Insulin preparations and methods of using insulin preparations in premature infants

Abstract

Insulin preparations and methods for preparing and using the insulin preparations. The insulin preparations include insulin, oligosaccharides, and sodium chloride in a ratio of 1:A:B (w:w:w, respectively), with A and B in the ranges of 1000-5000 and 10-50, respectively.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 173,442, filed April 11, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to insulin preparations formulated for use in infant milk feeds.Embodiments of the invention relate to insulin and methods of preparing infant milk feeds containing insulin, as well as methods of using insulin-containing infant milk feeds in preventing or reducing the severity of premature infant disorders such as late-onset sepsis or necrotizing enterocolitis / colitis / enterocolitis. [Background technology]

[0003] If delivery occurs before the 37th week of gestation, the birth is considered premature or preterm. The last few weeks in utero are crucial for healthy weight gain and adequate development of various vital organs.

[0004] Human milk is recognised as the milk nutrition of choice for all infants, including preterm infants, due to its proven health benefits to infants.

[0005] Human milk contains a significant number of hormones and growth factors, and studies have shown that some of these hormones (e.g., insulin, insulin-like growth factor 1, IGF-1, epidermal growth factor) have effects on the small intestine after oral administration.

[0006] Studies have also shown that adding insulin to preterm formulas improves growth and promotes intestinal maturation [Shulman, RJ, 2002;Archives of Disease in Childhood-Fetal and Neonatal Edition, 86(2), F131-F133].

[0007] Although insulin-containing infant milk nutritional formulas have been reported, when infants are born prematurely, their digestive systems may not be fully developed, and as a result, many of these premature infants experience feeding difficulties and disorders that are directly or indirectly related to malnutrition.

[0008] Late-onset sepsis (LOS), defined as sepsis with onset after 72 hours of life, is the leading cause of death in neonatal intensive care units. The incidence of LOS in preterm infants varies between 20% and 38% during the first 120 days of life, with mortality ranging from 13% to 19%.

[0009] Necrotizing enterocolitis (NEC) is a disease that primarily affects the intestines of premature infants. Bacteria invade the intestinal wall, causing local infection and inflammation that can eventually destroy the intestinal wall. Approximately 7% of premature infants develop necrotizing enterocolitis, and approximately 25% of affected infants die.

[0010] In reducing this invention to practice, the inventors have demonstrated for the first time that a solubilizable insulin preparation can be formulated in an infant milk diet (formula or breast milk) and that such a preparation can be used to prevent or reduce the severity of LOS and NEC. Summary of the Invention

[0011] According to one aspect of the present invention, there is provided a method for preparing insulin for use in an infant milk diet, the method comprising solubilizing insulin in hydrochloric acid to obtain solubilized insulin, and diluting the solubilized insulin with water to adjust the pH to 6.9-8.0.

[0012] According to an embodiment of the present invention, the method further comprises adding cyclodextrin to solution (b) thereby encapsulating insulin within the cyclodextrin cavity, and adjusting the pH to 6.9-8.0.

[0013] According to an embodiment of the present invention, the method further comprises combining the result of (b) or (d) with a sodium chloride solution and a maltodextrin solution to adjust the pH to 6.7-7.6.

[0014] According to an embodiment of the invention, the composition is combined with an oil-in-water nanodroplet emulsion, the nanodroplets containing docosahexaenoic acid (DHA) and coenzyme Q10.

[0015] According to an embodiment of the present invention, the method further comprises filtering the result of the blending and freeze-drying the filtrate to obtain an amorphous powder having a pH of 6.0-7.6.

[0016] According to another aspect of the present invention, there is provided a method of producing an infant milk food containing insulin, the method comprising reconstituting an amorphous powder as described herein in the infant milk food.

[0017] According to another aspect of the present invention, there is provided an insulin formulation comprising insulin, an oligosaccharide, and sodium chloride in a ratio of 1:A:B (w:w:w respectively), where A and B are in the ranges of 1000-100,000 and 10-50, respectively.

[0018] According to an embodiment of the invention, the insulin formulation has a pH of 6.0-7.6 when reconstituted in deionized water or 0.9% sodium chloride solution at a concentration of 0.03 g / ml.

[0019] According to an embodiment of the invention, the insulin formulation further comprises a cyclodextrin.

[0020] According to an embodiment of the present invention, the ratio of cyclodextrin to insulin is 10-1000.

[0021] According to an embodiment of the invention, 1 gram of the insulin preparation contains 2-40 IU.

[0022] According to another aspect of the invention there is provided a milk-based nutritional food for infants comprising the insulin formulation.

[0023] According to another aspect of the invention there is provided a method of preventing necrotising enterocolitis / enteritis / enterocolitis in a preterm infant comprising administering to the preterm infant a nutritious infant milk diet.

[0024] According to another aspect of the invention there is provided a method of reducing the prevalence and severity of necrotising enterocolitis / enteritis / enteritis in preterm infants comprising administering to the preterm infant a nutritious infant milk diet.

[0025] According to another aspect of the invention there is provided a method of reducing the prevalence of late onset sepsis in preterm infants comprising administering to the preterm infant a nutritious infant milk diet.

[0026] According to another aspect of the invention there is provided a method of reducing inflammation of the gastrointestinal tract in a premature infant comprising administering to the premature infant a nutritious infant milk diet.

[0027] According to another aspect of the invention there is provided a method of reducing the prevalence and severity of adverse events, life-threatening events and fatal complications in premature infants comprising administering to the premature infant a nutritious infant milk diet.

[0028] According to another aspect of the invention there is provided a method of reducing gastrointestinal (GI) tract permeability in a preterm infant comprising administering to the preterm infant a nutritious infant milk diet and measuring the lactose / lactulose ratio.

[0029] According to another aspect of the present invention, there is provided a method of increasing rapid maturation of the gut microbiota in preterm infants, the method comprising administering to the preterm infant a nutritious infant milk diet and measuring a reduction in the phylum Proteobacteria and an increase in the phylum Firmicutes and the family Clostridiaceae.

[0030] According to an embodiment of the present invention, the infant milk nutritional food contains 0.01-4 IU / ml.

[0031] According to another aspect of the invention there is provided a method of preventing late onset sepsis in a preterm infant comprising administering to the preterm infant a nutritious infant milk diet.

[0032] According to another aspect of the invention there is provided a method of reducing time to discharge from primary care of a premature infant comprising administering to the premature infant a nutritious infant milk diet as disclosed herein.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0034] The present invention is described herein by way of example only with reference to the accompanying drawings. With specific reference now made in detail to the drawings, it is emphasized that the particulars shown are presented merely by way of example and for the purpose of illustratively discussing preferred embodiments of the invention, in order to provide what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of the invention. In this regard, no attempt has been made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and it will become apparent to those skilled in the art from reading the description in conjunction with the drawings how some forms of the invention may be embodied in practice.

[0035] The drawings are as follows: [Brief description of the drawings]

[0036] [Figure 1]1 shows typical chromatograms of formulations I to IV of the present invention. [Diagram 2] 1 shows typical chromatograms of formulations V to VI of the present invention. [Diagram 3] 1 shows typical chromatograms of DHA and coenzyme Q10 in formulations IV to V of the present invention. [Figure 4] The probability of PN weaning by group (Kaplan-Meier survival curve) is shown. [Diagram 5] The mean daily PN intake by group is illustrated, showing a clear benefit of the treatment group compared with placebo, especially the enteral insulin preparation at 0.3 IU / kg / day (expressed as the percentage reduction in PN in total intake). [Figure 6] The percentage of enteral nutrition in total nutrition by group is illustrated. [Figure 7] The progress by group is illustrated in FIG. [Figure 8] 1 is a scanning electron microscope photograph showing the amorphous structure of Formulations I to IV of the present invention. [Figure 9] 1 is a graph showing thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Formulations I to IV of the present invention. [Figure 10] 1 is a graph showing thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of Formulations I to IV of the present invention. [Figure 11] 1 shows solid-state NMR spectra of formulations I to IV of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention relates to an insulin preparation that can be used in infant milk nutrition. Specifically, the present invention can be used to prevent or reduce the severity of premature infant disorders such as late-onset sepsis or necrotizing enterocolitis / colitis / enterocolitis.

[0038] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0039] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0040] Insulin-containing infant milk diets are known in the art and have been suggested for treating malabsorption in premature infants.

[0041] During the experimentation of various insulin preparations intended for use in infant milk nutrition, such as breast milk or formula, the inventors recognized that insulin, when solubilized in infant milk nutrition, exhibits a tendency to polymerize and aggregate, thus reducing the availability of insulin in the resulting milk nutrition formula. Vigorous mixing of the infant milk nutrition can solve this problem to some extent, but the inventors consider this to be a suboptimal solution and set out to improve the solubility of insulin in infant milk nutrition.

[0042] Thus, according to one aspect of the present invention, there is provided an insulin formulation that is readily soluble in an infant milk diet (e.g., breast milk or formula) and exhibits stability (structural and functional) in the milk diet without aggregation over an extended period of time.

[0043] The preparation of the present invention comprises insulin (e.g., human recombinant), carbohydrate (e.g., oligosaccharide such as maltodextrin), and sodium chloride in a ratio of 1:A:B (w:w:w, respectively), where A and B range from 1000 to 100,000 and 10 to 50, respectively. One gram of the insulin preparation may contain 2 to 40 IU. The insulin preparation may further comprise cyclodextrin in a ratio of 10 to 1000 to insulin. Carbohydrates such as monosaccharides (e.g., glucose), disaccharides (e.g., lactose, maltose), oligosaccharides (dextrins (e.g., maltodextrin), cyclodextrins (e.g., hydroxypropyl-beta-cyclodextrin (HPbCD)), and polysaccharides are used as stabilizers, bulking agents, and cryoprotectants. Buffers (e.g., sodium or potassium phosphates) may also be added to the composition.

[0044] The formulation can be combined with an oil-in-water nanoemulsion having nanodroplets containing docosahexaenoic acid (DHA) and coenzyme Q10.

[0045] The formulation may be in powder form, which when reconstituted in deionized water or 0.9% sodium chloride solution at a concentration of 0.03 g / ml has a pH of 6.0-7.6.

[0046] The inventors have found that by pre-treating insulin at pH 6.9-8.0, blending with carbohydrate at pH 6.7-7.6, and lyophilization, an amorphous insulin powder (Figure 8) is obtained that is readily soluble in human breast milk, thereby overcoming the aforementioned limitations of prior art insulin formulations.

[0047] The insulin preparation of the present invention is an amorphous powder in which insulin is incorporated into an amorphous structure composed mainly of bulking agents (e.g., maltodextrin). The water content of the preparation can be 6.5% or less, the osmolality is about 110-120 mosm / kg when reconstituted in deionized water at a concentration of 0.1 g / ml, and the pH is 6.0-7.6 when reconstituted in deionized water or 0.9% sodium chloride solution at a concentration of 0.03 g / ml. Typical thermogram curves obtained by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) are shown in Figures 9-10. The solid-state NMR spectrum of the preparation of the present invention is shown in Figure 11.

[0048] As mentioned herein, the insulin formulations of the present invention may be added as a powder (or liquid) to infant milk nutrition, such as human milk, which may be fresh homogenized mother's milk or donor breast milk.

[0049] When in powder form, the insulin preparations of the invention can be reconstituted in infant milk nutrition to a final insulin concentration of 0.1-8 IU / ml.

[0050] The formulations of the present invention can be packaged as powders and stored for extended periods of time (days / months).

[0051] The packaging may be, for example, a glass vial or an aluminum sachet filled with the formulation of the present invention in dry form. The packaging may contain 0.1 to 1.0 grams of the formulation in powder form, with 0.2 to 40 IU of insulin per gram. The headspace of the packaging unit may be air or an inert gas (e.g., nitrogen).

[0052] The moisture vapor transmission rate (MVTR) is approximately 0.1 g / m depending on the packaging. 2 / Less than 24 hours and oxygen transmission rate (OTR) of approximately 0.1 cm 3 / m 2 / A moisture barrier and / or oxygen barrier of less than 24 hours may be provided.

[0053] When packaged as disclosed herein, the formulations of the invention are highly stable and may be stored for extended periods of time without a decrease in insulin activity of more than about 1-10% when stored for up to 24 months at about 2° C. to about 8° C. Additionally, the moisture content of the packaged formulations is typically no more than about 4.5-6.5% by weight when stored for 24 months at about 2° C. to about 8° C.

[0054] The insulin formulation can be reconstituted in deionized water or 0.9% sodium chloride solution, and the resulting solution can be added to an infant milk diet (such as a mother's own milk, donor breast milk, or formula) to obtain the above insulin concentrations for the powder formulation.

[0055] As described herein and demonstrated in the Examples section, the insulin preparations of the invention and milk-based nutritional diets containing the insulin preparations of the invention can be used to facilitate weaning of preterm infants from parenteral nutrition and to substantially reduce overall adverse events and specific target conditions including necrotizing colitis (NEC) and late-onset sepsis (LOS).

[0056] Thus, according to another aspect of the present invention, there is provided a method of preventing or reducing the risk and severity of LOS in a subject in need thereof (e.g., a premature infant). The method is carried out by administering to a subject in need thereof a therapeutically effective amount of a nutritious infant milk food mixed with a medicament of the present invention. Such administration may be orally or via a feeding tube.

[0057] Thus, according to yet another aspect of the present invention, there is provided a method of preventing or reducing the risk and severity of NEC in a subject in need thereof (e.g., a premature infant). The method is effected by administering to a subject in need thereof a therapeutically effective amount of a nutritious infant milk food mixed with an agent of the present invention. Such administration may be orally or via a feeding tube.

[0058] The term "therapeutically effective amount" refers to a dose of an active ingredient, or of a milk-based nutritional supplement containing the active ingredient, that provides the therapeutic effect required for the active ingredient.

[0059] As used herein, the phrase "subject in need thereof" refers to a human infant. A human infant can be of any age (e.g., full-term or preterm) or of either gender. A subject in need thereof can be a preterm infant born at 24-37 weeks gestational age, or a low birth weight full-term infant, or an intrauterine growth retardation (IUGR) infant, or a small-for-gestational-age infant.

[0060] A typical dosing regimen established by the inventors may be administered by continuous enteral administration or by bolus ingestion, for example using 0.1-1 g of powder mixed in 1-3 ml of infant milk nutritional food, divided into 1-12 oral doses per day.

[0061] As described in the Examples section below, the regimen of the present invention reduced the number of NEC cases in the 0.3 IU / kg / day dose group compared to placebo. Necrotizing enterocolitis (Bell stage 2 or 3) occurred in 7 infants (6%) in the low dose group, 4 infants (5%) in the high dose group, and 10 infants (10%) in the placebo group. When looking at the most frail population (26-28 weeks gestational age stratum), the difference was even more pronounced, with 4.3% of subjects in the treatment group having NEC compared to 19.6% in the placebo group. There was a meaningful difference between the groups in the percentage of infants who experienced infections (including sepsis and other infections). When looking at the entire study population, the overall incidence was 30% in the 0.06 IU / kg / day dose group, 34.7% in the 0.3 IU / kg / day dose group, and 42.9% in the placebo group.

[0062] Simultaneous assessment of intestinal permeability and lactase activity by glucose absorption test (SAT) performed in a separate study using the regimen of the present invention showed decreased intestinal permeability (indicating rapid gastrointestinal (GI) maturation) and a trend toward higher lactase activity in the treatment group.

[0063] The efficacy of this study is also demonstrated by time to discharge (to home or secondary care) as demonstrated by the results shown in Example 4. As shown therein, the time to discharge preterm infants from primary care is reduced by at least 5% compared to untreated preterm infants (controls), which translates to discharge at least 5 days earlier than expected.

[0064] Another study was conducted to evaluate the effectiveness of two doses of enteral insulin compared to placebo on early differences in gut microbiota. This study showed significant differences in GI microbiota between treatment and placebo groups. At the end of treatment, the high insulin dose showed lower microbial diversity than the placebo group (p=0.0192). Insulin increases the phylum Firmicutes and decreases the phylum Proteobacteria. Enteral insulin administration reduced the phylum Proteobacteria and increased the phylum Firmicutes and the family Clostridiaceae. These results indicate that the formulation and regimen of the present invention promotes rapid maturation of the gut microbiota. The reduction in the phylum Proteobacteria and the increase in the phylum Firmicutes obtained in this study are changes previously observed in infants at low risk of NEC.

[0065] As used herein, the term "about" refers to ±10%.

[0066] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. EXAMPLES

[0067] Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non limiting fashion.

[0068] Example 1 Insulin and infant formulas An insulin formulation was prepared and tested for efficacy in treating preterm infants (in an infant milk diet).

[0069] Formulation I The active ingredient is insulin, the solubilizer is sodium chloride, and the bulking and stabilizing agent is an oligosaccharide (e.g., maltodextrin). The final product is a lyophilized powder filled into glass vials. The strength of the reconstituted solution is adjusted by the volume of diluent.

[0070] [Table 1]

[0071] Materials and Methods A 24.5 IU / ml rh-insulin solution was prepared as follows: 13 mg of insulin was solubilized in 1 ml of 0.1 N hydrochloric acid, diluted to 15 ml with water, and the pH was adjusted to 7.6 with 0.1 N sodium hydroxide.

[0072] Formulation Mixture: 40 grams of maltodextrin was dissolved in 100 ml of water and the pH of the resulting solution was adjusted to 7.2. 2 ml of 6% sodium chloride solution and 4 ml of insulin solution were added to the formulation mixture and the pH was adjusted to 7.0.

[0073] The resulting bulk was filtered through a 0.22 μm PES membrane, filled into glass vials (fill volume 1.7 ml) and lyophilized.

[0074] Each vial of lyophilized powder contained 1.25 IU of rh-insulin, 510 mg of maltodextrin, and 1.5 mg of sodium chloride.

[0075] Formulation II The active ingredient is insulin, the inclusion agent is a cyclodextrin (e.g., hydroxypropyl beta-cyclodextrin), the solubilizer is sodium chloride, and the bulking and stabilizing agent is an oligosaccharide (e.g., maltodextrin). The final product is a lyophilized powder filled into glass vials. The potency of the reconstituted solution is adjusted by the volume of diluent.

[0076] [Table 2]

[0077] Materials and Methods A 62 IU / ml rh-insulin solution was prepared as follows: 85.6 mg insulin was solubilized in 5 ml 0.1 N hydrochloric acid, diluted with 30 ml water and the pH was adjusted to 7.2 with 0.1 N sodium hydroxide.

[0078] Formulation Mixture: 200 grams of maltodextrin was dissolved in 600 g of water and the pH of the resulting solution was adjusted to 7.3. 2 g of sodium chloride pre-dissolved in 60 ml of water and insulin solution were added to the formulation mixture, the pH was adjusted to 7.0, and additional water was added until the bulk volume was 1 L.

[0079] The resulting bulk was filtered through a 0.22 μm PES membrane, filled into glass vials (fill volume 0.5 ml) and lyophilized.

[0080] Each vial of lyophilized powder contained 1.2 IU of rh-insulin, 100 mg of maltodextrin, and 1.0 mg of sodium chloride.

[0081] Preparation III A 243 IU / ml rh-insulin solution was prepared as follows: 84.4 mg insulin was solubilized in 1 ml 0.1 N hydrochloric acid, diluted to 10 ml with water and the pH was adjusted to 8.1 with 0.1 N sodium hydroxide.

[0082] Formulation Mixture: 30 grams of maltodextrin was dissolved in 60 ml of water and the pH of the resulting solution was adjusted to 7.1. 2.7 ml of 7.5% sodium chloride solution and 1 ml of insulin solution were added to the formulation mixture, the pH was adjusted to 7.3, and additional water was added to a bulk volume of 100 g.

[0083] The resulting bulk was filtered through a 0.22 μm PES membrane, filled into glass vials (fill volume 0.5 ml) and lyophilized.

[0084] Each vial of lyophilized powder contained 1.4 IU of rh-insulin, 172 mg of maltodextrin, and 1.2 mg of sodium chloride.

[0085] Formulation IV This example describes a commercial-scale preparation of 9 kg liquid bulk (0.9 kg lyophilized powder). Insulin as active ingredient, sodium chloride as solubilizer, and oligosaccharides (e.g., maltodextrin) as bulking and stabilizing agents. The final product is a lyophilized powder filled into glass vials. The potency of the reconstituted solution is adjusted by the volume of diluent.

[0086] [Table 3]

[0087] Materials and Methods A 33 IU / ml rh-insulin solution was prepared as follows: 0.39 g insulin was solubilized in 20 g 0.1 N hydrochloric acid, diluted with 300 g water for injection, and then neutralized with 20 g 0.1 N sodium hydroxide. The pH was adjusted to 7.2 with 0.1 N sodium hydroxide.

[0088] Formulation Mixture: 856 grams of maltodextrin and 8.6 grams of sodium chloride were dissolved in 6854 grams of water and the pH of the resulting solution was adjusted to 7.2. Insulin solution was added to the blended mixture and the weight of the resulting formulation was adjusted up to 9.0 kg and the pH was adjusted to 7.0.

[0089] The resulting bulk was filtered through a 0.22 μm PES membrane, filled into glass vials (fill volume 1 g) and lyophilized.

[0090] Each vial of lyophilized powder contained 1 IU of rh-insulin, 99 mg of maltodextrin, and 1 mg of sodium chloride.

[0091] The analytical characteristics of the resulting dry powder are: insulin (insulin + A21 desamido insulin) content 1.13 IU / vial, A21 desamido insulin content 0.5% (by area percent), total other related substances content 0.1% (by area percent), relative standard deviation (uniformity) of insulin content per vial 0.9%, water content 0.8%, pH 6.7, and reconstitution time of powder with water less than 5 minutes.

[0092] Formulation V The formulation was produced by forming an emulsion of DHA and coenzyme Q10 using the solvent displacement method, adding rh-insulin and a cryoprotectant (eg, dextrin, cyclodextrin), filtering and lyophilizing.

[0093] [Table 4]

[0094] Materials and Methods A 40 IU / ml rh-insulin solution was prepared as follows: 27.5 mg of insulin was solubilized in 2 ml of 0.1 N hydrochloric acid, diluted with 18 ml of water, and the pH was adjusted to 7.2 with 0.1 N sodium hydroxide.

[0095] An oil-in-water nanoemulsion (average droplet size 120 nm) was prepared as follows: 690 mg coenzyme Q10, 1533 mg DHA ethyl ester, 300 mg tyloxapol, and 300 mg Lipoid E80 were dissolved in 56 ml ethanol. The mixture was added dropwise to 300 ml of 0.1% PVA aqueous solution via a 21G needle and mixed continuously at 350 RPM at room temperature. The resulting emulsion was mixed for an additional 10 minutes, diluted with 0.1% PVA solution, and the organic solvent was removed by tangential flow filtration using a Hydrosart MWCO 30,000 membrane.

[0096] 10 grams of maltodextrin was dissolved in 160 ml of the resulting emulsion and then mixed with the insulin solution. The volume of the final bulk was brought to 300 ml and the pH was adjusted to 6.9. The emulsion was filtered through a 0.22 um PES membrane, filled into glass vials (fill volume 1 ml / vial) and lyophilized. Each vial contains 1.2 IU rh-insulin, 2.5 mg DHA, and 1.1 mg coenzyme Q10. The average droplet particle size in the reconstituted powder is 295 nm.

[0097] Formulation VI The formulation process is the same as that of Formulation V.

[0098] [Table 5]

[0099] Materials and Methods A 40 IU / ml rh-insulin solution was prepared as follows: 27.2 mg of insulin was solubilized in 2 ml of 0.1 N hydrochloric acid, diluted with 18 ml of water, and the pH was adjusted to 7.2 with 0.1 N sodium hydroxide.

[0100] An oil-in-water nanoemulsion (average droplet size 120 nm) was prepared as described for Formulation IV.

[0101] 10 grams of hydroxypropyl beta cyclodextrin was dissolved in 160 ml of the resulting emulsion and then mixed with the insulin solution. The volume of the final bulk was brought to 327 ml and the pH was adjusted to 6.9. The emulsion was filtered through a 0.22 μm PES membrane, filled into glass vials (filling volume 1 ml / vial) and lyophilized. Each vial contains 1.2 IU of rh-insulin, 2.5 mg of DHA, and 1.1 mg of coenzyme Q10. The average size of the droplet particles in the reconstituted powder is 143 nm.

[0102] Example 2 Testing of Formulations I-IV The content of rh-insulin was determined by reversed-phase high-performance liquid chromatography on a Hypersil Gold-C18 column (3 × 50 mm, 3 μm) using gradient elution with a mobile phase consisting of an aqueous phase acidified with 0.1% trifluoroacetic acid (TFA) and an organic phase (acetonitrile). The flow rate was 0.3 ml / min and UV detection at 205 nm. Typical chromatograms of formulations I-IV are shown in Figure 1.

[0103] Testing of Formulations V-VI The contents of rh-insulin, DHA, and coenzyme Q10 were determined by reversed-phase high-performance liquid chromatography on a Hypersil Gold-C18 column (3 × 50 mm, 3 μm) using gradient elution with a mobile phase consisting of an aqueous phase acidified with 0.1% trifluoroacetic acid (TFA) and an organic phase (a 75:15:10 mixture of acetonitrile:methanol:2-propanol). The flow rate was 0.6 ml / min, with UV detection at 205 nm for insulin and 214 nm for DHA and coenzyme Q10. Typical chromatograms for formulations V-VI are shown in Figure 2. Typical chromatograms for the determination of DHA and coenzyme Q10 in formulations V-VI are shown in Figure 3.

[0104] Example 3 Solubility of infant milk nutritional foods A study was designed to evaluate the solubility of the formulations of the present invention in the pH range of human breast milk. Three dry samples containing maltodextrin, sodium chloride, and insulin (1 IU) were prepared as follows.

[0105] Sample 1 is a crystalline heat-dried powder prepared by spraying a solution of maltodextrin, sodium chloride, and insulin onto solid maltodextrin particles, followed by drying at 35° C. The dried powder was mixed 1:3 with maltodextrin, with a final pH of 6.3.

[0106] Sample 2 is a variation of a formulation of the present invention prepared as described in Example 1, Formulations I-III, at pH 7.0 and presented as an amorphous powder.

[0107] Sample 3 was a physical mixture of dry ingredients (maltodextrin, sodium chloride, and insulin) that was dry blended without any pretreatment.

[0108] Aliquots of each formulation (equivalent to 1 IU insulin) were reconstituted in 1.8 ml of 0.1 M phosphate buffer at pH 6.3, 6.7, and 7.2 and visually inspected.

[0109] The results showed that Sample 2 (formulation of the present invention) was readily soluble over the broad pH range of breast milk (6.3-7.2) and gave clear solutions comparable to those of the reference buffer. Sample 1 (heat-dried formulation) and Sample 3 (physical blend of dry ingredients, no pretreatment) were partially soluble in the pH range tested and gave turbid solutions.

[0110] The dissolution profiles of the test samples were evaluated using HPLC in the following media: 0.01 N HCl, double deionized water, and 0.1 M phosphate buffer (pH 6.3 and 7.4).

[0111] For each sample, the percent dissolution in water and phosphate buffer was calculated relative to the amount of insulin determined in 0.01 N HCl. The resulting dissolution results (Table 6 below) demonstrate that the compositions of the present invention are highly soluble in the pH range of infant formula.

[0112] [Table 6]

[0113] In addition, Sample 1 and Sample 2 were reconstituted in milk (pH 6.89, fat content 3%). The results were consistent with the observations from the buffer recovery test. Sample 1 was only partially reconstituted in milk, resulting in visible clots that could adhere to or block the nasogastric tube through which the reconstituted drug passes. Sample 2 was completely reconstituted in milk, resulting in a homogenous mixture.

[0114] Example 4 Preterm Infant Trials - Efficacy A study was designed to evaluate the efficacy and safety of an enteral rh-insulin formulation in preterm infants.After a screening procedure, eligible infants born at 26 to a maximum of 32 weeks gestation and with a birth weight of at least 500 g were randomly assigned in a 1:1:1 ratio to one of three treatment groups.

[0115] Treatment began at least 6 hours after birth and up to 120 hours after birth. The treatment period for infants who were exclusively fed their own mother's milk (OMM) began within the first 72 hours after birth.

[0116] Treatment duration was defined as from the first day of dosing (day 1) for a maximum of 28 days or until discharge from the primary hospital if less than 28 days.

[0117] The tested formulations were administered at 0.3 IU / Kg / day for up to 28 days. Prior to administration, the powders were reconstituted in primarily human breast milk, donor breast milk, saline, or semi-saline.

[0118] result Infants were discharged from the primary hospital to either home or a secondary facility. When analysing time to discharge, two analyses are presented: 1) an analysis of discharge from the primary recruiting hospital (separate comparison of subjects transferred to a secondary hospital with those discharged to home), and 2) discharge to home (from either the primary or secondary hospital).

[0119] Time to discharge from primary hospital Infants were discharged from the primary hospital to either home or a secondary hospital, depending primarily on region. In the intention-to-treat analysis, there was one analysis target for each infant, thereby comparing each arm and strata. Both treatment arms demonstrated significantly shorter times to discharge and faster times to health stabilization compared with placebo.

[0120] The median number of days to discharge (either to home or a secondary hospital) was lowest in the 0.3 IU / Kg insulin group [median = 42.0 days, P = 0.055], followed by the 0.0406 IU / Kg insulin group [median = 43.0 days, p = 0.04*] compared with placebo [median = 49.5 days].

[0121] Time to discharge to secondary facility Some hospitals discharge infants to secondary facilities after they are stabilized and no longer in any immediately life-threatening condition. Infants are discharged to secondary facilities and then discharged home a few weeks later. In this study, subjects in the 0.3 IU / Kg insulin formulation group were discharged to secondary facilities sooner than patients in the placebo group, as shown by the median and difference from placebo (Table 7 below).

[0122] [Table 7]

[0123] Time to discharge to home Another type of analysis performed evaluated the time to discharge home for all infants, whether discharged directly from the primary hospital or after a secondary facility / unit. This analysis showed a similar trend in reducing time to discharge, but with much more variability compared to the main analysis, "discharge from primary hospital."

[0124] Treatment with 0.06 IU / Kg insulin significantly reduced the time to discharge to home compared with placebo (median = 48.0 days vs. placebo median = 60.0 days, P = 0.025*). Treatment with 0.3 IU / Kg insulin significantly reduced the time to discharge to home compared with placebo (median = 48.0 days vs. placebo median = 60.0 days, P = 0.025*). Treatment with 0.3 IU / Kg insulin significantly reduced the time to discharge to home compared with placebo (median = 55.0 days vs. placebo, P = 0.27).

[0125] When both treatment arms were analyzed together, patients were discharged sooner in the treatment group (median = 52 days, p = 0.046*) compared to placebo (median = 60 days).

[0126] Time to PN withdrawal A statistically significant difference was observed in time to PN weaning in the group receiving insulin-supplemented milk-fed diets at a concentration of 0.3 IU / kg / day compared with placebo (6 days vs. 8 days, p=0.0096**).

[0127] When evaluating time to PN weaning by gestational age group, the higher gestational age group (29-32 weeks) also had a shorter time to PN weaning in the 0.3 IU / kg / day group (4 days vs. 6 days). The results are summarized in Tables 8-10 below and shown graphically in Figure 4, which shows the probability of PN weaning by group (Kaplan-Meier survival curves).

[0128] [Table 8]

[0129] [Table 9]

[0130] [Table 10]

[0131] Other key endpoints of the study will be the percentage of PN in the infants' total intake and the tapering of the percentage of PN intake over time.

[0132] Figure 5 shows the mean daily PN intake by group and indicates a clear advantage of the treatment groups compared to placebo, especially the enteral rh-insulin formulation 0.3 IU / kg / day, in the percentage reduction in PN during total intake.

[0133] FIG. 6 shows the percentage of enteral nutrition in total nutrition by group, while FIG. 7 shows the progress by group.

[0134] Example 5 Preterm Infant Study-NEC and LOS The study population was also observed for adverse events such as necrotizing colitis (NEC) and late-onset sepsis (LOS).

[0135] Necrotizing colitis (NEC) There were meaningful differences in the percentage of infants experiencing NEC between the groups: looking at the entire study population, the overall incidence was 6.3% in the 0.06 IU / kg / day dose group, 4.3% in the 0.3 IU / kg / day dose group, and 10.2% in the placebo group, resulting in a 60% reduction in NEC cases in the 0.3 IU / kg / day dose group compared with placebo.

[0136] When looking at the lower gestational age, more prevalent study population (GA 26-28 weeks), the differences were even more pronounced, with 12.2% in the 0.06 IU / kg / day dose group, 4.3% in the 0.3 IU / kg / day dose group, and 19.6% in the placebo group, resulting in a nearly 80% reduction in NEC cases in the 0.3 IU / kg / day dose group compared to placebo.

[0137] Statistical analysis was performed using the non-parametric Kruskal-Wallis method to compare both the incidence and severity of NEC, which showed a significant statistical difference in the 26-28 GA group (p=0.077). The results of this study are shown in Tables 11 and 12 below.

[0138] [Table 11]

[0139] [Table 12]

[0140] A composite of adverse outcomes for the occurrence of one of the following: infection, NEC, and death was calculated and compared between groups and is shown in Table 13 below.

[0141] [Table 13]

[0142] The treatment groups were found to have a significantly lower percentage of subjects experiencing one of these adverse events of interest: 34.5% in the 0.06 IU / kg / day enteral Rh insulin group, 35.8% in the 0.3 IU / kg / day enteral Rh insulin group, and 50% in the placebo group. Pairwise comparisons of the rates were performed with p=0.034 for the difference between 0.06 IU / kg / day enteral Rh insulin and placebo, and p=0.065 for the difference between 0.3 IU / kg / day enteral Rh insulin and placebo (see Table 14 below).

[0143] [Table 14]

[0144] Late onset sepsis (LOS) There were meaningful differences between groups in the percentage of infants who experienced infections (including sepsis and other infections): looking at the entire study population, the overall incidence was 30% in the 0.06 IU / kg / day dose group, 34.7% in the 0.3 IU / kg / day dose group, and 42.9% in the placebo group.

[0145] Specifically, rates of clinically or culture-proven sepsis were lower in the treatment groups compared to placebo: incidence rates were 12% in the 0.06 IU / kg / day dose group, 11% in the 0.3 IU / kg / day dose group, and 15% in the placebo group (a nearly 30% reduction in the incidence of late-onset sepsis).

[0146] It is to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0147] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0148] It is the intention of the applicant(s) that all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference at the time of reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application are incorporated herein by reference in their entirety.

Claims

1. A composition comprising water-soluble amorphous insulin powder, the amorphous insulin powder comprising insulin bound to dextrin powder, having a gram / gram (g / g) insulin:dextrin ratio of 1:2500 to 1:10000, each gram of the amorphous insulin powder containing 0.2 IU to 40 IU of insulin, and the amorphous insulin powder, when reconstituted in deionized water to a concentration of 0.03 g / ml, has a pH of 6.0 to 7.

6.

2. The composition described in claim 1, further containing sodium chloride, wherein the g / g sodium chloride:dextrin ratio is 1:100 to 1:250 and the g / g insulin:sodium chloride ratio is 1:20 to 1:

50.

3. The composition described in claim 1, wherein the dextrin is maltodextrin.

4. The composition described in claim 1, wherein the dextrin is a cyclodextrin.

5. An infant nutritional powder comprising the composition of claim 1.

6. The composition described in claim 1, wherein the insulin is encapsulated within the voids of the dextrin particles.

7. A method for preparing a water-soluble amorphous insulin powder comprising: (a) solubilizing insulin in hydrochloric acid to obtain solubilized insulin; (b) diluting the solubilized insulin with water to adjust the pH to 6.9-8.0; (c) dissolving dextrin in water and adjusting the pH to 7.0-7.5; (d) adding the insulin solution of (b) to the dextrin solution of (c) to thereby encapsulate the insulin within the voids of the dextrin; (e) adjusting the pH of the solution of (d) to 6.9 to 8.0; and (f) freeze-drying the solution (e) to obtain amorphous insulin powder. A method comprising:

8. The method of claim 7, further comprising mixing the result of (b) or (e) with a sodium chloride solution and a maltodextrin solution at a pH of 6.7 to 7.5, and then adjusting the pH to 6.7 to 7.

6.

9. 9. The method of claim 8, further comprising filtering the result of said blending and lyophilizing the filtrate to obtain an amorphous insulin powder, said amorphous insulin powder having a pH of 6.0-7.6 when reconstituted in deionized water at a concentration of 0.03 g / ml.

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