Process for preparing a bioactive copolymer

Abstract

Process for preparing a bioactive polymer comprising acryaldehyde-derived segments and a polyalkylene glycol oligomer, said process comprising reacting a polyalkylene glycol with acryaldehyde in an aqueous solution at a temperature not greater than 15 °C to form a copolymer having a molecular weight not greater than 1000 daltons.

Description

Technical Field

[0001] The present invention relates to a process for preparing a bioactive copolymer comprising a segment derived from an acrolein monomer and a polyalkylene glycol segment. The present invention further relates to the use of the bioactive copolymer in the treatment of diseases, particularly in the treatment of bacterial infections, viral infections or cancer. Background Art

[0002] There is a growing need for effective antimicrobial, antiviral and anticancer agents. The evolution of antimicrobial resistance in pathogens has led to serious health risks, as infections cannot be controlled by many conventional antimicrobials. The emergence of new viral strains has led to a need for broad-spectrum treatments, particularly for severe viral infections. During 2020, the emergence of the global pandemic caused by SARS-CoV-2 has led to millions of infections worldwide and a large number of deaths in the elderly and those with comorbidities. This has led to an urgent need to effectively treat SARS-CoV-2 infections.

[0003] There is also an urgent need to effectively treat bacterial infections, particularly severe and life-threatening bacterial infections such as sepsis. In 2017, the global incidence and mortality of sepsis were estimated to be 48.9 million people, with 11 million sepsis-related deaths (The Lancet Vol 395, Issue 10219 pp200-211 (January 18, 2020)). Interventions targeting antimicrobial resistance are urgent for improving sepsis outcomes.

[0004] Our co-pending patent applications WO2016 / 077879 and WO2017 / 139849 disclose antimicrobial copolymers comprising a segment derived from an acrolein monomer and a polyalkylene glycol segment. The copolymers are formed by reacting polyacrolein with a polyalkylene glycol at a temperature of 25°C to 35°C to provide an antimicrobial agent having good activity against a wide range of bacteria and viruses. Polymeric antibiotics represent a significant advance in the treatment of infections such as sepsis, due to their activity and the reduced tendency for bacterial and viral strains to develop resistance due to the mode of action of the copolymer.

[0005] There has been a continuing need to provide bioactive compounds having improved activity, particularly antimicrobial activity. Summary of the Invention

[0006] We have now found that if a copolymer comprising an acrolein-derived segment and a polyalkylene glycol oligomer is formed at low temperature, the biological activity of the copolymer is significantly increased. Accordingly, we provide a process for preparing a bioactive copolymer comprising an acrolein-derived segment and a polyalkylene glycol oligomer segment, the process comprising reacting a polyalkylene glycol with acrolein in an aqueous solution at a temperature not greater than 15 °C, preferably not greater than 12 °C, such as not greater than 10 °C or not greater than 8 °C, to form a copolymer having a molecular weight not greater than 1000 daltons.

[0007] In a further embodiment, there is provided a method of treating a subject suffering from a disease selected from microbial infections, viral infections and cancer, the method comprising administering to the subject an effective amount of a bioactive copolymer prepared according to the process.

[0008] There is also provided the use of acrolein and a polyalkylene glycol in the manufacture of a medicament for treating a disease selected from bacterial infections, viral infections and cancer, wherein the use comprises the process.

[0009] The process is particularly suitable for treating cancer, bacterial infections or viral infections.

[0010] We have found that the activity of copolymers formed at low temperature is very significantly increased. In fact, the minimum inhibitory concentration (MIC), the lowest concentration that inhibits visible growth of bacteria (one or more bacteria), is very significantly lower at lower temperatures. For example, the MIC at 40 °C is generally at least three times higher than at 10 °C. In fact, for many bacteria, the MIC at 40 °C is four times higher and even at least six times higher than at 10 °C. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Examples of the invention are described with reference to the accompanying drawings. In the drawings:

[0012] Figure 1 is a graph having four line graphs mentioned in Example 2, which compares the colony forming unit counts (Log 10 CFU / swab) of a burn wound infection model treated with: (i) the copolymer composition (E1) of the process of the invention, (ii) the copolymer composition (CE3) of a comparative process not of the invention, (iii) using a known antimicrobial agent (Safromycin), and (iv) an untreated control.

[0013] Figure 2 is a graph having four line graphs mentioned in Example 2, which compares the wound contraction four days after infection after treatment with the composition mentioned in Figure 1 The wound contraction four days after infection after treatment with the composition mentioned in

[0014] Figure 3is a graph with the five line graphs mentioned in Example 3, which compares the colony forming unit counts (Log 10 CFU / g) of renal tissue after treating ascending urinary tract infections with each of the following four treatments: the copolymer composition (E1) of the process of the present invention at two different treatment rates (treatment rates); a positive control using the antibiotic meropenem, and early and late infection controls.

[0015] Figure 4 is a graph showing the five line graphs mentioned in Example 3, which compares the colony forming unit counts (Log 10 CFU / g) of bladder tissue after treating ascending urinary tract infections with each of the following four treatments: the copolymer composition (E1) of the process of the present invention at two different treatment rates; a positive control using the antibiotic meropenem, and early and late infection controls.

[0016] Figure 5 is a graph showing the nasal wash titer (log 10 genome s / μL) in hamsters infected with SARS-CoV-2 and treated with composition E1 and CE3 as described in Example 6.

[0017] Figure 6 is a bar graph showing the following: a concentration-dependent reduction of the SARS-CoV-2 virus in an in vitro study using organoids prepared from human respiratory epithelial cells as mentioned in Example 7.

[0018] Figure 7 is a bar graph showing the following: the percentage of maximum cytotoxicity observed for copolymer E1 at different concentrations (ppm) in the Vero cell protocol as mentioned in Example 7.

[0019] Figure 8 is a bar graph showing the following: the percentage of maximum cytotoxicity observed for copolymer CE1 at different concentrations (ppm) in the Vero cell protocol as mentioned in Example 7.

[0020] Figure 9 is a graph showing the bacterial load in vaginal swabs after treatment with the reference and the compositions of Example E1 and CE3 as mentioned in Example 8. Detailed Description

[0021] The term "body" means the body of a human and / or animal; the term "subject" means such a body which is the subject.

[0022] Intravenous therapy (abbreviated as IV therapy or iv therapy) is the direct infusion of a liquid substance into a vein.

[0023] As used herein, the term "parenteral" means entering (ingesting) the body in a manner different from through the intact digestive tract. That is, not in the normal stomach or intestine; non-enteral. Parenteral administration of the copolymer prepared by the process is preferred.

[0024] The term "parenteral infection" means an infection that is contracted by entering the body in a manner other than through the gastrointestinal tract. Such an infection can occur as follows: via the vascular (blood / lymph) system, the genitourinary tract, from the lungs, rupture of the skin or outer protective membrane, such as during surgery, a needle stick injury, a cut, a scrape, or any break in the skin or in the space between the skin and mucous membrane. It will be understood that a clear distinction is made between parenteral infection and parenteral administration of a drug, and that parenteral infection can potentially be treated by any method of drug administration, including oral administration (assuming an effective dose reaches the site of infection), and parenteral administration of a drug is limited to administration by a method different from the oral route.

[0025] As used herein when referring to a bacterial pathogen, the term "antibiotic-tolerant" or "superbug" refers to a bacterial pathogen that can withstand the antibiotics used in the art to treat that bacterial pathogen (i.e., the non-tolerant strain of that bacterial pathogen). For example, Staphylococcus aureus can be treated with methicillin; however, the antibiotic-tolerant strain of Staphylococcus aureus, Staphylococcus aureus USA 300, is methicillin-resistant Staphylococcus aureus (MRSA). Although this bacterial strain is common, Staphylococcus aureus:USA:300 typically infects those who are immunocompromised or in a susceptible environment. The infection will usually enter the body through a small cut or ulcer. Other symptoms associated with USA:300 are pneumonia, necrotizing fasciitis, endocarditis, and bone and joint infections.

[0026] The term "pulmonary administration" refers to administering the preparation of the present invention into the lungs by inhalation.

[0027] The term "polyalkylene glycol" includes linear and branched polyether homopolymers and copolymers of C2 to C4 alkylene glycol units. Preferred polyalkylene glycols are polyethylene glycol and polypropylene glycol and their copolymers, and most preferably polyethylene glycol.

[0028] The term "systemic" refers to a disease or disorder or the original site of injury that is distant from the original site of infection or involves the entire body (system) of an organism. Thus, the term "local" is used herein with respect to the site of the original infection. Accordingly, a systemic infection is an infection in which the pathogen is present in an organ or the blood (including bacteremia) and can be associated with a severe, potentially life-threatening disease such as sepsis. A local infection is an infection in which the pathogen has migrated only as far as the local tissues of the infection, such as the site of the lung or a wound.

[0029] As used herein, the term "inhalation" refers to the intake of air into the alveoli of the lungs. In a specific example, the intake can occur by self-administration of the formulation of the present invention while inhaling, or by administration via a respirator (e.g., administering to a patient on a respirator). The term "inhalation" as used with respect to the formulation of the present invention is synonymous with "lung administration".

[0030] The terms "treatment" and "treating" are intended to also encompass prophylaxis, therapy, and cure. Thus, in one aspect, treatment involves preventing or delaying or retarding the onset of a condition, disease, or disorder associated with antibiotic-tolerant bacteria (e.g., the symptoms associated with the disease, condition, or disorder). In another aspect, treatment involves treating (e.g., minimizing or reducing or delaying progression or regression) an existing condition, disease, or disorder associated with antibiotic-tolerant bacteria (e.g., the symptoms associated with the disease, condition, or disorder). In one embodiment, treatment provides a cure for the condition, disease, or disorder.

[0031] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting the subject copolymer and / or composition from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation (capsule) material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not causing undue harm to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffering solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0032] The term acryaldehyde-derived segment refers to a copolymer segment that contains one or more acryaldehyde monomer residues.

[0033] The terms oligomer, polyalkylene glycol oligomer, and polyacrolein oligomer refer to polymers composed of at least two monomer units, preferably at least three monomer units. Oligomers will typically contain from 2 to 20 monomer units; in one embodiment, the number of units is from 2 to 10.

[0034] The terms "monomer unit" and "monomer residue" refer to the units present in a copolymer derived from (resulting from) reaction monomers such as acryaldehyde and polyalkylene glycol. Polyalkylene glycol is considered a prepolymer that generally provides at least three diol monomer units.

[0035] The polydispersity index is the ratio of the weight-average molecular weight (M w ) of a polymer to the number-average molecular weight (M n ) of the polymer. The weight-average molecular weight and number-average molecular weight of a polymer can be determined by analytical methods such as high performance liquid chromatography. Once the weight-average molecular weight and number-average molecular weight are determined, the polydispersity index is calculated by dividing the weight-average molecular weight by the number-average molecular weight M w / M nThe polydispersity index can be easily calculated. It is assumed that a monodisperse polymer has a polydispersity index of 1.000. However, typical commercial polymers such as commercially available resins have a polydispersity index of 10 or more. Polymers with a broad molecular weight distribution have a higher polydispersity index, and polymers with a narrow molecular weight distribution have a lower polydispersity index.

[0036] The term "indirect heat exchange" means bringing fluids into a heat exchange relationship without any physical contact or intermixing between the fluids. The cooling fluid is external to the polymerization process.

[0037] Throughout the specification, the use of the term "comprising" or "including" or grammatical variants thereof shall be taken to specify the presence of the stated features, integers, steps or components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof not specifically mentioned.

[0038] The process for preparing a bioactive substance comprises reacting a polyalkylene glycol with acrolein in an aqueous solution at a temperature not greater than 15 °C, preferably not greater than 12 °C, such as not greater than 10 °C or not greater than 8 °C, to form a copolymer having a molecular weight not greater than 1000 daltons.

[0039] Without wishing to be bound by theory, it is believed that the formation of more active copolymers having hydrophobic pendant groups (side chain groups) is enhanced at low temperatures. The reaction between the functional groups within the polymer and the corresponding target bacteria, viruses or cancers (tumors, cancer) is generally enhanced by hydrophobic attraction therebetween. Hydrophobic attraction is measured by Gibbs free energy, which occurs with a decrease in enthalpy or an increase in entropy (as shown by the equation of the Gibbs thermodynamic relationship ΔG = ΔH - TΔS), which is especially the result of many interactions between the hydrophobic vinyl or alkyl groups in the polymer and the hydrophobic proteins in the target (bacteria, viruses or cancer).

[0040] The acrolein monomer (2-propen-1-one) can react with an alkylene glycol oligomer, either growing the chain through the hydrophobic vinyl and leaving a carbonyl pendant, or growing the chain through the carbonyl and leaving a hydrophobic vinyl pendant unhindered. The latter of these two alternatives has a pendant hydrophobic vinyl, enhancing the hydrophobic mechanism.

[0041] Without wishing to be bound by theory, it is believed that the occurrence of polymerization through the carbonyl group of the acrolein monomer rather than through the vinyl group of the acrolein monomer is more significant at low temperatures not greater than 15 °C, particularly not greater than 12 °C, such as not greater than 10 °C, or not greater than 8 °C, because the driving force for carbonyl polymerization (enthalpy decrease) is weaker and as a result depolymerization occurs at higher temperatures.

[0042] Those skilled in the art will appreciate that analogues (homologues) of the present monomers can be synthesized in an alternating or block configuration, thereby influencing hydrophilicity and thus bioreactivity in a manner governed by their respective monomeric reactivity ratios.

[0043] The process typically involves reacting a polyalkylene glycol with acrolein in an aqueous solution under basic catalyzed conditions. Preferably, the pH is not greater than 12.5, and preferably in a pH range greater than 7.0 to 12.5, such as 8 to 12.0.

[0044] Typically, the aqueous solution of polyalkylene glycol and acrolein contains water in an amount of at least 20% w / w, such as 20% w / w to 80% w / w, 20% w / w to 70% w / w, or 20% w / w to 60% w / w.

[0045] The weight ratio of polyalkylene glycol:acrolein is typically at least 4:1, preferably at least 5:1. For example, the weight ratio can be about 4:1 to 20:1, such as 5:1 to 20:1, or 10:1 to 15:1.

[0046] The molecular weight of the copolymer is not greater than 1000 daltons. The copolymer can have a molecular weight of 250 to 1000 daltons, particularly 300 to 1000 daltons, such as 400 to 1000 daltons. In the process, the polyalkylene glycol typically has a molecular weight of not greater than 600 daltons, such as 200 to 600 daltons.

[0047] The process can be conveniently carried out by adding acrolein to an aqueous solution of polyalkylene glycol. The aqueous solution of polyethylene glycol can contain at least 20% w / w water. The addition of polyacrolein to the aqueous polyethylene glycol can be controlled to minimize the effect of the exothermic reaction on increasing the solution temperature. A series of methods known in the art can be used to control the temperature during the reaction of acrolein monomer and polyethylene glycol. The aqueous solution can be brought into contact with a heat exchanger to provide cooling of the reaction mixture during the addition of acrolein monomer. For example, a computerized reactor system can be used to precisely control the temperature to maintain the temperature of the aqueous composition based on starting at the desired temperature.

[0048] Since the reaction is exothermic, the process preferably includes removing the heat of reaction from the reaction zone of acrolein monomer and polyalkylene glycol. The heat can be removed by adding additional solvent (such as additional water) or reactant to the reaction zone, or by transferring the heat from the reaction zone via commercially available and well-known heat exchange devices (such as shell and tube or spiral wound heat exchangers) provided with a cooling fluid stream such as a water stream.

[0049] In one embodiment, the exothermic reaction of acrolein and polyalkylene glycol can be controlled as follows and the temperature can be maintained within a desired range during the preparation of the copolymer: The polymerization reaction is carried out in a reaction vessel provided with indirect heat exchange to maintain the desired temperature. In one embodiment, the reaction vessel is a jacketed reaction vessel, and indirect heat exchange is provided by supplying a cooling fluid (such as water) flow in the jacket. The process can include maintaining the temperature at no greater than 15 °C, preferably no greater than 12 °C, by adjusting the cooling fluid flow and / or the rate of addition of reactants (such as acrolein monomer). The reaction can be carried out in a suitable reactor and is started at a desired temperature of no greater than 15 °C, preferably no greater than 12 °C, such as no greater than 10 °C, or no greater than 8 °C, and this temperature is maintained within the desired range.

[0050] In a preferred embodiment, the heat of polymerization can be removed from the reactor using indirect exchange by a cooling medium (such as water or other coolant fluid, depending on the desired temperature) in a jacket surrounding at least a portion of the reactor. The efficiency of heat removal can be computer-controlled to maintain the temperature within the desired range during the formation of the copolymer. The reactor can be a batch reactor having a jacket (for the cooling fluid flow), or can be a tubular reactor, such as a tubular loop reactor comprising one or more jackets (for a cooling fluid such as water) concentrically surrounding at least a portion of the tubular reactor.

[0051] Typically, the reaction vessel will include an agitator, such as a stirrer or other means for providing mixing, to minimize the occurrence of relatively hot or cold regions as a result of the exothermic reaction and / or heat exchange.

[0052] The process can be carried out in a set of embodiments at a temperature in the range of -20 °C to 15 °C, preferably no greater than 12 °C, such as -10 °C to 12 °C, or -10 °C to 10 °C. Typically, the temperature of the reactant solution will be at least -10 °C.

[0053] In a further set of embodiments, the temperature is in the range of -10 °C to 15 °C, such as -5 °C to 15 °C, or 0 °C to 15 °C. In a further set of embodiments, the temperature is in the range of -5 °C to 12 °C, or -5 °C to 10 °C.

[0054] In a set of embodiments, the temperature of the aqueous solution is maintained at no greater than 10 °C, such as no greater than 5 °C.

[0055] The process preferably involves adding acrolein to an aqueous solution of polyalkylene glycol. Acrolein can be added to an aqueous solution of polyethylene glycol containing at least 20% w / w water, where the acrolein is added in the form of an aqueous acrolein solution having a concentration of no greater than 50% w / w, preferably no greater than 30% w / w, of acrolein monomer.

[0056] The polyalkylene glycol may be a copolymer comprising one or more of ethylene glycol, propylene glycol, and butylene glycol monomers, and when more than one monomer is present, the copolymer may contain monomer units in a random or block distribution. A more preferred polyalkylene glycol is polyethylene glycol having a molecular weight of 200 to 600 daltons, such as polyethylene glycol having a molecular weight of 200 to 400 daltons. Those skilled in the art will understand that the term polyethylene glycol preferably does not include diethylene glycol. Polyethylene glycol having an average molecular weight of 200 to 600 daltons includes polyethylene glycol having a nominal molecular weight of 200 to 600 daltons, wherein the average molecular weight is not greater than 110% of the nominal value and not less than 90% of the nominal value (preferably, not greater than 105% and not less than 95%). The polyethylene glycol has the formula H--[OCH2CH2] n --OH. The average value of n is at least 3 and generally ranges from 3 to 10, such as 3 to 6 (although this average need not be an integer). Polyethylene glycol is widely available from commercial suppliers in pharmaceutical grade and is sold at specific nominal molecular weights, which generally indicates that the average molecular weight does not exceed 105% of the nominal value and is not less than 95% of the nominal value. Methods for determining viscosity and molecular weight are disclosed in the USPNF Official Compendium of Standards (Volume 11180 - 1182 [2007 Edition]). In one set of embodiments, the molecular weight of the polyethylene glycol is 200 to 400. In some embodiments, specific pure ethylene glycol oligomers may preferably be used, such as a compound of the following formula

[0057] H—[OCH2CH2] n —OH where n is 3 or 4.

[0058] The present invention also provides a method for treating a subject suffering from a disease selected from microbial infections, viral infections, and cancer, the method comprising administering to the subject an effective amount of a bioactive copolymer prepared according to the said process. The present invention may also be expressed as the use of acrolein and a polyalkylene glycol in the manufacture of a medicament for treating a disease selected from bacterial infections, viral infections, and cancer, wherein the use comprises the said process.

[0059] The copolymer prepared by the said process is particularly suitable for parenteral administration for treating diseases selected from cancer, viral infections, or bacterial infections.

[0060] The copolymer is typically administered systemically, for example by oral administration, inhalation, transdermal delivery or by injection such as into the bloodstream, or intramuscular injection or by intravenous therapy, such as by injection or infusion. It is generally believed that molecules with a molecular weight of no more than about 1000, particularly less than about 800 daltons, have reasonable freedom to cross the peritoneum. Oral administration requires the copolymer to be absorbed through the intestinal wall and enter the systemic circulation. In this embodiment, it is particularly preferred that the copolymer for oral administration has a molecular weight of no more than 1000 daltons, such as in the range of 250 to 800 daltons, such as 300 to 800 or 350 to 800. We have found that copolymers of this molecular weight are delivered into the systemic circulation upon oral administration to provide treatment of parenteral viral infections. For copolymers with a lower molecular weight in this range, the proportion of the copolymer absorbed through the intestinal wall is generally larger.

[0061] The copolymer can be administered as an aerosol, gel, topical foam or ointment or impregnated into a dressing for administration to the skin or mucosa for transdermal or transmucosal delivery. The copolymer can be administered as an inhalant via an aerosol, etc.

[0062] In a further embodiment, the copolymer is administered by transdermal delivery from a composition that may include a penetration enhancer for the polymer. A patch, microneedle or similar device can be used to enhance transdermal delivery.

[0063] The compositions of the present invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. The action of microorganisms can be ensured to be prevented by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include agents for adjusting tonicity, such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by including agents for delaying absorption, such as aluminum monostearate and gelatin.

[0064] In another preferred embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (s.c.) administration.

[0065] Pharmaceutical dosage forms suitable for oral administration include tablets (coated or uncoated), capsules (hard or soft shell), cachets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, dry-coated tablets (wafer) or patches such as oral patches.

[0066] Since the copolymer is soluble and remains soluble over the entire pH range of 1 to 14, it can be formulated in an aqueous composition. The copolymer can be administered in a composition with known pharmaceutically acceptable carriers and excipients; however, aqueous formulations offer significant advantages. Depending on the particular virus to be treated and the mode of administration, the composition can include a wide range of concentrations of the copolymer. In one set of embodiments, the concentration of the copolymer in the aqueous pharmaceutical composition ranges from 0.01 wt% to 20 wt% of the composition. Thus, in one set of preferred embodiments, the copolymer is administered as an aqueous solution.

[0067] The composition can be administered orally in the form of tablets, caplets, syrups or liquids, and the oral dosage will depend on the severity and type of the virus, but can range, for example, from 1 mg - 1000 mg / kg body weight / day, such as from 10 mg - 500 mg / kg body weight / day.

[0068] In a further aspect, the present invention provides a composition for treating a disease in a subject selected from bacterial infections, viral infections and cancer, wherein the composition is prepared by the process described herein.

[0069] One of the significant advantages of the copolymer and the treatment method is that they can be used against infections from a wide range of pathogens and, in particular, can be used to treat bacterial infections that can rapidly deteriorate and pose a serious threat, such as bacteremia or sepsis or pneumonia or meningitis or cellulitis. Specific examples of such bacterial infections can be selected from the group consisting of the following bacteria: Proteus spp, Serratia spp, Pseudomonas aeruginosa, Neisseria meningitidis, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Staphylococcus spp, Streptococcus pyogenes, Streptococcus pneumonia and Enterococcus spp.

[0070] Activity against a wide range of pathogens and, in particular, against a wide range of bacteria allows the copolymer to be used as a first line of treatment in severe or life-threatening infections, for example, in cases where the severity of the infection may not allow sufficient time to correctly identify the responsible bacteria.

[0071] Viral infections can be caused by a range of viruses, such as coated viruses (e.g., phospholipid-coated viruses), including herpes, HIV, cytomegalovirus, and influenza. Preferably, the viral infections treated and / or controlled by the methods of the present invention can be: HSV-1, HSV-2, Varicella Zoster Virus (in the form of chicken pox or shingles), HCMV, EBV, herpes 6, herpes 7, herpes 8, and SARS-CoV-2.

[0072] In another embodiment, the virus is an influenza virus, such as influenza A.

[0073] In yet a further embodiment, the virus is Ross River virus.

[0074] In a further embodiment, the virus is a coronavirus, including the coronavirus that causes severe acute respiratory syndrome.

[0075] In one embodiment, the viral infection is SARS-CoV-2, commonly also known as Covid-19.

[0076] In a set of preferred embodiments, the process for preparing the copolymer of the present invention comprises the following steps:

[0077] Providing an aqueous solution of a polyalkylene glycol (preferably polyethylene glycol with a molecular weight in the range of 200 to 600 daltons) that is weakly basic (preferably with a pH not greater than 12.5; more preferably with a pH of 8 to 12.5, such as 9.0 to 12.0);

[0078] Vigorously mixing the weakly basic solution to entrain air; adding acrolein as an aqueous solution (preferably slowly over a period of at least 2 minutes, more preferably at least 5 minutes) that is not more than 50% w / w of an aqueous acrolein solution (usually containing a preservative);

[0079] During the addition of acrolein, maintaining the solution at a temperature not greater than 15°C, preferably not greater than 12°C (such as not greater than 10°C, or not greater than 8°C) to form the copolymer;

[0080] And preferably, once the acrolein monomer has been depleted, adding an acid to provide a pH less than 9, and preferably not greater than 8.

[0081] Preferably, the temperature is controlled to no greater than 15 °C, preferably no greater than 12 °C, by conducting the reaction in a reaction vessel equipped with an indirect heat exchanger, such as a jacket having a coolant fluid stream (such as a water stream) surrounding at least a portion of the reaction vessel.

[0082] The molecular weight of the resulting copolymer is controlled by the molecular weight of the polyalkylene glycol and is directly proportional to its hydroxyl concentration. (The polymerization can be initiated at ambient temperature, and then the exotherm from the polymerization reaction is controlled by one or more of the following: the volume of the reaction mixture, the amount of solvent (such as water), the rate of addition of reactants (such as the rate of addition of acrolein to the aqueous polyalkylene glycol), and the use of a reaction vessel with indirect heat exchange.

[0083] During the reaction, stirring is preferably continued, and the pH is maintained weakly basic only as necessary (preferably pH no greater than 12.5, more preferably pH 9 to 12.5, 9 to 12, or 9 to 11). More base is added and its concentration is minimized to reduce degradation / side reactions and reduce the formation of carbonyl or carboxyl groups in the product.

[0084] Finally, the pH of the solution can be lowered. In a set of preferred embodiments, the pH is adjusted to near neutral by adding an acid. The extremely pungent odor of acrolein is no longer apparent in the copolymer product, which is typically formed in at least 99% yield.

[0085] The resulting acrolein-copolymer typically has a molecular weight in the range of 250 to 1000 daltons (e.g., 300 to 1000 daltons, 400 to 1000 daltons, or 400 to 800 daltons). The copolymer does not have the turbidity expected from any content of polyacrolein. The weight ratio of acrolein: polyethylene glycol used in its preparation is preferably between 1:4 and 1:40, more preferably between 1:8 and 1:20.

[0086] The preferred base is an aqueous solution of an alkali metal hydroxide; more preferably, the alkali metal hydroxide is sodium hydroxide.

[0087] The preferred acid is dilute hydrochloric acid, although acetic acid is useful for pH buffering purposes.

[0088] Preferably, it takes about 10 minutes to add acrolein to the aqueous solution of polyalkylene glycol, and the reaction to completion typically takes about 40 minutes and preferably no more than 90 minutes.

[0089] Typically, we have found that a reaction time of 50 minutes is suitable for obtaining substantially complete conversion to the copolymer product.

[0090] Acrolein is preferably added as an aqueous solution to the aqueous polyalkylene glycol, more preferably at a concentration of acrolein monomer in the range of 10 - 30% by weight, based on the weight of the acrolein aqueous solution to be added to the polyalkylene glycol aqueous solution.

[0091] The resulting copolymer has a reactive carbonyl content (plus any carboxyl content) of less than 10%, more preferably less than 5%, and even more preferably 0%.

[0092] The acrolein solution typically contains the inhibitor hydroquinone, for example not more than 0.5% and typically 0.01 - 0.5%, and more preferably 0.1% weight / weight.

[0093] It will be apparent to those skilled in the art that the copolymers herein can be included in a variety of compositions and physical forms. In particular, the compositions and methods of in vivo pharmaceutical use will be apparent, which utilize the slower clearance rate of the copolymers. Additionally, it is apparent that the change in molecular weight can be pharmacologically utilized to regulate the penetration rate (rate) through membranes, tissues, and organs, and the resulting absorption or distribution in the human or animal body; in this case, lower molecular weight copolymers such as, for example, 300 - 800 daltons are absorbed and distributed more rapidly than copolymers with a molecular weight exceeding 1000 daltons.

[0094] In view of the results herein, it is also conceivable to add proteins, particularly broth, to enhance the antimicrobial activity in the use of the copolymers.

[0095] The subject products herein are water-soluble and can be administered to humans / animals by conventional methods known in medicine (particularly orally or by injection), and may be capable of being used in any practical pharmaceutical manner, either alone or in the form of a composition, within organs and tissues, or in contact with or within the vascular system of a human or animal. When administering the copolymers to humans and animals, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0096] Those skilled in the art also know that bacterial infections can lead to cancer, either due to the infection causing chronic inflammation or due to the infection releasing metabolites that induce cancer (the example of Helicobacter pylori causing cancer is well-known). Thus, since the copolymers described within the present invention have been found to be useful as antibiotic drugs against bacteria, viruses, and cancer, the activities described herein should be advantageous and possibly synergistic. Additionally, since viral infections, particularly those caused by influenza viruses, are also often associated with bacterial infections, once again, the antiviral and antibiotic properties in a single drug are advantageously synergistic.

[0097] The present invention will now be further described with reference to the following examples. It will be understood that the examples are provided by way of illustration of the invention and in no way limit the scope of the invention.

[0098] Examples

[0099] Example 1 and Comparative Examples - Preparation of Copolymers of Approximately 500 Dalton MW

[0100] The following processes were carried out in a vessel in which the reaction temperature was computer-controlled in the range of 10 °C (Example E1 of the composition according to the invention) to 40 °C (Comparative Example 4), as shown in Table 1.

[0101] The reaction was carried out in a glass reactor equipped with a stirrer and a water-jacketed heat exchanger and automated computer feedback temperature control during the reaction.

[0102] Table 1

[0103]

[0104] A solution of freshly distilled acrolein (5 g; inhibited with 0.1% w / w hydroquinone) in water (20 g) was slowly added to a solution of water (20 g) and polyethylene glycol (60 g; MW 200) (pH 12 by addition of 1 M aqueous sodium hydroxide) over 10 minutes; during this 10-minute period, the yellow color of the oxidized hydroquinone rapidly appeared and then disappeared. During this process, the composition was continuously and vigorously stirred to provide ample contact with air. An exothermic and rapid polymerization occurred.

[0105] After an additional 50 minutes, the clear solution was adjusted to pH 7.5 by addition of 1 M aqueous hydrochloric acid; the product was a clear, almost colorless (very pale yellow) solution. All tests and results on the samples herein were carried out on samples without any purification.

[0106] The UV-visible, 200 - 600 nM spectrum of the product had significant absorption only at the far boundary of the 200 - 300 nM region.

[0107] HPLC indicated that the polymerization yield was 99 - 100% w / w and any residual acrolein monomer was less than 1 ppm w / w; the copolymer remained soluble when tested down to pH 1 (and up to pH 14).

[0108] For the copolymers of Example 1 and Comparative Examples 1 to 4, the MICs for Staphylococcus aureus and Escherichia coli were determined and are reported in Table 2 together with the UV peaks detected at the different wavelengths shown.

[0109] Table 2

[0110]

[0111] Example 2 - Treatment of Burn Infection

[0112] This example examined the efficacy (effectiveness) of the compositions of Example 1 and Comparative Example 3 with a moded compound in the treatment of wound infection, where the negative control received no treatment and the positive control used saphomycin.

[0113] Procedure

[0114] Experimental Design

[0115] Table 3

[0116]

[0117] Experimental Procedure

[0118] Preparation of Inoculum [Day - 1]

[0119] One day before inducing infection or on the day of creating the wound, the glycerol stock Staphylococcus aureus [ATCC 43300] culture was thawed and inoculated into fresh casein soy digest (CSD) broth and incubated overnight at 37°C in a shaking incubator (200 rpm) (preferably, the inoculation was done in the evening).

[0120] Generation of Burn Wounds

[0121] The back skin of 6 - 8 - week - old male Wistar rats was shaved. The animals were anesthetized by inhalation of 2 - 3% isoflurane anesthetic. The depth of anesthesia was checked by pinching the tail. Burn wounds were created on the shaved and disinfected surface using molten wax heated up to 80°C. The wax was poured onto the shaved back of the animal through a 2 cm × 2 cm plastic mold. The wax was allowed to remain on the skin until it solidified. Ketoprofen was administered subcutaneously at a dose of 10 mg / kg to reduce pain and stress. The animals were housed individually with provisions.

[0122] Induction of Infection

[0123] One day after injury, 100 μl of ~10 8 CFU / ml Staphylococcus aureus was used to infect the rats at the site of the skin wound, and the wound site was covered with sterile cotton gauze.

[0124] Formulations

[0125] The carrier of the copolymer was sterilized with 0.9% saline. The test doses of the copolymer were prepared as follows: Group 2 - 383 mg of Example 1 was added to 20 ml of the carrier (concentration = 19.15 mg / ml), and Group 3 - 383 mg of Comparative Example 3 was added to 20 ml of the carrier (concentration = 19.15 mg / ml).

[0126] Treatment

[0127] Treatment was started one day after infection. The animals were treated with the test and reference compounds for 3 consecutive days as shown in the experimental design.

[0128] A 100 μl volume of both Example 1 (concentration = 19.15 mg / ml) and Example 3 (concentration = 19.15 mg / ml) was applied to the wound infection area via a pipette. Subsequently, the solution was evenly diffused from the center to the peripheral ends on the wound surface using a sterile bent tip, and the wound was covered with a sterile cotton gauze.

[0129] Saphyromycin (30 mg b.i.d.) was applied to the wound surface using a sterile cotton swab.

[0130] Observation

[0131] Body weight: The body weight was recorded daily throughout the study.

[0132] Clinical signs: The clinical signs of the animals were monitored daily throughout the study.

[0133] Wound contraction

[0134] On Day 0 and Day 4, the change in wound area was measured by tracing the wound boundary on a transparent paper. Subsequently, the trace was transferred to a 1 mm 2 graph sheet, and the wound surface area was thus evaluated. Subsequently, the percentage of wound contraction was calculated using the following equation with the evaluated surface area:

[0135]

[0136] Epithelialization period

[0137] The epithelialization period was marked as the number of days after wound healing required for the scab to fall off without leaving the original wound.

[0138] Bacteriological evaluation of the wound

[0139] On the 1st and 4th days after injury, the entire wound was swabbed. Subsequently, the swab was cut off and placed in a tube containing 1 ml of sterile saline. After mixing using a vortex mixer to release bacterial cells from the swab into the saline, 100 μl of the undiluted cell suspension or its 100-fold dilution was plated on a CSD agar plate and incubated overnight (16 h) on the CSD agar plate at 37 °C for bacterial counting.

[0140] Data analysis

[0141] The body weight, wound contraction percentage, and bacterial load of each animal were estimated. The significant differences between the group and control means were evaluated using appropriate statistical tests with the software Graphpad Prism (v 5.0).

[0142] Results

[0143] The efficacy of the E1 and CE3 compositions was evaluated in a burn wound infection model in rats infected with Staphylococcus aureus [ATCC 4330].

[0144] Table 4. Bacterial load on the skin after treatment

[0145]

[0146] * Significantly lower than on the 1st day (p < 0.05, paired t-test)

[0147] Summary

[0148] ● The test dose of composition E1 (100 μl (19.15 mg / ml), topical, u.i.d, 3 days) showed a significant reduction in bacterial load on the 4th day compared to the 1st day, while in the vehicle control, there was no significant reduction in bacterial load (p > 0.05).

[0149] ● The test dose of CE3 (100 μl (19.15 mg / ml), topical, u.i.d, 3 days) showed significantly lower efficacy on the 4th day compared to E1.

[0150] ● Saffamycin (30 mg, topical, b.i.d, Q = 12 hr, 3 days) did not show significant efficacy on the 4th day compared to the 1st day, although the average load was lower.

[0151] The results are plotted in the figure shown in Figure 1 as follows.

[0152] Table 5. Wound contraction percentage

[0153]

[0154] *Significantly different from vehicle control (p < 0.05, one-way ANOVA)

[0155] Summary:

[0156] ● When compared to the vehicle control, the test doses of Composition E1 (100 μl (19.15 mg / ml), topical, u.i.d., 3 days), Composition CE3 (100 μl (19.15 mg / ml), topical, u.i.d., 3 days), and Saffromycin (30 mg, topical, b.i.d., Q = 12 hr, 3 days) showed that the wounds were significantly reduced on Day 4 compared to Day 1 (p < 0.05).

[0157] Results are plotted in Figure 2 .

[0158] Example 3 - Treatment of Ascending Urinary Tract Infection

[0159] This example compared the efficacy of Compositions E1 and CE3 against Escherichia coli in a rat model of ascending urinary tract infection.

[0160] Experimental Procedure

[0161] Catheterization of the jugular vein [2]

[0162] Twenty-four hours prior to infection, male Wistar rats, 6 - 8 weeks old, were anesthetized with ketamine and xylazine (70 + 10 mg / kg). Anesthesia was confirmed by tail pinch. The dorsal and ventral neck surfaces (3 - 4 cm) were shaved using a Wahl pet trimmer, and the surgical site was disinfected with 70% ethanol. The rats were placed in dorsal recumbency, and a 1 - 2 cm incision was made on the lateral ventral neck; the jugular vein was dissected and freed from the surrounding tissue. Two surgical ligatures were placed under the vein (one at the cranial end and the other at the caudal end). The cranial end was tightly tied, a small incision was made at the caudal end using spring scissors, and a catheter filled with heparinized saline (100 IU / ml) was inserted into the lumen of the vessel (approximately 25 - 35 mm). Sufficient blood was withdrawn into the cannula to ensure proper placement and patency.

[0163] Preparation of inoculum [Day - 1]

[0164] One day prior to infection (Day - 1), a glycerol stock of Escherichia coli [ATCC 25922] was inoculated into sterile casein soy digest broth and maintained at 37°C for incubation.

[0165] Day 0 (Infection Day)

[0166] On the day of infection, the overnight culture was examined, centrifuged, and the pellet was resuspended in sterile saline and serially diluted to obtain ~1 x 10 9 CFU / ml and used for infection. The inoculum was serially diluted 10-fold in sterile CSD broth, and for each strain, six dilutions of 0.05 ml were plated on pre-incubated CSD agar plates to determine the viable count (CFU / ml) of the inoculum.

[0167] Induce infection [3,4,5]

[0168] Animals with 100% patency were selected for the study and divided into different groups as specified in the experimental design. The caged animals were transported to the procedure room, near the biosafety cabinet. All infections were performed in the biosafety cabinet with appropriate personal protection. The animals were anesthetized by intraperitoneal injection of a cocktail of ketamine and xylazine (60 + 10 mg / kg i.p.). Once the animals were under sufficient deep anesthesia, as monitored by the pedal reflex, the abdominal wall of each rat was shaved with an electric razor and the skin was cleaned with 10% povidine iodine. After a 1.5 - to 2-cm abdominal wall incision, the abdominal wall muscles were separated by blunt dissection. The bladder was isolated and exposed, the urine in the bladder was removed, and 0.1 ml of sterile saline or the bacterial culture Escherichia coli (~1 x 10 8 CFU / animal) was injected into the bladder. After replacing the bladder to its original position, the abdominal muscles were approximated and the skin was closed using sutures. The surgical site was cleaned with 10% povidine iodine.

[0169] Formulation

[0170] The carrier for composition E1 was sterile 0.9% saline. Meropenem was prepared in MilliQ water.

[0171] Treatment

[0172] Four hours after infection, Groups 4, 5, and 6 were administered Composition E1 intravenously (jugular vein) as a single dose at a constant infusion rate (2 ml / kg / hr) over a 24-hour period to conscious animals, with a dose volume of 48 ml / kg. The dose levels of Composition E1 were 50, 500, and 4000 mg / kg. Group 2 was administered sterile 0.9% saline intravenously (jugular vein) as follows: as a single dose, at a constant infusion rate (2 ml / kg / hr), over a 24-hour period to conscious animals, with a dose volume of 48 ml / kg. Meropenem (Group 3) was administered as a single IV bolus of 30 mg / kg (dose volume of 5 ml / kg). The total duration of treatment was 24 hours.

[0173] Termination

[0174] Twenty-four hours after the start of treatment (28 hours after infection), the animals in each group were euthanized by overdose of CO2 in an appropriate exposure chamber, as specifically described in the experimental design. The animals in Group 1 were sacrificed 4 hours after infection.

[0175] Tissue collection and homogenization

[0176] The euthanized animals were immersed in 70% ethanol for surface decontamination. The organs were removed aseptically - the bladder was excised near the urethra, and the kidneys were removed by blunt dissection to avoid bleeding. The bladder and each kidney were homogenized separately in sterile saline using a homogenizer (Omni Tip (THB220 hand-held)), and viable counts were determined by the spread plate technique using CSD agar plates. After incubation at 37 °C for 18 - 24 hours, the CFU / ml of the homogenate of the bladder and kidneys was determined.

[0177] Data analysis

[0178] In each group, Log 10 CFU / g of bladder and Log 10 CFU / g of kidney were estimated. The significant differences between group means were analyzed as follows: one-way ANOVA analysis, followed by Dunnett's multiple comparison test, using Graphpad Prism at a 95% confidence level. A P-value of < 0.05 was considered significant.

[0179]

[0180] Kidney:

[0181] ● When compared to the vehicle control, meropenem showed significant bactericidal activity in the kidneys after a single IV bolus dose of 30 mg / kg.

[0182] ● Composition E1 [4000 mg / kg] showed toxicity - all animals died 12 hours after the start of infusion.

[0183] ● At 50 and 500 mg / kg, Composition E1 showed a dose-dependent antibacterial effect, with 500 mg / kg showing significant activity (p < 0.05) when compared to the vehicle control.

[0184] The results are plotted in Figure 3 .

[0185]

[0186] Bladder

[0187] ● When compared to the vehicle control, meropenem showed significant bactericidal activity in the bladder after a single IV bolus dose of 30 mg / kg.

[0188] ● When compared to the vehicle control, Composition E1 showed a significant dose-dependent effect (p < 0.05) in the bladder at 50 and 500 mg / kg.

[0189] The treatment results for bladder infection are shown in Figure 4 .

[0190] Example 4 - Influenza A virus infection

[0191] Overview

[0192] The aim of this study was to determine the efficacy of Composition E1 and Composition CE3 after intravenous (IV) administration (twice daily for 5 days in C57BL / 6 mice) against the influenza A virus (H1N1) strain: A / NWS / 33 ( VR-219 TM ). During the entire study period, mice treated with E1 and CE3 showed dose-dependent weight improvement. On day 4, animals in ribavirin, E1 (25 mg / kg, 50 and 100 mg / kg), and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly higher weights (p < 0.05) when compared to the vehicle control.

[0193] Ribavirin significantly reduced the viral growth rate and viral load in the lungs when compared to the vehicle control; E1 showed a significant dose-dependent reduction in viral growth rate and viral load when compared to the vehicle control. CE3 showed a significant dose-dependent reduction in viral growth rate and viral load when compared to the vehicle control. On day 4, animals in ribavirin E1 (25 mg / kg, 50, and 100 mg / kg) and CE3 (100 mg / kg, 500, and 1000 mg / kg) showed significantly lower viral titers (p < 0.05) when compared to the vehicle control.

[0194] In the provided solution, the concentration of composition CE3 and composition E1 was 1000 mg / ml.

[0195] Test system

[0196] Species: Mouse

[0197] Strain, sex: C57BL / 6, female

[0198] Age: 6 - 8 weeks

[0199] Number of groups: 9

[0200] Number of animals / group: 12

[0201] Total number of animals: 108

[0202] Virus: Influenza A virus (H1N1) strain: A / NWS / 33( VR - 219T M )

[0203] Table 8. Experimental design

[0204]

[0205] * Starting 4 h before infection

[0206] Induce infection

[0207] Mice were anesthetized intraperitoneally (i.p.) with ketamine 60 mg / kg IP + xylazine 10 mg / kg. Anesthetized mice were infected intranasally with 30 μl of virus inoculum (~1x10 4 ) IAV (plaque - forming units (PFU) / mouse) (15 μl / nare).

[0208] Formulation

[0209] Compositions E1, CE3, and ribavirin were formulated in sterile 0.9% saline.

[0210] Treatment

[0211] Four hours before infection, animals were dosed with the test and reference items as shown in the experimental design. Treat the animals for a duration of 6 days as shown in the experimental design.

[0212] Terminate

[0213] On days 1, 2, 4, and 6 post-infection, three animals from each group were sacrificed using an overdose of CO2.

[0214] Harvest tissues and homogenize

[0215] The euthanized animals were immersed in 70% ethanol for surface decontamination. The lungs were excised aseptically and homogenized in 1 mL of cold infection medium in a sterile homogenizer. The samples were centrifuged at 300 x g for 5 - 10 minutes at room temperature, the supernatant was collected in a fresh sterile tube, and stored at 4°C or at -80°C.

[0216] Clinical observations

[0217] Monitor the general clinical signs, morbidity, and mortality of the animals. Measure the body weight at the end of the study.

[0218] Data analysis

[0219] In each group, estimate the mean ± SD Log 10 PFU / g lung. Analyze the significant differences between the group means and the control as follows: one-way ANOVA, followed by Dunnett's multiple comparison test, using Graphpad Prism at a 95% confidence level. A P-value < 0.05 was considered significant.

[0220] Results

[0221] On day 6 PI, two out of three animals in the vehicle control group were found dead. In group E1 (25 mg / kg twice daily (IV bolus, q12h), 5 days), two out of three animals were weak and one out of three animals was found dead. During the entire study period, the animals in all other groups appeared normal.

[0222] The body weights of the animals during the entire study period are shown in Table 9.

[0223]

[0224] Animals in the uninfected control gained weight; animals in the vehicle control and E1 (25 mg / kg) groups lost weight significantly throughout the study period; animals in the positive control showed no significant weight loss. Animals in the E1 (50 mg / kg and 100 mg / kg) groups showed a decrease in mean body weight on day 2 but showed subsequent weight gain; animals in CE3 (100 mg / kg) showed weight loss throughout the study period, while 500 mg / kg and 1000 mg / kg showed no significant decrease on day 6. Statistical analysis of body weight could not be performed on day 6 because the number of mice in the vehicle control group was 1. Instead, this statistical analysis was completed on day 4. On day 4, animals in ribavirin, E1 (25 mg / kg, 50, and 100 mg / kg), and CE3 (100 mg / kg, 500, and 1000 mg / kg) showed significantly higher body weights (p < 0.05) when compared to the vehicle control.

[0225] Viral titers in the mouse lungs are shown in Table 10.

[0226]

[0227] Ribavirin significantly reduced the viral growth rate and viral load in the lungs when compared to the vehicle control; E1 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to the vehicle control. CE3 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to the vehicle control.

[0228] Statistical analysis of viral titers could not be performed on day 6 because the number of mice in the vehicle control group was 1. Instead, the statistical analysis was completed on day 4. On day 4, animals in ribavirin, E1 (25 mg / kg, 50, and 100 mg / kg), and CE3 (100 mg / kg, 500, and 1000 mg / kg) showed significantly lower viral titers (p < 0.05) when compared to the vehicle control.

[0229] Conclusion

[0230] In the use of influenza A virus (H1N1) strain: A / NWS / 33( VR-219 TMIn an infected murine influenza model, animals treated with the vehicle showed significant weight loss; mice treated with ribavirin showed no significant weight loss; mice treated with E1 and CE3 throughout the study showed dose-dependent weight improvement. On day 4, animals in ribavirin, E1 (25 mg / kg, 50, and 100 mg / kg), and CE3 (100 mg / kg, 500, and 1000 mg / kg) showed significantly higher body weights (p < 0.05) when compared to the vehicle control. Ribavirin significantly reduced the viral growth rate and viral load in the lungs when compared to the vehicle control; E1 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to the vehicle control. CE3 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to the vehicle control. On day 4, animals in ribavirin, E1 (25 mg / kg, 50, and 100 mg / kg), and CE3 (100 mg / kg, 500, and 1000 mg / kg) showed significantly lower viral titers (p < 0.05) when compared to the vehicle control.

[0231] Example 5 - Maximum Tolerated Dose

[0232] Example 5A - MTD after repeated intravenous bolus dosing in mice for 7 days.

[0233] Female BALB / c mice were dosed intravenously (bolus) with the vehicle, CE3 (5, 50, and 500 mg / kg q12h, 7 days), and E1 (5, 50, and 500 mg / kg q12h, 7 days). After dosing, the general clinical signs of the animals were observed. Briefly, when dosed intravenously twice daily (q = 12h) for 1 week, the MTD of CE3 in mice was found to be 500 mg / kg. When dosed intravenously twice daily (q = 12h) for 1 week, the MTD of E1 in mice was found to be 50 mg / kg.

[0234] Materials and Methods

[0235] Test System

[0236] Species: Mouse

[0237] Strain, Gender: BALB / c, Female

[0238] Age: 6 weeks

[0239] Number of Groups: 7

[0240] Number of Animals / Group: 5

[0241] Total Number of Animals: 35

[0242] The composition of E1 and CE3 is provided as a liquid formulation at a concentration of 1000 mg / ml.

[0243] Table 11. Experimental design

[0244]

[0245] Experimental procedure

[0246] Animals were dosed quantitatively by intravenous injection (bolus) with different doses of vehicle and test substance twice daily (q12 h) for 7 days as shown above. The animals were observed for 7 days and compared with the vehicle control group for the following parameters:

[0247] 1. Body weight, just before quantitative dosing and up to 7 days after quantitative dosing

[0248] 2. Clinical signs (gait, posture, morbidity), before quantitative dosing and immediately after quantitative dosing up to 7 days.

[0249] 3. Mortality of the observed animals.

[0250] Formulation

[0251] 0.9% normal saline was used as the vehicle for E1 and CE3.

[0252] E1 composition

[0253] 5 mg / kg: 2.0 μl of the formulation (1000 mg / ml) was diluted to 2 ml with the addition of 1.998 ml of 0.9% normal saline.

[0254] 50 mg / kg: 20 μl of the formulation (1000 mg / ml) was diluted to 2 ml with 1.980 ml of 0.9% normal saline.

[0255] 500 mg / kg: 200 μl of the formulation (1000 mg / ml) was diluted to 2 ml with 1.800 ml of 0.9% normal saline.

[0256] CE3 composition

[0257] 5 mg / kg: 2.0 μl of the formulation (1000 mg / ml) was diluted to 2 ml with the addition of 1.998 ml of 0.9% normal saline.

[0258] 50 mg / kg: 20 μl of the formulation (1000 mg / ml) was diluted to 2 ml with 1.980 ml of 0.9% normal saline.

[0259] 500 mg / kg: Dilute 200 μl of the formulation (1000 mg / ml) to 2 ml using 1.800 ml of 0.9% saline.

[0260] The formulation was freshly prepared daily before each dosing.

[0261] Quantitative dosing

[0262] Animals were dosed intravenously (bolus) with different doses of CE3 and E1 twice daily (q12h) for 7 days as shown above. The dose volume was 5 ml / kg.

[0263] Results

[0264] Clinical signs, gross pathology, and body weights of the animals are shown in Tables 12 and 13.

[0265]

[0266]

[0267]

[0268] Conclusions

[0269] Mice treated with low doses of E1 (5 and 50 mg / kg, i.v., q12 h for 7 days) appeared normal after dosing and were comparable to the vehicle group. Among the mice dosed with a high dose of E1 (500 mg / kg, i.v., q12 h for 7 days), three out of five mice were found dead after the 4th dose.

[0270] Animals in the CE3-treated groups (i.e., vehicle, low, medium, and high dose groups) appeared normal throughout the study.

[0271] In both the CE3- and E1-treated groups of mice (i.e., vehicle, low, medium, and high dose groups), the average body weight increased, except for the high-dose E1 group.

[0272] In summary, when dosed intravenously twice daily (q = 12 h) for 1 week, the MTD of CE3 in mice was found to be 500 mg / kg. When dosed intravenously twice daily (q = 12 h) for 1 week, the MTD of E1 in mice was found to be 50 mg / kg.

[0273] Example 5B - Determination of the maximum tolerated dose (MTD) of the compositions of Examples CE3 and E1 after repeated oral dosing in rats for 7 days

[0274] Summary

[0275] Male Sprague Dawley rats were dosed orally with vehicle, CE3 composition (5, 50, and 500 mg / kg q12h), and E1 composition (5, 50, and 500 mg / kg q12h) for 7 days. After dosing, the general clinical signs and body weights of the animals were observed. In rats, when dosed orally twice daily for 7 days, the maximum tolerated dose (MTD) of CE3 and E1 was found to be at least 500 mg / kg.

[0276] Test

[0277] The compositions of CE3 and E1 were supplied as solutions at a concentration of 1000 mg / ml in glass bottles.

[0278] Test subjects

[0279] Species: Rat

[0280] Strain: Sprague Dawley

[0281] Age: 6 - 8 weeks

[0282] Sex: Male

[0283] Experimental procedures

[0284]

[0285] The animals were divided into different groups as shown in the experimental design and were provided with food and water ad libitum. The rat groups were dosed orally with different doses of the test substances twice daily (q12h) for 7 days as described above.

[0286] Results

[0287] At all doses, the animals dosed with vehicle, CE3, and E1 appeared normal throughout the study (Table 1 below). In all groups, the mean body weight of the animals increased, and no significant abnormalities in gross pathology were observed in any of the groups (Table 2 below).

[0288]

[0289]

[0290] Conclusions

[0291] When dosed orally twice daily for 7 days, the maximum tolerated dose (MTD) of CE3 and E1 was found to be at least 500 mg / kg in rats.

[0292] Example 6 - SARS-CoV-2

[0293] In Syrian golden hamsters (a widely accepted infection model), the combination of E1 and CE3 demonstrated dose-dependent activity against SARS-CoV-2 in vivo.

[0294] Intranasal administration of both compounds supported multiple potential dosing regimens against SARS-CoV-2.

[0295] Methods

[0296] This study consisted of five groups of eight hamsters each, with each group receiving a different treatment - a placebo control of saline nasal lavage, low-dose CE3 (200 mg / kg), high-dose CE3 (400 mg / kg), low-dose E1 (100 mg / kg), and high-dose E1 (200 mg / kg). On day 0, all animals were infected with SARS CoV-2, treatment was administered twice daily on days 1 - 5, and viral titers were measured directly via qPCR on days 2, 4, and 6. In this model, viral titers typically peak between days 2 and 4.

[0297] Results in both CE3 and E1 demonstrated a positive reduction in COVID-19 viral load compared to the placebo group. The results are shown in the Figure 5 figure, which is a plot showing nasal lavage titers (log 10 genome / μL) in hamsters infected with SARS-CoV-2 and treated with the compositions E1 and CE3. In the figure, for each day, the figure refers from left to right to placebo, CE3 (200 mg / Kg), CE3 (400 mg / Kg), E1 (100 mg / Kg), and E1 (200 mg / Kg). The mean log reduction within the group on day 4, where the low E1 dose achieved a log reduction of approximately 1.5 logs, and the high dose of CE3 achieved a log reduction of 1.25 logs. On day 6, two of the five hamsters infected with COVID-19 in the high-dose E1 group exhibited poor clinical symptoms and were excluded from the study. The company considered the study particularly anomalous because E1 is generally well tolerated in vivo at relatively high intravascular infusion doses. The weights of the hamsters in all groups remained approximately equal at the start and end of the study.

[0298] This hamster study demonstrated the potential of nasal administration of E1, especially when used against the virus.

[0299] E1 demonstrated higher activity against SARS-CoV-2 at low doses. Thus, compared to CE3, the E1 composition exhibited superior in vivo antiviral efficacy, which was consistent with the lower MIC recorded for copolymers prepared at temperatures below 15 °C.

[0300] Example 7 - SARS-CoV-2 - Human airway epithelial (HAE) cells

[0301] Part A

[0302] COVID organoid protocol: Organoids containing human airway epithelial (HAE) cells were infected by inoculation with the SARS-CoV-2 virus and cultured at 37 °C with different concentrations of CE3 (H: 31 ppm, M: 10 ppm, L: 3 ppm) and E1 (H: 82 ppm, M: 41 ppm, L: 8 ppm), and the viral load was measured by the number of PFU (plaque-forming units of the virus) evaluated at time points. The control was polyethylene glycol (PEG) 200.

[0303] Data showed that compared to the control group, a concentration-dependent reduction from the SARS-CoV-2 (COVID-19) virus baseline was achieved with CE1 and E1. The SARS-CoV-2 virus is the cause of the global COVID-19 pandemic. The concentrations used were much lower compared to the set of preclinical data on the copolymer for the CE3 intravenous infusion procedure.

[0304] With different concentrations of the two copolymer compositions, a concentration-dependent reduction in virus infection was determined and is shown in Figure 6 the figure.

[0305] Data showed that composition E1 had higher activity against SARS-CoV-2 compared to CE3, especially at low doses.

[0306] Part B

[0307] In a separate study, CE3 and E1 showed an excellent toxicity profile, with less than 0.25% effect on Vero (monkey) cells at the concentrations tested.

[0308] Cytotoxicity testing in the Vero cell protocol: In the Vero cell luminescence assay, the cytotoxicity of a series of concentrations of CE3 (153 ppm, 76 ppm, 38 ppm, 19 ppm, 13 ppm, 6 ppm, 3 ppm, 2 ppm, 1 ppm) and E1 (82 ppm, 55 ppm, 41 ppm, 33 ppm, 27 ppm, 16 ppm, 12 ppm, 8 ppm, 4 ppm) was evaluated, and cell viability was measured at the time points of 1 hour, 24 hours, and 72 hours of incubation, using untreated healthy Vero cells as the control. At all measured time points and concentrations, both compounds demonstrated minimal cytotoxic effects, with greater than 99% of the tested cells remaining viable.

[0309] The results of the cytotoxicity testing are shown in Figure 7 (for E1) and Figure 8 (for CE3). For each concentration, the maximum percentage of cytotoxicity is reported (from left to right) for 1 hour, 24 hours, and 72 hours.

[0310] Example 8 - Efficacy of E1 and CE3 against Neisseria gonorrhoeae (ATCC 700825) in mice

[0311] Summary

[0312] The aim of this study was to evaluate the efficacy of the compositions of Example E1 and Comparative Example CE3 against Neisseria gonorrhoeae (ATCC 700825) in a murine vaginal infection model. When compared to the vehicle control, meropenem showed significant bactericidal activity in the vaginal load (p < 0.05) after a 7-day intravenous bolus administration of 50 mg / kg. E1 showed a dose-dependent antibacterial effect. When compared to the vehicle control, E1 at doses of 25 and 50 mg / kg (intravenous bolus, for 7 days) showed a dose-dependent decrease in the mean bacterial load, but they were not particularly significant; E1 at 100 mg / kg (intravenous bolus, for 7 days) showed a significant antibacterial effect (p < 0.05).

[0313] When compared to the vehicle control at 7 days post-infection (PI), CE3 at 100, 500, and 1000 mg / kg (intravenous bolus, for 7 days) showed a significant dose-dependent antibacterial effect in the vaginal load (p < 0.05).

[0314] Test items

[0315] CE3 and E1 were in an aqueous solution of 1000 mg / ml.

[0316] Test system species: Mouse

[0317] Strain, sex: BALB / c, female

[0318] Age: 4 - 6 weeks

[0319] Number of groups: 9

[0320] Total number of animals: 90

[0321] Neisseria gonorrhoeae inoculation

[0322] Animals in the estrus stage of the estrous cycle were implanted subcutaneously with 5 mg, 21 - day controlled - release estradiol tablets and treated with streptomycin and trimethoprim throughout the infection period to increase their susceptibility to Neisseria gonorrhoeae. Two days after implanting the tablets, the mice were inoculated intravaginally with Neisseria gonorrhoeae (~2×106 CFU / animal).

[0323] Formulation

[0324] The carriers of CE3, E1, and meropenem were sterilized 0.9% saline. The test compounds were diluted to the required concentrations using the carrier.

[0325] Treatment

[0326] Two days after infection, the animals were treated for 7 consecutive days as shown in the experimental design.

[0327] Table 14 - Clinical signs in infected mice treated with the test compounds.

[0328]

[0329] N: Appears normal

[0330] Results are depicted in Figure 9 which shows the bacterial load in vaginal swabs after treatment with reference and test items in mice. *P < 0.05, significantly different from the carrier control.

[0331] Conclusion

[0332] In a murine vaginal infection model infected with Neisseria gonorrhoeae (ATCC700825), after 7 - day intravenous bolus dosing at 50 mg / kg, meropenem showed significant bactericidal activity in terms of vaginal load (p < 0.05) when compared with the carrier control.

[0333] E1 showed a dose-dependent antibacterial effect. When compared to the vehicle control, E1 at the doses of 25 and 50 mg / kg (IV bolus, for 7 days) showed a mean dose-dependent reduction in bacterial load; E1 at 100 mg / kg (IV bolus, for 7 days) showed a significant antibacterial effect (p<0.05).

[0334] When compared to the vehicle control at 7 days PI, CE3 at 100, 500 and 1000 mg / kg (IV bolus, for 7 days) showed a significant dose-dependent antibacterial effect on vaginal load (p<0.05).

[0335] Thus, compared to CE3, the E1 composition presented superior in vivo efficacy, which was consistent with the lower MIC recorded for the copolymer prepared at temperatures below 15°C.

[0336] Example 9 - Determination of the MIC of the compositions of the invention prepared at 5°C and 10°C

[0337] Using the procedure of Example 1, the compositions of the invention were prepared at 5°C (Example 2 - E2) and 10°C (Example 1, E1).

[0338] The MICs of Staphylococcus aureus and Escherichia coli were determined for the copolymers in each of Example 1 and Example 2 and are reported in Table 15.

[0339] Table 15

[0340]

Claims

1. A process for preparing a bioactive copolymer comprising acryaldehyde-derived segments and a polyalkylene glycol oligomer, the process comprising reacting a polyalkylene glycol with acryaldehyde in an aqueous solution at a temperature in the range of -20 °C to 10 °C to form a copolymer having a molecular weight not greater than 1000 daltons, wherein the pH of the solution is alkaline and not greater than pH 12.

5.

2. The process according to claim 1, wherein the aqueous solution of the polyalkylene glycol and acryaldehyde contains water in an amount of at least 20% w / w.

3. The process according to claim 1 or claim 2, wherein the weight ratio of polyalkylene glycol:acryaldehyde is at least 4:

1.

4. The process according to claim 1 or claim 2, wherein the molecular weight of the polyalkylene glycol is not greater than 800 daltons.

5. The process according to claim 1 or claim 2, wherein the molecular weight of the polyalkylene glycol is from 200 to 600 daltons.

6. The process according to claim 1 or claim 2, wherein acryaldehyde is added to the aqueous solution of the polyalkylene glycol.

7. The process according to claim 1 or claim 2, wherein the temperature of the aqueous solution is at least -10 °C.

8. The process according to claim 1 or claim 2, wherein the temperature of the aqueous solution is from -5 °C to 10 °C.

9. The process according to claim 1 or claim 2, wherein the polyalkylene glycol is polyethylene glycol and acryaldehyde is added to an aqueous solution of polyethylene glycol containing at least 20% w / w water, wherein acryaldehyde is added in the form of an aqueous acryaldehyde solution having a concentration not greater than 50% w / w.

10. The process according to claim 9, wherein acryaldehyde is added in the form of an aqueous acryaldehyde solution having a concentration not greater than 30% w / w.

11. The process according to claim 1 or claim 2, which comprises the steps of: providing a weakly alkaline aqueous solution of the polyalkylene glycol having a pH not greater than 12.5; adding acryaldehyde as an aqueous solution having a concentration not greater than 50% w / w of the aqueous acryaldehyde solution; and maintaining the solution at a temperature not greater than 10 °C during the addition of acryaldehyde to form the copolymer.

12. The process according to claim 11, wherein the pH of the weakly alkaline aqueous solution is from 8 to 12.

5.

13. The process according to claim 11, wherein the pH of the weakly alkaline aqueous solution is from 9.0 to 12.

0.

14. The process according to claim 11, wherein the polyalkylene glycol is polyethylene glycol and the molecular weight of the polyethylene glycol is in the range of 200 to 600 daltons.

15. The process according to claim 11, wherein the step of adding acrolein as an aqueous solution is carried out slowly.

16. The process according to claim 15, wherein the acrolein is added over a period of at least 2 minutes.

17. The process according to claim 15, wherein the acrolein is added over a period of at least 5 minutes.

18. The process according to claim 11, wherein the acrolein aqueous solution contains a preservative.

19. The process according to claim 11, wherein once the acrolein monomer has been exhausted, an acid is added to provide a pH of less than 9.

20. The process according to claim 11, wherein once the acrolein monomer has been exhausted, an acid is added to provide a pH of not more than 8.

21. The process according to claim 1 or claim 2, wherein the reaction of the polyalkylene glycol with acrolein in an aqueous solution is carried out in a reaction vessel comprising a heat exchanger for controlling the temperature.

22. The process according to claim 19, wherein the reaction of the polyalkylene glycol with acrolein in an aqueous solution is carried out in a stirred reaction vessel provided with a jacket for flowing coolant liquid to control the temperature within the reaction vessel.

23. The process according to claim 19, wherein the reaction conditions are adjusted by computer control of one or more of the following reaction parameters: acrolein addition rate, water flow rate, temperature of the water in the jacket, and stirring speed of the aqueous solution.

24. Use of acrolein and a polyalkylene glycol in the manufacture of a medicament for the treatment of a disease selected from bacterial infections, viral infections, and cancers, wherein the cancer is mediated by a microbial infection or a viral infection, and wherein the use comprises the process according to any one of claims 1 to 23.

25. The use according to claim 24, wherein the disease is a parenteral disease.

26. A composition for the treatment of a disease in a subject selected from bacterial infections, viral infections, and cancers, wherein the cancer is mediated by a microbial infection or a viral infection, and wherein the composition comprises a bioactive copolymer prepared by the process according to any one of claims 1 to 23.

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