Abstract:
Disclosed herein are autoclavable formulations of cyclosporin A Form 2, methods of making such formulations, and methods of treating diseases of the eye with such formulations.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This patent application claims priority to U.S. Provisional Patent Application No. 61/559,849, filed Nov. 15, 2011, the entire contents of which are hereby incorporated by reference 
     
    
     BACKGROUND 
       [0002]    Aseptic processing of cyclosporin A suspensions in a hyaluronic acid media (a hydrogel used as a suspending agent), is complicated by the fact that both the drug and the hyaluronic acid need to be pre-sterilized. Pre-sterilized hyaluronic acid is extremely expensive, costing roughly $1 million dollars for a few kilograms (roughly $10,000 per ounce) of sterile raw material. Additionally, in the process of pre-sterilizing cyclosporin A, the drug is degraded upon irradiation, as shown below and in  FIGS. 1 and 2 : 
         [0000]                                          TABLE 1                   Impact o Irradiation on Cyclosporin Stability            Sterilization   Form 1 CsA   Form 2 CsA   Form 3 CsA   Amorph, CsA       Mode   (Potency and Imp.)   (Potency and Imp.)   (Potency and Imp.)   (Potency and Imp.)               None   98.4% w/w   94.6% w/w   97.7% w/w   96.5% w/w           Total Imp: 0.6%   Total Imp: 0.6%   Total Imp: 0.6%   Total Imp: 0.7%       15 kGy Gamma   93.9% w/w   91.8% w/w   94.3% w/w   92.1% w/w           % Rel. Change: 4.5%   % Rel. Change: 2.9%   % Rel. Change: 3.6%   % Rel. Change: 4.6%           Total Imp: 1.7%   Total Imp: 1.8%   Total Imp: 1.3%   Total Imp: 1.4%       30 kGy Gamma   90.7% w/w   88.5% w/w   91.0% w/w   87.7% w/w           % Rel. Change: 7.8%   % Rel. Change: 6.4%   % Rel. Change: 6.9%   % Rel. Change: 9.2%           Total Imp: 2.8%   Total Imp: 2.4%   Total Imp: 2.3%   Total Imp: 2.3%       E-Beam   92.6% w/w   90.3% w/w   93.4% w/w   92.0% w/w           % Rel. Change: 5.9%   % Rel. Change: 4.6%   % Rel. Change: 4.5%   % Rel. Change: 4.7%           Total Imp: 1.5%   Total Imp: 1.7%   Total Imp: 1.6%   Total Imp: 1.3%                    
Cooling the cyclosporin during irradiation does not significantly improve the results, as shown in Table 2, below:
 
         [0000]                                          TABLE 2                   Impact on Cyclosporin Stabilily after irradiation under Cold Conditions            Sterilization   Form 1 CsA   Form 2 CsA   Form 3 CsA   Amorph, CsA       Mode   (Potency and Imp.)   (Potency and Imp.)   (Potency and Imp.)   (Potency and Imp.)               None   99.4% w/w   97.6% w/w   98.4% w/w   96.5% w/w           Total Imp: 0.7%   Total Imp: 0.5%   Total Imp: 0.7%   Total Imp: 0.7%       Cold E-beam   94.6% w/w   91.1% w/w   94.6% w/w   92.3% w/w           % Rel. Change: 4.8%   % Rel. Change: 6.7%   % Rel. Change: 3.9%   % Rel. Change:           Total Imp: 1.5%   Total Imp: 1.5%   Total Imp: 1.8%   4.4%                       Total Imp: 1.3%       Regular E-Beam   % Rel. Change: 5.9%   % Rel. Change: 4.6%   % Rel. Change: 4.5%   % Rel. Change:       (from Previous   Total Imp: 1.5%   Total Imp: 1.7%   Total Imp: 1.6%   4.7%       Study) % Relative               Total Imp: 1.3%       Change in                       Potency on                       Sterilization                    
Additional levels of degradants need to be qualified in preclinical safety studies. Moreover, a suspension, prepared with only 90-95% of the labeled Cyclosporin A (due to the pre-sterilization process), has a substantial probability of failure to meet regulatory guidelines for shelf-life, since regulatory authorities generally prohibit shelf-lives below 90% of label.
 
         [0003]    The present invention solves these problems. Disclosed herein are formulations of cyclosporin A, combined with a parenterally-biocompatible suspending agent, which are sterile, exceptionally stable to heat sterilization, and have excellent long-term stability. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0004]      FIGS. 1 and 2  show change in cyclosporin A potency with change in crystal form and sterilization method. 
           [0005]      FIG. 3  shows x-ray powder diffraction pattern data of cyclosporin A Form 2 after autoclaving. 
           [0006]      FIG. 4  shows congestion seen on slit lamp examination with eight different formulations. 
           [0007]      FIG. 5  depicts characteristic X-ray powder diffraction (XRPD) patterns of CsA in a new crystalline form (designated as Form 2 herein), tetragonal form (designated as Form 1 herein), and orthorhombic form (designated as Form 3 herein). 
           [0008]      FIG. 6  depicts the XRPD diffractogram of CsA crystalline Form 2. 
           [0009]      FIG. 7  depicts the water sorption/desorption profile of CsA Form 2. 
           [0010]      FIG. 8  depicts MDSC analysis of CsA Form 2 recovered from 0.04% formulation with 1% PS80. 
           [0011]      FIG. 9  shows gross ocular congestion after an injection of 100 ul of CMC, HEC, HPMC, Pluronic and PVP in phosphate buffered saline was administered subconjunctivally to New Zealand white rabbits. The rabbits were observed for seven days. 
           [0012]      FIG. 10  shows gross ocular discharge in the experiment described in  FIG. 9 . 
           [0013]      FIG. 11  shows gross ocular swelling in the experiment described in  FIG. 9 . 
           [0014]      FIG. 12  shows the simulated XRPD pattern of cyclosporine A forms. 
       
    
    
     DETAILED DESCRIPTION 
     Cyclosporin A 
       [0015]    Cyclosporin A (CsA) is a cyclic peptide having the following chemical structure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    Its chemical name is cyclo[(E)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-Lleucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl]. It is also known by the names cyclosporin, cyclosporine A, ciclosporin, and ciclosporin A. It is the active ingredient in Restasis® (Allergan, Inc., Irvine, Calif.), an emulsion comprising 0.05% (w/v) cyclosporin. Restasis® is approved in the United States to increase tear production in patients whose tear production is presumed to be suppressed due to ocular inflammation associated with keratoconjunctivitis sicca. 
       Cyclosporin A Form 2 
       [0016]    Cyclosporin A is known to exist in an amorphous form, liquid crystal form, tetragonal crystalline form (form 1), and an orthorhombic form (form 3). A new crystalline form, cyclosporin A Form 2, has recently been discovered. 
         [0017]    The XRPD pattern of CsA Form 2 differs significantly from the tetragonal form and orthorhombic form ( FIG. 1 ). The major crystalline peaks for CsA form 2 appear at (2θ) when scanned by an X-ray diffractometer with X-ray source as Cu Kα radiation, λ=1.54 Å, at 30 kV/15 mA: 7.5, 8.8, 10.2, 11.3, 12.7, 13.8, 14.5, 15.6 and 17.5 (d-spacing in crystal lattice at about 11.8, 10.0, 8.7, 7.8, 7.0, 6.4, 6.1, 5.6 and 5.1 {acute over (Å)}, respectively,  FIG. 2 ). These major peaks are defined as those being unique to Form 2 relative to the orthorhombic or tetragonal forms; as well as, peaks having an intensity greater than 5 times the background. 
         [0018]    In one embodiment, the new crystalline form (Form 2) of CsA is a nonstoichiometric hydrate of Cyclosporin A. In another embodiment, the crystalline Form 2 is represented by the formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein X is the number of molecules of water and varies from 0 to 3. In one embodiment, X in the above formula is 2. 
         [0019]    Form 2 appears to be a kinetically stable form of CsA in aqueous suspensions. Suspensions containing Form 2 show no conversion to other known polymorphic or pseudomorphic forms upon storage. It has been found that Form 1 and the amorphous form convert to Form 2 in the presence of water. 
         [0020]    The single crystal structure of the hydrate form of CsA Form 2 has been determined and the crystal structure parameters are listed in Table 2. These results indicate that Form 2 is unique compared to other known crystalline forms of cyclosporine A. 
         [0000]                              TABLE 1               Crystal data and data collection parameters of crystal       structure solution of CsA Form 2.                                formula   C 62 H 115 N 11 O 14         formula weight   1238.67       space group   P 2 1  2 1  2 1  (No. 19)       a (Å)   12.6390(5)       b (Å)   19.7582(8)       c (Å)    29.568(2)       volume (Å 3 )    7383.8(7)       Z   4       d calc (g cm −3 )   1.114       crystal dimensions (mm)   0.27 × 0.18 × 0.12       temperature (K)   150       radiation (wavelength in Å)   Cu K 2  (1.54184)       monochromator   confocal optics       linear abs coef (mm −1 )   0.640       absorption correction applied   empirical 3         transmission factors (min, max)   0.80, 0.93       diffractometer   Rigaku RAPID-II       h, k, l range   −13 to 13 −21 to 21 −32 to 21       2θ range (deg)   5.38-115.00       mosaicity (deg)   1.31       programs used   SHELXTL       F 000     2704.0       weighting   1/[σ 2 (Fo 2 ) + (0.0845P) 2  + 0.0000P]           where P = (Fo 2  + 2Fo 2 )/3       data collected   37360       unique data   9964       R int     0.077       data used in refinement   9964       cutoff used in R-factor calculations   F o   2 &gt; 2.0s(F o   2 )       data with I &gt; 2.0s(I)   6597       number of variables   834       largest shift/esd in final cycle   0.00       R(F o )   0.061       R w (F o   2 )   0.145       goodness of fit   1.037       absolute structure determination   Flack parameter b (0.0(3))                    
The asymmetric unit of this CsA Form 2 contains one cyclosporine A molecule and two water molecules. It is possible that any small molecule that can hydrogen bond to water could play the role of space filler, which would give a range of potential structures running from the orthorhombic dihydrate to distorted monoclinic dihydrate The XRPD pattern calculated from the single-crystal structure is shown in  FIG. 12  and it matches the experimental pattern shown in  FIG. 2 . These matching patterns further corroborate that Form 2 is a unique and pure crystalline form of cyclosporine A.
 
         [0021]    Without wishing to be bound by theory, thermogravimetric analysis combined with KF titration and vapor sorption desorption analysis (VSA) suggest that CsA Form 2 is a non-stoichiometric hydrate of CsA. The vapor sorption analysis of Cyclosporine Form 2 indicates that water content in the new crystal form reversibly varies with relative humidity as shown in  FIG. 7 . Similar to the tetragonal form, the new CsA form undergoes a phase transition to a liquid crystal or amorphous form at 124.4° C. prior to melting as indicated by the modulated differential calorimetric (MDSC) analysis ( FIG. 8 ). 
         [0022]    Cyclosporin A Form 2 may be obtained by suspending amorphous 0.05% cyclosporin A (w/v) in 1% Polysorbate 80, heating the solution to 65° C., holding it at that temperature for 24 hours, and then recovering the precipitate by vacuum filtration. One can then use the cyclosporin A Form 2 thus obtained to generate additional amounts, using Cyclosporin A Form 2 as a seed crystal; in this method, one suspends about 30 g cyclosporin A in a solution of 900 ml water containing 1% (w/v) Polysorbate 80, heats the solution to 65° C., and then seeds it with 0.2 g of cyclosporin A Form 2 at a temperature of 52° C. The solution is then stirred for about 22 hours at a temperature of between about 61° C. and 65° C., and then recovers the precipitate that results. 
         [0023]    Further details regarding CsA Form 2 may be found in U.S. patent application Ser. No. 13/480,710, the entire contents of which are incorporated by reference herein. 
       Heat-Stable, Heat-Sterilized Suspensions of Cyclosporin a Form 2 
       [0024]    Compositions of the invention are ophthalmically acceptable suspensions of Cyclosporin A form 2. By “ophthalmically acceptable,” the inventors mean that the suspensions are formulated in such a way as to be non-irritating when administered to the eye of a mammal, such as a human. 
         [0025]    The suspensions of the invention comprise cyclosporin A form 2 and a vehicle comprising a suspending agent such as hyaluronic acid, a cellulose, polyvinylpyrrolidone (PVP), Pluronic® copolymers based on ethylene oxide and propylene oxide, and Carbopol® polymers. 
         [0026]    In one embodiment, the suspension comprises cyclosporin A Form 2 at a concentration of about 0.001% to about 10% (w/v). In one embodiment, the suspension comprises cyclosporin A form 2 at a concentration of about 0.001% (w/v) to about 0.01%, about 0.001% (w/v) to about 0.04% (w/v), about 0.001% (w/v) to about 0.03% (w/v), about 0.001% (w/v) to about 0.02% (w/v), or about 0.001% (w/v) to about 0.01% (w/v). In another embodiment, the suspension comprises cyclosporin A form 2 at a concentration of about 0.01% (w/v) to about 0.05%, about 0.01% (w/v) to about 0.04% (w/v), about 0.01% (w/v) to about 0.03% (w/v), about 0.01% (w/v) to about 0.02% (w/v), or about 0.01% (w/v) to about 0.01% (w/v). In another embodiment, the suspension comprises cyclosporin A form 2 at a concentration of about 0.01% (w/v) to about 0.1%, about 0.1% (w/v) to about 0.5% (w/v), about 0.01% (w/v) to about 1% (w/v), or about 1% (w/v) to about 10%. 
         [0027]    For example, the suspensions may comprise about 0.001% (w/v), about 0.002% (w/v), about 0.003% (w/v), about 0.004% (w/v), about 0.005% (w/v), about 0.006% (w/v), about 0.007% (w/v), about 0.008% (w/v), about 0.009% (w/v), about 0.01% (w/v), about 0.015% (w/v), about 0.02% (w/v), about 0.025% (w/v), about 0.03% (w/v), about 0.035% (w/v), about 0.04% (w/v), about 0.045% (w/v), about 0.05% (w/v), about 0.055% (w/v), about 0.06% (w/v), about 0.065% (w/v), about 0.07% (w/v), about 0.075% (w/v), about 0.08% (w/v), about 0.085% (w/v), about 0.09% (w/v), about 0.095% (w/v), about 0.1% (w/v), about 0.15% (w/v), about 0.2% (w/v), about 0.25% (w/v), about 0.3% (w/v), about 0.35% (w/v), about 0.4% (w/v), about 0.45% (w/v), about 0.5% (w/v), about 0.55% (w/v), about 0.6% (w/v), about 0.65% (w/v), about 0.7% (w/v), about 0.75% (w/v), about 0.8% (w/v), about 0.85% (w/v), about 0.9% (w/v), about 0.95% (w/v), or about 1.0% (w/v) cyclosporin A form 2. 
         [0028]    Examples are provided in Table 3, below: 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Autoclavable suspensions of cyclosporin A Form 2. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Autoclave 
               
               
                   
                 CsA 
                   
                   
                 Gelling 
                 Conditions 
               
               
                   
                 (Crystal 
                 CsA 
                 Gelling Agent 
                 Agent 
                 (Temp 
               
               
                 Formulation 
                 form) 
                 (%) 
                 (Type) 
                 (%) 
                 (° C.)/min) 
               
               
                   
               
             
          
           
               
                 1 
                 2 
                 20 
                 CMC 
                 5 
                 121/10 
               
               
                 2 
                 3 
                 20 
                 CMC 
                 3 
                 121/10 
               
               
                 3 
                 NA 
                 0 
                 Carbopol Ultrez 
                 1.5 
                 121/15 
               
               
                   
                   
                   
                 10 
                   
                   
               
               
                 4 
                 NA 
                 0 
                 Carbopol Ultrez 
                 2.0 
                 121/15 
               
               
                   
                   
                   
                 10 
                   
                   
               
               
                 5 
                 NA 
                 0 
                 Carbopol Ultrez 
                 2.5 
                 121/15 
               
               
                   
                   
                   
                 10 
                   
                   
               
               
                 6 
                 NA 
                 0 
                 Carbopol Ultrez 
                 1.0 
                 121/15 
               
               
                   
                   
                   
                 10 
                   
                   
               
               
                 7 
                 NA 
                 0 
                 Carbopol Ultrez 
                 4.0 
                 121/15 
               
               
                   
                   
                   
                 10 
                   
                   
               
               
                 8 
                 2 
                 5 
                 CMC 
                 3 
                 121/15 
               
               
                 9 
                 2 
                 5 
                 CMC 
                 2 
                 121/15 
               
               
                 10 
                 2 
                 20 
                 CMC 
                 10 
                 121/15 
               
               
                 11 
                 2 
                 0 
                 CMC 
                 10 
                 121/15 
               
               
                 12 
                 2 
                 5 
                 HPMC 
                 3 
                 121/15 
               
               
                 13 
                 2 
                 5 
                 HPMC 
                 6 
                 121/15 
               
               
                 14 
                 2 
                 20 
                 HPMC 
                 6 
                 121/15 
               
               
                 15 
                 2 
                 20 
                 HPMC 
                 10 
                 121/15 
               
               
                 16 
                 2 
                 5 
                 HPMC 
                 6 
                 121/15 
               
               
                 17 
                 2 
                 20 
                 HPMC 
                 3 
                 121/15 
               
               
                 18 
                 2 
                 5 
                 HPMC 
                 3 
                 121/15 
               
               
                 19 
                 2 
                 20 
                 HPMC 
                 3 
                 121/15 
               
               
                 20 
                 2 
                 10 
                 HPMC 
                 4.5 
                 121/15 
               
               
                 21 
                 2 
                 10 
                 HPMC 
                 4.5 
                 121/15 
               
               
                 22 
                 2 
                 10 
                 HEC 
                 3 
                 121/15 
               
               
                 23 
                 2 
                 10 
                 HEC 
                 3 
                 121/15 
               
               
                 24 
                 2 
                 30 
                 HEC 
                 1 
                 121/15 
               
               
                 25 
                 2 
                 10 
                 HA 
                 3.5 
                  121/15* 
               
               
                 26 
                 2 
                 10 
                 HA 
                 2.5 
                 121/15 
               
               
                 27 
                 2 
                 30 
                 HEC 
                 1 
                 121/15 
               
               
                 28 
                 2 
                 30 
                 HA 
                 1 
                  121/15* 
               
               
                 29 
                 2 
                 10 
                 HA 
                 2.5 
                 121/15 
               
               
                 30 
                 2 
                 10 
                 HA 
                 3.5 
                 121/15 
               
               
                 31 
                 2 
                 10 
                 HA 
                 4.5 
                 121/15 
               
               
                 32 
                 2 
                 30 
                 HA 
                 3.0 
                 121/15 
               
               
                 33 
                 2 
                 20 
                 HA 
                 1.5 
                 121/15 
               
               
                 34 
                 2 
                 20 
                 HA 
                 2.5 
                 121/15 
               
               
                 35 
                 2 
                 20 
                 HA 
                 3.5 
                 121/15 
               
               
                 36 
                 2 
                 10 
                 HA 
                 4 
                 121/15, 
               
               
                   
                   
                   
                   
                   
                 121/30, and 
               
               
                   
                   
                   
                   
                   
                 123/15 
               
               
                 37 
                 2 
                 10 
                 HA 
                 4 
                 121/15, 
               
               
                   
                   
                   
                   
                   
                 121/30, and 
               
               
                   
                   
                   
                   
                   
                 123/15 
               
               
                 38 
                 2 
                 10 
                 HA 
                 4 
                 121/15, 
               
               
                   
                   
                   
                   
                   
                 121/30, and 
               
               
                   
                   
                   
                   
                   
                 123/15 
               
               
                 39 
                 2 
                 35 
                 HA 
                 1 
                  121/15* 
               
               
                 40 
                 2 
                 5 
                 HA 
                 3.5 
                  121/15* 
               
               
                 41 
                 2 
                 10 
                 HA 
                 3.5 
                  121/15* 
               
               
                 42 
                 2 
                 20 
                 HA 
                 2.0 
                  121/15* 
               
               
                 43 
                 2 
                 20 
                 HA 
                 2.0 
                  121/15* 
               
               
                 44 
                 2 
                 10 
                 HA 
                 3.5 
                  121/15* 
               
               
                 45 
                 2 
                 10 
                 HA 
                 3.5 
                  121/15* 
               
               
                 46 
                 2 
                 25 
                 N/A 
                 0 
                 120/15 
               
               
                 47 
                 2 
                 25 
                 N/A 
                 0 
                 118/20 
               
               
                 48 
                 2 
                 25 
                 N/A 
                 0 
                 120/12 
               
               
                 HEC1 
                 2 
                 5 
                 HEC 
                 5 
                 121/15 
               
               
                 HEC2 
                 2 
                 20 
                 HEC 
                 5 
                 121/15 
               
               
                 HEC3 
                 2 
                 5 
                 HEC 
                 2 
                 121/15 
               
               
                 HEC4 
                 2 
                 20 
                 HEC 
                 2 
                 121/15 
               
               
                 HEC5 
                 2 
                 5 
                 HEC 
                 5 
                 121/15 
               
               
                 HEC6 
                 2 
                 20 
                 HEC 
                 5 
                 121/15 
               
               
                 HEC7 
                 2 
                 5 
                 HEC 
                 2 
                 121/15 
               
               
                 HEC8 
                 2 
                 20 
                 HEC 
                 2 
                 121/15 
               
               
                 HEC9 
                 2 
                 10 
                 HEC 
                 3 
                 121/15 
               
               
                 PVP1 
                 2 
                 10 
                 PVP 
                 25 
                 121/15 
               
               
                 PVP2 
                 2 
                 10 
                 PVP 
                 25 
                 121/15 
               
               
                 PVP3 
                 2 
                 10 
                 PVP 
                 15 
                 121/15 
               
               
                 PVP4 
                 2 
                 10 
                 PVP 
                 15 
                 121/15 
               
               
                 PVP5 
                 2 
                 25 
                 PVP 
                 25 
                 121/15 
               
               
                 PVP6 
                 2 
                 25 
                 PVP 
                 25 
                 121/15 
               
               
                 PVP7 
                 2 
                 25 
                 PVP 
                 15 
                 121/15 
               
               
                 PVP8 
                 2 
                 25 
                 PVP 
                 15 
                 121/15 
               
               
                 PVP9 
                 2 
                 10 
                 PVP 
                 25 
                 121/15 
               
               
                 PVP10 
                 2 
                 25 
                 PVP 
                 25 
                 121/15 
               
               
                   
               
               
                 CsA = cyclosporin A. 
               
               
                 CMC = carboxymethyl cellulose. 
               
               
                 HPMC = hydroxypropyl methyl cellulose. 
               
               
                 HEC = hydroxyethyl cellulose. 
               
               
                 HA = hyaluronic acid. 
               
               
                 PVP = polyvinylpyrrolidone. 
               
               
                 *= slurry autoclaved prior to addition of gelling agent. 
               
             
          
         
       
     
       Methods of Preparation 
       [0029]    Suspensions of the invention contain cyclosporin A Form 2 and a suspending agent. In another embodiment, the suspension also contains one or more of water, buffer, and salt, in sufficient quantities to provide a biocompatible formulation. By “biocompatible,” the inventors mean that the suspension is appropriate for administration to the eye (for example, by parenteral administration). 
         [0030]    The formulations of the invention may be manufactured by using either a heat-sterilized slurry of Form 2 cyclosporin mixed aseptically with a sterile parenterally-biocompatible suspending agent and other excipient; or by combining Form 2 cyclosporin with a parenterally-biocompatible suspending agent and other excipients and heat sterilizing the entire formulation. 
         [0031]    These methods address various important problems with cyclosporin formulation: 1) solid cyclosporin cannot be pre-sterilized by irradiation without significant drug degradation and formation of degradation products; 2) sterile filtration is also not feasible because the formulation is a suspension; and 3) terminal sterilization by heat will decrease gel viscosity. Also, in one embodiment, the final viscosity of the drug formulation is sufficiently high to keep the cyclosporin suspended throughout the product&#39;s shelf-life. In another embodiment, the viscosity is sufficiently low to permit the final formulation to flow through a narrow gauge syringe, such as a 22, 23, 24, 25, or 26 gauge needle or narrower. In still another embodiment, the formulation is sufficiently high to keep the cyclosporin suspended throughout the product&#39;s shelf-life, and also sufficiently low to permit the final formulation to flow through a syringe with a 22, 23, 24, 25, or 26 gauge needle or narrower. 
         [0032]    Methods 1 and 2, below, use hyaluronic acid as the suspending agent but, other suitable suspending agents may be substituted. 
         [0033]    It should be noted that sterile hyaluronic acid is very expensive and that method 2 provides a unique method of sterilization, which allows the use of non-sterile hyaluronic acid by heat-reducing the polymer to the correct molecular weight range, so that it reaches the target viscosity range. Method 2, therefore, requires precision manufacturing, where each new lot of hyaluronic acid may shift to a different viscosity range, under identical manufacturing conditions. Consequently, in order to assure the correct viscosity range is reached in every commercial batch, the heat cycle will need to be adaptive—that is—adjusted according to a set of guidelines and experiments on the raw material lot prior to manufacture of the drug product. 
         [0034]    Furthermore, it should be noted that Method 2 prepares all steps of the formulation in a single vessel. These two methods allow for the rapid production of the drug product and consequently, have substantial value in saving one day or more of valuable manufacturing time over Method 1. 
         [0035]    These methods depend on the inventors&#39; surprising discovery that cyclosporin A Form 2 may be autoclaved and still retain its potency and stability. Other forms of cyclosporin—amorphous, Form 1 and Form 3—cannot be autoclaved, without unacceptable loss of drug substance from the suspension. 
       Method 1—Aqueous Slurry Method 
       [0036]    The appropriate amount of cyclosporin A Form 2 is suspended and mixed in phosphate buffered saline solution and the slurry is heat sterilized by autoclave. In an aseptic environment, the appropriate amount of pre-sterilized hyaluronic acid is added to the sterile cyclosporin slurry, is mixed, and then dissolved. The drug product is brought to volume with sterile water for injection. The final product has a viscosity in the correct range to create a long-term stable suspension, while allowing the final formulation to flow through a syringe fitted with a narrow-gauge needle, such as 25 gauge needle or narrower. 
       Method 2—Single Vessel Method 
       [0037]    An excess of non-sterile hyaluronic acid is dissolved in phosphate buffered saline solution. Cyclosporin A Form 2 is suspended and mixed. The resulting suspension formulation is heat-sterilized by autoclave (using an “adaptive” heat cycle), at the appropriate temperature and for the appropriate amount of time, to both sterilize the formulation and bring the viscosity into the desired range. 
         [0038]    For parenteral formulations, it may be desirable to achieve a viscosity that is sufficiently high to keep the cyclosporin suspended throughout the product&#39;s shelf-life, and also sufficiently low to permit the final formulation to flow through a syringe with a 22, 23, 24, 25, or 26 gauge needle or narrower. While hydrogel solutions are generally recognized as safe for topical use, very few have been used for parenteral administration, and none have been demonstrated to be safely injected through a 25 gauge needle (or narrower) into subconjunctival tissue at high hydrogel concentrations. A high concentration of suspending agent (up to 25%) is necessary in order to maintain the suspendability of the 5-40% cyclosporin parenteral formulations described herein. In one embodiment, parenteral formulations for use in subconjunctival tissue are (1) injectable through a narrow-gauge needle, such as 25 gauge or narrower, in order to minimize tissue damage by the needle, to allow for quick healing of the needle entry-point, and to limit the back-flow of the injected formulation; (2) sterile; (3) biocompatible; and (4) sufficiently viscous to maintain suspendability throughout the shelf-life of the formulation and to prevent tissue reflux out of the subconjunctival space. In such formulations viscosity is sufficiently high to retain long-term suspendability of the drug but sufficiently low to allow the entire formulation to readily pass through a narrow gauge needle. 
         [0039]    In one embodiment of the invention, the formulations have a very high viscosity (e.g., ≧100,000 cps) yet may still able to be injected out of syringe through a narrow-gauge needle. The following table gives examples of such formulations. 
         [0000]    
       
         
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Formulation 
               
             
          
           
               
                   
                 5% CsA, 3.5% HA 
                 10% CsA, 3.5% HA 
                 20% CsA, 2.0% HA 
               
               
                   
                 (10203X) 
                 (10204X) 
                 (10205X) 
               
               
                   
                 Viscosity: TBD 
                 Viscosity: 1,300,000 cps 
                 Viscosity: 700,000 cps 
               
               
                   
                   
               
             
          
           
               
                 Needle size 
                 BD 
                 TSK Steriject 
                 BD 
                 TSK Steriject 
                 BD 
                 TSK 
               
               
                 and type 
                 Precision 
                 27G × 0.5″ 
                 Precision 
                 27G × 0.5″ 
                 Precision 
                 Steriject 
               
               
                   
                 Glide 
                 UTW (Ultra 
                 Glide 
                 UTW (Ultra 
                 Glide 
                 27G × 0.5″ 
               
               
                   
                 27G × 0.5″ 
                 Thin Wall) 
                 27G × 0.5″ 
                 Thin Wall) 
                 27G × 0.5″ 
                 UTW (Ultra 
               
               
                   
                 Needle 
                 Needle 
                 Needle 
                 Needle 
                 Needle 
                 Thin Wall) 
               
               
                   
                   
                   
                   
                   
                   
                 Needle 
               
               
                 Injectabiltiy 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
               
               
                   
               
             
          
         
       
     
       Methods of Treatment 
       [0040]    Compositions of the invention may be used to treat any condition of the eye which is known to be amenable to topical treatment with cyclosporin A (such as with Restasis®) at the concentrations stated here. For example, compositions of the invention may be used to treat patients suffering from dry eye, to treat blepharitis and meibomian gland disease, to restore corneal sensitivity that has been impaired due to refractive surgery on the eye, to treat allergic conjunctivitis and atopic and vernal keratoconjunctivitis, and to treat ptyregia, conjunctival and corneal inflammation, keratoconjuntivitis, graft versus host disease, post-transplant glaucoma, corneal transplants, mycotic keratitis, Thygeson&#39;s superficial punctate keratitis, uveitis, and Theodore&#39;s superior limbic keratoconjunctivitis, among other conditions. 
         [0041]    The International Dry Eye Workshop (DEWS) defines dry eye as “a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface, accompanied by increased osmolarity of the tear film and inflammation of the ocular surface.” It includes those conditions, such as keratoconjunctivitis sicca, that are caused by tear deficiency or excessive evaporation of tears. 
         [0042]    Blepharitis is a chronic disorder producing inflammation of the anterior and posterior lid margin, with involvement of skin and its related structures (hairs and sebaceous glands), the mucocutaneous junction, and the meibomian glands. It can also affect the conjunctiva, tear film, and the corneal surface in advanced stages and may be associated with dry eye. Blepharitis is commonly classified into anterior or posterior blepharitis, with anterior affecting the lash bearing region of the lids, and posterior primarily affecting the meibomian gland orifices. 
         [0043]    Meibomian gland disease most often occurs as one of three forms: primary meibomitis, secondary meibomitis, and meibomian seborrhea. Meibomian seborrhea is characterized by excessive meibomian secretion in the absence of inflammation (hypersecretory meibomian gland disease). Primary meibomitis, by contrast, is distinguished by stagnant and inspissated meibomian secretions (obstructive hypersecretory meibomian gland disease). Secondary meibomitis represents a localized inflammatory response in which the meibomian glands are secondarily inflamed in a spotty fashion from an anterior lid margin blepharitis. 
         [0044]    Impaired corneal sensitivity often occurs after refractive surgery, such as photorefractive keratectomy, laser assisted sub-epithelium keratomileusis (LASEK), EPI-LASEK, customized transepithelial non-contact ablation, or other procedures in which the corneal nerves are severed. Impaired corneal sensitivity may also occur after viral infection, such as by HSV-1, HSV-2, and VZV viruses. Patients with impaired corneal sensitivity often complain that their eyes feel dry, even though tear production and evaporation may be normal, suggesting that “dryness” in such patients is actually a form of corneal neuropathy that results when corneal nerves are severed by surgery or inflamed after viral infection. 
         [0045]    Allergic conjunctivitis is an inflammation of the conjunctiva resulting from hypersensitivity to one or more allergens. It may be acute, intermittent, or chronic. It occurs seasonally, that is, at only certain time of the year, or it occurs perennially, that is, chronically throughout the year. Symptoms of seasonal and perennial allergic conjunctivitis include, in addition to inflammation of the conjunctiva, lacrimation, tearing, conjunctival vascular dilation, itching, papillary hyperlasia, chemosis, eyelid edema, and discharge from the eye. The discharge may form a crust over the eyes after a night&#39;s sleep. 
         [0046]    Atopic keratoconjunctivitis is a chronic, severe form of allergic conjunctivitis that often leads to visual impairment. Symptoms include itching, burning, pain, redness, foreign body sensation, light sensitivity and blurry vision. There is often a discharge, especially on awakening from a night&#39;s sleep; the discharge may be stringy, ropy, and mucoid. The lower conjunctiva is often more prominently affected than the upper conjunctiva. The conjunctiva may range from pale, edematous, and featureless to having the characteristics of advanced disease, including papillary hypertrophy, subepithelial fibrosis, formix foreshortening, trichiasis, entropion, and madurosis. In some patients the disease progresses to punctate epithelial erosions, corneal neovascularization, and other features of keratopathy which may impair vision. There is typically goblet cell proliferation in the conjunctiva, epithelial pseudotubular formation, and an increased number of degranulating eosinophils and mast cells in the epithelium. CD25+T lymphocytes, macrophages, and dendritic cells (HLA-DR.sup.+, HLA-CD1+) are significantly elevated in the substantia propria. 
         [0047]    Like atopic keratoconjunctivitis, vernal keratoconjunctivitis is a severe form of allergic conjunctivitis, but it tends to affect the upper conjunctiva more prominently than the lower. It occurs in two forms. In the palpebral form, square, hard, flattened, closely packed papillae are present; in the bulbar (limbal) form, the circumcorneal conjunctiva becomes hypertrophied and grayish. Both forms are often accompanied by a mucoid discharge. Corneal epithelium loss may occur, accompanied by pain and photophobia, as may central corneal plaques and Trantas&#39; dots. 
       EXAMPLES 
       [0048]    The invention is further illustrated by the following examples. 
         [0049]    When the inventors autoclaved aqueous suspensions of cyclosporin A, the drug particles aggregated, making the product unacceptable. Additionally, the inventors found that hyaluronic acid also degrades upon autoclaving, causing a marked drop in viscosity. Lower viscosity, in turn, reduces the suspendability of the drug particles and causes them to settle. Formulations having drug particles in suspension that too rapidly settle, or irreversibly settle, may be useful for laboratory tests, but are not commercially viable. 
         [0050]    The inventors explored formulations of four cyclosporin A polymorphic forms, the amorphous form, the tetragonal crystalline form (form 1), the orthorhombic form (form 3), and cyclosporin A Form 2. 
         [0051]    A suspension of form 1 converts to the amorphous form and aggregates upon autoclaving; clumping of the cyclosporin is also observed. Consequently, neither form 1 nor the amorphous form is suitable for autoclave stabilization. Furthermore, an autoclaved suspension of F3 in water lost 11-28% of its potency during autoclaving (Table 4); this, too, is unacceptable. In contrast, a suspension of Form 2 in water was quite stable to autoclaving, resisting degradation when compared to a pre-sterilization control. X-ray analysis of filtered solid from the Form 2 formulation also confirms that Form 2 is polymorphically stable to autoclaving ( FIG. 3 ). These latter two findings are extremely surprising, considering the lack of either chemical or polymorphic stability of the other three forms. 
         [0052]    The inventors explored the autoclavability of a series of concentrated solutions of various polymers (no drug) which, when loaded in a syringe, will flow through a narrow-gauge needle (25 gauge or narrower). The polymers evaluated were as follows: cross-linked hyaluronic acid (Juvederm®), carbomer, carboxymethylcellulose-medium molecular weight, carboxymethylcellulose-high molecular weight, hydroxyethylcellulose, hydroxypropylcellulose, Pluronic F127 and polyvinylpyrrolidone K90. All of these are readily available from commercial suppliers. 
         [0053]    One hundred microliters of each of the autoclaved solutions was injected into rabbit conjunctiva, in order to evaluate the propensity for causing inflammation. Those polymers producing an inflammatory reaction were eliminated from consideration ( FIG. 4 , carbomer, both CMC&#39;s, and HPMC were eliminated). Additionally, Juvederm® was eliminated because it formed a long-lasting bleb which, in humans, might cause irritation as the eyelid moves over the site of injection. Both HPMC and Pluronic separated from the solution during/after autoclaving and consequently were also eliminated. Of the commercially viable hydrogels, only HEC and PVP demonstrated that they produced no inflammation in rabbit conjunctiva after autoclaving. These two hydrogels were used to formulate cyclosporin A suspensions for further evaluation. The results of the studies are shown in Table 5. 
         [0054]    Initially, the inventors explored the possibility of heat-sterilizing a slurry of cyclosporin A of Form 1 (which converts to the amorphous form). This approach resulted in agglomeration of the drug and consequently, the formulation was not viable. Further studies, adding PVP to suppress the agglomeration of Form 1/amorphous form, also failed. 
         [0055]    Since heat-sterilization of an aqueous suspension of cyclosporin did not appear to be viable, the inventors planned to prepare suspensions by aseptic technique, using pre-sterilize solid cyclosporin. Various solid cyclosporins (Forms 1, 2, and 3 and amorphous) were treated with gamma or e-beam irradiation. In all cases, significant loss of drug (3-9%) occurred ( FIG. 2  and Table 1). Furthermore, the substantial loss of drug indicates that high levels of degradation products (around 3-9%) are generated in the irradiation-sterilized material. These impurities may have negative toxicological and/or regulatory implications; consequently, this approach to sterilization appears to be undesirable. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Effect of Irradiation Sterilization on Cyclosporin (CsA) Drug Substance (solid) 
               
             
          
           
               
                 Sterilization 
                 Form 1 CsA 
                 Form 2 CsA 
                 Form 3 CsA 
                 Amorph. CsA 
               
               
                 Mode 
                 (Potency and Imp.) 
                 (Potency and Imp.) 
                 (Potency and Imp.) 
                 (Potency and Imp.) 
               
               
                   
               
               
                 None 
                 98.4% w/w 
                 94.6% w/w 
                 97.7% w/w 
                 96.5% w/w 
               
               
                   
                 Total Imp: 0.6% 
                 Total Imp: 0.6% 
                 Total Imp: 0.8% 
                 Total Imp: 0.7% 
               
               
                 15 kGy Gamma 
                 93.9% w/w 
                 91.8% w/w 
                 94.3% w/w 
                 92.1% w/w 
               
               
                   
                 % Rel. Change: 
                 % Rel. Change: 2.9% 
                 % Rel. Change: 3.6% 
                 % Rel. Change: 
               
               
                   
                 4.5% 
                 Total Imp: 1.8% 
                 Total Imp: 1.3% 
                 4.6% 
               
               
                   
                 Total Imp: 1.7% 
                   
                   
                 Total Imp: 1.4% 
               
               
                 33 kGy Gamma 
                 90.7% w/w 
                 88.5% w/w 
                 91.0% w/w 
                 87.7% w/w 
               
               
                   
                 % Rel. Change: 
                 % Rel. Change: 6.4% 
                 % Rel. Change: 6.9% 
                 % Rel. Change: 
               
               
                   
                 7.8% 
                 Total Imp: 2.4% 
                 Total Imp: 2.3% 
                 9.2% 
               
               
                   
                 Total Imp: 2.8% 
                   
                   
                 Total Imp: 2.3% 
               
               
                 E-Beam 
                 92.6% w/w 
                 90.3% w/w 
                 93.4% w/w 
                 92.0% w/w 
               
               
                   
                 % Rel. Change: 
                 % Rel. Change: 4.6% 
                 % Rel. Change: 4.5% 
                 % Rel. Change: 
               
               
                   
                 5.9% 
                 Total Imp: 1.7% 
                 Total Imp: 1.6% 
                 4.7% 
               
               
                   
                 Total Imp: 1.5% 
                   
                   
                 Total Imp: 1.3% 
               
               
                   
               
             
          
         
       
     
         [0056]    Subsequently, the inventors attempted to irradiate solid cyclosporin (Forms 1, 2, and 3 and amorphous), under the best conditions above, at cold temperatures. No significant improvement was noted with any of the Forms of cyclosporin (Table 2). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Effect of E-Beam Sterilization of Cyclosporins under Cold Conditions 
               
             
          
           
               
                 CsA Drug 
                   
                   
                   
                   
               
               
                 Substance Sample 
                 CsA Potency for 
                 CsA Potency 15 kGy 
                 CsA Potency 30 kGy Gamma 
                 CsA Potency 
               
               
                 Treatment 
                 Control Sample 
                 Gamma Treatment 
                 Treatment 
                 E-Beam 15 kGyTreatment 
               
               
                   
               
               
                 Dry Ice 
                 99.2% w/w 
                 96.7% w/w 
                 93.8% w/w 
                 93.8% w/w 
               
               
                   
                   
                 (% Rel. Change: 2.5%) 
                 (% Rel. Change: 5.4%) 
                 (% Rel. Change: 5.4%) 
               
               
                 Cold Pack 
                 96.5% w/w 
                 93.0% w/w 
                 92.1% w/w 
                 93.2% w/w 
               
               
                   
                   
                 (% Rel. Change: 3.6%) 
                 (% Rel. Change: 4.6%) 
                 (% Rel. Change: 3.4%) 
               
               
                   
               
             
          
         
       
     
         [0057]    After it became apparent that irradiation of solid cyclosporins produced too much degradation, the inventors attempted to irradiate an aqueous suspension of cyclosporin, using hyaluronic acid as a suspending agent. This approach resulted in 4-10% degradation of the drug within the formulation. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effect of Sterilization by Irradiation on Aqueous Suspensions of Cyclosporin [CsA] using 
               
               
                 Hyaluronic Acid [HA] as a Suspending Agent, at Various Temperatures 
               
             
          
           
               
                   
                 CsA Potency for 
                 CsA Potency Post- 
                 % Relative Change in 
               
               
                 Sterilization Treatment 
                 Control Sample 
                 Sterilization 
                 Potency 
               
               
                   
               
               
                 Cold Pack Control CsA 
                 103.2% w/w 
                 Not Applicable 
                 Not Applicable 
               
               
                 Hydrogel Sample 
                   
                   
                   
               
               
                 CsA-HA Sample (Cold Pack) 
                 103.2% w/w 
                 98.9% w/w 
                  4.2% 
               
               
                 Treated with 15 kGy Gamma 
                   
                   
                   
               
               
                 CsA-HA Sample (Cold Pack) 
                 103.2% w/w 
                 92.3% w/w 
                 10.6% 
               
               
                 Treated with 30 kGy Gamma 
                   
                   
                   
               
               
                 CsA-HA Sample (Cold Pack) 
                 103.2% w/w 
                 92.8% w/w 
                 10.1% 
               
               
                 Treated with E-Beam (15 kGy) 
               
               
                   
               
             
          
         
       
     
         [0058]    Finally, the inventor turned their focus on steam sterilization of slurries and full formulations of cyclosporins. Slurries of Form 1 (which converts to amorphous) agglomerate during heat-sterilization. Slurries of Form 3, while physically stable and more chemically stable than Form 1, degraded significantly during heat sterilization. But, to the inventors&#39; surprise, slurries of Form 2 were both physically and chemically stable (Tables 4 and 5). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Heat-Sterilization of Slurries of Cyclosporin (ScA) Form 2 (F-2) in Water 
               
             
          
           
               
                   
                 CsA-F2 Slurry % 
                 CsA-F3 Slurry % 
               
               
                   
               
             
          
           
               
                   
                 Initial 
                 96.86 
                 101.41 
               
               
                   
                 120 C. 15 min 
                 96.88 
                 88.61 
               
               
                   
                 108 C. 60 min 
                 106.69 
                 71.72 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Physical Stabiltiy of Forms 2 and 3 Before and After Heat Sterilization 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Formulation 
                 Material 
                 Spec. 
                 D90 
                 D50 
                 D10 
                 Conditions 
               
               
                   
               
               
                 A 
                 CsA-F2 
                 Slurry 
                 198.6313 
                 116.8544 
                 8.2711 
                 Slurry control for steam 
               
               
                   
                   
                 control 
                   
                   
                   
                 sterilization study 
               
               
                 A, autoclaved 
                 CsA-F2 
                 Autoclaved 
                 186.4431 
                 99.902 
                 7.0518 
                 Autoclaved at 120 C. for 15 
               
               
                   
                   
                 slurry 
                   
                   
                   
                 minutes 
               
               
                 A, autoclaved 
                 CsA-F2 
                 Autoclaved 
                 195.603 
                 112.532 
                 9.209 
                 Autoclaved at 108 C. for 60 
               
               
                   
                   
                 slurry 
                   
                   
                   
                 minutes 
               
               
                 B 
                 CsA-F3 
                 Slurry 
                 110.8281 
                 63.3348 
                 7.1711 
                 Slurry control for steam 
               
               
                   
                   
                 control 
                   
                   
                   
                 sterilization study 
               
               
                 B, autoclaved 
                 CsA-F3 
                 Autoclaved 
                 116.8761 
                 67.523 
                 12.1564 
                 Autoclaved at 120 C. for 15 
               
               
                   
                   
                 slurry 
                   
                   
                   
                 minutes 
               
               
                 B, autoclaved 
                 CsA-F3 
                 Autoclaved 
                 115.556 
                 65.3309 
                 10.5518 
                 Autoclaved at 108 C. for 60 
               
               
                   
                   
                 slurry 
                   
                   
                   
                 minutes 
               
               
                   
               
             
          
           
               
                   
                   
                   
                   
                 % potency 
               
               
                   
                   
                   
                   
                 compared to CsA 
               
               
                   
                 Formulation 
                 Material 
                 Conditions 
                 Form 2 standard 
               
               
                   
                   
               
               
                   
                 A 
                 CsA-F2 
                 Control 
                 96.9 
               
               
                   
                 A 
                 CsA-F2 
                 120° C., 15 min 
                 96.9 
               
               
                   
                 A 
                 CsA-F2 
                 108° C., 60 min 
                 106.7 
               
               
                   
                 B 
                 CsA-F3 
                 Control 
                 101.4 
               
               
                   
                 B 
                 CsA-F3 
                 120° C., 15 min 
                 88.6 
               
               
                   
                 B 
                 CsA-F3 
                 108° C., 60 min 
                 71.7 
               
               
                   
                   
               
             
          
         
       
     
       Ocular Congestion 
       [0059]    Parenterally-biocompatible suspending agents were identified by injecting sterile concentrated solutions into the subconjunctival space and evaluating the toxicological response. An injection of 100 ul of the following polymers in phosphate buffered saline was administered subconjunctivally to New Zealand white rabbits and observed for a period of seven days. 
         [0060]    2% Carbomer (Carbopol Ultrez 10NF, Lubrizol) 
         [0061]    8% Carboxymethyl Cellulose (low viscosity CMC, Lubrizol) 
         [0062]    6% Carboxymethyl Cellulose (high viscosity CMC, Lubrizol) 
         [0063]    6% HEC (Ashland) 
         [0064]    6% HPMC (Dow Chemical) 
         [0065]    Juvederm Ultra (Allergan, Inc) 
         [0066]    Pluronic F127 (BASF) 
         [0067]    Polyvinyl pyrrolidone (PVP K90, BASF) 
         [0000]                                                                                            tech               Alternative               type   name   source   Lot#   info   vendor   CoA   Grade   vendor   Grade               1   PVP   PVP K30   Sigma_Aldrich   BCBB7859   Mw 40K   Sigma_Aldrich   yes       BASF   PHEUR/                   81420-500G (or       (PSO:                   USP/                   PSO R14247)       5% in                   NF/JP                           water,                           pH 3.6)       2   PVP   PVP K90   Sigma_Aldrich   BCBB3954   Mw 360K   Sigma_Aldrich   yes       BASF   PHEUR/                   81440-250G                           USP/                                               NF/JP       3   PVP   PVP 10   Sigma-Aldrich   050M0039   Mw 10K   Sigma_Aldrich   yes       BASF   PHEUR/                   PVP10-500G                           USP/NF       4   HPMC   Hypromellose   PSO PM#   XB14012N11   Sigma   Dow   yes   USP/               (tested to JP)   1018       H3785:   Chemical       PHEUR                   (R19424)       4000 cP,                           2% in water       5   CMC   Carboxy   PSO   96413                   CMC from               methyl   R19716Q                       Ashland/               cellulose   pending                       Aqualon is               sodium                           NF/USP,       6   CMC   Carboxy   PSO   96077               methyl   R19717               cellulose               sodium       7   Hydroxyethyl   Natrosol   Kevin   F0854   Type 250-   Ashland           HEC from           cellulose   (Type   Warner       HHX pharm               Ashland./           (HEC)   250-HHX                           Aqualon is               pharm)                           USP/EP,       8   Acrylate/C10-   Carbopol   Kevin   EC742EK343   acrylate   Lubrizol       USP/           30 Alkyl   ETD   Warner       crosspolymer           NF           acrylate   2020NF           (Viscosity,                           47-77K cP                           0.5% wt at                           pH 7.5)       9   Carbomer   Carbopol   Kevin   CC83RZG726   type A   Lubrizol       USP/           Interpolymer   Ultrez 10   Warner       (Viscosity,           NF               NF           45-65K cP               polymer           0.5% wt at                           pH 7.5)       10   Carbomer-   Carbopol   Kevin   EC863CC625   type C   Lubrizol       USP/           Homopolymer   980 NF   Warner       (Viscosity,           PHEUR/               polymer           40-60K cP           JPE                           0.5% wt at                           pH 7.5)                                   tech               Alternative           type   name   source   Lot#   info   vendor   CoA   Grade   vendor   Grade               1   PVP   PVP K30   Sigma_Aldrich   BCBB7859   Mw 40K   Sigma_Aldrich   yes       BASF   PHEUR/                   81420-500G (or       (PSO:                   USP/                   PSO R14247)       5% in                   NF/JP                           water,                           pH 3.6)       2   PVP   PVP K90   Sigma_Aldrich   BCBB3954   Mw 360K   Sigma_Aldrich   yes       BASF   PHEUR/                   81440-250G                           USP/                                               NF/JP       3   PVP   PVP 10   Sigma-Aldrich   050M0039   Mw 10K   Sigma_Aldrich   yes       BASF   PHEUR/                   PVP10-500G                           USP/NF       4   HPMC   Hypromellose   PSO PM#   XB14012N11   Sigma   Dow   yes   USP/               (tested to JP)   1018       H3785:   Chemical       PHEUR                   (R19424)       4000 cP,                           2% in water       5   CMC   Carboxy   PSO   96413                   CMC from               methyl   R19716Q                       Ashland/               cellulose   pending                       Aqualon is               sodium                           NF/USP,       6   CMC   Carboxy   PSO   96077               methyl   R19717               cellulose               sodium       7   Hydroxyethyl   Natrosol   Kevin   F0854   Type 250-   Ashland           HEC from           cellulose   (Type   Warner       HHX pharm               Ashland./           (HEC)   250-HHX                           Aqualon is               pharm)                           USP/EP,       8   Acrylate/C10-   Carbopol   Kevin   EC742EK343   acrylate   Lubrizol       USP/           30 Alkyl   ETD   Warner       crosspolymer           NF           acrylate   2020NF           (Viscosity,                           47-77K cP                           0.5% wt at                           pH 7.5)       9   Carbomer   Carbopol   Kevin   CC83RZG726   type A   Lubrizol       USP/           Interpolymer   Ultrez 10   Warner       (Viscosity,           NF               NF           45-65K cP               polymer           0.5% wt at                           pH 7.5)       10   Carbomer-   Carbopol   Kevin   EC863CC625   type C   Lubrizol       USP/           Homopolymer   980 NF   Warner       (Viscosity,           PHEUR/               polymer           40-60K cP           JPE                           0.5% wt at                           pH 7.5)                    
Gross ocular congestion was shown to resolve within 7 days for CMC, HEC, HPMC, Pluronic and PVP. Ocular discharge was shown to resolve within three days. Ocular discharge resolved within 3 days for all groups except one. Results of the experiment are provided in  FIGS. 9-11 .
 
       Impurity and Potency Analysis 
       [0068]    The inventors prepared various formulations and evaluated their potency and purity, as well particle size distribution. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Impurities Analysis 
               
             
          
           
               
                   
                 Composition 
                   
                 Post- 
                   
               
             
          
           
               
                   
                 CsA 
                   
                 Pre-Autoclave 
                 Autoclave 
                   
               
             
          
           
               
                   
                 Particle 
                   
                 Potency (%) 
                 CsA Total 
                 CsA Total 
                 Absolute 
               
             
          
           
               
                   
                 Size 
                   
                 HEC 
                 No 
                   
                 Impurities 
                 Impurities 
                 Change 
               
               
                 Formulation 
                 (μm) 
                 CSA (%) 
                 (%) 
                 autoclave 
                 Autoclave 
                 (% a/a) 
                 (% a/a) 
                 (% a/a) 
               
               
                   
               
               
                 HEC-1 
                 10 
                 5 
                 5 
                 117.20% 
                 115.70% 
                 0.71% 
                 0.69% 
                 −0.02% 
               
               
                 HEC-2 
                 10 
                 20 
                 5 
                 103.60% 
                 116.60% 
                 0.61% 
                 0.61% 
                 0.00% 
               
               
                 HEC-3 
                 10 
                 5 
                 2 
                 116.40% 
                 118.80% 
                 0.78% 
                 0.70% 
                 −0.08% 
               
               
                 HEC-4 
                 10 
                 20 
                 2 
                 124.50% 
                 124.70% 
                 0.73% 
                 0.69% 
                 −0.04% 
               
               
                 HEC-5 
                 25 
                 5 
                 5 
                 126.70% 
                 116.60% 
                 0.58% 
                 0.58% 
                 0.00% 
               
               
                 HEC-6 
                 25 
                 20 
                 5 
                 140.00% 
                 147.40% 
                 0.56% 
                 0.56% 
                 0.00% 
               
               
                 HEC-7 
                 25 
                 5 
                 2 
                 137.50% 
                 142.50% 
                 0.63% 
                 0.59% 
                 −0.04% 
               
               
                 HEC-8 
                 25 
                 20 
                 2 
                 129.50% 
                 119.70% 
                 0.56% 
                 0.57% 
                 0.01% 
               
               
                 HEC-9 
                 10 
                 10 
                 3 
                 118.60% 
                 111.70% 
                 0.61% 
                 0.62% 
                 0.01% 
               
               
                   
               
             
          
           
               
                   
                 Composition 
                   
                   
               
             
          
           
               
                   
                 CsA 
                   
               
             
          
           
               
                   
                 Particle 
                   
                 Potency (%) 
                   
               
             
          
           
               
                   
                   
                 Size 
                   
                 PVP90 
                 No 
                   
               
               
                   
                 Formualtion 
                 (μm) 
                 CSA (%) 
                 (%) 
                 autoclave 
                 Autoclave 
               
               
                   
                   
               
               
                   
                 PVP-1 
                 10 
                 5 
                 25 
                 102.51 
                 101.01 
               
               
                   
                 PVP-2 
                 10 
                 20 
                 25 
                 113.81 
                 111.82 
               
               
                   
                 PVP-3 
                 10 
                 5 
                 15 
                 122.42 
                 114.04 
               
               
                   
                 PVP-4 
                 10 
                 20 
                 15 
                 120.28 
                 123.3 
               
               
                   
                 PVP-5 
                 25 
                 5 
                 25 
                 118.56 
                 118.46 
               
               
                   
                 PVP-6 
                 25 
                 20 
                 25 
                 114.55 
                 115.28 
               
               
                   
                 PVP-7 
                 25 
                 5 
                 15 
                 116.37 
                 115.66 
               
               
                   
                 PVP-8 
                 25 
                 20 
                 15 
                 120.9 
                 124.05 
               
               
                   
                 PVP-9 
                 10 
                 10 
                 25 
                 132.51 
                 136.36 
               
               
                   
                 PVP-10 
                 25 
                 10 
                 25 
                 118.03 
                 126.6 
               
               
                   
                   
               
             
          
           
               
                   
                 CsA 
                   
                 Autoclave Conditions 
                   
               
               
                   
                 Crystal 
                   
                 Temp (° C.)/ 
                 Particle size distribution 
               
             
          
           
               
                 Lot # 
                 Form 
                 Excipient 
                 Time (min.) 
                 D90 
                 D50 
                 D10 
               
               
                   
               
               
                 1 
                 2 
                 5% CMC 
                 None 
                 52.38 
                 10.80 
                 5.31 
               
               
                 2 
                 2 
                 5% CMC 
                 121/10 
                 18.02 
                 11.55 
                 5.74 
               
               
                 3 
                 3 
                 3% CMC 
                 None 
                 28.01 
                 12.09 
                 6.84 
               
               
                 4 
                 3 
                 3% CMC 
                 121/10 
                 20.31 
                 11.27 
                 6.56 
               
               
                 5 
                 2 
                 None 
                 None 
                 198.63 
                 116.85 
                 8.27 
               
               
                 6 
                 2 
                 None 
                 120/15 
                 186.44 
                 99.90 
                 7.05 
               
               
                 7 
                 2 
                 None 
                 108/60 
                 195.60 
                 112.53 
                 9.21 
               
               
                 8 
                 3 
                 None 
                 None 
                 110.83 
                 63.33 
                 7.17 
               
               
                 9 
                 3 
                 None 
                 121/15 
                 116.88 
                 67.52 
                 12.16 
               
               
                 10 
                 3 
                 None 
                 108/60 
                 115.56 
                 65.33 
                 10.55 
               
               
                 11 
                 2 
                 None 
                 None 
                 13.15 
                 9.12 
                 6.17 
               
               
                 12 
                 2 
                 None 
                 121/15 
                 14.15 
                 9.12 
                 6.42 
               
               
                 13 
                 2 
                 None 
                 None 
                 14.14 
                 9.66 
                 6.44 
               
               
                 14 
                 2 
                 None 
                 121/15 
                 14.30 
                 9.37 
                 5.95 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Key F2 Formulation Properties of Evaluated Polymers 
               
             
          
           
               
                   
                   
                   
                 In-vivo 
                   
                   
               
               
                   
                   
                 Autoclavability 
                 tolerability (1 wk 
                   
                 CSA-F2 
               
               
                   
                 Syringeability 
                 (121 C., 15 min.) 
                 sub-conj.) 
                 Settling 
                 Potency 
               
               
                   
               
               
                 Carbopol 
                 Max. conc. 4% 
                 No visible 
                 
                           
                 
                 na 
                 na 
               
               
                   
                 w/22 G 
                 change 
                 
                           
                 
                   
                   
               
               
                 Carboxymethyl Cellulose 
                 Max. conc. 9% 
                 No visible 
                 
                           
                 
                 na 
                 na 
               
               
                 (CMC) medium viscosity 
                 w/22 G 
                 change 
                 
                           
                 
                   
                   
               
               
                 Carboxymethyl Cellulose 
                 Max. conc. 6% 
                 No visible 
                 
                           
                 
                 na 
                 na 
               
               
                 (CMC) high viscosity 
                 w/22 G 
                 change 
                 
                           
                 
                   
                   
               
               
                 Hydroxyethyl Cellulose 
                 Max. conc. 6% 
                 No visible 
                 Well tolerated. 
                 No settling in 
                 No loss in 
               
               
                 (HEC) 
                 w/22 G 
                 change 
                 Slight congestion 
                 comparison 
                 potency 
               
               
                   
                   
                   
                 compared to 
                 with BDP gel 
                 post- 
               
               
                   
                   
                   
                 saline 
                 under same 
                 autoclave 
               
               
                   
                   
                   
                   
                 conditions 
                   
               
               
                 Hydroxypropyl Methyl 
                 Max. conc. 7% 
                 
                           
                 
                 Well tolerated 
                 na 
                 na 
               
               
                 Cellulose (HPMC) 
                 w/22 G 
                 
                           
                 
                 Comparable to 
                   
                   
               
               
                   
                   
                 
                           
                 
                 saline. 
                   
                   
               
               
                   
                   
                 
                           
                 
                   
                   
                   
               
               
                 Juvederm Ultra 
                 30G, as formulated 
                 Pre-sterilized by 
                 
                           
                 
                 na 
                 na 
               
               
                   
                 by manufacturer 
                 manufacturer 
                 
                           
                 
                   
                   
               
               
                   
                   
                   
                 
                           
                 
                   
                   
               
               
                 Pluronic F127 
                 Max. conc. 40% 
                 
                           
                 
                 Tolerated. Slight 
                 na 
                 na 
               
               
                   
                 w/22 G 
                 
                           
                 
                 congestion and 
                   
                   
               
               
                   
                   
                 
                           
                 
                 discharge 
                   
                   
               
               
                   
                   
                 
                           
                 
                 compared to 
                   
                   
               
               
                   
                   
                   
                 saline. 
                   
                   
               
               
                 Polyvinylpyrrolidone K90 
                 Max. conc. 27% 
                 No visible 
                 ok 
                 
                           
                 
                 No loss in 
               
               
                 (PVPK90) 
                 w/22 G 
                 change 
                   
                 
                           
                 
                 potency 
               
               
                   
                   
                   
                   
                 
                           
                 
                 post- 
               
               
                   
                   
                   
                   
                 
                           
                 
                 autoclave 
               
               
                   
                   
                   
                   
                 
                           
                 
                   
               
               
                   
                   
                   
                   
                 
                           
                 
               
               
                   
               
               
                             indicates data missing or illegible when filed