as summarized above , described herein are novel methods for providing liposomal formulations of camptothecins , camptothecin prodrugs , and analogs thereof , and compositions formulated thereby . it will be understood that the term camptothecin as used herein is intended to refer collectively to various camptothecins , camptothecin prodrugs , and camptothecin analogs as are well known in the art . the methods and compositions described herein may be accomplished by various means which are illustrated in the examples below . these examples are intended to be illustrative only , as numerous modifications and variations will be apparent to those skilled in the art . it is known that at ph ranges which favor the lactone - form camptothecin , during liposomal encapsulation the drug preferentially partitions into liposomal membranes . this minimizes exposure to the aqueous environment , resulting in a decrease in ring opening of the active lactone . accordingly , conventional liposomal formulations for camptothecins employ a low ph in liposomes to stabilize camptothecins in the active lactone form . however , this strategy requires use of an aqueous buffer to dissolve the drug prior to preparation of liposomes by conventional hydration - extrusion , sonication , or drug - lipid film techniques . because of the low water - solubility of neutral camptothecins and the concomitant reductions in retention time for liposomal formulations of neutral camptothecins ( see table 1 ), such conventional strategies are more favorable to liposomal encapsulation of cationic camptothecins than neutral camptothecins . camptothecins , including neutral camptothecins , exist predominantly in the active lactone form in aqueous solution at ph less than 6 . 0 , whereas the inactive carboxylate species dominates in aqueous solution at ph above 6 . 0 . at physiological ph ( 7 . 4 ), approximately 70 % of camptothecin is in the carboxylate form . thus , in plasma , approximately 70 % of the drug is in carboxylate ( inactive ) form and 30 % in lactone ( active ) form . the half - life of lactone - form camptothecin in rat plasma is approximately 40 min . encapsulating the lactone form of a representative neutral camptothecin , db - 67 , in pegylated liposomes at low ph ( 4 ) prolongs the half - life of the drug . however , at this ph it was not possible to retain the drug in liposomes in aqueous buffers for long periods of time . it has been found that preparing liposomes containing entrapped camptothecins , prodrugs , or analogs thereof , including neutral camptothecins , at a ph sufficient to keep substantially an entirety of the intraliposomal drug in the inactive carboxylate form significantly increased the half - life for retention in liposomes . this is because the release of the carboxylate form from the liposomes is negligible . surprisingly , however , slow conversion of the entrapped , ring - opened carboxylate occurs , providing a low , steady - state concentration of lactone form drug which is then slowly released from the liposome . thus , the present invention provides a slow and prolonged release of the active lactone form from the liposome . this release rate can be varied by changing the intraliposomal ph . that is , an increase in the intraliposomal ph slows the release of active lactone from the liposome , and vice - versa . the compositions contemplated herein may be formulated for delivery to patients in need thereof using methods and formulations well within the skill in the art . for example , the compositions may be prepared for direct delivery , or as pharmaceutical formulations along with suitable carriers or excipients as are well known to the skilled artisan . for example , one or more additives may be included with the compositions , such as one or more stabilizers , buffers , salts , preservatives , fillers , and the like . suitable buffers include without limitation phosphates , carbonates , citrates , and others . suitable preservatives include without limitation edta , egta , bha , bht , and others . the skilled artisan will also readily appreciate that pharmaceutical formulations comprising the present compositions will be highly dependent on the route of administration chosen . by way of non - limiting example , injectable formulations of the compositions may be provided as aqueous solutions , typically in physiologically compatible buffers such as hank &# 39 ; s solution , ringer &# 39 ; s solution , or physiological saline buffer . pharmaceutical formulations intended for parenteral injection , e . g ., by bolus injection or continuous infusion , may be provided in unit dosage form , e . g ., in ampoules or in multi - dose containers , typically with an added preservative as set forth above . set forth in greater detail below are specific details related to selected modes for carrying out the methods and compositions of the present invention . the examples set forth herein are in no way intended to limit the scope of the invention . those of skill in the art will realize that , given the teachings provided herein , many variations of the methods are possible that will fall within the scope of the present invention . unless otherwise indicated , all citations of literature are specifically incorporated by reference herein in their entirety . phospholipids and pegylated phospholipids were purchased as powders from avanti polar lipids ( alabaster , ala .). a representative neutral camptothecin , db - 67 , was from novartis pharmaceutical corp . ( east hanover , n . j .). sprague - dawley rat plasma was from bioreclamation , inc . ( east meadow , n . y .). centricon ® ( mwco : 100000 ) centrifugal filter devices from millipore ( billerica , mass .) and sephadex ® g - 25 m prepacked size exclusion columns ( ge healicare bio - sciences corp ., piscataway , n . j .) were used in liposome studies . all other reagents were from fisher scientific ( florence , ky .). liposomes were prepared by conventional hydration - extrusion technique . however , the skilled artisan will appreciate that any suitable method for preparing liposomes having a desired particle size and lamellarity is contemplated . in one aspect , liposomes having a particle size range of from about 50 to about 300 nm are contemplated for use in the present invention . methods for preparing liposomes falling within a predetermined size range are known in the art ( see drummond et al ., 1999 ). for the hydration - extrusion technique , films of the desired lipid mixtures were prepared in test tubes by dissolving weighed amounts of lipids in chloroform , evaporating the solvent under nitrogen , and drying overnight in vacuo . a stock solution of drug ( db - 67 ) in a buffer providing the desired ph was added to hydrate the lipids , followed by shaking and extrusion through polycarbonate membranes at 60 ° c . to obtain unilamellar vesicles containing entrapped drug . thus , intraliposomal ph of the final liposome - entrapped drug preparation prepared as described was substantially in accordance with the ph of the buffer / db - 67 solution in which phospholipid mixtures were hydrated . the liposomal permeability of db - 67 in aqueous solution was measured over a ph range of 4 . 5 to 9 . 5 using a dynamic dialysis method [ v . joguparthi and b . d . anderson , liposomal delivery of hydrophobic weak acids : enhancement of drug retention using a high intraliposomal ph , j . pharm . sci . 97 , 433 - 454 ( 2008 )]. liposome - encapsulated db - 67 solutions were prepared at varying ph values as described above . for liposome solutions at each ph evaluated , liposome - entrapped drug was separated from free drug by passing liposomes through a sephadex ® column followed by 5 ml of the same buffer added in 1 ml increments . the liposome - containing eluent was dialyzed at 37 ° c . in the same buffer . at intervals , 100 μl of liposome suspension was removed from the dialysis tube and added to 900 μl cold methanol / acetonitrile ( 2 : 1 , v / v ). these samples were dried under nitrogen and stored (− 25 ° c .) prior to analysis . with reference to table 2 , db - 67 was retained in liposomes ( prepared by hydration - extrusion ) in aqueous solution for increased periods of time when liposomes were prepared at high ph . in aqueous solution , the half - life for retention of db - 67 in liposomes increased from 3 hours at ph 4 to about 90 hours at ph 9 . 5 . without being restricted to any particular theory , this may be due to conversion of db - 67 lactone to the carboxylate form in the intraliposomal space . as shown in fig2 , when intraliposomal ph was maintained whereby the carboxylate species of db - 67 predominated , release of the drug from liposomes was negligible . for release to occur , ph conditions allowing formation of the active , lactone species were required . the efflux of db - 67 from liposomes in plasma was monitored . aliquots of liposome suspension containing db - 67 ( with unentrapped drug separated as described above ) were added to plasma and incubated at 37 ° c . at intervals , an aliquot of plasma was withdrawn , added to cold methanol / acetonitrile ( 2 : 1 v / v ), centrifuged ( 14000 rpm ) and stored frozen (− 25 ° c .) for hplc analysis . hplc analyses used herein have been previously described in detail ( joguparthi et al ., 2006 ). db - 67 carboxylate standards ( 10 - 100 nm ) were prepared in 10 mm carbonate buffer ( ph 10 . 4 ). db - 67 lactone standards ( 5 - 30 nm ) were prepared in acidified methanol . all standards were diluted into the desired concentration range using cold methanol / acetonitrile ( 2 : 1 v / v ). hplc retention times were 1 . 6 and 5 . 2 min for db - 67 carboxylate and lactone , respectively . fig3 shows liposomal release of db - 67 in plasma at ph 4 . 5 and ph 9 . 5 . in plasma , the half - life for retention in liposomes was 3 . 5 hours when intraliposomal ph was 4 . 5 and 6 . 3 hours when intraliposomal ph was 9 . 5 . the half - life for retention at intraliposomal ph 9 . 5 was less in plasma than in aqueous solution due to a decrease in intraliposomal ph observed after adding liposomes to plasma . however , even in plasma , preparing liposomes at ph 9 . 5 and maintaining intraliposomal ph at levels which preserve the carboxylate form of the entrapped neutral camptothecin prolonged intraliposomal retention . thus , the potential for exposure of healthy tissue to the drug was reduced . this improved intraliposomal retention enables the drug to remain in the liposomes while they are circulating in the bloodstream . after the liposomes collect in solid tumors due to their enhanced permeation across the tumor vasculature and their improved retention within the tumor tissue ( drummond et al ., 1999 ), the entrapped drug will be slowly released as the active , lactone form of the drug directly at the tumor site , providing a significant enhancement in efficiency of delivery . a 10 mg / ml solution of db - 67 was prepared in ph 9 . 5 sodium carbonate buffer and filtered through a 0 . 2 μm syringe filter . the drug solution was used to hydrate phospholipids ( dspc + 5 mol % m - peg dspe ) with shaking at 60 ° c . to form a 30 mg / ml suspension of multilamellar vesicles . the suspension was extruded through a high pressure extruder to form unilamellar vesicles . the vesicles were then cooled at room temperature and stored below 5 ° c . until use . prior to use , liposomes were separated from unentrapped db - 67 by passing through a gel filtration column which was pre - equilibrated with ph 7 . 4 phosphate buffered saline . 100 μl of liposomes collected from gel filtration were immediately added to 4 ml of plasma to study the release of liposome - entrapped db - 67 carboxylate from plasma as described above . samples were taken at various time intervals and db - 67 was extracted from 100 μl of plasma using 300 μl of ice - cold methanol solution and acetonitrile ( 2 : 1 v / v ) at − 9 ° c . the concentration of db - 67 was determined by hplc as described above . a 10 mg / ml solution of db - 67 was prepared in ph 9 . 5 sodium carbonate buffer and filtered through a 0 . 2 μm syringe filter . the drug solution was used to hydrate phospholipids ( dspc + 5 mol % m - peg dspe ) with shaking at 60 ° c . to form a 30 mg / ml suspension of multilamellar vesicles . the suspension was extruded through a high pressure extruder to form unilamellar vesicles . the vesicles were then cooled at room temperature and stored below 5 ° c . until use . prior to use , liposomes were separated from unentrapped db - 67 by passing through a gel filtration column which was pre - equilibrated with ph 9 . 5 sodium carbonate buffer . the liposomes collected from gel filtration were immediately loaded into a dialysis tube and dialyzed ( 37 c ) against 1000 ml of ph 9 . 5 sodium carbonate buffer . db - 67 analysis was by hplc as described above . a 20 mg / ml solution of db - 67 is prepared in ph 10 . 5 sodium carbonate buffer and filtered through a 0 . 2 μm syringe filter . the drag solution is used to hydrate phospholipids ( dspc + 5 mol % m - peg dspe ) with shaking at 60 ° c . to form a 30 mg / ml suspension of multilamellar vesicles . the suspension is extruded through a high pressure extruder to form unilamellar vesicles . the vesicles are then cooled at room temperature and stored below 5 ° c . until use . prior to use , liposomes are warmed to room temperature and separated from unentrapped db - 67 by gel filtration as described . 10 mg of db - 67 is added to 60 mg of a phospholipid mixture in 2 ml of a 2 : 1 mixture of chloroform : ethanol . the solution is evaporated under nitrogen to form a drug - lipid film . the film is hydrated with ph 10 . 5 carbonate buffer with shaking to form multilamellar vesicles . the suspension is extruded to form unilamellar vesicles . the vesicles are cooled to room temperature and unentrapped drug is separated from entrapped drug as described . the liposomes are then stored below 5 ° c . until use . drug is loaded in vesicles as described in examples 6 and 7 , except the drug and phospholipids are dissolved in pure chloroform , pure acetone , pure methanol , or a suitable combination of those solvents . subsequently , solvent is evaporated to form a drug - lipid film . unilamellar vesicles are prepared as described in examples 4 - 8 using ph 9 . 5 borate buffer . unilamellar vesicles are prepared as described in examples 4 - 8 using ph 9 . 5 tris - hcl buffer . unilamellar vesicles are prepared as described in examples 4 - 8 using ph 9 . 3 ammonium hydroxide . unilamellar vesicles are prepared as described in examples 4 - 8 using ph 9 glycine . unilamellar vesicles are prepared as described in examples 4 - 12 , with the exception that the vesicles are formed by sonication rather than extrusion . unilamellar vesicles are prepared as described in examples 4 - 13 , except the drug used is sn - 38 . unilamellar vesicles are prepared as described in examples 4 - 13 , except the drug used is karenitecan . unilamellar vesicles are prepared as described in examples 4 - 13 , except the drug used is gimatecan . unilamellar vesicles are prepared as described in examples 4 - 13 , except the drug used is 9 - nitro camptothecin . unilamellar vesicles are prepared as described in examples 4 - 17 , with the exception that during separation of entrapped from unentrapped drug , the extraliposomal buffer is exchanged for ph 7 . 4 phosphate with the proviso that intraliposomal ph is maintained the same as that used in liposome preparation . unilamellar vesicles are prepared as described in examples 4 - 17 , with the exception that during separation of entrapped from unentrapped drug , the extraliposomal buffer is exchanged for a desired concentration of nacl solution with the proviso that intraliposomal ph is maintained the same as that used in liposome preparation . unilamellar vesicles are prepared as described in examples 4 - 17 , with the exception that during separation of entrapped from unentrapped drug , the extraliposomal buffer is exchanged for a desired concentration of sucrose solution , with the proviso that intraliposomal ph is maintained the same as that used in liposome preparation . unilamellar vesicles are prepared as described in examples 4 - 20 , using a lipid mixture comprising 80 % dspc , 15 % cholesterol , and 5 % m - peg dspe . unilamellar vesicles are prepared as described in examples 4 - 20 , using a lipid mixture comprising 80 % hspc , 15 % cholesterol , and 5 % pegylated pe . unilamellar vesicles are prepared as described in examples 4 - 20 , using a lipid mixture comprising 55 % dspc , 40 % cholesterol , and 5 % m - peg dspe . the skilled artisan will readily appreciate that the present disclosure sets forth an efficient and efficacious method for preparing a liposomal formulation of a camptothecin , in one aspect being a neutral camptothecin , camptothecin prodrug , or analog thereof . advantageously , the compositions formulated by the present method provide a stable liposomal camptothecin in therapeutically effective amounts , wherein the drug is retained in the liposome under physiological conditions for increased periods of time . this allows accumulation of the liposomal camptothecin formulations at a tumor site , with limited side effects on healthy cells and tissue , and further allows in situ delivery of the active lactone form of the drug to directly to tumor tissue in adequate concentrations for effective tumor cell killing and / or growth inhibition . by increasing intraliposomal ph and maintaining that ph prior to administration and during in vivo delivery to a tumor site , it has been surprisingly found that liposomal retention of a camptothecin may be prolonged , reducing the potential for exposure of healthy tissue to the drug when administered in vivo . this improvement in retention allows the drug to accumulate at tumor tissue , i . e ., an in vivo enhanced permeation and retention effect ( drummond et al ., 1999 ). as the liposomes accumulate within the tumor tissue , over time the drug is released as the lactone form in situ , thus achieving release of drug in the active form directly at the tumor site , rather than drug residing in the bloodstream at physiological conditions favoring conversion to the inactive carboxylate form . accordingly , the problems of enhanced liposomal retention and delivery of the active lactone form camptothecin to a tumor site are simultaneously solved . the foregoing description of preferred embodiments has been presented for purposes of illustration and description . it is not intended to be exhaustive or limiting to the precise forms disclosed . obvious modifications or variations are possible in light of the above teachings . the embodiments were chosen and described to provide the best illustration of the principles described herein and their practical application to thereby enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated . all such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly , legally and equitably entitled .
a method for preparing a stable dispersion of a camptothecin , camptothecin prodrug , or analog thereof for administration to a patient in need thereof includes preparing a solution of the camptothecin , wherein the solution has a ph which places substantially an entirety of that camptothecin in a carboxylate form . the camptothecin may be a neutral camptothecin . the solution is next loaded into a liposome including at least one lipid , which may be a phospholipid . an intraliposomal ph is maintained which preserves substantially an entirety of the camptothecin in the carboxylate form . the liposomal dispersion is administered to an individual in need thereof , whereby the liposomes accumulate at and permeate into tumor tissue and an active , lactone form camptothecin is released in situ at the tumor site . compositions formulated in accordance with the described method for treatment of a cancer in an animal in need thereof are provided also .