Peptide stimulating antitumor immune response, pharmaceutical composition based on thereof, method for treatment of mammal and method for modulation of immune response

FIELD: biotechnology, medicine, oncology.

SUBSTANCE: invention proposes peptide of the structure Tyr-Ser-Leu and a pharmaceutical composition based on thereof that is used for stimulating antitumor immune response. Also, invention proposes methods for treatment of mammal and for modulation of the immune response. Proposed inventions expand assortment of agents used in treatment of cancer diseases.

EFFECT: valuable medicinal properties of peptide and pharmaceutical composition.

20 cl, 48 tbl

 

The present invention relates to the field of immunology. In particular, the present invention relates to peptides and pharmaceutical compositions capable of modulating immune responses.

Background of invention

It is known the use of peptides for the treatment of diseases and as pharmaceutical compositions. For example, in U.S. patent 6191113 described peptide, which has an inhibitory activity for the growth of smooth muscle cells and thus can be used for the prevention and treatment of pathological conditions associated with cell growth of smooth muscles, such as atherosclerosis, restenosis after angioplasty, the narrowing of the lumen after the transplantation of blood vessels and sarcoma of smooth muscles. In U.S. patent 6184208 described another peptide, which is found to modulate physiological processes, such as activity weight gain zone of epithelial growth and hair growth.

Summary of the invention

Thus, an object of the present invention is the identification of biologically active polypeptides. Standard chemical methods was synthesized many of peptides and screened for their biological activity. The peptides coded with letters CMS followed by the number. Just revealed 30 peptides with biologicheskii activity in vivo. Sequence and the relevant identification number (ID) of such biologically active peptides are shown in table A.

Table a
SEQ ID No:The title peptidePeptide sequence
1CMS001Pro Thr Thr Lys Thr Tyr Phe Pro His Phe
2CMS002Val Val Tyr Pro Trp Thr Gln Arg Phe
3CMS008Lys Ala Val Gly His Leu Asp Asp Leu Pro Gly Ala Leu
4CMS010Val Ala Pro Glu Glu His Pro Thr Leu Leu Thr Glu Ala Pro Leu Asn Pro Lys
5CMS012Leu Gly Met Glu Ala Cys Gly Ile His Glu Thr Thr Tyr
6CMS013Leu Arg Val Ala Pro Glu Glu His Pro Val Leu
7CMS014Ala Ala His His Pro Asp Asp Phe Asn Pro Ser Val
8CMS015Pro Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met
9CMS016Ile Gly Met Glu Ser Ala Gly Ile His Glu Thr Thr Tyr
10CMS018Val Gly Met Gly Glu Lys Asp Ser Tyr
11CMS019Val Gly Met Gly Gln Lys Asp Ser Tyr
12CMS020Val Gly Met Gly Gln Lys Asp Ser Tyr Val
13CMS021Met Ala Thr Ala Ala Ser Ser Se Ser Leu
14CMS022Tyr Ser Phe
15CMS023Ala Ala Phe
16CMS024Tyr Ser Leu
17CMS026Thr Thr Tyr Asn Ser Ile Met
18CMS027Phe Glu Glu Asn Met
19CMS028Phe Glu Pro Ser Phe
20CMS029Phe Asn Glu Glu
21CMS030Phe Glu Glu Met
22CMS032Phe Glu Glu Glu
23CMS033Phe Glu Ser Phe
24CMS034Pro Glu Asn Phe
25CMS035Phe Val Asn Asp
26CMS036Phe Gln Pro Ser Phe
27CMS003Phe Asn Phe Val Pro Pro
28CMS007Ala Gly Asp Asp Ala Pro Arg Ala Val Phe
29CMS009Leu Arg Val Ala Pro Glu Glu His Pro Thr Leu
30CMS011Arg Val Ala Pro Glu Glu His Pro Thr Leu

Accordingly, one aspect of the present invention relates to essentially pure peptides having the sequence identified as sequences of SEQ ID nos:1-30. Thus, this is sabreena also relates to essentially pure peptides comprising the amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also relates to essentially pure peptide consisting mainly of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30. In a specific embodiment, the peptides can modulate, but are not limited to modulating, one or more of the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

In another aspect, the present invention relates to essentially pure peptides that are functional derivatives of the peptides having the sequence identified as sequence ID No:1-30. Thus, the present invention also relates essentially to the pure peptide comprising the amino acid sequence, which is the functional derivative of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also relates to essentially pure peptide consisting mainly of the amino acid sequence which is a functional derivative of amino acid sequence selected from the group consisting the th of SEQ ID No:1-30. In a specific embodiment, the peptides that are functional derivatives, can modulate, but are not limited to modulating, one or more of the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

In another aspect, the present invention relates to nucleic acids that have sequences encoding the peptides identified above as sequences of SEQ ID nos:1-30. An additional aspect of the present invention relates to expression vectors that contain nucleic acid sequences of the peptides shown below as sequence SEQ ID No:1-30. Thus, this aspect of the present invention also relates to a genetic vector comprising the nucleotide sequence encoding the peptide comprising the amino acid sequence selected from the group comprising SEQ ID nos:1-30. It also refers to a genetic vector comprising a nucleotide sequence encoding a peptide consisting mainly of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30. The invention also relates to a genetic vector, include the him nucleotide sequence, encoding a peptide comprising a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also refers to a genetic vector comprising a nucleotide sequence encoding a peptide consisting mainly of functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

Another aspect of the present invention relates to hybrid peptides containing a leader or signal peptide adjacent to the peptide, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID nos:1-30. The present invention also relates to hybrid peptides containing a leader peptide adjacent to the peptide, the peptide includes a functional derivative of a peptide having an amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

The present invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide comprising amino acid leader sequence that is adjacent to a peptide comprising a functional amino acid sequence that not only is em a functional derivative of a biologically active amino acid sequence, selected from the group comprising SEQ ID nos:1-30. It also refers to a genetic vector comprising a nucleotide sequence encoding a peptide comprising amino acid leader sequence adjacent to the peptide, consisting primarily of a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

In a specific embodiment, the peptides produced in any of the above genetic vectors, can modulate, but are not limited to modulating, one or more of the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

In another aspect the present invention relates to a microorganism, the genome of which comprises a nucleotide sequence encoding a peptide consisting of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also refers to the organism, the genome of which comprises a nucleotide sequence encoding a peptide consisting mainly of the amino acid sequence of wybrand the th group, consisting of SEQ ID nos:1-30.

In another aspect the present invention relates to a micro-organism with genetic material that includes the nucleotide sequence encoding an exogenous peptide comprising a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also refers to an organism with a genetic composition that includes a nucleotide sequence that encodes an exogenous peptide, consisting primarily of a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID nos:1-30. The term "exogenous peptide", as used herein, refers to a peptide having an amino acid sequence that differs from any other peptide, usually expressed by the organism in its natural unmodified form.

In another aspect the present invention relates to a microorganism with a genetic composition that includes a nucleotide sequence that encodes an exogenous hybrid peptide comprising amino acid leader in sledovatelnot, adjacent to the peptide, the peptide includes an amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also refers to the organism, the genome of which comprises a nucleotide sequence encoding a hybrid peptide comprising amino acid leader sequence that is adjacent to a peptide consisting mainly of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

In another aspect, the present invention relates to a microorganism with a genetic composition that includes a nucleotide sequence that encodes an exogenous hybrid peptide comprising amino acid leader sequence adjacent to the peptide, the peptide includes a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also refers to an organism with a genetic composition that includes a nucleotide sequence that encodes an exogenous hybrid peptide comprising amino acid leader sequence adjacent to the peptide, consisting primarily of a functional amino acid sequence which is a functional derivative of the biological the ski active amino acid sequence, selected from the group consisting of SEQ ID nos:1-30.

In a specific embodiment, the peptides produced by any of the above microorganisms can modulate, but are not limited to modulating, one or more of the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

In the following aspect the present invention relates to pharmaceutical compositions comprising essentially pure peptide comprising amino acid sequence selected from the group consisting of SEQ ID nos:1-30. The invention also relates to pharmaceutical compositions comprising essentially pure peptide consisting mainly of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

The present invention also relates to pharmaceutical compositions comprising essentially pure peptide comprising a functional derivative of amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also relates to pharmaceutical compositions comprising essentially pure peptide consisting mainly of functional derived amino acid sequence selected from the group status is the present of SEQ ID No:1-30. In addition, the invention relates to a pharmaceutical composition consisting of essentially pure peptide consisting mainly of functional derived amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

In a specific embodiment, the peptides in the composition of any of the above pharmaceutical compositions can modulate, but are not limited to modulating, one or more of the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

In the following aspect the present invention relates to a method for producing a pharmaceutical composition, comprising obtaining essentially pure peptide comprising amino acid sequence selected from the group consisting of SEQ ID No:1-30, and mixing the specified essentially pure peptide with a pharmaceutically acceptable carrier. It also relates to a method for producing a pharmaceutical composition, comprising obtaining essentially pure peptide consisting mainly of the amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

In another aspect, the present invention relates to a method for Pharma is eticheskoi composition, including the production of essentially pure peptide comprising the amino acid sequence, which is the functional derivative of amino acid sequence selected from the group consisting of SEQ ID No:1-30; and mixing the specified essentially pure peptide with a pharmaceutically acceptable carrier.

The invention further relates to a method for producing a pharmaceutical composition, comprising obtaining essentially pure peptide consisting mainly of the amino acid sequence, which is the functional derivative of amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

In connection with any of the above methods, the peptide can modulate, but are not limited to modulating, one or more of the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

In another aspect the present invention relates to a method of treatment of a person, including the introduction of man pharmaceutically effective dose of essentially pure peptide comprising amino acid sequence selected from the group consisting of SEQ ID nos:1-30. It also relates to a method of treatment of man, includes introduction to the man pharmaceutically effective dose of essentially pure peptide comprising the amino acid sequence which is a functional derivative of amino acid sequence selected from the group consisting of SEQ ID nos:1-30.

In a specific embodiment, the above-described peptides used for treatment of humans, can be used to modulate, but are not limited to modulation of one or more of the following States: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancer cells, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and body weight.

In connection with any of the above sequences of nucleic acids, peptides and/or hybrid peptides expressed from the data sequences of nucleic acids can modulate, but are not limited to modulating, the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

Another aspect of the present invention relates to a method for treatment of diseases involving the introduction of a pharmaceutically effective dose of p is essentially pure peptide, with the sequence SEQ ID No:1 - SEQ ID No:30. In a particular embodiment, the input thus peptides can modulate, but are not limited to modulating, the following: immune activity; infection, hepatitis, including, but not limited to hepatitis b; jade; the growth of cancerous tumors, including, but not limited to, sarcoma, liver cancer, leukemia and melanoma; and the weight of the body.

As described above, another variant of implementation of the present invention is a peptide or polypeptide, consisting mainly of peptides of the present invention. As used herein, the term "consisting mainly of" refers to a peptide or polypeptide that includes the amino acid sequence of the peptides of the present invention together with additional amino acids at the carboxyl and/or amine ends, and which supports the activity presented here, the peptides of the present invention. Thus, as a non-limiting example, when the activity of the peptide of the present invention is the modulation of immune activity, peptide or polypetide, "consisting mainly" of the peptide of the present invention will possess activity modulation of immune activity, as presented here relative to the peptide, and will not be abled the th some characteristics, which significantly reduce the ability of the peptide or polypeptide to modulate immune activity, or which make a significant change in the basic and novel characteristics of the peptide as a modulator of immune activity. Thus, in the example above, the full-sized natural polypeptide, which has the primary activity that is different from the modulation of immune activity, and that somewhere contains the amino acid sequence of peptide according to the present invention, will not be a peptide or polypetide "consisting mainly" of the peptide of the present invention. Similarly, in the above example, genetically engineered peptide or polypeptide, which has the primary activity that is different from the modulation of immune activity, but somewhere in yourself amino acid sequence of the peptide of the present invention, will not be a peptide or polypeptide "consisting mainly" of the peptide of the present invention.

In addition to the above I used the example of modulating immune activity, the above definition also applies to all peptides of the present invention in regard to the types of activity that have such peptides. In particular, the above definition about what is worn to the peptides of the invention, possessing activity of modulating the degree of viral infection, the modulation degree of hepatitis b infection, the modulation degree of jade, to modulate the growth of cancer cells or modulation of body weight, as described below in the detailed description.

Specialists in this field will easily be able to determine, is or is not a peptide or polypeptide mainly from the peptide of the present invention within the definitions given above, by measuring the activity of the peptide or polypeptide using analyses of modulating immune activity, modulating the extent of viral infection, the modulation degree of hepatitis b infection, the modulation degree of jade, to modulate the growth of cancer cells or modulation of body weight, which are provided here for specific peptides of the present invention.

In a preferred embodiment, the term "consisting mainly of" refers to peptides or polypetides that have less than 20 amino acid residues in addition to the peptide according to the present invention. In a more preferred embodiment, the same terminology refers to peptides having less than 15 amino acid residues in addition to the peptide according to the present invention. In an even more preferred embodiment, the same terminology and refers to peptides, with less than 10 amino acid residues in addition to the peptide according to the present invention. In another preferred embodiment, the same terminology refers to peptides or polypeptides that have at least 6 amino acid residues in addition to one of the peptides according to the present invention. In another preferred embodiment, the same terminology refers to peptides or polypeptides having less than 4 amino acid residues in addition to one of the peptides according to the present invention. In the most preferred embodiment, the same terminology refers to peptides or polypeptides that have at least 2 amino acid residues in addition to one of the peptides according to the present invention.

Detailed description

Peptides can be synthesized by standard synthetic methods from L-amino acids, and can also be synthesized by genetic engineering methods using nucleic acid having a sequence encoding a specific peptides.

I. Biological activity

To study the possible biological activity of the peptides studied the immunological effect of the peptides on animal model using techniques appropriate to the "Principles of preclinical studies of new drugs", is commissioned by the Ministry of Health people's Republic of China [1].

To detect any possible effect of the peptides on specific cellular immune function used test transformation of T-lymphocytes, test the cytotoxic activity of NK cells and test secreting T lymphocytes IL-2 and IFN-γ. Test cleaning particles of coal used to detect any possible effect of the peptides on non-specific cellular immune function. Hemolysis of sheep erythrocytes (SRBC) were used to detect any possible effect of the peptides on humoral immune function. Test weighing immune organ used to detect any possible effect of the peptides on the organ level.

In this study, the group receiving saline was used as negative control, whereas the group receiving IL-2 and IFN-γused as positive controls, since IL-2 and IFN-γ are well known Immunostimulants[10]. This study used four arbitrary concentration samples of peptides to block 1000-fold range of doses. Due to the internal complexity of the immunological response in vivo and the lack of previous data on the response depending on the dosage, as a positive biological activity seen is any statistically significant difference relative to the negative control in any of the dosage groups.

The results of this study were as follows:

1. It is established that peptides S001, S002, S003, S007, S008, S009, S010, S011, S012, S015, S019, S021, S029 and S034 can enhance the transformation of T-lymphocytes, showing a statistically significant difference when compared with normal control group receiving saline. Also found that the peptides S014 and S036 able to inhibit the transformation of T lymphocytes, showing a statistically significant difference when compared with normal control group receiving saline.

2. It is established that peptides S001, S002, S003, S008, S009, S010, S011, S012, S013, S015, S016, S020, S021, S022, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 able to increase the cytotoxic activity of NK cells, showing a statistically significant difference when compared with normal control group receiving saline. Also found that the peptides S008 and S012 in a suitable concentration can reduce the cytotoxic activity of NK cells, showing a statistically significant difference when compared with normal control group receiving saline.

3. It is established that peptides S001, S003, S007, S009, S010, S011, S012, S015, S020, S022 and S034 able to increase the secretion of interleukin-2 (I-2) T-lymphocytes, showing a statistically significant difference when compared with normal control group receiving saline.

4. It is established that peptides S001, S003, S009, S010, S011, S012, S013, S016, S021, S022 and S028 can increase the secretion of IFN T-lymphocytes, showing a statistically significant difference when compared with normal control group receiving saline.

5. It is established that peptides S001, S002, S003, S007, S008, S009, S010, S011, S012, S013, S014, S015, S016, S018, S019, S020, S021, S022, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 can increase the synthesis of anti-SRBC antibodies with antigenic stimulation, showing a statistically significant difference when compared with normal control group receiving saline. Also found that the peptides S002, S003, S009, S010, S011, S013, S014, S015, S018, S019, S020, S026, S028, S029, S030, S034 and S036 in a suitable concentration able to inhibit the synthesis of anti-SRBC antibodies with antigenic stimulation, showing a statistically significant difference when compared with normal control group receiving saline.

6. It is established that peptides S003, S008, S009, S010, S011, S013, S016, S018, S019, S020, S022, S024, S027, S030, S035, S036 can enhance the phagocytic activity monoed the situations of phagocytes, showing a statistically significant difference when compared with normal control group receiving saline.

7. It is established that peptides S001, S002, S008, S010, S012, S013, S014, S015, S016, S018, S019, S020, S021, S022, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 able to increase the weight of the thymus gland, showing a statistically significant difference when compared with normal control group receiving saline.

8. It is established that peptides S019, S020 and S030 able to increase the weight of the spleen, showing a statistically significant difference when compared with normal control group receiving saline. It was also found that the peptides S001, S003, S007, S008, S009, S010, S011, S013, S014, S015, S021, S023, S024, S027, S029 and S036 in a suitable concentration can reduce the weight of the spleen, showing a statistically significant difference when compared with normal control group receiving saline.

The following describes the materials and methods used for impact analysis of peptides in mice:

Materials:

1. Experimental animal

Mouse BALB/c, weight 18-22 g, 50% females and 50% males, provided by the Center for experimental animals, national Institute of medical Sciences (National Institute of Medical Science, China.

2. BB is Denia

The group of recombinant murine IFN-γ(rmIFN-γ): 3×105IU/kg/day

The group of recombinant human IL (rhIL)-2: 3×105IU/kg/day

Group saline: 0.5 ml/every day

the dose of peptide in group I: 500 mcg/kg/day

the dose of peptide in group II: 50 mg/kg/day

the dose of peptide in group III: 5 mcg/kg/day

the dose of peptide in group IV: 0.5 mg/kg/day

All of the above substance was dissolved in 0.5 ml of saline and injected intraperitoneally (I.P. Pavlova.) continuously for 15 days, once a day.

3. Main reagents

Peptides were produced in the usual way American Peptide Company, Inc., USA

Fetal calf serum and cell culture medium RPMI-1640 from Gibco, USA

MTT and ConA, Sigma, USA

rmIFN-γ, Beijing Biotech Inc., China

rhIL-2, Shanghai Huaxin Biotech Inc., China

The solution for the separation of lymphocytes, research Institute of Hematologic diseases, national Institute of medical Sciences, China.

Vesicular stomatitis virus (VSV), IFN-γ and IL-2 standard samples, national Institute for control of pharmaceutical and biological products, China.

HT-2 cells and L929 cells, a gift of Professor Chen WF, Department of immunology, Peking University, Beijing, China.

Method

1. The effect of peptides on cell-mediated immunity

1.1. Obtaining suspensions of spleen cells[1,2]

<> BALB/c mice were randomly divided into groups of the peptide, IFN, IL-2 and saline solution. Ten mice per group. The day after the last injection of the analyte, the mice were killed by cervical vertebrae. The spleen was removed aseptically and manually dispersively in a cold solution of D-Hank using a needle for injection. Dispergirovannoyj cell suspension was additionally passed through a sieve of 100 gauge stainless steel with a diameter of 150 μm. After centrifugation at 200g for 10 minutes the supernatant (supernatant) was discarded. Sediment cells re-suspended in 10 volumes of buffer Tris-NH4Cl and then left for 10 minutes at room temperature. Suspended cells were collected by centrifugation at 150g for 10 minutes. Cells were washed 2-4 times with a cold solution of D-Hank by resuspendable and centrifugation under the above conditions. The washed cells were then diluted to the desired density of the cells using a culture medium RPMI-1640 containing 10% fetal calf serum.

1.2. The effect of peptides on the transformation of T-lymphocytes

Cells of the spleen density of 1×106ml was placed in 96-well plates to cell cultures, 100 μl/well, three parallel wells for each of the analyzed sample and control sample for ka the DOI mouse. In the analyzed wells were added 100 μl/well ConA solution 100 ág/ml in RPMI-1640 and 100 µl/well just RPMI-1640 was used for control. Cells were incubated for 66 hours at 37°C, 5% CO2. The cells were then besieged by centrifugation at 150g for 10 minutes. The supernatant was collected and stored at -20°to determine the cytokines IL-2 and IFN.

50 μl/well MTT solution 1 mg/ml in RPMI-1640 was added to the precipitate cells and cells re-suspended by shaking for 2 minutes. Incubation was continued for 4 hours. The supernatant was discarded after centrifugation at 150g for 10 minutes. 120 μl of 40 mm HCl in propanol-2 was added to the precipitate cells and was shaken for 3 minutes to obtain OPNmfor each hole, normalized to 630 nm. Used ELISA reader.

Calculations:

For each mouse was formed by three of the analyzed wells and three control wells. The stimulation index (SI) for each mouse received, initially obtain the average O.D. (optical density) for three parallel holes, and then dividing the value for the analyzed holes on the value for a key hole.

1.3. The effect of peptides on NK cell activity[3,4]

Received murine spleen cells by density 4×106ml, as described above in section 1.1. Target cells YAC-1 was taken in the logarithmic phase and brought up to 1×105Jr. With COI is whether the 96-well plates for cell cultures, 100 μl of cells in the murine spleen, and 100 µl of culture medium was added to control wells, containing only cells of the spleen; 100 μl of target cells and 100 μl of culture medium was added to control wells containing only target cells; 100 μl of cells of mouse spleen and 100 μl of target cells were added to the wells for analysis of NK activity. Prepared three parallel series of the above holes on the mouse.

Samples were centrifuged at 150 g for 10 minutes to collect cells. The supernatant was discarded and added 50 μl/well MTT solution 1 mg/ml, the Reaction mixture was then shaken for 2 minutes and incubated at 37°C, 5% CO2within 4 hours. The supernatant was discarded after centrifugation at 150g for 10 minutes. Added 120 μl of 40 mm HCl in propanol-2 and shaken for 3 minutes. The ELISA reader was used to obtain OPNmfor each hole, normalized to 630 nm.

Calculations:

For each mouse had 9 holes: three holes with spleen cells - only control, three with target cells only control and three of the analyzed wells with spleen cells and target cells. Index NK cell activity for each mouse received, initially receiving the O.D. value of three parallel wells each combination, and then using this average OD in the following formula:

The index of the activity of NK cells = [1-(mean OD of wells with cells of the spleen and cells-mission the mi - the average OD of the wells only with spleen cells)+(average OD of wells with only target cells)]×100%.

1.4. The effect of peptides on the activity of T-lymphocytes in the secretion of IL-2[5].

HT-2 cells in logarithmic phase were collected by centrifugation at 150g for 10 minutes and washed three times with a cold solution of Henk through re-suspension and centrifugation. Collected HT-2 cells re-suspended in RPMI-1640 and incubated at 37°C, 5% CO2within 30 minutes. Cells were additionally washed twice in RPMI-1640 with re-suspension and centrifugation and resuspendable to a final concentration of 2×105/ml in medium RPMI-1640.

Obtained in section 1.2 of the supernatant were diluted with medium RPMI-1640 to the following percentage concentrations: 100%, 50%, 25%, 12,5%, 6,25% and 3.125%.

rIL-2 were diluted with medium RPMI-1640 to the following concentrations: 500 IU/ml, 250 IU/ml, 125 IU/ml to 62.5 IU/ml, 31,25 IU/ml and 15.5 IU/ml

In 96-hole tablet for cell cultures used three parallel wells for combinations:

Negative control: 100 µl RPMI-1640+100 μl of the suspension of NT-2 cells

rIL-2 standard: 100 μl of a solution of rIL-2+100 μl of the suspension of NT-2 cells

Analyzed well: 100 μl of the diluted supernatant+100 μl of the suspension of NT-2 cells

Tablet incubated at 37°C, 5% CO2for 68 hours, then centrifuge is ovali at 150g for 15 minutes and supernatant was removed. To each well was added 100 μl of 0.5 mg/ml MTT in RPMI-1640 without phenolsulfonephthalein. After shaking for 3-4 minutes to resuspendable cells continued additional incubation for 4 hours. Then the samples were centrifuged at 150g for 15 minutes and supernatant was removed. To each well was added 120 μl of 40 mm HCl in 2-propanol was stirred for 3-4 minutes and using a tablet reader were analyzed by ELISA O.D. 570 nm, normalized to 630 nm.

Calculation:

Took the mean O.D. value for the three parallel wells for each dilution and built schedule regarding the concentration on semi-log scale by plotting the concentration axis X. Received the value of the concentration at 50% saturation of the OP how to test supernatant, and rIL-2.

IL-2 activity of the sample = (dilution of the sample at 50% of maximal activity ÷ standard breeding rIL-2 at 50% of maximal activity)(activity of rIL-2 standard at 50% of maximal activity (IU/ml).

1.5. The effect of peptides on the activity of T-lymphocytes in the secretion of interferon (IFN)[6]

The supernatant obtained in section 1.2, diluted culture medium RPMI-1640 to the following percentage concentrations: 100%, 50%, 25%, 12,5%, 6,25% and 3.125%.

Standard recombinant interferon (rIFN) diluted medium RPMI-1640 to the following concentrations: 500 IU/ml, MU/ml, 125 IU/ml to 62.5 IU/ml, 31,25 IU/ml and 15.5 IU/ml

Target cells L929 in the logarithmic phase was brought to a concentration of 2×105/ml medium RPMI-1640 with the same treatment as described in section 1.4 for cells HT-2. Source VSV also brought up to 100 TCID50medium RPMI-1640.

In 96-hole tablet for cell cultures used three parallel wells for combinations:

Negative control: 150 μl of RPMI-1640+100 ál of L929

Well of positive control: 100 μl of a solution of RPMI-1640+100 ál of L929+50 ál VSV

Hole activity of rIFN: 100 ál of rIFN standard+100 ál of L929+50 ál VSV

Analyzed well: 100 μl of the diluted supernatant+100 ál of L929+50 ál VSV

Samples were incubated at 37°C, 5% CO2within 24 hours. The positive control wells were periodically observed in the inverted microscope to confirm cell lysis, then harvested, washed and OD of the wells was read as described in section 1.4.

Calculation:

Concentration at 50% maximal activity was obtained in the same manner as in section 1.4. Expected IFN activity of the sample as follows:

IFN activity of the sample = (dilution of the sample at 50% of maximal activity ÷ standard breeding rIFN at 50% of maximal activity)(activity of rIFN standard at 50% of maximal activity (IU/ml).

2. The effect of peptides on the formation of antibodies[7]

Received Erie is rocity sheep (SRBC), collecting blood from the jugular vein and placing it in a sterile flask with glass beads. The flask was shaken for 3 minutes and then the blood was mixed with a solution of Alsever (Alsever) (glucose 2,05 g NaCl, 0.4 g, lemonade Na 0.8 g, brought to 100 ml with distilled water) and kept at 4°Caporetto before use, the samples were centrifuged at 130g for 5 minutes, collecting SRBC. Cells were twice washed with resuspending and centrifugation in normal saline solution. Then the precipitated cells were collected by centrifugation at 180g for 10 minutes and re-suspended in physiological solution, getting the final working SRBC suspension, 2% (V/V).

The complement was obtained by adding 10 volumes of fresh blood serum of the Guinea pig to a single compacted volume in the centrifuge SRBC and then gently shaking for 30 minutes at 4°C. SRBC was removed by centrifugation at 200g for 10 minutes. To get working Complementos solution was added 10 volumes of normal saline.

BALB/c mice were randomly divided into group of the peptide, IFN group, IL-2 group and the group receiving saline, 10 mice per group. The test substance was administered as described in section 1.1, plus intraperitoneal injection of 0.2 ml of SRBC in mice on day 12. The day after the last injection testero the constituent substances (16 day) collected blood from the angle of the palpebral fissure and left at room temperature for one hour to obtain a blood serum. After centrifugation at 200g for 10 minutes, the serum was diluted 500 times with normal saline.

To 1 ml of diluted mouse serum of each mouse was added 0.5 ml suspension of SRBC. Cooled on ice. Then added 1 ml of working solution of complement and incubated at 37°C in a water bath for 10 minutes. The reaction was stopped by cooling with ice. Then the samples were centrifuged at 200g for 10 minutes, obtaining the supernatant.

To 1 ml of this supernatant was added to 3 ml Drabkina (Drabkin) and left at room temperature for 10 minutes. Received OP540nm.

Calculation:

Standardized OP540nmwas obtained by mixing 0.25 ml SRBC suspension with a solution Drabkina to 4 ml and leaving the mixture on for 10 minutes before receiving OP540 nm.

The index of the serum sample = (OP540nmthe test sample (standardized OP540nm)×500

3. The effect of peptides on the phagocytic function managername of phagocyte and weight immune organ[8,9]

The next day after the last injection of the analyte (16 day) to mice did injection carcasses at a rate of 0.1 ml/kg body weight (5-fold dilution of normal saline) into the tail vein.

After one minute and five minutes after injection of the carcass were collected 20 µl of blood from the corner of the GLA is Noah cracks using a heparinised tube. The blood was mixed with 2 ml of 0.1% weight/about solution of Na2CO3and then got OPNm. The main index of deducing It was calculated by the following formula:

K=(lgA1-lgA2)+(t2-t1)

Key:

A1: OP680 nmin the first minute

A2: OP680 nmin the fifth minute

t2: 5 minutes

t1: 1 minute

The day after the last injection of the analyte (16 day) liver, spleen and thymus were separated, blotted dry with filter paper and weighed. The phagocytic index was calculated as shown below:

α=(3√)×(W÷WLS)

Key:

W: the weight of the body

WLS: the weight of the liver plus spleen

The index of thymus (%)=(weight of thymus/body weight)×100%

The spleen index (%)=(weight of the spleen/body weight)×100%

Results

Due to the large number of included original data presents only the compiled results. The group did not have statistically significant differences from the negative control group receiving saline solution, is also omitted.

1. The effect of peptides on the transformation of T-lymphocytes

Found that at a dose of 500 mcg/kg/day S002, S007, S008, S010, S012, S015, S019, S021 and CMS029 can enhance the transformation of T-lymphocytes, showing a statistically significant difference when compared with the group receiving the second physiological solution (P< 0,05). It is shown that among these peptides S010 and S015 statistically significantly different from group IFN-γ and IL-2 group (P<0,05), as shown below in table 1.

Table 1
GroupNX±SD (stimulation index)
CMS00281,8±0,3*
CMS00791,6±0,1*
CMS00891,7±0,1*
CMS009101,7±0,2*
CMS01092,0±0,3*@∧
CMS01291,6±0,2*
CMS01591,9±0,3*@∧
CMS01991,8±0,3*
CMS021101,6±0,1*
CMS02991,7±0,3*
IFN-γ101,6±0,2*
IL-2101,7±0,2*
Saline101,3±0,1
*compared with a group receiving physical and the ideological solution, P<0,05

@comparison with IFN-γ group P<0,05

comparison with IL-2 group P<0,05

Found that at a dose of 50 mg/kg/day S001, S002 and CMS003 able to stimulate the transformation of T-lymphocytes, showing a statistically significant difference when compared with the group receiving saline, IFN-γ group and IL-2 group (P<0,05). It is shown that S014 and S036 able to inhibit the transformation of T lymphocytes, showing a statistically significant difference when compared with the group receiving saline (P<0,05). Detailed data are given below in table 2.

Table 2
GroupNX±SD (stimulation index)
CMS001102,2±0,5*@∧
CMS002102,6±0,3*@∧
CMS00382,2±0,5*@∧
CMS01491,0±0,1*
CMS03691,0±0,1*
IFN-γ91,7±0,2*
IL-2101,8±0,2*
Saline 101,3±0,1
*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S001, S003, S007 and CMS034 able to stimulate the transformation of T-lymphocytes, showing a statistically significant difference when compared with the group receiving saline (P<0,05), as shown in table 3.

Table 3
GroupNX±SD (stimulation index)
CMS001101,7±0,2*
CMS003101,6±0,2*
CMS00781,7±0,1*
CMS03491,5±0,2*
IFN-γ101,6±0,2*
IL-291,6±0,1*
Saline101,3±0,1
*compared with a group receiving saline, P<0,05

Found that at a dose of 0.5 mcg/kg/day S008, S10 and CMS011 able to stimulate the transformation of T-lymphocytes, showing a statistically significant difference when compared with the group receiving saline (P<0,05), as shown in table 4.

Table 4
GroupNX±SD (stimulation index)
CMS008101,7±0,3*
CMS01091,7±0,3*
CMS011101,6±0,4*
IFN-γ101,6±0,2*
IL-2101,6±0,1*
Saline101,3±0,1
*compared with a group receiving saline, P<0,05

2. The influence of the peptide on the cytotoxic activity of NK cells

Found that at a dose of 500 mcg/kg/day S010, S013, S016, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and CMS036 able to increase the cytotoxic activity of NK cells, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is shown that among these peptides S010, S016 and S030 show a statistically significant difference with IFN-γ GRU who sing and IL-2 group (P< 0,05), as shown in table 5.

Table 5
GroupNX±SD (%)
CMS010991±4*@∧
CMS013884±9*
CMS016991±7*@∧
CMS0231079±12*
CMS0241089±8*
CMS0261089±7*
CMS0271088±8*
CMS0281090±5*
CMS0291087±4*
CMS0301091±5*@∧
CMS0321087±5*
CMS033989±8*
CMS0341185±9*
CMS035890±10*
CMS0361088±7*
IFN-γ1077±8*
IL-28 77±8*
Saline1063±9

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 50 mg/kg/day S001, S003, S015, S021, S026 and CMS035 able to increase the cytotoxic activity of NK cells, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is shown that among these peptides S021 shows a statistically significant difference with IFN-γ group and IL-2 group (P<0,05). It is shown that S012 able to inhibit the cytotoxic activity of NK cells, showing a statistically significant difference when compared with the group receiving saline (P<0,05). Detailed data are given below in table 6.

Table 6
GroupNX±SD (%)
CMS0011085±10*
CMS0031085±6*
CMS012940±9*
CMS015878±8*
CMS021 888±12*@∧
CMS0261076±9*
CMS0351072±9*
IFN-γ1073±10*
IL-21074±8*
Saline1056±8

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S008, S009, S010, S011, S012, S020, S024, S034 and CMS036 able to increase the cytotoxic activity of NK cells, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is shown that among these peptides S008 and S009 show a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 7.

Table 7
GroupNX±SD (%)
CMS0081092±4*@∧
CMS009892±6*@∧
CMS01010 82±9*
CMS0111076±10*
CMS0121085±7*
CMS020991±6*
CMS024978±3*
CMS034890±5*
CMS0361075±9*
IFN-γ1080±8*
IL-21080±8*
Saline1060±9

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 0.5 mcg/kg/day S002, S011, S012, S022, S028 and CMS035 able to increase the cytotoxic activity of NK cells, showing a statistically significant difference when compared with the group receiving saline (P<0,05). Found that S008 able to inhibit the cytotoxic activity of NK cells, showing a statistically significant difference when compared with the group receiving saline (P<0,05). Detailed data are given below in table 8.

Table 8
GroupNX±SD (%)
CMS002876±9*
CMS0081046±12*
CMS011979±3*
CMS012977±6*
CMS0221073±11*
CMS028879±3*
CMS0351076±10*
IFN-γ1072±9*
IL-21074±10*
Saline1158±7

*compared with a group receiving saline, P<0,05

3. The effect of peptides on the activity of T-lymphocytes with IL-2 secretion

Found that at a dose of 500 mcg/kg/day S007, S009, S010 and CMS015 able to stimulate the secretion of IL-2 by T lymphocytes, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is shown that among these peptides S007 and S015 statistically significantly different from the group of IL-2 (P<0,05). Also found that S009 and S010 prowl who have a statistically significant difference when compared with IFN-γ group, and IL-2 group, as shown in table 9.

Table 9
GroupNX±SD (IU)
CMS007986±15*∧
CMS00910114±13*@∧
CMS0109125±17*@∧
CMS015985±17*∧
IFN-γ10100±18*
IL-21070±13*
Saline1039±10

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 50 mg/kg/day S001 and CMS003 able to stimulate the activity of T-lymphocytes to IL-2 secretion, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is established that among these peptides S003 statistically significantly different from the group of IL-2 (P<0,05), as shown in table 10.

The table is 10
GroupNX±SD (IU)
CMS0011060±10*
CMS003886±9*∧
IFN-γ999±16*
IL-21072±12*
Saline1039±10

*compared with a group receiving saline, P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S007, S012 and CMS020 capable of stimulating T-lymphocytes to IL-2 secretion, showing a statistically significant difference when compared with the group receiving saline (P<0,05), as shown in table 11.

Table 11
GroupNX±SD (IU)
CMS007864±12*
CMS012965±16*
CMS020863±11*
IFN-γ1096±14*
IL-21077±13*
Physiologically the solution 1037±9

*compared with a group receiving saline, P<0,05

Found that at a dose of 0.5 mcg/kg/day S010, S011, S012, S022 and CMS034 capable of stimulating T-lymphocytes to IL-2 secretion, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is established that among these peptides S034 is a statistically significant difference with the group of IL-2 (P<0,05). Also found that S011 and S022 show a statistically significant difference when compared with IFN-γ group and IL-2 group, as shown in table 12.

Table 12
GroupNX±SD (IU)
CMS010966±11*
CMS01110101±19*@∧
CMS012859±13*
CMS0229109±14*@∧
CMS0341085±10*∧
IFN-γ1087±15*
IL-21073±13*
Saline38±13

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

4. The effect of peptides on the activity of T-lymphocytes secreting IFN

Found that at a dose of 500 mcg/kg/day S010, S013 and CMS016 capable of stimulating T cells to release IFN (interferon), showing a statistically significant difference when compared with the group receiving saline, IFN-γ group, and IL-2 group (P<0,05), as shown in table 13.

Table 13
GroupNX±SD (IU)
CMS0109167±13*@∧
CMS0139154±15*@∧
CMS0166162±19*@∧
IFN-γ10139±16*
IL-210120±13*
Saline1065±11

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

The mouth is attached, what dose of 50 mg/kg/day S001, S003 and CMS021 capable of stimulating T cells to release IFN, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is established that among these peptides S021 is a statistically significant difference when compared with IFN-γ group and with IL-2 group, as shown in table 14.

Table 14
GroupNX±SD (IU)
CMS00110110±15*
CMS0038106±16*
CMS0218143±17*@∧
IFN-γ9125±18*
IL-210113±17*
Saline1061±11

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S009 and CMS012 able to stimulate the secretion of IFN T-lymphocytes, showing a statistically significant difference when compared with the group receiving saline (P<, 05). It is shown that among these peptides S009 statistically significantly different from the group of IL-2 (P<0,05), as shown in table 15.

Table 15
GroupNX±SD (IU)
CMS00910121±15*@∧
CMS012986±9*@∧
IL-29105±14*
Saline1066±10

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 0.5 mcg/kg/day S010, S011, S022 and CMS028 able to stimulate the secretion of IFN T-lymphocytes, showing a statistically significant difference when compared with the group receiving saline (P<0,05). It is shown that among these peptides S010 and S022 statistically significantly different from IFN-γ group and IL-2 group (P<0,05), as shown in table 16.

Table 16
GroupNX±SD (IU)
CMS0109142±18*@∧
CMS0111089±18*
CMS0229145±13*@∧
CMS0281096±13*
IFN-γ10124±16*
IL-210107±13*
Saline1064±13

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

5. The effect of peptides on the formation of antibodies

Found that at a dose of 500 mcg/kg/day S002, S003, S007, S008, S009, S010, S011, S012, S013, S014, S015, S016, S018, S019, S020, S022, S023, S024, S029, S033 and S035 able to stimulate the formation of anti-SRBC antibodies, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is shown that among these peptides S002, S003, S007, S008, S013, S019, S024 and S035 statistically significantly different from IFN-γ group (P<0,05). It was also found that S009, S010, S011, S012, S014, S015, S016, S020, S023, S029 and S033 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 17.

Table 17
GroupNX±SD (Units)
CMS0021087±18*@
CMS0031096±18*@
CMS0071069±17*@
CMS0081082±15*@
CMS00910113±22*@∧
CMS01010112±30*@∧
CMS0118188±16*@∧
CMS0128141±21*@∧
CMS0131080±16*@
CMS01410130±24*@∧
CMS01510136±22*@∧
CMS0168143±38*@∧
CMS0181066±16*
CMS0191091±26*@
CMS0206155±35*@∧
CMS022868±31*
CMS023 9110±45*@∧
CMS024875±29*@
CMS0298115±22*@∧
CMS03310143±27*@∧
CMS0351088±16*@
IFN-γ937±10
IL-21071±11*
Saline1032±7

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

It is shown that the dose of 50 mg/kg/day S003, S011, S012, S013, S015, S021, S022, S023, S026, S027, S029, S030, S032, S033, S034, S035 and S036 able to stimulate the formation of anti-SRBC antibodies, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is established that among these peptides S011, S013 and S015 statistically significantly different from IFN-γ group (P<0,05). It was also found that S021, S022, S023, S026, S027, S029, S030, S032, S033, S034, S035 and S036 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05). It is established that S009 to inhibit the formation of anti-SRBC antibodies showing a statistically significant difference compared with the group receiving saline (P<0,05). Detailed data are given below in table 18.

Table 18
GroupNX±SD (Units)
CMS0031052±11*
CMS009813±5*
CMS011967±9*@
CMS012850±14*
CMS013870±9*@
CMS0151054±9*@
CMS021994±20*@∧
CMS0229110±16*@∧
CMS023884±11*@∧
CMS026998±9*@∧
CMS027993±11*@∧
CMS02910143±13*@∧
CMS03010141±33*@∧
CMS0329131±24*@∧
CMS0338112±15*@∧
CMS03410136±11*@∧
CMS035897±10*@∧
CMS03610118±11*@∧
IFN-γ937±10
IL-21071±11*
Saline1032±7

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S001, S003, S007, S008, S009, S011, S012, S013, S015, S016, S019, S020, S021, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 able to stimulate the formation of anti-SRBC antibodies, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is shown that among these peptides S003, S008, S009, S012, S015, S016, S020 and S021 statistically significantly different from IFN-γ group (P<0,05). It was also found that S001, S007, S011, S019, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 have a statistically significant difference with IFN-γ group, the AK and IL-2 group (P< 0,05), as shown in table 19.

CMS027
Table 19
GroupNX±SD (Units)
CMS0019110±24*@
CMS003991±24*@
CMS0079122±12*@∧
CMS008997±26*@
CMS009879±18*@
CMS01110115±27*@∧
CMS0121081±22*@
CMS0131093±28*@
CMS015894±37*@
CMS016993±32*@
CMS01910118±20*@∧
CMS0201089±24*@
CMS021982±30*@
CMS02310166±27*@∧
CMS0247171±39*@∧
CMS0269191±17*@∧
9117±45*@∧
CMS02810121±48*@∧
CMS0299147±23*@∧
CMS0309158±37*@∧
CMS0329157±37*@∧
CMS0337128±39*@∧
CMS0348172±37*@∧
CMS0359176±39*@∧
CMS0368179±34*@∧
IFN-γ937±10
IL-21071±11*
Saline1032±7

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

comparison with IL-2 group P<0,05

Found that at a dose of 0.5 mcg/kg/day S021, S023, S024, S027 and S033 able to stimulate the formation of anti-SRBC antibodies, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is shown that among these peptides S021 and S033 significantly different from IFN-γ group (P<0,05). It was also found that S023, S024 and S027 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05). In addition, it is shown that S002, S003, S009, S010, S011, S013, S014, S015, S018, S019, S020, S026, S028, S029, S030, S034 and S036 able to inhibit the formation of anti-SRBC antibodies, showing a statistically significant difference compared with the group receiving saline (P<0,05). Detailed data are given below in table 20.

Table 20
GroupNX±SD (Units)
CMS00294±1*
CMS00392±1*
CMS00992±1*
CMS0101010±3*
CMS011105±3*
CMS013107±1*
CMS0141015±6*
CMS015913±4*
CMS01893±1*
CMS019912±3*
CM020 910±3*
CMS021957±9*@
CMS02310108±21*@∧
CMS0241098±6*@∧
CMS0261019±6*
CMS0271099±14*@∧
CMS0281018±5*
CMS029918±7*
CMS030919±7*
CMS033978±12*@
CMS0341020±2*
CMS036920±6*
IFN-γ937±10
IL-21071±11*
Saline1032±7

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

6. The effect of peptides on the phagocytic activity managername of phagocyte.

Found that at a dose of 500 mcg/kg/day S003, S008, S020, WITH the S022 and S024 can enhance the phagocytic activity managername of phagocyte, having a statistically significant difference compared with the group receiving saline (P<0,05). It is established that among these peptides S022 is a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 21.

Table 21
GroupNX±SD (phagocytic index)
CMS003106,6±0,7*
CMS008106,5±1,2*
CMS020106,4±0,6*
CMS022107,4±0,6*@∧
CMS024106,4±1,0*
IFN-γ106,4±0,9*
IL-295,7±0,8
Saline105,1±0,6

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 50 mg/kg/day S019, S024 and S030 can enhance the phagocytic activity managername of phagocyte, having statistically testing imoe difference compared with group, receiving saline (P<0,05). It is established that among these peptides S019 is a statistically significant difference from the IL-2 group (P<0,05), as shown in table 22.

Table 22
GroupNX±SD (phagocytic index)
CMS01996,7±0,9*∧
CMS02486,6±0,7*
CMS030106,3±0,5*
IFN-γ106,4±0,9*
IL-295,7±0,8
Saline105,1±0,6

*compared with a group receiving saline, P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S003*, S008, S009, S010, S011, S013, S016, S018, S019 and S035 can enhance the phagocytic activity managername of phagocyte, having statistically significant difference compared with the group receiving saline (P<0,05). It is established that among these peptides S003, S009, S010, S016, S019 and S035 have a statistically significant difference from the IL-2 group (P<0,05), as shown in the tables is 23.

Table 23
GroupNX±SD (phagocytic index)
CMS00396,9±0,9*∧
CMS00896,4±0,5*
CMS00996,9±0,9*∧
CMS010107,1±0,7*∧
CMS011106,4±1,1*
CMS013106,7±0,2*
CMS01696,9±0,8*∧
CMS01886,7±1,2*
CMS01986,8±0,6*∧
CMS03596,9±0,9*∧
IFN-γ106,4±0,9*
IL-295,7±0,8
Saline105,1±0,6

*compared with a group receiving saline, P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 0.5 mcg/kg/day S024, S027 and S036 can enhance the phagocytic act shall want to make managername of phagocyte, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is established that among these peptides S024 is a statistically significant difference from the IL-2 group (P<0,05), as shown in table 24.

Table 24
GroupNX±SD (phagocytic index)
CMS024106,7±0,5*∧
CMS027106,4±0,6*
CMS03696,2±0,3*
IFN-γ106,4±0,9*
IL-295,7±0,8
Saline105,1±0,6

*compared with a group receiving saline, P<0,05

^ comparison with IL-2 group P<0,05

7. The effect of peptides on the weight of immune organ

Found that at a dose of 500 mcg/kg/day S008, S010, S016, S019, S020, S022, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 able to increase the weight of the thymus gland, showing a statistically significant difference compared with the group receiving saline (P<0,05). There Yes the different peptides S027 and S034 have a statistically significant difference from the IL-2 group (P< 0,05). Also found that S008, S022, S029, S030, S032, S033 and S035 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 25.

Table 25
GroupNX±SD (%)
CMS00880,21±0,03*@∧
CMS010100,19±0,04*
CMS01690,18±0,05*
CMS019100,19±0,02*
CMS020100,19±0,04*
CMS02290,26±0,05*
CMS026100,20±0,03*
CMS02780,20±0,03*∧
CMS028100,19±0,02*
CMS029100,22±0,04*@∧
CMS03080,30±0,03*@∧
CMS03280,25±0,03*@∧
CMS03390,25±0,04*@∧
CMS0349 0,20±0,05*∧
CMS035100,21±0,03*@∧
CMS03690,18±0,02*
IFN-γ100,15±0,04
IL-290,14±0,03
Saline90,12±0,02

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 500 mcg/kg/day S019 able to increase the weight of the spleen with the statistically significant difference compared with the control group receiving saline (P<0,05), as shown in table 26. It is established that S001, S003, S007, S009, S011, S013, S014, S015, S021, S023, S024, S027 and S036 able to reduce the weight of the spleen with the statistically significant difference compared with the control group receiving saline (P<0,05). Detailed data are given below in table 26.

td align="center"> 0,40±0,04*
Table 26
GroupNX±SD (%)
CMS001100,43±0,07*
CMS0038
CMS00790,32±0,05*
CMS00990,41±0,03*
CMS01190,41±0,04*
CMS013100,44±0,07*
CMS014100,40±0,03*
CMS01590,36±0,07*
CMS01990,63±0,08*
CMS02190,36±0,04*
CMS02390,36±0,06*
CMS02490,34±0,05*
CMS027100,37±0,03*
CMS036100,40±0,03*
Saline100,53±0,05

*compared with a group receiving saline, P<0,05

Found that at a dose of 50 mg/kg/day S002, S008, S012, S014, S016, S018, S019, S020, S022, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034 and S036 able to increase the weight of the thymus gland, showing a statistically significant difference compared with the group receiving fisiologicas the th solution (P< 0,05). It is established that among these peptides S034 is a statistically significant difference from the IL-2 group (P<0,05). Also found that S002, S008, S012, S014, S016, S018, S019, S020, S022, S023, S024, S026, S027, S030, S032 and S036 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 27.

Table 27
GroupNX±SD (%)
CMS002100,21±0,02*@∧
CMS008100,20±0,04*@∧
CMS012100,26±0,02*@∧
CMS014100,21±0,02*@∧
CMS016100,20±0,03*@∧
CMS018100,23±0,02*@∧
CMS019100,20±0,03*@∧
CMS020100,27±0,03*@∧
CMS022100,30±0,03*@∧
CMS023100,20±0,02*@∧
CMS024100,27±0,02*@∧
CMS026100,27±0,02*@∧
CMS02780,21±0,03*@∧
CMS028100,18±0,04*
CMS02990,18±0,05*
CMS030100,25±0,04*@∧
CMS032100,27±0,03*@∧
CMS03390,18±0,03*
CMS03480,19±0,04*∧
CMS03690,22±0,02*@∧
IFN-γ100,15±0,04
IL-290,14±0,04
Saline90,12±0,02

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 50 mg/kg/day S008, S010 and S029 able to reduce the weight of the spleen with statistically significant difference compared to the group receiving saline (P<0,05). Detailed data are given below in table 28.

Table 28
GroupNX±SD (%)
CMS008100,39±0,08*
CMS010100,38±0,05*
CMS029100,42±0,04*
IFN-γ100,50±0,04
IL-290,62±0,07
Saline90,53±0,05

*compared with a group receiving saline, P<0,05

Found that at a dose of 5 mcg/kg/day S001, S002, S010, S011, S012, S013, S014, S015, S016, S018, S019, S020, S021, S022, S023, S024, S026, S028, S029, S030, S32, S033, S034 and S036 able to increase the weight of the thymus gland, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is established that among these peptides S002, S014, S024 and S030 have a statistically significant difference from the IL-2 group (P<0,05). Also found that S010, S012, S018, S019, S020, S022, S026, S028, S032, S034 and S036 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 29.

Table 29
GroupNX±SD (%)
CMS001100,22±0,05*
CMS00290,24±0,05*∧
CMS01090,27±0,05*@∧
CMS011100,22±0,04*
CMS012100,27±0,06*@∧
CMS013100,21±0,05*
CMS014100,23±0,06*∧
CMS01590,20±0,08*
CMS016100,22±0,06*
CMS018100,24±0,04*@∧
CMS019100,24±0,02*@∧
CMS020100,24±0,07*@∧
CMS02190,20±0,06*
CMS02290,25±0,04*@∧
CMS023100,23±0,06*
CMS02490,23±0,06*∧
CMS026100,31±0,05*@∧
CMS028100,28±0,06*@∧
CMS029100,21±0,03*
CMS030100,23±0,07*∧
CMS032100,29±0,04*@∧
CMS033100,20±0,02*
CMS03490,27±0,06*@∧
CMS035100,21±0,04*
CMS036100,25±0,04*@∧
IFN-γ100,15±0,04
IL-290,14±0,04
Saline90,12±0,02

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 5 mcg/kg/day S030 able to increase the weight of the spleen, with a statistically significant difference between the group receiving saline (P<0,05). It is established that S015 able to reduce the weight of the spleen, with a statistically significant difference between the group receiving saline (P<0,05). Detailed data are given below in table 30.

Table 30
GroupNX±SD (%)
CMS01590,38±0,15*
CMS030100,64±0,09*
Saline100,53±0,05

*compared with a group receiving saline, P<0,05

Found that at a dose of 0.5 mcg/kg/day S002, S008, S010, S012, S014, S018, S020, S022, S026, S028, S030 and S032 able to increase the weight of the thymus gland, showing a statistically significant difference compared with the group receiving saline (P<0,05). It is established that among these peptides S008 and S012 have a statistically significant difference from the IL-2 group (P<0,05). Also found that S002, S020 and S030 have a statistically significant difference with IFN-γ group and IL-2 group (P<0,05), as shown in table 31.

Table 31
GroupNX±SD (%)
CMS00280,26±0,06*@∧
CMS008100,22±0,07*∧
CMS010 90,21±0,03*
CMS012100,22±0,06*∧
CMS014100,20±0,04*
CMS018100,20±0,03*
CMS02090,23±0,05*@∧
CMS022100,21±0,06*
CMS02690,21±0,05*
CMS028100,20±0,06*
CMS03080,24±0,05*@∧
CMS032100,21±0,06*
IFN-γ100,15±0,04
IL-290,14±0,04
Saline90,12±0,02

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

Found that at a dose of 0.5 mcg/kg/day S020 able to increase the weight of the spleen, with a statistically significant difference between the group receiving saline, IFN-γ group and IL-2 group (P<0,05). It is established that S001 able to reduce weight Selezen is when a statistically significant difference with group, receiving saline (P<0,05). Detailed data are given below in table 32.

Table 32
GroupNX±SD (%)
CMS001100,40±0,05*
CMS02080,68±0,09*@∧
IFN-γ100,53±0,05
IL-2100,50±0,04
Saline100,62±0,07

*compared with a group receiving saline, P<0,05

@comparison with IFN-γ group P<0,05

^ comparison with IL-2 group P<0,05

In conclusion, the authors of the present invention have found that peptides S001, S002, S003, S007, S008, S009, S010, S011, S012, S013, S014, S015, S016, S018, S019, S020, S021, S022, S023, S024, S026, S027, S028, S029, S030, S032, S033, S034, S035 and S036 possess biological activity tested in vivo in animal models.

II. Antiviral activity of the peptides in vivo

In order to establish whether these peptides possible therapeutic effect on viral infections, the authors of the present invention used in this study, the stomach is th model duck hepatitis b, to study the influence of the above peptides in vivo in infected animals.

Received model of hepatitis b In ducks Chongqing, which were treated with peptides via intraperitoneal injection (50 mg/kg/day, once daily) for 4 weeks. Analyzed the level of DHBV DNA hybridization serum by the method of the dot-blotting. Treatment with lamivudine and normal saline were used as positive and negative control, respectively. It is established that the peptide CMS001 able to reduce the level of DHBV DNA in serum at 4 weeks of treatment with a statistically significant difference with the control group receiving saline (P<0,05). We can conclude that CMS001 at an appropriate dose level can be used as part of or by itself for the treatment of infections of viral hepatitis.

Materials and methods

Bepridil were synthesized in the usual way American Peptide company, Inc., USA, L-amino acids.

2. Animal model of[1]

Ducks Chongqing at the age of one day was inoculable using intraperitoneal injection of 0.1 ml of the original serum positive for DNA of the virus duck hepatitis b virus (DHBV) (5x107copies/ml). A week later collected samples of blood from the external jugular vein and confirmed the infection by the hybridization method of the dot-blotting using DHBV DNA probe, labeled Digox what Mr. [2]. Ducks were grown to 2 weeks of age for the start of the study.

3. Grouping and processing

DHBV infected ducks were divided randomly into the following groups

A) negative control Group (n=9): Normal saline solution was injected at a rate of 1 ml per duck once a day.

b) the lamivudine Group (n=8): as a group of positive control. Lamivudine[4]was given at a dose of 50 mg/kg/day by oral administration once a day

(C) Peptide group (n=9): 50 mcg/kg/day peptide (brought up to a final volume of 0.5-1 ml of normal saline) was administered once daily via intraperitoneal injection.

Treatment continued for 4 weeks and observation continued for one week after termination of treatment. Blood samples of 1 ml were collected from the external jugular vein ducks on days 0, 7, 14, 21, 28 and 35 after the start of treatment. Serum from blood samples immediately identified[3]and kept at -20°until analysis.

4. Determining the level of DHBV DNA in serum.

DHBV DNA probe was fluorescently labeled in accordance with the Protocol set of labels from the kit manufacturer (Amersham Pharmacia Biotech Co.). 40 μl of duck blood serum were subjected to dot-blot (1 duplicate spot on the sample) on nitrocellulose membrane and hybridisable fluorescently-labeled DHBV DNA Zonda is for the quantitative determination of [2]. After hybridization blotty showed in the CDP-Star fluorimetric reagent RPN2690 and scanned using scanner Vuego Scan Brisa-620st). The software ImageMaster TotalLab v.1.11.Ink used for the quantitative analysis of blotto. Statistical analysis was performed in accordance with paired t-test using the software SPSS.

Results

Table II.1

The titer of DHBV DNA in serum before and after processing
The level of DHBV DNA (mean (standard deviation, 103units)
0 dayday 7day 1421 days28 day35 day
Normal saline26±1245±3149±23102±6660±3850±43
Lamivudine21±96±4*7±6*8±7*8±5*20±19
CMS00121±1811±1320±1814±45±3*18±16

Paired t-test, compared to day 0 for the same animals: *P<0,05

A negative (normal saline) and positive (lamivudine) of the control group confirms the successful creation of animal models of hepatitis. Found that at a dose of 50 mg/kg/day peptide CMS001 able to reduce the titer of DHBV DNA in serum after 4 weeks of treatment with a statistically significant difference (p<0,05) with the value for the same animals before treatment. At the end of processing the titer of DHBV DNA in serum were returned to the value, no statistical differences with the value before treatment, showing that the effect of the peptide CMS001 may be reversible and/or required a longer period of treatment to suppress the virus.

Discussion

Animal model of duck hepatitis[1]represents a model is developed to study the pathogenesis of hepatitis b in humans and for screening therapeutic agents for the treatment of hepatitis C. this study found that CMS001 way to reduce the titer of DHBV DNA in serum after 4 weeks of treatment, indicating that the peptide CMS001 at an appropriate dose level and at a suitable scheme may be useful by itself or in combination with other substances as agent for treatment of hepatitis b in humans.

This study tested the introduction is via intraperitoneal injection, but this does not exclude the possible effectiveness of the peptide with its introduction of other alternative ways. The peptides can also be administered by intravenous injection, intramuscular injection, subcutaneous injection, and subcutaneous implantation, with or without using a device to facilitate the delivery of drugs, such as liposomes, protection for slow release, etc. Peptide can also be entered in any form for oral administration such as tablet, capsule, suspension, solution, etc. in a suitable form without modification or form with delayed release, or with or without the use of gastrointestinal protection. In addition, the peptide can be used in any form for local use, such as ointment, cream, gel, etc. with or without using a device to facilitate the transcutaneous penetration, or inhalation powder dissolved or liposome-protected form. The peptide can also be transferred in its genetic sequence and cloned into the expression system or by itself or in combination with other peptide sequences for the formation of the final peptide molecules for use in the activity of the peptides, as described in this invention, cleaning or no cleaning of the final peptide.

Table II.2.

The effect of peptides on hepatitis b
Code CMSSEQ ID No
CMS0011

References

1. Yaxi Chen, Guo shuhua Zhang Dingfeng, et al. Foundation and application of Chongqing duck hepatitis B model. Chinese Journal of Hepatology. 1993; 1(2): 89-91

2. Yaxi Chen, Guo shuhua, Chen Xuehua. Preparation and application of DHBV DNA probe labeled with digoxin. Journal of Chongqing University of Medical Sciences. 1994; 19(4): 295-297

3. Tang Ni, Huang Ailong, Guo shuhua, et al. Systemic foundation and application of serological parameters of humoral immunity to duck hepatitis B virus. Chinese Journal of Hepatology. 2001; 9(1): 13-15

4. Yaxi Chen, Guo shuhua, Qi Zhenyuan, et al. An experimental study of lamivudine against duck hepatitis B virus in combination with famciclovir. Chinese Journal of Hepatology. 2001; 9(4): 209-211

III. The effect of peptides on jade

In order to find out whether these peptides possible therapeutic effect on the jade, the authors of the present invention used in this study, an animal model Masugi (Masugi) nephritis in rats in order to test in vivo the influence of the above peptides for sick animals[3,4].

The objective of this research was to study therapeutic effect of the peptides in vivo in chronic glomerulonephritis. Rat model jade Masugi created by injection of rabbit IgG Anticriminal renal cortical substance healthy rats Sprague Dawley, and then immediately injected them intraperitoneally injection of peptides in a dose of 50 µg/to whom/day once daily for 3 weeks. As a positive control was used hydrocortisone. Found that proteinuria, creatinine levels in the serum and spleen index in rats treated S014, S018, S030 and S036 were reduced compared to the control group with statistical significance (p<0,05). The anatomic study under the microscope kidney showed that therapeutic action of these peptides was similar to treatment with hydrocortisone. We can conclude that S014, S018, S030 and S036 can be used as a tool for the treatment of chronic nephritis.

Materials.

Rat Sprague Dawley (SD)male, weighing 120±20 g obtained from the center of experimental animals of the University of Guangzhou Traditional Chinese medicine, Guangzhou University of Traditional Chinese Medicine), as well as the First Military medical University. Rabbits breed "chinchilla", weight 3 kg, obtained from the center of experimental animals of the University of Guangzhou Traditional Chinese medicine

Peptides derived from L-amino acids, were synthesized in the usual way American Peptide Company, Inc., USA and diluted to a concentration of 10 μg/ml of sterile normal saline. Hydrocortisone received from Yangzhou Pharmaceutical Factory, China.

Kit for determination of creatinine from Shanghai Rongsheng Biological technique Company, the PRC.

Methods:

1. Getting a rabbit and is taylored Anticriminal renal cortical substance [1]. 20 healthy SD rats were anestesiologi using intravenous injection of 3%pentobarbital sodium, 40 mg/kg of the Abdominal aorta was opened and kidneys were filled with normal saline until then, until they have become clean from the blood. Was isolated renal cortical substance, homogenized in 5 volumes of 0,01M buffer Tris-HCl, pH 8,1, and filtered through a stainless steel sieve 140 caliber. The filtrate was collected and mixed with either supplemented with GIBCO-RPE or incomplete adjuvant's adjuvant (Freund) in the ratio of 1:1 and mulgirigala, getting work immunizing solution.

10 healthy rabbits were immunized immunizing solution, the first time using the full adjuvant's adjuvant, and then incomplete. The antigen was injected hypodermal injection in 6 randomly selected points 0.1 ml on the point once in 10 days. After 6 weeks collected blood from the ear vein and determined the titer of antibodies by double-diffusion method[2]. 8 weeks after the start of immunization, rabbits were anestesiologi using intravenous injection of 3%pentobarbital sodium, 20 mg/kg and collected blood from the carotid artery. Then anticigarette filtered and allocated.

Whole blood was collected from 15 healthy SD rats. Isolated erythrocytes and washed three times it normal saline. Net erythrocytes were mixed with 250 ml of Crawley is ia antisera and incubated at 4° With during the night. After incubation, erythrocytes were removed by centrifugation and the supernatant additionally iactiveaware at 56°C for 30 minutes. After centrifugation to remove any precipitate from the supernatant were isolated crude IgC and partially purified three times precipitating (NH4)2SO4(50%, 33% and then 33%)[5]. Untreated IgC was re-dissolved in 125 ml of double-distilled water and conducted clearing dialysis ammonium sulfate. The titer of antibodies Anticriminal renal cortical substance was determined by a double-diffusion method[2].

2. Induction of glomerulonephritis in rats[5]: to 0.25 ml of the seed solution (containing approximately 4 mg of nonspecific rabbit IgG with complete adjuvant's adjuvant) was administered via intraperitoneal injection of healthy SD rats for premirovany rats for five days before induction. Except for the normal healthy control group, which received normal saline, group subsequently induced with intraperitoneal injection of 1 ml of rabbit IgG Anticriminal renal cortical substance, obtained as described in "Method 1".

3. Divide into groups and introduction: male SD rats were randomly divided into the following groups: normal healthy control group, the control nerita group processing p is acebo (placebo), nerita group by treatment with peptide and control nerita group by treatment with hydrocortisone (hydrocortisone), 12 animals per group. Treatment started on the day of induction. Peptides - 50 µg/kg/day hydrocortisone - 3,3 mg/kg/day and normal saline was used as placebo, in all cases, the introduction was performed intraperitoneally, once daily for 3 weeks.

4. Controlled parameters[4]:

(a) the level of protein in the urine: each rat was kept in an individual cage. Provide adequate water for drinking. Urine was collected once a week for 24 hours. The protein content in the urine was determined by the method comasina blue.

(b) the level of creatinine in serum: blood was collected after three weeks of treatment and were used to define the level of creatinine in the serum. Set for creatinine obtained from Shanghai Rongsheng Biological Technique Company.

(c) the index of the weight of the spleen: the index of the weight of the spleen was determined by the following formula:

Index of spleen weight = average weight of the spleen÷the average weight of the body

(d) Pathological microscopic examination of the kidney: a pathological study in each group chose the 6 most severe renal

Statistical analysis:

Used t-test for comparisons between groups were separated set at p<0,05. For all groups of two rats with the lowest the values of protein in the urine were excluded from statistical analysis.

Result

1. The treatment effect of the peptide on the protein level in urine

Table III.1.

The treatment effect of the peptide on the protein level in the urine (unit:mg)
GroupnWeek 1Week 2Week 3
CMS014105,5±4,0*6,3±6,8*2,8±1,9*
CMS018107,0±3,7*5,5±7,9*3,3±3,4*
CMS030105,4±3,4*a 9.5±16,22,4±1,5*
CMS036107,9±5,8*5,0±7,1*1,3±0,9*
Hydrocortisone103,1±2,0*7,5±and 7.77,6±7,1*
Placebo1022,9±2217,2±14,520,2±29
Normal91,7±1,3*2,3±1,1*0,4±0,2*

Compared with placebo *p<0,05

It is established that peptides CMS014, CMS018, CMS030 and CMS036 dose 50 mg/kg/day once a day is able to reduce the level of protein in the urine in a rat model of Masugi nephritis when a statistically significant difference with the control group, receiving the placebo treatment. Also noted that the rate of growth in groups CMS014, CMS030 and CMS036 and hydrocortisone was lower than the normal growth rate. The growth rate in the hydrocortisone group had decreased to such an extent that after the first week, the dose was halved in order to avoid severe intolerance.

2. The treatment effect of the peptide on the level of creatinine in serum

Table III.2.

The effect of peptides on the level of creatinine in serum
GroupnThe level of creatinine in serum (µmol/l)
CMS01410159±1
CMS0181067±1*
CMS0301093±1*
CMS0361080±1*
Placebo10265±212
Hydrocortisone10239±107
Normal980±1*

Comparison with rats, patients with nephritis treated with placebo treatment (placebo), *p<0,05

The level of creatinine in rats with Masugi nephritis receiving treatment with placebo, was us who CSOs higher level in the normal control group, demonstrating that renal function in rats with jade was abnormal. It is established that peptides CMS014, CMS018, CMS030 and CMS036 dose of 50 mg/kg/day once a day is able to reduce creatinine levels in a statistically significant difference with rats suffering from nephritis, when processing a placebo.

3. The effect of peptides on the indexes of the spleen

Table III.3.

The effect of peptides on the spleen index (×10-3)
GroupnIndex of spleen
CMS014103,6±1,3*
CMS018103,3±0,8*
CMS030103,0±0,5*
CMS036103,4±0,8*
Placebo104,8±1,1
Hydrocortisone104,5±1,4
Normal92,4±0,1*

Comparison with rats, patients with nephritis treated with placebo treatment (placebo), *p<0,05

The spleen of all induced rats was increased compared with normal rats, indicating that the induction of nephritis associated with immune response. It is established that PE is Chida CMS014, CMS018, CMS030 and CMS036 dose of 50 mg/kg/day once a day is able to reduce the index of the spleen with a statistically significant difference with rats suffering from nephritis, when processing placebo, p<0,05. On this basis it is assumed that the peptides can have a corrective action in respect of jade through immunosuppressing (immunosuppressive) mechanism.

4. The effect of peptides on pathological microscopic examination of kidneys

When compared with normal rats rats suffering from nephritis, when processing placebo (placebo group) showed signs of formation of fibrous tissue in the glomerular capsule, hyperplasia of the epithelium of the glomeruli, education, growth, expansion and blockage of the capillaries of the glomeruli, swelling of the longitudinal tubules epithelium and formation of curvature in the peripheral tubule and collecting duct. Such pathological changes confirmed the successful induction of nephritis. Observed that in the group CMS014 there was only one rat that had signs of the formation of fibrous tissue in the glomerular capsule and hyperplasia of the epithelium of the glomeruli. Other options for the same rats and other rats in the same group had essentially the same histology of the kidney, and normal rats. In groups CMS018, CMS030 and CMS036 all pathological parameters for all the rats were within the normal range.

Conclusion

we Can conclude, the peptides CMS014, CMS018, CMS030 and CMS036 dose of 50 mg/kg/day once daily intraperitoneal injection can have a therapeutic effect in an experimental rat model of Masugi nephritis. Proteinuria, excretion of creatinine and kidney histology were corrected by these peptides in the treated group, with a statistically significant difference with the control group receiving the placebo treatment. These peptides may act through an immunological mechanism, which is indicated by their effect on the index weights of the spleen, but we cannot exclude the possibility of their actions through other mechanisms.

Discussion

Peptides CMS014, CMS018, CMS030 and CMS036 can be used as part or by themselves for the treatment of nephritis in humans. For example, the peptides can be used for the correction of proteinuria or to restore urinary function in patients with nephritis. The peptides can also be used to prevent further deterioration of kidney function in patients with nephritis. The peptides can be used by themselves or in combination of two or more peptides, or in combination with other pharmaceuticals or food additives, as a complete treatment of nephritis.

Table III.4.

The effect of peptides on jade
SEQ ID No
CMS0147
CMS01810
CMS03021
CMS03626

References

1. SDA(State Drag Administration, P.R.China). The guideline of preclinical researches of new drugs. 1994, the 1st edition, Page 96

2. Xu Shuyun, et al. The methodology of pharmacological experiment, the People's Sanitation Publishing Company; Beijing, the 2nd edition, 1991:1071.

3. Chen Qi, et al. The methodology of pharmacological researches of traditional Chinese medicine, the People's Sanitation Publishing Company, Beijing, the 1st edition. 1993:390.

4. Du Guanhua. The guideline for pharmacological experiment-the discovery and pharmacological evaluation of new drugs, Science Publishing Company, Beijing, the 1st edition, 2001:598.

5. Wang Shuxian. Nephrology, the People's Sanitation Publishing Company, Beijing, the 11thedition, 1987:244.

IV. The impact of CMS peptides on cancer

In order to establish whether these peptides medicines for cancer, the authors of the present invention used a variety of standard animal models of cancer in this research to study the biological activities of the above mentioned peptides on infected animals.

Materials

1. Experimental animals

Mouse BALB/c, C57BL/6 and DBA/2, weighing 18-22 g Institute of medical Sciences of China Medical Science Institute), China.

2. Cell line

Cells of mouse sarcoma S180the cells In16and cells L1210from the Department of cancer research Institute of medical Sciences (Cancer Research Department, China Medical Science nstitute).

Cells YAC-1 donated by Professor Yao Zhi, medical University of Tangina (Tianjin Medical University).

3. Essential drugs and reagents

The peptides used in this analysis were produced in the usual way American Peptide Company, Inc., USA.

Fetal calf serum, RPMI-1640 cell culture medium from Gibco, USA.

MTT, ConA from Sigma, USA.

Recombinant murine interferon-((rmIFN-γ) from Beijing Biotech Inc., China

Recombinant interleukin-2 (rhIL-2) from Shanghai Huaxin Biotech Inc., China

The solution for the separation of lymphocytes, research Institute of Hematologic diseases, national Institute of medical Sciences, China.

Cyclophosphamide received from the 12th pharmaceutical factory of Shanghai, China.

Methods

1. The introduction of the investigated substances

Intraperitoneal injection once a day. Except for the group of cyclophosphamide in all groups started treatment within 5 days prior to transplantation of the cancer cells. A group of cyclophosphamide they began to work the next day after the transplantation of cancer cells. The processing of all groups studied substances was carried out for 30 days or until such time as the animal did not die, unless otherwise stated.

2. The effect of peptides on the growth rate of the transplanted cells sarcoma S180mice BALB/c and on the immunological function is ozaena.

BALB/c mice were randomly divided into a peptide group, a group of cyclophosphamide, a group of rmIFN-γgroup rhIL-2 and the group of saline, 20 animals per group.

The original cell sarcoma S180incubated in DMEM/F12 supplemented with 10% fetal calf serum, 37°C, 5% CO2within 72 hours, then washed 3-4 times with a solution of Henk (Hank) at room temperature. The cell concentration was brought to 1-2×109per liter of solution, Henk. 0.2 ml of cell suspension was implanted several BALB/c mice in the armpit for 10-12 days. Mice were killed by displacement of the cervical vertebrae. Intensively reared and not destroyed tumor mass was collected and washed clean sterile saline. The fabric has dispersible in physiological solution until a homogeneous cell suspension at a ratio of 1 g tissue in 4 ml of saline. Mouse model of sarcoma was obtained by injection of 0.2 ml of cell suspension through the armpit[1]. The introduction of the analyte started, as described in "Method 1".

2.1. The effect of peptides on the phagocytic function managername of phagocyte in mice with sarcoma S180[2,3]analyzed by conducting carcass into the tail vein at a rate of 0.1 ml/10 g body weight (dilution 1:5 normal saline) on the second day after the last is the first introduction of the analyte. After one minute and five minutes after injection of the carcass were collected 20 µl of blood from the angle of the palpebral fissure using a heparinised tube. The blood was mixed with 2 ml of 0.1% weight/about Na2CO3and then got OP680 nm. The main explicit index was calculated by the following formula:

K=(lgA1-lgA2)+(t2-t1)

Key:

A1: OP680 nmin the first minute

A2: OP680 nmin the fifth minute

t2: 5 minutes

t1: 1 minute

Then after researching the phagocytic index, the mouse was killed by displacement of the cervical vertebrae. The liver, spleen and cancer tissue was dissected, blotted dry and weighed.

The phagocytic index was calculated as shown below):

α=(3√)×(W÷WLS)

Key:

W: the weight of the body

WLS: the weight of the liver and spleen

2.2. The index of inhibition of tumor growth was calculated by the following formula:

The index of inhibition of tumor growth = (average weight of tumors in the control group the average weight of the tumors in the group handling)÷the average weight of tumors in the control group.

3. The effect of peptides on the survival of BALB/c mice with transplantirovannam liver cancer N22ascitic liquid type

BALB/c mice were randomly divided into a peptide group, a group of cyclophosphamide, a group of rmIFN-γgroup rhIL-2 and the group of saline, 20 animals per group.

IP is adnie cells N 22incubated in DMEM/F12 supplemented with 10% fetal calf serum, 37°C/5% CO2within 72 hours, then washed 3-4 times with a solution of Henk (Hank) at room temperature. The cell concentration was brought to 1-2×109per liter of solution, Henk. 0.2 ml of cell suspension was implanted several BALB/c mice in the abdominal cavity for 6-8 days[1]. Mice were killed by displacement of the cervical vertebrae. Ascitic fluid of mice were collected aseptic manner, and the cell concentration was brought to 1×106per ml solution of Henk. 0.2 ml of cell suspension was implanted into the peritoneal cavity of normal mice to obtain mice model bearing H22, liver cancer ascitic liquid type. The introduction of the analyte started, as described in "Method 1". Recorded data on the survival of mice. If the animal lived longer than continued the experiment, survival was recorded as the duration of the experiment. Average day survival were obtained using the method of Kaplan-Meier in "Survival" software SPSS. The survival index was calculated according to the following formula:

The survival index = (average number of days of survival in the group of treatment - the average number of days of survival of the control group)÷the average number of days of survival of the control group (100%)

4. The effect of peptides is and cellular immunity in BALB/c mice with transplantirovannam liver cancer N 22ascitic liquid type

4.1. Obtaining suspensions of spleen cells[1,4]

Healthy BALB/c mice were randomly divided into the peptide group, the group rmIFN-γgroup rhIL-2 and the group of saline, 15 mice per group and received carrier H22murine model, as described in "Method 3". After implantation of cancer cells test substance was administered for 15 days, mice were killed by displacement of the cervical vertebrae. The spleen was separated and was manually dispersively in a cold solution of D-Hank using a needle for injection. Dispergirovannoyj cell suspension was additionally passed through a sieve of 100 gauge stainless steel with a diameter of 150 μm. After centrifugation at 200g for 10 minutes the supernatant was discarded. Sediment cells re-suspended in 10 volumes of buffer Tris-NH4Cl and then left for 10 minutes at room temperature. Suspended cells were collected by centrifugation at 150g for 10 minutes. Cells were washed 2-4 times with a cold solution of D-Hank by resuspendable and centrifugation under the above conditions. The washed cells were then diluted to the desired density of the cells using a culture medium RPMI-1640 containing 10% fetal calf serum.

4.2. The effect of peptides on the transformation of T-lymphocytes in mice with transplanted ofanim liver cancer N 22ascitic liquid type[1,4]

Cells of the spleen density of 1×106ml was placed in 96-well plates to cell cultures, 100 μl/well, three parallel wells for each of the analyzed sample and the control sample for each mouse. In the analyzed wells were added 100 μl/well ConA solution 100 ág/ml in RPMI-1640 and 100 µl/well of the RPMI-1640 medium was used for control. Cells were incubated for 66 hours at 37°C, 5% CO2. The cells were then besieged by centrifugation at 150g for 10 minutes. The supernatant was collected and stored at -20°to determine the cytokines IL-2 and IFN.

50 μl/well MTT solution 1 mg/ml in RPMI-1640 was added to the precipitate cells and cells re-suspended by shaking for 2 minutes. Incubation was continued for 4 hours. The supernatant was discarded after centrifugation at 150g for 10 minutes. 120 μl of 40 mm HCl in propanol-2 was added to the precipitate cells and was shaken for 3 minutes. Used ELISA reader for receiving OP570 nmfor each hole, normalized to 630 nm.

For each mouse was formed by three of the analyzed wells and three control wells. The stimulation index (SI) for each mouse received, initially receiving the O.D. value of three parallel holes, and then dividing the value for the analyzed holes on the value for a key hole.

4.3. Influence is of peptides on NK cell activity in mice with transplantirovannam liver cancer N 22ascitic liquid type[5,6]

Received murine spleen cells by density 4×106ml, as described above in section 4.1. Target cells YAC-1 was taken in the logarithmic phase and brought up to 1×105Jr. using 96-well plates for cell cultures, 100 μl of cells in the murine spleen, and 100 µl of culture medium was added to control wells containing only cells of the spleen; 100 μl of target cells and 100 μl of culture medium was added to control wells containing only target cells; 100 μl of cells of mouse spleen and 100 μl of target cells were added to the wells for analysis of NK activity. Prepared three parallel series of the above holes on the mouse.

Samples were centrifuged at 150g for 10 minutes to collect cells. The supernatant was discarded and added 50 μl/well MTT solution 1 mg/ml, the Reaction mixture was then shaken for 2 minutes and incubated at 37°C, 5% CO2within 4 hours. The supernatant was discarded after centrifugation at 150g for 10 minutes. Added 120 μl of 40 mm HCl in propanol-2 and shaken for 3 minutes. The ELISA reader was used to obtain OP570 nmfor each hole, normalized to 630 nm.

For each mouse had 9 holes: three holes with spleen cells - only control, three with target cells - only controli analyzed three wells with cells of the spleen, and with target cells. Index NK cell activity for each mouse received, initially receiving the O.D. value of three parallel wells each combination, and then using this average OD in the following formula:

Index NK cell activity = [1-(mean OD of wells with spleen cells and target cells - the average OD of the wells only with spleen cells)÷(average OD of wells with only target cells)]×100%

5. The effect of peptides on the survival of DBA/2 mice with transplantirovannam L1210leukemia

Mice DBA/2 randomly distributed on the peptide group, the group of cyclophosphamide, a group of rmIFN-γgroup rhIL-2 and the group of saline, 20 animals per group.

The original cells L1210cells were then incubated in DMEM/F12 medium, supplemented with 10% fetal calf serum, 37°C/5% CO2within 72 hours, then washed 3-4 times with a solution of Henk and brought up to 1×105cells per liter. 0.1 ml of cell suspension was implanted into the abdominal cavity more healthy mice DBA for 6-8 days. Then the mice were killed by displacement of the cervical vertebrae and collected aseptic way ascitic fluid. The concentration of cells in ascitic fluid collected was brought to 1×106per ml solution of Henk. 0.1 ml of cell suspension was implanted each of the test animals and recorded the data in the rapidly grow ing animals. The processing is started, as described in "Method 1". Average day survival were obtained using the method of Kaplan-Meier in "Survival" software SPSS. If the animal lived longer than continued the experiment, survival was recorded as the duration of the experiment. The survival index was calculated according to the following formula:

The survival index = (average number of days of survival in the group of treatment - the average number of days of survival of the control group)÷the average number of days of survival of the control group

6. The effect of peptides on humoral immunity of mice C57BL/6 bearing transplantirovannam16melanoma and metastatic ability inoculated with melanoma cells

Mice C57BL/6, age 6-8 weeks, weight 18-22 g, were randomly divided into a peptide group, a group of cyclophosphamide, a group of rmIFN-γgroup rhIL-2 and the group receiving saline, 20 animals per group.

The original cell16murine melanoma cells were then incubated in DMEM/F12 supplemented with 10% fetal calf serum, 37°C/5% CO2within 72 hours, then washed 3-4 times with a solution of Henk. The cell concentration was brought to 1×105cells per liter and 0.1 ml of cell suspension was administered by injection into the tail vein of the tested mice to generate animal models of In16mesland what we [7,8]. The processing of the investigated substance is started, as described in "Method 1".

6.1. The effect of peptides on humoral immunity of mice C57BL/6 bearing transplantirovannam16melanoma[9]

Got sheep erythrocytes (SRBC), collecting blood from the jugular vein and placing it in a sterile flask with glass beads. The flask was shaken for 3 minutes and then the blood was mixed with a solution of Alsever (Alsever) (glucose 2,05 g NaCl, 0.4 g, lemonade Na 0.8 g, brought to 100 ml with distilled water) and kept at 4°C. Immediately before use, the samples were centrifuged at 130g for 5 minutes, collecting SRBC. Cells were twice washed with resuspending and centrifugation in normal saline solution. Then the precipitated cells were collected by centrifugation at 180g for 10 minutes and re-suspended in physiological solution, getting the final working SRBC suspension, 2% (V/V).

The complement was obtained by adding 10 volumes of fresh blood serum of the Guinea pig to a single compacted volume in the centrifuge SRBC, and then gently shaking for 30 minutes at 4°C. SRBC was removed by centrifugation at 200g for 10 minutes. To get working Complementos solution was added 10 volumes of normal saline.

Test animals on the 27th day of processing test ve what estom were administered 0.2 ml of the working suspension of cells SRBC each animal to induce antibodies. The day after the last injection of the analyte collected blood from the angle of the palpebral fissure and left at room temperature for one hour exudation of serum. After centrifugation at 200g for 10 minutes, the collected serum was diluted 500 times with normal saline.

To 1 ml of diluted mouse serum of each mouse was added 0.5 ml suspension of SRBC. Cooled on ice. Then added 1 ml of working solution of complement and incubated at 37°C in a water bath for 10 minutes. The reaction was stopped by cooling with ice. Then the samples were centrifuged at 200g for 10 minutes, obtaining the supernatant.

To 1 ml of this supernatant was added to 3 ml Drabkina (Drabkin) and left at room temperature for 10 minutes. Received OP540 nm.

Standardized OP540 nmwas obtained by mixing 0.25 ml SRBC suspension with a solution Drabkina to 4 ml and leaving the mixture on for 10 minutes before receiving OP540 nm.

Index of hemolysis = (OP540 nmthe test sample (standardized OP540 nm)×500

6.2. After investigation of the humoral immunity of mice were killed by displacement of the cervical vertebrae. Spent the anatomic study of animals. Registered pathological changes and counted the number metastannic PTS is the total of melanoma in the lungs.

Results

1. The influence of the peptide on the cell phagocytosis in BALB/c mice transplanted with sarcoma S180

Table IV.1.

The influence of the peptide on the index of phagocytosis in BALB/c mice transplanted with sarcoma S180
GroupDoseNThe index of phagocytosis
CMS00150 ág/kg206,24±0,33*
CMS0015 mcg/kg196,67±0,43*
CMS0345 mcg/kg196,20±0,44*
CMS0340.5 ág/kg206.35mm±1,02*
IL-23×105IU/kg19of 6.96±1,37*
IFN-γ3×105IU/kg17the 5.45±0,71
Cyclophosphamide20 mg/kg19of 5.92±2,47
Saline0.5 ml195,38±0,85

*: comparison with saline solution, P<0,05

compared with IFN-γ, P<0,05

It is established that CMS001 dose of 50 mg/kg/day and 5 µg/to whom/day and CMS034 in a dose of 5 mcg/kg/day and 0.5 mg/kg/day can increase the index of phagocytosis when a statistically significant difference with group, receiving saline.

2. The influence of the peptide on the growth rate of transplanted sarcoma S180in BALB/c mice

Table IV.2.

The influence of the peptide on the growth of transplanted tumor S180
GroupDoseNWeight of tumor (g)Inhibition of tumor
CMS010500 ug/kg200,67±0,35*48,4
CMS0340.5 ág/kg200,83±0,48*35,9
CMS0355 mcg/kg200,71±0,37*44,6
IL-23×105IU/kg200,69±0,37*46,2
IFN-γ3×105IU/kg180,96±0,45to 25.3
Cyclophosphamide20 mg/kg200,68±0,32*47,3
Saline0.5 ml201,29±0,50

*: compared with a group of saline, P<0,05

It is established that CMS010 at a dose of 500 mcg/kg/day, CMS034 at a dose of 0.5 mcg/kg/day, CS035 in a dose of 5 mcg/kg/day can reduce the growth of transplanted sarcoma S 180when a statistically significant difference between the group receiving saline (P<0,05).

3. The influence of the peptide on survival of BALB/c mice with transplantirovannam liver cancer N22ascitic liquid type (Method 3)

Table IV.3.

The influence of the peptide on the index of the survival of BALB/c mice with transplantirovannam liver cancer N22ascitic liquid type
GroupDoseNDays of survivalThe survival index (%)
CMS0085 mcg/kg2050,7±20,9*&67,8
CMS0115 mcg/kg2036,4±22,2*&60,2
CMS02450 ág/kg2036,3±12,7*&$38,4
CMS0245 mcg/kg1940,6±14,6*&$of 54.8
CMS0240.5 ág/kg1946,4±14,8*∧&$76,9
CMS0320.5 ág/kg2042,8±12,2*∧&$63,3
rhIL-23×105IU/kg8 13,6±0,5
rmIFN-γ3×105IU/kg2027,8±7,56,1
Cyclophosphamide20 mg/kg2024,7±10,2
Saline0.5 ml1926,2±6,8

*: comparison with saline solution, P<0,05

compared with rmIFN-γ, P<0,05

&comparison with rhIL-2, P<0,05

$: comparison with cyclophosphamide, P<0,05

It is established that CMS008 in a dose of 5 mcg/kg/day, CMS011 in a dose of 5 mcg/kg/day, CMS024 dose of 50 mg/kg/day, CMS024 at a dose of 0.5 mcg/kg/day and CMS032 at a dose of 0.5 mcg/kg/day can prolong the survival of BALB/c mice with transplantirovannam liver cancer N22ascitic liquid type with a statistically significant difference between the group receiving saline (P<0,05). It was also observed that in the group CMS024 0.5 μg/kg/day, more than 30% (n=6) mice lived longer than 90 days (two months after the end of the experiment). The anatomic study of mice showed no signs of tumor development. Thus, CMS024 at a dose of 0.5 mcg/kg/day may prevent the growth of transplanted H22preventing its development or inducyruya complete recovery from developed RA is A.

4. The influence of the peptide on the transformation of T lymphocytes in BALB/c mice with transplantirovannam liver cancer N22ascitic liquid type (4.2 Method)

Table IV.4.

The influence of the peptide on the transformation of T-lymphocytes
GroupDoseNThe stimulation index
CMS010500 ug/kg201,45±0,21*$
CMS0190.5 ág/kg191,50±0,19*$
CMS0240.5 ág/kg191,46±0,19*$
CMS0245 mcg/kg201,45±0,21*$
CMS0340.5 ág/kg201,37±0,10*$
CMS0350.5 ág/kg201,40±0,13*$
CMS0355 mcg/kg201,46±0,16*$
rhIL-23×105IU/kg191,46±0,21*
rmIFN-γ3×105IU/kg181,27±0,14
Cyclophosphamide20 mg/kg191,01± 0,23*
Saline0.5 ml201,25±0,07

*: comparison with saline solution, P<0,05

$: comparison with cyclophosphamide, P<0,05

It is established that CMS010 at a dose of 500 mcg/kg/day, CMS019 at a dose of 0.5 mcg/kg/day, CMS024 at a dose of 0.5 mcg/kg/day and 5 mg/kg/day and CMS035 at a dose of 0.5 mcg/kg/day and 5 mg/kg/day can increase the stimulation index T-lymphocyte with a statistically significant difference between the group receiving saline (P<0,05).

5. The influence of the peptide on the activity of NK cells in BALB/c mice with transplantirovannam liver cancer N22ascitic liquid type (Method 4.3)

Table IV.5.

The influence of the peptide on the index of the cytotoxic activity of NK cells
GroupDoseNIndex of NK activity
CMS003500 ug/kg1737,9±14,5*∧$
CMS0140.5 ág/kg1740,7±19,7*$
CMS0240.5 ág/kg1839,3±18,7*$
CMS0245 mcg/kg2034,9±12,1*∧$
CMS0240 mcg/kg 2043,6±13,9*∧$&
CMS0325 mcg/kg2052,6±12,5*∧S&
CMS03250 ág/kg1941,0±18,7*∧$&
CMS03450 ág/kg2057,3±17,9*∧$&
IL-23×105IU/kg1926,0±9,0
IFN-γ3×105IU/kg1820,9±3,3
Cyclophosphamide20 mg/kg1916,5±7,2*
Saline0.5 ml2024,0±8,2

*: when compared with saline, P<0,05

: when compared with IFN-γ, P<0,05

&: when compared with IL-2, P<0,05

$: when compared with cyclophosphamide, P<0,05

It is established that CMS003 at a dose of 500 mcg/kg/day, CMS014 at a dose of 0.5 mcg/kg/day, CMS024 at a dose of 0.5 mcg/kg/day, 5 mg/kg/day and 50 mg/kg/day and CMS034 dose of 50 mg/kg/day can increase the cytotoxic activity of NK cells in a statistically significant difference between the group receiving saline (P<0,05).

6. The influence of the peptide on the survival of DBA/2 mice with transplantirovannam L1210l is icotom (method 5)

Table IV.6.

The influence of the peptide on the survival index of the investigated animals
GroupDoseNDays of survivalThe survival index (%)
CMS0190.5 ág/kg2021,1±5,8*26,8
CMS0350.5 ág/kg2029,3±15,4*76,1
IL-23×105IU/kg2032,0±13,7*92,3
IFN-γ3×105IU/kg2015,6±2,2
Cyclophosphamide20 mg/kg2024,0±5,3*44,2
Saline0.5 ml2116,6±5,6

*: compared with a group of saline, P<0,05

It is established that CMS019 at a dose of 0.5 mcg/kg/day and CMS035 at a dose of 0.5 mcg/kg/day is able to prolong the survival of DBA/2 mice with transplantirovannam L1210leukemia when a statistically significant difference between the group receiving saline (P<0,05).

7. The influence of the peptide on humoral immunity C57BL/6 m the necks with transplanted In 16melanoma (method 6.1)

Table IV.7.

The influence of the peptide on the index of hemolysis C57BL/6 mice with transplanted In16melanoma
GroupDoseNIndex of hemolysis
CMS0010.5 ág/kg2051,0±16,2*$
CMS0015 mcg/kg2041,3±17,7*$
CMS00150 ág/kg1945,0±31,9*$
CMS001500 ug/kg2036,0±10,2*$
CMS0030.5 ág/kg2061,6±26,9*$
CMS0035 mcg/kg2037,2±15,9*$
CMS00350 ág/kg2038,7±13,5*$
CMS003500 ug/kg2035,9±13,0*$
CMS0080.5 ág/kg1942,7±18,4*$
CMS0085 mcg/kg2038,9±12,0*$
CMS00850 ág/kg2037,1±16,7* $
CMS008500 ug/kg2050,1±17,8*$
CMS0100.5 ág/kg1834,9±10,5*$
CMS0105 mcg/kg2051,0±14,6*$
CMS01050 ág/kg2039,6±7,7*$
CMS010500 ug/kg2050,1±16,7*$
CMS0110.5 ág/kg2032,0±14,7*
CMS011500 ug/kg2034,4±19,4*
CMS016500 ug/kg2043,3±29,9*
CMS0190.5 ág/kg2042,0±12,0*$
CMS0195 mcg/kg2035,4±15,1*$
CMS01950 ág/kg2028,3±7,6*
CMS0240.5 ág/kg2043,0±10,7*$
CMS0245 mcg/kg2042,2±11,8*$
CMS02450 ág/kg2027,8±9,1*
CMS024500 ug/kg 1830,1±10,0*
CMS0340.5 ág/kg1850,8±18,4*$
CMS0345 mcg/kg1943,0±11,7*$
CMS03450 ág/kg2030,2±10,9*
CMS0355 mcg/kg2038,9±21,2*$
CMS03550 ág/kg2044,7±22,7*$
CMS035500 ug/kg1940,5±25,8*
rhIL-23×105IU/kg1949,3±24,7*
rmIFN-γ3×105IU/kg1960,5±17,4*
Cyclophosphamide20 mg/kg1920,7±19,1
Saline0.5 ml2019,0±9,1

*: comparison with saline solution, P<0,05

$: comparison with cyclophosphamide, P<0,05

It is established that CMS001, CMS003, CMS008, CMS010, CMS011, CMS016, CMS019, CMS024, CMS034 and CMS035 can enhance the humoral response (increase in the index of hemolysis) C57BL/6 mice with transplanted In16melanoma in the dosages shown in table IV.7, prostatitious significant difference with group, receiving saline (P<0,05).

8. The influence of the peptide on the survival of inoculated cells B16 melanoma in C57BL/6 mice (6.2 method)

The anatomic study of animals after finishing the test substance did not reveal any signs of lesions of B16 metastases in the lung of mice that were treated CMS008 in doses of 0.5 μg/kg/day, 5 mg/kg/day and 50 mg/kg/day and CMS016 at doses of 5 mcg/kg/day and 500 mg/kg/day.

Conclusions

In accordance with the Guidelines for preclinical studies of new drugs, issued by the Department of administration of drugs of the Department of health of the people's Republic of China in 1993, the influence of the peptide in mice with transplanted cancer cells. In the end concluded that

1. CMS010, CMS034 and CMS035 in proper dosage can significantly inhibit the development of the transplanted S180sarcoma in mice;

2. CMS001 and CMS034 in proper dosage can enhance the phagocytic immune activity in mice transplanted with S180sarcoma;

3. CMS008, CMS011, CMS024 and CMS032 in proper dosage can prolong the survival of mice that transplanted liver cancer ascitic liquid type n;

4. CMS010, CMS019, CMS024, CMS034 and CMS035 in proper dosage can enhance the transformation of T lymphocytes in mice, which TRANS is antiroman liver cancer ascitic liquid type n;

5. CMS003, CMS014, CMS024, CMS032 and CMS034 in proper dosage can increase the cytotoxic activity of NK cells in mice, which transplanted liver cancer ascitic liquid type n;

6. CMS019 and CMS035 in proper dosage can prolong the survival of mice that transplanted L1210 leukemia;

7. CMS008 and CMS016 in proper dosage can inhibit the development of the transplanted B16 melanoma in mice;

8. CMS001, CMS003, CMS008, CMS010, CMS011, CMS016, CMS019, CMS024, CMS034 and CMS035 in proper dosage can enhance the humoral immune response in mice, which transplanted B16 melanoma.

CMS001, CMS003, CMS008, CMS010, CMS011, CMS014, CMS016, CMS019, CMS024, CMS032, CMS034, CMS035 can be used as part or by themselves to treat cancer in humans. For example, CMS001, CMS003, CMS008, CMS010, CMS011, CMS014, CMS016, CMS019, CMS024, CMS032, CMS034, CMS035 can be used to enhance immunity in patients with cancer. CMS008, CMS010, CMS016, CMS034 and CMS035 can be used to prevent the growth of cancer cells in patients. CMS008, CMS011, CMS019, CMS024, CMS032 and CMS035 can be used for prolonging the life expectancy of cancer patients. The peptides can be used alone, in combination of two or more peptides or in combination with other pharmaceuticals or food additives as a complete treatment of cancer.

Table IV.8.

Peptides effective in cancer
Code CMSSEQ ID No
CMS0011
CMS00327
CMS0083
CMS0104
CMS01130
CMS0147
CMS0169
CMS01911
CMS02416
CMS03222
CMS03424
CMS03525

References

1. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7: 137-143

2. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7: 128-129

3. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition). People's Health Publishing House. 1998, 137-138

4. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 1991, 1221-1234

5. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7: 140

6. Jinsheng He, Ruizhu Li Tingyi Zong. The study on MTT reduction method of testing NK cell activity. China Immunology Journal. 1996, 1(6): 356-358

7. Yang Yaoqin, Huchuan Yang, Huihong Tao, et al. The synergic effect of Tween-80 on the antitumor of hyperfhenm'a-Experimental studies of mouse melanoma. Cancer Research on Prevention and Treatment. 1999, 26(4): 8-12

8. Jian Fu, Jie Zheng, Weigang Fang, et al. Interleukin-12 gene transfection into murine B16 melanoma cells suppresses tumorigenicity and decreases metastatic potential. National Medical Journal of China. 1998, 78(8): 627-629

9. Qian Wang. Modern medical experiment method. People's Health Pulishing House. 1998, 482-483

10. Qichao Pan, Bin Xu. Cancer pharmacology and chemotherapy, Henan medical university Publishing House. 2000, 66-69

11. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition). People's Health Publishing House. 1998, 131

V. Impact on the weight of the body

Healthy rats were fed a highly nutritious diet for 5 weeks using simultaneous treatment with or without peptide treatment (intramuscular injection of 300 mg/kg/day). As a negative control was used rats with normal diet, which was administered injections of saline. After 5 weeks of treatment the injection was stopped and maintained the same diet for three weeks. Data of body weight were collected simultaneously with a weekly interval. Also observed the behavior of rats. It was found that in the processing of peptide in rats treated with the peptide S015, there was a statistically significant smaller increase in body weight compared with control. This tendency to reduce body weight gradually decreased after cessation of treatment S015. Concluded that S015 at an appropriate dose level can reversibly control the development of obesity-induced overeating.

Materials

Rats Sprague-Dawley (SD)weight 145±10 g supplied from the Center of experimental animals of the University of Guangzhou Traditional Chinese medicine, Guangzhou University of Traditional Chinese Medicine), China (certificate No. A). Peptides were with nasirovna in the usual way (on the basis of L-amino acids) American Peptide company, Inc., USA, and diluted to a concentration of 10 μg/ml in normal saline solution. Highly nutritious and regular meals were prepared in accordance with the guidelines for preclinical testing of drugs against obesity, published by the SDA (State drug administration tools)PRC[1].

Methods

Healthy rats statistically distributed on experimental, positive control and negative control group of 10 rats per group, half males and half females. The experimental group of rats was fed a highly nutritious diet for 5 weeks with the simultaneous introduction of the peptide at a dose of 300 mg/kg/day once daily by intramuscular injection. A positive control group received the same high-nutrient diet, but "placebo" injection of saline, whereas the negative control group, used to prove the successful establishment of models of obesity, received regular meals with "placebo" injection of saline. After 5 weeks of treatment the injection was stopped and maintained the same pattern of power for another 3 weeks. Rats were weighed at the same time with one week interval. Also observed the behavior of rats.

Data are presented as mean value (standard deviation. Paired t criterion or detectory ANOVA was used to compare between groups and within groups. The limit of statistical significance was P≤0,05.

Results

1. The influence of the peptide on body weight in rats SD

Table V.1

The influence of the peptide on body weight in rats SD
The positive control group (g)CMS015 processing Group (g)
Males n=5Females n=5Males n=5Females n=5
Before processing145,6±13,6133,6±4,6145,6±8,5to 129.2±3,3
Week 1194,4±14,5164,4±8,7183,8±10,6157,8±8,3
Week 2239,6±13,4188,0±6,4br220.6±12,2*176,0±11,2*
Week 3265,0±11,8208,8±8,2239,0±16,0*196,0±10,5*
Week 4287,4±17,7227,2±8,2258,2±18,1*212,0±13,5*
Week 5299,4±21,2236,6±10,9268,8±17,7*221,4±13,2*
Week 6333,4±27,1249,4±16,3 299,4±21,2*235,6±16,3
Week 7349,2±28,9261,2±13,4310,4±25,9*242,2±18,8*
Week 8374,4±37,2255,6±11,5337,4±30,6252,8±22,5

When compared with positive control group: p<0,05

It was found that at a dose of 300 mg/kg/day S015 able to limit weight gain in greasy rats caused by overfeeding, showing a statistically significant difference when compared with the control group (p<0,05). The difference between the processing group and the control group increased with increasing duration of treatment. Upon termination of the processing of peptide S015 in the processing group the difference between the processing group and the control group gradually decreased and became statistically insignificant after three weeks, showing that the effect of the peptide on body weight of SD rats is reversible.

Throughout the experiment it was observed that the appetite and activity of all groups of rats remained normal.

Discussion

Concluded that CMS015 under suitable dosage level may limit the development of obesity-induced overeating. The peptide can be used in humans to combat obesity. The peptides can be used are is be, as a combination of two or more peptides or in combination with other pharmaceuticals or food additives as a complete treatment of obesity.

This study tested the introduction using intramuscular injections, but this does not exclude the possible effectiveness of the peptide with its introduction of other alternative ways. The peptides can be administered by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and subcutaneous implantation, with or without using a device to facilitate the delivery of drugs, such as liposomes, protection for slow release, etc. Peptide can also be entered in any form for oral administration such as tablet, capsule, suspension, solution, etc. in the usual form as appropriate without modification or form with delayed release, with or without the use of gastrointestinal protection. In addition, the peptide can be used in any form for local use, such as ointment, cream, gel, etc. with or without using a device to facilitate the transcutaneous penetration, or inhalation powder dissolved or liposome-protected form. The peptide can also be converted into its genetic follower of the awn and cloned into the expression system, or by itself or in combination with other peptide sequences for the formation of the final peptide molecules for use in the activity of the peptides, as described in this invention, cleaning or no cleaning of the final peptide.

Table V.2.

The effectiveness of the peptides in obesity
Code CMSSEQ ID No
CMS0158

Literature

1.SDA PR China, The guideline for pre-clinical research of new drugs, 1993.

It should be understood that it is possible to add additional amino acids to the amino or carboxyl end of the above described peptides as another method, the practical implementation of the invention. For example, one or two amino acids may be added to the peptide without affecting its biological activity. You can also add three or four amino acids, and still retain the function of the peptides. They are referred to as variants of the same peptide. Alternatively, one or two amino acids may be deleted from the peptide without affecting its biological activity. In addition, it is possible to remove three or four amino acids, without affecting the biological function of the peptide. They are referred to as fragments of this peptide. In addition, for the practical implementation of the Oia another aspect of the present invention can be used derivative peptide, for example obtained by conservative substitution of one amino acid to another amino acid of the same functional class. For example, peptides with non-polar or hydrophobic side chains, possible substitution of one side of the group to another without compromising biological activity. As an additional example, the linker/spacer can be introduced into the peptide with education options, but options still retaining its active fragment as the initial peptide used in this study. They are also considered variants of the peptides. Similar peptide, as used herein, includes peptides having the amino acid molecules that mimic the structure of natural amino acids, for example similar with a different skeletal structure or a D-amino acid substitution. As an additional example, although the amino acids used for peptide synthesis, represent the L-optical isomer forms, peptides with one or more amino acids in the sequence are replaced by D-form, can have similar biological activity. It is implied that the term "functional derivative", as used in the claims, includes fragments, variants, analogs, or chemical derivatives of the peptide.

As used in the nom description the term "hybrid peptide" is used to refer to peptides that contain additional peptides embedded in the original biologically active peptides with SEQ ID nos: 1-30, or their functional derivatives, but still retains essentially the same activity. Additional peptides include leader peptides, which contain, for example, amino acid sequence that is recognized by one or more prokaryotic or eukaryotic cells as a signal for secretion of the hybrid protein into the environment or into the cell. The secretion may be a directly secretion or indirect secretion by secretory of vesiculo.

"Essentially pure peptide" refers to peptides which have at least 10% weight/weight purity, more preferably 20%, more preferably 40; and much more preferably 60% and even more preferably more than 90% purity. In the most preferred embodiment, the purity is greater than 99%. Essentially pure peptide can be used for pharmaceutical and food compositions, which can be a complex mixture, as described below.

The application of the aforementioned peptides in the pharmaceutical compositions can be used for the possible treatment of immunological violated the th or diseases, having a secondary effect on the immune system such as cancer or infection or any of the above illnesses. The composition can contain one of the identified peptides, mixed with other active or inactive ingredients, including other peptides. For example, two or more (e.g., 3-5) of these peptides can be added to the same composition or without other ingredients. Alternatively, one of these peptides can be used to obtain compositions together with peptides that are not listed in this invention. They can be administered intravenously, intramuscularly, intracutaneously, subcutaneously or intradermally. The method of introduction may also be an intra-arterial injection, which leads directly to the patient body. Other methods of introduction is percutaneous introduction, inhalation in the form of a powder or spray and other types of delivery known in this field. The composition may also be an orally taken drug, and may contain carriers, which are used to prevent cleavage of the peptide in the stomach after oral administration or any other media known in the art (for percutaneous introduction such as liposomes).

The pharmaceutical composition may include any of the known FA the pharmaceutical carriers. Examples of suitable carriers include any of the standard pharmaceutically acceptable carrier, known to specialists in this field. They include, but are not limited to, saline solution, water, emulsions, including oil and water mixture or emulsion of triglycerides, and other types of agents, fillers, film-coated tablets and capsules. Suitable carrier may be selected based on the method of administration of the pharmaceutical composition.

The peptides can be entered via an intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and subcutaneous implantation. The peptide can also be entered in any form for oral administration such as tablet, capsule, suspension, solution, etc. in the usual form as appropriate without modification or form with delayed release, with or without the use of gastrointestinal protection. In addition, the peptide can be used in any form for local use, such as ointment, cream, gel, etc. with or without using a device to facilitate the transcutaneous penetration. The peptide can also be converted into its genetic sequence and cloned into the expression system or by itself or in combination with other peptide sequences to form a final PEP is IGNOU molecules for use in the activity of the peptides, as described in this invention.

The dose of each peptide can be 1 ng to 10 g per kg of body weight. The preferred dose of 10 ng-10 mg / kg and more preferably 1 μg to 1 mg per kg for injection route of administration. However, the effective dose can be as low as 1 ng per kg of body weight, because one or more of the peptides may act through receptors that induce a cascade of physiological responses. Alternatively, one or more peptides can only be initiated for a whole cascade of reactions. For oral administration, the number can be 1 ng to 10 g per day per kg of body weight, more preferably 0.1 mg to 1 g per day per kg of body weight and even more preferably 1 μg to 10 mg per day.

VI. Gene therapy and treatment method

Gene-therapy based on open peptide sequences carried out by generating a sequence of nucleic acids that encodes one of these peptides. The nucleic acid may be synthesized chemically or functionally Legerova with the promoter and cloned into the expression vector. The expression vector is then introduced into the human body in a form of gene therapy for expression in human cells. The term "genetic vectors", as used herein, includes such expression vectors. Vectors, caloriemate be used for gene therapy, include adeno-associated virus (Mizuno M., et al., 1998), Jpn J Cancer Res., 89, 76-80), the vector LNSX (A.D. Miller et al., (1993), Methods Enzymol., 217, 581-599) and lentivirus (Goldman, M.J. et al., (1997) Hum Gene Ther., 8, 2261-2268).

Other media for the delivery of peptide include expression vectors encoding the desired peptide, which can be transferred into an organism that can replicate in the body-master, which it is desirable to introduce the peptide, without significant adverse effects on the health of the host body. For example, expression vectors can be transferred into the body, which is not pathogenic to the host body, which it is desirable to introduce the peptide. In some embodiments, the implementation of the expression vector produces the target peptide in the body, bacteria or fungus, which does not have a significant adverse impact on the health of the host body, which is inserted peptide. For example, the expression vector encoding the desired peptide can be an expression vector that produces the desired peptide in the body, such as lactic acid bacteria, E.coli or yeast. In one embodiment, the expression vector produces the desired peptide in the microbe, usually found in the gastrointestinal tract of a mammal, or germ, which are tolerant of the gastro-intestinal tract of the mammal. Some of the varieties is Ikramov, which can be expressed desired peptide include, but are not limited to, species of Lactobacillus, such as L.acidophilus, L.amylovorus, L.casei, L.crispatus, L.gallinarum, L.gasseri, L.johnsonii, L.paracasei, L.plantarum, L.reuteri, L.rhamnosus or other; Bifidobacterium species, such as B.adolescentis, B.animalus, B.bifidum, B.breve, B.infantis, B.lactis, B.longum or other; Enterococcus faecalis or Ent.facium; Sporolactobacillus inulinus; Bacillus subtilis or Bacillus cereus; Escherichia coli; Propionibacterium freudenreichii; or Saccharomyces cerevisitae or Saccharomyces boulardii.

Nucleic acid sequences that encode the peptides of the present invention, chemically synthesized or produced by other means, including, but not limited to, reverse transcription of mRNA for the production of DNA molecules included in the expression vectors for gene transfer into the target organisms by methods of genetic engineering, familiar to specialists in this field. The expression vectors can be DNA vectors or RNA vectors. For example, expression vectors can be based on plasmid or viral genetic elements. The expression vectors can be represented as vectors that replicate extrachromosomal, or vectors, integrated into the chromosome.

The expression vectors include a promoter functionally linked to a nucleic acid that encodes a peptide of the present invention. The promoter may be a regulated promoter, such as the Indus is Zemelny promoter or a constitutive promoter. In some embodiments, the implementation of a promoter can be selected to provide the desired level of expression of the peptide. Additionally, if desired, the expression vectors may include other sequences to stimulate the production, presentation and/or secretion of peptides. In some embodiments, the implementation of the nucleic acid encoding the peptide of the present invention, functionally linked to a sequence of nucleic acids, which regulates the secretion of the peptide. For example, nucleic acid encoding the peptide of the present invention can be functionally linked to a nucleic acid that encodes a signal peptide.

In some embodiments, the implementation of the expression vectors which are designed to encode the peptides of the present invention, can be a expression vectors adapted for expression of the peptide of the present invention in the types of bacteria that create the normal flora in the gastrointestinal tract of mammals, such as species of Lactobacillus and Bacillus subtilis. Examples of such expression vectors can be found in U.S. patent No. 6100388, Casas, and No. 5728571, Bellini, respectively. These documents are specifically incorporated in this description by reference in its entirety. You should understand that you can use any expression vector, which which facilitates the expression of the peptide of the present invention in the body, which does not have a negative impact on the health of the host body, which enter the peptide.

In some embodiments, the implementation of the expression vectors which are designed to encode the peptides of the present invention, can be a expression vectors adapted for expression of the peptide of the present invention in the types of yeast that are well tolerated by the gastrointestinal tract of mammals, such as Saccharomyces cerevisiae; or, preferably Saccharomyces boulardii, which can colonize the gastrointestinal tract of a human or used to treat some forms of diarrhea. You can use yeast expression vectors that constitutively Express heterologous proteins and peptides, and are highly stable, so well transmitted to progeny cells during mitosis and meiosis and may include the coding sequence of the signal peptide or peptides that regulate high levels of secretion of the recombinant protein. An example of such a yeast vector is shown in U.S. patent No. 6391585, Jang et al., included in this description by reference in its entirety.

The expression vectors encoding the peptides of the present invention can be introduced into the body, which is suitable for expression of the peptides, methods, known in the village is authorized area. Such methods include traditional methods of transformation of bacteria, yeast or other microorganisms through the use of chemically competent bacterial cells, electroporation or lithium acetate transformation (for yeast), also, for example, using the latest achievements in the transformation of bacterial species resistant to such methods. In some embodiments, the implementation of the expression vectors introduced into the lactic acid bacteria, known as resistant to transformation, using the method described Leer at el. (WO 95/35389), which is included in this description by reference in its entirety. The input sequence can be introduced into microbial chromosomal DNA, or they can remain extrachromosomal DNA elements.

Such genetically-engineered microbe containing the expression vector can then be inoculated in the digestive tract, vagina, trachea, etc. to realize prolonged immunotherapy. In some embodiments, the implementation of organisms expressing the peptides of the present invention, swallowed in an inactive form or preferably in a live form. In the digestive tract, these microorganisms produce these peptides, releasing them into the lumen by secretion or lysis of the microorganism or in some other way providing eptide owner, this implies that produced when the peptides have an effect on the body is the master. In other embodiments, implementation of the peptides provided to the owner on the mucous membrane of the nasal passages, vagina, or small intestine.

Another method of treatment is the use of liposomes as delivery vehicles of a particular nucleic acid into the cells of the body. Nucleic acid (such as an expression vector containing a nucleotide sequence that encodes the peptides of sequences SEQ ID No: 1-30) is delivered in an environment that supports cellular uptake and chromosomal fusion, as described X. Gao and L. Huang (1995), Gene Ther., 2, 710-722, and U.S. patent 6207456. Alternatively, the peptide can be encapsulated in the liposome and directly delivered using the method described in U.S. patent 6245427. All of the above scientific publications and patents are included in this description by reference.

Nucleic acid sequences that are suitable for the above gene therapy and treatment method, include sequences that encode these peptides and their functional derivatives. Any of the numerous sequences of nucleic acids can be used to encode these peptides and derivatives on the basis of the degeneracy of Genet the ical code.

The following links are included in this description in its entirety.

1. Principles of Pre-clinical Research of New Drugs, People's Republic of China. 1993, 7: 134-135

2. Shuyun Xu, Rulian Bian, Chen Xiu. Methodology of pharmacological experiment. People's Health Publishing House. 1991, 1221-1234

3. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7: 140

4. Jinsheng He, Ruizhu Li Tingyi Zong. The study on MTT reduction method of testing NK cell activity. China Immunology Journal. 1996, 1(6): 356-358

5. Qian Wang. Modem medical experiment method. People's Health Publishing House. 1998, 482-483

6. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7: 141

7. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7: 132-133

8. Principle of new drug research in pre-clinic issued by Ministry of Health, People's Republic of China. 1993, 7: 128-129

9. Yuanpei Zhang, Huaide Su. Pharmacological experiment (second edition). People's Health Publishing House. 1998, 137-138

10. Li Jiatai, Clinical pharmacology (second edition). People's Health Publishing House. 1998, 1338-1339.

Example 1

Delivery of peptides using genetically engineered bacteria Lactobacillus

Below is one of illustrative methods of delivery of the peptides according to the invention to the owner, as described above. The DNA sequence encoding one of the peptides listed above in table a, synthesized by chemical methods, and this DNA sequence is integrated into the expression vector using standard genetic engineering techniques known to experts in this field. The selected expression vector contains Constitution the th promoter, functional in Lactobacillus, a multiple cloning site for introduction of DNA sequences into specific 5'-3' position, as well as the selective marker gene, which confers resistance to the antibiotic (to facilitate cloning) and may include other sequences, to facilitate production and/or secretion of peptides, such as the signal peptide sequence. An example of such a vector is described in U.S. patent 5592908, Pavla, which is included in this description by reference in its entirety. Briefly, this patent discusses several well-known promoters that function in the species Lactobacillus, as well as the detection of new promoters in the specified bacteria, any of which may be functionally linked to a nucleic acid that encodes a peptide of the present invention for expression of the peptide Lactobacillus. Nucleic acid encoding a signal peptide, such as peptides containing 16-35 most hydrophobic amino acids, which are active in Lactobacillus lactis, described in the above U.S. patent 5529908, is placed between the promoter and the nucleic acid that encodes a peptide of the present invention so that the nucleic acid encoding the signal peptide is in frame with nucleic acid that encodes a peptide truly is obreteniyu.

In addition to the coding sequence of the peptide synthesized DNA sequence may include sequences to facilitate ligation and cloning of the specified DNA in an expression vector. For example, the website pattern recognition restricts, which correspond to the sites found in the multiple cloning site of the vector, can be embedded in the synthesized DNA on the 5' and 3' ends of the sequence so that the sequence can be cloned in the correct orientation in the vector. As a vector, and the synthesized DNA, broken down by specific enzymes and then purified. After reactions ligation of vector and synthesized DNA spend transform into a suitable strain of E. coli. The transformed bacterium is placed in a medium containing the antibiotic to which the given vector resistance. The colony of transformed bacteria are selected for the growth of crops and procedures for obtaining plasmids; confirm the presence of the synthesized DNA in the correct orientation.

This expression vector is then transformed into a bacterial cell host species of Lactobacillus, such as L.acidophilus. Transformed cells are selected on the basis of the selective marker detected in the sequence of the vector, and the secretion of the peptide can be confirmed by conducting Vester the blot, conducting gel electrophoresis of peptides present in the environment for growth, or other standard methods. Select transformed colony of bacteria and is used to produce in large scale cultures of bacteria obtained by genetic engineering methods. The culture is grown genetically engineered bacteria expressing the desired peptide, and at least a portion of its type in the digestive tract, the vagina, the trachea or other areas of the host body where bacteria are able to replicate. If desired, the bacterial culture can be treated in different ways to obtain additives consumption in the intestine of the host. Such processing include lyophilization or other means of preserving bacteria, in addition to combining bacteria with agents-carriers, such as solutions, solvents, dispersing medium, delaying the release agents, emulsions and the like. The use of such agents to obtain additives well known in this field. For example, bacteria can be used for containing culture dairy products or other foods for human consumption, so that the body expressing the peptide, forming colonies in the digestive tract of the host body. A number of different ways of introducing specific strains of molochnokislykh bacteria in food, such as yogurt, kimchi, cheese and butter, are described in U.S. patent 6036952, Oh, which is included in this description by reference in its entirety. When the consumption of bacteria through one of the many ways engineered organisms can colonize in the digestive tract and be given the opportunity to provide and/or to absorb peptides according to this invention through the mucus layer of the digestive tract.

Example 2

Delivery of peptides using genetically engineered forms of Bacillus subtilis

Below is another illustrative method of delivery of the peptides according to the invention to the owner, as described above. The DNA sequence encoding one of the peptides listed above in table a, synthesized by chemical methods, and this DNA sequence inserted in the expression vector using standard genetic engineering techniques; all the methods known to experts in this field. The selected expression vector includes a Shuttle vector, such as pTZ18R (Pharmacia, Piscataway, NJ), are able to multiply in E.coli and B.Subtilis and containing the gene for antibiotic resistance for selection of transformed colonies of bacteria. Such a vector may contain a constitutive promoter, active in B.Subtilis, such as a promoter obtained from a Sac B B.Subtilis gene, and the nucleotide sequence encoding a signal or ederny peptide, active in B.Subtilis, which regulates the effective export of expressed heterologous protein from the bacterial cells. An example of such a vector is described in U.S. patent 6268169, Fahnestock, the description of which is incorporated by reference in its entirety. Briefly, as described above, DNA encoding a peptide according to this invention will be synthesized using enzymatic restriction sites and/or other sequences to facilitate cloning DNA using methods familiar to specialists in this field. After transformation in E. coli growing culture on the plate, selection and propagation of plasmids to create a source of plasmids, plasmid transform in B.Subtilis and select transformants on the basis of resistance to the antibiotic in the culture medium of the culture.

Production of peptide and secretion of genetically engineered B.Subtilis confirmed using methods well known to specialists in this field, such as the tagging of peptides isotopic label for autoradiographical detection after SDS-PAGE analysis or Western blotting.

The culture is grown genetically engineered bacteria and at least a portion of its type in the digestive tract, the vagina, the trachea or other areas of the host body where bacteria are able to reproduce.

Example 3

<> Delivery of peptides using genetically engineered species of Saccharomyces

Below is another illustrative method of delivery of the peptides according to the invention to the owner, as described above. The DNA sequence encoding one of the peptides listed above in table a, synthesized by chemical methods, and this DNA sequence inserted in the expression vector using genetic engineering techniques; all the methods known to experts in this field. The selected expression vector includes stably supported yeast protein expression vector comprising a constitutive yeast promoter, such as pADH1, sites for replication of the vector in yeast and in E. coli., gene, giving phototropy of auxotrophic yeast mutants for the selected objectives, a multiple cloning site (MCS) and, if desired, sequences that encode a signal peptide. Vectors, such as what is specified, are commercially available and well known in this area or can easily be constructed using standard methods. After embedding the synthesized DNA into a yeast vector, transformation in E. coli cultivation of transformed E. coli on the selective medium, selection of transformed bacterial colonies and obtain plasmid DNA from cultures of the bacteria the bacilli from this colony, vector transform in Saccharomyces cerevisiae using well known methods, such as lithium acetate transformation or electroporation. The Saccharomyces cerevisiae strain selected for transformation, is an auxotrophic mutant strain that requires gene on the plasmid for growth on minimal medium plates. Transformed yeast colonies produce by growing yeast on medium for growth without the gene, the built-in vector. Only such a yeast that has received vector and its selective gene and Express this gene product, will be able to grow into colonies on minimal medium. Confirmation of the secretion of the peptide can be obtained by carrying out Western blotting, conducting gel electrophoresis of peptides present in the growth medium, or other standard methods.

Select transformed colony of yeast and is used to obtain large-scale cultures. The culture is grown genetically engineered yeast and at least a portion of its type in the digestive tract, the vagina, the trachea or other areas of the host body where bacteria are able to reproduce. If desired, yeast culture can be treated in different ways to obtain additives consumption in the intestine of the host. Such processing include lyophilization or other ways saved what I yeast in addition to combining bacteria with agents-carriers, such as solutions, solvents, dispersing medium, delaying the release agents, emulsions and the like. The use of such agents to obtain additives well known in this field. In another embodiment, the transformed yeast is used for producing food products, such as fermented dairy products such as yogurt and kefir, using technology well known to specialists in this field. As in the case of live lactic acid bacterial cultures in such foods, the transformed yeast form colonies in the digestive tract, at least temporarily, and are used to deliver peptides to the owner through the lumen of the digestive tract.

1. The selected peptide that stimulates antitumor emanny response, characterized by amino acid sequence SEQ ID No: 16.

2. The peptide according to claim 1, characterized in that the response is due to the modulation of immune activity in a patient.

3. The peptide according to claim 1, characterized in that the specified response modulates the development of cancer.

4. The peptide according to claim 3, characterized in that said cancer is a cancer of the liver.

5. The peptide according to claim 3, characterized in that the cancer is a melanoma.

6. Headlight is aseptically composition, stimulating an antitumor immune response, consisting of an effective amount of a peptide according to claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that the response is due to the modulation of immune activity in a patient.

8. The pharmaceutical composition according to claim 6, characterized in that the specified response modulates the development of cancer.

9. The pharmaceutical composition of claim 8, characterized in that said cancer is a cancer of the liver.

10. The pharmaceutical composition of claim 8, characterized in that the cancer is a melanoma.

11. A method of treating a mammal suffering from a disease, the treatment of which requires the stimulation of an antitumor immune response, involving the introduction of a pharmaceutically effective dose of the pharmaceutical composition according to claim 6.

12. The method of treatment according to claim 11, characterized in that the specified mammal diagnosed with cancer,

13. The method of treatment according to claim 11, characterized in that said cancer is a cancer of the liver.

14. The method of treatment according to claim 11, characterized in that the cancer is a melanoma.

15. A method of modulating an immune response in a mammal suffering from a disease, the treatment of which requires the stimulation of an antitumor immune response, enabling the th introduction of a pharmaceutically effective dose of the pharmaceutical composition according to claim 6.

16. The modulation of the immune response by § 15, characterized in that the immune response is to increase the cytotoxic activity of NK cells.

17. The modulation of the immune response by § 15, characterized in that the immune response is to strengthen the synthesis of anti-SRBC antibodies with antigenic stimulation.

18. The modulation of the immune response by § 15, characterized in that the immune response is to strengthen the phagocytic activity managernew phagocytes.

19. The modulation of the immune response by § 15, characterized in that the immune response is to increase the weight of the thymus gland.

20. The modulation of the immune response by § 15, characterized in that the immune response is to reduce the weight of the spleen.



 

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12 cl, 6 dwg, 8 tbl, 9 ex

FIELD: medicine, chemistry of peptides, amino acids.

SUBSTANCE: invention relates to novel biologically active substances. Invention proposes the novel composition comprising peptides of the formula: H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys. The composition shows ability to inhibit proliferative activity of mononuclear cells, to induce suppressive activity and their ability for secretion of cytokines TNF-1β (tumor necrosis factor-1β) and IL-10 (interleukin-10 ).

EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

The invention relates to novel soluble synthetic polimersvarka the anthracyclines, exhibiting antitumor activity, to a method of receiving and containing pharmaceutical compositions

FIELD: synthesis of biologically active compounds.

SUBSTANCE: invention provides 1,5-benzothiazepines of general formula I (formulae presented below), in which Rv and Rw are independently selected from hydrogen and C1-C5-alkyl; one of Rx and Ry represents hydrogen or C1-C6-alkyl and the other hydroxy or C1-C6-alkoxy; Rz is selected from halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1 of invention; v is a number from 0 to 5; one of R4 and R5 represents group of general formula IA; R3 and R6 and the second from R4 and R5 are independently selected from hydrogen, halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1; R3 and R6 and the second from R4 and R5 being optionally substituted by one or several R16 groups at their carbon atoms; D represents -O-, -N(Ra)-, -S(O)b- or -CH(Ra)-, wherein Ra is hydrogen or C1-C6-alkyl; and b=0-2; ring A represents aryl or heteroaryl and is optionally substituted by one or several substituents selected from R17; R7 represents hydrogen, C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R18; R8 represents hydrogen or C1-C4-alkyl; R9 represents hydrogen or C1-C4-alkyl; R10 represents hydrogen or C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R19; R11 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORc)(ORd), -P(O)(OH)(ORc), -P(O)(OH)(Rd), or -(O)(ORc)(Rd), wherein Rc and Rd are independently selected from C1-C6-alkyl; or R11 represents group of general formula IB, in which X is -N(Rq)-, N(Rq)C(O)-, -O-, or -S(O)a, wherein a=0-2; and Rq is hydrogen or C1-C4-alkyl; R12 represents hydrogen or C1-C4-alkyl; R13 and R14 are independently selected from hydrogen, C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23 can be optionally independently substituted by one or several substituents selected from R20; R15 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORe)(ORf), -P(O)(OH)(ORe), -P(O)(OH)(Re), or -P(O)(ORe)(Rf), wherein Re and Rf are independently selected from C1-C6-alkyl; or R15 represents group of general formula IC, in which R24 is selected from hydrogen and C1-C4-alkyl; R24 is selected from hydrogen, C1-C4-alkyl carbocyclyl, heterocyclyl, and R27, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R27 can be optionally independently substituted by one or several substituents selected from R28; R26 is selected from carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORg)(ORh), -P(O)(OH)(ORg), -P(O)(OH)(Rg), or -P(O)(ORg)(Rh), wherein Rg and Rg are independently selected from C1-C6-alkyl; p=1-3; wherein meanings for R13 can be the same or different; q=0-1; r=0-3; wherein meanings for R14 can be the same or different; m=0-2; wherein meanings for R10 can be the same or different; n=1-3; wherein meanings for R7 can be the same or different; z=0-3; wherein meanings for R25 can be the same or different; R16, R17, and R18 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N,N-(di-C1-C4-alkyl)amino, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, and N,N-(di-C1-C4-alkyl)sulfamoyl; wherein R16, R17, and R18 can be optionally independently substituted by one or several of R21 at their carbon atoms; R19, R20, R23, R27, and R28 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N.N-(di-C1-C4-alkyl)amino, C1-C4-alkanoylamino, N-(C1-C4-alkyl)carbamoyl, N,N-(di-C1-C4-alkyl)carbamoyl, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, N,N-(di-C1-C4-alkyl)sulfamoyl, carbocyclyl, heterocyclyl, sulfo, sulfino, amidino, phosphono, -P(O)(ORa)(ORb), -P(O)(OH)(ORa), -P(O)(OH)(Ra), or -P(O)(ORa)(Rb), wherein Ra and Rb are independently selected from C1-C6-alkyl and wherein R19, R20, R23, R27, and R28 can be optionally independently substituted by one or several of R22 at their carbon atoms; R21 and R22 are independently selected from halogen, hydroxy, cyano, carbamoyl, mercapto, sulfamoyl, trifluoromethyl, trifluoromethoxy, methyl, ethyl, methoxy, ethoxy, vinyl, allyl, ethynyl, methoxycarbonyl, formyl, acetyl, formamido, acetylamino, acetoxy, methylamino, dimethylamino, N-methylcarbamoyl, N,N-dimethylcarbamoyl, methylthio, methylsulfinyl, mesyl, N-methylsulfamoyl, N,N-dimethylsulfamoyl; or pharmaceutically acceptable salt thereof, solvate, or salt solvate. Described are also method for preparing compounds of formula I, pharmaceutical compositions based on compounds I, and a method for achieving inhibiting effect relative to interscapular brown adipose tissue (IBAT), and intermediates. (I), (IA), (IB), (IC).

EFFECT: expanded synthetic possibilities in the 1,5-benzothiazepine series.

36 cl, 121 ex

The invention relates to a series peptidergic heterocyclic compounds, intermediates used in their receiving and containing pharmaceutical compositions

FIELD: synthesis of biologically active compounds.

SUBSTANCE: invention provides 1,5-benzothiazepines of general formula I (formulae presented below), in which Rv and Rw are independently selected from hydrogen and C1-C5-alkyl; one of Rx and Ry represents hydrogen or C1-C6-alkyl and the other hydroxy or C1-C6-alkoxy; Rz is selected from halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1 of invention; v is a number from 0 to 5; one of R4 and R5 represents group of general formula IA; R3 and R6 and the second from R4 and R5 are independently selected from hydrogen, halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1; R3 and R6 and the second from R4 and R5 being optionally substituted by one or several R16 groups at their carbon atoms; D represents -O-, -N(Ra)-, -S(O)b- or -CH(Ra)-, wherein Ra is hydrogen or C1-C6-alkyl; and b=0-2; ring A represents aryl or heteroaryl and is optionally substituted by one or several substituents selected from R17; R7 represents hydrogen, C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R18; R8 represents hydrogen or C1-C4-alkyl; R9 represents hydrogen or C1-C4-alkyl; R10 represents hydrogen or C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R19; R11 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORc)(ORd), -P(O)(OH)(ORc), -P(O)(OH)(Rd), or -(O)(ORc)(Rd), wherein Rc and Rd are independently selected from C1-C6-alkyl; or R11 represents group of general formula IB, in which X is -N(Rq)-, N(Rq)C(O)-, -O-, or -S(O)a, wherein a=0-2; and Rq is hydrogen or C1-C4-alkyl; R12 represents hydrogen or C1-C4-alkyl; R13 and R14 are independently selected from hydrogen, C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23 can be optionally independently substituted by one or several substituents selected from R20; R15 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORe)(ORf), -P(O)(OH)(ORe), -P(O)(OH)(Re), or -P(O)(ORe)(Rf), wherein Re and Rf are independently selected from C1-C6-alkyl; or R15 represents group of general formula IC, in which R24 is selected from hydrogen and C1-C4-alkyl; R24 is selected from hydrogen, C1-C4-alkyl carbocyclyl, heterocyclyl, and R27, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R27 can be optionally independently substituted by one or several substituents selected from R28; R26 is selected from carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORg)(ORh), -P(O)(OH)(ORg), -P(O)(OH)(Rg), or -P(O)(ORg)(Rh), wherein Rg and Rg are independently selected from C1-C6-alkyl; p=1-3; wherein meanings for R13 can be the same or different; q=0-1; r=0-3; wherein meanings for R14 can be the same or different; m=0-2; wherein meanings for R10 can be the same or different; n=1-3; wherein meanings for R7 can be the same or different; z=0-3; wherein meanings for R25 can be the same or different; R16, R17, and R18 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N,N-(di-C1-C4-alkyl)amino, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, and N,N-(di-C1-C4-alkyl)sulfamoyl; wherein R16, R17, and R18 can be optionally independently substituted by one or several of R21 at their carbon atoms; R19, R20, R23, R27, and R28 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N.N-(di-C1-C4-alkyl)amino, C1-C4-alkanoylamino, N-(C1-C4-alkyl)carbamoyl, N,N-(di-C1-C4-alkyl)carbamoyl, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, N,N-(di-C1-C4-alkyl)sulfamoyl, carbocyclyl, heterocyclyl, sulfo, sulfino, amidino, phosphono, -P(O)(ORa)(ORb), -P(O)(OH)(ORa), -P(O)(OH)(Ra), or -P(O)(ORa)(Rb), wherein Ra and Rb are independently selected from C1-C6-alkyl and wherein R19, R20, R23, R27, and R28 can be optionally independently substituted by one or several of R22 at their carbon atoms; R21 and R22 are independently selected from halogen, hydroxy, cyano, carbamoyl, mercapto, sulfamoyl, trifluoromethyl, trifluoromethoxy, methyl, ethyl, methoxy, ethoxy, vinyl, allyl, ethynyl, methoxycarbonyl, formyl, acetyl, formamido, acetylamino, acetoxy, methylamino, dimethylamino, N-methylcarbamoyl, N,N-dimethylcarbamoyl, methylthio, methylsulfinyl, mesyl, N-methylsulfamoyl, N,N-dimethylsulfamoyl; or pharmaceutically acceptable salt thereof, solvate, or salt solvate. Described are also method for preparing compounds of formula I, pharmaceutical compositions based on compounds I, and a method for achieving inhibiting effect relative to interscapular brown adipose tissue (IBAT), and intermediates. (I), (IA), (IB), (IC).

EFFECT: expanded synthetic possibilities in the 1,5-benzothiazepine series.

36 cl, 121 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to method for production of acetylamidiniophenylalanylcyclohexylglycilpypidinioalanin amides of formula I , wherein X anions are physiologically acceptable anions, and analogous thereof. Said compounds are effective inhibitors of fibrillation factor Xa and are useful, for example, in prevention of thrombosis. Claimed method includes coupling of 2-[2-acetylamino-3-(4-amidinophenyl)-propionylamino]-2-cyclohexylacetic acid, obtained from 2-[2-acetylamino-3-(4-cyanophenyl)acryloylamino]-2-cyclohexylacetic acid by assimetric hydration and converting of cyano group to amidine, or salt thereof with 3-(2-amino-2-carbamoylethyl)-1-methylpyridinic acid or salt thereof. Also are disclosed starting materials and intermediated used in this method, process for production the same and acetyl-(S)-4-amidiniophenylalanyl-(S)- cyclohexylglycil-(S)-(1-methyl-3-pypidinio)alanin amide in form of ditosylate.

EFFECT: simplified method; increased commercial availability of compounds with applicable anion.

14 cl, 16 ex

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