Biological Activity of Urease Formulated in Poloxamer 407 after lntraperitoneal Injection in the Rat E. A. PEC, 2. G.WOUT, AND T. P. JOHNSTON' Received December 5, 1990, from the Department of Pharmaceutics (M/C 880), Coll e of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612. Accepted for publication August 21, 3 9 1 . Abstract 0 The advent of genetic engineering has resulted in a prolif-
eration of protein pharmaceuticals available for a variety of therapeutic needs. However, the formulation and delivery of these proteins remain an intriguing challenge. Polymer-based protein drug delivery systems continue to be investigated, although many of the fabrication techniques used to incorporate proteins into the polymer matrix or device result in irreversible inactivation (denaturation) of the proteins. A wellcharacterized model enzyme, urease, was formulated in 33% (wlw) poloxamer 407 (PluronicF-127) vehicle and injected intraperitoneally (ip) into rats in an attempt to achieve both preservation of biological activity and sustained release of the protein. The resulting ammonia concentration in plasma-time profileswere compared with those for rats injected with an identical dose (27.6 units of activity per 200 g of body weight) of urease dissolved in pH 7 phosphate buffer. Neither a pH 7 phosphate buffer solution nor poloxamer 407 (33%, w/w) dissolved in pH 7 phosphate buffer, when injected ip into rats, resulted in elevated ammonia levels in plasma. The time to reach a maximum ammonia level in plasma was increased approximatelythreefold following the injection of the urease-poloxamer 407 formulation,compared with that In control rats administered an identical dose of urease in solution. In addition, hyperammonernia was extended almost threefold in treated rats cornpared with control rats, without untoward effects. However, prolonged hyperammonemia in animals receiving an ip injection of the ureasepoloxamer 407 formulation may have potentially resulted from the reduced clearance of ammonia and ammonium ion in the proximal tubules of the rats. Thus, it was not possible to definitively assign the threefold increase in the time to the maximum ammonia level in plasma to either the sustained release of urease from the semisolid ureasepoloxamer 407 matrix in the peritoneal cavity or a poloxamer 407induced decrease in the elimination of ammonia by the rat kidney. The half-lifeof elimination for poloxamer 407 in the urine of rats following an ip injection of poloxamer 407 vehicle alone (1.7 g/kg) was estimated to be 20.9 f 0.9 h. On the basis of a 1-g dose of a potential proteinpoloxamer 407 formulation containing 33% (w/w) poloxamer 407 being injected intramuscularly or subcutaneously into a 70-kg patient (dose of g/kg), it is anticipated that poloxamer 407 poloxamer 407, 4.7 x would not interfere with the renal elimination of a protein cleared predominantly by the kidney. Thus, our preliminary results suggest that poloxamer 407 may potentially be a useful vehicle for preserving the biological activity of select recombinant-derived protein pharmaceuticals that are administered extravascuiarly. In addition,the potential exists for such a protein-poloxamer407 formulation to sustain the rate of input of the therapeutic protein into the systemic circulation following subcutaneous or intramuscular injection.
Owing primarily to significant advances in recombinant DNA techniques, a vast array of protein pharmaceuticals are currently being developed. Most of these genetically engineered drugs will be designed for parenteral administration because of the inherent limitations to the delivery of these biologically active molecules across biomembranes.13 Proteins administered by the intravenous (iv) route tend to be cleared from the circulation quite rapidly because of renal elimination and equilibration with the extravascular interstitial fluids.' The therapeutic efficacy of several recombinant-derived proteins could be improved if the blood levels of 626 I Journal of Pharmaceutical Sciences Vol. 81, No. 7, July 1992
the proteins could be maintained within some predetermined therapeutic range.4 In addition, many of the toxic effects of these potent proteins and hormones could be avoided by administering less total drug less frequently. The requisite of frequent injections of any protein or nonprotein formulation is less acceptable to the patient.6 The integrity of the injection site with repeated administration must also be considered. As an example, frequent injections of a protein formulation or a formulation containing a low-molecular-weight drug substance into the muscle tissue may potentially result in acute andlor chronic myotoxicity.6~7 Polymeric carriers have been used with several proteins to maintain blood levels within a desired therapeutic range.8 However, if a polymeric carrier is to be used in the formulation and subsequent controlled delivery of a protein, then one of the primary concerns is whether the polymeric vehicle or matrix causes irreversible denaturation (inactivation) of the protein. Presently, many formulation approaches used to incorporate proteins into polymeric systems often result in irreversible inactivation of the proteins because of the presence of organic solvents, pH changes, and thermal effects. The pharmaceutical literature concerned with formulation strategies for newer protein pharmaceuticals is rather limited. Approaches that have been reported to enhance the physical stability of recombinant proteins include the use of low-ionicstrength buffers; the avoidance of transition metals; the use of carbohydrates, polyalcohols, and certain amino acids; and the incorporation of surfactants.9 However, the last approach is generally reserved for nonionic surfactants. In addition, it has been reported that nonionic detergents have often been used as pharmaceutical additives for the stabilization of proteins.'+-'* In particular, Tween and Pluronic detergents have been investigated for their potential to prevent the adsorption of proteins to surfaces,12J3 to inhibit aggregation and precipitation,loJl and to hinder denaturation.14J6 It has also been reported that many proteins can be refolded and thus stabilized by the use of appropriate detergents. Nonionic detergents have also been reported to facilitate the reactivation of denatured enzymes. Thus, the purpose of the present study was to evaluate the ability of a commercially available block copolymer, namely, a Pluronic polyol (poloxamer407; Pluronic F-127),to preserve the biological activity of a model enzyme following intraperitoneal (ip) administration to rats. It was hypothesized that poloxamer 407 might serve the dual role of preserving the activity of the enzyme in a physiological environment (elevated temperature, presence of proteases, etc.) and allowing for the controlled release of the enzyme from a semisolid poloxamer 407 matrix formed immediately after ip injection of a urease-poloxamer 407 formulation into rats. Prior research lended support for such a hypothesis, because it had been demonstrated that urease incubated in vitro in pH 7 phosphate buffer containing poloxamer 407 (14%, w/w) for 8 h at 4, 22, and 37 "C lost negligible biological activity.16 In OO22-3~9/92/07OO-0626$02.50/0 0 1992, American Pharmaceutical Association
addition, the release of urease from a poloxamer 407 gel matrix (35%, w/w) in vitro a t 37 "C proceeded at a constant (zero-order) rate.16 Further support for the hypothesis that the urease (protein) might potentially diffuse from the semisolid polymer matrix in a sustained fashion after ip administration was provided by results demonstrating that poloxamer 407 was able to sustain the blood levels of a biologically inactive macromolecule (inulin) following intramuscular (im) administration to rats." In addition to testing our hypothesis that the biological activity of urease might be retained if urease were formulated with poloxamer 407 and injected ip into rats, we also wished to estimate the rate of renal elimination of poloxamer 407 in the rat. The enzyme (protein) selected for incorporation into the block copolymer was urease. Urease was selected as the model protein for inclusion into the poloxamer 407 vehicle because it was inexpensive, it has been extensively studied in animal models, and it is highly specific and efficient in catalyzing the hydrolysis of urea to ammonia. Poloxamer 407was selected as the parenteral vehicle because of its reverse thermal gelation properties.18 This property allowed for the ip administration via syringe of a urease-poloxamer 407 formulation as a cool solution, with gelation occurring immediately &r ip injection because of an increase in temperature.
Experimental Section Materials-Urease (from jack beam; type IV; lot 55F-9357) having an activity of 69 000 unitdg and dibasic sodium phosphate were purchased from Sigma (St.Louis, MO). Poloxamer 407, NF (National Formulary XVII) grade, was a girt from the BASF Corporation (Parsippany, NJ) and was used as received. Monosodium potaasium phosphate, sodium hydroxide, and tetrasodium EDTA were obtained from Fisher (Fair Lawn, NJ). Sodium chloride injection solution, USP, was obtained from Abbott Laboratories (North Chicago, IL). Heparin sodium injection solution, USP, was purchased from ElkinsSinn, Inc. (Cherry Hill, NJ). The animals used were male SpragueDawley rats (200-225 g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis, IN). Injections were performed with a 1-mL tuberculin syringe fitted with a 19-gauge, 1.5-in. (ca. 3.8-cm) needle (Becton Dickinson & Co., Rutherford, NJ). Urine samples were filtered with 0.22-pm-pore-size disposable filters obtained from Gelman Sciences (Ann Arbor, MI). Filtered urine was placed in 15-mL disposable polypropylene centrifuge tubes (Corning Laboratory Sciences, Park Ridge, IL). Deoxycholate (DOC), trichloroacetic acid (TCA), and sodium azide were purchased from Sigma and used as received. Blood samples were collected into disposable glass culture tubes obtained from VWR Scientific (San Francisco, CA). Ammonia levels in plasma were determined with a model 700 Kodak EKTACHEM clinical blood analyzer purchased from the Eastman Kodak Company (Rochester, NY). Polymer quantitation was done with the aid of a total-protein kit containing bicinchoninic acid (microprotein assay kit 23235; Pierce Scientific, Rockford, IL). All weighing was performed with an analytical balance (model AE240; Metler, Hightstown, NJ). Methods-Preparation of Urease Formulation-The ureasepoloxamer 407 formulation for ip administration was prepared by adding 10 mg (ca. 690 unite) of urease to an appropriate volume of phosphate buffer. The phosphate buffer consisted of 0.05 M monobasic potassium phosphate and 0.015 M dibasic sodium phosphate adjusted to pH 7.0 k 0.01 with 5 N NaOH. The buffer system also contained EDTA a t a concentration of 1 mM.19 Following complete dimlution of the urease in the phosphate buffer, an appropriate amount of poloxamer 407 was added so that, when additional phosphate buffer was added, the final polymer concentration would be 33% (w/w). The veesel was placed on ice overnight to facilitate dissolution of the polymer by the cold method.18 On the following day, the vessel was gently swirled (25 rpm) a t 4°C on an orbital shaker (Lab Line Instruments, Melrose Park, IL) to ensure a uniform and homogeneous solution. Urease Administration a n d Blood Sampling-The ureasepoloxamer 407 formulation was administered ip as a cold solution to the rate. The volume of the formulation selected for injection was 1
mL. The weight of each of the syringes containing the formulation was determined on an analytical balance prior to and after ip injection. Four groups of four rats each were studied. The first group of rats received a 1-mL injection of pH 7 phosphate buffer. To as8e88 any changes in ammonia levels in plasma that could potentially result from the administration ofpoloxamer 407, we injected a second group of rats with a 33% (w/w) solution of poloxamer 407 diseolved in phosphate buffer (pH 7.0). The remaining two groups of rate received identical doses of urease (400 pg of ureaae per 200 g of body weight; equivalent to approximately 27.6 units of activity per 200 g of body weight). However, the third group received an injection of ureaae dissolved in sterile phosphate buffer (pH 71, whereas the fourth group received an injection of the urease-poloxamer 407 formulation. The concentration of the polymer used in the fourth group of rate was 33% (w/w). Following ip injection, the rats were placed in cages and periodically removed at various time points for blood collection. Blood samples (1 mL) were collected by tail clippingm into h e p arinized glass test tubes. The blood samples were placed on ice and centrifuged at 1500 rpm (centrifuge model Centra-7R, International Equipment Company) within 30 min of blood collection. The plasma was stored at -70 "C until the time of analysis. Determination of Blood Ammoniu-The concentration of ammonia in plasma for each rat was determined with an EKTACHEM clinical blood analyzer and expressed as micromoles per liter. The EKTACHEM blood analyzer determines ammonia levels in blood by use of a colorimetric assay based on reflectance spectroscopy.21 Urinary Excretion of PoloMmer 407-To assess the renal elimination kinetics of the polymeric vehicle, we iqjected each of three rats with 1g of a chilled solution of poloxamer 407 (33%, w/w). The rats were placed in individual metabolism cages for 4 days and allowed food and water ad libitum. Urine samples were collected over the following time intervals for each r a t 0-24, 24-48, 48-72, and 72-96 h. At the end of each collection interval, the volume of urine obtained was determined, and the samples were frozen at -70 "C until the time of polymer analysis. Analysis of urine samples for polymer content was done by first vortexing the tubes containing the thawed urine specimens for 1min. Approximately 5 mL of urine from each time interval for each rat was filtered through a 0.22-jm-pore-diameter filter and added to a tared 15-mL centrifuge tube. The tubes were weighed again on the analytical balance to determine the weight of the filtered urine sample. To each filtered urine sample was added 0.7 mL of DOC, the tube contents were mixed, and the tubes were allowed to stand at room temperature for 15 min. Afbr 15 min of incubation, 0.8 mL of TCA was added to each tube, and the tubes were vortexed and then centrifuged at 2800 rpm (centrifuge model Centra-7R, International Equipment Company) for 15 min at 10 "C. The DOC-TCA protein precipitation procedure was previously observed in our laboratory (unpublished data) to precipitate poloxamer 407 aRer centrifugation. Sodium azide was added to each tube containing a urine sample to yield a final sodium azide concentration of 0.1% (w/v). All tubes were stored at room temperature until the supernatant had completely evaporated. To correct for any additional weight contributed by DOC, TCA, and sodium azide added to each urine sample, we treated a set of three control tubes each containing an accurately weighed volume of doubly deionized water (-5 g) in the same manner as t u b containing urine samples. Following evaporation of the supernatant, all tubes were placed in a desiccator for 3 days to remove any residual moisture. After the tubes were removed from the desiccator, they were weighed again. The amount of polymer excreted in a given 24-h time interval was calculated by first subtracting the mean residual weight of DOC, TCA, and sodium azide (52.9 f 4.6 mg) added to the control tubes from the net weight of the dried protein-polymer precipitate from each urine sample. Next, the weight of protein and polymer contained in the original volume of urine collected over each 24-h time interval for each rat was calculated (normal urine output, 10-15 muday). Lastly, the weight of protein that would normally be excreted by a control rat (0.4-1.0 mg/mL)22 into the original volume of urine collected from each rat over each 24-h time interval was subtracted from the weight of protein and polymer that was calculated to be present in the original volume of urine collected from each rat over each 24-h urine collection period. The percentage of the total dose (330 mg) of polymer excreted over each 24-h urine collection interval was then calculated. The percentage of polymer excreted in the urine over any 24-h time interval was expressed as a range because of the variation in the
Journal of Pharmaceutical Sciences I 627 Vol. 81, No. 7, JuM 1992
amount of protein normally excreted in rat urine. Polymer Detection in Urine-To determine the percentage of polymer retained in the supernatant after the DOC-TCA-ntrifugation procedure, we developed a method to quantitate the amount of polymer present in an aqueous-based biological matrix such as urine. To quantitate the amount of polymer in an aqueous sample, we added 0.015 g of poloxamer 407 to each of three separate glassfials. Phosphate buffer (pH 7.0) was added to each vial to yield a final weight of 3.0 g or a polymer concentration of 0.5% (w/w). Three polymer solutions were prepared in a similar manner at the following concentrations: 1.0,2.0,4.0,6.0, and 8.0% (w/w). All vials were placed on ice overnight to facilitate dissolution of the polymer. On the next day, 1mL of a given polymer solution was combined with 1ml of the working reagent contained in the microprotein assay kit, and the tubes were vortexed and placed in a 60 "C water bath for 1h. After cooling to room temperature, each solution was analyzed spectrophotometrically at 562 nm in accordance with the manufacturer's instructions. Similar analyses were performed on 1-mL samples of supernatants obtained from urine specimens that had been treated with DOC-TCA and tentrifuged. Data Analys&All ammonia concentrations in plasma were determined at each time,point for each rat in the four study groups. The mean values of the ammonia concentrations in plasma determined for rate contained in the same group were calculated and plotted against sampling time. No attempt was made to fit the concentration-time data for each rat to a polyexponential equation because of the limited blood samples obtained during the absorption phase. However, the terminal elimination phase of each ammonia concentration in plasma-time profile w& plotted on semilogarithmic paper to obtain an estimate of the elimination rate constant for ammonia for the two groups to which urease was administered. The t- (time to reach a maximum ammonia concentration in plasma) values listed in Table I are simply the time points at which the maximum ammonia concentration in plasma was observed relative to those at all other sampling time points, not the t,, values predicted from a mathematical fit of the data. Similarly, the C, (maximum ammonia concentration in plasma) values are the ammonia concentrations in plasma corresponding to the t- values listed in Table I, not the C, values predicted from a mathematical fit of the concentration-time data. The area under the curve (AUC) for each ammonia concentration in plasma-time profile was calculated by trapezoidal integration to time infinity. The apparent clearance of ammonia in plasma was not included in Table I, because the concentration of an end product of a biochemical reaction, namely, NH,, not that of the administered urease, was determined in plasma over time. Hence, the exact dose or quantity of NHS produced after ip iqjection of a given quantity of urease was unknown. Estimates of the percentage of polymer excreted in the urine over each 24-h urine collection interval were expreased as ranges because of the variation in the amount of protein normally excreted in rat urine. Statistical significance of differences between mean values of the pharmacokinetic parameters listed in Table I was determined by use of the t test.
Results Plasma Ammonia Levels following ip Injection of UreFigure 1, ammonia levels in plasma after a urease dose of 400 pgl200 g of body weight (27.6 units per 200 g of body weight) administered as an aqueous solution reached a maximum of -2200 #I at 2 h postinjection. An identical dose of the enzyme administered as a ureasepoloxamer 407 formulation gave rise to an ammonia concena s ~ b shown , in
3000 -
2500
i
M
I Z
eration of protein pharmaceuticals available for a variety of therapeutic needs. However, the formulation and delivery of these proteins remain an intriguing challenge. Polymer-based protein drug delivery systems continue to be investigated, although many of the fabrication techniques used to incorporate proteins into the polymer matrix or device result in irreversible inactivation (denaturation) of the proteins. A wellcharacterized model enzyme, urease, was formulated in 33% (wlw) poloxamer 407 (PluronicF-127) vehicle and injected intraperitoneally (ip) into rats in an attempt to achieve both preservation of biological activity and sustained release of the protein. The resulting ammonia concentration in plasma-time profileswere compared with those for rats injected with an identical dose (27.6 units of activity per 200 g of body weight) of urease dissolved in pH 7 phosphate buffer. Neither a pH 7 phosphate buffer solution nor poloxamer 407 (33%, w/w) dissolved in pH 7 phosphate buffer, when injected ip into rats, resulted in elevated ammonia levels in plasma. The time to reach a maximum ammonia level in plasma was increased approximatelythreefold following the injection of the urease-poloxamer 407 formulation,compared with that In control rats administered an identical dose of urease in solution. In addition, hyperammonernia was extended almost threefold in treated rats cornpared with control rats, without untoward effects. However, prolonged hyperammonemia in animals receiving an ip injection of the ureasepoloxamer 407 formulation may have potentially resulted from the reduced clearance of ammonia and ammonium ion in the proximal tubules of the rats. Thus, it was not possible to definitively assign the threefold increase in the time to the maximum ammonia level in plasma to either the sustained release of urease from the semisolid ureasepoloxamer 407 matrix in the peritoneal cavity or a poloxamer 407induced decrease in the elimination of ammonia by the rat kidney. The half-lifeof elimination for poloxamer 407 in the urine of rats following an ip injection of poloxamer 407 vehicle alone (1.7 g/kg) was estimated to be 20.9 f 0.9 h. On the basis of a 1-g dose of a potential proteinpoloxamer 407 formulation containing 33% (w/w) poloxamer 407 being injected intramuscularly or subcutaneously into a 70-kg patient (dose of g/kg), it is anticipated that poloxamer 407 poloxamer 407, 4.7 x would not interfere with the renal elimination of a protein cleared predominantly by the kidney. Thus, our preliminary results suggest that poloxamer 407 may potentially be a useful vehicle for preserving the biological activity of select recombinant-derived protein pharmaceuticals that are administered extravascuiarly. In addition,the potential exists for such a protein-poloxamer407 formulation to sustain the rate of input of the therapeutic protein into the systemic circulation following subcutaneous or intramuscular injection.
Owing primarily to significant advances in recombinant DNA techniques, a vast array of protein pharmaceuticals are currently being developed. Most of these genetically engineered drugs will be designed for parenteral administration because of the inherent limitations to the delivery of these biologically active molecules across biomembranes.13 Proteins administered by the intravenous (iv) route tend to be cleared from the circulation quite rapidly because of renal elimination and equilibration with the extravascular interstitial fluids.' The therapeutic efficacy of several recombinant-derived proteins could be improved if the blood levels of 626 I Journal of Pharmaceutical Sciences Vol. 81, No. 7, July 1992
the proteins could be maintained within some predetermined therapeutic range.4 In addition, many of the toxic effects of these potent proteins and hormones could be avoided by administering less total drug less frequently. The requisite of frequent injections of any protein or nonprotein formulation is less acceptable to the patient.6 The integrity of the injection site with repeated administration must also be considered. As an example, frequent injections of a protein formulation or a formulation containing a low-molecular-weight drug substance into the muscle tissue may potentially result in acute andlor chronic myotoxicity.6~7 Polymeric carriers have been used with several proteins to maintain blood levels within a desired therapeutic range.8 However, if a polymeric carrier is to be used in the formulation and subsequent controlled delivery of a protein, then one of the primary concerns is whether the polymeric vehicle or matrix causes irreversible denaturation (inactivation) of the protein. Presently, many formulation approaches used to incorporate proteins into polymeric systems often result in irreversible inactivation of the proteins because of the presence of organic solvents, pH changes, and thermal effects. The pharmaceutical literature concerned with formulation strategies for newer protein pharmaceuticals is rather limited. Approaches that have been reported to enhance the physical stability of recombinant proteins include the use of low-ionicstrength buffers; the avoidance of transition metals; the use of carbohydrates, polyalcohols, and certain amino acids; and the incorporation of surfactants.9 However, the last approach is generally reserved for nonionic surfactants. In addition, it has been reported that nonionic detergents have often been used as pharmaceutical additives for the stabilization of proteins.'+-'* In particular, Tween and Pluronic detergents have been investigated for their potential to prevent the adsorption of proteins to surfaces,12J3 to inhibit aggregation and precipitation,loJl and to hinder denaturation.14J6 It has also been reported that many proteins can be refolded and thus stabilized by the use of appropriate detergents. Nonionic detergents have also been reported to facilitate the reactivation of denatured enzymes. Thus, the purpose of the present study was to evaluate the ability of a commercially available block copolymer, namely, a Pluronic polyol (poloxamer407; Pluronic F-127),to preserve the biological activity of a model enzyme following intraperitoneal (ip) administration to rats. It was hypothesized that poloxamer 407 might serve the dual role of preserving the activity of the enzyme in a physiological environment (elevated temperature, presence of proteases, etc.) and allowing for the controlled release of the enzyme from a semisolid poloxamer 407 matrix formed immediately after ip injection of a urease-poloxamer 407 formulation into rats. Prior research lended support for such a hypothesis, because it had been demonstrated that urease incubated in vitro in pH 7 phosphate buffer containing poloxamer 407 (14%, w/w) for 8 h at 4, 22, and 37 "C lost negligible biological activity.16 In OO22-3~9/92/07OO-0626$02.50/0 0 1992, American Pharmaceutical Association
addition, the release of urease from a poloxamer 407 gel matrix (35%, w/w) in vitro a t 37 "C proceeded at a constant (zero-order) rate.16 Further support for the hypothesis that the urease (protein) might potentially diffuse from the semisolid polymer matrix in a sustained fashion after ip administration was provided by results demonstrating that poloxamer 407 was able to sustain the blood levels of a biologically inactive macromolecule (inulin) following intramuscular (im) administration to rats." In addition to testing our hypothesis that the biological activity of urease might be retained if urease were formulated with poloxamer 407 and injected ip into rats, we also wished to estimate the rate of renal elimination of poloxamer 407 in the rat. The enzyme (protein) selected for incorporation into the block copolymer was urease. Urease was selected as the model protein for inclusion into the poloxamer 407 vehicle because it was inexpensive, it has been extensively studied in animal models, and it is highly specific and efficient in catalyzing the hydrolysis of urea to ammonia. Poloxamer 407was selected as the parenteral vehicle because of its reverse thermal gelation properties.18 This property allowed for the ip administration via syringe of a urease-poloxamer 407 formulation as a cool solution, with gelation occurring immediately &r ip injection because of an increase in temperature.
Experimental Section Materials-Urease (from jack beam; type IV; lot 55F-9357) having an activity of 69 000 unitdg and dibasic sodium phosphate were purchased from Sigma (St.Louis, MO). Poloxamer 407, NF (National Formulary XVII) grade, was a girt from the BASF Corporation (Parsippany, NJ) and was used as received. Monosodium potaasium phosphate, sodium hydroxide, and tetrasodium EDTA were obtained from Fisher (Fair Lawn, NJ). Sodium chloride injection solution, USP, was obtained from Abbott Laboratories (North Chicago, IL). Heparin sodium injection solution, USP, was purchased from ElkinsSinn, Inc. (Cherry Hill, NJ). The animals used were male SpragueDawley rats (200-225 g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis, IN). Injections were performed with a 1-mL tuberculin syringe fitted with a 19-gauge, 1.5-in. (ca. 3.8-cm) needle (Becton Dickinson & Co., Rutherford, NJ). Urine samples were filtered with 0.22-pm-pore-size disposable filters obtained from Gelman Sciences (Ann Arbor, MI). Filtered urine was placed in 15-mL disposable polypropylene centrifuge tubes (Corning Laboratory Sciences, Park Ridge, IL). Deoxycholate (DOC), trichloroacetic acid (TCA), and sodium azide were purchased from Sigma and used as received. Blood samples were collected into disposable glass culture tubes obtained from VWR Scientific (San Francisco, CA). Ammonia levels in plasma were determined with a model 700 Kodak EKTACHEM clinical blood analyzer purchased from the Eastman Kodak Company (Rochester, NY). Polymer quantitation was done with the aid of a total-protein kit containing bicinchoninic acid (microprotein assay kit 23235; Pierce Scientific, Rockford, IL). All weighing was performed with an analytical balance (model AE240; Metler, Hightstown, NJ). Methods-Preparation of Urease Formulation-The ureasepoloxamer 407 formulation for ip administration was prepared by adding 10 mg (ca. 690 unite) of urease to an appropriate volume of phosphate buffer. The phosphate buffer consisted of 0.05 M monobasic potassium phosphate and 0.015 M dibasic sodium phosphate adjusted to pH 7.0 k 0.01 with 5 N NaOH. The buffer system also contained EDTA a t a concentration of 1 mM.19 Following complete dimlution of the urease in the phosphate buffer, an appropriate amount of poloxamer 407 was added so that, when additional phosphate buffer was added, the final polymer concentration would be 33% (w/w). The veesel was placed on ice overnight to facilitate dissolution of the polymer by the cold method.18 On the following day, the vessel was gently swirled (25 rpm) a t 4°C on an orbital shaker (Lab Line Instruments, Melrose Park, IL) to ensure a uniform and homogeneous solution. Urease Administration a n d Blood Sampling-The ureasepoloxamer 407 formulation was administered ip as a cold solution to the rate. The volume of the formulation selected for injection was 1
mL. The weight of each of the syringes containing the formulation was determined on an analytical balance prior to and after ip injection. Four groups of four rats each were studied. The first group of rats received a 1-mL injection of pH 7 phosphate buffer. To as8e88 any changes in ammonia levels in plasma that could potentially result from the administration ofpoloxamer 407, we injected a second group of rats with a 33% (w/w) solution of poloxamer 407 diseolved in phosphate buffer (pH 7.0). The remaining two groups of rate received identical doses of urease (400 pg of ureaae per 200 g of body weight; equivalent to approximately 27.6 units of activity per 200 g of body weight). However, the third group received an injection of ureaae dissolved in sterile phosphate buffer (pH 71, whereas the fourth group received an injection of the urease-poloxamer 407 formulation. The concentration of the polymer used in the fourth group of rate was 33% (w/w). Following ip injection, the rats were placed in cages and periodically removed at various time points for blood collection. Blood samples (1 mL) were collected by tail clippingm into h e p arinized glass test tubes. The blood samples were placed on ice and centrifuged at 1500 rpm (centrifuge model Centra-7R, International Equipment Company) within 30 min of blood collection. The plasma was stored at -70 "C until the time of analysis. Determination of Blood Ammoniu-The concentration of ammonia in plasma for each rat was determined with an EKTACHEM clinical blood analyzer and expressed as micromoles per liter. The EKTACHEM blood analyzer determines ammonia levels in blood by use of a colorimetric assay based on reflectance spectroscopy.21 Urinary Excretion of PoloMmer 407-To assess the renal elimination kinetics of the polymeric vehicle, we iqjected each of three rats with 1g of a chilled solution of poloxamer 407 (33%, w/w). The rats were placed in individual metabolism cages for 4 days and allowed food and water ad libitum. Urine samples were collected over the following time intervals for each r a t 0-24, 24-48, 48-72, and 72-96 h. At the end of each collection interval, the volume of urine obtained was determined, and the samples were frozen at -70 "C until the time of polymer analysis. Analysis of urine samples for polymer content was done by first vortexing the tubes containing the thawed urine specimens for 1min. Approximately 5 mL of urine from each time interval for each rat was filtered through a 0.22-jm-pore-diameter filter and added to a tared 15-mL centrifuge tube. The tubes were weighed again on the analytical balance to determine the weight of the filtered urine sample. To each filtered urine sample was added 0.7 mL of DOC, the tube contents were mixed, and the tubes were allowed to stand at room temperature for 15 min. Afbr 15 min of incubation, 0.8 mL of TCA was added to each tube, and the tubes were vortexed and then centrifuged at 2800 rpm (centrifuge model Centra-7R, International Equipment Company) for 15 min at 10 "C. The DOC-TCA protein precipitation procedure was previously observed in our laboratory (unpublished data) to precipitate poloxamer 407 aRer centrifugation. Sodium azide was added to each tube containing a urine sample to yield a final sodium azide concentration of 0.1% (w/v). All tubes were stored at room temperature until the supernatant had completely evaporated. To correct for any additional weight contributed by DOC, TCA, and sodium azide added to each urine sample, we treated a set of three control tubes each containing an accurately weighed volume of doubly deionized water (-5 g) in the same manner as t u b containing urine samples. Following evaporation of the supernatant, all tubes were placed in a desiccator for 3 days to remove any residual moisture. After the tubes were removed from the desiccator, they were weighed again. The amount of polymer excreted in a given 24-h time interval was calculated by first subtracting the mean residual weight of DOC, TCA, and sodium azide (52.9 f 4.6 mg) added to the control tubes from the net weight of the dried protein-polymer precipitate from each urine sample. Next, the weight of protein and polymer contained in the original volume of urine collected over each 24-h time interval for each rat was calculated (normal urine output, 10-15 muday). Lastly, the weight of protein that would normally be excreted by a control rat (0.4-1.0 mg/mL)22 into the original volume of urine collected from each rat over each 24-h time interval was subtracted from the weight of protein and polymer that was calculated to be present in the original volume of urine collected from each rat over each 24-h urine collection period. The percentage of the total dose (330 mg) of polymer excreted over each 24-h urine collection interval was then calculated. The percentage of polymer excreted in the urine over any 24-h time interval was expressed as a range because of the variation in the
Journal of Pharmaceutical Sciences I 627 Vol. 81, No. 7, JuM 1992
amount of protein normally excreted in rat urine. Polymer Detection in Urine-To determine the percentage of polymer retained in the supernatant after the DOC-TCA-ntrifugation procedure, we developed a method to quantitate the amount of polymer present in an aqueous-based biological matrix such as urine. To quantitate the amount of polymer in an aqueous sample, we added 0.015 g of poloxamer 407 to each of three separate glassfials. Phosphate buffer (pH 7.0) was added to each vial to yield a final weight of 3.0 g or a polymer concentration of 0.5% (w/w). Three polymer solutions were prepared in a similar manner at the following concentrations: 1.0,2.0,4.0,6.0, and 8.0% (w/w). All vials were placed on ice overnight to facilitate dissolution of the polymer. On the next day, 1mL of a given polymer solution was combined with 1ml of the working reagent contained in the microprotein assay kit, and the tubes were vortexed and placed in a 60 "C water bath for 1h. After cooling to room temperature, each solution was analyzed spectrophotometrically at 562 nm in accordance with the manufacturer's instructions. Similar analyses were performed on 1-mL samples of supernatants obtained from urine specimens that had been treated with DOC-TCA and tentrifuged. Data Analys&All ammonia concentrations in plasma were determined at each time,point for each rat in the four study groups. The mean values of the ammonia concentrations in plasma determined for rate contained in the same group were calculated and plotted against sampling time. No attempt was made to fit the concentration-time data for each rat to a polyexponential equation because of the limited blood samples obtained during the absorption phase. However, the terminal elimination phase of each ammonia concentration in plasma-time profile w& plotted on semilogarithmic paper to obtain an estimate of the elimination rate constant for ammonia for the two groups to which urease was administered. The t- (time to reach a maximum ammonia concentration in plasma) values listed in Table I are simply the time points at which the maximum ammonia concentration in plasma was observed relative to those at all other sampling time points, not the t,, values predicted from a mathematical fit of the data. Similarly, the C, (maximum ammonia concentration in plasma) values are the ammonia concentrations in plasma corresponding to the t- values listed in Table I, not the C, values predicted from a mathematical fit of the concentration-time data. The area under the curve (AUC) for each ammonia concentration in plasma-time profile was calculated by trapezoidal integration to time infinity. The apparent clearance of ammonia in plasma was not included in Table I, because the concentration of an end product of a biochemical reaction, namely, NH,, not that of the administered urease, was determined in plasma over time. Hence, the exact dose or quantity of NHS produced after ip iqjection of a given quantity of urease was unknown. Estimates of the percentage of polymer excreted in the urine over each 24-h urine collection interval were expreased as ranges because of the variation in the amount of protein normally excreted in rat urine. Statistical significance of differences between mean values of the pharmacokinetic parameters listed in Table I was determined by use of the t test.
Results Plasma Ammonia Levels following ip Injection of UreFigure 1, ammonia levels in plasma after a urease dose of 400 pgl200 g of body weight (27.6 units per 200 g of body weight) administered as an aqueous solution reached a maximum of -2200 #I at 2 h postinjection. An identical dose of the enzyme administered as a ureasepoloxamer 407 formulation gave rise to an ammonia concena s ~ b shown , in
3000 -
2500
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