The subject matter described herein relates to an endogenously-formed conjugate comprised of a therapeutic agent and endogenous albumin, and to methods of providing a therapeutic agent in the form of a conjugate comprised of the therapeutic agent and endogenous albumin.
Human serum albumin is a multifunctional protein found in the bloodstream. It is an important factor in the regulation of plasma volume and tissue fluid balance through its contribution to the colloid osmotic pressure of plasma. Albumin normally constitutes 50-60% of plasma proteins and because of its relatively low molecular weight (66,500 Daltons), exerts 80-85% of the colloidal osmotic pressure of the blood. Albumin regulates transvascular fluid flux and hence, intra and extravascular fluid volumes, and transports lipid and lipid-soluble substances. Albumin solutions are frequently used for plasma volume expansion and maintenance of cardiac output in the treatment of certain types of shock or impending shock including those resulting from burns, surgery, hemorrhage, or other trauma or conditions in which a circulatory volume deficit is present.
Albumin has a blood circulation half-life of approximately two weeks and is designed by nature to carry lipids and other molecules. A hydrophobic binding pocket and a free thiol cysteine residue (Cys34) are features that enable this function. Due to its low pKa (approx. 7) Cys34 is one of the more reactive thiol groups appearing in human plasma. The Cys34 of albumin also accounts for the major fraction of thiol concentration in blood plasma (over 80%) (Kratz et al., J. Med. Chem., 45(25):5523-33 (2002)). The ability of albumin through its reactive thiol to act as a carrier has been utilized for therapeutic purposes. For example, attachment of drugs to albumin to improve the pharmacological properties of the drugs has been described (Kremer et al., Anticancer Drugs, 13:(6):615-23 (2002); Kratz et al., J. Drug Target., 8(5):305-18 (2000); Kratz et al., J. Med. Chem., 45(25):5523-33 (2002); Tanaka et al., Bioconjug. Chem., 2(4):261-9 (1991); Dosio et al., J. Control. Release, 76(1-2):107-17 (2001); Dings et al., Cancer Lett., 194(1):55-66 (2003); Wunder et al., J. Immunol., 170(9):4793-801 (2003); Christie et al., Biochem. Pharmacol., 36(20):3379-85 (1987)). The attachment of peptide and protein therapeutics to albumin has also been described (Holmes et al., Bioconjug. Chem., 11 (4):439-44 (2000), Leger et al., Bioorg. Med. Chem. Lett., 13(20):3571-5 (2003); Paige et al., Pharm. Res., 12(12):1883-8 (1995)). Conjugates of albumin and interferon-alpha (Albuferon™) and of albumin and human growth hormone (Albutropin™) and of albumin and interleukin-2 (Albuleukin™) are being tested for therapeutic effectiveness. The art also describes the use of standard recombinant molecular biology techniques to generate an albumin-protein fusion (U.S. Pat. No. 6,548,653). All but the latter conjugates with albumin involve ex vivo conjugate formation with an exogenous albumin. Potential drawbacks to using exogenous sources of albumin are contamination or an immunogenic response.
In vivo attachment of therapeutic agents to albumin has also been described, where, for example, a selected peptide is modified prior to administration to allow albumin to bind to the peptide. This approach is described using dipeptidyl peptidase IV-resistant glucagon-like-peptide-1 (GLP-1) analogs (Kim et al., Diabetes, 52(3):751-9 (2003)). A specific linker ([2-[2-[2-maleimido-propionamido-(ethoxy)-ethoxy]-acetamide) was attached to an added carboxyl-terminal lysine on the peptide to enable a cysteine residue of albumin to bind with the peptide. Others have investigated attaching specific tags to peptides or proteins in order to increase their binding to albumin in vivo (Koehler et al., Bioorg Med. Chem. Lett., 12(20):2883-6 (2002); Dennis et al., J. Biol. Chem., 277(38):35035-35043 (2002)); Smith et al., Bioconjug. Chem., 12:750-756 (2001)). A similar approach has been used with small molecule drugs, where a derivative of the drug was designed specifically to have the ability to bind with a cysteine residue of albumin. For example, this pro-drug strategy has been used for doxorubicin derivatives where the doxorubicin derivative is bound to endogenous albumin at its cysteine residue at position 34 (Cys34; Kratz et al., J Med. Chem., 45(25): 5523-33 (2002)). The in vivo attachment of a therapeutic agent to albumin has the advantage, relative to the ex vivo approach described above, in that endogenous albumin is used, thus obviating problems associated with contamination or an immunogenic response to the exogenous albumin. Yet, the prior art approach of in vivo formation of drug conjugates with endogenous albumin involves a permanent covalent linkage between the drug and the albumin. To the extent the linkage is cleavable or reversible, the drug or peptide released from the conjugate is in a modified form of the original compound.
It would be desirable to provide a conjugate of a therapeutic agent with endogenous albumin where the conjugate is (i) formed in vivo and (ii) reversible in vivo to yield the therapeutic agent in its native form.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
Accordingly, in one aspect, a method for delivering a therapeutic agent in the form of a conjugate with albumin is provided. The method comprises administering to a subject a compound of the form polymer-disulfide-therapeutic agent, wherein said therapeutic agent comprises at least one amine moiety. Administration of the compound achieves formation of a conjugate comprised of the subject's endogenous albumin and the therapeutic agent.
In one embodiment, the polymer-disulfide-therapeutic agent conjugate is a polymer-dithiobenzyl-therapeutic agent conjugate having the structure:
where orientation of CH2-therapeutic agent is selected from the ortho position and the para position.
In another embodiment, the amine-containing therapeutic agent is selected from a protein and a drug. In preferred embodiments, the therapeutic agent is a protein having a drug or a protein having a molecular weight of less than about 45 kDa, more preferably of less than 30 kDa, and still more preferably of 15 kDa or less.
The polymer, in a preferred embodiment, is polyethylene glycol or a modified polyethyleneglycol.
In another aspect, a prodrug for treatment of a subject is described, the prodrug being comprised of the subject's endogenous albumin and a therapeutic agent comprising at least one amine moiety, the albumin and the therapeutic agent joined by a disulfide.
In yet another aspect, a method for extending the blood circulation lifetime of a therapeutic agent is contemplated, the method involving administering a polymer-disulfide-therapeutic agent conjugate as described above to achieve formation of a prodrug conjugate comprised of endogenous albumin and the therapeutic agent.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.
“Protein” as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, polypeptide, oligopeptide, and enzyme are included within the definition of protein. This term also includes post-expression modifications of the protein, for example, glycosylations, acetylations, phosphorylations, and the like.
“Amine-containing” intends any compound having a moiety derived from ammonia by replacing one or two of the hydrogen atoms by alkyl or aryl groups to yield general structures RNH2 (primary amines) and R2NH (secondary amines), where R is any therapeutic moiety.
“Polymer” as used herein refers to a polymer having moieties soluble in water, which lend to the polymer some degree of water solubility at room temperature, i.e., the polymer is a hydrophilic polymer. Exemplary hydrophilic polymers include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropyl-methacrylamide, polymethacrylamide, polydimethyl-acrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, copolymers of the above-recited polymers, and polyethyleneoxide-polypropylene oxide copolymers. Properties and reactions with many of these polymers are described in U.S. Pat. Nos. 5,395,619 and 5,631,018. A preferred polymer is poly(ethyleneglycol) (PEG) and modified versions of PEG, such as methoxyPEG (mPEG). The molecular weight of the polymer is widely variable, and a typical range for mPEG is from 1,000 Daltons to 50,000 Daltons, more preferably, from 1,500 Daltons to 30,000 Daltons. In other embodiments, an mPEG molecular weight of less than about 30,000 Daltons is contemplated.
Reference to a polymer, drug, or therapeutic agent in the form of a “polymer-DTB-therapeutic agent conjugate” or to a “polymer-DTB-drug conjugate” or to an “albumin-therapeutic agent conjugate” or “albumin-drug conjugate” intends that the polymer, drug, or therapeutic agent is modified in some manner for conjugate formation, the modification including but not limited to addition of a functional group or loss of one or more chemical entities upon reaction with to form the conjugate.
Abbreviations: PEG, poly(ethylene glycol); mPEG, methoxy-PEG; DTB, dithiobenzyl; mDTB, methoxyDTB; EtDTB, ethoxyDTB; Epo, Erythropoietin; HSA, human serum albumin; BSA, bovine serum albumin; Cys, cysteine; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; MALDI-TOF MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; kDa, kilodaltons; EDTA, ethylenediaminetetraacetic acid; NBD, (7-nitrobenz-2-oxa-1,3-diazole).
In one aspect, a method for the in vivo formation of a compound comprised of endogenous albumin and a therapeutic agent is provided. The therapeutic agent can be any entity with an amine group, and exemplary entities are given below. It will be appreciated that conjugate formation between the two species, endogenous albumin and the therapeutic agent, results in modification of the endogenous albumin and/or the agent. Use of the terms “endogenous albumin” and “therapeutic agent” in the context of the conjugate intends residues of these species that comprise the conjugate. Formation of the in vivo adduct achieves an increased blood circulation lifetime of the therapeutic agent by virtue of its coupling with endogenous albumin. Thus, the method provides a solution to the problems associated with the short blood circulation time often observed with macromolecular biological therapeutics, and in particular, polypeptides, as well as low molecular weight drugs common in the pharmaceutical industry. By attaching endogenous albumin for use as a carrier protein, the lifetime of the polypeptide or drug can be extended, with the additional benefit of little, if any immunogenic response, since the patient's own albumin is used in formation of the conjugate.
As noted above, the therapeutic agent can be virtually any amine-containing compound. The compound can be a therapeutic agent or a diagnostic agent or a compound with neither therapeutic nor diagnostic activity but desirous of in vivo administration. In preferred embodiments, the amine-containing therapeutic agent is a drug or a protein. A wide variety of therapeutic drugs have a reactive amine moiety, such as mitomycin C, bleomycin, doxorubicin and ciprofloxacin, and the method contemplates any of these drugs with no limitation. The molecular weight of such drugs is typically less than 2 kDa, often less than 1 kDa. Most proteins contain reactive amino groups, and proteins for therapeutic purposes or for targeting purposes are known in the art. Exemplary proteins can be naturally occurring or recombinantly produced polypeptides. Small, human recombinant polypeptides are preferred, and polypeptides in the range of 0.1-45 kDa, more preferably 0.5-30 kDa, still more preferably of 1-15 kDa are preferred. Molecular weights of polypeptides are reported in the literature or can be determine experimentally using routine methods.
A general reaction scheme for preparation of a polymer-DTB-therapeutic agent conjugate is shown in
When mPEG-DTB-therapeutic agent conjugate is exposed to plasma, the free thiol of albumin Cys-34 attacks the DTB moiety of the conjugate, resulting in its decomposition, as illustrated in
Example 2 describes a study to illustrate an embodiment of the method, where conjugates comprised of methoxypolyethylene glycol (mPEG) and of lysozyme as a model therapeutic agent were prepared. Synthesis of the mPEG-DTB-lysozyme conjugates is described in Example 1A and conjugates with mPEG molecular weights of 5 kDa and 12 kDa (designated herein as mPEG5K-DTB-lysozyme and mPEG12K-DTB-lysozyme, respectively) were prepared. The conjugates were incubated with cysteine or with bovine serum albumin for 47 hours. Aliquots were withdrawn at times of 10 minutes, 30 minutes, 2 hours, 6 hours, 23 hours, and 47 hours for analysis via HPLC (Example 2). The results are shown in
The data in
In a study designed to identify the newly formed peaks, described in Example 3, a 1:1 conjugate of mPEG5K-DTB-lysozyme was prepared. The conjugate was incubated with BSA for two days and the incubation mixture was then analyzed by HPLC and by MALDI-TOFMS. The HPLC trace is shown in
Fractions obtained by ion-exchange chromatography (HPLC shown in
The BSA migration on SDS gels corresponds to molecular weight of approximately 55 kilodaltons (kDa) (Lane 8), although the theoretical molecular weight of albumin is 66.5 kDa. Fractions E2 and E3 (Lanes 1, 2) contained a major band having a molecular weight of approximately 60 kDa. The anticipated migration of an albumin-lysozyme (theoretical molecular weight 81 kDa) product would be 69 kDa, the sum of BSA (55 kDa) and lysozyme (14 kDa). The fractions loaded onto Lanes 1 and 2 having a molecular weight of 65 kDa are in good agreement with the molecular weight for an albumin-lysozyme conjugate. Fraction F1 (Lane 3) contains mPEG-lysozyme conjugate and some BSA contaminant. Fraction G2 (Lane 4) contains lysozyme only. Fraction G4 (Lane 5) contains lysozyme and another band that appears to be of approximate molecular weight of 24 kDa.
When the fractions identified from the HPLC E2, G2, and G4 were analyzed by both reducing (with β-mercaptoethanol) and non-reducing SDS-PAGE the following picture emerged. The gel is shown in
mPEG-DTB-lysozyme conjugates were also fluorescently labeled and examined in the presence of rat plasma or bovine serum albumin (BSA) over a timecourse at 37° C. As detailed in Example 4, the conjugates were labeled with ALEXA FLUOR 488, which labels free lysine residues in the lysozyme, and then incubated with rat plasma or with bovine serum albumin. Samples were collected as a function of time and analyzed by SDS PAGE. The fluorophore image was quantitated using a fluorescence imager. The SDS gel was also stained with SYPRO red to visualize total protein. The results are shown in
The data in
With respect to
The studies described above using lysozyme as a model therapeutic agent illustrate formation of a prodrug conjugate of albumin-lysozyme, subsequent to administration of a polymer-DTB-lysozyme conjugate. In a preferred embodiment, at least about 35% of the polymer-DTB-therapeutic agent conjugate that is administered is converted to a prodrug conjugate comprised of endogenous albumin and the therapeutic agent. In other words, of the total amount of therapeutic agent administered in the form of a polymer-DTB-therapeutic agent conjugate, at least about 35%, more preferably at least about 50%, still more preferably at least about 70%, is found in the blood two hours after administration in the form of an albumin-therapeutic agent conjugate.
Additional studies were conducted using erythropoietin (Epo) as a model therapeutic agent. A conjugate comprised of mPEG12K-DTB-Epo was prepared, as described in Example 5. For comparison, a non-cleavable conjugate of mPEG-Epo was also prepared. The conjugates were incubated in the presence of human serum albumin. In order to ensure all reaction products were visualized by SDS-PAGE, the concentration of HSA was significantly lower than physiological conditions and small molecule thiols were not included in the reaction, to prevent subsequent cleavage of the newly formed albumin-Epo conjugates. The albumin-Epo product is generated through a thiolytically cleavable bond as was observed when the reaction was treated with cysteine (data not shown).
According to prestained molecular weight markers in the gels, the apparent molecular weights of the molecules of interest by SDS-PAGE are as follows:
Fluorescently-labeled mPEG-DTB-Epo conjugates were observed in the presence of rat plasma or bovine serum albumin over a timecourse at 37° C., similar to the study discussed above for the mPEG-DTB-lysozyme conjugates (Example 4). The data for the mPEG-DTB-Epo conjugates (mPEG molecular weights of 12 kDa and 30 kDa) is shown in
The data in
Notably, and in comparison to the data described above on the lysozyme-containing conjugates, only about 25% of the Epo in the form of an mPEG-DTB-Epo conjugate was converted into an albumin-Epo conjugate, considerably less than observed for the lysozyme conjugates. Incubation of mPEG-DTB-Epo conjugate in plasma for two hours and longer resulted in 25-30% of the Epo appearing in the plasma in the form of an Epo-albumin conjugate.
Table 2 is a summary of the cleavage rates (T1/2 values) determined from the data presented in
*BSA = bovine serum albumin
The blood circulation half-life of the PEG12K-DTB-lysozyme conjugate was about five-fold less than the blood circulation half-life of the PEG12K-DTB-Epo conjugate, indicating a faster rate of cleavage of the disulfide linkage and formation of a conjugate with albumin.
The results above for the conjugates prepared with the model proteins Epo and lysozyme shows that an albumin-protein conjugate is formed when a polymer-DTB-protein conjugate interacts with albumin, with the smaller molecular weight protein yielding a greater amount of albumin-protein conjugate. Potentially, hindrance caused by the therapeutic protein charge or structure near the site of DTB attachment contributes to the yield of albumin-protein conjugate formed. The studies also show that the albumin-protein conjugate is cleaved in the presence of a reducing thiolytic agent, indicating that the linker is disulfide, likely to be the thiobenzyl linker.
Additional studies examining the cleavage rate of the disulfide-linker were performed, as described Example 6. Rather than a protein as in Examples 4 and 5, a small molecule, fluorescent amino acid derivative, lysine-NBD (7-nitrobenz-2-oxa-1,3-diazole), having a molecular weight of 344.79 Daltons, was used. Briefly, mPEG30K-DTB-NPC was conjugated to the fluorescent lysine-NBD. As a control, a non-cleavable conjugate of MPEG and lysine-NBD was prepared using mPEG-succinimidyl carbonate. The conjugates were incubated in bovine serum albumin with aliquots withdrawn at specified times for analysis by SDS-PAGE. The gels are shown in
A similar study was conducted where mPEG30K-DTB-Lysine-NBD conjugate was incubated with an equimolar concentration of BSA. The corresponding SDS-PAGE gels are shown in
In another study, described in Example 7, a Micrococcus luteus turbidity assay was used to analyze mPEG5K-DTB-lysozyme activity after treatment with 4% BSA or cysteine, or with saline as a control.
In vivo administration of the polymer-DTB-therapeutic agent was studied by administering a conjugate comprised of mPEG12K-DTB-lysozyme to rats. As described in Example 8, the mPEG12K-DTB-lysozyme was administered intravenously to a group of three rats. Additional rats were treated with a noncleavable mPEG-lysozyme conjugate or with free lysozyme as comparative control. Blood samples were taken at selected intervals over a 24 hour time period and analyzed for lysozyme concentration. The results are shown in
From the foregoing, it can be seen how various objects and features of the invention are met. The polymer-disulfide-therapeutic agent conjugate that is prepared ex vivo can be administered to a subject to achieve formation of an albumin-therapeutic agent conjugate that has a long drug circulation lifetime. While the studies above use a dithiobenzyl linkage, it will be appreciated that other disulfide linkages are equally applicable. The therapeutic agent in its native form is recovered after thiolytic cleavage of the albumin-therapeutic agent conjugate in vivo. The albumin-therapeutic agent conjugate is formed in situ using endogenous albumin. The long circulation time of albumin, and thus of the albumin-therapeutic agent conjugate, provides the ability of targeting the drug to tissues, such as tumors or to the synovium for treatment of rheumatoid arthritis. Those of skill in the art can appreciate the variety of disease conditions that would benefit from an extended blood circulation lifetime of a therapeutic agent. By increasing the circulation time of therapeutics such as protein molecules, less therapeutic agent may be required for treatment, thus reducing costs per dose. In addition, less frequent dosing is possible, therefore improving patient compliance. The technology described herein can be utilized with any therapeutic agent having an amine group.
The following examples further illustrate the invention described herein and are in no way intended to limit the scope of the invention.
This reaction scheme is illustrated in part in
A. mPEG-DTB-Lysozyme
mPEG-methylDTB-nitrophenylcarbonates of various molecular weights (5-30 kDa) were prepared as described in Example 2A of U.S. Pat. No. 6,605,299, which is incorporated by reference herein. The structure of the mPEG-Me-NPC conjugate is shown in
Lysozyme (at final concentration of 10 mg/mL) was allowed to react in borate buffer (0.1 M, pH 8.0) at 25° C. for 2-5 h with either mPEG-DTB-NPC or mPEG-NPC, using the feed molar ratio of 3.5 PEG/lysozyme (0.5 PEG/amino group). The conjugation reactions were quenched by the addition of 10-fold excess of glycine.
PEG-lysozyme conjugates were purified on a carboxymethyl HEMA-IEC Bio 1000 semi-preparative HPLC column (7.5×150 mm) purchased from Alltech Associates, Deerfield. IL. First, the conjugation reaction was injected into the HPLC column in 10 mM sodium acetate buffer pH 6. The elution with this buffer was continued until all unreacted PEG was removed. Then 0.2 M NaCl in 10 mM sodium acetate pH 6 was applied for 15 minutes in order to elute the PEGylated-lysozyme. Finally, the native lysozyme was eluted by increasing the salt concentration to 0.5 M NaCl over 20 min. Fractions (1 mL) were collected and assayed for protein and PEG contents. Thus aliquots (25 μL) of each fraction were reacted with BCA protein assay reagent (200 μL, Pierce Chemical Company, Rockford, Ill.) in microtiter plate wells at 37° C. for 30 min, and the absorbance was read at 562 nm. Similarly, for PEG determination, 25 μL aliquots were reacted with 0.1% polymethacrylic acid solution in 1 N HCl (200 μL) [S. Zalipsky & S. Menon-Rudolph (1997) Chapter 21, in Poly(ethylene Glycol): Chemistry and Biological Applications (J. M. Harris & S. Zalipsky, eds.), ACS Symposium Series 680, Washington, D.C., pp. 318-341], in microtiter plate wells, followed by absorbance reading at 400 nm. Fractions containing both protein and PEG were pooled. For the isolation of the PEG-lysozyme containing only one PEG moiety, the same cation exchange chromatography protocol was used, and the collected fractions were analyzed by the HPLC reversed-phase assay. Fractions containing the single peak of 1:1 PEG per lysozyme conjugate species were pooled.
B. mPEG-DTB-EPO
mPEG-MeDTB-nitrophenylcarbonates of various molecular weights (5-30 kDa) were prepared as described in Example 2A of U.S. Pat. No. 6,605,299, which is incorporated by reference herein.
Stock solutions of 16 mM mPEG-DTB-NPC (199.6 mg/mL) and mPEG-NPC (195.3 mg/mL) in acetonitrile were prepared.
Recombinant, human erythropoietin (EPO, EPREX®) was obtained preformulated at a protein concentration of 2.77 mg/mL in 20 mM Na citrate, 100 mM NaCl buffer pH 6.9.
mPEG-DTB-NPC was mixed with Epo at a 6:1 molar ratio in 50 mM MOPS, pH 7.8 for 4 hours at room temperature (approximately 25° C.). The reaction was further incubated at 4° C. overnight and then quenched by dialyzing in 10 mM Tris buffer, pH 7.5.
Prior to purification, the conjugates were dialyzed in 20 mM Tris pH 7.5 buffer and filtered through 0.2 μm Acrodisc® HT Tuffryn low protein binding syringe filter. The purification was done on a 1 mL Q XL anion exchanger column obtained from Amersham Biosciences Corp. (Piscataway, N.J.), using a step gradient elution profile from mobile phase A containing 20 mM Tris pH 7.5 buffer, to mobile phase B containing 500 mM NaCl in 20 mM Tris pH 7.5 buffer. The gradient was: 100% A for 8 minutes, 18% B for 25 minutes, then 70% B for 10 minutes. Elution fractions were collected in polypropylene tubes at 1 mL per fraction. The fractions eluting at 18% of mobile phase B (90 mM NaCl) were identified as the purified conjugates fractions (10 fractions), pooled in one tube, and stored at 2-8° C.
The purified mPEG-DTB-EPO conjugates were dialyzed in 20 mM sodium citrate, 100 mM NaCl buffer pH 6.9 (4 exchanges of 4 L buffer), using a Spectra/Por 6000-8000 MW cutoff dialysis tubing. A 10 mL Amicon concentrator with a YM10 membrane were used to bring down each sample volume from 10 to approximately 4.5 mL, under 45-50 psi nitrogen pressure.
Conjugates of PEG-DTB-lysozyme were prepared as described in Example 1A. The conjugates (100 μg/mL=0.066 mM) were incubated in 0.6 mM cysteine or with 4% BSA at room temperature (22-24° C.), in 10 mM phosphate buffer pH 7.4 containing 2 mM EDTA. Aliquots were taken at various time points, reactions were stopped with 20 mM iodoacetamide, and stored at 2-8° C. until analysis.
For the conjugates incubated with cysteine, analysis of the aliquots was as done follows. The samples were diluted 1/10 in 10 mM NaPO4 pH 7.4 and analyzed on a carboxymethyl (CM) cation exchanger column.
For the conjugates incubated with BSA, analysis of the samples was done by diluting the samples 1/10 in 10 mM PO4 pH 7.4, passing through Q spin columns (Vivascience) in order to trap the albumin and any of its related products, and then analyzing on the same CM column.
HPLC was performed with the following conditions: Column: TOSOH TSK CM-5PW 10 micron (7.5 mm×7.5 cm); Mobile phase: (A) 10 mM NaPO4 pH 7.4 and (B) 500 mM NaCl in 10 mM NaPO4 pH 7.4; Gradient: 5 min 100% A, 20 min 0% B to 100% B; Flow rate: 1 mL/min; Fluorescence detector: λex 295 nm, λem 360 nm (slit 30 nm); and injection volume, 100 μL.
The results are shown in
A. Analysis by HPLC
mPEG5k-DTB-lysozyme 1-1 conjugate (100 μg/mL), prepared as described in Example 1, was incubated with 4% bovine serum albumin in 10 mM NaPO4, 2 mM EDTA buffer, pH 7.4, for 2 days, at room temperature (22-24° C.). The reaction was then injected on a carboxymethyl (CM) cation exchanger column, and 0.5 mL fractions were collected and analyzed. The ion exchange separation conditions were: Column: HEMA CM 6.6 mL; Mobile Phase: A) 10 mM NaPO4 pH 7.4, B) 500 mM NaCl in 10 mM NaPO4 pH 7.4; Gradient: 10 min 100% A, 40 min 0% B to 100% B, then 1 min at 100% B; Flow rate: 1 mL/min; UV detector: 215 nm and 280 nm; injection volume, 3.3 mL. The HPLC trace is shown in
B. Analysis by SDS-PAGE and by MALDI-TOFMS
Polyacrylamide gel electrophoresis under denaturing conditions was performed for conjugates characterization. Pre-cast NuPAGE® Bis-Tris gels (4-15%), NuPAGE® MES running buffer, molecular weight protein standards (Mark12™), and Colloidal Coomassie® G-250 staining kit, were all obtained from Invitrogen, Carlsbad, Calif. In a typical electrophoresis, 1 to 3 μg of protein containing sample were loaded per well on the gel, then electrophoresed at constant voltage of 200 mV, and stained for protein according to the manufacturer instructions. For PEG detection, a duplicate gel was stained with iodine according to Kurfürst. M., Anal. Biochem., 200(2):244-248 (1992). Fractions collected from the CM column separation were analyzed by SDS-PAGE gel as shown in
The fractions collected from the CM column separation also incubated with 50 mM β-mercaptoethanol and then analyzed by SDS-PAGE again. The gel is shown in
The purified albumin-lysozyme adduct (fraction E2 in
mPEG-DTB-lysozyme and mPEG-DTB-erythropoietin conjugates derived from mPEG of molecular weight 5, 12 and 30 kDa were prepared as described above. The conjugates were labeled with Alexa Fluor™ 488 and free dye was removed. Labeled conjugates (0.05-0.1 mg/mL) were incubated with 75% rat plasma or with 3.55% bovine serum albumin (BSA) in the presence of phosphate buffered saline, pH 7.4. Samples withdrawn for analysis at a specified time point were treated with 50 mM iodoacetamide to terminate the cleavage of the disulfide and then placed on ice. Collected samples were analyzed by SDS PAGE and the Alexa Fluor™ 488 fluorophore image was quantitated using a fluorescence imager. The results are shown in
A. Cleavage of Conjugate in Cysteine and in HSA
mPEG-DTB-Epo (prepared as described above), mPEG-Epo, or Epo (0.2 mg/mL) was incubated with 0.05% human serum albumin (HSA) in 100 mM HEPES, 2 mM EDTA, pH 7.5 buffer for 21 hours at 37° C. To ensure visualization of the reaction products by SDS-PAGE, the concentration of HSA was significantly lower than physiological conditions and small molecule thiols were not included in the reaction, to prevent subsequent cleavage of any formed albumin-Epo. The SDS-PAGE gel stained with SYPRO™ red protein stain is shown in
B. Cleavage of Fluorescent Conjugates in Rat Plasma and in BSA
Fluorescently labeled mPEG-DTB-protein conjugates were also observed in the presence of rat plasma or bovine serum albumin over a timecourse at 37° C. mPEG-DTB-Epo conjugates were labeled and purified using the Alexa Fluor™ 488 labeling kit from Molecular Probes (Eugene, Oreg.), essentially according to kit instructions. Plasma from Sprague Dawley rats was collected with EDTA as the anticoagulant and stored in aliquots at −20° C. Bovine serum albumin from Proliant (Ankeny, IA) was resuspended in 50 mM NaPO4/2 mM EDTA, pH 7.4. Reactions contained 75% plasma or 3.5% BSA, 0.05-0.1 mg/mL labeled conjugate protein (1.6-3.3 μM for Epo; 3.5-7 μM for lysozyme) and phosphate buffered saline, pH 7.4 in tubes with o-ring caps. Samples were taken from each reaction mixture and stopped with 50 mM iodoacetamide (150 mM stock concentration in 50 mM NaPO4/2 mM EDTA), and placed on ice, protected from light. For time zero samples, plasma or BSA was quenched with iodoacetamide prior to addition of fluorescent mPEG-DTB-protein.
Collected samples were separated on NuPAGE™ 4-12% gels (Invitrogen, Carlsbad, Calif.) with MOPS or MES running buffer in presence of excess NuPAGE™ loading buffer. Prestained molecular weight markers were from Invitrogen (Carlsbad, Calif.). Imaging and quantitation was done using the Typhoon™ 9400 and ImageQuant™ (Amersham Biosciences) at λex=488 nm, λem=520 nm band pass 40. Following Alexa Fluor™ 488 quantitation, total protein signal was imaged (at λex=488 nm, λem=610 nm band pass 30) after staining with SYPRO™ red (Amersham Biosciences). The percent of each species compared to the total Alexa Fluor™ 488 labeled material was determined for each lane. Results are shown in
mPEG30K-DTB-Lysine-NBD prepared similarly to Example 1 above using 2 mM mPEG30K-DTB-nitrophenylcarbonate and 5-fold molar excess H-Lys-(ε-NBD)-NH2 (custom synthesized by Anaspec, San Jose, Calif.) in the presence of 60 mM hydroxysuccinimide, 60 mM HEPES, pH 7.5. Non-cleavable mPEG30K-Lysine-NBD was prepared using PEG30K-succinimidyl carbonate. In both preparations, free H-Lys-(ε-NBD)-NH2 was removed by Sephadex G-25 in PBS, pH 7.4. Cleavage reactions with BSA and analysis were essentially as described in Example 5B using 3.3% BSA in an equimolar ratio to the PEG reagent. Higher ratios of PEG reagents led to high background from the PEG reagent. When lower ratios of PEG reagent were used, the reagent was completely consumed in the reaction with time, but detection was low. An equimolar ratio allowed optimal visualization for quantifying the NBD (7-nitrobenz-2-oxa-1,3-diazole) fluorophore by SDS-PAGE and fluorescence imaging at λex=488 nm, λem=555 nm band pass 20. The results are shown in
A conjugate of mPEG5k-DTB-lysozyme was purified and prepared as a stock solution of 2.56 mg/mL. The solution contained 96% of pure 1-1 mPEG-protein conjugate, 1.6% of 2-1 conjugate, and approximately 2% of unconjugated lysozyme. A Micrococcus luteus turbidity assay was used to measure the amount of active lysozyme regenerated after cleavage of the conjugate.
mPEG5k-DTB-lysozyme (50 μg/mL in protein concentration) was incubated with 0.6 mM cysteine and with 4% BSA (containing approximately 0.45 mM free thiol, assuming that 75% of the albumin was in free SH form), at 37° C., in 10 mM NaPO4/140 mM NaCl/2 mM EDTA pH 7.4 buffer. At various time points, aliquots from the incubation vials were added to iodoacetamide to a final concentration of 20 mM, in order to stop the cleavage reaction. Samples were stored at 2-8° C. prior to analysis.
Micrococcus luteus stock solution was prepared at 0.3 mg/mL in 100 mM KPO4 pH 7. Lysozyme standards solutions were prepared at 1, 2, 4, 6, 8, and 10 μg/mL in PBS and a lysozyme standard curve was constructed (not shown). The samples from the cleavage reactions were diluted 1/10 in PBS. For the assay, 50 μL of standard, sample, or control were added per well to 96-well microtiter plates. To each well, 200 μL of Micrococcus luteus were added, and without delay, plates were read at 450 nm at 25° C. in a plate reader of a period of 10 min, in 30 second reading intervals.
The slopes (ΔA/min) were calculated for the first 5 minutes of the reading, and the corresponding lysozyme concentrations were extrapolated from the lysozyme standard curve. The results are shown graphically in
A. Preparation of 125I PEG-Lysozyme
Lysozyme (66 mg in 100 mg/ml in 0.1 M sodium phosphate buffer pH 7.3) was mixed with 605 μCi of Na125I (ICN Biomedicals, Irvine, Calif.), in Iodo-Gen® coated tube (Pierce Chemical Company, Rockford, Ill.), and allowed to react for 1 hour at room temperature with 20 min intervals mixing. The iodination reaction was stopped by removing the free 125I on a Sephadex G-25F gel filtration column (17 mL), and collecting the 125I-lysozyme, which was then reacted with either mPEG-DTB-NPC and mPEG-NPC, and purified by cation exchange chromatography as described above.
B. Pharmacokinetic Experiments
Male Sprague-Dawley rats (250-330 g each, 3 animals per formulation per experiment) were dosed either by intravenous (via a lateral tail vein) or by subcutaneous (dorsally above the right rear leg) with 125I labeled lysozyme or its PEG conjugates (0.35 mL, 0.4 mg protein/mL, 4.6×106 cpm/mL). Blood samples (0.4 mL) were collected via the retro-orbital sinus. All injections blood collections were performed while the animals were under inhaled anesthesia (isoflurane/O2). Samples were collected on heparin into polypropylene tubes and stored on ice for no longer than one hour before being pipetted in triplicate (0.100 mL) into fresh polypropylene tubes. Blood samples were collected at the following times after dosing (no single rat had blood collected at all of the following times): 30 sec, 15 min, 30 min and 1, 2, 3, 4, 6, 8, 24, 48, 72, 96, 120 and 168 hours post-dose. Note that the last 4 time points were added for the longer subcutaneous experiments. The samples were then counted for 125I in a Packard™ 5000 gamma counter. The cpm counts were converted to concentration according to the specific activity of the samples.
The results are shown in
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
This application claims the benefit of U.S. Provisional Application No. 60/607,110, filed Sep. 3, 2004, incorporated herein by reference in its entirety.
Number | Date | Country | |
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60607110 | Sep 2004 | US |