Process for the preparation of bioactive copolymers

Abstract

A process for the preparation of a bioactive polymer comprising acrolein-derived segments and polyalkylene glycol oligomers, the process comprising reacting polyalkylene glycol with acrolein in an aqueous solution to form a copolymer with a molecular weight not exceeding 1000 daltons at a temperature not exceeding 15°C.

Description

[Technical field]

[0001] The present invention relates to a process for the preparation of biologically active copolymers comprising segments derived from acrolein monomers and polyalkylene glycol segments. The present invention further ... in the treatment of diseases, in particular in the treatment of bacterial infections, viral infections or cancer. polymer Regarding the use of. 2. Background of the Invention Summary of the Invention [Problem to be solved by the invention]

[0002] There is an increasing need for effective antibiotic, antiviral and anticancer drugs. The evolution of antibiotic resistance in pathogens creates serious health risks due to the inability of many conventional antibiotics to control infections. Also, the emergence of new viral strains leads to a demand for broad-spectrum treatments, especially for severe viral infections. The outbreak of a global pandemic caused by SARS-CoV-2 during 2020 has infected millions worldwide and resulted in high mortality rates, especially among the elderly and people with comorbidities. This has led to an urgent need for effective treatment of SARS-CoV-2 infections.

[0003] There is also an urgent need for effective treatment of bacterial infections, especially severe and life-threatening bacterial infections such as sepsis. The incidence and mortality of sepsis worldwide in 2017 was estimated at 48.9 million, with 11 million sepsis-related deaths (The Lancet, Vol 395, Issue 10219, pp 200-211 (18 Jan 2020)). Interventions targeting antibiotic resistance are a challenge to improve sepsis outcomes.

[0004] Our co-pending patent applications WO2016 / 077879 and WO2017 / 139849 disclose antibiotic co-polymers comprising segments derived from acrolein monomers and polyalkylene glycol segments. The copolymers are formed by reaction of polyacrolein with polyalkylene glycols at temperatures between 25°C and 35°C, providing antibiotics with good activity against a broad range of bacteria and viruses. Polymeric antibiotics provide significant advances in the treatment of infections, such as sepsis, due to their activity and the reduced tendency of bacterial and viral strains to acquire resistance due to the copolymer's mode of action.

[0005] There is a continuing need to provide bioactive compounds with improved activity, particularly antibiotic activity. Summary of the invention [Means for solving the problem]

[0006] We have now found that the biological activity of polymers containing acrolein-derived segments and polyalkylene glycol oligomers is significantly increased when the copolymers are formed at low temperatures. Accordingly, we provide a process for the preparation of bioactive polymers containing acrolein-derived segments and polyalkylene glycol oligomer segments, which comprises reacting polyalkylene glycol with acrolein in an aqueous solution to form a copolymer of molecular weight not exceeding 1000 Daltons at a temperature not exceeding 15°C, preferably not exceeding 12°C, such as not exceeding 10°C or not exceeding 8°C.

[0007] In a further embodiment, a method is provided for treating a subject suffering from a disease selected from microbial infection, viral infection and cancer, comprising administering to said subject a biologically active agent prepared according to the process. polymer The method includes administering to a subject an effective amount of

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

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

[0010] We found that the activity of the copolymers formed at low temperatures increases very significantly. Indeed, the minimum inhibitory concentration (MIC), the lowest concentration that prevents appreciable growth of a bacterium or group of bacteria, is very significantly lower at much lower temperatures. For example, the MIC is generally at least three times higher at 40°C than at 10°C. In fact, for many bacteria, the MIC is four times higher at 40°C than at 10°C, and even at least six times higher.

[0011] Examples of the invention will now be described with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a graph having four plots as referred to in Example 2, comparing colony forming unit counts (Log10 CFU / swab) in a burn wound infection model treated with (i) a copolymer composition of the inventive process (E1), (ii) a copolymer composition of a comparative process that is not the inventive process (CE3), (iii) a positive control using a known antibiotic (Safromycin), and (iv) an untreated control. [Diagram 2] 2 is a graph with four plots as referred to in Example 2, comparing wound contraction four days after infection following treatment with the composition referred to in FIG. [Diagram 3]FIG. 1 is a graph with five plots as referred to in Example 3, comparing the number of colony forming units (Log10 CFU / g) in kidney tissue after treatment of ascending urinary tract infections with four treatments: the copolymer composition of the process of the present invention (E1) at two different treatment rates; a positive control using the antibiotic meropenem, and early and late infection controls, respectively. [Figure 4] FIG. 1 is a graph showing the five plots mentioned in Example 3, comparing the number of colony forming units (Log10 CFU / g) in bladder tissue after treatment of ascending urinary tract infections with four treatments: the copolymer composition of the process of the present invention (E1) at two different treatment rates; a positive control using the antibiotic meropenem, and early and late infection controls, respectively. [Diagram 5] 1 is a graph showing nasal wash titers (log10 genomes / μL) of hamsters infected with SARS-CoV-2 and treated with compositions E1 and CE3 as described in Example 6. [Figure 6] Column chart showing concentration-dependent reduction of SARS-CoV-2 virus in an in vitro study using organoids made from human airway epithelial cells mentioned in 7. [Figure 7] 1 is a column chart showing the percent of maximum cytotoxicity observed in the Vero cell protocol for copolymers of E1 at different concentrations (ppm) mentioned in Example 7. [Figure 8] 1 is a column chart showing the percent of maximum cytotoxicity observed in the Vero cell protocol for copolymers of CE1 at different concentrations (ppm) mentioned in Example 7. [Figure 9] FIG. 1 is a graph showing the bacterial load in vaginal swabs after treatment with the compositions of Examples E1 and CE3 mentioned in the reference and Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The term "body" refers to the human and / or animal body, and the term "subject" refers to such things as, for example, a body, that is an object.

[0014] Intravenous therapy (briefly IV therapy or iv therapy) is the infusion of liquid substances directly into a vein.

[0015] As used herein, the term "parenteral" means taken into the body in a manner other than through the intact digestive tract, i.e., not in the normal stomach or intestines; other than the digestive system. Parenteral administration of the copolymers prepared by the present process is preferred.

[0016] The term "parenteral infection" refers to such infection by introduction into the body other than in the gastrointestinal tract. Such infection may occur via the vascular (blood / lymphatic) system, the genital-urinary tract, through the lungs, through a breach of the skin or outer protective membrane, such as in surgery, through a needlestick injury, a cut, an abrasion, or any breach of the skin or gap between the skin and a mucous membrane. It will be understood that a clear distinction is made between parenteral infections that can potentially be treated via any method of drug administration, including oral administration (assuming an effective dose reaches the site of infection) and parenteral administration of drugs that is restricted to administration other than oral.

[0017] As used herein when referring to bacterial pathogens, the term "antibiotic resistant" or "superbug" refers to a bacterial pathogen that can withstand the effects of antibiotics used in the art to treat bacterial pathogens (i.e., non-resistant strains of bacterial pathogens). For example, Staphylococcus aureus can be treated with methicillin; however, an antibiotic resistant strain of Staphylococcus aureus, S. aureus USA 300, is methicillin-resistant Staphylococcus aureus (MRSA). Although subspecies are common, S. aureus USA 300 typically infects people who are usually immunocompromised or in susceptible environments. Infection often enters the body through a small cut or sore. Other conditions associated with USA:300 are pneumonia, necrotizing fasciitis, endocarditis, and bone and joint infections.

[0018] The term "pulmonary administration" refers to administration of a formulation of the present invention to the lungs by inhalation.

[0019] The term "polyalkylene glycol" includes linear and branched polyether homopolymers and copolymers of C2 through C4 alkylene glycol units. Preferred polyalkylene glycols are polyethylene glycol and polypropylene glycol and copolymers thereof, and most preferred is polyethylene glycol.

[0020] The term "systemic" refers to disease or injury distant from the original site of infection or to the involvement of the entire body of the organism. Thus, the term "local" is used herein with respect to the original site of infection. Thus, a systemic infection is one in which the pathogen is found in the organs or blood (including bacteremia) and may be associated with serious and potentially life-threatening diseases, such as sepsis. A local infection is one in which the pathogen only travels to the infected local tissue, such as the lungs or wound site.

[0021] As used herein, the term "inhalation" refers to the taking in of air into the alveoli of the lungs. In particular embodiments, the taking in can occur while inhaling by self-administering a formulation of the invention, or via the respiratory tract, for example, by administration to a ventilated patient. The term "inhalation" as used with respect to a formulation of the invention is synonymous with "pulmonary administration."

[0022] The terms "treatment" and "treating" are also intended to encompass prophylaxis, therapy, and cure. Thus, in one aspect, treatment encompasses preventing or delaying or slowing the onset of a condition, disease, or disorder (e.g., symptoms associated with a disease, condition, or disorder) associated with antibiotic-resistant bacteria. In another aspect, treatment encompasses treating (e.g., minimizing or reducing, or slowing the onset or reversing) an existing condition, disease, or disorder (e.g., symptoms associated with a disease, condition, or disorder) associated with antibiotic-resistant bacteria. In one embodiment, treatment provides a cure for a condition, disease, or disorder.

[0023] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which is involved in carrying or transporting the subject copolymer and / or composition from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not unduly harmful to the patient. Some examples of materials that can function as pharma-ceutically 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes. waxes, and the like; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical preparations.

[0024] The term acrolein-derived segment refers to a copolymer segment that includes one or more (one or more also means one or more) acrolein monomer residues.

[0025] The terms oligomer, polyalkylene glycol oligomer and polyacrolein oligomer refer to polymers consisting 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.

[0026] The terms "monomer unit" and "monomer residue" refer to units present in copolymers derived from reactive monomers such as acrolein and polyalkylene glycols. The polyalkylene glycols are generally considered to be prepolymers providing at least three glycol monomer units.

[0027] The polydispersity index is the ratio of the weight average molecular weight (Mw) of a polymer to the number average molecular weight (Mn) 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 n A hypothetical 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 higher. 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.

[0028] The term "indirect heat exchange" means bringing fluids into a heat exchange relationship without any physical contact or intermixing with one another. The cooling fluid is external to the polymerization process.

[0029] Throughout this specification, use of the terms "comprise" or "comprising" or grammatical variations thereof should be taken to specify the presence of stated features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof that are not specifically stated.

[0030] The process for preparing the biologically active form involves reacting a polyalkylene glycol with acrolein in an aqueous solution to form a copolymer of molecular weight not exceeding 1000 Daltons at a temperature not exceeding 15°C, preferably not exceeding 12°C, such as not exceeding 10°C or not exceeding 8°C.

[0031] Without wishing to be bound by theory, it is believed that the formation of more active copolymers with hydrophobic pendant groups is promoted at low temperatures. The reaction between the functional groups in the polymer and the respective target bacteria, virus or cancer is generally enhanced by the hydrophobic attractions between them. The hydrophobic attractions, measured in terms of Gibbs free energy, arise as a decrease in enthalpy or an increase in entropy (as equated in the Gibbs thermodynamic relationship ΔG=ΔH-TΔS) as a result of the many interactions between the particularly hydrophobic vinyl or alkylene groups in the polymer and the hydrophobic proteins in the target (bacteria, virus or cancer).

[0032] Acrolein monomer (2-propen-one) can be reacted with alkylene glycol oligomers either chaining up through the hydrophobic vinyl and leaving a carbonyl pendant, or alternatively chaining up through the carbonyl and leaving a hydrophobic vinyl pendant and unhindered. The latter of the two options with a pendant hydrophobic vinyl enhances the hydrophobic mechanism.

[0033] 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 polymerization through the vinyl group of the acrolein monomer, is more significant at relatively low temperatures not exceeding 15°C, particularly not exceeding 12°C, such as not exceeding 10°C, or not exceeding 8°C, because the driving force for carbonyl polymerization (decrease in enthalpy) is weaker and, as a result, depolymerization occurs at higher temperatures.

[0034] Those skilled in the art will recognize that analogs of the constituent monomers of the present invention can be synthesized in alternating or block configurations in a manner dictated by their respective monomer reactivity ratios to affect hydrophilicity, and hence biological reactivity.

[0035] The process typically involves reacting a polyalkylene glycol with acrolein in an aqueous solution under alkaline catalyzed conditions, preferably at a pH not exceeding 12.5, and preferably within a pH range of from 7.0 to 12.5, such as from 8 to 12.0.

[0036] Typically, the aqueous solution of polyalkylene glycol and acrolein contains water in an amount of at least 20% w / w, such as from 20% w / w to 80% w / w, from 20% w / w to 70% w / w, or from 20% w / w to 60% w / w, etc.

[0037] The weight ratio of polyalkylene glycol to acrolein is typically at least 4:1, and preferably at least 5:1. For example, the weight ratio can be from about 4:1 to 20:1, such as from 5:1 to 20:1, or from 10:1 to 15:1, and the like.

[0038] The molecular weight of the copolymer does not exceed 1000 Daltons. The copolymer may have a molecular weight of from 250 to 1000 Daltons, particularly from 300 to 1000 Daltons, such as from 400 to 1000 Daltons. In the process, the polyalkylene glycol is typically of a molecular weight of not more than 600 Daltons, such as from 200 to 600 Daltons.

[0039] 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 increasing the temperature of the solution due to exothermic reaction. Various methods known in the art can be used to control the temperature during the reaction of acrolein monomer with polyethylene glycol. The aqueous solution can be contacted 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 a desired temperature.

[0040] Because the reaction is exothermic, the process preferably includes removing the heat of reaction from the acrolein monomer and polyalkylene glycol reaction zone. Heat removal can be accomplished by adding additional solvent, such as additional water, or reactants to the reaction zone, or by transferring heat from the reaction zone through commercially available and well-known indirect heat exchange devices, such as shell-and-tube or spiral wound heat exchangers with a flow of a cooling fluid, such as water.

[0041] In one embodiment, the effect of the exothermic reaction of acrolein with polyalkylene glycol can be controlled in the required range during the preparation of the copolymer by carrying out the polymerization reaction in a reaction vessel equipped with indirect heat exchange to maintain the required temperature and the temperature was maintained. In one embodiment, the reaction vessel is a jacketed reaction vessel, and the indirect heat exchange is provided by the flow of a cooling fluid, such as water, in the jacket. The process can include maintaining the temperature at not more than 15°C, preferably not more than 12°C, by adjusting the flow of the cooling fluid and / or the rate of addition of the reactants, such as acrolein monomer. The reaction can be carried out in a suitable reactor and can be started at the required temperature, such as not more than 15°C, preferably not more than 12°C, such as not more than 10°C or not more than 8°C, and the temperature can be maintained in the required range.

[0042] In a preferred embodiment, the heat of polymerization may be removed from the reactor using indirect exchange with a cooling medium, such as water or other coolant fluid, depending on the temperature required, in a jacket surrounding at least a portion of the reactor. The efficiency of heat removal may be computer controlled to maintain the temperature within the required range during the formation of the copolymer. The reactor may be a batch reactor having a jacket for the flow of a cooling fluid, or may be a tubular reactor, such as a tubular loop reactor, including one or more jackets for a cooling fluid, such as water, concentrically surrounding at least a portion of the tubular reactor.

[0043] Typically, the reaction vessel will include an agitator, such as a stirrer or other means of providing mixing, to minimize the development of relatively high or low areas as a result of reaction exotherms and / or heat exchange.

[0044] The process may be carried out in one set of embodiments at a temperature in the range of from -20°C to 15°C, preferably not exceeding 12°C, such as from -10°C to 12°C, or from -10°C to 10°C, etc. Typically, the temperature of the reactant solution will be at least -10°C.

[0045] In a further set of embodiments, the temperature is in the range of -10°C to 15°C, such as, for example, -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.

[0046] In one set of embodiments, the temperature of the aqueous solution is maintained at no more than 10°C, such as no more than 5°C.

[0047] The process preferably involves the addition of acrolein to an aqueous solution of a polyalkylene glycol. The acrolein may 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 solution of acrolein monomer at a concentration not exceeding 50% w / w, preferably not exceeding 30% w / w.

[0048] The polyalkylene glycol may be a copolymer containing one of more of ethylene glycol, propylene glycol and butylene glycol monomers, which when more than one, may contain the monomer units in a random or block distribution. More preferably, the polyalkylene glycol is a polyethylene glycol having a molecular weight of from 200 to 600 daltons, such as from 200 to 400 daltons. It will be understood by those skilled in the art that the term polyethylene glycol preferably does not include diethylene glycol. Polyethylene glycol having an average molecular weight of from 200 to 600 daltons includes polyethylene glycols having a nominal average molecular weight of from 200 to 600 daltons, where the average molecular weight is not more than 110% and not less than 90% (preferably not more than 105% and not less than 95%) of the nominal value. Polyethylene glycol has the formula H--[OCH2CH2] n --OH. The average value of n is at least 3 and generally from 3 to 10, such as from 3 to 6, etc. (although the average value need not be an integer). Polyethylene glycol is widely available in pharmaceutical grades from commercial suppliers and sold at specific nominal molecular weights, which generally represent an average molecular weight not more than 105% and not less than 95% of the nominal value. Viscosities and methods for the determination of molecular weight are disclosed in the USP NF Official Compendium of Standards, Volume 11180-1182 [2007 Edition]. In one set of embodiments, the polyethylene glycol is of molecular weight from 200 to 400. In some embodiments, certain pure oligomers of ethylene glycol, such as those of the formula H-[OCH2CH2] n -OH, where n is 3 or 4 Compounds such as those listed below may be used.

[0049] The present invention also provides a method of treating a subject suffering from a disease selected from microbial infection, viral infection and cancer, comprising administering to said subject a biologically active agent prepared according to the present process. polymer The method includes administering to a subject an effective amount of

[0050] The invention may also be expressed as the use of acrolein and a polyalkylene glycol in the manufacture of a medicament for the treatment of a disease selected from bacterial infection, viral infection and cancer, wherein the use comprises the present process.

[0051] The copolymers prepared by the present process are particularly suitable for administration for the treatment of a parenteral disease selected from cancer, viral infections or bacterial infections.

[0052] The copolymers are typically administered systemically, for example, by oral administration, inhalation, transdermal delivery, or by injection, such as into the bloodstream or intramuscularly, or by intravenous therapy, such as by injection or infusion. It is generally accepted that molecules with molecular weights not exceeding about 1000, particularly less than about 800 daltons, pass reasonably freely across abdominal membranes. Oral administration requires that the copolymer is absorbed through the intestinal wall and into the systemic circulation. In this embodiment, it is particularly preferred that the orally administered copolymer has a molecular weight not exceeding 1000 daltons, such as a molecular weight 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, when administered orally, are carried into the systemic circulation to provide parenteral treatment of viral infections. The proportion of the copolymer absorbed through the intestinal wall is generally greater for the lower molecular weight copolymers in this range.

[0053] The copolymers may be applied as an aerosol, gel, topical foam or ointment for transdermal or transmucosal delivery, or may be impregnated into a dressing for application to the skin or mucosa. The copolymers may be applied as inhalation, such as via aerosol or the like.

[0054] In a further embodiment, the copolymer is administered by transdermal delivery from a composition, which may include a penetration enhancer for the polymer. Patches, microneedles or similar devices may be used to enhance transdermal delivery.

[0055] The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibiotic and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include agents for adjusting tonicity, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

[0056] In another preferred embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (sc) administration.

[0057] Suitable pharmaceutical dosage forms for oral administration include such things as tablets (coated or uncoated), capsules (hard or soft shell), caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches, e.g., buccal patches, and the like.

[0058] The copolymer can be formulated in an aqueous composition such that it is soluble and remains soluble over the entire range of pH from 1 to 14. The copolymer can be administered in compositions with known pharma- ceutically acceptable carriers and excipients; however, aqueous formulations offer important advantages. The composition can contain a wide range of concentrations of the copolymer, depending on the particular virus being treated and the mode of administration. In one set of embodiments, the concentration of the copolymer in the aqueous pharmaceutical composition ranges from 0.01% to 20% by weight of the composition. Thus, in a preferred set of embodiments, the copolymer is administered as an aqueous solution.

[0059] The composition may be administered orally in the form of a tablet, caplet, syrup or liquid, and the dose administered orally will depend on the severity and type of virus, but may be in the range, for example, from 1 mg to 1000 mg per kg of body weight per day, such as from 10 mg to 500 mg per kg of body weight per day.

[0060] In a further aspect, the present invention provides a composition for the treatment of a disease in a subject selected from a bacterial infection, a viral infection and cancer, wherein said composition is prepared by the process described herein.

[0061] One of the key benefits of the copolymers and treatment methods is that they can be used against infections from a wide range of pathogens, and are particularly useful in treating bacterial infections that can rapidly spread to pose serious threats such as bacteremia or septicemia or pneumonia or meningitis or cellulitis. Specific examples of such bacterial infections are Proteus spp, Serratia spp, Pseudomonas aeruginosa, Neisseria meningitidis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, coagulase-negative Staphylococcus spp, Streptococcus pyogenes, Streptococcus pneumoniae and Enterococcus spp (Enterococcus spp., Enterococcus spp.).

[0062] Activity against a broad range of pathogens, and especially a broad range of bacteria, allows the copolymers to be used as a first line of treatment in severe or life-threatening infections, where, for example, the severity of the infection may not allow sufficient time to adequately identify the causative organism.

[0063] Viral infections can be caused by a range of viruses, such as enveloped viruses (e.g. lipid-enveloped viruses) including herpes, HIV, cytomegalovirus and influenza. Preferably, the viral infections treated and / or controlled by the methods of the invention can be HSV-1, HSV-2, Varicella Zoster Virus (in chickenpox or varicella shingles form), HCMV, EBV, herpes 6, herpes 7, herpes 8 and SARS-CoV-2.

[0064] In another embodiment, the virus is an influenza virus, such as, for example, influenza A.

[0065] In yet another embodiment, the virus is Ross River virus.

[0066] In further embodiments, the virus is a coronavirus, including the coronavirus that causes Severe acute respiratory syndrome.

[0067] In one embodiment, the viral infection is SARS-CoV-2, also commonly referred to as Covid-19.

[0068] In a preferred set of embodiments, the process for preparing the copolymers of the invention comprises the following steps: providing a mildly basic (preferably pH not exceeding 12.5; even more preferably pH from 8 to 12.5, such as, for example, from 9.0 to 12.0) aqueous solution of a polyalkylene glycol (preferably polyethylene glycol of molecular weight in the range of from 200 to 600 daltons); vigorously mixing the mildly basic solution to entrain air; adding acrolein (preferably slowly over a period such as, for example, at least 2 minutes, more preferably at least 5 minutes) as an aqueous solution at a concentration not exceeding 50% w / w of the acrolein in water solution (usually containing a preservative); maintaining the solution at a temperature of not more than 15° C., preferably not more than 12° C. (e.g., not more than 10° C., not more than 8° C., etc.) during the addition of acrolein to form the copolymer; and preferably, after the acrolein monomer has been consumed, adding acid to provide a pH of less than 9, and preferably not greater than 8. Includes.

[0069] Preferably, the temperature is controlled to not exceed 15°C, preferably not exceed 12°C, by carrying out the reaction in a reaction vessel equipped with an indirect heat exchanger, such as a jacket having a coolant fluid flow (e.g., a water flow) about at least a portion of the reaction vessel.

[0070] 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 amount of volumetric solvent, such as water, of the reaction mixture, the rate of addition of reactants (such as the rate of addition of acrolein to aqueous polyalkylene glycol), and the use of a reaction vessel with indirect heat exchange.

[0071] Stirring is preferably continued during the reaction and only as necessary to maintain the pH mildly basic (preferably a pH not exceeding 12.5, more preferably a pH between 9 and 12.5, between 9 and 12, or between 9 and 11). The addition of more base and its concentration is minimized to reduce decomposition / side reactions and to reduce carbonyl or carboxyl formation in the product.

[0072] Finally, the pH of the solution can be lowered. In a preferred set of embodiments, the pH is adjusted to near neutral by the addition of acid. The extremely pungent odor of acrolein is no longer evident in the copolymer product, which is usually formed in at least 99% yield.

[0073] The resulting acrolein copolymers typically have molecular weights in the range of 250 to 1000 Daltons (such as, for example, 300 to 1000 Daltons, 400 to 1000 Daltons, or 400 to 800 Daltons, etc.). The copolymers are free of the haze that would be expected from any content of polyacrolein. The weight ratio of acrolein:polyethylene glycol used in the preparation of the copolymer is preferably between 1:4 and 1:40, and more preferably between 1:8 and 1:20.

[0074] A preferred base is an aqueous solution of an alkali hydroxide; even more preferably, the alkali hydroxide is sodium hydroxide.

[0075] The preferred acid is dilute hydrochloric acid - although acetic acid is useful for pH buffering purposes.

[0076] Addition of the acrolein to the aqueous solution of the polyalkylene glycol preferably takes about 10 minutes--and the reaction to completion usually takes about 40 minutes, and preferably does not exceed 90 minutes.

[0077] We have typically found that a reaction time of 50 minutes is adequate to obtain substantially complete conversion to the copolymer product.

[0078] The acrolein is preferably added to the aqueous polyalkylene glycol as an aqueous solution - even more preferably at a concentration ranging from 10% to 30% by weight acrolein monomer, based on the weight of the acrolein solution added to the aqueous polyalkylene glycol solution.

[0079] The resulting copolymers have a reactive carbonyl group content (plus any carboxyl group content) of less than 10%, more preferably less than 5%, and even more preferably zero%.

[0080] The acrolein solution usually contains an inhibitor, such as hydroquinone, at not more than 0.5%, and typically from 0.01 to 0.5%, and even more preferably 0.1% w / w.

[0081] It will be apparent to those in the art that the copolymers herein may be included in a variety of compositions and physical forms. In particular, pharmaceutical methods of compositions and use in vivo that take advantage of the slower clearance of the copolymers will be apparent. It will also be apparent that variations in molecular weight may be utilized pharmaceutical to adjust the rate of penetration through membranes, tissues and organs--and the resulting absorption or distribution within the human or animal body; in this regard, lower molecular weight copolymers, such as those of 300 to 800 daltons, will be absorbed and distributed more rapidly than copolymers of molecular weights over 1000 daltons.

[0082] In view of the results herein, it is also conceivable to add proteins, especially broth, to enhance the antimicrobial activity of the copolymer during use.

[0083] Here, the subject products are water-soluble and administration to humans / animals can be by conventional methods known in medicine, in particular by mouth or injection, and can be used in any practical pharmaceutical manner, either alone or in compositions, in organs and tissues, or in contact with or in the in vivo vascular system of either humans or animals. When administered to humans and animals, the copolymers can be given by themselves or, for example, in combination with a pharma- ceutically acceptable carrier, as pharmaceutical compositions containing from 0.1% to 99.5% (and more preferably from 0.5% to 90%) of the active ingredient.

[0084] It is also known by those in the art that bacterial infections can lead to cancer, either because the infection causes chronic inflammation or because the infection releases cancer-inducing metabolites (Helicobacter pylori leading to cancer is a well-known example). Thus, the copolymers described within this invention should advantageously, and perhaps synergistically, be found to be useful as antibiotic drugs against bacterial, viral and cancer activity as described herein. Also, since viral infections, particularly those caused by influenza viruses, are often accompanied by bacterial infections as well - again, the simultaneous antiviral and antibiotic properties in one drug are advantageously synergistic.

[0085] The 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 that they in no way limit the scope of the invention. EXAMPLES

[0086] Example 1 and Comparative Example - Preparation of a Copolymer with MW of about 500 Daltons

[0087] The following process was carried out in vessels with computer-controlled reaction temperatures ranging from 10° C. (composition example E1 according to the invention) to 40° C. for Comparative Example 4, as shown in Table 1.

[0088] The reaction was carried out in a glass reactor equipped with a stirrer and water jacket heat exchange and automatic computer feedback temperature control during the reaction.

[0089] [Table 1]

[0090] A solution of freshly distilled acrolein (5 g; inhibited with hydroquinone 0.1% w / w) in water (20 g) was slowly added over 10 minutes to a solution of water (20 g) and polyethylene glycol (60 g; MW 200) at pH 12 by addition of 1 M aqueous sodium hydroxide; during the 10 minutes the yellow color of the oxidized hydroquinone quickly appeared and then disappeared. The composition was continuously and vigorously stirred during the process to provide abundant contact with air. An exothermic and rapid polymerization occurred.

[0091] After another 50 minutes, the clear solution was adjusted to pH 7.5 by the addition of 1 M aqueous hydrochloric acid; the product was a clear, almost colorless (very pale yellow) solution. All tests performed on samples and results here were performed on samples without any purification.

[0092] Only the UV-visible, 200-600 nM spectrum of the product had substantial absorbance at the far end of the 200-300 nM region.

[0093] HPLC indicated that the polymerization yield was 99-100% w / w and that any residual acrolein monomer was less than 1 ppm w / w; the copolymer remained soluble when tested up to pH 1 (and up to pH 14).

[0094] The MICs for S. aureus and E. coli were determined for each of the copolymers in Example 1 and Comparative Examples 1 to 4 and are reported in Table 2 with the UV peaks detected at different wavelengths indicated.

[0095] [Table 2] Example 2 - Treating burn infection

[0096] This example examines the effectiveness of the compositions of Example 1 and Comparative Example 3 in treating wound infection moded compounds with a negative control of no treatment and a positive control of saflomycin.

[0097] procedure

[0098] Experimental design

[0099] [Table 3]

[0100] Experimental procedure

[0101] Inoculum preparation [Day 1]

[0102] One day prior to induction of infection or on the day of wounding, a Glycerol Stock Staphylococcus aureus [ATCC 43300] culture was thawed and inoculated into fresh casein soyabean digest (CSD) broth and incubated overnight in a 37°C shaking incubator (200 rpm) (preferably inoculation was performed in the evening).

[0103] Burn wound creation

[0104] The dorsal skin of 6-8 week old male Wistar rats was shaved. The animals were anesthetized by inhalation of 2-3% isoflurane anesthesia. The depth of anesthesia was confirmed by tail pinch. A burn wound was created on the shaved and disinfected surface with molten wax heated to 80°C. The wax was poured onto the shaved back of the animals through a 2 cm x 2 cm plastic mold. The wax was left on the skin until it hardened. Ketoprofen was administered subcutaneously at a dose of 10 mg / kg to reduce pain and stress. The animals were housed individually with enrichment.

[0105] Induction of infection

[0106] One day after wound creation, the rats were placed in a dermal wound site for ∼10 min. 8 The wounds were infected with 100 μl of CFU / ml of Staphylococcus aureus and the wound site was covered with sterile cotton gauze.

[0107] Preparations

[0108] The vehicle for the copolymer was sterile 0.9% saline. Test doses of copolymer were prepared as follows: Group 2 - 383 mg of Example 1 was added to 20 ml of vehicle (concentration (conc) = 19.15 mg / ml), and Group 3 - 383 mg of Comparative Example 3 was added to 20 ml of vehicle (concentration = 19.15 mg / ml).

[0109] process

[0110] Treatment began on day 1 post-infection. Animals were treated for 3 consecutive days with test and reference compounds as indicated in the experimental design.

[0111] Both Example 1 (concentration=19.15mg / ml) and Example 3 (concentration=19.15mg / ml) were applied to the wound infected area by pipette in an amount of 100μl, then the solution was evenly spread on the wound surface from the center to the peripheral edge using a sterile bent tip, and covered with sterile cotton gauze.

[0112] Saphromycin (30 mg bid) was applied to the wound surface with a sterile cotton swab.

[0113] observation

[0114] Body weight: Body weight was recorded daily throughout the study.

[0115] Clinical Signs: Animals were monitored daily for clinical signs throughout the study period.

[0116] Wound Contraction

[0117] Changes in wound area were measured by tracing the wound borders on transparent paper on days 0 and 4. The traces were then removed by 1 mm 2 The measured wound surface area was then transferred to a graph sheet from which the wound surface area was estimated. The estimated surface area was then employed to calculate the wound contraction percentage by using the following formula: Initial wound size - Wound size on a specific date % Wound Contraction Rate = ----------------------------------- x 100 Initial wound size

[0118] Epithelialization period

[0119] The duration of epithelialization was recorded as the number of days required for the eschar to fall off without leaving a fresh wound after wound healing.

[0120] Bacteriological evaluation of the wound

[0121] On the 1st and 4th days after wounding, the entire wound was wiped with a cotton swab. The swab was then cut off and placed in a tube containing 1 ml of sterile physiological saline. After vortex mixing to release the bacterial cells from the swab into the saline, 100 μl of undiluted cell suspension or a 100-fold dilution thereof was plated and incubated overnight (16 h) at 37° C. on CSD agar plates for bacterial enumeration.

[0122] Data analysis

[0123] Body weight, wound contraction rate, and bacterial load were assessed for each animal. Significant differences between group and control means were assessed using appropriate statistical tests using the software Graphpad Prism (v 5.0).

[0124] result

[0125] Evaluation of the efficacy of E1 and CE3 compositions in a burn wound infection model in rats infected with Staphylococcus aureus [ATCC 4330].

[0126] [Table 4]

[0127] overview Composition E1 test dose (100 μl (19.15 mg / ml), topical, uid, 3 days) showed a significant reduction in bacterial load on day 4 when compared to day 1, while the vehicle control had no significant reduction in bacterial load (p>0.05). The CE3 test dose (100 μl (19.15 mg / ml), topical, uid, 3 days) showed significantly lower efficacy than E1 on the 4th day. Safromycin (30 mg topically, bid, Q=12 hr, 3 days) showed lower mean weights but no significant efficacy on day 4 when compared with day 1.

[0128] The results are plotted in the graph shown in FIG.

[0129] [Table 5]

[0130] overview Composition E1 test dose (100 μl (19.15 mg / ml), topical, uid, 3 days), Composition CE3 test dose (100 μl (19.15 mg / ml), topical, uid, 3 days) and Safromycin (30 mg, topical, bid, Q=12 hr, 3 days) showed significant wound reduction (p<0.05) when compared to day 1 and when compared to vehicle control on day 4.

[0131] The results are plotted in FIG.

[0132] Thus, the E1 composition exhibited superior in vivo efficacy compared to CE3, consistent with the lower MICs recorded for copolymers prepared at temperatures below 15°C.

[0133] Example 3 – Treating ascending urinary tract infection

[0134] This example compares the efficacy of E1 and CE3 compositions against E. coli in a rat model of ascending urinary tract infection.

[0135] Experimental procedure

[0136] Jugular vein catheterization [2]

[0137] 24 hours before infection, 6-8 week old male Wistar rats were anesthetized with ketamine and xylazine (70 + 10 mg / kg). Anesthesia was confirmed by tail pinch. The dorsal and ventral neck (3-4 cm) were shaved with a Wahl pet trimmer, and the surgical site was disinfected with 70% alcohol. The rats were placed in dorsal recumbence and a 1-2 cm incision was made on the ventral neck side; the jugular vein was dissected and freed from the surrounding tissue. Two surgical sutures were placed under the vein (one at the cranial end and another at the caudal end). The cranial end was tied tightly, a small incision was made with spring scissors at the caudal end, and a heparinized saline (100 IU / ml) filled catheter was inserted into the vessel lumen (approximately 25-35 mm). Sufficient blood was drawn into the cannula to ensure proper placement and patency.

[0138] Preparation of inoculum [day-1]

[0139] One day prior to infection (day -1), a glycerol stock of E. coli [ATCC 25922] was inoculated into sterile Casein Soyabean Digest Broth and kept at 37°C for incubation.

[0140] Day 0 (day of infection)

[0141] On the day of infection, overnight cultures were checked, centrifuged, and pelleted cells were resuspended in sterile normal saline and diluted to ∼1 × 10 9 Serial dilutions were made to obtain CFU / ml and used for infection. The inoculum was serially diluted 10-fold in sterile CSD broth and 0.05 ml of six dilutions for each strain was plated onto pre-incubated CSD agar plates to determine the viable cell count (CFU / ml) of the inoculum.

[0142] Induction of infection [3,4,5]

[0143] Animals with 100% patency were selected for the study and divided into different groups as specified in the experimental design. The cages of grouped animals were transported to a procedure room close to a biological safety cabinet. All infections were performed in a biological safety cabinet with appropriate personnel protection. The animals were anesthetized by intraperitoneal injection of a cocktail of ketamine and xylazine (60 + 10 mg / kg ip). Once the animals were in a deep enough plane of anaesthesia as monitored by pedal reflex, the abdominal wall of each rat was shaved with electric clippers and the skin was cleaned with 10% povidine iodine. After a 1.5 to 2 cm incision in the lower abdominal wall, the abdominal wall muscles were separated by blunt dissection. The bladder was isolated and exposed, urine in the bladder was removed and cultured in 0.1 ml of sterile saline or bacterial culture E. coli (~1 × 10 8 CFU / animal) was instilled into the bladder. After the bladder was replaced, the abdominal muscles were attached with sutures and the skin was closed. The surgical site was cleaned with 10% povidine iodine.

[0144] Preparations

[0145] The vehicle for Composition E1 was sterile 0.9% saline. Meropenem was prepared in MilliQ water.

[0146] process

[0147] Four hours after infection, groups 4, 5 and 6 were administered composition E1 as a single dose intravenously (jugular vein) in conscious animals at a dose volume of 48 ml / kg at a constant rate of infusion (2 ml / kg / hr) over a 24 hour period. The dose levels of composition E1 were 50, 500 and 4000 mg / kg. Group 2 was administered sterile 0.9% saline as a single dose at a dose volume of 48 ml / kg at a constant rate of infusion (2 ml / kg / hr) over a 24 hour period in the jugular vein in conscious animals. Meropenem (group 3) was administered as a single IV bolus dose of 30 mg / kg (dose volume of 5 ml / kg). The total duration of treatment was 24 hours.

[0148] termination

[0149] Twenty-four hours after the start of treatment (28 hours post-infection), the animals were euthanized by CO2 overdose in appropriate exposure chambers as specified in the experimental design. Group 1 animals were sacrificed 4 hours post-infection.

[0150] Tissue harvesting and homogenization

[0151] Euthanized animals were immersed in 70% ethanol for superficial decontamination. Organs were removed aseptically - the bladder was cut close to 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 cell counts were determined by the spread plate technique using CSD agar plates. CFU per ml of bladder and kidney homogenates were determined after 18-24 h incubation at 37°C.

[0152] Data analysis

[0153] Log 10 CFU / gram bladder and Log10 CFU / gram kidney was assessed in each group. Significant differences between group means were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test using Graphpad Prism at the 95% confidence level. A P value of <0.05 was considered significant.

[0154] [Table 6] kidney: Meropenem demonstrated significant bactericidal activity in the kidney after a single IV bolus dose of 30 mg / kg when compared with vehicle control. Composition E1 [4000 mg / kg] showed toxicity - all animals died within 12 hours after the start of injection. Composition E1 demonstrated a dose-dependent antibacterial effect at 50 and 500 mg / kg, with 500 mg / kg demonstrating significant activity when compared to vehicle control (p<0.05).

[0155] The results are plotted in FIG.

[0156] [Table 7] bladder Meropenem demonstrated significant bactericidal activity in the bladder after a single IV bolus dose of 30 mg / kg when compared with vehicle control. Composition E1 showed significant dose-dependent effects at 50 and 500 mg / kg when compared to vehicle control in the bladder (p<0.05).

[0157] The results of treatment of the bladder infection are shown in FIG.

[0158] Example 4 - Influenza A virus infection

[0159] overview

[0160] The purpose of this study is to determine the composition of the E1 and CE3 compositions, and the influenza A virus (H1N1) strain: A / NWS / 33 (ATTCC (R) VR-219 TM )( (R) is a registered trademark in the United States, etc. TM The efficacy of ribavarin (trademark in the United States and elsewhere) was determined in C57BL / 6 mice after intravenous (IV) administration twice daily for five days. Mice treated with E1 and CE3 showed a dose-dependent improvement in body weight throughout the study. Animals on Ribavarin, E1 (25 mg / kg, 50 and 100 mg / kg) and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly higher body weights when compared to vehicle controls on day 4 (p<0.05).

[0161] Ribavarin significantly reduced the viral growth rate and viral load in the lungs when compared to vehicle control; E1 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to vehicle control. CE3 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to vehicle control. Animals in ribavarin E1 (25 mg / kg, 50 and 100 mg / kg) and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly lower viral titers when compared to vehicle control on day 4 (p<0.05).

[0162] Compositions CE3 and E1 in the provided solutions had a concentration of 1000 mg / ml.

[0163] Test Systems Species: Mouse Strain, sex: C57BL / 6, female Age: 6-8 weeks Number of groups: 9 Number of animals / groups: 12 Total number of animals: 108

[0164] Virus: Influenza A virus (H1N1) Strain: A / NWS / 33 (ATTCC (R) VR-219 TM )

[0165] [Table 8]

[0166] Induction of infection

[0167] Mice were anesthetized with ketamine 60 mg / kg IP + xylazine 10 mg / kg intraperitoneally (ip). Anesthetized mice received 30 μl of virus inoculum (~1 × 10 4 ) Infection was performed intranasally by instillation (15 μl / nostril) of IAV (plaque forming units (PFU) / mouse).

[0168] Preparations

[0169] Compositions of E1, CE3 and Ribavarin were formulated in sterile 0.9% saline.

[0170] process

[0171] Four hours prior to infection, animals were administered the test and reference articles as indicated in the experimental design. Animals were treated for a duration of 6 days as indicated in the experimental design.

[0172] termination

[0173] Three animals from each group were sacrificed by CO2 overdose on days 1, 2, 4, and 6 postinfection.

[0174] Tissue harvesting and homogenization

[0175] Euthanized animals were immersed in 70% ethanol for surface decontamination. Lungs were aseptically removed and homogenized in 1 mL of cold infection medium with a sterile homogenizer. Samples were centrifuged at 300×g for 5-10 min at RT, and the supernatants were collected in fresh sterile tubes and stored at 4°C or at -80°C.

[0176] Clinical Observations

[0177] Animals were monitored for general clinical signs, morbidity and mortality. Body weights were measured at the end of the study.

[0178] Data analysis

[0179] Mean±SD Log 10 PFU / g lung was assessed in each group. Significant differences between group means and controls were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test using Graphpad Prism at the 95% confidence level. P values ​​<0.05 were considered significant.

[0180] result

[0181] On day 6 PI, two 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 of three animals were found to be wasting and a third animal had died. Animals in the other groups were apparently normal throughout the study. The body weights of the animals throughout the study are shown in Table 9.

[0182] [Table 9] Animals in the uninfected control gained weight; animals in the vehicle control and E1 (25 mg / kg) groups lost weight significantly throughout the study; animals in the positive control group showed no significant decrease in body weight. Animals in the E1 (50 mg / kg and 100 mg / kg) groups showed a drop in average body weight on day 2, but showed an increase in body weight thereafter; animals in the CE3 (100 mg / kg) showed a decrease in body weight throughout the study, while the 500 mg / kg and 1000 mg / kg showed no significant decrease on day 6. Because the number of mice in the vehicle control group was 1, it was not possible to perform statistical analysis on body weight on day 6. Instead, statistical analysis was performed on day 4. Animals in Ribavarin, E1 (25 mg / kg, 50 and 100 mg / kg) and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly higher body weights when compared to vehicle controls on day 4 (p<0.05).

[0183] The virus titers in the lungs of mice are shown in Table 10.

[0184] [Table 10] Ribavarin significantly reduced the viral growth rate and viral load in the lungs when compared to vehicle control; E1 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to vehicle control. CE3 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to vehicle control.

[0185] Because the number of mice in the vehicle control group was one, it was not possible to perform statistical analysis on the viral titers on day 6. Instead, statistical analysis was performed on day 4. Animals in ribavarin, E1 (25 mg / kg, 50 and 100 mg / kg) and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly lower viral titers when compared to vehicle controls on day 4 (p<0.05).

[0186] conclusion

[0187] Influenza A virus (H1N1) strain: A / NWS / 33 (ATCC (R) VR-219 TM In a mouse influenza model infected with CE3, animals treated with vehicle lost weight significantly; Ribavirin treated mice showed no significant loss in body weight; E1 and CE3 treated mice showed a dose-dependent improvement in body weight throughout the study. Animals on Ribavarin, E1 (25 mg / kg, 50 and 100 mg / kg) and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly higher body weights when compared to vehicle control on day 4 (p<0.05). Ribavarin significantly reduced the viral growth rate and viral load in the lungs when compared to vehicle control; E1 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to vehicle control. CE3 showed a significant dose-dependent reduction in the viral growth rate and viral load in the lungs when compared to vehicle control. Animals in ribavarin, E1 (25 mg / kg, 50 and 100 mg / kg) and CE3 (100 mg / kg, 500 and 1000 mg / kg) showed significantly lower virus titers on day 4 when compared to vehicle controls (p<0.05).

[0188] Example 5 - Maximum Tolerated Dose

[0189] Example 5A - MTD after repeated intravenous bolus dosing for 7 days in mice.

[0190] Female BALB / c mice were intravenously dosed (bolus) with vehicle, CE3 (5, 50 and 500 mg / kg q12h for 7 days) and E1 (5, 50 and 500 mg / kg q12h for 7 days). After dosing, animals were observed for general clinical signs. CE3 The MTD of was found to be 500 mg / kg when administered intravenously twice daily (q=12 h) for 1 week. E1 The MTD of was found to be 50 mg / kg when administered intravenously twice daily (q=12 h) for 1 week.

[0191] material and method

[0192] Test Systems Species: Mouse Strain, sex: BALB / c, female Age: 6 weeks Number of groups: 7 Number of animals / groups: 5 Total number of animals: 35

[0193] Compositions E1 and CE3 were provided as liquid formulations at a concentration of 1000 mg / ml.

[0194] [Table 11]

[0195] Experimental procedure

[0196] Animals were treated with vehicle and different doses of test substance twice daily (q12h) for 7 days as indicated above. Intravenous (bolus) The animals were observed for 7 days and compared to the vehicle control group for the following parameters: 1. Body weight immediately before and 7 days after administration 2. Clinical signs (gait, posture, pathology) before and immediately after dosing for up to 7 days 3. Animals were observed for mortality.

[0197] Preparations

[0198] 0.9% normal saline was used as the vehicle for E1 and CE3.

[0199] E1 composition

[0200] The 5 mg / kg:2.0 μl preparation (1000 mg / ml) was diluted to 2 ml with the addition of 1.998 ml of 0.9% normal saline.

[0201] The 50 mg / kg:20 μl preparation (1000 mg / ml) was diluted to 2 ml with 1.980 ml of 0.9% normal saline.

[0202] 500 mg / kg: 200 μl of the preparation (1000 mg / ml) was diluted to 2 ml with 1.800 ml of 0.9% normal saline.

[0203] CE3 composition

[0204] The 5 mg / kg:2.0 μl preparation (1000 mg / ml) was diluted to 2 ml with the addition of 1.998 ml of 0.9% normal saline.

[0205] The 50 mg / kg:20 μl preparation (1000 mg / ml) was diluted to 2 ml with 1.980 ml of 0.9% normal saline.

[0206] 500mg / kg: 200μl of the preparation (1000mg / ml) was diluted to 2ml with 1.800ml of 0.9% normal saline.

[0207] The formulations were prepared fresh each day prior to each dose.

[0208] dosage

[0209] Animals were dosed intravenously (bolus) with CE3 and E1 at different doses twice daily (q12h) for 7 days as indicated above. Dose volume was 5ml / kg.

[0210] result

[0211] The clinical signs, gross appearance and body weights of the animals are shown in Tables 12 and 13.

[0212] [Table 12-1] [Table 12-2]

[0213] [Table 13]

[0214] conclusion

[0215] E1 Mice treated with low doses of (5 and 50 mg / kg, iv, q12 h days, for 7 days) appeared normal after dosing and were comparable to the vehicle group. E1 In mice dosed with the high dose (500 mg / kg, iv, q12h for 7 days), 3 out of 5 mice were found to die after the 4th dose.

[0216] CE3 Animals in the treatment groups, ie, vehicle, low, medium and high dose groups, appeared normal throughout the study.

[0217] Both CE3 and E1 treated mouse groups, i.e.E1 Mean body weight increased in the vehicle, low, medium and high dose groups, but not in the high dose group.

[0218] In summary, when administered intravenously twice daily (q=12h) for one week, CE3 The MTD for was found to be 500 mg / kg. E1 The MTD of was found to be 50 mg / kg when administered intravenously twice daily (q-12h) for 1 week.

[0219] Example 5B - Identification of the maximum tolerated dose MTD of compositions of Examples CE3 and E1 after 7 days of repeated oral dosing in rats.

[0220] overview

[0221] Male Sprague Dawley rats were orally dosed with vehicle, The Composition of CE3 (5, 50 and 500 mg / kg q12h) and Composition of E1 (5, 50 and 500 mg / kg q12h) for 7 days. After dosing, animals were observed for general clinical signs and body weight. The maximum tolerated dose (MTD) of CE3 and E1 was found to be at least 500 mg / kg in rats when repeatedly orally administered twice daily for 7 days.

[0222] test

[0223] The CE3 and E1 compositions were supplied as solutions in glass vials at a concentration of 1000 mg / ml.

[0224] What to test Species: Rat Stock: Sprague Dawley Age: 6-8 weeks Gender: male

[0225] Experimental procedure

[0226] The animals were divided into different groups as indicated in the experimental design and were provided with food and water ad libitum. Groups of rats were orally dosed with the test substances twice a day (q12h) for 7 days at different doses as indicated above.

[0227] result

[0228] Animals dosed with vehicle, CE3 and E1 at all doses appeared normal throughout the study (Table 1). The mean body weight of the animals increased in all groups and no obvious abnormalities were observed macroscopically in any group.

[0229] conclusion

[0230] The maximum tolerated dose (MTD) of CE3 and E1 was determined when administered orally twice daily for 7 days. It was found to be at least 500 mg / kg in rats.

[0231] Example 6 - SARS-CoV-2

[0232] E1 and CE3 The composition demonstrated dose-dependent activity in vivo against the SARS-CoV-2 virus in Syrian golden hamsters, a well-accepted model of infection.

[0233] Intranasal administration of both compounds supports multiple potential modes of administration against SARS-CoV-2.

[0234] method

[0235] The study consisted of five groups of eight hamsters, each receiving a different treatment - a placebo control of saline nasal wash, a low dose of CE3 (200mg / kg), a high dose of CE3 (400mg / kg), a low dose of E1 (100mg / kg), and a high dose of E1 (200mg / kg). All animals were infected with SARS CoV-2 on day 0, with treatments administered twice daily on days 1-5, and viral titers measured directly via qPCR on days 2, 4, and 6. In this model, viral titers typically peak between days 2 and 4.

[0236] The results demonstrated a positive reduction in COVID-19 viral load in both CE3 and E1 compared to the placebo group. The results are shown in Figure 5 of the drawings, where the graph shows the nasal wash titers (log10 genomes / μL) in hamsters infected with SARS-CoV-2 and treated with compositions E1 and CE3. In the graph, the plots from left to right for each day relate 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 groups on day 4, where the low E1 dose achieved a log reduction on the order of 1.5 logs, and the high dose CE3 achieved a log reduction of 1.25 logs. Two of the five hamsters infected with COVID-19 on day 6 showed adverse clinical symptoms in the high dose E1 group and were removed from the study. The Company considers this to be a study-specific anomaly since E1 is routinely well tolerated at significantly higher intravenous infusion doses in vivo. Hamster weights across all groups at the start and end of the study remained approximately the same.

[0237] This hamster study demonstrates the potential of intranasal administration of E1, particularly for use against viruses.

[0238] E1 demonstrated higher activity against SARS-CoV-2 at lower doses. The E1 composition thus exhibited superior in vivo antiviral efficacy compared to CE3, consistent with the lower MICs recorded for copolymers prepared at temperatures below 15°C.

[0239] Example 7 - SARS-CoV-2 - Human Airway Epithelial (HAE) Cells

[0240] Part A

[0241] COVID Organoid Protocol: Organoids containing human airway epithelial (HAE) cells were infected by inoculation with SARS-CoV-2 virus and incubated at 37°C with varying concentrations of CE3 (H: 31 ppm, M: 10 ppm, L: 3 ppm) and E1 (H: 82 ppm, M: 41 ppm, L: 8 ppm) and viral load measured by number of PFUs (viral plaque forming units) was assessed at time points. The control was polyethylene glycol (PEG) 200.

[0242] The data indicate a concentration-dependent reduction of SARS-CoV-2 (COVID-19) virus from baseline by CE3 and E1 when compared to the control group. The SARS-CoV-2 virus is the cause of the global COVID-19 pandemic. The concentrations utilized were much lower than the preclinical data series for the copolymer in the CE3 intravenous infusion program.

[0243] The concentration-dependent reduction in viral infectivity was determined at varying concentrations of the two copolymer compositions and is shown in Figure 6 of the accompanying drawings.

[0244] The data indicated greater activity of composition E1 against SARS-CoV-2 compared to CE3, especially at low doses.

[0245] Part B

[0246] In separate studies, CE3 and E1 demonstrated excellent toxicity profiles with less than 0.25% effect on Vero (monkey) cells at the concentrations tested.

[0247] Cytotoxicity Testing Protocol in Vero Cells: Cytotoxicity over a range 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 assessed in a Vero cell luminescence assay and cell viability was measured at 1 hour, 24 hours and 72 hours of incubation with untreated healthy Vero cell controls. At all time points and concentrations measured, both compounds demonstrated minimal cytotoxic effects, with greater than 99% of the cells tested retaining their viability.

[0248] The results of the cytotoxicity tests are shown in Figure 7 for E1 and in Figure 8 for CE3. For each concentration, the percent of maximum cytotoxicity for 1 h, 24 h and 72 h is reported (from left to right).

[0249] Example 8 - Efficacy of E1 and CE3 against Neisseria gonorrhoeae (ATCC 700825) in mice

[0250] overview

[0251] The purpose of the study was to evaluate the efficacy of the compositions of Example E1 and Comparative Example CE3 against Neisseria gonorrhoeae (ATCC700825) in a mouse vaginal infection model. Meropenem showed significant bactericidal activity in vaginal load after 7 days of IV bolus dosing at 50 mg / kg when compared to vehicle control (p<0.05). E1 showed a dose-dependent antibacterial effect. At doses of 25 and 50 mg / kg, E1 showed a mean dose-dependent reduction in bacterial load, but these were not particularly significant; at a dose of 100 mg / kg, E1 showed a significant antibacterial effect when compared to vehicle control (p<0.05).

[0252] CE3 demonstrated significant dose-dependent antibacterial effects in vaginal challenges at 100, 500 and 1000 mg / kg (7-day IV bolus) when compared to vehicle control 7-day PI (p<0.05).

[0253] Test items

[0254] CE3 and E1. were in aqueous solution at 1000 mg / ml. Test system type: Mouse Strain, gender: BALB / c, female Age: 4-6 weeks Number of groups: 9 Total number of animals: 90

[0255] Inoculation with N. gonorrhoeae

[0256] Animals in the diestrus stage of the estrous cycle were implanted subcutaneously with 5 mg, 21-day controlled-release estradiol pellets and treated with streptomycin and trimethoprim to increase their susceptibility to N. gonorrhoeae during infection. Two days after pellet implantation, mice were inoculated intravaginally with N. gonorrhoeae (~2 × 106 CFU / animal).

[0257] Preparations

[0258] The vehicle for CE3, E1 and meropenem was sterile 0.9% saline. Test compounds were diluted in vehicle to the required concentrations.

[0259] process

[0260] Two days after infection, animals were treated for 7 consecutive days as indicated in the experimental design.

[0261] [Table 14]

[0262] The results are depicted in Figure 9, showing the bacterial load in vaginal swabs after treatment with reference and test articles in mice. *P<0.05 significantly different from vehicle control.

[0263] conclusion

[0264] In a mouse vaginal infection model with N. gonorrhea (ATCC700825), meropenem demonstrated significant bactericidal activity in vaginal challenge after IV bolus dosing at 50 mg / kg for 7 days when compared with vehicle control (p<0.05).

[0265] E1 demonstrated a dose-dependent antibacterial effect. At doses of 25 and 50 mg / kg (IV bolus for 7 days), E1 demonstrated a mean dose-dependent reduction in bacterial load; at 100 mg / kg (IV bolus for 7 days), E1 demonstrated a significant antibacterial effect when compared to vehicle controls (p<0.05).

[0266] CE3 demonstrated significant dose-dependent antibacterial effects in vaginal challenges at 100, 500 and 1000 mg / kg (IV bolus for 7 days) when compared to vehicle control 7 days PI (p<0.05).

[0267] Thus, the E1 composition exhibits superior in vivo efficacy compared to CE3, consistent with the lower MICs recorded for copolymers prepared at temperatures below 15°C.

[0268] Example 9 - MIC Determination of Compositions of the Invention Prepared at 5°C and 10°C

[0269] Compositions of the invention were prepared using the procedure of Example 1 at 5° C. (Example 2-E2) and 10° C. (Example 1-E1).

[0270] The MICs for S. aureus and E. coli were determined for each of the copolymers of Examples 1 and 2 and are reported in Table 15.

[0271] [Table 15]

Claims

1. A method for producing a product, comprising the steps of: The product is a bioactive polymer comprising an acrolein-derived segment and a polyalkylene glycol oligomer, The method comprises reacting a polyalkylene glycol with acrolein in an aqueous solution to form a copolymer having a molecular weight not exceeding 1000 Daltons at a temperature between −5° C. and 10° C. and an alkaline pH not exceeding pH 12.5; The reaction of the polyalkylene glycol with acrolein in aqueous solution is carried out in a reaction vessel containing a heat exchanger to control the temperature; The solution is maintained at a temperature of −5° C. to 10° C. during the reaction to form the copolymer; and The bioactive polymer is for use in a method of treating a subject suffering from a disease.

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

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

1.

4. 4. The method of claim 1, wherein the molecular weight of the polyalkylene glycol does not exceed 800 Daltons.

5. 5. The method of claim 4, wherein the polyalkylene glycol has a molecular weight of from 200 to 600 daltons.

6. 6. The process of any one of claims 1 to 5, wherein the acrolein is added to an aqueous solution of the polyalkylene glycol.

7. 7. The process according to any one of claims 1 to 6, wherein the acrolein is added to an aqueous solution of polyethylene glycol containing at least 20% w / w of water, wherein the acrolein is added in the form of an aqueous solution of acrolein not exceeding a concentration of 50% w / w.

8. The method of claim 7, wherein the acrolein is added to an aqueous solution of polyethylene glycol containing at least 20% w / w water, wherein the acrolein is added in the form of an aqueous solution of acrolein not exceeding a concentration of 30% w / w.

9. Providing a basic aqueous solution of a polyalkylene glycol; adding acrolein as an aqueous solution at a concentration not exceeding 50% w / w of an aqueous acrolein solution; adding an acid after the acrolein monomer has been consumed to provide a pH of less than 9; The method of any one of claims 1 to 8, comprising:

10. The method of claim 9, wherein adding the acrolein as an aqueous solution having a concentration not exceeding 50% w / w of the aqueous acrolein solution is carried out slowly over a period of at least 2 minutes or at least 5 minutes.

11. The method of claim 9 or 10, wherein an acid is added to provide a pH of 8 or less after the acrolein monomer has been consumed.

12. The method of any one of claims 9 to 11, wherein the aqueous acrolein solution contains a preservative.

13. 13. The process of any one of claims 1 to 12, wherein the reaction of the polyalkylene glycol with acrolein in aqueous solution is carried out in a stirred reaction vessel equipped with a jacket of flowing coolant liquid to control the temperature inside the reaction vessel.

14. 14. The method of claim 13, wherein the reaction conditions are regulated by computer control of one or more reaction parameters selected from the rate of acrolein addition, the flow rate of water, the temperature of the water in the jacket, and the speed at which the aqueous solution is stirred.

15. Use of an effective amount of a bioactive agent comprising an acrolein-derived segment and a polyalkylene glycol oligomer, prepared according to the method of any one of claims 1 to 14, in the manufacture of a medicament for the treatment of a disease selected from bacterial infection, viral infection and cancer.

16. 15. 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 cancer, comprising the method of any one of claims 1 to 14.

17. 17. The use of claim 15 or claim 16, wherein the disease is a parenteral infection.

18. A composition for the treatment of a disease in a subject selected from bacterial infection, viral infection and cancer, said composition being prepared by a process comprising the method of any one of claims 1 to 14.

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