JP2769114B2 - Pharmaceutical composition for treating minimal hepatitis B infection - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to minimal hepatitis B (min)
B) The present invention relates to a pharmaceutical composition for treating infectious diseases. More specifically, the present invention provides a hepatitis B virus
("HBV"). More particularly, the present invention relates to a pharmaceutical composition for treating a minimal type B hepatitis carrier using thymosin α-1, or a biologically active fragment or analog of thymosin α-1.

[0002]

BACKGROUND OF THE INVENTION HBV infection is the second most common infection worldwide, with various pathologies caused by infection with the hepatitis B virus. Hepatitis B virus ("HBV") is a DNA virus. HBV is known to be transmitted through blood transfusions, contaminated hypodermic needles and sexual intercourse, as well as from mother to child (vertical transmission). Many people are infected by unknown means.

A description of the hepatitis B virus and its modes of transmission, as well as the pathologies associated with HBV infection, can be found in the following literature: for example, JH Hoofnagle (1990). New England Journal of Medicine (New Eng)
land Jour. Med.), Volume 323, pp. 337-39;
JH Hoofnagle
(1990a), Vaccine, Volume 8, Sup
pl .; H. Popper et al. (1987), Hepatology, 7, 762-72.
P .; Zakim and Boyer, eds., (19)
90), Hepatology, a Textbook of River Disease
r Disease), WW Sauders Co., 2nd edition; FB Hollinger et al. (1991), Viral
Viral Hepatitis, Raven Press, 2nd edition.

[0004] In adults, about 5% of humans infected with HBV develop long-term infections. That is, they become the "carrier" of the virus. A very large number (perhaps 90%) of humans infected in the perinatal period, which is responsible for most hepatitis B infections worldwide, are carriers. About 5% of the world's population is a carrier of HBV, and many carriers are carriers. That is, they may transfer HBV infection to other humans through sex, through blood or blood products, or vertically (at birth).

[0005] A variety of clinically distinct pathologies can be exhibited in humans infected with hepatitis B, including chronic and minimal hepatitis. About 50% of humans infected with HBV
Show chronic inflammatory changes in the liver, and about 50% of them
It has histopathological changes that can induce fibrosis and eventually cirrhosis and progressive liver failure (referred to as "chronic active hepatitis").

[0006] Carriers of HBV that do not have chronic inflammatory changes in the liver may develop chronic active hepatitis;
And about 10% to 30% of humans infected with HBV
Develop liver cancer. It is estimated that about 4 million hepatitis B carriers die each year from liver cancer or cirrhosis.

[0007] Persistent infections in individuals infected with chronic hepatitis are due to an incomplete or physiologically immature immune response that impairs the ability to clear the virus. Although the mechanisms responsible for liver damage are poorly understood, in most individuals it is likely that such damage results from stroke by the body's immune system on infected hepatocytes rather than from liver destruction by HBV. Cytotoxic T cells appear to be responsible for immune-mediated liver damage. The balance between suppression of immune system activity against normal tissues and the immunological response prepared against the virus appears to be compromised in chronic hepatitis B. Many specific immunodeficiencies, including defective production of α-interferon by HBV-infected hepatocytes and inhibition of cytotoxic T cell responses, have been described in chronic hepatitis [M. Peters et al. 19
May 1991), Hepatology (United States)
(Hepatology (United States)), Vol. 13, No. 5,
977-94].

[0008] Patients with severe chronic active hepatitis account for a very small percentage of the total number of people whose liver biopsy is judged to exhibit chronic active hepatitis.

[0009] Approximately 40% of hepatitis B carriers account for "minimal disease", often referred to as "asymptomatic" hepatitis.
l disease). Minimal disease carriers have detectable hepatitis B antigen or viral DNA in their sera and have no clinical signs or symptoms of liver damage, or almost no clinical signs of liver damage. Or a person who has only symptoms.
Minimal disease carriers develop chronic active hepatitis and, as noted above, can induce cirrhosis with liver failure. Clinical progression is slow in asymptomatic hepatitis, and the factors contributing to the development of active disease in patients initially listed as minimal disease carriers are currently unknown.

[0010] The reason why some people infected with HBV do not have inflammation or liver damage, while others do, is currently not understood. These are 2
It appears to represent a type of medical condition. In the minimal state, the carrier does not show any obvious signs of immunological imbalance, as evidenced by the lack of readily observable destruction of liver tissue.

Although there is currently no effective treatment for minimal disease carriers, it is highly desirable to treat them before such carriers exhibit more severe forms of the disease or transfer them to others. . In addition, minimal disease carriers are eligible for treatment without relying on liver biopsy, as the condition of the carrier can be diagnosed by measuring hepatitis B antigen or HBV DNA in the blood. B
Although vaccines are available to prevent hepatitis B, they are not used on a large scale and do not alleviate disease in living carriers. Thus, the need for effective treatment of individuals suffering from hepatitis B virus infection, even when existing vaccines are given on a large scale,
Due to the large number of carriers and the highly susceptible nature of the disease, it will be maintained for at least several decades.

Thymosin α-1 is a 28 amino acid peptide. This peptide, originally isolated from bovine thymus thymosin fraction 5, is one of several polypeptides present in thymosin fraction 5, which participate in the regulation, differentiation and function of thymus-derived lymphocytes (T-cells). It is. The isolation, characterization and use of thymosin α-1
For example, it is described in U.S. Pat. No. 4,079,127.

Although the mechanism by which thymosin α-1 mediates its effects is unknown, evidence suggests that thymosin α-1.
Seems to function through regulation of the immune system. Thymosin alpha-1 has been found to be responsible for the maturation of lymphocytes, increases T-cell function and promotes the reconstitution of immune deficiencies [TLK Lowe (TLK. Low) et al.
(1984), Thymus, Vol. 6, No. 27-4.
2 pages]. Thymosin has been found to be responsible for increased lymphocyte counts and enhanced production of γ-interferon in individuals suffering from chronic active hepatitis B [M.
MG Mutchnick et al. (1991),
Hepatology, Vol. 14, No. 409-1
5 pages].

[0014] MG Matchnick (MG Mutchn)
(ick) et al. (1991) disclose the use of thymosin alpha-1 in the treatment of chronic active hepatitis B, ie, in patients with evidence of liver damage on biopsy. Patients to which the peptide was administered were converted from serum to hepatitis B virus DNA
Were cleaned and tested negative for serum HBV DNA after treatment was completed. Asymptomatic carriers without histopathological evidence of liver injury were not included in this study.

The present inventors have developed thymosin α-1 and biologically active fragments and analogs of thymosin α-1 with minimal B
It can be used to treat hepatitis B infection. That is, by administering an appropriate amount of thymosin α-1 to a patient suffering from minimal B-type hepatitis, the parameter characteristics of minimal B-type hepatitis in the patient can be reduced.

[0016]

The present invention relates to (1) at least one thymosin α-1, a biologically active fragment of thymosin α-1, and a biologically active analog of thymosin α-1. Minimal B, comprising an effective therapeutic amount of the peptide and a pharmaceutically acceptable excipient
(2) the pharmaceutical composition according to (1), wherein the peptide is thymosin α-1, and (3) the pharmaceutical composition, wherein the peptide is a biologically active fragment of thymosin α-1. )
And (4) the peptide is thymosin α-1.
(5) the pharmaceutical composition according to (1), which is for subcutaneous administration, and (6) the amount of peptide per 1 m ^{2 of} body surface area. 60
The pharmaceutical composition according to the above (1), wherein the amount of the peptide is from about 60 to about 3000 μg per 1 m ^{2 of} body surface area;
0 to about 1500 μg of the pharmaceutical composition according to the above (1), (8) a dose of about 90 peptide / m ^{2 of} body surface area.
0 to about 1200 μg of the pharmaceutical composition according to the above (1).

The present invention further provides for the use of thymosin alpha-1 (and biologically active analogs and fragments of thymosin alpha-1) in a pharmaceutical composition for the treatment of minimal hepatitis B hepatitis, ie, seropositivity for HBsAg. Test and / or test positive for HBV DNA in serum but are asymptomatic and show no biochemical or histopathological evidence of liver disease (or very weak biochemical (Indicating only statistical or histopathological evidence).

Treatment of such a minimal carrier of hepatitis B virus results in the removal of HBV from the subject as evidenced by the loss of HBV DNA from the serum, whereby the subject no longer has active hepatitis. Or the risk of developing liver cancer or HBV in other humans
The danger of transferring can be eliminated.

Accordingly, in one embodiment, the present invention provides a method for treating thymosin alpha-1, in a pharmaceutically acceptable excipient, comprising:
A biologically active fragment of thymosin alpha-1, and thymosin alpha-1
Treating a minimal hepatitis B infection in a subject by administering to the subject a composition comprising at least one peptide selected from the group consisting of:

In a particularly preferred embodiment, the peptide is
Thymosin alpha-1, and the composition is administered subcutaneously.

[0021] These and other embodiments of the present invention will be readily apparent to those skilled in the art in light of the description herein.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of protein chemistry, molecular biology, microbiology and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. For example, Scopes, RK, Protein Purification Principles and
Practice (Protein Purification Principles an
d Practice), 2nd edition, (Springer-Blaglag (Spr
inger-Verlag) 1987); Methods in Enzymology (S. Colowick and N. Kaplan).
Hen, Academic Press Inc. (Ac
ademicPress, Inc.); Sambrook, Fritsch & Maniati
s), Molecular Cloning: A Laboratory
Manual (Molecular Cloning: A Laboratory M
anual), 2nd edition (Cold Spring Harbor Laboratory Press)
ry Press) 1989); Oligonucleotide Synthesis (MJ Gait, eds., 1984);
Of ixpermental immunology (Handbook o
f Experimental Immunology, Volumes I-IV (Edited by DM Weir and CC Blackwell), 1986, Blackwell Scientific Publications (1986) Blac
kwell Scientific Publications).

[0023] All patents, patent applications and publications mentioned herein are cited herein.

Definitions The following terms are used herein and are defined as follows:

The term "minimal hepatitis B hepatitis" refers to having hepatitis B serum antigen or having HBV DN in serum.
Hepatitis B defined as having A but having normal or slightly elevated serum enzyme levels, or having no or very little biochemical or histopathological evidence of liver disease Means the condition present in the carrier. Minimal disease states are easily understood and identifiable by a skilled practitioner.

As used herein, “thymosin α-
"1" indicates a 28-mer described below with or without an N-acetyl group, a biologically active fragment of this sequence substantially corresponding to the peptide sequence shown below, and a biological activity of the sequence. It is meant to include analogs (ie, deletion, substitution and addition mutants).

A peptide "substantially identical" to thymosin alpha-1 has at least about 30% of the amino acids, preferably at least about 85% to 90%, and most preferably at least about 95%, Over the following sequence.

A "biologically active" fragment or analog of thymosin alpha-1 retains a significant amount of the activity of the natural molecule, that is, it is capable of expressing serum HBV DNA and / or hepatitis B surface antigens as further described below. Can be reduced,
It is a fragment or analog of thymosin α-1.

As used herein, the term "treatment" refers to (i) preventing (preventing) infection or re-infection, or (ii) reducing or eliminating (treating) an indicator of minimal B-type hepatitis. Show.

An "effective therapeutic amount" of thymosin alpha-1 is the amount of a peptide capable of altering a measurable parameter of a hepatitis infection. Since patients falling within the category of minimal disease have normal liver enzyme biochemistry, the parameters usually monitored are serum hepatitis B envelope antigen (HBe
Ag) and serum viral DNA (HBV DNA). Response is defined as a significant decrease in any of these parameters. The amount of the peptide capable of eliciting a response is considered an "effective therapeutic amount". HBV DNA was obtained from MG Mutchnick et al.
1), Hepatology, Vol. 14, No. 409
-15 page and H.M. Lieberman (HMLi)
eberman) et al. (1983), Hepatology,
3, pages 285-91. Alternatively, the presence of HBV DNA in the blood can be measured using standard PCR techniques. See, for example, U.S. Patent Nos. 4,683,202 and 4,683,194, which are incorporated herein by reference. Serum HBeAg levels are, for example, MG
Matchnick, MG (M.G.Mutchnick, MG)
(1991), Hepatology, 14th.
Vol. Pp. 409-15.
Or can be monitored by standard ELISAs.

General Methods The invention relates to the use of thymosin alpha-1 for the treatment of minimal hepatitis B infection. Thymosin alpha-1 is one of several peptides present in thymosin fraction 5 from the thymus. The natural molecule is a 28-mer with the following amino acid sequence:

Ac-Ser-Asp-Ala-Ala-Val-As
p-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-
Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-V
al-Glu-Glu-Ala-Glu-Asn-OH.

Thymosin α-1, and fragments and analogs thereof, are readily synthesized using standard methods of peptide synthesis known to those skilled in the art. US Patent 4,1
48,788 and 4,844,407 disclose liquid and solid phase synthesis of thymosin alpha-1, respectively, which are incorporated herein by reference. For a discussion of solid phase peptide synthesis methods, see JD Young, Solid Phase Peptide
Solid Phase Peptide Synthesis, 2nd Edition (Pierce Chemical Company)
l Company) 1984); and G. Barany
any) et al., The Peptidedis: Analysis, Synthesis,
Biology (The Peptides: Analysis, Synthesi
s, Biology), Volume 12 (E. Gross and J. Meienhofer, eds., Academic Press 1980); and for liquid phase peptide synthesis, M. Bodansky
(M. Bodansky), Principles of Peptides
Synthesis (Principles of Peptide Synthesis) (Springer-Verlag 1984);
And E. Gross, E. and J. Meienhofer, eds., The Peptidedis: Analysis, Synthesis, Biology (The P
eptides: Analysis, Synthesis, Biology), Volume 1
(Academic Press 1980).

[0034] Thymosin alpha-1 can also be isolated directly from a suitable tissue expressing thymosin alpha-1, using techniques readily known in the art. this is,
Generally, this is done by first preparing a crude tissue extract that lacks cellular components and some exogenous proteins. The thymosin α-1 can then be further purified, for example, by column chromatography, HPLC, immunoadsorption techniques or other conventional methods known in the art. U.S. Pat. No. 4,079,127 discloses a method for purifying thymosin alpha-1 from bovine thymus (cited herein).

Finally, thymosin alpha-1 and fragments or analogs thereof are produced recombinantly using methods well known to those skilled in the art. For example, Sambrook,
Fritsch and Maniatis (Sambrook, Fritsc
h & Maniatis), molecular cloning:
Laboratory Manual (Molecular Cloning: AL
aboratory Manual), 2nd edition (Cold Spring
Harbor Laboratory Press (Cold Springer Ha)
rbor Laboratory Press) 1989); and MJ Gate (ed., 1984), Oligonucleotise Synthesis.
See

Thymosin α-1, or an active fragment thereof, or an analog thereof, can be administered to a subject diagnosed or suspected of having minimal hepatitis B infection. Thymosin α-1 may be administered alone or in admixture with pharmaceutically acceptable excipients or excipients.

Typically, the thymosin alpha-1 composition is prepared for injection as a liquid solution or suspension: a solid form suitable for dissolution or suspension in a liquid vehicle prior to injection. May be prepared. Emulsify the preparation or
Alternatively, the active ingredients can be encapsulated in a liposome excipient. As mentioned above, the active ingredient is often mixed with excipients containing excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the excipient may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. For example,
Remington's Pharmaceutical Sciences (Mack Publishing Co.)
mpany), Easton, PA, 15th edition, 19
75). The composition or formulation to be administered is in each case determined from the serum of the subject to be treated by HBV
Contains an appropriate amount of peptide to reduce or eliminate DNA and / or HBsAg.

Injectable compositions are preferably administered by the subcutaneous route; intramuscular or intravenous injections are also acceptable. Injectable preparations contain an effective amount of the active ingredient in the excipient. The exact amount is readily determined by one skilled in the art. The active ingredient typically comprises about 1% of the composition
９５95% (w / w), higher or lower as desired. The amount to be administered depends on factors such as the age, weight and health of the subject to be treated. For the formulation of the present invention,
Thymosin α-1 per m ^{2 of} body surface about 600 to about 30
Of between about 400 and about 3200 μg / m ^{2 of} body surface area for the formulations of the present invention.
Or an amount between about 600 and about 1500 μg, or between about 900 and about 1200 μg is effective. Other effective doses can be readily established by those skilled in the art through routine trials by establishing dose-response curves.

Additional formulations suitable for other modes of administration include suppositories, and in some cases, aerosol, intranasal, oral and sustained release formulations. For suppositories, the excipient composition will include conventional binders and carriers such as polyalkaline glycols or triglycerides. Such suppositories are about 0.
5% to about 10% (w / w), preferably about 1% to about 2%
Formed from a mixture containing the active ingredients in the range of As an oral excipient, for example, pharmaceutical mannitol,
Commonly used excipients such as lactose, starch, magnesium, stearate, sodium saccharin cellulose, magnesium carbonate and the like can be mentioned. These oral compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 10% to about 95%, preferably about 10%, of the active ingredient. 25
% To about 70%.

Intranasal formulations generally include excipients that do not irritate the nasal mucosa or significantly interfere with ciliary function. Diluents such as water, saline or other known substances can be used in the present invention. Nasal preparations
Preservatives such as chlorobutanol and benzalkonium chloride can also be included. Surfactants may be present to enhance absorption of the major protein by the nasal mucosa.

The controlled release or sustained release formulations, liposomes, ethylene - such as non-absorbent impermeable polymers, swellable polymers such as hydrogels or collagen and certain polyacids, such as vinyl acetate copolymers and Hytrel ^R copolymers Formulated by incorporating the peptide into a carrier or excipient such as a polyester, such as those used to make absorbable polymers or absorbable sutures. The peptide can also be provided using an implanted minipump, well known in the art.

Further, the peptide may be formulated into a composition in a neutralized or salt form. Pharmaceutically acceptable salts include acid addition salts (formed at the free amino group of the active peptide), for example, inorganic salts such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, mandelic acid Formed with organic acids such as Salts formed from free carboxyl groups include, for example, inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxide, and isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine,
It may be derived from an organic base such as procaine.

The subject is treated by administration of the thymosin alpha-1 peptide, or a fragment thereof, or an analog thereof, in at least one administration. Preferably, thymosin α
-1 is administered once to several times a week for several weeks to several months. More preferably, the composition is administered once to seven times a week, most preferably twice a week, for at least about 3 months to about 12 months or more, more preferably for at least 6 months. Is done. The composition can be administered frequently in the early weeks and then less frequently in the later weeks (eg, two weeks daily, then biweekly thereafter). The progress of the treatment, as described above,
It can be monitored by measuring the presence of HBV DNA and / or HBeAg.

The following examples are specific examples relating to the implementation of the present invention. The examples are provided for illustrative purposes only and in no way limit the scope of the invention.

[0045]

【Example】

Example 1 This example describes an illustrative protocol used to clinically test the efficacy of thymosin alpha-1 or a biologically active analog or fragment of thymosin alpha-1 in the treatment of minimal hepatitis B hepatitis. I will provide a. The study involves 33 patients each divided into three groups.

Criteria for Subject Selection The candidates included in the test are men and women between the ages of 18 and 64 at the start of the test. The subject is selected for inclusion in the study using criteria established to maximize safety and ensure a link between the subject's symptoms and its course of treatment.

There are some restrictions on subject inclusion. Included subjects were seropositive for hepatitis B surface antigen ("HBsAg") for at least 6 months prior to the start of the study. Subject serum alanine aminotransferase
("ALT") levels are no more than 2.0 times the upper normal limit, as determined according to the description below. The subject was "HBeA
g "is seropositive and has HBV DNA in serum as determined as described below.

The subjects included were hematocrit ≧ 3
0%, platelet count ≧ 80 × 10 ⁹ / L, leukocyte count ≧ 3 × 1
0 ⁹ / L and polymorphonuclear leukocyte count ≧ 1.5 × 10 ⁹ / L. Kidney function, serum creatinine ≤180μ
at least normal as indicated by the mol / L. Prothrombin time is less than 5 seconds relative to controls, serum albumin ≧ 30 g / L, bilirubin ≦
68 μmol / L. Included subjects have no history of hepatic encephalopathy or hemorrhagic esophageal venous vein. Liver biopsies of included subjects show either "minimal lesions" or "chronic persistent hepatitis".

A candidate is excluded from the test if he has any of several indicators.

The most recently tested serum HBV DNA
Serum HBV DN before the start of the study as indicated by the level being less than 50% of the level previously tested
If any reduction in A-level progression is noted, the candidate is excluded. After at least one month, further testing is performed and if serum HBV DNA levels are stable, such candidates may be included.

[0051] Candidates are identified if they have been receiving interferon or any other type of immunotherapy within one year prior to the start of the study, or have used corticosteroids within six months prior to the start of the study. exclude. Toxic hepatitis,
Evidence for alcoholic liver disease, Wilson's disease, primary biliary cirrhosis, hepatitis C, or hepatitis C virus ("H
CV "), delta hepatitis, hemochromatosis, or autoimmune hepatitis, if there is evidence of the presence of antibodies;
Alternatively, if diagnosed by HIV seropositivity and has an HIV infection confirmed by Western blot; or a malignancy other than a curatively treated skin cancer or a surgically treated neck cancer in situ. If there is a concomitant medical history or previous history of the disease; or if there is any active infectious process other than HBV which is not self-limiting such as tuberculosis "TB" or acquired immunodeficiency syndrome "AIDS". Or rheumatoid arthritis or other autoimmune disease, or serum ANA>
If it has a 1:40, exclude the candidate. Have a history of intravenous substance abuse within 5 years prior to study initiation;
Alternatively, if any drug known to be hepatotoxic is additionally used, the candidate is excluded.
Excludes women who showed pregnancy by urine pregnancy tests and includes female subjects who have agreed to restrict birth during the study.

Those who have a low risk of drug or psychiatric illness,
Or, even if the subject has any non-malignant systemic symptoms, the person conducting the test states that the candidate is unlikely to be able to complete the protocol, or the person conducting the test is Anyone who makes an opinion that the protocol conditions cannot be met is excluded from the candidates.

Screening Step Preferably, the subject is a candidate screened from a large number of candidates according to the following scheme.

In general, a candidate is identified as being suitable for treatment according to the present invention by a pattern of criteria for minimal hepatitis B hepatitis. In particular, preferred screening protocols include primary and secondary screening diagnoses that are separated by 4 weeks to 2 months, and the first two diagnoses may not be clear for inclusion in the test. Shall perform a tertiary screening diagnosis between 4 weeks and 2 months after the second diagnosis. Candidates for treatment meet the criteria set forth above.

With respect to serum ALT for subject restriction, preferably, serum ALT levels are above the upper normal limit of 2.50 in each of the primary and secondary screening diagnoses.
0-fold or less; or alternatively, the mean of serum ALT from three screening diagnoses is 2.0 above the upper normal limit
It is assumed that it is less than twice.

HBeAg and H for subject restriction
For BV DNA, the test result must be positive in each of the first two screens. If the HBV DNA level in the second diagnosis is less than 50% of the first diagnosis level, to include the candidate,
The third diagnostic level must be at least 50% of the first.

Liver biopsies are preferably evaluated by the so-called Knodell scale [Knodell et al., (1)
981), Hepatology, Volume 4, Volume 5
No., page 434 and subsequent pages]. The majority of subjects with minimal B-type hepatitis as defined above have a total Knodel score ("HAI" or "Histological Activity Index") of less than 4, preferably 3 or less or 2 or less. As indicated herein, candidates with a HAI of 4 or more must not be properly classified as having minimal hepatitis B hepatitis.

For the treatment study, individual subjects are divided into thirds. Group 1 subjects are administered thymosin alpha-1 over a 6 month treatment period and then observed for 6 months.
A second group of subjects is administered thymosin alpha-1 over a treatment period of 12 months and then observed for 6 months. Subjects in the third group receive no treatment and are observed for 12 months.

Following the observation period, the HB of the third group of subjects
Subjects whose V DNA status is checked and who are still HBV DNA positive will be randomly subjected to a 6 or 12 month treatment period. That is, after the first 12 months of observation, subjects in the third group are divided into thirds. Subjects in Group 3A (HBV DNA negative at 12 months) receive no treatment. Subjects in Group 3B are administered thymosin alpha-1 over a 6 month treatment period and then observed for 6 months. Subjects in group 3C are administered thymosin alpha-1 over a 12 month treatment period and then observed for 6 months.

The subject's liver function and hepatitis B markers are examined every month during the treatment period and every three months during the observation period. Preferably, each subject will undergo a liver biopsy at the end of the treatment period.

Dosage Formulation For testing, thymosin alpha-1 is injected subcutaneously to all treated patients, twice a week, 1.6 mg each per injection. 1.45mg to 1.75mg per injection
Is considered safe and effective.

Efficacy Virological Response: The primary test of efficacy in a preferred test is the virological response of the subject. Three virological response indicators can be used. That is, 1. HB becoming undetectable (negative) using standard assays such as Abbott's HBV DNA kit
V. 2. Impairment of HBeAg from serum (detected by EIA). 3. Impairment of HBsAg from serum.

The response of the subject may be classified as follows. 1. Complete: serum HBV DNA that becomes negative and remains negative at the end of the test (with loss of HBeAg).
HBsAg may or may not be present at the end of the test. 2. Partial: 50% or greater loss of serum HBeAg and reduction of serum HBV DNA. But not negative. 3. Non-response: less than 50% serum HBV D at the end of the study
NA decrease. 4. Reactivation: serum HBV DNA by the end of the study
And HBeAg disappear and then recover. 5. Inconclusive: HBeA from start to finish
g, at least 50% serum HB at the end of the study
With a decrease in V DNA. In addition, serum HBV DNA levels generally do not show a constant decreasing tendency, but decrease while repeating up-and-down fluctuations even when effective.

Clinical response: Since minimally affected HBV carriers generally show no or only a few signs specific to their carrier status, clinical response is a significant measure of response to treatment. Not a measure. However, subjects may or may not show improved clinical symptoms (malaise, fatigue, anorexia, daily activities).

Histological Response: Using the method of Knodel et al. And the HAI index, an improvement of at least 25% in the histological stage of liver biopsy specimens and the replication of HBV DNA forms in liver tissue at the final liver biopsy If there is a decrease or decrease in hepatitis B core antigen, the subject is classified as a responder by histological response; if the histological score does not significantly improve or worsens, Classify.

The results are shown in Tables 1 to 4 below.
"Table 1" and "Table 2" show B in the first group (treatment group 1) and the second group (treatment group 2), respectively.
Changes in hepatitis B virus DNA levels are shown as percent of data per month, with data at month 0 taken as 100%. Table 3 shows the change in hepatitis B virus DNA levels in group 3 (untreated group) patients as a percentage of the monthly data, with the data at month 0 as 100%. "Table 4"
Changes in ALT levels are indicated by the percentage of data per month, with the data at month 0 taken as 100%.

[0067]

[Table 1]

[0068]

[Table 2]

[0069]

[Table 3]

[0070]

[Table 4]

Example 2 This example describes another form of test. Total sample size is 4
0 patients (individual patients are at least 18 years old at the start of treatment), randomized release trial
(open trial). Candidates to be included in the study are hepatitis B surface antigen ("HBsA
g ") Seropositive and have HBV DNA in serum for at least 3 months prior to treatment (measured by PCR or any other standard DNA detection method). Only minimal disease patients were included in the study. These patients have normal plasma liver enzyme levels and show minimal or no inflammatory changes when liver biopsies are performed, especially no evidence of scar formation or cirrhosis.

An HIV-infected patient who had been treated with interferon during the previous year and had HIV seropositivity as confirmed by Western blot, and was hepatitis C virus (“HCV”) antibody seropositive. , Or subjects who have abused intravenous drugs during the previous five years are excluded from the study.

Prior to the treatment protocol, the patient is subjected to a monthly pretreatment test for at least three months. The test consists of: 1. FBC and prothrombin time by discrimination count
["PT"], glucose, creatinine and electrolytes,
Liver function test [“LFT”], α-fetoprotein [“AF
P "], HIV antibody, HCV antibody, hepatitis delta antibody, HB
Blood test consisting of markers (HBeAg and HBV DNA) and thymosin alpha-1 levels. 2. Liver biopsy and liver HBV DNA analysis. 3. Normal urine analysis. 4. Pregnancy test for women of childbearing age.

The selected patients are randomized into two groups. The first group of patients receives thymosin alpha-1 treatment and the second group of patients receives a placebo. Group 1 patients, 6 months, 2 weeks
Thymosin α-1 is administered by subcutaneous injection at a dosage of 1600 μg per injection. A second group of patients will be injected with a placebo by the same prescription. All patients are observed at 2 and 4 weeks from the start of treatment and then at 1 month intervals for 6 months of treatment. After treatment is complete,
Monitor patients monthly for 6 months to 2 years.

The response to treatment was determined by measuring serum HBV DNA
Alternatively, it is determined by the loss of serum HBeAg. Responders are those who have achieved and maintain reduced serum HBV DNA or HBeAg levels during the 12 month study period. Non-responders received serum H at the end of the study (month 12).
Those who do not show any changes in BV DNA or HBsAg. Patients who lost HBV DNA at the beginning of the study but have recovered the DNA by the end of the study are considered recurrent.

The group mean was calculated as
The comparison is made by a two-tailed t test. The change in measurement between the intervening value and the subsequent time point is compared by Student's two-tailed paired t-test or Wilcoxon paired sample test.

The patient is monitored for any significant side effects or allergic episodes resulting from the treatment.

The results are shown in Tables 5 and 6.

[0079]

[Table 5]

[0080]

[Table 6]

Example 3 To treat minimal B-type hepatitis, the serum of the patient to be treated is tested for the presence of HBsAg and / or HBV DNA. Further appropriate testing of seropositive individuals for HBV infection indicates that treatment does not result in unacceptably high-risk iatrogenic disorders and that the patient is indeed a minimal carrier of hepatitis B hepatitis Determine that both. A liver biopsy may or may not form part of a further test. Additional blood chemistry tests (eg, analysis for ALT or other enzymes) may or may not be part of the additional tests.

The hepatitis B carrier diagnosed as minimal hepatitis B may then be used in an individual patient, using appropriate dosages and dosage regimens for the individual thymosin α-1 or analog or fragment thereof. And treated according to the present invention.

Other Embodiments The preferred embodiments of the present invention have been described in detail. It is understood that obvious variations can be made without departing from the spirit and scope of the invention as defined by the appended claims.

[0084]

EFFECT OF THE INVENTION By administering the pharmaceutical composition of the present invention containing an appropriate amount of thymosin α-1 to a patient suffering from minimal B-type hepatitis, the minimal B-type hepatitis in the patient can be reduced. Parameter characteristics can be reduced.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor John Di Baxter, San Pablo 131, San Francisco, California 94127 United States (72) Inventor David El Holwitz United States 94010 Hillsborough, West Hillsboro, California 94010 Ainez 220 (56) References JP-A-54-11220 (JP, A) JP-A-7-188049 (JP, A) JP-A-7-2693 (JP, A) Journal of Medical Virology, VOL. 21, No. 1 (1987) p. 25-37 (58) Field surveyed (Int. Cl. ⁶ , DB name) A61K 38/00-38/58 CA (STN)

Claims (8)

(57) [Claims] 1. An effective therapeutic amount and a pharmaceutically acceptable amount of at least one peptide selected from the group consisting of thymosin α-1, a biologically active fragment of thymosin α-1, and a biologically active analog of thymosin α-1. A pharmaceutical composition for treating minimal hepatitis B infection, comprising: a vehicle. 2. The pharmaceutical composition according to claim 1, wherein the peptide is thymosin α-1. 3. The pharmaceutical composition according to claim 1, wherein the peptide is a biologically active fragment of thymosin α-1. 4. The pharmaceutical composition according to claim 1, wherein the peptide is a biologically active analog of thymosin α-1. 5. The pharmaceutical composition according to claim 1, which is for subcutaneous administration. 6. The pharmaceutical composition according to claim 1, wherein the dose is 600 to 3000 μg of peptide per m ^{2 of} body surface area. 7. The pharmaceutical composition according to claim 1, wherein the dose is 600 to 1500 μg of peptide per 1 m ^{2 of} body surface area. 8. The pharmaceutical composition according to claim 1, wherein the dose is 900 to 1200 μg of peptide per m ^{2 of} body surface area.