bioflavonoids are a 
 group of polyphenolic compounds that are common 
 throughout the plant kingdom . they are widely studied and have been 
 found to promote healthy living in epidemiologic studies .
much of the attention that bioflavonoids have attracted is due mainly 
 to the  french paradox  .
this is the 
 dietary anomaly in which people in the mediterranean culture have 
 a higher fat intake but a lower incidence of cardiovascular disease 
 and increased longevity .
one of the most widely studied bioflavonoids is , epigallocatechin-3-gallate 
 ( egcg ) , the most abundant polyphenol in green tea .
it has been studied 
 extensively during the past decade for its therapeutic potential in 
 various cancers , alzheimer s disease , obesity , and diabetes . despite these promising therapeutic uses , the plasma concentrations 
 of egcg that are required are prohibitively high .
thus , novel methods 
 for improving the oral bioavailability of egcg are desirable . in a 
 previous study
, we demonstrated that proprietary nanolipidic particles 
 could be used to improve the bioactivity of egcg for reducing amyloid 
 beta production in a cell model of alzheimer s disease while 
 also more than doubling the oral bioavailability .
although this could potentially overcome the problems thwarting 
 the clinical translation of egcg , the technology is limited because 
 it requires an alcohol suspension .
until now there have been 
 no reports of any of the crystalline 
 forms of egcg in the cambridge structural database ( csd ) including 
 the crystal structure of pure egcg which makes it worth studying from 
 a structural perspective .
therefore , we undertook the synthesis of 
 cocrystals of egcg and also crystallized its pure form .
this was accomplished 
 by taking advantage of crystal engineering concepts and several crystallization 
 techniques .
cocrystal formers ( ccfs )  , has emerged over the 
 past decade as a materials science approach to generate novel solid 
 forms of active pharmaceutical ingredients with improved physicochemical 
 properties .
we recently reported four new cocrystal forms of another flavonoid , 
 quercetin , which improved solubility by up to 14-fold and oral bioavailability by up to 10-fold.(27 ) unlike quercetin , egcg is highly soluble in 
 water .
previous pharmacokinetics experiments 
 have shown that egcg is absorbed rapidly in the gut following oral 
 administration .
thus , egcg would likely 
 fall into the biopharmaceutics classification system ( bcs ) 3 : high 
 solubility , low permeability .
we hypothesized that new cocrystals 
 of egcg with reduced water solubility might exhibit pharmacokinetic 
 profiles that are more desirable for drug development .
moreover , we 
 hypothesized that reducing the solubility would decrease the rate 
 of dissolution and slow the absorption of egcg in vivo .
we report herein the results of our structural , solubility , and 
 pharmacokinetic studies .
green tea - derived egcg ( > 95% 
 purity by hplc ) was purchased from http://www.herbs-tech.com .
isonicotinamide ( inm , > 99% purity ) , isonicotinic acid ( ina , 
 > 99% 
 purity ) , nicotinamide ( nic , > 99% purity ) , and nicotinic acid ( nac , 
 98% pure ) were purchased from sigma - aldrich corporation ( st . louis , 
 mo , usa ) .
all crystallization 
 and cocrystallization experiments were either conducted at room temperature 
 or at 4 c .
egcg ( 65.0 
 mg , 0.14 mmol ) was dissolved in 
 1 ml of acetonitrile ( 99% pure ) .
the resulting solution was layered 
 on 2.5 ml of dichloromethane and was allowed to stand in the refrigerator . 

egcg ( 200.0 mg , 0.436 mmol ) was dissolved 
 in 2.5 ml of acetonitrile ( 99% pure ) .
the solution was layered onto 
 6 ml of dichloromethane and 2 ml of nitrobenzene and then allowed 
 to stand in a refrigerator .
yellow crystalline needles were harvested 
 after 24 h. the pure crystal line form 
 of egcg , form iv , 
 was obtained by heating forms ii or iii at 120 for 2025 
 min .
form iv crystals can also be reproduced by dissolving egcg ( 45.0 
 mg 0.098 mmol ) in 1 ml of acetonitrile ( 99% pure ) .
the solution was 
 layered on 2.5 ml of dichloromethane and seeded with form iv and was 
 allowed to stand in the refrigerator .
egcg 
 ( 45.80 mg , 0.099 
 mmol ) and iso - nicotinamide ( 99% pure , used as received , 
 12.2 mg , 0.0998 mmol ) were dissolved in 5 ml of water .
colorless block - like 
 crystals of egcginm5h2o were harvested in less than 
 5 min . however
to further grow better quality crystals , 1:1 water / methanol 
 ( v / v % ) solvent was used , and the crystals were obtained in one day .
egcg ( 45.80 mg , 0.099 
 mmol ) and nicotinamide ( 99% pure , used
as received , 12.2 mg , 0.0998 
 mmol ) were dissolved in 2 ml of water .
looking carefully under the microscope 
 a colorless block like single crystal was separated from the cluster 
 and was used for single crystal x - ray diffraction analysis .
egcg ( 45.80 mg , 0.099 
 mmol ) and iso - nicotinic acid ( 99% pure , used as received , 
 12.3 mg , 0.0998 mmol ) were dissolved in 5 ml of water .
egcg 
 ( 458.0 mg , 0.99 mmol ) and nicotinic acid ( 99% pure , used as received , 
 123.0 mg , 0.998 mmol ) were slurried in 2 ml of water overnight in 
 a vial .
the vial was left in the hood undisturbed , and after two days 
 a cluster of block shaped crystals was obtained .
open alumina 
 crucibles were used for analysis from 30 to 300 c at 5 c / min 
 heating rate under nitrogen purge .
thermal analysis 
 was carried out employing a ta instruments dsc 2920 differential scanning 
 calorimeter .
an empty pan , sealed in the same way as the sample , was 
 used as a reference pan . a typical sample ( 28 mg )
was heated 
 in a dsc from 30 to 300 c at a 5 c / min heating rate .
the 
 experimental data were analyzed using commercially available software 
 ( ta universal analysis 2000 ; ta instruments ) .
all of the forms of egcg 
 were characterized by infrared spectroscopy using a nicolet avatar 
 320 ft - ir instrument .
the samples were placed on 
 the sample holder with the help of vacuum grease and exposed , at room 
 temperature , to cu k radiation (  = 1.54056  ; 40 
 kv  30 ma , bruker axs d8 advance ) .
for all the samples , the 
 angular range was 30 2 , with a step size of 0.05 
 2. intensity counts were accumulated for 0.5 s at each step . 

single crystals of forms ii , iii , 
 and iv and cocrystals egcginm5h2o , egcgnic9h2o , egcgina3h2o , and egcgnacxh2o were examined 
 under a microscope , and suitable crystals were selected for single 
 crystal x - ray crystallography .
single crystal x - ray diffraction data 
 on forms iii and iv were collected on a bruker - axs smart apex ccd 
 diffractometer with monochromatized mo k radiation ( (  
 = 0.71073  ) , whereas for form ii and cocrystals egcginm5h2o , egcgnic9h2o , egcgina3h2o , and egcgnacxh2o , data were collected on a bruker - axs 
 smart apex 2 ccd diffractometer with monochromatized cu k radiation 
 (  = 1.54178  ) .
both of the diffractometers are connected 
 to a kryo - flex low temperature device .
the structure was solved using shelxs-97 ( direct methods ) 
 and refined using shelxl-97 ( full - matrix least - squares on f2 ) contained 
 in apex2 and wingx v1.70.01 programs packages .
hydrogen atoms of ch and 
 ch2 groups were placed in geometrically calculated positions 
 and included in the refinement process using a riding model with isotropic 
 thermal parameters : uiso(h ) = 1.2ueq ( ch2 , ch ) .
hydrogen atoms 
 of oh , nh groups and water molecules have been found 
 from difference fourier map and refined using dfix ( d(o  h ) = 0.84  for hydroxyl groups , dfix 2.2 
 antibumping restraints for two hh distances ) and 
 sadi restraints for water molecules ( o  h and hh 
 distances ) with uiso(h ) = 1.5ueq(oh ) . some of water molecules were observed 
 to be disordered in the structure .
hydrogen atoms of ch and ch2 groups were placed in geometrically calculated positions 
 and included in the refinement process using riding model with isotropic 
 thermal parameters : uiso(h ) = 1.2ueq(ch2 , ch ) .
hydrogen 
 atoms of oh and nh groups were placed in geometrically 
 calculated position and refined using afix 147 and afix 93 correspondingly . 

positions of hydrogen atoms h4 , h15 , and h25a were further refined 
 using a distance restraint ( ho{h - bond acceptor } ) 
 of 1.865(8 )  as found from a csd analysis .
some of the water 
 molecules were observed to be disordered , and it was not possible 
 to locate hydrogen atoms for those molecules .
hydrogen atoms of ch and ch2 groups were placed in geometrically calculated positions 
 and included in the refinement process using a riding model with isotropic 
 thermal parameters : uiso(h ) = 1.2ueq(ch2 , ch ) .
hydrogen 
 atoms of oh groups were placed in geometrically calculated 
 position and refined using afix 147 or afix 83 .
the observed residual electron 
 density 1.258 el/  can probably be attributed to 
 the presence of small satellite crystals and/or disordered chains 
 of ina .
the 
 cocrystals egcginm5h2o , egcgnic9h2o , egcgina3h2o , and egcgina were made in bulk for 
 dissolution studies using the slurry method .
egcgnacxh2o was not included in the dissolution study 
 due to difficulties in reproducing it in bulk powder form .
slurry 
 experiments to reproduce egcgnacxh2o resulted in a glue - like material rather than a powder .
for the 
 remaining cocrystals , stoichiometric amounts of the starting materials 
 were stirred overnight in 23 ml of water with the help of 
 a magnetic stir bar on a stir plate that produced the cocrystals with 
 100% yield .
the purity of the bulk material was tested by powder x - ray 
 diffractometry ( pxrd ) and differential scanning calorimetry ( dsc ) . 
 a uniform particle size ( between 53 and 75 m ) for the bulk 
 powder was obtained for all the cocrystals and egcg by sieving using 
 standard astm sieves .
solubility studies 
 were performed on egcg , egcginm5h2o , egcgnic9h2o , egcgina3h2o , and egcgina using uv / vis / nir 
 spectrophotometry in water at room temperature .
the wavelength used 
 for the determination of egcg was 305 nm since there is no interference 
 with the ccfs at this wavelength .
the dissolution studies were conducted 
 by taking approximately 3 g of the cocrystal in 50 ml of water and 
 stirring with a magnetic stir bar at ca .
125 rpm for 4 h. aliquots 
 were drawn from the slurry at regular time intervals ( 5 , 10 , 15 , 20 , 
 25 , 30 , 45 , 60 , 75 , 90 , 120 , 150 , 180 , and 240 min ) and filtered using 
 a 0.45 m nylon filter .
the filtrates were diluted appropriately 
 and analyzed to measure the concentration of egcg ( at 305 nm ) by using 
 a uv / vis / nir spectrometer .
the remaining undissolved solid was analyzed 
 by pxrd and dsc to confirm phase stability .
all animal studies were conducted 
 in accordance with a university 
 of south florida iacuc - approved protocol .
dawley 
 rats ( n = 3 per group ) weighing 200250 g 
 were purchased from harlan laboratories ( indianapolis , in ) .
the rounded tip catheters 
 were surgically implanted into the jugular vein of the rats making 
 multiple , precise blood draws painless to the animal .
the rats were 
 food ( not water ) deprived for 18 h prior to the start of the experiment . 

corn oil was selected as the gavage vehicle because all crystal forms 
 were observed to be insoluble in it .
all egcg forms were sieved to 
 attain a particle size between 53 and 75 m prior to suspending 
 in corn oil at 20 mg of egcg per ml .
the egcg formulations were delivered 
 via oral gavage at a dosage of 100 mg egcg per kg body weight .
blood 
 was collected at the following time points : 0 , 5 , 10 , 30 , 60 , 120 , 
 240 , and 480 min . because heparin was kept in the catheter lines to 
 prevent clotting , a small amount of blood was drawn and discarded 
 before collecting each sample .
approximately 300 l of blood 
 was collected in edta tubes for each time point .
the samples were 
 kept on ice to preserve their integrity and then centrifuged at 4000 
 rpm for 10 min , after which the plasma was transferred to sterile 
 centrifuge tubes .
a preservative solution was added to each plasma 
 sample at 10% ( v / v ) concentration to ensure the integrity of the egcg 
 during storage .
this preservative was 
 comprised of 20% ascorbic acid ( to prevent oxidation ) and 0.1% edta 
 ( to scavenge any metal contaminants ) .
the samples were stored at 80 
 c until they were analyzed for egcg content .
to accurately 
 quantify the concentration of egcg in the plasma , a previously described 
 method was employed using liquid chromatography with tandem mass spectrometry .
the standard spiking solutions were prepared 
 by diluting the stock solution to 1000 and 100 g / ml using acetonitrile  water 
 ( 1:1 , v : v ) .
both solutions were protected from light using amber vials , 
 and all solutions were stored at 20 c . for this analysis two standard 
 curves were prepared : one with a higher ( 100.100 g / ml ) 
 dynamic range , and the other a lower range ( 100010 ng / ml ) . 

the results indicated that the standard 
 curve performance was within acceptable range for bioanalytical method 
 acceptance ( r > 0.99 ) .
mean plasma egcg concentrations 
 and the standard error in the mean ( sem ) were graphed using graphpad 
 prism software ( graphpad software , inc . ) .
phoenix winnonlin version 
 6.3 ( pharsight corporation , mountain view , ca ) was used to conduct 
 a noncompartmental analysis of the pharmacokinetic data and generate 
 the pharmacokinetic parameters .
the reported pharmacokinetic parameters 
 included cmax , tmax , area under curve ( auc ) , relative bioavailability ( frel ) , and apparent terminal half - life ( hl_lambda_z ) . 

relative bioavailability was determined by dividing the mean auc of 
 each egcg formulation by the control .
two - tailed t - tests 
 were used to assess the statistical significance at each time point 
 for the pharmacokinetic curves .
the 
 ccfs were selected based on the supramolecular synthon approach . according 
 to this approach
the ccfs were identified based on the functional 
 groups present on egcg and analyzing the frequency for the occurrence 
 of supramolecular synthons ( homo and hetero ) with other functional 
 moieties .
this analysis was carried out via the csd , an archive of 
 over 600  000 organic crystal structures .
the csd also 
 offers a software platform that facilitates statistical analysis of 
 packing motifs , thereby providing empirical information on common 
 functional groups and how they associate at the molecular level .
several 
 fragments of egcg were identified , and a csd analysis was conducted 
 to determine if these fragments and/or the whole molecule ( egcg ) are 
 susceptible to form cocrystals with carboxylic acids , alcohols , or 
 weak bases .
furthermore , the selected ccfs should be suitable for 
 use in drug products ( e.g. , fda approved , gras or eafus listed ) .
several 
 potential ccfs were selected based on the above - mentioned criteria . 
 however , we were unable to obtain cocrystals with any other ccfs except 
 those presented herein .
in addition , efforts to obtain pure crystalline 
 forms of egcg via different crystallization techniques led to the 
 isolation of two solvates which upon desolvation resulted in a pure 
 crystalline form of egcg .
interestingly , all of the isolated cocrystals are in the 
 form of 
 hydrates .
egcg is a large and flexible molecule containing a number 
 of hydrogen bond donors and acceptors .
furthermore , the raw material used is egcg monohydrate , 
 and dehydration of the starting material is reversible . except for 
 egcgina3h2o , all cocrystals became amorphous following 
 dehydration . egcgina3h2o upon dehydration yields 
 an anhydrous cocrystal of egcg and ina .
we believe that water plays 
 a pivotal role in crystallizing these cocrystals , but it is difficult 
 to say whether the water molecules are playing a space - filling role 
 in channels or they are an integral part of the crystal lattice .
form ii crystallizes 
 in the monoclinic c2 space 
 group and is a high z structure consisting 
 of two egcg , three acetonitrile , three water , and a disordered dichloromethane 
 molecule in the asymmetric unit .
the two symmetry - independent egcg 
 molecules form sheets with voids that accommodate the solvent molecules , 
 as shown in figure 2a and b. ( a ) egcg molecules in 
 form ii forming sheets with channels .
( b ) 
 a typical channel being occupied by solvent molecules in form ii .
form iii , a solvated form of 
 egcg with nitrobenzene and water , crystallized 
 in the orthorhombic p21 space group with 
 one molecule of each component in the asymmetric unit . in the crystal 
 structure the egcg and water molecules form sheets .
( a ) illustration of a 
 representative sheet formed by egcg and water 
 molecules in form iii .
( b ) nitrobenzene molecule sandwiched between 
 sheets of egcg and water molecules in form iii .
pure egcg , form iv , crystallizes in monoclinic p21 space group with one egcg molecule in asymmetric 
 unit .
the voids 
 are interpenetrated with other sheets to exhibit 2-fold interpenetration 
 as shown in figure 4a and b. ( a ) crystal packing of 
 form iv ; crinkled sheets with cavities were 
 created via several o 
the single crystal x - ray 
 structure analysis reveals that egcginm5h2o is a 
 pentahydrate of the 1:1 cocrystal of egcg and inm .
egcg molecules 
 and inm molecules interact through one point hydrogen bonds ( o  hn , 
 on : 2.756 ( 2 )  ) between a hydroxyl group of 
 egcg molecules and the aromatic nitrogen of inm molecules .
ho ( oo : 
 2.662 ( 4 )  ) hydrogen bonds formed between hydroxyl moieties 
 of egcg and carbonyl moieties of inm molecules and thereby form zigzag 
 chains as illustrated in figure 5 .
the crystallization of 
 egcg and nic in water results in the formation of a 1:1 cocrystal 
 nonahydrate .
it crystallizes in p1 space group with 
 two molecules each of egcg and nic molecules and nine water molecules 
 in the asymmetric unit ( figure 6a ) .
the n  h 
 functionality of the nic molecules interact with one of the o  h 
 moieties present of ring a of egcg with an no bond 
 distance of 2.570 ( 2 )  ( figure 6b ) .
the cocrystallization 
 of egcg and ina resulted in the formation of a cocrystal trihydrate 
 which crystallizes in the orthorhombic space group p21p21p21 .
the ina and egcg molecules interact with each other through 
 water molecules ( figure 7a ) .
one of the three 
 water molecules interacts with two egcg and one ina molecule through 
 the following h - bonds : ( a ) the o  h of the water molecule interacts 
 with the c  o moiety of the ina molecule with a bond distance 
 of 2.739 ( 3 )  ; ( b ) the o  h of the water molecule interacts 
 with two neighboring egcg molecules with h - bond distances of 2.704 
 ( 5 ) and 2.752  ( 3 )  .
the other water molecule forms a 
 trifurcated bond with three different egcg molecules as presented 
 in figure 7b through o  ho 
 bonds of 2.667 ( 3 ) , 2690 ( 2 ) , and 2.712 ( 2 )  .
ho bifurcated 
 hydrogen bonds ( 2.540 ( 2 ) and 2.986 ( 3 )  ) ( figure 7a ) .
the 1:1 
 cocrystal of egcg and nac crystallized as a hydrate in monoclinic 
 space group c2 . unlike the other cocrystals the stiochiometry 
 of water in the egcg - nicotinic acid ( nac ) is indefinite .
we were unable 
 to determine the number of water molecules in the crystal lattice 
 of egcg - nac cocrystals due to poor quality crystals and single crystal 
 data ( see table 1 ) . from the crystal structure 

c bond angle is 
 122.76 , and the c  o bond distances are 1.237 and 1.252 
  .
each of the nac 
 zwitterions interacts two neighboring egcg molecules via the following 
 h - bonds : ( a ) an h - bond formed between one of the o 
h groups 
 on ring b with one of the carboxylates of the nac molecules at a distance 
 of 2.588 ( 3 )  ; ( b ) an h - bond between the n  h of the nac 
 molecule and one of the o  h functionalities present on ring 
 a of another egcg molecule ( n  ho : 2.902 ( 3 )  ) . 

the intermolecular h - bonding between egcg and nac zwitterions is illustrated 
 in figure 8 .
illustration hydrogen bonding between 
 egcg and nac molecules in 
 egcgnacxh2o cocrystal ( water molecules 
 are not shown in the figure for clarity ) .
egcg is relatively 
 soluble in water compared to many other flavonoids such as quercetin . 

we determined the solubility profiles of the four new egcg cocrystals 
 reported herein and observed that all of the cocrystals exhibit reduced 
 solubility .
the solubility studies were conducted for 4 h because 
 after 1 h it was observed that the solubilities remained almost constant 
 and did not change for the next 3 h. this suggests that the solubilities 
 had reached equilibrium after 1 h. figure 9 shows the dissolution profiles of egcg and the egcg cocrystals . 

the maximum solubility of egcg determined experimentally was approximately 
 23.6 mg / ml during the 4 h dissolution experiment . the egcg cocrystals 
 were found to exhibit far lower aqueous solubilities when compared 
 to egcg as illustrated in figure 9 .
the cocrystal 
 that exhibited the highest aqueous solubility was egcgnic9h2o ( 2.95  0.11 mg / ml ) .
egcgina ( anhydrous ) , egcginm5h2o , and egcgina3h2o were observed to exhibit 
 solubilities of 1.227  0.036 , 1.401  0.122 , and 0.97  
 0.07 mg / ml , respectively .
after the dissolution 
 experiment has concluded ( 4 h ) , the identity of the powders was confirmed 
 by pxrd and dsc .
egcgina anhydrous had converted to the hydrate form , 
 egcgina3h2o , while the remaining cocrystals were 
 found to be stable up to at least 4 h .. dissolution profiles 
 of ( a ) egcg and its cocrystals and ( b ) the 
 cocrystals alone in water .
the pharmacokinetic 
 curves for egcg and the egcg cocrystals are presented in figure 10 .
each cocrystal is shown separately ( solid line ) 
 against the egcg control ( dashed line ) .
egcg peaked rapidly at 
 7.50  2.74 min ( tmax ) due to its 
 high solubility and consequent high rate of dissolution .
the cocrystals 
 peaked in the plasma in the following order : egcgnic9h2o , egcginm5h2o and egcgina3h2o , 
 egcgina .
surprisingly , only two of the cocrystal forms resulted in 
 improved bioavailability : egcgina3h2o and egcgina . 

these cocrystals had modest improvements in relative bioavailability 
 at frel of 1.37 and 1.05 , respectively
. 
 egcg peaked more rapidly than all of the cocrystal forms and exhibited 
 the shortest apparent terminal half - life , 249.35  60.73 min . 

this was most apparent with the egcgina3h2o and egcgina cocrystals , which exhibited drastic changes 
 in the time to reach maximal plasma concentrations ( tmax ) at 30.00  24.49 and 160  61.97 min , respectively . 
 in some cases
, these pharmacokinetic profiles might be advantageous 
 to the rapid peak and elimination profile of free egcg .
< 0.05 . due to 
 an insufficient number of 
 data points in the elimination phase of the pharmacokinetic profile ,
egcg research increased drastically when researchers reported that 
 epidemiological evidence indicated lower rates of certain cancers 
 in populations that consumed the most green tea . since then , numerous 
 molecular mechanisms for these beneficial properties have been discovered . 

not surprisingly , egcg has become one of the most popular nutraceutical 
 ingredients in the world .
egcg is highly susceptible to oxidation , first pass metabolism , 
 and rapid efflux .
numerous groups have 
 evaluated delivery systems to overcome these limitations with egcg , 
 but none have tried cocrystallization .
cocrystallization has been 
 used extensively to improve the solubility and/or bioavailability 
 of poorly soluble apis .
this study is the first report on the bioavailability effects of 
 reducing the solubility of a highly soluble api .
we evaluated 
 the structure , solubility , and pharmacokinetics of 
 four new cocrystals of egcg , all of which were found to exhibit reduced 
 water solubility .
we hypothesized that these changes in solubility 
 would change the pharmacokinetics of egcg , perhaps improving its bioavailability . 
 when interpreting our data , it is important to note that we used a 
 nonconventional gavage vehicle ( corn oil ) to prevent presolubilizing 
 of the sieved crystalline forms .
our egcg control peaked approximately 
 16.5 min more rapidly than previously published pharmacokinetic data 
 at the same dose in rats using a saline gavage vehicle .
furthermore , we previously assessed the pharmacokinetics 
 of an egcg nanolipidic particle suspension in 10% ethanol . for an accurate comparison
this resulted in lower levels of free egcg 
 measured in the plasma ( cmax = 116.57 
 ng / ml ) .
nonetheless , 
 the results presented in this paper should be considered a stand - alone 
 comparison of egcg and egcg cocrystals in a common vehicle . at first glance
, it seems that cocrystallization might not be the 
 most useful method for improving the bioavailability of egcg , since 
 only two cocrystals exhibited improved relative bioavailability and 
 the increases were very modest ( table 3 ) .
although 
 we did not monitor the metabolic profile of the circulating egcg , 
 it is likely that the blunted bioavailability increases were due to 
 the well - known metabolism issues with egcg , which is left unchecked in these cocrystal forms .
an additional 
 contributor to the observed relative bioavailability for this experiment 
 is apparent when considering the variability for the egcg control 
 ( table 3 ) .
one of the experimental rats achieved 
 three times higher plasma levels of free egcg than observed in the 
 other rats .
however , 
 if this animal had been excluded as an outlier , the relative bioavailability 
 for the cocrystal forms would have increased .
this decision was 
 driven by our desire to minimize the use of animals and based on previous 
 pharmacokinetic studies performed in our lab .
however , the pharmacokinetic study suffered from unexpected levels 
 of variability , which should be considered when drawing conclusions 
 from this data .
the pharmacokinetic parameters presented in table 3 include the means with standard deviations . for 
 the egcgina , an asterisk marks the hl_lambda_z , because there were 
 insufficient data points in the elimination phase such that the cmax had to be included in the noncompartmental 
 analysis in winnonlin .
this only occurred with the egcgina cocrystal 
 because we did not expect such a robust change in the tmax . even though our new forms of egcg did not lead 
 to large increases 
 in oral bioavailability
, this does not mean that cocrystallization 
 can not benefit the commercialization of egcg .
the ability to have 
 multiple solid forms of egcg with different physicochemical properties 
 and pharmacokinetic profiles could be very useful
. further examination 
 of figure 10 indicates that some of the egcg 
 cocrystals exhibited different pharmacokinetic profiles .
the egcgina3h2o and egcgina cocrystals not only had the highest relative 
 bioavailabilities of the cocrystals that we evaluated but also resulted 
 in higher concentrations of free egcg after 60 and 120 min , respectively
. 
 this plateau effect could be useful for achieving more sustained levels 
 of egcg in the blood .
furthermore , because pharmaceutical and nutraceutical 
 products often contain combinations of ingredients with synergistic 
 effects , cocrystallization could be used to select forms with similar 
 pharmacokinetics as the other ingredients .
our results generally support 
 our hypothesis that reducing the 
 solubility reduces the rate of dissolution and delays the time at 
 which maximal plasma levels are reached ( tmax ) .
egcgnic9h2o was found to be the most water - soluble 
 cocrystal form and had the most rapid tmax after free egcg . egcginm5h2o and egcgina3h2o
however , egcgina peaked at 
 160 min ( table 3 ) and was found to exhibit 
 similar solubility to that of egcginm5h2o and egcgina3h2o ( figure 9 ) .
normally , it might be reasonable 
 to assume that other pharmacokinetic parameters are being changed , 
 like elimination rate or metabolism .
however , this can not be the case for egcgina 
 and egcgina3h2o since they have the same ccf .
as 
 expected , the hydrate exhibited slightly lower water solubility and 
 peaked later in comparison to the other more soluble egcg forms .
because 
 anhydrous and hydrous forms were evaluated , we were able to conclude 
 that the presence of ina does not explain the discrepancy observed 
 with the egcgina cocrystal .
nonetheless , it is very clear that cocrystallization 
 can be used to generate novel forms of egcg with modulated dissolution 
 and pharmacokinetic profiles ( see also supporting 
 information ) . in conclusion ,
the present study demonstrates 
 how crystal engineering 
 concepts can be utilized for isolating new cocrystals of egcg and 
 that with the aid of several crystallization techniques we were able 
 isolate the first pure ( i.e. , nonsolvated , nonhydrated ) crystal form 
 of egcg
. however , cocrystallization might not be the best option for 
 improving the bioavailability of bcs 3 compounds like egcg , but it 
 could be very useful when absorption kinetics is critical such as 
 it is in synergistic and/or combination products .
for example , selection 
 of a cocrystal form that exhibits a dissolution rate - limited pharmacokinetic 
 profile could achieve sustained plasma levels of free active ingredient 
 similar to other sustained release preformulation techniques like 
 transdermal delivery .
sustained plasma 
 levels are preferred so that the therapeutic bioactivity of the active 
 ingredient is maintained for a longer duration .
even though transdermal 
 delivery can also accomplish sustained plasma levels , it has numerous 
 disadvantages to oral formulation such as complicated production , 
 increased cost , and reduced patient compliance due to common side 
 effects like skin irritation at the adhesion site .
this study 
 therefore lends credence to the concept of using cocrystallization 
 to modulate the pharmacokinetics of highly water - soluble compounds . 
 in this study , we evaluated four new solid forms of egcg with different 
 solubility properties and consequent pharmacokinetic profiles .
although 
 we confirmed that a single cocrystal with reduced solubility relative 
 to a highly soluble pure form is unlikely to produce improved bioavailability , 
 the ability to modulate the pharmacokinetic curve using crystal engineering 
 could be very useful in the clinical translation of active ingredients . 
 in theory
 the 
 most abundant polyphenol in green tea , epigallocatechin-3-gallate 
 ( egcg ) , has recently received considerable attention due to the discovery 
 of numerous health - promoting bioactivities . despite reports of its 
 poor oral bioavailability 
 , egcg has been included in many dietary 
 supplement formulations . 
 conventional preformulation methods have 
 been employed to improve the bioavailability of egcg . 
 however , these 
 methods have limitations that hinder the development of egcg as an 
 effective therapeutic agent . in this study 
 , we have utilized the basic 
 concepts of crystal engineering and several crystallization techniques 
 to screen for various solid crystalline forms of egcg and evaluated 
 the efficacy of crystal engineering for modulating the pharmacokinetics 
 of egcg . 
 we synthesized and characterized seven previously undescribed 
 crystal forms of egcg including the pure crystal structure of egcg . 
 
 the aqueous solubility profiles of four new egcg cocrystals were determined . 
 
 these cocrystals were subsequently dosed at 100 mg egcg per kg body 
 weight in rats , and the plasma levels were monitored over the course 
 of eight hours following the single oral dose . 
 two of the egcg cocrystals 
 were found to exhibit modest improvements in relative bioavailability . 
 
 further , cocrystallization resulted in marked effects on pharmacokinetic 
 parameters including cmax , tmax , area under curve , relative bioavailability , and apparent 
 terminal half - life . 
 our findings suggest that modulation of the pharmacokinetic 
 profile of egcg is possible using cocrystallization and that it offers 
 certain opportunities that could be useful during its development 
 as a therapeutic agent . 
Introduction
Experimental Section
Results
Discussion and Conclusions