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WO2019220088A1 - Polymères, nanoparticules formées à partir des polymères et compositions pharmaceutiques comprenant un agent actif encapsulé dans les nanoparticules - Google Patents

Polymères, nanoparticules formées à partir des polymères et compositions pharmaceutiques comprenant un agent actif encapsulé dans les nanoparticules Download PDF

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Publication number
WO2019220088A1
WO2019220088A1 PCT/GB2019/051307 GB2019051307W WO2019220088A1 WO 2019220088 A1 WO2019220088 A1 WO 2019220088A1 GB 2019051307 W GB2019051307 W GB 2019051307W WO 2019220088 A1 WO2019220088 A1 WO 2019220088A1
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polymer
nanoparticle
general formula
nps
exenatide
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Mark Gumbleton
Muthanna ABDULKARIM
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University College Cardiff Consultants Ltd
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University College Cardiff Consultants Ltd
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/30—Prediction of properties of chemical compounds, compositions or mixtures
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/34—Introducing sulfur atoms or sulfur-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/44—Preparation of metal salts or ammonium salts
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/50—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for simulation or modelling of medical disorders
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F120/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F120/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F120/10—Esters
    • C08F120/12—Esters of monohydric alcohols or phenols
    • C08F120/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
    • C08F120/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]

Definitions

  • the present invention relates to novel polymers, in particular to block copolymers, and more specifically to block copolymers comprising a lipophilic block and a zwitterionic 5 block, which is suitably a polysulfobetaine block.
  • the invention also relates to nanoparticles formed from these polymers, to the nanoparticles loaded with biologically active substances and to the loaded nanoparticles for use in delivering the biologically active substance through a layer of a biopolymer such as mucous or microbial biofilm.
  • a drug is formulated for delivery via a route such as oral, rectal, nasal, bronchial (inhaled), topical (including eye drops, buccal and 15 sublingual), vaginal administration than via a parenteral route, for example subcutaneous, intramuscular, intravenous and intradermal administration.
  • orally administered therapeutic peptides are highly susceptible to degradation in the intestinal mucous layer by various protease enzymes such trypsin, chymotrypsin, and 25 carboxypeptidase.
  • Polymers comprising sulfobetaine-modified monomer units are known and have been used to confer anti-bioadherent properties and as coatings to prevent bio-adhesion.
  • the present inventors have developed a novel block copolymer comprising alkyl (meth)acrylate units and a zwitterionic block comprising betainised monomer units.
  • the polymer can form particles suitable for loading with a biologically active material and for transporting that material through a biopolymer.
  • a polymer comprising a polymer chain of general formula (I):
  • R 1 is H or methyl
  • R 2 is C1 -6 alkyl
  • R 3 is H or methyl
  • R 4 is -(CH 2 ) P -N + (CH3)2-(CH 2 )3-S(0)20- or -(CH 2 ) P -N(CH 3 )2;
  • p is an integer of 1 to 4.
  • n is an integer of 15 to 30;
  • n:m is an integer wherein the ratio of n:m is from 80:20 to 20:80;
  • R 4 groups are -(CH 2 ) P -N + (CH 3 ) 2 -(CH 2 ) 3 -S(0) 2 0 ⁇
  • the polymers of the invention are particularly advantageous because they contain a lipophilic block and a zwitterionic hydrophilic block, wherein, in the zwitterionic block, each monomer unit carries both a positive and a negative charge. Moreover, the ratio of lipophilic to zwitterionic monomer residues is such that polymer particles can be formed.
  • the particles formed from the polymers of the present invention are of uniform size, do not aggregate when suspended in a solvent and can be loaded with biologically active molecules. The particles also show excellent penetration of biopolymers such that they have utility for transporting biologically active molecules across layers of mucous or of bacterial biofilms.
  • Ci-e alkyl refers to a straight or branched chain fully saturated hydrocarbon group having from one to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl. Terms such as C1-4 alkyl and C1-3 alkyl have similar meanings but the number of carbon atoms in the alkyl group is different (1 to 4 and 1 to 3 carbons respectively in these examples).
  • (meth)acrylate refers to an acrylate and a methacrylate.
  • the polymer consists essentially of the polymer chain of general formula (I).
  • the polymer may be terminated at each end by small organic groups.
  • the polymer may be of general formula (Iz):
  • R 1 , R 2 , R 3 and R 3 are as defined in general formula (I);
  • X is a residue of a chain transfer agent
  • Y is a residue of a monomer.
  • chain transfer agent is a reagent suitable for use in a reversible addition-fragmentation chain-transfer (RAFT) polymerisation method.
  • Suitable chain transfer agents include compounds of the structure:
  • R is a free radical leaving group which can re-initiate radical polymerisation, such as Ci-e alkyl optionally substituted with CN or C(0)0H, for example cyanomethyl, 2-cyano-2- propyl, 4-cyano-pentanoic acid, or 2-methylpropionic acid.
  • chain transfer agents examples include 2-cyano-2-propyl dodecyl trithiocarbonate, cyanomethyl dodecyl trithiocarbonate, 4-cyano-4-[(dodecylsulfanylthio- carbonyl)sulfanyl]pentanoic acid and 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid.
  • 2-Cyano-2-propyl dodecyl trithiocarbonate is particularly suitable.
  • X may be represented by the formula:
  • a“residue of a monomer” refers to the remaining portion of the monomer unit after polymerisation has taken place.
  • the monomer residue Y may be the residue after polymerisation of a (meth)acrylate monomer of formula (II):
  • R 1 and R 2 are as defined for general formula (I).
  • the monomer may be of formula (lx) or (ly)
  • R 1 , R 2 , R 3 and R 4 are as defined for general formula (I) and X is as defined for general formula (Iz).
  • R 1 and/or R 3 is methyl. More suitably, both R 1 and R 3 are methyl.
  • R 2 is C3-6 alkyl, for example a propyl, butyl or pentyl group and, in particular, a straight chain alkyl group such as n-propyl, n-butyl or n-pentyl. Polymers in which R 2 is n-butyl are particularly suitable.
  • n is from 15 to 29, for example 20 to 28, 22 to 27.
  • n is from 15 to 28, 15 to 27, 15 to 26 or 15 to 25. In some cases, n is from 18 to 28, 18 to 27, 18 to 26 or 18 to 25. In other cases, n may be from 20 to 28, 20 to 27, 20 to 26 or 20 to 25.
  • n may be from 22 to 26 or 24 to 26, typically about 25.
  • the ratio of n:m may be from 30:70 to 70:30 or from 40:60 to 60:40.
  • Example values for the ratio of n:m are 30:70, 40:60, 50:50, 60:40 and 70:30.
  • suitable values for p are 1 to 3, for example 1 or 2 and particularly 2.
  • At least 95%, 96%, 97%, 98%, 99% or 99.5% of the R 4 groups are -(CH 2 )p-N + (CH 3 )2-(CH 2 )3- S(0) 2 0 ⁇
  • 100% of the R 4 groups are -(CH 2 )p-N + (CH 3 )2-(CH 2 )3-S(C>) 2 C>- such that the polymer comprises a polymer chain of general formula (la):
  • the polymer may consist essentially of the polymer chain of general formula (la).
  • the polymer may have the formula (laz):
  • a particularly suitable example of a polymer of the present invention is a betainised block copolymer of n-butyl methacrylate with 2-(dimethylamino)ethyl methacrylate comprising a polymer chain of general formula (lb):
  • R 1 , R 3 , m and n are as defined above for general formula (I) and
  • R 4 is -(CH 2 ) 2 -N + (CH 3 ) 2 -(CH 2 ) 3 -S(0) 2 0- or -(CH 2 )2-N(CH 3 )2, wherein at least 90%, 95%,
  • R 4 groups 96%, 97%, 98%, 99% or 99.5% of the R 4 groups are -(CH 2 ) 2 -N + (CH 3 ) 2 -(CH 2 ) 3 -S(0) 2 0 ⁇
  • the polymer may consist essentially of the polymer chain of general formula (lb).
  • the polymer may be of formula (Ibz):
  • R 1 , R 2 , R 4 , m and n are as defined above for general formula (I); and X and Y are as defined above for general formula (Iz).
  • the polymer is fully betainised and the polymer comprises a polymer chain of formula (lc):
  • R 1 , R 3 , m and n are as defined above for general formula (I).
  • the polymer consists essentially of the polymer chain of general formula (lc).
  • the polymer may have the formula (lcz)
  • R 1 , R 3 , m and n are as defined above for general formula (I) and X and Y are as defined above for general formula (Iz).
  • X and Y groups for the polymers of formulae (laz), (Ibz) and (lcz) are as discussed above for the polymers of general formula (Iz).
  • X may be a residue of a (meth)acrylate monomer of general formula (II).
  • the polymers of general formula (I) suitably have a narrow size distribution, with a polydispersity index of not greater than 1.4 (low polydispersity) [1 ,2], typically from about 1.1 to 1.4.
  • Polydispersity index in the polymers is defined by the equation:
  • M w is the weight average molecular weight and M n is the number average molecular weight.
  • Polymers comprising a polymer chain of general formula (I) are suitably prepared by RAFT polymerisation. Therefore, in a further aspect of the invention there is provided a process for the preparation of a polymer comprising a polymer chain of general formula (I), the process comprising:
  • R 1 and R 2 are as defined for general formula (I);
  • R 1 , R 2 and n are as defined for general formula (I);
  • R 3 is as defined in general formula (I) and
  • R 5 is -(CH 2 ) P -N(CH 3 ) 2 , where p is as defined in general formula (I);
  • R 1 , R 2 , R 3 , n and m are as defined for general formula (I) and R 5 is as defined for general formula (IV)
  • step (iii) Reacting the polymer comprising the chain of general formula (V) with 1 ,3- propane sultone to form a polymer comprising a chain of general formula (I).
  • the ratio of the monomer of general formula (II) to CTA is from about 15:1 to 35: 1 , more suitably from 20:1 to 30:1 , for example 22:1 to 28:1 , 23:1 to 27:1 or 24:2 to 26: 1 , typically about 25: 11.
  • Suitable CTAs are as described above.
  • Any suitable radical polymerisation initiator may be used.
  • One example of such an initiator is 2,2'-azobis(2-methylpropionitrile) (AIBN).
  • AIBN 2,2'-azobis(2-methylpropionitrile)
  • the polymerisation of step (i) may be carried out in any suitable organic solvent, for example dioxane, typically under an inert atmosphere, for example under nitrogen.
  • the reaction temperature is suitably about 60-80°C, typically about 70°C.
  • the polymerisation reaction is stopped when n reaches the desired value, typically after about 5 to 7 hours.
  • the value of n may be determined by NMR.
  • gas permeation chromatography GPC may be used to determine the molecular weight and polydispersity index of the polymer comprising the polymer chain of general formula (III).
  • the polymer comprising the polymer chain of general formula (III) may be a polymer of general formula (lllz):
  • R 1 and R 2 are as defined for general formula (I); X is as defined for general formula (Iz) and Y is a residue of the monomer of general formula (II).
  • Step (ii) is suitably carried out using the dried polymer comprising a chain of general formula (III).
  • the polymer comprising the chain of general formula (III) serves as a macro-CTA for the RAFT polymerisation and therefore it is not necessary to add a CTA at this stage.
  • Trithiocarbonate as a macro-CTA agent was widely reported as a good source for di-block and tri-block control copolymerization [3,4]
  • the RAFT polymerisation conditions are similar to those used for step (i).
  • the amount of the monomer of general formula (IV) is selected such that the molar ratio of monomer of general formula (II) to monomer of general formula (IV) used to form the polymer comprising the chain of general formula (V) is from 80:20 to 20:80, in order to give a ratio of n:m as defined above for general formula (I).
  • the polymer comprising the chain of general formula (V) is a polymer of general formula (Vz)
  • R 1 , R 2 , R 3 , n and m are as defined for general formula (I); X is as defined for general formula (Iz), Y is a residue of the monomer of general formula (II) and R 5 is as defined for general formula (IV).
  • step (iii) the polymer having the chain of general formula (V) is reacted with 1 ,3- propane sultone.
  • the reaction is carried out in an organic solvent such as tetrahydrofuran.
  • the degree of reaction can be determined using IR and NMR spectral analysis and the reaction terminated once betainisation is complete.
  • the reaction time is 60 to 84 hours, for example about 72 hours.
  • the polymers of general formula (I) may be used to form nanoparticles. Therefore, in a further aspect of the invention, there is provided a nanoparticle comprising a compound of general formula (I), wherein the particle has an average diameter of about 20 to 70 nm, more suitably 25 to 60 nm, for example about 40-50 nm.
  • average particle diameter is determined on an intensity basis such that the z-average value is obtained, and is suitably measured using photon correlation spectroscopy.
  • a method for particle size measurement is provided in the examples below. Suitable instruments for making the measurement are well known to those of skill in the art.
  • the particles are relatively monodisperse, i.e. they have a polydispersity index of about 0.2 to 0.6, more usually about 0.3 to 0.5, typically about 0.4.
  • the nanoparticles of the invention have a hydrophilic surface which is densely charged and zwitterionic but, because the positive and negative charges are carried on the same monomer unit, the surface has an overall net neutral zeta potential when suspended in an aqueous solvent.
  • the nanoparticles of the present invention exhibit relatively small particle size and show minimal aggregation.
  • the nanoparticles are bioinert and have also shown an ability to pass rapidly through biopolymers such as mucous layers and bacterial biofilms. Furthermore, the nanoparticles of the invention have the ability to avoid non-specific surface based cellular uptake processes by the body and are therefore suitable as vehicles for the targeted delivery of biologically active molecules to tissues.
  • the nanoparticles may be loaded with a biologically active agent.
  • Suitable active compounds include peptides and proteins such as exenatide, insulin, leucine enkephalin, proapoptotic peptide and cilengitide as well as other active agents such as antimicrobial agents, for example antibacterial agents such as polymyxin B, tobramycin, and benzyl penicillin, anti-viral agents such as zanamivir, sialidases and oseltamivir , anti-fungal agents such as Econazole nitrate, itraconazole and pimaricin or anti-protazoal agents such as nitazoxanide and chloroquine.
  • antimicrobial agents for example antibacterial agents such as polymyxin B, tobramycin, and benzyl penicillin, anti-viral agents such as zanamivir, sialidases and oseltamivir , anti-fungal agents such as Econazole nitrate, itraconazole and pimaricin or anti-protazoal agents such as
  • the sulfobetaine serves as the particle shell and the lipophilic polymer (BMA) as a core.
  • BMA lipophilic polymer
  • the biologically active agent is encapsulated in the lipophilic core of the nanoparticle.
  • the nanoparticles may be formed by a nano-precipitation technique comprising
  • Suitable solvents include methanol, ethanol or mixtures thereof or mixtures of methanol or ethanol with aqueous solvents, for example containing salts.
  • a suitable solvent is a mixture of aqueous sodium chloride solution and methanol in a ratio which may be determined experimentally and which is dependent on the ratio of n:m in the polymer comprising the chain of general formula (I).
  • the rate of addition of the polymer solution to the solvent may be from about 10 to 30 pi ⁇ per minute, more usually 15 to 25 pL per minute, for example about 20 pL per minute.
  • the volume of the aqueous phase may be from about 1 to 10 ml_, suitably 3 to 7 ml_, typically about 5 ml_.
  • the aqueous phase to which the polymer solution is added may have a pH from 5.5 to 7.5, typically pH 6.5 to 7.5, for example pH 6.8 to 7.0.
  • a phosphate buffer may be used.
  • mixing may be continued for 10 to 60 minutes to allow evaporation of non-aqueous solvent (methanol).
  • the process for forming the nanoparticles may include the additional step of dialysing the suspension of nanoparticles against a further aqueous solution, for example phosphate buffered saline at a pH from 5.5 to 7.5, typically pH 6.5 to 7.5, for example pH 6.8 to 7.0.
  • a further aqueous solution for example phosphate buffered saline at a pH from 5.5 to 7.5, typically pH 6.5 to 7.5, for example pH 6.8 to 7.0.
  • the process may include the additional step of loading the nanoparticles with a biologically active agent. This may be achieved by modifying step (i) above by solubilising the active agent in the suitable solvent.
  • a co-solvent may be employed.
  • particles are formed in which the biologically active agent is encapsulated in the lipophilic core of the particle.
  • the nanoparticles of the invention are of use in medicine for delivering biologically active agents to a chosen site which might be less accessible using conventional formulations.
  • “medicine” comprises both human and veterinary medicine.
  • a nanoparticle of the invention for use in delivering a biologically active agent across a mucous membrane or a bacterial biofilm.
  • nanoparticle of the invention in the preparation of an agent for use in delivering a biologically active agent across a mucous membrane or a bacterial biofilm.
  • the invention provides a method for delivering a biologically active agent across a mucous membrane or a bacterial biofilm, the method comprising administering to a patient in need of such treatment an effective amount of nanoparticle according to the invention.
  • the mucous membrane is the lining of the gastro-intestinal tract, the lungs or the genito-urinary tract.
  • the bacterial biofilm may comprise a biofilm found in a wound.
  • the biologically active agent is suitably an anti-bacterial agent.
  • the biologically active agent may comprise a biologically active peptide or other pharmacologically active molecule which it is desirable to deliver across the mucous membrane.
  • biologically active peptides include exenatide, insulin, leucine, enkephalin, proapoptotic peptides and cilengitide and particles loaded with these peptides may be used for treating type II or type I diabetes, pain, or cancer.
  • the nanoparticles may be delivered orally such that they are absorbed through the mucous membrane comprising the lining of the gastrointestinal tract.
  • the biologically active agent When the biologically active agent is delivered across a bacterial biofilm, it may be an anti-bacterial agent, for example an agent suitable for the treatment of a bacterial infection e.g. Pseudomonas aeruginosa infection.
  • the nanoparticles may be delivered topically to a wound or lesion infected with the bacteria.
  • nanoparticle according to the invention for use in medicine wherein:
  • the biologically active agent is exenatide or insulin and the nanoparticle is for use in the treatment of diabetes; or
  • the biologically active agent is leucine encephalin and the particle is for use in the treatment of pain;
  • the biologically active agent is a proapoptotic peptide or cilengitide and the nanoparticle is for use in the treatment of cancer.
  • a particle according to the invention loaded with exenatide or insulin for use in the treatment of diabetes.
  • nanoparticle according to the invention loaded with exenatide or insulin in the preparation of an agent for the treatment of diabetes.
  • a method for the treatment of diabetes comprising administering to a patient in need of such treatment an effective amount of exenatide or insulin encapsulated in a particle according to the invention.
  • the particles of the invention are suitably formulated for oral administration.
  • the biologically active agent may be an anti-microbial agent, e.g. an anti bacterial such as polymyxin B, tobramycin, and benzyl penicillin, anti-viral such as Zanamivir, sialidases and oseltamivir , anti-fungal such as econazole nitrate, itraconazole and pimaricin or anti-protazoal agent such as nitazoxanide and chloroquine.
  • an anti-microbial agent e.g. an anti bacterial such as polymyxin B, tobramycin, and benzyl penicillin
  • anti-viral such as Zanamivir, sialidases and oseltamivir
  • anti-fungal such as econazole nitrate, itraconazole and pimaricin
  • anti-protazoal agent such as nitazoxanide and chloroquine.
  • a particle according to the present invention loaded with an anti-microbial agent for use in treating a microbial infection.
  • a method for the treatment of a microbial infection comprising administering to a patient in need of such treatment an effective amount of a particle according to the present invention loaded with an anti-microbial agent.
  • the microbial infection may be a bacterial, viral, fungal or protozoal infection and the antimicrobial agent will be selected accordingly.
  • the microbial infection is a topical infection or an infection of a wound.
  • the nanoparticles may be formulated for topical administration.
  • the infection may be a bacterial infection, for example an infection of a bacteria which produces a bacterial biofilm, for example Pseudomonas aeruginosa.
  • the particles of the present invention may be formulated for topical administration to a wound or lesion infected with the bacteria.
  • the particles will generally be formulated as part of a pharmaceutical composition and therefore in a further aspect of the invention there is provided a pharmaceutical composition comprising a particle of the present invention loaded with a biologically active agent.
  • pharmaceutical composition refers to a composition for use either in human or veterinary medicine. The composition may therefore be adapted for administration to humans or animals, more suitably mammals.
  • Suitable biologically active agents are as outlined above.
  • composition will be formulated for oral, rectal, nasal, bronchial (inhaled), topical (including eye drops, buccal and sublingual) or vaginal administration.
  • topical including eye drops, buccal and sublingual
  • vaginal administration The particular route of administration selected will depend on the nature of the biologically active agent.
  • the biologically active agent when the biologically active agent is insulin or exenatide, it may be appropriate to formulate the composition for oral administration.
  • the biologically active agent when the biologically active agent is an antibacterial agent, the composition may be formulated for administration by the most convenient route to reach the site of infection. This may be, for example topical administration.
  • the composition may be prepared by bringing into association the above defined particles with the carrier.
  • the formulations are prepared by uniformly and intimately bringing into association the particles with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the invention extends to methods for preparing a pharmaceutical composition comprising bringing particles of the present invention in conjunction or association with a pharmaceutically acceptable carrier or vehicle.
  • Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid; or as a bolus etc.
  • the term“acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate, stearic acid, silicone fluid, talc waxes, oils and colloidal silica.
  • Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
  • compositions suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
  • the particles may be made up into a cream, ointment, jelly, solution or suspension etc.
  • Cream or ointment formulations that may be used for the drug are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia.
  • Particles loaded with an appropriate biologically active agent may be used for the treatment of the respiratory tract by nasal, bronchial or buccal administration of, for example, aerosols or sprays which can disperse the particles loaded with pharmacologically active ingredient in the form of a powder or in the form of drops of a solution or suspension.
  • compositions with powder-dispersing properties usually contain, in addition to the active ingredient, a liquid propellant with a boiling point below room temperature and, if desired, adjuncts, such as liquid or solid non-ionic or anionic surfactants and/or diluents [5]
  • Pharmaceutical compositions in which the pharmacological active ingredient is in solution contain, in addition to this, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser.
  • compressed air can also be used, it being possible for this to be produced as required by means of a suitable compression and expansion device.
  • FIGURE 1 is a schematic representation of the method used to synthesise the polymer and form the nanoparticles.
  • FIGURE 2 is the NMR spectrum of butyl methacrylate (BMA) polymer polymerised for 6 hours.
  • FIGURE 3 is the GPC profile of BMA polymer showing the molecular weight and the polydispersity index (PDI) of the BMA polymerized for 6 hours.
  • PDI polydispersity index
  • FIGURE 4 is the 1 H-NMR spectrum of the BMA:DMAEMA di-block copolymer in which the peaks showing the ratios of number of units of BMA to DMAEMA was highlighted.
  • A (P1) BMA:DMAEMA (70:30).
  • B (P2) BMA:DMAEMA (60:40).
  • C (P3) BMA:DMAEMA (50:50).
  • D (P4) BMA: DMAEMA (40:60).
  • E) (P5) BMA:DMAEMA (30:70). (Solvent: CDC ).
  • FIGURE 5 is an infra-red (IR) spectrum showing the structural formation of BMA- sulfobetaine step by step where step (A) shows the functional groups of the BMA, step (B) shows the functional groups of BMA-DMAEMA while step (C) shows the functional groups of BMA-sulfobetaine. (Note: only the main functional groups were highlighted each IR spectrum).
  • IR infra-red
  • FIGURE 6 is a histogram comparison of diffusivities of sulfobetaine NPs (defined by PXS abbreviation) as compared to 113 other NPs comprising various surface chemistries and permeation strategies.
  • A ⁇ Deff> of various sulfobetaine NPs.
  • B % ratio ⁇ Deff>/D° of various sulfobetaine NPs.
  • FIGURE 7 is a plot showing the in vitro release profile of Lumogen® red from sulfobetaine NPs (P2S and P5S).
  • A Percent of Lumogen® release.
  • B Cumulative amount of Lumogen® release in pg.
  • FIGURE 8 is a series of plots showing the in vitro release profile of exenatide by the dialysis method in PBS buffer pH 6.8 at 37 °C
  • 8A Cumulative release profile in pg of exenatide from sulfobetaine NPs (P2S, P3S, P4S and P5S) for 8 hours [lower panel] Cumulative release profile in pg of free exenatide versus its release from sulfobetaine NPs over the first 2 hours.
  • FIGURE 9 is a series of plots showing the in vitro release profile of exenatide acetate from P5S NPs assessed by the dialysis method in which the first 2 hr release was conducted in buffer pH 1.2 and remaining 2 hr to 24 hr release in PBS buffer pH 6.8 at 37 °C.
  • FIGURE 10 is a schematic representation showing the treatment arms for the in vivo studies on exenatide in rats.
  • LDH lactate dehydrogenase
  • FIGURE 16 Minimum Inhibitory Concentration (MIC) of polymyxin on non-mucoidal P. aeruginosa (black bars) versus mucoidal P. aeruginosa (grey bars) grown overnight and treated with (A) Free polymyxin (8 pg/ml - 0.007813 pg/ml); (B) Polymyxin loaded into sulfobetaine NPs (8 pg/ml - 0.007813 pg/ml) and (C) Free NPs (unloaded) at the same concentrations range of NPs used to load polymyxin in (B). NPs alone had no effect on bacteria and bacterial biofilm growth.
  • MIC Minimum Inhibitory Concentration
  • polymyxin loaded into sulfobetaine NPs had substantial impact on the P. aeruginosa compared with free polymyxin especially with mucoidal P. aeruginosa where MIC was significantly different for polymyxin loaded into NP versus free polymyxin at concentrations of 0.25, 0.5 and 1 pg/ml
  • Example 1 Synthesis of Polymer and Formation of Nanoparticles
  • the polymer was synthesised and nanoparticles formed according to the method shown schematically in Figure 1.
  • the first step of the synthesis of the zwitterionic polymer was the polymerization of the lipophilic block, which was carried out as shown in Scheme 1 , where, in the BMA-CTA polymer Y is the residue of the butyl methacrylate monomer and CTA is the residue of the chain transfer agent.
  • RAFT technique was used to control the molecular weight and the chain length of the BMA polymer.
  • BMA monomer was used at the ratio of (25:1) to the chain transfer agent (CTA) (2-Cyano-2- propyl dodecyl trithiocarbonate).
  • CTA chain transfer agent
  • AIBN 2,2'-Azobis(2-methylpropionitrile)
  • the ingredients were dissolved in Dioxane and the solution was flushed with nitrogen gas for 30 min before the reaction; the container was kept under the nitrogen flushing during the polymerization process.
  • the NMR spectrum ( Figure 2) was used to detect the number of BMA units (24 units) in each polymer chain by comparing the peak signal of the BMA monomer (highlighted with symbol A) to the peak signal of the CTA agent.
  • PBMA lipophilic block polybutylmethacrylate
  • DMAEMA 2- (dimethylamino)ethyl methacrylate
  • BMA polymer holding CTA agent served as a macro-initiator for the Raft polymerization as shown in Scheme 2, where Y and CTA are as defined in Scheme 1.
  • Figure 4 shows the BMA-DMAEMA amphiphilic copolymers at the ratios of (P1) 70:30, (P2) 60:40, (P3) 50:50, (P4) 40:60 and (P5) 30:70. It can be seen that the synthesized ratios were similar to the calculated ones indicating a successful control polymerization.
  • Table 2 shows the calculated versus the detected by the NMR ratios of BMA: DMAEMA (n:m).
  • Table 2 Calculated and detected ratios of BMA block polymer to DMAEMA block polymer and the molecular weights of each BMA-DMAEMA di-block copolymer.
  • Y and CTA are as defined in Schemes 1 and 2.
  • BMA-sulfobetaine The synthesis of BMA-sulfobetaine was confirmed by IR and NMR spectra analysis. Indeed, IR spectral analysis was used to identify the structural transformation of the BMA polymer (Figure 5A) to BMA-DMAEMA copolymer ( Figure 5B) then to BMA-sulfobetaine ( Figure 5C) through identifying the main functional groups in each polymer.
  • the complete betainisation (100% betainisation of DMAEMA amino group) was confirmed (in the next step) by the measurement of the zeta potential of sulfobetaine NPs which showed a slightly negatively charged NPs surface ( ⁇ -2) indicated a complete betainisation of the DMAEMA to form electrically neutral betainised block polymer.
  • the yield of P1S (BMA (70: 30) Sulfobetaine) was very low and it was excluded from further studies (nanoparticles formation).
  • Sulfobetaine nanoparticles were formed by the nano-precipitation technique where the polymer is solubilised in a proper solvent then added dropwise into excess aqueous phase with mixing to allow the formation of nanoparticles by self-assembly.
  • the solubilisation media consisted of solution of 2 M NaCI + methanol where different ratios of each solvent was used to dissolve each of the BMA-Sulfobetaine polymers (P2S, P3S, P4S and P5S) (l.e., the ratio of methanol to 2 M NaCI was varied depending on the ratios of BMA to the Sulfobetaine in each copolymer).
  • Table 3 shows the ratios of solvents solubilisation where 5 g of each polymer was dissolved in 500 pi solubilisation media then added dropwise at a rate of 20 mI/min into a 5 ml pH 6.8 phosphate buffer. The media was further mixed for 45 minutes to allow the methanol evaporation then NPs suspension was dialysed using dialysis tube (molecular weight cut off (MWCO): 20000) against 500 ml PBS 6.8 for 4 hrs.
  • MWCO molecular weight cut off
  • Lumogen® red dye was loaded into the NPs following the same nano precipitation method except that Lumogen® red was dissolved in methanol during the polymer solubilisation. After preparation, these NPs was freeze dried for clinical use and the particles size and zeta potential were measured.
  • Particle size was measured by photon correlation spectroscopy (Malvern Zetasizer NANO ZS, UK) with data collected in uni-modal setting. The instrument was standardised prior to each experiment by use of calibration standards. The Malvern system allows defining particle size distribution in form of intensity, volume and number distributions. In Table 4, particle sizes represent the Z-average particle sizes which are the intensity-based overall average sizes while PDI represents the polydispersity. Z- average particle size is the standard method to present particle sizes since the size distribution by intensity is obtained from an entirely different fitting scheme (instead of a force fit to one average size) where a simple Gaussian distribution is obtained in which the Z-average is the mean and the PDI is related to the width of this simple distribution.
  • the PDI value depends on the type of nanoparticle and method of preparation with most nanoparticles falling within the range of 0.1- 0.6.
  • a PDI value greater than 0.7 indicates sample has a very broad size distribution (ISO 13321 :1996, ISO 22412:2008 and ISO 22412:2017).
  • the Malvern Zetasizer NANO ZS system identifies the quality of results as good if the polydispersity is acceptable.
  • the Malvern system allows the distribution to be shown in volume or number form to obtain more detail about the sample.
  • the size and type of NPs formed from pre-synthesized di-block copolymers is highly affected by the molecular weight of the lipophilic and the hydrophilic block polymers [9] If the lipophilic block polymer is smaller than 9000 Da, micelles-like NPs are formed which are characterised by particle sizes as small as micelles [10,11] This is in agreement with the synthesized BMA- sulfobetaine in this study in which the molecular weight of the BMA lipophilic block polymer is 3500 Da (much less than 9000 Da). Hence, these sulfobetaine NPs should have small particle sizes (less than 50 nm).
  • a Malvern Zetasizer NANO ZS (Malvern, UK) was used to measure the zeta potential of the NP samples.
  • the instrument was standardised prior to each experiment by use of calibration standards.
  • Table 4 shows the particle size and zeta potential of each sulfobetaine NPs in phosphate buffer pH 6.8 and in response to loading of Lumogen® red, freeze drying and storage of NP suspension up to 6 hrs after formation at 25 °C and 37 °C. It can be seen that all particles showed sizes lower than 50 nm with slightly negative to neutral surface charge. Also, the particle sizes and zeta potential did not change for all particles throughout the storage time scale up to 6 hours at room temperature and 37 °C indicating the stability of these particles for the time enough to exert biological action after oral administration.
  • this table shows that the freeze dried NPs retain their physicochemical properties after re-suspension in phosphate buffer aqueous phase indicating the re-suspendability of these particles and ease of storage of these NPs for future use and testing. Moreover, loading of Lumogen® red showed no impact on the particle sizes and zeta potential of sulfobetaine NPs (More loading studies were carried out in Examples 4 and 5.1).
  • Table 4 Particle sizes and zeta potentials of sulfobetaine NPs at external phase PBS pH6.8 and after Lumogen® loading, freeze drying, at PBS pH 7.4, and after 6 hr aggregation studies at 37 °C and 25 °C.
  • Example 2 Diffusion coefficient of Sulfobetaine NPs through the mucous Barrier
  • Diffusion coefficient of particles was measured by the multiple particle tracking (MPT) technique.
  • MPT multiple particle tracking
  • Epifluorescence microscopy is used to capture the videos of the movements of the fluorescently labelled particles (Lumogen® red) inoculated into the mucous. Videos were analysed by special software (ImageJ) and particles’ movements were tracked and these trajectories were converted into mathematical values representing the displacements of particle in pixel. These displacements were converted into metric value to allow the measurement of particles diffusion in cm 2 per seconds.
  • Table 5 shows the calculated diffusion coefficient in water by Stock-Einstein equation (D°), ensemble effective diffusion coefficients in mucous ( ⁇ Deff>), percent ratio of diffusion coefficient in mucous to that freely diffusion in water (% ratio of ⁇ Deff> / D°) and the percent of diffusive particles of various formulae of BMA-sulfobetaine NPs.
  • Table 5 Diffusion coefficients in mucous, ratio of diffusion coefficient in mucous to that freely diffusion in water and the percent of diffusive particles of various formulae of BMA- sulfobetaine NPs. (D° is diffusion in water, ⁇ Deff> is ensemble diffusion coefficient in mucous).
  • these NPs showed direct correlation between diffusivity through the mucous and the degree of the shell hydrophilicity of these NPs represented by the ratio of the hydrophilic sulfobetaine to lipophilic BMA core (Table 5). I.e., while these NPs have close zeta potential and particle size ( ⁇ 0 mV, ⁇ 50 nm), their diffusivities are ranked based on the ratio of the muco-inert highly hydrophilic sulfobetaine to BMA.
  • Slippery-PEGylated strategy involving particles coated or copolymerized with PEG
  • Slippery-Amphiphilic polymer strategy involving particles comprising amphiphilic polymers with the hydrophilic polymer at the surface and the lipophilic polymer at the core
  • Slippery polyelectrolyte strategy involving particles comprising +ve and -ve charged polymers
  • SMEDD Self-microemulsifying drug delivery systems: involving microemulsion systems in which the effects of the various ingredients were studied
  • Mucolytic NPs strategy involving particles loaded with mucolytic agents
  • Thiolated NPs strategy involving particles loaded with thiomers.
  • Figure 6 shows that sulfobetaine NPs out-performed by up to x100-fold all of the current‘gold-standard’ particles, i.e. pegylated solid NPs or nanoemulsions.
  • the capacity of sulfobetaine NP to rapidly penetrate the rate-limiting intestinal mucous layers will facilitates the delivery of cargo directly to the intestinal absorption surface (underlying epithelial surface).
  • sulfobetaine NPs were synthesised to mimic some muco-inert viruses with highly dense, hydrophilic and zwitterionic (electrically neutral) surface completely devoid of hydrophobic domains so the particles can slip through mucous in a non-destructive manner.
  • These NPs should induce a relatively high bioavailability of the orally administered peptide/cargo if these cargos are well protected in the core of NPs. Hence, loading and release of various molecules were studied in the coming sections.
  • Example 3 BMA-Sulfobetaine NPs to Treat Chronic Wound Infections (Microbial Biofilm) Following the successful performance of these novel NPs in mucous barrier, sulfobetaine NPs were tested as a delivery system through microbial biofilm for clinical unmet condition of chronic wound infection. Specifically, MPT technique was used where the diffusion coefficients of the all particles were confirmed to be measured within the bacterial biofilm by staining the biofilms and tracking the particles within the biofilm.
  • sulfobetaine NPs was faster by 1100 times compared with the +ve charged amino polystyrene NPs (Table 6) and 2.5 times faster than the 40 nm negatively charged fluosphere.
  • This small difference between our novel NPs and negatively charged particles is due to the negative nature of the Pseudomonas aeruginosa biofilm leading to the high repulsion with the negatively charged fluosphere and apparent high diffusion of these particles.
  • the diffusion coefficient of sulfobetaine NPs through the highly dense multi-resistant Staphylococcus aureus (MRSA) biofilm is shown in Table 7 where it is illustrated in comparison with 200 nm +ve charged amino fluosphere and different sizes -ve charged carboxylate fluosphere.
  • Table 6 Diffusion coefficient of 40, 100, 200 and 500 nm negatively charged carboxylate fluosphere, positively charged amine fluosphere and neutrally charged sulfobetaine NPs in water calculated by Stoke-Einstein equation versus their effective diffusion coefficients through Pseudomonas aeruginosa bacterial biofilm (PA01) measured by the MPT technique.
  • Table 7 Diffusion coefficient of 40, 100, 200 and 500 nm negatively charged carboxylate fluosphere, positively charged amine fluosphere and neutrally charged sulfobetaine NPs in water calculated by Stoke-Einstein equation versus their effective diffusion coefficients through Methicillin-Resistant Staphylococcus aureus (MRSA) bacterial biofilm measured by the MPT technique.
  • MRSA Methicillin-Resistant Staphylococcus aureus
  • the very large lipophilic molecules (Lumogen® red) with molecular weight 1079 gm was selected to study the maximum loading capacity of sulfobetaine NPs to lipophilic cargo.
  • the loading method was carried out as described in the NPs formation section on sulfobetaine NPs with the smallest hydrophilic sulfobetaine ratio (40% P2S) and NP with the biggest hydrophilic sulfobetaine ratio (70%, P5S) with aimed loading capacity of 50%.
  • the Lumogen® red weight is 50% of the NPs weight so 500 pg Lumogen® red was used for each 1mg polymeric NPs.
  • the entrapment efficiency (EE%) and the loading capacity (LD%) were calculated by the following equations:
  • Table 8 shows EE% and LD% of these NPs toward the Lumogen® red (i.e., the capacity of incorporation of the lipophilic agent into the BMA core. While P2S showed very high EE% (82.26%) and LD% (40.33%), P5S showed lower EE% and LD% for Lumogen® red (4.53 % and 2.26 % respectively). It can be seen that loading capacity for sulfobetaine NPs was associated with the ratio of the lipophilic BMA core, i.e., the loading capacity increased from 2.26% to 40.33% when the BMA ratio increased from 30% to 60%.
  • the NPs with high lipophilic BMA content showed a very high loading capacity to load the lipophilic cargo due to the larger content of the BMA lipophilic core. Accordingly, P5S showed acceptable loading capacity toward a large lipophilic molecule like Lumogen® red indicating the suitability of these novel NPs for loading of lipophilic cargo.
  • Table 8 Entrapment efficiency and loading capacity of sulfobetaine NPs to Lumogen® red lipophilic dye at concentrations of 0.1 % and 50%.
  • the in vitro release of the Lumogen® red was studied using the dialysis method. Briefly, freeze dried NPs loaded with Lumogen® red was re-suspended in phosphate buffer pH 6.8 (1 mg NPs per 1 ml buffer). Then, suspension was divided into 1 ml aliquots, each aliquot was added into dialysis tube (1 ml, MWCO: 20000) then dialysed against 500 ml PBS 6.8. In vitro release was studied up to 24 hrs where samples were collected at time intervals of 30 min, 1 , 2, 3, 4, 6, 8, 16 and 24 hr.
  • Figure 7 shows the in vitro release profile of Lumogen® red from formulae P2S and P5S where 7A shows the percent of release while 7B shows the cumulative amount in pg.
  • both particles exhibited incomplete release profile of Lumogen® red after 24 hr, where P5S and P2S showed 80% and 48% release after 24 hr.
  • P5S NP exhibited fast release (33%) within the first 2 hr followed by a gradual release within the time intervals between 2 and 8 hr.
  • P2S showed a gradual release reaching to 30% after 8 hr. Both formulae showed almost a plateau release profile after 8 hr up to 24 hr.
  • Figure 7B shows that the total cumulative release of Lumogen® red from P5S was 18 pg after 24 hrs which is equal to the amount released within the first 30 minutes for P2S.
  • the observed huge difference between the release profiles in the 2 figures is associated with the high content of Lumogen® red in P2S (40.33) versus the content in P5S (2.26).
  • the slow release profile of Lumogen red from these NPs is related to the high solubility of the lipophilic dye in the lipophilic core of the NP versus the low solubility in the hydrophilic external medium. This slow release is required for the delivery of drug through various routes since it gives prolonged protection of drug throughout the delivery process.
  • the loading and in vitro release data show the suitability of these novel particles for the delivery of lipophilic agents.
  • the candidate peptide was exenatide which is a hormone used for the treatment of type 2 diabetes (non-insulin dependent) which global prevalence is ca 7-8%. Due to the very low stability in the intestinal environment, exenatide is administered in injectable form to control the blood glucose level and promote central satiety leading to weight loss benefits. Hence, patient adherence to medication is critical and will be enhanced by a proper oral medication. Thus, these NPs were studied for their effectiveness to improve the in vivo pharmacokinetic and pharmacodynamics characteristics of exenatide. This technology should be applicable to a wide range of alternative markets demanding the oral delivery of peptides.
  • exenatide was modified into a more lipophilic molecule by ion-pairing with sodium dodecyl sulphate (SDS) to generate lipophilic exenatide laurate, where the sulphate ion of SDS have four potential ion-pairing sites within the exenatide molecule, (i.e. amino acids Hi, R20, Ki2,K27).
  • SDS sodium dodecyl sulphate
  • exenatide acetate were mixed at molar ratios of 3:1 , 4:1 and 5:1.
  • Table 9 The % of exenatide laurate yield as a result of ion pairing with SDS.
  • the encapsulation of the lipophilic exenatide laurate into the BMA lipophilic core of the NPs was carried out similarly to the method of Lumogen® red loading where exenatide laurate was dissolved in methanol aliquot prior to the mixing with 2M NaCI: methanol to solubilise the BMA-sulfobetaine polymer.
  • NP suspensions were then dialysed (dialysis tubing, MWCO: 20000) against PBS 6.8 for 4 hr and the exenatide EE% and LD% were measured.
  • Table 10 shows the entrapment efficiency (EE%) of the NPs which exceeded 90% and loading capacity (LD%) exceeding over 13.5%.
  • the loading capacity is significantly in excess of the acceptable industrial level (ca. 5%) and is an indication of the commercial potential of the NPs. Further data obtained from the in-vitro dialysis release studies showed no burst release of the exenatide into the external media ( Figure 8) indicating the suitability of these NPs for the oral delivery of these peptides.
  • Figure 8 shows the in vitro release profile of exenatide either free or from NPs (pH 6.8 throughout).
  • Figure 8A shows the 8 hr cumulative release (pg) profile for P2S, P3S, P4S and P5S versus time with the lower panel showing the release over the first 2 hrs.
  • the release rates over the first 8 hr varied between 4.21 to 5.08 pg/hr for formulations P2S to P4S while the NP formulation with the smallest BMA hydrophobic core, P5S, showed a significantly greater release rate of 8.85 pg/hr.
  • Figure 8B shows the corresponding data represented as % of loaded exenatide released. Over 8 hr the NPs P2S to P4S released 10-14% of the loaded material, while P5S had released 26% of the original exenatide loaded into the NPs ( Figure 8B).
  • the lower panels in both Figures 8A and 8B also show the transfer of free exenatide across the dialysis tubing over the first 2 hrs with recovery of approximately 90% of the material by this time.
  • Sulfobetaine P5S NP formulation showed an extended release throughout the 24 hr period. This release profile was characterised by a clear lag phase of ca 4 hr prior to the more linear pseudo-steady-state release of exenatide with only 1.2 pg (0.8% of loaded material) released after 2 hours and overall release of 43% after 24 hrs. Moreover, Exposing the P5S NP formulation to pH 1.2 for the first 2 hrs of release showed no significant difference to release at pH 6.8 indicating no effect of low pH (1.2) upon exenatide release characteristics. Thus, based on the loading and release data, P5S formulation was the most efficient carrier for exenatide oral delivery.
  • PK-PD studies involve four treatment arms ( Figure 10).
  • PD studies include the measurement of glucose level in response to intraperitoneal (I.P) glucose challenge test (GCT) where 2 g/kg glucose is administered by I.P into rats then blood glucose level is measured up to 5 hrs after glucose administration by Glucometer AccuCheck Active.
  • GCT glucose challenge test
  • PK studies involve the measurement of exenatide plasma glucose level up to 10 hrs after exenatide administration without glucose administration (No GCT).
  • the 4 arms in the PK-PD studies are ( Figure 10): (i) I.P GCT PD studies alone (no treatment), (ii) PD studies in response to GCT and S.C. administration of exenatide solution and PK studies after S.C.
  • Pilot study was conducted to confirm the suitability of the measurement technique to detect the glucose level at time intervals of 0, 30 min, 1 hr, 1.5 hr, 2 hr, 3hr, 4 hr and 5 hr after I.P administration of 2 g/kg glucose to the experimental rats.
  • This study showed that a volume of 40 pi blood samples was appropriate to measure glucose levels by Glucometer AccuChek® Active.
  • pilot study showed that the peak glucose level was reached 20 min after the I.P administration of glucose.
  • the peak glucose level is important to identify the time at which exenatide should be administered orally or S.C. ii. Arm 2:“SC exenatide + I.P. GCT” PK and PD study:
  • Exenatide was administered S.C. at a dose of 20 pg at different time before the GCT. Pilot studies showed exenatide S.C. dosing can show significant effect on the glucose level if it is administered 10 min before the I.P glucose challenge test. Thus, a 10 min delay between injecting exenatide SC and the IP administration of glucose was set for the following full-scale studies. iii. Arm 3:“Oral Trust NPs with exenatide + I.P. GCT” PK and PD study.
  • Figure 12 shows the absolute blood glucose levels at time interval 0, 0.16, 0.5, 1 , 1.5, 2, 3, 4 and 5 hrs of rats subjected to the 4 treatment arms (time 0 is the time immediately before glucose administration by I.P route, while remaining time intervals are post glucose administration). It can be seen that exenatide oral solution given 4 hrs before the GCT (green line) showed no effect on the glucose levels compared with the control GCT alone (blue line). On the other hand, both of S.C. exenatide solution (red line) and sulfobetaine exenatide NPs (black line) showed significant effect on the blood glucose levels compared with GCT alone.
  • glucose AUC was used to identify the pharmacological effect of orally administered sulfobetaine exenatide NPs compared with the commercial S.C. route.
  • the relative bioavailability was 12.3% which is much higher than the industrially acceptable bioavailability (5%) This is another indication about the effectiveness of these particles in oral delivery of peptides.
  • exenatide plasma concentration was measured for orally administered exenatide sulfobetaine NP versus S.C exenatide solution to calculate exenatide bioavailability and to add value and substantiate the data obtained from PD studies.
  • blood samples were collected at time intervals of 0, 0.5, 1 , 1.5, 2, 3.5, 6 and 10 hrs. Blood samples were processed to separate plasma for measurement of exenatide and stored at -70 ° C. Extraction of exenatide from plasma samples was carried out following the protocol from phoenix pharmaceuticals, where plasma samples were acidified by equal volume of acidic buffer (supplied by Phoenix) and centrifuged for 20 minutes at 17,000 x g (4°C).
  • sulfobetaine NPs were shown through the loading, in vitro and in vivo studies on oral delivery of exenatide peptide.
  • the safety of these NPs for biological application is important requirement to be proven by studying the cytotoxicity of sulfobetaine NP.
  • MDCK Cells (passage 7) were grown in standard conditions (humidified incubator at 37°C with 5% C02) using DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. MDCK cells were seeded in 96-well plates at a density of 5*10 4 cells/well (100 pi culture medium per well) and cultured for 24 hrs. Next day, well plate was examined under microscopy for cells confluency.
  • Freeze dried sulfobetaine NPs sample was re-suspended in the same MDCK culture medium and diluted serially to prepare a concentration range of NPs suspensions between 1 mg/ml to 10 ng/ml. 100 mI of each NPs suspension was added in triplicate by replacing the 100 mI medium in each well. Besides NPs, three controls were used, these are: (i) Cells without NP to determine the 100% cells viability (ii) Medium without cells or NP (to determine the background) (iii) Cells without NP but with 1% Triton (positive control for 0% cells viability).
  • the absorbance of well plate was measured at wave length between 550 and 600 nm and reference wavelength of 650 nm using Elisa reader (LT-5000 MS, Taiwan).
  • the absorbance value represents the concentration of formazen solution that is formed by viable cells.
  • the % of viable cells for each NPs concentration versus cells alone (the 100% viable cells) was determined by the following equation:
  • CNP represents the cells with NPs suspension
  • M is the media only without cells or NPs to subtract the background
  • C is the cells with media (no particles) representing 100% viability.
  • Figure 14 shows the viability of cells in response to a range of NPs concentrations versus no particles (cells only, 100% viability) and 1 % triton (0% viability).
  • the maximum NPs concentration was selected depending on the maximum possible concentration that these NPs can reach in the body. It can be seen that there is no significant difference in cell viability among all the concentrations of NPs versus the 100% cells viability (no particle in the media) indicating that these NPs are nontoxic and can be administered safely.
  • EU-NCL carried out lactate dehydrogenase (LDH) assays which measures the membrane integrity since LDH enzyme is released into the cytoplasm upon cell lysis.
  • LDH assay was performed on 2 cell types: hepatocarcinoma cells (Hep G2) (Liver carcinoma cell line is commonly used as model for liver toxicity) and porcine proximal tubule cells (LLC-PK1).
  • both cell types were seeded in 96- well plates (100 pi of 2.5-5 x 10 5 cells /ml and incubated for 24 hr at 5% C02, 37°C and 95% humidity).
  • Cells were treated with sulfobetaine NPs for up to 48 hrs by adding 100 mI of NPs suspensions at range concentrations of 0.01-650 pg/ml (cytotoxicity was measured for up to 48 hrs). After the incubation period, 100 mI was transferred from each well plate into another 96 well plates to which LDH reagent was added and incubated for 20 minutes followed by reading the absorbance (plate reader) at 490 nm using a reference wavelength of 680 nm.
  • Example 7 Loading and release of a hydrophilic agent across bacterial biofilms
  • Polymyxin B is an antibacterial agent used as a model for treatment of bacterial biofilms in chronic wounds.
  • This hydrophilic agent consists of 9 amino acid with a molecular weight of 1 ,301 g/mole which makes it a proper candidate to study the loading capacity of sulfobetaine NPs’ shell toward hydrophilic agents and their delivery across biological barriers.
  • a different loading strategy was selected to enable the separation of loaded polymyxin from unloaded free agent (i.e.
  • the strategy was selected to employ the centrifugation method to separate the unloaded polymyxin from the centrifuged NPs loaded with polymyxin).
  • all trials to load polymyxin into sulfobetaine NPs using dialysis tubes or ultra-centrifugation method resulted in failure to separate unloaded polymyxin since polymyxin is small peptide that can stick to the dialysis membranes and ultra-centrifugation filter.
  • sulfobetaine polymer was dispersed firstly in 2 M NaCL (1 mg/50 pi) then overnight in PBS (1 mg/ml) to form clear suspension of NPs.
  • BCA bicinchoninic acid
  • the antibacterial activity of loaded polymyxin versus free polymyxin was carried out by SINTEF (Norway) where the minimum inhibitory concentration (MIC) was calculated.
  • MIC minimum inhibitory concentration
  • measuring the antibacterial activity of polymyxin reflects its delivery across the bacterial biofilm since bacteria were grown overnight which is enough time for bacteria to grow biofilms.
  • This study reflects the antibacterial/anti-biofilm activity of loaded polymyxin into the NPs.
  • the studied bacterial strains were P. aeruginosa ATCC15692 (PA01 , non-mucoid) and mucoidal P. aeruginosa ATCC39324.
  • TLB Tryptic soy broth
  • OD600 optical density 600 nm
  • 30 pi were then inoculated to 96-well plates containing 120 mI Muller-Hinton medium with free polymyxin, nanoparticles and nanoparticles loaded with polymyxin.
  • Figure 16A, 16B and 16C shows the effect of free polymyxin, loaded polymyxin into sulfobetaine NPs and unloaded NPs correspondingly on non-mucoidal (black bars) versus mucoidal P. aeruginosa (grey bars).
  • Figure 16 shows NPs alone had no effect on bacterial and bacterial biofilm growth.
  • polymyxin loaded into sulfobetaine NPs had substantial impact on the P. aeruginosa compared with free polymyxin especially with mucoidal P. aeruginosa where MIC was significantly different for polymyxin loaded into NP versus free polymyxin at concentrations of 0.25, 0.5 and 1 pg/ml.
  • thermoresponsive diblock copolymer assemblies Temperature directed morphology transformations, J. Polym. Sci. Part A Polym. Chem. 50 (2012) 4879 ⁇ 1887. doi: 10.1002/pola.26313.
  • PEG PEG nanoparticles: NMR studies of the central solidlike PLA core and the liquid PEG corona, Langmuir. (2002) 3669-3675. http://pubs.acs.Org/doi/abs/10.1021/la011393y.

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Abstract

La présente invention concerne un polymère comprenant une chaîne polymère de formule générale (I) : où R1 est H ou un groupe méthyle ; R2 est un groupe alkyle en C1-6 R3 est H ou un groupe méthyle ; R4 est -(CH2)p-N+ (CH3)2-(CH2)3-S(O)2O- ou -(CH2)p-N(CH3)2 ; p est un nombre entier d'une valeur de 1 à 4 ; n est un nombre entier d'une valeur de 15 à 30 ; et m est un nombre entier dans lequel le rapport de n:m est de 80:20 à 20:80 ; à condition qu'au moins 90 % des groupes R4 soient -(CH2)P-N+ (CH3)2-(CH2)3-S(O)2O-. L'invention concerne également des nanoparticules formées à partir des polymères et des compositions pharmaceutiques comprenant un agent actif encapsulé dans les nanoparticules.
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US12441996B2 (en) 2023-12-08 2025-10-14 Battelle Memorial Institute Use of DNA origami nanostructures for molecular information based data storage systems
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WO2021110735A1 (fr) * 2019-12-03 2021-06-10 Universite De Strasbourg Nanoparticules polymères zwittérioniques luminescentes
CN114981384A (zh) * 2019-12-03 2022-08-30 斯特拉斯堡大学 发光两性聚合物纳米颗粒
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