JP2003201248A - Secernent from exocrine tissue including prolactin, and transgenic animal having transduced prolactin gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicinal composition for treating or preventing a disease accompanied with the decrease of external secretion caused by the disorder, the inflammatory or the like of the exocrine tissue, and to make the mechanism of the action of the medicinal composition, the regulation of the secretion from the exocrine tissue, and the mechanism of the promotion of development of the exocrine tissue clear. <P>SOLUTION: The medicinal composition containing prolactin is used for the promotion of the secretion from the exocrine tissue. The transgenic animal having a prolactin gene introduced thereto is created. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating or preventing diseases associated with a decrease in exocrine secretion due to disorders of exocrine gland tissues, inflammation, etc., such as dry eye and dry mouth. In addition, the mechanism of action of the above pharmaceutical composition,
The present invention relates to a model animal for analyzing the mechanism of control of secretion from exocrine gland tissue and the promotion of development of exocrine gland tissue.

[0002]

2. Description of the Related Art Dry eye (dry eye, dry eye) is a disease in which the surface of the eye, that is, the corneal conjunctiva is damaged by qualitative and quantitative abnormalities of tear fluid. In addition, various dry eye diseases such as autoimmune disorders such as Sjogren's syndrome, keratoconjunctivitis sicca, Steven-Jenson syndrome, eyelid margin inflammation, dry eye associated with post-cataract surgery and allergic conjunctivitis, and in recent years OA Due to the rapid increase in VDT work (video screen terminal work) accompanying this, and the drying of the room due to air-conditioning, etc., tear-reducing diseases without systemic abnormality are increasing.
The number of dry eye patients is 800 in Japan, including potential patients.
It is said that the number of dry eyes is increasing, and it has become a social problem.

The lacrimal fluid is a thin liquid layer having a thickness of about 7 μm existing at the boundary where the eyeball and the atmosphere come into contact, and is composed of three layers, from the outside, an oil layer, a water layer, and a mucin layer. The oil layer forming the outermost layer of the tear fluid has a role of covering the entire water layer and preventing water evaporation. The oil layer is mainly composed of secretions from the glands that exist around the eye called the meibomian glands. When the secretions from the meibomian glands are reduced due to inflammation or the like, the water in the water layer easily evaporates, which is one of the causes of dry eye.

In recent years, the number of patients with dry mouth called dry mouth has been increasing. A patient with dry mouth may have a problem that chewing and swallowing are difficult due to a decrease in the amount of saliva, and that it is difficult to make a pronunciation, and that a patient is likely to have a tooth decay. Dry mouth is also caused by hypertension, Sjogren's syndrome, diabetes, salivary gland, and other side effects of drugs.

On the other hand, prolactin (PR)
L) is a simple protein hormone secreted from the anterior pituitary gland, which not only stimulates the mammary gland to promote the secretion of milk protein, but also has various physiological actions such as immunoregulatory action.

The following are known to date as facts suggesting that prolactin is deeply involved in the immune system. 1) Essential for T cell proliferation and functional expression. 2) Inhibits thymus retraction. 3) Prolactin receptor is present on lymphocytes. 4) Lymphocytes produce prolactin. 5) Induce INF-γ production. 6) Induce the expression of IL-2 receptor. 7) Antagonizes cyclosporin A. 8) Insufficient prolactin secretion reduces cell-mediated immunity. 9) Hyperprolactinemia reduces the activity of NK cells. 10) Secretion is remarkably enhanced during transplant rejection. 11) Prolactin is C which is a T cell activation marker
Enhances D69 expression. 12) Prolactin stimulation enhances the expression of IL-6 and IL-8, increases MMP-3, and suppresses TIMP-1.

It has been known that hyperprolactinemia is widely observed in patients with autoimmune diseases. In addition, prolactin has an action of activating T cells as described above, its secretion is remarkably enhanced at the time of rejection reaction, and the expression of IL-6 and IL-8 is enhanced by stimulation with prolactin, and MMP- 3 is increased and TIMP-1 is suppressed.
The relationship between the effects on the immune system and pathological conditions such as autoimmune diseases and the effects on the exocrine glands such as the lacrimal gland, salivary gland tissue and meibomian gland are not known.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the above prior art, and includes a lacrimal gland, a meibomian gland,
It is intended to provide a composition for promoting secretion from exocrine gland tissues such as salivary glands or promoting activation of exocrine gland tissues after injury. Furthermore, the present invention aims to provide a model animal useful for studying disorders of exocrine gland tissue and treatment thereof.

[0009]

[Means for Solving the Problems] The present inventors investigated various compositions for promoting secretion from exocrine gland tissue or activation after injury, and found that prolactin promotes secretion from exocrine gland tissue. The inventors have found that it is effective and completed the present invention. Furthermore, the present inventors succeeded in creating a transgenic non-human animal in which secretion from the exocrine gland was promoted by overexpressing prolactin in glandular tissue.

That is, the present invention provides an agent for promoting secretion from exocrine tissue containing prolactin. The present invention also provides a therapeutic agent containing prolactin such as so-called dry eye (dry eye) and dry mouth (dry mouth). The therapeutic agent of the present invention can also be used to prevent or treat dry eye or dry mouth-like symptoms due to side effects of the drug.

The present invention provides a transgenic animal which can be used as a research model of exocrine tissue characterized by having a prolactin gene introduced therein. Further provided is a transgenic animal into which a gene containing a gland-specific promoter has been introduced upstream of the prolactin gene.
This gland-specific promoter includes Lama6 (Parotidsec
retory protein (PSP) signal peptide coding sequence
s) can be used. Further provided is a transgenic animal in which a gene containing the poly A addition site of the SV40 late gene is introduced downstream of the prolactin gene. This transgenic animal can be any mammal except humans, but rat can be preferably used because the transgenic operation and the method for measuring tear volume have been established. In this case,
The rat prolactin gene can be used as the prolactin gene.

In addition, Lama6, prolactin gene, SV
A transgenic animal can be prepared using a DNA containing the poly A addition site of the 40 late poly gene arranged in this order.

Such a transgenic animal into which the prolactin gene has been introduced can be used as a model animal for studying glandular tissue, and the mechanism of action of the pharmaceutical composition containing the prolactin of the present invention, control of secretion from exocrine glandular tissue, It is also useful for analyzing the mechanism of exocrine gland tissue development promotion.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The agent for promoting secretion from the exocrine gland and the therapeutic agent for dry eye or dry mouth of the present invention include prolactin, and the dosage form is not particularly limited, and examples thereof include eye drops and sprays. It can be administered as an oral drug or an ointment. Oral administration,
It can be appropriately selected from various administration forms such as topical administration to the eye and mouth, intravenous administration, transdermal administration, intramuscular administration. In addition to prolactin, the pharmaceutical composition of the present invention contains additives such as known excipients, carriers, coating agents, stabilizers, buffers, preservatives, and fragrances in addition to the dosage form. Is also good. The prolactin contained in the pharmaceutical composition administered to humans is preferably human prolactin, but is not limited thereto.

The exocrine gland tissues targeted by the pharmaceutical composition of the present invention include parotid gland, submandibular gland, sublingual gland, oral salivary glands (labial gland, tongue gland, palate gland, cheek gland), lacrimal gland. , Meibomian glands and pancreatic β cells.

So-called dry eyes which can be treated by the pharmaceutical composition of the present invention include simple dry eyes (decreased tears), dry eye, lacrimation, Sjögren's syndrome, keratoconjunctivitis sicca, Steven-Jenson syndrome, ophthalmology. Examples include pemphigus, blepharitis, cataract surgery and dry eye associated with allergic conjunctivitis. In addition to the treatment of dry eye, it can be widely used for the purpose of alleviating symptoms called so-called tired eyes and itchy eyes by enhancing lacrimal secretion and protecting the eyes after surgery.

So-called dry mouth (dry mouth) that can be treated by the pharmaceutical composition of the present invention includes Sjogren's syndrome, hypertension, diabetes, adverse effects of radiation therapy, renal disease, salivary atrophy in the elderly, salivary gland, salivary glands. In addition to related central and nervous system disorders, side effects of the drug and a temporary decrease in women's menopause may be mentioned.

In the present invention, a transgenic animal in which secretion from the glandular tissue was promoted was prepared by overexpressing prolactin in the glandular tissue. Specifically, a prolactin gene is incorporated into a vector, a transgene is prepared, and the transgene is injected into the male pronucleus of a fertilized egg by the microinjection method, cultured, and then returned to the oviduct to transform the transgenic non-human animal. To overexpress the prolactin gene in such transgenic animals. Prolactin (PRL)
CDNA has already been isolated in various animals, and its nucleotide sequence is known (Cooke, NE et al., Structure of cloned DNA co
mplementary to rat prolactin messenger RNA, J. Bio
l. Chem. 255 (13), 6502-6510 (1980)), SEQ ID NO: 1
Indicated by.

[0019] Preferably, in order to overexpress prolactin in a gland-specific manner, a prolactin gene linked under the control of a lacrimal gland and salivary gland-specific promoter is incorporated into a vector to adjust the transgene. Such gland-specific promoters include, for example, parotid secretory protein.
La, the promoter of (Parotide secretory protein)
ma6 is given. The lama6 promoter is a promoter already reported to be specifically expressed in salivary glands and lacrimal glands. (Nucleic Acids Research, Vol. 20, No. 9, 2249-2
255, 1992.). Furthermore, S is located downstream of the prolactin gene.
Examples include those containing the poly A addition site of the V40 late gene.

The procedure for producing a transgenic non-human animal using a gene construct containing these genes can be carried out by a conventional method. Hereinafter, an example of a method for producing a transgenic animal using a rat will be described.

A microinjection method can be mentioned as a method for introducing a DNA containing prolactin into a fertilized egg. In the microinjection method, a micromanipulator is used to microinject into the male pronucleus of a 1-cell stage fertilized egg under a microscope. When the fertilized egg injected with the DNA is returned to the oviduct of a pseudopregnant female rat and allowed to develop, a baby rat is born.

As a method other than the above-mentioned microinjection method, a method using a retrovirus vector containing the above DNA or a method using ES cells can be used. According to the viral vector method, 4-8 cell stage embryos from which the zona pellucida has been removed are soaked in a concentrated viral solution, infected with the viral vector, and then developed into blastocysts and returned to the uterus of a pseudopregnant female rat. In the method using ES cells, the above DNA is introduced into cultured ES cells by electroporation, the transformed ES cells are screened by an arbitrary method, and then ES cells are introduced into the blastocoel of the blastocyst of another embryo. Transgenic rats are prepared by mating the chimeric rats obtained by injection.

Whether or not it has the transgene is confirmed by Southern blot analysis, dot blot hybridization or PCR, and the resulting pups are screened. The transgenic rat having the transgene integrates the exogenous prolactin gene into its own chromosome in somatic cells and germ cells, and overexpresses prolactin under the control of a promoter. In particular, Lama
When under the control of a gland-specific promoter such as 6, it overexpresses prolactin specifically in the parotid and lacrimal glands. By obtaining a homozygous rat individual by mating such transgenic rats, it is possible to obtain a rat strain in which the foreign prolactin gene is inherited in the next generation and the foreign prolactin gene is specifically overexpressed in glandular tissue. You can

[0024]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the present invention.

Preparation of transgenic rat The rat prolactin gene was ligated downstream of Lama6,
The polyA addition site of the SV40 late gene was ligated to the downstream of the prolactin gene (FIG. 1), and an EcoRI-BstI linker primer was further added. Lama6 was provided by J. Peter Hjorth, the reporter of this promoter. The rat prolactin cDNA was used as provided.

The thus prepared cDNA clone (lam
a6 promoter-rat PRL cDNA clone)
It was introduced into the male pronucleus of the fertilized egg (supplied by CLEA Japan, Inc.) by the microinjection method. Then, about 20 surviving fertilized eggs were transplanted into the uterus of a pseudopregnant rat (temporary parent) to give birth. Successful introduction of the gene was confirmed at 4 weeks after delivery by performing PCR with a primer for prolactin using DNA extracted from cells at the tip of the tail. These methods are described in JHGordon et al., Proc.
Natl. Acad. Sci. USA, 77, p. 7380, 1980.

Confirmation of gland-specific expression of transgene Regarding transgenic rats in which the prolactin gene was confirmed to be introduced, submandibular gland, parotid gland,
The expression of prolactin in the lacrimal gland, lung, liver, pancreas and kidney was examined by Northern blotting analysis. The results are shown in Figure 2. From the results of Northern blotting analysis, it was confirmed that the introduced prolactin was significantly expressed in the parotid gland and lacrimal gland, and was not clearly expressed in other organs. That is, it was confirmed that the foreign prolactin gene was specifically expressed in glandular tissue.

Phenotype of transgenic rats The amount of tears and the amount of saliva of the transgenic rats confirmed to have the prolactin gene introduced were measured. In the transgenic rat, both tears and saliva were enhanced as compared with the control rat which was not subjected to the transgenic manipulation.

To this transgenic rat,
The radiation of y was applied to damage the glandular tissue, and the activation of secretory capacity was measured. Figure 3 shows the results of measurement of tear secretion.
FIG. 4 shows the results of measurement of salivary secretion. Although the amount of saliva was significantly decreased by the irradiation of radiation until 3 weeks after the irradiation, the transgenic rat gradually increased thereafter, and at 16 weeks after the irradiation, the amount of secretion was recovered to the same level as that before the irradiation. On the other hand, the control rat, which was not subjected to the transgenic operation, showed a significant decrease after irradiation and showed almost no recovery.

Effects of salivary secretion by prolactin
Hibiki prolactin (1 μg / ml) was added to the salivary gland cell line HSY, and salivary amylase, which is an index of salivary secretion ability, was detected by Western blot. 0 minutes after adding prolactin,
The results of Western blotting of the samples after 15, 30, 45, 60 minutes are shown in FIG. Apparent amylase protein expression was observed 30 minutes after the addition of prolactin. From this, it became clear that the addition of prolactin promotes salivary secretion from the salivary glands.

For phosphorylation of CREB by prolactin
Effect on cAMP response element (CR
The control mechanism via E) has been clarified, and it is known that the response to various stimuli is regulated by phosphorylating CREB, which is a transcription factor binding to the cAMP response element. It was investigated whether promotion of salivary secretion by prolactin is mediated by such phosphorylation of CREB. Prolactin 1 μg / m in salivary gland cell line HSY
1 was added, and phosphorylated CREB was detected 0 minutes, 10 minutes, and 30 minutes after the addition. For detection, an antibody specific for CREB in which the serine at position 133 was phosphorylated was used for detection by immunoblotting. In addition, CR
It is known that the one in which fibroblast growth factor (FGF), which is known to be phosphorylated on EB, is added can be detected by the above-mentioned antibody in the same manner as phosphorylated CREB by using it as a positive control for phosphorylation. Phosphorylated ATF-1 was used as a positive control for the detection system. Results are shown in FIG. CREB phosphorylation was caused by the addition of prolactin, and this phosphorylation was observed about 15 minutes after the addition of prolactin and tended to increase over time. Therefore, stimulation with prolactin phosphorylates CREB via intracellular signal transduction, resulting in phosphorylated CRE.
It was suggested that B promotes transcription of a gene having a cAMP response element, thereby increasing secretion from salivary glands and lacrimal glands.

Exocrine gland by local administration of prolactin
Enhancement of secretion from the rats The prolactin extracted from the pituitary gland of the rat was instilled into both eyes of male 6-week-old wister rats (supplied by CLEA Japan, Inc.). Prolactin was administered 3 times a day at 1 μg / ml for the low dose group and 1 for the high dose group.
0 μg / ml was applied to 7 animals each. In the control group, PBS was administered to 7 animals 3 times a day. Such administration is performed 5 days a week for 6 weeks, and 6 days after the start of administration.
After a week, the amount of lacrimal secretion was measured as the amount of secretion from the exocrine glands. Six weeks after the start of administration, rats were anesthetized by intraperitoneal administration of Nembutal (65 mg / kg body weight), and the amount of lacrimal secretion for 5 minutes was measured using a cotton thread. After the measurement, the rat was allowed to stand for 5 minutes, and pilocarpine (0.05 mg / 10
0g body weight and isoprotenol (0.05mg / 100)
g body weight) was further administered. Five minutes after the administration, the tear volume was measured using a cotton thread for 5 minutes. These measurements were repeated 3 times for 30 minutes and used as the amount of tear secretion. The results are shown in the table below. The numerical values in the table are mean tear volume ± standard deviation mm /
It is expressed in minutes.

[0033]

[Table 1]

It was shown that the rat administered with prolactin had increased tear volume in both eyes as compared to the rat administered with PBS as a control. Therefore, prolactin has a secretagogue effect from exocrine gland tissue,
It has been found that a drug containing prolactin is effective as a secretagogue for exocrine gland tissue.

[Sequence list] SEQUENCE LISTING <110> TUBOTA Ltd.,       SAITO, Ichiro <120> A medicine for promoting of secretion from exocrine gland tissues,  comprising prolactin and an transgenic animal introduced with a prolact ine gene. <130> 011111 <140> <141> <160> 1 <170> PatentIn Ver. 2.1 <210> 1 <211> 823 <212> DNA <213> Rattus rattus <400> 1 agtggttctc ttaggacttc ttggggaagt gtggtcccag tggtcatcac catgaacagc 60 caggtgtcag cccggaaagg gacactcctc ctgctgatga tgtcaaacct tctgttctgc 120 caaaatgtgc agaccctgcc agtctgttct ggtggcgact gccagacacc tctcccggag 180 ctgtttgacc gtgtggtcat gctttctcac tacatccata ccctgtatac agatatgttt 240 attgaatttg ataaacagta tgtccaagat cgtgagttta ttgccaaggc catcaatgac 300 tgccccactt cttccctagc tactcctgaa gacaaggaac aagcccagaa agtccctccg 360 gaagttcttt tgaacctgat cctcagtttg gtgcactcct ggaatgaccc tctgtttcaa 420 ctaataactg gactaggtgg aatccatgaa gctcctgatg ctatcatatc aagagccaaa 480 gagattgagg aacaaaacaa gcggcttctc gaagggatcg aaaagataat tggccaggcc 540 tatcctgaag ccaaaggaaa tgagatctac ttggtttggt cacaactccc atccctgcaa 600 ggagttgatg aagaatccaa agacttggct ttttataaca acattcggtg cctgcgcagg 660 gattcccaca aggttgacaa ttatctcaag ttcctgaggt gccaaattgt ccataaaaac 720 aactgctaag cctacattca ttccatgtac atccgagatg ttcttaaaag tctatttctt 780 caaaggttct atttgcatta caactttcag cacatgctta aga 823

[0035]

INDUSTRIAL APPLICABILITY The secretory promoting agent from the exocrine gland and the therapeutic agent for dry eye or dry mouse which contain prolactin of the present invention exhibit an excellent effect of promoting secretion from exocrine gland tissue. In particular, prolactin itself is a hormone that exists in the living body, and thus is a highly safe therapeutic drug. This pharmaceutical composition can treat and prevent disorders of exocrine glands such as dry eye and dry mouth. Further, a transgenic non-human animal into which the prolactin gene of the present invention has been introduced, and D for preparing the transgenic animal
NA is useful for analyzing the mechanism of action of a pharmaceutical composition containing prolactin, the regulation of secretion from exocrine tissue, and the mechanism of promotion of development of exocrine tissue.

[Brief description of drawings]

FIG. 1 shows a schematic diagram of the structure of the transgene used in Examples.

FIG. 2 shows the results obtained by confirming the expression of prolactin in each tissue of the transgenic rats of the example by Northern blotting analysis.

FIG. 3 shows activation of lacrimal secretion capacity when glandular tissue is damaged by irradiation with 15 Gy of radiation.

[Fig. 4] Fig. 4 shows activation of salivary secretory function when glandular tissue is damaged by irradiation with 15 Gy of radiation.

FIG. 5 shows the results of Western blot analysis of amylase secretion of HSY to which prolactin was added.

FIG. 6 shows CR of HSY to which prolactin was added.
The result of having analyzed the phosphorylation of EB by the immunoblotting is shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 105 A61K 37/32 C12N 5/10 C12N 15/00 ZNAA 15/09 ZNA 5/00 B F Terms (reference) 4B024 AA01 CA04 DA02 FA02 GA11 GA12 4B065 AA91X AA91Y AB01 AC14 BA02 BA04 CA24 CA44 CA60 4C084 AA01 AA02 BA01 BA08 BA22 CA23 CA53 DB23 MA13 MA28 MA52 MA57 MA58 MA63 NA14 ZA332 ZA672 ZB212

Claims (10)

[Claims] 1. An agent for promoting secretion from exocrine gland tissue, which comprises prolactin. 2. A therapeutic agent for dry eye containing prolactin. 3. A therapeutic agent for dry mouth (dry mouth) containing prolactin. 4. A model animal for transgenic research into which the prolactin gene has been introduced. 5. The model animal according to claim 4, wherein a DNA containing a gland-specific promoter upstream of the prolactin gene has been introduced. 6. The model animal according to claim 5, wherein the gland-specific promoter is Lama6. 7. SV40 is provided downstream of the prolactin gene.
The model animal according to claim 5, wherein a DNA containing a poly A addition site of a late gene is introduced. 8. The model animal according to any one of claims 4 to 7, which is a rat. 9. The model animal according to claim 8, wherein the prolactin gene is a rat prolactin gene. 10. Lama6, prolactin gene, SV4
0 A DNA in which the poly A addition site of the late poly gene is linked in this order from the 5 ′ side to the 3 ′ side.