WO2022002008A1 - Quick-acting insulin composition and medical use thereof - Google Patents

Abstract

Disclosed are a quick-acting insulin composition and the medical use thereof, the composition containing: 1) insulin aspart, and 2) at least one of iloprost and tafluprost. The composition has faster pharmacokinetics, better stability, and causes less irritation to an injection site than commercial preparations of existing insulin analog products.

Description

Rapid-acting insulin composition and medical use thereof technical field

The present disclosure belongs to the field of biomedicine, and relates to a pharmaceutical formulation comprising 1) insulin aspart, and 2) at least one of iloprost and tafluprost.

Background technique

Diabetes Mellitus (DM, Diabetes Mellitus) is a common chronic non-communicable disease, a clinical syndrome caused by the interaction of genetic and environmental factors, and a metabolic and lifelong disease characterized by elevated blood sugar. According to data released by IDF, the number of people with diabetes in China in 2019 was about 116 million, and China has become the country with the largest number of people with diabetes in the world; at the same time, the number of diabetic patients continues to grow rapidly. IDF predicts that the number of people with diabetes in China will reach 151 million in 2040. The growth rate of my country's insulin market has also exceeded the global average growth rate.

的原料药；门冬胰岛素，

的原料药)与改变胰岛素聚集体存在状态进而加速胰岛素起效(例如赖谷胰岛素APIDRA，缺乏Zn而降低六聚体存在)。

是目前唯一获批的新一代更快起效的餐时胰岛素注射剂，包含门冬胰岛素，增加吸收速度的维生素B3(烟酰胺)，以及确保稳定性的精氨酸。即使如此，仍需一种更快的胰岛素组合物，其可在进餐时间或进餐后使用，比现有胰岛素产品更快摄取，更快起效；同时，保证长期储存的理化稳定性与注射时的安全及可耐受性。 In order to help relieve/avoid high blood sugar levels, diabetic patients need to implement multiple long-term injection therapy; insulin is divided into rapid-acting insulin, intermediate-acting insulin, and long-acting insulin according to the duration of action. For diabetic patients, their blood sugar rises rapidly due to food absorption after a meal. If the onset of insulin is too slow or the release of insulin is insufficient at this time, it will lead to postprandial hyperglycemia. In response to this phenomenon, insulin preparation products have been developed to reduce the injection-onset interval, and the mechanism of action includes changing the sequence of the amino acid chain in human insulin and thereby changing the onset time of insulin (such as insulin lispro,

API; Insulin Aspart,

APIDRA) and change the existing state of insulin aggregates to accelerate the onset of insulin (such as insulin glulisine APIDRA, lack of Zn and reduce the existence of hexamers).

It is the only approved next-generation, faster-acting mealtime insulin injection that contains insulin aspart, vitamin B3 (niacinamide) to increase absorption, and arginine to ensure stability. Even so, there is still a need for a faster insulin composition, which can be used at mealtime or after a meal, is ingested faster than existing insulin products, and has a faster onset of action; safety and tolerability.

SUMMARY OF THE INVENTION

The present disclosure provides a pharmaceutical composition comprising:

1) insulin aspart, and

2) At least one of iloprost and tafluprost.

In the context of this disclosure, insulin aspart refers to human insulin in which amino acid 28 of the B chain is mutated to aspartic acid, CAS number 116094-23-6, and molecular weight 5826.

In some optional embodiments, the pharmaceutical composition further comprises arginine.

In some embodiments, the pharmaceutical composition further comprises:

buffer;

metal ions, such as zinc ions;

isotonic agents, such as glycerol and/or sodium chloride; and/or

preservative.

In some embodiments, the buffer is selected from the group consisting of phosphate buffer, acetate buffer, and citrate buffer.

In some embodiments, the buffer is a phosphate buffer, such as disodium hydrogen phosphate.

In some embodiments, the preservative is selected from the group consisting of phenol, m-cresol, and benzyl alcohol.

In some embodiments, the preservatives are phenol and m-cresol.

In some embodiments, the concentration of insulin aspart in the pharmaceutical composition is 0.3 mM-2.4 mM, 0.4 mM-2 mM, 0.5 mM-1.5 mM, 0.5 mM-1 mM, or 0.5 mM-0.8 mM, eg, about 0.5 mM, about 0.51 mM, about 0.52 mM, about 0.53 mM, about 0.54 mM, about 0.55 mM, about 0.56 mM, about 0.57 mM, about 0.58 mM, about 0.59 mM, about 0.6 mM, about 0.61 mM, about 0.62 mM, about 0.63 mM, about 0.64 mM, about 0.65 mM, about 0.66 mM, about 0.67 mM, about 0.68 mM, about 0.69 mM, or about 0.7 mM.

In some embodiments, the concentration of iloprost in the pharmaceutical composition is 0.01 μM-60 μM, eg, the concentration of iloprost is 0.05 μM-60 μM, 0.05 μM-55 μM, 0.05 μM-50 μM, 0.05 μM-45 μM, 0.05μM-40μM, 0.05μM-35μM, 0.05μM-30μM, 0.05μM-25μM, 0.05μM-20μM, 0.1μM-18μM, 0.5μM-15μM, 0.5μM-10μM, 1μM-5μM, or 2μM-4μM, specific about 0.05 μM, about 0.1 μM, about 0.5 μM, about 1 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3.0 μM, about 5.0 μM , about 6.0 μM, about 7.0 μM, about 8.0 μM, about 9.0 μM, or about 10 μM.

In some embodiments, the concentration of tafluprost in the pharmaceutical composition is 0.005 μM-10 μM, eg, the concentration of tafluprost is 0.01 μM-10 μM, 1 μM-10 μM, or 5 μM-10 μM, specifically, about 6 μM, about 7 μM, about 8 μM, about 8.1 μM, about 8.2 μM, about 8.3 μM, about 8.4 μM, about 8.5 μM, about 9 μM, or about 10 μM.

In some embodiments, the concentration of arginine in the pharmaceutical composition is 10mM-100mM, such as 20mM-80mM, 20mM-40mM, 40mM-60mM, or 60mM-80mM, specifically, about 10mM, about 15mM, about 20mM, About 25 mM, about 30 mM, about 50 mM, about 70 mM, about 90 mM, or about 100 mM.

In some embodiments, the zinc ions are present in the pharmaceutical composition in a ratio of about 2-4 zinc ions per 6 insulin molecules. In some embodiments, the zinc ions are present in the pharmaceutical composition in a ratio of about 2.5-3.5 zinc ions per 6 insulin molecules. In some embodiments, the concentration of zinc ions in the pharmaceutical composition is 0.1 mM-0.5 mM, such as 0.2 mM-0.4 mM, such as 0.3 mM. By complexing Zn ²⁺ , insulin depolymerization is promoted under the premise of stability.

In some embodiments, the pharmaceutical composition comprises:

0.3mM-2.4mM insulin aspart, such as 0.5mM-1.5mM insulin aspart or 0.5mM-1mM insulin aspart;

0.01 μM-65 μM iloprost, such as 0.05 μM-65 μM iloprost, 0.05 μM-50 μM iloprost, 0.1 μM-15 μM iloprost, or 1 μM-10 μM iloprost; and

10 mM-100 mM arginine, eg 20 mM-80 mM arginine.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

0.05μM-15μM iloprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

0.05μM-15μM iloprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

0.05μM-15μM iloprost;

20mM-80mM arginine;

1mM-5mM disodium hydrogen phosphate;

0.1mM-0.5mM zinc acetate;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

0.05μM-15μM iloprost;

20mM-80mM arginine;

1mM-5mM disodium hydrogen phosphate;

0.1mM-0.5mM zinc acetate;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

2.5μM-3μM iloprost;

about 20mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

About 19.6 mg/mL glycerol;

about 1.5 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

2.5μM-3μM iloprost;

about 20mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

About 3.3 mg/mL glycerol;

about 1.5 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

2.5μM-3μM iloprost;

about 40mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

About 19.6 mg/mL glycerol;

about 1.5 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

2.5μM-3μM iloprost;

about 40mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

About 3.3 mg/mL glycerol;

about 1.5 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

2.5μM-3μM iloprost;

about 80mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

About 19.6 mg/mL glycerol;

about 1.5 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

2.5μM-3μM iloprost;

about 80mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

About 3.3 mg/mL glycerol;

about 1.5 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.3mM-2.4mM insulin aspart, for example 0.5mM-1.5mM or 0.5mM-1mM insulin aspart;

0.005 μM-10 μM tafluprost, such as 0.01 μM-10 μM tafluprost or 1 μM-10 μM tafluprost; and

10 mM-100 mM arginine, eg 20 mM-80 mM arginine.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

1 μM-10 μM tafluprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

1 μM-10 μM tafluprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

1 μM-10 μM tafluprost;

20mM-80mM arginine;

1mM-5mM disodium hydrogen phosphate;

0.1mM-0.5mM zinc acetate;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

0.5mM-1.5mM insulin aspart;

1 μM-10 μM tafluprost;

20mM-80mM arginine;

1mM-5mM disodium hydrogen phosphate;

0.1mM-0.5mM zinc acetate;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

8μM-9μM tafluprost;

about 20 mM, about 40 mM, or about 80 mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

about 0.6mM insulin aspart;

8μM-9μM tafluprost;

about 20 mM, about 40 mM, or about 80 mM arginine;

3.5mM-4mM disodium hydrogen phosphate;

about 0.3mM zinc acetate;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the concentration of insulin aspart in the pharmaceutical composition is 1.75 mg/mL-14 mg/mL, 2.33 mg/mL-11.65 mg/mL, 2.91 mg/mL-8.74 mg/mL, 2.91 mg/mL- 5.83 mg/mL, or 2.91 mg/mL-4.67 mg/mL; in some embodiments, the concentration of insulin aspart in the pharmaceutical composition is 1.75 mg/mL-14 mg/mL, such as 3.5 mg/mL-7 mg/mL ; In some embodiments, the concentration of insulin aspart in the pharmaceutical composition is about 3.5 mg/mL.

In some embodiments, the concentration of iloprost in the pharmaceutical composition is 0.0036 μg/mL-21.6 μg/mL, eg, the concentration of iloprost is 0.018 μg/mL-21.6 μg/mL, 0.018 μg/mL- 19.8μg/mL, 0.018μg/mL-18μg/mL, 0.018μg/mL-16.2μg/mL, 0.018μg/mL-14.4μg/mL, 0.018μg/mL-12.6μg/mL, 0.018μg/mL-10.8 μg/mL, 0.018μg/mL-9μg/mL, 0.018μg/mL-7.2μg/mL, 0.036μg/mL-6.48μg/mL, 0.18μg/mL-5.4μg/mL, 0.18μg/mL-3.6μg /mL, 0.36 μg/mL-1.8 μg/mL, or 0.72 μg/mL-1.44 μg/mL; in some embodiments, the concentration of iloprost in the pharmaceutical composition is 0.1 μg/mL-10 μg/mL, For example, 0.3 μg/mL to 5 μg/mL; in some embodiments, the concentration of iloprost is 0.6 μg/mL to 3 μg/mL, such as about 1 μg/mL.

In some embodiments, the concentration of tafluprost is 0.00225 μg/mL-4.5 μg/mL, eg, the concentration of tafluprost is 0.0045 μg/mL-4.5 μg/mL, 0.45 μg/mL-4.5 μg/mL , or 2.25 μg/mL-4.5 μg/mL; in some embodiments, the concentration of tafluprost is about 3.75 μg/mL.

In some embodiments, the concentration of arginine is from 10 mM to 100 mM, eg, 20 mM to 80 mM, eg, about 20 mM.

In some embodiments, the pharmaceutical composition comprises:

1.75mg/mL-14mg/mL insulin aspart;

0.0036μg/mL-21.6μg/mL iloprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

1.75mg/mL-14mg/mL insulin aspart;

0.0036μg/mL-21.6μg/mL iloprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

3.5mg/mL-7mg/mL insulin aspart;

0.6μg/mL-3μg/mL iloprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

3.5mg/mL-7mg/mL insulin aspart;

0.6μg/mL-3μg/mL iloprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

1.75mg/mL-14mg/mL insulin aspart;

0.00225μg/mL-4.5μg/mL tafluprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

1.75mg/mL-14mg/mL insulin aspart;

0.00225μg/mL-4.5μg/mL tafluprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

3.5mg/mL-7mg/mL insulin aspart;

2.25μg/mL-4.5μg/mL tafluprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-25mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

3.5mg/mL-7mg/mL insulin aspart;

2.25μg/mL-4.5μg/mL tafluprost;

20mM-80mM arginine;

1mM-5mM phosphate buffer;

0.1mM-0.5mM zinc ion;

1mg/mL-5mg/mL glycerol;

1 mg/mL-3 mg/mL phenol; and

1 mg/mL-3 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

About 3.5mg/mL insulin aspart;

About 3 μg/mL iloprost;

about 20mM arginine;

About 0.53mg/mL disodium hydrogen phosphate;

About 65.8μg/mL zinc acetate;

About 19.6 mg/mL glycerol;

about 1.50 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

About 3.5mg/mL insulin aspart;

About 3 μg/mL iloprost;

about 20mM arginine;

About 0.53mg/mL disodium hydrogen phosphate;

About 65.8μg/mL zinc acetate;

About 3.3 mg/mL glycerol;

about 1.50 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

About 3.5mg/mL insulin aspart;

About 1 μg/mL iloprost;

about 20mM arginine;

About 0.53mg/mL disodium hydrogen phosphate;

About 65.8μg/mL zinc acetate;

About 19.6 mg/mL glycerol;

about 1.50 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

About 3.5mg/mL insulin aspart;

About 1 μg/mL iloprost;

about 20mM arginine;

About 0.53mg/mL disodium hydrogen phosphate;

About 65.8μg/mL zinc acetate;

About 3.3 mg/mL glycerol;

about 1.50 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

About 3.5mg/mL insulin aspart;

About 0.6 μg/mL iloprost;

about 20mM arginine;

About 0.53mg/mL disodium hydrogen phosphate;

About 65.8μg/mL zinc acetate;

About 19.6 mg/mL glycerol;

about 1.50 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition comprises:

About 3.5mg/mL insulin aspart;

About 0.6 μg/mL iloprost;

about 20mM arginine;

About 0.53mg/mL disodium hydrogen phosphate;

About 65.8μg/mL zinc acetate;

About 3.3 mg/mL glycerol;

about 1.50 mg/mL phenol; and

About 1.72 mg/mL m-cresol.

In some embodiments, the pharmaceutical composition is in the form of a solution or a lyophilized formulation. In some embodiments, the pharmaceutical composition is an injectable solution. In some embodiments, the pharmaceutical composition is formulated into a prefilled needle.

Routes of use of the pharmaceutical compositions of the present disclosure include, but are not limited to, subcutaneous injection by self-administration, such as by use of a syringe or pen device, or by continuous subcutaneous insulin infusion therapy with an insulin pump device, intravenous, intradermal or intraperitoneal route.

In some embodiments, the pharmaceutical composition is stable at 25±2°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the pharmaceutical composition is stable at 40±2°C for at least 5 days, at least 7 days, at least 14 days, or at least 28 days, and after low temperature cycling, freeze-thaw cycling and good stability.

In an optional embodiment, the pharmaceutical composition is placed at 25°C ± 2°C for 3 months, 6 months, 9 months, 12 months or 18 months, and the high molecular protein content is not more than 1%, and can be 0.9% , 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less.

In an optional embodiment, the pharmaceutical composition is placed at 40±2°C for 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months or 6 months, and the high molecular protein content is not greater than 1%, for example, can be 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less.

In an alternative embodiment, the pharmaceutical composition is placed at 25°C ± 2°C for 3 months, 6 months, 9 months, 12 months or 18 months, and the known impurity content is not greater than 1.75%, for example, it can be 1.7 %, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1% or less.

In an alternative embodiment, the pharmaceutical composition is placed at 40±2°C for 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months or 6 months, and the known impurity content is not greater than 1.75%, for example, can be 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1% or less.

In an optional embodiment, the pharmaceutical composition is placed at 25°C±2°C for 3 months, 6 months, 9 months, 12 months or 18 months, and the other total impurity content is not more than 0.75%, for example, it can be 0.74 %, 0.73%, 0.72%, 0.71%, 0.7%, 0.69%, 0.68%, 0.67%, 0.66%, 0.65%, 0.64%, 0.63%, 0.62%, 0.61%, 0.6% or less.

In an optional embodiment, the pharmaceutical composition is placed at 40±2°C for 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months or 6 months, and other total impurities are not greater than 0.75%, can be 0.74%, 0.73%, 0.72%, 0.71%, 0.7%, 0.69%, 0.68%, 0.67%, 0.66%, 0.65%, 0.64%, 0.63%, 0.62%, 0.61%, 0.6% or lower.

In an optional embodiment, the pharmaceutical composition is placed at 25°C ± 2°C for 3 months, 6 months, 9 months, 12 months or 18 months, and the purity of the main peak is not less than 97%, which can be 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98% or higher.

In an optional embodiment, the pharmaceutical composition is placed at 40±2°C for 5 days, 7 days, 14 days, 28 days, 1 month, 2 months, 3 months or 6 months, and the purity of the main peak is not less than 97 %, which can be 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98% or higher.

The chemical stability of protein formulations can be assessed by measuring the amount of chemical degradation products at various time points after exposure to different environmental conditions (often by accelerating the formation of degradation products, for example, by increasing temperature). The amount of each individual degradation product is often determined by separating the degradation products according to molecular size and/or charge using various chromatographic techniques (eg, SEC-HPLC and/or RP-HPLC), such as are typical of the stability of pharmaceutical compositions in the present disclosure. The accepted criterion is that generally no more than about 10%, preferably no more than about 5%, of the active ingredient is degraded as measured by HPLC.

In alternative embodiments, the pharmaceutical compositions of the present disclosure are non-irritating to the injection site and surrounding tissues. According to the skin irritation part of the technical guidelines for drug irritation, allergy and hemolysis research, the skin irritation intensity score of 0-0.49 is regarded as non-irritating.

In an optional embodiment, the irritation score of the pharmaceutical composition of the present disclosure to the injection site and surrounding tissues is lower than 0.49, lower than 0.45, lower than 0.4, lower than 0.35, lower than 0.34, lower than 0.33, Below 0.32, Below 0.31, Below 0.3, Below 0.29, Below 0.28, Below 0.27, Below 0.26, Below 0.25, Below 0.24, Below 0.23, Below 0.22, Below 0.21, Below 0.2, below 0.19, below 0.18, below 0.17, below 0.16, below 0.15, below 0.14, below 0.13, below 0.12, below 0.11, below 0.1 or 0.

In alternative embodiments, the pharmaceutical composition of the present disclosure has a rapid onset of action after injection, and the onset time of subcutaneous injection is 3-30 minutes, 5-25 minutes, 5-20 minutes, 5-15 minutes, or 5-10 minutes minutes, or subcutaneous injection within 30 minutes, within 25 minutes, within 20 minutes, within 18 minutes, within 15 minutes, within 12 minutes, within 10 minutes, within 8 minutes, within 5 minutes, or within 3 minutes; Peak times were 20 minutes-2 hours, 30 minutes-2 hours, or 30 minutes-1 hour; durations were 1-6 hours, 2-6 hours, 3-6 hours, or 4-6 hours.

The present disclosure also provides a method of preparing the aforementioned pharmaceutical composition, comprising the step of mixing insulin aspart with at least one of iloprost and tafluprost; in an optional embodiment, the method further Include a step for adding arginine.

The present disclosure provides the use of a pharmaceutical composition in the manufacture of a medicament for the treatment and/or prevention of hyperglycemia.

The present disclosure provides a method of treating hyperglycemia comprising administering to a subject in need thereof an effective amount of any of the aforementioned pharmaceutical compositions.

The present disclosure also relates to the aforementioned pharmaceutical compositions for use in the treatment of hyperglycemia.

In some embodiments, the hyperglycemia is selected from the group consisting of type I diabetes, type II diabetes, gestational diabetes, and other diseases that cause hyperglycemia.

The present disclosure relates to pharmaceutical composition formulations comprising 1) insulin aspart, and 2) at least one of iloprost and tafluprost, for rapidly lowering blood glucose levels at mealtime or after meals, preventing possible occurrence of Glucagon. The pharmaceutical compositions provided by the present disclosure have equivalent or better physical and/or chemical stability, lower irritation, and faster pharmacokinetics compared to commercially available formulations of existing insulin analog products.

Description of drawings

C为门冬胰岛素+伊洛前列素+精氨酸；H为门冬胰岛素+他氟前列素+精氨酸。 Figure 1 shows the whole blood glucose test results, where: A is the listed reference substance insulin aspart

C is insulin aspart + iloprost + arginine; H is insulin aspart + tafluprost + arginine.

C为门冬胰岛素+伊洛前列素+精氨酸；H为门冬胰岛素+他氟前列素+精氨酸。 Figure 2 shows the results of serum blood glucose testing, where: A is the listed reference substance insulin aspart

C is insulin aspart + iloprost + arginine; H is insulin aspart + tafluprost + arginine.

样品C为门冬胰岛素+伊洛前列素+精氨酸；样品H为门冬胰岛素+他氟前列素+精氨酸。 Figure 3 shows the detection results of serum blood glucose, wherein: a is the detection result of serum blood glucose at 0 min, b is the detection result of serum blood glucose at 15 min, and c is the detection result of serum blood glucose at 18 min. Sample A is the listed reference substance insulin aspart

Sample C is insulin aspart + iloprost + arginine; sample H is insulin aspart + tafluprost + arginine.

Figures 4A-4C show the results of hypoglycemic effect after administration of the pharmaceutical formulations R, S, and T of the present disclosure, respectively, in the LYD pig model; Figures 4D-4F show the administration of the pharmaceutical formulations of the present disclosure in the LYD pig model, respectively Changes in plasma insulin concentration after R, S, and T.

Figures 5A-5B show the percent change in blood glucose after administration of Formulation R or S in the LYD pig model; Figure 5C shows the results of the change in plasma insulin concentration after administration of Formulation S.

Figure 6 shows the results of the hypoglycemic effect of Formulation S in human subjects.

detailed description

definition

A "pharmaceutical composition" of the present disclosure means a mixture comprising insulin aspart in combination with at least one of iloprost and tafluprost described herein, and other chemical components, such as physiological/ Pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of insulin aspart and iloprost and/or tafluprost, promote administration to the organism, facilitate the absorption of active ingredients and exert biological activity. Herein, "pharmaceutical composition" and "preparation" are not mutually exclusive and are sometimes used interchangeably.

"Chemical stability" of a composition is used herein to refer to changes in covalent protein structure that result in the formation of chemical degradation products that have potentially less biological potential and/or potentially increased potential compared to the native protein structure. Immunogenic properties. Various chemical degradation products can be formed, depending on the type and properties of the native protein and the environment to which the protein is exposed. Increased amounts of chemical degradation products are often seen during storage and use of protein formulations. Most proteins are susceptible to deamidation, which is the hydrolysis of side chain amide groups in glutamyl or asparagine residues to form free carboxylic acids or the hydrolysis of asparagine residues to form IsoAsp (isoaspartic acid) derivatives the process of. Other degradation pathways include the formation of high molecular weight products in which two or more protein molecules are covalently bound to each other through transamidation and/or disulfide interactions, resulting in the formation of covalently bound dimers, oligomers and polymer degradation products (Stability of Protein Pharmaceuticals, Ahern. TJ & Manning MC, Plenum Press, New York 1992). Oxidation (eg of methionine residues) may be mentioned as another variant of chemical degradation. Additionally, low HMWP levels are advantageous for the pharmaceutical compositions of the present disclosure because high molecular weight protein (HMWP) products are potentially immunogenic and biologically inactive.

The term "type I diabetes", also known as insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is caused by B-cell destruction, often resulting in absolute insulin deficiency. The term "type II diabetes", also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is primarily associated with insulin resistance and thus relative insulin deficiency and/or insulin secretion deficiency predominately accompanied by insulin resistance.

The expression "about" in this disclosure has a range of error of ±10% to ±20%.

The term "onset time" refers to the time from administration (eg, subcutaneous administration) of a pharmaceutical composition of the present disclosure to the first detection of insulin aspart in the blood.

The term "absorption rate" refers to the slope of the PK curve.

The term "arginine" or "Arg" includes arginine and/or salts thereof.

The pharmaceutical excipients or reagents involved in the present disclosure can all come from commercial sources.

The present disclosure is further described below in conjunction with the examples, but these examples do not limit the scope of the present disclosure. The experimental methods that do not specify specific conditions in the embodiments of the present disclosure are generally carried out in accordance with conventional conditions; or in accordance with conditions suggested by raw material or commodity manufacturers. The reagents without specific sources are the conventional reagents purchased in the market.

The preparation of embodiment 1 pharmaceutical preparation

(1) prepare a series of preparations of insulin aspart according to the preparation composition of Table 1, the preparations are prepared as a mixture containing active ingredients and zinc ions (for example, zinc acetate or zinc chloride can be added), buffer salts, preservatives, isotonic agents aqueous solution. These formulations were used for stability, irritation and pharmacodynamic (PD)/pharmacokinetic (PK) testing in subsequent examples.

Table 1. Formulation composition of insulin aspart

(2) Increase the arginine concentration to 6.96mg/ml (40mM) and 13.92mg/ml (80mM) to further investigate the stability; in the betahistine prescription, betahistine sets two concentrations of 5mg/ml ml and 10 mg/ml; in the tafluprost prescription, the concentration of tafluprost was kept at 3.75 μg/ml; in the iloprost prescription, iloprost set two concentrations of 10 μg/ml and 1 μg/ml ml. The specific formulation composition is shown in Table 2.

Table 2. Formulation composition of insulin aspart

Example 2 Analysis of chemical stability of pharmaceutical preparations

All formulations prepared in Research Example 1 were set up with or without arginine, and placed in a stability test. The chemical stability was characterized by the content of degradation products (%) measured by the difference between the contents after 5 days, 10 days and 30 days of preparation at 40°C, and the detection of impurities was carried out according to the following detection conditions.

Related Substance Testing Conditions

Column model: Sepax Bio-C18, 4.0×250mm, 5μm;

Column temperature: 40℃; Detection wavelength: 214nm; Flow rate: 1.0ml/min; Injection volume: 10μl;

Mobile phase-dissolve with 0.2mol/L sodium sulfate buffer (weigh 28.4g of anhydrous sodium sulfate in 800ml water, add 2.7ml phosphoric acid, adjust the pH value to pH3.6 with 4mol/L sodium hydroxide solution, add water to 1000ml , passed through a 0.45 μm filter membrane)-acetonitrile (90:10) as mobile phase A; 50% acetonitrile as mobile phase B, carry out gradient elution according to Table 3:

table 3

From the high molecular weight proteins shown in Table 4, known impurities (B3Asp+A21Asp+B3isoAsp+B28isoAsp impurities, it means that the 3rd position of the B chain is aspartic acid, the 21st position of the A chain is aspartic acid, and the 3rd position of the B chain is aspartic acid. It is the sum of the impurities of isoaspartic acid and the 28th position of the B chain as the sum of the known impurities, and is called other total impurities) and other total impurities. The stability of L (betahistine) is relatively good, but other total impurities increase rapidly, and the color changes at the same time. It is speculated that during the placement process, the protein degrades or reacts with other substances in the prescription, resulting in the increase of other total impurities. ; It can be seen from the liquid phase that the protein degradation in formulation J (nicorandil) was obvious and was excluded in the follow-up study; formulation B (iloprost) and formulation F (tafluprost) were relatively stable Well, the main peak purity decreased a little more, but was not considered to constitute a significant difference.

After adding arginine, the stability of the formulation was improved, and the stability of formulations C, G, K, M, and U were better than the corresponding formulations B, F, J, and L without arginine.

Table 4. Chemical stability data for insulin aspart formulations

Note: 1. Known impurities: the impurities whose chemical structure has been clarified, this product mainly refers to B ₂₈ isoAsp insulin aspart, A ₂₁ Asp insulin aspart, B ₃ Asp insulin aspart, B ₃ isoAsp insulin aspart; 2 、Other total impurities: that is, the sum of impurities other than known impurities; 3. High molecular protein: also called high molecular polymer, mainly refers to the polymer formed by the polymerization of two or more units of main components or impurities .

The effect of arginine concentration on formulation stability was further studied, and the results are shown in Table 5. For formulations D and E (insulin aspart + tafluprost), when the arginine concentration was increased to 40 mM, the formulations were more stable, and continued to increase the arginine concentration to 80 mM, from the analysis of the 5-day results, the formulation stability was not obvious change. For preparations N, O, P, Q (insulin aspart + betahistine), the stability of the preparation was poor, and the content of other total impurities increased significantly, and increasing the concentration of arginine could not improve the stability of the preparation.

Example 3 Irritation test of pharmaceutical preparations

New Zealand rabbits (n=5, purchased from Pizhou Dongfang Breeding Co., Ltd.) were given a single subcutaneous injection of preparations C and H, 3 days after a single injection of 100 μL injection (10 units), gross anatomical observation and histopathological examination , observe the irritant response to the injection site and surrounding tissues. Tables 6 and 7 are the skin irritation part in the technical guidelines for drug irritation, allergy and hemolysis, and the irritation of the preparations in this study was scored with reference to this standard. Table 8 shows the irritation score results.

Table 6. Skin irritation response scoring criteria

Table 7. Evaluation criteria for skin irritation intensity

Score Evaluation 0～0.49 non-irritating 0.5～2.99 mild irritation 3.0～5.99 Moderately irritating 6.0～8.00 severe irritation

Table 8. Irritation results and scores for insulin formulations C and H

preparation Score Evaluation C 0.33 non-irritating H 0 non-irritating

The irritation score results showed that formulation C and formulation H could be regarded as non-irritant, and formulation H containing iloprost had a better irritation score than formulation C containing tafluprost.

Example 4 Animal PK/PD experiments of pharmaceutical preparations

Pharmacokinetic/pharmacodynamic studies and plasma/serum analysis were carried out in LYD (Land race, Yorkshire and Duroc) pig model (about 50kg, single sex, n=10, purchased from Chengdu Shepherd Village Agricultural Development Co., Ltd.) .

During the adaptation period, blood glucose monitoring and fasting blood glucose monitoring were performed at any time. In the experimental period, the fasting blood glucose was measured in each round of the test (fasting and water overnight), subcutaneous injection, and the test sample was not diluted. The control group was given the preparation A, the experimental group was given the preparation C or H, and the dosage: a single subcutaneous injection of 1 nmol/kg using a micro-syringe. Blood glucose was measured again 12 times within 1 hour after administration (before administration and 3, 6, 9, 12, 15, 18, 24, 30, 36, 48, 60 min after administration).

Blood collection method: Take a long rope as thick as chopsticks, make one end of the rope into a looper, put it into the back of the upper canine teeth of the pig's mouth, and then tighten the rope and lift it up. At this time, the pig's attention is focused on the nose. The upper part is mostly in a backward posture, so as to maintain a stable standing state. Sterilize the lowest part of the right chest cavity with an alcohol cotton ball and insert the needle. If there is a negative pressure and there is blood returning, it means that the anterior vena cava has been pierced, and the inner core of the syringe is gently pulled to collect blood. After the blood was collected, the needle was pulled out and the bleeding was stopped by pressing with a cotton ball.

Blood glucose meter detects blood glucose in real time: after blood collection, drop a drop of whole blood on a clean surface and use a blood glucose meter for real-time measurement.

Determination of serum blood glucose by biochemical method: The collected whole blood was injected into a coagulation tube, and after blood coagulation, centrifuged at 2500 × g for 10 min at 4°C, and serum was collected, and blood glucose was measured with a biochemical analyzer.

12 time points were set after each round of administration (before administration and 3, 6, 9, 12, 15, 18, 24, 30, 36, 48, 60 min after administration), the blood collection method was the same as above, and blood was collected and injected. In an EDTA-2K anticoagulation tube, centrifuge the plasma at 10,000 × g for 6 min at 4°C to obtain 100 μL of plasma, which is stored at -80°C.

Figures 1 and 2 show the results of the whole blood glucose test and serum blood glucose test results at 12 time points, respectively, and Figure 3 shows the results before administration (0min, a) and 15min (b) and 30min (c) after administration ), it can be seen that preparations C and H show a faster hypoglycemic effect than control group A, and preparation H containing iloprost lowers blood sugar faster than preparation C containing tafluprost.

Example 5 Animal PK/PD experiments of pharmaceutical preparations

Formulations R, S, T and U were prepared according to the formulation composition of Table 9. Pharmacokinetic/pharmacodynamic studies and plasma/serum assays were performed in LYD pig model (around 50 kg, single sex) according to the method of Example 4.

Table 9. Formulation composition of insulin aspart

的趋势，血浆门冬胰岛素浓度AUC优于

As shown in Figure 4A-Figure 4F and Figure 5A-Figure 5C, the absorption rate of insulin aspart of formulation R (3 μg/mL iloprost) was significantly better than that of ordinary insulin aspart, with better than

trend, plasma insulin aspart concentration AUC is better than

相当，血浆门冬胰岛素浓度AUC略优于

The absorption rate of insulin aspart in formulation S (1 μg/mL iloprost) was significantly different from the

equivalent, plasma insulin aspart concentration AUC slightly better than

的趋势，但血浆门冬胰岛素浓度AUC优于

The rate of absorption of insulin aspart in formulation T (0.6 μg/mL iloprost) was inferior to

trend, but plasma insulin aspart concentration AUC was better than

Formulation U was comparable to Formulation S.

Example 6 Study on the hypoglycemic effect of pharmaceutical preparations

组(6例)：研究护士用1ml注射器吸取

注射液于上臂三角肌下缘部位皮下注射给药，1min内给药完毕，研究医生现场监督实施。制剂S组(6例)：研究护士用1ml注射器吸取制剂S于上臂三角肌下缘部位皮下注射给药，1min内给药完毕，研究医生现场监督实施。各受试者每次给药后间隔2天进行下一个周期的给药。 The 12 male subjects were equally divided into 2 groups, with 6 people in each group, and a double-crossover test within each group was performed (see Table 10 for the protocol). The subjects entered the Phase I clinical trial ward on the day before the trial, and were provided with standard meals and drinking water on time. Before administration, the research doctor and research nurse double check the random code of the subjects and the drug code, and the administration can only be done after confirmation. A standard breakfast was eaten at 7:00 in the morning on the day of administration, and administered two hours later (9:00 in the morning), with no food or water within 4 hours after administration. The administered dose is: 0.2 U/kg (0.002 mL/kg), wherein 1 ml of insulin solution contains 100 U of insulin aspart (equivalent to 3.5 mg).

Group (6 cases): the research nurse sucked with a 1ml syringe

The injection was administered subcutaneously at the lower border of the deltoid muscle of the upper arm, and the administration was completed within 1 min, and the implementation was supervised by the research doctor on site. Preparation S group (6 cases): The research nurse sucked the preparation S with a 1ml syringe and injected it subcutaneously at the lower border of the deltoid muscle of the upper arm. Each subject was given 2 days after each administration for the next cycle of administration.

Table 10. Crossover Experimental Protocol

和普通

的起效时间分别为19min和29min，对应的门冬胰岛素起效血药浓度约为250pM；制剂S在15min时的平均血药浓度约为380pM，已超过了门冬胰岛素的起效浓度(或称有效浓度；参见图6)； According to reports,

and ordinary

The onset time of insulin aspart was 19min and 29min respectively, and the corresponding plasma concentration of insulin aspart was about 250pM; the average plasma concentration of preparation S at 15min was about 380pM, which had exceeded the onset concentration of insulin aspart (or called the effective concentration; see Figure 6);

为16.7min，达峰时间相当； As shown in Table 11, the time for formulation S to reach 50% Cmax (t _{50% Cmax} ) was 19.7 min,

is 16.7min, and the peak time is equivalent;

相当(236pmol×h/L Vs 226pmol×h/L)；制剂S在注射后0-60min的暴露量高于

(623pmol×h/L Vs 544pmol×h/L)。 The exposure of Formulation S from 0 to 33 min after injection was similar to

Equivalent (236pmol×h/L Vs 226pmol×h/L); the exposure of Formulation S at 0-60min after injection was higher than

(623pmol×h/L Vs 544pmol×h/L).

Table 11. Hypoglycemic Results of Pharmaceutical Formulations

Although the foregoing invention has been described in detail with the aid of the drawings and examples for a clear understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (16)

A pharmaceutical composition comprising: 1) insulin aspart, and 2) At least one of iloprost and tafluprost. The pharmaceutical composition of claim 1, further comprising arginine. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of insulin aspart is 1.75 mg/mL to 14 mg/mL, preferably 3.5 mg/mL to 7 mg/mL, more preferably about 3.5 mg/mL. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of the iloprost is 0.018 μg/mL to 21.6 μg/mL, preferably 0.6 μg/mL to 3 μg/mL, more preferably about 1 μg/mL. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of tafluprost is 0.00225 μg/mL-4.5 μg/mL, preferably 2.25 μg/mL-4.5 μg/mL, more preferably About 3.75 μg/mL. The pharmaceutical composition of any one of claims 1 to 5, wherein the arginine is present at a concentration of 10 mM to 100 mM, preferably 20 mM to 80 mM, more preferably 20 mM to 40 mM, most preferably about 20 mM. The pharmaceutical composition of any one of claims 1 to 6, further comprising: buffer; metal ions, such as zinc ions, preferably zinc acetate; isotonic agents, such as glycerol and/or sodium chloride; and/or preservative. The pharmaceutical composition of claim 7, wherein the buffer is selected from the group consisting of phosphate buffer, acetate buffer, and citrate buffer. The pharmaceutical composition according to claim 7, wherein the preservative is selected from phenol, m-cresol and benzyl alcohol; preferably, the preservative is phenol or m-cresol. The pharmaceutical composition of claim 1, comprising: 1.75mg/mL-14mg/mL insulin aspart; 0.0036 μg/mL-21.6 μg/mL iloprost; and 20mM-80mM Arginine. The pharmaceutical composition of claim 1, comprising: 3.5mg/mL-7mg/mL insulin aspart; 0.6μg/mL-3μg/mL iloprost; 20mM-80mM arginine; 1mM-5mM disodium hydrogen phosphate; 0.1mM-0.5mM zinc acetate; 1mg/mL-25mg/mL glycerol; 1 mg/mL-3 mg/mL phenol; and 1 mg/mL-3 mg/mL m-cresol. The pharmaceutical composition of claim 1, comprising: 1.75mg/mL-14mg/mL insulin aspart, preferably 3.5mg/mL-7mg/mL insulin aspart; 2.25 μg/mL to 4.5 μg/mL tafluprost, preferably about 3.75 μg/mL tafluprost; and 20 mM-80 mM arginine, preferably about 20 mM arginine. The pharmaceutical composition of claim 1, comprising: About 3.5mg/mL insulin aspart; About 1 μg/mL iloprost; about 20mM arginine; About 0.53mg/mL disodium hydrogen phosphate; About 65.8μg/mL zinc acetate; About 3.3 mg/mL glycerol; about 1.50 mg/mL phenol; and About 1.72 mg/mL m-cresol. The pharmaceutical composition of claim 1, comprising: About 3.5mg/mL insulin aspart; About 1 μg/mL iloprost; about 20mM arginine; About 0.53mg/mL disodium hydrogen phosphate; About 65.8μg/mL zinc acetate; About 19.6 mg/mL glycerol; about 1.50 mg/mL phenol; and About 1.72 mg/mL m-cresol. A method of preparing a pharmaceutical composition according to any one of claims 1 to 14, comprising the step of mixing insulin aspart with at least one of iloprost and tafluprost; preferably also comprising Steps for adding arginine. Use of the pharmaceutical composition according to any one of claims 1 to 14 in the preparation of a medicament for the treatment and/or prevention of hyperglycemia, preferably, the hyperglycemia is selected from type I diabetes, type II diabetes Diabetes and gestational diabetes.

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