Patent Application
20160060305
The Sequence Listing in an ASCII text file, named 30742_SEQ_revised_ST25.txt of 13 KB, created on Sep. 23, 2015, and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.
This invention belongs to the new application of recombinant protein in medicine and drug, in particular to the new application of a flagellin derivative from Salmonella, named CZLC331, in the prevention and treatment of inflammatory bowel disease (IBD).
Ulcerative colitis (UC), also known as idiopathic ulcerative colitis or chronic nonspecific ulcerative colitis, is a disease of large intestine with infiltration of the mucosa and idiopathic chronic non-specific inflammation. The main clinical symptoms are abdominal pain, diarrhea, mucus and bloody stools, and associated with optic neuritis, neuritis, and osteoporosis. It has been characterized as a hardy disease by the World Health Organization (WHO) because treatment of the disease is difficult and time-consuming and the disease relapses easily. Although currently there are a variety of drugs for curing IBD, such as amino salicylic acid, glucocorticoids and immunosuppressive agents, but they have many shortcomings, such as poor efficacy (especially to severe UC), slow onset, long course, and large side effects. Therefore, the drug with an effective, rapid onset, short course, high safety, little side effects, convenient for IBD (especially chronic nonspecific ulcer colitis) is urgently needed.
It has been shown that CBLB502, one of the flagellin derivatives from Salmonella, has a protective effect on the hematopoietic system, and can extend the survival time of mice to the high-dose radiation injury and improve the survival rates of low-dose irradiated mice (Lyudmila G. Burdelya, et al,. An Agonist of Toll-Like Receptor 5 Has Radioprotective Activity in Mouse and Primate Models. Science 2008; 320 (5873): 226-230). The study found that the flagellin protein derivatives from Salmonella and other derivants containing the N-and C-terminal conserved domain have the radial protection, they can effectively improve the number of hematopoietic stem cells in the bone marrow of mice and effective preventing the death of mice caused by lethally irradiation. Its mechanism may be the anti-apoptotic role through NF-κB signal pathway. It means that this protein can be applied to development of anti-radiation drugs. There is no report on Salmonella flagellin derivatives except for the anti-radiation drug.
The object of this invention is to provide a flagellin derivative of Salmonella, named CZLC331, in the prevention and treatment for IBD.
The CZLC331 has a good preventive and therapeutic effect for IBD such as ulcerative colitis (UC) and Crohn's disease (CD) in this invention. Therefore, this protein can be developed as a candidate drug for the prevention and treatment of IBD.
The CZLC331 protein of this invention having the amino acid sequence shown in SEQ ID NO: 3 includes a Tat protein transduction peptide and N-terminal amino acid residues 1-176 and 402-505 of Salmonella flagellin. The fusion protein may be connected by a flexible linker peptide.
The CZLC331 can be prepared using conventional prokaryotic expression, and the purity is more than 95%. The preparation methods of CZLC331 includes following steps:
The prokaryotic expression vectors of carrying coding sequence of Tat transduction peptide is one prokaryotic expression vectors of E. coli in the preparation of CZLC331, pET-22b, pET-28, or pET-15, the best is pET28b-Tat. The prokaryotic expression vector of CZLC331 is pET28b-Tat-CZLC331 which carries the coding sequence of Tat transduction peptide.
The host bacteria is E. coli BL21(DE3), E. coli ER2566(DE3), E. coli BL21(DE3) plysS, E. coli JM109, E. coli HB101 or E. coli Top10 in step 3). The best host bacterium is E. coli BL21 (DE3).
The drug containing CZLC331 protein can be prepared into injection, oral, enema, capsules, enteric-coated tablets, powders or tablets, and the better is injection, enema, or enteric-coated tablets in this invention.
The above-described drugs can be administered in various dosage forms according with the conventional method in the pharmaceutical field.
The active pharmaceutical ingredient of CZLC331 can be 1-35% in capsules, enteric-coated tablets, powders, granules.
The concentration of CZLC331 can be 2-64 g/L in injection, oral and enema liquid dosage forms.
When necessary, one or more pharmaceutically acceptable excipients can also be added to the above drugs, including absorption accelerators, surfactants, lubricants, stabilizers, diluents, bonding agents, wetting agents, disintegrants, diluents, adsorption carrier, and excipient. If necessary, colorings, sweeteners and flavoring agent may also be added.
Specifically, the drug is a protein injection, and the component of this drug is CZLC331 what ratio of weight/volume is 5% (5 g/100 ml), sodium chloride (0.85%, 0 85 g/100 ml) and water for injection.
Alternatively, the drug is coated tablets, and the component is CZLC331 100, lactose 60, microcrystalline cellulose 20, sodium carboxymethyl starch 20, K30 10 granulation 20. And then mixed evenly tablet was made by dry particles 2500: talc tableting 50, and spray coated enteric film-coated layer is made in the table side.
The dosage of this drugs is generally 0. 2-6. 4 mg CZLC331 protein/kg body weight, and is administered 1-2 times a day, the course is 5-10 days. The injection can be given by intramuscular injection, intraperitoneal injection or intravenous injection.
It is shown the new application of CZLC331 in IBD in this invention. CZLC331 has the obvious prevention and therapeutic effects for IBD through the simulate experiment of human ulcerative colitis by giving 2,4, 6-trinitrobenzene sulfonic acid (TNBS). The results are 1) model group: the feeding/drinking water of mice significantly reduced, the activities reduced and accompanied by blood in the stool. There are visible congestion, edema, bleeding and ulcers in the colon. And it is visible about the cell structural disorder, disappearance of goblet cells, lymphocytes, and neutrophils infiltration under the microscope. 2) intraperitoneal administration group: the feeding/water intake of mice return to normal, the activities increased significantly and reduce blood in the stool. The visible congestion, edema, bleeding and ulcers in the colon are significantly reduced. And the cell structural disorder, disappearance of goblet cells, lymphocytes, and neutrophils infiltration under the microscope are significantly reduced. The experimental results show that intraperitoneal injection CZLC331 may be effective against colon inflammation on intestinal damage, and has played a protective role in inflammation in mice. Thus, it is possible that CZLC331 protein was made as active drugs for IBD. The drug has the following advantages: 1) significant effect (effective rate is 100%, the cure rate is 70%), while the effective rate of clinical drug sulfasalazineis only about 90%, and the markedly effective rate was only 50%; 2) rapid onset (24 hours after administration to onset), sulfasalazine is about two weeks, and then the symptoms was improved; 3) short course (treatment is usually 5-10 days), sulfasalazine treatment is usually about 6 weeks; 4) safe (non-toxic), and patients who taking sulfasalazine alanine have elevated aminotransferase (ALT) and aspartate aminotransferase (AST), these suggesting that there is liver toxicity; 5) small side effects (no significant side effects), but there are are nausea, rash, neutropenia after taking sulfasalazine; 6) medication convenient (intraperitoneal injection once daily), and sulfasalazine is 3-4 g/d, 3-4 times a day orally. In summary, the CZLC331 can solve the poor efficacy, slow onset, long course of treatment, side effects of existing treatment drugs for IBD, and it can significantly reduce the pain of patient, promote physical rehabilitation, and improve the patient's quality of life. It will be play an important role in the prevention and treatment of IBD in this invention, and it has broad application prospects.
In order to study the new features of Salmonella flagellin and to develop as a new drug, it was found that CZLC331 may be cut TLRs expression in IBD, such as UC and Crohn's disease, and has a good preventive and therapeutic effect.
It was reported that intestinal epithelial cells can maintain intestinal immune tolerance by down-regulating the expression of TLR4 (Abreu M T, Vora P, Faure E, Thomas L S, Arnold E T, Ardit i M. Decreased expression of Toll like receptor—4 and MD—2 correlates with intest inal epithelial cell protect ion against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol, 2001 Aug 1;167(3):1609-16). It was unexpectedly found that Salmonella flagellin has a role in the prevention and treatment of UC, and the further evidence suggested that this effect is achieved by down-regulating the expression of TLR in colon.
Toll-like receptor (TLR) family is a receptor protein which has a homology with the Drosophila Toll protein, it belongs to the pattern recognition receptors. The main function is identifying the conservative structure of pathogenic microorganisms, improve immune system through a variety of signal transduction ways. The TLR family plays a key role in the anti-infective immunity and innate immunity, and it is also an important to acquired immune regulatory factors. The activation signal is delivered to the intracellular after TLR identifying pathogens, and activate NF-κB transcription factor. TL-1, IL-6, IL-8 are induced, upregulation of IL-12, TNF. IL-12 is a key regulator for inducting T and B lymphocytes cells in the cell-mediated immune response. It can be used as biological adjuvants specific T cell responses against pathogens. Therefore, it can be said TLR family is the early signal for IL-12 and Thl cells involved in the immune response. When the balance between TLRs and the normal flora is broken, it will cause pathological intestinal inflammation. In particular, Salmonella flagellin has the effect for down-regulating the expression of TLR family in colon tissue, and it may be the important mechanism for its role in the treatment of UC.
The coding sequences of amino acids 1-176 and amino acids 402-505, respectively, of Salmonella flagellin were synthesized, and a protein of the total length of 331 amino acids is prepared by prokaryotic expression, and named as CZLC331 (SEQ ID NO: 3). It has been proved that it has a good prevention and treatment for IBD, such as UC and Crohn's disease. It will provide important reference for the prevention and treatment of IBD.
The detailed embodiments and specific procedure are given as follows, but the scope of protection of this invention is not limited to the following embodiments. The methods used in this invention are conventional methods.
1. The construction of prokaryotic expression vector pET28b-Tat-CZLC331 for expressing CZLC331
b) The digestion of the target gene CZLC296 and pET28b-TAT vector using restriction endonuclease.
CZLC296 and pET28b-TAT vector were digested using restriction endonuclease BamH I and Xho I. The construction of pET28b-TAT vector is synthesizing the TAT sequence for having the upstream of Ncol and the downstream of NdeI, then digested the TAT sequence and pET28b using restriction endonuclease. The recovered fragment was recycled by T4 DNA ligase and restriction digestion. The sequence was confirmed by Invitrogen Co., Ltd. And then the digested production was transformed into competent cells E. coli DH5α using T4 DNA ligase at 16° C.
The sequencing results show that the obtained sequence and the insertion position were correct, and the recombinant expression vector included the coding sequence of the Tat protein transduction peptide and CZLC296. The vector was named pET28b-Tat-CZLC331. The protein included the Tat protein transduction peptide at the N terminal end of CZLC296 and was named CZLC331. The nucleotide sequence encoding the protein is shown in SEQ ID NO: 2.
The prokaryotic expression vector pET28b-Tat-CZLC331 what was constructed correctly was transformed into E. coli BL21 (DE3), and then coating to the LB plate what contain the Kana+ 100 μg/ml. Then the clone was inoculated into 5 mL LB containing Kana+ 100 μg/mL, the bacteria was shake at 220 rpm for 16 h to be fully recovered bacteria.
The bacteria was recovered, and it was diluted to OD600=0.8. Then the bacteria (5 ml) were inoculated into 150 ml LB containing Kana+ (final concentration is 100 μg 1 ml). The bacteria were shaking at 37° C. approximately 4-5 hours at 220 rpm. The inducing agent IPTG (final concentration 1 mM) was added rapidly to the bacteria when the OD600 is 0.6-1.0, and it was induced and expressed at 30° C., 220 rpm by 8 hours.
Experimental Animal: Male BALB/c mice (7-8 weeks old, 25±5 g) were obtained from Experimental Animal Center of Academy of Military Medical Sciences (Beijing, China). Mice were provided sterile food and water, and 12 h light/12 h dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee at Academy of Military Medical Sciences.
Reagents: 2,4,6-trinitrobenzene sulfonic acid (TNBS, were purchased from Sigma Chemical Co.), CZLC331 (prepared in Example 1), RT PCR kit (ID isFSK100, purchased from Toyobo Biotechnology Co., Ltd.).
Model preparation: Mice (hunger for 36 h) were anesthetized with isoflurane using anesthesia machine. To induce IBD, 150 mg/kg of TNBS (Sigma-Aldrich) in 38% ethanol (to break the intestinal epithelial barrier) was administered by the mouse gavage needle equipped with a 1 ml syringe, what the diameter of silicone tube is 2.0 mm and the length is about 10 cm, the gavage needle was advanced into the rectum until the tip was 4 cm proximal to the anal verge. The total injection volume was 100 μl.
Experimental animal groups: model group, treatment group and control group (n=8). The control mice received 38% ethanol alone by using the same method described above, and normal feeding two days. The treatment group: BALB/c mice were treated therapeutically with CBLB502 (3.2 mg/kg) at 0.5 h after TNBS administration. At the same time, BALB/c mice were treated therapeutically with CBLB502 (0.2-6.4 mg/kg) from 0.5 h to 8 h after TNBS administration, and normal feeding two days. The mice of model group were normal feeding two days after TNBS administration.
Mice were monitored for the appearance of diarrhea, changes of body weight, and overall mortality. At the end of the experiment after TNBS administration two days, surviving mice were killed, blood samples were collected by cardiac puncture, and a 7-cm segment of the colon was excised for macroscopic (
1. The results shown that feeding/water and activity were significantly reduced, and there were blood in the stool in model mice. There were significant congestion, edema, hemorrhage, and ulceration in modeling. The colon appearance and colonic mucosa of mice with ulcerative colitis after 0.5 h treating with 0.2mg/kg CZLC331 protein in TNBS modeling is shown in
The food intake and water intake is normal, and there were no blood in the stool and reduction activities in control mice. The cells arranged in neat rows, no decrease in goblet cells and infiltration of lymphocytes and neutrophils.
2. Histological examination: The colonic samples were fixed in 4% buffered formalin overnight and then transferred to 70% ethanol, embedded in paraffin for sectioning, and then stained with hematoxylin and eosin (H&E) to examine the histological differences.
The colonic mucosa HE staining of mice with ulcerative colitis after 0.5 h treating with 0.2 mg/kg CZLC331 protein in TNBS modeling is shown in
3. The Expression of TLR Family
The colon samples were removed at the indicated times, washed with phosphate-buffered saline (pH 7.2), and cut in small pieces. Total RNA of colon samples what were control group, modeling group and treatment group were extracted by Total RNA kits II (Omega, Japan) according to the manufacturers' instructions, and then reverse-transcribed by the Reverse Transcription System (Sigma, USA). Subsequently, the TLRs and β-actin were amplified by PCR using the following primers (Table 1). The reaction system of PCR was 50 μl, Ex Taq 0.25 μl, 10×buffer 5 μl, dNTP 5 μl, template 2 μl, the upstream and downstream primers 1 μl, ddH2O 35.75 μl. The reaction condition of PCR is 95° C. 5 min, 95° C. 45 sec, 62° C. 45 sec, 72° C. 1 min, total 25 cycles, and then 72° C. 5 min. PCR products were identified on 2% agarose-gel electrophoresis followed by image analysis software.
The results were that the expression of TLR family has significant differences with the model group after CZLC331 treatment in
The treatment and mechanism may also be related to reduce expression of TLR family. And it suggested that the therapy of CZLC331 for IBD was effective through the pathway of TLR family, it can be become the drug for treating IBD.
Experimental Animals: the same as in Example 2.
Reagents: the same as in Example 2.
Model preparation: the same as in Example 2.
Experimental animal groups: model group, prevention group and control group (n=8). The prevention group: BALB/c mice were treated therapeutically with CBLB502 (3.2 mg/kg) before TNBS administration, and then the mice were given TNBS and normal feeding two days. The model group was normal feeding two days after TNBS administration. The control mice received 38% ethanol alone by using the same method described above, and normal feeding.
Test Contents and Test Methods: the Same as in Example 2.
The results shown that feeding/water and activity were significantly reduced, and there were blood in the stool in model mice. There were significant congestion, edema, hemorrhage, and ulceration in modeling (
1. Composition: the CZLC331 injection (5% by weight/volume (mg/mL) CZLC331 protein), 0.85% sodium chloride and water for injection.
2. The Preparation Method of CZLC331 Protein Injection:
1) The prokaryotic expression vector pET28b-Tat-CZLC331 what was constructed correctly was transformed into E. coli BL21 (DE3), and then coating to the LB plate what contain the Kana+ 100 μg/ml. Then the clone was inoculated into 5m1 LB containing Kana+ 100 μg/ml, the bacteria was shake at 220 rpm for 16 h.
2) The bacteria was recovered, and it was diluted to OD600=0.8. Then the bacteria (5 ml) were inoculated into 150 ml LB containing Kana+ (final concentration is 100 μg/ml). The bacteria were shaking at 37° C. approximately 4-5 hours at 220 rpm. The inducing agent IPTG (final concentration 1mM) was added rapidly to the bacteria when the OD600 is 0.6-1.0, and it was induced and expressed at 30° C., 220rpm by 8 hours.
3) The induced and expressed bacteria were collected at 12000 rpm for 10 minutes at 4° C. The bacteria were broke by ultrasound using 20 mM sodium phosphate buffer, and was prepared a CZLC331 protein. The unpurified CZLC331 was directly loaded onto the HisTrap HP 5 ml column (purchased from GE Corporation), and then the peak baseline was washed using 4-5 column volumes of the above buffer. Finally, the protein was washed using elution buffer (20 mM Na3PO4+0.5M NaCl+0.5M imidazole, pH7.4), and then CZLC331 was obtained.
4) The CZLC331 protein was dissolved in injection water, and then sodium chloride was added, and uniformly stirred. The activated carbon what was treated by dry heat at 0.3% (weight/volume) was added to the solution for adsorbing 15 min, the solution was filtered to clarity and safekeeping. The filter includes three filters: a titanium filter for decarburizations, a 0.45 μm cartridge filter filtering and a 0.22 μm drum filter cleaning strainer.
1. The CZLC331 protein 100 g is weight, and it is through the 100-mesh sieve. The lactose is 60 g, microcrystalline cellulose is 20 g, carboxymethyl starch sodium is 20 g, povidone K30 10 g through a 80-mesh sieve. The main drugs and accessories were mixed in accordance to the principle of equal increments, and then other materials is added for a 30% volume ratio. Finally, the drug is granulated through the 20 mesh and gets the wet granules.
2. The CZLC331 protein was dried for 3 hours at 55-65° C., and got dry pellets after 20-mesh sieve. The 50 g talcs were added to 2500 g pellets of CZLC331, and was mixed. The content of mixed protein was determinated. The determinated pellets were sending to a tablet machine for fitting speed and compress, and were suppressed into platode CZLC331 protein tablet what is a diameter of 1.2 cm.
3. The enteric coating was painting on platode CZLC331 tablet for about 1.0 mm. The coating material is EOBS68, the enteric coating is airtight and moisture, it has strongly coat strength and resistance to the gastric juice. The effective and safe release of drug is done through changing the pH of solution.
Efficacy Analysis:
The CZLC331 protein as the active ingredient was prepared to the drug for preventing and curing IBD. It has the following characteristics:
1) High efficiency: the experimental data show that all animals less fooding and activity, and blood in the stool have improved significantly in the treatment group and the prevention group in example 2 and example 3. These results indicated that the effective rate is 100%. The effective rate of recovered colon mucosa is 75% in treatment group. The clinical drugs sulfasalazine has the effective rate of only about 90%, and the markedly effective rate was only 50%.
2) Rapid onset: the experimental data show that all animals have effective after treating at 24 h, while the sulfasalazine take is about two weeks in example 2 and example 3.
3) The course of treatment is short (usually 5-10 days): The sulfasalazine treatment is generally about 6 weeks. The course of treatment using this drug is 1-2 days, the effect of sulfasalazine is a week or so, and the clinical onset is six weeks. Therefore, the course of treatment is 5-10 days in this invention.
4) High safe (non-toxic): The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of patients who take sulfasalazine increased, it suggested that there is liver toxicity. The CZLC331 belongs to the protein drugs, it has been used as anti-radiation drug, and it has non-toxic to the body.
5) Side effects (no significant side effects): There have nausea, rash, neutropenia after taking the sulfasalazine, this is a protein drug, it has been used as anti-radiation drug, and it has no significant side effects to the body.
6) Convenient (only once daily intraperitoneal injection): The usage of sulfasalazine is 3-4 g/d orally, and needed divided into 3-4 times a day.
The invention provides a flagellin derivative of Salmonella CZLC331 in the prevention and treatment for IBD. It has high efficacy, fast onset, short course, non-toxic, small side effects, and medication convenient. The CZLC331 can solve the poor efficacy, slow onset, long course of treatment, side effects of existing treatment drugs for IBD, and it can significantly reduce the pain of patient, promote physical rehabilitation, and improve the patient's quality of life. It will be play an important role in the prevention and treatment of IBD in this invention, and it has broad application prospects.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201110186065.6 | Jul 2011 | CN | national |
This application is a continuation of U.S. application Ser. No. 14/131,050 filed Feb. 28, 2014, which is the national phase of International Application No. PCT/CN2012/000097 filed Jan. 19, 2012, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14131050 | Feb 2014 | US |
| Child | 14940648 | US |
