Formulation and Characterization of Wound Care Products

Formulation and Characterization of Wound Care Products
Project Review from Formulation Development to Analysis

1. PRAYAS ACHARYA Master of Pharmacy (Pharmaceutics) 18PHP207 GUIDE: Prof. Purnima D. Amin Dept. of Pharmaceutical Sciences and Technology, Institute of Chemical Technology (ICT), Deemed University, Elite Status and Centre of Excellence(GOM),Mumbai 400019 E-mail:prayasacharya10 @gmail.com Formulation and Characterization of Wound Care Products

2. PART A PART B 2

3. Objectives of the project To formulate stable silver sulfadiazine (AgSD) nanosuspension and incorporate it into topical dosage forms with a view to increase bactericidal activity in burn therapy. To make a topical gel of AgSD in combination with Salibact (a novel patented antimicrobial agent synthetically obtained from an organic acid derived from nature and a well-known guanidine base). To elaborate an antibacterial silver sulfadiazine wound dressing. 3

4. Introduction Wound is defined as an interruption of the defensive role of skin to protect the internal organs from harmful environment. It usually results in the failure of skin in performing normal anatomical and physiological functions. Injuries/Wounds can vary from simple disruption of superficial epithelial layer of skin or may show deeper injury reaching to subcutaneous layer and hampering other important structures such as muscles, ligaments, tendons and even bones . 4

5. Classification of wounds Depending on aetiology 5

6. Wound Healing Healing is a complex process involving co-ordinated interactions between diverse immunological and biological systems. It involves a cascade of carefully and precisely regulated steps and events that correlate with the appearance of various cell types in the wound bed during distinct phases of the healing process. 6

7. Phases of Wound Healing Step 1- Coagulation and haemostasis, beginning immediately after injury Step 2- Inflammation, which begins shortly thereafter Step 3- Proliferation, which starts within days of the injury and encompasses the major healing processes Step 4- Wound remodelling, in which scar tissue formation takes place, and which may last up to a year or more   7

8. Phases of Wound Healing 8

9. Selection of Active Ingredient 1. Silver Sulfadiazine Silver sulfadiazine (AgSD) is the selected drug as it is used for the prevention and treatment of wound sepsis in patients with second and third-degree burns. It is bactericidal for many gram- negative and gram-positive bacteria as well as being effective against yeast. It achieves bacterial inhibition by binding to the base pairs in the DNA helix and thus inhibits transcription. 9

10. Drug Profile- Silver sulfadiazine Parameters Description Chemical Name Silver sulfadiazine IUPAC Name Silver(4-aminophenyl) sulfonyl-pyrimidin-2-ylazanide Appearance White crystalline powder Molecular Weight 357.137260 [g/mol] Molecular Formula C 10 H 9 AgN 4 O 2 S Stability Stable in air but turns yellow on exposure to light. Log P 0.19 Water Solubility Highly insoluble in water. Melting point (°C) 285 °C Absorption Very limited penetration through the skin. Indications Indicated as an adjunct for the prevention and treatment of wound sepsis in patients with second and third degree burns. Storage Conditions Away from light and moisture under inert atmosphere. 10

11. Selection of Active Ingredient 2. Salibact ® Salibact® is an antibacterial and antifungal active ingredient Effective against gram positive and gram negative bacteria, fungi, yeast and molds Biodegradable Compatible with most of the other personal care ingredients Near zero probability of bacterial resistance Effective on pH range 3.5 to 10 Salibact can be applied around 0.05% to 0.15% in the finished formulation 11

12. Drug Profile- Salibact Product Name Salibact INCI Name Chlorhexidine Di-Undecylenate Molecular Formula C 44 H 70 CI 2 N 10 O 4 Molecular Weight 873 g/mol Physical Appearance White powder Solubility Freely soluble in alcohols and solvents 12 Time Kill Test of Salibact

13. Drug Characterization Studies IR Graph of Silver sulfadiazine 13

14. Drug Characterization Studies 14 The absorption maximum was observed at 252 nm due to the presence of conjugated bonding system which contains delocalized electrons. The reported wavelength for maximum absorption is 252nm.

15. Analytical Method Development of AgSD Parameters Silver Sulfadiazine Solvent Methanol Linearity 1-10 μg/ml Detection wavelength 252 nm Slope 0.0831 Correlation coefficient 0.999 Y-intercept +0.0068 15 Calibration curve of Silver sulfadiazine Standard curve parameters of AgSD in methanol

16. Analytical Method Development of AgSD Concentration ( μg /ml) Absorbance*mean; n=3 %RSD 1 0.08±0.0925 19.11099 2 0.14±0.1705 25.29825 4 0.298±0.3455 19.44288 6 0.423±0.4995 21.65913 8 0.565±0.6625 20.81295 10 0.715±0.846 21.89858 16 Standard curve reading of AgSD in methanol *The analytical method was found to be linear in the concentration range of 1-10 μg /ml with a coefficient of correlation of 0.999. Solution (6 μg/ml) Absorbance 1 0.423 2 0.421 3 0.425 Mean 0.423 Standard Deviation 0.002 %RSD 0.472 System precision data of AgSD

17. Analytical Method Development of AgSD Sample Absorbance Mean SD %RSD 1 0.424   0.425   0.00152   0.357 2 0.427 3 0.426 Sample Absorbance Mean SD %RSD 1 0.298   0.298   0.00208   0.697 2 0.301 3 0.297 Inter-day precision of AgSD Intra-day precision of AgSD 17

18. Research Objectives- Part A To formulate and optimize nanosuspension of silver sulfadiazine and incorporate it into gel and cream bases for topical delivery. To formulate and optimize stable silver sulfadiazine gel with improved AgSD solubility with the combination of Salibact To compare the activities of the prepared formulations with the conventional dosage forms. 18

19. Experimental Part Selection of excipients Drug-excipient compatibility Selection of stabilizers Selection of compatible polymers Optimization of formulation considerations Stability analysis 19

20. Selection of Excipients and compatibility The drug-excipient compatibility studies deliver the scheme for the drugs combination with excipients in the formulation of the dosage form. The study aimed to establish that, the therapeutically active drug has not undergone any changes after it has been subjected to processing steps during the formulation of topical products. Compatibility study of silver sulfadiazine was done by mixing definite proportion of active substance and different grades of HPMC, Poloxamer 188, Poloxamer 407, PVP K30, Propylene glycol, Tween 80, PEG 6000, methyl paraben, Carbopol 934, Carbopol 940, Carbopol 980, Disodium EDTA, Triethanolamine in the ratio 1:1 at 45 C for one month. 20

21. PART-A Formulation of AgSD Gel 21

22. Formulation Trials and Optimization Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) A1 10000 5 1550 0.850 5.05 A2 10000 10 1342 0.779 1.06 A3 5000 5 2130 1.20 -0.296 A4 5000 10 1988 1.18 1.089 Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) B1 10000 10 1590 0.899 -0.510 B2 10000 5 834 0.666 0.909 B3 5000 10 1225 0.709 3.98 B4 5000 5 2035 1.00 7.05 22 Stabilizer solution of 0.1% HPMC E5 and 0.1% Tween 80 Stabilizer solution of 0.2% HPMC E5 and 0.2% Tween 80

23. Formulation Trials and Optimization Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) C1 10000 10 1350 0.702 8.6 C2 10000 5 1742 0.622 7.2 C3 5000 10 1556 0.585 3.6 C4 5000 5 1593 0.420 3.2 23 Stabilizer solution of 0.1% HPMC E5 and 1% PVP K30

24. Formulation Trials and Optimization Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) D1 10000 10 1026 0.520 -3.7 D2 10000 5 1223 0.423 2.5 D3 5000 10 1256 0.455 6.2 D4 5000 5 1450 0.780 6.8 24 Stabilizer solution of 1% Poloxamer 407

25. Formulation Trials and Optimization Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) E1 10000 10 1024 0.423 -2.6 E2 10000 5 1136 0.589 -6.3 E3 5000 10 1188 0.523 -3.1 E4 5000 5 1220 0.686 2.5 25 Stabilizer solution of 1% PVP K30

26. Formulation Trials and Optimization Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) F1 10000 10 750.5 0.450 -5.05 F2 10000 5 680.4 0.321 -3.7 F3 5000 10 824 0.420 4.6 F4 5000 5 1036 0.521 2.5 26 Stabilizer solution of 1% Poloxamer 407 and 1% PVP K30

27. Formulation Trials and Optimization Batch No. Speed (rpm) Time (min) Size (nm) PDI Zeta Potential (mV) F5 10000 10 853 0.650 -7.05 F6 10000 5 897.2 0.411 -6.8 F7 10000 10 723.8 0.524 -4.6 F8 10000 5 951.6 0.479 -5.5 27 Optimization batches in triplicates

28. Inference In the current study, the non-ionic surfactant (Poloxamer 407) and non-ionic polymer (PVP K30) were the optimized stabilizers after several trials in existence of a specified amount of AgSD. PVP K30 acts as a capping agent for the nanoparticles which aids in reduction of particle size as well as prevents nanoparticle aggregation. PDI values within range further hints to the good dispersion of silver sulfadiazine in the stabilizer solution. 28

29. Inference The possible mechanism of action of P407 as a stabilizer in this study was that the nanocrystals were stabilized by the surface adsorption of P407. In other words, the absorbance of P407 onto the surfaces of AgSD nanocrystals provided a steric stabilization effect. Moreover, the viscosity of P407 might also contribute to the stability of AgSD nanosuspensions. To the mixture of stabilizer solution, 1% of silver sulfadiazine was dispersed to the solution and subjected to particle size reduction homogenization under IKA homogenizer. Time and speed are the critical process parameters during the process. 29

30. Evaluation of AgSD Nanosuspension Particle size, polydispersity index and zeta potential The average particle size of all the prepared formulas were calculated using Malvern Zetasizer . Not all of the prepared AgSD nanosuspension formulas showed a particle size result within nano range. The formula F2 showed lowest PDI (0.321), as seen in the data, indicates good uniformity of nanoparticle size. The range of PDI values (0-0.05) means (monodisperse system), 0.05-0.08 (nearly monodisperse), 0.08-0.7 (mid-range polydispersity), and >0.7 (very polydisperse). 30

31. Effect of using co-stabilizers PVP K-30, Poloxamer 407, HPMC E5 LV and Tween 80 were used as stabilizers in trial formulas. PVP K-30 and Poloxamer 407 are polymeric non-ionic stabilizers for nanosuspensions, they form physical barrier on the surface and interrupt the contact of the close particles. The non-ionic stabilizers with amphiphilic moieties are usually employed to give steric stabilization, which is dominated by wetting effect. Stabilizers can efficiently decrease the surface activity energy to inhibit aggregation that decrease dissolution rate. The critical parameter of stabilizer is the stabilization ability by enhancement of the physical stability of nanoparticulate system and maintaining the smallest size of particles. According to the above results, a smaller particle size was achieved in a combination between PVP K30 and Poloxamer 407. Since this combination show significant reduction in particle size as when compared with other combinations, and it may be because of the highest affinity to the drug molecules. Evaluation of AgSD Nanosuspension Effect of using one stabilizer 31

32. Evaluation of AgSD Nanosuspension Stability of AgSD Nanosuspension The aver­age particle size (Z-average) and polydispersity indexes (PDIs) were detected using Malvern Zetasizer to investigate the short-term stability of AgSD nanosuspensions. The samples were collected on days 1, 3, 5, 7, and 30 for determining AgSD/NS stability. Day 1 was the day of AgSD/NS production and the change in the average particle size and polydispersity indexes was found to be negligible which suggests its long-term stability. 32 Sample Particle Size (nm) Day 1 Day 3 Day 5 Day 7 Day 30 F5 853 854.6 856.3 856.4 863.7 F6 897.2 898.1 898.8 899.8 921.3

33. Nano AgSD Topical Gel Ingredients Quantity (%) Function Silver Sulfadiazine 1 API (Anti-microbial agent) Poloxamer 407 1 Stabilizer PVP K30 1 Stabilizer Disodium EDTA 0.1 Chelating agent Propylene Glycol 4 Humectant Methyl Paraben 0.1 Preservative HPMC K100M/PVA/P407 q.s. Gelling Agents Purified Water q.s. Vehicle 33

34. Optimized Formula for AgSD and Salibact Topical Gel Phase Ingredients Quantity (%) Function A Silver sulfadiazine 1 Antibacterial (API) Stabilizer solution (in purified water) Optimised concentration in q.s. water Stabilizer B Salibact 0.1 Preservative + Antibacterial Propylene Glycol 3.0 Humectant Tween 80 2.0 Solubilizer Methyl paraben 0.2 Preservative C HPMC K100M 1.5% Gelling agent 34

35. Evaluation of Optimized Gels Test A1 A2 A3 Spreadability Easy Easy Easy Homogeneity Yes Yes Yes Colour White White White Odour No No No Phase separation No No No Feel upon application Smooth Smooth Smooth Test B1 B2 B3 Spreadability Easy Easy Easy Homogeneity Yes Yes Yes Colour White White White Odour No No No Phase separation No No No Feel upon application Smooth Smooth Smooth Physical Evaluation of AgSD and Salibact Gel Physical Evaluation of AgSD Gel 35

36. Evaluation of Optimized Gels pH of optimized gels Viscosity of optimized gels Formulation pH B1 6.78 B2 6.42 B3 7.20 Formulation pH A1 7.23 A2 7.11 A3 6.95   AgSD Gel AgSD and Salibact Gels A1 A2 A3 B1 B2 B3 Viscosity 151383 153475 152375 162319 163215 160157 pH of AgSD Gels pH of AgSD + Salibact Gels 36

37. Evaluation of Optimized Gels   AgSD Gel AgSD and Salibact Gels A1 A2 A3 B1 B2 B3 Viscosity before centrifugation 151383 153475 152375 162319 163215 160157 Viscosity after centrifugation 150989 152825 151982 161782 162565 159923 This test is done to check whether the formulation is capable of withstanding the stress of handling and transportation. 37

38. Stability Studies  Formulations 0 month 1 month 3 months pH Viscosity (cps) pH Viscosity (cps) pH Viscosity (cps) A1 7.23 151383 7.21 151302 7.22 151212 A2 7.11 153475 7.11 153120 7.13 153212 A3 6.95 152375 6.97 152297 6.98 152123 B1 6.78 162319 6.79 162002 6.80 162156 B2 6.42 163215 6.49 162993 6.45 162986 B3 7.20 160157 7.21 160032 7.20 159952 Stability studies of optimized gels for storage conditions 25 C/ 40% RH 38

39. Stability Studies  Formulations 0 month 1 month 3 months pH Viscosity (cps) pH Viscosity (cps) pH Viscosity (cps) A1 7.23 151383 7.20 151026 7.28 151285 A2 7.11 153475 7.13 153219 7.13 153254 A3 6.95 152375 6.92 152145 6.99 152198 B1 6.78 162319 6.76 162306 6.81 162150 B2 6.42 163215 6.45 163157 6.43 163125 B3 7.20 160157 7.21 160029 7.25 159925 Stability studies of optimized gels for storage conditions 40 C/ 75% RH 39

40. Stability Studies Inference In case of semisolids, the appearance, pH and viscosity are the 3 major parameters which should checked because these are the ones which can change over a period of time when the sample is kept at accelerated storage conditions and hence also throughout its shelf life. It was found that the appearance of none of the products changes over the time when kept for stability studies. From the above results, it is clear that there is not much difference in the pH and viscosity of the formulations when kept at accelerated storage conditions. Thus, it can be said that the products are stable. 40

41. Microbiological Studies The antimicrobial analysis of both the AgSD gel and AgSD and Salibact combination gels were studied and more than 99.99% reduction was observed in antimicrobial efficacy test within 10 to 90 sec for E. coli and C. albicans . 41

42. Microbiological Studies Time (sec) Initial Count (CFU/ml) 10 sec (CFU/ml) 20 sec (CFU/ml) 30 sec (CFU/ml) 40 sec (CFU/ml) 50 sec (CFU/ml) 60 sec (CFU/ml) 75 sec (CFU/ml) 90 sec (CFU/ml) Culture E. coli ATCC8739 2x10 6 <10 <10 <10 <10 <10 <10 <10 <10 % Reduction NA >99.99 >99.99 >99.99 >99.99 >99.99 >99.99 >99.99 >99.99 C. albicans ATCC10231 1.9 x 10 5 26 <10 <10 <10 <10 <10 <10 <10 % Reduction NA >99.99 >99.99 >99.99 >99.99 >99.99 >99.99 >99.99 >99.99 Microbial Analysis of AgSD gel C/o INTERTEK LAB 42

43. Microbiological Studies Microbial Analysis of AgSD and Salibact combination gel C/o INTERTEK LAB 43

44. PART-B Preparation of AgSD wound dressing 44

45. Research Objectives- PART B To prepare an antibacterial silver sulfadiazine wound dressing to enhance wound healing. Evaluation of prepared medicated textile dressings. 45

46. Rationale for wound dressings 46 The topical solutions and ointments create a cream-fibrin adherent layer onto the burn wound, making it difficult to monitor the evolution of the wound and create a very good environment for bacteria like pseudomonas, thus promoting in some degree the colonization of the burn, after the silver core becomes inactive.  Dressings, being a net-like structure they allow a proper drainage of the exudate produced by the burn lesions, while still keeping the burn moist.  To prevent contamination from bodily discharge. To protect the wound from mechanical trauma. During the wound healing process, wound dressings protects the injury and contributes to the recovery of dermal and epidermal tissues.

47. Selection of Textile dressing Medical gauze is the most widely used wound dressing product. Gauze is made from woven or nonwoven fabrics based on natural or synthetic fibers , such as cotton yarns and polyester fibers . Gauzes can absorb exudate from the wounds and can keep the environment moist. T he type of dressing or fabric selected for initial trials was the gauge dressing as well as non- woven textile dressing. Nonwoven fabrics are selected because they possess light weight, flexibility, higher blending options, and high strength. The use of nonwoven fabrics in medical textiles provides essential features such as high absorption, porosity, versatility in the sense of bulk and area densities. 47

48. Why gauge-based dressings? 48

49. AgSD impregnated dressings 49 Silver sulfadiazine works by stopping the growth of bacteria that may infect an open wound which helps to decrease the risk of the bacteria spreading to surrounding skin, or to the blood where it can cause a serious blood infection (sepsis). Silver sulfadiazine is absorbed into the skin, where it forms a reservoir of silver ions, which are then released into the tissues. Antimicrobial to reduce bioburden of wound through slow release of silver ion into the wound. AgSD dressings release charge silver atoms (ionic Ag+) on contact with the wound fluid. One free radical kills one pathogen. Silver ion has property to penetrate biofilms (resistant layer formed by microbes to antibiotics) Initial release of high levels followed by sustained release appears to aid reduction in bacterial numbers and a wide spectrum of activity.

50. Processing Steps 50

51. Impregnation of AgSD suspension on dressings Pre-treatment of Non-woven Textiles Non-woven t extile samples are pre-treated with a polysaccharide, cyclodextrin crosslinked with citric acid by a pad/dry/cure process. It presents an anionic character thanks to the presence of residual carboxylate groups carried by the citrate crosslinks. This eventually improves swelling properties to promote wound healing with high level of exudate absorption. The goal of the study was to elaborate an antibacterial silver wound dressing covered by a protective coating that would prevent silver diffusion toward skin without losing its biocide properties. 51 A. Mogrovejo-Valdivia, O. Rahmouni, N. Tabary, M. Maton, C. Neut, B. Martel, N. Blanchemain , In vitro evaluation of drug release and antibacterial activity of a silver-loaded wound dressing coated with a multilayer system, International Journal of Pharmaceutics (2018), doi : https://doi.org/10.1016/j.ijpharm. 2018.12.018

52. Impregnation of AgSD suspension on dressings 52 A. Mogrovejo-Valdivia, O. Rahmouni, N. Tabary, M. Maton, C. Neut, B. Martel, N. Blanchemain , In vitro evaluation of drug release and antibacterial activity of a silver-loaded wound dressing coated with a multilayer system, International Journal of Pharmaceutics (2018), doi : https://doi.org/10.1016/j.ijpharm. 2018.12.018

53. Preparation of slurry of AgSD Batch No. AgSD % Poloxamer 407 % PVP K30% Propylene Glycol % PVA % HPMC K4M % HPMC K100M % 1 1 1 1 5 10 - - 2 1 2 - 5 10 - - 3 1 1 1 5 - 1 - 4 1 2 - 5 - 1 - 5 1 1 1 5 - - 1 6 1 2 - 5 - - 1 Formula for trial batches for slurry of Silver sulfadiazine 53

54. Preparation of slurry of AgSD 54

55. Textile Machines Employed Laboratory Padding Machine Textile coating machine 55

56. Future scopes FTIR Studies Stability Studies Silver release and silver loading profiles SEM Analysis Antimicrobial analysis of the dressings In-vivo Studies 56

57. References M. M. et. al. Ueda, Clarence T., Vinod P. Shah, Kris Derdzinski , Gary Ewing, Gordon Flynn, Howard Maibach , “Validation of Compendial Methods Section,” United State Pharmacopeial/National Formul . , p. 2256, 2009, doi : 10.1787/9789264071087-pt. A. Adhya et al. , “Healing of burn wounds by topical treatment: A randomized controlled comparison between silver sulfadiazine and nano-crystalline silver,” J. Basic Clin. Pharm. , vol. 6, no. 1, p. 29, 2015, doi : 10.4103/0976-0105.145776. A. C. Miller, R. M. Rashid, L. Falzon , E. M. Elamin , and S. Zehtabchi , “Silver sulfadiazine for the treatment of partial-thickness burns and venous stasis ulcers,” Journal of the American Academy of Dermatology . 2012, doi : 10.1016/j.jaad.2010.06.014. P. M. Kumar and A. Ghosh, “Development and evaluation of silver sulfadiazine loaded microsponge based gel for partial thickness (second degree) burn wounds,” Eur. J. Pharm. Sci. , 2017, doi : 10.1016/j.ejps.2016.09.038. H. Hoeksema, D. Vandekerckhove , J. Verbelen , A. Heyneman , and S. Monstrey , “A comparative study of 1% silver sulphadiazine ( Flammazine ®) versus an enzyme alginogel ( Flaminal ®) in the treatment of partial thickness burns,” Burns , 2013, doi : 10.1016/j.burns.2012.12.019. S. Joshua, H. Kerr, N. Howard, and M. Guillian , “Wound healing dressings and drug delivery systems,” Journal of Pharmaceutical Sciences , vol. 97, no. 8. pp. 2892–2923, 2008, doi : 10.1002/ jps . J. Boateng and O. Catanzano , “Silver and Silver-Nanoparticle Based Antimicrobial Dressings,” in Therapeutic Dressings and Wound Healing Applications , First., John Wiley & Sons Ltd., 2020, pp. 157–184. Venkataraman et al. Silver Sulfadiazine Nano systems for Burn Therapy. AAPS Pharm Sci Tech, Vol. 14, No. 1, March 2013 (# 2012) DOI: 10.1208/s12249-012-9914-0 57

58. Acknowledgements I would like to convey my heartfelt gratitude to my respected guide, Prof. Dr. P. D. Amin, for her constant support, guidance, love and care both on and off academics throughout my post-graduation. I would also like to thank Prof. A.B. Pandit, Vice Chancellor, Institute of Chemical Technology, Prof. G. D. Yadav, former Vice Chancellor, for providing me with all research associated infrastructure and facilities required for my research. I would like to pay my respects to Prof. S. V. Joshi, Head of Department, Department of Pharmaceutical Sciences and Technology as well as Prof. P. R. Vavia , Prof. P. V. Devarajan, and Prof. V. B. Patravale , as well as all teaching and non-teaching staffs at ICT. My lab members- Mr. Dilipkumar , Mr. Umesh Shinde, Mr. Durgesh Jha, Mr. Tousif , Mr. Rahul, Ms. Devanshi Shah, Mr. Sunny, Mrs. Sharda, Divya , Shrushti , Apoorva, Sharvari , Akash , Tamil , Madhuri , Manindra , Mahak , Shrikant, and Nikita for their valuable help during the project and for being the best people to work with in the lab. I would also like to express my gratitude to MHRD India and EdCil India for providing me this wonderful opportunity to study in ICT. Abbott Healthcare, Mumbai for providing me the platform for my internship. Finally, I take this opportunity to thank India and all my Indian friends who made me feel at home in this beautiful country. 58

59. Thank You! 59