WO2024133782A1 - Clay nanoparticles for medical use - Google Patents

Clay nanoparticles for medical use Download PDF

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Publication number
WO2024133782A1
WO2024133782A1 PCT/EP2023/087424 EP2023087424W WO2024133782A1 WO 2024133782 A1 WO2024133782 A1 WO 2024133782A1 EP 2023087424 W EP2023087424 W EP 2023087424W WO 2024133782 A1 WO2024133782 A1 WO 2024133782A1
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WIPO (PCT)
Prior art keywords
composition
therapeutic
gel
typically
composition according
Prior art date
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PCT/EP2023/087424
Other languages
French (fr)
Inventor
Richard Oreffo
Jonathan Dawson
Mohamed MOUSA
Allison Shaw
Agnieszka JANECZEK
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Renovos Biologics Limited
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Priority claimed from US18/491,443 external-priority patent/US12036328B1/en
Application filed by Renovos Biologics Limited filed Critical Renovos Biologics Limited
Publication of WO2024133782A1 publication Critical patent/WO2024133782A1/en

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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/20—Silicates
    • C01B33/36—Silicates having base-exchange properties but not having molecular sieve properties
    • C01B33/38—Layered base-exchange silicates, e.g. clays, micas or alkali metal silicates of kenyaite or magadiite type
    • C01B33/40—Clays
    • C01B33/405—Clays not containing aluminium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention relates to compositions comprising clay nanoparticles, methods for making such compositions and their use in medical and cosmetic methods.
  • the nanoclays may be used alone or in combination with therapeutic or diagnostic agents.
  • the nanoclays may be used in the delivery of therapeutic or diagnostic agents or in regenerative medicine.
  • Clay nanoparticles are nanoparticles of layered mineral silicates that have numerous applications including in cosmetics and paints. Clay nanoparticles have also been used in the pharmaceutical industry as excipients and active agents. Depending on chemical composition, crystallographic structures and nanoparticle morphology, clay nanoparticles are organised into several classes such as montmorillonite, bentonite, smectite, kalinite, hectorite, and halloysite.
  • Certain clay nanoparticles self-assemble into a gel in an aqueous environment, for example in the body, and the resulting nanoclay gels have been found to be particularly well suited to providing a regenerative microenvironment, or a platform for drug delivery.
  • An example of a nanoclay that has been used in medical and cosmetic formulations is LaponiteTM (a synthetic smectite clay manufactured by BYK).
  • LaponiteTM is a discshaped synthetic hectorite type clay which has been cited as a potential nanomedicine for use in drug delivery, bio-imaging, tissue engineering and regenerative medicine.
  • nanoclay gels when used as a nanomedicine include ease of administration and high stability in vivo, post administration.
  • a high stability is desired such that, once the gel is situated at the target site, it provides a stable matrix structure into which new cells can grow, or from which drugs can be delivered. Poor stability at the target site can lead to the nanoclay gel becoming dispersed throughout the subject, meaning it does not serve its intended purpose in the correct location, and can cause potentially harmful effects elsewhere in the body.
  • clay nanoparticle gels which can be effectively localised at a target site and have high stability under physiological conditions (i.e. pH 7.35 to 7.45, and 36.1 °C to 37.5 °C) and which can be safely and conveniently administered and retained to a subject.
  • physiological conditions i.e. pH 7.35 to 7.45, and 36.1 °C to 37.5 °C
  • Current known clay nanoparticles do not meet these requirements.
  • the present inventors have surprisingly found that clear gels which have a high stability in vivo can be produced from clay nanoparticles having an increased ratio of magnesium ions to lithium ions within the clay nanoparticles, compared to existing nanoclays.
  • Nanoclay gels produced from such particles may have good rheological properties, and in particular exhibit a change in rheological properties in physiological conditions, thus facilitating administration of the gels by injection, whilst providing a clear and stable gel at physiological pH.
  • the formulations comprising the clay nanoparticles of the invention can self-assemble, and can easily be manufactured.
  • the present inventors have developed a composition comprising a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I): [(Si 8 MgbLi c )02o(OH) 4 ] (I) wherein 5.5 ⁇ b ⁇ 6, and wherein c > 0 and b/c > 12.
  • the gel compositions described herein have high stability at physiological pH. Such gels can be easily administered, typically by a syringe, without causing significant discomfort or distress to a subject. Further, the gels stabilise in physiological conditions, ensuring effective localisation at the target site and reducing undesired effects caused by delocalisation of the gel.
  • composition of the invention for use in the treatment of the human or animal body by therapy or diagnosis, in particular by therapy.
  • the composition may be for use in a method of tissue repair and/or regeneration, and/or a method of cell delivery.
  • the composition may also be for use in the treatment or prevention of infection.
  • composition of the invention may be used in cosmetic methods.
  • the composition is a cosmetic composition, in particular a tissue filler or a topical gel or cream.
  • the invention also provides a composition of the invention for use in a method of delivery of one or more therapeutic or diagnostic agents to a target site in the human or animal body, wherein said method comprises administering said composition to the human or animal body.
  • a composition comprising a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I):
  • composition according to any one of paragraphs 1 to 7 which further comprises one or more therapeutic or diagnostic agents.
  • composition according to paragraph 8 wherein the one or more therapeutic or diagnostic agents are therapeutic or diagnostic agents suitable for use in regenerative medicine.
  • composition according to paragraph 8 or paragraph 9 wherein the one or more therapeutic or diagnostic agents are selected from small molecules, growth factors, antibodies, biological tissue substitutes, synthetic bone grafts, antimicrobial agents, antibiotics and cells.
  • composition according to any one of paragraphs 1 to 10 wherein the composition comprises BMP -2.
  • a pharmaceutical composition comprising a composition according to any one of paragraphs 1 to 14, a pharmaceutically acceptable carrier or diluent, and optionally one or more pharmaceutically acceptable excipients.
  • a kit comprising a syringe or a vial (e.g. a sprayable vial), wherein the syringe or vial contains a composition according to paragraph 13 or paragraph 14, or a composition according to paragraph 15 wherein the composition is in the form of a gel.
  • a syringe or a vial e.g. a sprayable vial
  • a kit comprising separately a composition according to any one of paragraphs 1 to 7; and one or more therapeutic or diagnostic agents as defined in any one of paragraphs 8 to 11, preferably wherein (a) the composition is in powder form or in the form of a gel; and/or (b) the therapeutic or diagnostic agent is in a powder form.
  • composition according to any one of paragraphs 1 to 15, or 23 for use in a method of regenerative medicine is provided.
  • composition according to any one of paragraph 1 to 15, or 23 for use in the treatment or prevention of infection is provided.
  • a cosmetic composition comprising a composition according to any one of paragraphs 1 to 14 and optionally one or more additives, preferably wherein the cosmetic composition is a tissue filler or a topical gel or cream.
  • FIG. 1 shows the comparison of elemental ratios between LaponiteTM XLG and the compositions of the invention set out in Table 1 (Renovite).
  • Empirically determined Mg, Li and Na are presented relative to empirically determined Si scaled according to (Na+ a [(SisMgbLid)02o(OH)4]' a . Bars represent mean and SD. P values calculated using an unpaired T-test.
  • Figure 2 shows comparison of elemental ratios between LaponiteTM XLG and a range of compositions of the invention as set out in Table 2 (Renovite analogues).
  • Mg content calculated from empirical Si, Li and Na values on basis of the formula: (Na+ a [(SisMgbLid)02o(OH)4]' a . Bars represent mean.
  • Figure 3 shows enhanced gelation of Renovite (AS-19, Table 1) in response to blood serum.
  • Figure 4 shows enhanced gelation of compositions of the invention (Renovite: mean of all clay batches listed in Table 1) in response to blood serum.
  • Renovite mean of all clay batches listed in Table 1
  • Renovite displayed equivalent gel strength (yield stress (Pa)), stiffness (storage modulus (G’)) and viscosity (storage modulus (G”)) to XLG in its native state.
  • yield stress Pa
  • stiffness stiffness
  • viscosity storage modulus
  • FIG. 6 shows Zeta Potential Half Maximum (PHM) and its significance for nanoclay gelation.
  • ZP negative zeta potential
  • AS-14 negative zeta potential
  • pH/>// ⁇ / represents the pH at which this sharp decrease in negative ZP occurs by measuring the pH at which negative ZP is 50% of its recorded mV prior to addition of titrant. Titrations and dynamic ZP measurements were performed on the StabinoTM II device programmed to add 30 pl of IM HCL every 20 seconds.
  • Figure 7 shows Zeta Potential Half Maximum (PHM) for a composition of the invention (AS-14, labelled Renovite 1.0) versus LaponiteTM.
  • the PHM for Renovite occurs near physiological pH7.4 (shaded grey) compared with pH 5.9 in LaponiteTM.
  • Titrations and dynamic ZP measurements were performed on the StabinoTM II programmed to add 30 pl of IM HCL every 20 seconds to a suspension of 1 wt% nanoclay.
  • FIG 8 shows Zeta Potential Half Maximum (PHM) correlates with Mg and Li content.
  • pH at PHM PHM
  • PHM pH at PHM
  • XLG reference sample AS-33 vs LaponiteTM
  • PHPHM for Renovite 1.0 occurred at pH values approximating physiological pH 7.4 (shaded grey).
  • Mg content calculated from empirical Si, Li and Na values on basis of the formula: (Na+ a [(SisMgbLid)02o(OH)4]' a .
  • P values report Pearson’s correlation coefficients where a significant correlation is P ⁇ 0.05.
  • a clay nanoparticle as described herein is an inorganic nanoparticle.
  • a clay nanoparticle may comprise or consist of a silicate.
  • the silicate may comprise layered silicate.
  • the clay nanoparticles may have an average size of from about 15 nm to about 50 nm in the longest dimension.
  • the clay nanoparticles may have an average size of from about 20 nm to about 40 nm in the longest dimension.
  • the clay nanoparticles may have an average size of from about 15 nm to about 40 nm, or from about 20 nm to about 50 nm, or from about 24 nm to about 36 nm in the longest dimension.
  • the clay nanoparticles may have an average thickness (shortest dimension) of from about 1 nm to about 3 nm.
  • the clay nanoparticles may have an average thickness of from about 1 nm to about 2.5 nm, or from about 1.5 nm to about 2.5 nm, or from about 1.3 nm to about 2.2 nm.
  • the dimensions of the clay nanoparticles may be determined by any appropriate method known to the skilled person. Typically the dimensions are determined using smallangle X-ray scattering. The dimensions of the clay nanoparticles may be determined when dispersed in an aqueous environment.
  • the clay nanoparticles may have an aspect ratio of at least 1 :5.
  • the clay nanoparticles may have an aspect ratio of at least 1 : 10, or at least 1 :20, or at least 1:25.
  • the clay nanoparticles may have an aspect ratio of less than 1 : 100.
  • the clay nanoparticles may have an aspect ratio of less than 1 :50, or less than 1 :30.
  • the clay nanoparticles may have an aspect ratio of from 1 : 10 to 1 :50.
  • the clay nanoparticles may have an aspect ratio of from 1 :5 to 1 :30 or from 1 : 10 to 1:30.
  • Each clay nanoparticle of the invention comprises an anionic component and a cationic component.
  • the anionic component has the formula (I): [(Si 8 Mg b Li c )02o(OH) 4 ] (I).
  • the anionic component of the clay nanoparticle carries a negative charge.
  • this negative charge is a-, wherein a is equal to the positive charge carried by the cationic component, a is greater than 0, and is not necessarily an integer value.
  • b in the above formula (I) represents the number of Mg atoms present per unit of the anionic component, b is not necessarily an integer value, b is typically ⁇ 6.0. More typically, b ⁇ 5.9. b is typically > 5.6, preferably 5.5 ⁇ b > 6.0, for example 5.6 ⁇ b > 6.0 or 5.6 ⁇ b > 5.9.
  • c in the above formula (I) represents the number of Li atoms present per unit of the anionic component, c is greater than 0, and is not necessarily an integer value, c is typically ⁇ 2, more typically ⁇ 1. Preferably c is ⁇ 0.5, more preferably c ⁇ 0.45, for example c may be ⁇ 0.4. c is typically > 0.2, more typically > 0.3.
  • 0.2 ⁇ c ⁇ 0.5 in particularly preferred embodiments 0.3 ⁇ c ⁇ 0.5.
  • the ratio b/c is greater than 12. Typically the ratio is less than 100, such that typically 12 ⁇ b/c ⁇ 100. In some embodiments, b/c is > 13. In some embodiments, b/c > 14. b/c is typically ⁇ 18. In some preferred embodiments b/c ⁇ 16. Therefore preferably, 12 ⁇ b/c ⁇ 18, preferably from 12 ⁇ b/c ⁇ 18. In some embodiments, 13 ⁇ b/c ⁇ 18, 13 ⁇ b/c ⁇ 16, 14 ⁇ b/c ⁇ 18 or 14 ⁇ b/c ⁇ 16.
  • the figures b and c are preferably determined using elemental analysis using inductively coupled plasma-optical emission spectrometry (ICP-OES).
  • ICP-OES inductively coupled plasma-optical emission spectrometry
  • the cationic component of each clay nanoparticle typically comprises one or more metal cations.
  • the cationic component comprises one or more metal cations selected from alkali metal cations, alkaline earth metal cations, transition metal cations and combinations thereof.
  • metal cations may include one or more cations of lithium, sodium, calcium, copper, potassium or magnesium. Sodium is preferred.
  • the metal cation may be of the formula M n+ , wherein M is a metal and n is an integer, typically an integer from 1 to 6, more typically from 1 to 4. Preferably n is i or 2, more preferably 1. M n+ is preferably Na + .
  • the clay nanoparticle comprises an anionic component and a cationic component such that the clay nanoparticle has the formula: [(M n+ ) x (M2P + )y][(Si 8 Mg b Li c )0 2 o(OH) 4 ] a - wherein b, c and a are as defined anywhere herein, and wherein M n+ and M2 p+ are different metal cations, and n and p are the same or different and are integers of from 1 to 6, typically 1 or 2.
  • x + y is ⁇ 2, more typically ⁇ 1.
  • x + y is ⁇ 0.7 more preferably x + y is ⁇ 0.5.
  • x + y is > 0.3, more typically > 0.4.
  • 0 ⁇ (x + y) ⁇ 2 such as 0 ⁇ (x + y) ⁇ 1, 0 ⁇ (x + y) ⁇ 0.7, 0 ⁇ (x + y) ⁇ 0.5, 0.2 ⁇ (x + y) ⁇ 0.7, 0.3 ⁇ (x + y) ⁇ 0.7, 0.4 ⁇ (x + y) ⁇ 0.7, 0.3 ⁇ (x + y) ⁇ 0.5, or 0.4 ⁇ (x + y) ⁇ 0.5.
  • the cationic component of the clay comprises only one type of metal cation, such that the clay nanoparticle has the formula: (M n+ ) z [(Si 8 Mg b Li c )0 2 o(OH) 4 ] a - wherein n is an integer from 1 to 4, typically 1 or 2, more typically 1, and z is equal to a/n.
  • M n+ is Na +
  • the clay nanoparticles has the formula: (Na + )a[(Si 8 Mg b Li c )02o(OH) 4 ] a - wherein a, b, and c are as defined anywhere herein.
  • the nanoparticles of the invention may contain impurities and/or by-products.
  • the nanoparticles are preferably 90% pure, more preferably 95% pure, more preferably 98% or 99% pure. Most preferably, the nanoparticles are substantially pure particles of formula [(Si8MgbLi c )02o(OH) 4 ] as defined herein.
  • compositions of the invention are provided.
  • the composition of the invention comprises a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I): [(Si 8 MgbLi c )0 2 o(OH) 4 ] (I) wherein 5.5 ⁇ b ⁇ 6, and wherein c > 0 and b/c > 12.
  • a plurality of clay nanoparticles refers to two or more clay nanoparticles.
  • a composition of the invention may contain up to 100 wt% of the clay nanoparticles of the invention. More typically, it contains up to 85 wt% of the clay nanoparticles of the invention, or up to 50 %wt of the clay nanoparticles of the invention. Typically, the composition contains at least 0.1 wt% clay nanoparticles, e.g. from 0.1 to 100 wt%, 0.1 to 85 wt% or 0.1 to 50 wt%.
  • the clay nanoparticles described herein may comprise coordinated water.
  • Reference herein to clay nanoparticles of the invention is therefore intended to encompass clay nanoparticles having coordinated water.
  • reference herein to dried compositions is intended to encompass compositions comprising clay nanoparticles which comprise coordinated water. Therefore, a dried composition may contain up to 10 wt% water. More typically it contains up to 7 wt% water. Typically the composition contains at least 3 wt% water, or at least 5 wt% water. Typically, therefore, the composition may comprise from 3 wt% to 10 wt% water, such as from 5 wt% to 7 wt% water. In such embodiments, the remainder of the composition may contain up to 100 wt% clay nanoparticles of the invention, such that the composition contains, for example, 93 wt% to 95 wt% clay nanoparticles of the invention.
  • the composition may consist of, or consist substantially of, the clay nanoparticles of the invention.
  • the composition is typically in dried form, e.g. a powder, a film, granules, a coating, porous sponge, or fibres.
  • the composition may for example comprise at least 90% clay nanoparticles, at least 95% clay nanoparticles, at least 98% or 99% clay nanoparticles or about 99.5% clay nanoparticles.
  • additional materials may be present in the composition, for example including carriers or diluents (e.g. water or saline), excipients and/or therapeutic or diagnostic agents.
  • the composition may further comprise additional materials such as ions, proteins, and polymers.
  • the composition of the invention is in dried form, e.g. in the form of a powder.
  • the composition may be lyophilised, e.g. a lyophilised powder.
  • An alternative dried form is a film.
  • the composition is a liquid-solid fluid mixture such as a slurry, or a paste.
  • the composition is in the form of a gel.
  • a gel is a preferred form of the composition of the invention and the compositions are typically administered in this form.
  • a gel of the invention may be formed, for example, by casting the clay nanoparticles in water.
  • a gel of the invention may be dried to form a film, or lyophilised. Where the composition is provided as a film or in lyophilised form, it may be administered by application to a surface. Alternatively, it may be reconstituted with a diluent, such as water or saline, before use.
  • the composition may be a pharmaceutical composition, which is a composition suitable for pharmaceutical use.
  • a pharmaceutical composition typically comprises one or more pharmaceutically acceptable excipients.
  • Preferred pharmaceutical compositions are sterile and pyrogen free.
  • Suitable carriers and/or diluents for use in the invention include pharmaceutically acceptable carriers such as water and aqueous solutions, in particular saline. Isotonic solutions, in particular isotonic saline, are preferred.
  • the carrier or diluent is typically sterile. Thus, sterile, isotonic saline is a preferred carrier.
  • Such carriers can be used in the case of compositions which are in the form of a slurry or a gel.
  • compositions may also be included in the composition, as needed.
  • a second carrier material for the therapeutic or diagnostic agent may be used.
  • the second carrier may be a polymeric material.
  • the second carrier is selected from carboxymethylcellulose, gelatine and collagen.
  • diluents e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch
  • rheology modifiers such as carboxymethylcellulose, gelatine and collagen
  • lubricants e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols
  • binding agents e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone
  • disaggregating agents e.g.
  • starch alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; wetting agents, such as lecithin, polysorbates, laurylsulphates; dispersants, such as sodium polyacrylate and pyrophosphates; and, in general, non toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • rheology modifiers such as carboxymethylcellulose, gelatine and collagen
  • carriers for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • carriers for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • rheology modifiers such as carboxymethylcellulose, gelatine and collagen; olive oil; ethyl oleate; glycols, e.g. propylene glycol; and if desired, a suitable amount of lidocaine hydrochloride.
  • compositions of the invention comprise the nanoparticles of the invention and water, wherein the nanoparticles are present in an amount of from about 0.1 wt% to about 50 wt% based on the total weight of the composition.
  • Preferred compositions comprise from about 0.5 wt% to about 40 wt% nanoparticles of the invention, based on the total weight of the composition.
  • the composition may comprise from about 1 wt% or from about 2 wt% and up to about 40 wt% nanoparticles of the invention.
  • the composition typically also comprises water or saline.
  • the composition may optionally comprise one or more further additives.
  • the composition may consist of, or consist essentially of, the nanoparticles of the invention and water or saline.
  • a preferred composition of the invention is a gel, particularly a pharmaceutically acceptable gel.
  • a gel typically comprises the nanoparticles of the invention in an amount of from about 2 wt% to about 12 wt%, based on the total weight of the gel.
  • the gel may further comprise water in an amount of from about 80 wt% to about 98 wt %, for example from about 85 wt% to about 96 wt% or from about 88 wt% to about 94 wt%.
  • a gel comprises the nanoparticles of the invention in an amount above 7 wt%
  • the nanoparticles may not adequately disperse.
  • the gel may also comprise a dispersant such as pyrophosphate or sodium polyacrylate.
  • the gel may comprise the nanoparticles of the invention in an amount of up to around 12 wt% of the total weight of the gel.
  • the gel may comprise the nanoparticles of the invention in an amount of from about 2 wt% to about 12 wt%, from about 4 wt% to about 10 wt%, or from about 7 wt% to about 10 wt%.
  • Such a gel may further comprise water in an amount of from about 80 wt% to about 98 wt %, for example from about 85 wt% to about 96 wt% or from about 88 wt% to about 93 wt%.
  • compositions which do not comprise a dispersant typically forms a gel when the nanoparticles are present in an amount of up to about 7 wt% based on the total weight of the gel.
  • a dispersant e.g. a composition which consists of or substantially consists of nanoparticles of the invention and water
  • the gel typically comprises nanoparticles of the invention in an amount of about 2 wt% to about 7 wt% of the total weight of the gel.
  • the gel comprises the nanoparticles of the invention in an amount of from about 2 wt% to about 4 wt% of the total weight of the gel.
  • the gel comprises the nanoparticles of the invention in an amount of from about 4 wt% to about 5 wt%. In another embodiment, the gel comprises the nanoparticles of the invention in an amount of from about 5 wt% to about 6 wt%.
  • the water content of the gel may in this case be from about 93 wt% to about 98 wt%, such as from about 96 wt% to about 98 wt%, based on the total weight of the gel.
  • composition of the invention may alternatively be a paste.
  • the composition comprises the nanoparticles of the invention in an amount of from 7 wt% to 50 wt% of the total weight of the composition, such as from 10 wt% to 40 wt%, or from 20 wt% to 30 wt%.
  • composition of the invention may alternatively be a sol.
  • the composition of the invention comprises the nanoparticles of the invention in an amount of from 0.1 wt% to 2% of the total weight of the composition, such as from 0.5 wt% to 2 wt%, or from 1 wt% to 2 wt%.
  • compositions of the invention are typically biodegradable.
  • biodegradable is understood to mean that the composition has the ability to break down over time in the tissue or body of a human or animal and/or in the environment.
  • the time for complete degradation may be at least 1 week, at least one month, at least 2 months, at least 6 months or at least 12 months.
  • the time for complete degradation may be no more than 12 months or no more than 6 months.
  • the time for complete degradation may be from 1 week to 12 months, 1 week to 6 months, 1 month to 12 months, 1 month to 6 months, 2 months to 12 months or 2 months to 6 months.
  • the degradation time may affect the speed at which drug is released from the gel when in situ in the body. Therefore, the degradation time be desirably varied depending on any therapeutic or diagnostic agent which is present in the composition.
  • the gel When the composition is a gel, the gel preferably demonstrates a 50% change in zeta potential within a pH range of from pH 6.5 to pH 8.
  • a 50% change in zeta potential is defined as a change of measured zeta potential of at least 50% compared with the baseline zeta potential, on titration with acid.
  • Baseline zeta potential is the zeta potential of the gel without pH modification, i.e. before addition of acid.
  • Baseline zeta potential is typically that measured at pH 9 to pH 11, more typically from pH 9.5 to pH 10.5, more typically about pH 10.
  • the change in zeta potential is typically a reduction of the magnitude of the zeta potential value, i.e. the absolute value of the zeta potential moves towards zero. This indicates an increase in stability of the gel.
  • the zeta potential value may be positive or negative. A reduction in the absolute value in either case indicates that the value is closer to zero.
  • the 50% change in zeta potential occurs at a pH range of from pH 7 to pH 7.8, more preferably at a pH range of from pH 7.2 to pH 7.8.
  • the 50% change in zeta potential of the gel occurs in a pH range of from pH 7.3 to pH 7.5.
  • the change in zeta potential is measured via titration of the gel with an acid, such as hydrochloric acid.
  • Zeta potential change can be determined using any appropriate method known in the prior art. For example it can be determined using equipment designed for measuring zeta potential, such as a Stabino ZetaTM kit.
  • the zeta potential is measured using a solution of nanoparticles in water, typically a 1 wt% solution of nanoparticles in ultrapure water.
  • the zeta potential is a measure of the electrokinetic potential of particles in a colloidal system and can be used as an indication of the stability of the gel. Particles having a large negative zeta potential will tend to repel one another.
  • the particles of the invention therefore preferably have a negative zeta potential, wherein the value is ⁇ 50% of the absolute value, at a pH in physiological range.
  • the gels of the invention typically have a pH of around 10 in the absence of rheological or pH modifiers. At this pH, the gels typically have very large negative zeta potential, meaning the particles repel one another and are more flowable, and therefore relatively easy to administer, for example by injection.
  • a change in zeta potential occurs, typically reducing the repelling forces between particles.
  • the stability or stiffness of the gel therefore increases, thus providing a stiffer, more stable gel at the site at which it is delivered. This further gelation may be a result of new attractive interactions as a result of increased ionicity. Further, when in contact with bodily fluids, proteins that diffuse into the gel may bind and form bridges between particles to further stabilise the gel. The increase in stability helps to retain the gel at the site of administration and avoid off-target side effects.
  • composition of the invention may further comprise one or more therapeutic or diagnostic agents.
  • a therapeutic or diagnostic agent may be a small molecule therapeutic or diagnostic agent, including small molecule drugs, prodrugs, or dyes or colourants.
  • a therapeutic or diagnostic agent may alternatively be a biologic, wherein a biologic may be an amino acid, signalling molecule, peptide, protein (wherein the protein may be a recombinant protein or a native protein), antibody, nucleic acid, oligonucleotide (such as an aptamer), or cell.
  • a therapeutic or diagnostic agent may alternatively be a polymeric material. Combinations of one or more therapeutic or diagnostic agent may be used.
  • the nanoclay compositions of the invention may be used for the delivery of a wide variety of different therapeutic or diagnostic agents and the nature of the agent(s) used is not particularly limited.
  • exemplary therapeutic agents include antimicrobial agents (for example antibiotics), growth factors, antibodies, nutrients, enzymes, hormones, steroids, biological tissue substitutes, synthetic tissue substitutes, aptamers and cells.
  • exemplary diagnostic agents include dyes, colourants, radioisotopes (which may be for X-ray detection and or monitoring of degradation), contrast agents (such as contrast agents for CT, such as iodine containing contrast agents and barium sulfate; contrast agents for MRI, such as gadolinium (III), iron oxide, iron platinum and manganese; or contrast agents for ultrasound), or fluorescent molecules.
  • contrast agents such as contrast agents for CT, such as iodine containing contrast agents and barium sulfate
  • contrast agents for MRI such as gadolinium (III), iron oxide, iron platinum and manganese
  • contrast agents for ultrasound or fluorescent molecules
  • the one or more therapeutic or diagnostic agents are agents which are useful in regenerative medicine.
  • the one or more therapeutic or diagnostic agents are selected from growth factors, antibodies, biological tissue substitutes, synthetic bone grafts and cells.
  • Antimicrobial agents may be included in the composition in addition or instead of such therapeutic agents. Any antimicrobial agent may be used. Examples include antibiotics such as vancomycin, gentamycin, tobramycin and chlorhexidine.
  • a biological tissue substitute may be a substitute for a soft tissue such as bone marrow or plasma or a substitute for a hard tissue such as bone.
  • biological tissue substitutes include platelet rich plasma (PRP) and biological bone-graft substitute.
  • a synthetic tissue substitute may be a synthetic bone graft.
  • Synthetic bone grafts include calcium salts such as calcium sulfates or calcium phosphates, alone or in combination.
  • P-tricalcium phosphate P-TCP
  • HA Hydroxyapatite
  • a cell may be a somatic cell, such as a chondrocyte; a progenitor cell; or a stem cell, such as a mesenchymal stem cell.
  • Cells may also be provided as bone marrow aspirate (BMA).
  • a protein is a recombinant protein. Any protein for use in regenerative medicine may be used.
  • a recombinant protein is a growth factor, such as a bone morphogenetic protein (BMP), platelet derived growth factor (PDGF), transforming growth factor (TGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or insulin like growth factor (IGF).
  • BMP bone morphogenetic protein
  • PDGF platelet derived growth factor
  • TGF transforming growth factor
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • IGF insulin like growth factor
  • An aptamer is a short synthetic ssDNA or ssRNA sequence.
  • An aptamer may be a therapeutic, or a diagnostic aptamer, such as an aptamer which interacts with a biomarker.
  • Such an aptamer may further comprise a diagnostic feature, such as a fluorescent marker, or a radioisotope.
  • the composition comprises BMP, or a BMP analogue such as AMP.
  • BMP -2, BMP-3, BMP-4, BMP-6, BMP-7, and BMP-8 or heterodimers of thereof may be mentioned.
  • AMP -2 is an example of a suitable analogue of BMP -2.
  • the composition comprises BMP-2 and/or BMP-7 and analogues thereof, in particular BMP-2 and/or BMP-7.
  • the composition comprises BMP -2 and analogues thereof, in particular BMP-2.
  • the one or more therapeutic or diagnostic agents may alternatively be for use in treating or diagnosing diseases and disorders other than those relevant to regenerative medicine.
  • the composition of the invention may be for use in delivering therapeutic and/or diagnostic agents suitable for treating or diagnosing cancer.
  • diagnostic agents which exhibit chemiluminescence, fluorescence, or contain radioactive isotopes may be used.
  • Suitable therapeutic agents include one or a combination selected from doxorubicin, P-lapachone, or methotrexate.
  • composition typically comprises a diagnostically visible and/or therapeutically effective amount of the one or more diagnostic or therapeutic agents respectively. This amount could be determined by the skilled person in the art based on the nature of the drug and the size and weight of the subject.
  • the BMP may be provided at a dose of from about 0.01 pg to about 50mg, for example from about 0.1 pg to about 20mg, or from about 1 pg to about 15mg total dose.
  • the dose may be dependent on the clinical context, such as the size of the area to be treated or the total volume of the composition.
  • clay nanoparticles for BMP delivery provides dosing control, allowing low doses of BMP (e.g. from 0.1 to 300 pg) to be used to encourage bone formation at a defect, or allowing higher doses (e.g. from 10 pg to about 25 mg, or from 300 pg to about 15mg) to be used with reduced risk of off-target effects.
  • BMP e.g. from 0.1 to 300 pg
  • higher doses e.g. from 10 pg to about 25 mg, or from 300 pg to about 15mg
  • Studies have demonstrated significant adverse effects with higher dose BMP use and its poor local retention such as: heterotopic ossification, osteolysis, and swelling. Therefore, allowing low dosages of BMP or ensuring retention of the protein at the target site, facilitates fracture healing and arthrodesis without precipitation of serious adverse effects.
  • Targeted administration of BMP as facilitated by the high stability and stiffness of gels provided by the present invention, also reduces off-target, adverse side
  • the therapeutic and/or diagnostic agent(s) may be provided in combination with a pharmaceutically acceptable carrier, referred to herein as a second carrier.
  • a pharmaceutically acceptable carrier referred to herein as a second carrier.
  • Any suitable drug carrier may be used and the skilled person would be familiar with the selection of suitable carriers and excipients for the therapeutic and diagnostic agents which can be used in the present invention.
  • a carrier may be selected from carboxymethylcellulose, gelatine and collagen. Further excipient(s) may optionally be incorporated.
  • the composition does not comprise a further diagnostic and/or therapeutic agent.
  • kits comprising the compositions or pharmaceutical compositions according to the invention.
  • a kit of the invention comprises a syringe, e.g. a pre-filled syringe wherein the syringe contains a composition as described herein wherein the composition is in the form of a gel.
  • a kit may comprise separately a syringe and a composition of the invention.
  • the composition may be in a gel form, stored in a container such as a pouch.
  • the composition may be in a form which may be a precursor to a gel form, typically solid form, such as a powder, typically a lyophilised powder, which may be hydrated to form a gel.
  • a kit of the invention comprises a vial, which may be a sprayable vial, e.g. a pre-filled vial containing a composition of the invention as described herein.
  • kits of the invention may comprise one or more therapeutic or diagnostic agents as defined anywhere herein.
  • the therapeutic and/or diagnostic agent(s) may be provided separately from the composition of the invention, or they may be provided within the composition of the invention.
  • the composition is in powder form or in the form of a gel; and/or the therapeutic or diagnostic agent(s) are in a powder form.
  • the composition is in a powder form
  • use of the kit may involve the powder being hydrated to form a gel.
  • composition of the invention and the therapeutic and/or diagnostic agent(s) may be provided in a form suitable for separate administration.
  • the one or more therapeutic and/or diagnostic agent(s) may be provided in a form suitable for addition to the gel prior to administration to a subject.
  • the drug and the composition may be pre-mixed in dried form and use of the kit may involve hydration of the mixture to form a gel suitable for administration.
  • a kit may comprise a biological implant, and the composition of the invention.
  • the biological implant is pre-coated with a gel or a dried film, as described herein.
  • the composition e.g. a gel or a dried film
  • the implant may be provided separately.
  • a biological implant, as referred to herein may be a medical device, such as a pace maker, implantable defibrillator or contraceptive implant; or a prosthetic implant, such as a knee, hip, or shoulder replacement, or medical screw or other fixator.
  • a kit may comprise the gel of the invention in the form of a dehydrated film.
  • the kit may further comprise equipment for hydrating the film.
  • the use of such a kit may involve administration of the film, for example by application onto the skin, and subsequent hydration of the film.
  • kits may comprise the composition in the form of granules, or microparticles.
  • the kit may also comprise instructions for use.
  • composition as described anywhere herein may be for use in the treatment of the human or animal body.
  • the subject is a mammal, in particular a human.
  • it may be non-human.
  • Preferred non-human animals include, but are not limited to, primates, such as marmosets or monkeys, commercially farmed animals, such as horses, cows, sheep or pigs, and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters.
  • the composition is for use in a method of regenerative medicine.
  • the invention also provides the use of a composition of the invention for the manufacture of a medicament for use in a method of regenerative medicine.
  • regenerative medicine comprises tissue repair and regeneration, wherein tissue repair and regeneration may include tissue formation and/or fusion.
  • tissue repair and regeneration may include tissue formation and/or fusion.
  • This can include repair or regeneration of soft tissues, wherein soft tissues include muscle (smooth muscle, skeletal muscle and cardiac muscle), fat, fibrous tissue (connective tissue, cartilage, tendons and ligaments), synovial tissue, blood vessels (arteries, veins and capillaries), lymph vessels, skin, and nerves.
  • Regenerative medicine also comprises repair and regeneration of hard tissues, wherein hard tissues include bone, tooth enamel, dentin and cementum.
  • Tissue repair and regeneration also comprises tissue replacement, such as the filling of a void left by a tumour.
  • the method of regenerative medicine is the repair or regeneration of skin, bone and/or cartilage.
  • a method of regenerative medicine may be a method of wound repair.
  • the one or more drugs which may comprise the composition may be for use in wound repair, and to prevent or treat concurrent conditions often present in those who are in need of wound repair, for example inflammation, infection and pain.
  • regenerative medicine may comprise cell delivery, such as delivery of cells to regenerate tissues, such as the delivery of somatic cells, such as chondrocytes; progenitor cells; or stem cells such as mesenchymal stem cells.
  • somatic cells such as chondrocytes; progenitor cells; or stem cells such as mesenchymal stem cells.
  • composition of the invention may be for use in a method of tissue repair or regeneration, and/or cell delivery.
  • the invention also provides the use of a composition of the invention for the manufacture of a medicament for use in tissue repair or regeneration, and/or cell delivery.
  • the invention also provides a method of tissue repair or regeneration, and/or cell delivery, wherein said method comprises administering to a subject an effective amount of a composition of the invention.
  • composition of the invention may be for use in the treatment or prevention of infection.
  • the invention also provides the use of a composition of the invention for the manufacture of a medicament for use in the treatment or prevention of infection.
  • the invention also provides a method of treating or preventing infection, wherein said method comprises administering to a subject an effective amount of a composition of the invention.
  • the composition of the invention may also be for use in a method of delivery of one or more therapeutic or diagnostic agents to a target site in the human or animal body, wherein said method comprises administering said composition to the human or animal body, wherein the composition comprises one or more therapeutic or diagnostic agents and/or wherein one or more therapeutic or diagnostic agents are administered separately into the composition in vivo at the target site.
  • the therapeutic or diagnostic agent for delivery may be any therapeutic or diagnostic agent as described herein.
  • the therapeutic or diagnostic agent is a cell.
  • the therapeutic or diagnostic agent is an agent useful in regenerative medicine.
  • the therapeutic or diagnostic agent is useful in the treatment of cancer, i.e. a cancer therapeutic such as doxorubicin, P-lapachone, or methotrexate.
  • the target site may typically be a tumour.
  • compositions and pharmaceutical compositions of the invention may be administered in a variety of dosage forms.
  • the compositions and pharmaceutical compositions of the invention may for example be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrastemally, or transdermally.
  • the compositions of the invention are administered by injection or are applied to an exposed target site.
  • the composition may be administered directly to the surface of a subject to be treated, i.e. the surface is coated with a composition of the invention (e.g. by spray-coating a surface to be treated); or the composition is administered directly into a void in the subject’s tissue, i.e. to fill or partially fill the void.
  • the composition of the invention may be delivered via a fixed entry point, for example, via a cannula.
  • composition of the invention When the composition of the invention is in the form of a gel or dried film, it may be administered directly to a wound, e.g. a skin wound, or a bum, by covering the wound with the gel or film. Alternatively, the composition of the invention may be applied to a wound by spraying. A further dressing, such as a gauze and/or a bandage may then be applied. Alternatively, the gel or film may be incorporated in to the dressing, i.e. a bandage may comprise a gel or dried film of the invention.
  • the composition may also be provided as a coating on a medical device and provided to a subject together with the device. Rehydration of the composition may be carried out after the device is inserted, in the case of a composition provided as a dried film. Suitable medical devices include a pace maker, implantable defibrillator, or contraceptive implant; or a prosthetic implant, such as a knee, hip, or shoulder replacement, or medical screw or other fixator.
  • the one or more therapeutic or diagnostic agent may administered as a single composition comprising the nanoparticles described herein and the therapeutic and/or diagnostic agent(s).
  • the one or more therapeutic or diagnostic agents may be administered separately, subsequently or sequentially to administration of the gel.
  • the separate, subsequent or sequential administration of the one or more therapeutic or diagnostic agents may comprise administration to the gel on a target surface or injection into the gel at the target site.
  • the one or more therapeutic or diagnostic agents may be added in a therapeutically effective amount.
  • the one or more therapeutic or diagnostic agents may be added in a single administration or via multiple administrations.
  • the one or more therapeutic or diagnostic agents may be added in situ at a target site, to provide sustained delivery of the one or more therapeutic or diagnostic agents.
  • the one or more therapeutic or diagnostic agents may be added in from 1 to 10 administrations, typically from 1 to 6 administrations, more typically from 1 to 4 administrations. There is no limit to the number of administrations that can be provided, provided the composition is not saturated. Therefore administration may continue until such a point as a subject’s treatment has been completed.
  • composition of the invention does not comprise any further therapeutic and/or diagnostic agents.
  • the composition may be administered via multiple administrations, such as from 1 to 10 administrations, typically from 1 to 6 administrations, more typically from 1 to 4 administrations.
  • the one or more therapeutic or diagnostic agents are added to the composition in multiple administrations, there is no specific time lapse between each administration.
  • the time lapse between each administration may be judged by the skilled person administering the one or more therapeutic or diagnostic agents and will depend on aspects such as the half-life of the therapeutic or diagnostic agent in question and the rate of release from the gel.
  • a therapeutically effective amount of a composition of the invention is administered to a patient.
  • a typical dose may be determined by the skilled person, according to the activity of the specific composition, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
  • compositions as described anywhere herein may be used in cosmetic methods. Suitable methods include a method of hydrating the skin, improving the evenness of skin complexion, preventing or reducing wrinkles or skin embellishments, as a cosmetic topical cream or gel, or as a cosmetic tissue filler, such as a lip filler, or a dermal filler. In some preferred embodiments, the composition is used as a cosmetic tissue filler. Alternatively, it may be used in a cosmetic topical cream.
  • composition as described anywhere herein may be a cosmetic composition, which is a composition suitable for cosmetic use.
  • a cosmetic composition typically comprises one or more additives useful in formulating cosmetic compositions.
  • the cosmetic composition may be a tissue filler, or a topical cream or gel.
  • composition of the invention is a cosmetic composition, or used in a cosmetic method, it does not comprise a further diagnostic or therapeutic agent.
  • a cosmetic composition of the invention may optionally comprise an antiinflammatory agent to reduce swelling.
  • anti-inflammatory agents are not present in a cosmetic composition.
  • a cosmetic composition does not comprise any therapeutic agent.
  • Also provided herein is a process for making the clay nanoparticles and compositions of the invention.
  • the process comprises combining a lithium salt solution, a magnesium salt solution, a solution comprising the cationic component and a silicate solution.
  • salt solutions to be used in the process of the invention may be any suitable aqueous solutions known to the skilled person.
  • the molar ratio of magnesium to lithium which is input into the combined solutions is from 2 to 30, such as from 5 to 25, or from 10 to 25.
  • lithium salt solution lithium sulfate solution, lithium chloride solution, lithium hydroxide solution, lithium nitrate solution and combinations thereof, can be used. Typically lithium sulfate solution is used.
  • the process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving a lithium salt in water to form an aqueous solution.
  • the salt may be a hydrated salt, such as a monohydrate.
  • the process involves a step of dissolving lithium sulfate monohydrate in water.
  • magnesium salt solution magnesium sulfate solution, magnesium chloride solution, magnesium nitrate solution, and combinations thereof, can be used. Typically, magnesium sulfate solution is used.
  • the process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving a magnesium salt in water to form an aqueous solution.
  • the salt may be a hydrated salt, such as a monohydrate or a heptahydrate.
  • the process involves a step of dissolving magnesium sulfate heptahydrate in water.
  • the process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving a silicate in water to form an aqueous solution. In some embodiments the process involves a step of dissolving sodium silicate in water.
  • any appropriate solution may be used. These include, for example, a carbonate solution, a phosphate solution, a sulfate solution, a nitrate solution, or a combination thereof.
  • a carbonate solution is used, such that when the cationic component comprises sodium, a sodium carbonate solution may be used.
  • the process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving the cationic component salt in water to form an aqueous solution. In some embodiments the process of the invention comprises a step of dissolving sodium carbonate to form an aqueous solution. Once the solutions have been combined the process of the invention typically comprises heating the combined solutions together.
  • the weight % of the solids in the slurry compared to the total weight of the slurry is typically from around 1 wt% to 15 wt%, typically around 3 wt% to 12 wt%. Sometimes, the weight % of the solids in the slurry compared to the total weight of the slurry is from around 3 wt% to around 5 wt%. Sometimes, the weight % of the solids in the slurry compared to the total weight of the slurry is from around 8 wt% to around 10 wt%.
  • the combined solutions are typically heated to the boiling point of the slurry (i.e. to about 100 °C). Heating at this temperature, typically under reflux, is typically continued for a period of from around 30 minutes to around 2 hours, typically about an hour.
  • the process of the invention may comprise a further step comprising hydrothermal treatment of the slurry.
  • a hydrothermal treatment as referred to herein, is any treatment comprising heating of a composition (in this instance a slurry) in the presence of water.
  • hydrothermal treatment may comprise autoclaving.
  • hydrothermal treatment may occur at a temperature of from 60 to 500 °C.
  • the hydrothermal treatment may occur at an elevated pressure (i.e. above standard atmospheric pressure (about 100 000 Pa)). Hydrothermal treatment at elevated pressure may be referred to as autoclaving.
  • the process of the invention may comprise an autoclaving step.
  • the hydrothermal treatment step occurs at a temperature of from around 100 °C to around 300 °C, typically around 150 °C to around 250 °C, preferably around 180 °C to around 220 °C.
  • the hydrothermal treatment step occurs at a pressure of from 30 psi to around 1800 psi, typically from around 60 psi to around 600 psi, preferably from around 100 psi to around 400 psi.
  • the hydrothermal treatment step lasts for approximately 1 hour to 24 hours, preferably 2 hours to 12 hours, 4 hours to 8 hours, most preferably around 6 hours.
  • the process of the invention may further comprise a drying step, wherein the slurry is dried to form a solid. Drying of the slurry may be carried out using any appropriate method known to the skilled person, for example rotoevaporation, oven drying or vacuum filtration. Preferably vacuum filtration is used (e.g. using a Buchner funnel).
  • the process may also comprise a step of washing and re-drying the dried product.
  • Any appropriate solvent may be used to wash the product. Typically deionised water is used. Vacuum filtration (e.g. using a Buchner funnel) may be used in this step.
  • the washing step is especially useful to remove soluble salt impurities, such sulfates.
  • BaCh may be added to the filtrate. If a precipitate is formed, typically further washing is required.
  • more than one drying method is used, such as vacuum filtration in combination with oven drying.
  • Oven drying typically occurs at a temperature not too far about standard temperature.
  • oven drying occurs at a temperature of from around 40 °C to around 80 °C, more typically around 55 °C to around 65 °C.
  • a drying step may last for around 1 hour to 24 hours, typically around 6 hours to 14 hours.
  • the process of the invention may further comprise a step of grinding the dried product to form a powder. Grinding may occur using any appropriate method, such as use of a pestle and mortar.
  • the process of the invention may further comprise a step of adding water to the composition to form a gel.
  • the composition When the water is added to the composition, the composition may be subjected to rigorous stirring, i.e. stirring with a magnetic stirrer at a rate of 300 rpm to 1000 rpm, such as 500 rpm to 800 rpm.
  • the stirring can occur at any temperature, such as from 0 °C to 100 °C, or 10 °C to 40 °C.
  • the stirring occurs as standard atmospheric temperature and pressure (SATP), such as around 25 °C, and around 15 psi.
  • SATP standard atmospheric temperature and pressure
  • the gel may then be sterilised, optionally via autoclaving.
  • Autoclaving typically occurs at from around 100 °C to around 140 °C, such as around 120 °C.
  • Autoclaving typically occurs for between 10 minutes and 1 hour, such as 30 minutes.
  • Autoclaving typically occurs at from 13 psi to 20 psi, such as from 15 psi to 20 psi.
  • the process of the invention may further comprise a step of adding one or more therapeutic or diagnostic agents to the composition.
  • the one or more therapeutic or diagnostic agents may be added to the composition at any step during the process of the invention.
  • the one or more therapeutic or diagnostic agents may also be added to the composition prior to formation of the gel.
  • the one or more therapeutic or diagnostic agents may be added after the composition when it is in the form of a gel, but prior to administration to the subject.
  • the one or more therapeutic or diagnostic agents may alternatively or additionally be administration to the subject separately from the gel, e.g.
  • the one or more therapeutic or diagnostic agents are administered by administration of the one or more therapeutic or diagnostic agents into the gel in vivo at the target site, or by adding the one or more therapeutic or diagnostic agents as a coating to the gel, in particular when the gel itself is coated on the surface of a medical device, such as an implant, a stent, or a balloon.
  • the one or more therapeutic or diagnostic agents are added after the composition when it is in the form of a gel, but prior to administration to the subject.
  • Li2SO4.H2O was dissolved into 170 mL of de-ionised water at room temperature using a stirrer. Once the Li2SO4.H2O was dissolved, 56.10 g of MgSO4. H2O was added. The mixed salt solution was then transferred to a round bottom flask (RBF) in an assembled reactor which was heated to 60 ⁇ 10 °C.
  • the Na2CO 3 solution was added using a stoppered self-equalising dropping funnel at roughly 1 drop a second.
  • a reflux condenser was then fitted to the reactor and the temperature of the reactor increased to 100°C.
  • the mixed salt solution was then allowed to boil under reflux at atmospheric pressure for 1 hour.
  • reaction mixture was then reduced to 60 °C, keeping all other conditions unchanged. A resultant slurry has been formed.
  • An oven was pre-heated to 200 °C, with the fan set to maximum and the damper to 40%.
  • the slurry is transferred into autoclave vessels (120 mL per vessel).
  • the autoclave vessels were then placed in the oven for hydrothermal treatment (HT) at 200 °C for 6 hrs.
  • HT hydrothermal treatment
  • the oven was then switched off and the vessels allowed to cool. Once cooled the slurry was washed from the autoclave vessels.
  • the contents of each autoclave vessel was then transferred individually into a Buchner funnel, and the filter cake washed with de-ionised water. 400 mL of de-ionised water were used per autoclave vessel.
  • the Buchner flask was regularly emptied and the filtrate tested with BaCh solution.
  • the filter cake was then transferred into a vessel suitable for the drying procedure.
  • the filter cake was then left to dry for at least 8 to 12 hours, but could be left for up to 72 hours. Drying occurred in an oven set at 60 °C at 100% fan, and 40% damper.
  • the dried filter cake was then ground for about 10 minutes into a powder using a mortar and pestle to achieve fine particles.
  • Example 2 To exemplify further variation in elemental composition of the nanoclay of the invention, the method of Example 1 was repeated with variation in the synthesis protocol as set out in Table 2 below. For each modified synthesis, ICP-OES analysis data for of the resulting nanoclay is provided and elemental ratios for Mg/Si and Mg/Li in contrast to LaponiteTM are presented in Figure 2. TABLE 2:
  • Gels of the invention are prepared as a weight percentage (wt.%) of nanocl ay/ water where 1% is equivalent to 1g / 100 ml or lOmg / ml. Gels were prepared using the nanoclays of the invention at concentrations in the range 2 to 7 wt%. Reference gels were also prepared using corresponding amounts of LaponiteTM as the nanoclay, for comparison purposes. The mixing procedure is designed to ensure complete wetting out and dispersion of the clay particles.
  • Deionised water (18.2 MQ, pH 7) is added into a glass bottle with a magnetic stirrer bar and place on a stirrer to generate a stable vortex that extends close to the base of the bottle but without exposing the stirrer to air.
  • Powdered nanoclay is added gradually into the vortex over the course of 5-10 seconds.
  • the total weight of the bottle containing the gel is measured before undergoing autoclave sterilisation for ⁇ 30min at 121 °C and 15 psi using a bench top autoclave suitable for liquid sterilization. Once it has cooled, the bottle is weighed again and any lost water replaced followed by mixing on a vortex. To keep the gel of the invention sterile, handling is under sterile conditions and sterile deionized water used to replace lost water.
  • the rheological properties of gels of the invention were characterised both in their native state and following addition to a bath of simulated blood serum (40 mg/ml bovine serum albumin in phosphate buffered saline, pH 7.4) to assess their response in physiological solutions (such as, for example, following injection into the body).
  • simulated blood serum 40 mg/ml bovine serum albumin in phosphate buffered saline, pH 7.4
  • Figure 4 shows that, in its native state, a gel of the invention can possess equivalent gel strength (yield stress (Pa)), stiffness (storage modulus (G’)) and viscosity (storage modulus (G”)) to a LaponiteTM gel having a corresponding nanoclay concentration (Fig 4a, top), but in simulated blood serum, the increase in gel strength, stiffness and viscosity was significantly enhanced in gels of the invention (Fig 4b, bottom).
  • Gel strength yield stress
  • G stiffness
  • G viscosity
  • Figure 5 reports storage and loss modulus of a range of high and low Li / Mg gels of the invention and their analogues vs LaponiteTM suspensions in simulated blood serum and shows a significant positive correlation with Mg/Si (a) and a significant negative correlation with Li/Si (b) molar ratios across the various nanoclays.
  • Example 5 Protocol for measuring Zeta Potential Half Maximum (PHM) and gelation response to pH
  • Zeta potential is the electrical potential at the slipping plane. This plane is the interface which separates mobile fluid from fluid that remains attached to the surface. Zeta potential describes the electrokinetic potential in colloidal dispersions and is a function of the specific surface chemistry of a dispersion which are affected by changes in pH, salt, and surfactant concentration. At its minimum zeta potential (i.e. its absolute lowest value) a colloid is a its most stable as a dispersion due to the dominance of repulsive over attractive interactions between particles. As the zeta potential approaches the isoelectric point attractive interactions increase resulting in aggregation of particles.
  • FIG 6 shows the sharp decrease in negative zeta potential (ZP) occurring between pH8 and pH5 over the course of titration with HC1. This was a characteristic feature of all nanoclays tested and coincided with an increase in particle (aggregate) size indicating a transition from repulsive to attractive particle interactions. At high nanoclay concentrations (> 2 wt%) an increase in attractive interactions yields a gel.
  • the pH at which this sharp decrease in potential occurred corresponded with the zeta potential half maximum (PHM) defined as the point at which negative ZP is 50% of its recorded mV prior to addition of titrant.
  • PHM zeta potential half maximum
  • Figure 7 shows that the pH at which the PHM occurs (PHPHM) for gels of the invention averages at 7.33 - close to physiological pH.
  • FIG. 8 shows, as with the improved rheological response to serum, the physiologically optimised PHPHM of gels of the invention correlates with its higher Mg and lower Li content compared with LaponiteTM and other high Li analogues of the gels of the invention.

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Abstract

The invention relates to a composition comprising a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I): [(Si8MgbLic)O20(OH)4] (I) wherein 5.5 < b ≤ 6, and wherein c > 0 and b/c > 12.

Description

CLAY NANOPARTICLES FOR MEDICAL USE
FIELD OF THE INVENTION
The invention relates to compositions comprising clay nanoparticles, methods for making such compositions and their use in medical and cosmetic methods. The nanoclays may be used alone or in combination with therapeutic or diagnostic agents. For example, the nanoclays may be used in the delivery of therapeutic or diagnostic agents or in regenerative medicine.
BACKGROUND
Clay nanoparticles are nanoparticles of layered mineral silicates that have numerous applications including in cosmetics and paints. Clay nanoparticles have also been used in the pharmaceutical industry as excipients and active agents. Depending on chemical composition, crystallographic structures and nanoparticle morphology, clay nanoparticles are organised into several classes such as montmorillonite, bentonite, smectite, kalinite, hectorite, and halloysite.
Certain clay nanoparticles self-assemble into a gel in an aqueous environment, for example in the body, and the resulting nanoclay gels have been found to be particularly well suited to providing a regenerative microenvironment, or a platform for drug delivery. An example of a nanoclay that has been used in medical and cosmetic formulations is Laponite™ (a synthetic smectite clay manufactured by BYK). Laponite™ is a discshaped synthetic hectorite type clay which has been cited as a potential nanomedicine for use in drug delivery, bio-imaging, tissue engineering and regenerative medicine.
Important features of some nanoclay gels when used as a nanomedicine include ease of administration and high stability in vivo, post administration. A high stability is desired such that, once the gel is situated at the target site, it provides a stable matrix structure into which new cells can grow, or from which drugs can be delivered. Poor stability at the target site can lead to the nanoclay gel becoming dispersed throughout the subject, meaning it does not serve its intended purpose in the correct location, and can cause potentially harmful effects elsewhere in the body.
There is a need therefore, for clay nanoparticle gels which can be effectively localised at a target site and have high stability under physiological conditions (i.e. pH 7.35 to 7.45, and 36.1 °C to 37.5 °C) and which can be safely and conveniently administered and retained to a subject. Current known clay nanoparticles do not meet these requirements.
Some attempts have been made to address the issues relating to gel stability by creating organo-nanoclay composites. However, these employ cross-linkable polymer chemistry which is complex, hard to regulate, and extremely time sensitive. For clinical viability, more reliable formulations, which can easily be manufactured are desirable.
The present inventors have surprisingly found that clear gels which have a high stability in vivo can be produced from clay nanoparticles having an increased ratio of magnesium ions to lithium ions within the clay nanoparticles, compared to existing nanoclays. Nanoclay gels produced from such particles may have good rheological properties, and in particular exhibit a change in rheological properties in physiological conditions, thus facilitating administration of the gels by injection, whilst providing a clear and stable gel at physiological pH. In addition, the formulations comprising the clay nanoparticles of the invention can self-assemble, and can easily be manufactured.
SUMMARY OF THE INVENTION
The present inventors have developed a composition comprising a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I): [(Si8MgbLic)02o(OH)4] (I) wherein 5.5 < b < 6, and wherein c > 0 and b/c > 12.
Surprisingly, it has been found that the gel compositions described herein have high stability at physiological pH. Such gels can be easily administered, typically by a syringe, without causing significant discomfort or distress to a subject. Further, the gels stabilise in physiological conditions, ensuring effective localisation at the target site and reducing undesired effects caused by delocalisation of the gel.
Also provided by the present invention is a process for making a composition of the invention wherein the process comprises combining a lithium salt solution, a magnesium salt solution, a solution comprising the cationic component, and a silicate solution, and heating the solutions together until a slurry is formed. Further provided is a composition of the invention for use in the treatment of the human or animal body by therapy or diagnosis, in particular by therapy. In particular, the composition may be for use in a method of tissue repair and/or regeneration, and/or a method of cell delivery. The composition may also be for use in the treatment or prevention of infection.
The composition of the invention may be used in cosmetic methods. In some embodiments, the composition is a cosmetic composition, in particular a tissue filler or a topical gel or cream.
The invention also provides a composition of the invention for use in a method of delivery of one or more therapeutic or diagnostic agents to a target site in the human or animal body, wherein said method comprises administering said composition to the human or animal body.
The invention in particular the features set out in the following numbered paragraphs:
1. A composition comprising a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I):
[(Si8MgbLic)02o(OH)4] (I) wherein 5.5 < b < 6, and wherein c > 0 and b/c > 12.
2. A composition according to paragraph 1 wherein 12 < b/c < 100.
3. A composition according to paragraph 1 or paragraph 2 wherein 12 < b/c < 18, preferably wherein 13 < b/c < 18.
4. A composition according to any one of paragraphs 1 to 3, wherein 0.2 < c < 0.5.
5. A composition according to any one of paragraphs 1 to 4 wherein the cationic component comprises Na+ 6. A composition according to any one of paragraphs 1 to 5, wherein the figures b and c are determined by elemental analysis using inductively coupled plasma-optical emission spectrometry (ICP-OES).
7. A composition according to any one of paragraphs 1 to 6 wherein the average size of the longest dimension of the clay nanoparticles is from about 15 nm to about 50 nm, and wherein the average size of the shortest dimension of the clay nanoparticles is from about 1 nm to about 3 nm.
8. A composition according to any one of paragraphs 1 to 7 which further comprises one or more therapeutic or diagnostic agents.
9. A composition according to paragraph 8 wherein the one or more therapeutic or diagnostic agents are therapeutic or diagnostic agents suitable for use in regenerative medicine.
10. A composition according to paragraph 8 or paragraph 9 wherein the one or more therapeutic or diagnostic agents are selected from small molecules, growth factors, antibodies, biological tissue substitutes, synthetic bone grafts, antimicrobial agents, antibiotics and cells.
11. A composition according to any one of paragraphs 1 to 10 wherein the composition comprises BMP -2.
12. A composition according to any one of paragraphs 1 to 11 wherein the composition is in a solid form, preferably in a powder form.
13. A composition according to any one of paragraphs 1 to 11 wherein the composition is in the form of a gel or a film. 14. A composition according to paragraph 13 wherein the gel demonstrates a 50% change in zeta potential within a pH range of from pH 6.5 to pH 8, when zeta potential is measured using acid titration.
15. A pharmaceutical composition comprising a composition according to any one of paragraphs 1 to 14, a pharmaceutically acceptable carrier or diluent, and optionally one or more pharmaceutically acceptable excipients.
16. A kit comprising a syringe or a vial (e.g. a sprayable vial), wherein the syringe or vial contains a composition according to paragraph 13 or paragraph 14, or a composition according to paragraph 15 wherein the composition is in the form of a gel.
17. A kit comprising separately a composition according to any one of paragraphs 1 to 7; and one or more therapeutic or diagnostic agents as defined in any one of paragraphs 8 to 11, preferably wherein (a) the composition is in powder form or in the form of a gel; and/or (b) the therapeutic or diagnostic agent is in a powder form.
18. A process for making a composition as described in any one of paragraphs 1 to 15, wherein the process comprises combining a lithium salt solution, a magnesium salt solution, a solution comprising the cationic component and a silicate solution, and heating the solutions together until a slurry is formed.
19. A process according to paragraph 18 wherein the process further comprises a hydrothermal treatment step.
20. A process according to paragraph 18 or paragraph 19 wherein the process further comprises a step of drying the slurry to form a solid, optionally wherein the process further comprises a step of grinding the solid to form a powder.
21. A process according to paragraph 20, wherein the process further comprises adding water to the composition to form a gel. 22. A process according to any one of paragraphs 18 to 21 wherein the process further comprises a step of adding one or more therapeutic or diagnostic agents to the composition, wherein the one or more therapeutic or diagnostic agents are as defined in any one of paragraphs 8 to 11.
23. A composition according to any one of paragraphs 1 to 15 for use in the treatment of the human or animal body.
24. A composition according to any one of paragraphs 1 to 15, or 23 for use in a method of regenerative medicine.
25. A composition for use according to paragraph 24, wherein the composition is for use in a method of tissue repair or regeneration, and/or cell delivery.
26. A composition according to any one of paragraph 1 to 15, or 23 for use in the treatment or prevention of infection.
27. A composition according to any one of paragraphs 1 to 15, or 23 for use in a method of delivery of one or more therapeutic or diagnostic agents to a target site in the human or animal body, wherein said method comprises administering said composition to the human or animal body, wherein the composition comprises one or more therapeutic or diagnostic agents and/or wherein one or more therapeutic or diagnostic agents are administered separately into the composition in vivo at the target site.
28. A cosmetic composition comprising a composition according to any one of paragraphs 1 to 14 and optionally one or more additives, preferably wherein the cosmetic composition is a tissue filler or a topical gel or cream.
BRIEF DESCRIPTION OF THE FIGURES
References in the Figures to Renovite, Renovite 1.0 or Renovite analogues refer to compositions of the invention, unless otherwise specified. Figure 1 shows the comparison of elemental ratios between Laponite™ XLG and the compositions of the invention set out in Table 1 (Renovite). Empirically determined Mg, Li and Na are presented relative to empirically determined Si scaled according to (Na+a[(SisMgbLid)02o(OH)4]'a. Bars represent mean and SD. P values calculated using an unpaired T-test.
Figure 2 shows comparison of elemental ratios between Laponite™ XLG and a range of compositions of the invention as set out in Table 2 (Renovite analogues). Mg content calculated from empirical Si, Li and Na values on basis of the formula: (Na+a[(SisMgbLid)02o(OH)4]'a. Bars represent mean.
Figure 3 shows enhanced gelation of Renovite (AS-19, Table 1) in response to blood serum. Rheometric isotherm of changes in gel stiffness (storage modulus) in Renovite vs Laponite™ (XLG) suspensions following exogenous exposure to simulated blood serum (40 mg/ml bovine serum albumin in phosphate buffered saline, pH 7.4). With exposure to simulated blood serum, the observed increase in gel stiffness was enhanced in Renovite. Measurements taken at 25°C.
Figure 4 shows enhanced gelation of compositions of the invention (Renovite: mean of all clay batches listed in Table 1) in response to blood serum. Rheometric analysis of Renovite vs Laponite™ (XLG) suspensions in their native state (a) and after 1-hour exogenous exposure to simulated blood serum (b; 40 mg/ml bovine serum albumin in phosphate buffered saline, pH 7.4). Renovite displayed equivalent gel strength (yield stress (Pa)), stiffness (storage modulus (G’)) and viscosity (storage modulus (G”)) to XLG in its native state. With exposure to simulated blood serum, the observed increase in gel strength, stiffness and viscosity was significantly enhanced in Renovite. Yield stress was calculated as the point of inflection (>5%) from the linear dependence of shear stress on shear strain. Histograms plot mean values. Error bars = SD, N= 6, **, and **** indicate P < 0.01 and E < 0.0001, respectively (unpaired t-test). Figure 5 shows enhanced gelation of compositions of the invention (AS-13, 14, 15, 18, 19, 21, 22, 33) in response to blood serum correlates with Mg and Li content. Storage and loss modulus of high and low Mg / Li compositions of the invention and reference samples as set out in Table 2 vs Laponite™ (XLG) suspensions after 1-hour exogenous exposure to simulated blood serum shows a significant positive correlation with Mg/Si (a) a significant negative correlation with Li/Si (b) and a slight positive trend association with Mg/Li molar ratios. Mg content calculated from empirical Si, Li and Na values on basis of the formula: (Na+a[(SisMgbLid)02o(OH)4]'a. P values report Pearson’s correlation coefficients where a significant correlation is P<0.05. ns = not significant.
Figure 6 shows Zeta Potential Half Maximum (PHM) and its significance for nanoclay gelation. A sharp decrease in negative zeta potential (ZP) of nanoclays (here, AS-14) is observed between pH8 and pH5 over the course of titration with HCL. This sharp decrease in negative ZP coincides with an increase in particle (aggregate) size. This indicates a phase-transition from repulsive to attractive particle interactions which under diffusion and at high nanoclay concentrations (> 2 wt%) yields a gel. pH/>//\/ represents the pH at which this sharp decrease in negative ZP occurs by measuring the pH at which negative ZP is 50% of its recorded mV prior to addition of titrant. Titrations and dynamic ZP measurements were performed on the Stabino™ II device programmed to add 30 pl of IM HCL every 20 seconds.
Figure 7 shows Zeta Potential Half Maximum (PHM) for a composition of the invention (AS-14, labelled Renovite 1.0) versus Laponite™. The PHM for Renovite occurs near physiological pH7.4 (shaded grey) compared with pH 5.9 in Laponite™. Titrations and dynamic ZP measurements were performed on the Stabino™ II programmed to add 30 pl of IM HCL every 20 seconds to a suspension of 1 wt% nanoclay.
Figure 8 shows Zeta Potential Half Maximum (PHM) correlates with Mg and Li content. pH at PHM (PHPHM) for 1 wt% high and low Mg / Li compositions of the invention (AS- 13, 14, 15, 18, 19, 21, 22, labelled Renovite 1.0), and reference sample AS-33 vs Laponite™ (XLG) suspensions. A significant positive correlation with Mg/Si and Mg/Li and a significant negative correlation with Li/Si was observed. PHPHM for Renovite 1.0 occurred at pH values approximating physiological pH 7.4 (shaded grey). Mg content calculated from empirical Si, Li and Na values on basis of the formula: (Na+a[(SisMgbLid)02o(OH)4]'a. P values report Pearson’s correlation coefficients where a significant correlation is P<0.05.
DETAILED DESCRIPTION OF THE INVENTION
Clay nanoparticles for medical use
A clay nanoparticle as described herein is an inorganic nanoparticle. A clay nanoparticle may comprise or consist of a silicate. The silicate may comprise layered silicate.
The clay nanoparticles may have an average size of from about 15 nm to about 50 nm in the longest dimension. The clay nanoparticles may have an average size of from about 20 nm to about 40 nm in the longest dimension. For example, the clay nanoparticles may have an average size of from about 15 nm to about 40 nm, or from about 20 nm to about 50 nm, or from about 24 nm to about 36 nm in the longest dimension.
The clay nanoparticles may have an average thickness (shortest dimension) of from about 1 nm to about 3 nm. For example, the clay nanoparticles may have an average thickness of from about 1 nm to about 2.5 nm, or from about 1.5 nm to about 2.5 nm, or from about 1.3 nm to about 2.2 nm.
The dimensions of the clay nanoparticles may be determined by any appropriate method known to the skilled person. Typically the dimensions are determined using smallangle X-ray scattering. The dimensions of the clay nanoparticles may be determined when dispersed in an aqueous environment.
The clay nanoparticles may have an aspect ratio of at least 1 :5. For example, the clay nanoparticles may have an aspect ratio of at least 1 : 10, or at least 1 :20, or at least 1:25. The clay nanoparticles may have an aspect ratio of less than 1 : 100. For example, the clay nanoparticles may have an aspect ratio of less than 1 :50, or less than 1 :30. The clay nanoparticles may have an aspect ratio of from 1 : 10 to 1 :50. For example, the clay nanoparticles may have an aspect ratio of from 1 :5 to 1 :30 or from 1 : 10 to 1:30.
Each clay nanoparticle of the invention comprises an anionic component and a cationic component. The anionic component has the formula (I): [(Si8MgbLic)02o(OH)4] (I).
The anionic component of the clay nanoparticle carries a negative charge. Typically this negative charge is a-, wherein a is equal to the positive charge carried by the cationic component, a is greater than 0, and is not necessarily an integer value. b, in the above formula (I) represents the number of Mg atoms present per unit of the anionic component, b is not necessarily an integer value, b is typically < 6.0. More typically, b < 5.9. b is typically > 5.6, preferably 5.5 < b > 6.0, for example 5.6 < b > 6.0 or 5.6 < b > 5.9. c in the above formula (I) represents the number of Li atoms present per unit of the anionic component, c is greater than 0, and is not necessarily an integer value, c is typically < 2, more typically < 1. Preferably c is < 0.5, more preferably c < 0.45, for example c may be < 0.4. c is typically > 0.2, more typically > 0.3. Therefore, typically, 0 < c < 2, 0 < c < l, 0 < c < 0.5, 0 < c < 0.4, 0.2 < c < 2, 0.2 < c < 1, 0.2 < c < 0.5, 0.2 < c < 0.45, 0.3 < c < 0.5, or 0.3 < c < 0.45. In some preferred embodiments 0.2 < c < 0.5, in particularly preferred embodiments 0.3 < c < 0.5.
The ratio b/c is greater than 12. Typically the ratio is less than 100, such that typically 12 < b/c < 100. In some embodiments, b/c is > 13. In some embodiments, b/c > 14. b/c is typically < 18. In some preferred embodiments b/c < 16. Therefore preferably, 12 < b/c < 18, preferably from 12 < b/c < 18. In some embodiments, 13 < b/c < 18, 13 < b/c < 16, 14 < b/c < 18 or 14 < b/c < 16.
The figures b and c are preferably determined using elemental analysis using inductively coupled plasma-optical emission spectrometry (ICP-OES). Example 2 described herein provides further details of a suitable method for determining these values using elemental analysis.
The cationic component of each clay nanoparticle typically comprises one or more metal cations. In some embodiments the cationic component comprises one or more metal cations selected from alkali metal cations, alkaline earth metal cations, transition metal cations and combinations thereof. Such metal cations may include one or more cations of lithium, sodium, calcium, copper, potassium or magnesium. Sodium is preferred.
Typically the metal cation may be of the formula Mn+, wherein M is a metal and n is an integer, typically an integer from 1 to 6, more typically from 1 to 4. Preferably n is i or 2, more preferably 1. Mn+ is preferably Na+. Therefore, typically the clay nanoparticle comprises an anionic component and a cationic component such that the clay nanoparticle has the formula: [(Mn+)x(M2P+)y][(Si8MgbLic)02o(OH)4]a- wherein b, c and a are as defined anywhere herein, and wherein Mn+ and M2p+ are different metal cations, and n and p are the same or different and are integers of from 1 to 6, typically 1 or 2. Typically the charge is balanced such that nx + py = a. x and y need not be integers, and may be the same or different. The sum of x and y is greater than 0. Typically the sum of x and y (i.e. x + y) is < 2, more typically < 1. In some preferred embodiments x + y is < 0.7 more preferably x + y is < 0.5. Typically x + y is > 0.3, more typically > 0.4. Therefore, in some embodiments, 0 < (x + y) < 2, such as 0 < (x + y) < 1, 0 < (x + y) < 0.7, 0 < (x + y) < 0.5, 0.2 < (x + y) < 0.7, 0.3 < (x + y) < 0.7, 0.4 < (x + y) < 0.7, 0.3 < (x + y) < 0.5, or 0.4 < (x + y) < 0.5. In some preferred embodiments 0.2 < (x + y) < 0.7, more preferably wherein 0.3 < (x + y) < 0.5.
In some embodiments the cationic component of the clay comprises only one type of metal cation, such that the clay nanoparticle has the formula: (Mn+)z[(Si8MgbLic)02o(OH)4]a- wherein n is an integer from 1 to 4, typically 1 or 2, more typically 1, and z is equal to a/n. In some preferred embodiments Mn+is Na+, such that the clay nanoparticles has the formula: (Na+)a[(Si8MgbLic)02o(OH)4]a- wherein a, b, and c are as defined anywhere herein.
The nanoparticles of the invention may contain impurities and/or by-products. The nanoparticles are preferably 90% pure, more preferably 95% pure, more preferably 98% or 99% pure. Most preferably, the nanoparticles are substantially pure particles of formula [(Si8MgbLic)02o(OH)4] as defined herein.
Compositions of the invention
The composition of the invention comprises a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I): [(Si8MgbLic)02o(OH)4] (I) wherein 5.5 < b < 6, and wherein c > 0 and b/c > 12. Herein a plurality of clay nanoparticles refers to two or more clay nanoparticles.
A composition of the invention may contain up to 100 wt% of the clay nanoparticles of the invention. More typically, it contains up to 85 wt% of the clay nanoparticles of the invention, or up to 50 %wt of the clay nanoparticles of the invention. Typically, the composition contains at least 0.1 wt% clay nanoparticles, e.g. from 0.1 to 100 wt%, 0.1 to 85 wt% or 0.1 to 50 wt%.
The clay nanoparticles described herein may comprise coordinated water. Reference herein to clay nanoparticles of the invention is therefore intended to encompass clay nanoparticles having coordinated water. Further, reference herein to dried compositions is intended to encompass compositions comprising clay nanoparticles which comprise coordinated water. Therefore, a dried composition may contain up to 10 wt% water. More typically it contains up to 7 wt% water. Typically the composition contains at least 3 wt% water, or at least 5 wt% water. Typically, therefore, the composition may comprise from 3 wt% to 10 wt% water, such as from 5 wt% to 7 wt% water. In such embodiments, the remainder of the composition may contain up to 100 wt% clay nanoparticles of the invention, such that the composition contains, for example, 93 wt% to 95 wt% clay nanoparticles of the invention.
The composition may consist of, or consist substantially of, the clay nanoparticles of the invention. In such embodiments, the composition is typically in dried form, e.g. a powder, a film, granules, a coating, porous sponge, or fibres. The composition may for example comprise at least 90% clay nanoparticles, at least 95% clay nanoparticles, at least 98% or 99% clay nanoparticles or about 99.5% clay nanoparticles. Alternatively, additional materials may be present in the composition, for example including carriers or diluents (e.g. water or saline), excipients and/or therapeutic or diagnostic agents. In embodiments wherein the composition is in a powder form, for example, the composition may further comprise additional materials such as ions, proteins, and polymers.
In some preferable embodiments the composition of the invention is in dried form, e.g. in the form of a powder. The composition may be lyophilised, e.g. a lyophilised powder. An alternative dried form is a film. In some embodiments, e.g. where water or saline is present in the composition, the composition is a liquid-solid fluid mixture such as a slurry, or a paste. In some embodiments, e.g. where water or saline is present in the composition, the composition is in the form of a gel. A gel is a preferred form of the composition of the invention and the compositions are typically administered in this form.
A gel of the invention may be formed, for example, by casting the clay nanoparticles in water. A gel of the invention may be dried to form a film, or lyophilised. Where the composition is provided as a film or in lyophilised form, it may be administered by application to a surface. Alternatively, it may be reconstituted with a diluent, such as water or saline, before use.
The composition may be a pharmaceutical composition, which is a composition suitable for pharmaceutical use. A pharmaceutical composition typically comprises one or more pharmaceutically acceptable excipients. Preferred pharmaceutical compositions are sterile and pyrogen free.
Suitable carriers and/or diluents for use in the invention include pharmaceutically acceptable carriers such as water and aqueous solutions, in particular saline. Isotonic solutions, in particular isotonic saline, are preferred. The carrier or diluent is typically sterile. Thus, sterile, isotonic saline is a preferred carrier. Such carriers can be used in the case of compositions which are in the form of a slurry or a gel.
Excipients may also be included in the composition, as needed. The skilled person would be familiar with suitable excipients that may be useful to include. In one embodiment, where the composition further comprises a therapeutic and/or diagnostic agent, for example, a second carrier material for the therapeutic or diagnostic agent may be used. The second carrier may be a polymeric material. In some preferred embodiments the second carrier is selected from carboxymethylcellulose, gelatine and collagen.
For solid form drugs: diluents e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; rheology modifiers, such as carboxymethylcellulose, gelatine and collagen; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents, e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; wetting agents, such as lecithin, polysorbates, laurylsulphates; dispersants, such as sodium polyacrylate and pyrophosphates; and, in general, non toxic and pharmacologically inactive substances used in pharmaceutical formulations. For liquid forms such as syrups: rheology modifiers, such as carboxymethylcellulose, gelatine and collagen; carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
For suspensions and emulsions: carriers, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
For injectable forms, as well as sterile water which is mentioned already above: rheology modifiers, such as carboxymethylcellulose, gelatine and collagen; olive oil; ethyl oleate; glycols, e.g. propylene glycol; and if desired, a suitable amount of lidocaine hydrochloride.
Preferred compositions of the invention comprise the nanoparticles of the invention and water, wherein the nanoparticles are present in an amount of from about 0.1 wt% to about 50 wt% based on the total weight of the composition. Preferred compositions comprise from about 0.5 wt% to about 40 wt% nanoparticles of the invention, based on the total weight of the composition. For example, the composition may comprise from about 1 wt% or from about 2 wt% and up to about 40 wt% nanoparticles of the invention. The composition typically also comprises water or saline. The composition may optionally comprise one or more further additives. The composition may consist of, or consist essentially of, the nanoparticles of the invention and water or saline.
A preferred composition of the invention is a gel, particularly a pharmaceutically acceptable gel. A gel typically comprises the nanoparticles of the invention in an amount of from about 2 wt% to about 12 wt%, based on the total weight of the gel. The gel may further comprise water in an amount of from about 80 wt% to about 98 wt %, for example from about 85 wt% to about 96 wt% or from about 88 wt% to about 94 wt%.
Where a gel comprises the nanoparticles of the invention in an amount above 7 wt%, the nanoparticles may not adequately disperse. In this case, the gel may also comprise a dispersant such as pyrophosphate or sodium polyacrylate. In embodiments where a dispersant is present in the gel, the gel may comprise the nanoparticles of the invention in an amount of up to around 12 wt% of the total weight of the gel. For example, where a dispersant is present, the gel may comprise the nanoparticles of the invention in an amount of from about 2 wt% to about 12 wt%, from about 4 wt% to about 10 wt%, or from about 7 wt% to about 10 wt%. Such a gel may further comprise water in an amount of from about 80 wt% to about 98 wt %, for example from about 85 wt% to about 96 wt% or from about 88 wt% to about 93 wt%.
Compositions which do not comprise a dispersant, e.g. a composition which consists of or substantially consists of nanoparticles of the invention and water, typically forms a gel when the nanoparticles are present in an amount of up to about 7 wt% based on the total weight of the gel. Accordingly, in embodiments where a dispersant is not present, for example where the composition consists of, or substantially consists of the nanoparticles of the invention and water, the gel typically comprises nanoparticles of the invention in an amount of about 2 wt% to about 7 wt% of the total weight of the gel. Often the gel comprises the nanoparticles of the invention in an amount of from about 2 wt% to about 4 wt% of the total weight of the gel. In one embodiment, the gel comprises the nanoparticles of the invention in an amount of from about 4 wt% to about 5 wt%. In another embodiment, the gel comprises the nanoparticles of the invention in an amount of from about 5 wt% to about 6 wt%. The water content of the gel may in this case be from about 93 wt% to about 98 wt%, such as from about 96 wt% to about 98 wt%, based on the total weight of the gel.
The composition of the invention may alternatively be a paste. Typically when the composition is a paste, the composition comprises the nanoparticles of the invention in an amount of from 7 wt% to 50 wt% of the total weight of the composition, such as from 10 wt% to 40 wt%, or from 20 wt% to 30 wt%.
The composition of the invention may alternatively be a sol. Typically when the composition of the invention is a sol, the composition comprises the nanoparticles of the invention in an amount of from 0.1 wt% to 2% of the total weight of the composition, such as from 0.5 wt% to 2 wt%, or from 1 wt% to 2 wt%.
The compositions of the invention are typically biodegradable. The term biodegradable is understood to mean that the composition has the ability to break down over time in the tissue or body of a human or animal and/or in the environment. The time for complete degradation may be at least 1 week, at least one month, at least 2 months, at least 6 months or at least 12 months. The time for complete degradation may be no more than 12 months or no more than 6 months. For example, the time for complete degradation may be from 1 week to 12 months, 1 week to 6 months, 1 month to 12 months, 1 month to 6 months, 2 months to 12 months or 2 months to 6 months. The degradation time may affect the speed at which drug is released from the gel when in situ in the body. Therefore, the degradation time be desirably varied depending on any therapeutic or diagnostic agent which is present in the composition.
When the composition is a gel, the gel preferably demonstrates a 50% change in zeta potential within a pH range of from pH 6.5 to pH 8. A 50% change in zeta potential is defined as a change of measured zeta potential of at least 50% compared with the baseline zeta potential, on titration with acid. Baseline zeta potential is the zeta potential of the gel without pH modification, i.e. before addition of acid. Baseline zeta potential is typically that measured at pH 9 to pH 11, more typically from pH 9.5 to pH 10.5, more typically about pH 10.
The change in zeta potential is typically a reduction of the magnitude of the zeta potential value, i.e. the absolute value of the zeta potential moves towards zero. This indicates an increase in stability of the gel. The zeta potential value may be positive or negative. A reduction in the absolute value in either case indicates that the value is closer to zero.
Preferably, the 50% change in zeta potential occurs at a pH range of from pH 7 to pH 7.8, more preferably at a pH range of from pH 7.2 to pH 7.8. In particularly preferred embodiments, the 50% change in zeta potential of the gel occurs in a pH range of from pH 7.3 to pH 7.5.
Typically, the change in zeta potential is measured via titration of the gel with an acid, such as hydrochloric acid. Zeta potential change can be determined using any appropriate method known in the prior art. For example it can be determined using equipment designed for measuring zeta potential, such as a Stabino Zeta™ kit. Typically the zeta potential is measured using a solution of nanoparticles in water, typically a 1 wt% solution of nanoparticles in ultrapure water. The zeta potential is a measure of the electrokinetic potential of particles in a colloidal system and can be used as an indication of the stability of the gel. Particles having a large negative zeta potential will tend to repel one another. However, a low negative zeta potential indicates that there is little repulsion between the particles. The particles of the invention therefore preferably have a negative zeta potential, wherein the value is <50% of the absolute value, at a pH in physiological range. This means that the gel is at its most stable in the region of physiological pH. The gels of the invention typically have a pH of around 10 in the absence of rheological or pH modifiers. At this pH, the gels typically have very large negative zeta potential, meaning the particles repel one another and are more flowable, and therefore relatively easy to administer, for example by injection. However, on contact with bodily fluids or other physiological pH environment, a change in zeta potential occurs, typically reducing the repelling forces between particles. The stability or stiffness of the gel therefore increases, thus providing a stiffer, more stable gel at the site at which it is delivered. This further gelation may be a result of new attractive interactions as a result of increased ionicity. Further, when in contact with bodily fluids, proteins that diffuse into the gel may bind and form bridges between particles to further stabilise the gel. The increase in stability helps to retain the gel at the site of administration and avoid off-target side effects.
Therapeutic or Diagnostic Agents
The composition of the invention may further comprise one or more therapeutic or diagnostic agents. A therapeutic or diagnostic agent may be a small molecule therapeutic or diagnostic agent, including small molecule drugs, prodrugs, or dyes or colourants. A therapeutic or diagnostic agent may alternatively be a biologic, wherein a biologic may be an amino acid, signalling molecule, peptide, protein (wherein the protein may be a recombinant protein or a native protein), antibody, nucleic acid, oligonucleotide (such as an aptamer), or cell. A therapeutic or diagnostic agent may alternatively be a polymeric material. Combinations of one or more therapeutic or diagnostic agent may be used.
The nanoclay compositions of the invention may be used for the delivery of a wide variety of different therapeutic or diagnostic agents and the nature of the agent(s) used is not particularly limited. Exemplary therapeutic agents include antimicrobial agents (for example antibiotics), growth factors, antibodies, nutrients, enzymes, hormones, steroids, biological tissue substitutes, synthetic tissue substitutes, aptamers and cells. Exemplary diagnostic agents include dyes, colourants, radioisotopes (which may be for X-ray detection and or monitoring of degradation), contrast agents (such as contrast agents for CT, such as iodine containing contrast agents and barium sulfate; contrast agents for MRI, such as gadolinium (III), iron oxide, iron platinum and manganese; or contrast agents for ultrasound), or fluorescent molecules. A combination of two or more therapeutic agents may be used. A combination of two or more diagnostic agents may be used. One or more therapeutic agents may be used in combination with one or more diagnostic agents.
In one embodiment, the one or more therapeutic or diagnostic agents are agents which are useful in regenerative medicine. In some embodiments, the one or more therapeutic or diagnostic agents are selected from growth factors, antibodies, biological tissue substitutes, synthetic bone grafts and cells. Antimicrobial agents may be included in the composition in addition or instead of such therapeutic agents. Any antimicrobial agent may be used. Examples include antibiotics such as vancomycin, gentamycin, tobramycin and chlorhexidine.
Typically a biological tissue substitute may be a substitute for a soft tissue such as bone marrow or plasma or a substitute for a hard tissue such as bone. Examples of biological tissue substitutes include platelet rich plasma (PRP) and biological bone-graft substitute.
Typically a synthetic tissue substitute may be a synthetic bone graft. Synthetic bone grafts include calcium salts such as calcium sulfates or calcium phosphates, alone or in combination. P-tricalcium phosphate (P-TCP) is a preferred calcium phosphate. Hydroxyapatite (HA) is a further preferred calcium phosphate.
Typically a cell may be a somatic cell, such as a chondrocyte; a progenitor cell; or a stem cell, such as a mesenchymal stem cell. Cells may also be provided as bone marrow aspirate (BMA).
Typically, a protein is a recombinant protein. Any protein for use in regenerative medicine may be used. Typically a recombinant protein is a growth factor, such as a bone morphogenetic protein (BMP), platelet derived growth factor (PDGF), transforming growth factor (TGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or insulin like growth factor (IGF). A combination of two or more such growth factors may be used.
An aptamer, as referred to herein, is a short synthetic ssDNA or ssRNA sequence. An aptamer may be a therapeutic, or a diagnostic aptamer, such as an aptamer which interacts with a biomarker. Such an aptamer may further comprise a diagnostic feature, such as a fluorescent marker, or a radioisotope.
In some preferred embodiments the composition comprises BMP, or a BMP analogue such as AMP. BMP -2, BMP-3, BMP-4, BMP-6, BMP-7, and BMP-8 or heterodimers of thereof may be mentioned. AMP -2 is an example of a suitable analogue of BMP -2. More preferably, the composition comprises BMP-2 and/or BMP-7 and analogues thereof, in particular BMP-2 and/or BMP-7. Most preferably the composition comprises BMP -2 and analogues thereof, in particular BMP-2.
The one or more therapeutic or diagnostic agents may alternatively be for use in treating or diagnosing diseases and disorders other than those relevant to regenerative medicine. For instance, the composition of the invention may be for use in delivering therapeutic and/or diagnostic agents suitable for treating or diagnosing cancer. For example, diagnostic agents which exhibit chemiluminescence, fluorescence, or contain radioactive isotopes may be used. Suitable therapeutic agents include one or a combination selected from doxorubicin, P-lapachone, or methotrexate.
The composition typically comprises a diagnostically visible and/or therapeutically effective amount of the one or more diagnostic or therapeutic agents respectively. This amount could be determined by the skilled person in the art based on the nature of the drug and the size and weight of the subject.
In an embodiment where BMP is provided as a therapeutic agent the BMP may be provided at a dose of from about 0.01 pg to about 50mg, for example from about 0.1 pg to about 20mg, or from about 1 pg to about 15mg total dose. The skilled person will understand that the dose may be dependent on the clinical context, such as the size of the area to be treated or the total volume of the composition.
The use of clay nanoparticles for BMP delivery provides dosing control, allowing low doses of BMP (e.g. from 0.1 to 300 pg) to be used to encourage bone formation at a defect, or allowing higher doses (e.g. from 10 pg to about 25 mg, or from 300 pg to about 15mg) to be used with reduced risk of off-target effects. Studies have demonstrated significant adverse effects with higher dose BMP use and its poor local retention such as: heterotopic ossification, osteolysis, and swelling. Therefore, allowing low dosages of BMP or ensuring retention of the protein at the target site, facilitates fracture healing and arthrodesis without precipitation of serious adverse effects. Targeted administration of BMP, as facilitated by the high stability and stiffness of gels provided by the present invention, also reduces off-target, adverse side-effects of BMP.
When the composition comprises one or more therapeutic and/or diagnostic agent(s), the therapeutic and/or diagnostic agent(s) may be provided in combination with a pharmaceutically acceptable carrier, referred to herein as a second carrier. Any suitable drug carrier may be used and the skilled person would be familiar with the selection of suitable carriers and excipients for the therapeutic and diagnostic agents which can be used in the present invention. Typically a carrier may be selected from carboxymethylcellulose, gelatine and collagen. Further excipient(s) may optionally be incorporated.
In some embodiments, the composition does not comprise a further diagnostic and/or therapeutic agent.
Kits
Also provided herein are kits comprising the compositions or pharmaceutical compositions according to the invention.
In some embodiments a kit of the invention comprises a syringe, e.g. a pre-filled syringe wherein the syringe contains a composition as described herein wherein the composition is in the form of a gel. In some embodiments a kit may comprise separately a syringe and a composition of the invention. In such embodiments the composition may be in a gel form, stored in a container such as a pouch. In other such embodiments, the composition may be in a form which may be a precursor to a gel form, typically solid form, such as a powder, typically a lyophilised powder, which may be hydrated to form a gel.
In some embodiments, a kit of the invention comprises a vial, which may be a sprayable vial, e.g. a pre-filled vial containing a composition of the invention as described herein.
In some embodiments a kit of the invention may comprise one or more therapeutic or diagnostic agents as defined anywhere herein. The therapeutic and/or diagnostic agent(s) may be provided separately from the composition of the invention, or they may be provided within the composition of the invention.
Preferably the composition is in powder form or in the form of a gel; and/or the therapeutic or diagnostic agent(s) are in a powder form. When the composition is in a powder form, use of the kit may involve the powder being hydrated to form a gel.
The composition of the invention and the therapeutic and/or diagnostic agent(s) may be provided in a form suitable for separate administration. Alternatively, the one or more therapeutic and/or diagnostic agent(s) may be provided in a form suitable for addition to the gel prior to administration to a subject. Alternatively, the drug and the composition may be pre-mixed in dried form and use of the kit may involve hydration of the mixture to form a gel suitable for administration.
A kit may comprise a biological implant, and the composition of the invention. In one embodiment, the biological implant is pre-coated with a gel or a dried film, as described herein. Alternatively, the composition (e.g. a gel or a dried film) and the implant may be provided separately. A biological implant, as referred to herein may be a medical device, such as a pace maker, implantable defibrillator or contraceptive implant; or a prosthetic implant, such as a knee, hip, or shoulder replacement, or medical screw or other fixator.
A kit may comprise the gel of the invention in the form of a dehydrated film. The kit may further comprise equipment for hydrating the film. The use of such a kit may involve administration of the film, for example by application onto the skin, and subsequent hydration of the film.
In some embodiments a kit may comprise the composition in the form of granules, or microparticles.
The kit may also comprise instructions for use.
Therapeutic uses
The composition as described anywhere herein may be for use in the treatment of the human or animal body.
In one aspect, the subject is a mammal, in particular a human. However, it may be non-human. Preferred non-human animals include, but are not limited to, primates, such as marmosets or monkeys, commercially farmed animals, such as horses, cows, sheep or pigs, and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters.
In one embodiment, the composition is for use in a method of regenerative medicine.
Therefore the invention also provides the use of a composition of the invention for the manufacture of a medicament for use in a method of regenerative medicine.
The invention also provides a method of regenerative medicine, comprising administering to a subject an effective amount of a composition of the invention. As described herein regenerative medicine comprises tissue repair and regeneration, wherein tissue repair and regeneration may include tissue formation and/or fusion. This can include repair or regeneration of soft tissues, wherein soft tissues include muscle (smooth muscle, skeletal muscle and cardiac muscle), fat, fibrous tissue (connective tissue, cartilage, tendons and ligaments), synovial tissue, blood vessels (arteries, veins and capillaries), lymph vessels, skin, and nerves. Regenerative medicine also comprises repair and regeneration of hard tissues, wherein hard tissues include bone, tooth enamel, dentin and cementum.
Tissue repair and regeneration also comprises tissue replacement, such as the filling of a void left by a tumour.
Preferably, the method of regenerative medicine is the repair or regeneration of skin, bone and/or cartilage.
A method of regenerative medicine may be a method of wound repair. For instance, the one or more drugs which may comprise the composition may be for use in wound repair, and to prevent or treat concurrent conditions often present in those who are in need of wound repair, for example inflammation, infection and pain.
In some instances, regenerative medicine may comprise cell delivery, such as delivery of cells to regenerate tissues, such as the delivery of somatic cells, such as chondrocytes; progenitor cells; or stem cells such as mesenchymal stem cells.
Therefore, the composition of the invention may be for use in a method of tissue repair or regeneration, and/or cell delivery.
Therefore the invention also provides the use of a composition of the invention for the manufacture of a medicament for use in tissue repair or regeneration, and/or cell delivery.
The invention also provides a method of tissue repair or regeneration, and/or cell delivery, wherein said method comprises administering to a subject an effective amount of a composition of the invention.
In some instances, the composition of the invention may be for use in the treatment or prevention of infection.
Therefore, the invention also provides the use of a composition of the invention for the manufacture of a medicament for use in the treatment or prevention of infection. The invention also provides a method of treating or preventing infection, wherein said method comprises administering to a subject an effective amount of a composition of the invention.
The composition of the invention may also be for use in a method of delivery of one or more therapeutic or diagnostic agents to a target site in the human or animal body, wherein said method comprises administering said composition to the human or animal body, wherein the composition comprises one or more therapeutic or diagnostic agents and/or wherein one or more therapeutic or diagnostic agents are administered separately into the composition in vivo at the target site. The therapeutic or diagnostic agent for delivery may be any therapeutic or diagnostic agent as described herein. In some embodiments, the therapeutic or diagnostic agent is a cell. In some embodiments, the therapeutic or diagnostic agent is an agent useful in regenerative medicine. In some embodiments, the therapeutic or diagnostic agent is useful in the treatment of cancer, i.e. a cancer therapeutic such as doxorubicin, P-lapachone, or methotrexate. In such embodiments, the target site may typically be a tumour.
The compositions and pharmaceutical compositions of the invention may be administered in a variety of dosage forms. The compositions and pharmaceutical compositions of the invention may for example be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrastemally, or transdermally. Typically the compositions of the invention are administered by injection or are applied to an exposed target site. For example the composition may be administered directly to the surface of a subject to be treated, i.e. the surface is coated with a composition of the invention (e.g. by spray-coating a surface to be treated); or the composition is administered directly into a void in the subject’s tissue, i.e. to fill or partially fill the void. Alternatively, the composition of the invention may be delivered via a fixed entry point, for example, via a cannula.
When the composition of the invention is in the form of a gel or dried film, it may be administered directly to a wound, e.g. a skin wound, or a bum, by covering the wound with the gel or film. Alternatively, the composition of the invention may be applied to a wound by spraying. A further dressing, such as a gauze and/or a bandage may then be applied. Alternatively, the gel or film may be incorporated in to the dressing, i.e. a bandage may comprise a gel or dried film of the invention. The composition may also be provided as a coating on a medical device and provided to a subject together with the device. Rehydration of the composition may be carried out after the device is inserted, in the case of a composition provided as a dried film. Suitable medical devices include a pace maker, implantable defibrillator, or contraceptive implant; or a prosthetic implant, such as a knee, hip, or shoulder replacement, or medical screw or other fixator.
In embodiments where one or more therapeutic or diagnostic agents are used, the one or more therapeutic or diagnostic agent may administered as a single composition comprising the nanoparticles described herein and the therapeutic and/or diagnostic agent(s). Alternatively, the one or more therapeutic or diagnostic agents may be administered separately, subsequently or sequentially to administration of the gel. The separate, subsequent or sequential administration of the one or more therapeutic or diagnostic agents may comprise administration to the gel on a target surface or injection into the gel at the target site.
In such embodiments, the one or more therapeutic or diagnostic agents may be added in a therapeutically effective amount. The one or more therapeutic or diagnostic agents may be added in a single administration or via multiple administrations. In some embodiments the one or more therapeutic or diagnostic agents may be added in situ at a target site, to provide sustained delivery of the one or more therapeutic or diagnostic agents. The one or more therapeutic or diagnostic agents may be added in from 1 to 10 administrations, typically from 1 to 6 administrations, more typically from 1 to 4 administrations. There is no limit to the number of administrations that can be provided, provided the composition is not saturated. Therefore administration may continue until such a point as a subject’s treatment has been completed.
In some embodiments the composition of the invention does not comprise any further therapeutic and/or diagnostic agents. In such embodiments, the composition may be administered via multiple administrations, such as from 1 to 10 administrations, typically from 1 to 6 administrations, more typically from 1 to 4 administrations.
When the one or more therapeutic or diagnostic agents are added to the composition in multiple administrations, there is no specific time lapse between each administration. The time lapse between each administration may be judged by the skilled person administering the one or more therapeutic or diagnostic agents and will depend on aspects such as the half-life of the therapeutic or diagnostic agent in question and the rate of release from the gel.
A therapeutically effective amount of a composition of the invention is administered to a patient. A typical dose may be determined by the skilled person, according to the activity of the specific composition, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration.
Cosmetic uses
The composition as described anywhere herein may be used in cosmetic methods. Suitable methods include a method of hydrating the skin, improving the evenness of skin complexion, preventing or reducing wrinkles or skin embellishments, as a cosmetic topical cream or gel, or as a cosmetic tissue filler, such as a lip filler, or a dermal filler. In some preferred embodiments, the composition is used as a cosmetic tissue filler. Alternatively, it may be used in a cosmetic topical cream.
Accordingly, the composition as described anywhere herein may be a cosmetic composition, which is a composition suitable for cosmetic use. A cosmetic composition typically comprises one or more additives useful in formulating cosmetic compositions. The cosmetic composition may be a tissue filler, or a topical cream or gel.
Typically when the composition of the invention is a cosmetic composition, or used in a cosmetic method, it does not comprise a further diagnostic or therapeutic agent.
A cosmetic composition of the invention may optionally comprise an antiinflammatory agent to reduce swelling. In some embodiments, anti-inflammatory agents are not present in a cosmetic composition. In some embodiments, a cosmetic composition does not comprise any therapeutic agent.
Process for making clay nanoparticles and compositions
Also provided herein is a process for making the clay nanoparticles and compositions of the invention.
Typically, the process comprises combining a lithium salt solution, a magnesium salt solution, a solution comprising the cationic component and a silicate solution. Such salt solutions to be used in the process of the invention may be any suitable aqueous solutions known to the skilled person.
Typically the molar ratio of magnesium to lithium which is input into the combined solutions (Mginput/Li input) is from 2 to 30, such as from 5 to 25, or from 10 to 25.
As the lithium salt solution, lithium sulfate solution, lithium chloride solution, lithium hydroxide solution, lithium nitrate solution and combinations thereof, can be used. Typically lithium sulfate solution is used. The process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving a lithium salt in water to form an aqueous solution. The salt may be a hydrated salt, such as a monohydrate. In some embodiments the process involves a step of dissolving lithium sulfate monohydrate in water.
As a magnesium salt solution, magnesium sulfate solution, magnesium chloride solution, magnesium nitrate solution, and combinations thereof, can be used. Typically, magnesium sulfate solution is used. The process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving a magnesium salt in water to form an aqueous solution. The salt may be a hydrated salt, such as a monohydrate or a heptahydrate. In some embodiments the process involves a step of dissolving magnesium sulfate heptahydrate in water.
As a silicate solution, sodium silicate solution, potassium silicate solution, or a combination thereof can be used. Typically, sodium silicate solution is used. The process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving a silicate in water to form an aqueous solution. In some embodiments the process involves a step of dissolving sodium silicate in water.
As a salt solution comprising the cationic component, any appropriate solution may be used. These include, for example, a carbonate solution, a phosphate solution, a sulfate solution, a nitrate solution, or a combination thereof. Typically a carbonate solution is used, such that when the cationic component comprises sodium, a sodium carbonate solution may be used. The process of making the clay nanoparticles and compositions of the invention may first involve a step of dissolving the cationic component salt in water to form an aqueous solution. In some embodiments the process of the invention comprises a step of dissolving sodium carbonate to form an aqueous solution. Once the solutions have been combined the process of the invention typically comprises heating the combined solutions together. Heating typically continues until a slurry is formed. The weight % of the solids in the slurry compared to the total weight of the slurry is typically from around 1 wt% to 15 wt%, typically around 3 wt% to 12 wt%. Sometimes, the weight % of the solids in the slurry compared to the total weight of the slurry is from around 3 wt% to around 5 wt%. Sometimes, the weight % of the solids in the slurry compared to the total weight of the slurry is from around 8 wt% to around 10 wt%. The combined solutions are typically heated to the boiling point of the slurry (i.e. to about 100 °C). Heating at this temperature, typically under reflux, is typically continued for a period of from around 30 minutes to around 2 hours, typically about an hour.
The process of the invention may comprise a further step comprising hydrothermal treatment of the slurry. A hydrothermal treatment as referred to herein, is any treatment comprising heating of a composition (in this instance a slurry) in the presence of water. For instance, hydrothermal treatment may comprise autoclaving. Typically, hydrothermal treatment may occur at a temperature of from 60 to 500 °C. The hydrothermal treatment may occur at an elevated pressure (i.e. above standard atmospheric pressure (about 100 000 Pa)). Hydrothermal treatment at elevated pressure may be referred to as autoclaving. Thus, the process of the invention may comprise an autoclaving step.
Typically, the hydrothermal treatment step occurs at a temperature of from around 100 °C to around 300 °C, typically around 150 °C to around 250 °C, preferably around 180 °C to around 220 °C. Typically the hydrothermal treatment step occurs at a pressure of from 30 psi to around 1800 psi, typically from around 60 psi to around 600 psi, preferably from around 100 psi to around 400 psi. Typically the hydrothermal treatment step lasts for approximately 1 hour to 24 hours, preferably 2 hours to 12 hours, 4 hours to 8 hours, most preferably around 6 hours.
The process of the invention may further comprise a drying step, wherein the slurry is dried to form a solid. Drying of the slurry may be carried out using any appropriate method known to the skilled person, for example rotoevaporation, oven drying or vacuum filtration. Preferably vacuum filtration is used (e.g. using a Buchner funnel).
The process may also comprise a step of washing and re-drying the dried product. Any appropriate solvent may be used to wash the product. Typically deionised water is used. Vacuum filtration (e.g. using a Buchner funnel) may be used in this step. The washing step is especially useful to remove soluble salt impurities, such sulfates. To assess if all impurities have been removed, BaCh may be added to the filtrate. If a precipitate is formed, typically further washing is required.
Typically, more than one drying method is used, such as vacuum filtration in combination with oven drying. Oven drying typically occurs at a temperature not too far about standard temperature. Typically oven drying occurs at a temperature of from around 40 °C to around 80 °C, more typically around 55 °C to around 65 °C.
A drying step may last for around 1 hour to 24 hours, typically around 6 hours to 14 hours.
The process of the invention may further comprise a step of grinding the dried product to form a powder. Grinding may occur using any appropriate method, such as use of a pestle and mortar.
The process of the invention may further comprise a step of adding water to the composition to form a gel.
When the water is added to the composition, the composition may be subjected to rigorous stirring, i.e. stirring with a magnetic stirrer at a rate of 300 rpm to 1000 rpm, such as 500 rpm to 800 rpm. The stirring can occur at any temperature, such as from 0 °C to 100 °C, or 10 °C to 40 °C. Typically the stirring occurs as standard atmospheric temperature and pressure (SATP), such as around 25 °C, and around 15 psi.
In some embodiments no further steps are required to obtain the gel. In some embodiments of the process, the gel may then be sterilised, optionally via autoclaving. Autoclaving typically occurs at from around 100 °C to around 140 °C, such as around 120 °C. Autoclaving typically occurs for between 10 minutes and 1 hour, such as 30 minutes. Autoclaving typically occurs at from 13 psi to 20 psi, such as from 15 psi to 20 psi.
The process of the invention may further comprise a step of adding one or more therapeutic or diagnostic agents to the composition. The one or more therapeutic or diagnostic agents may be added to the composition at any step during the process of the invention. The one or more therapeutic or diagnostic agents may also be added to the composition prior to formation of the gel. The one or more therapeutic or diagnostic agents may be added after the composition when it is in the form of a gel, but prior to administration to the subject. The one or more therapeutic or diagnostic agents may alternatively or additionally be administration to the subject separately from the gel, e.g. by administration of the one or more therapeutic or diagnostic agents into the gel in vivo at the target site, or by adding the one or more therapeutic or diagnostic agents as a coating to the gel, in particular when the gel itself is coated on the surface of a medical device, such as an implant, a stent, or a balloon. Typically, the one or more therapeutic or diagnostic agents are added after the composition when it is in the form of a gel, but prior to administration to the subject.
Examples
Example 1 - Synthesis of clay nanoparticles
2.65g of Li2SO4.H2O was dissolved into 170 mL of de-ionised water at room temperature using a stirrer. Once the Li2SO4.H2O was dissolved, 56.10 g of MgSO4. H2O was added. The mixed salt solution was then transferred to a round bottom flask (RBF) in an assembled reactor which was heated to 60 ± 10 °C.
21.95g of Na2CO3 was dissolved in a separate vessel into 130 mL DI water, at room temperature and under stirring.
When the RBF contents had reached 60 °C (±10°C), the Na2CO3 solution was added using a stoppered self-equalising dropping funnel at roughly 1 drop a second.
Using a fresh dropping funnel, 69.7 g of sodium silicate solution was then added to the RBF at roughly 1 drop a second.
A reflux condenser was then fitted to the reactor and the temperature of the reactor increased to 100°C. The mixed salt solution was then allowed to boil under reflux at atmospheric pressure for 1 hour.
After one hour, the reaction mixture was then reduced to 60 °C, keeping all other conditions unchanged. A resultant slurry has been formed.
An oven was pre-heated to 200 °C, with the fan set to maximum and the damper to 40%.
The slurry is transferred into autoclave vessels (120 mL per vessel). The autoclave vessels were then placed in the oven for hydrothermal treatment (HT) at 200 °C for 6 hrs. The oven was then switched off and the vessels allowed to cool. Once cooled the slurry was washed from the autoclave vessels. The contents of each autoclave vessel was then transferred individually into a Buchner funnel, and the filter cake washed with de-ionised water. 400 mL of de-ionised water were used per autoclave vessel. The Buchner flask was regularly emptied and the filtrate tested with BaCh solution.
If a precipitate formed, it indicated that sulfate was still being washed from the filter cake. Further washing was continued until no precipitate formed.
The filter cake was then transferred into a vessel suitable for the drying procedure. The filter cake was then left to dry for at least 8 to 12 hours, but could be left for up to 72 hours. Drying occurred in an oven set at 60 °C at 100% fan, and 40% damper.
The dried filter cake was then ground for about 10 minutes into a powder using a mortar and pestle to achieve fine particles.
Example 2 - Chemical composition of the clay nanoparticles
The composition of the clay nanoparticles prepared using the method of Example 1 was then analysed by elemental analysis using inductively coupled plasma-optical emission spectrometry (ICP-OES). Laponite™ XLG was also assessed using the same method. The results are summarised in the below Table 1 where empirically determined Mg, Li and Na are presented relative to empirically determined Si which is scaled according to the following chemical formula ratio (Na+)a[(SisMgbLic)02o(OH)4]'a . The results are summarised in the below Table 1 :
TABLE 1 :
Figure imgf000032_0001
The comparison of elemental ratios between Laponite™ and the compositions set out in Table 1 (collectively referred to as Renovite) are also displayed graphically in Figure 1.
To exemplify further variation in elemental composition of the nanoclay of the invention, the method of Example 1 was repeated with variation in the synthesis protocol as set out in Table 2 below. For each modified synthesis, ICP-OES analysis data for of the resulting nanoclay is provided and elemental ratios for Mg/Si and Mg/Li in contrast to Laponite™ are presented in Figure 2. TABLE 2:
Figure imgf000033_0001
Example 3 Protocol for producing gels
Gels of the invention are prepared as a weight percentage (wt.%) of nanocl ay/ water where 1% is equivalent to 1g / 100 ml or lOmg / ml. Gels were prepared using the nanoclays of the invention at concentrations in the range 2 to 7 wt%. Reference gels were also prepared using corresponding amounts of Laponite™ as the nanoclay, for comparison purposes. The mixing procedure is designed to ensure complete wetting out and dispersion of the clay particles.
1) Deionised water (18.2 MQ, pH 7) is added into a glass bottle with a magnetic stirrer bar and place on a stirrer to generate a stable vortex that extends close to the base of the bottle but without exposing the stirrer to air. 2) Powdered nanoclay is added gradually into the vortex over the course of 5-10 seconds.
3) The suspension is left to stir for 1-2 mins at room temperature before being briefly mixed on a vortex to remove any powder stuck on the sides or forming clumps. Stirring is continued for a further 25 mins, until the colloidal solution is clear.
4) The total weight of the bottle containing the gel is measured before undergoing autoclave sterilisation for ~30min at 121 °C and 15 psi using a bench top autoclave suitable for liquid sterilization. Once it has cooled, the bottle is weighed again and any lost water replaced followed by mixing on a vortex. To keep the gel of the invention sterile, handling is under sterile conditions and sterile deionized water used to replace lost water.
Example 4: Rheometric measurements and gelation response to blood serum
The rheological properties of gels of the invention were characterised both in their native state and following addition to a bath of simulated blood serum (40 mg/ml bovine serum albumin in phosphate buffered saline, pH 7.4) to assess their response in physiological solutions (such as, for example, following injection into the body).
All rheological measurements were conducted on an MCR 92 rheometer (Anton Paar, UK) using a 12mm parallel plate geometry (PPI 2 probe) where 117pl gel suspensions were loaded onto the rheometer plate pre-set to 25 °C with a 1 mm gap. Isotherm measurements of storage and loss modulus in response to blood serum over time were taken over 60 min at a constant oscillatory strain of 0.005 % and angular frequency of 1 rad s'1. After 5 minutes a serum reservoir was applied up to the level of the lower surface of the upper plate. Figure 3 shows that the observed increase in storage modulus in response to serum addition was enhanced in gels of the invention compared with Laponite™ gels having a corresponding nanoclay concentration.
Amplitude sweeps covering the range of 0.01-100 % strain were applied to measure storage and loss modulus within the linear viscoelastic range (LVR) and to define yield strain and stress, again at a constant angular frequency of 1 rad s'1. To calculate yield stress and yield strain, elastic stress (T) was plotted against shear strain (y) and yield stress and yield strain for each treatment calculated as the point of inflection (>5%) from the linear dependence of T on y. Figure 4 shows that, in its native state, a gel of the invention can possess equivalent gel strength (yield stress (Pa)), stiffness (storage modulus (G’)) and viscosity (storage modulus (G”)) to a Laponite™ gel having a corresponding nanoclay concentration (Fig 4a, top), but in simulated blood serum, the increase in gel strength, stiffness and viscosity was significantly enhanced in gels of the invention (Fig 4b, bottom). Figure 5 reports storage and loss modulus of a range of high and low Li / Mg gels of the invention and their analogues vs Laponite™ suspensions in simulated blood serum and shows a significant positive correlation with Mg/Si (a) and a significant negative correlation with Li/Si (b) molar ratios across the various nanoclays.
Example 5: Protocol for measuring Zeta Potential Half Maximum (PHM) and gelation response to pH
Gels of the invention prepared according to Example 3 were measured to assess zetapotential kinetics in response to pH. Zeta potential is the electrical potential at the slipping plane. This plane is the interface which separates mobile fluid from fluid that remains attached to the surface. Zeta potential describes the electrokinetic potential in colloidal dispersions and is a function of the specific surface chemistry of a dispersion which are affected by changes in pH, salt, and surfactant concentration. At its minimum zeta potential (i.e. its absolute lowest value) a colloid is a its most stable as a dispersion due to the dominance of repulsive over attractive interactions between particles. As the zeta potential approaches the isoelectric point attractive interactions increase resulting in aggregation of particles.
Titrations using IM HCL (30 ul at 20 second intervals) were performed on the Stabino™ II kit allowing dynamic measurement of zeta potential (P) with falling pH. All titrations were performed on 1 wt. % solutions of clay in filtered ultrapure water, prepared on the day of testing.
Figure 6 shows the sharp decrease in negative zeta potential (ZP) occurring between pH8 and pH5 over the course of titration with HC1. This was a characteristic feature of all nanoclays tested and coincided with an increase in particle (aggregate) size indicating a transition from repulsive to attractive particle interactions. At high nanoclay concentrations (> 2 wt%) an increase in attractive interactions yields a gel. The pH at which this sharp decrease in potential occurred corresponded with the zeta potential half maximum (PHM) defined as the point at which negative ZP is 50% of its recorded mV prior to addition of titrant. Figure 7 shows that the pH at which the PHM occurs (PHPHM) for gels of the invention averages at 7.33 - close to physiological pH. This is in contrast to Laponite™ which had a much lower average PHPHM of 5.9. Figure 8 shows, as with the improved rheological response to serum, the physiologically optimised PHPHM of gels of the invention correlates with its higher Mg and lower Li content compared with Laponite™ and other high Li analogues of the gels of the invention.

Claims

Claims:
1. A composition comprising a plurality of clay nanoparticles, wherein each clay nanoparticle comprises an anionic component and a cationic component wherein the anionic component has the formula (I):
[(Si8MgbLic)02o(OH)4] (I) wherein 5.5 < b < 6, and wherein c > 0 and b/c > 12.
2. A composition according to claim 1 wherein 12 < b/c < 18, preferably wherein 13 < b/c < 18.
3. A composition according to claim 1 or claim 2, wherein 0.2 < c < 0.5.
4. A composition according to any one of claims 1 to 3 wherein the cationic component comprises Na+.
5. A composition according to any one of claims 1 to 4 which further comprises one or more therapeutic or diagnostic agents.
6. A composition according to claim 5 wherein the one or more therapeutic or diagnostic agents are therapeutic or diagnostic agents suitable for use in regenerative medicine.
7. A composition according to claim 5 or claim 6 wherein the one or more therapeutic or diagnostic agents are selected from small molecules, growth factors, antibodies, biological tissue substitutes, synthetic bone grafts, antimicrobial agents, antibiotics and cells.
8. A composition according to any one of claims 1 to 7 wherein the composition comprises BMP -2.
9. A composition according to any one of claims 1 to 8 wherein the composition is in the form of a gel or a film.
10. A composition according to claim 9 wherein the gel demonstrates a 50% change in zeta potential within a pH range of from pH 6.5 to pH 8, when zeta potential is measured using acid titration.
11. A kit comprising a syringe or a vial, wherein the syringe or vial contains a composition according to claim 9 or claim 10.
12. A process for making a composition as described in any one of claims 1 to 10, wherein the process comprises combining a lithium salt solution, a magnesium salt solution, a solution comprising the cationic component and a silicate solution, and heating the solutions together until a slurry is formed; optionally subjecting the slurry to hydrothermal treatment; optionally drying the slurry to form a solid; and optionally grinding the solid to form a powder.
13. A process according to claim 12, wherein the process further comprises adding water to the composition to form a gel.
14. A composition according to any one of claims 1 to 10 for use in a method of regenerative medicine, a method of tissue repair or regeneration, a method of cell delivery, or for use in the treatment or prevention of infection.
15. A composition according to any one of claims 1 to 10 for use in a method of delivery of one or more therapeutic or diagnostic agents to a target site in the human or animal body, wherein said method comprises administering said composition to the human or animal body, wherein the composition comprises one or more therapeutic or diagnostic agents and/or wherein one or more therapeutic or diagnostic agents are administered separately into the composition in vivo at the target site.
PCT/EP2023/087424 2022-12-22 2023-12-21 Clay nanoparticles for medical use WO2024133782A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP22216031 2022-12-22
EP22216031.9 2022-12-22
US18/491,443 2023-10-20
US18/491,443 US12036328B1 (en) 2022-12-22 2023-10-20 Clay nanoparticles for medical use

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