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Journal of Sci entifi c & Industri al Research Vol. 59 , December 2000, pp 1027-103 1 Dextran - HPMC Eye Drops As Artificial Tears Milind Redkar, B Srividya, P Ushasree and P D Amin* University Department of Che mi cal Technology (Autonomous), Matun ga, Mumbai 400019 , lndi a. Recei ved: 24 Janu ary 2000; accepted 2 1 August 2000 In the present study, a formula ti on containing a combinati on of dextran and hydroxypropyl methylcellul ose (HPMC ) was developed where HPM C, a vi scosity-imparting agent , forms a thin film over precornea l tear film while dex tran provides mechani cal strength to thi s film. The formul ation was developed by studyin g various polymers. A buffer system was selected to ac hieve an alkaline pH . Various preserv atives were evalu ated and J suit able antim icrobial preservati ve hav ing suitab le concentration was selected. Eye drops were evaluated for pH , viscosity, clarity, light stabilit y, dex tran, and HPMC content , and steril it y. Ocular irrit ation studies and Schirmer 's test we re carried out on rabbits. The final formulation was subj ected to accelerated thermal studi es . The developed formulation WJS non-irrit ant and showed optimum vi scosity. It had beller film forming properties and improved welling characteri sti cs than the marketed formulation MOISOL®. The developed formulation has potenti al as artifici al tears in the treatment of dry eye syndrome. Introduction Dry eye syndrome is a condition which results from a deficiency of either the aq ueou s or mucin component of the precoineal tear fi 1m. Patients complain of severe ocular pain due to a sensation of dryness in the eyes . The terms dry eye and kerato conjunctivitis sicca (KCS) are most commonly used to indicate problems of the ocular surface connected with the reduction or instability of the precorneal tear filml. The dry eye syndrome is most commonly found in individuals who spend most of their tim e in front of Video Display Terminal s (VDTs) , e .g ., PCs and computer games. The prolonged use of VDTs is associated with a decreased frequency of blinking and an increased rate of tear evaporation. Thi s leads to ocular fatigue which is one of th e major symptoms of dry eye. The underlyin g causes of dry eye can be as follows:(i) aqueous tear deficiency, (ii) mucin abnormalities, (iii) impaired lid function . Due to persi stent disruption of tear fi Im, the symptoms of KCS develop . Tn KCS , mild de g ree of conjuctival infection is associated with considerable irritation, photophobia, foreign body sensation , and burning2. * For co rrespondence Artificial tear formulations fulfill the physicochemical role of a normal tear. Artificial tears effectively lower the surface tension of the tear fi lm , aid the formation of hydrophilic layer that is compatible with absorbed mucin , and enhance tear vo lume when necessary. Thus the mainstay of treatme nt of dry eye conditions remains the supplementation of tear production by artificial preparati o ns. Almost all the artificial tears available in the market contain hydrophilic polymer system . The objective of the present work was to develop artificial tears with hydroxypropyl methylcellulose (HPMC) and dextran, which are hydrophilic polymers. The rationale for their use is that HPMC is a viscosity-imparting agent and it forms a thin f ilm over precorneal tear film , while dextran pro vides the mechanical strength to thi s film. Materia Is and Methods For the selection of raw mate rial s, based on the req uirements of ideal artificial tears , diffe rent pH and tonicity adjusting agents were studied , with spec ia l emphasis o n hydrophili c polyme ri c syste m , J SCI IND RES VOL. 59 DECEMBER 2000 1028 Table I Formulation C larit y pH Eva lu ati on of formula ti ons Sp. V i scos it y gr~vity (c P) A C lear H. 16 I. OO<)l) 26.75 B Clear X.I I 1.0 I 06 6S. 02 C Clea r H. 16 1.00% 1.10 D Clear X. 15 I .009X 25.4<) E Clear S. 17 1.0098 7 1.46 MOISOL® Clear IUO 1.0079 42.30 whi ch decides the tear film characteristics, as well as the viscosi ty. (AJ Forlllulation Development Based on tear studies the requirements of an idea l tea r substitutes are : (a) Shou ld be isoto ni c, (b) Should have pH 01'7.4 ± 0. 1 which is equivalent to tear fluid pH , (c) Should contain preservatives to prevent mi crobi al growth , (d) Shou ld have viscosity in between 25 to 50 cP, and (e) Sho ul d be steril ized by suitabl e method. Initially, isoto ni c buffer veh ic le was se lected. Though the eye is tolerant to toni city variation, given a choi ce, isotonicity is always desirab le. 1n th e present study, sodium chl oride (0.45 % w/v) was usee! as a tonicity adjust ing agent. Freezing poi nt method and mo lec ul ar concentration meth od we re used to ca lculate the amount of NaCI requi red . The buffered veh icle for the eye drops was prepared in order to prepare alkaline so luti on havin g pH about 7.4. However, pH of th e formulation was se lec ted to be 8. 1, for achieving the stabi lity or the polymeric system. Borax and boric acid buffer were used as th e veh icle for th e form ulati on. It was prepared by disso lvi ng 0.19 g of boric ac id , 0.19 g of bo ra x, and 0.35 g of potassium chloride in 100 1111 o r puri fied water. The nex t step was to select a suitab le antimi crobial preservative. Benzalkonium chlorid e (BKC), pheny lmercuri c nitrate, and chl orbutanol were con sidered. PMN was rejected as the use of orga nic mercuri als is restri cted to neutral to alkalin e so lu ti o ns and as PMN has been repor ted to cause mercurial entes. Chlorbutanol was avo ided because of its reported slow action , and formul at ion and packaging difficulti es. BKC (0.0 I percent w /v) was selected as the prese rvati ve for the system . Fin all y the selec tion of the polymeri c system was made. Viscosity of artificia l tears may be achieved between 2.5 to 50 cP by viscosity imparting agen ts such as polyvi ny l alcohol (PVA ) and ce llul osic polyme rs s uch as me th ylce llu lo se (MC), hydrox ypro pylcellul ose (HPC), and hydroxypropy l met hylcellul ose (HPMC) . To stud y different viscosity impartin g age nts, roll ow in g fo rmulati ons with varyin g polymers and thei r combin atio ns were prepared (Tab le I) Formula A = HPMC K4M alone (0.7 per cent w/v) in borate buffe r pH 8.0. Formula B = HPC MFF (0.7 pe r cent w/v) alone in bo rate buffer pH 8.0. Formula C = Dex tran 70 (0.1 er cent w/v) alon e in borate bu ffe r pH 8.0. Formul a D = HPM C K4M (0.7 percent w/v ) and dex tran 70 (0.1 pC I' cent "v/v) in borate buffer pH 8.0. Formu la E = HPC MFF (0.7 percent w/v ) and REDKAR ('/ 01.: DEXTRAN - I-IPMC EYE DROPS AS ARTIFICIAL TEA RS 0.5 dextran 70 (0.1 per cent w/v) in borate buffer pH 8.0. 1029 ~---------------:;c------, 0.4 (B) Evaluation ol Opht/w/Illic SoluTions The prepared formulations were eva luated for the following : b .;; Clarity "§'" 'oa The ophthalmi c formu lation fill ed into vial s was observed for the presence of mctal/glass particles, precipitates, etc., against different background in a well-lit cabinet. OJ ·c t.) 0.2 y ~ O.0002x - 0.0033 0.1 R2 ~ 0.998 O ~-~---r--.---.---.-~ 500 1000 1500 2000 2500 3( -0.1 L _ _ _ _ _ _ _ _ _ _ _ _ _ _-.--J pH Adequate volume of the formulation was taken in a 50 ml beaker and the pH was recorded on a standardized Systroni c pH mcter. Specific Gravity concentration (mcglml) Figure I - Standard cur ve of de xtran 70 Content of Dextran 70 and HPMC Specific gra vity of the formulation was measured by using specific gravity bottle of 10 ml capacity using purified water as standard . We ight or 10111 101' rorlllulatioll Sp. gra vity of formul ati on = Weight of 10 1111 of purified water Viscosity Viscosity of the formulation was measured usin g Osw ald 's Vi sco meter o. 3. The viscos ity studies were ca rri ed at constant temperature of 20°C and purifi ed water was used as a sta ndard. Form ula used to determine the viscos ity was: = where 11 I = viscosity of the forll1ula l iU Il. 11 0_ = viscosity of purified water at 20" C = 1.008 6, PI =Sp. gra vity offormulati on, p ~ = Sp. :;raYlly of purified water at 20 0 C, I I = limc lakcll h) i"(lrIl1Ulali on in scconds , Jnd 12 = time l~l kcn hy purified waler ill seconds. Dextran 70 is an opticall y ac tive compound . Its optical activity was used to analyze its content. A standard curve of dextran 70 was plotted using a Carl Zeiss polarimeter ~md the percent content of dextran in the formulation was found out by ex trapolati on (Figure I). Since HPMC and dextran are carbohydrates the anth rone method was used to find out total content of ca rbohydrates in the formulation. The principl e behind anthrone reaction is th at all carbohyd rates , onlreatment with concentrated H 2S0 4 , undergo acid hyd roly sis to fo rm glucose, which on heat in g undergoes dehyd rati on and rin g formation to produ ce a i"urfu ral deri vative. Thi s derivative produ ces a co lour in the presence of anthrone, which can be detected by UV spectrophotometry. A standard curve was first establi shed usin g the same ratio of HPMC an e! dextran prese nt in th e fo rmulati on. The tOlal carbohydrate co nten t in thc formu lat ion was determin ed by ex trapolati on from th e sta ndard cur ve (Table 2). From th e total ca rbohydrate content the aillount of dex tran obta ined from polarimetry experiment was subtracted to give th e content of HPM C in the fo rmulati on. J SCI IND RES VOL. 59 DECEMBE R 2000 1030 Table 2 - Standard clirve or HPMC alld dextran HPMC Dex tran 70 Total carbohydra te ( ~ g/m l ) ~g/m l ) ( ~ g/ m l ) 42 6 48 84 12 126 Abso rba nce 0.326 Regress ion Ana lys is 96 0 .594 r = 0.9954 18 144 0.864 Slope = .005708 168 24 192 1.070 2 10 30 240 1.478 (C) Stability Studies Stability of the fo rmul ation was studied under the fo ll ow in g: Acceler ated Thermal Studies The selected fo rmul ati on of dex tran-HPMC eye drops was subj ected to stability studi es at vari ous temperatures, i.e., room temperature, 37°C and 5°C. The eye drops we re evalu ated at every 15 d fo r a peri od of six months fo r cl arity, pH, viscosity, specific gravity, per cent content of dex tran and HPMC. Light Sta bility Studies The effect of li ght on the ph ys ica l and chem ical stabili ty was examined by ex pos ing the fina l formul ati on in clear glass vi als to direc t sunli ght and checkin g fo r cl ari ty and di sco lorat ion. Autoclaving Studies To check fo r the stability of the fo rmul ati on during terminal sterilization, i.e., autoc lav in g, it was ex posed to autoc lavi ng condi tions at 12 1°C and 15 psi fo r 15,20 and 30 min , respecti vely. The hi gher time po in ts were used in an attempt to force degradation. Sterility Tes tin g The devc loped fo rmul ation was tested for the presence or absence of fungi and aerobic and anaerobic bacteria by membrane filt ration techniquc in the presence of posit ive and negative controls. Y -in tercept = 0.0404 (D) Animal Studies To determine the efficacy of the form ul ati on, studies we re carri ed out on rabbits usin g the marketed preparation MOISOL® for comparative purpose. (i) Schirmer 's Tes t " The test was performed by usi ng Whatman fil te r paper No.4. I strips of 35 mm by 5 mm dimensio n. The first 5 mm at one end of the paper stri p was bent to work as a hook for eyeli ds (Thi s porti on was not included when the length of the mo istened stri p was measured). The paper notch was in serted in to the conjunctival fo rni x fo lded, whe re it was left in pos iti on. The amoun t of wetting of stri p was measured after 5 min and the strip was re moved. Since the instill ati on of pape r may produce some reflex tears the eye was anaes theti zed by in stillin g a local anaetheti c so lu tion (xy locaine 0.5 per cent) 10 min pri or to the in se rti on of stri p. (ii) Ocular Irrita tion Studies Assessment of ocu lar irritation potenti al of ophthalmic so lu tion is an extreme ly important step in the development of ophth almic product. Six rabb its we ighin g 2-3 kg each were used for the study. Each ti me, 2 drops of the fo rmu lation were ins til led into the cul -de-sac of left eye of rabbit. Observations were made aftcr 5. 10, and IS min , respecti ve ly of instillation and eyes were observed for redness , swel lin g, wateri ng, ctc. Th e ri ght eye served as control. REDKAR el at.: DEXTR AN - HPM C EYE DROPS AS ARTI FICIAL TEA RS Results And Discussion Borax and boric acid buffer were used as the vehicle with sodium chloride as the tonicity adju stin g agent. Formul ati ons containing HPC (Formul ati ons B and E) were found to have relati ve ly hi gh viscos iti es, which made in still ati on into the eye di fficul t. Formul ation containin g onl y dex tran (Formu lati on C) had very low viscos ity, whi ch was in creased in combin ati on with HPM C. Formulation D was se lected because its vi scos ity was optimum as compared to other formulati ons. Al so, formul ation D was abl e to produce a stabl e tear film . As seen in Table I the ch osen fo rmul ati on passed the evalu ation tests for clarity, pH , viscos ity, and spec ifi c grav ity. In terms of clarity of the fo rmul ati on, pH, specific gra vity, viscosity and per cent drug content, the fo rmulati on was fo und to be stabl e durin g acce lerated therm al studi es for the test peri od of six months. It was observed th at li ght had no detrimental effect on the formul ation. It is reported th at so me fracti ons of dextran may adopt certain degree of crystallinity when stored at accelerated temperatu re. However, none of the vial s showed any type of precipi tation or fl ake fo rmati on. After autocl av in g the HPMC was fo und to precipitate at hi gh temperature. Hence the vials mu st be shaken in order to redi sperse the HPMC. At roo m temperature the HPMC goes into so luti on and vials remain clear. I Oj l The results obtained with Schirmer's test revealed that the wettin g effect by MOISOL® on eye was maintained for one-and- a-h alf hour, whereas the wettin g effect produced by deve loped formul ation was maintained fo r more th an 2 h. Al so the amoun t of wettin g was more with deve loped formul ation th an that with MOISOL®. Ocul ar irritati on studi es revea led th at none of th e rabbits showed any type of ocul ar irritati on towards fo rmulati on as well as towards MOISOL®. Conclusion Thi s study was aimed at developin g a suitab le artificial tear fo rmul ati on fo r the treatment of dry eye syndrome. An ophthalmic soluti on was prepared by usin g HPM C and dext ran po lymers, which showed better fi1m formin g properti es th an the marketed preparati on. The in stillati on of normal saline for dry eyes is of very limited va lue. Other tear substitutes include vitamin A deri vati ves, viscoelastic age nts, mucolyti c agents, and lipid containing fo rmul ati on. However, arti ficial tears, due to better tear film fo rming capac ity, ease of fo rmul ati on, ease of insti Il ation and better pati ent co mpli ance may be considered as th e mos t suitabl e treatment for dry eye syndrome. References I Kul karni S P , Bhanum alhi B , T hoppi l S O , B hoglc C P. Vanak 2 D & A min P D. Dmg Dev Illd Pha rlll , 23 ( 1997) 405. Holl y F J & L emp M A , SlI r Oplllha llll ol, 22 ( 1997) (iC) . :\ Haberg G P & Berens C, Alii } Ophthallll ol, 51 ( 1961 ) X-HJ.