Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
  • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
  • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/5115—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy

Definitions

• Paste comprising magnetic alkoxysilane-coated metal containing nanoparticles
• Magnetofluids comprising magnetic alkoxysilane-coated nanoparticles are known in the art.
• aqueous suspensions comprising magnetic alkoxysilane-coated metal containing nanoparticles, methods of producing them and their use in the treatment of proliferative diseases have been described in WO 2013/020701.
• Magnetofluids comprising magnetic alkoxysilane-coated nanoparticles are injected into tumors for treating the tumors by hyperthermia upon applying an alternating magnetic field.
• the nanoparticles agglomerate and form a depot, a kind of implant. Since more than one depot can be generated by this process, the entirety of all depots in their shape, position and distribution determines the temperature distribution that is achieved when the particles are subsequently activated in the tumor by an alternating magnetic field.
• the tumor is covered exactly by the therapeutically necessary minimum temperature and the areas of high temperature in which the tissue is directly killed lie exclusively within the tumor.
• the present invention relates to a magnetic particle composition which overcomes the disadvantage of deferred solidification after injection into the target site and the further disadvantage of spreading and scattering of the nanoparticles or agglomerates of nanoparticles into neighboring tissue.
• a particular pasty composition i.e. a composition in the form of a paste
• a composition in the form of a paste can be provided by thermal treatment when taking an aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles as a starting material.
• a pasty composition or paste is characterized by the presence of yield stress and by shear thinning behavior; allowing the paste on the one hand to be drawn up into e.g. a syringe, e.g. via a needle, or into a cannula.
• the present invention provides a paste comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles which can be injected in the daily routine precisely into tissue and remains at the injection site, without spreading or substantially spreading to surrounding tissue. This allows a more accurate hyperthermia treatment particularly of small tumors and of tumorous tissue in the vicinity of channels, where the suspension of magnetic nanoparticles would otherwise leave the treatment area. Furthermore, the present invention provides a manufacturing process for such paste.
• the present invention provides a paste comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles obtainable by a method, wherein the method comprises the step of thermally treating an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles.
• the paste is characterized by plastic behavior. ln a further preferred embodiment of the invention, the paste is characterized by the presence of yield stress.
• the yield stress is preferably between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa.
• the paste has a shear thinning behavior.
• the paste is characterized by a viscosity, wherein the viscosity of the paste is between 0.03 and 7 Pa s, more preferably between 0.03 and 5 Pa s, still more preferably between 0.08 and 3.5 Pa s, still more preferably between 0.1 and 3.5 Pa s, still more preferably between 0.1 and 3.0 Pa s, still more preferably between 0.5 and 3.0 Pa s, still more preferably between 0.7 and 3.0 Pa s, still more preferably between 1 and 3.0 Pa, measured at a shear rate of 50/s.
• the step of thermally treating is carried out at a temperature between 25 and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60 °C.
• the step of thermally treating is carried out for a time period of between 0.1 to 96 hours (hrs), preferably for a time period of between 0.5 to 72 hours (hrs), more preferably for a time period of between 1 to 48 hours (hrs).
• the aqueous suspension comprises a concentration of at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M, as determined by its metal content.
• the method further comprises the step of incubating the aqueous suspension of magnetic metal containing nanoparticles with an alkoxysilane before thermally treating the aqueous suspension.
• the step of incubating is carried out in the absence of an added organic solvent.
• the alkoxysilane is a trialkoxysilane, preferably selected from the group consisting of 3-(2-aminoethylamino)-propyl-trimethoxysilane, 3- aminopropyltriethoxysilane, trimethoxysilylpropyl-diethylenetriamine and N-(6- aminohexyl)-3-aminopropyltrimethoxysilane, especially 3-(2-aminoethylamino)-propyl- trimethoxysilane, and/or 0.3 to 0.6 x 10 3 mol, preferably 0.4 to 0.5 x 10 3 mol, more preferably 0.43 to 0.45 x 10 3 mol, alkoxysilane per 0.9 mol metal is added in the incubating step.
• a trialkoxysilane preferably selected from the group consisting of 3-(2-aminoethylamino)-propyl-trimethoxysilane, 3- aminopropyltrieth
• the magnetic metal containing nanoparticles comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, preferably iron salts, more preferably wherein the iron salt is an iron oxide, preferably magnetite and/or maghemite.
• the iron oxide nanoparticles are provided.
• the method further comprises the step of ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles before thermally treating the aqueous suspension.
• the specific absorption rate (SAR) of the nanoparticles is larger or equal than 2 W/g Me, preferably larger or equal than 3 W/g Me, more preferably 4 to 12 W/g Me, as determined at a magnetic field strength of 3.5 kA/m and a frequency of 100 kHz.
• the present invention relates to a medical device comprising the paste of the present invention.
• the invention relates to a pharmaceutical composition comprising the paste of the present invention.
• the paste of the invention is for use in a method for treating or preventing proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection in a patient.
• the pharmaceutical composition comprising the paste of the invention is for use in a method for treating or preventing proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection in a patient.
• the present invention relates to a method of preventing or treating a proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection in a subject in need thereof, comprising administering the paste or the pharmaceutical composition of the invention to the subject in need thereof.
• the present invention provides a method for producing the paste of the invention, wherein the method comprising any of the steps as detailed above.
• the method comprises the step of thermally treating an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles.
• This thermal treating step may be carried out at a temperature between 25 and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60°C, and/or for a duration of time of between 0.1 to 96 h, preferably 0.5 to 72 h, more preferably 1 to 48 h.
• the aqueous suspension comprises a concentration of at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M, as determined by its metal content.
• the method may further comprise the step of incubating the aqueous suspension comprising magnetic metal containing nanoparticles with an alkoxysilane before the step of thermally treating the aqueous suspension.
• the incubating step may be carried out in the absence of an added organic solvent.
• the alkoxysilane may be a trialkoxysilane, preferably selected from the group consisting of 3- (2-aminoethylamino)-propyl-trimethoxysilane, 3-aminopropyltriethoxysilane, trimethoxysilylpropyl-diethylenetriamine and N-(6-aminohexyl)-3- aminopropyltrimethoxysilane, especially 3-(2-aminoethylamino)-propyl-trimethoxysilane.
• the magnetic metal containing nanoparticles may comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, preferably iron salts, more preferably wherein the iron salt is an iron oxide, preferably magnetite and/or maghemite, whereby the iron oxide nanoparticles may be provided by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide, or by thermal decomposition of an iron salt or an iron complex compound.
• ..viscosity refers to the quotient of shear stress t and shear rate
• the first is a measure for the applied force F on the liquid in direction of flow, divided by the area A in direction of the flow (F/A).
• the latter is a measure for the velocity-gradient dv/dy in the liquid perpendicular to the flow of the liquid (see Fig. 1).
• “Newtonian behavior” means that for such a liquid, h is a constant depending only on the temperature. That is, a graph of stress t versus shear rate g yields a straight line through the origin with h as its slope (see Figure 2a).
• non-Newtonian behavior means, that h is also a function of shear stress t and shear rate
• h is also a function of shear stress t and shear rate
• T T 0 + K ⁇ n
• K consistency (slope at zero shear rate)
• n flow index (curvature of the graph; referring to shear thinning (n ⁇ 1) or shear thickening (n > 1))
• Yield stress refers to the necessary shear stress, above which a paste is movable, e.g. behaves like a liquid and/or adopts a liquid state.
• the yield stress is the value of the intersection of the graph and the y-axis (see Figure 2a).
• Yield stress is a property of the paste of the present invention, adopted by thermal treatment, allowing the paste to adopt a movable state by application of a shear stress above the yield stress, e.g. to behave like a liquid and/or to become a liquid, and to adopt a non-movable state by application of a shear stress below the yield stress or by not applying a shear stress.
• the paste may e.g. behave like a solid and/or is a solid.
• the paste of the present invention shows non-Newtonian behavior, thus, the yield stress is above zero.
• “Movable”, as used herein, refers to the adoption of a state of a paste, in which adopted state the paste is able to move or flow. This state is adopted by the influence of shear stress which converts the non-movable paste into a state, in which the paste can move or flow.
• Non-movable refers to a state of the paste, in which the paste is not able to move or flow by itself. In the non-movable state, the paste may behave like a solid or is a solid. However, in the non-movable state, the paste can be deformed by applying shear stress in an elastic way only -in other words: is reversible-, as long as the applied shear stress is lower than the yield stress. Applying a shear stress higher than the yield stress transfers the paste from the non-movable to the movable state. “Solid”, as used herein, means that the paste is in a firm and stable shape.
• Shear thinning behavior refers to a composition, where the viscosity decreases with increasing shear stress or shear rate. This results in lower necessary force if, e.g. the composition is forced through a small opening such as present in a syringe, needle or cannula.
• “Plastic behavior” refers to a composition which is non-movable, e.g. behaves like a solid or adopts a solid state, below a certain value of shear stress (yield stress), but is movable, e.g. behaves like a liquid or adopts a liquid state (see Figure 2a).
• Paste means a composition showing plastic behavior.
• a paste can have a non-movable state, (below its yield stress) or a paste can have a movable state (above its yield stress).
• “Pasty” means the state adopted by a paste.
• agitation is meant any strong impact that is applied to the paste of the present invention. This includes strong shaking, preferably over a long time such as 6 to 24 hours, heavy vibration such as vortexing or application of a high kinetic impulse. Agitation changes the consistency of the paste having been thermally treated from a non movable composition such as a solid back to the original liquid consistency of the aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles, from which the paste is formed by thermal treatment.
• shearing is meant any flowing movement of the material, e.g. paste, whereby a force in the direction of flow is applied on the material, e.g. paste.
• the changes made by this type of influence are reversible, i.e. if the shear stress falls under the yield stress, the movement of the paste stops and the paste adopts its non-movable state, e.g. behaves like a solid or is a solid, without that thermal treatment is applied. If the shear stress rises over the yield stress, the paste gets into movement.
• SAR Specific Absorption Rate
• Zero potential refers to measured electrical potential of a colloidal nanoparticle in aqueous environment, measured with an instrument such as a Malvern ZetaSizer 3000 HSA at pH 5.2 and a conductivity of 2.0 mS/cm (each determined at 25°C).
• the zeta potential describes the potential at the boundary between bulk solution and the region of hydrodynamic shear or diffuse layer.
• “Hydrodynamic diameter” describes the size of the agglomerates as found in the suspension or in the paste. It is determined by light scattering. In this context, the average size is determined in water according to example 3. With this light scattering measurement, the size of agglomerates of nanoparticles is determined - in contrast to the size of the ball-shaped or cubic electron-dense single nanoparticles (“primary particles”) which are forming such agglomerates.
• Z-average with respect to the size of agglomerates means the readout of the light scattering size determination as carried out in example 3. Z-average values above the provided ranges lead to sedimentation of the nanoparticles and are therefore generally not suitable for the foreseen applications of these nanoparticles.
• Hydrodynamic volume means the average space needed for an average agglomerate. It can be calculated from the hydrodynamic diameter by the formula:
• V H 1/6 nd wherein V H is the hydrodynamic volume and d H is the hydrodynamic diameter.
• the hydrodynamic volume still includes water.
• the hydrodynamic volume of the agglomerates is not exclusive. Nevertheless, it restricts the space for movement of the agglomerates.
• the agglomerates are seen as spheres, there is a theoretical maximum for filling a space with spheres. This is about 74 % in case of ideal spheres of same size in a hexagonal or cubic dense packing in a perfect crystal lattice (Hollemann, 1985).
• the agglomerates will interact with each other even at much lower space filling, starting with thickening, shear thinning and at higher space filling plastic behavior (Mueller, 2010).
• the maximum for the growth of the agglomerates seems to be due to maximum space filling.
• the term “about” means a deviation from the given number or value of ⁇ 10 %, preferably of ⁇ 5 % and especially of ⁇ 1 %.
• magnetic incorporates magnetic, paramagnetic, ferromagnetic, anti ferromagnetic, ferrimagnetic, antiferrimagnetic and superparamagnetic.
• the nanoparticles are paramagnetic, more preferably ferromagnetic, ferrimagnetic, antiferrimagnetic or superparamagnetic, particularly preferably, the nanoparticles are superparamagnetic.
• nanoparticles shall mean nanoparticles in the nanometer range, meaning nanoparticles from 1 to 100 nm with respect to its metal core, as can be determined by electron microscopy.
• the nanoparticles have a size of 5 to 25 nm, more preferably 7 to 20 nm and still more preferably 9 to 15 nm.
• Metal nanoparticle refers to a magnetic nanoparticle, which contains metal or metal ions.
• alkoxysilane coating refers to a coating resulting from the polycondensation of alkoxysilanes, a process which is also referred to as “aminosilane coating”.
• polycondensation as used herein generally means any condensation reaction of a monomer with two functional groups which leads to the formation of a polymer and water.
• Agglomerating means that several individual nanoparticles form agglomerates or clusters of nanoparticles. “Agglomerates” refer to agglomerated nanoparticles or clusters of nanoparticles.
• the paste is characterized by the presence of a yield stress and by a shear thinning behavior, meaning that the paste can adopt a non-movable, such as a solid, state and a movable, such as a liquid, state. If a shear stress higher than said yield stress is applied to the paste, the paste adopts a movable state, e.g. the paste may show the characteristics of a liquid, e.g. it may become a viscous liquid, whereby the viscosity decreases with increasing shear stress or shear rate.
• the paste of the present invention is characterized by a yield stress of between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa.
• the paste of the present invention shows a “non-Newtonian behavior”, preferably a “plastic behavior”, as defined above.
• the paste Upon agitation, as defined herein, the paste is converted a liquid suspension with nearly the same rheological properties as the suspension from which it is formed by thermal treatment, without plastic behavior.
• the viscosity and the yield stress can be newly set to obtain desired values by thermal treatment. This means, the conversion of the liquid suspension to the paste is reversible.
• yield stress and/or viscosity can be adjusted to any levels which are desired.
• yield stress can be adjusted to between 0.03 and 80 Pa, more preferably between 0.06 and 70 Pa, still more preferably between 0.1 and 40 Pa and most preferably between 1 and 35 Pa and/or viscosity can be adjusted to values between 0.03 and 7 Pa s, more preferably between 0.03 and 5 Pa s, still more preferably between 0.08 and 3.5 Pa s, still more preferably between 0.1 and 3.5 Pa s, still more preferably between 0.1 and 3.0 Pa s, still more preferably between 0.5 and 3.0 Pa s, still more preferably between 0.7 and 3.0 Pa s, still more preferably between 1 and 3.0 Pa s, as determined by rotational rheometry at 20°C, measured at a shear rate of 50/s.
• the paste Upon shearing, as defined herein, the paste leaves its non-movable state, however, only to an extent that reversibility from the adopted movable state to the non-movable state is achieved by itself, i.e. if the shear stress is below the yield stress and without thermal treatment.
• yield stress and shear thinning behavior have also the effect that the paste is processable, as, due to the adoption of a movable state by applying shear stress, the paste can be for example drawn up into a cannula or into a syringe, e.g. through a needle, as shown in example 7.
• the shear stress is diminished or stopped, the movable state of the paste is reversed into the non-movable state, e.g. solid state.
• the paste when drawn up into a cannula or syringe, adopts its movable state as long as moving through narrow openings or through a cannula, so that it can easily be handled.
• the paste upon injection into a desired site, the paste gets non movable, due to the drop of shear stress between hard cannula and soft tissue after leaving the narrow cannula.
• This process of adoption of the previous state regarding movability and/or viscosity is much quicker than the agglomeration/coagulation process which occurs in a non-thermally treated suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles at contact with a physiological saline solution, as e.g. present in a body, leading to solidification.
• the paste from the thermally treated suspension regains its solid character directly after leaving the cannula and therefore keeps the form of the droplets, the droplets of the non- thermally treated suspension entering the salt solution as a liquid are spread and scattered. In the latter case, the formation of a solid mass is too slow to maintain the droplet form during the way to the bottom of the beaker. Consequently, the paste of the present invention is highly suitable for forming a solid depot or implant upon injection into a body, as the paste regains its non-movable, e.g. solid character, immediately after leaving the injection cannula or needle. Thus, flowing off nanoparticles and formation of undesired off-target depots are prevented.
• a suspension comprising magnetic agglomerates of alkoxysilane- coated metal containing nanoparticles which is not thermally treated has a delayed solidification, resulting in spreading and/or scattering of the agglomerated nanoparticles and escape from the injection site into the surrounding tissue.
• the paste of the present invention is associated with a more precisely controllable injection into tumorous tissue leading to a much better control over the heat distribution in the tumor and a lower occurrence of depots outside the treatment area lowering the efficacy of a hyperthermia treatment, as compared to a suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles which is not thermally treated.
• thermally treating means subjecting the aqueous suspension comprising agglomerates of alkoxysilane- coated magnetic metal containing nanoparticles to a temperature of between 25°C and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60°C.
• the temperature may be 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or 121 °C.
• the viscosity By heating the aqueous suspension, the viscosity is increased. Thereby, the level of increase of viscosity depends on the temperature. Higher temperatures result in higher viscosities.
• the temperatures may be between 25°C and 130°C, preferably between 30 and 120°C, more preferably between 40 and 100°C, still more preferably between 40 and 80°C, still more preferably between 40 and 60°C, and may be 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or 121 °C.
• the increase of the viscosity also depends on the duration of the thermal treating step. The longer the thermal treating is, the higher the viscosity becomes.
• the thermal treating step is carried out for a time period of between 0.1 to 96 hours (hrs), preferably for a time period of 0.5 to 72 hours (hrs), more preferably for a time period of 1 to 48 hours (hrs).
• a maximum viscosity can be reached during thermal treating, whereby still longer thermal treating may result in a decrease of viscosity, as can be taken from Table 5.
• the viscosity of the paste also of the aqueous suspension also depends on the molarity or concentration, as determined by the metal content. Thereby, higher molarities result in higher viscosities.
• the molarity may be at least about 2 M, preferably of at least about 3 M, more preferably of at least about 4 M, even more preferably of at least about 5 M, and especially of at least about 6 M, to about 8, 9 or 10 M.
• the desired metal concentration can be adjusted, e.g. by evaporating water from the aqueous suspension in a rotation evaporator. Samples can be analyzed regarding solids content and metal- concentration using the method disclosed below (see, for example, Example 2).
• the concentration of metal can be determined by photometry of certain metal complexes, e.g. iron can be determined after transformation into an iron(ll) phenanthroline complex, as described in Example 2.
• the viscosity is increased.
• the z-average value and the hydrodynamic volume per particle are increased.
• the viscosity of the paste is between 0.03 and 7 Pa s, more preferably between 0.03 and 5 Pa s, still more preferably between 0.08 and 3.5 Pa s, still more preferably between 0.1 and 3.5 Pa s, still more preferably between 0.1 and 3.0 Pa s, still more preferably between 0.5 and 3.0 Pa s, still more preferably between 0.7 and 3.0 Pa s, still more preferably between 1 and 3.0 Pa s, as determined by rotational rheometry at 20°C, measured at a shear rate of 50/s.
• Rotational rheometry according to the present invention is, for example, exemplified in Example 6.
• a maximum viscosity may be reached during thermal treating, as can be taken from Table 3 and as depicted in Figure 3a.
• the size of the agglomerates of the aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles is increased, as exemplarily presented in Table 1.
• the agglomerates in the paste have an average size of 40 to 460 nm, more preferably of 60 to 360 nm, still more preferably of 80 to 310 nm, still more preferably of 100 to 200 nm, still more preferably of 140 to 160 nm, as determined by light scattering.
• a maximum size of agglomerates may be reached during thermal treating.
• the size of agglomerated nanoparticles can be measured, as for example described in Example 3.
• the size of the metal cores of the nanoparticles, the shape of the nanoparticles and the SAR values of the nanoparticles in the paste are the same as in the aqueous suspension, from which the paste is formed by thermal treatment.
• the paste of the present invention can be injected (e.g. via syringe or cannula) in the daily routine into tissues such as tumors, remains within the tissue at the site of injection and therefore can be used for precisely placed hyperthermia and/or thermoablation. It has surprisingly been found that thermal treatment of an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles leads to the increase of viscosity to become a paste which can, due to the presence of yield stress and its shear-thinning properties, be easily moved, e.g. drawn up in a syringe, allowing injection into tissues where the suspension immediately thickens due to its plastic behavior to achieve the solidification state of the original paste.
• the agglomerates stay in close proximity within the injection site without escaping to neighboring environment.
• an aqueous suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles may be produced by any suitable method. Methods of producing such aqueous suspensions are known to the skilled person and have been described, for example, in WO 2013/020701.
• an aqueous suspension comprising metal containing magnetic nanoparticles is incubated with an alkoxysilane to produce agglomerates of the magnetic alkoxysilane-coated metal containing nanoparticles.
• incubating means any experimental setup, experimental condition(s) or reaction mixture(s) which allow for the polycondensation of alkoxysilanes and thereby for the aminosilane coating of nanoparticles.
• the incubation of an aqueous suspension comprising magnetic metal containing nanoparticles with alkoxysilanes is preferably carried out essentially in the absence of an organic solvent, more preferably in the absence of an added organic solvent.
• “Essentially in the absence” in the context of organic solvents means that small traces of organic solvents may be present, preferably the amount of organic solvents is smaller than 10 % by volume, more preferably smaller than 5 % by volume, still more preferably smaller than 1 % by volume, especially smaller than 0.5 % by volume.
• minor amounts of methanol may be produced during the reaction and, therefore, to some extent may remain in the product.
• the coating is carried out in absence of an organic solvent, especially the coating is carried out in the absence of an added organic solvent.
• the preferred solvent for the coating reaction is water.
• An organic solvent may be a liquid organic compound, i.e. a carbohydrate, with the power to dissolve solids, gases, or liquids.
• organic solvents include, but are not limited to, ethylene glycol, acetone, toluene and equivalents.
• the aqueous suspension comprising the agglomerates of magnetic alkoxysilane- coated metal containing nanoparticles is essentially free of organic solvents.
• “Essentially free of organic solvents” in this context means that the small traces of organic solvents may be present, e.g. the amount of organic solvents is smaller than 5% by volume, preferably 1 % by volume, more preferably smaller than 0.5 % by volume, especially smaller than 0.1 % by volume.
• no organic solvent can be detected in the nanoparticle preparation by customary methods.
• the production method of the agglomerates is preferably carried out in the absence of ethylene glycol.
• Ethylene glycol interferes with the coating reaction.
• it is at least very difficult if not impossible to remove it completely from the nanoparticle preparation as usually relatively large amounts of ethylene glycol remain attached to the coating of the nanoparticles and due to its high boiling point of 197 °C.
• European Pharmacopeia only 600 ppm of ethylene glycol are allowed in the final medical product, which makes nanoparticle preparations with higher amounts of ethylene glycol inacceptable for commercial clinical use.
• the metal nanoparticles comprise iron, iron complex compounds, iron carbonyl compounds or iron salts, whereas iron salts are preferred. Iron comprising nanoparticles are preferred due to their low toxicity compared to other magnetic metals such as cobalt or nickel.
• the iron complex compounds, the iron carbonyl compounds or iron salts are essentially free of other metals and other contaminants in order to avoid toxicities. It is well known in the art that chemicals may contain traces of contaminants. Therefore, “essentially free” in this context means preferably that less than 1% by weight, preferably less than 0.1% by weight of other contaminants is comprised within the iron complex compounds, iron carbonyl compounds or iron salts. Especially preferred are iron salts essentially free of other contaminants.
• the iron salt is an iron oxide, preferably magnetite and/or maghemite.
• Such iron nanoparticles made of iron oxide can be manufactured by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide.
• “Iron nanoparticles” as used herein are nanoparticles containing Fe atoms or Fe ions. Accordingly, in a preferred embodiment, the iron oxide nanoparticles are provided by precipitating a solution containing a mixture of iron(lll) chloride and iron(ll) chloride with sodium hydroxide.
• the ratio between iron(lll) chloride and iron(ll) chloride is preferably of about 2:1.
• iron nanoparticles and “iron oxide nanoparticles” are equivalently used.
• Suitable precipitation reactions and conditions have been described by Massart (1981) and reviewed by Mohapatra and Anand (2010).
• Preferred conditions for the precipitation reaction are (i) a ratio of Fe(lll)chloride and Fe(ll)chloride of about 2:1; (ii) pouring the Fe(lll)chloride and Fe(ll)chloride solution into a sodium hydroxide solution with a concentration of about 2.13 M; (iii) precipitation temperature of about 25°C; and (iv) time for the precipitation reaction of about 52 min.
• the sodium hydroxide solution is poured into the iron chloride solution (instead of vice e versa) during a period of about 39 min at about 15°C.
• a method for producing coated iron oxide nanoparticles by means of precipitating iron salts in solution is, for example, exemplified in Example 1.1.
• the iron oxide nanoparticles can be provided by thermal decomposition of an iron salt or an iron complex compound.
• iron complex compound generally means any complex containing iron, preferably any compound comprising complexed iron. Suitable methods have been described by Waldoefner and Stief (2011). Briefly, an iron-containing compound and an organic solvent are kept for 10 min at a temperature between 50°C and 50°C below the reaction temperature. Next, the solution is heated to 200 to 400°C to yield nanoparticles. The nanoparticles are oxidized with oxygen, peroxide or a tertiary amineoxide, and treated with nitric acid and ironnitriate resulting in maghemite nanoparticles.
• the iron oxide nanoparticles are provided by thermal decomposition of an iron salt or an iron complex compound.
• Iron salts and iron complexes which are applicable in the method of producing iron oxide nanoparticles are well known to the person skilled in the art and include, but are not limited to, iron(lll) chloride, iron(ll) chloride, iron (III) acetylacetonate, iron carbonyls and equivalents.
• the metal nanoparticles may be treated with H2O2 prior to the incubation with alkoxysilane.
• This optional step is preferred as the iron is fully oxidized to Fe 2 0 3 (maghemite) under defined conditions and, as a consequence, subsequent reaction steps can be conducted in the absence of a protective gas (e.g. argon). Otherwise in the absence of H2O2, it is preferred to work under protective gas such as argon in order to control reaction conditions.
• a protective gas e.g. argon
• the alkoxysilan is preferably a trialkoxysilane. It is more preferably selected from the group consisting of 3-(2-aminoethylamino)-propyl-trimethoxysilane (DIAMO), 3- aminopropyltriethoxysilane (APTES), trimethoxysilylpropyl-diethylenetriamine (TRIAMO) and N-(6-aminohexyl)-3-aminopropyltrimethoxysilane.
• the alkoxysilane is 3-(2-aminoethylamino)-propyl-trimethoxysilane.
• the coating reaction is carried out by adding 0.3 to 0.6 x 10 3 mol, preferably 0.4 to 0.5 x 10 3 mol and especially 0.43 to 0.45 x 10 3 mol trialkoxysilane per 0.9 mol of the metal.
• the incubation with alkoxysilane is performed at a pH of between 2 and 6 (which means that also a pH of 2 or 6 is included into this range), more preferably of between 2.5 and 5.5, still more preferably of 4.5 ⁇ 1.
• the pH may be adjusted to said values, if required.
• Acetic acid can be used to adjust the pH accordingly.
• the metal magnetic nanoparticles are disintegrated prior to the incubation with alkoxysilane.
• the nanoparticles are disintegrated preferably by ultrasound treatment in order to generate a suspension of ball-shaped or cubic electron-dense nanoparticles which can then be subjected to the coating reaction.
• Ultrasound treatment may be done in an ultrasonic bath at 45 kHz 30 min to 2 h, especially for about 1 h.
• This disintegration method preferably is carried out at acidic conditions, preferably between pH 2.5 and 3.0. Disintegration of nanoparticles according to the present invention is, for example, described in Example 1.1.
• Another suitable method for disintegrating nanoparticles is laser-based deagglomeration / laser fragmentation technique (Schnoor et al. 2010).
• the method of producing agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles may further comprise the step of disintegrating the agglomerates in the aqueous suspension after starting the incubation with alkoxysilane, which can be carried out as described above.
• disintegration of the nanoparticles starts with or after the coating step.
• the disintegration step may start prior to the coating step and may be further carried out simultaneously with and/or after the coating step.
• disintegration is started prior to the coating step and is continued during and after the coating step.
• disintegration is carried out for a total of about 24 h or more.
• a suspension Upon possible disintegration and coating, a suspension can be generated that can stably be stored at room temperature. It is assumed that most individual nanoparticles are completely coated with the alkoxysilane, easily adhere to neighboring nanoparticles and form agglomerates. Still, the suspension is an aqueous fluid suspension which is fluent enough to easily pass through syringes and to be injectable into tissue such as tumor tissue.
• an additional step for removing incompletely coated and/or very large agglomerates (e.g. agglomerates of more than 2,000 nanoparticles) from the suspension is carried out.
• Suitable methods for this step are centrifugation (e.g. for 10 min at 2,000 rpm) and filtration (e.g. through a pleated filter with a pore size of 12-25 pm).
• both centrifugation and filtration are carried out. It has been observed that predominantly and completely alkoxysilane-coated nanoparticles do not sediment from the suspension e.g. if centrifuged for 10 min at 2,000 rpm.
• the supernatant of the centrifugation and/or the flow-through of the filtration is/are a suspension which does/do not show sedimentation over one day, preferably one week, especially one month, and therefore can be stored over a long time.
• incompletely coated nanoparticles can be removed to a large extent from the suspension e.g. by such centrifugation. Such removal of incompletely coated nanoparticles is preferred, as incompletely coated nanoparticles have a reduced SAR which therefore reduce the volume SAR of a suspension.
• the disintegration step(s) and optionally the removal step is/are preferably carried out, until the agglomerates of the metal nanoparticles have an average size (z-average) of 30 to 450 nm, preferably of 50 to 350 nm and more preferably of 70 to 300 nm, as determined by light scattering.
• the average size is determined in water according to example 3.
• the size of agglomerates of nanoparticles is determined - in contrast to the size of the ball-shaped or cubic electron-dense single nanoparticles which are forming such agglomerates.
• “Z-average” with respect to the size of agglomerates means the readout of the light scattering size determination as carried out in example 3.
• Z-average values above the provided ranges lead to sedimentation of the nanoparticles and are therefore generally not suitable for the foreseen applications of these nanoparticles. Even if the dispersion may be reconstituted prior to instillation of a tumor, larger agglomerates may lead to serious problems, as the dispersion may partially separate into buffer and agglomerates while passing through the needle leading to an uneven distribution of the nanoparticles within the tissue.
• the agglomerates in the aqueous suspension have an average size of 30 to 450 nm, more preferably of 50 to 350 nm and still more preferably of 70 to 300 nm, as determined by light scattering.
• the size of agglomerated nanoparticles can be measured, as for example described in Example 3.
• the metal nanoparticles are preferably nanoparticles having a metal core with a size of 5 to 25 nm, preferably with a size of 7 to 20 nm, more preferably with a size of 9 to 15 nm, as determined by electron microscopy.
• the agglomerates of the suspension are preferably composed of dozens to hundreds of such individual nanoparticles, whereas any or only very few are small agglomerates of less than ten nanoparticles, as may be determined in transmission electron microscopy (TEM) e.g. according to the method described by Jordan et al. (1996, on page 712, 3.2.2), preferably less than 3 agglomerates of 10 or less nanoparticles in a representative TEM picture displaying 700 nm by 700 nm and at least 1000 nanoparticles.
• TEM transmission electron microscopy
• nanoparticle in this context is one basically ball-shaped or cubic electron-dense nanoparticle visible in transmission electron micrographs.
• a single nanoparticle is a nanoparticle which is not attached to at least one other nanoparticle.
• the shape of the single nanoparticles is ball-shaped or cubic. Size and shape of the nanoparticles can be tailored by adjusting pH, ionic strength, temperature, nature of the salts (perchlorates, chlorides, sulfates, and nitrates), or the Fe(ll)/Fe(l II) concentration ratio (reviewed by Mohapatra and Anand 2010).
• the suspension comprising agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles may have a zeta potential of 25 to 80 mV, preferably of 35 to 70 mV, especially of 45 to 60 mV.
• the zeta potential is determined as described in Example 4 at pH 5.2 and at a conductivity of 2.0 mS/cm (each determined at 25°C).
• the zeta potential is dependent on the successful coating of the nanoparticles as it depends on the amino groups of the alkoxysilanes. Lower zeta potentials indicate an insufficient coating of the nanoparticles
• the correct zeta potential within the provided ranges contributes to the properties of the nanoparticles upon injection into tissue, i.e.
• the zeta potential in the provided ranges ensures optimal colloidal stability and therefore extends the shelf life of the nanoparticle composition.
• the agglomerates of magnetic alkoxysilane-coated metal containing nanoparticles are suspended in a water-based physiologically acceptable buffer.
• physiologically acceptable buffers include for example acetate, citrate, carbonate or phosphate at a pH (at 25 °C) between 5 and 8, preferably between 5 and 6, and especially between 5.1 and 5.8 and a conductivity (at 25 °C) of 1.5 to 2.5 mS/cm, preferably 1.7 to 2.3 mS/cm.
• the osmolality of a suitable suspension is 0.01 to 0.09 Osmol/kg, preferably 0.02 to 0.07 Osmol/kg.
• the specific absorption rate (SAR) of the nanoparticles is larger or equal than 2 W/g of the respective metal (e.g. iron), more preferably larger or equal than 3 W/g of the respective metal and still more preferably 4 to 50 W/g of the respective metal, as determined at a magnetic field strength of 4 kA/m and a frequency of 100 kHz according to the method as described by Jordan et al. (1993).
• the SAR value is determined as described in Example 5.
• high SAR values are preferred, as consequently higher temperatures can be achieved during exposure to an alternating magnetic field. If the SAR value of the nanoparticles is too low, i.e.
• the method further comprises the step of ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles before thermally treating the aqueous suspension.
• this step of ultrasound treatment is performed after nanoparticle preparation and optional coating and before thermal treatment.
• the aqueous suspension after its preparation and before thermal treatment and instillation is stored at temperatures lower than room temperatures.
• the step of ultrasound treatment may be performed after storage and before thermally treating the aqueous suspension in order to ensure that the suspension after storage is still an homogeneously dispersed aqueous fluid suspension, which then by thermal treatment forms a homogeneous paste.
• a homogeneous paste passes more easily through syringes and is injectable into tissue such as tumor tissue.
• the aqueous solution is filled into a syringe for further instillation, it may be objected to ultrasound treatment and then thermally treated in the syringe.
• Ultrasound treatment may be done in an ultrasonic bath at any common frequency. Using a higher frequency may be required for shorter treatment times and using a lower frequency may be sufficient for longer treatment times.
• ultrasound treatment may be performed at 45 kHz for 30 min to 4 h, preferably for 1 h to 3 h, more preferably for 1.5 h to 2.5 h, most preferably for about 2 h.
• ultrasound treatment is performed at room temperature. This ultrasound treatment of nanoparticles according to the present invention is, for example, described in Example 8.
• the present invention relates to a medical device comprising the paste of the present invention.
• the magnetic nanoparticles exert their therapeutic effect upon exposure to an alternating magnetic field through generation of heat as a physical mode of action and do not directly interact with the metabolism of the patient, these nanoparticles are classified in multiple jurisdictions as medical devices. Still, they can be used as powerful tools for the treatment or prophylaxis of tumor diseases and other diseases through hyperthermia and/or thermoablation, where cells are malfunctioning in a certain region of the body.
• Tumor diseases which can be treated with the paste of the present invention are preferably solid tumors, especially local or locally advanced tumors or systemic tumor diseases which cause local problems such as inoperable metastasis.
• Examples are brain tumors, e.g. glioblastoma and astrocytoma, brain metastasis, prostate cancer, prancreatic cancer, hepatocellular carcinoma, head and neck cancer, bladder cancer, gastric cancer, renal cell carcinoma, ovarian carcinoma, cervical carcinoma, sarcoma, basal cell carcinoma and melanoma.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising the paste of the present invention.
• the aqueous suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles can be formulated with active pharmaceuticals such as anti-cancer agents, e.g. chemotherapeutic agents (which can be grouped into alkylating agents, antineoplastic antibiotics, anti-metabolites, natural source derivatives), hormones/growth factors or hormone/growth factor analogues or inhibitors, signal transduction inhibitors and immune therapeutics.
• active pharmaceuticals such as anti-cancer agents, e.g. chemotherapeutic agents (which can be grouped into alkylating agents, antineoplastic antibiotics, anti-metabolites, natural source derivatives), hormones/growth factors or hormone/growth factor analogues or inhibitors, signal transduction inhibitors and immune therapeutics.
• active pharmaceuticals such as anti-cancer agents, e.g. chemotherapeutic agents (which can be grouped into alkylating
• the present invention relates to the paste of the present invention or the medical device or the pharmaceutical composition for use in a method of treating or preventing the diseases, as referred to above, including proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection, in a patient.
• diseases including proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection, in a patient.
• the present invention relates to a method of treating or preventing the diseases, as referred to above, including proliferative disease, cancer, tumor, rheumatism, arthritis, arthrosis or bacterial infection, comprising the step of administering the paste of the present invention to a human or animal patient.
• the paste of the present invention for use in a method of treating or preventing the diseases, as referred to above, or the method of treating or preventing the diseases further includes exposing the patient to an alternating magnetic field.
• the alternating magnetic field is applied hours or days after injecting the paste into the target region, e.g. tumor, of the patient (Johannsen et al. 2007; Thiesen and Jordan 2008; Maier- Hauff et al. 2011).
• the paste of the present invention can further be used in a method for increasing the activity of an anti-cancer agent comprising the steps of administering to a patient in need thereof a pharmaceutical composition comprising the paste of the present invention and administering at least one anti-cancer agent together with at least one pharmaceutically acceptable excipient, carrier and/or solvent.
• the two administrations may be simultaneous, e.g. as separate formulations or the aqueous suspension is formulated with active pharmaceuticals, or one after the other (first nanoparticles, second anti-cancer agent or vice versa), however, in such a way that nanoparticles and anti-cancer agent are present at the same time within the patient’s body, in order to be able to act together and enhance each other’s therapeutic effect.
• the nanoparticle agglomerates remain within the tissue for months or years within the target area and can generate heat upon exposure to an alternating magnetic field
• an administered anti-cancer agent typically acts for hours or days. “Act together” in this context therefore means, that still sufficient pharmacologically active levels of the anti cancer agent are present in the tissue.
• the present invention relates to the paste of the present invention for use in a method for the prophylaxis and/or treatment of tumor diseases, wherein the paste is administered together with an anti-cancer agent in such a way, that nanoparticles and anti-cancer agent are present at the same time within the patient’s body.
• the agglomerates of nanoparticles may be complexed with or covalently coupled to an active pharmaceutical agent or to a targeting agent such as antibodies, antibody fragments or ligands, as known in the art.
• a targeting agent such as antibodies, antibody fragments or ligands
• the coupling of active pharmaceuticals and/or ligands to nanoparticles is described in Jordan et al. (2008), Gao et al. (2011), Waldoefner and Stief (2011) and Ivkov et al. (2005).
• compositions or medical devices of the present invention can easily be combined with conventional therapies used for the respective treatment or prophylaxis of the disease, such as chemotherapy or radiation. They can be used either to increase the effectiveness of the individual treatment and/or reduce side effects of the conventional therapy by lower their dose if combined with the pharmaceutical compositions or medical devices of the present invention.
• Figure 1 Schematic drawing showing the principles of viscosity. Force F is applied on the liquid in direction of flow; A is the area in direction of the flow; v is velocity; y the axis of the distance between the fluid layers parallel to the surface A, r is the distance between the surface of the streaming liquid and the mid of the stream.
• Figure 3a Impact of thermal treatment on viscosity of 6 M suspensions comprising magnetic alkoxysilane-coated metal containing nanoparticles.
• Figure 3a shows viscosity versus duration of heating at 60°C. The viscosity increases with increasing duration of heating. After 48 hours, the maximal viscosity is nearly reached.
• Figure 3b Impact of thermal treatment on yield stress of a 6 M suspension comprising alkoxysilane-coated magnetic metal containing nanoparticles.
• Figure 3b shows viscosity versus the duration of heating at 60°C. The yield stress increases with increasing duration of heating. After 48 hours, the maximal yield stress is nearly reached.
• Figure 4 Photograph showing the impact of thermal treatment of a 6 M suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles on the behavior in a physiological sodium chloride solution.
• the left beaker shows what happens to the paste of the present invention, exemplified by the thermally treated 6 M suspension of magnetic alkoxysilane-coated metal containing nanoparticles, heated at 60°C for 1 h.
• the paste is dropped from a syringe into the solution. Droplets are on the bottom of the beaker.
• the right beaker contains a non-thermally treated 6 M suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles from the same production batch dropped from a syringe into the solution. Rough distributed heaps of solid matter are scattered on the bottom of the beaker.
• Precipitation and Washing NaOH is weighted out into a flask, is solved in purified water to a concentration of 2.13 M and is subsequently chilled to 25°C.
• Fe(lll)chloride and Fe(ll)chloride ratio 2:1 are filled into a glass bottle and solved in purified water to get a 0.48 M Fe(lll)chloride / 0.24 M Fe(ll)chloride solution.
• the iron chloride solution is poured into the NaOH solution and is mixed during a period of about 53 min, while the temperature is constantly held at 25 °C. The generated nanoparticles are left to sediment and the supernatant is removed.
• the nanoparticles are washed with degassed water until the supernatant reaches a conductivity of ⁇ 5mS/cm.
• the nanoparticles used in the following are prepared by this method.
• the NaOH solution may be poured into the iron chloride solution (instead of vice e versa) during a period of about 39 min at 15°C.
• the nanoparticle suspension from above is adjusted with diluted HCI until pH is between 2.5 and 3.0. Afterwards the flask is positioned in an ultrasonic bath and treated with ultrasound at 45 kHz for 1 h while stirring. Now over a time of 90 min 3-(2- aminoethylamino)propyl)trimethoxysilane (Fluka, 48 ml per 1,2 I nanoparticle suspension) is added dropwise, while the pH is kept below a threshold of 5.5 by adding drops of acidic acid, but the pH shall not get lower than 5.0. After this step, the pH is adjusted to 4.65 with diluted HCI and the suspension is further treated with ultrasound for 23 hours.
• 3-(2- aminoethylamino)propyl)trimethoxysilane Fruka, 48 ml per 1,2 I nanoparticle suspension
• the nanoparticles may be treated with H2O2 for two days prior to the coating in order to achieve a finer dispersion of the nanoparticles and a better colloidal stability.
• H2O2 may be used in order to completely oxidize Fe under controlled conditions to Fe 2 0 3 .
• subsequent reactions can be performed in the absence of a protections gas (e.g. argon). This optional step has not been performed to the nanoparticles used herein.
• Dialysis The suspension is purified with a blood dialysis cartridge (Fresenius F8 HPS) against degassed ultrapure water until a conductivity of 400 pS/cm is reached.
• a blood dialysis cartridge Frsenius F8 HPS
• Nanoparticles may also be produced similar to the methods described in Waldoefner and Stief (2011). Briefly, iron(lll) chloride sodium acetate, diaminohexane and ethyleneglycole were combined in a three necked flask and stirred until a homogeneous solution was obtained. Then the mixture was heated strongly until near boiling and was refluxed for five hours. After washing and collecting the particles via centrifugation the dried particles were mixed with trimethyleneoxide in ethylene glycol and heated to 130°C and kept for 2 h. Then the mixture was heated under reflux for 1 h. For the following oxidation step the washed particles were resuspended in nitric acid and treated with iron nitride.
• Nanoparticles may also be produced similar to the methods described by Guardia et al. (2010 a; 2010 b; 2012).
• the suspensions individually filled in 15 ml injection vials with stopper and metal cap, were heated in a water bath at a fixed temperature for the specified time. Designation of sample, its production batch as well as time and temperature for the thermal treatment are given in the respective tables.
• Determination of the iron concentration within a suspension is based on the photometric measurement of the extinction of an iron(ll) phenanthroline complex.
• the complex is generated by extraction of the nanoparticles with hydrochloric acid until the extraction is complete as determined by visual inspection. All iron contained is reduced to iron (II) using hydroxylamine-hydrochloride and transformed into the phenanthroline complex in acetic acid/acetate buffer. Extinction of the complex is determined at 513 nm using a Shimadzu UV-1700 Pharmaspec against an iron(ll) ethylendiammonium sulfate standard (Merck, Darmstadt). The solid content of a suspension is determined by weighing e.g. 1 ml of the suspension prior to and after evaporation of the solvent (e.g. water).
• solvent e.g. water
• a light scattering procedure is used to determine the hydrodynamic size of the nanoparticle preparation (e.g. Malvern ZetaSizer 3000 HSA or Malvern Zetasizer Nano ZS).
• the primary parameter is the z-average value, which is weighted by the scattering intensity. Therefore, in case of a polydisperse distribution, larger nanoparticles are weighted stronger than smaller ones. Furthermore, this method determines the average size of the nanoparticle agglomerates, and not the size of the single or primary nanoparticles.
• a sample is vortexed for 30 sec.
• 75 ml of a 1:1000 dilution of the solution with a concentration of about 0.11 mg/ml for Fe (or other metal) in ultrapure water is prepared and treated for 15 min with ultrasound.
• 20 ml of the solution are injected in the measuring cell of the Malvern ZetaSizer 3000 HSA (or Malvern Zetasizer Nano ZS) and measured according to the recommendations of the manufacture.
• the pH of the solution is determined with a separate pH meter.
• the viscosity of the nanoparticle samples was determined by Malvern material characterization services using rotational rheometry at 20°C, wherein the shear viscosity (in Pa s) was determined in dependence of the shear rate (from 7 to 1500 /s).
• the samples were measured in a rotational rheometer (Bohlin GEMIN 200, Malvern) with cone-plate geometry (2°, 60mm 0), whereby the samples were kept at 20°C each time.
• Table 3 shows that the viscosity of the suspension increases with increasing duration of heating. After 48 hours, the maximal viscosity does not seem to have been reached. A graph of the viscosity values is shown in Figure 3a and the yield stress values are shown in Figure 3b.
• Table 4 Measured data for a 6 molar suspension heated at 40°C or 50°C
• Table 3 shows that the viscosity of the suspension increases with increasing heating temperature. After 48 hours, the maximal viscosity does not seem to have been reached.
• Table 5 Measured data for a 2 molar suspension heated at 60°C Table 5 shows that the viscosity of the suspension increases with increasing duration of heating. After 48 hours, the maximal viscosity seems to have been reached. The viscosity of the suspension is lower as compared to a 6 molar suspension (see Table 3). The behavior of the suspension seems to be similar to the 6-molar suspension.
• Each of the samples was dropped from the syringe via the 19 G-cannula into one of the beakers with the physiological saline.
• the distance between the end of the cannula and surface of the saline solution was approximately 3 to 5 cm.
• the result is shown in Figure 4.
• the left beaker shows what happens to the paste of the present invention, the thermally treated 6 M suspension of magnetic alkoxysilane-coated metal containing nanoparticles, heated at 60°C for 1h. It is readily visible that the form of the drops leaving the syringe is maintained during their way sinking through the physiological saline solution. Obviously, the material regained immediately its pasty consistency before entering the salt solution. Thus, it was not scattered before completion of agglomeration.
• the right beaker contains a non-thermally treated 6 M suspension comprising magnetic alkoxysilane-coated metal containing nanoparticles from the same production batch dropped from a syringe into the solution. It is readily visible that the drops leaving the syringe are scattered by the impact into the salt-solution, before they can solidify by agglomeration. 8. Ultrasound treatment of the aqueous suspension of magnetic metal containing nanoparticles
• the prostate used in this study was collected from a patient on Day 1 of the study and received on Day 2. The study was performed on the day the prostate was received.
• the aqueous suspension of magnetic metal containing nanoparticles used in this study was filled in vials and had been stored therein for about 3 years and 10 months.
• the vial of Group I was put into an ultrasonic water bath at room temperature for 30 minutes. Subsequently, 5ml of the aqueous solution were filled into each of the two syringes and incubated for 2 hours at 60°C.
• the vial of Group 2 was not treated in an ultrasonic water bath. Subsequently, also in this case 5ml of the aqueous solution were filled into each of the two syringes and incubated for 2 hours at 60°C.
• Instillation i.e. introduction of the paste into the prostate tissue
• IV extension tubing fill volume of 2.1ml
• 18G 10" Chiba needles Pumps were run at 0.7ml/hr for a total infusion volume of 0.25ml.
• the right side of all prostates was a control using Group 2 aqueous solution (60°C for 2 hours).
• Tubing and needle were primed in each channel before insertion.
• the pump was started as soon as the needle was placed.
• the channels were primed one by one and only right before the needle was placed.
• the pump was run to dispense volume of 0.25ml from each channel. It was waited for 10 min after instillation was complete in each channel before the needles were removed.
• Pretreating the aqueous suspension of magnetic metal containing nanoparticles in the sonicator had a significant effect on how the aqueous suspension of magnetic metal containing nanoparticles behaves in tubing and needle. There was much less resistance when priming the tubing. However, pre-treating the aqueous suspension of magnetic metal containing nanoparticles via ultrasound treatment helped to make the viscosity more consistent and allowed for easier flow in the tubing while still maintaining higher concentrations in the tissue and less loss due to efflux through the urethra.

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