Patent Application
20240335373
The presently disclosed subject matter is directed to the development and optimization of post-fabrication drug loading processes, methods, systems and devices. The presently disclosed subject matter is also directed to medical devices configured with improved release kinetics of pharmaceutical compounds.
Extended-drug delivery systems have long been a part of the overall strategy for controlled therapeutic administration, the primary aim of which is to achieve sustained delivery an active pharmaceutical ingredient (API) within a therapeutically relevant range [1]. These systems provide unique advantages over conventional dosage forms such as oral or direct injection [2] including improved absorption rates, preservation of the API from degradation, and targeted delivery [3]. Polymeric systems in particular allow for tunable therapeutic encapsulation and controlled release through selection of molecular backbone, formation method and drug delivery mechanism [2]. Polymer-based delivery devices can be characterized by their drug release mechanisms as either diffusion-driven, as with silicones and polyurethanes [4], or biodegradable, such as poly (vinyl alcohol) (PVA) or polycaprolactone (PCL) [5]. Appropriate polymer selection is guided by API choice and target indication, which in turn dictates method of device manufacturing [3]. There are numerous device fabrication platforms developed to achieve sustained drug delivery, ranging from electro spun scaffolds [6], in-situ forming implants [7, 8], and hydrogels [9]. Each method has an optimal processing window which may only be suitable for a sub-class of APIs. Therefore, a platform that allows for compatibility to a larger range of drugs, from small molecule hydrophobic to large molecular weight biologics, while maintaining the precision of targeted and sustained release that polymeric devices provide, has long been the target.
The relationship between device geometry and release rate has been well-studied. However, because there is often a relationship between achievable geometry and material, each polymer fabrication or formation is contained within a limited design space. Additive manufacturing (AM), or more commonly 3D printing, is one method of polymer fabrication that enables theoretical design freedom by computationally defining a specific device geometry and replicating through the layerwise assembly or deposition of material [10]. The selective layerwise addition of material can occur through several mechanisms, such as extrusion of a heated filament as with fused deposition modeling (FDM) or with a rastering laser to join polymer or metal particles, as with selective laser sintering (SLS) [11, 12]. AM has been and continues to be explored for its potential toward the design, fabrication, and implementation of polymeric medical devices to achieve greater control and precision of drug delivery [14, 15]. Specifically, the freedom of design principle has been systematically investigated in several AM platforms to tune drug release in a way that could not have been achieved using conventional methods, as the case with designing channeled tablets [16-18], lattice architectures [19, 20], and hydrogels [21].
3D printing, while promising, still presents significant challenges to overcome to achieve application toward medical devices for the sustained and controlled release of medical devices. The layerwise assembly method, across nearly all AM platforms, is not only time-consuming but also imparts directional mechanical strains [22, 23] that can result in part failure and require elaborate methods to overcome [24]. There is a tradeoff between layer thickness, part resolution and total fabrication time, where increased resolution (smaller layer thicknesses), increases the total number of layers and results production times that are prohibitive to manufacturing scale up [25, 26]. Finally, as with conventional methods, there is matter of drug incorporation or encapsulation [13]. Incorporation of API at the point of device fabrication varies by AM method. If melting polymeric material to achieve layerwise assembly, as in the case of FDM or SLS, API incorporation is limited by thermal exposure and solubility just as with injection molding [15]. Polymer materials can be selectively bound as in the case of material jetting or binder jetting methods which unify material through UV exposure or solidification, limiting the potential drug candidates to those that are soluble or not photosensitive [13]. Vat polymerization methods utilize a rastering laser or UV light to selectively cure via free radical polymerization mechanism and is similarly constrained by light exposure and solubility as well as potential degradation in the presence of free radicals [12, 14]. API incorporation post-fabrication is similarly challenging and often results in surface adsorption as opposed to true matrix penetration via absorption.
Digital light synthesis (DLS), also known as Continuous Liquid Interface Production (CLIP), is a novel AM method that utilizes the selective exposure of ultraviolet (UV) light through an oxygen permeable membrane onto a photosensitive resin [27]. DLS offers significant benefits over conventional AM methods such as stereolithography (SL) or fused deposition modeling (FDM) through the comparably fast and layerless fabrication of parts [28]. Compatible resins with the DLS platform include two-part resins containing both a UV-curable component, activated during the 3D-printing step, and a thermally-curable component, activated during a post-fabrication heating step [29], The dual curing allows for the formation of complex polymer matrices which display unique compression and extension properties beyond what is observed with traditional (meth) acrylate chemistries [30]. While translational from a part manufacturing perspective in terms of speed and physical properties [31, 32], DLS is not without its drawbacks when applied to drug delivery, particularly in the case of the dual-cure resins. Incorporation of the API in the resin prior to cure ensures a homogeneous distribution within the part however exposure of UV light and subsequent heat severely restricts the number of compatible APIs that could survive the process without degradation.
Therefore, there is a need for new technologies that utilize efficient and cost effective methods to load medical devices, e.g. intravaginal rings (IVRs), with active pharmaceutical ingredients (APIs). More particularly, there is an unmet need to develop alternative loading routes, systems, methods and techniques for APIs that may not be compatible with the fabrication processes due to thermal or UV-light sensitivity. Moreover, there is a need for streamlined precision controls over loading particularly as it relates to influencing changes in drug release rate from such medical devices. The presently disclosed subject matter addresses these long-felt needs. We present a method to address this by utilizing a post-fabrication absorption process to load a variety of therapeutics into a 3D printed silicone material. Using a controlled system, we systematically investigated the effect of a geometry on the swelling, drug uptake, and drug release in simulated vaginal fluid and correlated these performance parameters to the fundamental characteristics of the part, namely the specific surface area.
This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
In some embodiments, provided are post-fabrication methods for drug loading a medical device with an active pharmaceutical ingredient (API), the methods comprising providing a medical device comprising a polymer matrix, exposing the medical device to a loading solution comprising the API for a time sufficient to cause the API to be integrated within the polymer matrix, wherein the polymer matrix, after exposure to the loading solution with the API, exhibits a degree of swelling in a range of about 100% to about 1100% of the polymer matrix relative to an unswollen state of the polymer matrix prior to exposure to the solution comprising the API, and/or a degree of swelling in which the polymer matrix increases in a dimension from about 60% to about 500% along an axis. In some embodiments, the medical device comprises an intravaginal ring (IVR), optionally wherein the IVR is 3D printed. The degree of polymer swelling can be influenced by a factor selected from the group consisting of network crosslinking density of the polymer matrix, polymer backbone properties (e.g. MW, charge, polarity), presence of side chains in the polymer matrix, polymer structure (e.g. linear versus branched), dimensions of the medical device (e.g. surface area, volume, thickness), and/or combinations thereof. In some aspects, the degree of polymer swelling is influenced by an interaction of the polymer matrix with a solvent in the solution. In some aspects, a percent solvent uptake and swelling is solvent dependent (i.e. interaction of matrix with solvent).
In some embodiments, a geometry of the medical device influences the degree of swelling and percent drug incorporation/loading, optionally wherein the geometry comprises a part volume defining an amount of macro space within the polymer matrix. In some aspects, a total loaded amount of API is tuned by an initial concentration of the loading solution comprising the API. The role of diffusion distance in drug delivery duration and the role of specific surface area (SSA) in the prediction of drug release can be defined and used to fine-tune drug release. In some aspects, a degree of crosslinking of the polymer matrix is substantially proportional to the degree of swelling, optionally wherein the degree of crosslinking defines an accessibility of a micro space within the polymer matrix. In some embodiments, the degree of swelling is substantially directly proportional to the degree of API loading. At a given degree of swelling of the polymer matrix there can be a substantially linear correlation between API concentration in the loading solution and percent API loaded in the polymer matrix. The degree of swelling of the polymer matrix can substantially increase with increasing diffusion distance in the polymer matrix.
In some aspects, the medical device is exposed to a loading solution comprising more than one API. In some embodiments, the methods further comprise removal of extractables and/or leachables (i.e. unreacted or unincorporated monomers or oligomers) from the polymer matrix of the device.
In some embodiments, API loaded medical devices produced by the disclosed methods are provided. The API loaded medical devices can in some aspects comprise substantially controlled drug release kinetics, optionally wherein the release kinetics can be optimized based on swelling duration, solvent type, API concentration, rate controlling additives, release rate controlling membranes and combinations thereof.
Provided in some aspects are medical devices comprising a polymer matrix and an active pharmaceutical ingredient (API), wherein the API is loaded into the polymer matrix by adsorption and/or swelling after fabrication of the polymer matrix, wherein the medical device is configured to achieve release kinetics in a range of about one day to about 360 days, optionally wherein the release kinetics comprise a substantially sustained release for at least about 30 days or more, optionally for at least about 60 days or more, optionally for at least about 90 days or more, optionally for at least about 120 days or more. The device comprises a geometrical distance and a volume, wherein a release rate of the API from the polymer matrix is controlled by a diffusion distance and a part volume. A release rate of the API from the polymer matrix is controlled by an interaction between the API and polymer matrix, and/or an interaction between the API and a surrounding environment, wherein the surrounding environment comprises one or more of: swelling of polymer matrix parts that impact accessibility to API, a surface area of the device that impacts accessibility to API, another API or release rate controlling additive that impacts accessibility to API, a polymeric membrane that surrounds the device and impacts accessibility to API, and an initial loaded concentration of API and changes thereto as API is released. In some aspects, the medical devices can comprise an intravaginal ring (IVR), wherein the IVR comprises one or more APIs.
Provided in some embodiments are methods of treating a subject, wherein the method of treatment, prevention or diagnostic comprises providing a subject in need of treatment, prevention or a diagnostic, providing a medical device as disclosed herein, and administering, placing and/or applying the medical device to/in the subject in need of treatment, prevention or diagnostic. The medical devices can comprise an intravaginal ring (IVR), wherein the IVR comprises one or more APIs. The medical devices can further comprise a release rate controlling additive. In some aspects, the medical device in such methods can comprise an intravaginal ring (IVR), wherein the IVR comprises a release rate controlling polymeric membrane. The release kinetics of the one or more APIs can be in a range of about one day to about 360 days, and any range in between. In some aspects, the one or more APIs comprises a therapeutic compound selected from an antiviral, antiretroviral, microbicide, contraceptive, antibiotic, hormone, pre-exposure prophylaxis, small molecule drug, macromolecule drug, biopharmaceutical, chemotherapeutic, monoclonal antibody, protein, peptide, diagnostic marker, other pharmaceutical compound, and combinations thereof. In some embodiments, the subject in need of treatment is in need of HIV pre-exposure prophylaxis (PrEP), HIV treatment, contraception, and/or prevention of sexually transmitted diseases (STDs), women's health indication (e.g. infertility, hormone replacement, gynecology oncology, diagnostic). In some embodiments, the subject in need of treatment can be a female human subject or transgender.
These and other objects are achieved in whole or in part by the presently disclosed subject matter. Other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.
The presently disclosed subject matter can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figures, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the presently disclosed subject matter.
For a more complete understanding of the presently disclosed subject matter, reference is now made to the following figures.
The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.
All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.
Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a unit cell” includes a plurality of such unit cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of a composition, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.
As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
As used herein, the term “and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
As used herein, the term “subject” refers to an individual (e.g., human, animal, or other organism) to be assessed, evaluated, and/or treated by the methods, devices, systems or compositions of the presently disclosed subject matter. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and includes humans. As used herein, the terms “subject” and “patient” are used interchangeably, unless otherwise noted.
As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount that provides a therapeutic effect, e.g., an amount of a composition or active pharmaceutical ingredient that is effective to treat or prevent pathological conditions in a subject.
As used herein, the term “adjuvant” as used herein refers to an agent which enhances the pharmaceutical effect of another agent.
The terms “compound” and “active pharmaceutical ingredient” can be used interchangeably, and as used herein, refer to any type of substance or agent that is commonly considered a chemical, drug, or a candidate for use as a drug, pharmaceutical, therapeutic agent, and the like, as well as combinations and mixtures of the above.
A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
The term “modulate”, as used herein, refers to changing the level of an activity, function, or process. The term “modulate” encompasses both inhibiting and stimulating an activity, function, or process.
As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in an animal. In some embodiments, a pharmaceutically acceptable carrier is pharmaceutically acceptable for use in a human.
The term “standard”, as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered or added to a control sample and used for comparing results when measuring said compound in a test sample. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured.
The term “symptom”, as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a sign is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.
As used herein, the term “treating” includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
Provided herein are post-fabrication methods for drug loading a medical device with an active pharmaceutical ingredient (API), or other desired drug compound or therapeutic composition. Such methods can include providing a medical device comprising a polymer matrix, exposing the medical device to a solution comprising the API for a time sufficient to cause the API to be integrated within the polymer matrix, wherein the polymer matrix, after exposure to the solution with the API, exhibits a degree of swelling in a range of about 100% to about 1100% of the original polymer matrix, and/or a degree of swelling in which an object increases in dimension from about 60% to about 500% along an axis. The medical device can comprise an intravaginal ring (IVR), optionally wherein the IVR is 3D printed or otherwise additively manufactured.
In such methods the degree of polymer swelling can be influenced by a factor selected from the group consisting of network crosslinking density, polymer backbone properties (e.g. molecular weight (MW), charge, polarity), presence of side chains, polymer structure (e.g. linear vs. branched), device dimensions (e.g. surface area, volume, thickness), and/or combinations thereof. In some aspects, the degree of polymer swelling can be influenced by an interaction of the polymer matrix with a solvent in the solution. Sometimes a percent solvent uptake and swelling is solvent dependent (i.e. interaction of matrix with solvent).
In some embodiments the geometry of the medical device can influence the degree of swelling and percent drug incorporation/loading, optionally wherein the geometry comprises a part volume defining an amount of macro space within the polymer matrix. A degree of crosslinking of the polymer matrix can be substantially proportional to the degree of swelling, optionally wherein the degree of crosslinking defines an accessibility of a micro space within the polymer matrix. The degree of swelling can be substantially directly proportional to the degree of API loading. At a given degree of swelling of the polymer matrix within the specified range there is a substantially linear correlation between API concentration in the loading solution and percent API loaded in the polymer matrix.
In some embodiments, the medical device is exposed to a solution comprising more than one APIs. In some aspects, such methods further comprise removal of extractables/leachables (i.e. unreacted or unincorporated monomers or oligomers) from the polymer matrix of the device.
Also provided herein are API loaded medical devices produced by the methods disclosed herein. The API loaded medical devices can comprise substantially controlled drug release kinetics, optionally wherein the release kinetics can be enhanced or optimized based on swelling duration, solvent type, API concentration, rate controlling additives, release rate controlling membranes and combinations thereof.
Also provided herein are medical devices comprising a polymer matrix and an API, wherein the API is loaded into the polymer matrix after fabrication of the polymer matrix by adsorption and/or swelling, wherein the medical device is configured to achieve release kinetics in a range of about one day to about 360 days. Such devices can comprise a geometrical distance and a volume, wherein a release rate of the API from the polymer matrix is controlled by a diffusion distance and a part volume. In some embodiments, a release rate of the API from the polymer matrix is controlled by an interaction between the API and polymer matrix, and/or an interaction between the API and a surrounding environment, wherein the surrounding environment comprises one or more of: swelling of polymer matrix parts that impact accessibility to API; a surface area of the device that impacts accessibility to API; another API or release rate controlling additive that impacts accessibility to API; a polymeric membrane that surrounds the device and impacts accessibility to API; and an initial loaded concentration of API and changes thereto as API is released. Such medical devices can comprise, for example, an intravaginal ring (IVR), wherein the IVR comprises one or more APIs.
Finally, provided herein in some embodiments are methods of treating a subject, wherein the method of treatment, prevention or diagnostic can comprise providing a subject in need of treatment, prevention or a diagnostic, providing a medical device of any of the above claims, and administering, placing and/or applying the medical device to/in the subject in need of treatment, prevention or diagnostic. The medical device in such methods can in some embodiments comprise an IVR, wherein the IVR comprises one or more APIs. The medical device in such methods can further comprise a release rate controlling additive. The medical device in such methods can comprise an IVR, wherein the IVR comprises a release rate controlling polymeric membrane. Release kinetics of the one or more APIs are in a range of about one day to about 360 days, about 15 days to about 360 days, about 30 days to about 360 days, about 100 days to about 300 days, greater than about 30 days, greater than about 60 days, greater than about 90 days, greater than about 120 days, greater than about 200 days, greater than about 300 days, or greater than about 360 days or more. The one or more APIs can comprise a therapeutic compound selected from an antiviral, antiretroviral, microbicide, contraceptive, antibiotic, hormone, pre-exposure prophylaxis, small molecule drug, macromolecule drug, biopharmaceutical, chemotherapeutic, monoclonal antibody, protein, peptide, diagnostic marker, other pharmaceutical compound, and combinations thereof. In such methods the subject in need of treatment can be in need of HIV pre-exposure prophylaxis (PrEP), HIV treatment, contraception, and/or prevention of sexually transmitted diseases (STDs), women's health indication (e.g. infertility, hormone replacement, gynecology oncology, diagnostic). In some aspects, the subject in need of treatment can be a female human subject or transgender.
The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many 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 presently disclosed subject matter.
Diffusion Blocks. Diffusion blocks were defined as prismatic rectangles of specified length (Z, constant), width (Y, constant), and height (X, variable). Blocks were defined by the X (variable) dimension. Blocks were generated in SolidWorks (Dassault Systèmes). As described in
Unit Cells. All unit cell used were generated in SolidWorks (Dassault Systèmes). The cylinder unit cell was generated to have a 5:4 outer to inner diameter ratio. The Honeycomb, Trident, and Diamond unit cells were generated to have internally consistent strut thicknesses. All unit cells were arrayed to yield approximately 0.5 mm struts when fabricated in SIL 30 using Digital Light Synthesis (DLS). As such, the unit cell sizes (defined as a cube of X, Y, and Z) were set as follows: cylinder, trident and diamond at 3.80×3.80×3.80 mm and honeycomb at 2.53×2.53×2.53 mm. These values represent integer distances within the 7.60 mm cross-section of the IVR (3.80 mm is 1:2 and 2.53 mm is 1:3)
Geometrically Complex IVRs. Geometrically complex intravaginal rings (IVR) designs were generated using methods as disclosed in PCT International Application Serial No. PCT/US2017/023777 (published as WO 2017/165624), the entirety of which is incorporated herein by reference. Briefly, the multistep process begins with a ‘template ring’ of given outer diameter (54 mm) and cross-sectional diameter (7.6 mm) generated in SolidWorks (Dassault Systèmes) and converted to a .STL. The template was imported into Magics (Materialise) and a selected unit cell arrayed into the template using the ‘Scaffold’ feature. Rings were exported as a .STL. Separately, a ring band was generated in SolidWorks (Dassault Systèmes) to encase the ring with dimensions of 4.0 mm height and 0.6 mm thickness and exported as a .STL. Both the geometrically complex IVR and band were imported into MeshMixer (AutoDesk), cenetered on the absolutely origin, combined and exported as a unified .STL. Rings were imported into Magics to correct any tessellation errors accumulated during the file transfer processes. Fully banded geometrically complex rings were exported as a .STL for fabrication.
Printer. Parts were fabricated with an M1 DLS (Digital Light Synthesis) printer (Carbon, Inc.). Diffusion blocks were fabricated with the XY plane of the block oriented to the build platform. Geometrically complex IVRs were fabricated vertically and manually supported using the support feature within the Carbon user interface. Each ring was supported using approximately 40 supports covering the bottom third of the IVR closest to the build platform.
Blocks in SIL 30. Diffusion blocks were fabricated in SIL 30 using two methods: ‘combined’ and ‘separated’. Using the same project as the prototyped blocks, ‘combined’ batches were fabricated n=20 of each type spaced 2.5 mm apart. Batches fabricated as ‘separated’ consisted of each individual block type, replicated n=40, and spaced 3.75 mm apart. This distance was selected to represent an integer value of the projected pixel of the DLS printer (75 μm). Approximately 100 mL of SIL 30 was dispensed from a static mixer into the build reservoir. Each print was 34 min. long using standard slicing of 100 μm.
Blocks in Prototyping Resin. Upon completion of the print, blocks fabricated in UMA prototyping resin were removed from the build platform and placed in a sealed container with 200 mL of isopropyl alcohol (IPA). The container was placed on a shaker table for 5 min. The container was removed and using tweezers, the solvent stirred to ensure no blocks were adhered to the container or each other. Blocks were then removed from the solvent and air-dried for 1 hr. Parts were treated to a UV post-cure with a FireJet Fj800 Controller (Phaseon Technology) in a chamber purged with N2 for 30 s prior to a 2 min exposure of 20 mW/cm2 385 nm light per side.
Blocks in SIL 30. Blocks fabricated in SIL 30 were treated to a post-fabrication cleaning procedure modified from the guidance provided by Carbon. Blocks were removed from the build platform with a razor and placed into a sealed container with 300 mL isopropyl alcohol (IPA). The container was placed on a shaker table from 1 minute then the blocks were removed. Blocks were laid individually on WipeAll towels to ensure no blocks were adhered to one another and allowed to air-dry for approximately 1 hr. Parts were then placed in a programmable oven to initiate a secondary thermal post cure. The program followed recommended curing, beginning at 31° C. and ramping up to 120° C. over 15 min, holding at 120° C. for 8 hr and finishing by ramping down to 31° C. in 15 min. Parts were removed from the oven for immediate further testing or stored at −4° C.
Geometrically Complex IVRs in SIL 30. IVRs fabricated in SIL 30 were treated to a post-fabrication cleaning procedure modified from the guidance provided by Carbon, as previously described. Briefly, parts were removed from the build platform, the supports removed, and the rings placed in a sealed container with 200 mL of IPA. The container was placed on a shaker table for 30 s and then the rings were removed from the solvent. The band of each ring was smoothed using a razor and all rings were placed in a manual spinner to remove excess solvent for 1 min. Rings were then pressed between two Teflon plates from approximately 45 min. The washing and spinning steps were repeated. Finally, parts were placed in a programmable oven to initiate a secondary thermal post cure. The program followed recommended curing, beginning at 31° C. and ramping up to 120° C. over 15 min, holding at 120° C. for 8 hr and finishing by ramping down to 31° C. in 15 min. Parts were removed from the oven for immediate further testing or stored at −4° C.
Block Fabrication. All block types were fabricated in prototyping resin (UMA) under combined conditions (n=3 for each distance) as well as in silicone resin (SIL 30) under combined and separate conditions (n=3 for each distance).
Dimensional Analysis. Azimuthal axis dimensions were taken for each block as a function of type, resin, and fabrication condition.
Dimensional Accuracy. Percent deviation from CAD was calculated using the following equation: % Deviation from CAD=100×(DExp−DCAD)/DCAD where DExp is the experimentally determined distance (X, Y, or Z) and DCAD was the input distance in the CAD file. All values were calculated individually for each block and reported as average and standard deviation.
Blocks fabricated in SIL 30 were assessed for swelling compatibility under placebo loading conditions in methanol and acetone. Placebo testing was conducted under conditions mimicking the post-loading process and under conditions to completely remove the soluble fraction within the SIL 30 matrix. In all processes, the metrics of mass and distance in the X, Y, and Z azimuthal dimensions were obtained during the initial, swollen and dried stages. Distance values were obtained using calipers. For each treatment and block type n=3 blocks were used and average and standard deviation values determined.
Swell Testing as a Function of Time. Initial metrics for blocks (n=3) of each type were taken and then blocks were batch immersed in either acetone or methanol. Swelling metrics were taken at days 1, 2, 3, and 7. Following Day 7 assessment, blocks were dried for approximately 48 hrs. and dried metrics were collected. Blocks were tracked individually throughout the process based on unique tag.
Post-Loading Mimic. The post-loading process was mimicked using either methanol or acetone in which blocks (n=3 of each type) were immersed in 30 mL of solvent for 24 hr., removed, and air-dried for approximately 48 hrs. Metrics were collected in the initial, swollen, and dried state.
Gel Fraction. The complete soluble fraction removal was conducted in either methanol or acetone in which a specified block type was immersed individually in 30 mL of solvent for 7-days, removed, and air-dried for approximately 48 hrs.
Calculations. Degree of swelling was calculated as a function of time using the following equation: Degree of Swelling (%)=100×(MS−MI)/MI where Ms is the mass in the swollen state and MI was the mass in the initial state. Surface area and volume were experimentally determined in the initial, swollen (Day 1, 2, 3, and 7), and dried states using standard formulas for rectangles. Specific surface area (SSA) was calculated as:
Degree of swelling (%) for each azimuthal axis was calculated as: Degree of Swelling (%)=100×(DS−DI)/DI where DS is the swollen dimension and DI is the initial dimension. Degree of shrinkage for each azimuthal axis was calculated as: Degree of Swelling (%)=100×(DF−DI)/DI where DFis the final dimension and DI is the initial dimension. The matrix fraction, that is the fraction of both the residual soluble and insoluble material remaining in the matrix, was calculated using the following equation: Matrix Fraction=MD-24 Hr./MI where MD-24 Hr. is the mass of the dried block following immersion in solvent for 24 hr. and MI is the initial mass. Gel fraction was calculated as: Gel Fraction=MD-7 Day./MI where MD-7 Days is the mass of the dried block following immersion in solvent for 7 days. All values were calculated individually for each block and reported as average and standard deviation.
The post-loading absorption process was tested with both hydrophobic and hydrophilic dyes. A hydrophobic solution was prepared with rhodamine B (RhB, Sigma Aldrich) in methanol at a concentration of 0.08 mg/mL. This stock solution was serial diluted to obtained solutions at 0.04, 0.02, 0.01, and 0.005 mg/mL. A similar method was used to obtain a hydrophilic solution set containing Nile Blue A (NBA, Sigma Aldrich) in methanol. A stock solution of both dyes was prepared in methanol with 0.04 mg/mL RhB and 0.04 mg/mL NBA and serial diluted to obtain solutions at individual dye concentrations of 0.02, 0.01, 0.005, and 0.0025 mg/mL, with the total dye loading held constant.
Block Preparation and Swelling in Simulated Vaginal Fluid (SVF). Blocks (n=3 of each type) were fabricated in SIL 30 as previously described and treated to a 24 hr post-loading mimic cycle in acetone. Placebo blocks were assessed for initial metrics and batch immersed in jar containing 400 mL simulated vaginal fluid (SVF) and placed in an incubator at 37° C. The SVF consisted of 25 mM sodium acetate buffer (pH 4.2) plus 2% Solutol (Kolliphor HS 15). Blocks were assessed for metrics at the following time points: day 1, 2, 3, 4, 7, 8, 9, 10, 11, 14, 21 and 28.
Calculations. Block metrics were used to calculate volume, surface area, density, mass increase (%), increase in the z-axis (%), volume increase (%), surface area increase (%) and specific surface area (SSA). Plots were generated as either a function of time or a function of block distance and values reported as an average and standard deviation. SSA plots were generated as a function of time for each block type, a log curve fitted and an equation and coefficient of variation determined.
Post-Loading with Model Drugs
Loading. Two small-molecule model drugs were selected to investigate the post-loading absorption method: β-estradiol (hydrophobic, log p=3.75) and 2-fluoro-2′-deoxyadenosine (FdA) (hydrophilic, log p=−0.57). Solutions of 5 mg/mL of drug in acetone were prepared and validated via HPLC. Blocks (n=8 of each type) were batch immersed for 24 hr, removed and air dried for approximately 48 hrs. Blocks were then divided into an extraction set and a release set.
Extraction. Drug-loaded blocks were extracted using methods previously described in Section 2.6 using acetone as the extraction solvent. Aliquots were collected and analyzed with HPLC. Weight percent loading was determined as the total drug extracted relative to the mass of the dried blocks.
Release in SVF. Post-loaded blocks were placed individually into sealed jars containing 60 mL of simulated vaginal fluid (SVF). The SVF consisted of 25 mM sodium acetate buffer (pH 4.2) plus 2% Solutol (Kolliphor HS 15). Jars were placed in incubator at 37° C. Aliquots of 1 mL were taken daily and replaced with fresh media. Sink conditions were maintained and monitored with media changes occurring when saturation had been reached. The saturation solubility of β-estradiol in SVF was determined to be 108 μg/mL The concentration of β-estradiol in the aliquots was determined using an Agilent 1260 HPLC with a Diode Array Detector, on an Inertsil ODS-3 column (4.6×150 mm, 5 um) maintained at 40° C., with a flow rate of 1.0 mL/min, 25 μl sample injection, and an acetonitrile/water mobile phase, each modified with 0.1% trifluoroacetic acid. A gradient method was utilized to achieve separation (0-20 min: 5%-100% acetonitrile; 20-22 min: 100% acetonitrile; 23-25 min: 5% acetonitrile). β-Estradiol was eluted at 13.8 min and measured at 280 nm. FdA was eluated at 5.3 min and measured at 265 nm. Area under the curve (AUC) was computed using Chemstation software, and concentrations were derived from a calibration curve generated using β-Estradiol or FdA standards prepared in 100% acetonitrile (250 ug/ml-61 ng/ml). Release was determined as complete when additional drug was no longer detected in the SVF medium. Blocks were then removed and placed in acetone for extraction of residual or trapped drug. Aliquots were analyzed via HPLC.
Calculations. Release of model drugs were reported as either cumulative amount (μg) or percent (%) over time. Burst release was determined as the cumulative amount (μg) or percent (%) of drug released within 24 hr. The release of drug over time was plotted for each block and linear curves fitted from Day 2 onward to obtain equations describing zero order release and associated coefficients of variation. Values were averaged for each block type and reported as average and standard deviation. To account for changes in block geometry associated with SVF swelling, the SSA equations determined in Section 2.7 were used to calculate specific surface areas (mm−1) for each block using the initial dimensions of the release blocks. Calculated values were then used to normalize the release data by computing either μg/mm−1 or %/mm−1 as a function of release duration. Burst release values were similarly normalized using the Day 1 SSA values obtained during placebo swelling analysis in SVF (Section 2.7).
Block types 6.0 and 7.6 mm (n=8 of each type) were fabricated in SIL 30. A solution containing 8.562 mg/mL of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) in acetone was prepared and validated via HPLC. Blocks were batch immersed in post-loading solution for up to 24 hr. Blocks were divided into extraction (n=4) and release (n=4) sets. EFdA was extracted from blocks in acetone as previously described and quantified with HPLC. Release blocks were individually immersed in 60 mL of SVF and placed in an incubator at 37° C. Aliquots and analysis were completed using methods previously described. EFdA was quantified with HPLC, eluting at 5.31 min at 265 nm. Concentrations were derived from a calibration curve generated using EFdA prepared in 100% acetonitrile (250 ug/ml-61 ng/ml) with the LOQ determined as 0.21 μg/mL. Values were reported as cumulative release amount (μg) or percent (%) as a function of time and normalized by SSA as previously described for the model drugs. All values reported represent average and standard deviation of n=4 samples.
Loading. Geometrically complex IVRs with varying unit cells but constant strut thickness were fabricated in SIL 30 and cleaned as described in Section 2.3 and 2.4, respectively (n=5 per ring design). Rings were assessed for metrics of mass, outer diameter (mm), and cross-sectional diameter (mm) and batch immersed in a solution of 22 mg/mL β-Estradiol in methanol for 24 hrs. Rings were removed and air dried for approximately 48 hrs.
Extraction. One ring of each design was designated for extraction using methods previously described. A representative weight percent loading for each design was determined.
Release in SVF. Rings were individually placed in jars with 60 mL SVF in an incubator at 37° C. Aliquots of 1 mL were taken and analyzed via HPLC as previously described. Rings were removed at Day 28 and underwent a full extraction to determine total loading. Values were reported as cumulative release amount (μg) or percent (%) as a function of time. Metrics associated with burst release and zero order release were determined. All values reported represent average and standard deviation of n=4 samples.
DLS IVRs fabricated were imaged with a Zeiss Stemi 508 Stereo Microscope Labscope. Images were captured and dimensional analysis conducted with Zeiss Labscope software. Strut thickness and band thickness measurements were conducted using ImageJ. Measurements were n=4 for each ring for n=4 samples resulting in 16 total measurements per design. Average and standard deviations were reported.
Intravaginal rings (IVRs) can be fabricated with geometric complexity to leverage the design freedom associated with additive manufacturing [33]. However, these systems would be too complex to systematically investigate the underlying fundamental aspects of the post-loading process. Therefore, a simplified system defined as ‘diffusion blocks’ was utilized. An overview of the design and fabrication of the simplified system can be seen in
The blocks were fabricated using Digital Light Synthesis (DLS) or as previously published, Continuous Liquid Interface Production (CLIP). This stereolithography system utilizes the interplay of ultra-violet (UV) light and oxygen to selectively polymerize and solidify a photo-active resin. A schematic of this process is shown in
The photo-active SIL 30 resin used in these studies is a silicone-based resin. The resin consists of two parts, a UV-active component and a thermal-active component, described in
The fabrication of SIL 30 resin with the DLS system has been assessed for geometric fidelity and minimum thresholds established. This includes a minimum wall thickness of 1.5 mm that can be fabricated within acceptable tolerances. The projected pixel, or the area of light illuminated by a single micro-mirror and dictated by the distance between the DMD and the window, of the Ml system used is 75×75 μm. It is possible then to fabricate features below the 1.5 mm threshold however the dimensions of the part may not be as accurate or within acceptable tolerances. To assess the fidelity of the blocks produced using DLS, particularly those falling below the 1.5 threshold, blocks were fabricated both as a function of resin (prototyping and SIL 30) and as a function of placement (combined and separated). The blocks were fabricated in a fast-reacting urethane methacrylate prototyping resin (UMA) to assess the underlining effects of the interplay between the DMD and the fabricated dimension. Because the SIL 30 wall thickness threshold and the interplay between the projected pixel and the fabricated part, two placement orientations were investigated: combined and separated (shown in
Blocks were assessed for part fidelity in the variable X dimension both as a function of resin and placement. Analysis of absolute distance in the X dimension is shown in
The proposed post-fabrication absorption process involves the exposure of the parts to solvent to swell the SIL 30 matrix and allow for drug intercalation. There are several experimental factors to determine including choice of solvent and exposure duration. Solvents were preliminarily screened and selected based on solvent class, logP, and boiling point, resulting in the selection of methanol (MeOH, Class II) and acetone (Ace, Class III). Exposure duration was determined by immersing all block types in either Ace or MeOH and assessing part metrics (mass and azimuthal distances) on Day 1, 2, 3, and 7. By tracking the degree of swelling as a function of time (
The collection of azimuthal dimensions enabled the calculations of surface area (SA, mm2) and volume (V, mm3) as a function of swelling time. This is shown for Ace,
The solvent exposure duration analysis suggested that swelling and drying were uniform based on V and SA tracking however this can be further investigated by tracking the dimensions of each azimuth during the post-loading cycle. This was assessed by immersing all block types in either Ace or MeOH for 24 hr and tracking initial, swollen, and dried metrics in the X, Y and Z dimension. A degree of swelling can be computed for each dimension, shown in
The SSA tracking shown in
The motivation for the development of post-fabrication drug loading methods was two-fold. First, one goal was to develop an alternative loading route for active pharmaceutical ingredients (APIs) that may not be compatible with the fabrication process due to thermal or UV-light sensitivity. Second, was to streamline precision control over loading particularly as it relates to influencing changes in drug release rate.
At least two aspects of the invention that were assessed included elucidating the relationship between degree of swelling and diffusion distance, and establishing why it exists, and how it influences loading.
A test part described as a diffusion block with dimensions in X, Y, and Z of 10 mm, 20 mm, and 4 mm, respectively, was primarily used. The general flow of the post-loading process is outlined in
Equation 1 Degree of swelling calculation based on mass. Conventional calculation using the swollen mass (Ms) and the dried mass (MD).
Equation 2 Degree of swelling calculation based on dimension. Modified calculation where A represents a measurement along a given axis (X, Y, or Z) in the swollen state (AS) and the dried state (AD).
Following the post-loading procedure with drug, blocks were either treated to EtOH for extraction of β-estradiol or to SVF for release of β-estradiol as a function of time, shown in
There are several potential advantages to post-fabrication loading. First, the swelling of the 3D printed parts in an organic solvent can serve as a removal route of unincorporated extractables and leachables as a step in the device preparation process. Second, it was hypothesized that there exists a region of linearity in which post-loading solvent API concentration correlates with total incorporated amount within the device. This region would enable precision and bespoke loading, adding a second degree of customization to the device. Finally, several drugs can be simultaneously solubilized within a single loading solution, enabling co-formulation of drugs and the preparation of a multipurpose device.
A more detailed breakdown of the potential steps to achieve targeted post-loading amounts and release profiles is shown in
The process flow depicted in
The effectiveness of liquid post-loading method (as opposed to supercritical fluids) lies with its ability to penetrate and swell the polymer network of the fabricated part, an aspect largely dictated by solvent-polymer interactions. The resin utilized in IVR fabrication was SIL 30, a highly hydrophobic resin. Therefore, it was likely that solvents with large positive logP values would penetrate and expand the polymer network, quantified via degree of swelling. To investigate this, an exploratory panel of solvents was selected based on FDA solvent classification system (with class III defined as least toxic in residual amounts), logP, and boiling point (° C.), described in Table 1.
The polymerization process is known to result in structures that contain components covalently bonded to the matrix and components trapped during the solidification process, or the soluble fraction. The post-loading process swells the matrix in a manner that allows for the soluble fraction to be removed. In the presence of a drug-solvent solution, the part swelling would represent a two-way street in which the soluble fraction was leached out and the drug was absorbed in. Therefore, it was necessary to understand what fraction remained following post-loading mimic exposure (24 hr.) and full soluble fraction removal (7-day exposure). The calculation of the ratio of the dried mass to the initial mass to determine the matrix fraction (24 hr. exposure) and the gel fraction (7-day exposure) as a function of block type and solvent are shown in Table 1. Both methanol and acetone were observed to have a similar capacity for soluble fraction removal with resulting gel fractions near 0.90. For all blocks except the 7.6 mm type, the 24 hr. exposure appears to be sufficient in removing the bulk of the soluble fraction as evidenced by the similarities between the matrix and gel fractions. This correlates with what was observed during the solvent swelling analysis as a function of time, where swelling equilibration was achieved within the first 24 hrs. The 7.6 mm block types, requiring an additional day to achieve equilibration, exhibited the largest difference between matrix fraction (0.90±0.03) and gel fraction (0.83±0.01), particularly in the case of methanol, which was shown to swell parts upwards of 400%. Collectively these data indicate that 24 hrs. is a sufficient exposure duration for most distances to remove a significant portion of the unincorporated material within the device, which would be left unremoved, would serve as a potential leachable and extractable source.
The exploratory solvent study with whole rings indicates that there was a tradeoff between degree of swelling and imparted defects on the ring that must be considered when moving forward. It should be noted that this panel was specific to the SIL 30 resin utilized in fabrication. The solvents carried forward for the pilot post-loading study were methanol, acetone, isopropyl acetate and chloroform. Acetone and methanol both yielded rings of comparable gel fractions, comparable swelling, and did not severely damage the ring in the process. Isopropyl acetate is a known class III solvent and damaged the ring after 4 days of immersion, a duration exceeding the targeted loading time. Chloroform was observed to be the most aggressive solvent in terms of degree of swelling and therefore will be carried forward as a ‘best case scenario’ solvent. The solvents selected from this panel have varying degrees of swelling, log P as well as drug solubility and therefore, performance in terms of total loading and loading efficiency can only be partly attributed to solvent uptake.
The exploratory solvent panel yielded four solvents of potential interest to investigate further in terms of soak duration of a solvent loaded with a model drug, β-Estradiol. To further simplify the system and enable quantitative check points (as opposed to previous qualitative observations), whole IVRs were replaced with unloaded diffusion blocks, with dimensions outlined in Table 2. Blocks utilized in soak duration study were fabricated from a single batch file and the initial metrics of dimensions in each direction and mass were taken. The average and standard deviations as well as the percent relative standard deviations (% RSD) are shown for each metric in Table 2. Collectively, the initial metrics suggest there is minimal variability in the sample set prior to solvent treatments.
Diffusion blocks were incubated in respective solvents super saturated with β-Estradiol (20 mL per block). Soak durations were set at 8, 24 and 48 hr. A control block was soaked in a drug-free solvent for 48 hr. A sectioned NuvaRing (EVA) segment of comparable mass was concurrently tested to serve as a known benchmark. Blocks were immersed for indicated durations and swollen metrics taken. Blocks were dried, metrics assessed and placed in EtOH for three days to facilitate extraction of the loaded β-Estradiol. Ethanol was selected as an extraction solvent given previous observation of degree of swelling as well as high solubility of β-Estradiol.
Parts (diffusion block and NuvaRing sections) were incubated in respective solvents for the prescribed time. Images of the swollen parts at the conclusion of the immersion are shown in
Diffusion blocks were quantitatively assessed throughout the post-loading process by documenting dimensions via calipers and mass. The dried masses of the blocks were evaluated relative to the initial masses as a function of solvent type and sample condition, shown in Table 3. This is a similar assessment as gel fraction however does not completely describe the samples when the solvents are loaded with β-estradiol. The true gel fraction of the parts at 48 hr is highlighted in green with the unloaded sample condition. These values are similar and slightly elevated relative to the values obtained previously with rings following a 6-day incubation, as to be expected. Values above 1 indicate mass was gained following the post-loading processes. It should be noted that all solvents are capable of removing the soluble fraction (to varying degrees) and therefore mass can be acquired via drug uptake without achieving a fraction above 1. The NuvaRing sections that were recovered had a gel fraction of 1.
The unloaded 48 hr sample was compared to the loaded 48 hr sample for all solvent types at all stages of the post-loading process. The masses are shown in Table 4 and compared. Percent increase was calculated of the loaded sample relative to the unloaded. Samples were initially similar in masses prior to treatment. Additionally, samples exhibited similar swelling behavior for all solvents, with the potential exception of isopropyl acetate. Methanol, acetone, and isopropyl acetate yielded samples with an increased mass of the dried loaded part relative to the unloaded control. Chloroform yielded a comparative mass loss.
The solutions used for post-loading were evaluated via HPLC to determine concentration of β-Estradiol. The values averaged were samples collected following the post-loading process which were then compared to the original (unused) post-loading solutions. The total loading concentrations vary depending on the solubility of β-Estradiol within each solvent therefore this analysis is best served by calculating the % RSD within each sample set, shown in Table 5. These values are all acceptably low expect within the case of chloroform. It should be noted where other solvents were clear after β-Estradiol incorporation, chloroform was cloudy. This could be a factor in the variability of the loading concentration.
To demonstrate reproducibility of the process, diffusion blocks were treated to the optimized post-loading procedure using methanol, acetone, and isopropyl acetate. The initial metrics obtained for each solvent condition are compiled in Table 6. It can be seen that as with the pilot study, little sample variation exists between the blocks, represented by the low % RSD values.
The post-loading process was conducted utilizing the 24 hr time point and the swollen metrics of the blocks taken following removal from solvent treatment. The obtained values are shown in Table 7. Given the different logP values associated with each solvent and therefore the different propensities to swell the SIL 30 matrix, similar values between solvent conditions was not expected. Rather, to assess the reproducibility of the solvent, the variation observed within each sample condition was determined to be of interest. As shown in Table 7, there exists little sample variation within each solvent condition as denoted by the low % RSD values. While these values are slightly elevated relative to the initial, they are still acceptably low. This suggests that the blocks are behaving similarly during the solvent swelling and drug uptake step of the process and can be confirmed through quantification of total extracted drug.
The slightly more controlled method of drying was utilized following removal from loading solvent, enabling the quantification of metrics after the drying step was completed due to lack of distortions observed in the samples. The obtained values are compiled in Table 8. As with the previous checkpoints, little sample variation was observed within each solvent condition. Furthermore, the blocks appeared to have returned to remarkably similar values in terms of dimension and mass, regardless of sample treatment.
Results of total drug loaded, weight percent loading and loading efficiency were quantified and calculated, shown in Table 9. The total drug loaded and weight percent loaded (determined by normalizing the extracted drug to the mass of the dried block) yielded values in line with those observed during the pilot study. The pre-loaded maximum for β-Estradiol was determined to be 10 wt. % and the below values indicate that the post-loading method was roughly on par. Importantly, the loading procedure yielded minimal sample variability, particularly with methanol and acetone, suggesting the developed method to be robust and reproducible. Finally, the loading efficiencies, calculated utilizing the [β-Estradiol] of the loading solution following immersion, indicated similar uptake between solvents. The post-loading solution was super saturated with β-Estradiol; therefore, it is possible that these efficiencies could improve with an optimized concentration of loaded drug.
The release properties of parts both pre-and post-loaded with β-Estradiol were investigated in simulated vaginal fluid (SVF). All parts were between 800-900 mg and therefore the volume of SVF used for all release studies was 60 mL. Aliquots of 1 mL were removed for HPLC analysis and the medium replenished. Analysis was conducted against β-Estradiol standards prepared in ACN and peaks quantified at 280 nm.
Diffusion blocks fabricated with a pre-loaded SIL 30 resin containing 1 wt. % β-Estradiol. Blocks were placed in SVF and aliquots analyzed via HPLC. The resulting profiles are shown in
The release kinetics of diffusion blocks post-loaded as a function of solvent type were quantified similar to the pre-loaded blocks. The resulting analyses are shown in
The percent cumulative release of the post-loaded blocks was plotted against the pre-loaded average for the first four days, shown in
In a similar post-loading study was conducted on Estring sections of comparable mass to the diffusion blocks (n=1). The cumulative release by μg and cumulative percent release is shown in
The ability to successively load a series of blocks utilizing the same loading solution was investigated. The post-loading solvent procedure utilizes 20 mL of solution per block, which depending on the solvent, will uptake between 1-2.5 mL of solvent. Therefore, the 20 mL, while reduced, is not fully used up in a single loading cycle. Given the potential monetary savings, it would be ideal to recycle or reuse the post-loading solvent for successive loading.
The procedure of successive loading is outlined in
Throughout the post-loading process, blocks were assessed utilizing methods established in Section 2 and 3. To identify differences in loading during the iterative process, blocks were evaluated based on uptake and extraction.
Solvent uptake was calculated following removal from the post-loading solution and values compiled as degree of swelling in Table 10. No true trend in terms of decreased degree of swelling was observed for the dimensions of the blocks as a function of loading order. However, there was a notable decrease in percent mass increase as loading order progresses. It should be noted, however, that the variability is percent mass uptake is fairly small at 3.74%. Overall, there was minimal indication that the behavior of the blocks in the loading solution varies significantly as a function of loading order.
Blocks were further assessed by quantifying the extracted drug via HPLC. Values obtained are compiled in Table 11. The total amount of drug extracted, weight percent loading and loading efficiency were calculated and averaged, with variation described by % RSD. Overall, a slight decrease in loading metrics was observed as a function of loading order. However, once averaged out, these values yielded minimal variability. Additionally, values shown are in-line with those obtained during the reproducibility study (Section 3.2) for methanol.
Within error, minimal differences were observed in drug loading of successively loaded blocks as a function loading order were observed. This study was conducted using a single solution of 20 mL for four cycles. This represents approximately 8.5-10 mL of solvent uptake leaving 10 mL necessary to fully coat the bottom of the sample container. Given the residual volume, more iterations could be possible however is constrained by the sample container. Thus, it could be possible to recycle the post-loading solution for several cycles rather than reloading the same sample container.
The successive loading study iteratively loaded four diffusion blocks with a single loading solution and represents a pilot concept to reuse loading solution. Samples yielded similar uptake and total loading suggesting that the successive loading is feasible. Additionally, the values obtained are in-line with the reproducibility study for methanol. A likely application would be toward the recycling of post-loading solutions given the consistent loading between cycles.
Solvent swelling analysis indicated the uniform swelling and shrinking of the matrix but it was necessary to understand if API uptake would be homogenous therefore blocks were exposed to dyes to allow for visualization of the loading process. Blocks (Xi=4.0 mm) were exposed to a rhodamine B (RhB) methanol solution for 24 hrs as shown in schematic
To further investigate the visualization of the post-loading process, three exposure conditions were generated in methanol: hydrophobic dye (rhodamine B, RhB), hydrophilic dye (nile blue A, NBA) and hydrophobic/hydrophilic combination (RhB/NBA). These solutions, starting at 80 μg/mL were serial diluted down to 5 μg/mL, resulting in 5 solutions per dye condition. It should be noted that the combination solution held total dye amount constant resulting in half of the initial individual dye concentration. Blocks of the 4.0 mm type (n=3 condition) were exposed to these solutions for 24 hrs. and removed. Upon removal, blocks were bisected, as indicated by the dashed line in
Initial tests investigated post-loading parts as a function of material (EstRing, NuvaRing, SIL 30 block) in a Rhodamine B/Methanol solution. The analysis was further expanded to an investigation as a function of RhB concentration in methanol for SIL 30 blocks. Swelling metrics and extracted RhB were quantified as a function of loading concentration.
To aid in the understanding and explanation of post-loading, it was necessary to take a step back and utilize a dye to visually track the penetration of the drug. Therefore, rhodamine B (RhB) was used in place of the model drug in the post-loading process using previously optimized parameters. As shown in
The SIL 30 resin, due to its unique crosslinking process, was hypothesized to react differently to the post-loading process compared to standard IVR materials such as silicone and EVA. Therefore, as a proof of concept, post-loading was conducted as a function of material in an 80 μg/mL RhB/MeOH solution using previously optimized loading parameters. Materials included placebo EstRing sections, NuvaRing sections, and SIL 30 4 mm blocks. All samples were sectioned to have similar masses with initial materials shown in
Parts were quantified for gel fraction and degree of swelling following the post-loading process in RhB, with results shown in Table 12 as a function of material type. It should be noted that in this case gel fraction represents the ratio of the dry mass to initial mass as the post-loading process is not a true soluble fraction removal. The degree of swelling was calculated using swollen and dry masses of the parts. Both the NuvaRing section and Estring section yielded minimal gel fractions and minimal swelling. The post-loading process relies on swelling the structure to incorporate API, and therefore these results concur with the lack of visually observable RhB on the parts. Conversely, the SIL 30 block yielded a lower gel fraction on par with previous results and a high degree of swelling, corroborating the intensity of RhB observed in the swollen part.
The variability in the Estring sample was concerning. It was previously observed that the RhB, following removal from the post-loading solution, was easily wiped away, suggesting an adsorption process. Previous experiments with EstRing sections in MeOH found a gel fraction of 0.97±0.00 and a degree of swelling of 2.89±0.28%. These previous observations of low variability clash with the present experiment. All three Estring sections following post-loading are shown in
Experiments with post-loading as a function of material type indicate that RhB in MeOH serves as a useful tool to visualize the post-loading process. Commercially available materials such as silicone and EVA were found to be incompatible with the post-loading process. The Estring sections at best exhibit surface adsorption of API that was unpredictable and wildly variable. Conversely, the SIL 30 blocks exhibited complete and homogenous incorporation of RhB, as shown in the cross-section. Collectively, these findings corroborate what was previously observed in post-loaded release studies. These data suggest that SIL 30, likely due to its crosslinking process, is uniquely amenable to the post-loading process developed.
To further investigate the visualization of post-loading, SIL 30 blocks were post-loaded as a function of RhB concentration in MeOH. The process is shown in
It should be noted that the initial intention of these samples was to image using fluorescence microscopy. Therefore, initial RhB concentration values were conservative due to concerns of saturating the detector. However, the uptake of RhB was so clearly visual and worked so well, that fluorescence detection would not have been compatible. Therefore, samples were assessed during the post-loading process and the RhB extracted for quantification.
Metrics of the blocks treated as a function of post-loading solution concentration were tracked enabling the calculation of gel fraction* and degree of swelling. As previously mentioned, the gel fraction in this case merely represents the ratio of dry mass to initial mass. Given that both dimensional and mass metrics are tracked during the loading process, degree of swelling can be calculated both on the primary azimuthal axis's as well as traditionally with mass. These values are shown in Table 13 for each loading concentration as well as pooled. The homogenous absorption of RhB was observed in the nearly uniform gel fractions*. The minimally variable degree of dimensional and mass swelling suggests that increasing RhB concentration does not affect the post-loading mechanism of SIL 30. That is, the blocks behave similarly regardless of loading concentration.
Finally, blocks loaded as a function of RhB concentration were extracted in EtOH and 200 μL aliquots were analyzed for fluorescence via plate reader. A standard curve of RhB was prepared in EtOH. Each standard and sample were run in triplicate. The quantification of RhB was done using the averaged standard curve (R2=0.992, EtOH evaporation contributed to error). The results are shown in
A simplified system of diffusion blocks was investigated for properties during the post-loading process. Three types of block preparation were investigated: those treated to a simulated loading process in the absence of drug, those pre-loaded with β-estradiol, and those post-loaded with β-estradiol. Swelling properties were compared between simulated and actual post-loaded blocks. Quantification metrics of pre-loaded and post-loaded blocks were evaluated and release profiles in SVF were obtained.
The diffusion distance within the unit cell of the IVR may dictate in part the rate of release. The overarching goal of this research was to obtain a better understanding of the contribution of diffusion distance on release rate to enabled more targeted designs. Therefore, a simplified system involving diffusion blocks fabricated as a function of distance was developed. Shown in
It was necessary to establish the swelling characteristics of blocks fabricated as a function of distance in the absence of drug to determine baseline behavior. Outlined in
The quantification metrics collected throughout the experiment including dimensional and mass information are compiled in Tables 14-16 for initial, swollen, and dried states, respectively. Average and standard deviation values were calculated within each condition. For the X and Y dimensions, which were not varied during fabrication, the entire series for each solvent was compiled, resulting in a ‘combined’ value, shown in italics. The initial values in Table 14 include a percent deviation in Z from CAD calculation. This value describes the difference in the Z axis of the fabricated structure from the specified CAD dimension. It can be seen that as diffusion distance decreases, both deviation and variability increase. Because these blocks were taken from the same batch and then divided, the variability and deviation are expected to be similar for each solvent group. Accounting for error finds this to be true. Therefore, it can be concluded that the sample sets, while deviating and variable, do so similarly and with a predictable trend.
The swollen metrics were combined and calculated in a similar manner in Table 15. Again, values for the X and Y dimensions were combined within each solvent type. The blocks appear to swell slightly more in methanol than acetone however this could be a productive of part geometry. The swelling dimensions in X and Y appear to trend with the decreasing diffusion distance. Interestingly, when combined, the values indicate that the blocks, regardless of Z distance, swell similarly in the X and Y dimensions. This was encouraging as it suggests a device could potentially have multiple distances (i.e. band distances and unit cell distances) and swelling behavior would not be lopsided or uneven. It should be noted that the degree of swelling by percent decreases with decreasing diffusion distance. It is likely that this is a product of the decreasing surface area to volume ratio. Calculations are currently being conducted to test this theory.
Finally, the dried metrics are shown in Table 16 as a function of diffusion distance. Here, percent deviation from CAD represents the degree of deviation of the dried structures from the original CAD dimensions. For methanol, the final dimensions in X are similar to the initial dimensions and the Y dimensions are collectively smaller. For acetone, both the X and Y dimensions are collectively smaller. Within error, the deviation from CAD was largely the same for both methanol and acetone.
The above data can be used to calculate degrees of swelling for the X, Y and Z dimension using the following equation:
where A represents values in a given dimension. These values were plotted as a function of diffusion distance shown in
The purpose of this study was to gage gel fraction as a function of diffusion distance with methanol as a swelling solvent. This does not represent a true soluble fraction removal as the soak time was limited to 24 hours as opposed to the previously determined 4-6 days. The calculation of gel fraction instead allows the inference of how much potential extractables can be removed from the part during the post-loading process. This is a critical metric to understand given that the parts do contain a sizable percentage of unreacted/unincorporated monomer/oligomers. Minimizing leachables remains a primary concern. Therefore, understanding what ‘comes out’ during the post-loading process can be helpful. These values are shown in
It was important to understand how the metrics obtained during the simulated loading process compared to metrics obtained during the actual post-loading of blocks as a function of diffusion distance. Shown in
Finally, the gel fractions of unloaded and loaded samples were compared as a function of diffusion distance and compiled in Table 17. The percent increase in gel fraction from the presence of drug was also calculated. The gel fraction values for the loaded series are higher, which was to be expected given the calculation does not represent a true soluble fraction removal. It was interesting that the percent increase, while positive for all distances, was not constant nor does a trend emerge. Rather, the values range from 2-10%.
Blocks as a function of diffusion distance were fabricated both pre-loaded and unloaded (for post-processing loading). Pre-loaded blocks were fabricated with 4.0 wt. % β-estradiol. Previous post-loading experiments found 4 mm blocks could be loaded between 4-6 wt. % however β-estradiol solubility was limited in SIL 30, therefore, to be conservative, the lower loading limit was selected. Initial metric values for pre-and un-loaded blocks are shown in
Unloaded diffusion blocks were post-loaded with a super saturated solution of β-estradiol in three individual batches, designated by loading round. The differences in the loading rounds are identified in Table 18. The original post-loading method utilized glass jars to prepare the samples. However, for the large number of samples used in the diffusion distance set, 50 mL falcon tubes were used in Round I. It was observed that the tubes inhibited the swelling of the blocks. Therefore, Round II and Round III were prepared according to the original protocol. Round I and Round II were used for extraction and Round III used in a release study. The gel fraction and degree of swelling metrics were pooled for each block dimension and compiled in Table 18. The compiled values represent a total of 11 samples. Gel fraction values simply represent the ratio of dried to initial mass. The degree of swelling was calculated using the previously mentioned equation. Collectively there was a decreasing trend in gel fraction and degree of swelling with decreasing diffusion distance. This was previously observed with the unloaded samples. It was interesting to see the variability, as shown by the percent relative standard deviation, increases in the Z dimension as the diffusion distance decreases. The variability was previously noted as being initially significant and therefore it can be seen that the error compounds during the post-loading process.
Both pre-loaded and post-loaded blocks were extracted in 20 mL of EtOH for 72 hours. Aliquots were taken and analyzed via HPLC. The weight percent loadings are shown in
Weight percent loadings between pre-and post-loaded blocks are compared in
A proposed utility of the post-loading method is as a drug incorporation process for 3D-printed intravaginal rings (IVRs). Given the matrix can be purposely swollen to allow for drug uptake, it was necessary to investigate the swelling behavior of the SIL 30 material under relevant in vitro release conditions. All block types (0.5-7.6 mm, n=3 of each) were immersed in acetone for 24 hrs, removed and allowed to air dry, mimicking the proposed post-loading process in the absence of API. Once dried, metrics (mass and dimensions) were taken, and blocks were immersed in simulated vaginal fluid (SVF) at 37° C. Blocks were tracked daily for the first two weeks of exposure and weekly following for 28 days. Given the aqueous nature of the SVF, comparable swelling to acetone or methanol was not anticipated, however because of the unique curing conditions of the resin and the elevated temperature, some swelling was expected. The experimental mass and calculated volume of the blocks as function of exposure time in SVF are shown in
To assess the extent of the swelling in SVF, percent increases were computed for each block type from day 0 (initial) to day 28. The percent mass increase and diffusion distance (X dimension) are shown in
Finally, total loading values of β-estradiol were determined as a function of diffusion distance for both pre-and post-loaded blocks. These values are compiled in
Blocks prepared both by pre-and post-loading methods were evaluated for release characteristics in simulated vaginal fluid S(VF). Blocks were placed in 60 mL of SVF and 1 mL aliquots taken as a function of time. Aliquots were analyzed for β-estradiol via HPLC. Representative chromatograms obtained are shown for both pre-and post-loaded samples in
The release profile for the first four days was obtained for pre-loaded samples, shown in
The release profiles were obtained for post-loaded samples, shown in
The post-loading process was investigated by incorporating small molecule model drugs or drug surrogates into the SIL 30 matrix. Two model drugs were selected: β-Estradiol (β-Est) a hydrophobic (log p of 3.75) hormone used in the treatment of menopause and commercially available in an IVR (EstRing) and 2′fluoro-2′-deoxyadenosine (FdA), a hydrophilic (log p of −0.57) analog to the nucleoside reverse transcriptase inhibitor (NRTTI) Islatravir. Blocks of all types were immersed into acetone solutions containing 5 mg/mL of either β-Est or FdA for 24 hrs. Blocks were then extracted using two cycles of acetone to determine total drug loading, shown in
To investigate model drug loading further, the weight percent loading per block type was investigated. In
It has been demonstrated that post-loading results in a generally homogenous distribution within the SIL 30 polymer matrix, however as with all absorption methods, the possibility that API will release in one initial burst rather than a sustained delivery. In vitro release testing in simulated vaginal fluid (SVF) was conducted to assess the capacity of blocks loaded with a model drugs for sustained release. Cumulative release profiles in micrograms are shown for β-Est (
Initial analysis indicates the release rates appear to be driven by block distance however these blocks are not infinite planes and therefore additional geometric factors must be accounted for. For a drug eluting toroidal device, release is often normalized by the cross-sectional diameter of the torus, particularly when utilizing the mathematical relationships between diffusion coefficient (Df) and drug solubility (Cs) established in the Higuchi Model [36, 37]. Therefore, as with analysis of total drug uptake, it was necessary to account for the geometry of the block given that they are not and were not intended to mimic infinite planes as seen in theoretical modeling [37]. Cumulative release was normalized per block type using the equations derived from
Cumulative release profiles were quantitatively assessed for β-Est (Table 20) and FdA (Table 21) by experimental diffusion block distance (as opposed to theoretical block type distance). Drug loading and weight percent loading from
A defining feature of any drug-delivery device is the percent burst release and for long-acting devices, it is necessary that this value is low to sustain delivery performance. Blocks loaded with model drugs were assessed for burst release, defined as amount eluted from the device in the first 24 hrs. Microgram and percent burst for β-Est and FdA are shown in
The role of specific surface area in the behavior of the blocks has been previously demonstrated as important, and therefore must be accounted for in the swollen SVF state. This was done for β-Est (
To demonstrate the utility of post-fabrication drug absorption, and by way of example only and not limitation, the post-loading method was translated into a medical device using the model drug β-Estradiol (β-Est). As previously described, geometrically complex IVRs represent an attractive next-generation medical device for sustained drug delivery [33]. The incorporation of design complexity via CAD has been proposed to enable the control of release characteristics such as release rate and duration. Therefore, to assess the value of controlling for diffusion distance via geometric complexity, four unit cells were developed and explored. The theoretical values associated with the resulting geometrically complex IVRs are shown in
Rings were loaded with a model drug, β-Est, at 22 mg/mL in methanol for 24 hrs. Methanol was selected for ring exposure given the higher boiling point relative to acetone and thus, a slower evaporation from the SIL 30 matrix. There is a risk of interior architecture disruption if the swelling and drying of the polymer matrix is too rapid. Therefore, the selection of MeOH was a precautionary step and as with previous results, the post-loading method should be investigated for extension to other Class II/III solvents such as ethanol. IVRs were tested for in vitro release in SVF medium. The cumulative release profiles are shown in
Rings were fabricated on an M1 DLS (Carbon, Inc.) 3D-printer. Rings were uploaded to the Carbon UI, orientated vertically and supported with approximately 40 supports per ring. Rings were fabricated in a silicone-based polyurethane resin (SIL 30). This is a two-part resin that was dispensed immediately prior to printing using the Carbon-supplied static mixer. Each print consisted of 16 rings, vertically oriented, fabricated using approximately 150 mL of SIL 30. Carbon's pre-optimized and standardized exposure parameters for SIL 30 were used and yielded a fabrication time of 3 hr. 9 min. Rings were removed with a razor following fabrication and the supports separated. Rings were exposed to two 30 s cycles of approximately 200 mL of IPA per ring using a previously optimized cleaning process. SIL 30 rings were allowed to air dry for 45 min and then placed in a programmable oven for 8 hr. at 120° C., as prescribed by Carbon (
In the preloaded IVRs, a pre-determined amount of β-estradiol was mixed in with the resin prior to the printing process. An aliquot of the resin was used to determine analytical drug concentration in the resin. Placebo IVRs were printed using identical parameters as the drug loaded IVRs. Masses and dimensions were recorded before drug incorporation using absorption. A pre-determined amount of β-estradiol was dissolved in acetone and the placebo IVRs were placed in this solution for 24 hours. These were later removed and dried in a fume hood for 24 hours. Once the mass has plateaued, the dimensions and final masses were recorded.
The concentration of β-estradiol+acetone solution was determined by the use of the loading equation. There is a linear relationship between the API-Solvent concentration and the amount of API incorporated in the SIL30 3D printed IVR. This linear relationship has been used to derive the loading equation. The maximum loading achieved is 13.6 mg β-estradiol/1000 mg of SIL30 3D CLIP printed IVR, using acetone as the loading solvent, and the loading duration of 24 hours. The IVRs were removed after 24 hours and dried in a fume hood until IVR mass has plateaued; the dimensions and final masses were recorded.
Studies to measure in vitro release of β-estradiol into a simulated vaginal fluid (SVF) were carried out on 3D-IVRs (n=4). The SVF consisted of 25 mM sodium acetate buffer (pH 4.2) plus 2% Solutol (Kolliphor HS 15). The IVRs were placed in straight-sided glass jars containing 200 mL SVF at 37±2° C. 1 ml aliquots of the release medium were removed at specified time intervals and complete media changes were carried out to maintain sink conditions. The concentration of β-Estradiol in the aliquots was determined using an Agilent 1260 HPLC with a Diode Array Detector, on an Inertsil ODS-3 column (4.6×150 mm, 5 μm) maintained at 40° C., with a flow rate of 1.0 mL/min, 25 ul sample injection, and an acetonitrile/water mobile phase, each modified with 0.1% trifluoroacetic acid. A gradient method was utilized to achieve separation (0-20 min: 5%-100% acetonitrile; 20-22 min: 100% acetonitrile; 23-25 min: 5% acetonitrile). β-Estradiol was eluted at 13.8 min. Data collected at 280 nm was computed using Chemstation software, and concentrations were derived from a calibration curve generated using β-Estradiol standards prepared in 100% acetonitrile (250 ug/ml-61 ng/ml).
The preload process most closely resembles the matrix IVR design used in many marketed IVRs. Keeping the API loading the same in preload and postload IVRs, we did a head-to-head comparison of the two different loading techniques. A zero-order release kinetics pattern was observed in both groups, as can be observed in
Having established that both these methods provide the same in-vitro release profiles, we wanted to further investigate the post loading method. The Cyl-3.80 and HC-2.53 IVR designs were post-loaded with β-estradiol, and their release profile is shown in
MPT IVRs for prevention of HIV, HSV-2 and unplanned pregnancy are being developed using our 3D printed IVR technology.
Three (3) APIs were loaded in a single IVR using our post-loading method. First, loading equations were developed and validated for each API (
Another MPT IVR for prevention of HIV and unplanned pregnancy has been developed with an anti-HIV drug (Islatravir, EFdA) and the NuvaRing contraceptive combination (etonogestrel/ethinylestradiol, ENG/EE). All drugs were successfully loaded in a single IVR using our post-loading method to achieve target loading for each drug and target release rates for over 4 months (last time point analyzed) (
Macaque size (25 mm OD) 3D printed IVRs post-loaded with EFdA at 45 mg/IVR or 62 mg/IVR were administered to female pigtailed macaques and plasma, PBMCs, vagina and rectal fluids, and vaginal and rectal biopsies were collected over 28 days and analyzed for EFdA concentration and EFdA-TP (triphosphorilated EFdA, EFdA-TP) in PBMCs. Results showed that PK levels were dose dependent with higher levels achieved with the 62 mg EFdA IVR compared to the 45 mg EFdA IVR. More importantly EFdA-TP levels in PBMCs and EFdA levels in tissue and fluid samples were above the known efficacious levels based on preclinical studies in NHP and human clinical trials with oral EFdA (
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It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application is a divisional of U.S. patent application Ser. No. 18/026,969 filed on Mar. 17, 2023, which is a 371 of International Application No. PCT/US2021/050949 filed on Sep. 17, 2021, which claims benefit of and priority to U.S. Provisional Patent Application Ser. No. 63/079,953, filed Sep. 17, 2020, the disclosures of which are incorporated herein by reference in their entireties.
This invention was made with government support under Grant Numbers HD100190, AI136002 and AI150358 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63079953 | Sep 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18026969 | Mar 2023 | US |
| Child | 18591127 | US |
