INSULIN
Insulin is a hormone produced polipetida in β cells of the pancreas gland Langerhaens. Insulin plays an important role in the regulation of blood sugar (blood glucose 3.5 to 8.0 is maintained mmol / liter). Insulin deficiency can cause diseases such as insulin-dependent diabetes mellitus (type 1 diabetes).
This form of therapy for insulin deficiency may include:
Providing Insulin: Insulin is given directly to the patient by injection
Transplantation: patients who already damaged pancreas replaced with a new one via transplant.
Gene therapy
INSULIN PROPERTIES
Insulin Function:
Can stimulate glucose trasport (also amino acids, ions K, and other nutrients) into the cell
Can stimulate intracellular biosynthetic pathways (anabolic pathway) such as glycogen synthesis
Inhibit catabolic pathway (breakdown of large molecules into smaller molecules) as the process of glycogenolysis is glycogen into glucose, which resulted in blood glucose levels rise
Stimulates the synthesis of DNA and protein
Insulin will spur a wide range of gene expression through the activation of a wide variety of genes, DNA synthesis and protein production.
In general, insulin can control the metabolic pathways mealui:
Deposporilasi induces several regulators of enzymes that play a role in the pathways of anabolism and katablisme
Regulation induces transcription levels of several genes that encode metabolic enzymes
Insulin consists of 51 amino acids. Insulin molecule composed by two polypeptide chains A and B are connected by disulfide bonds. A chain consists of 21 amino acids and the B chain consists of 30 amino acids (Figure 1, 2).
Figure 1. Amino acid sequence in humans and conformation chain A and chain B.
Figure 2. Amino acid sequence in humans and conformation chain A and chain B. Variations in the type of amino acid residues and their potential involvement in primary and or hexamer has been identified. (Modified from Brange and Langkjaer, 1993)
There are some sites on the insulin that is susceptible to degradation by an enzyme such as carboxypeptidase A, leucin aminopeptidase, trypsin, and Glu Glu C. C is a microbial enzyme produced by Staphylococcus bacteria. Glu C cut of insulin in 4 places. Site where the enzyme trypsin, namely the introduction of the amino acids glycine and arginine (Figure 3).
Figure 3. Places insulin by enzyme cuts
Undergo proteolysis of proinsulin into insulin and amino acid residue 34 C peptide. Mature insulin consists of two polypeptide chains linked two disulfide bonds in the chain. Proinsulin was actually already have activity as an insulin without having to turn into insulin (aktivtasnya 10% of the activity of insulin). Therefore, if the expected long acting then given in the form of proinsulin has been a long while since proinsulin proteolysis of proinsulin into insulin action is still running so time will be longer (Figure 4).
Figure 4. Proteolytic processing of proinsulin yielding mature insulin
Insulin varies from one organism to another, but this does not distinguish activity. For example, insulin in humans, pigs, and cattle have differences in amino acid composition, but its activity remained the same (Table 1).
Table 1
The differences in the amino acid composition of human insulin, pork (pork), and cattle (beef)
Species
A8
A10
B28
B29
B30
Human
Thr
Ile
Pro
Lys
Thr
Pig
Thr
Ile
Pro
Lys
In the style of
Cattle
In the style of
Val
Pro
Lys
In the style of
Human insulin and porcine insulin only 1 amino acid difference is in B30, whereas human insulin and bovine insulin 3 different amino acids that is on the A8, A10, and B30 so that the use of pig insulin is less immunogenic than bovine insulin. But the thing is, 1 pig insulin extracted just enough for one person for 3 days ± whereas there are currently 60 million people in the world who suffer from insulin-dependent diabetes and is thought to increase 5-6% per year. Thus it is now widely developed recombinant technology to get insulin.
Advantages of insulin production by recombinant methods:
Reliability of supply
Eliminsi risk of pathogen presence of animals
Cost effective
INSULIN PRODUCTION WITH recombinant DNA technology
There are 2 ways of production of insulin by recombinant DNA technology.
As in the process of making insulin using lactose operon (lac Z gene), the manufacture of insulin using a β-galactosidase also make chains A and B separately. Remember, separate first and later combined. Initially, the genes that encode both chains A and B chains combined with β-galactosidase gene. β-galactosidase was used as a promoter, with only IPTG (a lactose analog) β-galactosidase gene then be synthesized. A chain genes and B chain that kept his company would also be synthesized. This is the way how the A chain genes into proteins synthesized A.
When the β-galactosidase gene inducer is met with IPTG, then this gene will be synthesized. A chain of methionine and B which are close mauoun will participate teersintesis, the next process, the same as using the lactose operon (Remember, the same function with β-lac galactosidase).
In the last stage, chains A and B will use your combined oxidation process. The oxidation process takes place as follows:
Oxidized A and B chains, if desired protein is not formed then reduced again. Oxidation-reduction process is time consuming and inefficient. So that actually produced insulin in a way that nudah, conducted a second recombinant techniques.
Figure 5.
The second technology is a recombinant insulin use proinsulin. As we know that proinsulin be directly converted into insulin by enzyme proteoltik (beneficial and effective). How:
proinsulin mRNA is converted into cDNA using reverse transcriptase enzyme proinsulin. Also required for the synthesis of codons encode proteins. What is the purpose converted into cDNA and methionine sisntesis?
Then methionine codon in the cDNA and ligation with the plasmid. Previously, in front of the proinsulin cDNA gene β-galactosidase there are functioning ......
In the E. coli gene would be the protein that is synthesized peptides A, C (an internal fragment) and B (remember peptides A, C, and B is proinsulin, without chain C is called insulin).
CNBr will memeotong meth and β-galactosidase separates and proinsulin
Proinsulin perpetually altered by the enzymatic proteolytic enzymes into insulin.
Figure 6. Procedure INVOLVED in the peroduction of bioengineered human insulin in the special strains of E. coli using recombinant DNA technology semisynthetic proinsulin gene (Method II). (From Watson et al., 1993 with permision)
Purification INSULIN
In general, the purification of insulin can be done in 3 ways:
Gel filtration chromatographic system (using sephadex G-50 column)
Ion exchange step
Reverse-Phase HPLC
First of all, cells are taken from the fermenter and then crushed to obtain proinsulin in rough shape (crude proinsulin). Further purification is done by crystallization stage 1 produces proinsulin which has been partially purified. Then use traditional enzymatic process resulting crude protrolisis insulin. Then purified by ion exchange and gel filtration. RP-HPLC last use would be associated with insulin with a high degree of purity.
Figure 7. A Likely purification scheme for human insulin prb *. A final polishing step RP-HPLC yields a highly pure product. * Thus, the various kinds of human insulin: No human insulin crb, emp, prb, pyr, Insulin BP / Eur. Ph / USP and USP Insulin
In the process of separation of insulin:
- Made pH <5.3 (below pH insulin) insulin in order to stay in solution
- With gradient elution using acetonitrile
- With the starting material purity is almost 92% it will get up to 99% purity.
The following figure is an example of the pemurniam insulin. Gel filtration chromatography to separate the high molecular weight fractions such as proinsulin, insulin forms dimers, and especially the proteolytic enzymes of insulin products. Some pancreatic polypeptide that has insulin-like BM can also be separated (Figure 8).
Figure 8. Chromatographic purification of recrystallized on a Sephadex G-50 gel-filtration column. Separation of high-molecular-mass proteolytic enzymes, proinsulin and some very low-molecular-mass material is obvious. Insulin elutes from the column as a single peak, hence the term "single-peak insulin '.
Chemical stability of insulin
Chemical degradation of insulin can occur due to:
1. Transfer hydrolytic acid amide group into a shape that will lead to inactivation of insulin.
Example: Glutamine into glutamate
Asparagine into aspartic
2. Formation of dimers or oligoner resulting in aggregation and difficult to dissolve in the medium
The rate of degradation of insulin is affected by pH and temperature are:
- Easy digredasai insulin in pleasant base (stable at pH 2.3 to 3.5 insulin)
- At temperatures> 26oC, insulin will be degraded
In kondsisi acid (low pH): Asn (A21) = Asparagine number 21 on chain A will be aspartate, under these conditions, the potential of insulin will be lost. While on konsisi neutral Asn (B3) = Asparagine at number 3 B chain will undergo deamination into aspartic acid and acid isospartat. However, this amino acid change does not affect insulin activity.
If insulin is stored at a temperature of ± 5 ° C then the insulin will have only 2% per year, so it is still active (fully bioactive).
Physical stability of insulin
In physics indulin may experience:
1. Aggregation noncovalent
This happens because adanaya:
- Hydrophobic interactions
- Interaction elektostatik
Hydrophobic interactions that have occurred due to the nature of hydrophobic molecules tend to gather, separate from other molecules.
Elektostatik interaction will cause repulsion or attraction attractive intermolecular charged.
Temperatures> 25 ° C will accelerate due to protein aggregation at temperatures denatured san tinffi will consequently not water soluble. Should also avoid excessive shaking to prevent degradation due to physical factors.
2. Changes in physical
For example, change the color or kejarnihan.
INSULIN FORMULATIONS
Monomeric form of insulin is the most active form but not stable in storage. Therefore, products that do modifkasi insulin dosage remained stable in storage before use.
Insulin can form dimers because there are some amino acids that are responsible for the formation. Dimers can form irregular polymers that can lead to aggregation. For it is in storage plus Zn 2 + which will cause the regularity of the molecular form of insulin into hexamers (Hexamer T6) are stable. Further added excipients like phenolic phenol and m-cresol as a preservative that can be functioning and stabilizer (Figure 9).
Figure 9. A schematic of the self-association of insulin
With the phenol Penamabahan Maacah hexamer form T6 R6 hexamer form will be more stable. Keep in mind that the phenol is karrsinogenik potent because it can lead to changes bases AT -> GC or GC into AT. However, because the levels of phenol used small it can be ignored. To minimize damage and pain during jaringn injection because the addition of Zn 2 + or preservative, then the dosage of insulin is often added isotonic agents (such as glycerol and NaCl) as well as physiological buffers (such as sodium phosphate).
R6 insulin hexamer although stable but does not affect the insulin because they can not penetrate biological memberan (Figure 10).
Figure 10. Diagram that shows the association and dissociation of insulin. At neutral pH, insulin-associate into dimers and hexamers form. Dimeric and monomeric insulin is absorbed into the blood faster than the hexamer. (Adapted with permission from Thieme Medical Publishers)
Therefore, in the body, insulin R6 hexamer will be broken down into the active form of insulin that forms monomers (Figure 11). Insulin hexamer will berkesetimbangan the T6 hexamer form a less stable, dimer, and monomer. But it turns out even if given large doses of insulin heksamaer R, the effects are relatively small. This happens because not all forms of hexamer smoothly split into monomeric form so that only a few monomer produced. Dimeric and monomeric forms of insulin will mempu penetrate biological membranes and cause effects although small concentration (10-8 M).
Figure 11. A schematic of the dissociation of a soluble human insulin after a subcutaneous injection Hexamer.
Currently has developed a formulation of insulin that can provide fast action is insulin lispro (insulin LysB28ProB29). This will split insulin hexamers into monomers directly from monomers that can be found in greater concentrations (10-3 M) so that rapid effect (Figure 12).
Figure 12. Hypothecal A schematic of the dissociation of soluble insulin lispro after a subcutaneous injection Hexamer.
Examples of formulations of insulin can be listened to in the following table (Table 2).
Table 2
A list of neutral human U100 insulin formulations
What distinguishes insulin preparations are given additional compounds in the formulation. For example in NPH (Neutral Protamine Hagendorn) is no more protamine can stabilize the R6 hexamer forms that release more insulin underlayer (action much longer).
Lente: a mixture of 70% ultralente (rhombohedral) and 30% semilente (amorphous).
MODIFICATION OF INSULIN
Modifications can be done by making the insulin analogue insulin for example, by site directed mutagenesis. There are several ways you can do:
1. Easy Insulin binds to its receptor on a modified amino acid that is bound to its receptor binding site (A1, A5, A21, B10, B16, B23-25 is a binding site on the insulin receptor). Example: glutamate on chain B number 10 was changed to histidine it will increase the activity of 5-fold.
2. Changing the amino acids that can affect the pharmacokinetic profile of insulin that acts rapidly there is a long-acting insulin, and others. Example: dimer-oligomer formation by altering the amino acid B8, B9, B12-13, B16, and B23-28 can increase the t ½ to 35 hours, so that one may insulin injections for 1 day.
For other modifications, see in Table 3 below!
Table 3
Human insulin and analogues
Insulin
A21
B3
B28
B29
B31
B32
Human
Asn
Asn
Lys
Pro
-
-
Lispro
Asn
Asn
Pro
Lys
-
-
Aspart
Asn
Asn
Asp. Acid
Pro
-
-
Glulisine
Asn
Lys
Lys
Asp. Acid
-
-
Glargine
Gly
Asn
Lys
Pro
Arg
Arg
Detemir
Fatty acid
at B29
and B30
removed
Factors affecting insulin action:
1. Dose
The higher the dose, the faster the action.
2. Place of injection
In general, insulin administered by injection through the skin. Fast action on intravenous administration, subcutaneous or transdermal pad then on muscle insulin degradation occurs 20-25%. So should be taken into account to get the correct dosage. Most insulin is injected in the abdomen (intrperional). Insulin injection needles to small and short (0.5-1 cm). Can also use breast implants that can supply pad bit by tiny fraction of insulin.
3. Presence of insulin antibodies
It is mainly on the use of animals as insulin. If used outside feared insulin antigen antibody reaction or destruction of another, except in patients with autoimmune.
4. Physical activity
The more physical activity we do then we need energy (of glucose) is greater so it does not need extra insulin action to convert glucose into glycogen (the less insulin is needed).
Table 4
Pharmacokinetic characteristics: short, intermediate and long-acting insulin preparations
Category
Onset (hours after administration)
Activity peak (hours after administration)
Duration (hours)
Short action
0.5-1
2-5
6-8
Intermediate action
2
4-12
Up to 24
Long action
4
10-20
Up to 36
Insulin administration:
- Short-acting: given 0.5-1 hours before maakan
- Intermediates acring: given 2 hours before eating
- Long acting: given 4 hours before eating
Giving insulin preparations need to be arranged as above so that when high glucose levels in the body (reach the top), the insulin levels are too high already, so it should be balanced.
- If high levels of insulin low blood glucose levels there will be a shock.
- If insulin levels are low but high blood glucose kada then there is excess sugar (diabetes)
Insulin is a hormone produced polipetida in β cells of the pancreas gland Langerhaens. Insulin plays an important role in the regulation of blood sugar (blood glucose 3.5 to 8.0 is maintained mmol / liter). Insulin deficiency can cause diseases such as insulin-dependent diabetes mellitus (type 1 diabetes).
This form of therapy for insulin deficiency may include:
Providing Insulin: Insulin is given directly to the patient by injection
Transplantation: patients who already damaged pancreas replaced with a new one via transplant.
Gene therapy
INSULIN PROPERTIES
Insulin Function:
Can stimulate glucose trasport (also amino acids, ions K, and other nutrients) into the cell
Can stimulate intracellular biosynthetic pathways (anabolic pathway) such as glycogen synthesis
Inhibit catabolic pathway (breakdown of large molecules into smaller molecules) as the process of glycogenolysis is glycogen into glucose, which resulted in blood glucose levels rise
Stimulates the synthesis of DNA and protein
Insulin will spur a wide range of gene expression through the activation of a wide variety of genes, DNA synthesis and protein production.
In general, insulin can control the metabolic pathways mealui:
Deposporilasi induces several regulators of enzymes that play a role in the pathways of anabolism and katablisme
Regulation induces transcription levels of several genes that encode metabolic enzymes
Insulin consists of 51 amino acids. Insulin molecule composed by two polypeptide chains A and B are connected by disulfide bonds. A chain consists of 21 amino acids and the B chain consists of 30 amino acids (Figure 1, 2).
Figure 1. Amino acid sequence in humans and conformation chain A and chain B.
Figure 2. Amino acid sequence in humans and conformation chain A and chain B. Variations in the type of amino acid residues and their potential involvement in primary and or hexamer has been identified. (Modified from Brange and Langkjaer, 1993)
There are some sites on the insulin that is susceptible to degradation by an enzyme such as carboxypeptidase A, leucin aminopeptidase, trypsin, and Glu Glu C. C is a microbial enzyme produced by Staphylococcus bacteria. Glu C cut of insulin in 4 places. Site where the enzyme trypsin, namely the introduction of the amino acids glycine and arginine (Figure 3).
Figure 3. Places insulin by enzyme cuts
Undergo proteolysis of proinsulin into insulin and amino acid residue 34 C peptide. Mature insulin consists of two polypeptide chains linked two disulfide bonds in the chain. Proinsulin was actually already have activity as an insulin without having to turn into insulin (aktivtasnya 10% of the activity of insulin). Therefore, if the expected long acting then given in the form of proinsulin has been a long while since proinsulin proteolysis of proinsulin into insulin action is still running so time will be longer (Figure 4).
Figure 4. Proteolytic processing of proinsulin yielding mature insulin
Insulin varies from one organism to another, but this does not distinguish activity. For example, insulin in humans, pigs, and cattle have differences in amino acid composition, but its activity remained the same (Table 1).
Table 1
The differences in the amino acid composition of human insulin, pork (pork), and cattle (beef)
Species
A8
A10
B28
B29
B30
Human
Thr
Ile
Pro
Lys
Thr
Pig
Thr
Ile
Pro
Lys
In the style of
Cattle
In the style of
Val
Pro
Lys
In the style of
Human insulin and porcine insulin only 1 amino acid difference is in B30, whereas human insulin and bovine insulin 3 different amino acids that is on the A8, A10, and B30 so that the use of pig insulin is less immunogenic than bovine insulin. But the thing is, 1 pig insulin extracted just enough for one person for 3 days ± whereas there are currently 60 million people in the world who suffer from insulin-dependent diabetes and is thought to increase 5-6% per year. Thus it is now widely developed recombinant technology to get insulin.
Advantages of insulin production by recombinant methods:
Reliability of supply
Eliminsi risk of pathogen presence of animals
Cost effective
INSULIN PRODUCTION WITH recombinant DNA technology
There are 2 ways of production of insulin by recombinant DNA technology.
As in the process of making insulin using lactose operon (lac Z gene), the manufacture of insulin using a β-galactosidase also make chains A and B separately. Remember, separate first and later combined. Initially, the genes that encode both chains A and B chains combined with β-galactosidase gene. β-galactosidase was used as a promoter, with only IPTG (a lactose analog) β-galactosidase gene then be synthesized. A chain genes and B chain that kept his company would also be synthesized. This is the way how the A chain genes into proteins synthesized A.
When the β-galactosidase gene inducer is met with IPTG, then this gene will be synthesized. A chain of methionine and B which are close mauoun will participate teersintesis, the next process, the same as using the lactose operon (Remember, the same function with β-lac galactosidase).
In the last stage, chains A and B will use your combined oxidation process. The oxidation process takes place as follows:
Oxidized A and B chains, if desired protein is not formed then reduced again. Oxidation-reduction process is time consuming and inefficient. So that actually produced insulin in a way that nudah, conducted a second recombinant techniques.
Figure 5.
The second technology is a recombinant insulin use proinsulin. As we know that proinsulin be directly converted into insulin by enzyme proteoltik (beneficial and effective). How:
proinsulin mRNA is converted into cDNA using reverse transcriptase enzyme proinsulin. Also required for the synthesis of codons encode proteins. What is the purpose converted into cDNA and methionine sisntesis?
Then methionine codon in the cDNA and ligation with the plasmid. Previously, in front of the proinsulin cDNA gene β-galactosidase there are functioning ......
In the E. coli gene would be the protein that is synthesized peptides A, C (an internal fragment) and B (remember peptides A, C, and B is proinsulin, without chain C is called insulin).
CNBr will memeotong meth and β-galactosidase separates and proinsulin
Proinsulin perpetually altered by the enzymatic proteolytic enzymes into insulin.
Figure 6. Procedure INVOLVED in the peroduction of bioengineered human insulin in the special strains of E. coli using recombinant DNA technology semisynthetic proinsulin gene (Method II). (From Watson et al., 1993 with permision)
Purification INSULIN
In general, the purification of insulin can be done in 3 ways:
Gel filtration chromatographic system (using sephadex G-50 column)
Ion exchange step
Reverse-Phase HPLC
First of all, cells are taken from the fermenter and then crushed to obtain proinsulin in rough shape (crude proinsulin). Further purification is done by crystallization stage 1 produces proinsulin which has been partially purified. Then use traditional enzymatic process resulting crude protrolisis insulin. Then purified by ion exchange and gel filtration. RP-HPLC last use would be associated with insulin with a high degree of purity.
Figure 7. A Likely purification scheme for human insulin prb *. A final polishing step RP-HPLC yields a highly pure product. * Thus, the various kinds of human insulin: No human insulin crb, emp, prb, pyr, Insulin BP / Eur. Ph / USP and USP Insulin
In the process of separation of insulin:
- Made pH <5.3 (below pH insulin) insulin in order to stay in solution
- With gradient elution using acetonitrile
- With the starting material purity is almost 92% it will get up to 99% purity.
The following figure is an example of the pemurniam insulin. Gel filtration chromatography to separate the high molecular weight fractions such as proinsulin, insulin forms dimers, and especially the proteolytic enzymes of insulin products. Some pancreatic polypeptide that has insulin-like BM can also be separated (Figure 8).
Figure 8. Chromatographic purification of recrystallized on a Sephadex G-50 gel-filtration column. Separation of high-molecular-mass proteolytic enzymes, proinsulin and some very low-molecular-mass material is obvious. Insulin elutes from the column as a single peak, hence the term "single-peak insulin '.
Chemical stability of insulin
Chemical degradation of insulin can occur due to:
1. Transfer hydrolytic acid amide group into a shape that will lead to inactivation of insulin.
Example: Glutamine into glutamate
Asparagine into aspartic
2. Formation of dimers or oligoner resulting in aggregation and difficult to dissolve in the medium
The rate of degradation of insulin is affected by pH and temperature are:
- Easy digredasai insulin in pleasant base (stable at pH 2.3 to 3.5 insulin)
- At temperatures> 26oC, insulin will be degraded
In kondsisi acid (low pH): Asn (A21) = Asparagine number 21 on chain A will be aspartate, under these conditions, the potential of insulin will be lost. While on konsisi neutral Asn (B3) = Asparagine at number 3 B chain will undergo deamination into aspartic acid and acid isospartat. However, this amino acid change does not affect insulin activity.
If insulin is stored at a temperature of ± 5 ° C then the insulin will have only 2% per year, so it is still active (fully bioactive).
Physical stability of insulin
In physics indulin may experience:
1. Aggregation noncovalent
This happens because adanaya:
- Hydrophobic interactions
- Interaction elektostatik
Hydrophobic interactions that have occurred due to the nature of hydrophobic molecules tend to gather, separate from other molecules.
Elektostatik interaction will cause repulsion or attraction attractive intermolecular charged.
Temperatures> 25 ° C will accelerate due to protein aggregation at temperatures denatured san tinffi will consequently not water soluble. Should also avoid excessive shaking to prevent degradation due to physical factors.
2. Changes in physical
For example, change the color or kejarnihan.
INSULIN FORMULATIONS
Monomeric form of insulin is the most active form but not stable in storage. Therefore, products that do modifkasi insulin dosage remained stable in storage before use.
Insulin can form dimers because there are some amino acids that are responsible for the formation. Dimers can form irregular polymers that can lead to aggregation. For it is in storage plus Zn 2 + which will cause the regularity of the molecular form of insulin into hexamers (Hexamer T6) are stable. Further added excipients like phenolic phenol and m-cresol as a preservative that can be functioning and stabilizer (Figure 9).
Figure 9. A schematic of the self-association of insulin
With the phenol Penamabahan Maacah hexamer form T6 R6 hexamer form will be more stable. Keep in mind that the phenol is karrsinogenik potent because it can lead to changes bases AT -> GC or GC into AT. However, because the levels of phenol used small it can be ignored. To minimize damage and pain during jaringn injection because the addition of Zn 2 + or preservative, then the dosage of insulin is often added isotonic agents (such as glycerol and NaCl) as well as physiological buffers (such as sodium phosphate).
R6 insulin hexamer although stable but does not affect the insulin because they can not penetrate biological memberan (Figure 10).
Figure 10. Diagram that shows the association and dissociation of insulin. At neutral pH, insulin-associate into dimers and hexamers form. Dimeric and monomeric insulin is absorbed into the blood faster than the hexamer. (Adapted with permission from Thieme Medical Publishers)
Therefore, in the body, insulin R6 hexamer will be broken down into the active form of insulin that forms monomers (Figure 11). Insulin hexamer will berkesetimbangan the T6 hexamer form a less stable, dimer, and monomer. But it turns out even if given large doses of insulin heksamaer R, the effects are relatively small. This happens because not all forms of hexamer smoothly split into monomeric form so that only a few monomer produced. Dimeric and monomeric forms of insulin will mempu penetrate biological membranes and cause effects although small concentration (10-8 M).
Figure 11. A schematic of the dissociation of a soluble human insulin after a subcutaneous injection Hexamer.
Currently has developed a formulation of insulin that can provide fast action is insulin lispro (insulin LysB28ProB29). This will split insulin hexamers into monomers directly from monomers that can be found in greater concentrations (10-3 M) so that rapid effect (Figure 12).
Figure 12. Hypothecal A schematic of the dissociation of soluble insulin lispro after a subcutaneous injection Hexamer.
Examples of formulations of insulin can be listened to in the following table (Table 2).
Table 2
A list of neutral human U100 insulin formulations
What distinguishes insulin preparations are given additional compounds in the formulation. For example in NPH (Neutral Protamine Hagendorn) is no more protamine can stabilize the R6 hexamer forms that release more insulin underlayer (action much longer).
Lente: a mixture of 70% ultralente (rhombohedral) and 30% semilente (amorphous).
MODIFICATION OF INSULIN
Modifications can be done by making the insulin analogue insulin for example, by site directed mutagenesis. There are several ways you can do:
1. Easy Insulin binds to its receptor on a modified amino acid that is bound to its receptor binding site (A1, A5, A21, B10, B16, B23-25 is a binding site on the insulin receptor). Example: glutamate on chain B number 10 was changed to histidine it will increase the activity of 5-fold.
2. Changing the amino acids that can affect the pharmacokinetic profile of insulin that acts rapidly there is a long-acting insulin, and others. Example: dimer-oligomer formation by altering the amino acid B8, B9, B12-13, B16, and B23-28 can increase the t ½ to 35 hours, so that one may insulin injections for 1 day.
For other modifications, see in Table 3 below!
Table 3
Human insulin and analogues
Insulin
A21
B3
B28
B29
B31
B32
Human
Asn
Asn
Lys
Pro
-
-
Lispro
Asn
Asn
Pro
Lys
-
-
Aspart
Asn
Asn
Asp. Acid
Pro
-
-
Glulisine
Asn
Lys
Lys
Asp. Acid
-
-
Glargine
Gly
Asn
Lys
Pro
Arg
Arg
Detemir
Fatty acid
at B29
and B30
removed
Factors affecting insulin action:
1. Dose
The higher the dose, the faster the action.
2. Place of injection
In general, insulin administered by injection through the skin. Fast action on intravenous administration, subcutaneous or transdermal pad then on muscle insulin degradation occurs 20-25%. So should be taken into account to get the correct dosage. Most insulin is injected in the abdomen (intrperional). Insulin injection needles to small and short (0.5-1 cm). Can also use breast implants that can supply pad bit by tiny fraction of insulin.
3. Presence of insulin antibodies
It is mainly on the use of animals as insulin. If used outside feared insulin antigen antibody reaction or destruction of another, except in patients with autoimmune.
4. Physical activity
The more physical activity we do then we need energy (of glucose) is greater so it does not need extra insulin action to convert glucose into glycogen (the less insulin is needed).
Table 4
Pharmacokinetic characteristics: short, intermediate and long-acting insulin preparations
Category
Onset (hours after administration)
Activity peak (hours after administration)
Duration (hours)
Short action
0.5-1
2-5
6-8
Intermediate action
2
4-12
Up to 24
Long action
4
10-20
Up to 36
Insulin administration:
- Short-acting: given 0.5-1 hours before maakan
- Intermediates acring: given 2 hours before eating
- Long acting: given 4 hours before eating
Giving insulin preparations need to be arranged as above so that when high glucose levels in the body (reach the top), the insulin levels are too high already, so it should be balanced.
- If high levels of insulin low blood glucose levels there will be a shock.
- If insulin levels are low but high blood glucose kada then there is excess sugar (diabetes)
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