CN1352567A - A means for the prophylactic and therapeutic treamtent of streptococcal infections - Google Patents

Abstract

The present ivnention relates to compositions containing Group C streptococcal phage associated lysin enzyme for the prophylactic and therepeutic treatment of Streptococcal infections, including the infection commonly known as strep throat. Methods for therapeutically and prophylactically treating such infection also are described.

Description

Methods of prophylactic and curative treatment of streptococcal infection

Background of the Invention

1. Field of invention

The present invention relates to a composition comprising a group C streptococcal phage-associated lysinase and a carrier for releasing the group C streptococcal phage-associated lysinase into the mouth, throat or nasal cavity of a mammal . The compositions are particularly useful in the prophylactic and therapeutic treatment of streptococcal infections, including infections commonly referred to as strep throat.

2. Description of existing technology

Group A Streptococcus has been shown to be an important pathogen capable of being a carrier in asymptomatic individuals or symptomatic individuals with mild sore throat, tonsillitis or impetigo. If left untreated, these streptococcal infections can lead to glomerulonephritis, rheumatic fever, and possibly permanent rheumatic heart disease. With the advent of antibiotics, especially penicillin antibiotics, pathogenic organisms can be easily eliminated in accordance with appropriate antibiotic treatment prescriptions.

Because infected individuals (usually children and young adults) can transmit group A strep to others, especially in nurseries and schools, known infections must be passed on at least 24-72 hours after initiation of antimicrobial Individuals are separated from these environments. In controlled studies it has been shown that early diagnosis and appropriate treatment can reduce the overall circulating infection of the nuisance pathogen and can reduce or reduce the sequelae of class A infection (arthritis or nephritis).

US Patent No. 5,604,109 (Fischetti et al.) teaches that rapid and sensitive detection of group A streptococcal antigens can be performed by diagnostic assays employing group C streptococcal phage-associated lysinases. Such testing can facilitate rapid identification of infected individuals, who can then be placed on routine antibiotic therapy.

However, there is also the question of whether it will be effective against strep infections. Patients with strep throat are highly contagious and can pass the pathogen on to close contacts, resulting in strep throat epidemics. This problem is particularly prominent in schools and the military where people come into close contact with each other with a large population density. Furthermore, there is a need for prophylaxis against streptococcal infections because the usual prophylactic use of antibiotics is inappropriate for a number of reasons.

Summary of Invention

The present invention (which incorporates by reference the contents of U.S. Patent No. 5,604,109) provides a composition comprising a group C streptococcal phage-associated cytolysinase and a carrier for converting the group C streptococcal phage-associated Cytolysin enzymes are released into the mouth, throat or nasal cavities of animals such as humans. Such compositions are particularly useful as prophylactic treatment to prevent infection after exposure to an infected individual, or therapeutic treatment of Strep A throat infection in an already infected patient to alleviate the infection.

Due to the presence of cytolysins in the oral or nasal cavity, at least some of the invading strep are killed when strep is introduced from an infected individual, thus achieving a prophylactic effect by preventing infection. This rapid and specific (lethal) activity of lysinase against pre-existing streptococcal infections has a therapeutic effect by reducing the infection and facilitating the reduction of streptococcal transmission to other individuals.

In one embodiment of the present invention, the cytolysin enzyme is administered in the form of candy, chewing gum, lozenge, lozenge, tablet, powder, aerosol, liquid (eg gargle) or liquid spray. In another embodiment of the invention, the lysinase is administered as a nasal spray.

The method of treating streptococcal A infection comprises: administering an effective dose of a group C streptococcal bacteriophage-associated cytolysinase in an effective amount to the oral or nasal cavity of a mammal in need of treatment, allowing the cytolysinase to contact the oral mucosa And stay for a period of time necessary to saturate the oral mucosa; then administer another dose of this cytolysinase in the same manner until the treatment is completed. Depending on the carrier containing the lysinase, the enzyme can be administered to the mucosal lining of the throat or nose.

In addition to being effective against Group A Streptococcus infections, the composition can also be used in a similar manner against Group C Streptococcus infections. Also, the enzyme is also resistant to group E Streptococci in a similar manner.

Detailed description of the invention

It has been found that when streptococcus invades from an infected individual, cytolysin stays in the oral cavity for a period of time to kill the invading streptococci, thereby preventing infection and providing prophylactic treatment to prevent streptococcal infection. This rapid and specific (lethal) activity of lysinase against streptococcal infection also produces a therapeutic effect in infected individuals by reducing the infection and the transmission of streptococcal bacteria to other individuals.

The amidase muralytic (cytolysin) enzyme produced by group C streptococcal organisms upon infection with a specific bacteriophage (called C1 ) is isolated and harvested as described in US Patent No. 5,604,109. Alternatively, this group C streptococcal enzyme (also known as lysinase), which has unique specificity for the cell walls of group A, C and E streptococci, can also be isolated and collected by any other known method .

Compositions for the prophylactic and therapeutic treatment of streptococcal infections comprise: a lysinase and an application means such as a carrier system or an oral mode of administration for delivery to the oral or nasal mucosal walls.

The enzyme is preferably placed in a stable buffered environment for maintaining a pH in the range of about 4.0 to about 8.0, more preferably about 5.5 to about 7.5 and Most preferably about 6.1.

The stabilizing buffer should allow optimal activity of the lysinase. The buffering agent may be a reducing agent such as dithiothreitol. The stabilizing buffer may also contain or may be a metal chelating agent such as ethylenediaminetetraacetic acid disodium salt, or it may also contain a phosphate or citrate phosphate buffer.

To prevent deterioration, the stabilizing buffer may also contain an antibacterial or bacteriostatic agent as a preservative, for example it may contain a small amount of sodium benzoate.

Application methods include but are not limited to direct, indirect, carrier and special methods or combinations of any methods. Cytolysin enzymes can be applied directly to the nasal mucosa through nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal fillers or by applying the ointment directly to the nostrils, nasal bridge or face Or any combination of the above, or by similar application methods or other known nasal vehicles. Administration to the mouth or throat can be by way of application of throat lozenges or by application of mouthwashes, gargles, solutions, sprays, candies, gums and the like. Thus, the forms in which cytolysin may be administered include, but are not limited to, lozenges, lozenges, candies, injections, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols.

The lozenges, tablets or gums to which the lysin enzyme is added may contain sugar, corn steep liquor, various dyes, non-sugar sweeteners, flavoring agents, any binding agent or a combination of these substances. Similarly, any gum base product may contain gum arabic, carnauba wax, citric acid, corn starch, food coloring, flavoring, non-sugar sweeteners, gelatin, dextrose, glycerin, gum base, shellac, sodium saccharin , sugar, water, white wax, cellulose, other binders and combinations of the above.

Lozenges may also contain sugar, corn starch, acacia, tragacanth, anethole, flaxseed, oleoresins, mineral oil and cellulose, other binders and combinations of these substances. In another embodiment of the invention, glucose, sucrose or other sugars are replaced with sugar substitutes.

The enzyme can also be placed in a nasal spray, wherein the nasal spray is the carrier. The nasal sprays may be long-acting sprays or temporary release sprays and may be prepared by means well known in the art.

All these carriers for lysinase can be prepared in conventional manner, but it is preferred that any mouthwash or similar product does not contain ethanol to prevent enzyme denaturation. Similarly, when placing cytolysin in a lozenge, gum, candy, or lozenge during the manufacturing process, the cytolysin should be added to the lozenge before the lozenge or candy hardens but after the lozenge or candy has cooled slightly. Set aside so that enzyme denaturation is avoided.

The enzyme can be added to these materials in liquid form or in a lyophilized state so that the enzyme dissolves when it encounters the liquid. The enzymes may also be in, for example, micellar or liposome form, or otherwise encapsulated, or administered in prodrug or sustained release form to provide prolonged shelf life and/or release.

The effective dose or amount of lysinase to treat an infection will depend in part on the following factors: whether the lysinase is used prophylactically or therapeutically, the duration of the user's exposure to streptococcal bacteria, and/or the nature of the infection, the size of the individual and weight etc. Determination of appropriate dosages is readily determined by those skilled in the art. The duration of application of the composition containing the enzyme also depends on the following factors: whether the application is for prophylactic purposes, in which case the application can be, for example, daily or weekly, for a defined period of time, such as 1 week, 1 months or longer; or whether the application is for therapeutic purposes, when it is for therapeutic purposes, a more intensive regimen such as application for 2, 3, 4, 5, 6, 7, 10 or 14 days or longer may be applied. Thus, therapeutic treatment may span days or weeks, may be on a day basis, and may have multiple intervals during the day.

To be effective, the enzyme should be present in a sufficient amount to provide an effective number of enzyme units to contact the mouth, throat or nasal cavity. If too few enzyme units contact the mouth, throat or nasal cavities, even prolonged exposure will not produce the desired beneficial effect. Thus, all dosage forms used should provide approximately the minimum number of units over a period of time such that the dosage form will deliver the enzyme to the mouth, throat, or nasal cavities. In the nasal or oral environment, enzyme concentrations in the range of about 100 units to about 100,000 units of active units are considered to provide effective levels or dosages. A wider range from about 100 units to about 10,000 units is believed to be acceptable. Such units may be present in a small volume carrier, eg 1 ml or less, eg liquid or saliva (eg lozenge), or in a larger volume, eg several milliliters of gargle. Thus, generally, the greater the volume of the carrier, the greater the total number of units required to achieve an effective concentration of active enzyme. Thus, acceptable concentrations may be from about 100 units/ml to about 100,000 units/ml of fluid in the nose or mouth. Within this range, concentrations of about 100 units/ml to about 10,000 units/ml are acceptable.

Thus, in practice, the time of exposure of the active enzyme units can affect the required concentration of active enzyme units per milliliter of dose. For example, carriers contemplated to provide sustained release (certain nasal sprays, lozenges, and encapsulated enzymes) are able to provide lower concentrations of active enzyme units per milliliter, but for longer periods of time. In contrast, a shorter duration of treatment (such as a gargle) can provide a higher concentration of active enzyme units per milliliter. Any dosage containing sufficient lysinase to provide an effective concentration of active enzyme at the site of infection or to provide a sufficient prophylactic effect is well within the range of routine experimentation and is therefore fully within the scope of the present invention.

The composition of the invention may also contain at least one other additive effective for the therapeutic or prophylactic treatment of streptococcal infection or the alleviation of the symptoms thereof. In addition, these agents may also be effective in certain other therapeutic and/or prophylactic applications, such as pain relief. Some agents may be topically or systemically active and may contain antibiotics or pain relieving medications including, for example, topical antiseptics for the relief of sore throats. Alternatively, such agents may be administered before or after treatment with a composition of the invention. The dose of antibiotic should be effective for the curative or prophylactic treatment of streptococcal infections or for the treatment of symptoms of these infections.

While these treatments are used in any animal or mammal, it is preferred to apply these products to humans.

The carrier containing each dose of cytolysin is allowed to contact the oral mucosa for the time necessary to provide the desired prophylactic or therapeutic effect. The mode of administering the cytolysin enzyme to the oral mucosa can be any mode, for example: gargles, mouth rinses (mouth rinses), lozenges, lozenges, chewing gums, candies, powders, and nasal or oral sprays, Provided that it is safe and that it provides the desired prophylactic or therapeutic effect. Other possible methods and compositions for administering the lysinase are known to those skilled in the art according to the present invention.

Example 1

Group C Streptococcus strain 26RP66 (ATCC #21597) or any Group C Streptococcus strain was grown in an 18mm tube in Todd Hewitt's medium at 37°C until it was at 650nm The OD was 0.23. Group C phage (C1) (ATCC #21597-B1) was added at a titer of 5 x ¹⁰⁶ at a ratio of 1 part phage to 4 parts cells. The mixture was then maintained at 37°C for 18 minutes, during which time the infected cells were poured onto ice to reduce the temperature of the solution to below 15°C. Infected cells were then harvested in a refrigerated centrifuge and suspended at 1/300 of the original volume in 0.1 M phosphate buffer, which had a pH of 6.1 and contained 5 x 10 ^-3 M disulfide Threitol and 10 μg DNase. These cells will lyse releasing phage and lysinase. After centrifugation at 100,000Xg for 5 hours to remove most cell debris and phage, the enzyme solution was aliquoted and tested for its ability to lyse group A streptococci.

The number of units/ml of enzyme determined was the reciprocal of the highest dilution of enzyme that reduced the OD650 of a suspension of group A Streptococci from 0.3 to 0.15 within 15 minutes. In a typical enzyme preparation, 4 x ¹⁰⁵ to 4 x ¹⁰⁶ units are produced in a single 12 liter batch.

Enzyme application requires a minimal amount of lysinase units, depending on the desired incubation time. The enzyme is diluted with a suitable modulator containing a stabilizing buffer substance which stabilizes the enzyme and allows for maximal enzymatic activity. A preferred protocol is the use of freeze-dried cytolysin enzymes. The stable buffer substance may contain a reducing agent, and the reducing agent may be dithiothreitol at a concentration of 0.001M-1.0M, preferably 0.005M. The stabilizing buffer substance may contain a metal chelator, which may be ethylenediaminetetraacetic acid disodium salt at a concentration of 0.00001M to 1.0M, preferably 0.005M. The stable buffer substance may contain citrate phosphate buffer at a concentration of 0.001M-1.0M, preferably 0.05M. The pH of the stabilizing buffer is in the range of about 4.0-8.0, preferably 6.1. Stabilizing buffer materials can contain bactericides or antibacterial agents as preservatives. Such a preservative may be sodium azide at a concentration of 0.001% to 0.1%, preferably 0.02%.

The preparation of phage material for lysin production can be carried out in the same manner as described above for the infection of phage and group C streptococci in the preparation of lysin enzymes. However, this preparation does not pour the infected cells onto ice, and the incubation time at 37°C lasts a total of 1 hour to dissolve and release the phage and enzymes in the total volume. In order to use phage for subsequent cytolysin production, residual enzymes must be inactivated, or removed to prevent lysis from cells without group C phage but not from phage-infected cells.

Example 2

Dilute the enzyme prepared according to Example 1 to a concentration of 100 units/ml buffer solution, the composition of the buffer solution is: 0.05M citrate phosphate buffer (its pH is 6.1 and contains 0.1% rabbit immunoglobulin Protein, 0.005M (ethylene dinitro) tetraacetic acid disodium salt (EDTA), 0.005M dithiothreitol, 0.02% sodium azide, 0.01% N-acetylglucosamine. Group A Streptococcus antibody-labeled colloidal gold sol (OD ₅₂₀ 1.5) was suspended in 0.02M Tris buffer (its pH was 8.2, containing 1.0% bovine serum albumin, 0.02% sodium azide, 300K unit heparin), and 1 part This suspension was added to 3 parts of the enzyme reagent, mixed, and filtered through a 0.22 micron filter, and each tube contained 200 microliter aliquots and freeze-dried. The freeze-dried enzyme ) can be stable for a short time (2 weeks), and can remain stable for a long time (more than 1 year) at room temperature.

The method of embodiment 3

1. Cultivate group A streptococcus strain S43/192/39R (streptomycin resistant) for 1 day (from frozen blood broth); place 500 μl in 50ml Todd containing 1% yeast extract and 100pl streptomycin/ml Hewitt (TH) broth.

2. Culture until OD ₆₅₀ is 0.59.

3. Centrifuge at 3000 rpm for 15 minutes to pellet the bacteria.

4. Resuspend the organisms in a 1 ml volume of TH w/o antibiotics (3 x ^105/100 [mu]l, determined by plate count).

5. Add 0.5 of the cells at these concentrations to 1.5ml of pH 6.1 phosphate buffer as a control.

6. 5 minutes before administration to the mice, mix 0.5 ml of the concentrated cell suspension with 0.5 ml of a solution of phage somatolysin in pH 6.1 phosphate buffer, wherein said phage somatolysin Solutions were pre-diluted to 10,000 units/ml.

Five mice receiving 60 [mu]l of the "control" solution were divided and equal amounts were administered orally and nasally.

Five mice receiving 60 μl of cytolysin and bacteria mixture were divided into oral and nasal administrations in equal amounts. Throat scrubs were performed on 5% sheep blood, peptone agar plates containing With 500 μg/ml streptomycin. Incubate the watch plate overnight at 37°C. The following results are obtained:

7/22 7/23 7/24 7/28

  1 day 2 days 3 days 7 days

Plouting forms a unit cell soluder L1 0 0 0 0L2 0 0 0 0L3 0 0 0L4 0 0L5 0L5 01 0 control C1 26 14 7 0c2> 400 17 100 83C3 9 0 0c4> 400> 400> 400 220C5 2 2 2 30 0

These results show that in this model system, exposure of phage somatolysin to group A streptococci for as little as 5 minutes prevents colonization of the upper airways of mice by said streptococci.

Example 4

In order to replicate what might have happened, the cytolysin was placed in the oral cavity (i.e. released via lozenge) before the streptococci entered the environment, animals, bacteria, and then the cytolysin was prepared in the same manner as in Example 3 (1-5), However, in this experiment, the animals were treated as follows:

Cytolysin group: 5 mice received 25 μl of cytolysin followed immediately by 50 μl of bacterial suspension.

Control group: 5 mice received 25 μl of buffer immediately followed by 50 μl of bacterial suspension.

  24-hour cytolysin

L6 -

L7 -

L8 -

L9 -

L10 - control group

C6 -

C7 +

C8 -

C9 +

C10+

Results: When Streptococci were added, 0/5 of the animals with Cytolysin in the oral cavity were colonized, whereas 3/5 of the animals receiving buffer were colonized with Streptococci. Therefore, if cytolysin is present before adding group A streptococci, colonization can be prevented.

Example 5

Group AM group 6 streptococci were grown overnight at 37°C in Todd Hewitt medium. Organisms were washed once with sterile cytolysin buffer (50 ml phosphate buffer, pH 6.1). The cell pellet was then suspended in 5 ml of the same buffer.

The bacteriophage cytolysin was diluted to two times the appropriate unit in cytolysin buffer containing 5 mM DTT, and the mixture was subjected to sterile filtration.

1.0 ml of the bacterial suspension was added to 1.0 ml of the appropriate cytolysin dilution, and the mixture was incubated at 37°C. Samples were removed at regular intervals, diluted appropriately, and placed on blood agar dishes to determine bacterial numbers.

A control sample consisting of 1.0 ml of bacterial suspension was added separately to 1.0 ml of cytolysin buffer. Aliquots were removed, then diluted in 10-fold diluent, and aliquots were plated on blood agar plates to determine bacterial numbers. Results Number of bacteria containing cytolysin Initial number of cytolysin units 5 seconds 30 seconds 60 seconds 5 minutes 10 minutes 1000 5×10 ⁶ 0 0 0 0 0 (average 2 exp) 100 8.6×10 ⁶ 1530 1196 771 64 6 (4 exp on average) 10 9.8×10 ⁶ >3000 >3000 >3000 >3000 >3000 (3 exp on average)

Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it should be understood that the scope of the present invention is determined by the claims rather than limited to the solutions specifically described in the specification.

Claims (31)

1. Compositions for the prophylactic or therapeutic treatment of streptococcal infections, comprising: an effective amount of group C streptococcal phage-associated lysinase; and A carrier for releasing the lysinase in the mouth, throat or nasal cavity. 2. The composition of claim 1, wherein said carrier is selected from the group consisting of: candy, chewing gum, lozenge, lozenge, tablet, powder, aerosol, liquid, liquid spray, nasal spray, and nasal ointment. 3. The composition of claim 1, further comprising a buffer substance to maintain the pH of said composition from about 4.0 to about 9.0. 4. The composition of claim 3, wherein said buffer substance maintains the pH of the composition from about 5.5 to about 7.5. 5. The composition of claim 3, wherein said buffer substance contains a reducing agent. 6. The composition of claim 5, wherein the reducing agent is dithiothreitol. 7. The composition of claim 3, wherein said buffer substance comprises a metal chelator. 8. The composition of claim 7, wherein the metal chelator is ethylenediaminetetraacetic acid disodium salt. 9. The composition of claim 3, wherein the buffer substance is citrate phosphate buffer. 10. The composition of claim 1, further comprising antibacterial and bactericidal agents as preservatives. 11. The composition of claim 1, wherein the lysinase is lyophilized. 12. The composition of claim 1, wherein said carrier further comprises a sweetening agent. 13. The composition of claim 1, wherein said carrier provides a concentration of about 100 to about 100,000 active enzyme units per milliliter of fluid in a nasal or oral environment. 14. The composition of claim 13, wherein the carrier provides a concentration of about 100 to about 10,000 active enzyme units per milliliter of fluid in a nasal or oral environment. 15. The composition of claim 1, wherein said composition is used for the therapeutic treatment of streptococcal infection. 16. The composition of claim 1, wherein said composition is used for prophylactic treatment of streptococcal infection. 17. The composition of claim 15, wherein the streptococcal infection is a streptococcal throat infection. 18. The composition of claim 1, wherein the carrier is a candy. 19. The composition of claim 1, wherein said carrier is chewing gum. 20. The composition of claim 1, wherein said carrier is a lozenge. 21. The composition of claim 1, wherein said carrier is a lozenge. 22. The composition of claim 1, wherein said carrier is a powder. 23. The composition of claim 1, wherein said carrier is an aerosol. 24. The composition of claim 1, wherein said carrier is a liquid spray. 25. The composition of claim 1, wherein said carrier is a nasal spray. 26. The composition of claim 1, wherein said mammal is a human. 27. The composition of claim 1, wherein said carrier is adapted to deliver said lysinase to the mouth and throat. 28. The composition of claim 1, wherein said carrier is adapted to deliver said lysinase to the nasal cavity. 29. The composition of claim 1, further comprising at least one agent effective for the therapeutic or prophylactic treatment or alleviation of symptoms of streptococcal infection. 30. A method for prophylactic or therapeutic treatment of streptococcal infection, comprising: administering the composition of any one of 1-29 to a mammal in need. 31. Use of group C streptococcal phage-associated cytolysin in the preparation of the pharmaceutical composition according to any one of claims 1-29.