent, colorless, elastomeric polymer, see
For orally administered drugs, dissolution methodologies should examine the drug product in an in vivo Relevant (ivR) in vitro experimental system that accurately simulates the critical parameters of the in vivo environment and kinetic processes of the human GI tract. The ivR hypothesis is that using physiologically relevant fluids (e.g., pH, volumes, temperature, buffer, buffer capacity, surfactants), hydrodynamic conditions (e.g., shear, advection), and mass transfer rates (e.g., diffusion, permeation to simulate the absorption process) in an in vitro system can better simulate in vivo performance than current and common compendial dissolution methodologies.1-4 This disclosure establishes that PDMS can be used to replicate the passive absorption kinetics of the human GI so that it can be applied to a device that meets the criteria for ivR dissolution. PDMS was characterized using a rotating membrane diffusion cell (
The data disclosed herein indicate that it is feasible to construct physically viable membranes with permeation properties adequate to simulate oral absorption for a wide variety of drug molecules, typically those considered to be high permeability (BCS Class 1 and 2).
Mudie et al. laid the experimental ground work for ivR absorption6, the goal of which is to construct an in vitro system in which the partition rate constant (kabs
Renaming the variables to the membrane-based absorption system, Sinko et al propose:
kabs
kabs
Pin vivo=the measured/predicted permeability coefficient in humans
AG-I=surface area available for absorption in the human GI
=the area of contact between the organic and aqueous phases
Vin vivo=the effective aqueous volume of dissolution within the human GI
PPDMS=the permeability coefficient of drug in PDMS
Amembrane=surface area of membrane available for transport
Vin vitro=the effective volume in the dissolution apparatus which dissolution occurs (volume of the drug-donating phase)
Where Equation (3) allows for human absorption kinetics, either measured or predicted, to be replicated in vitro by scaling the Ain vitro/Vin vitro to make kabs in vivo kabs in vitro.
Ten molecular probes were used to evaluate the transport pathways and properties used to predict human oral absorption rates. The transport pathways through PDMS (bulk/pore) are analogous to transcellular (TCDT) and paracellular (PCDT) drug transport pathways. PDMS PCDT was assessed using positronium annihilation life-time spectroscopy (PALS) and partition experiments; TCDT using diffusion and partition experiments. PALS determined that PDMS pores were uniform (D˜0.85 nm), isolated, and void volume was unaffected by drug accumulation after equilibrium partitioning. Therefore, there is no PCDT or convective flow through PDMS. A strong linear correlation exists between predicted octanol-water partition coefficients and PDMS partition coefficients (Log PPDMS=0.736×Log PO-W−0.971, R2=0.981). The pH-partition hypothesis is confirmed in PDMS using ibuprofen over pH 2-12. Diffusivity through PDMS is a function of lipophilicity and polar surface area
Varying the mass % of curing agent changed the lipophilicity and diffusivity (p<0.02), but not practically (K×D=2.23×10−5 cm2s−1 versus 2.60×10−5 cm2s−1), and does affect elastic modulus (3.2%=0.3 MPa to 25%=3.2 MPa).
As the experiments described in the following examples show, PDMS does not have interconnected porosity as measured by beam PALS. No drug was quantified in the PALS void volume (as measured by a change in lifetime), nor was any change in mass of the membrane measured when soaked in pure water. The effective diffusive flow of drug appears to transport within the densely packed-domains in the polymer network. PDMS is pH stable, as shown in the Log DPDMS experiments for ibuprofen over a pH range of 2.0-12.0. The K×D product successfully predicted ibuprofen permeability over a 500 μm difference in thickness of PDMS membrane. The use of the K×D product (Diffusivity) to predict PDMS-drug permeability is valid at any thickness at which PDMS membranes are currently produced. Large area (>5 cm2), ultra-thin (1 μm) membrane fabrication is possible and is an exemplary type of geometry useful for characterizing absorption rates of pharmaceuticals that are comparable to human GI absorption rates.47
PDMS, however, can be fabricated to have a 3 dimensional surface area, which is capable of accommodating even larger surface area to volume ratios without sacrificing the physiologically relevant volumes required by the ivR methodology.48 The pure diffusion coefficients in PDMS are significantly slower (about 102) than those in water, but the true diffusion coefficients for PDMS must account for the partitioning behavior into PDMS and the polar surface area of the solute molecule
Knowing the thickness-independent permeability (i.e., the diffusivity) (K×D) behavior allows for ivR modeling of the absorption kinetics using an in vitro test. PALS characterization at room and physiologic temperature of the PDMS membrane shows that the physical structure of the membrane is not significantly affected by any processing or experimental parameters that a membrane would be exposed to in ivR dissolution and absorption experiments. Dissolving the PDMS components in hexane produces softer (lower Young's modulus) membranes than PDMS that is fabricated with no solvent. The Young's modulus can be modulated approximately between 0.3 MPa and 2.3 MPa by changing the amount of curing agent added to the base material during fabrication. Even though high temperature curing is limited to about 60° C. in hexane during polymerization (TBoiling hexane=about 70° C.), exposing the polymer solution to temperatures above RT can result in up to a 0.5 MPa increase in the Young's modulus. While these mechanical differences may be significant in fabrication of the in vitro absorption component, the mechanical differences do not affect the drug permeation performance significantly.
The following examples are included to demonstrate embodiments of the disclosed subject matter. Those of skill in the art will, in light of the present disclosure, appreciate that changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Materials and Methods
Materials
Materials used in the experiments disclosed herein include an Agilent 1100 High Performance Liquid Chromotography (HPLC), an Eclipse Plus C 18 Column (3.5 μm×4.6 μm×150 mm), Acetonitrile (EMD Millipore, HPLC grade), deionized water (Milli-Q purified), trifluoroacetic acid (Fisher Scientific, Optima Grade), triethylamine (Fisher Scientific, Optima Grade), methanol (Fisher Scientific, HPLC grade), Ibuprofen (Albemarle Lot No. 2050-0032F), Progesterone (Sigma Aldrich, CAS 57-83-0), Benzoic Acid (Fisher Scientific CAS 65-85-0), Metoprolol Tartrate (Sigma Aldrich, CAS 56392-17-7), Caffeine (Sigma Aldrich, CAS 58-08-2), Atenolol (Sigma Aldrich, CAS 29122-68-7), Ketoprofen (TCI Tokyo, Japan, CAS 22071-15-4), Hydrochloric acid buffer pH 2.0 (USP guideline), Acetate Buffer 5.0 (USP guideline), Phosphate buffer pH 6.5 (USP guideline), Sodium Hydroxide Buffer pH 12 (NaOH+KCl), Poly(dimethyl siloxane), (Sylgard 184 elastomer kit, Dow Corning), Hexane (Fisher, reagent grade), Instron uniaxial press, Fisher-Scientific accuSpin micro17 Centrifuge, Wenesco Inc., and HP1212-D cure plates with glass covers.
Generic Membrane Casting Procedure
Sylgard 184 base was weighed in a glass container and moved to a vacuum chamber where a −750 mbar vacuum was pulled for 25 minutes to remove gas. Separately, an appropriate amount of Sylgard 184 curing agent was weighed on an analytical balance. A 1:1 ratio (total mass:volume) of hexane was measured in a graduated cylinder. The hexane was used to dissolve the catalyst component and then was added to the container containing the base polymer. Manual mixing was done until the base was completely dissolved (easily observed by a change in the index of refraction). An appropriate volume of solution was drop cast into polyethylene weigh boats using a pipette. The PDMS solution cured at the desired temperature and time with solvent evaporating into a lab hood.
Strain Rate Effect in PDMS Mechanical Samples
Three strain rates were tested: 1, 0.1, 0.01 mm s−1 strain rates in uniaxial compression.
PDMS Elastic Modulus and Cure Temperature Sample Preparations
PDMS cylinders were prepared at 3 mm thickness and 6 mm diameter. For the elastic modulus samples, the ratio of the polymer base to curing agent was varied (3, 7, 10, 15, 20, 30):1. For cure temperature samples, the base to curing agent ratio was 10:1. Each sample was cured at a different temperature for 9 days. Samples cured above 40° C. were allowed to cure at RT until the hexane was evaporated (<1 day) until the film was semi-solid, and then the remainder of the nine-day cure was completed. This prevented boiling hexane from forming bubbles within the sample.
Effect of Curing Temperature on Elastic Modulus
The curing temperatures studied were 20° C., 40° C., and 60° C. Five samples were prepared using a 6 mm diameter surgical punch (L/D (length/diameter) about 0.5). The modulus was calculated from the linear slope on the compression stress-strain curve at a strain rate of 0.01 mm s−1.
Elastic Modulus Versus Composition Ratio
Five samples were prepared using a 6 mm diameter surgical punch (L/D about 0.5). The modulus was calculated from the linear slope on the compression stress-strain curve.
HPLC Methods
For acidic drugs, 0.085% v/v trifluoroacetic acid was used in both water and acetonitrile. For basic drugs, 0.1% v/v triethylamine was used in both water and acetonitrile. See Table 2 for details. For HPLC Standard Curves, limit of detection (LOD) and limit of quantitation (LOQ), a five-point standard curve (two-fold dilution per step) was created for each drug in the buffer used for the experiment. See Example 11 for LOD and LOQ.
Partition Coefficient Measurements
For each drug, five membranes were prepared. Each membrane was prepared with 10 parts base to 1 part curing agent and cured at 20° C. for at least 72 hours. Once cured, the membranes were sectioned using a template and razor blade. The dimensions of the perimeter and thickness were measured using a caliper to determine the volume of membrane. After determining membrane density, subsequent volume measurements were made using the density relationship. Stock solution was distributed to 5 sample vials with 1 membrane-free vial to serve as a control. The time zero point was measured from the blank vial and time points and 1 mL samples were taken at 12 and 24 hours. The collected samples were assayed in duplicate by HPLC.
Distribution Partition Coefficient Measurements
This experiment was conducted in a manner similar to the experiment described in “Partition Coefficient Measurements”, above. The model drug, ibuprofen, was exposed to RT 13 mM HCl at pH 2.00, 50 mM acetate at pH 5.00, 50 mM phosphate at pH 6.50, and 39 mM NaOH at pH 12.00.
Non-Ionized Thermodynamic Solubility Determination at 37° C.
Five 1.5 mL centrifuge tubes were labeled and prepared with 1 mL of the appropriate non-ionizing buffer (see Table 5). Solid drug in powder form was added to each individual vial and then vortexed. The addition of drug and vortex mixing was repeated until there was visible undissolved drug powder present. The centrifuge tubes were then put into a hot box where they remained at an aqueous temperature of 37° C. (set point 43° C.). The internal temperature was determined by measuring the temperature of a “blank” tube in the rack. Once the internal temperature reached 37° C., the tubes were held at 37° C. for 48 hours. The tubes were removed from the hot box and centrifuged at 17,000G for 3 hours. The tubes cooled below 37° C. during the centrifugation so the tubes were re-inserted into the hot box and allowed to reach 37° C. over the course of 1 hour. Supernatant was then extracted directly from the tube in the hot box without disturbing the pellet. The supernatant was appropriately diluted for HPLC analysis. The limit of quantification and limit of detection were calculated to ensure the validity of the dilution scheme and use of the standard curves (Equation S49-S51, see Example 11).
Rotating Membrane Diffusion Cell Experiments
Thickness of the sample membrane was measured using a caliper at the center of the membrane and then at four additional points within the circumference of the membrane in the region which was exposed to the drug-saturated aqueous phase. The initial mass of the membrane was weighed prior to drug exposure. A recirculating bath warmed the beaker containing the donor aqueous suspension (0.9 mM sodium dodecyl sulfate) of drug to 37° C. A rotating membrane diffusion cell was utilized, as shown in
Positron Annihilation Lifetime Spectroscopy (PALS)
Radioactive 22Na deposited and sealed in a thin kapton film was used as the positron source. This source was placed between two 41 mm×41 mm×1.3 mm sheets of PDMS. This configuration was found to effectively stop the majority of positrons (excluding the 8% stopped in the kapton film) in the sample PDMS. Lifetime measurements were initially taken in both air and vacuum. There was a lower event acquisition rate in the vacuum setup due to the increased distance necessary to fit the vacuum chamber between the detectors. With the ability to mathematically compensate for the pick-off annihilation, the characterization of the free volume voids was primarily run in air at and above 20° C., while the sub 20° C. was run under vacuum.
The lifetime of the particle called positronium (Ps) is most important in analyzing the pore properties of PDMS. Ps is analogous to a hydrogen atom, but with no nucleus and a positron (anti-matter electron) that orbits with an electron in a triplet state energy configuration. Since Ps can trap in open volume voids, this positronium is directly sensitive to the pore size in which it resides. The other two short lifetimes are related to singlet Ps and positrons that annihilate with an electron without forming Ps and will not be considered further. All fitting of the PALS spectra were done using a customized version of the Posfit program.34
PDMS membranes were not returned into hexane to remove any non-crosslinked material nor was the cured membrane put into vacuum to attempt to remove any latent hexane, as proposed by others.31 However, high vacuum was used during a PALS measurement to see if there was any change in the lifetime as any hexane was “extracted” from the membrane. There was no irreversible change in positronium lifetime when the vacuum and air samples (after compensating for known air effects on the positronium life) were compared.
PALS Thermal Expansion Series
A PDMS membrane was sectioned for PALS analysis. The RT positronium lifetime was measured. The same sample was then heated to a target temperature and held at that temperature until sufficient data was gathered for a positronium measurement at the target temperature. The same sample was then brought back to RT, where the positronium lifetime was measured again. This cycle was repeated until all the temperature values were measured. For measurements at cryogenic temperatures, the sample was cycled between RT & −230° C. with data taken at selected temperatures in between.
Error Bars
All error bars are reported as the standard error of the mean unless n<5, in which no summary statistic is given (mean, SEM).
Paracellular Type (Pore) Drug Transport in PDMS
Pore transport in PDMS was measured by PALS to evaluate whether pores play a significant role in the overall conduction of drug molecules from donor to receiver phases. Positron Annihilation Spectroscopy has been used for 40-50 years to characterize single vacancies and vacancy clusters. Positronium Annihilation Lifetime spectroscopy (PsALS or PALS), over the same time course, has been used to measure sub-nanometer and intermolecular voids in polymers, making this technique a robust method for probing the porous part of the PDMS polymer network. When a positron is injected into materials, it will eventually annihilate with an electron with the complete conversion of the pair's combined mass, m, into high-energy photons with total energy E=mc2. There are two types of particles that are examined during PALS analysis, i.e., free positron annihilation (with electrons in the target material) and Positronium (Ps) annihilation. Both positrons and Ps seek out and localize in vacancies/voids in metals and insulators. Simple coulomb attraction forces positrons into electron-decorated vacancies in metals, whereas in insulators the reduced dielectric interaction in a void energetically favors trapping neutral Ps in low-density regions. Ps has two states, singlet (para-) and triplet (ortho-), depending on the relative spin state of the positron and electron. The self-annihilation lifetime of para-Ps is short, i.e., 125 ps, and this rapid singlet annihilation occurs with the emission of two back-to-back gamma rays of 511 keV. However, ortho-Ps (o-Ps) in vacuum is required to annihilate into at least three photons to conserve angular momentum, and this slower, triplet process has a long, characteristic lifetime of 142 ns. Lifetime spectroscopy can easily distinguish this long-lived triplet state of Ps; therefore, o-Ps plays the key role in probing porous materials.34-35
To determine whether the voids in PDMS form an interconnected porous network or are isolated voids, a “beam PALS” spectrometer, in which a low energy focused beam of positrons is used to shallowly implant positrons and form Ps close to the PDMS sample surface, was used to resolve pore connectivity.34 Ps can diffuse in an interconnected porous network and escape into vacuum producing a readily distinguishable approximately 142 ns lifetime component. Using beam energies (mean positron implant depths) of 1.2 keV (40 nm), 3.2 keV (180 nm) and 4.2 keV (280 nm), the telltale 140 ns vacuum component was not found, indicating no Ps diffusion. It is conclusive that the voids of PDMS are isolated. Positronium lifetimes were converted into a spherical pore diameter over the range of interest using the Tao-Eldrup model (assuming a simple spherical pore model).34, 36, 37 In
To see whether drug accumulated in the pores of the membrane, PALS was used to measure a PDMS membrane before and after equilibrium partitioning with ibuprofen (
Transcellular Type (Bulk) Drug Transport in PDMS
Partitioning of Molecular Probes into PDMS
Bulk transport properties of PDMS were measured to evaluate PDMS fitness for the mimicking human oral absorption. The partition coefficient was calculated according to Equation 4.
K=Partition coefficient
Cmem*=Equilibrium concentration of drug in membrane
Caq*=Equilibrium concentration of drug in the aqueous media
The first study conducted was to determine if process variations in fabrication would lead to differences in the equilibrium partition of the model drug, ibuprofen (
The Log D relationship is given in Equation 5 for a monoprotic acid (model 1) (see Supporting Information for derivation).39 In Table 3 the Log D was also calculated using the Wagner model which accounts for ionized drug partitioning (Equation 6, model 2) and the pKa of ibuprofen was back calculated to confirm the validity of the fit for both models (for model 2, Equation 8 was used to transform the shifted pKa from Equation 6 back to the true pKa).40
log D=the log10 of the distribution coefficient
log K=the log10 of the intrinsic non-ionized partition coefficient
Pu=the intrinsic non-ionized partition coefficient
Pi=the intrinsic ionized partition coefficient
fE=fraction extracted by the membrane
fu=fraction of non-ionized drug in solution
PHfrom fit=input pKa in the Log D equation
pKaactual=the un-shifted pKa from fit, the true pKa of the molecule
Iexperimentally determined at pH 11.81
IIusing the experimental data at pH 11.81 and fu~0, Pi was calculated to fit equation 6
Permeability is a function of the partition coefficient (Equation 9).
K=partition coefficient (unitless)
D=diffusion coefficient [cm2 s−1]
h=thickness of the membrane [cm]
Therefore, it was important to see if the partition coefficient could be predicted for any drug in PDMS. A simple correlation was created between the predicted partition coefficient of drugs in the octanol-water system (the standard reference system for partitioning) and the PDMS system.
Diffusion of Molecular Probes through PDMS Membranes
Membrane permeability was calculated from the linear slope of the concentration versus time curve for each experiment. This pseudo-steady-state linear region was determined by calculating the linear regression coefficient of the slope and optimizing the range to achieve a R2 as close to 1 as possible. The slope of this line, when multiplied with the receiver volume, gave the mass transfer coefficient (Equation 10).
{dot over (m)}=m
pseudo-steady
×V
receiver (10)
{dot over (m)}=mass transfer coefficient [μg s−1]
mpseudo-steady=slope of concentration versus time plot in the pseudo-steady state region [μg mL-1 s−1]
Vreceiver=volume of the drug receiving phase [mL]
The effective permeability was calculated using permeability layer theory (Equation 11) and, as a check, diffusion coefficient was calculated using Crank's uniform initial distribution and surface concentration different for diffusion in a plane sheet (Equation 9, 12-13).42 Permeability layer theory considers every layer in the diffusion system (solid and liquid interfaces), while Crank's approach examines diffusion only within the membrane itself.
Peff=permeability of molecule through the membrane & aqueous boundaries [cm s−1]
Amembrane=area of membrane available to transport [cm2]
S=the solubility of the molecule [μg mL−1]
{dot over (m)}=mass transfer coefficient [μg s−1]
QT=mass per unit Area [μg cm−2] (from the slope of the receiver concentration profile multiplied by Vaq/Amembrane
D=the diffusion coefficient of the molecule through the membrane [cm2 s−1]
C2=concentration of the molecule at the inner surface of the membrane (Caq total*K) [μg cm−3]
t=time [s]
and the membrane permeability from permeability layer theory is Equation 14 and Crank's membrane permeability is Equation 15 (both derived in Example 11).
haq=Levich boundary layer thickness [cm]
Daq=drug's aqueous diffusion coefficient [cm2 s−1]
mdC/dt=slope of the concentration versus time curve in the receiver compartment [μg cm−3 s−1]
PPDMS=the permeability of the PDMS membrane [cm s−1]
The difference in the permeability when the aqueous boundary permeation is assumed to be of negligible importance, and when the contribution is accounted for, was examined. For drugs with a Log KPDMS<1.5, the permeability difference is <10% and for higher partitioning drugs (1.5<Log KPDMS<2.1), results were found to have up to a 25% difference. The lag time (time to steady-state transport) was calculated by solving the pseudo-steady-state linear regression equation for Y=0, where Y=concentration and X=time. This lag time can be predicted by Equation 16.4243
tpseudo-s.s.=time to pseudo steady state transport [s]
hmembrane=membrane thickness [cm]
D=diffusion coefficient of the molecule through the membrane [cm2 s−1]
It was hypothesized that modulating the elastic modulus of PDMS could modulate the drug permeability.
The K×D product was measured for the same set of drugs for which the partition coefficient was measured (
The permeation of model drug, ibuprofen, behaved according to Equation 12, which demonstrates that permeability can be predicted over a wide range membrane thicknesses, as seen in
In each permeation experiment, the donor-phase-containing drug was at a non-ionizing pH if the drug was ionizable and the receiver phase was at a completely ionizing (>99%) pH. The values of the non-ionized thermodynamic solubility at 37° C. that were used in the permeation calculations were reported in Table 5. Progesterone solubility was determined twice, the second time as an analytical check. This check shows that the method used to determine solubility was unaffected by the amount of dilution used to obtain the solubility (diluted 3.5× and 10.5×, respectively).
Table 6 shows the experimentally measured permeability, diffusion coefficient, and lag time values for each drug, along with experimental conditions used to generate those values. These permeability measurements are in the intrinsic ionization state (completely non-ionized), but it is expected that ivR testing will occur at pH values where many drugs will have some fraction of ionized molecules. The Wagner models and Winne models for pH-dependent absorption show that significant absorption occurs in vivo even under pH conditions were there is a large fraction of ionized drug.40, 44 From a characterization standpoint, however, the use of the relationship between Log KPDMS and PDMS permeability is valid for any Log DPDMS. An experimental compound may be partially ionized under physiologic pH conditions, but if the Log DPDMS can be measured or predicted, so can the correct PDMS permeability. PDMS reflects the in vivo situation, where ionized drug is present in the absorption pathway and absorption rate will be a function of pH.
6.02 × 10−10
Mechanical Properties and Microstructural Analysis
The stiffness of the polymer (crosslinking) was expected to govern the transport of drug molecules. Additionally, modulating the stiffness of the network was expected to provide a secondary method of modulating the permeability of PDMS membranes. It was also necessary to understand the material's mechanical properties for fabrication of an in vitro absorption material, such as a membrane, e.g., an ultra-thin membrane. Before any mechanical testing was conducted, a study of the strain rate effect was conducted. The initial studies were performed in tension, but the material was found to be too soft for use in the testing cell. Therefore, all mechanical measurements disclosed herein were completed in compression.
To evaluate RT pore stability, the PDMS void structure was quantified via PALs at 3, 103, and 193 days after casting (
Additional Materials
The materials and equipment used in the following experiments included Agilent 1100 high-performance liquid chromatography (HPLC), Extend C 18 column (3.5 μm×4.6 μm×150 mm), acetonitrile (EMD Millipore, HPLC grade), deionized water (Milli-Q purified), trifluoroacetic acid (Fisher Scientific, Optima grade), methanol (Fisher Scientific, HPLC grade), Crospovidone (UM2012-085 Lot # K-H09074), Croscarmellose Sodium Non-GMP (Material #10127157), HPMC-AS, LF (UM 2012-091 Lot #007), Microcrystalline Cellulose (PH102 UM2012-004), Mannitol, NF (Glaxo-Smith Klein UM2009-010), Dibasic Calcium Phosphate (JRS Lot #2059X UM2011-049), Lactose Monohydrate-310-NF (UM2001-018), Magnesium Stearate (UM2009-013), Sodium Dodecyl Sulfate for Electrophoresis 99% (Sigma-Aldrich), Citric Acid Anhydrous (Fisher A940-500), Ibuprofen (Albemarle Lot No. 2050-0032F), hydrochloric acid buffer pH 2.0 (USP guideline), phosphate buffer pH 6.5 (USP guideline), sodium hydroxide buffer pH 12 (NaOH+KCl), 1-Octanol, 99% pure (Acros Organics Lot # A0358670 CAS 111-87-5), polyvinylalcohol MW=25 K, 88% hydrolyzed (Poly Sciences, Inc. #02975 Lot #652279), poly(dimethylsiloxane), Sylgard 184 elastomer kit (Dow Corning), hexane (Fisher, reagent grade), Model WS-650MZ-23NPPB spin processor (Laurell Technologies), vacuum drying oven (Yamato ADP300C), 100 mm Test Grade Silicon Wafers with native silicon oxide layer (Encompass Distribution Services), Stratasys J750 printer build size: 490×390×200 mm, VEROCLEAR RGD810 (material for J750), Dimension Elite 3-D printer build size 203×203 mm, and a Tescan MIRA3 FEG Scanning Electron Microscope.
Design, Simulation, & Evaluation of the UTLAM Diffusion Cell
Design—Dissolution Bowl
Historically, dissolution apparatuses have suffered from fluid and particle distribution heterogeneity. The standard round-bottom USP 2 vessel has been modeled in the past and has been shown to have large volumes of low or no velocity and shear, coupled with areas of intense velocity & shear. The advantage of the round-bottom vessel is that the dosage form can reproducibly sit in the same spot in the vessel and experience consistent hydrodynamic forces across many studies, however, it has been reported that dosage forms do not sit at the apex position. The weakness of this built-in feature is that the USP 2 round-bottom vessel suffers from a large dead volume immediately below the impeller, and this is where the tablet sits.80-84 During disintegration and dissolution of the solid dosage form, disintegrated particles also accumulate in this dead volume, known as “coning”. Decreasing the dead volume and increasing the homogeneity of the fluid flow to reduce the “coning” problem became the primary concerns when developing the dissolution component of the UTLAM diffusion cell. For the dissolution vessel part of the UTLAM, flat-bottom, round-bottom, and cone-shaped vessel geometries were considered (
Impeller
The UTLAM dissolution bowl and impeller were designed to balance the need for analytical robustness while maintaining physiologic hydrodynamic conditions. One unique aspect of this approach was to consider the two components pieces (vessel and impeller) together when designing, rather than considering each component as a separate entity, as has been the approach in the past. Since the UTLAM was used to disintegrate and dissolve intact drug products, the impeller's main function was to keep drug particles suspended homogeneously and to keep fluid homogeneously distributed within the dissolution vessel while maintaining lower bulk fluid shear rates. Two types of impellers were investigated using the COMSOL, the hydrofoil and the anchor, to see which impeller produced low shear and sufficient velocity profiles in the dissolution bowl while maintaining particle suspension and a homogeneous distribution of particles.
A comparison of the flow fields between two candidate impeller configurations and a traditional USP 2 paddle is shown in
Parameter Study of the Impeller
The main criterion for the impeller is to generate physiologic hydrodynamic conditions. From the CFD simulations of the human gastrointestinal tract it is known that the Shear-Peclet Number from peristalsis is about 10-25. The in vivo environment has an order of magnitude lower shear-peclet range than the USP 2 paddle apparatus where Shear-Peclet number is at least 150.63-65,88,89 The Shear-Peclet number is dependent on particle size, so the intent of this design was to achieve the minimum possible shear imparted via the impeller to give flexibility in accommodating a wider range of particle sizes.
Where: S*=Shear-Peclet number, S=shear rate, Rparticle=radius of the dissolving particle, Dm=diffusion coefficient of the dissolving solid.
The ratio of the impeller diameter to the vessel diameter was also investigated to see their effect on bulk shear rate, fluid velocity, and the axial/radial mixing time scale (
Based on the results shown in
Absorption Compartment
The absorption compartment resulted from many design decisions concerning the impeller and the dissolution vessel. For the UTLAM diffusion cell absorption chamber, a simple planar membrane at the bottom of the dissolution bowl was used. This geometry and orientation were simple to integrate into the design decisions discussed above. However, the disclosure contemplates configurations that take advantage of the side walls, or non-2D planar configurations. A mesh support was built at the interface between the dissolution vessel and the absorption chamber to support the membrane from below. Because the mesh support is only located underneath the membrane, the full surface area is accessible for transport from the dissolving drug particles. To select the best design for the dissolution bowl and the membrane surface area, the dissolution volume and surface area/dissolution volume were plotted against the fill height of the cylinder created by the aqueous fluid.
Prototype Fabrication and Evaluation
Fabrication of Ultra Thin Large Area PDMS Membranes
Prior to the spin casting technique, PDMS membranes produced by drop casting were only as thin as about 150 micron due to the propensity for the PDMS to self-adhere and tear under the applied stresses during the separation process. Another method of fabrication was required to achieve thicknesses below 150 micron while maintaining uniform thickness, and so the spin coating process was chosen. Even though PDMS is not a delicate material, at the cross sectional length scale targeted for fabrication (1-60 μm, E1 μm, =8 MPa, E60 μm=2 MPa)47, it would be time consuming to remove such a high aspect ratio structure from its casting substrate as the shear modulus appeared to be much lower than the elastic modulus, rendering the membrane susceptible to mechanical failure under shear stresses. Therefore, an accelerated removal process was implemented using a water-soluble sacrificial layer composed of PVA, which also removed any significant mechanical force needed to separate the membrane from the casting substrate. A nanoscopic layer of PVA was deposited from a 3% w/w PVA in deionized water solution at 5000 rpm onto a silicon wafer that had been rinsed with deionized water and dried thoroughly prior to casting. The solution fully coated the stationary silicon wafer but was not allowed to sit stationary for more than a few seconds to avoid adherence of the polymer to the surface, which could lead to heterogeneity. The ellipsometer was used to characterize the thickness of the PVA layer on the silicon. Twenty wafers were measured at five consistent points (approximately 5, 33, 50, 67, and 95% of the distance along the major diameter).
Ellipsometry proved to be a fast and convenient tool to measure the PVA films, but the tool cannot reliably measure films thicker than about 3 microns. Therefore, another technique was required to measure the thickness of PDMS. One technique that was investigated was atomic force microscopy (AFM) in the soft tapping configuration (
Once the preparation method of freeze-fracturing the wafer composite was confirmed not to influence the sample, samples were re-processed by forming new edges on the original AFM samples, which were then examined with a scanning electron microscope (SEM). The SEM results demonstrated that the liquid nitrogen freeze-fracture technique prevented any unintentional plastic deformation of the sample edge and allowed for a clean brittle fracture to propagate from the silicon wafer through the PDMS layer. The results of the SEM study showed similar trends in thickness change with changing solution concentrations and rotational speeds as reported in the literature for spin coating polymer solutions. This study led to two conclusions. The first was that samples would have to be prepared under plastic conditions to prevent modification of the PDMS membrane during preparation. The second conclusion was that even though the wafer composite was vacuum-annealed for 24 hours at 65° C., residual compressive stresses remain in the PDMS from casting, causing significant diameter reduction in the membrane.
Plackett-Burman DOE for Blended Uncompacted Solid Oral Dosage Forms Containing Standard Excipients at Commonly Used Levels
The UTLAM diffusion cell was evaluated for solvent compatibility and partitioning affinity so that the chemical performance of the SL printed VeroClear material could be established. It was established that the VeroClear material had significant partitioning ability and that the device could withstand exposure to aqueous buffers and cleaning solvents (e.g., methanol) (
Experiments were designed to assess whether absorption kinetics are important for API with significant lipophilicity and significant differences in in vitro parameters that could be better measured by dissolution systems with absorption components. It was important to obtain these experimental results because the UTLAM method of incorporating absorption is easier, more cost effective (no need for filtering receiver phase samples, could be compatible with UV-dip probes which provide more data, real-time, and reduce HPLC throughput demands), more environmentally friendly, and more pleasant to work with as compared to the biphasic aqueous-organic solvent test. The experimental design incorporated 11 common ubiquitous excipients in solid oral dosage forms, with the typical levels of each excipient identified. The marker was ibuprofen and ibuprofen's dissolution rate, area under the curve (AUC), and absorption rate (where applicable) were measured for each formulation in a standard USP 2 900 mL dissolution test, a 200 mL/200 mL aqueous/1-octanol Biphasic dissolution test in USP 2 vessel, and the 130 mL donor/100 mL receiver UTLAM dissolution test in 50 mM phosphate buffer, pH 6.5. This design allowed for a Plackett-Burman 11 factor in 12 runs analysis with the addition of a 13th run to serve as a negative control (drug only, no excipients). Plackett-Burman partial factorial arrays are efficient experimental designs for screening in which main interactions between factors can be studied rapidly; however, the second order and higher interactions are confounded.90-92 In such cases, a different experimental design must be used once the main factors of interest are identified and further investigation of the higher order interactions becomes necessary.
The excipients chosen from this study represent most major excipient functions in modern solid oral dosage forms at typical compositional levels (
To conduct the experiment, 5.4 L of phosphate buffer was initially degassed and then heated to 37° C. in the USP 2 six-station apparatus. During the heating process, the motor was engaged, allowing for the fluid to be stirred at 50 rpm for no less than 30 minutes prior to dosing the formulations. Formulations were weighed on an analytical balance prior to dosing and were administered through ports in lids on the USP 2 bowl. The experiments ran for one hour and at the end of the experiment, 100 mL of acetonitrile was added to solubilize any undissolved ibuprofen. The acetonitrile/phosphate was then allowed to run for an additional hour, at which time the mass balance sample was taken. Two mL samples were drawn and media was not replaced for the USP 2 monophasic experiments. One mL of sample was discarded through a 0.45 μm PVDF syringe filter and then one mL of sample was captured and diluted 1:1 in acetonitrile for HPLC analysis.
The HPLC data was converted from peak area to concentration with a standard curve and then the time course data was input to a MATLAB program that fit the data with a spline function. This allowed for better estimations of Cmax and tmax when the Cmax and tmax did not fall within the first 10 minutes, as well as allowing for rapid calculation of the AUC0-60 min via a numerical trapezoidal method. This program was applied to the data of all three apparatuses and a comparison of the manual and MATLAB method can be found in
USP 2 200 mL/200 mL Biphasic 1-Octanol/Water Dissolution Experiment
In preparation for the dissolution experiment, 200 mL of phosphate buffer was degassed and heated in a single, water jacketed USP 2 bowl. A custom lid cover was created to house the sampling ports for the aqueous and organic phases, as well as house a large diameter tube to dose the solids directly to the aqueous phase after the organic phase had been poured on top. The advantage of this approach was that the two phases equilibrated prior to the dosing of the solid, as opposed to having to rapidly fill the organic phase immediately post-dose. The tip of the dosing tube was far enough under the aqueous surface that no organic could enter the tube (even under the pressure applied by the 1-octanol) and the tip was close enough to the top edge of the USP 2 paddle that a large shear could pull powder down into the vessel without any experimenter assistance. The dosing tube was constructed from two 10 mL pipette tips, which were wide enough to prevent any “rat holing” or other powder-flow concerns. The aqueous media was gently poured down the sides of the dosing tube to replenish media removed from sampling to catch any solid that may have stuck to the tube during initial dosing, and the 1-octanol was carefully injected through the organic sampling cannula to avoid disturbing the organic-water interface with bubbles. Two milliliter samples were drawn from each phase and filtered with a 0.45 μm PVDF syringe filter. The respective media were replaced in each phase post-sampling. The dissolution and partitioning profiles are shown for each formulation in
The biphasic device had significantly varied performance when compared to the counterpart USP 2 experiments (
Ultra Thin, Large Area Membrane (UTLAM) Conventional Dissolution Experiments
Experiments using the VeroClear SL printed prototype UTLAM diffusion cell used the standard 162 mg dose mass of ibuprofen. The parts were soaked in deionized water (milliQ), then vigorously scraped to remove residual support material. To measure the partition coefficient of the VeroClear resin, 10 mm diameter spheres were printed, cleaned, and then exposed to 19.5 mL of 400 μg mL−1 solution of ibuprofen in a 50 mM phosphate buffer at pH 6.5 for 48 hours. The dry mass and diameter of the spheres were recorded prior to the beginning of the partitioning study. The results of the equilibrium partitioning experiments are presented in
According to the SEM freeze-fracture-determined thickness based on the terminal rotational speed and mass fraction of PDMS, the membranes tested in the following experiments were 57 microns thick. 130 mL of 50 mM phosphate buffer pH 6.5 was degas sed and used as the donor phase, while 100 mL of 50 mM phosphate buffer pH 8.0 was degas sed and used as the receiver phase. The rationale behind this is that PDMS has poor ability to transport ions (a significant pH-partition relationship) and the more ionized the drug is, the less driving force the drug will provide for reverse transport out of the receiver phase. However, with this set of pHs, the pH-distributed partition coefficient (referred to as Log D) of ibuprofen in both compartments will not have a large difference, leading to a nearly equivalent permeability on both sides. Ultimately, this leads to a significant transport rate of ibuprofen returning to the donor compartment. Even with this bi-directional flux, the net flux yields an absorption rate for ibuprofen that is within the same order of magnitude of ibuprofen absorption in human beings. It is understood that the measured absorption rate is increasing due the additional partitioning kinetics introduced by the resin that forms the UTLAM device.
The PDMS UTLAM is floated in deionized water and in about two hours the PVA is dissolved enough to tease the PDMS UTLAM to the surface of the water. The membrane is then moved across the water surface to the support mesh and the water is drained from the vessel allowing the PDMS UTLAM to settle onto the mesh support structure without handling the UTLAM or the support is brought beneath the UTLAM and positioned with tweezers or other handling device, then lifted from the water. This support structure screws into the central hub of the UTLAM diffusion cell and then the dissolution bowl and absorption chamber can be assembled. The receiver phase is filled first, with the aid of an accessory design to compress the membrane during filling to prevent mechanical failure. Then, the receiver phase is cleared of bubbles and the donor phase is poured into the dissolution bowl. The hydrofoil is rotated at 50 rpm to be consistent with the impeller rotational speeds in the monophasic and biphasic dissolution experiments. Equivalent stirring rates could be calculated for the USP 2 paddle using CFD measurements made in COMSOL. The UTLAM diffusion cell was then placed in a water bath with immersion heater (sous vide heater) to adjust the aqueous phase temperature to 37° C.
Re-use of membranes was also examined. Experiment 1 provided a control in assessing the behavior of virgin PDMS UTLAM and the expected j-shaped curve (indicating a small lag time) was observed. Experiments 2 and 3 were conducted using the same experimental conditions except for the membrane, which was the membrane that was used in experiment 1, but washed with deionized water and methanol in between experiments. The permeability measured from the pseudo-steady state region decreased with re-use, but the initial concentration decreased as well (
The permeability of ibuprofen in the UTLAM was consistent with predictions in
Simulations of ibuprofen dissolution in the UTLAM were run in COMSOL and in MATLAB to confirm that all the experimental data and the mechanistic models were consistent, in addition to being able to apply the hydrodynamic parameters measured via CFD. See
The simulations show that there is a mismatch between the experiment and the simulation when unidirectional flux is assumed from donor to receiver phase. After bi-directional flux was added to the code and the predicted mass at the test end point was more accurate (there were still solid particles at the end point), but simulation as a whole were still very inaccurate (see
The data disclosed herein establish that, not only does the resin partition significant quantities of ibuprofen, but that the process water used to warm the device was acting like a drug sink. The USP 2 vessel was modeled in CAD and COMSOL and the MATLAB simulation of the dissolution experiment was conducted for both the compendial vessel and 3D printed vessel (
Ultra Thin, Large Area Membrane (UTLAM) In Vivo Relevant Dissolution Experiments
Once the UTLAM is fabricated in a partition resistant material, experiments with pH 4.95 and 5.5 in low buffer capacity phosphate buffer are performed to more closely mimic the pH conditions in the duodenum and jejunum. The impeller speed is reduced to reduce the bulk shear rate so that the Shear Peclet and Shear Reynolds numbers are more consistent with in vivo values. With the decrease in pH, it is expected that the absorption rate will increase significantly. This would indicate that the UTLAM device will still perform within expectations and prove useful as in in vitro model of in vivo drug absorption behavior.
Derivation of Log D formula for monoprotic acid
Write the ionization reaction and define the reaction rate constant.
The total amount of drug in the mass balance is the summation of all the forms of the drug present in the acid-base reaction.
Using the rate constant to solve for the ionized form of the drug, establish the total amount of drug in the system as a function of the non-ionized form.
The partition coefficient is defined as the ratio of non-ionized drug in the non-aqueous phase and the non-ionized drug in the aqueous phase.
The distribution coefficient is defined as the ratio non-ionized drug in the non-aqueous phase to the sum of the ionized and non-ionized form of the drug in the aqueous.
Rearranging the partition coefficient equation:
Substitute all the values into the distribution coefficient formula.
Factor out the RT term and divide it out
Divide out the (ka/[H]+1) term
We now know the distribution partition coefficient as a function of the hydrogen ion concentration. To make this more useful we convert ka/[H] into pH and pKa.
Take the logarithm of both sides of the equation
Simplify the logarithm and obtain the pH distributed partition coefficient
Derivation of Levich rotating disk aqueous boundary layer thickness
Dividing by d
Therefore:
ν=is the viscosity in centipoise
ρ=fluid density g/cm3
haq=is the boundary layer thickness in centimeters
ω=(2*π*Rotations per min)/60
D=diffusion coefficient in cm2/s
Derivation of the Permeability equation using Permeability Layer Theory
Peff=effective membrane permeability
Paq=Permeability of the aqueous boundary layer
PPDMS=Permeability of the membrane
Vreceiver=drug receiving phase volume
Am=area of membrane for transport
haq=Levich boundary layer thickness
Dag=diffusion coefficient of the drug through the aqueous medium
hm=thickness of membrane
DPDMS=diffusion coefficient of the drug through PDMS membrane
KPDMS=partition coefficient of the drug in PDMS
Under sink conditions:
Csol limit=S=Drug solubility & Creceiver=0
Where m=slope of the concentration versus time curve from experiment with units [μg/(mL*s)]
Substituting in permeability components
Divide by the solubility
Simplify
Because:
Isolating experimental and aqueous diffusion components from the membrane components:
Derivation of the Permeability Equation Using Crank's Approach to Calculating Diffusion Coefficient
QT=[Mass/Area] flowing through the membrane
D=diffusion coefficient of the transporting molecule
C2=the surface concentration at the inner surface of the membrane
h=thickness of the membrane
t=time
P=permeability of the molecule through the membrane
mdC/dt=the slope of the pseudo-steady state transport region on the concentration versus time plot
Vreceiver=aqueous volume in the receiver compartment
KPDMS=the partition coefficient of the molecule in PDMS
Caq total=total aqueous concentration of the molecule in the bulk donor phase
First we measure the slope of the concentration versus time curve in the pseudo steady state region and convert it into mass per area.
Then we calculate the concentration at the inner surface of the membrane, which is the total concentration in the aqueous donor phase multiplied by the partition coefficient of the molecule in the material.
C
2
=K
PDMS
×C
aq Total (S42)
The definition of permeability:
Rearrange D1:
Fully substituting all variables for experimental measurements:
As a check: Since D1=D2 we can compare the measured time to steady state from the rotating membrane diffusion cell experiment with the time predicted in D2.
Each of the references listed above and cited throughout the disclosure is incorporated by reference herein in its entirety, or in relevant part, as would be apparent from context. The disclosed subject matter has been described with reference to various specific embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the spirit and scope of the disclosed subject matter.
This application claims the priority benefit of provisional U.S. Patent Application No. 62/560,552, filed Sep. 19, 2017, herein incorporated by reference.
This invention was made with government support under grant no. HHS F223201510157C, awarded by the U.S. Food and Drug Administration. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/51605 | 9/18/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62560552 | Sep 2017 | US |
