New insulin formulations for use in insulin pumps

Abstract

It is provided an insulin formulation comprising insulin and antimicrobial excipients for use in insulin pumps, connected to subcutaneous tissue via a catheter, a method also known as continuous subcutaneous insulin infusion, wherein the antimicrobial excipient inhibits growth of bacteria in insulin formulations and biofilm bacterial population formation on the internal surfaces of a catheter, allowing extending the time of use of the catheter.

Description

NEW INSULIN FORMULATIONS FOR USE IN INSULIN PUMPSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is claiming priority from U.S. Provisional Application No. 63 / 652260 filed May 28, 2024, the content of which is hereby incorporated by reference in their entirety.TECHNICAL FIELD

[0002] It is provided insulin formulations comprising insulin and new antimicrobial excipients.BACKGROUND

[0003] The continuous subcutaneous insulin infusion (CSIls, also called insulin pumps) and the continuous glucose monitoring (CGM) are both modern portable medical devices making up the artificial pancreas (AP). The AP technology can optimize glycemic control in patients with type 1 and type 2 diabetes. The CSII is a medical device that delivers insulin subcutaneously via a catheter.

[0004] CSIls are associated with better glycemic control when compared with multiple daily injections of insulin and are increasingly used in clinical practice. It is still recommended that infusion sets (catheters) for CSIls be worn for 2 to 3 days while CGMs are now worn for 10-14-day period. However, frequent catheter replacement is costly, out of synch for catheter vs. CGM sites, unpleasant, and increases the environmental footprint.

[0005] Increasing catheter lifetime without significant side effects is, therefore, an important practical goal to simplify CSII / CGM therapy and to reduce associated costs and burden. Nevertheless, longer use of catheters could increase the risk of adverse events including bacterial skin colonization, local inflammation and infections, which can lead to cellulitis and even toxic shock syndrome. Above all, inflammation and infection at catheter sites may cause incrustation and blockade of catheter lumen, which alters both absorption and therefore insulin actions.

[0006] Antibiotics cannot reasonably be envisioned for life-long insulin therapy as they may cause both toxicity (such as kidney failure, of particular importance in people with diabetes) and emergence of antibiotic-resistant bacteria. Moreover, they were shown to be rather inactive against bacterial biofilms, which account for up to 80% of human chronic infections according to the National Institutes of Health. The use of antimicrobialimpregnated catheters was suggested to minimize the risk of infection of any medical devices. However, such method is still to be developed for life-long conditions, such as diabetes. Hence, new, effective, and safe antimicrobial additives for use in insulin formulations are needed to be active at the catheter injection site and to avoid the untoward medical occurrence of catheters-associated local inflammation / infections and consequences.

[0007] It is thus highly desired to be provided with means to extend catheter use.SUMMARY

[0008] It is provided an insulin formulation comprising insulin and an antimicrobial excipient.

[0009] In an embodiment, the formulation comprises at least 0.5 mM of the antimicrobial excipient; more preferably the formulation comprises at least 1 mM of the antimicrobial excipient.

[0010] In another embodiment, the insulin formulation comprises between 0.5-30 mM of the antimicrobial excipient.

[0011] In an embodiment, the antimicrobial excipient is a metal.

[0012] In a further embodiment, the metal is copper, zinc, nickel, cobalt, cadmium, a salt thereof, or a combination thereof.

[0013] In an embodiment, the antimicrobial excipient is a salt of CuSO4, CuCI2, ZnSO4, ZnCI2, ZnO, NiSO4, NiCI2or a combination thereof.

[0014] In a further embodiment, the antimicrobial excipient is an antioxidant excipient, or a combination of an antioxidant excipient and at least one metal.

[0015] In an embodiment, the antimicrobial excipient is N-acetylcysteine or cysteamine.

[0016] In a further embodiment, the insulin formulation comprises between 1-10 mg / mL of the antimicrobial excipient.

[0017] In an embodiment, the formulation being free or comprising a phenolic compound or a derivative thereof.

[0018] In a further embodiment, the phenolic compound is phenol or metacresol.

[0019] In an embodiment, the insulin is a regular insulin or a transformed insulin.

[0020] In a further embodiment, the insulin is a short-acting insulin analog, a biosimilar insulin, or a (ultra)fast-acting mealtime insulin.

[0021] In an embodiment, the short-acting insulin analogs is insulin lispro, insulin glulisine, or insulin aspart.

[0022] In an embodiment, the biosimilar insulin is Admelog®, Trurapi®, or Adipra®

[0023] In a further embodiment, the ultra fast-acting mealtime insulin is ultra-rapid aspart or ultra-rapid lispro.

[0024] In an embodiment, the ultra fast-acting mealtime insulin is Fiasp®, URLi® or Lyumjev®.

[0025] In another embodiment, the insulin formulation encompassed herein further comprises one or more vasoactive agents.

[0026] It is also provided a kit comprising the insulin formulation as defined herein and a device for administering the insulin formulation to a subject. In an embodiment, the subject is an animal or a human patient.

[0027] In an embodiment, the kit comprises an insulin pump configured for administering the insulin formulation to the subject through a catheter.

[0028] In another embodiment, the kit further comprises a hardware processor in communication with the insulin pump for monitoring the insulin administering.

[0029] It is further provided a method of treating diabetes in a subject comprising administering the insulin formulation as defined herein to said subject.

[0030] In a further embodiment, the insulin formulation is administered to the subject using the kit as described herein.

[0031] It is also provided the use of the insulin formulation orthe kit as encompassed herein for treating diabetes in a subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Reference will now be made to the accompanying drawings.

[0033] Fig. 1 illustrates pictures of scanning electron microscopy examination of Teflon CSIl-catheters worn by patients with type 1 diabetes (n= 2 patients). Transversal (upper) and circular (bottom) cross-sections views. Left panels: Typical scanning electron microscopy images revealing the presence of intraluminal bacterial biofilm on and in catheters samples. Middle panels: Higher magnification views of the catheters shown in left panels. Numbers of days of wearing are indicated in parenthesis. Right panels: Control, not-worn catheters showing the absence of bacterial colonization.

[0034] Fig. 2 illustrates stability of insulin solutions (LisPro - Humalog is the brand name of lispro) with and without antimicrobial agents at room temperature determined by high-performance liquid chromatograph (HPLC). Representative chromatograms (Abs at 214nm) are shown. Note the similar peak area values between insulin formulations in the presence or absence of antimicrobial agents.

[0035] Fig. 3 illustrates the inhibitory effects of antimicrobial agents on planktonic growth of CSIl-catheters colonized bacterial species in BHIg medium after 24h treatments, as determined by O.D.600 readings. Results are means ± SEM of technical duplicates from two independent experiments.

[0036] Fig. 4 illustrates the effects of agents, alone or in combination, on biofilm formation of bacterial colonizers of CSIl-catheters over a period of 24h assessed by electrochemical impedance spectroscopy. The impedance signal produced by a bacterial biofilm is a consequence of both the cells and the extracellular polymeric substances. Fig. 4A shows the stimulatory effects of insulin on biofilm formation. Fig.4B-F shows the concentration-dependent inhibitory effects of metal salts on biofilm formation in the presence of insulins. Fig. 4G depicts the area under the curves calculated from the different cell index curves using GraphPad Prism 10.1.2. Fig. 4H shows the antibiofilm effects of CuCI2persisting over a 96h (4-day) period. Fig. 4I shows an additive or synergistic effect of combining low concentrations of CuCI2and ZnCI2against biofilm formation. Values are presented as mean ± SEM of triplicates from one representative experiment of three independent experiments.

[0037] Fig. 5 illustrates inhibitory effects of / V-acetylcysteine (NAC) on biofilm formation of CSIl-catheters colonized bacterial species in presence or absence of bovine insulin over a period of 24h by electrochemical impedance spectroscopy. Fig. 5A showsimpedance analyses of biofilm formation following NAC treatment (left panel). Area under the curves calculated from the different cell index curves (right panel). Fig. 5B shows quantification of biofilm formation from the same set of experiments presented in Fig. 5A by crystal violet staining performed at the end of the 24h incubation period. Values are expressed as mean ± SEM of three replicates from one representative experiment of three independent experiments.

[0038] Fig. 6 illustrates acute toxicity testing of antimicrobial agents in cultured human / murine normal cell lines after 24h drug exposure determined by colorimetric crystal violet assays. Data are presented as mean ± SEM, n= 2-3 assays from 3-8 technical replicates.DETAILED DESCRIPTION

[0039] In accordance with the present description, there is provided insulin formulations comprising insulin and antimicrobial excipients.

[0040] It is provided new insulin formulations specifically designed for use in insulin pumps or CSII, which are increasingly used for the treatment of people with type 1 and type 2 diabetes. To prevent bacterial colonization of catheters and therefore possible infections at catheter insertion site, it is provided the use of additional antimicrobial excipients in insulin formulations.

[0041] As intended herewith, an antimicrobial excipient means an excipient that inhibits growth of bacteria in insulin formulations, and biofilm bacterial population formation on inner and outer surfaces of a catheter.

[0042] In an embodiment, the antimicrobial excipient is a metal, such as copper, zinc, nickel, cobalt, cadmium, a salt thereof or a combination thereof. In a further embodiment, the antimicrobial excipient can be an excipient with anti-inflammatory properties, e.g. / V- acetylcysteine or cysteamine. The encompassed antimicrobial excipients are effective in countering bacterial growth free-floating in a fluid medium and / or the development of biofilms in and around catheters.

[0043] As provided herewith, the development of a wide range of planktonic (free- floating) and biofilm (surface-attached) bacteria can be blocked by various metal salts, such as copper (CuSO4 / CuCI2), zinc (ZnSO ZnO / ZnCh), and nickel (NiSO NiCh) salts. The addition of copper-, zinc-, or nickel-salts into insulin solutions allows to bring the anti-biofilm weapon directly where it is needed, inhibiting the genesis of biofilms locally, and extending the lifetime of catheters more than the current recommendation of 2 to 3 days.

[0044] The insulin formulation described herein comprises, in an embodiment, of the antimicrobial excipient, preferably between 0.5-30 mM of the antimicrobial excipient, more preferably 1-10 mM, more preferably at least 0.5 mM or at least 1 mM of metal salts and between 1-10 mg / mL (or 3-30 mM) for / V-acetylcysteine or cysteamine.

[0045] It is demonstrated the variable extent of bacterial colonization of catheters among CSII users. As demonstrated herewith, metal excipients are biocompatible with fast-acting insulin formulations. The efficacy of metallic excipient agents in inhibiting growth of multi-species planktonic and biofilm populations of CSII catheters worn by patients with type 1 diabetes is demonstrated. As encompassed herein, a patient can be a pediatric or adult patient.

[0046] In this context, the potential of oligodynamic metals, especially those considered essential for human health, e.g., copper, zinc, and nickel, has been tested herewith. Indeed, minute quantities of these essential metals are highly effective at preventing growth of a wide range of planktonic (free-floating) and biofilm (surface- attached) bacteria. Of note, they have lower toxicity to human cells than bacteria due to divergent regulatory mechanisms involved in the uptake / efflux transport, compartmentation and / or detoxification of these metals.

[0047] Using high resolution scanning electron microscopy, systematic presence of complex microbial communities of biofilms (surface-attached bacteria) within catheters worn 1 to 6 days by patients with type 1 diabetes (n=13), was found (see representative illustrations in Fig. 1). The degree of bacterial colonization within catheters worn by CSII users appears to be highly variable and seems unrelated to catheters wearing time patients. There was virtually no sign of bacterial colonization detected on the external surface of catheters.

[0048] Each milliliter of Humalog U-100 contains insulin lispro 100 units, glycerin, dibasic sodium phosphate, metacresol (m-cresol), zinc oxide, phenol, and water for injection. Similarly, NovoRapid® contains insulin aspart, as well as glycerol, phenol, metacresol, zinc chloride, sodium chloride, disodium phosphate dihydrate, sodium hydroxid, hydrochloric acid, and water for injection. Although manufacturers add considerable amounts (~30 mM; 3.15 mg / mL) of antimicrobial preservative excipients, e.g., either phenol, metacresol, or both, into insulin formulations, this strategy isineffective to control bacterial colonization of catheters as provided herewith. Accordingly, it is provided the use of an additional antimicrobial excipient in an insulin formulation allowing better control against biofilm formation on CSIl-catheters. The new selected excipients could also be used individually, in combination with others, or as alternative substitutes to traditional phenolic based excipients that are suspected to be a major cause of inflammatory reactions in subcutaneous tissues, limiting the use of CSIl- catheters for a prolonged period.

[0049] Accordingly, it is provided a formulation comprising a therapeutically effective amount of one or more insulin(s) and an antimicrobial preservative excipient as described herein, the formulation being free or not of phenolic compound or derivative such as metacresol.

[0050] In one embodiment, the insulin can be any regular mammalian insulin(s) or any transformed (genetically or by any other means) insulin. It is encompassed any insulin that is typically used in a pump to treat diabetes such as currently available shortacting insulin analogs (e.g., insulin lispro (Humalog®), insulin glulisine (Apidra®), insulin aspart (Novolog®), biosimilar insulins of lispro and aspart (e.g., Admelog®, Trurapi®), or ultra fast-acting insulins (e.g., ultra-rapid aspart (Fiasp®), ultra-rapid lispro (URLi® or Lumjiev®)), and whichever the composition of excipients or dilution liquid.

[0051] In another embodiment, the insulin can be in a formulation as know and as described e.g. in US 2018 / 221385. Accordingly, the insulin can be formulated with one or more vasoactive agents. An example of a suitable class of vasoactive agent includes prostacyclin IP1 receptor agonists such as iloprost (llomedine®, Ventavis®), epoprostenol (Flolan® and Veletri®) or treprostinil (Remodulin® & Tyvaso®). Other suitable classes of very powerful vasoactive agents and agonists of protein G-coupled receptors are: purinergic class 2 receptor agonists such as regadenoson (Lexiscan®), which is an adenosine A2A receptor agonist; tachykinin receptor agonists such as Homspera®, which is a substance P NK1 receptor agonist; histaminergic class 2 receptor agonists such as betazole, which is an histamine H2 receptor agonist; and kinin B2 receptor agonist such as labradimil, which is a bradykinin B2 receptor agonist. Another suitable class of vasoactive agents is the potassium channel openers, like minoxidil, nicorandil and maxipost.

[0052] In one embodiment of the methods, composition or combination described herein, said insulin is transformed insulin, preferably a short-acting insulin analog suchas, insulin lispro, insulin glulisine or insulin aspart, i.e. those mostly utilized in daily practice. In one embodiment, the said insulin is insulin lispro.

[0053] Whole-genome microbial sequencing analyses of extracted biofilms’ bacterial communities within worn Teflon CSII catheters (5 subjects) was performed using Illumina NGS method (Table 1), wherein respective catheter wearing time is indicated in parenthesis.Table 1 : Participants with diabetes type 1 and characteristics of CSIl-catheters used

[0054] Table 2 provides an overview of the strain diversity of bacterial species from catheters (at least 15 strain types) and showed the expected predominance of S. epidermidis and S. aureus strains (-84%). The values indicated in the table represent the amounts of reads.Table 2: Strain profiling of bacterial species from catheters

[0055] Conversely, a very low degree of bacterial colonization was observed with the use of a steel CSII catheter (see results of patient #7); however, the latter may be more likely to cause local tolerability issues with long-term use. As expected, there was no sign at all of bacterial contamination in unused, unworn Teflon catheters processed under the same conditions (see results of patient #8).

[0056] HPLC results indicated that the addition of CuCI2(10 mM) or the antioxidant / V-acetylcysteine (NAC; 5 mg / mL) did not affect stability of lispro insulin (Humalog®) at room temperature for up to 7 days (Fig. 2). Similar results were obtained with two other selected agents, ZnCI2and NiCI2.

[0057] It is further provided that planktonic growth of CSII catheters colonized bacterial species in standard lab milieu (brain heart infusion media (BHI) containing 0.5% (w / v) glucose) was strongly inhibited following treatment with the selected antimicrobial agents over a 24h period (Fig. 3A).

[0058] Adding insulin (3.14 mg / mL) to BHI media at 37°C significantly increases biofilm forming capability of CSII catheters colonized bacterial species (Fig. 5A; ~1.75- fold increase in AUC). When tested individually, all selected antimicrobial agents exerted concentration-dependent anti-biofilm effects over a 24h period (see Figs. 4 and 5). The ability of the excipients to suppress the in vitro biofilm development persisted for at least 96h (4 days) (Fig. 4H). Moreover, combining the selected antimicrobial agents may broaden their spectrum of activity through additive or synergistic effects, enabling the use of lower individual doses to achieve comparable anti-biofilm efficacy while potentially reducing the risk of toxicity (Fig. 4I).

[0059] Thus, in lab, addition of a preservative metal excipient, such as copper (CuSO CuCh), zinc (ZnSO ZnO / ZnCh), or nickel (NiSO4 / NiCI2) salt, or antiinflammatory / V-acetylcysteine into commercial insulin solutions avoids catheters colonization by directly affecting bacterial viability, growth, and adhesion as well as biofilm formation. In clinic, such insulin formulation may improve catheters wearing time and thereby improve the safety and quality of care of patients using CSIls, as well as costs and environmental footprint.

[0060] As seen in Fig. 6, in contrast to insulin’s conventional m-cresol excipient, which reduced viability in some but not all human and mouse normal cell lines, none of the selected antimicrobial agents induced in vitro acute cytotoxic effects at concentrations inhibiting bacterial biofilm formation.

[0061] As defined herein “patient” refers to both human and non-human subjects (e.g., dog, cat, horse, other). The subject is preferably human (pediatric and adult).

[0062] In any embodiment of the methods, composition or combination, the subcutaneous administration of the formulation described herein is by means of an automated infusion pump.

[0063] In an embodiment, said composition and combination defined herein are for use in any method defined herein, in particular the treatment of diabetes in a subject in need thereof.

[0064] The excipient(s) for use in pharmaceutical compositions in accordance with the disclosure must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not being deleterious to the recipient thereof. The pharmaceutical composition, or for instance each component of the combination, in particular the composition comprising insulin, may optionally comprise excipients such as preservatives, chelating agents, tonicity modifiers, bulking agents, stabilizers, antioxidants, polymers and surfactants, metal ions, oleaginous vehicles and proteins (e.g., human serum albumin, gelatine or proteins).

[0065] Examples of buffer include sodium acetate, sodium carbonate, citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.

[0066] Examples of preservative in the composition comprising insulin include those of conventional insulin compositions, such as phenol, m-cresol, methylparaben, and zinc or other ions.

[0067] As encompassed herein is a device for administering the insulin formulation described herein and and / or a system for administering the insulin formulation described herein. In some configurations, the system comprises the device for administering the insulin formulation described herein, an insulin pump configured to administer contents from the device to a subject through a subcutaneous catheter, and optionally one or more hardware processors in communication with the insulin pump.EXAMPLE IReagents

[0068] Copper(ll) chloride (CuCI2anhydrous), copper (II) sulfate (CuSO4anhydrous), nickel (II) chloride (NiCI2*6H2O), nickel (II) sulfate (NiSO4’6H2O), zinc chloride (ZnCI2), / V-acetylcysteine (NAC), cysteamine, human insulin and bovine insulin were all obtained from Millipore-Sigma (Oakville, On, CA). The zinc oxide (ZnO) was purchased from Fisher Scientific (Ottawa, On, CA). Stock solutions of antimicrobial agents were prepared in nanopure water and filtered through a 0.22 pm syringe filter prior to use.Recruitment of -CS 11 users with type 1 diabetes and collect of catheters.

[0069] Research protocol was approved by the ethics committee of each participating site (Comite d’ethique de la recherche du Centre integre universitaire de sante et de services sociaux de I’Estrie - Centre hospitalier universitaire de Sherbrooke (CIUSSSE - CHUS), study no. 2019-2046, and Comite d’ethique de la recherche de I'lnstitut de recherches cliniques de Montreal (IRCM), study no. 2020-1048). Patients were informed and gave their consent to participate in the study prior to initiating any research activities.

[0070] Patients were eligible if they were: 18 years old or older, using a tethered insulin pump, able to read and understand French or English, not using systemic antiinflammatory, not having uncontrolled active disease (investigator judgement), and otherwise in good health status.

[0071] Patients installed their insulin pump catheter as they usually do. They wore their catheter as long as their blood sugar values remained as their usual ones, the insertion site of their catheter remained comfortable, and there was no objective infection sign. When one or many of these situations occurred, patients removed their catheter. Once the catheter had been removed, patients would grasp the distal part of the catheter using very fine forceps. They then cut the proximal part of the catheter with scissors. The forceps and scissors were cleaned with an alcohol swab and air dried just prior to catheter removal. Patients then placed the cut catheter in a sterilized plastic microtube. The microtube was kept at room temperature and returned to the research team within 24h of catheter removal. The date and time of insertion and removal of the catheter were recorded on a sheet which was given to the research team at the same time as the catheter. The date and time of catheter removal were subtracted from the date and time of insertion to obtain the duration of catheter wear.Scanning electron microscopy examination of Teflon CSIl-catheters worn by patients with type 1 diabetes.

[0072] Catheters collected from CSII users (n=13 patients) were cut with fine scissors and plunged rapidly into a fixative solution (0.5 mL of glutaraldehyde 2.5% in sodium cacodylate buffer pH 7.4) in a sterile microtube. They were kept at 4°C in refrigerator until analysis by field emission scanning electron microscopy (Hitachi S-4700 and ThermoScientific™ Phenom XL) using institutional core facilities.Characterization of bacterial biofilms within used Teflon CSIl-catheters by 16S rRNA gene and metagenome sequencing.

[0073] DNA extraction from catheters associated bacterial biofilms (n=5 patients) was performed using DNeasy Powerbiofilm columns (cat no: 2400 Qiagen). One unworn catheter served as a negative control. Bacterial 16S ribosomal RNA sequencing (an amplification of the V4 region of the 16S rRNA gene using the 515F and 806R universal primers) was performed, and the fragments were sequenced on the MiSeq platform (Illumina) using the MiSeq Reagent Kit v2 500 cycles (24-30 million reads). Wholegenome microbial sequencing analyses (sequenced on the NextSeq 500 using the NextSeq® 500 / 550 High Output Kit v2 (75 cycles) reagent kit) were performed using Illumina next-generation sequencing (NGS) technology. These experiments and the subsequent analysis were conducted at the RNomics platform at the Cancer Applied Research Pavilion of Universite de Sherbrooke. For the whole-genome sequencing analysis, Trimmomatic (V0.39) was used to trim the reads and the quality was assessed using FastQC (V0.11.9). Bowtie2 (V2.2.5) was used to remove as much human reads as possible and Kraken2 (V2.1 .2) was employed to generate the final classification report for every catheter.Stability of insulin solutions determined by HPLC analysis

[0074] The commercial rapid-acting insulin analog lispro (Humalog 100 U / mL, equivalent to 600 pM), in 10 mL vials, were purchased from local hospital pharmacy, and stored at 4°C until use. The impact of antimicrobial agents on insulin stability was assessed by HPLC. For this purpose, Humalog solutions, previously incubated with or without antimicrobial agents for 1 week at 25°C, were loaded on an XDB-C18 column (5 pm, 150 x 4.6 mm; Agilent) and analyzed at 214nm with an Agilent 1100 system equipped with a UV detector, using a gradient method with mobile phases of 0.10% trifluoroacetic acid (TFA) in water and 0.10% TFA in acetonitrile.Extraction of intraluminal biofilms from Teflon CSIl-catheters

[0075] Catheters were collected from CSII users (n=2 patients; patients #1 and 6, see Table 1) and placed into sterile Eppendorf tubes filled with 1 ml BHIg medium (BHI + 0.5% glucose). The catheters lumens were flushed 3 times (with quick up-and-down motions) with the BHIg medium contained in the Eppendorf using insulin syringes with needle 28G x % inch (U-100 Insulin syringe BD Micro Fine 0.5 ml_). The Eppendorf tubes with catheters were then vortexed thoroughly. The catheters and bacteria in BHIg media were transferred into conical bottom tubes and then placed in a 37°C humidified incubator on a gyratory shaker (ThermoForma orbital shaker) at 250 rpm for either 24h or 48h. Prior to the beginning of the experiment, a fraction of the bacterial suspension was diluted to a O.D.600 = 0.2. The remaining bacterial culture was used to prepare a glycerol stock solution of bacteria consisting of a 50 / 50 mix of the bacterial suspension and a sterile 25% glycerol solution. The glycerol stock solution was aliquoted and stored at -80°C for later use.

[0076] The remaining bacterial culture was mixed in equal proportion (50 / 50) with a sterile 25% glycerol solution, aliquoted and stored at -80°C for future use.Measurement of planktonic growth of bacterial colonizers of CSIl-catheters via absorbance at 600 nm

[0077] The bacterial culture was diluted in BHIg medium to a final optical density (O.D.) at 600nm of 0.2. Ten microliters of a 50 / 50 mix of each individual culture coming from two CSII users (patients #1 and 6, see Table 1), was added to each test tube containing 190pL of BHIg media in presence or absence of the antimicrobial agents at the indicated concentrations. Samples were next incubated at 37°C for 24h under constant shaking. At the end of the incubation time, 100pL of each sample was aliquoted in a 96-well plate and bacterial growth was evaluated by measuring the O.D. at 600nm using a Tecan Infinite M200 plate reader. Data were obtained from background corrected O.D. values.Monitoring of biofilm formation of bacterial colonizers of CSIl-catheters by impedance analyses

[0078] The impedance measurements (Z values in Ohms) of biofilm growth were recorded with an ECIS ZOlinked to a 96-well array station (Applied Biophysics, Troy, NY, USA). The cysteine-stabilized 96-well plates (Applied Biophysics, USA) were filled withBHIg medium (190pL / well) containing or lacking insulin (either human recombinant or bovine), with or without the antimicrobial agents, and then incubated at 37°C. Impedance measurements were initiated by the addition of mixed biofilm cultures of catheters from two CSII users (O.D.600 of 0.2; 10pL / well; patients #1 and 6, see Table 1) and monitored over a 24h- and 96h- (4-day) periods, with readings taken every 10 min. All experiments were performed at a single frequency of 16 kHz. For each experimental condition, a cell index was determined by the ratio of the electrical impedance Z value at a specific time (Zt) over the Z value at time t=0 (Zt I Zt=O).Quantification of bacterial biofilms by crystal violet staining

[0079] At the end of the electrical impedance assays, bacterial biofilms were washed with phosphate-buffered saline, stained / fixed in a crystal violet solution (0.5% crystal violet / 30% ethanol / 3% formaldehyde) then washed again with water. Stained biofilms were solubilized in a 1% sodium dodecyl sulfate solution and the intensity of coloration was evaluated at 570nm using a Tecan M200 spectrophotometer.Acute toxicity testing of antimicrobial agents in cultured normal human and mouse cell lines determined by crystal violet staining

[0080] Cells were seeded in 96-well plates and cultured to 70-90% confluency in complete media (DMEM: HEK293, differentiated 3T3-L1 ; RPMI: RWPE1 ; EGM-2: HDMVEC; EMEM: L929) containing 10% fetal bovine serum. On the day of the treatment, cells were washed and incubated in respective serum free medium for 24h in presence or absence of tested compounds. At the end of the incubation period, cells were washed with phosphate-buffered saline, stained / fixed in a crystal violet solution (0.5% crystal violet / 30% ethanol / 3% formaldehyde) then washed again with water. Stained cells were solubilized in a 1% sodium dodecyl sulfate solution and the intensity of coloration was evaluated at 570nm using a Tecan M200 spectrophotometer.

[0081] While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An insulin formulation comprising insulin and an antimicrobial excipient.

2. The insulin formulation of claim 1 , wherein the formulation comprises at least 0.5 mM of the antimicrobial excipient.

3. The insulin formulation of claim 1 or 2, wherein the formulation comprises at least 1 mM of the antimicrobial excipient.

4. The insulin formulation of any one of claims 1-3, comprising between 0.5-30 mM of the antimicrobial excipient.

5. The insulin formulation of any one of claims 1 -4, wherein the antimicrobial excipient is a metal.

6. The insulin formulation of claim 5, wherein the metal is copper, zinc, nickel, cobalt, cadmium, a salt thereof, or a combination thereof.

7. The insulin formulation of claim 5 or 6, wherein the antimicrobial excipient is a salt of CuSO4, CuCI2, ZnSO4, ZnCI2, ZnO, NiSO4, NiCI2or a combination thereof.

8. The insulin formulation of any one of claims 1 -4, wherein the antimicrobial excipient is an antioxidant excipient, or a combination of an antioxidant excipient and at least one metal.

9. The insulin formulation of claim 8, wherein the antimicrobial excipient is N- acetylcysteine or cysteamine.

10. The insulin formulation of claim 8 or 9, comprising between 1-25 mg / mL of the antimicrobial excipient.

11. The insulin formulation of any one of claims 1-10, said formulation being free or comprising a phenolic compound or a derivative thereof.

12. The insulin formulation of claim 10, wherein the phenolic compound is phenol or metacresol.

13. The insulin formulation of any one of claims 1-12, wherein said insulin is a regular insulin or a transformed insulin.

14. The insulin formulation of any one of claims 1-13, wherein said insulin is a shortacting insulin analog, a (ultra)fast-acting mealtime insulin, or any biosimilar of regular, rapid or ultra rapid insulins.

15. The insulin formulation of claim 14, wherein the short-acting insulin analogs is insulin lispro, insulin glulisine, or insulin aspart.

16. The insulin formulation of claim 14, wherein the biosimilar insulin is Admelog® or Trurapi®, or Adipra®17. The insulin formulation of claim 14, wherein the ultra fast-acting mealtime insulin is ultra-rapid aspart or ultra-rapid lispro.

18. The insulin formulation of claim 15, wherein the ultra fast-acting mealtime insulin is Fiasp®, URLi® or Lyumjev®.

19. The insulin formulation of any one of claims 1-18, further comprising one or more vasoactive agents.

20. A kit comprising the insulin formulation of any one of claims 1-19 and a device for administering the insulin formulation to a subject.

21. The kit of claim 20, comprising an insulin pump configured for administering the insulin formulation to the subject through a catheter.

22. The kit of claim 21 , further comprising a hardware processor in communication with the insulin pump for monitoring the insulin administering.

23. A method of treating diabetes in a subject comprising administering the insulin formulation of any one of claims 1-19 to said subject.

24. The method of claim 23, wherein the insulin formulation is administered to the subject using the kit of any one of claims 19-21.

25. Use of the insulin formulation of any one of claims 1-19 or the kit of any one of claims 20-22 for treating diabetes in a subject.

26. The kit of any one of claims 20-22, the method of claim 23 or 24, or the use of claim 25, wherein the subject is an animal or a human patient.

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