CN1310674C - Medicinal composition for treating serious acute respiratory syndrome - Google Patents

Abstract

The present invention relates to the production field of a medical composition for treating patients with severe acute respiratory syndrome (SARS) or suspected patients with severe acute respiratory syndrome (SARS). Especially, when the severe acute respiratory syndrome is caused by SARS virus infection and the patients and the suspected patients are in late phase and in the SARS danger at the lethal stage, the present invention provides an application of at least one gene regulatory peptide or an object with similar functions to produce the medical composition for treating patients with severe acute respiratory syndrome (SARS) or suspected patients with severe acute respiratory syndrome (SARS).

Description

Pharmaceutical composition for treating severe acute respiratory syndrome

technical field

The invention relates to the field of production of a composition for treating subjects suffering from or suspected of suffering from severe acute respiratory syndrome (SARS).

Background technique

In late 2002 and early 2003, many countries were alarmed by reports of a rapidly spreading viral pneumonia with very high mortality. The disease quickly gained a name - severe acute respiratory syndrome (SARS) - based on its most prominent feature, which sometimes causes death. The disease causes high fever, myalgias, and chills, followed by dry cough and dyspnea. Another stage is followed by dry cough, dyspnea and tissue hypoxia in nearly 10% of patients. The currently reported mortality rate is 5-7%. Many countries are now actively trying to save lives and their healthcare systems from collapse. A single individual is sometimes enough to shut down a hospital; rapidly infecting healthcare workers and threatening other patients and family members.

SARS has been confirmed to be transmitted by droplets produced by sneezing and coughing, and its symptoms include high fever, chills, dry cough, nasal congestion, myalgia and dyspnea. According to the report of the World Health Organization, it has been reported that 300 people have died of SARS and more than 5,000 people have suffered from this mysterious pneumonia since November. The first known outbreak was in southern China's Guangdong province, which borders Hong Kong, but outbreaks of the disease have been reported from Vietnam to Canada, but most confirmed cases are in China, Hong Kong and Singapore.

It is currently believed that SARS is caused by a virus, also referred to herein as SARS virus, which is considered to be a variant of coronavirus, which has approximately 50% homology with human coronaviruses known so far. The incubation period after SARS infection is usually short, 1-3 days (but also close to 16 days), followed by headache, sore throat, myalgia, chills and nasal congestion. Then usually profuse clear nasal discharge, which gradually thickens and becomes mucopurulent, decreases in quantity. In some cases, the infection resolves within about a week, however, in others, the illness typically persists with a fever of more than 100.4 degrees Fahrenheit. Fever is sometimes accompanied by chills, headache, malaise, and generalized headache. Some patients also experience mild dyspnea at the onset of illness. After about 3-7 days, the patient may develop a dry cough, accompanied or further developed into hypoxemia. At this stage, patches of pneumonia are usually seen on X-ray or CAT scan.

Most patients started with usually flu-like symptoms in the first phase and then apparently recovered, and some patients suddenly deteriorated in the second phase even after apparently getting better. Clinical signs and laboratory abnormalities that indicate serious problems are as follows: increased LDH, increased creatine kinase, neutrophils, platelets are usually inhibited, prolonged prothrombin time and accelerated systemic blood clotting can be treated with d2 Aggregate analysis reflects. Other indicative clinical signs include migratory pneumonia (confirmed by X-ray or CT scan), and tissue hypoxia in many patients. In 10%-20% of the initial SARS cases, the problem was so severe that the patient required mechanical ventilation. The histopathology of this overpowering pneumonia shows few or no viral inclusions and generally paints a pathology nearly identical to that of other ADRS or acute respiratory distress syndrome and BOOP or bronchiolar disappearing organizing pneumonia. Both syndromes are usually caused by damage produced by pro-inflammatory cytokines and other inflammatory mediators.

Scientists participating in the WHO laboratory collaboration network have revealed some diagnostic tests for SARS. These include so-called "PCR" tests, which can detect specific genetic information of a virus and can determine the number of copies of said virus in any particular sample. Crucial for PCR are the primers, which are publicly available via Weblab on the WHO website ( http://www.who.int/csr/sars/primers/en/ ), which opened on 4 April. The primers have been used by many countries in the world. WHO scientists are now increasingly optimistic that the current PCR diagnostic test for SARS can be fine-tuned rapidly. Fine-tuning is required because existing tests have significant limitations in their ability to rule out the presence of other coronaviruses in SARS.

The ability to detect people infected with the SARS virus early on is a key measure. Existing PCR tests are very specific but cannot detect all patients who shed the coronavirus. Early and reliable detection of SARS virus in samples is very helpful for medical workers to determine that patients with fever and other suspected symptoms should be immediately isolated and controlled according to strict measures to control infection. This procedure then significantly reduces the likelihood of the infection spreading to other people.

However, diagnostic technology seems to be developing rapidly, but there is currently no specific treatment for SARS infection, and the second stage of SARS is only symptomatic or sloppy treatment. Complications that can worsen stage 2 conditions include, for example, bacterial or chlamydial infections that require antibiotic treatment. Other complications include acute respiratory distress syndrome (ARDS), multiple organ disorder syndrome (MODS) or multiple organ failure (MOF), systemic inflammatory response syndrome (SIRS), which is a virus-induced immune activation in the host and thus The immediate secondary effects of immunoregulatory dysregulation occurred, and sepsis, septic shock, and death, partly due to primary infection or co-infection. Doctors try to determine what management is best. The CDC recommends treatment whenever a patient is suspected of having "population-acquired pneumonia of unknown etiology." They should be isolated in a negative pressure room and given antibiotics and other care. They were also given some standard antiviral drugs such as ribavirin or tamiflu and globulin preparations from recovering or surviving SARS-infected patients. About 90% of patients recover after 6 days. Half of the remaining 10% require ventilators. However, there is no specific drug for the treatment of SARS.

Contents of the invention

The present invention provides the use of at least one gene regulatory peptide or its functional analogue in the production of a pharmaceutical composition for treating subjects, preferably humans, suffering from or suspected of suffering from severe acute respiratory syndrome (SARS) or related diseases. In particular, the present invention provides the use of at least one gene regulatory peptide or its functional analog in the production of a pharmaceutical composition for the treatment of subjects suffering from or suspected of suffering from severe acute respiratory syndrome (SARS) or related diseases, especially when severe acute respiratory syndrome (SARS) or related diseases The respiratory syndrome is caused by the SARS virus, and particularly when the subject is at risk of having or suspected of having an advanced and possibly fatal stage of SARS.

The present invention provides an unexpected treatment mode that has never been disclosed, including cytokine gene regulation targeting the immune activity of SARS patients, and has made significant progress in the treatment of patients suffering from or suspected of suffering from SARS-related diseases.

The present invention relates to the body's innate regulatory pathways for important immune functions. In a preferred embodiment, the present invention provides the use of at least one gene regulatory peptide or its functional analog in the production of a pharmaceutical composition for the treatment of subjects suffering from or suspected of suffering from severe acute respiratory syndrome (SARS) or related diseases purposes, wherein the syndrome is caused by a viral infection, such as by influenza virus or other pathogens causing severe respiratory disease with signs of immunological pathogenesis of ARDS, with or without DIC, SIRS (systemic inflammatory response syndrome) or mods signs.

The present invention particularly provides the use of at least one gene-regulating peptide or its functional analogues to produce a medicament for treating subjects suffering from or suspected of suffering from SARS caused by SARS virus infection. Strikingly however, in the second phase of SARS, the SARS virus is usually not found to be active in the pulmonary system, and the main factor in the deterioration of the patient's condition is a significant inflammatory response, for which the pharmaceutical composition of the present invention Treatment is most preferred. We provide insight that most patients can control this virus by immunization and that a minority may develop immune dysfunction complications of ARDS with some signs of DIC and MODS; in said minority of patients, there is no modulation of the host immune system pro-inflammatory Impaired capacity, which is a key pathogenesis of ARDS, DIC, and MODS. The abnormal immune response is regulated by the drug treatment of the present invention, and the pro-inflammatory damage is reduced or eliminated, and then the disease of the host patient presents a benign process, and the patient recovers and controls the virus infection by itself.

The use of the present invention is particularly effective in those situations in which the patient is at risk of having or suspected of having such a secondary or late and possibly lethal stage of SARS characterized by one or more clinical signs or laboratory findings indicative of the presence of such an immune response, for example, characterized by clinical signs or laboratory findings selected from the group consisting of elevated lactate dehydrogenase levels, elevated hormone kinase levels, neutrophil Cytosis, prolonged prothrombin time, platelet inhibition, increased d-dimer, migratory pneumonia, and progressive tissue hypoxia, progression to ARDS, with or without DIC, SIRS, or MODS.

In the present application, the use of small gene regulatory peptides or their functional analogues, which are naturally present in pregnant women, and derived from placental gonadotropins such as human chorionic gonadotropin (hCG) produced during pregnancy is described. ) proteolysis. These peptides, which in their active state are generally only about 4-6 amino acids long, have been shown to have very remarkable immunological activity, acting by modulating the expression of genes encoding inflammatory mediators such as cytokines. Surprisingly, it was found that the breakdown of hCG provides a series of peptides that help maintain immune homeostasis in pregnant women. These peptides or their functional analogues are natural self-substances that balance the immune system to ensure that the mother maintains a stable immune status and that her fetus is not prematurely rejected during pregnancy, but spends it safely in the mother's body until birth, these substances It is particularly effective in the treatment of patients suffering especially from the second stage of SARS, which is characterized by severe complications as described above. The peptides or functional analogues thereof can be obtained, for example, from urine or other organic product sources such as serum, whey, placental extracts, plants, cells or tissues comprising gonadotropins or functionally equivalent compounds thereof. Here, "obtainable" means that the peptide or its functional analogue can be directly or indirectly obtained from the source. The peptide or its functional analogue can also be obtained by chemical synthesis, or obtained from animal or plant materials in nature, for example. Peptides or functional analogues thereof can be found, for example, in urine fractions obtained from pregnant women or in commercial preparations of hCG or other gonadotropins; at least in those breakdown products which contain sufficient amounts of hCG or other gonadotropins and have In preparations for gene regulation and especially immunomodulatory activity.

In one embodiment, the present invention provides the use of at least one gene-regulating peptide or its functional analogue in the manufacture of a medicament for the treatment of severe acute respiratory syndrome (SARS) or related diseases , wherein said peptide or analogue thereof is obtainable from the urine of a pregnant woman, particularly wherein said woman is within the first trimester of her pregnancy. This drug is conveniently prepared from other discarded urine residues used for the isolation and chemical preparation of human gonadotropins.

In another embodiment, the present invention provides the use of at least one gene regulatory peptide or its functional analog in the manufacture of a medicament for the treatment of patients with or suspected of severe acute respiratory syndrome (SARS) or related Diseases wherein said peptide or analogues thereof are especially proteolytically producible from a gonadotropin preparation, especially wherein said gonadotropin or a fraction thereof comprises natural or recombinant human chorionic gonadotropin (hCG) or its fraction.

In another embodiment, the present invention provides the use of at least one gene regulatory peptide or its functional analog in the manufacture of a medicament for the treatment of patients with or suspected of severe acute respiratory syndrome (SARS) or related Disease, wherein said peptide or its analog is obtained by chemical synthesis, especially wherein said peptide or its analog is selected from LQG, AQG, LQGV, AQGV, LQGA, VLPALPQVVC, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ, LQGVLPALPQVVC, VCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, RPRCRPINATLAVEK, EGCPVCITVNTTICAGYCPT, SKAPPPSLPSPSRLPGPS, SIRLPGCPRGVNPVVS, LPGCLPGVNVN, or derivatives thereof.

The most preferred use of the invention is wherein the composition comprises a mixture of peptides LQGV, AQGV and VLPALP. Additionally, the invention provides any use wherein said composition or a representative sample thereof is tested for immunomodulatory activity in an in vivo bioassay, such as in the animal experiments provided in the detailed description of the invention, or wherein The composition is tested for its immunomodulatory activity in an in vitro bioassay or for the presence or absence of an immunomodulatory compound for the treatment of SARS according to the guidelines provided, such as in the NF-κB translocation assay or gene in the array. In that manner, guidance for the preparation of an appropriate pharmaceutical composition for the treatment of SARS may be found at one or more stages during or after the manufacturing process, testing all of the composition or one or more of its compounds or representative samples. Immunomodulatory or gene modulatory functional activity is required, for example in in vivo bioassays with animal experiments, in in vitro bioassays to determine the functional activity of modulating immune processes under the control of cytokine genes, or by determining the composition of the test or The physiological presence or absence of the desired gene-regulating peptide or its functional analog in the compound. Of course, when purifying from organic sources such as urine, from preparations of (recombinant) gonadotropins that can be subjected to proteolysis with e.g. leukocyte elastase activity, or from crudely synthesized compositions or compounds such as batch or chemically synthesized peptides , the purification process can be easily manipulated to select the most desired immunomodulatory active fraction for further purification or inclusion in a pharmaceutical composition for the treatment of SARS.

Description of drawings

Figure 1 : This figure shows that in LPS-induced shock the fractions with anti-inflammatory activity in the early stages of the disease are III and V, whereas in the late stages of the disease the fractions with anti-inflammatory activity are IV and VI.

Figure 2: Fractions 1, 2 and pooled fractions 3, 4 and 5 were tested in an in vivo bioassay, demonstrating that only pooled fractions 3, 4 and 5 had anti-inflammatory activity in LPS-induced shock.

Figure 3: Separation of Pregnyl (206nm) on G25 column.

Figure 4: Pregnyl elution profile on GPC 300A column.

Figure 5: Elution profile of fraction 3 from a GPC 300A column separated on a GPC 60A column.

Figure 6: GPC 60A chromatogram of fraction 3 of the GPC 300A column obtained from the urine of a 3-month pregnant woman with anti-inflammatory activity. The figure shows that the ratio between fractions 3.2 and 3.3 is about 1:2.2.

Figure 7: GPC 60A chromatogram of fraction 3 obtained from a GPC 300A column of Pregnyl without anti-inflammatory activity. The figure shows that the ratio between fractions 3.2 and 3.3 is about 1:3.4.

Figure 8: GPC 60A chromatogram of fraction 3 obtained from a GPC 300A column of Pregnyl with anti-inflammatory activity. The figure shows that the ratio between fractions 3.2 and 3.3 is approximately 1:1.

Figure 9: GPC 60A chromatogram of Pregnyl.

Figure 10: Chromatogram of Bio-gel P2 (urine in the first trimester) (206nm).

Figure 11: Chromatogram of Bio-gel P2(pregnyl)(206).

Figure 12: This figure shows the results of BEAS 2B cell line: Dexamethasone (DX) can inhibit TNF-α-induced IL-6 and RANTES production in BEAS 2B cell line. The active fraction can also inhibit TNF-α-induced production of inflammatory cytokines. In addition, dexamethasone restored the TNF-α-induced downregulation of the anti-inflammatory TGF-β cytokine, while the active fraction not only restored TNF-β production but further elevated this anti-inflammatory cytokine. In addition, both dexamethasone and the active fraction were able to inhibit IFN-γ-induced RANTES production. Flow cytometry analysis of the BEAS 2B cell line showed that both dexamethasone and the active fraction could negatively regulate the expression of HLA-DR and ICAM-1 induced by TNF-α.

Detailed ways

The minimal breakdown products of proteins are generally considered to have no specific biological function on their own (other than as immune system antigens), but it has now been shown that the body routinely utilizes the normal proteolytic process of proteins to produce important gene-regulatory compounds that control Short peptides expressed by the body's own genes. Apparently, the organism uses precisely a genetic control system controlled by small breakdown products of proteins encoded by its own genes.

It has long been known that during pregnancy, the mother develops a state of transient immunoregulation that results in suppression of the maternal rejection response against the fetus. Paradoxically, maternal infection resistance is often increased during pregnancy and has been found to be better protected against clinical symptoms of various autoimmune diseases such as rheumatism and multiple sclerosis. Fetal protection should therefore be interpreted not only as a result of immunosuppression, but also as a result of the immune balance between maternal and fetal protection.

Some small breakdown products of hCG (i.e., small peptides that can be readily synthesized chemically or recombinantly and used as pharmaceutical compositions if desired) are known to exert pro- or anti-inflammatory cytokine cascades controlling important transcription factor families The main regulatory activity of the NF-κB family plays an important role in regulating the expression of genes that shape the body's immune response.

Most of the hCG produced during pregnancy is produced by cells of the placenta, the site of intimate contact between cells and tissues of the mother and fetus and where immunomodulation is most needed in an effort to avoid rejection. Locally produced gene regulatory peptides broken down from placental hCG immediately balance the pro- or anti-inflammatory cytokine cascades found in the no-man's land between the mother and fetus. The trophoblast produced by typical placental cells, through which the peptide penetrates to the extracellular space; enters cells of the immune system and exerts its immunomodulatory activity by regulating NF-κB- and AP-1-mediated cytokine gene expression, Thereby suppressing the immune response in the placenta.

The present invention provides for the beneficial effects on the development and severity of autoimmune diseases in pregnant women resulting from the excess entry of hCG-derived peptides into the body in general; however, these effects cannot be overestimated because it is readily understood that the further , the less immunomodulatory activity directed at preventing fetal rejection, simply because the peptides produced by the placenta in general are diluted throughout the body. However, the immunomodulatory and gene modulatory activity of the peptide should by no means occur only during pregnancy and in the placenta; men and women produce hCG in the same way, for example in their pituitary gland, and nature does largely utilize the peptide's Gene regulatory activity.

Therefore, the present invention provides a new therapeutic approach using gene regulatory peptides and their derivatives with pharmacological potential. Indeed, after treatment of immune cells with the gene-regulating peptides in vitro and experimental animals in vivo, individual and consistent immune responses to the body driven by NF-κB or AP-1 were found in the silicosis gene array by expression profiling studies Signs of specific positive or negative regulation of pro- or anti-inflammatory cytokine cascades. NF-κB (and AP-1) are also considered to be the main effectors of the disease, and the use of hCG-derived gene regulatory peptides has important potential for the treatment of SARS, thereby identifying the exact factors that help balance the mother's immune system to safely maintain her pregnancy status. The drug potential of the substance.

The present invention provides a treatment for a subject suffering from or suspected of suffering from a disease caused by SARS virus infection, by administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a pharmaceutically effective amount of a gene regulating peptide or its Functional analogs and a pharmaceutically appropriate diluent. Particularly useful pharmaceutical diluents are sterile water or isotonic saline solutions such as 0.9% saline or phosphate buffered saline (PBS). In a preferred embodiment, the present invention provides the treatment of a subject suffering from or suspected of suffering from a disease caused by SARS virus infection, by administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a pharmaceutically effective amount of Two or more gene regulating peptides or their functional analogues and a pharmaceutically appropriate diluent.

The present invention provides the use of the regulatory peptide pharmaceutical composition for a subject suffering from or suspected of suffering from SARS virus infection by systemically regulating the gene expression in the body cells of the subject. Such gene-regulating peptides or functional analogs thereof are for example selected from the group consisting of oligopeptides LQG, AQG, LQGV, AQGV, LQGA, VLPALPQVVC, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA , GVLPALP, GVLPALPQ, LQGVLPALPQVVC, VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, RPRCRPINATLAVEK, EGCPVCITVNTTICAGYCPT, SKAPPPSLPSPSRLPGPS, SIRLPGCPRGVNPVVS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, QVVC and their functional analogs or derivatives. Functional analogues can be found, for example, in urine fractions obtained from pregnant women or in commercial preparations of hCG or other gonadotropins; at least in those preparations which contain sufficient hCG or other gonadotropic breakdown products and possess modulatory activity.

The preferred size of the modulatory peptides included in the pharmaceutical compositions of the invention is up to 15 amino acids, although smaller molecules have also shown particular potency. Surprisingly, the present invention provides the insight that gene expression in SARS-infected patients can be systematically regulated or controlled by small peptides and their functional analogues. Preferred peptides are breakdown products of larger polypeptides such as chorionic gonadotropin (CG) and growth hormones or growth factors such as fibroblast growth factor, EGF, VEGF, phosphate cyclization of the 3' end of RNA Enzymes and CAP18, or their synthesis. In principle, such regulatory peptide sequences can be derived from any polypeptide molecular protein produced by prokaryotic and/or eukaryotic cells, and the present invention provides the insight that breakdown products of polypeptides, preferably oligopeptides, of the sizes provided can be used to produce Systemic and beneficial effects, the breakdown products are naturally involved in the regulation of gene expression as gene regulatory peptides. In particular, the present invention provides a (synthetic) gene regulatory peptide obtainable or derived from β-human chorionic gonadotropin (β-hCG), preferably from or derived from nicked β-hCG . It was previously believed that the breakdown products of β-hCG were involved in immune regulation by regulating gene expression (WO99/59671, WO01/72831, PCT/NL02/00639), or in the treatment of wasting syndrome (WO97/49721), but in these publications The relationship between systematic regulation of cytokine gene expression in SARS-infected subjects is not reflected in the study. Of course, such gene-regulating peptides or functional equivalents or derivatives thereof may be obtained or derived from other proteins undergoing cleavage or proteolysis and access to the gene regulatory cascade.优选地，所述肽分子得自具有至少10个氨基酸的肽，如具有以下氨基酸序列的肽：MTRVLQGVLPALPQVVC、SIRLPGCPRGVNPVVS、VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL、RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT、CALCRRSTTDCGGPKDHPLTC、SKAPPPSLPSPSRLPGPS、CRRSTTDCGGPKDHPLTC、TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ。

Without being bound by theory, the present invention reveals an unexpected pattern of gene regulation for the purpose of treating subjects with SARS-related diseases. Breakdown of polypeptides such as endogenous CG, EGF, etc., but also pathogenic polypeptides such as viral, bacterial or protozoan polypeptides, into unique oligopeptides, eg by intracellular proteolysis. Unique proteolytic enzymes are widely available in cells, for example in the lysosomal or proteasome systems of eukaryotic cells. Some of the resulting breakdown products are oligopeptides of 3-15, preferably 4-9, most preferably 4-6 amino acids in length, which surprisingly do not have any function or effect on the cell, suggesting here that they may be mediated by endogenous Participation in the regulation of gene expression by feedback mechanisms as signaling molecules in the case of polypeptide breakdown, as evidenced by, for example, the following peptides regulating the activity or translocation of gene transcription factors such as NF-κB, said peptides being LQGV, VLPALPQVVC, LQGVLPALPQ, LQG, GVLPALPQ, VLPALP, VLPALPQ, GVLPALP, VVC, MTRV, and MTR. The present invention provides the synthesis of these above peptides, and the functional analogues or derivatives of these breakdown products, to regulate gene expression in cells and to be used in methods for treating SARS-related diseases. Oligopeptides such as LQG, AQG, LQGV, AQGV, LQGA, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ, LQGVLPALPQVVC, SIRLPGCPRVNPVVS, SKNAPPPRVSPLPGPSLP, , MTRV, MTR, VVC, or longer sequence functional analogs or derivatives thereof (including breakdown products), are particularly effective.

In a preferred embodiment, the present invention provides the treatment of an object suffering from or suspected of suffering from an inflammatory disease caused by SARS virus infection by administering a pharmaceutical composition therefor, the pharmaceutical composition comprising a drug A pharmacologically effective amount of a gene-regulating peptide capable of regulating the expression of genes encoding inflammatory mediators such as cytokines. Potent gene-regulating peptides for inclusion in drugs for the treatment of SARS-associated diseases, especially the lethal inflammatory disease common in the second stage of SARS infection, are those peptides or functional analogues thereof, which are naturally present in pregnant women and derived from placental gonadotropins The proteolytic products of hormones, such as human chorionic gonadotropin (hCG) produced during pregnancy, the placental gonadotropin, however, those skilled in the art can readily synthesize the synthesis of functionally identical or similar activity of these peptides (for example Recombinantly produced) variants and modifications, and tested for their activity using, for example, animal experiments, such as experiments with mice as described in the present invention. In another embodiment, the present invention provides a pharmaceutical composition for treating SARS-related diseases, said pharmaceutical composition comprising a gene regulatory peptide or its functional analogue, and providing a gene regulatory peptide or its functional analogue The purposes of the compound in the production of the pharmaceutical composition for the treatment of SARS-associated diseases. The term "pharmaceutical composition" refers to both an active functional compound alone and a composition comprising said compound and a pharmaceutically appropriate carrier, diluent or excipient. Suitable diluents for the pharmaceutical composition are, for example, physiological saline solution or phosphate buffered saline solution. In one embodiment of the present invention, the pharmaceutical composition is administered systemically to an animal or a human in an effective concentration, for example by intravenous, intramuscular or intraperitoneal administration.

Example

review

Patients who are in the onset or period of late stage immunodeficiency syndrome of SARS are preferably treated with the pharmaceutical composition for treating SARS provided by the present invention. Such patients usually have evidence of progressive tissue hypoxia, as evidenced by low partial pressure and oxygen saturation in their arterial blood and by oximetry. These patients typically present with the previously described confluence of signs and symptoms characteristic of a SARS-like illness with laboratory findings that include the following: lymphopenia, thrombocytopenia, positive d-dimer, partial prothrombin Prolonged time, LDH increased, and CK increased. These patients are in great need of supplemental oxygen or even artificial ventilation support. These patients can be treated with antiviral drugs, such as ribavirin 400 mg orally three times a day, or intravenously administered antibiotics, such as cefotaxime and clarithromycin (or levofloxacin), according to established regimens to combat the causes of mass-acquired pneumonia common pathogens, corticosteroids (prednisolone, 1mg/kg body weight/day), or other anti-inflammatory drugs, or drugs (such as vaccines) specifically against SARS virus.

Appropriate patients for treatment usually have progressive pneumonia in the form of ARDS, as evidenced by X-ray or CT findings. They are given a pill every 8 hours or the full therapeutic dose is continued until treatment is deemed no longer necessary. Results were measured by survival, hypoxic resolution time, and resolution time and progression of pneumonia and other immune dysfunction, including normalization of clotting time, CK, LDH, and TH1 and TH2 cytokine/chemokine cascades.

Example 1

In one embodiment, the present invention provides a pharmaceutical composition, which has been set forth in the detailed description of the invention, wherein said active ingredient is present as an eluted fraction having a molecular weight of significantly less than 15 kD by gel permeation chromatography Determined, for example, wherein the active ingredient is present as an eluted fraction with a molecular weight apparently less than 2 kD, by gel permeation chromatography. Although the pharmaceutical composition of the present invention is readily obtained from urine, for example where the preparation of mammalian chorionic gonadotropin is obtained from urine, or from a fraction of urine in which the active hormone is only insignificantly present, as in Urine fractions discarded during commercial gonadotropin production processes, but other sources such as serum, cells or tissues containing gonadotropins or their breakdown products can also be used. The present invention also provides a pharmaceutical composition obtained from said raw material, which can, for example, regulate the activity of Th1 and/or Th2 cells, and/or can regulate the differentiation of dendritic cells.

Preferably, the pharmaceutical composition provided by the invention is obtained from a pregnant mammal, preferably a human, e.g. by preparing a pharmaceutical preparation containing (placental) gonadotropins such as those found in pregnant mare serum hormone (PMSG), or pregnant mouse uterus extract (PMUE), which is extracted from the uterus of pregnant mice, or human chorionic gonadotropin (hCG or HCG), which is found in the placenta, blood, or urine of pregnant mice. The pharmaceutical compositions provided herein may be with or without gonadotropins, such as those found in the urine of pregnant women at 3 months of gestation or in commercial hCG preparations.

The present invention provides a method of treating an immune-mediated disease comprising treating the animal with at least one pharmaceutical composition obtained from a pregnant mammal. Said treatment may be direct, for example comprising providing said individual with a pharmaceutical composition, such as hCG or PMSG preparation, which comprises an immunomodulator provided by the present invention. Said pharmaceutical composition may also be provided with a fraction obtained from a pregnant animal, for example by sampling urine or serum or placenta (maternal or fetal origin) or other tissue or cells, and fractionating by methods known in the art ( For example, gel permeation chromatography) to prepare the immunoregulatory factor containing the active ingredient from the urine or serum or tissue or cells, and test the active ingredient.

Example 1

Preparation of pharmaceutical composition for treating SARS from urine fraction of pregnant women

Urine Purification (Method 1): Maternal urine (2 L) collected from healthy volunteers at 3 months of pregnancy was bottled and chilled until transported to the laboratory within two days. During transport, 1 g/L sodium azide was added and the pH was adjusted to 7.2-7.4 with sodium hydroxide and precipitated at room temperature (RT) for 1 hour. Approximately 75% of the supernatant was decanted, and the near-precipitated residue was centrifuged (10 minutes, 25000 rpm, 4°C) to remove the precipitate and added to the remaining supernatant. Concentrate the supernatant by one of the following two methods. In the first method, the supernatant was filtered in a 0.45 μm Minitan (Millipore) transverse filter device. Subsequently, the filtrate (2 L) was concentrated in an Amicon ultrafiltration device equipped with a YM Dipole membrane of 10 kD pore size. The final volume (250ml) was dialyzed twice against 10L of Milli Q water. The samples were then ultrafiltered in an Amicon through a 10 kD pore size to a final volume of 3 ml. In the second method, the supernatant (2 L) was concentrated in a Pellicon ultrafiltration device equipped with a 5 kD pore size Pellicon XL filter membrane (Millipore, cat. No. PXC010C50). The final volume is 150ml.

Using one of the following methods, the raw material is further used in gel filtration to obtain a fraction for treating SARS.

1. Desalting column G25: Sephadex G-25 Fine (Pharmacia) equipped with HPLC was used. The size of the column is 5×60 cm, the volume of sample added is 6-10 ml, and the flow rate is 3 ml/min. The running buffer used was 50 mM ammonium bicarbonate plus 5% methanol. Sephadex G-25 is a desalting column for the separation of small peptides and small proteins. The fractionation range of the column is 100Dalton-5000Dalton. Fractions (typically 50ml) were collected and lyophilized (see Figure 1 for separation diagram). The freeze-dried fraction with molecular weight lower than 1000Dalton is dissolved in 2-4ml PBS (pH 7.4), and test in vivo bioassay to determine the adaptability of pharmaceutical composition for SARS treatment. This bioassay indicated that the fractions with anti-inflammatory activity in the early stages of LPS-induced shock disease were III and V, whereas those with anti-inflammatory activity in the later stages of the disease were IV and VI (Figure 1).

2. Gel filtration column Superdex 75: use a Superdex 75 (Pharmacia) column equipped with a 10×300mm column bed FPLC. The amount of sample added was 50 μl, and the flow rate was 1 ml/min for a total of 52 minutes. The running buffer used was 50 mM ammonium bicarbonate plus 5% methanol. Superdex 75 columns are excellent equipment for the separation of lower molecular weight recombinant proteins and peptides in monomeric and dimer form. The fraction range for this column is 3,000 Dalton-70,000 Dalton for globular proteins. Fractions (typically 1 ml) were collected and pooled according to the separation scheme (see Figure 2) eluting at molecular weights below 15,000 Dalton (fractions 3, 4 and 5; Figure 2). Fractions from a collection of at least 20 different runs were lyophilized. The lyophilized fraction was dissolved in 1-2 ml of 10 mM phosphate buffered saline pH 7.4 to determine the suitability of the pharmaceutical composition for use in the treatment of SARS in an in vivo bioanalytical test, or in 2-4 ml of 50 mM ammonium bicarbonate and further fractionated as described in point 1.

Fractions 1, 2 and pooled fractions 3, 4 and 5 were tested in an in vivo bioassay, indicating that pooled fractions 3, 4 and 5 had anti-inflammatory activity in the early stages of LPS-induced shock disease.

3. Gel Permeation Column (GPC) 300A: We use a Shimadzu HPLC system equipped with an Alltech large spherical size exclusion (GPC) column 300 Ȧ (250×4.6mm or 300×7.5mm), using 50mM ammonium bicarbonate running buffer liquid. The separation range of this column is 1,200,000-7,500 Dalton. The sample loading volume is 10-50 μl concentrated urine of pregnant women in the third month of pregnancy. The flow rate was 0.5ml/min and the run was 45 minutes. Use objective molecular weight standards to calibrate the column elution position. 50 μl fractions were collected and pooled according to the elution profile (see Figure 3). A collection of fractions eluted at least 20 times at a molecular weight lower than 7,000 Dalton was lyophilized and dissolved in 10 mM phosphate buffered saline pH 7.4, and the pharmaceutical composition was determined to be useful in the treatment of SARS in an in vivo bioanalytical test or dissolved in 1-2 ml of 50 mM ammonium bicarbonate and further fractionated as described in points 1 and 4.

All fractions were tested in an in vivo bioassay and showed that fraction 3, which eluted at a molecular weight below 7,000 Dalton, had anti-inflammatory activity.

4. Gel Permeation Column (GPC) 60A: In another experiment, lyophilized pooled fractions (see point 3) with a molecular weight below 7,000 Dalton obtained from a GPC300A column were further separated on a GPC60A column. We used a Shimadzu HPLC system equipped with an Alltech large spherical size exclusion (GPC) column 60A (250 x 4.6 mm or 300 x 7.5 mm), run with 50 mM ammonium bicarbonate. The separation range of this column is 28,000-250 Dalton. Sample injection volumes were 10-50 [mu]l concentrated pooled fractions with a molecular weight below 7,000 Dalton from a GPC300A column. The flow rate was 0.3ml/min and the run was 25 minutes. The column elution position was also calibrated using objective molecular weight standards. The concentrated collection fraction 3 obtained from the GPC300A column was separated on the GPC60A column, and 3 regions were selected for fractionation, called fraction 3.1, which was eluted with a molecular weight > 2000Da, and fraction 3.2, which was eluted with a molecular weight between 2000-300Da. , and fraction 3.3, which eluted at a molecular weight below 300 Da (Figure 3.). All these fractions obtained from at least 20 different runs were lyophilized and dissolved in 1-2 ml PBS (pH 7.4) and the suitability of the pharmaceutical composition for use in the treatment of SARS was determined in an in vivo bioanalytical test .

All fractions were tested in an in vivo bioassay, showing anti-inflammatory activity in fraction 3.2 eluted at molecular weights between 2000-300 Da and anti-inflammatory activity in fraction 3.3 eluted at molecular weights below 300 Da. Concentrated 10-50ml 3-month pregnant women's urine was directly separated on a GPC60A column, 3 regions were selected for fractionation, called 1, 2 and 3 (see Figure 4), and collected from at least 20 runs ( For a separation diagram, see Figure 4). Pooled fractions 1, 2 and 3 were lyophilized and dissolved in 1-2 ml PBS. All fractions were tested in an in vivo bioassay, showing that fraction 2 has anti-inflammatory activity and fraction 3 has anti-inflammatory activity in late stages of LPS-induced shock. Both fractions elute at molecular weights below 1,000 Dalton.

5.Bio-Gel P-2: The lyophilized collection fraction eluted from the superdex G25 column at a molecular weight lower than 5,000 Dalton and the fraction 3 from the GPC60A column were placed on the Bio-Gel P-2 column (96× 1.5 cm) was further isolated by gel filtration chromatography. The material (13-17mg) was suspended in double distilled water (8-12ml). The material did not completely dissolve. The precipitate (8-11 mg) was separated from the supernatant by centrifugation (Sigma201, 10 min, 3000 rpm). The supernatant was fractionated by gel filtration on a Bio-Gel P-2 column. The column was eluted with water at a flow rate of 15 ml/min. Elution was monitored with LKB 2142 differential refractometer and LKB 2238 Uvicord SII (206nm). Fractions were collected (20 minutes) by means of a Pharmacia Frac 100 Fraction Collector (see Figure 10 for separation diagram) and certain fractions were pooled and lyophilized. Each fraction was dissolved in 1 ml PBS and tested for anti-inflammatory activity. These experiments showed that fractions with a molecular weight below 1,000 Dalton had anti-inflammatory activity (see Figure 10; arrows indicate the elution position of the anti-inflammatory activity).

Example 2

Preparation of pharmaceutical compositions for treating SARS from commercial gonadotropin preparations, recombinant gonadotropin preparations or their smaller fractions

In all isolation methods below, 30,000 IU of a commercial hCG preparation (Pregnyl, Profasi, etc.) was dissolved in 2 ml of 50 mM ammonium bicarbonate.

1. Desalting column G25: Sephadex G-25Fine (Pharmacia) equipped with HPLC was used. The size of the column is 5×60 cm, the volume of sample loaded is 2-4 ml (15,000 IU/ml), and the flow rate is 3 ml/min. The running buffer used was 50 mM ammonium bicarbonate plus 5% methanol. Sephadex G-25 is a desalting column for the separation of small peptides and small proteins. The fractionation range of the column is 100Dalton-5000Dalton. Fractions (typically 50ml) were collected and lyophilized (see Figure 3 for separation diagram). The freeze-dried fraction with molecular weight lower than 1000Dalton is dissolved in 2-4ml PBS (pH 7.4), and test in vivo bioassay to determine the adaptability of pharmaceutical composition for SARS treatment. This bioassay indicated that the fractions with anti-inflammatory activity in the early stages of LPS-induced shock disease were III and V, whereas those with anti-inflammatory activity in the later stages of the disease were IV and VI (Figure 3).

2. Gel filtration column Superdex 75: use a Superdex 75 (Pharmacia) column equipped with a 10×300mm column bed FPLC. The amount of sample added was 50 μl (15,000 IU/ml), and the flow rate was 1 ml/min for a total of 52 minutes. The running buffer used was 50 mM ammonium bicarbonate plus 5% methanol. Superdex 75 columns are excellent equipment for the separation of lower molecular weight recombinant proteins and peptides in monomeric and dimer form. The fraction range for this column is 3,000 Dalton-70,000 Dalton for globular proteins. Fractions (typically 1 ml) were collected and pooled according to the separation scheme (see Figure 2) eluting at molecular weights below 15,000 Dalton (fractions 3, 4 and 5; Figure 2). Pooled fractions from at least 20 different runs were lyophilized. The lyophilized fraction was dissolved in 1-2 ml of 10 mM phosphate buffered saline pH 7.4 to determine the suitability of the pharmaceutical composition for use in the treatment of SARS in an in vivo bioanalytical test, or in 2-4 ml of 50 mM carbonic acid ammonium hydrogen and further fractionated as described in point 1.

Fractions 1, 2 and pooled fractions 3, 4 and 5 were tested in an in vivo bioassay, indicating that pooled fractions 3, 4 and 5 had anti-inflammatory activity in the early stages of LPS-induced shock disease.

3. Gel Permeation Column (GPC) 300A: We use a Shimadzu HPLC system equipped with an Alltech large spherical size exclusion (GPC) column 300 Ȧ (250×4.6mm or 300×7.5mm), using 50mM ammonium bicarbonate running buffer . The separation range of this column is 1,200,000-7,500 Dalton. Sample injection volumes were 10-50 μl of a commercial hCG preparation (30,000 IU/ml). The flow rate was 0.5ml/min and the run was 45 minutes. Use objective molecular weight standards to calibrate the column elution position. The markers used were: aprotinin (6,500 Da), cytochrome C (12,400), carbonic anhydrase (29,000), albumin (66,000) and blue dextran (2,000,000). 50 μl fractions were collected and pooled according to the elution profile (see Figure 4). Will obtain at molecular weight lower than 7,000Dalton and fraction collection elution at least 20 times is freeze-dried, is dissolved in the 10mM phosphate buffered saline pH7.4, and confirms in vivo bioanalytical test that the pharmaceutical composition is used for the treatment of SARS Adapt, or dissolve in 1-2 ml of 50 mM ammonium bicarbonate and further fractionate as described in points 1 and 4.

All fractions were tested in an in vivo bioassay and showed that fraction 3, which eluted at a molecular weight below 7,000 Dalton, had anti-inflammatory activity.

4. Gel Permeation Column (GPC) 60A: In another experiment, lyophilized pooled fractions (see point 3) with a molecular weight below 7,000 Dalton obtained from a GPC300A column were further separated on a GPC60A column. We used a Shimadzu HPLC system equipped with an Alltech large spherical size exclusion (GPC) column 60A (250 x 4.6 mm or 300 x 7.5 mm), run with 50 mM ammonium bicarbonate. The separation range of this column is 28,000-250 Dalton. Sample injection volumes were 10-50 [mu]l concentrated pooled fractions with a molecular weight below 7,000 Dalton from a GPC300A column. The flow rate was 0.3ml/min and the run was 25 minutes. The column elution position was also calibrated using objective molecular weight standards. The concentrated collection fraction 3 obtained from the GPC300A column was separated on the GPC60A column, and 3 regions were selected for fractionation, called fraction 3.1, which was eluted with a molecular weight > 2000Da, and fraction 3.2, which was eluted with a molecular weight between 2000-300Da. , and fraction 3.3, which eluted at a molecular weight below 300 Da (Figure 5). All these fractions obtained from at least 20 different runs were lyophilized and dissolved in 1-2 ml PBS (pH 7.4) and the suitability of the pharmaceutical composition for use in the treatment of SARS was determined in an in vivo bioanalytical test .

Ratio of fractions 3.2 and 3.3: Next, we further purified fraction 3 from the GPC300A column on a GPC60A column from inactive and active hCG preparations in the early stages of the disease. The same analysis was performed on maternal urine at 3 months of gestation and the ratio of fractions 3.2 and 3.3 was determined. We found a ratio of about 1:2.2 (fraction 3.2:fraction 3.3) in the urine of 3-month-old pregnant women with anti-inflammatory activity in the early stage of the disease (Fig. The ratio was 1:3.4 in the hCG preparation (Pregnyl) (Fig. 7) and 1:1 in the hCG preparation with anti-inflammatory activity in the early stages of the disease (Fig. 8).

Separate 10-50 μl of a commercial hCG preparation (15,000 IU/ml) directly on a GPC 60A column, select 3 regions for fractionation, termed 1, 2 and 3 (see Figure 9), and pool from at least 20 runs Fractions (see Figure 9 for separation diagram). Pooled fractions 1, 2 and 3 were lyophilized and dissolved in 1-2 ml PBS. All fractions were tested in an in vivo bioassay, showing that fraction 2 has anti-inflammatory activity in the early stages of the disease and fraction 3 has anti-inflammatory activity in the late stages of the disease. Both fractions elute at molecular weights below 1,000 Dalton.

5.Bio-Gel P-2: The lyophilized collection fraction eluted from the superdex G25 column at a molecular weight lower than 5,000 Dalton and the fraction 3 from the GPC60A column were placed on the Bio-Gel P-2 column (96× 1.5 cm) was further isolated by gel filtration chromatography. The material (13-17mg) was suspended in double distilled water (8-12ml). The material did not completely dissolve. The precipitate (8-11 mg) was separated from the supernatant by centrifugation (Sigma201, 10 min, 3000 rpm). The supernatant was fractionated by gel filtration on a Bio-Gel P-2 column. The column was eluted with water at a flow rate of 15 ml/min. Elution was monitored with LKB 2142 differential refractometer and LKB 2238Uvicord SII (206nm). Fractions were collected (20 minutes) by means of a Pharmacia Frac 100 Fraction Collector (see Figure 11 for separation diagram) and certain fractions were pooled and lyophilized. Each fraction was dissolved in 1 ml PBS and tested for anti-inflammatory activity. These experiments showed that fractions with a molecular weight below 1,000 Dalton had anti-inflammatory activity (see Figure 11; arrows indicate the elution position of the anti-inflammatory activity).

Example 3

Preparation of pharmaceutical composition for treating SARS from commercially synthesized peptides

In another embodiment, wherein the gene regulating peptide provided by the present invention is used as a regulator of NF-κB for treating diseases, the peptide of the present invention or its functional derivatives or analogs are used to produce a pharmaceutical composition to Treat or reduce levels of inflammatory cytokines (TNF-α, IL-1 isoforms, IL-6, IL-8, I1-12, IL-23) commonly seen in patients following SARS virus infection Responses and responses to compounds such as nitric oxide (NO) and arachidonic acid metabolites. Effective NF-κB negative regulatory peptides contained in such pharmaceutical compositions are, for example, VLPALPQVVC, LQGVLPALPQ, LQG, LQGV, GVLPALPQ, VLPALP, VVC, MTR and cyclic LQGVLPALPQVVC, which are, for example, effective for treating severe acute respiratory syndrome (SARS In the treatment of the IL-6 hyperresponse seen in ). Preferably including treatment with concomitant antiviral agents such as ribavirine and gene regulatory peptides to reduce eg NF-κB mediated IL-6 activity.

In another embodiment, wherein the gene regulating peptide provided by the present invention is used as a NF-κB regulating factor for the treatment of diseases, the present invention provides that the NFκB regulating peptide or its derivatives are used in the production of medicaments for treating inflammatory diseases in primates The application in the composition, and a method for treating primate inflammatory SARS disease is provided. Preferably said treatment comprises administering to the patient a pharmaceutical composition comprising NF-κB negative regulatory peptide or a functional analogue thereof. Potent NF-κB negative regulatory peptides are VLPALPQVVC, LQGVLPALPQ, LQG, LQGV, GVLPALPQ, VLPALP, VVC, MTR and cyclic LQGVLPALPQVVC. Using the method provided by the invention can find more negative regulatory peptides and their functional analogues.

Peptide Synthesis: The peptides were prepared by solid phase synthesis (Merrifield, 1963) using a fluorenylmethoxycarbonyl (Fmoc)/tert-butyl based method (Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos, 1991) as a solid support . The side chain of glutamine is functionally protected with a trityl group. The peptides were synthesized artificially. Each coupling involved the following steps: (i) removal of the α-amino protection by piperidine in dimethylformamide (DMF), (ii) conversion of the diisopropyl group in DMF/N-methylformamide (NMP) Coupling of carbodiimide (DIC)/l-hydrozybenzotriazole (HOBt) with Fmoc amino acids (3eq.) and (iii) capping of remaining amino groups with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP Function. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS) 95:2.5:2.5. After 30 minutes, TIS was added until decolorization. The solution was evaporated in vacuo and the peptide was precipitated with diethylethyl ester. The crude peptide was dissolved in water (50-100 mg/ml) and purified by reverse phase high performance chromatography (RP-HPLC). The HPLC conditions are as follows: Chromatographic column: Vydac TP21810C18 (10×250mm) (the selection of chromatographic column is very critical, because the peptide has a short retention time); Elution system: 0.1% TFA v/v (A) and acetonitrile in water 0.08-1% TFA v/v (B) gradient system in (ACN); flow rate 6ml/min; absorbance at 190-370nm. Different gradient systems can be used. For example for peptide LQGV: 100% A for 10 minutes, followed by a linear gradient 0-10% B for 50 minutes. For peptide VLPALP: 5 min 5% A followed by a linear gradient of 1% B/min. The collected fractions were concentrated to about 5 ml by rotary membrane evaporation at 40 °C under reduced pressure. The remaining TFA was exchanged with acetic acid in the acetate form by two elutions through a column with anion exchange resin (Merck II). The eluate was concentrated and lyophilized within 28 hours. The lyophilized peptides were then tested in an in vivo bioassay to determine the suitability of the pharmaceutical composition for use in the treatment of SARS by testing its effect on LPS-induced inflammation in a BALB/c mouse model.

Table 1 ID sequence early activity late activity Peptide-1 Peptide-2 Peptide-3 Peptide-4 Peptide-5 Peptide-6 Peptide-7 Peptide-8 Peptide-9 Peptide-10 Peptide-11 Peptide-12 Peptide-13 Peptide-14 Peptide-43 Peptide-44 Peptide- 45 peptide-46 peptide-47 peptide-48 peptide-49 peptide-50 peptide-51 peptide-52 peptide-53 peptide-54 peptide-55 peptide-56 peptide-57 peptide-58 peptide-59 peptide-60 peptide-61 peptide -62 peptide-63 peptide-64 peptide-65 peptide-66 peptide-67 peptide-68 peptide-69 peptide-70 peptide-71 peptide-74 peptide-75 peptide-76 peptide-78 VLPALPQVVCLQGVLPALPQLQGLQGVGVLPALPQVLPALPVLPALPQGVLPALPVVCQVVCMTRVMTRLQGVLPALPQVVC环-LQGVLPALPQVVCAQGLAGLQAAQGVLAGVLQAVLQGAALPALPVAPALPVLAALPVLPAAPVLPALAALPALPQVAPALPQVLAALPQVLPAAPQVLPALAQVLPALPAVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQSIRLPGCPRGVNPVVSLPGCPRGVNPVVSCPRGVNPVVSLPGCCPRGVNPPGCPRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQGVLPALPQMTRVLPGVLPALPQVVCCALCRRSTTDCGGPKDHPLTCSKAPPPSLPSPSRLPGPSTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQCRRSTTDCGGPKDHPLTC ++++++++++++++++++++++++-+-++++-+-++-+ ++++++++++++++++++++++++-++-+-+-++++

Table 1: Beneficial peptide activities. From ± to + indicates minimal activity to full activity in LPS-induced shock

Example 4

Treatment programs

Treatment with the pharmaceutical composition of the present invention may, for example, include injecting Ringer's lactic acid in the first 24 hours with a pharmaceutical composition provided by the present invention, preferably 1-1000 mg/l peptide, such as VLPALPQ, GVLPALP or MTRV, Or a mixture of two or three of these peptides. During the treatment of severely infected patients, e.g. with the aid of mechanical respiration, it is often important to keep the volume stable and, if necessary, to provide the pharmaceutical composition (e.g. a peptide or a functional analogue thereof) in an even less osmolar solution, As in 0.3-0.6% saline.

The gene modulating peptides can be administered in the same infusion solution, the peptides (or similar) are preferably concentrated from about 1-1000 mg/l, but the peptides can also be administered by bolus injection. The recommended dose is 1-5 mg/kg body weight in bolus injection or each infusion, every 8-12 hours until the patient's condition is stable.

Example 4

Treatment options for SARS

Pharmaceutical composition 1 (Superdex G25): For the single treatment of SARS patients, the following scheme can be used.

1 L of 3-month pregnant woman's urine was filtered and concentrated 13 times (according to Example 1). This provides approximately 75ml of concentrated 3-trimester urine (desalted). The 75ml of concentrated 3-month pregnant women's urine was fractionated on Superdex G25 with a sample volume of 6ml per run. Active fractions were pooled and lyophilized.

Pharmaceutical composition 2 (Superdex G25): For the single treatment of SARS patients, the following scheme can be used.

Dissolve 150,000 IU of commercial hCG preparation in 2-4 ml of 50 mM ammonium bicarbonate. This 2-4 ml hCG preparation was fractionated on Superdex G25, and the active fractions from at least 3 runs were pooled and lyophilized.

Pharmaceutical composition 3 (GPC 300A): For the single treatment of SARS patients, the following scheme can be used.

1 L of 3-month pregnant woman's urine was filtered and concentrated 13 times (according to Example 1). This provides approximately 75ml of concentrated 3-trimester urine (desalted). This 75ml concentrated 3-month pregnant urine was fractionated on a GPC 300A with a sample volume of 50-100μl per run. Active fractions 3 were pooled and lyophilized. This lyophilizate was used for treatment or further fractionated on a GPC 60A column and fractions 3.2 and 3.3 were lyophilized and used for treatment.

Pharmaceutical composition 4 (GPC 300A): For the single treatment of SARS patients, the following scheme can be used.

150,000 IU of commercial hCG preparation was dissolved in 6 ml of 50 mM ammonium bicarbonate. This 6ml hCG preparation was fractionated on a GPC 300A column, and the active fraction 3 was pooled and lyophilized. This lyophilizate was used for treatment or further fractionated on a GPC 60A column and fractions 3.2 and 3.3 were lyophilized and used for treatment.

Pharmaceutical composition 5 (GPC 60A): for the monotherapy of SARS patient, following scheme can be used.

1 L of 3-month pregnant woman's urine was filtered and concentrated 13 times (according to Example 1). This provides approximately 75ml of concentrated 3-trimester urine (desalted). This 75ml concentrated 3-month pregnant urine was fractionated on a GPC 60A with a sample volume of 50-100μl per run. Active fractions 3 were pooled and lyophilized.

Pharmaceutical composition 6 (GPC 60A): for the monotherapy of SARS patient, following scheme can be used.

150,000 IU of commercial hCG preparation was dissolved in 6 ml of 50 mM ammonium bicarbonate. This 6 ml hCG preparation was fractionated on a GPC 60A column with a sample volume of 50-100 μl per run, and the active fraction 3 was pooled and lyophilized.

Pharmaceutical composition 7 (Superdex 75): For the monotherapy of SARS patient, following scheme can be used.

1 L of 3-month pregnant woman's urine was filtered and concentrated 13 times (according to Example 1). This provides approximately 75ml of concentrated 3-trimester urine (desalted). The 75ml concentrated 3-month pregnant urine was fractionated on Superdex 75 with a sample volume of 50-100μl per run. Active fractions 3-5 were pooled and lyophilized.

Pharmaceutical composition 8 (Superdex 75): For the single treatment of SARS patients, the following scheme can be used.

150,000 IU of commercial hCG preparation was dissolved in 6 ml of 50 mM ammonium bicarbonate. This 6 ml hCG preparation was fractionated on a Superdex 75 column with a sample volume of 50-100 μl per run, and active fractions 3-5 were pooled and lyophilized.

Pharmaceutical composition 10 (peptide): For the monotherapy of SARS patients, the following scheme can be used.

Treatments were performed with single peptides (see Table in Example 3) or peptide mixtures. The following are examples of peptide mixtures for use in therapy: Peptide 1 and 2, Peptide 1, 2 and 4, Peptide 1, 2 and 3, Peptide 3 and 5, Peptide 4 and 6, Peptide 4, 6 and 7, Peptide 3, 6 and 7, peptides 1, 2, and 6, peptides 1, 2, and 7, peptides 1, 2, and 9, peptides 1, 2, and 10, peptides 1, 2, and 11, peptides 1, 212, peptides 4, 5 , and 6, peptides 4, 5, and 46, peptides 4, 5, and 48, peptides 4, 6, and 46, peptides 4, 6, and 48, peptides 66 and 6, peptides 66 and 7, peptides 66 and 5, Peptide 64 and 65, Peptide 4 and 68, Peptide 66 and 76, Peptide 9 and 11, Peptide 10 and 12, Peptide 46, 49 and 59, Peptide 48, 49 and 59, Peptide 46, 48, 49 and 59, Peptide 48 and 60, peptides 46, 48, 49, 59 and 60, peptides 14 and 48, peptides 14 and 46, peptides 14 and 7, peptides 14 and 6, peptides 14 and 3, peptides 14 and 4. Single peptides or peptide mixtures can also be used in combination or in combination with third trimester urine fractions, commercial hCG preparations or recombinant hCG preparations. Alternatively, the 3-month pregnant urine fraction can also be mixed with the commercial hCG fraction. In a more preferred embodiment, a 1:1:1 mixture of the peptides AQGV, LQGV and VLPALP is administered to the patient in a bolus injection at 5 mg/kg body weight during the challenge treatment phase, followed by regular and continuous infusions of the aforementioned Ringers lactic acid 4 hours later Or a hypotonic solution containing 100 mg/l of each of the three peptides at a flow rate of 20-200 ml/hour. It is preferable to monitor the cytokine profile in the plasma of treated patients, such as TNF-α, IL-10 or IL-6 levels, NO, and arachidonic acid levels, and to terminate treatment only when these levels are considered to be within the normal range.

Example 5

In vivo bioassays to determine the suitability of pharmaceutical compositions for use in the treatment of SARS

Animal experiment

Animals were maintained under specific pathogen-free conditions according to the protocol described in the report of the research group on animal health in the European Association for Laboratory Animal Science (FELASA) (Experimental Animals 28: 1-24, 1994).

Example 5

In vivo bioassays to determine the suitability of pharmaceutical compositions for use in the treatment of SARS

1. LPS-induced inflammation: To determine the anti-inflammatory activity, female BALB/c mice (Harlan) aged 8-10 weeks were i.p. injected with 30mg/kg LPS (E.coli 055:B5; DifcoLab, Detroit, MI, USA ) or 9-10mg/kg LPS (E.coli 026:B6; Difco Lab, Detroit, MI, USA). Control group mice (10) were treated with 100 μl PBS (pH 7.4) 2 hours after LPS injection, while mice in other groups (10) were treated with 100 μl (1 mg/ml) fraction, 100-600 IU commercial hCG (Pregnyl, Profasi ) or peptide (0.05-5 mg/kg) was i.p. treated. In other experiments, mice were treated with fractions, commercial hCG preparations or peptides 24 or 36 hours after LPS injection. During the experiment, disease status was observed from time 0 (before LPS injection). Afterwards, the mice were only observed for further recovery. In this experiment, LPS caused the PBS group to die within 48-60 hours after LPS injection. Fractions, commercial hCG preparations or peptides were considered functional when the mortality rate was reduced from 100% to at least 40%.

2. TSST-1-induced inflammation: To determine the anti-inflammatory activity, 8-10 week-old female BALB/c mice (Harlan) were injected intraperitoneally with 20 mg D- Galactosamine. They were then administered subcutaneously with 4 µg of TSST-1 dissolved in 100 µl of sterile saline solution (9%) at two sites approximately 0.5 cm below the scapula on each side. Control groups were injected subcutaneously with 4 μg TSST-1 without D-galactosamine, or treated with D-galactosamine alone. The Balb/c mouse group sensitized to D-galactosamine was also treated with 100 μl (1 mg/ml) fraction after TSST-1 injection, 100-600 IU commercial hCG products (Pregnyl, Profasi) or peptide (0.05-5 mg/ml) kg) for processing. In other experiments, mice were treated with fractions, commercial hCG preparations or peptides 24 or 36 hours after injection of TSST-1. During the experiment, disease status was observed from time 0 (before TSST-1 injection). Afterwards, the mice were only observed for further recovery. In this experiment, TSST-1 caused the PBS group to die within 48-60 hours after injection of TSST-1. Fractions, commercial hCG preparations or peptides were considered functional when the mortality rate was reduced from 100% to at least 40%.

3. Experiments in monkeys: Only naive monkeys were used in this preclinical study to exclude any interaction with previous treatments. The animals were anesthetized with ketamine. Animals were orally intubated and allowed to breathe freely. Animals were kept under anesthesia with _O2 / _N2O /isoflurane. Animals were given atropine as a pretreatment drug for O ₂ /N ₂ O/isoflurane anesthesia. The level of surgical anesthesia was maintained during the 2-hour infusion of E. coli, and 6 hours after the E. coli challenge, the endotracheal cannula was removed and the animals were euthanized. A 1-hour pre-infusion with heart rate and blood pressure monitoring was performed prior to bacterial introduction.

4. Two rhesus monkeys were infused with 10 ¹⁰ CFU/kg Gram-negative bacteria Escherichia coli to produce fatal septic shock. One monkey received placebo and was sacrificed within 7 hours of infusion of the bacteria without recovery from anesthesia and a second monkey was treated with the test compound and sacrificed at the same time point. For example, in one experiment the test compound was a mixture of 3 peptides; 30 minutes after infusion of E. coli, animals were given a single intravenous bolus injection, a mixture containing the following peptides: FLQGV (5mg/kg) , AQGV (5mg/kg) and VLPALP (5mg/kg). These peptides were dissolved in 0.9% sodium chloride solution for injection (NPBI, Emmer Compascuum, The Netherlands). A fraction, commercial hCG preparation or peptide, was considered active when the mortality rate dropped from 100% to at least 40%.

Example 6

In vitro bioassays to determine the suitability of pharmaceutical compositions for SARS treatment

1. NF-κB analysis: the mouse macrophage cell line (RAW 264.7 mouse macrophage) was incubated at 37°C, 5% CO ₂ , in the presence of 10% or 2% FBS, penicillin, streptomycin and glutamine cultured in DMEM. Cells in a total volume of 1 ml were seeded in 12-well plates for 2 hours (3×10 ⁶ cells/ml), and then treated with LPS (E. coli 026:B6; DifcoLaboratories, Detroit, MI, USA) and/or peptide (1 μg/ml). mL), fraction (1 μg/mL) or hCG preparation (100-300 IU/mL) stimulation. After 30 minutes of incubation, the plates were centrifuged and cells were harvested for nuclear extraction.

2. Nuclear extraction: Nuclear extracts and EMSA were prepared according to the method described by Schreiber et al. (Schriber et al., 1989, Nucleic Acids Res. 17). Cells were collected in tubes and centrifuged at 2000 rpm (revolutions per minute) for 5 minutes at 4°C (Universal 30RF, Hettich Zentrifuges). Rinse the pellet with ice-cold Tris-buffered saline (TBS pH 7.4) and resuspend in 400 μl hypotonic buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and protease Inhibitor cocktail (Complete ^™ Mini, Roche) was placed on ice for 15 minutes. 25 μl of 10% NP-40 was added and the sample was centrifuged (2 minutes, 4000 rpm, 4° C.). The supernatant (cytoplasmic fraction) was collected and stored in -70°C. The pellet containing nuclei was washed with 50 μl buffer A and resuspended in 50 μl buffer C (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail and 10% glycerol). The samples were shaken at 4°C for at least 60 minutes. Finally, the samples were centrifuged and the supernatant (nuclear fraction) was stored at -70°C.

Final protein concentrations in extracts were determined using Bradford reagent (Sigma).

3. EMSA: For electrophoretic mobility shift analysis, an oligonucleotide was synthesized to express the NF-κB binding sequence (5’-AGC TCA GAG GGG GAC TTT CCG AGA G-3’).

100 pl of sense and antisense oligos were annealed and labeled with γ- ^32P -dATP (Promega, Madison, WI) using T4 polynucleotide kinase according to the manufacturer's instructions. Nuclear extracts (5-7.5 μg) were incubated with 75000 cpm probes in a binding reaction mixture (20 μl) containing 0.5 μg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB ( 25mM _MgCl2 , 5mM _CaCl2 , 5mM DTT and 20% Ficoll). DNA-protein complexes were dissociated from free oligonucleotides by electrophoresis in a 4-6% polyacrylamide gel (150V, 2-hours). The gel is then dried and exposed to X-rays.

4. NO determination: The RAW 264.7 mouse macrophage cell line obtained from the American Type Culture Collection (Manassas, VA, USA) was cultured at 37° C. in 5% CO ₂ with DMEM containing 10% Fetal calf serum (FCS), 50 U/ml penicillin, 50 μg/ml streptomycin, 0.2 M sodium pyruvate, 2 mM glutamine and 50 μM 2-mercaptoethanol (Bio Whittaker, Europe). Medium was changed every 2 days.

Nitrite assay: Nitrite production was assayed in RAW 264.7 macrophage supernatants. Cells (7.5×10 ⁵ /ml) were cultured in 500 μl medium in 48-well plates. Cells were stimulated with LPS (10 μg/ml) and/or peptides or fractions (1 pg/ml, 1 ng/ml, 1 μg/ml) for 24 hours, and then the medium was collected. Nitrite was determined by adding 100 μl Griess reagent (Sigma) to 100 μl medium samples. The _OD540 was determined using a microplate reader and the nitrite concentration was calculated by comparison with the _OD540 generated in the medium using a standard sodium nitrite solution.

PM1T-cell line or RAW 264.7 was obtained from American Type Culture Collection

(ATCC, Manassas, VA) and cultured at 37°C in 5% CO ₂ . For genomic experiments, PM1T-cells (2×10 ⁶ /ml) were treated with 2 ml of phytohemagglutinin (PHA, 10 μg/mL) and IL-2 (200 IU/ml) or PHA, IL-2 in a 6-well plate Incubate with a peptide such as LQGV (10 μg/mL). After 4 h of culture, 10 × ¹⁰ cells were washed and prepared for gene chip probe array experiments. For macrophages, RAW 264.7 cells were incubated with LPS (1 μg/mL) or LPS and a peptide such as LQGV (10 μg/mL). After incubation for 1 hour, 10×10 ⁶ cells were washed and prepared for gene chip probe array experiments. Gene chip expression analysis was performed according to the manufacturer's instructions (Technical Guide for Expression Analysis, Affymetrix Gene Chip). The following major steps summarize gene chip expression analysis: 1) target preparation, 2) target hybridization, 3) experimental and applied fluidics equipment, 4) probe array washing and staining, 5) probe array scanning and 6) data analysis.

5. Th1/Th2 polarization analysis: Mice were treated ip with PBS or test compounds (fraction: 1-100 μg, peptide: 1-100 μg, hCG: 100-300 IU) for 3 days. Splenocytes were then removed under aseptic conditions and single cell suspensions were prepared. Purified CD4+ T cells from the spleen were obtained by complement depletion with heat stable antigen (HAS), CD16/32, MHC class II (BALB/c, M5/114; NOD, M10/216) and antibodies to GR-1. Cells were further purified using magnetic activated cell sorting with a mixture of biotinylated mAbs against CD11b (M1/70), B220 (RA3 6B2), CD8 (YTS-169) and CD40 (FCK-45.5 ) antibody followed by incubation with streptavidin-conjugated microbeads (MiltenyBiotech, Bergisch Gladbach, Germany). CD4 ⁺ cells used in experiments were typically 90-95% pure as determined by flow cytometry.

For polarization assays, purified CD4 ⁺ were initially stimulated by culturing 1×10 ⁵ cells/well in 96-well flat-bottom plates (Nalge Nunc Int., Naperville, IL., USA) and treating them with plate-bound anti-CD3 mAb ( 145-2C11; 25 μg/ml) were stimulated in the presence of soluble CD28 mAb (37-51; 10 μg/ml) and IL-2 (50 U/ml). For Th1 cell differentiation, anti-IL-4 mAb (11B11; 10 ng/ml) and IL-12 (10 ng/ml) were added to the culture. Th2 cells were primed with IL-4 (35 ng/ml) and anti-IFN-γ mAb (XMG 1.2, 5 microg/ml). Non-polarized cultures contained only anti-CD3, anti-CD28 and IL-2.

All doses were optimized in preliminary experiments. After 4 days in culture, cells were washed 3 times and transferred to new anti-CD3-coated 96-well plates and restimulated in the presence of IL-2 (50 U/ml) and anti-CD28 (10 μg/ml) . After 48 hours, supernatants were collected and analyzed by ELISA for IL-4, IFN-γ and IL-10 production as a readout for Th1/Th2 polarization.

6. TNF-α, IFN-γ and MHC II expression: isolate splenocytes obtained from, for example, BALB/c or NOD mice, and use plate-bound anti-CD3 mAb (145-2C11, 25 μg/ml) in the presence of IL-2 ( 50U/ml) or IL-2 and fractions or peptides. After 48 hours, culture supernatants were collected and analyzed for IFN-γ by ELISA. In other experiments, splenocytes, isolated macrophages, DCs or antigen presenting cells (APCs) were stimulated for 24 hours with LPS or LPS and fractions, peptides or hCG. TNF-α was then determined by ELISA or MHC II expression by flow cytometric analysis (FACS).

Human bronchial epithelial cell line BEAS 2B

Diseases characterized by inflammation of the airways affect a large portion of the population. These diseases include SARS. Cytokines and growth factors, produced in response to stimuli, infection, and inflammatory mediators, play an important role in the initiation, persistence, and suppression of acute and chronic airway inflammation.

Airway inflammation is associated with the overproduction and activity of several mediators and cytokines released by inflammatory and resident cells in the airways. It is now clear that epithelial cells are not only an important target of inflammatory mediators but also an active participant in autoinflammatory processes. Bronchial epithelial cells can replenish inflammatory cells in the respiratory tract by releasing chemoattractants, guide inflammatory cells to migrate through the epithelium through the expression of cell adhesion molecules, and regulate the inflammatory activity of other cells by releasing mediators, such as cytokines, chemokines, Arachidonic acid metabolites and relaxation and contraction factors.

The bronchial epithelium not only forms a passive barrier but also plays a role in the immune response. They can produce many mediators with pro-inflammatory or anti-inflammatory effects. In addition, bronchial epithelial cells can express adhesion molecules of many different cell types, thereby facilitating their recruitment.

TNF-α produced by inflammatory cells present in the airway can trigger other inflammatory cytokines and chemokines such as RANTES and IL-6. It can also negatively regulate the production of anti-inflammatory cytokines and thereby disrupt the barrier function of epithelial cells. Glucocorticoids inhibit transcription of most cytokines and chemokines associated with asthma, including IL-6, RANTES, IL-4. This suppression is at least partly related to the therapeutic effect of glucocorticoids.

BEAS 2B cells were stimulated with TNF-α or TNF-γ in the presence of test compounds. Here we show the results for the active fraction from pregnyl as an example. As shown in Figure 12, our results showed that dexamethasone could inhibit TNF-α-induced IL-6 and RANTES production in the BEAS 2B cell line. As tested here, a pharmaceutical composition for the treatment of SARS provided by the present invention can also inhibit TNF-alpha and IL-6 induced inflammatory cytokines. In addition, dexamethasone restored TNF-α-induced negative regulation of the anti-inflammatory TGF-β cytokine, while fractions not only restored TGF-β production but also promoted this anti-inflammatory cytokine. In addition, both dexamethasone and the composition can inhibit the production of RANTES induced by IFN-γ. TNF-α can also induce cell adhesion markers such as HLA-DR and ICAM-1 on the surface of epithelial cells, which then recruit inflammatory cells. In this way, epithelial cells can also act as antigen presenting cells (APCs). Our results show that both dexamethasone and the pharmaceutical composition for treating SARS can negatively regulate the expression of HLA-DR and ICAM-1 induced by TNF-α.

Claims (12)

1. Use of at least one gene-regulating peptide or functional analogue thereof in the production of a pharmaceutical composition for the treatment of a subject suffering from or suspected of suffering from severe acute respiratory syndrome (SARS), wherein said peptide or analogue is selected from LQG 、AQG、LQGV、AQGV、LQGA、VLPALPQVVC、VLPALP、ALPALP、VAPALP、ALPALPQ、VLPAAPQ、VLPALAQ、LAGV、VLAALP、VLPALA、VLPALPQ、VLAALPQ、VLPALPA、GVLPALP、GVLPALPQ、LQGVLPALPQVVC、VCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL、RPRCRPINATLAVEK、EGCPVCITVNTTICAGYCPT、SKAPPPSLPSPSRLPGPS、SIRLPGCPRGVNPVVS , LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, and QVVC. 2. The purposes of claim 1, wherein said severe acute respiratory syndrome is caused by SARS virus infection. 3. The use according to claim 1 or 2, wherein the subject is at risk of having or suspected of having an advanced and possibly lethal stage of SARS characterized by the group consisting of One or more of the following clinical signs or laboratory findings: elevated lactate dehydrogenase levels, elevated creatine kinase levels, neutropenia, thrombocytopenia, prolonged prothrombin time, migratory pneumonia, and tissue hypoxia . 4. The use according to any one of claims 1-3, wherein the peptide or analogue thereof is obtainable from the urine of a pregnant woman. 5. The use of claim 4, wherein the woman is in the first trimester of her pregnancy. 6. The use according to any one of claims 1-3, wherein the peptide or analogue thereof is obtainable from a gonadotropin preparation. 7. The use of claim 6, wherein the gonadotropin comprises human chorionic gonadotropin (hCG). 8. The use according to any one of claims 1-3, wherein the peptide or analogue thereof is obtainable by chemical synthesis. 9. The use according to claim 8, wherein the composition comprises a mixture of peptides LQGV, AQGV and VLPALP. 10. The use according to any one of claims 1-9, wherein the immunomodulatory activity of the composition is tested in an in vivo bioassay. 11. The use according to any one of claims 1-10, wherein said composition is tested for immunomodulatory activity in an in vitro bioassay. 12. The use according to any one of claims 1-11, wherein the composition is tested for the presence or absence of a compound having immunomodulatory activity.

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