Patent Application
20240197894
This U.S. Utility Patent application claims the benefit of priority under 35 U.S.C. § 119(a) of Chinese Patent Application No. 202211485727.4, filed on Nov. 24, 2022. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
The present invention generally related to biopharmaceuticals, and particularly provides to a drug conjugate based on recombinant human serum albumin and a therapeutic drug.
The therapeutic efficacy of a drug depends on its availability at the target site with a specific concentration and dosing frequency, thus it is very important to maximize the therapeutic effects and minimize side effects for the patients in new drug development. Drugs such as proteins and polypeptides are basically completely inactivated after oral administration because they cannot tolerate the destruction of the digestive system, thus requiring larger doses. Meanwhile, drugs with small molecular weights are easily cleared by the kidneys or hydrolyzed by proteases even when injected, resulting in a short half-life in the body and requiring frequent administration. Conventional small molecule drugs are usually distributed non-specifically in the body, which results in low bio-availability at the target site in that to require high dose; meanwhile, due to their small molecular weights to rapid clearance by the kidneys, these need to be administered frequently. The conventional administration method and frequency cause more inconvenience and painful to patients.
Human Serum Albumin (HSA) is a most abundant protein in human plasma, accounting for more than 60% of total serum protein. HSA has a half-life of approximately 19 days. It consists of 585 amino acids, including 17 disulfide bonds and a free cysteine at position 34 (Cys34) with a molecular weight of approximately 66.5 kDa. HSA not only has the physiological function of maintaining plasma osmolarity, but also is an important non-specific transport protein in the body, which can reversibly bind to many insoluble organic small molecules or inorganic ions in the body to form the soluble complexes, which become the transport form of these substances in the blood circulation.
In the physiological environment, HSA exists as a negative charge and each HSA molecule is capable of carrying more than 200 negative charges, avoiding to be phagocytosed by the cells of the endothelial system. Meanwhile, it is very stable in the blood since it can disperse inside and outside the blood vessels. The characters of long half-life of HSA is attribute to a recycling mechanism of FcRn receptor-mediated (pH-dependent, preventing degradation by the lysosomal pathway) and to its ability to avoid renal clearance due to HSA has a large molecular weight than the renal threshold and can be recycled by the receptor-mediated endocytosis in the renal proximal tubule, thus avoiding renal clearance.
HSA also is tumor- and inflammation-targeted. The increased vascular permeability and poor lymphatic drainage at the tumor or inflammatory site makes drug molecules easier to permeate out of the tumor or inflammation vessels, and difficult to reenter the body circulation through the lymphatic route leading accumulation in the tumor or inflammation tissue. This phenomenon is known as enhanced permeability and retention effect (EPR). The concentration of HSA in the blood is about 45-55 mg/ml, while is about 14 mg/ml in the tumor stroma. At this concentration difference, HSA shows good passive tumor targeting through the EPR effect. In addition, HSA is dependent on the lymphatic system to circulation from the extracellular space, making it easily to accumulate in tumors with poor lymphatic drainage. Furthermore, the rapid metabolism and growth of cells at the tumor or inflammatory site requires actively uptake large amounts of extracellular proteins as a source of amino acids including HSA, which makes HSA more likely to be distributed in tumor or inflammatory tissues. MTX-HSA is the first chemical conjugate of HSA to be applied in clinical practice and is derived from the covalent coupling of methotrexate (MTX) to the lysine residue of HSA. Compared to MTX, data show that MTX-HSA has a significantly longer half-life and produces significant tumor targeting due to the introduction of HSA (Burger A M, 2001).
Human serum albumin is an ideal drug carrier due to its stability in vivo, safety, non-toxicity, low immunogenicity, biodegradability and tumor- or inflammatory-targed. HSA-based drug long-lasting technology mainly includes the construction of HSA fusion protein, coupling to HSA through covalent chemical bonding, and reversible binding to HSA through non-covalent bonding, and so on. HSA fusion technology is fused with a target protein/polypeptide drug either directly or by a linker. The first FDA-approved HSA fusion protein drug, Albiglutide, is a fusion protein of GLP-1 analogue (8Gly) connecting with HSA in series, with a half-life of up to 5 days, and can be administered once a week. The advantage of HSA fusion links directly to the target protein/polypeptide at the DNA level without additional chemical modification, however, it needs simple purification and preparation process. It is easier to quality control. However, the disadvantages of HSA fusion proteins have less biological activity in vitro, and heterogeneity, easy degradation and low expression levels (Zhao H L, 2008).
In contrast to fusion techniques, coupling HSA to a drug molecule by chemical coupling techniques does not alter the drug molecule structure, especially in the case of proteins and polypeptides, and has less impact on the active structural domains of the drugs. The lysine residue (Lys) is an important functional group for establishing the molecular linkage between HSA and the drug. The current HSA:drug conjugates as a carrier are mostly covalently linked by the drug itself or its derivatives to the amino group of the Lys residue of HSA in the presence of a cross-linking agent. Small molecule drugs bound to HSA by amide bonds using this approach include methotrexate, curcumin and adriamycin etc. (Hoogenboezem and Duvall 2018). The advantage of this coupling approach is that multiple Lys of HSA can be used. However, the lack of selectivity may affect the binding of HSA to FcRn and the consequent pharmacokinetics of HSA (Kuhlmann et al. 2017). Furthermore, this approach may make it difficult to control the number of modifications and sites for each HSA. For example, MTX-HSA is the earliest HSA chemical conjugate in clinical practice, and its biggest drawback is that MTX does not bind to HSA in equal amounts, since there are many Lys residues of HSA, only the average binding ratio of the reaction is 1:1, and the chemical structure of this conjugate is not clear, and its decomposition rate decomposition products are not yet clear (Fiehn et al. 2004).
Cys34 in HSA is another important functional group for molecular linkage between HSA and drugs. The free sulfhydryl group may be specifically coupled to a reactive-to-sulfhydryl group such as maleimide, and the Michael addition reaction between them is highly selective, rapid and under mild conditions. Since Cys34 is located on the surface of HSA, which is far from other ligand binding sites. Furthermore, the Cys34-coupled compounds do not interfere with the affinity and biological activity of HSA with other ligands. For this reason, Cys34 is an ideal site for targeted coupling of drugs in HSA.
The HSA currently used in the market has a potential risk of contamination by viruses and prion because of derived from plasma. More importantly, plasma-derived HSA may have low free sulfhydryl level of HSA since its Cys34 may bind certain heavy metal ions, glutathione or cysteine, whereas recombinant human serum albumin does not have these problems. Characterization of plant-derived recombinant human serum albumin (OsrHSA) shows the same as primary and tertiary structures of HSA. Non-clinical and clinical studies have shown that OsrHSA is safe, well tolerated and efficacy non-interfering to pHSA. It demonstrated that OsrHSA can replace pHSA with advantages of unique, non-plasma-dependent and virus-free. More importantly, Cys34 in OsrHSA is not reduced by glutathione, in that it has a higher content of free sulfhydryl group, which is more beneficial for the preparation of conjugates.
In alternative embodiments, provided herein is a drug conjugate of human serum albumin and a therapeutic drug.
In alternative embodiments, provided herein are methods of preparing the drug conjugate of recombinant human serum albumin and a therapeutic drug.
Recombinant human serum albumin has a plurality of modifiable sites, and the free sulfhydryl group on Cys34, as used in drug conjugates as provided herein, is more efficient in the coupling to a therapeutic drug. The coupling of a therapeutic drug to recombinant human serum albumin as provided for in exemplary drug conjugates as provided herein can significantly increase the molecular weight of the coupled drug, an reduce renal clearance and can prolong half-life in vivo and prevent from the serum clearance rate, moreover, exemplary drug conjugates as provided herein can be tumor- and inflammation-targeted and reduce the toxic effects of the drug.
In alternative embodiments, provided are human serum albumin conjugates, characterized in that the conjugate has the following structure of:
In alternative embodiments, the recombinant human serum albumin is derived from yeast or rice endosperm cell, optionally recombinant human serum albumin (OsrHSA) is expressed in rice endosperm cells.
Further, the recombinant human serum albumin is a high purity, clinical grade recombinant human serum albumin.
In the recombinant human serum albumin conjugate of the present invention, the linker has a heterobifunctional group.
In alternative embodiments, the bifunctional group has a reactive-to-sulfhydryl group and a group reactive to one of amino, hydroxyl, carbonyl and carboxyl, wherein the reactive-to-sulfhydryl group is covalently coupled to the 34th cysteine of the recombinant human serum albumin; and the group reactive to amino, hydroxyl, carbonyl, or carboxyl is covalently coupled to the drug molecules.
In the recombinant human serum albumin conjugate of the present invention, the drug molecule is a protein, polypeptide or small molecule compound with a molecular weight of less than 80 kDa and without free sulfhydryl groups.
Further, the drug molecule includes, but is not limited to, lactoferrin, growth hormone, insulin, Teriparatide, Amphotericin B and a derivative thereof.
In a second aspect, the present invention provides a method of preparing the drug conjugate of recombinant human serum albumin and a therapeutic drug, which comprises the following steps of:
The present invention also discloses the advantages of the recombinant human serum albumin conjugate over human serum albumin in terms of coupling efficiency regarding to the oxidated/reduced status of Cys 34. The advantages are mainly reflected in the fact that the coupling efficiency of recombinant human serum albumin to the drug molecule is increased by approximately 50% compared to human serum albumin derived from plasma (pHSA), which can significantly reduce the cost of the coupled drug. Further analysis reveals that the content of free sulfhydryl groups in the recombinant human serum albumin is approximately three times higher than that of pHSA.
In order to visually demonstrate the method of preparing the drug conjugate according to the present invention and the advantages of the drug conjugate prepared by recombinant human serum albumin over that prepared by pHSA conjugate, the drugs with different molecular weights, such as lactoferrin, growth hormone, insulin, Teriparatide and Amphotericin B are used as target drugs and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS) is used as a representative linker. The present invention describes in detail the preparation of the recombinant human serum albumin conjugate and compares its coupling efficiency to that of pHSA, showing that the coupling efficiency of the recombinant human serum albumin to the drug is significantly higher than that of pHSA.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.
Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, provided are drug-linker-albumin conjugates comprising:
In alternative embodiments of drug-linker-albumin conjugates as provided herein:
In alternative embodiments, provided are methods of preparing the drug-linker-albumin conjugate as provided herein, comprising the following steps of:
In alternative embodiments of methods as provided herein:
and/or
In alternative embodiments, conditions for practicing methods as provided herein comprise:
In alternative embodiments, for polypeptides or protein drugs, the drugs are were dissolved in an appropriate buffer, optionally deionized water, distilled water, or PBS, and then the heterobifunctional linker dissolved in a solvent (for example, DMSO, DMF, or equivalent) was slowly added dropwise at a molecule molar ratio of drugs to linker of about 1:1 to 1:10, and stirred at room temperature (RT) for at least 15 min.
In alternative embodiments, for chemical drugs, the drugs (1 eq. (equivalent)) were dissolved in appropriate solvent (DMF, DMSO, etc.), then base (2 eq., DIEA, TEA) was added and the mixture was stirred at appropriate temperature (15° C. at approximately 60° C.) for 10 min. Then the linker (1 eq. approximately 1.2 eq.) was added to the mixture. Stirring was allowed to continue for an additional 2 h approximately 16 h. TLC confirmed the reaction was completed.
In alternative embodiments, methods comprise removing, or substantially removing, excess uncoupled heterobifunctional linker by desalting or chemical separation, and exemplary protocols and conditions comprise:
In alternative embodiments, for polypeptides or protein drugs, due to the low molecular weight of the linker, the excess uncoupled linkers can be removed by ultrafiltration, dialysis, gel filtration, and other methods. For ultrafiltration or dialysis, the molecular weight cut-off of the membrane should be close to 1000 Da. Under this condition, the solvent and linker present in the aqueous phase of the reaction mixture pass through the membrane. Desalting Columns can also be used in buffer exchange and removal of low-molecular weight compounds.
In alternative embodiments, for chemical drugs, the solvent was removed under reduced pressure to give crude product. The crude product was purified by prep-TLC (petroleum ether:ethyl acetate=3:1 approximately dichloromethane:methanol=10:1) or flash (acetonitrile:water, 1% TFA, flow rate: 20 approximately 100 ml/min, elution gradient was between about 20% to approximately 80%). The solvent was removed with appropriate method to give the desired product.
Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.
Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.
As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples. The contents of the present invention are further illustrated by the following specific examples. The examples are merely provided to illustrate the present invention and do not in any way limit the rest of what is disclosed by the invention.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
Human serum albumin derived from plasma (pHSA): commercially available from Grifols, lot No. A3AFE01332;
Recombinant human serum albumin (OsrHSA) derived from rice seeds: prepared according to the patents CN100540667C and CN103880947A;
Recombinant human lactoferrin (OsrhLF, molecular weight 78.6 kDa): prepared according to the patent application publication WO 2020/088207 A1;
Recombinant human growth hormone (OsrhGH, molecular weight 22 kDa): prepared according to patent CN111057138A;
Teriparatide (molecular weight 4252 Da): commercially available from Thinheal Pharmaceuticals Co., Ltd.;
Recombinant human insulin (Insulin, molecular weight 5.8 kDa): commercially available from Hefei Tianmai Biotechnology Development Co., Ltd.
All other materials were purchased commercially.
Recombinant human lactoferrin (OsrhLF) with a molecular OsrhLF weight 78.6 kDa (prepared according to the patent WO2020088207A1) was dissolved in phosphate buffer solution (PBS), and then EMCS solution (100 mmol/L) was slowly added dropwise at a molecule molar ratio of OsrhLF to EMCS of 1:5, and stirred at room temperature for 30 min. The reaction was terminated by adding Glycine solution (1 mol/L), which was 5 folds of the molar concentration of EMCS.
After the reaction, excess EMCS was removed using a Bestdex G-25 M desalting column. OsrhLF-EMCS after desalination was divided into 2 parts and added with OsrHSA (C001202010002, 20%) and pHSA (Grifols, lot No. A3AFE01332, 20%), respectively, at a molecular molar ratio of 1:0.5; and reacted at room temperature for 1 h. An appropriate amount of each reaction solution was used for the analysis of the coupling efficiency of OsrHSA or pHSA to OsrhLF by 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As shown in
Recombinant human growth hormone (OsrhLF) with molecular weight 22 kDa was prepared according to the patent CN111057138A, and was dissolved in phosphate buffer solution (PBS). And then EMCS solution (100 mmol/L) was slowly added dropwise at a molecule molar ratio of OsrhGH to EMCS of 1:5 and stirred at room temperature for 30 min. The reaction was terminated by adding Glycine solution (1 mol/L), which was 5 folds the molar concentration of EMCS.
After the reaction, excess EMCS was removed using a Bestdex G-25 M desalting column. OsrhGH-EMCS after desalination was divided into two parts and added with OsrHSA or HSA injection, respectively, with a molecular molar ratio of 1:1. The reaction was conducted at room temperature for 1 h. Then the coupling efficiency of OsrHSA or pHSA to OsrhGH was analyzed by 4-20% SDS-PAGE. As shown in
22 mg of teriparatide (Thinheal Pharmaceuticals, molecular weight 4252 Da) was dissolved in ultrapure water (4.25 mg/ml). EMCS stock solution (100 mmol/L) was added at a molar ratio of Teriparatide to EMCS of 1:2, and then stirred at room temperature for 30 min. The reaction was terminated by adding 1 mol/L Glycine.
After the reaction, excess EMCS was removed using a Bestdex G-25 M desalting column. Teriparatide-EMCS after desalination was divided into two parts, and added with OsrHSA and pHSA, respectively, with a molecular molar ratio of 1:1. The reactions were conducted at room temperature for 1 h. Then the coupling efficiency of OsrHSA or pHSA to Teriparatide was analyzed by 4-20% SDS-PAGE. As shown in
100 mg of Amphotericin B (AmB, MW 924.09) was dissolved in 5 ml of N,N-dimethylformamide at 25° ° C. under dark, then N,N-diisopropylethylamine (37 μl, 0.216 mmol), hydroxybenzotriazole (5 mg, 32.5 μmol) and maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (Mc-Val-Cit-PABC-PNP, 96 mg, 0.13 mmol) were stepwisely added with stirring, respectively. The reaction solution was stirred at 25ºC for 5 h under dark. Thin layer chromatography (TLC, dichloromethane:methanol=5:1) and LC-MC showed that Amphotericin B was completely reacted. The coupled products was concentrated, and then 10 ml of methanol was added to dissolve the concentrated product. After then, 30 ml of methyl tert-butyl ether was slowly added dropwise to the reaction flask with stirring. The suspension was stirred at room temperature for 10 min, the filter cake was collected by filtration through negative pressure. The coupled products were dried with a yellow solid product, AmB-Mc-Val-Cit-PAB-PNP (AmB-MVCPP, MW: 1521.76).
AmB-MVCPP was dissolved in DMSO (10 mmol/L), divided into two parts, and then added OsrHSA and HSA at a molecular molar ratio of 1:0.2, respectively. The reaction was carried out at room temperature overnight. Then the coupling efficiency of OsrHSA or pHSA to AmB was analyzed by 8% SDS-PAGE. As shown in
To investigate whether the half-life of OsrHSA-EMCS-OsrhGH was significantly longer than that of OsrhGH, the OsrHSA-EMCS-OsrhGH prepared in Example 2 further was purified by Phenyl HP and Nano 50Q to remove unreacted OsrHSA and OsrhGH. The final product was injected subcutaneously into Kunming mice with a dose of 5 mg/kg (based on OsrhGH). Blood was then taken from the orbital vein of the mice at different timepoints after administration. The OsrhGH content in serum was determined using the Human Growth Hormone (GH) DuoSet ELISA (DY1067) kit. The pharmacokinetic parameters of OsrhGH were calculated based on the OsrhGH content in serum at different timepoints.
As shown in Table 1, the half-life (t1/2) of OsrhGH in mice was 0.47 h, while the half-life of OsrHSA-EMCS-OsrhGH was 7.39 h, approximately 16 times longer than that of OsrhGH, indicating that the half-life of OsrhGH coupled to OsrHSA was significantly prolonged. The peak time (Tmax) of OsrhGH was 1 h, while the peak time of OsrHSA-EMCS-OsrhGH was 9 h, namely, the action duration of OsrHSA-EMCS-OsrhGH was significantly longer than that of OsrhGH. The peak concentration (Cmax) of OsrHSA-EMCS-OsrhGH was approximately twice that of OsrhGH, while the area under the drug-time curve (AUClast) of OsrHSA-OsrhGH was approximately 12 times that of OsrhGH. In summary, OsrhGH coupled with OsrHSA had a significantly longer half-life, peak time, peak concentration and AUClast than that of OsrhGH alone (
In order to verify whether OsrHSA-EMCS-OsrhGH has biological activity, OsrHSA-EMCS-OsrhGH prepared in Example 2 was subjected to carried out biological activity assay in vitro using mouse lymphoma NB2-11 cell. The results are shown in
42 ml of Insulin solution (0.4 mmol/L dissolved in coupling solution) was slowly added dropwise with 8 ml of N-succinimidyl 3-(2-pyridinedithio)propionate (SPDP) solution (100 mmol/L, dissolved in DMSO) with a molecular molar ratio of Insulin to SPDP of 1:5, then stirred at room temperature for 1 h. And then the reaction was terminated by adding Glycine solution, which was 5-folds of the molar amount of SPDP.
After the completion the reaction, excess SPDP and DMSO were removed with a Bestdex G-25 M desalting column. The Insulin-SPDP after desalting was added with 3.88 ml of OsrHSA solution (3 mmol/L) at a molecular molar ratio of Insulin-SPDP to OsrHSA of 1:1, then mixed thoroughly and left to react at 4ºC for 18 h.
First, the conductance of the coupled product solution was adjusted with 3M ammonium sulfate to consistence to the equilibrium buffer (10 mM PB, 0.2 M ammonium sulfate, pH 6.5). And then the equilibrium buffer was used to dilute the coupled product to final concentration approximately 1 mg/ml (based on OsrHSA). The coupled product was loaded onto a column filled with 176 ml of Phenyl Bestrose HP chromatography resin with a column height of 25 cm at a flow rate of 15 ml/min. After the sample loading, the chromatographic column was equilibrated again with the equilibration buffer until the UV was the same as the baseline. The dimer of the coupled product was removed using a washing buffer (10 mM PB, 0.18M ammonium sulfate, pH 6.5). Finally the coupled product was eluted using an elution buffer (10 mM PB, pH 7.2). The eluate was concentrated and ultrafiltrate using a 50 kDa ultrafiltration membrane pack. The stock solution of OsrHSA-SPDP-Insulin conjugate was obtained. The purity of the final OsrHSA-SPDP-Insulin as shown in chromatogram (A) and SDS-PAGE assay results (B) of the OsrHSA-SPDP-Insulin coupling product are shown in
To demonstrate the long-lasting efficacy of OsrHSA-SPDP-Insulin for hyperglycemia, a male BSK-DB diabetic mouse model was used for the study. OsrHSA-SPDP-Insulin was prepared as described in Example 7. After the mice were fasted overnight without water deprivation, five mice each group were received single dose by subcutaneous infusion of OsrHSA-SPDP-Insulin with doses of of 25 IU/kg and 50 IU/kg, positive control Insulin with a dose of 10 IU/kg, and negative control (an equal volume of saline. Blood samples were collected from the tail vein of the mice at before dosing and 8 h after the dosing. The blood glucose level was detected using a Roche blood glucose meter and testing strips. The blood glucose change curves were plotted according to the change value (Tn/T0) in blood glucose value (Tn) relative to the initial blood glucose value (T0) at different timepoints. As shown in
In order to elucidate the mechanism by which the coupling efficiency of OsrHSA is higher than that of pHSA, the content of free sulfhydryl groups was determined by the DTNB method. OsrHSA or pHSA was diluted to 50 mg/ml (752 μmol/L) using a reaction buffer (100 mmol/L sodium phosphate, 1 mmol/L EDTA, pH 8.0) for in use. In a 5 ml reaction tube, 2.5 ml of reaction buffer and 50 μl of DTNB reaction solution (4 mg/ml) were added first, and then added 250 μl of cysteine standard solution (0.25 approximately 1.5 mmol/L) or the sample to be tested, respectively. The reaction was carried out at room temperature for 15 min. And then the OD412 values were measured using a spectrophotometer. A standard curve was plotted based on the absorbance of the cysteine standard solution, and then the sulfhydryl content was calculated. The results indicated that the free sulfhydryl content in OsrHSA of 541 μmol/L of 752 μmol/L was sulfhydryl free, accounting for 72% sulfhydryl free, while the free sulfhydryl content in pHSA was 183 μmol/L out of 752 μmol/L, accounting for 24% only. The content of free sulfhydryl groups in OsrHSA is three folds higher than that of pHSA.
Example 10 Preparation of exemplary OsrHSA-EMCS-Insulin at high loading capacity 400 mg of Insulin dry powder (purchased from Eastern Antibiotics, Calt No. GMP-045) was dissolved in 20 ml of 4 mmol/L dilute hydrochloric acid (pH 2.0), and slowly adjusted to pH 7.2 (pH should not exceed 8.0) using 0.5 mol/L NaOH. And then supplemented with a coupling buffer (20 mmol/LPB, 2 mmol/LEDTA, pH 7.2) to 100 ml. After then 3.43 ml of EMCS solution (31 mg/ml) was slowly added dropwise with stirring at a mass ratio of Insulin to EMCS of 1:0.266 (molar ratio 1:5), and stirred at 25° C. for 30 min. The reaction was terminated by adding Glycine, which was 5 times the molar amount of EMCS.
Excess EMCS was removed using a G-25 desalting column (column volume 490 ml, flow rate 25-40 ml/min). The desalted Insulin-EMCS was diluted to approximately 1600 ml using the coupling buffer. Then 50.9 ml of OsrHSA (153 mg/ml) was added with a mass ratio of Insulin-EMCS:OsrHSA of 1:22.9 (molar ratio 1:2), the final concentration of OsrHSA in the system was 5 mg/ml). The reaction was carried out at 25° C. for 50 min. The reaction was terminated by adding Cys, which was 5 times the molar amount of OsrHSA.
The coupling product was diluted to approximately 1 mg/ml (based on OsrHSA) and purified using a Phenyl HP chromatography column. The eluant was concentrated and ultrafiltrated using a 50 kDa ultrafiltration membrane pack, then at least two times the volume of 10 mmol/LPB (pH 7.2) was added, concentrated for dialysis and repeated 7 times, the resulting of OsrHSA-EMCS-Insulin stock solution was obtained.
The coefficient of variation of the chromatograms and SDS-PAGE indicated that the yield from the three validated batches were less than 10%, showing that it is excellent consistence and the monomer of OsrHSA-EMCS-Insulin purity are 94.94% (Table 1, Table 2 and
An intraperitoneal glucose tolerance tests (IPGTT) in rat was performed for OsrHSA-EMCS-Insulin that was prepared according to Example 10. The test was divided into three groups, the control and two experimental groups with 6 SD rats for each group. The experimental group was administered once by subcutaneous injection at a single dose of 125 nmol/kg and the control group was given the same volume of placebo saline. 16 h before the glucose tolerance test, the rats were fasted without water deprivation. Blood sample was collected from the orbital plexus of rats at −0.5 h (baseline fasting blood glucose concentration before administration), 0 h (blood glucose concentration before intraperitoneal injection of 20 g/kg glucose at 0.5 h after administration), 0.5 h, 1 h, 2 h, 4 h and 6 h. The blood glucose level were measured at 0.5 h, 1 h, 2 h, 4 h and 6 h after glucose dosing. The results showed that the efficacy of OsrHSA-EMCS-Insulin could last up to 45-49 h compared with the Insulin group and the negative control group, demonstrating that OsrHSA-EMCS-Insulin has a longer efficacy (
In order to demonstrate the longevity and efficacy of OsrHSA-EMCS-Insulin, SD rats were selected as the study subjects for testing. Seven weeks old SD male rats with 200 g-250 g body weigh were randomly grouped as non-fasting conditions. OsrHSA-EMCS-Insulin and an equal volume of saline (negative control) were administered once by subcutaneous injection with different doses. Then blood was taken from the orbital plexus of the rats at 4 h, 8 h, 24 h, 32 h and 48 h after administration and treated at 37° C. for 30 min, and then centrifuged at 4000 rpm for 15 min. The supernatant was stored at −80° ° C. for use. The blood glucose and insulin levels were measured using blood glucose test strips (Roche) and Insulin ELISA kit (R&D, DY8056-05). As shown in in
Data provided herein demonstrates the obvious advantages of the exemplary recombinant human serum albumin over pHSA in terms of more free sulfhydryl-based coupling sites and the preparation method for coupling therapeutic drug.
However, embodiments as provided herein, or the invention, are not limited to the detailed methods described above, i.e., it does not mean that embodiments as provided herein, or the present invention, must rely on the detailed preparation methods described above in order to be implemented. It should be clear to those skilled in the art that any improvements to embodiments as provided herein, or the present invention, substitutions of the drugs coupled to OsrHSA in the present invention, etc., fall within the scope of protection and disclosure of the present invention.
A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211485727.4 | Nov 2022 | CN | national |
