Insulin in tablets: details

Insulin "in tablets" has been tested on rats

Anastasia Pashutova, "Elements"

Attempts to replace insulin injections with pills began several decades ago. However, so far not a single drug has gone into mass production. Scientists from Harvard University (USA) and the University of California at Santa Barbara has developed capsules with insulin in the form of an ionic liquid that reduce blood glucose levels for a long period. Now the researchers want to get approval for clinical trials involving humans.

In living organisms, a huge number of chemical reactions are constantly taking place, which are aimed at maintaining life. The combination of these reactions is commonly called metabolism, or metabolism. All chemical transformations can be divided into two types: reactions during which complex organic substances are split into simpler ones, and reactions resulting in the formation of complex molecules from simple ones.

One of the regulators of metabolism is the protein hormone insulin (Fig. 1), whose main function is to reduce blood glucose levels.

Fig. 1. A model of a human insulin molecule obtained by X-ray diffraction analysis. The red spiral in the foreground is the B–chain, behind it, also red, is the A-chain. Green are the N- and C-ends of the polypeptide chains (the N-end is the part of the molecule where the amino group of the first amino acid sticks out, the C–end is the end of the molecule where the carboxyl group sticks out). Image from the article: V. I. Timofeev et al., 2010. X-ray investigation of gene-engineered human insulin crystallized from a solution containing polysialic acid.

Insulin triggers the mechanism of glucose uptake by cells by binding to receptors on their surface. Later, this glucose will be used by the cells for their intended purpose. For example, in the process of glycolysis, two molecules of pyruvic acid will be obtained from one glucose molecule. Insulin is also able to start the process of fat formation from glucose in special fat cells – adipocytes (G. Dimitriadis et al., 2011. Insulin effects in muscle and adipose tissue).

Insulin is synthesized by beta cells - special cells located in the pancreas. The structure of insulin is very simple: 51 amino acids form two polypeptide chains – A and B. The A-chain contains 21 amino acid residues, and in the B-chain there are 30 such residues.

About this hormone, we can safely say "small, yes, it is", because insulin, one of the smallest among protein hormones in size and number of amino acids, participates in a large number of processes in the body (for comparison: growth hormone – somatotropin – has 191 amino acids in its composition, and the hormone of the anterior pituitary prolactin – 199 amino acids). One of these processes is very well known to everyone: insulin is able to reduce the amount of glucose in the blood. The fact is that a large amount of sugar in the blood is fraught with consequences: excessive sugar can glycosylate blood proteins, for example hemoglobin, which causes hemoglobin to perform its functions worse, that is, it is worse to transport oxygen and carbon dioxide. With increased sugar, the viscosity of the blood also changes.

Well, so that the reader is finally convinced that high blood sugar is bad, I suggest searching the Internet for the expression "diabetic foot". We constantly get micro-injuries on the foot. With uncontrolled diabetes mellitus, the integrity of the vessels of the extremities is violated, and sensitivity in these areas decreases. What do we have at the output? A small amount of blood accumulates in micro-injuries. And if bacteria don't like ordinary blood very much, because there are a lot of cells that kill them, then in the situation with sweet blood, the situation changes. As a result, we get wounds on the feet that are teeming with bacteria, and since the sensitivity has decreased, the person does not even feel pain.

How does insulin reduce the amount of glucose in the blood? Together with blood, insulin and glucose come to various cells of our organs. Next, the hormone binds to receptors on the surface of the cell membrane. This binding triggers the work of glucose transporters – a large group of membrane proteins whose task is to transfer glucose through the membrane from the outer space into the cell. Insulin also activates the conversion of sweet monomeric glucose into glycogen. Glycogen is a polymer molecule that consists of interconnected glucose residues. New structure – new properties. The polymer molecule is unsweetened and insoluble in water, which means it does not affect the chemical reactions that occur around. The polysaccharide can lie quietly on the sidelines until better times. It is in the form of glycogen that glucose is stored in the body for a long time, creating a reserve for the future. By analogy with starch in plants, glycogen can form granules in the cytoplasm. As soon as the cell needs energy, and free glucose is not "at hand", the bonds in the polysaccharide will be cut by enzymes and a monomer will appear.

With disorders in the work of the insulin system, diabetes mellitus develops – a chronic disease of the endocrine system. There are two types of diabetes mellitus:

Type 1: the pancreas produces insufficient amounts of insulin;
Type 2: the interaction of the body's cells with insulin is disrupted.

Pathologies in carbohydrate metabolism are a serious problem of our time. The number of patients with diabetes mellitus is growing every year. According to WHO, the number of people with diabetes has increased from 108 million in 1980 to 422 million in 2014. In 2015 alone, diabetes claimed the lives of about 1.6 million people; for comparison, in the same year, about 1.3 million people died in road accidents.

Approximately 10-15% of cases of diabetes mellitus – that is, approximately 40 million people worldwide – are type 1 diabetes. As already mentioned, type 1 diabetes occurs when the pancreas produces insufficient insulin. This is due to the death of beta cells of the pancreas. The cause may be stress, viral infections and autoimmune disorders (which occur when the immune system begins to perceive its own cells as an enemy and simply destroys them). The only way out for patients with type 1 diabetes is lifelong insulin injections and adherence to a special diet (with type 2 diabetes, it's enough to simply follow a diet, because there is insulin, but it takes more for the cells to respond to it).

If you reduce the amount of carbohydrates in the diet, there is more insulin left to affect the cells. Special tables have been developed for diabetics (Fig. 2), thanks to which they calculate the required amount of insulin for injection. Nutritionists have proposed a unit of measurement – the bread unit, or HE. One HE is about 10-12 grams of digestible carbohydrates; for example, one piece of white bread weighing 20 g is one bread unit. Now there are already mobile applications that make counting easier.

Fig. 2. Table of bread units

The amount of insulin is calculated by the doctor individually, but it is important for the patient to follow a diet: if he has consumed carbohydrates more or less than the norm, he can adjust the amount of insulin himself. When the insulin dose is calculated, it is necessary to make an injection. The injection is made into the skin fold, so that the insulin ends up in the fatty tissue (Fig. 3), from where it will gradually move into the blood.

Fig. 3. Scheme of subcutaneous administration of insulin

Injection sites can be different: abdomen, hip, shoulder, etc. However, it should be borne in mind that the rate of insulin absorption in different places is different (Fig. 4).

Fig. 4. Areas for insulin injections with indication of the rate of absorption

But where can I get insulin for injections? At the beginning of the XX century, cow's insulin was used for this, which differs from human by several amino acids, but nevertheless is able to reduce blood glucose levels. With the development of genetic engineering, cow insulin has been replaced by human insulin, which is synthesized by E. coli or yeast. In short, the human insulin gene is inserted into the DNA of E.coli or yeast. The selection method selects those cells in which the gene is successfully embedded. Then they are grown on a nutrient medium, they multiply, calmly and unpretentiously synthesize human insulin in addition to their proteins. Eventually, the cells are destroyed, and the resulting mixture is purified. Human insulin is ready! This method of obtaining is still used today.

And although the problem with obtaining insulin for injection has been solved, such therapy is not always successful: patients may miss injections due to fear of needles or pain during the injection of a syringe, especially if it is about children. In addition, if you incorrectly calculate the required amount of insulin and inject significantly more, hypoglycemia may occur – a condition when the amount of glucose in the blood decreases to a critical level (below 2.2–2.8 mmol / l). In this state, a tremor begins in a person, blood pressure increases, general weakness and impaired consciousness are observed. The worst outcome is coma and even death. That is why finding an alternative way to deliver insulin is an urgent problem.

The way out of the situation would be the use of insulin orally (through the mouth). However, the gastrointestinal tract is a barrier to such delivery: hydrochloric acid in the stomach denatures protein molecules, enzymes in the intestine break down proteins to amino acids, and epithelial cells, which make up the villi of the small intestine, absorb only small molecules. In addition, the villi are covered with a layer of mucus, which makes it difficult to absorb.

Researchers from Harvard University (USA) and the University of California at Santa Barbara has developed an ionic liquid-based drug for delivering insulin through the gastrointestinal tract, which can help patients with type 1 diabetes.

What is an ionic liquid and what is it for?

Protein molecules are not particularly stable: they need certain conditions so that they do not collapse into irregular shapes. If storage conditions are violated, the protein may become inactive. And then an ionic liquid comes to the rescue, which has recently been often used as a solvent. As you can guess from the name, ionic liquids consist of ions: cations and anions. In fact, these are salts in a liquid state. The complex of the drug with an ionic liquid makes it highly soluble in water and allows it to remain in the same active form. Since insulin is a protein, in their work, scientists mixed insulin in an ionic liquid consisting of choline and geranium acid (Fig. 5) – these ingredients, according to the US Food and Drug Administration, are safe for humans.

Fig. 5. On the left – geranium acid, C ₁₀ H ₁₆ O ₂; on the right – choline, C ₅ H ₁₄ NO. From the article by M. Zakrewsky et al.

This composition has already proven itself well: in previous studies of the same scientific group, it was shown that the choline – geranium acid complex improves the penetration of the drug into the skin by almost 5 times (M. Zakrewsky et al., 2014. Ionic liquids as a class of materials for transdermal delivery and pathogen neutralization). The intestinal villi, as well as the skin, consist of epithelial tissue, so it was decided to develop a drug for oral administration based on ionic fluid.

To overcome the first obstacle – the acidic environment of the stomach – the preparation of insulin in an ionic liquid was placed in a capsule with an acid-resistant coating (Fig. 6), which has long been used in pharmacology. This coating dissolves when the capsule enters the alkaline environment of the intestine, as a result of which the ionic liquid and insulin are released.

Fig. 6. Capsules with insulin in ionic liquid for oral administration (for rats). The capsule capacity is 0.025 ml, the outer length is 8.4 mm. Photos from additional materials to the article under discussion in PNAS.

As mentioned earlier, the next obstacle is enzymes in the intestine that break down protein molecules. It turned out that the ionic liquid protects insulin from the action of enzymes, which allows the insulin molecule to remain unchanged. In addition, the choline – geranium acid complex is able to dilute intestinal mucus, thereby facilitating the absorption of the drug.

The last barrier is the intestinal epithelial cells themselves. The fact is that these cells perform a transport and barrier function, so they are able to pass some substances and delay others. Selective permeability is provided by cell membranes, in which proteins are embedded that pass certain molecules. For example, amino acids are transported via transporter proteins. In the presence of sodium ions, the amino acid binds to the protein, which changes its shape and passes the molecule inside. Each type of molecule has its own transport proteins. No protein – no transport. There are no such proteins for insulin in the small intestine, because normally it gets from the pancreas directly into the bloodstream. However, there are small gaps between the cells, through which the so-called paracellular transport is carried out. As a rule, such a passage is open to ions and hydrophilic molecules, but not for such large molecules as insulin. However, the ionic liquid makes the whole complex hydrophilic, so the molecule easily passes through this barrier. After passing through the cell layer, insulin enters the blood.

As the researchers themselves note, their approach is like a Swiss knife, where one drug has all the tools to eliminate each of the obstacles.

Another obvious advantage of this drug is that its manufacture does not require a lot of investment, and the medicine is stable enough and can be stored at room temperature for two months – this is significantly longer than the shelf life of insulin for injection. To check whether the shape of the protein changes during prolonged exposure to ionic liquid, the scientists stored samples of the drug at room temperature and at a temperature of 4 ° C. For four months, using the circular dichroism spectroscopy method, freshly prepared samples and those that were stored were compared. All this time, the structure of insulin remained unchanged (Fig. 7).

Fig. 7. A graph based on the results of the circular dichroism spectroscopy method. It is clearly seen that insulin remained in the same alpha-helical conformation throughout the entire storage period. On the y–axis is mdeg (milligradus of rotation of polarized light), on the x-axis is the wavelength in nm. An image from the article under discussion in PNAS.

The biological activity of the drug was tested on male rats: they were injected with samples with different shelf life, and then the blood glucose level was checked – it decreased in the same way. The researchers also tested the effectiveness of oral administration. Rats were given ready-made capsules of size 9 (with a capacity of 0.025 ml and a length of 8.4 mm), and then their blood glucose levels were measured. Two hours after taking the capsule, the glucose level decreased by 38%, and after 10 hours – by 45%.

To make sure that the capsules with ionic liquid are biocompatible with the tissues of the small intestine, histological preparations were prepared. The rats that received the capsules were opened, the small intestine was removed and a series of sections were prepared. The sections were stained with hematoxylin and eosin, classic dyes for histological preparations. There were no changes in intestinal morphology, and villi were found on all sections.

The advantages do not end there. The researchers note that it is oral delivery, unlike injections, that mimics the physiological pathway of insulin. In the body, insulin from the pancreas is transferred through the portal vein to the liver, where about 80% of the hormone is retained and only the remaining part enters the blood. Insulin injected subcutaneously enters the blood unevenly and at different rates depending on the injection site.

Attempts to invent a drug for delivering insulin through the gastrointestinal tract began many decades ago. Several strategies have been developed to overcome obstacles, but to date, none of the approaches has allowed us to create a cheap and non-toxic medicine that copes with all obstacles. For example, Austrian scientists have proposed a complex that includes proteins that stop the action of enzymes in the intestine. (A. H. Krauland et al., 2004. Oral insulin delivery: the potential of thiolated chitosan-insulin tablets on non-diabetic rats). However, enzyme inhibitors can cause side effects such as intoxication and pancreatic hypertrophy.

Japanese scientists have proposed a pill coated with three shells, which dissolves only in the large intestine, where enzymes that break down proteins to amino acids are no longer so active. (M. Katsuma et al., 2006. Effects of absorption promoters on insulin absorption through colon-targeted delivery). However, the area of the large intestine is smaller than the small intestine, and there is less water in it, so it is not so well suited for the absorption of medications.

Many of the proposed drugs require large production costs or have a complex formulation. Thus, the drug considered in this article is the most preferable for mass production: it is effective, stored for a long time, easy to prepare, safe and cheap enough. The researchers plan to continue working and hope to get approval for clinical trials involving humans.

A source: Banerjee et al., Ionic liquids for oral insulin delivery // PNAS, 2018.

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