CN104784679A - Safe dose of neuregulin applied to people - Google Patents

Abstract

The present invention discloses a safe dose of neuregulin applied to people. After 2.99-35.83U / kg (protein / body weight) of neuregulin is intravenously injected into healthy people one time, negative reactions or ECG (electrocardiogram) changes of some people are found, the negative reactions all disappear eight hours after, and the electrocardiogram also returns to normal 7 days after. In all research objects, heart function and breathing are always normal. An attempt is further made to inject 2.99-17.92U / kg of neuregulint into healthy people once in one day for continuous five days, the negative reactions or ECG (electrocardiogram) changes of some people are also found, and the protein dose is higher, the frequency of abnormal electrocardiogram is higher. But the negative reactions quickly disappear, and the electrocardiogram also returns to normal 7 days after the last injection. According to results of clinical trials, 2.99-35.83U / kg of neuregulin can be used as a safe dose for human body.

Description

Neuregulin is applied to the safe dose of people

Technical field

The present invention relates to the safe administration dosage to human body, especially, relate to safe dose when neuregulin is applied to people.

Background technology

Neuregulin (neuregulin, NRG; Heregulin, HRG), be again glial growth factor (glial growth factor, GGF), neu differentiation factor (new differentiationfactor, NDF), for molecular weight is at the glycoprotein of about 44KD, they are at intercellular trafficking signal, are the parts of tyrosine kinase receptor ErbB family.Neuregulin family is containing 4 members: NRG1, NRG2, NRG3, NRG4.(Falls et al., Exp Cell Res.284:14-30,2003) know little about it comparatively speaking to the biological function of rear three.NRG1 plays an important role in nervous system, heart and mammary gland, also evidence show that NRG1 signal transmission works in the pathogeny of the growth of some other tract, function and human diseases (comprising schizophrenia and breast carcinoma).NRG1 has a lot of isomer.The research of mutant mouse (knock out mice) is illustrated that it is also different in body function in the different isomer in N-terminal district or epidermal growth factor (EGF) similar district.The present invention is based on neuregulin 1 β (NRG1 β).

Neuregulin 1 β is a transmembrane protein (Holmes et al., Science 256,1205-1210,1992).Film outward part is N-terminal, comprises the similar district of immunoglobulin (Ig-likedomain) and the similar district of EGF (EGF-like domain), and in film, part is C-terminal.Under the metalloproteases effect of extracellular matrix, the outer part of film of neuregulin digested can be got off and be free state, thus is conducive to the ErbB receptors bind with peripheral cell surface, activates corresponding cell signal transmission.

ErbB receptor family is also divided into four classes, ErbB1, ErbB2, ErbB3 and ErbB4, and they are all transmembrane proteins, and molecular weight is near 180-185KD.Except ErbB2, their N-terminal outside film all contain ligand binding domain; Except ErbB3, their C-terminal in film all contain protein tyrosine kinase activity.Wherein ErbB1 is the receptor of epidermal growth factor, ErbB3 and ErbB4 is the receptor of neuregulin.In the receptor of neuregulin, only have ErbB2 and ErbB4 higher at heart expression.(Yarden et al.，Nat Rev Mol CellBiol，2：127-137，2001)

When neuregulin and ErbB3 or ErbB4 film outward part in conjunction with time, ErbB3, ErbB4 and other ErbB receptors (usually comprising ErbB2) will be caused to form heterodimer, or ErbB4 self forms homodimer, then part in the film of receptor is caused to be phosphorylated (Yarden et al., Nat Rev Mol Cell Biol, 2:127-137,2001).In the film of phosphorylation, part can be combined with intracellular multi-signal transferrin further, thus activate downstream ERK or AKT signal path, cause a series of cell effect: comprise stimulation or antiproliferative effect, apoptosis, cell migration, cell differentiation or cytoadherence.

The growth of neuregulin to heart is even more important (WO0037095, CN1276381, WO03099300, WO9426298, US 6444642, WO 9918976, WO 0064400, Zhao et al., J.Biol.Chem.273,10261-10269,1998).Early stage at fetal development, the expression of neuregulin is mainly limited to endocardium, be discharged into surrounding myocardium cell by paracrine approach subsequently and with the protein tyrosine kinase receptor ErbB4 film on cell membrane outward part combine, ErbB4 and then form heterodimer with ErbB2.It is necessary that the formation of ErbB4/ErbB2 complex and activation form girder to early stage cavernous transformation heart.Any one disappearance in neuregulin, ErbB4 and ErbB2 tri-protein gene all can make embryo not have girder and die from uterus in early days in growth.WO0037095 shows certain density neuregulin sustainable activation ERK signal path, promote growth and the differentiation of myocardial cell, guide the reconstruction of myocardial cell and cytoadherence place muscle segment and cytoskeleton, improve the structure of myocardial cell, strengthen the contraction of myocardial cell.WO0037095 and WO03099300 also points out that neuregulin can be used for detection, the various cardiovascular disease of Diagnosis and Treat.

List some prior art documents relevant with the present invention below:

1、Cardiac muscle function and manipulation，WO0037095

2, the new opplication of rhNRG-1BETA S1Q237 and analog thereof, CN1276381

3、Neuregulin based methods and compositions for treating cardiovasculardiseases，WO03099300

4、You-yang Zhao，Douglas R.Sawyer，Ragavendra R.Baliga，Douglas J.Opel，Xinqiang Han，Mark A.Marchionni，and Ralph A.NeuregulinsPromote Survival and Growth of Cardiac Myocytes.Kelly J.Biol.Chem.273，10261-10269(1998)

5、Methods for treating muscle diseases and disorders，WO9426298

6、Methods of increasing myotube formation or survival or muscle cellmitogenesis，differentiation or survival using a neuregulin，US 6444642

7、Therapeutic methods comprising use of a neuregulin，WO 9918976

8、Methods for treating congestive heart failure，WO 0064400

9、William E.Holmes，Mark X.Sliwkowski，Robert W.Akita，William J.Henzel，James Lee，John W.Park，Daniel Yansura，Nasrin Abadi，HelgaRaab，Gail D.Lewis，H.Michael Shepard，Wun-Jing Kuang，William I.Wood，David V.Goeddel，Richard L.Wandlen.Identification of heregulin，a specific activator of p185(erbB2).Science 256，1205-1210(1992)

10、Douglas L.Falls.Neuregulins：functions，forms，and signalingstrategies.Experimental Cell Research 284，14-30(2003)

11、Yosef Yarden，Mark X.Sliwkowski.Untangling the ErbB signallingNetwork.Nature Reviews：Molecular Cell Biology 2127-137(2001)。

We have found that the neuregulin injecting for several times a certain amount of (149.3U/kg: protein content/body weight) continuously to Heart Failure Wistar Rats can activate ERK and the AKT signal path in its body, and be conducive to the recovery of heart failure.

In order to find effective, the safe dose by neuregulin treatment popular feeling disease of ZANG-organs, needing first to do toxicological experiment to Healthy People, finding the safe dose of Healthy People without toxic side effect.The present invention, by great many of experiments and analysis, has found that neuregulin is applied to the safe dose of people, has met this kind of needs.

Summary of the invention

The invention provides safe dose when neuregulin is applied to people.In one embodiment of the present invention, described safe dose is when neuregulin one or many is administered to healthy population, the safe dose that the Healthy People physical ability obtained is born.Particularly, described safe dose in the scope of about 2.99 to about 35.83U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg, approximately 17.92U/kg, approximately 23.89U/kg or approximately 35.83U/kg (albumen/body weight).More preferably, described safe dose is in the scope of about 2.99 to about 17.92U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg or approximately 17.92U/kg (albumen/body weight).

Present invention also offers method human-body safety being used to neuregulin dosage, it comprises the neuregulin one or many of doses is administered to healthy population, and can not cause negative response or only cause the negative response that can be accepted degree.Wherein, described dosage in the scope of about 2.99 to about 35.83U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg, approximately 17.92U/kg, approximately 23.89/kg or approximately 35.83U/kg (albumen/body weight).More preferably, described dosage is in the scope of about 2.99 to about 17.92U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg or approximately 17.92U/kg (albumen/body weight).In preferred embodiments, the such as neuregulin one or many of about 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg or about 17.92U/kg (albumen/body weight) in about 2.99 to about 17.92U/kg (albumen/body weight) scope is administered to healthy population, and negative response can not be caused or only cause the negative response that can be accepted degree.Or, will more than 17.92U/kg in about 35.83U/kg (albumen/body weight) scope such as approximately the neuregulin applied once of 23.89/kg or about 35.83U/kg (albumen/body weight) to healthy population, and negative response can not be caused or only cause the negative response that can be accepted degree.

Present invention also offers for human body is safe drugs preparation, wherein comprises the neuregulin of safe dose.Described safe dose in the scope of about 2.99 to about 35.83U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg, approximately 17.92U/kg, approximately 23.89/kg or approximately 35.83U/kg (albumen/body weight).More preferably, described safe dose is in the scope of about 2.99 to about 17.92U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg or approximately 17.92U/kg (albumen/body weight).

Accompanying drawing is sketched

Fig. 1 is experimenter A302 (35.83U neuregulin/kg body weight) electrocardiogram before administration.

Fig. 2 is experimenter A302 (35.83U neuregulin/kg body weight) electrocardiogram of 2 hours upon administration.

Fig. 3 is experimenter A302 (35.83U neuregulin/kg body weight) electrocardiogram of 24 hours upon administration.

Fig. 4 is that experimenter A302 (35.83U neuregulin/kg body weight) is at the electrocardiogram of administration after 7 days.

Fig. 5 is that administration 17.92U/kg (neuregulin/body weight) organizes experimenter C405 electrocardiogram before administration.

Fig. 6 is the electrocardiogram that administration 17.92U/kg (neuregulin/body weight) organizes experimenter C405 after first time administration 24 hours.

Fig. 7 is the electrocardiogram that administration 17.92U μ g/kg (neuregulin/body weight) organizes experimenter C405 after second time administration 2 hours.

Fig. 8 is the electrocardiogram that administration 17.92U/kg (neuregulin/body weight) organizes experimenter C405 after second time administration 24 hours.

Fig. 9 is the electrocardiogram that administration 17.92U/kg (neuregulin/body weight) organizes experimenter C405 after the 5th administration 2 hours.

Figure 10 is that administration 17.92U/kg (neuregulin/body weight) organizes the electrocardiogram of experimenter C405 after the 5th administration when 7 days.

Detailed Description Of The Invention

Term used herein " neuregulin (neuregulin; NRG) " refers to the molecule that can activate the different bigeminy protein tyrosine kinase of ErbB2/ErbB4 or ErbB2/ErbB3, comprise the EGF region in neuregulin isomer, neuregulin, neuroregulation protein mutant, and any gene outcome that can activate the neuregulin class of above-mentioned receptor.As an example, but without limitation, neuregulin of the present invention is a fragment of neuregulin β 2 isomer, i.e. 177-237 amino acids fragment, wherein contains EGF-class district, receptor binding domain.The aminoacid sequence of this fragment is:

SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ。

Neuregulin of the present invention can be separated from natural source and obtain, or is obtained by recombinant technique or other means.

Term used herein " unit of activity " or " 1U " refer to the neuregulin dosage of the half inducing maximum activation.Particularly, our definition is a unit of activity with the sample of neuregulin EC50 (μ g/ml) equivalent.A unit of activity of the present invention's neuregulin used is 0.067 μ g, and namely 1 μ g is 14.93U.The mensuration of protein sample EC50 is a kind of basic fundamental, for the people in this field knows.

Term used herein " safety " or " safe dose ", relate to represent such dosage, that is, after the neuregulin of described dosage is expelled to healthy population, negative response can not be produced, or a few peoples there will be negative response in the short time, and/or groups of people's electrocardiogram occurs abnormal, but negative response recovers mostly very soon, such as recovered in 8 hours, electrocardiogram also recovers normal within short-term, such as, just all recover normal after 7 days.Therefore, the negative response in these situations is considered to can acceptance level, in other words, is not the order of severity.Particularly, the neuregulin of safe dose of the present invention in the scope of about 2.99 to about 35.83U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg, approximately 17.92U μ g/kg, approximately 23.89U/kg or approximately 35.83U/kg (albumen/body weight).More preferably, described safe dose is in the scope of about 2.99 to about 17.92U/kg (albumen/body weight), such as approximately 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg or approximately 17.92U/kg (albumen/body weight).

Term used herein " negative response ", comprise Nausea and vomiting, appetite poor, tired, to feel dizzy or irritated, or its combination in any.Negative response also comprises other symptom that may occur over the course for the treatment of well known by persons skilled in the art, but does not comprise serious symptom, and such as cardiac function is not normal, dyspnea and other severe reaction.When using neuregulin with safe dose of the present invention or comprising the preparation of neuregulin, the negative response that may occur is all can the negative response of acceptance level or the non-order of severity.

Neuregulin or its preparation of safe dose of the present invention once or repeatedly can be administered to healthy population, and do not occur negative response or do not occur the negative response of the order of severity.Here " once " used refers to discrete using, such as every seven days once.Here " repeatedly " used refers to and can use continuously, such as once a day, and continuous five days or one week or longer time.According to dosage of the present invention, neuregulin or its preparation can applied onces or repeatedly use.Such as, dosage range such as neuregulin one or many of about 2.99U/kg, approximately 5.97U/kg, approximately 11.94U/kg or about 17.92U/kg (albumen/body weight) in about 2.99 to about 17.92U/kg (albumen/body weight) scope is administered to healthy population.Or, will more than 17.92U/kg in about 35.83U/kg (albumen/body weight) scope such as approximately the neuregulin applied once of 23.89/kg or about 35.83U/kg (albumen/body weight) to healthy population.Further, such dosage application can be repeated, thus forms periodic dosage regimen.

The neuregulin of safe dose of the present invention can be mixed with various compositions or preparation, such as, and solution, suspension, Emulsion, tablet, tincture, unguentum or spray, or other any forms being suitable for using.Therefore, dosage of the present invention can be given with various different approach, such as, by intravenous injection, inculcate, oral, Intraperitoneal medication, intranasal administration, etc.Concrete route of administration partly depends on adopted concrete dosage form, and this is well known by persons skilled in the art.Usually, safe dose of the present invention is all applicable for any dosage form and/or route of administration.

Embodiment

The determination of neuregulin safe dose

To the neuregulin of Healthy People 1 time or 5 times intravenous injection various dose, the physiological reaction of time sight object, electrocardiogram and other physiologic index, as heart rate, blood pressure, cardiac enzymes etc., thus to determine 2.99-35.83U/kg neuregulin be drug dose to human-body safety, more preferably, be 2.99-17.92U/kg scope.

Neuregulin used in the examples below is 177-237 human glial growth factors Beta2 (the 177-237 amino acid fragment of NRG1 β 2, Ze Sheng Science and Technology Development Co., Ltd., lot number: 200503002).

Embodiment 1:1 injection

Healthy volunteer is divided at random A (4 people), B (4 people), C (4 people), D (6 people), E (6 people), F (6 people) 6 groups, slowly evenly (2ml/min injects 10 minutes) neuregulin 2.99U/kg, 5.97U/kg, 11.94U/kg, 17.92U/kg, 23.89/kg, 35.83U/kg is injected respectively with micro pump.Inquiry before injection, record experimenter medical history, detect that its physique, hematuria, blood sample are biochemical, cardiac enzymes, also need in addition to have an electro-cardiogram and the heart superly to detect (indices eligible totally 28 people, the age 20-45 year between, 15 male 13 female).Observe the negative response of volunteer at certain hour after injection neuregulin and detect its electrocardiogram.The grouping of final experimenter and detection case are as table 1, and visible all experimenters safety successfully complete experiment, even 5 people of injection maximum dose level 35.83U/kg are no exception.

After administration, in the short time, groups of people have the negative responses (table 2) such as nauseating, tired, appetite is poor or feel dizzy, but all recover in administration 8 hours; The recovery of electrocardiographic abnormality (as T ripple moves down or is inverted) then needs the longer time, and minority experimenter is about 7 days ability normal (table 2, table 3) upon administration.

Fig. 1-Fig. 4 be respectively experimenter A302 (35.83U/kg) before administration, 2 hours, 24 hours and the electrocardiogram of administration after 7 days after administration after administration.2 hours experimenter's electrocardiograms are all normal as seen from the figure before administration, after administration, and within 24 hours, occur obvious II upon administration, and III, avF, V4-V6T ripple is inverted, administration after 7 days electrocardiogram recover again normal.

Table 4-table 7 lists the time dependent situation of experimenter's electrocardiogram of injection various dose medicine (2.99,17.92,23.89 or 35.83U/kg) respectively.The electrocardiogram of visible most people all recovers normal in administration after 7 days.All are normal to do cardiac function (cardiac output, blood pressure, heart rate etc.), cardiac enzymes, breathing and hematochemistry detection display to the experimenter of electrocardiographic abnormality, and experimenter's electrocardiogram QT and QT after administration _cconstant, prompting sinus rate is normal.

Embodiment 2:5 injection

32 experimenters are divided into 4 groups at random, often organize 8 people.Inject 2.99U/kg, 5.97U/kg, 11.94U/kg or 17.92U/kg neuregulin respectively, once a day, continuous five days.Before per injection and injection within latter 2 hours, all to monitor experimenter's electrocardiogram, observe and record the negative response of experimenter.In addition also will after the 5th administration after 24 hours, 7 days and monitor experimenter's electrocardiogram and negative response afterwards in 14 days.Experimenter's distribution is as table 8.Visible when 17.92U/kg dosage, there is a people to drop by the wayside.

As can be seen from Table 9, when successive administration, many people have negative response, symptom comprise Nausea and vomiting, appetite poor, tired, to feel dizzy and irritated, and dosage higher negative reactor ratio is higher.But these negative responses all only continue 2-6 hour, automatically recover subsequently.Also have some experimenters to occur electrocardiographic abnormality in addition, as ST-T section move down, T ripple moves down or T ripple is inverted, and the persistent period is longer, and when high dose, incidence rate is higher, but all recovers after 7 days in last administration, as shown in table 10-13.T wave (width and the degree of depth) not change when data also show electrocardiographic abnormality, and women occurs that the ratio of electrocardiographic abnormality is higher than male.In 8 experimenters of 17.92U/kg group, there is electrocardiographic abnormality in 4 women, only has 2 to occur exception in 4 male.

Fig. 5-10 for administration 17.92U/kg group experimenter C405 before administration, 24 hours, 2 hours, electrocardiogram after second time administration after 24 hours, the 5th time administration after 2 hours, the 5th time administration when 7 days after second time administration after first time administration.As can be seen from Figure, after first time administration there is electrocardiographic abnormality in 24 hours experimenters, shows as II, III, avF, V3-V6 T ripple moves down, be inverted, bipolarity, after second time administration, 2 hours electrocardiograms still show as II, III, avF, V3-V6T ripple moves down, is inverted, and after second time administration, 24 hours Electrocardiogram Features are II, III, avF, V3-V6T ripple is low flat, within 2 hours, shows as V5-V6T ripple lower after the 5th administration, and the 5th administration after 7 days electrocardiogram just recover normal completely.

Table 11-13 lists the time dependent situation of part experimenter electrocardiogram of five injection 5.97U/kg, 11.94U/kg and 17.92U/kg medicines respectively.Visible experimenter there will be T ripple and moves down, is inverted or the electrocardiographic abnormality such as bipolarity, but all within 7 days, automatically recovers afterwards the 5th administration.All experimenters do not occur that chest pain, cardiac function are not normal, dyspnea symptom from start to finish, and heart enzyme and hematochemistry detect also all normal.

Below by specific embodiments, present invention is described.However, it is to be understood that, multiple change can be carried out, as long as it does not deviate from the spirit and scope of the present invention to the various examples described in it.Therefore, other embodiment is still within the scope of claims.

Claims (9)

1. be applied to the safe drugs preparation of people, it contains for healthy human body is the neuregulin of safe dose.

2. pharmaceutical preparation according to claim 1 is safe when wherein said preparation one or many is administered to healthy human body.

3., there is negative response in the short time when wherein said preparation is administered to healthy human body in pharmaceutical preparation according to claim 2, but negative response can disappear very soon.

4. the pharmaceutical preparation in claim 1-3 described in any one, wherein said neuregulin is the function amino acid fragment of NRG1, namely comprises any NRG1 fragment in the similar district of EGF.

5. pharmaceutical preparation according to claim 4, wherein said safe dose is in 2.99-35.83U neuregulin/kg weight range.

6. pharmaceutical preparation according to claim 5, wherein said safe dose is in 2.99-17.92U neuregulin/kg weight range.

7. pharmaceutical preparation according to claim 5, wherein said safe dose is 23.89 or 35.83U neuregulin/kg body weight.

8. pharmaceutical preparation according to claim 6, wherein said safe dose is 2.99,5.97,11.94 or 17.92U neuregulin/kg body weight.

9. pharmaceutical preparation according to claim 1, it is injectable form.