WO2019236653A1

WO2019236653A1 - System and method for removing phenolic preservations from insulin formulations

Info

Publication number: WO2019236653A1
Application number: PCT/US2019/035494
Authority: WO
Inventors: Francis Buan HONG LIM; Obdulio PILOTO; Ian Shen-Yi CHEONG
Original assignee: Entopsis Llc
Priority date: 2018-06-06
Filing date: 2019-06-05
Publication date: 2019-12-12

Abstract

The present invention comprises a method for removing or depleting phenolic preservatives found in insulin formulations in order to prevent inflammation at the site of infusion in a diabetic patient. Particularly, the disclosed embodiments include the separation device, system for removing phenolic preservatives, and separating phenolic preservatives from insulin, and methods of making and using the same. A phenolic preservative separation device comprises: a vessel able to store a liquid insulin formulation comprising a phenolic preservative and an insulin; and, a physical substrate stored within the vessel, the substrate comprising at least one macromolecule able to selectively bind and remove from the liquid about 60-99% of the phenolic preservative within the insulin formulation, wherein the insulin does not bind to, or binds 10% or less to, or about 1-5% to, the physical substrate.

Description

System and Method for Removing Phenolic Preservations from Insulin Formulations

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to United States provisional application Serial No. 62/681,167 filed on June 6, 2018, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to a device and method for removing or depleting phenolic preservatives from insulin formulations.

COPYRIGHT NOTICE

[0003] A portion of the disclosure of this provisional patent application document contains material that is subject to copyright protection. The copyright owner-inventor-assignee has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever. All copyrights disclosed herein are the property of their respective owners.

TRADEMARKS DISCLAIMER

[0004] The product names used in this document are for identification purposes only. All trademarks and registered trademarks are the property of their respective owners.

BACKGROUND OF THE INVENTION

[0005] For patients afflicted with diabetes, continuous subcutaneous insulin infusion (CSII) therapy is the most advanced form of insulin administration because it provides superior control over a patient’s fasting glucose, post-prandial blood glucose (BG) and HbAlC values. However, current CSII sets have lifetimes of only two or three days due to increased likelihood of adverse events that include variable insulin uptake, inadequate control over BG levels, intermittent hyperglycemia and ketosis, scarring and thinning or thickening of subcutaneous fat [1-6]. Insulin formulations require phenolic preservatives such as phenol or meta-cresol to maintain insulin stability and sterility [7]. Data however suggests that (i) phenolic preservatives induce local inflammation at the site of administration and (ii) this inflammation at the site of infusion significantly alters insulin pharmacokinetics [1-6]. Consequently, in order to reduce site inflammation, materials and methods are required which selectively remove phenolic preservative compounds prior to insulin delivery. By thus extending the functional lifetime of CSII infusion sets, insulin infusion biocompatibility and patient outcomes are hence improved.

SUMMARY OF THE INVENTION

[0006] At the heart of the present invention lies the observation that polymeric acrylates and acrylamides may be designed to bind phenolic preservatives with high binding affinities, but also to have a low binding affinity to insulin.

[0007] In accordance with the invention, the problem of removing or depleting phenolic preservatives of phenol, or meta-cresol, or both, found in insulin formulations is solved by using a physical substrate that selectively binds to phenolic compounds.

Phenolic Preservative Binder or Separation Device

[0008] The present disclosure comprises various embodiments of a phenolic preservative binder, e.g. a separation device, comprising: 1) a vessel or receptacle for holding an insulin sample; and, 2) a physical substrate within the vessel/receptacle, wherein the physical substrate comprises at least one macromolecule for selectively binding and removing about 60-99%, or 70-99% of a phenolic preservative from the insulin sample; and, wherein the insulin does not bind to, or binds to 10% or less of, or about 1-5% of, the physical substrate.

[0009] The vessel or receptacle comprises one or more of: a plastic microcentrifuge tube, a plastic 96 well plate, a plastic test tube, a glass test tube, and a glass vial, or any similar container able to hold the insulin sample and the macromolecule.

[0010] In an embodiment, the insulin sample is Insulin Lispro, e.g. the drug known as Humalog™.

[0011] The phenolic preservative comprises one of: a phenol; a meta-cresol; or a phenol and a meta-cresol. [0012] In an embodiment, the physical substrate is a macromolecule comprising one or more of: acrylamide, glycerol and a crosslinker, wherein the crosslinker comprises N,N’- Methylenebisacrylamide.

[0013] In another embodiment, the physical substrate is a macromolecule comprising at least one copolymer, wherein the copolymer comprises one or more monomers selected from a group consisting of: glycerol, acrylamide, hydroxypropyl acrylate isomers, 4-hydroxybutyl acrylate, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate.

[0014] In another embodiment, the physical substrate is a macromolecule comprising a polymer consisting of hydroxypropyl acrylate isomers and N-isopropyl acrylamide, and in a ratio of about 50:50 to 30:70.

[0015] Additionally, the physical substrate is in the form of a solid hydrogel that is polymerized by Ultra-Violet exposure with the photo-initiator DMPA.

Physical substrates

[0016] In all embodiments, phenolic preservatives, such as phenol or meta-cresol in the insulin sample, bind to the physical substrate located within the vessel/receptacle of the separation device, but the insulin does not bind to the physical substrate.

[0017] In an embodiment, the physical substrate comprises a macromolecular matrix; and/or is in the shape of: a dome, sphere, sheet, pieces, slurry or any similar configuration.

[0018] In an embodiment, the physical substrate is a crosslinked copolymer comprising: acrylamide, N,N’-Methylenebisacrylamide, glycerol and one or more derivatives of acrylamide and acylate.

[0019] In another embodiment, the physical substrate is a macromolecule comprising at least one copolymer, wherein the copolymer comprises one or more monomers selected from a group consisting of: glycerol, acrylamide, hydroxypropyl acrylate isomers, 4-hydroxybutyl acrylate, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate.

[0020] In another embodiment, the physical substrate is made by mixing hydroxypropyl acrylate isomers and N-isopropyl acrylamide in a ratio of about 50:50 to 30:70 in a container such as a 2mL microcentrifuge tube, or a 96 well plate, and adding one volume of DMPA photo-initiator, and subsequently polymerizing under UV light.

[0021] In another embodiment, the physical substrate is made by mixing N-isopropyl acrylamide, hydroxypropyl acrylate isomers and 4-hydroxybutyl acrylate in a ratio of about 20:40:40 to 25:37.5:37.5 in a container, such as a 2m 1. microcentrifuge tube or 96 well plate; and then adding one volume of DMPA photo-initiator, and subsequently polymerizing under UV light.

[0022] In another embodiment, the physical substrate is made by mixing hydroxypropyl acrylate isomers, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate, and in a ratio of about 85:7.5:7.5 to 90:5:5 in a container, such as a 2mL microcentrifuge tube or 96 well plate, and adding one volume of DMPA photo-initiator, and subsequently polymerizing under UV light.

Method of Removing Phenolic Preservatives from Insulin

[0023] The present invention further comprises a method for removing phenolic preservatives from a liquid insulin formulation, comprising the steps of: 1) providing a liquid insulin formulation comprising at least one phenolic preservative and an insulin; and 2) binding the physical substrate specifically to the phenolic preservative in such a manner that the phenolic preservative is removed from the liquid insulin formulation without binding the insulin.

Method of Reducing Inflammation in Insulin Infusion

[0024] The present disclosure comprises a method of reducing inflammation at the dermal site of an insulin infusion in a diabetic patient, comprising the steps of: 1) providing a phenolic preservative separation device, comprising a vessel/receptacle able to store a liquid insulin formulation, and a physical substrate within the vessel or receptacle that is able to bind at least one phenolic preservative with binding insulin; 2) combining the physical substrate and the liquid insulin formulation in the vessel, in a ratio of 1:1, at room temperature for at least 5 minutes, wherein the phenolic preservatives are bound (or bind) to the physical substrate; 3) removing a supernatant from the vessel comprising the insulin formulation without the phenolic preservatives; and 4) infusing the supernatant into a diabetic patient at a dermal site, wherein no inflammation of the dermal site occurs from the infusion due to the absence of phenolic preservatives in the supernatant.

[0025] In one embodiment, phenol or meta-cresol is dissolved in IX PBS at concentrations of 10 mg/mL, 5 mg/mL, 3 mg/mL, 1 mg/mL and Omg/mL, and reacted with 10% FeCh to form colored solutions. The standard curve is constructed by determining the relative intensity units of the colored solutions with ImageJ analysis. [0026] In another embodiment, phenol or meta-cresol binds to a physical substrate. The recovered supernatant is reacted with 10% FeCh to form colored solutions, and the amount of binding is then ascertained by Image J and Standard curve analyses.

[0027] In another embodiment, meta-cresol in the insulin formulation LISPRO is dissolved in IX PBS at concentrations of 3.l5mg/mL, 2 mg/mL, l.575mg/mL, 1 mg/L, 0.5mg/mL and Omg/mL, and reacted with 10% FeCh to form colored solutions. The standard curve is constructed by determining the relative intensity units of the colored solutions with ImageJ analysis.

[0028] In another embodiment, the phenolic preservative of meta-cresol found in the insulin formulation LISPRO binds to a physical substrate. The recovered supernatant is reacted with 10% FeCh to form colored solutions, and the amount of binding then ascertained by Image J and Standard curve analyses.

[0029] Various objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] For a more complete understanding of the invention, reference is made to the following description and accompanying color photographs, in which:

[0031] FIG. 1 depicts an exemplary Phenolic Preservative Binder Device comprising a plastic microcentrifuge tube vessel with a macromolecule at or near the bottom of the vessel in a solution of phenol, meta-cresol, or the Insulin formulation LISPRO, and the macromolecule is able to bind and remove the phenolic preservatives from the solution. lOOuL of polymer is located at the bottom of the tube. For the binding studies, lOOuL of phenol, meta-cresol or the Insulin formulation LISPRO is added to the polymer and incubated at room temperature for 5 minutes. Samples are then recovered by transferring 80uL to new tubes containing 8uL of 10% FeCh to develop the color, and the relative intensity units are then determined by Image J analysis.

[0032] FIG. 2 depicts the phenol standard curve. 80uL of phenol at concentrations of lOmg/mL, 5mg/mL, 3mg/mL, lmg/mL and Omg/mL were reacted with 8uL of 10% FeCh to develop colored solutions. The varying colored intensities (ranging from purple to yellow) were then ascertained in relative intensity units by using Image J analytical software to construct the standard curve.

[0033] FIG. 3 depicts the meta-cresol standard curve. 80uL of meta-cresol at concentrations of lOmg/mL, 5mg/mL, 3mg/mL, lmg/mL and Omg/mL were reacted with 8uL of 10% FeCL to develop colored solutions. The varying colored intensities (ranging from purple to yellow) were then ascertained in relative intensity units by using Image J analytical software to construct the standard curve.

[0034] FIG. 4 depicts the meta-cresol standard curve of the Insulin formulation LISPRO. 80uL of LISPRO at concentrations of 3.l5mg/mL, 2mg/mL, l.575mg/mL, lmg/mL, 0.5mg/mL and Omg/mL were reacted with 8uL of 10% FeCL to develop colored solutions. The varying colored intensities (ranging from purple to yellow) were then ascertained in relative intensity units by using Image J analytical software to construct the standard curve.

[0035] FIG. 5 depicts the Human Insulin ELISA standard curve. The ELISA kit (cat no. ab2000l l) and standard protocol was obtained from abeam® (Cambridge, MA).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0036] Unless otherwise defined herein, all terms used in this application are to be afforded the usual meaning in the art, as they would be understood by a person of ordinary skill at the time of invention. It should be understood that throughout this application singular forms, such as“a”,“an”, and“the” are often used for convenience; however, singular forms are intended to include the plural unless specifically limited to the singular either explicitly or by context.

[0037] The term“about” as used herein refers to plus or minus 5 percent of the stated amount, or range of values.

[0038]“Macromolecules” include oligomers, polymers, dendrimers, nanospheres, nanotubes and the like.

[0039]“Small molecules” include biologically or environmentally relevant molecules having a molecular weight lower than that of macromolecules.

[0040]“Phenol” is phenol dissolved in PBS at concentrations of lOmg/mL, 5mg/mL, 3mg/mL and lmg/mL.

[0041]“Meta-Cresol” is meta-cresol dissolved in PBS at concentrations of lOmg/mL, 5mg/mL, 3mg/mL and lmg/mL.

[0042]“LISPRO” is an Insulin formulation used in this study and contains the phenolic preservative meta-cresol at a concentration of 3.l5mg/ml. For the standard curve determination, LISPRO was diluted in PBS at concentrations of 3.l5mg/mL (undiluted), 2mg/mL, l.575mg/mL, lmg/mL, 0.5mg/mL and Omg/ml.

[0043]“DMPA” is 2,2-dimethoxy-2-phenylacetophenone and is a photo-initiator that produces free radicals upon UV light exposure resulting in polymerization of acrylamide-based polymers.

[0044]“DWG solvent” consists of DMSO, water and glycerol in a ratio of 88:10:2 and is the solvent used to dissolve the DMPA photo-initiator powder.

[0045]“DMPA Photo-Initiator Solution” is 20 to 50mg of DMPA dissolved in DWG solvent. One volume of this solution is then added to the acrylamide-based monomer mix to enable polymerization under UV light exposure.

[0046]“PBS’ is Phosphate buffered saline (lOmM Na₂HP0₄, 2mM KH2PO4, l37mM NaCl, 2.7mM KC1, pH 7.4)

[0047]“FeCL” is Iron (III) Chloride that is dissolved in PBS to a concentration of 10%. It reacts with phenol or meta-cresol to form colored solutions, whose relative intensities can be ascertained by Image J analytical software.

[0048]“Human Insulin ELISA” is a kit purchased from abeam® (cat no: ab2000ll) to determine the amount of insulin in the LISPRO insulin product that is bound by the polymers.

[0049]“Image J’ is an image processing and analytical program developed at the National Institutes of Health. It supports standard image processing functions such as logical and arithmetical operations between images, contrast manipulation, convolution, Fourier analysis, sharpening, smoothing, edge detection, and median filtering. It can create density histograms and line plots. As disclosed herein, the ability of Image J is utilized to calculate area and pixel value statistics of user-defined selections and intensity-threshold objects to obtain the standard curves of phenol and meta-cresol, and the amount of phenol, or meta-cresol or phenol and meta-cresol in LISPRO that is or are bound respectively by the various polymers.

[0050] Various embodiments of the invention are described more fully hereinafter with reference to the accompanying figures, in which some, but not all embodiments are shown in the figures. Indeed, these inventions may be embodied in 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.

Phenolic Preservation Binder Device

[0051] FIG. 1 is a perspective view on representative but is not limited to other versions of the phenolic preservation binder device 100, otherwise known as the phenolic binding polymer separation device. Device 100 comprises a vessel 120, such as a plastic microcentrifuge tube, with a physical substrate, i.e. the macromolecule 130, which is located near the bottom of the vessel in a solution 140 that comprises the purified insulin. The macromolecule physical substrate is bound to the phenol preservatives that were in the original liquid insulin formulation, comprising: phenol, or meta-cresol, or the both. In all embodiments, the macromolecule is able to bind and remove about 70% or 60%, to about 99% of the phenolic preservatives from the insulin solution 140, while removing 0 to 10% of the insulin, such as 1- 5%.

[0052] In an embodiment, physical substrate 130 is a crosslinked copolymer comprising: acrylamide, N,N’-Methylenebisacrylamide, glycerol and one or more derivatives of acrylamide and acrylate.

[0053] In another embodiment, physical substrate 130 is a macromolecule comprising at least one copolymer comprising one or more monomers selected from a group consisting of glycerol, acrylamide, hydroxypropyl acrylate isomers, 4-hydroxybutyl acrylate, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate.

[0054] In another embodiment, physical substrate 130 is a macromolecule comprising a polymer consisting of hydroxypropyl acrylate isomers and N-isopropyl acrylamide, and in a ratio of about 50:50 to 30:70 with 1 volume of DMPA photo-initiator then added and subsequently polymerized under UV light.

[0055] In another embodiment, physical substrate 130 is a macromolecule comprising N- isopropyl acrylamide, hydroxypropyl acrylate isomers and 4-hydroxybutyl acrylate in a ratio of about 20:40:40 to 25:37.5:37.5 with 1 volume of DMPA photo-initiator then added and subsequently polymerized under UV light.

[0056] In another embodiment, physical substrate 130 is a macromolecule comprising hydroxypropyl acrylate isomers, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate, and in a ratio of about 85:7.5:7.5 to 90:5:5 with 1 volume of DMPA photo-initiator then added and subsequently polymerized under UV light.

[0057] KIT: The various embodiments of the present disclosure further comprise a kit comprising the Phenolic Preservation Binder Device 100, with printed instructions on how to use the kit, and/or a printed link to a website comprising the instructions. The kit may comprise a one-time use for at home, over-the-counter, or in a clinical setting; or for multiple uses. Method of Reducing Inflammation in Insulin Infusion Patients

[0058] The present disclosure comprises various embodiments of a method of reducing inflammation at the dermal site of an insulin infusion in a diabetic patient, comprising:

[0059] A method of reducing inflammation at the dermal site of an insulin infusion in a diabetic patient, comprising the steps of: 1) providing a phenolic preservative separation device, comprising a vessel able to store a liquid insulin formulation, and a physical substrate within the vessel, able to bind at least one phenolic preservative with binding insulin; 2) combining the physical substrate and the liquid insulin formulation in the vessel, in a ratio of 1: 1, at room temperature for at least 5 minutes, wherein the phenolic preservatives are bound to the physical substrate; 3) removing a supernatant from the vessel comprising the insulin formulation without the phenolic preservatives; and 4) infusing the supernatant into a diabetic patient at a dermal site, wherein no inflammation of the dermal site occurs from the infusion due to the absence of phenolic preservatives in the supernatant.

[0060] In another embodiment, the various embodiments of the separation device of the present disclosure may be part of a medical device used to prepare and/or infuse insulin into a patient, wherein the separation device is used to remove the phenolic preservatives from the insulin before it is infused to the patient.

EXEMPLIFICATIONS

[0061] In one embodiment, the vessel 120 of the separation device 100 is a plastic microfuge tube containing lOOuL of the physical substrate polymer 130 at the bottom and sides of the tube. lOOuL of 3mg/mL Phenol or meta-Cresol was incubated with lOOuL polymer in the plastic microfuge tube for 5 mins at room temperature. 80uL of the supernatant was then transferred to a new tube containing 8uL of 10% FeCb to develop the color, and the relative intensity units subsequently determined by Image J analysis. The relative intensity units for the incubation with the polymers correspond to the amount of phenol or meta-cresol that is left in the supernatant, which are then determined from the standard curve equations in FIGS. 2 and 3, respectively. The amount of phenol or meta-cresol that is bound by each polymer is determined by l-(Phenol or meta-Cresol Polymer Supernatant/ 3mg/mL Input) x 100% (Tables 1 and 2).

[0062] Table 1 shows the amount of phenol that is bound by the polymers. lOOuL of 3mg/mL Phenol was incubated with lOOuL polymer in the plastic microfuge tube for 5 mins at room temperature. 80uL of the supernatant was then transferred to a new tube containing 8uL of 10% FeCF to develop the color, and the relative intensity units subsequently determined by Image J analysis. Finally, the relative intensity units for each polymer incubation corresponds to the amount of phenol that is left in the supernatant, which is then determined from the standard curve equation in FIG. 2. The amount of phenol that is bound is ascertained as shown in Table

1.

Table 1. Proportion of Phenol bound to Polymers

[0063] Table 2 shows the amount of meta-cresol that is bound by the polymers. lOOuL of 3mg/mL meta-cresol was incubated with lOOuL polymer in the plastic microfuge tube for 5 mins at room temperature. 80uL of the supernatant was then transferred to a new tube containing 8uL of 10% FeCh to develop the color, and the relative intensity units subsequently determined by Image J analysis. Finally, the relative intensity units for each polymer incubation corresponds to the amount of meta-cresol that is left in the supernatant, which is then determined from the standard curve equation in FIG. 2. The amount of meta-cresol that is bound is ascertained as shown in Table 2. Table 2. Proportion of meta-cresol bound to polymers.

[0064] In another embodiment, the vessel of the separation device is a plastic microfuge tube containing lOOuL of the physical substrate polymer at the bottom and sides of the tube. lOOuL of the insulin formulation LISPRO containing meta-cresol was incubated with lOOuL polymer in the plastic microfuge tube for 5 mins at room temperature. 80uL of the supernatant was then transferred to a new tube containing 8uL of 10% FeCL to develop the color, and the relative intensity units subsequently determined by Image J analysis. The relative intensity units for the incubation with the polymers correspond to the amount of meta-cresol that is left in the LISPRO supernatant, which is then determined from the standard curve equation in FIG. 4. The amount of meta-cresol that is bound by each polymer is determined by l-(meta-Cresol Polymer LISPRO Supernatant/ 3.l5mg/mL Input meta-Cresol LISPRO) x 100% (Table 3).

[0065] Table 3 shows the amount of meta-cresol in the insulin formulation LISPRO that is bound by the polymers. lOOuL of LISPRO was incubated with lOOuL of polymer in a plastic microfuge tube for 5 mins at room temperature and 80uL of the supernatant then transferred to a new tube containing 8uL of 10% FeCL to develop the color, and the relative intensity units subsequently determined by Image J analysis. The control was lOOuL LISPRO incubated in an empty plastic microfuge tube (no polymer) for 5 mins at room temperature, and 80uL of the supernatant then transferred to a new tube containing 8uL of 10% FeCL to develop the color, and the relative intensity units subsequently determined by Image J analysis. Finally, the relative intensity units for each polymer incubation corresponds to the amount of meta-cresol that is left in the LISPRO supernatant, which is then determined from the standard curve equation in FIG. 2. The amount of meta-cresol that is bound is ascertained as shown in Table 3. Table 3. Proportion of meta-cresol in LISPRO which is bound to polymers.

[0066] In another embodiment, the standard curves of phenol and meta-cresol were obtained by using 80uL of phenol or meta-cresol at concentrations of lOmg/mL, 5mg/mL, 3mg/mL, lmg/mL and Omg/mL, and reacted with 8uL of 10% FeCL to develop colored solutions. The varying colored intensities (ranging from purple to yellow) were then ascertained in relative intensity units by using Image J analytical software to construct the standard curves and determine the standard curve equations (FIGS. 2 and 3).

[0067] In another embodiment, the standard curve of meta-cresol in the insulin formulation LISPRO was obtained by using LISPRO meta-cresol at concentrations of 3.l5mg/mL, 2mg/mL, L575mg/mL, lmg/mL, 0.5mg/mL and Omg/mL, and reacted with 8uL of 10% FeCL to develop colored solutions. The varying colored intensities (ranging from purple to yellow) were then ascertained in relative intensity units by using Image J analytical software to construct the standard curves and determine the standard curve equations (FIG. 4).

[0068] In another embodiment, the vessel of the separation device is a plastic microfuge tube containing lOOuL of the physical substrate polymer at the bottom and sides of the tube. lOOuL of the insulin formulation LISPRO control (no polymer) was incubated in an empty plastic microfuge tube for 5 mins at room temperature and the supernatant subsequently transferred to a new tube. lOOuL of the insulin formulation LISPRO was incubated with lOOuL of polymer in the plastic microfuge tube for 5 mins at room temperature and the supernatant then transferred to a new tube. The LISPRO supernatants were then diluted 1/10 with PBS and 50uL was used in the ELISA assay according to the company’s (abeam®) protocol. The Human Insulin ELISA standard curve was also determined by using the provided purified human insulin in the ELISA kit according to abeam’ s protocol. The amount of Insulin remaining in each sample (LISPRO control and LISPRO with polymer) was determined from the standard curve equation in FIG. 5. The amount of insulin that is bound by each polymer was then ascertained by 1 -(LISPRO Polymer/ LISPRO No Polymer) x 100% (Table 4).

[0069] Table 4 is the ELISA study showing the amount of Insulin from the insulin product LISPRO that are bound by the polymers. lOOuL of LISPRO control (no polymer) was incubated in an empty plastic microfuge tube for 5 mins at room temperature and the supernatant subsequently transferred to a new tube. lOOuL of LISPRO was incubated with lOOuL of polymer in a plastic microfuge tube for 5 mins at room temperature and the supernatant then transferred to a new tube. The LISPRO supernatants were then diluted 1/10 with PBS and 50uL was used in the ELISA assay according to the company’s (abeam®) protocol. The amount of Insulin remaining in each sample (LISPRO control and LISPRO with polymer) was determined from the standard curve equation in Fig.4. The amount of insulin that is bound by each polymer was ascertained as shown in Table 4.

Table 4. Proportion of Insulin in LISPRO bound to polymers.

[0001] It is understood that the configuration of the Phenolic Preservative Binder Device as illustrated herein in but one of number of the potential embodiments of this invention; and, the absence of description of such alternatives is not intended as limiting the scope of this invention which has been reserved for the following claims.

[0002] Although the present disclosure has been described in detail for the purpose of illustration based on what is currently considered to be one of the most practical and preferred implementations, it is to be understood that such detail is solely for that purpose and that the various embodiments of the disclosure are not limited to the disclosed implementations, but, on the contrary, are intended to cover modifications and equivalent arrangements that are within the scope of the appended claims. For example, it is to be understood that the present disclosure contemplates that, to the extent possible, one or more features of any embodiment can be combined with one or more features of any other embodiment.

[0003] As used herein, the terms,“comprises” and“comprising” are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms,“comprises” and“comprising” and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0004] As used herein, the term“exemplary” means“serving as an example, instance, or illustration,” and should not be construed as preferred or advantageous over other configurations disclosed herein.

[0005] As used herein, the term“substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result.

[0006] As used herein, the term“about” means plus or minus 10 percent of the stated value, and any value in between.

[0007] Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood to one of ordinary skill in the art.

LIST OF REFERENCES CITED

1. Heinemann L, Krinelke L. Insulin infusion set: the Achilles heel of continuous subcutaneous insulin infusion. J Diabetes Sci Technol. 20l2;6: 954-964.

2. Johansson U-B, Adamson U, Lins P-E, Wredling R. Patient management of long-term continuous subcutaneous insulin infusion. J Adv Nurs. 2005;5l: 112-118.

3. Schober E, Rami B. Dermatological side effects and complications of continuous subcutaneous insulin infusion in preschool-age and school-age children. Pediatr Diabetes. 2009; 10: 198-201.

4. Ponder SW, Skyler JS, Kruger DF, Matheson D, Brown BW. Unexplained hyperglycemia in continuous subcutaneous insulin infusion: evaluation and treatment. Diabetes Educ. 2008;34: 327-333. 5. Weber C, Kammerer D, Streit B, Licht AH. Phenolic excipients of insulin formulations induce cell death, pro-inflammatory signaling and MCP-l release. Toxicology Reports. 20l5;2: 194-202.

6. Jeitler K, Horvath K, Berghold A, Gratzer TW, Neeser K, Pieber TR, et al. Continuous subcutaneous insulin infusion versus multiple daily insulin injections in patients with diabetes mellitus: systematic review and meta-analysis. Diabetologia. 2008;5l: 941-951.

7. Teska BM, Alarcon J, Pettis RJ, Randolph TW, Carpenter JF. Effects of phenol and meta-cresol depletion on insulin analog stability at physiological temperature. J Pharm Sci. 20l4;l03: 2255-2267.

Claims

What is claimed is:

1. A method for removing phenolic preservatives from a liquid insulin formulation, comprising,

- providing a liquid insulin formulation comprising at least one phenolic preservative and an insulin;

- binding the physical substrate specifically to the phenolic preservative in such a manner that the phenolic preservative is removed from the liquid insulin formulation without binding the insulin.

2. The method of claims 1, wherein the physical substrate is a macromolecule comprising one or more of: polymers, surfactants, nanospheres, nanotubes, dendrimers, microspheres, and polymerized microspheres.

3. The method of claim 2, wherein the physical substrate is a macromolecule comprising at least one polymer.

4. The method of claims 2, wherein the physical substrate is a macromolecule comprising one or more of: acrylamide; one or more derivatives of acrylamide and acrylate; and glycerol and a crosslinker, wherein the crosslinker comprises N,N’-Methylenebisacrylamide.

5. The method of claims 2, wherein the physical substrate is a macromolecule comprising at least one copolymer comprising one or more monomers selected from a group consisting of glycerol, acrylamide, hydroxypropyl acrylate isomers, 4-hydroxybutyl acrylate, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate.

6. The method of claim 5, wherein the physical substrate is a macromolecule comprising a polymer consisting of hydroxypropyl acrylate isomers and N-isopropyl acrylamide, and in a ratio of about 50:50 to 30:70.

7. The method of claim 5, wherein the physical substrate is a macromolecule comprising N- isopropyl acrylamide, hydroxypropyl acrylate isomers and 4-hydroxybutyl acrylate in a ratio of about 20:40:40 to 25:37.5:37.5.

8. The method of claim 5, wherein the physical substrate is a macromolecule comprising hydroxypropyl acrylate isomers, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate, and in a ratio of about 85:7.5:7.5 to 90:5:5.

9. The method of any one of claim 1-8, wherein the macromolecule is contained within a phenolic preservation separation device comprising a plastic microcentrifuge tube, a plastic 96 well plate, a plastic test tube, a glass test tube, a glass vial, or any similar vessel that is able to hold the macromolecule and the insulin formulations.

10. The method of any one of claims 1-8, wherein the macromolecule comprises a polymer consisting of a gel, gel pieces, gel particles, slurry or any similar gel configuration that is able to selectively bind phenolic preservatives out of the insulin formulations.

11. The method of any one of claims 1-8, wherein the phenolic preservative is phenol.

12. The method of any one of claims 1-8, wherein the phenolic preservative is meta-cresol.

13. The method of any one of claims 1-8, wherein the phenolic preservatives are phenol and meta-cresol.

14. The method of any one of claims 1-8, wherein the insulin formulation is Lispro.

15. The method of any one of claims 1-8, wherein 10% or less of the insulin within the insulin formulation binds to the physical substrate.

16. A phenolic preservative separation device comprising,

-a vessel able to store a liquid insulin formulation comprising a phenolic preservative and an insulin; and,

-a physical substrate stored within the vessel, the substrate comprising at least one macromolecule able to selectively bind and remove from the liquid about 60- 99% of the phenolic preservative within the insulin formulation, wherein the insulin does not bind to, or binds 10% or less to, or about 1-5% to, the physical substrate.

17. The phenolic preservative separation device of claim 16, wherein the vessel comprises one or more of: a plastic microcentrifuge tube, a plastic 96 well plate, a plastic test tube, a glass test tube, and a glass vial, or any similar container able to hold the liquid insulin formulation sample.

18. The phenolic preservative separation device of claim 16, wherein the phenolic preservative comprises one of: a phenol; a meta-cresol; or a phenol and a meta-cresol.

19. The phenolic preservative separation device of claim 16, wherein the liquid insulin formulation comprises Insulin Lispro.

20. The phenolic preservative separation device of claim 16, wherein the physical substrate is a macromolecule comprising one or more of: acrylamide, one or more derivatives of acrylamide and acrylate, glycerol and a crosslinker, wherein the crosslinker comprises N,N’- Methylenebisacrylamide.

21. The phenolic preservative separation device of claim 16, wherein the physical substrate is a macromolecule comprising at least one copolymer comprising one or more monomers selected from a group consisting of glycerol, acrylamide, hydroxypropyl acrylate isomers, 4- hydroxybutyl acrylate, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate.

22. The phenolic preservative separation device of claim 16, wherein the physical substrate is a macromolecule comprising a polymer consisting of hydroxypropyl acrylate isomers and N- isopropyl acrylamide, and in a ratio of about 50:50 to 30:70.

23. The phenolic preservative separation device of claim 16 or 21, wherein the physical substrate is a macromolecule comprising N-isopropyl acrylamide, hydroxypropyl acrylate isomers and 4-hydroxybutyl acrylate in a ratio of about 20:40:40 to 25:37.5:37.5.

24. The phenolic preservative separation device of claim 16 or 21, wherein the physical substrate is a macromolecule comprising hydroxypropyl acrylate isomers, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate, and in a ratio of about 85:7.5:7.5 to 90:5:5.

25. A method of reducing inflammation at the dermal site of an insulin infusion in a diabetic patient, comprising:

- providing a phenolic preservative separation device, comprising a vessel able to store a liquid insulin formulation, and a physical substrate within the vessel, able to bind at least one phenolic preservative with binding insulin;

- combining the physical substrate and the liquid insulin formulation in the vessel, in a ratio of 1:1, at room temperature for at least 5 minutes, wherein the phenolic preservatives are bound to the physical substrate;

- removing a supernatant from the vessel comprising the insulin formulation without the phenolic preservatives; and

- infusing the supernatant into a diabetic patient at a dermal site, wherein no or minimal inflammation of the dermal site occurs from the infusion due to the absence of phenolic preservatives in the supernatant.

26. The method of reducing inflammation of claim 25, wherein the vessel comprises one or more of: a plastic microcentrifuge tube, a plastic 96 well plate, a plastic test tube, a glass test tube, and a glass vial, or any similar container able to hold the liquid insulin formulation sample.

27. The method of reducing inflammation of claim 25, wherein the phenolic preservative comprises one of: a phenol; a meta-cresol; or a phenol and a meta-cresol.

28. The method of reducing inflammation of claim 25, wherein the liquid insulin formulation comprises Insulin Lispro.

29. The method of reducing inflammation of claim 25, wherein the physical substrate is a macromolecule comprising one or more of: acrylamide, one or more derivatives of acrylamide and acrylate, glycerol and a crosslinker, wherein the crosslinker comprises N,N’- Methylenebisacrylamide.

30. The method of reducing inflammation of claim 25, wherein the physical substrate is a macromolecule comprising at least one copolymer comprising one or more monomers selected from a group consisting of glycerol, acrylamide, hydroxypropyl acrylate isomers, 4- hydroxybutyl acrylate, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate.

31. The method of reducing inflam ation of claim 25 or 30, wherein the physical substrate is a macromolecule comprising a polymer consisting of hydroxypropyl acrylate isomers and N- isopropyl acrylamide, and in a ratio of about 50:50 to 30:70.

32. The method of reducing inflammation of claim 25 or 30, wherein the physical substrate is a macromolecule comprising hydroxypropyl acrylate isomers, N-isopropyl acrylamide, and ethylene glycol phenyl ether acrylate, and in a ratio of about 85:7.5:7.5 to 90:5:5.

33. The method of reducing inflammation of claim 25 or 30, wherein the physical substrate is a macromolecule comprising a polymer consisting of N-isopropyl acrylamide, hydroxypropyl acrylate isomers and 4-hydroxybutyl acrylate in a ratio of about 20:40:40 to 25:37.5:37.5.