|Title of Invention||
"INTERLEUDIN-6 POLYETHYLENE GLYCOL CONJUGATE AND ITS PREPARING METHOD AND USE"
|Abstract||The invention provides an interleukin-6 polyethylene glycol conjugate, and medical compositions comprising the said conjugate and pharmaceutically acceptable excipients, as well as the application of said interleukin-6 polyethylene glycol conjugate for preparing medicines treating thrombocytopenia, medicines for chemotherapy adjuvants and for immunoenhancement. The mono-modified PEG- IL-6 has significantly better biostability, longer in vivo half-life, and lower plasma clearance. Its usage dose and frequency are reduced significantly, as well as the side-effects, thus facilitating the use by the subject, reducing cost, and improving the safety during use, lessening the pain on the part of the subject significantly. Moreover, the mono-modified human interleukin-6 polyethylene glycol conjugate of the present invention has good uniformity, can meet the requirement of safety, effectiveness and controllable quality for clinical administration.|
INTERLEUKIN-6 POLYETHYLENE GLYCOL CONJUGATE AND ITS PREPARING
METHOD AND USE
FIELD OF THE INVENTION
The present invention relates to an interleukin-6 polyethylene glycol conjugate; the invention also relates to the method for the preparation and use thereof.
BACKGROUND OFTHE INVENTION
Interleukin-6 (IL-6), which is also called Interferon ß2, is a multi-functional cytokine. IL-6, mainly acting on immune system, can promote the differentiation of T cells and nerve cells, stimulate the growth of T cells and stem cells, and induce the differentiation of multinucleate cells, thus promoting the production of platelets. The medical applications of IL-6 mainly comprise three aspects: (1) IL-6 has a certain therapeutic effect on thrombocytopenia induced by chemotherapy and radiotherapy, and was used as a substitute of platelet preparations, being placed great expectation for treating anticarcinogen-induced hematopoietic insufficiency; (2) IL-6 can enhance the immunity of cancer patients, clear the in vivo residual tumor cells of patients after operation, and prevent the recurrence of malignant tumor; (3) IL-6 can enhance humoral and cellular immunity, hence having some therapeutic effect on certain patients with immunodeficiency diseases, hi the past, the research and development on IL-6 for treating thrombocytopenia had already entered clinical trial (phase II), but its practical application was not realized due to its short plasma half-life, large usage dose and the tendency to cause adverse reactions (such as fever, headache, and the drop of albumin).
Polyethylene glycol (PEG) can form conjugate with protein and polypeptide drugs. Such PEG-modified protein and polypeptide drugs can change the properties of protein drugs, such as enhancing the solubility and stability thereof, decreasing or eliminating immunogenicity, antigenicity and toxicity, increasing the therapeutic index of drugs, expanding the scope of clinical applications, as well as improving the in vivo pharmacokinetics properties and prolonging the in vivo half-life of drugs, etc. The pharmacokinetics properties of PEG type
modifiers differ as a result of the differences in the properties of the proteins being modified, in the relative molecular weights of the modified products and in the administration approaches. The modifying approaches of PEG with regard to proteins and polypeptides mainly include amino modification (including acetylization of N-terminal amino group, acetylization of lysine e-amino group, and alkylation of N-terminal amino group), carboxyl-modification, sulphydryl-modification, etc. To control and determine the modification degree and modification site has been a difficulty in the modification of proteins and polypeptides with PEG, for there are many amino groups in the structure of proteins and polypeptides, making their structures complex and diversified. There are many other problems found in the PEGylation of polypeptides; for example: (1) the acylation ammoniation of tyrosine residues on the protein can also result in the lowering of bioactivity of the modified products; (2) certain PEGylated proteins are extremely unstable, thus having no medical value; (3) certain reagents used for modification are difficult to select, for some agents used in reaction are insufficiently reactive, thus costing longer time during which pro-modified or post-modified protein denaturation can occur, and some are totally without reaction; (4) the hydrolysis of proteins in aqueous solution at physiological pH is prone to resist the PEGylation of proteins, thus causing many modification difficulties.
To overcome such difficulties, researchers have done tremendous research work on the PEGylation technology of protein drugs, and progress is made in the modification of IL-6 with PEG too.
U.S. Patent No. 5,264,209 discloses a PEG-IL-6. PEG with molecular weights of 4500, 10000 and 12000 are used to modify human IL-6, wherein PEG 4500 and PEG 12000 are linear PEGs, and bis-PEG 5000 (total molecular weight being 10000) is branched PEG The modified product of this patent is a mixture of products with different PEGylation degree (2-9 PEG moieties). Result of animal experiments indicates that the platelet producing activity of modified IL-6 is superior to that of unmodified IL-6 with the same dosages (4 εg-10mg/kg), and it is believed that at least two and preferably more than five PEG modities are required to ensure the in vivo activity of the modified product.
Tsunoda S, Tsutsumi Y et al. compared the in vivo thrombopoietic activity of polyethylene glycol (PEG 5000)-modified interleukin-6 (MPEG-IL-6) to that of native IL-6 (IL-6) (PEGylation of interleukin-6 Effectively increases Its Thrombopoietic Potency. Tsunoda S,
Tsutsumi Y .Thrombopoietic and heamostasis; 77(1)168-173,1997). MPEG-IL-6 was obtained from the reaction of PEG 5000 with IL-6, in which 54% of the 14 lysine amino groups of IL-6 were coupled with PEG, but only about 51% of the bioactivity of IL-6 was shown. Pharmacodynamics tests indicated that by respectively administering IL-6 and MPEG-IL-6 subcutaneously to mice every 2 day for 7 days, IL-6 increased not only the peripheral platelet count, but also the plasma-IgGl level. In comparison with IL-6, MPEG-IL-6 does not enhance IgGl level the same time as it enhances platelet count. MPEG-IL-6 significantly stimulates platelet recovery in mice treated with 5-fluorouracil, whereas IL-6 has a negligible effect. The plasma half-life of MPEG-IL-6 is about 100 times longer than that of IL-6.
Subsequently, Tsunoda S et al. employed a pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), to the modification of IL-6 with PEG (Selective enhancement ofthrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol. 2001 Jan;112(l):181-8), wherein IL-6 was still modified with PEG-5000. In this research, mono-modified product fraction Fr3 with molecular weight of 26,500 was obtained. Although it retained much in vitro activity, when administered subcutaneously into mice, it was found to have an ultrashort half-life (the elimination half-life was about 12 hours), and the in vivo residence time was about 25 hours, showing little difference to unmodified IL-6. The in vivo activity of Fr3 was also inferior to that of fractions with higher degree of modification, and its in vivo activity was lower than IL-6 of 50-fold dosage. The mono-modified DmPEG-IL-6 prepared by DMMAn as amino protectant not only showed no significant difference to the mono-modified PEG-IL-6 in pharmacokinetics parameters as well as in the in vivo activity, but also required three reaction steps to be obtained, which meant a too complicated procedure for production.
Most of the PEG-modified IL-6 products disclosed above were a mixture of products of different modification sites and modification degrees. Since the product was heterogeneous, and effective quality control was impossible, it was hard to ensure safety, efficiency and controllable quality, hence hard to put into practical application; and the mono-modified IL-6 polyethylene glycol conjugate obtained in few cases had defects such as short half-life, low activity, poor efficacy, low yield, etc. To date, there exists an urgent need in the field of art for the development of a human mono-modified PEG-IL-6 that can overcome the defects of present products, a drug
with good uniformity, clinical safety, efficiency, controllable quality, low cost, and suitable for mass production
SUMMARY OF THE INVENTION
One object of the present invention is to provide an interleukin-6 polyethylene glycol conjugate (hereafter referred to as IL-6 polyethylene glycol conjugate or PEG-IL-6). Such IL-6 polyethylene glycol conjugate is obtained by covalent mono modification of IL-6 with polyethylene glycol, wherein each IL-6 molecule is covalently bonded by a polyethylene glycol molecule, the molecular weight of PEG being 15000-30000Da.
In the IL-6 polyethylene glycol conjugate of the present invention, the preferable modification site of IL-6 with PEG lies in the side chain amino group of lysine residue of IL-6 molecule or in the N-terminal amino group of the peptide of IL-6 molecule.
The PEG molecule for modification can be a linear PEG molecule, i.e., a PEG chain attached to said lysine amino groups of IL-6 molecule or said peptide N-terminal amino group of IL-6 molecule. It can also be a branched PEG molecule, which means that two or more of PEG chains are attached to the active group, the molecular weight being the total molecular weight of the two chains. These two kinds of PEG molecules may show difference in the selection of binding site to IL-6 molecule during modification due to their differences in spatial structure.
For the IL-6 polyethylene glycol conjugate modified by branched PEG, it has the structure shown in formula (I) below:
Wherein "m" represents methyl, and "i" and "j" are integers between 100 and 1000. The
sum of i and j makes the molecular weight of PEG in the conjugate being 15000-30000.
As a preferred embodiment of the invention, the PEG molecules that may be used as modifier are such as mPEG2-NHS of branched structure, and mPEG-aldehyde of linear structure.
An IL-6 molecule is reacted with a PEG molecule at a certain temperature, pH and reaction time to prepare the IL-6 polyethylene glycol conjugate of the present invention. Therefore the invention also provides a method to prepare the abovementioned IL-6 polyethylene glycol conjugate, which comprises the following steps:
1) preparing an IL-6 protein solution at a concentration of 0.05-20 mg/ml and pH 6.5-10.0
2) reacting the IL-6 solution prepared with activated PEG at 15-35°C for 5-100 minutes to
obtain an IL-6 polyethylene glycol conjugate, the amount of the said PEG is 1-100 times of the
weight of IL-6;
3) isolating and purifying the IL-6 polyethylene glycol conjugate obtained to obtain a pure
mono-modified IL-6 polyethylene glycol conjugate.
Wherein, the SDS-PAGE purity of IL-6 in step 1) of the above method is over 95%, and a solution with protein concentration of 0.5-1 mg/ml and pH 8.7-9.3 is prepared.
Wherein, the purification procedure in step 3) of the above method comprises: treating the reactant solution obtained in step 2) with a G-25 gel filtration column for desalinization, and then loading on a cation exchange column for primary separation. Finally, purifying the mono-modified product obtained with a Superdex 200 gel filtration column.
The present invention also provides a pharmaceutical composition, in which way the pharmaceutically acceptable excipients are added to an IL-6 polyethylene glycol conjugate of the invention to get the preparation.
The present invention yet still provides a use of said IL-6 polyethylene glycol conjugate in preparing medicines treating thrombocytopenia, medicines for chemotherapy and radiotherapy adjuvants and for immunoenhancement.
The said IL-6 of the invention can have many sources. Any homologous peptide similar to natural human IL-6, whether synthetically produced, or expressed in prokaryotic and eukaryotic expression system, or even modified, can be the material for PEG modification of the invention,
so as to obtain a PEG-modified IL-6.
The modifiers used in the invention can be activated PEG esters. Alternatively, other methods can be used to couple the lysine amino groups of IL-6 with the said PEG of the invention covalently. Using PEGs having other activated groups or activating the corresponding lysine residue of IL-6 peptide is within the scope of the present invention too.
Since the characteristics and in vivo pharmacokinetic properties of each protein are different, the molecular weights and properties of PEG agent vary a lot, and there are many ways of PEGylation of protein, which makes it very complicated for the PEG modification of protein drugs. Meanwhile, PEG modification requires highly specific but moderate reaction conditions. It is almost impossible to anticipate how a protein can be PEGylated successfully to obtain high-yield and homogeneous target modified products. The content of mono-modified PEG-IL-6 (monoPEG-IL-6) in the product prepared according to the method of the invention can be over 85%. In addition, the pharmaceutically acceptable excipients can be added to the IL-6 polyethylene glycol conjugate of the invention to prepare various preparations, such as injection, freeze dried formulation and the like.
Since PEG with appropriate molecular weight is used, the mono-modified PEG-IL-6 not only has the physiologic activity of IL-6, but also a greatly improved stability. It has longer in vivo half-life and lower plasma clearance. The usage dose and frequency are significantly lowered, and side-effect reduced. Its properties in all aspects are obviously better than IL-6 and mono-modified IL-6 presently disclosed, which can facilitate the use by patients, reduce the use cost, and improve the safety during use, lessening the pain of patients significantly. Meanwhile, the mono-modified PEG-IL-6 obtained in the present invention is superior to multi-modified PEG-IL-6 in that it has good uniformity and is easy for quality control, being able to meet the requirements of clinical administration for safety, efficiency and controllable quality, and suitable for mass production. It makes it possible for the industrial production and practical application of PEGylated recombinant human IL-6, thus guaranteeing a great market prospect.
BRIEF EXPLANATION OF THE DRAWINGS
FIG. 1 demonstrates the profile of SDS-PAGE of IL-6 products after PEG modification and purified products thereof; wherein, 1 and 9 are protein molecular weight markers; 10 and 13 are
PEG-modified products; 2-8, 11-12 and 15 are elution fractions after purified by SP Sepharose
High Performance cation exchange chromatography, wherein 15 is mono-modified product; 14 is mono-modified product after isolation and purification by Superdex 200 gel filtration chromatography, the main band of modified products (MW60,000) being more than 85%. The mass spectrometric analysis shows that the molecular weight of such modified protein is about 46,000, which indicates that only one PEG 20,000 molecule is modified;
FIG. 2 compares the in vivo bioactivities of four PEG-modified rhIL-6s in mice; wherein, PBS: negative control; rhIL-6: 2.5 ug/mouse; mPEG2-NHS: 0.05ug/mouse; mPEG-aldehyde: 1 .0ug/mouse;PEG-SPA: 1 .0ug/mouse; mPEG-SPA:2.5ug/mouse;
FIG. 3 shows the process flow diagram for the freeze dried formulation of PEG-IL-6.
DETAILED DESCRIPTION OF THE INVENTION
Exemplary embodiments of the invention are explained in detail with reference to the drawings to describe the invention in a non-limited way.
Unless specifically pointed out, all the materials used in the embodiments below are commercially available.
Selection of the process conditions for modifying PEG with rhIL-6
1. Screening for modifying agents (PEG)
1.1 Comparison of the structural features of four modifying agents (PEG)
During the research period before the formal animal tests, four PEG modifying agents are tested successively. Table 1 shows various features of the said four modifying agents.
Table 1. Comparison of four PEG modifiers
The molecular formulas of the four modifiers are as follow:
1.2 Comparison of the molecular weight distributions of four PEG-modified rhIL-6s, in vitro activities and their therapeutic activities in mice
The purified rhIL-6 products are modified with these four PEG modifiers respectively, and purification is carried out on modified products to remove unmodified rhIL-6 and the by-products of modification. The molecular weight distribution, in vitro activities retained and in vivo therapeutic activities in mice are measured respectively.
1.2.1 The molecular weight distribution of four PEG-modified rhIL-6s The molecular weight distribution of PEG-modified rhIL-6s is measured by SDS-PAGE, and Gel Scanning and Imaging System is used to calculate the molecular weight and content of each band after electrophoresis. Since long chain PEG is a linear macromolecule, its apparent molecular weight in SDS-PAGE usually being 2-4 times of its real molecular weight, the molecular weight of modified rhIL-6 cannot be calculated accurately. All the molecular weights listed in table 2 are the estimated apparent molecular weights.
Table 2. The apparent molecular weights distribution of four PEG-modified rhIL-6s
1.2.2 Comparison of in vitro activities of four PEG-modified rhIL-6s Similar to rhIL-6, the in vitro activities of PEG-modified rhIL-6s are also measured by MTT assay. The specific activity of positive control rhIL-6 is defined as 100%, against which the activity of each PEG-modified rhIL-6 is compared to calculate the percentage content. All the literatures published and our experiments indicate that after PEG modification, as a result of the masking and barrier effect of the long chain PEG on protein surface, the combination of protein with receptor, coenzyme, prosthetic group and the like is restricted, thus lowering both the enzyme activity and bioactivity to some extent. The activities of post-modified rhIL-6s are lower than that of pre-modified. The results are shown in table 3.
Table 3. Comparison of in vitro activities of four PEG-modified rhIL-6s
1.2.3 Comparison of in vivo bioactivities of four PEG-modified rhIL-6s in mice The in vivo activity of rhIL-6 and PEG-modified rhIL-6 can be indirectly reflected by the in vitro cell activity measured by MTT assay, but the platelet producing activity should still be verified by animal experiment. The mice injected with cyclophosphamide are used to simulate the thrombocytopenia after radiothrapy (radiotherapy) or chemotherapy, and then rhIL-6 and PEG-modified rhIL-6 are injected to monitor the platelet recovery. The detailed scheme of the experiments is as follows: 0.5 ml/mouse of test sample is administered subcutaneously once a day for five days; 2 mg/mouse cyclophosphamide is administered intraperitoneally per day for 3 consecutive days from the 3rd day of test sample injection; 10 ul blood sample is taken from the
tail of each mouse before the first injection and on the 8-17th day of injection, diluted 6 times to count platelets using Cell-DYN1600 blood-cell counter. Since the animal experiments are carried out with four PEG-modified rhIL-6s successively, relative ratio is used to reflect platelet counts. The platelet counts before sample injection is defined as 100%, against which the relative percentage ratios of the platelet counts thereafter are calculated, and the statistical analysis is conducted. For the purpose of comparison, the minimum dose in dose group having significant difference in each PEG-modified rhIL-6 is compared with that in rhIL-6. The results are shown in Fig. 2. 1.3 Conclusions
After comprehensive analysis of the results above, following conclusions are made:
(1) After modified by various PEGs, the bioactivities of rhIL-6s are retained more or less.
However, the in vitro activities do not conform to those of in vivo, the former of mPEG2-NHS
being only 5-15%, but only 0.05 ug/mouse can reach the platelet producing activity of rhIL-6 at a
dose of 2.5 ug/mouse. Accordingly, it may be believed that the post-modified activity (in vivo)
increased 50 times as compared with the pre-modified activity. That maybe because the
modification of PEG decreases the clearance of drugs in plasma, prolonging the retention time
and increasing the bioavailability, thus ensuring the drugs to act longer in vivo. At the same time,
as PEG modification can also delay the degradation of protease, the in vivo activity of rhIL-6 is
kept in this way too.
(2) Considering the in vivo activities of these PEG modifiers, mPEG2-NHS is apparently
superior to the other three. With reference to various literatures and patent data, it is believed that
due to the spacial steric effect caused by the high molecular weight and branched structure,
mPEG2-NHS of 20 kDa can only get close to modifiable amino site on the surface of rhIL-6 in
modification reactions; moreover, after combined with such a molecule, other PEG molecules are
hard to get close to rhIL-6 molecule due to the spacial steric effect, so it is possible to obtain a
modified product mainly comprising monoPEG-rhIL-6. Although, in the in vitro activity assay,
the two 10 kDa PEG chains of mPEG2-NHS attached to the protein surface prevent rhIL-6 to
combine with cell surface receptor, which makes the activity become only 5-15% of rhIL-6, its
effect on prolonging retention time and delaying protease hydrolysis is superior to the other three
(3) PEG-aldehyde has high selection on protein N-terminal amino acid, and most of the
modified products are N-terminal mono-modified. Its in vitro activity is good, and the in vivo
activity is lower than that of mPEG2-NHS. However, because the PEG is of linear molecule, its
apparent molecular weight is lower than the branched type, it is easy to be cleared by kidney in
(4) The molecular weights of PEG in PEG-SPA and mPEG-SPA are small, and the plasma
clearance speed is fast, hence higher dose is needed to obtain notable platelet producing activity.
Similarly, it is disadvantageous for subsequent purification verification and quality control due to
the lower molecular weight, large amount of multi-modified products, such as diPEG-rhIL-6,
triPEG-rhIL-6 and polyPEG-rhIL-6 and the continuous distribution of molecular weights.
PEG-SPA is a bifunctional modifier that can also cause the coupling reaction between molecules.
To sum up, the inventors believe that it is ideal to select mPEG2-NHS (20 kDa) as PEG modifying agent of rhIL-6, which can not only achieve the expected changes in the efficacy and pharmacodynamics of rhIL-6 after modification, but also make it easy to control the quality of modified products, thus guaranteeing the quality and effect of final products.
2. Optimization for PEG chemical modification process of rhIL-6
After choosing mPEG2-NHS as PEG modifier, the modification reaction conditions should be optimized to obtain the optimal yield as well as the optimal physiochemical properties and bioactivities of modified products. The reaction between mPEG2-NHS and protein molecule can be described briefly as:
The hydrolytic rate of mPEG2-NHS molecule in aqueous solution is fast, in which the half-life is only 4.9 minutes at pH 8.0 and 25 "C, therefore the modification reaction is substantially achieved within 45 minutes, leaving the residual mPEG2-NHS molecules less than
0.1%. The invalid reaction will also occur between the modifier and water molecule at the same time of reacting with amino group, so the optimization of reaction conditions is necessary, reducing invalid reaction and improving modification yield. Described below is the optimal screening for three important conditions as pH of reaction system, reaction time, and the molar ratio of modifier to protein.
2.1 Optimization of pH in reaction system
rhIL-6 samples complying with the purification standard are divided into 5 groups. The pH of the buffers are adjusted to 7.5, 8.0, 8.5, 9.0 and 9.5 respectively, and equal volumes of mPEG2-NHS are added respectively, mixed and reacting in a 45 °C water-bath for 20 minutes. Samples are taken for SDS-PAGE electrophoresis. After scanning and imaging, the ratio of various products is calculated. The results are shown in table 4.
Table 4. The effects of rhIL-6 modified by mPEG2-NHS in different pH conditions
Since high pH value makes the amino residue more nucleophilic and easy to combine with electrophilic modifier, high yield of mono-modified PEG-rhIL-6 (monoPEG-rhIL-6) can be obtained when the pH of reaction system is over 9.0. However, rhIL-6 may become unstable due to an exorbitant pH, so no higher pH is used. Considering that the yields at pH 9.0 and pH 9.5 are similar, pH 9.0 is selected as the optimal pH value of the modification reaction. 2.2 Optimization of reaction time
rhIL-6 samples complying with the purification standard are selected and the pH is adjusted to 9.0. Then mPEG2-NHS is added and mixed, reacting in a 25 °C water-bath. 25min, Ih, 2h, 4h, and 7h after modifier is added, samples are taken respectively for FPLC detection, integrally
calculating the ratio of modified rhIL-6 to unmodified rhIL-6. Since the reaction rate increases as the pH increases, over 98% mPEG2-NHS has reacted or hydrolyzed 25 minutes after reaction starts, and the ratio of modified rhIL-6 remains constant then. With the reaction time being controlled at 30-45 minutes, the whole reaction process is able to be completed. 2.3 Optimization of the ratio of protein and modifier
rhIL-6 samples complying with the purification standard are divided into 5 groups. The pH of the buffers is adjusted to 9.0 and the concentration of protein is adjusted to 0.8 mg/ml. mPEG2-NHS is added respectively to obtain the molar ratios of rhIL-6 to modifier of 1:1, 1:3, 1:5,1:10 and 1:20 respectively, mixed and reacting in a 25 °C water-bath for 45 min. The samples are taken for SDS-PAGE electrophoresis. After scanning and imaging, the percentage of products with different modification degree in the total modified products is calculated. The results are shown in table 5.
Table 5. The effects of rhIL-6 modified by different protein/modifier ratios
It can be seen from table 5 that when the ratio of rhIL-6 to modifier is between 1:1 to 1:3, mono-modified product is the main component in modified products obtained, i.e. monoPEG-rhIL-6. Such product has higher homogeneity, which is beneficial for subsequent purification.
2.4 Determination of PEG modification process
After the optimal screening for the reaction conditions, the following modification process that is stable and efficient is confirmed:
Sample: pure rhIL-6 with SDS-PAGE purity more than 95%, protein concentration between 0.5 and 1 mg/ml, pH 9.0, and PB as buffer, wherein no other amino compounds are present.
Modification agent: mPEG2-NHS MW20kDa, preserved in a dry place at -20 °C
a): hIL-6 is heated in the water bath to 25 °C. mPEG2-NHS of 1-2 times of the total amount of rhIL-6 is measured and put in a dry, clean, nontoxic and apyrogenic container;
b): hIL-6 is poured into a container with mPEG2-NHS, mixed immediately to allow mPEG2-NHS solve completely, reacting in a 25 "C water-bath for 45 minutes; Glycine is added to 0.45M to stop the reaction;
c): After reaction, the resultant is stored at 4°C, sampled and detected, leaving other samples for column chromatography purification.
Preparation of IL-6 polyethylene glycol conjugates (PEG-IL-6)
Example 1. Preparation of PEG-IL-6
The chemical modification equation for PEG-IL-6 preparation is as follows: p
Wherein i and j are integers between 100 and 1000, and the sum of i and j makes the molecular weight of PEG in the conjugate beingl 5000-30000, preferably (preferably) 20000. The amino of-NH-IL-6 in the structure of equation is a ε-amino group of lysine residue. Modification process:
Sample: pure rhIL-6 (Sigma Corporation) with SDS-PAGE purity more than 95%, protein
concentration between 0.5 and 1 mg/ml, pH 9.0, and PB as buffer, wherein no other amino compounds are present.
Modification agent: mPEG2-NHS MW20kDa, preserved in a dry place at -20°C
a): hIL-6 was heated in the water bath to 25 °C. mPEG2-NHS of 1-2 times of the total amount of rhIL-6 is measured and put in a dry, clean, nontoxic and apyrogenic container;
b): hIL-6 was poured into a container with mPEG2-NHS, mixed immediately to allow mPEG2-NHS solve completely, reacting in a 25 °C water-bath for 45 minutes;
c): After reaction, the resultant was stored at 4°C, sampled and detected, leaving other samples for column chromatography purification.
FPLC detection showed that there were 40-60% of the modified products (including PEG-IL-6 of various modification degrees); SDS-PAGE showed that the mono-modified product (monoPEG-IL-6) was more than 60% of modified products (results were showed in Lane 10 and 13 in Fig 1.).
Example 2. Purification of PEG-IL-6
First, homogeneously mix the three batches of products obtained according to the method of example 1, desalt with G-25 gel column which has been balanced to pH 5.0 by 10 mM HAc buffer, and adjust the pH of sample buffers (buffer formulation: Na2HPO12H2O, 15.04g/L; NaH2PO4-2H2O, 1.25g/L; NaCl 8.77g/L. pH 9.0) from 9.0 to 5.0. Then isolate the mixture of modification reaction by one-step SP Sepharose High Performance cation exchange chromatography (elution formulation: solution A: NaAc 0.82 g/L, with pH adjusted to 5.0 by HAc; solution B: NaAc 0.82 g/L, NaCl 29.25 g/L, with pH adjusted to 5.0 by HAc). Under this condition, the hydrolytic mPEG molecules get through because they cannot be adsorbed to the column as a result of not carrying charge or carrying negative charge. Then, by elution of gradual increase of salt concentration, multi-modified mPEG-IL-6 was eluted first, and the mono-modified mPEG-IL-6 second, and the unmodified IL-6 in the end.
Collect all the elution fractions respectively for SDS-PAGE test. The results are shown in Lane 2-8 and Lane 11-12 in Fig 1. In the mono-modified PEG-IL-6 by cation exchange chromatography, there remains a little residual multi-modified mPEG-IL-6 and unmodified IL-6.
Since these three vary greatly in molecular weight (more than 20 kDa), Superdex 200 gel
filtration chromatography column is used for separation.
The fineness of the target sample separated by Superdex 200 gel filtration chromatography column is tested by SDS-PAGE, and the results indicate that the content of mono-modified PEG-IL-6 (monoPEG-IL-6) in the sample after purification is over 85% (Lane 14 in Fig 1), the total contents of PEG-IL-6 with different modification degrees being over 95%, reaching or exceeding the quality requirement of other PEG-modified proteins and polypeptides at home and abroad. Meanwhile, the purification process above should be carried out under nontoxic and apyrogenic condition so as to ensure that the products are up to the state's relative requirements of biochemical drugs.
Example 3. Preparation prescription and process
1 Preparation prescription
1.1 Preparation prescription (based on 1000 bottles)
PEG-rhIL-6 prepared according to the above methods
PEG-rhIL-6 as main component 15mg
Human serum albumin lOg
1.2 Preparation type
Freeze-dried injection prescribed in the "Pharmacopoeia of Peoples Republic of China", 2005 Edition, Third Section, Appendix I "General rules on preparations".
1.3 Specification of preparation
15µg/0.5ml/bottle (200,000 units)
2 For process flow of preparation, please refer to Fig 3.
Pharmacodynamic test security experiment and pharmacokinetic experiment
Test l. Screening the effective dose of PEG-IL-6 on thrombocytopenia induced by
cyclophosphamide on mice
Experimental animals: 18-20g female Balb/c mice
Experimental scheme: the mice were randomly divided into 10 groups, 5 mice in each group; 0.5ml test sample (PEG-IL-6 prepared according to the method of example above) is subcutaneously injected every day into each mouse on the l-5th day; from the 3rd day of injection, 2mg/mouse cyclophosphamide is injected intraperitoneally every day into each mouse for three consecutive days; 10 ul blood sample is collected before the first injection and on the 8-17th day from the tail of each mouse, diluted 6 times for platelet counts with Cell-DYN1600 blood-cell counter.
Experimental grouping scheme:
AO 0.5ml/mouse of PBS is injected intraperitoneally every day;
Al 0.5ml (0.20ug/ml) of test sample IL-6 is injected intraperitoneally every day; 0. lug/mouse;
A2 0.5ml (lug/ml) of test sample IL-6 is injected intraperitoneally every day; 0.5ug/mouse;
A3 0.5ml (2ug/ml) of test sample IL-6 is injected intraperitoneally every day; l.Oug/mouse;
A4 0.5ml (5ug/ml) of test sample IL-6 is injected intraperitoneally every day; 2.5ug/mouse;
Bl 0.5ml (0.lug/ml) of test sample PEG-IL-6 is injected intraperitoneally every day; O.OSug/mouse;
B2 0.5ml (0.2ug/ml) of test sample PEG-IL-6 is injected intraperitoneally every day; 0. lug/mouse;
B3 0.5ml (lug/ml) of test sample PEG-IL-6 is injected intraperitoneally every day; 0.5ug/mouse;
B4 0.5ml (2ug/ml) of test sample PEG-IL-6 is injected intraperitoneally every day; l.Oug/mouse;
B5 0.5ml (5ug/ml) of test sample PEG-IL-6 is injected intraperitoneally every day. 2.5ug/mouse.
The experimental results are shown in table 6:
Table 6. Platelet counting on chemotherapeutic mice injected intraperitoneally
with IL-6 and PEG-IL-6
The starting value (normal value) is based on the average platelet count of 1092xl09/L of all
mice, wherein the excessive counts are deducted and the insufficient counts are added to
eliminate errors. The starting value of each group is defined as 100%, and the percentage of the
average value to the starting value in each group every day is calculated to indicate the recovery rate of platelet counts. The results are shown in table 7.
Table 7. Recovery rates of platelet counts in different groups (%)
Statistics analysis: the whole value calculated on the percentage of average value: to.os=2.179 (n=7)
Comparison between preparation-taken groups and the control group (the first seven data are analyzed):
Al-AO: t=2.13 No significant difference
A2-AO: t=1.85 No significant difference
A3-AO: t=1.63 No significant difference
A4-AO: t=2.17 Significant difference
Bl-AO: t=2.3 Significant difference
B2-AO: t=2.29 Significant difference
B3-AO: t=1.24 No significant difference
B4-AO: t=l.ll No significant difference
It can be concluded from the experimental results that the pre-modification (IL-6) effective dose is 2.5ug/mouse. And the post-modification effective dose is in the range of 0.01-0.5ug/mouse. Within the effective doses, Bl (PEG-IL-6) is significantly lower than A4 (IL-6), the maximum being 250 times lower, which makes the application dose decrease greatly. Test 2. Pharmacodynamic test of PEG-IL-6 on thrombocytopenia induced by cyclophosphamide in mice and Beagle dogs.
Two experimental animal models of hemopoietic system destroyed by cyclophosphamide, mouse and Beagle dog, are used to test three doses of high, middle and low respectively. The commercial drug IL-ll(Jijufen, Yixing, et al., 3ml/tube) is used as the drug for positive control. Results show that the duration time of thrombocytopenia in each dose group is shorter than in the model group, and recovery is faster and the degree of thrombocytopenia is less serious than in the model group, which indicate that the product of this invention can increase the platelet counts of two animal models significantly, lessen the degree of platelet decrease, reduce the duration time of thrombocytopenia, and improve the recovery rate. For the same effect reached as the positive drug, the usage dose is much lower than that of the positive drug. Meanwhile the product can improve canine leukocytes and lymphocyte transiently but has no significant effects on erythrocyte, hemoglobin and reticulocyte. Test 3. The test results of in vitro bioactivities of PEG-IL-6
The in vitro bioactivities of PEG-IL-6 are measured by 7TDlcell/MTT assay; the results are shown in table 8.
Table 8. The results of in vitro bioactivities
The results of activity experiment indicate that the in vitro activity of PEG-IL-6 of present invention is lower than that of unmodified IL-6, which is consistent with the present research. Test 4. Security experiments on PEG-IL-6
1. Acute toxicity test on mice: PEG-IL-6 prepared according to the example above is
administrated via subcutaneous injection, tail vein injection, and intraperitoneal injection in mice,
the doses corresponding to 1000 times of routine dose for clinical use (15 ug/tube, 0.3ug/kg). 14
consecutive days of observation reveals no toxic reactions. LD50 of subcutaneous, tail vein and
intraperitoneal injection on mice is more than 4000µg/kg.
2. Chronic toxicity test on Beagle dogs: PEG-I1-6 of present invention is injected
subcutaneously into Beagle dog daily in high, middle and low doses (30.0, 12.0, 6.0µg.kg~1) for
32 consecutive days, and observation lasts for 15 days after injection stops. The results indicate
that a dose under 12.0µg/kg of present invention injected subcutaneously into Beagle dog is safe.
The experimental results indicate that the PEG-IL-6 of present invention is of fine safety. Test 5. Pharmacokinetic experiments on PEG-IL-6
The 125I-PEG-rhIL-6 up to the requirement of routine pharmacokinetic testing is obtained by labelling PEG-rhIL-6 (prepared according to the aforesaid example) with 125I. The following experiments are carried out according to the requirement for routine pharmacokinetic testing.
125I-PEG-rhIL-6 is injected subcutaneously on rat. Its metabolism is in accordance with one compartment model, the distribution half-life t1/2(a) being 1.4-5.Ih, elimination half-life t1/2 (P) being 58.3~236.5h, peak time being 7.9~13.3h, and in vivo retention time being 42~52h.
125I-PEG-rhIL-6 is intravenously injected on rat. Its metabolism is in accordance with two compartment model, the distribution half life being about l.l~2.5h, elimination half-life being 13~18h.
125I-PEG-rhIL-6 is injected subcutaneously on Beagle dogs. The dogs are divided into three dose groups: 20µg/kg, lOµg/kg, and 5µg/kg. Venous blood is taken from forelimb after administration. The results indicate that their metabolisms are in accordance with one compartment model, the distribution half-life being 0.1~2.2h, elimination half-life being
70.8-247. Ih, peak time being 0.9~10.4h, and in vivo retention time being 69.7~91.7h.
The pharmacokinetic experimental results indicate that several indexes of the mono-modified PEG human IL-6 of the present invention such as retention time, peak time, half-life and the like are much longer than those of the mono-modified IL-6 reported in current literature (see Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol.2001 Jan;112
The stability of IL-6 after mono-modified with PEG has been greatly improved; the mono-modified PEG-IL-6 has longer in vivo half-life and lower plasma clearance. Its usage dose and frequency are reduced significantly, as well as the side-effect, thus facilitating the use by the subject, reducing cost, and improving the safety during use, lessening the pain on the part of the subject significantly. Moreover, the human mono-modified IL-6 polyethylene glycol conjugate of the present invention has good uniformity, and can meet the requirement of safety, effectiveness and controllable quality for clinical administration, suitable for mass production and having a great application prospect.
The detailed illustration as described above does not limit the present invention. All kinds of modifications and variations can be made by those skilled in the art. As long as they do not go beyond the spirit of the invention, such will be within the scope defined by the claims.
AMENDED CLAIMS (Article 41)
1. An interleukin-6 polyethylene glycol conjugate, characterized in that said
interleukin-6 polyethylene glycol conjugate is obtained through covalent mono
modification of IL-6 with polyethylene glycol (PEG), wherein each IL-6 molecule is
covalently bonded with a PEG molecule, the molecular weights of PEG being
2. The interleukin-6 polyethylene glycol conjugate of claim 1, characterized in
that said PEG molecule is bonded to the side chain amino groups of lysine residue of
IL-6 molecule or to the peptide N-terminal amino group of IL-6 molecule.
3. The interleukin-6 polyethylene glycol conjugate of claim 2, characterized in
that said PEGs are branched PEGs, wherein said branched PEGs are two or more PEG
chains attached to said lysine amino groups of IL-6 molecule or peptide N-terminal
amino group of IL-6 molecule.
4. The interleukin-6 polyethylene glycol conjugate of claim 3, characterized in
that said interleukin-6 polyethylene glycol conjugate has the structure of formula (I)
Wherein m represents methyl, i and j are integers between 100 and 1000, the sum of i and j making the molecular weights of PEG in the conjugate being 15000-30000 Da.
5. The interleukin-6 polyethylene glycol conjugate of claim 4, wherein the PEG
molecule used for covalent modification of IL-6 is mPEG2-NHS.
6. The interleukin-6 polyethylene glycol conjugate of claim 2, characterized in
that said PEG is linear PEG, and the said linear PEG is a PEG chain attached to said
lysine amino groups of IL-6 molecule or said peptide N-terminal amino group of IL-6 molecule.
7. The interleukin-6 polyethylene glycol conjugate of claim 6, wherein the PEG
molecule used for covalent modification of IL-6 is mPEG-aldehyde.
8. The interleukin-6 polyethylene glycol conjugate of claim 1, wherein said
interleukin-6 polyethylene glycol conjugate is prepared as follows:
1) preparing an IL-6 solution having protein concentration of 0.05-20 mg/ml and
pH 7.5-10.0 with IL-6;
2) reacting the IL-6 solution prepared with PEG at 15-35°C for 5-100 minutes to
obtain an IL-6 polyethylene glycol conjugate, wherein said PEG is 1-100 times of
IL-6 by weight;
3) isolating and purifying the IL-6 polyethylene glycol conjugate obtained in step
2) to obtain a mono-modified IL-6 polyethylene glycol conjugate.
9. The interleukin-6 polyethylene glycol conjugate of claim 8, wherein said
interleukin-6 polyethylene glycol conjugate is prepared as follows:
i) preparing a solution with IL-6 of SDS-PAGE purity over 95% by using phosphate buffer, in which the protein concentration is between 0.5 and 1 mg/ml, and pH is 9.0;
ii) heating the solution prepared in the water bath to 25 °C, and then reacting in a 25 °C water-bath for 45 minutes with mPEG2-NHS, wherein said mPEG2-NHS is 1-3 times of the total amount of IL-6; adding glycine to 0.45M to stop the reaction and obtain an IL-6 polyethylene glycol conjugate;
iii) isolating and purifying the IL-6 polyethylene glycol conjugate obtained in step ii) to obtain a mono-modified IL-6 polyethylene glycol conjugate.
10. A method for preparing the IL-6 polyethylene glycol conjugate of claim 1,
comprising the steps of:
1) preparing an IL-6 solution having protein concentration of 0.05-20 mg/ml and
pH 7.5-10.0 with IL-6;
2) reacting IL-6 solution thus prepared with PEG at 15-35°C for 5-100 minutes to
obtain an IL-6 polyethylene glycol conjugate, said PEG being 1-100 times of IL-6 by
3) isolating and purifying the IL-6 polyethylene glycol conjugate thus obtained to obtain a mono-modified IL-6 polyethylene glycol conjugate.
11. The method of claim 10, wherein the SDS-PAGE purity of said IL-6 in step 1)
is over 95%, and the solution prepared has a protein concentration of 0.5-1 mg/ml and
pH of 8.7-9.3.
12. The method of claim 10, wherein said purification in step 3) comprising the
treating the IL-6 polyethylene glycol conjugate obtained in step 2) with a G-25 gel filtration column for desalinization, and then loading on a cation exchange column for primary separation;
purifying the resultant with a Superdex 200 gel filtration column to obtain a mono-modified IL-6 polyethylene glycol conjugate.
13. The method of claim 10, comprising the following steps:
i) preparing a solution is prepared having IL-6 of SDS-PAGE purity over 95% by using phosphate buffer, in which the protein concentration is between 0.5 and 1 mg/ml, and pH is 9.0;
ii) heating the solution prepared in the water bath to 25 °C, and then reacting in a 25 °C water-bath for 45 minutes with mPEG2-NHS, wherein said mPEG2-NHS is l-3times of the total amount of IL-6; adding glycine is added to 0.45M to stop the reaction and obtain an IL-6 polyethylene glycol conjugate;
iii) isolating and purifying the IL-6 polyethylene glycol conjugate thus obtained to obtain a mono-modified IL-6 polyethylene glycol conjugate.
14. A pharmaceutical composition comprising the IL-6 polyethylene glycol
conjugate of claim 1 and pharmaceutically acceptable excipients.
15. A use of IL-6 polyethylene glycol conjugate of claim 1 in preparing
Pharmaceuticals for treating thrombocytopenia, Pharmaceuticals for chemotherapy
adjuvants and for immunoenhancement..
|Indian Patent Application Number||9196/DELNP/2007|
|PG Journal Number||42/2013|
|Date of Filing||29-Nov-2007|
|Name of Patentee||CHENGDU INSTITUTE OF BIOLOGICAL PRODUCTS CO., LTD.|
|Applicant Address||CHENGDU JINHUA ROAD 379 #, CHENGDU, SICHUAN PROVINCE 610023,CHINA.|
|PCT International Classification Number||C07K 14/54|
|PCT International Application Number||PCT/CN2006/000891|
|PCT International Filing date||2006-04-30|