JPH02306920A - Preventive and treating agent for disease by herpesvirus - Google Patents

Abstract

PURPOSE:To obtain the subject preventive and treating agent, containing a nonapeptide known as a blood serum thymic factor or a derivative thereof as an active ingredient and capable of exhibiting effects by activating immunological or biophylactic systems of a host. CONSTITUTION:A preventive and treating agent, containing a nonapeptide having an amino acid sequence expressed by the formula or an ester or amide at the carboxyl group of aspartic acid at the C-terminal thereof or a pharmaceutically acceptable salt thereof as an active ingredient and useful for diseases by infection with human herpesvirus, such as herpes simplex virus-I or II or Epstein-Barr virus, and various animal-infected herpesvirus. The above-mentioned agent is safe with ultralow toxicity and can be administered for a long period.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel drug for humans or animals, and more specifically, to a novel drug for treating diseases caused by human herpesviruses or various animal-infected herpesviruses. This relates to preventive and therapeutic agents.

[Prior art]

In recent years, findings regarding various peptides have been reported as having efficacy against various viral diseases. for example,
JP-A-59-5124'7 and JP-A-59-9
Publication No. 8049 describes the synthesis of various novel peptides and the efficacy of these synthetic peptides against E. coli or herpes virus infected animal models. Furthermore, Japanese Patent Application Laid-open No. 104019/1983 describes that certain alipeptides containing D-unit amino acids have the effect of increasing the survival rate of mice infected with influenza virus or herpes virus. There is. Additionally, JP-A-57-192349 describes that six types of repeptides consisting of three amino acids, leucine, alanine, and phenylalanine, exhibit antiviral effects on mice infected with hebatitis virus MHV3. has been done. Also, JP-A-58-9264
Publication No. 2 describes Arg-Lys-Asp-Va 1-T
A basic pentapeptide having the amino acid sequence of yr is
There are descriptions that it is useful for treating viral infections.

Also, Nature, 321.439-441 (19
86) and 441-443 (1986) describe the anti-herpesvirus effect based on the enzymatic inhibitory effect of certain synthetic nonapeptides on herpesvirus polynucleotide reductase.

Many of the peptides in these known documents are synthetic and new, but they have only been reported as mentioned above and are still
Not only have they not been put into practical use as therapeutic or prophylactic drugs for viral diseases, but many of these synthetic peptides have not been used as pharmaceuticals for humans or animals.
In the case of application, toxicity, side effects, antigenicity, ana7raxine yoke, etc., have hardly been studied.

[Disclosure of the invention]

The present inventors have developed various highly safe naturally derived peptides that can be used to treat viral infections, with the aim of developing drugs that, unlike chemotherapeutic agents, enhance the body's natural defense ability. , especially herpes simplex virus-f
f, cytomegalovirus, varicella zoster virus,
As a result of intensive searches for agents useful as prophylactic and therapeutic agents for diseases caused by infection with human herpesviruses such as EpsLein-Barr virus and various animal-infected herpesviruses, we discovered that serum thymus factor
ique Serique, hereinafter abbreviated as FTS. )
It has been discovered that nonapeptide known as . The present invention has been made based on this knowledge.

That is, the present invention relates to viruses belonging to the herpesviridae family, such as human herpesvirus, chimpanzee herpesvirus, orangutan herpesvirus, human herpesvirus, squirrel monkey herpesvirus, spider monkey herpesvirus, cottontail rabbit herpesvirus, guinea pig herpesvirus, and Marek's disease virus. The purpose of the present invention is to provide drugs that can prevent and treat various infectious diseases caused by various viruses such as cotton chinensis virus and cotton cilia virus, as well as various disorders associated therewith.

One of the inventors previously demonstrated that nonapeptides, known as FTS, can treat immune deficiencies such as multiple sclerosis, Guillain-Barre syndrome, inflammatory neuritis, multiple sclerosis, and other immune-mediated demyelinating diseases. They found that it is suitable as a therapeutic agent for various diseases associated with the disease, and provided such a therapeutic agent (Japanese Patent Application Laid-Open No.
2225).

In addition, the present inventors have provided a preventive/therapeutic agent for diseases and disorders caused by infection with various viruses, which contains this nonapeptide or its derivative as an active ingredient (Japanese Patent Application No. 63-294678).

The present invention further provides a prophylactic/therapeutic agent for diseases caused by herpes virus infection, which contains this nonapeptide or a derivative thereof as an active ingredient.

The fact that the nonapeptide known as FTS has prophylactic and therapeutic effects against diseases caused by infection with human herpesviruses such as herpes simplex virus-I, II, Epstein-Barr virus, and various animal-transmitted herpesviruses has been proven from the prior art. This is completely unexpected and was discovered for the first time by the present inventors. Thus, the present invention provides a nonapeptide pGIu-Ala-Lys-Ser-G In-GI having the following amino acid sequence:
y-Gly-5ar-Asn or an ester, amide, or a pharmaceutically acceptable salt thereof at the carboxyl group of asparagine at the C-terminus as an active ingredient. Prevention of herpesvirus disease.・Provides therapeutic agents.

The above-mentioned 7-napeptide used in the present invention can be produced without difficulty by a liquid phase or in-phase peptide synthesis method commonly used for peptide synthesis (
Regarding these methods, please refer to Japanese Patent Application Laid-Open No. 16425/1983, USP 4301065). Alternatively, it can also be prepared by genetic engineering or cell engineering techniques.

The ester at the carboxyl group of asparagine at the C-terminus of the nonapeptide used in the present invention is
pharmaceutically acceptable esters of carboxylic acids;
Examples include methyl ester, ethyl ester, flobyl ester, isopropyl ester, n-butylgostyl, isobutyl ester, tert-butyl ester, n-pentyl ester, isopentyl ester, neopentyl ester, tart-pentyl ester,
-hexyl ester, 5ec-hexyl ester, heptyl ester, octyl ester, 56C-octyl ester, tert-octyl ester, nonyl ester, decyl ester, undecyl ester, dodecyl ester, tridecyl ester, tetradecyl ester,
Hexadecyl ester, octadecyl ester, nonadecyl ester, eicosyl ester, cyclopentyl ester, cyclohexyl ester, cyclohegetyl ester, cyclooctyl ester, allyl ester, impropenyl ester, benzyl ester, o, m-1 or p-chlor Benzyl ester, O-, m% or p-fluorobenzyl ester, o-, m-1 or p-
Bromobenzyl ester, O-, lTl-1 or p-iodobenzyl ester, o-1m-, or p-methylbenzyl ester, o, m-1 or p-ethylbenzyl ester, o-1m-1 or p-isopropyl benzyl ester, cinnamyl ester, amide/ethyl ester,
0-lm- or p-aminobenzyl ester, 0-1
m-1 or p-nitrobenzyl ester, 0-1m-1
or p-methoxybenzyl ester, 0-, m-, or p-ethoxybenzyl ester, 0-1m-1 or p-
aminophenethyl ester, σ-furfuryl ester,
Examples include σ-chenyl methyl ester, α-pyridyl methyl ester, a-pyridylethyl ester, piperidinomethyl ester, a-piperidyl methyl ester, morpholinoethyl ester, and a-morpholinyl methyl ester.

Further, the amide in the carboxyl group of asparagine at the C-terminus of the nonapeptide used in the present invention is a pharmaceutically acceptable carboxylic acid amide,
Examples include amide, methylamide, ethylamide, pro-butylamide, isopropylamide, n~butyramide, inbutyramide, tert-butyramide, n
-Pentylamide, isopentylamide, neopentylamide, LerL-pentylamide, n-hexylamide, 5ec-hexylamide, heptylamide, octylamide, 56C-octylamide, tart-octylamide, nonylamide, decylamide, undecylamide , dodecylamide, tridecylamide, tetradenylamide, hexadecylamide, octadecylamide,
Nonadecylamide, eicosylamide, cyclopentylamide, cyclohexylamide, cyclohebutylamide, cyclooctylamide, allylamide, isopropylamide, benzylamide %0-1m+, or p-chlorobenzylamide, o-1m-1 or p-fluor benzylamide, '''-, m-% or p-bromobenzylamide, o-1m-1 or p-iodobenzylamide,
o-1m-1 or p-methylbenzylamide, o-
1m- or p-ethylbenzylamide, O% m
% or p-inpropylbenzylamide, cinnamylamide, aminoethylamide, o-, m+, or p-aminobenzylamide, O-5m-% or p-nitrobenzylamide % O-% m-% or p- Methoxybenzylamide, Os m s or p-ethoxybenzylamide, Os m-1 or p-aminophenethylamide,
σ-furfurylamide, a-thenylmethylamide, σ
-pyridylmethylamide, a-pyridylethylamide,
Piperidinomethylamide, α-piperidylmethylamide, morpholinoethylamide, α-morpholinylmethylamide, methoxycarbonyl-(σ-mercaptomethyl)
Examples include methylamide, etquincarbonyl-(σ-mercaptomethyl)methylamide, and the like.

Further, the above-mentioned pharmaceutically acceptable salts include acid addition salts at the amino group and base salts at the carboxyl group of HE nonapeptide. Examples of M addition salts include polysalts with organic or inorganic acids, such as formic acid, acetic acid, propionic acid, trifluoroacetic acid, tartaric acid, fumaric acid, malic acid, maleic acid,
Salts with carboxylic acids such as oxalic acid and naphthoic acid; salts with sulfonic acids such as methanesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid; salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; can be given.

Examples of the above-mentioned base salts include salts with inorganic bases, such as alkali metal salts, alkaline earth metal salts, ammonium salts, and salts with organic bases, such as amines. salt, sodium salt,
Examples include potassium salt, calcium salt, ammonium salt, triethylamine salt, ethanolamine salt, tris salt, dicyclohexylamine salt, and the like.

The prophylactic/therapeutic agent for diseases caused by herpesvirus infection according to the present invention is prepared using conventional formulation methods depending on its dosage form. That is, the active ingredients selected from the above-mentioned 7 nabebutide, its ester or amide derivatives, or their salts are prepared in an appropriate dosage form using a pharmaceutically acceptable carrier, excipient, diluent, etc. niR
M. The dosage form can be various dosage forms suitable for administration routes such as external use, oral administration, parenteral administration, etc.

The effects of the prophylactic/therapeutic agent according to the present invention have been confirmed by the following experiments. That is, using ICR mice of each week of age, herpes simplex virus (he
rpes simpler virus.

Hereinafter, it will be abbreviated as H5V. ) was intraperitoneally inoculated to cause infection, and a predetermined amount of the active ingredient was administered parenterally or orally before and after the day of infection, and the survival or death of the mice was observed orally. The results of this experiment showed that all or almost all of the control group mice died 4 to 5 days or 9 to 10 days after virus infection, whereas the mice in the drug administration group according to the present invention died on each evaluation day. All cases survived or the number of deaths decreased dramatically, symptoms became milder, and the date of death was significantly delayed. Also,
Even in animals such as mice and rabbits that had been induced with corneal herpesosis, lacrimal herpesosis, or herpes encephalitis, remarkable improvement effects on symptoms and prevention effects on recurrence were observed in the group administered with the drug according to the present invention.

Sarah was given acyclovir or arabinofuranosyladenine (Ar), which are commercially available anti-herpesvirus drugs.
When the above-mentioned 7napeptides or their derivatives were simultaneously administered parenterally or orally to mice administered with aA), it was observed that the number of mouse deaths further decreased or the date of death was delayed. Ta.

Thus, it has been revealed that the above-mentioned 7nabeptide or its derivatives have a herpesvirus inhibiting effect when administered alone, and also have an effect of enhancing the anti-herpesvirus effect of chemotherapeutic agents and a combined effect.

In order to examine the toxicity of the active ingredient in the drug according to the present invention, 1001 g/9 of the active ingredient was subcutaneously administered to mice every day for 14 days, but no abnormalities were observed in appearance. In addition, the active ingredient substance 3
01g/hg was administered subcutaneously for 21 days, but no abnormalities were found in appearance, serum biochemistry, or pathological autopsy. As described above, the drug according to the present invention is a safe drug with extremely low toxicity, and can be administered for a long period of time.

Examples of target animals to which the drug according to the present invention can be administered include humans, livestock such as cows, horses, pigs, sheep, goats, rabbits, dogs, and cats, lions, elephants, giraffes, bears, gorillas, monkeys, Examples include mammals kept in zoos such as chimpanzees, mice, rats, guinea pigs, and other experimental animals, poultry such as chickens, birds for beds, reptiles, amphibians, and fish. The dosage is usually 0 per kg of body weight in these animals.
．． 1 μg to 50011 g/day, these are e.g.
The drug may be administered in divided doses from once to six times a day, and the dose can be increased or decreased as appropriate depending on the age, medical condition, etc. of the subject. The route of administration is not particularly limited, but intravenous, intramuscular, subcutaneous, or subcutaneous injections are also possible.

In addition, by preparing an ointment, it can be applied to the eyes, oral cavity, nasal cavity, skin, etc.;
It can also be administered as eye drops, nasal drops, nasal or oral absorption preparations, aerosols, sprays, oral preparations, and the like. In order to prevent the active ingredient from being rapidly degraded or inactivated in the body, the active ingredient may be mixed with a suitable formulation ingredient, such as an alcoholic, oily or fatty solid such as lecithin, or a physiologically harmless solid. It is also possible to prepare a formulation using liquid materials, suspensions thereof, liposomes, etc., and the activity can be maintained for a long time.

The drug according to the present invention can be used in combination with other drugs, such as chemotherapeutic agents with antiviral effects, antibiotics, vaccines for various viruses, various drugs such as antibodies, or biological res-ponse modifiers such as immunostimulants.
fier (BRM), or they can be added to the formulation to enhance clinical efficacy as a combination drug. Examples of the above chemotherapeutic agents include acyclovir, arabinofuranosyl adenine (AraA), arabinofuranosyl thymine (^raT), and the like.

The present invention will be explained in more detail by Examples and Experimental Examples below, but the present invention is not limited by these. In the examples, FTS stands for the nonapeptide described above.

Example 1 Vial preparation for injection FTS-CH, C0OH・2H20 (manufactured by Mitsui Pharmaceutical Industries, Ltd.) 1119 was dissolved in distilled water, passed through a sterilized mouth, filled into a vial, and freeze-dried.

Example 2 Ampoule preparation for injection FTS-CH3COOH・2oto (manufactured by Mitsui Pharmaceutical Industries, Ltd.) 5IIIg was dissolved in physiological saline, passed through a sterilized mouth, and filled into the ampoule.

Example 3 Subcutaneous injection FTS-C! (, C0OH・2Hio (manufactured by Mitsui Pharmaceutical Industries, Ltd.) per unit dose of 2TR9 was suspended in a 2% carboxymethyl cellulose PBS (phosphate buffered saline) solution, and L i pom consisting of soybean phosphatide was prepared.
a l(Huhtamaki Oy/Leiras P
pharmaceuticals) or intravenous oil-in-water emulsion Intralipid (Cut
er Labora (manufactured by Ories). L
When using i poma l, FTS-dissolved PBS
Equal amounts of the solution and Lipomal were mixed together.

When using InLralipid, FTS-dissolved PB
S solution'1.5mQ1Tveen 80 (Sigma
Chemicals) 0.11112 and Int
ralipid 4.6m (! mixed together.

Example 4 Liposome preparations There are three types of liposome preparations with different charges, and these are further classified into 4a1 type based on their structure.

There are three types of charges: neutral, positive, and negative, and the structure is that of multilamellar liposomes (MLV).
vssic le), small unilamellar liposomes (SU
V.

small unilamellar vesicle
), and large unilamellar liposomes (LUV, lar
ge unilanallarvesicle), and a structure similar to LUV but with several membranes (REV
, reverse-phase evapora-t
Four types of ion vesicles are known.

■ Neutral charge liposome encapsulated with FTS A chloroform solution of phospholipid such as phosphatidylcholine sphingomyelin and cholesterol was mixed at a molar ratio of 2:1, 4:1, or 2:1, and the solvent was once distilled off under reduced pressure. l/1 relative to the amount of fat
00-1/1000 equivalent of FTS in PBS (!J acid buffered saline) solution was added and Vortex tar
After thorough mixing using an xer, MLV was obtained.

Furthermore, sUv was obtained by ultrasonication at a temperature above the phase transition temperature of phospholipids (C). A calcium chloride aqueous solution was added to the obtained SUV, and it was incubated at 37°C for 1 hour to fuse. After that, add EDTA and heat at 37°C.
After incubating for 30 minutes to remove Ca'', L[I
V was obtained.

The method for preparing REV is as follows. That is, after removing the solvent from a chloroform solution of lipids under reduced pressure, an appropriate amount of diethyl ether was added to fully dissolve the solution, and FTS (
7) A PBS solution was added and subjected to ultrasonic treatment, resulting in a homogeneous single-phase solution. After the obtained solution was concentrated under reduced pressure at room temperature, a PBS solution was added and thoroughly mixed using a Vortex m1xer to obtain REV.

(2) FTS-encapsulated positively charged liposomes The preparation method is the same as that for the above-mentioned neutrally charged liposomes, except that the constituent components of the lipids are different.

Phosphatidylcholines, phospholipids such as sphingomyelin, cholesterol, and positively charged higher aliphatic amines such as stearylamine in a molar ratio of 7:2:l or 4.
:l:l to obtain a lipid component, and FT in the same manner.
S was enclosed.

■ F Ding S Fujin negative! The loaded posomes are mixed with phospholipids such as phos-7 atidylcholines, sphingomyelin, cholesterol and negatively charged higher aliphatic esters such as dicetyl phos-7ate and sulfatide in a molar ratio of 7:2:1 or 4:1:I. , as a lipid component, and FTS was encapsulated in the same manner.

Example 5 Ointment FTS-CH, C0OH・2LO (manufactured by Mitsui Pharmaceutical Industries, Ltd.)
2TRg was dissolved in purified water. Next, 25g of white petrolatum
, 20 g of stearyl alcohol, HCO-6049 and 1 g of glyceryl monostearate were taken and mixed to add 12 g of propylene glycol prepared in advance, methyl hydroxybenzoate O, h, propyl hydroxybenzoate 0.
1 g of an aqueous solution (containing FTS) was added and thoroughly mixed to obtain an emulsion, which was then mixed by continuing the mixing operation until it cooled and solidified.

Example 6 Suppository FTS-CJCooH・2HzO (manufactured by Mitsui Pharmaceutical Industries, Ltd.)
10 m9 was dispersed in a pre-warmed hard 7at, making the total amount 29.

Example 7 Nasal capsule FTS O,05 9 was prepared under sterile conditions.
Migdiol 812 neutral oil (manufactured by Dynamite Nobel). The solution is filled into a conventional unit dose dispenser and placed into the drive capsule before use.

Example 8 Nasal Drop The following amounts of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and EDTA-disodium salt were dissolved in distilled water at room temperature. This solution 1: FTS was dissolved and filtered through a membrane filter.

FTS 0.10tsg
Phosphoric acid-sodium hydrogen 2H! 0 00-30a
Sodium dihydrogen phosphate・12HzO10,10mg Falconium bechloride 0.10mg Sodium chloride 4.501119 Steamed
Water 987.601
++9p11 value 5.0±0.3 Example 9 Nasal spray formulation FTS-CH3COOH-2H,O (manufactured by Mitsui Pharmaceutical Industries, Ltd.) 2 mg was suspended in hydroquinopropyl cellulose or hydroxypropyl methyl cellulose and sprayed with a spray formulation machine. It was made into a spray agent.

Examples of pharmacological experiments and toxicity experiments regarding the drug of the present invention are listed below.

Experimental example 1 Herpes simplex virus type l (HSV-
1) Effect on infected mice Groups of 10 to 20 male ICR mice, 4 weeks old, were prepared. HSV-I CHR-3 strain with various titers was injected into mice 1.
0.5 rQ per animal was administered intraperitoneally (viral titer: 5X 10' PFU/mouse ~ 5X 10' PF
U/fish).

(14 mice in the active ingredient administration group) F
TS was administered at the beginning. That is, 2 days before and 1 day before infecting mice with H5V-1, and the day of infection (day 0), 1 day after infection, 2 days after infection, and 4 days after infection, or the day of infection, 1 day after infection, and 1 day after infection. F on day 2 and day 4 post-infection.
TS was administered.

In addition, to the mice in the control group, only physiological saline was administered intraperitoneally on a schedule similar to that of FTS administration.

The survival and death of the mice were observed daily for 10 days after infection. The results are shown in Table I.

When administered to H5V-1 infected mice, both intracavitary and oral administration of FTS delayed the death date of the mice and reduced the number of deaths compared to the control group. Thus, it was revealed that FTS, when administered alone, has a life-prolonging effect and a death-preventing effect on H5v-1 infected mice.

Similar effects were confirmed when 2-week-old or 6-week-old mice were infected with different virus titers.

Experimental Example 2 In the same manner as Experimental Example 1, H5 was administered to 4-week-old male ICR mice.
II, and each dose of acyclovir was administered intraperitoneally on day 1, day 2, and day 4 postinfection.

Furthermore, each dose of FTS shown in Table 2 was administered intraperitoneally or orally at the same time as acyclovir administration, and FT
The effect of combined use of S with acyclovir was investigated. The results are shown in Table 2.

As shown in Table 2, FTS can be administered in combination with
It was shown to potentiate the effects of acyclovir alone. Thus, when FTS is used in combination with a chemotherapeutic agent to reduce the dose of the chemotherapeutic agent, which generally has a relatively high incidence of side effects, the dose is equivalent to the administration of the chemotherapeutic agent alone.
It becomes possible to obtain the effect.

Experimental Example 3 Effect on humans infected with herpes When the ointment preparation prepared in Example 5 was applied for one day to the rash around the mouth of a human infected with OSV-I (a 36-year-old male), 5 days after the start of administration. The symptoms improved.

Experimental Example 4 1B sex test 5 week old male mice of the ddY strain were given 5 mice per group as active ingredients.
0+*9/b9 and 1ook/kg each day 14
The drug was administered subcutaneously for several days, but no toxicity was observed.

Experimental Example 5 Toxicity Test 30rs9/kg of the active ingredient was subcutaneously administered to 10 Wistar rats of 5-week old group every day for 21 days, but no toxicity was observed.

The drug according to the present invention has excellent preventive and curative effects against diseases caused by infections such as single herpes virus (Harpes 5ivaplex virus), which could not be obtained by conventional vaccines or chemotherapeutic agents alone, with almost no side effects. It is revolutionary in that it is expressed by activating the host's immune system or biological defense system. In addition, the nonapeptide (FTS) used in the present invention is an animal-derived bezutide and is a natural substance, so unlike FTS analogs (analobes) with similar amino acid sequences reported in the literature, it is It is also completely non-toxic, and there are no problems such as antigenicity or anaphylactic shock. Therefore, the drug according to the present invention can be used as a safe and useful medicine for humans and animals.

Claims (1)

[Claims] 1) The following amino acid sequence pGlu-Ala-Lys-Ser-Cln-Gly-
A disease caused by infection with a herpesvirus characterized by containing a nonapeptide having Gly-Ser-Asn, or an ester, amide, or a pharmaceutically acceptable salt thereof at the carboxyl group of asparagine at the C-terminus as an active ingredient. preventive and therapeutic agent. 2) The above virus is herpes simplex virus-I,
The prophylactic/therapeutic agent according to claim 1, which is a human herpesvirus such as II, Epstein-Barr virus, or various animal-infected herpesviruses.