WO2019177475A1 - The use of copolymers for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the hsv-1 virus - Google Patents
The use of copolymers for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the hsv-1 virus Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/74—Synthetic polymeric materials
- A61K31/785—Polymers containing nitrogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
Definitions
- copolymers for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus
- the invention relates to the use of block copolymers for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
- HSV-1 human herpesvirus type 1
- herpes simplex virus type 1 also known as herpes simplex virus type 1.
- Reactivation of the virus, coinciding with weakened immune system responses leads to the formation of characteristic ulcers at the sites of primary infection (mainly lips and mucous membranes), but in some cases the virus replicates at other sites of the body, causing e.g., eyeball or brain disease.
- the use of certain polymers for the treatment of herpes caused by HSV-1 is known in the art.
- the invention described in international patent application W018015465A1 relates to virucidal compounds, virucidal compositions containing them and their uses in the treatment of viral infections, sterilization and disinfection.
- These compounds are sulfonylalkyl cyclodextrins substituted with polyethylene glycol).
- the compounds of the invention are applicable to the treatment of viral infections caused, e.g. by herpes virus.
- polish patent application P.416749 made by the present inventors discloses the use of dextran, modified cationically by means of quaternary ammonium groups, for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
- the invention described in international application W015082172A1 relates to the use of polydimethylsiloxane, also referred to as dimethicone, for the topical treatment of viral infections, such as herpes virus (HSV) type 1 and type 2 infections.
- HSV herpes virus
- the above use of polydimethylsiloxane is particularly suitable for the treatment of facial and genital herpes.
- the above invention further relates to topical formulations and kits containing polydimethylsiloxane in combination with copper for the treatment of facial herpes or genital herpes.
- polymers based on polyethylene glycol) (PEG) or polymers containing polycationic groups that have been used to inhibit viral replication.
- U.S. patent application US2009074721A discloses a pharmaceutical composition comprising compounds and/or compositions useful for inhibiting viral replication.
- the compositions contain a combination of polyethylene glycol), e.g. PEG-400, and an active substance derived from sterols.
- U.S. patent application US2002025919A discloses an antiviral compound comprising a linear polymer having many side chains wherein at least one of the side chains is substituted with anionic or cationic moiety.
- block polymers based on PEG containing a polycationic block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus is not known. Therefore, it is an object of the present invention to provide a new use of PEG-based block polymers and containing a polycationic block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by HSV-1 virus, which acts by inhibiting HSV-1 replication - for which the polycation block is responsible, while ensuring its low cytotoxicity - for which the PEG block is responsible.
- PEGx-PMAPTACy symbols PEG - polyethylene glycol
- PMAPTAC - poly(3- (methacryloylamino)propyl) trimethylammonium chloride) as used herein describe a block copolymer according to the invention consisting of a PEG block with degree of polymerization x and a PMAPTAC block with degree of polymerization y.
- treatment means, but is not limited to, all activities that lead to improving the state of health of the patient, restoration of health, and removal of the effects of HSV- 1 activity.
- prophylaxis means, but is not limited to, preventing the occurrence of the effect of HSV-1 virus.
- the subject of the invention is the use of block copolymers with chains composed of a polyethylene glycol) block (PEG) and poly(3-(methacryloylamino)propyl)trimethylammonium chloride block (PMAPTAC) for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
- PEG polyethylene glycol
- PMAPTAC poly(3-(methacryloylamino)propyl)trimethylammonium chloride block
- the treatment and prophylaxis of diseases caused by the HSV-1 virus consists in inhibiting the replication of the HSV-1 virus. More preferably, the diseases caused by the HSV-1 virus are herpes, herpetic conjunctivitis, herpetic keratitis and herpetic encephalitis.
- the use is preferably characterized in that the polyethylene glycol) (PEG) block copolymer and poly (3-(methacryloylamino)propyl)trimethylammonium chloride (PMAPTAC) - PEG x -PMAPTAC y - is a copolymer in which the degree of polymerization x of the PEG block is 45-47, while the degree of polymerization y of the PMAPTAC block is in the range of 22-102.
- PEG polyethylene glycol)
- PMAPTAC poly (3-(methacryloylamino)propyl)trimethylammonium chloride
- the polyethylene glycol) (PEG) and the poly (3-(methacryloylamino)propyl)trimethylammonium chloride (PMAPTAC) block copolymer is PEG47-PMAPTAC53, PEG 47 -PMAPTAC7 4 , PEG 45 -PMAPTAC 51 or PEG 45 -PMAPTAC5 8 .
- the use is characterized in that the formulation is administered topically. More preferably, the formulation is applied to the skin, lips, mucous membrane of: nose, genital region or to the eye.
- the use is characterized in that the formu lation is in a form selected from the group comprising a solution, gel, stick, lipstick, cream, serum, powder, dusting powder, ointment, eye drops or adhesive plaster.
- the use is characterized in that the concentration of the PEG-PMAPTAC block copolymer in the formulation is from 10 pg/ml to 100 mg/ml.
- block copolymers with chains composed of a polyethylene glycol) (PEG) block and a poly(3-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus allows achieving high treatment efficiency with very low toxicity of the agents used. Furthermore, it is possible to use the cationic block copolymers indicated in the description in the treatment of infections caused by strains which are resistant to active substances currently used in medical practice.
- the polymers used according to the present invention are very well defined (they have a well-controlled molecular weight and molecular weight dispersity) as opposed to other polymers, especially those obtained by conventional radical polymerization, and natural polymers. It is therefore possible for a person skilled in the art - a physician - to choose PEG x -PMAPTAC y polymer with appropriate degrees of polymerization so that it has the appropriate ratio of effectiveness to toxicity for treatment for a particular patient, i.e. a specific case of herpetic disease, and for a specific route of administration.
- PEG-PMAPTAC polymers used in the invention were prepared according to a controlled radical polymerization (CRP) of the RAFT type, i.e.
- the degrees of polymerization of the obtained PEG x -PMAPTAC y polymers were determined by gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy ⁇ H-NM R).
- the copolymer to be used for inhibiting the HSV-1 virus must have an appropriate combination of individual block lengths, so not all PEG-PMAPTAC polymers exhibit similar efficacy. Without wishing to be bound by any theory, it seems that the length of the PEG block must have a certain minimum value to effectively reduce the toxicity of the PMAPTAC block, whereas the PMAPTAC block length must be large enough to interact with the cell/virus, but it cannot be too long so that the polymer is not toxic (polycations are usually toxic and their toxicity increases with molecular weight).
- the polymers used according to the invention have a very high ability of HSV-1 virus replication inhibition, with very low cytotoxicity even at high concentrations (1 mg/ml). At a polymer concentration of 500 pg/ml, a reduction in the virus titer by approximately 6 orders of magnitude (99.9999%) was observed.
- the IC90 values (90% inhibitory concentration, the concentration of the drug (formulation) that inhibits 90% virus replication) calculated for the PEG47-PMAPTAC53 and PEG 46 - PMAPTAC73 polymers are 4.12 pg/ml and 34.66 pg/ml, respectively. The functional tests carried out showed that the polymer interacts with the cells, creating a protective layer limiting the ability of the virus to adsorb, and consequently to infect.
- Fig. 1 illustrates a graph showing the cytotoxicity of block polymers. Cell viability was reported as a % of the value of the control sample, not treated with polymers.
- Fig. 2 illustrates graphs showing the antiviral activity of block copolymers.
- the amount of viral DNA in the medium was determined by quantitative PCR (A), while the number of functional viral particles was determined by plaque assay (B).
- Fig. 3 illustrates a graph showing the mechanism of activity of block copolymers. A series of functional tests involving the addition of an inhibitor at various stages of the viral infection allows to determine at which stage the inhibition occurs. Antiviral activity is presented as the logarithmic decrease in the number of copies of viral DNA in a milliliter of medium.
- Fig. 4 illustrates images obtained by confocal microscopy showing the inhibition of HSV-1 infection by block copolymers. The images represent maximum projections.
- Fig. 5 shows the antiviral activity of the PEG46-PMAPTAC52 block copolymer in a mouse model. The animals were infected by scratching the skin. The medical formulations were then administered every 12 hours for 4 days, and observation of animal health was carried out for the next 3 days. Progression of the disease was assessed according to the following scale: 0 - no symptoms, 1 - blister formation, 2 - light ulceration, 3 - large ulcer patches, 4 - confluent ulceration, 5 - hind limb paralysis, 6 - death.
- Groups 0 - no treatment, no infection; NaCI - placebo formulation, no infection; mock - no treatment, mock infection; HSV-1 - no treatment, HSV-1 infection; HSV-1 + ACV - acyclovir treatment, HSV 1 infection; HSV-1 + PEG46-PMAPTAC52 - treatment with PEG46-PMAPTAC52, HSV-1 infection.
- the method of preparation of PEG47-PMAPTAC53 block polymer for use according to the patent application is as follows. (3-(methacryloylamino)propyl)trimethylammonium chloride (MAPTAC, 5.53 g, 25.0 mmol), initiator (4,4'-azobis(4-cyanopentanoic acid), V-501, 69.2 mg, 0.247 mmol) ) and the macro chain transfer agent (PEG47-CTA, 1.13 g, 0.501 mmol) were dissolved in 41 mL of water. The mixture was degassed by passing argon through it for 30 minutes. The polymerization was carried out at 70 °C for 5 hours. The post-reaction mixture was dialyzed against pure water for 2 days. PEG47- PMAPTAC53 was isolated by lyophilization to obtain 5.58 g of material (83.8% yield).
- MATAC 3-(methacryloylamino)propyl)trimethylammonium chloride
- Example 1 Effect of block copolymers on cell viability.
- Cytotoxicity of the block copolymers was checked using the XTT test.
- the analysis was carried out on the Vero E6 cell line (Cercopithecus aethiops renal epithelial cells, ATCC CRL-1586), which is permissive for HSV 1 virus. This test is based on the study of mitochondrial enzyme activity by determining their ability to reduce the substrate to a colored product. Cell viability was determined by measuring the absorbance at 450 nm relative to the control, which were cells cultured in medium without the addition of polymers.
- the cells were cultured for 2 days in DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 3% heat inactivated fetal bovine serum (FBS) and penicillin and streptomycin, with the addition of the tested polymer.
- DMEM medium Dulbecco's Modified Eagle's Medium
- FBS heat inactivated fetal bovine serum
- penicillin and streptomycin penicillin and streptomycin
- Fig. 1 depict the low cytotoxicity of the studied polymers even at high concentrations (500 pg/ml).
- the CC50 of both compounds is above 2,000 pg/ml.
- Example 2 Effect of block copolymers on herpes simplex type 1 virus (HSV-1) replication.
- HSV-1 virus replication After determination of the concentrations of compounds that did not have a toxic effect on the cells, their effect on HSV-1 virus replication was examined.
- viral DNA was isolated from the cell media and used as a template in the real-time quantitative PCR reaction (forward sequences 5'- CAT CAC CGA CCC GGA GAG GGA C-3 ', reverse 5'-GGG CCA GGC GCT TGT TGG TGT A-3 ', 5'-FAM CCG CCG AAC TGA GCA GAC ACC CGC GC BHQ1-3’ probe).
- the ability of polymers to inhibit HSV-1 replication was determined as the logarithmic decrease in viral DNA copy number in a milliliter of the medium ( Figure 2).
- the amount of functional virus particles in the medium was determined using a standard plaque assay. The amount of virus particles in the medium is shown as the number of plaque forming units (PFU).
- Tested block copolymers show antiviral activity and inhibit the multiplication of HSV-1 virus.
- the calculated IC90 values determined by the real-time PCR method were 4.115 pg/ml for PEG47- PMAPTAC53 and 34.66 pg/ml for PEG46-PMAPTAC73, respectively, while the values determined by plaque assay were 1.4 pg/ml for PEG47-PMAPTAC53 and 1.685 pg/ml for PEG46-PMAPTAC73, respectively.
- the values of the selectivity index determined by the real-time PCR method are > 121.5 for PEG47-PMAPTAC53 and >14.43 for PEG46-PMAPTAC73, respectively, while the values determined by plaque assay are >357.14 for PEG47-PMAPTAC53 and >296.74 for PEG46-PMAPTAC73 .
- Test I inactivation test
- Test II interaction test
- Test III absorption test
- Test IV folding, packaging and release test
- DAPI chromosomal DNA
- f-actin phalloidin conjugated with Alexa 633 fluorescent dye
- virus antibodies specific for VP5 viral protein capsid
- Example 5 PEG-PMAPTAC block copolymer toxicity study in a mouse model.
- the aim of the experiment was to determine the maximum non-toxic dose of the PEG- PMAPTAC block copolymer in the mouse model.
- the test material was 6-week-old female Balb/c mice treated with different amounts of PEG 46 -PMAPTAC 5 2 block polymer. Only healthy individuals selected at random were qualified for the experiment. The animals were divided into groups.
- the test material was applied directly to the damaged skin in a volume of 100 mI/mouse, 2 times a day for 4 days.
- the animals were shaved on the lateral dorsal side, the skin was scratched with a needle and the polymer containing formulation in a volume of 100 mI per mouse was applied to the scarified area.
- the treatment was repeated every 12 hours for 4 consecutive days.
- the animals were examined daily for skin anomalies, general health and weighed for 7 consecutive days. Detailed clinical observations were made daily from the day the compound was administered. Body weight measurement was carried out before the test material was administered and daily during the observation. After the experiment was completed, organs (liver, kidneys, spleen) were removed from the animals to compare organ weights in particular groups.
- PEG46-PMAPTAC52 block copolymer at concentrations of 10%, 5%, 1%, 0.1% w/v did not cause clinical symptoms. After completing the necropsy, no macroscopic changes in the organs were observed. As a result, it was found that the tested polymer in concentrations from 0.1% to 10% w/v is not toxic to animals.
- Example 6 Study of PEG46-PMAPTAC52 block copolymer activity in a mouse model.
- the purpose of the experiment was to determine the activity of PEG46-PMAPTAC52 block copolymer and to check its antiviral effect on infection of mice induced by HSV-1 virus. Only healthy individuals selected at random were qualified for the experiment. The animals were divided into groups.
- mice Six-week-old female BALB/c mice were shaved on the lateral side of the back, the skin was scratched with a needle and the polymer containing formulation was applied in a volume of 100 mI per mouse to the scarified area and left for 30 min. Then, HSV-1 at 1 x 10 7 PFU in a volume of 100 mI was applied to the wound on the adhesive tape along with the formulation for treatment. After 2 hours, the adhesive tape was removed and the formulation was applied again. The treatment was repeated every 12 hours for 4 consecutive days.
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Abstract
The invention relates to the use of block copolymers with chains composed of a polyethylene glycol) (PEG) block and poly(3-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
Description
The use of copolymers for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus
The invention relates to the use of block copolymers for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
Human herpesviruses are among the most common causes of infections in humans. One of the most prevalent species of this family is human herpesvirus type 1 (HSV-1; herpes simplex 1), also known as herpes simplex virus type 1. Already a single contact with this pathogen leads to life-long infection due to ability of the virus to remain dormant in its latent form. It is estimated that up to 80% of the world's human population is infected with the virus, while about 40% develops symptomatic disease. Reactivation of the virus, coinciding with weakened immune system responses leads to the formation of characteristic ulcers at the sites of primary infection (mainly lips and mucous membranes), but in some cases the virus replicates at other sites of the body, causing e.g., eyeball or brain disease. The use of certain polymers for the treatment of herpes caused by HSV-1 is known in the art.
For example, the invention described in international patent application W018015465A1 relates to virucidal compounds, virucidal compositions containing them and their uses in the treatment of viral infections, sterilization and disinfection. These compounds are sulfonylalkyl cyclodextrins substituted with polyethylene glycol). The compounds of the invention are applicable to the treatment of viral infections caused, e.g. by herpes virus.
In turn, the Polish patent application P.416749 made by the present inventors discloses the use of dextran, modified cationically by means of quaternary ammonium groups, for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
The invention described in international application W015082172A1 relates to the use of polydimethylsiloxane, also referred to as dimethicone, for the topical treatment of viral infections, such as herpes virus (HSV) type 1 and type 2 infections. The above use of polydimethylsiloxane is particularly suitable for the treatment of facial and genital herpes. The above invention further relates to topical formulations and kits containing polydimethylsiloxane in combination with copper for the treatment of facial herpes or genital herpes.
Also known are polymers based on polyethylene glycol) (PEG) or polymers containing polycationic groups that have been used to inhibit viral replication.
U.S. patent application US2009074721A discloses a pharmaceutical composition comprising compounds and/or compositions useful for inhibiting viral replication. The compositions contain a combination of polyethylene glycol), e.g. PEG-400, and an active substance derived from sterols.
U.S. patent application US2002025919A discloses an antiviral compound comprising a linear polymer having many side chains wherein at least one of the side chains is substituted with anionic or cationic moiety.
The use of block polymers based on PEG containing a polycationic block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus is not known. Therefore, it is an object of the present invention to provide a new use of PEG-based block polymers and containing a polycationic block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by HSV-1 virus, which acts by inhibiting HSV-1 replication - for which
the polycation block is responsible, while ensuring its low cytotoxicity - for which the PEG block is responsible.
PEGx-PMAPTACy symbols (PEG - polyethylene glycol), PMAPTAC - poly(3- (methacryloylamino)propyl) trimethylammonium chloride) as used herein describe a block copolymer according to the invention consisting of a PEG block with degree of polymerization x and a PMAPTAC block with degree of polymerization y.
The term "treatment", as used herein, means, but is not limited to, all activities that lead to improving the state of health of the patient, restoration of health, and removal of the effects of HSV- 1 activity. In turn, the term "prophylaxis", as used herein, means, but is not limited to, preventing the occurrence of the effect of HSV-1 virus.
The subject of the invention is the use of block copolymers with chains composed of a polyethylene glycol) block (PEG) and poly(3-(methacryloylamino)propyl)trimethylammonium chloride block (PMAPTAC) for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus.
Preferably, the treatment and prophylaxis of diseases caused by the HSV-1 virus consists in inhibiting the replication of the HSV-1 virus. More preferably, the diseases caused by the HSV-1 virus are herpes, herpetic conjunctivitis, herpetic keratitis and herpetic encephalitis.
The use is preferably characterized in that the polyethylene glycol) (PEG) block copolymer and poly (3-(methacryloylamino)propyl)trimethylammonium chloride (PMAPTAC) - PEGx-PMAPTACy - is a copolymer in which the degree of polymerization x of the PEG block is 45-47, while the degree of polymerization y of the PMAPTAC block is in the range of 22-102. More preferably, the polyethylene glycol) (PEG) and the poly (3-(methacryloylamino)propyl)trimethylammonium chloride (PMAPTAC) block copolymer is PEG47-PMAPTAC53, PEG47-PMAPTAC74, PEG45-PMAPTAC51 or PEG45-PMAPTAC58.
Preferably, the use is characterized in that the formulation is administered topically. More preferably, the formulation is applied to the skin, lips, mucous membrane of: nose, genital region or to the eye. Preferably the use is characterized in that the formu lation is in a form selected from the group comprising a solution, gel, stick, lipstick, cream, serum, powder, dusting powder, ointment, eye drops or adhesive plaster.
Preferably, the use is characterized in that the concentration of the PEG-PMAPTAC block copolymer in the formulation is from 10 pg/ml to 100 mg/ml.
The use of block copolymers with chains composed of a polyethylene glycol) (PEG) block and a poly(3-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus allows achieving high treatment efficiency with very low toxicity of the agents used. Furthermore, it is possible to use the cationic block copolymers indicated in the description in the treatment of infections caused by strains which are resistant to active substances currently used in medical practice. The polymers used according to the present invention, obtained by the RAFT technique, are very well defined (they have a well-controlled molecular weight and molecular weight dispersity) as opposed to other polymers, especially those obtained by conventional radical polymerization, and natural polymers. It is therefore possible for a person skilled in the art - a physician - to choose PEGx-PMAPTACy polymer with appropriate degrees of polymerization so that it has the appropriate ratio of effectiveness to toxicity for treatment for a particular patient, i.e. a specific case of herpetic disease, and for a specific route of administration.
PEG-PMAPTAC polymers used in the invention were prepared according to a controlled radical polymerization (CRP) of the RAFT type, i.e. radical polymerization with addition-fragmentation chain transfer as described in the publication "Synthesis of Oppositely Charged Block Copolymers of Polyethylene glycol) via Reversible Addition-Fragmentation Chain Transfer Radical Polymerization and Characterization of Their Polyion Complex Micelles in Water ", Shin-ichi Yusa, Yuuichi Yokoyama, and Yotaro Morishima, Macromolecules 2009, 42, 376-383, which is referenced in the present description. The above-mentioned method consists in reacting polyethylene glycol), functionalized at one end with a methoxy group, with 4-cyanopentanoic acid dithiobenzoate (CPD), acting as a chain transfer agent (CTA). To the thus obtained PEG-CTA, PMAPTAC block was then attached in the polymerization reaction initiated with 4,4'-azobis (4-cyanopentanoic acid) (V-501). The polymer was purified by two precipitations with methanol in the presence of excess acetone.
The degrees of polymerization of the obtained PEGx-PMAPTACy polymers were determined by gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy ^H-NM R).
The inventors have determined, within the context of the present invention, that the copolymer to be used for inhibiting the HSV-1 virus must have an appropriate combination of individual block lengths, so not all PEG-PMAPTAC polymers exhibit similar efficacy. Without wishing to be bound by any theory, it seems that the length of the PEG block must have a certain minimum value to effectively reduce the toxicity of the PMAPTAC block, whereas the PMAPTAC block length must be large enough to interact with the cell/virus, but it cannot be too long so that the polymer is not toxic (polycations are usually toxic and their toxicity increases with molecular weight).
The polymers used according to the invention have a very high ability of HSV-1 virus replication inhibition, with very low cytotoxicity even at high concentrations (1 mg/ml). At a polymer concentration of 500 pg/ml, a reduction in the virus titer by approximately 6 orders of magnitude (99.9999%) was observed. The IC90 values (90% inhibitory concentration, the concentration of the drug (formulation) that inhibits 90% virus replication) calculated for the PEG47-PMAPTAC53 and PEG46- PMAPTAC73 polymers are 4.12 pg/ml and 34.66 pg/ml, respectively. The functional tests carried out showed that the polymer interacts with the cells, creating a protective layer limiting the ability of the virus to adsorb, and consequently to infect.
The subject of the invention is illustrated by means of the following figures, supplementing the information contained in the embodiments:
Fig. 1 illustrates a graph showing the cytotoxicity of block polymers. Cell viability was reported as a % of the value of the control sample, not treated with polymers.
Fig. 2 illustrates graphs showing the antiviral activity of block copolymers. The amount of viral DNA in the medium was determined by quantitative PCR (A), while the number of functional viral particles was determined by plaque assay (B).
Fig. 3 illustrates a graph showing the mechanism of activity of block copolymers. A series of functional tests involving the addition of an inhibitor at various stages of the viral infection allows to determine at which stage the inhibition occurs. Antiviral activity is presented as the logarithmic decrease in the number of copies of viral DNA in a milliliter of medium.
Fig. 4 illustrates images obtained by confocal microscopy showing the inhibition of HSV-1 infection by block copolymers. The images represent maximum projections.
Fig. 5 shows the antiviral activity of the PEG46-PMAPTAC52 block copolymer in a mouse model. The animals were infected by scratching the skin. The medical formulations were then administered every 12 hours for 4 days, and observation of animal health was carried out for the next 3 days. Progression of the disease was assessed according to the following scale: 0 - no symptoms, 1 - blister formation, 2 - light ulceration, 3 - large ulcer patches, 4 - confluent ulceration, 5 - hind limb paralysis, 6 - death. Groups: 0 - no treatment, no infection; NaCI - placebo formulation, no infection; mock - no treatment, mock infection; HSV-1 - no treatment, HSV-1 infection; HSV-1 + ACV - acyclovir treatment, HSV 1 infection; HSV-1 + PEG46-PMAPTAC52 - treatment with PEG46-PMAPTAC52, HSV-1 infection.
Embodiments
The method of preparation of PEG47-PMAPTAC53 block polymer for use according to the patent application is as follows. (3-(methacryloylamino)propyl)trimethylammonium chloride (MAPTAC, 5.53 g, 25.0 mmol), initiator (4,4'-azobis(4-cyanopentanoic acid), V-501, 69.2 mg, 0.247 mmol) ) and the macro chain transfer agent (PEG47-CTA, 1.13 g, 0.501 mmol) were dissolved in 41 mL of water. The mixture was degassed by passing argon through it for 30 minutes. The polymerization was carried out at 70 °C for 5 hours. The post-reaction mixture was dialyzed against pure water for 2 days. PEG47- PMAPTAC53 was isolated by lyophilization to obtain 5.58 g of material (83.8% yield).
It is within the skill of the person skilled in the art to determine the method of obtaining the remaining polymers covered by this patent specification.
Example 1. Effect of block copolymers on cell viability.
Cytotoxicity of the block copolymers was checked using the XTT test. The analysis was carried out on the Vero E6 cell line (Cercopithecus aethiops renal epithelial cells, ATCC CRL-1586), which is permissive for HSV 1 virus. This test is based on the study of mitochondrial enzyme activity by determining their ability to reduce the substrate to a colored product. Cell viability was determined by measuring the absorbance at 450 nm relative to the control, which were cells cultured in medium without the addition of polymers. The cells were cultured for 2 days in DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 3% heat inactivated fetal bovine serum (FBS) and penicillin and streptomycin, with the addition of the tested polymer.
The results presented in Fig. 1 depict the low cytotoxicity of the studied polymers even at high concentrations (500 pg/ml). The CC50 of both compounds is above 2,000 pg/ml.
Example 2. Effect of block copolymers on herpes simplex type 1 virus (HSV-1) replication.
After determination of the concentrations of compounds that did not have a toxic effect on the cells, their effect on HSV-1 virus replication was examined. Cells were pre-incubated with polymers of appropriate concentrations for 30 min and then, still in the presence of these polymers, infected with HSV 1, strain 17+ with TCIDS0 (tissue culture infective dose, infecting 50% of the culture) = 400 per milliliter. After 2 hours, unbound virus particles were removed by 3 washes with PBS and incubated, also in the presence of polymers, for fu rther 2 days. After this time, viral DNA was isolated from the cell media and used as a template in the real-time quantitative PCR reaction (forward sequences 5'- CAT CAC CGA CCC GGA GAG GGA C-3 ', reverse 5'-GGG CCA GGC GCT TGT TGG TGT A-3 ', 5'-FAM CCG CCG AAC TGA GCA GAC ACC CGC GC BHQ1-3’ probe). The ability of polymers to inhibit HSV-1 replication was determined as the logarithmic decrease in viral DNA copy number in a milliliter of the medium (Figure 2). In addition, the amount of functional virus particles in the medium was determined using a standard plaque assay. The amount of virus particles in the medium is shown as the number of plaque forming units (PFU).
Tested block copolymers show antiviral activity and inhibit the multiplication of HSV-1 virus. The calculated IC90 values determined by the real-time PCR method were 4.115 pg/ml for PEG47- PMAPTAC53 and 34.66 pg/ml for PEG46-PMAPTAC73, respectively, while the values determined by plaque assay were 1.4 pg/ml for PEG47-PMAPTAC53 and 1.685 pg/ml for PEG46-PMAPTAC73, respectively. The values of the selectivity index determined by the real-time PCR method are > 121.5 for PEG47-PMAPTAC53 and >14.43 for PEG46-PMAPTAC73, respectively, while the values determined by plaque assay are >357.14 for PEG47-PMAPTAC53 and >296.74 for PEG46-PMAPTAC73 .
Example 3. Mechanism of antiviral activity of block copolymers.
The mechanism of antiviral activity of block copolymers was examined using a series of functional tests based on the use of an inhibitor at various stages of a viral infection:
• Test I (inactivation test) - does the polymer interact directly with the virus, making it unable to infect cells?
• Test II (interaction test) - does the polymer interact directly with the cell, making it resistant to viral infection?
• Test III (adsorption test) - does the polymer interfere with the interaction of the virus with its adhesive factor on the cell surface, preventing the virus from attaching to the cell?
• Test IV (folding, packaging and release test) - Does the polymer interfere with the late stages of viral infection (e.g. assembly, packaging, release), preventing the generation of infectious progeny virions?
It has been found that inhibition of HSV-1 infection by block copolymers occurs at the stage of adsorption of the virus to the cell and by the interaction of the polymer with the cell. Based on Fig. 3, a significant decrease in the number of viral DNA copies can be found, evident in the folding, packaging and release test, which is probably a consequence of inhibiting the infection at an earlier stage, however, additional inhibitory effects at the late stages of infection cannot be ruled out.
Example 4. Imaging of the effect of block copolymers on HSV-1 infection by means of confocal microscopy (Figure 4)
To illustrate the effect of block copolymers on viral infection, cells were preincubated with polymers and then infected with HSV-1. One hour later, the cells were fixed and labeled for the presence of chromosomal DNA (DAPI), f-actin (phalloidin conjugated with Alexa 633 fluorescent dye) and virus (antibodies specific for VP5 viral protein capsid).
In Fig. 4, a reduction in the amount of virus present in cells treated with block copolymers is clearly visible.
Example 5. PEG-PMAPTAC block copolymer toxicity study in a mouse model.
The aim of the experiment was to determine the maximum non-toxic dose of the PEG- PMAPTAC block copolymer in the mouse model. The test material was 6-week-old female Balb/c mice treated with different amounts of PEG46-PMAPTAC52 block polymer. Only healthy individuals selected at random were qualified for the experiment. The animals were divided into groups.
Control grou p - not treated (n = 5)
Control group - hydrogel containing physiological saline (n = 5)
Experimental grou p - a hydrogel containing a block copolymer at a concentration of 10% w/v
Experimental group - a hydrogel containing a block copolymer at a concentration of 5% w/v (n = 5)
Experimental group - a hydrogel containing a block copolymer at a concentration of 1% w/v (n = 5)
Experimental group - a hydrogel containing a block copolymer at a concentration of 0.1% w/v
(n = 5).
The test material was applied directly to the damaged skin in a volume of 100 mI/mouse, 2 times a day for 4 days. The animals were shaved on the lateral dorsal side, the skin was scratched with a needle and the polymer containing formulation in a volume of 100 mI per mouse was applied to the scarified area. The treatment was repeated every 12 hours for 4 consecutive days. The animals were examined daily for skin anomalies, general health and weighed for 7 consecutive days. Detailed clinical observations were made daily from the day the compound was administered. Body weight measurement was carried out before the test material was administered and daily during the observation. After the experiment was completed, organs (liver, kidneys, spleen) were removed from the animals to compare organ weights in particular groups.
In the study of animals after direct application to injured skin, PEG46-PMAPTAC52 block copolymer at concentrations of 10%, 5%, 1%, 0.1% w/v did not cause clinical symptoms. After completing the necropsy, no macroscopic changes in the organs were observed. As a result, it was found that the tested polymer in concentrations from 0.1% to 10% w/v is not toxic to animals.
Example 6. Study of PEG46-PMAPTAC52 block copolymer activity in a mouse model.
The purpose of the experiment was to determine the activity of PEG46-PMAPTAC52 block copolymer and to check its antiviral effect on infection of mice induced by HSV-1 virus. Only healthy individuals selected at random were qualified for the experiment. The animals were divided into groups.
Control group - mock (n = 5)
Control group - saline (n = 5)
Control group - zero (n = 5)
Copolymer† HSV-1 group (n = 5)
Acyclovir† HSV-1 group (n = 5)
HSV-1 group (n = 5)
Six-week-old female BALB/c mice were shaved on the lateral side of the back, the skin was scratched with a needle and the polymer containing formulation was applied in a volume of 100 mI per mouse to the scarified area and left for 30 min. Then, HSV-1 at 1 x 107 PFU in a volume of 100 mI was applied to the wound on the adhesive tape along with the formulation for treatment. After 2 hours, the adhesive tape was removed and the formulation was applied again. The treatment was repeated every 12 hours for 4 consecutive days. Every day an assessment of the disease progression was made (0 - no symptoms, 1 - blister formation, 2 - light ulceration, 3 - large ulcer patches, 4 - confluent ulceration, 5 - hind limb paralysis, 6 - death) and general health (0 - good condition, no signs of disease, 1 - apathy, ruffled fur, tanning, 2 - weight loss not more than 20% of the initial value, 3 - weight loss greater than 20% of the initial value; 4 - agony; 5 - death).
On day 7, most of the HSV-1 infected mice that did not receive any treatment died, and therefore the remaining experimental animals were euthanized and dissected. Until the last day of the study, all animals from the control groups as well as animals infected with HSV-1 receiving ACV treatment showed no signs of disease and their overall health was good. Of the PEG46-PMAPTAC52 treated group, 3 animals developed small blisters or minor ulcerations in the infected area of the skin and general apathy until the end of the study. The sequestration results revealed an enlarged stomach and heterogeneous pancreatic tissue in two cases.
The results of the experiment are summarized in Fig. 5.
Claims
1. Use of block copolymers with chains composed of a polyethylene glycol) (PEG) block and a poly(3-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) block for the preparation of a formulation for the treatment and prophylaxis of diseases caused by the HSV- 1 virus.
2. Use according to claim 1, characterized in that the treatment and prophylaxis of diseases caused by the HSV-1 herpes virus consists in the inhibition of the HSV-1 virus replication.
3. Use according to claim 1, characterized in that the diseases caused by the HSV-1 virus are herpes, herpetic conjunctivitis, herpetic keratitis and herpetic encephalitis.
4. Use according to claim 1, characterized in that the polyethylene glycol) (PEG) and poly(3- (methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) block copolymer - PEGx-PMAPTACy - is a copolymer in which the degree of polymerization x of the PEG block is 45-47, while the degree of polymerization y of the PMAPTAC block is in the range of 22-102.
5. Use according to claim 4, characterized in that the polyethylene glycol) (PEG) and poly(3- (methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) block copolymer is PEG47-PMAPTAC53, PEG47-PMAPTAC74, PEG45-PMAPTAC51 or P EG45-P M APT AC58.
6. Use according to any of the preceding claims, characterized in that the formulation is administered topically.
7. The use according to claim 6, characterized in that the formulation is applied to the skin, lips, nasal mucous membrane, genital region or to the eye.
8. Use according to any of the preceding claims, characterized in that the formulation is in a form selected from the group comprising a solution, gel, stick, lipstick, cream, serum, powder, dusting powder, ointment, eye drops or adhesive plaster.
9. Use according to any of the preceding claims, characterized in that the concentration of the PEG-PMAPTAC block copolymer in the formulation is from 10 pg/ml to 100 mg/ml.
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| Application Number | Priority Date | Filing Date | Title |
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| DE112019001290.8T DE112019001290T5 (en) | 2018-03-13 | 2019-03-13 | Use of copolymers for the manufacture of a formulation for the treatment and prophylaxis of diseases caused by the HSV-1 virus |
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| PL424871A PL235423B1 (en) | 2018-03-13 | 2018-03-13 | Application of copolymers for production of a preparation intended for treatment and prevention of diseases induced by HSV-1 virus |
| PLP.424871 | 2018-03-13 |
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| WO2024019626A1 (en) * | 2022-07-17 | 2024-01-25 | Uniwersytet Jagielloński | Cationic polymers with selective antifungal properties |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015082172A1 (en) * | 2013-12-03 | 2015-06-11 | Zzip Ag | Polydimethylsiloxane for the treatment of viral infections |
| WO2016200284A1 (en) * | 2015-06-10 | 2016-12-15 | Uniwersytet Jagiellonski | Use of a block polymer comprising a block of poly(3-(methacryloylamino)propyltrimethylammonium chloride) (pmaptac) for the neutralization of heparin |
| WO2017175132A1 (en) * | 2016-04-04 | 2017-10-12 | Uniwersytet Jagiellonski | The use of cationic derivative of dextran for inhibition of herpes simplex viruses |
-
2018
- 2018-03-13 PL PL424871A patent/PL235423B1/en unknown
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2019
- 2019-03-13 WO PCT/PL2019/050015 patent/WO2019177475A1/en active Application Filing
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015082172A1 (en) * | 2013-12-03 | 2015-06-11 | Zzip Ag | Polydimethylsiloxane for the treatment of viral infections |
| WO2016200284A1 (en) * | 2015-06-10 | 2016-12-15 | Uniwersytet Jagiellonski | Use of a block polymer comprising a block of poly(3-(methacryloylamino)propyltrimethylammonium chloride) (pmaptac) for the neutralization of heparin |
| WO2017175132A1 (en) * | 2016-04-04 | 2017-10-12 | Uniwersytet Jagiellonski | The use of cationic derivative of dextran for inhibition of herpes simplex viruses |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024019626A1 (en) * | 2022-07-17 | 2024-01-25 | Uniwersytet Jagielloński | Cationic polymers with selective antifungal properties |
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