formulation and evaluation of delivery system of protein and peptide.pptx

the presention gives idea about various formulation and evaluation of various delivery system based on the delivery routes for protein and peptide drug delivery in the body.

1. Formulation and evaluation of delivery system of protein and other macromolecule 04-Jul-23 1 Submitted to: Dr. Sateesha SB Professor Department of Pharmaceutics ABMRCP, Bengaluru Prepared by: S Kishor Kumar Singha ACP22PHCE005 Department of Pharmaceutics ABMRCP, Bengaluru

2. Content Introduction. Structural complexity of protein and peptide molecule. Delivery of peptide-based pharmaceuticals for systemic medication (Formulation Approach). Evaluation of protein and peptide drug formulations. 04-Jul-23 2

3. Introduction: Protein and peptide are the most abundant material of living system and biological cell. It acts a hormones, enzymes, structural elements and immunoglobulin. Protein are compounds having linear chain amino acids which are held together by the covalent linkages called peptide bonds. Currently there are more than 100 approved peptide based therapeutics on the market. The market for peptide and protein drugs are growing faster because protein and peptide can be highly selective as they have multiple points of interaction with the target. 04-Jul-23 3

4. Structural complexity of protein and peptide molecule: Peptides and proteins are the most abundant components of biological cells. These naturally-occurring macromolecules exist not only as the structural components of cells but also as functioning moieties (e.g., enzymes, antibodies, hormones, and transport mediators). Each peptide or protein molecule is a polymer chain with α -amino acids linked together in a sequential manner by peptide bonds, which are peptide linkages formed by interaction between the α -carboxyl and α -amino groups of the adjacent amino acids. The resulting polymers are generally called peptides. The term "polypeptide" refers to the peptide molecule that contains about eight or more units of amino acids, whereas an "oligopeptide“ is a peptide molecule with the peptide chain of fewer than eight amino acids. An amino acid unit in a polypeptide is called a residue. A polypeptide that contains from about 50 to as many as 2500 units of amino acids in a peptide chain is called a protein. 04-Jul-23 4

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6. Delivery of peptide-based pharmaceuticals for systemic medication: Parenteral systemic delivery Non-parenteral systemic delivery Nasal delivery. Ocular delivery Pulmonary delivery Buccal delivery Rectal delivery Oral delivery Vaginal delivery Transdermal delivery 04-Jul-23 6

7. A. Parenteral systemic delivery It refers to administration of therapeutic protein and peptide through injection or infusion directly into the blood stream. This route of administration is often used because they are typically degraded in gastrointestinal tract and are not absorbed efficiently. Some common approaches in parenteral systemic delivery are: Intravenous injection, Subcutaneous injection, Intramuscular injection, Implantation, Interperitoneal injection. 04-Jul-23 7

8. Several delivery techniques and systems have been developed for parenteral controlled and sustained delivery of insulin. Long-acting insulin injectables: An early approach to the development of long acting insulin preparations included the complexation of insulin with zinc salt and basic proteins; for e.g., the formation of protamine-Zn-insulin suspension. This approach used a buffer medium with controlled pH in which insulin can be precipitated by zinc ion to yield a water-insoluble Zn-insulin complex in either a crystalline or an amorphous form. The crystallinity of the complex can be controlled by varying the pH of the buffer medium. The crystalline Zn-insulin complex formed is an extremely long-acting insulin, whereas the amorphous Zn-insulin complex is a moderately long-acting insulin. 04-Jul-23 8

9. New injection devices for insulin delivery: 04-Jul-23 9

10. The main components of the Preci -Jet 50 and the Adapt-o-Jet (vial holder). For filling with insulin, the nozzle is unscrewed and replaced with the Adapt-o-Jet and an insulin vial. The injector is filled with insulin by turning the power rack counterclockwise until the number on the small window indicates the number of insulin units required. The nozzle is then screwed back into position, and pressure is generated by turning power pack clockwise until a click is heard. 04-Jul-23 10

11. 3. Infusion pumps for insulin delivery: 04-Jul-23 11

12. An implantable controlled-release micropump was recently developed for the intraperitoneal modulated delivery of insulin. It is an open-loop control system characterized by operation at two levels: basal delivery for the between-meal period and augmented delivery for short periods following the ingestion of meals. The rate of delivery is adjusted to meet the insulin requirement of the respective meal. With an adequate supply of insulin to the pump, the concentration gradient from the reservoir produces the delivery of insulin at the basal rate (when no external power source is applied). Augmented delivery is achieved by repeated compression of the foam membrane above the steel piston, which applies a current to the solenoid cell. 04-Jul-23 12

13. 4. Self regulating delivery system: 04-Jul-23 13

14. T he development of an artificial beta cell that consists of a glucose-sensitive hydrogel membrane for the feedback-controlled delivery of insulin. The glucose-sensitive membrane is fabricated by entrapping a glucose oxidase in a hydrogel polymer with pendant amine groups. As glucose diffuses into the polymer glucose oxidase catalyzes its conversion to gluconic acid, thereby lowering the microenvironmental pH in the membrane. The reduced pH results in increased ionization of the pendant amine groups. Electrostatic repulsion between the ionized amine groups increases the swelling and thus the permeability of the hydrogel membrane to insulin contained in the reservoir. Ultimately, the membrane permeability to insulin is thus a function of the glucose concentration surrounding the membrane, and the release of insulin is accelerated by the increase in the glucose level. 04-Jul-23 14

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16. B iochemical approach based on the principle of the competitive and complementary binding behavior of concanavalin A ( ConA ) with glucose and glycosylated insulin (G-insulin). The ConA -G-insulin complex is encapsulated inside a device with a polymeric membrane that is permeable to glucose and G-insulin but not to ConA or its complex. As the glucose level increases, the influx of glucose to the device increases, resulting in the displacement of Ginsulin from the ConA complex and its efflux to the body. 04-Jul-23 16

17. B. Non-parenteral Systemic Delivery: 04-Jul-23 17

18. 1. Nasal Delivery: U nderneath the nasal mucosa, an extensive network of microcirculation that enables the effective nasal absorption of orally inactive drugs, which are highly susceptible to gastrointestinal degradation when administered orally, for systemic delivery. Furthermore, it is also known that drug molecules absorbed nasally can directly enter the systemic circulation before passing through the hepatic circulation. 04-Jul-23 18

19. Various approaches for Nasal Delivery of peptide/protein drugs are: Viscosity modification: The clearance time from the nasal cavity can be delayed by using solutions with higher viscosity. E.g. the half time of clearance could be increased significantly with 0.6% of HPMC. pH modification: Peptides and proteins usually exhibit the lowest solubility at their isoelectric point. Thus, by adjusting the pH farther away from the isoelectric point of a particular peptide, its solubility can be increased. Satisfactory nasal absorption of insulin was observed with sodium deoxycholate (adsorption enhancer, bile salts), that insulin is capable of crossing nasal membrane in an acidic medium. 04-Jul-23 19

20. P eptides have been successfully developed and marketed as nasal pharmaceutical products in the United States: oxytocin ( Syntocinon , Sandoz) and N afarelin acetate ( Synarel , Syntex ). Synarel spray: Nafarelin acetate, is a gonadotropin-releasing hormone agonist (GnRH agonist) medication which is used in the treatment of endometriosis. Oxytocin nasal spray generic brands in India are Evatocin ( Neon Labs), Genox ( Intas ). 04-Jul-23 20

21. 2. Pulmonary Delivery: Lungs are attractive site for systemic delivery of proteins and peptides because of their enormous surface area (70 sq. m) and high vascularization. Alveoli and lungs are the absorption sites and drugs are absorbed through lungs by carrier mediated transport. 04-Jul-23 21

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25. 3. Buccal delivery: By buccal delivery, drugs are absorbed rapidly into the reticulated vein, which lies under the oral mucosa, and enter the systemic circulation directly, bypassing the liver. Strategies employed for buccal delivery include addition of adhesive promoters, absorption promoters like Sodium glycocholate (bile salt), chelating agents ( EDTA), surfactants ( sodium lauryl sulphate) 04-Jul-23 25

26. Novel formulation approaches: Particulate system such as emulsions, liposomes, nanoparticles have been widely used and remain as a promising approach for transmucosal delivery of protein and peptide. Chitosan appears to be the most suitable polymer that can retain the peptide in buccal mucosa. E.g., insulin loaded chitosan-ethylenediaminetetraacetic acid hydrogel films are used as mucoadhesive resulted in pronounced hypoglycemic effect in healthy rats. The use of alginates and poloxamers as encapsulation materials also represents promising approach overcome poor stability, low bioavailability. They can swell and retain significant fraction of water leading to formation of hydrogels. 04-Jul-23 26

27. Various approaches for transdermal drug delivery system: Iontophore sis: Phonophoresis: Penetration enhancers: Prodrug approach: 04-Jul-23 27 4. Transdermal delivery:

28. 04-Jul-23 28 Macroflux patch technology is a patch application system to ensure consistent dosing and ease of use.

29. 5. Oral delivery: The problems that make the oral route unsuitable for the systemic delivery of therapeutic protein and peptides are: Poor intrinsic permeability of peptides and proteins across biological membranes. Susceptibility to enzymatic attack by intestinal proteases and peptidases. Rapid post absorptive clearance. Physical instability like aggregation and adsorption. 04-Jul-23 29

30. Approaches to overcome the barriers in oral peptide and protein drug delivery Penetration enhancer. Cell penetrating peptide. Chemical modification. PEGylation. Cyclization. 04-Jul-23 30

31. Formulation Approach: Micro emulsion. Nano emulsion. Liposome. Mucoadhesive polymeric system. 04-Jul-23 31

32. Advance technologies to overcome the barriers in oral drug delivery system: Peptelligence. Gastrointestinal permeation enhancement technology. Transient permeation enhancer-based technology. Gastro retentive mucoadhesive patch system. Gastro retentive innovative device. 04-Jul-23 32

33. Penetration enhancer: 04-Jul-23 33 Intestinal epithelial cell barriers Comprehensive mechanistic approach to overcome the epithelial barriers.

34. C hitosan and its derivative are class of amino polysaccharide, which have potential improving the mucoadhesive and opening tight junctions between intestinal epithelial cells. The nano-complex of insulin with amino clay was designed for enhanced the oral absorption of insulin via spontaneous co-assembly approach, which was then coated with glycol-chitosan and Eudragit®S100 (EGAC-Ins) excipients 04-Jul-23 34

35. 2 . Cell Penetrating Peptide: 04-Jul-23 35 Fig: illustration of cellular uptake of peptide drug via CPP carrier to enhance the permeability.

36. CPPs are usually defined as 5–30 amino acids comprising cationic and amphipathic peptides, which are primarily derived from natural proteins. The electrostatic interaction or bonding effect of hydrogen between the groups CPPs (e.g. arginine guanidine of some CPPs) and cell membrane surface lipid components (e.g. heparin sulphate proteoglycans) that are responsible for direct CPP transduction or translocation. Endocytosis is normally expected to be the most common absorption. Therefore, the mode of uptake mechanism of many cationic CPPs is dependent on the concentration of CPPs, that is, rapid cytosolic uptake leading to direct penetration is observed. 04-Jul-23 36

37. 3 . PEGylation: PEGylation can be extensively used which includes covalent conjugation of peptide drugs with polyethylene glycol. PEGylation of protein confers benefit regarding the protection against proteases as well as increased the intestinal permeability. covalently cross-linked chitosan-poly (ethylene glycol) (CSPEG-H-CS) derivatives used as controlled release profile of protein bovine serum of albumin as model protein from hydrogel-based matrix. 04-Jul-23 37

38. Direct PEGylation can aid in the stability of proteins for delivery, mainly leading to an increase in circulation time. PEG molecules are highly hydrated, and this increased size leads to decreased glomerular filtration. Due to the size of PEG, steric hindrance may decrease the activity of the protein. Also, increased protein aggregation after PEGylation has been noted. 04-Jul-23 38

39. 4. Microemulsion: Micro emulsion is an isotropic, thermodynamically safe clear liquid, water and surfactant solution, frequently delivered with a co-surfactant. Scientists are interested in it due to its lipophilic and hydrophilic domain which can incorporate both hydrophilic and hydrophobic drug. These adaptable delivery systems protect from oxidation, enzymatic hydrolysis and hence improve bioavailability. Novartis designed cyclosporine oral delivery by formulating it in a micro emulsion that contains a hydrophilic component. The hydrophilic state produced a small molecular weight mono- or poly-oxy-alkane diol ( e.g.Transcutol ®, Glycofurol ®); or 1,2-propyleneglycol. This micro emulsion composition has the particle size 0.15 micron. Favor's the cyclosporine better drug delivery and improved efficacy. 04-Jul-23 39

40. 5 . Nano emulsion: Nano emulsions are nano-sized emulsions which are developed to enhance active pharmaceutical product distribution. These are the thermodynamically stable isotropic structures in which two immiscible liquids are blended by an emulsifying agent, that is, surfactant and co surfactant. SNEDDS formulation for oral insulin delivery containing a hydrophobic ion pair of insulin/ dimyristoyl phosphatidylglycerol (INS/DMPG) excipients. The therapeutic activity of protein was successfully protected against the intestinal enzymes (i.e., trypsin, α- chymotrypsin) in enzymatic degradation. The SNEDDS based formulation effectively improved the permeability and also stability of peptide across the mucosal intestinal barrier. 04-Jul-23 40

41. 6. Liposomes: Liposome (phospholipids-based vesicles) is colloidal carriers used in drug delivery systems that use biodegradable materials to entrap aqueous media in bilayer lipids. Steroids, antimicrobials, and vaccines are mostly encapsulated in liposome. Lactoferrin is a peptide that has gained a lot of attention because of its essential role in the immune system, as well as its antioxidant, anti-inflammatory, antibacterial, and antiviral properties. However, susceptible to gastrointestinal enzyme hydrolysis, which limits its oral bioavailability of Lactoferrin. 04-Jul-23 41

42. 7. Mucoadhesive polymeric systems : 04-Jul-23 42

43. 8. Peptelligence: Oral formulation based on peptelligence technology integrates a permeation-enhancing an acid excepients to lower the local pH of the intestinal fluids in order to decrease protease activity. This technology was initially developed by Unigene and then Enteris Biopharma based on enteric-coated tablets, where formulating material comprises peptide, organic acid enzyme inhibitors (citric acid) and permeation enhancer (acylcarnitine). 9. Gastrointestinal permeation enhancement technology: The oral formulation developed by Merrion pharmaceuticals used as enteric coated technology. The strategy aimed to guard the peptide in the gastric acidic fluid supports its entry into the small intestine. The technique is focused on the use of fatty acid from the medium chain or its variants coupled with salts. 04-Jul-23 43

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45. 10. Gastrointestinal mucoadhesive system: 04-Jul-23 45

46. (i) The backing layer is made of water insoluble polymer, ethyl cellulose (EC) to protect protein drugs from enzymatic hydrolysis. (ii) The surface layer is made of an enteric pH sensitive polymer such as hydroxypropyl methylcellulose phthalate (HP-55), Eudragit L-100 or S-100 and coated with an adhesive layer. (iii) The middle layer is the drug containing layer, and (iv) lastly, an adhesive layer between the middle and surface layer between the patch and intestinal enterocytes. These patches provide unique platform for oral drug delivery of drugs in facilitating their intestinal absorption through localized drug depot at the delivery site. 04-Jul-23 46

47. 12. Gastro retentive innovative device: 04-Jul-23 47

48. Oleotec ™ and Soctec ™ gastro-retentive technology has been developed by Skyepharma . In the field of oral drug delivery, this system has gained immense popularity. It is a commonly used technique for retaining the drug in the stomach for a long period of time and eventually releasing the medication in controlled way. This innovative system is based on polymer coating with specialized mechanism, Upon ingestion of the formulated dosage along with food, it floats instantaneously in the gastric content. Encapsulated drug activated by GIT fluid. It gradually leads to swelling of the polymer between 8 and 10 times its original size, guaranteeing its preservation in the stomach. even after 6–8 h of gastric emptying and released drugs in a controlled manner. The Accordion Pill™ is a unique gastro retentive formulation composed of pharmaceutical biodegradable polymeric films. 04-Jul-23 48

49. Evaluation of protein and peptide drug formulations: Stability testing: The capability of a particular formulation in a specific container/closure system to remain within its physical, chemical, microbiological, toxicological and protective specifications. Evaluates the effect of environmental factors on the quality of the drug substance or the formulated product which is utilized for prediction of its shelf life and proper storage conditions. 04-Jul-23 49

50. 2. Bioassay: Due to complexity of proteins, bioassay are required to assess potency of the formulation. Bioassay are of two types: in vitro and in vivo. In case of in vitro bioassay response of cells to hormones and growth factors is monitored. In case of in vivo bioassay pharmacological response of animals to proteins is monitored. 04-Jul-23 50

51. 3 . UV spectroscopy: Proteins containing aromatic amino acid residues such as phenyl alanine, tyrosine, tryptophan can be detected by UV spectroscopy. It can be used as analytical determination method. 4. Bradford Assay: This assay employs the principle that in presence of protein in absorption medium, absorption maximum of coomasie brilliant blue G-250 dye changes. If there is no protein to bind, the solution remains brown. The dye forms a complex with carboxyl end of proteins by Van der Waals forces to form a blue colored solution. The intensity of colored solution can be measured using a spectrophotometer to determine the concentration of protein in the sample. 04-Jul-23 51

52. Reference: Novel drug delivery system. Chien, Yie W. Challenges of peptide and protein drug delivery by oral route: Current strategies to improve the bioavailability. Verma S,. et al. Basics and recent advances in peptide and protein drug delivery , Benjamin J Bruno, Geoffrey D Miller, and Carol S Lim. Protein and Peptide Drug Delivery , Nitai Charan Giri . 04-Jul-23 52