Patent Application: US-55932004-A

Abstract:
the failure of many anticancer drugs to control growth of solid cancers may stem in part from inadequate delivery to tumor regions distant from vasculature . however , extricating the effect of tissue penetration from the many other factors that affect a drug &# 39 ; s efficacy in tumors with distance from vasculature is generally not possible . the present invention relates to an effect - based assay which employs a multilayered cell culture , a tissue - engineered disc grown from tumor cells on a permeable membrane . one side of the culture may be exposed to a drug or other test substance while the other side is temporarily sealed off . cells adjacent both the exposed side and the sealed side , of the mcc may then be compared since they have similar rates of proliferation and other biochemical properties . for example , the effect of the drug or other the test substance may be evaluated by immunohistochemical or other labeling techniques . the invention may be applied to discover drugs that exhibit desirable penetration properties .

Description:
throughout the following description , specific details are set forth in order to provide a more thorough understanding of the invention . however , the invention may be practiced without these particulars . in other instances , well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention . accordingly , the specification and drawings are to be regarded in an illustrative , rather than a restrictive , sense . as shown in fig1 ( a ), the present invention may employ a cell culture insert 10 comprising a permeable or semi - permeable membrane 12 held in a cylindrical support 14 . a multi - layered cell culture ( mcc ) 16 having a first side 18 and a second side 20 is grown on membrane 12 as described in u . s . pat . no . 5 , 602 , 028 issued feb . 11 , 1997 which is hereby incorporated by reference . cell culture 16 may comprise , for example , layers of cells approximately 200 μm in total thickness ( fig4 ). as described in the &# 39 ; 028 patent , cell culture 16 is grown while submerged in a liquid growth medium contacting both sides 18 , 20 of membrane 12 . accordingly , cells toward both sides 18 , 20 of culture 16 are typically proliferating at approximately the same rate . a gradient in cellular proliferation may develop between the exposed sides 18 , 20 of culture 16 ( which are better supplied with oxygen and nutrients ) and a central portion of the culture ( where the cells may be primarily non - cycling ). the mcc essentially provides a three - dimensional tissue culture model of solid tumors . fig1 ( b ) illustrates a growth vessel 21 which includes a teflon frame 23 designed to hold multiple mccs suspended in stirred media . for example , the mccs may be grown in batches of 8 separate cultures . fig2 depicts an apparatus 22 for receiving and temporarily supporting the cell culture insert 10 of fig1 ( a ). apparatus 22 includes a back plate 24 coupled to a forward housing 26 defining a reservoir 28 . reservoir 28 contains a liquid growth media 30 . a magnetic stir bar 31 is provided within reservoir 28 to stir media 30 . apparatus 22 allows media 30 to be gassed , stirred and maintained at optimal temperature and permits samples to be taken from reservoir 28 . apparatus 22 could , for example , be made of plastic or plexiglas , machined or molded . it could be sterilized or provided as a sterile “ one use ” item . housing 26 also includes a slot 32 adjacent back plate 24 and in fluid communication with reservoir 28 for removably receiving an insert 10 . an o - ring 34 bears against frame supports 14 when insert 10 is positioned within slot 32 to maintain insert 10 snugly in position . when insert 10 is inserted as shown in fig2 , only one side 18 of culture 16 is exposed to reservoir 28 . the other side 20 of culture 16 adjacent membrane 12 bears against back plate 24 and is sealed off . in particular , in one embodiment membrane 12 supporting side 20 may be clamped sideways against back plate 24 with a layer of parafilm sandwiched in between to ensure a complete seal . as will be apparent to a person skilled in the art , other means for temporarily sealing off one side 20 of culture 16 could be envisaged . in alternative embodiments of the invention , housing 26 could include multiple slots 32 . this would permit housing 26 to receive multiple inserts 10 thereby enabling multiple assays to be performed simultaneously . in accordance with the applicant &# 39 ; s invention , each multi - layered cell culture 16 is grown as described above on an insert 10 such that cells proximate at least the exposed sides 18 , 20 of culture 16 are actively proliferating . each insert 10 is then placed within a corresponding slot 32 of apparatus 22 as aforesaid . a test material , such as a drug or other substance , is then inoculated into reservoir 28 . the test material is allowed to pass through exposed side 18 and penetrate within culture 16 for a predetermined length of time ( which may depend on the test material and the specific type of cell culture employed ). preferably the length of the test period is selected so that cells proximate to the sides 18 , 20 of culture 16 remain proliferating throughout . that is , cells proximate to sides 18 , 20 are in a similar metabolic state and have similar intrinsic rates of proliferation and sensitivities . insert 10 is then removed from apparatus 22 and later analyzed , for example immunohistochemically , to determine the extent of penetration of the test drug or other substance . in particular , the invention may be used to identify cells proliferating ( or not proliferating ) at a location removed from the side 18 of culture 16 which is in fluid contact with the test material . the invention may be employed as a rapid screening method for assessing drug penetration . this technique differs from multicellular spheroid based cell survival experiments which cannot isolate drug effects from gradients of intrinsic sensitivity . fig3 illustrates as dual - reservoir apparatus 40 having a first reservoir 42 and a second reservoir 44 . as shown in fig3 , an insert 10 may be disposed between reservoirs 42 , 44 to permit diffusion of media therethrough . apparatus 40 enables measurement of flux through multi - layered cell cultures 16 and is useful for experimental controls as discussed below . for example , the growth media and oxygenation conditions may be selected in each reservoir 42 , 44 to simulate the effect of sealing one side 20 of culture 16 as described above . the following examples will illustrate the invention in further greater detail although it will be appreciated that the invention is not limited to the specific examples . fig5 and 6 illustrate test results relating to a multi - layered cell culture 16 exposed to the anthracycline doxorubicin ( dox ) ( i . e . the test material ). cultures 16 were exposed to doxorubicin ( at differing concentrations ) from one side 18 only for one hour and then incubated for 24 hours in fresh media . cultures 16 were then exposed to bromodeoxyuridine ( brdurd ) for two hours to label proliferating cells . brdurd is a thymidine analogoue that is incorporated into dna during the s phase of the cell cycle and is therefore an effective marker of cellular proliferation . following brdurd exposure , cultures 16 were frozen and cyrosections taken from them . cryosections were then immunostained to detect brdurd labeled s - phase cells . fig6 illustrates the distribution of brdurd - labelled cells as a function of position within the cultures . the results clearly indicate that tissue penetration limits doxorubicin effectiveness . in particular , results indicate that at low concentrations doxorubicin only affects cells close to the site of exposure ( i . e . exposed side 18 ) while proliferating cells at the far side of the cultures ( i . e . sealed side 20 ) are only affected at high concentrations . in this example four different anthracyclines clinically used as chemotherapeutic agents were studied , namely doxorubicin ( dox ), epirubicin ( epi ), daunorubicin ( dau ) and the related compound mitoxantrone ( mit ). these anthracyclines have a similar mechanism of action but differing physicochemical properties and clinical activities . in addition , the first three agents fluoresce and hence allow for direct comparison of drug effect and drug fluorescence within tissue . the agents are weak bases with pka values of ˜ 8 . 3 for dox , dau and mit and ˜ 8 . 1 for epi ( 10 , 11 ) and at ph 7 . 4 are between 85 - 90 % charged . entry into cells is believed to occur via passive uptake of the neutral species for all four drugs . of the three , dau is most lipophilic , entering cells 3 - 10 times more quickly than dox ( 12 , 13 ), while epi exhibits an uptake rate approximately twice that of dox ( 14 ). all four drugs bind readily to dna and accumulate in cells ( 15 ). it is believed that the more rapid rate of cellular uptake of dau may compromise its ability to penetrate into tissue . hct - 116 human colorectal carcinoma cells were purchased from american type culture collection . cells were grown in monolayers using minimum essential media ( gibco / brl , burlington , on , canada ) supplemented with 10 % foetal bovine serum ( gibco / brl ) and passaged every 3 - 4 days . standard tissue culture inserts 10 ( cm 12 mm , pore size 0 . 4 μm , millipore , nepean , on , canada ) were coated with 150 μl collagen ( rat tail type i , sigma ), dissolved in 0 . 01 m hcl and diluted 1 : 4 with 60 % ethanol to 0 . 75 mg / ml , and allowed to dry over night . hct - 116 , 0 . 75 × 10 6 in 0 . 5 ml growth media , were then added to the inserts 10 and incubated for 15 hours to allow the cells to attach . the cultures 16 were then incubated for 2 days in custom built growth vessels 21 ( fig1 ( b )) to form multilayered cell cultures ( mccs ) 16 which are ˜ 150 μm in thickness . each growth vessel 21 includes a frame 23 that held the inserts 10 completely immersed in 130 ml of stirred media ( 700 rpm , 25 mm stir bar ) under continual gassing ( 5 % co 2 , balance air ) at 37 ° c . the mccs 16 were grown in batches of 8 cultures of which 6 were treated with drug and 2 were kept as untreated controls . mccs 16 were exposed to anthracyclines from one exposed side 18 using a polyarcylate apparatus 22 ( fig2 ) designed such that the bottom of each insert 10 is clamped against a flat block 24 of apparatus 22 with a layer of parafilm ( american national can , chicago , ill ., usa ) sandwiched in between to ensure a complete seal . each mcc 16 was exposed to a 7 . 5 ml stirred reservoir 28 kept under controlled gassing and temperature . a silicone o - ring 34 was used to seal the gap between the mcc 16 and the orifice in the reservoir 28 . once placed in the apparatus 22 , mccs 16 were allowed to equilibrate for 45 minutes , drug was then added to the growth medium at concentrations of 0 . 3 , 1 , 3 and 10 μm . after 1 - hour drug exposure , the reservoirs 28 were rinsed twice with fresh media . mccs 16 were then incubated for a second hour prior to removal from the apparatus 22 to allow initial drug wash out from the exposed side 18 only . mccs 16 were then removed , rinsed in fresh media three times for 15 seconds and placed in a growth vessel 21 containing 2 untreated control mccs 16 . media was replaced at 1 and 4 hours following return to the growth vessel 21 , to reduce residual drug levels . incubation with control mccs 16 ruled out the possibility that drug wash out into the media from the exposed sides 18 of the mccs 16 was affecting results . mccs 16 were left for 1 or 3 days from the time of beginning drug exposure to allow manifestation of drug effect . following this , 100 μm brdurd ( 5 - bromo - 2 - deoxyuridine , sigma chemical , oakville , on , canada ) was added to the media and cultures incubated for 4 hours to label s - phase cells . mccs 16 were then removed , frozen in o . c . t . medium ( tissue - tek , torrance , calif ., usa ) and stored at − 80 ° c . until sectioning . control experiments were carried out using a dual - reservoir apparatus ( fig3 ) in which equal drug exposure was made to both sides 18 , 20 of mccs 16 but under 20 % oxygen , 1 g / l glucose in the first reservoir 42 versus 0 % oxygen , 0 . 1 g / l glucose in the second reservoir 44 . media was gassed overnight and then re - equilibrating for 1 - hour upon insertion of the mccs within the dual - reservoir apparatus . for these experiments , the media used was glucose free dmem gibco / brl , burlington , on , canada ) supplemented with 10 % foetal bovine serum ( gibco / brl ). sodium bicarbonate levels were set to produce ph 7 . 4 under 5 % carbon dioxide . glucose was then added to the oxygenated side to match physiological levels , 1 g / l . on the anoxic side , the 0 . 1 g / l glucose was derived from the 10 % foetal bovine serum . drug exposure and wash out were carried out in the manner described in the previous section . mcc cryosections ( 10 μm ) were air dried for 24 hours and then fixed in a 1 : 1 mixture of acetone - methanol for 10 minutes at room temperature . slides were immediately transferred to distilled water for 10 minutes and then treated with 2 m hcl at room temperature for 1 hour followed by neutralization for 5 minutes in 0 . 1 m sodium borate . slides were then washed in distilled water and transferred to a pbs ( phosphate buffered saline ) bath . subsequent steps were each followed by a 5 minute wash in pbs . brdurd incorporated into dna was detected using a 1 : 200 dilution of monoclonal mouse anti - brdurd ( clone bu33 , sigma ) followed by 1 : 100 dilution of anti - mouse peroxidase conjugate antibody ( sigma ) and 1 : 10 dilution of metal enhanced dab substrate ( pierce , rockford , ill .). slides were then counterstained with haematoxylin , dehydrated and mounted using permount ( fisher scientific , fair lawn , n . j ., usa ). the imaging system consisted of a fluorescence microscope ( zeiss iii rs , oberkochen , germany ), a cooled , monochrome ccd video camera ( model 4922 , cohu , san diego , calif ., usa ), frame grabber ( scion , frederick , md ., usa ), a custom built motorized x - y stage and customized nih - image software ( public domain program developed at the u . s . national institutes of health , available at http :// rsb . info . nih . gov / nih - image /) running on a g4 macintosh computer . the motorized stage allowed for tiling of adjacent microscope fields of view . using this system , images of mcc sections , 8 mm in length , were captured at a resolution of 1 pixel / μm2 . two cryosections were imaged from each mcc . for mccs immunostained for brdurd incorporation , bright field images were captured . for drug fluorescence visualization , mccs were imaged using unmounted cryosections 1 - hour after sectioning using a 510 - 555 nm excitation filter and a 575 - 640 nm emission filter . using the nih - image software application and user supplied algorithms , digital images of brdurd staining and drug fluorescence within mcc cryosections were analyzed in the following manner . pixels making up the cryosection were first sorted based on their distance relative to either edge of the tissue . for brdurd images , the fraction of positively stained pixels at each position relative to the two edges was then calculated . pixels two and a half standard deviations above tissue background levels were classified as brdurd positive . for fluorescence images , the average value of pixels from each group relative to the two edges was calculated . detection of tissue edges was facilitated by capturing high - contrast , bright - field images of the mccs ( prior to staining and mounting the slides in the case of the brdurd sections ). the penetration of the four anthracyclines was evaluated over a range of concentrations via detection of their effect on s - phase labeling using hct - 116 mccs grown under controlled conditions in specialized growth vessels , fig1 ( a ). drug exposures were made to one side of the mccs using the apparatus shown in fig2 ; the two sides of the mccs were defined as the near side 18 ( exposed ) and far side 20 ( sealed off ). exposure from one side enabled a comparison of drug effect on cells that display similar rates of proliferation that are either directly in contact with the drug in media or separated from the drug by the thickness of the culture itself . following 1 hour drug exposures , cultures were returned to their growth vessels and incubated for 1 to 3 days in fresh media to allow manifestation of drug effect within the tissue . data from experiments where mccs were exposed for 1 hour to 0 . 3 , 1 , 3 and 10 μm epi and then incubated for 1 day to allow time for manifestation of drug effect are shown in fig7 . results show a gradual increase in depth of the effect of epi from 0 . 3 to 10 μm . only at the 10 μm exposure is epi found to exert an equal effect to either edge of the mcc . the mcc exposed to 10 μm epi is approximately the same thickness it was at the time of treatment while the other cultures exposed to lower concentrations have grown increasingly thicker during the day following treatment . fig8 shows a summary graph comparing the effect of the four drugs on the near and the far side of mccs 1 day following a 1 - h drug exposure . brdurd labeling in the first 30 μm of tissue on either side of the mccs is expressed as the fraction of labeling seen on the respective sides of untreated mccs from each experiment . shaded regions on each panel indicate typical drug exposures achieved in humans ( 16 - 19 ). in all cases the relevant human exposures are not high enough to achieve an equal effect on both sides of the cultures . for dox , epi and dau data from both near and far sides exhibit a general trend of decreasing labeling with increasing drug concentration . however , the mit data shows an increase in labeling on the near side with increasing drug concentration . since mit already exhibits a large effect at the lowest concentration , this is likely due either to non - proliferation related brdurd labeling or to cell loss on the near side at the higher concentrations . from the data , estimates of the decrease in drug exposure seen by cells on the far side relative to the near side were ˜ 12 - fold for dox , ˜ 10 - fold for epi , ˜ 30 - fold for dau and greater than 30 - fold for mit . these values were arrived at through determination of the drug concentration which produced an effect on the near side that matched that seen at 10 μm drug on the far side . the average fraction of brdurd positive tissue in untreated mccs was 0 . 40 ± 0 . 06 ( s . d .) on near side , versus 0 . 46 ± 0 . 04 ( s . d .) on far sides . this difference is thought to be mainly due to differences in edge detection at the tissue to media transition , near side , in comparison with the more well defined tissue to membrane transition , far side . middle regions of the mccs exhibited a significantly lower average brdurd stained fraction , 0 . 28 ± 0 . 05 ( s . d .). control experiments were carried out using a dual - reservoir apparatus 40 ( fig3 ) where 1 h drug exposure was made to both sides 18 , 20 of mccs 16 with normal conditions on one side versus low oxygen and glucose on the other . results are shown in fig8 . the low oxygen and glucose levels were chosen to simulate levels seen on the far sealed side 20 of the mccs 16 during the closed - off experiments . results were used to estimate what effect an equal drug exposure would have had on the two sides 18 , 20 of the mccs 16 during the closed - off experiments . no significant difference in drug effect between the two sides was observed . these experiments were carried out separately from previous work and performed in different media , which may explain their slightly lower brdurd labeling values as compared with the other data presented in fig8 . the effect of 1 h drug treatment on mccs 16 thickness 1 day following exposure is summarized in fig9 . data are expressed relative to the average thickness of control mccs 16 at the time of exposure , 160 ± 20 μm ( s . d . ), as shown by the solid horizontal lines . untreated cultures 16 grown for 1 day from time of treatment reached 225 ± 30 μm ( s . d .) in thickness , indicated by - dashed horizontal lines . drug treated cultures 16 are seen to lie between their starting thickness and that of untreated cultures left for the additional 24 hours . mccs 16 exposed at the higher drug concentrations do not grow as thick , as expected from the reduction in cell proliferation . based on this reduction in proliferation , cell loss due to removal of cells on the near side is likely no more than 2 - 3 cell layers . in separate experiments , mccs 16 exposed to 100 μm dox were only 1 - 2 cell layers thinner than mccs 16 exposed to 10 μm dox , indicating no significant change in cell loss . mccs 16 cultured for 3 days following clinically relevant exposures showed a similar distribution of s - phase cells as seen after 1 day , fig1 , ( a ) 3 μm × h dox , ( b ) 3 μm × h epi , ( c ) 1 μm × h dau and ( d ) 1 μm × h mit . cultures continued to increase in thickness and in all cases there was an increase in proliferation towards the far sides indicating penetration limited drug effectiveness . the profile of anthracycline derived fluorescence within cultures was examined using the closed - mcc apparatus 22 immediately after the end of a 1 - h . 10 mm drug exposure and 1 day following incubation in fresh media . fluorescence intensity of the drugs in tissue sections serves as a rough indicator of relative drug levels . only dox , epi and dau , which all posses the same fluorophore , could be examined . emission intensities of the three drugs in aqueous solution were within 10 % of each other . fig1 shows average tissue fluorescence as a function of distance into the cultures from the near sides . of the three drugs , dox appears to accumulate the least over the 1 - h exposure with epi and dau reaching ˜ 1 . 5 and ˜ 7 times higher levels respectively on the near sides , panels a - c . the much higher accumulation of dau in the tissue can be related to its higher partition coefficient , leading to a higher rate of entry in cells . interestingly , the drugs show similar cellular accumulation from the middle regions towards the far sides of each culture , see panels d - f which show the data on the same scale . position within mccs 16 is presented as a fraction between 0 and 1 to allow comparison between mccs 16 taken immediately after drug exposure , which measured 140 ± 15 μm in thickness , and mccs 16 incubated for the additional day , which were 185 ± 20 μm in thickness . a comparison of effect based evaluation of drug distribution versus drug derived fluorescence is shown in the table of fig1 . results from the two methods indicated that the effect based data reasonably predicts drug distribution as seen from fluorescence data . effect based results were estimated from data in fig8 . the decrease in fluorescence on the far , sealed side 20 relative to the near , exposed side 18 , was calculated by averaging fluorescence over the first 30 μm of tissue on each side of the mccs 16 shown in fig1 . in this example mccs 16 were used in accordance with the invention to isolate the role of drug penetration from other factors that determine drug efficacy with depth into tissue . by temporarily closing off one side 20 of the mccs 16 during drug exposure , a comparison was made between a drug &# 39 ; s effect on proliferating cells that were directly exposed to drug and those that lay on the far , sealed side 20 of the mccs 16 . results showed that limited tissue penetration of the drugs resulted in a 10 - 30 fold difference in drug exposure between cells on the near , exposed side 18 versus far , sealed side 20 of the cultures . fluorescence based detection of the distribution of dox , epi and dau accumulation in mccs immediately after exposure was consistent with the effect - based assay results for drug penetration . interestingly , drug fluorescence on the far sides of the cultures was not significantly different for the three drugs . drug fluorescence following 1 day of wash out was much reduced on the near , exposed side 18 of the cultures but still present towards the central region of the cultures . the relative fluorescence on the exposed side 18 of the mccs 16 matched the expected ranking of drug uptake rates from cell suspension data , determined mostly by lipophilicity . a main concern of closing off the mccs 16 during drug exposure was that deprivation of oxygen and glucose and build - up of lactate on the far ( closed - off ) side 20 would result in a reduction in cell proliferation and potentially lead to a change in the intrinsic sensitivity of the cells to the drugs . we have previously shown that proliferation continues on the far side 20 of the mccs during a 1 - h exposure preceded by a 45 - minute equilibration period ( 20 ). in this experiment , we simulated the effect of oxygen and nutrient deprivation on the far side 20 of the cultures 16 in control experiments ( using the dual - reservoir apparatus of fig3 ) where equal drug exposure was made to both sides 18 , 20 of mccs 16 but with one side under normal oxygen and glucose and the other under low oxygen and glucose . comparison of drug effect on the two sides 18 , 20 were found to be within experimental error for all four drugs . the distribution of brdurd labeling 3 days after 1 h exposures were similar to that seen after 1 day . due to continued growth of the cultures 16 , especially on the far side 20 , it was not feasible to perform an in - situ cell survival assay by leaving the cultures for longer periods . however our results are not inconsistent with published work with v79 multicellular spheroids , in which cell survival assays were performed . following dox exposure of ˜ 3 . 5 μm × h , spheroids that were incubated in drug free medium for an additional 24 hours prior to disaggregation exhibited a surviving fraction of ˜ 0 . 2 on the exposed outer layers versus ˜ 0 . 6 , 130 μm into the tissue ( 7 ). in the spheroid study , reduced intrinsic sensitivity of cells in the central area of the spheroids and time of disaggregation were shown to play key roles in determining drug toxicity . in comparison with spheroids , mccs 16 pose a greater barrier to penetration due to their planar rather than spherical geometry . in contrast , the outward - radial dilution experienced by a drug moving out of a blood vessel and into the surrounding tissue of a tumor likely poses an even greater barrier to penetration . as will be apparent to those skilled in the art in the light of the foregoing disclosure , many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof . accordingly , the scope of the invention is to be construed in accordance with the substance defined by the following claims . 1 . rubin , p . and casarett , g . microcirculation of tumors — part i : anatomy , function and necrosis . clin . radiol ., 17 : 220 - 229 , 1966 . 2 . thomlinson , r . h . and gray , l . h . the histological structure of some human lung cancers and the possible implications for radiotherapy . br . j . cancer , 9 : 539 - 549 , 1955 . 3 . jain , r . k . transport of molecules in the tumor interstitium : a review . cancer research , 47 : 3039 - 3051 , 1987 . 4 . jain , r . k . delivery of molecular and cellular medicine to solid tumors . adv drug deliv rev , 26 : 71 - 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