Microencapsulation of Human Growth Hormone

Microencapsulation of Human Growth Hormone Group 8: Nadia Bibi Nada El Hanna Melanie Lamontagne Outline • • • • • • • Introduction Background Problem Statement Process Description Problem Analysis Results and Discussion Conclusion Introduction • hGH is administered to patients with hGH deficiencies as daily injections. • Typical Dosage: 0.35 rhGH/kg/week for children and 0.1-0.2 rhGh/kg/week for adults • An alternative would be monthly or bimonthly injections of sustained release microcapsules. Less painful, more convenient. Background Method of encapsulation • Hormone is freeze-dried and encapsulated in a non-toxic polymer PLGA (poly-lactic/glycolic acid). • Another way of measuring the levels of hGH is to measure the level of IGF-1 (Insulin Growth Factor-1) • hGH is converted to IGF-1 which remains in the body for 24-36 hours. Problem Statement • hGH is exposed to several stresses during production that can cause denaturation • We want to find a method that will retain the activity of the hGH when it is encapsulated. • This is also crucial during the hormone’s eventual release into the bloodstream. S/O/W Double Emulsion Solid in Oil emulsion Oil in Water emulsion Solid/Oil/Water Solvent Evaporation Technique Ammonium Acetate+hGH (23,000 rpm, 30 sec. Creates S/O dispersion) DCM evaporates during stirring, PLGA-ZnO hardens Solid hGH remains (AA evaporates) PVA (aq phase) (Hgh is freeze-dried) PLGA-DCM-ZnO Solution (organic phase) (6,000 rpm, 3 hrs. Solid hormone (solid phase) Creates S/O/W emulsion) Rinsed with water Capsules freeze-dried again >74 μ Sieve <=74 μ →Solid Capsules are now ready for injection Solvent discarded Problems that can arise • Denaturation & Aggregation when Freeze Drying -Use volatile salt that disappears after lyophilization: Ammonium Acetate -Less static electricity when handling; Inhibits interaction of rhGH molecules during lyophilization; Prevents aggregation • Denaturation at Organic/Water interface -Protein denaturation at hydrophobic interface. Protein unfolds to expose its hydrophobic regions. Aggregation occurs -Solidifying the hormone prior to emulsifying bypasses this entire situation • Initial burst and hormone stability in vivo. -Zn(2+) from ZnO ensures stability of hormone after injection. -Dimerizes hormone and reduces solubility. -Also complexes with PLGA which improves erosion kinetics PLGA-ZnO microsphere Encapsulated rhGH In Vivo Initial Burst Sustained release Day 0 Day 14 Time (days) Problems that can arise • Denaturation & Aggregation when Freeze Drying -Use volatile salt that disappears after lyophilization: Ammonium Acetate -Less static electricity when handling; Inhibits interaction of rhGH molecules during lyophilization; Prevents aggregation AMMONIUM ACETATE • Denaturation at Organic/Water interface -Protein denaturation at hydrophobic interface. Protein unfolds to expose its hydrophobic regions. Aggregation occurs -Solidifying the hormone prior to emulsifying bypasses this entire situation SOLIDIFYING HORMONE • Initial burst and hormone stability in vivo. -Zn(2+) from ZnO ensures stability of hormone after injection. -Dimerizes hormone and reduces solubility. -Also complexes with PLGA which improves erosion kinetics ZINC OXIDE Results and Discussion Non-release pattern of hGH from microcapsules produced using the W/O/W method 70 In vitro hGH Release (%) 60 50 40 30 20 10 0 0 5 10 Tim e (Day) 15 20 A B C Microencapsulation of Human Growth Hormone within Biodegradable Polyester Microspheres: Protein Aggregation Stability and Incomplete Release Mechanism PLGA-ZnO microsphere Encapsulated rhGH Aggregation? Denaturation? In Vitro Non-release Initial Burst Sustained release Day 0 Day 14 Time (days) Lyophilizing hGH with ammonium acetate Mean particle Mean particle AA/ rhGH size Entrapment size rhGH (molar ratio) microcapsules (%) (µm) (µm) 10 20 2.8 3 35.3 35.0 100 92.0 Loading amounts of rhGH and zinc oxide were 7% and 0.5%, respectively. PLGA with L/G (lactic acid/glycolic acid ratio) of 50/50 and MW of 12800 was used. Addition of ZnO in the PLGA-DCM Location and structure of the Zn binding site on hGH molecule Addition of ZnO in the PLGA-DCM 1000 Serum hGH (ng/ml) 100 10 1 0 5 Tim e (Days) 10 15 0% Zinc Oxide 0.5% Zinc Oxide Reference: Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique Addition of ZnO in the PLGA-DCM 6 5 4 3 2 1 0 0 Zinc Oxide (%) 0.5 AUC (µg*hr/ml) Ensuring hGH stability by measuring activity Two methods: • Nb2 lymphoma cell proliferation assay • IGF-1 produced in vivo (rats) Nb2 lymphoma cell proliferation assay rhGH Intact Lyophilized rhGH-SR-2W IU/mg 3.43 3.34 ± 0.364 3.56 ± 0.294 n (number of samples) 2 6 6 Reference: Poly(lactic-co-glycolic acid) and functional hydrogels for drug delivery applications Serum rIGF-1 levels in immunosuppressed rats 1600 1400 1200 1000 800 600 400 200 0 0 3 6 9 Tim es (days) 12 15 Serum rIGF-1 (ng/ml) Daily x 14 Microcapsule - 1 injection Control Serum rIHG-1 levels in immunosuppressed rats comparison Conclusions • Encapsulation of hGH in polymer matrix using the solvent evaporation technique with incorporation of lyophilization of hGH, ZnO and AA was successful. • The hGH was sustained release and activity of the hGH was retained throughout the process. Questions? Improvements? • Addition of base to neutralize acidic environment when PLGA is broken into lactic/glycolic acid • Freeze drying with Zn to form hGH-Zn complex. • Niche for this product is there but needs more long term studies to ensure acceptance. (must also reduce cost) Lyophilization • Freezing: Product is first converted to a frozen state. (product must not be altered as a result of the process) • Solvent ( water) is crystallized. • Separation of the solutes and the solvent • Formation of ice forces the solutes into a region between the crystals known as the “interstitial” • Sublimation: Placing the frozen material into a chamber and removing the gases to create a vacuum. • Desorption: greater that 5% moisture in the product. Moisture contained on the surface of the cake is defined as adsorbed water. • Vacuum System: The freeze-dryer is equipped with a mechanical pumping system that removes the noncondensable gases. Homogenizers used Polytron Homogenizer (Kinematica GmBh) Autohomomixer (Tokushu Kika Kogyo Co.) Coulter Multisizer Creates size distribution and PLGA-ZnO microsphere Encapsulated rhGH Aggregation? Denaturation? In Vivo Non-release Initial Burst Sustained release Day 0 Day 14 Time (days) • Backup with ZnO and hGH graph from sustained release to prove that ZnO gets released at same time, and dolubilty stuff… Alternative:Cryogenic & Nonaqueous 1. Micronization of Zn-hGH into liquid N2 and then freeze-dried 2. Added to DCM-PLGA polymer solution and sonicated. 3. Homogenized then atomized into vessel containing frozen ethanol 4. DCM evaporates Similar to our process except aqueous phase is avoided and involves liquid N2. Nutropin® Depot Number of Injections Per Dose Patient Weight (kg) <15 0.75 mg /kg twice monthly 1 1.5 mg/kg once monthly Nutropin ® Depot has been discontinued. Nutropin ® daily injections are still available and cost $219.99 per 5 mg vial (contains 3.33mg hGH). Price quote from www.drugstore.com for Saizen, a Nutropin equivalent. 1 >15–30 >30–45 >45–60 >60 1 2 2 3 2 3 * * Nutropin® is used to treat children and adults with hGH deficiencies, as well as patients with Turner’s Syndrome (Dosages will always depend on the doctor’s prescription. These are approximate values.) *Twice-monthly dosing recommended Prescription Information: http://www.nutropin.com/patient/u_1_prescribing_information.jsp Each 1.5 mL single-use vial of Diluent for Nutropin Depot contains 30 mg/mL carboxymethylcellulose sodium salt, 1 mg/mL polysorbate 20, 9 mg/mL sodium chloride, and sterile water for injection; pH 5.8-7.2 Other Sustained Release Drugs PLGA Structure http://www.drugdeliverytech.com/cgi-bin/articles.cgi?idArticle=58 W/O/W results 70 In vitro hGH Release (%) 60 50 40 30 20 10 0 0 5 10 Tim e (Day) 15 20 A B C Batch A B C Co-solvent volume Loading % Average ratio (methylene Encapsulation (rhGH/microspere Particle chloride / ethyl efficiency (%) (w/w)) size (µm) acetate) 3/0 2/1 1/2 12.2 11.6 11.5 90.4 85.9 85.3 45.3 50.1 46.3 Lyophilizing hGH with AA X 10 X 20 Static Electricity and AA/hGH molar ratio Reference: Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique Ammonium acetate/ rhGH (molar ratio) Mean Mean particle size particle size of microcapsules of rhGH(µm) (µm) Entrapment (%) 0 10 20 30 70 10.5 2.8 3 4.7 7.2 40.9 35.3 35.0 33.7 35.9 86.6 100 92.0 89.9 83.3 100 11.8 36.5 70.1 Primary Structure of hGH Disulphide bonds Metal-catalyzed Oxidation of Histidine in Human Growth Hormone MECHANISM, ISOTOPE EFFECTS, AND INHIBITION BY A MILD DENATURING ALCOHOL* Protein Structures • Primary: amino acid sequence in a protein. • Secondary and tertiary: threedimensional (3D) conformation of a protein chain. • Quaternary: interaction between separate chains in a multi-chain protein. The types of interactions may be any of those found in secondary and tertiary structures. Results and Discussion • Lyophilizing hGH with ammonium acetate 3000 2500 Serum hGH (ng/ml) 2000 1500 1000 500 0 0 5 10 15 20 Time (hours) 25 30 AA / hHG = 1 AA / hHG = 30 AA / hHG = 10 AA / hHG = 70 AA / hHG = 20 AA / hHG = 100 Lyophilizing hGH with ammonium acetate 50 Initial release (%) 40 30 20 10 0 0 10 20 30 70 100 Am m onium acetate / hGH (m olar ratio) Area under the curves