WO2001028587A2

WO2001028587A2 - Magnetic targeted carrier

Info

Publication number: WO2001028587A2
Application number: PCT/US2000/028615
Authority: WO
Inventors: Scott Raymond Rudge; Terri Lynn Kurtz; Gilles Hugues Tapolsky; Yuhua Li
Original assignee: Ferx Incorporated
Priority date: 1999-10-18
Filing date: 2000-10-13
Publication date: 2001-04-26
Also published as: JP2005200426A; AU1090501A; CN1420787A; EP1221974A2; CZ20021357A3; WO2001028587A3; NZ518311A; AU772755B2; CZ293919B6; JP2003512336A; DE1221974T1; CA2387925A1; IL149004A0; CN1287859C; MXPA02003891A; KR20020064299A; BR0014877A; ES2182731T1; JP2003321348A; HK1045110A1

Abstract

The invention relates to magnetically responsive compositions comprising iron-ceramic particles used to carry substances for in vivo medical diagnosis and/or treatment. The particles are formed by joint deformation of iron and ceramic powders. Diagnostic or therapeutic substances may be adsorbed thereon. The particles may be produced by mechanical milling of a mixture of iron and ceramic powders.

Description

MAGNETIC TARGETED CARRIER COMPOSED OF IRON AND POROUS MATERIALS FOR THE TARGETED DELIVERY OF BIOLOGICALLY ACTIVE

AGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the United States national phase of International Patent

Application No. PCT/US00/ , filed October 13, 2000, which claims priority to United

States Provisional Application Serial No. 60/160, 293, filed October 18, 1999.

INTRODUCTION

This invention relates to compositions, methods of manufacture and methods for delivery of biocompatible particles to a selected location in a body and, more particularly, relates to particles capable of carrying biologically active compounds and which provide for targeted magnetic transport ofthe particles and their maintenance in a predetermined place as a localized therapeutic treatment for disease, diagnostic aid, or bifunctional composition capable of acting as both a diagnostic and therapeutic agent.

The site-specific delivery of biologically active agents would enable enhancement of therapeutic activity of chemotherapeutics while minimizing systemic side effects. Magnetic carrier compositions for treating various disorders have been previously suggested and utilized, and include compositions which are guided or controlled in a body in response to an externally applied magnetic field. (See Lieberman et al., U.S. Patent 4,849,209; Schroder et al., U.S. Patent 4,501,726; Chang, U.S. Patent 4,652,257; and Mirell, U.S. Patent 4,690,130). One such known composition, deliverable by way of intravascular injection, includes microspheres made of a ferromagnetic component covered with a biocompatible polymer (albumin, gelatin, and polysaccharides) which also contains a drug (Driscol, CF. et al., Prog.

Am. Assoc. Cancer Res., 1980, p. 261).

It is possible to produce albumin microspheres up to 3.0 μm in size containing a magnetic material (magnetite Fe₃0 ) and the anti-tumoral antibiotic doxorubicin (Widder, K. et al, J. Pharm. Sci., 68:79-82, 1979). Such microspheres are produced through thermal and/or chemical denaturation of albumin in an emulsion (water-in-oil), with the disperse phase containing a magnetite suspension in a medicinal solution. A similar technique has been used to produce magnetically controlled, or guided, microcapsules covered with ethylcellulose containing the antibiotic mitomycin-C (Fujimoto, S. et al, Cancer, 56: 2404- 2410,1985). Magnetically controlled liposomes, 200 nm to 800 nm in size, capable of carrying preparations that can dissolve atherosclerotic formations are also known. This method is based upon the ability of phospholipids to create closed membrane structures in the presence of water (Gregoriadis G., Ryman B.E., Biochem. J., 124:58, 1971).

Such previously known compositions have not always proven practical and/or effective. Often, there is ineffective drug concentration delivered to the targeted site. Many of the compositions lack adequate transport capacity, exhibit weak magnetic susceptibility, and/or require extremely high flux density magnetic fields for their control. In some cases, there is no real localization ofthe particles enabling a precise local therapy. Other shortcomings include non-specific binding and toxicity to untargeted organs for compositions incorporating antibodies and peptides, and drug diffusion outside ofthe desired site for intra- tumoral injection based technologies. Some compositions are difficult to manufacture or prepare consistently, sterilize, and store without changing their designated properties.

Thus, there remains a need for an effective biocompatible composition that is capable of being transported magnetically and that is relatively easy to manufacture, store and use. One suggested composition comprises ferrocarbon particles for use as magnetically susceptible material for magnetically controlled compositions. These particles have a major dimension (i.e., largest diameter) of about 0.2 μm to about 5.0 μm (and preferably from 0.5 μm to 5.0 μm) and contain from about 1.0% to about 95.0% (by mass) of carbon, with the carbon strongly connected to iron. The particles are obtained by jointly deforming (i.e., milling) a mixture of iron and carbon powders. See U.S. Patents 5,549,915; 5,651,989;

5,705,195 and U.S. Patent Application Serial Nos. 09/003,286, and 09/226,818, which are incorporated herein by reference.

Previous applications of this technology arose from a desire to make alloys that were not achievable through smelting processes. Not all conceivable alloys can be made by smelting, as the solubility of one molten metal in another limits the concentrations that the mixtures can achieve Milled ferrocarbon particles were derived as an aαaptation ot a technique for making alloys The milling technique is fine tuned to produce a αurable connection between the two matenals without intimately mixing them as an alloy, which would result in reduction or elimination of both the magnetic moment and/or the drug carrying capacity The idea of combining iron and carbon by milling arose from their natural mixability, as in the smelting process for forming alloys

SUMMARY OF THE INVENTION

It has now been found that iron-ceramic particles can be produced by the milling method This is surprising because alloys using these matenals have not previously been demonstrated Thus, it was not thought that a durable interface between the iron and the ceramic mateπal could be formed

Iron-ceramic composite particles show great versatility to bind to various drugs that adsorb at the particle surface for easy incorporation of the active agent Additionally, lron- ceramic particles utilize metallic iron with a higher magnetic susceptibility than iron oxides, thereby facilitating and expediting mobility to the treatment site Furthermore, the biocompatibility properties of ceramics are well known

Biocompatible and biodegradable ceramic matenals, based on hydroxyapatite and other calcium phosphate deπvative matenals have been used as bone replacement matenal m dental and skeletal procedures However, the concept of magnetically targeting a ceramic matenal used as a earner is completely novel This invention provides a magnetically responsive composition that carnes biologically active substances Generally, iron-ceramic composite particles can be used to target the delivery of a number of biologically active agents, diagnostics, or bifunctional compositions Methods of production and use thereof are also provided

The aim of this invention is to improve some parameters of magnetically controlled compositions used for the targeted transport of a biologically active substance, including enabling use of natural bone constituents in the carrier particle, expanding the categones of therapeutics and diagnostics for which this technology can be used, increasing relative absorption capacity and magnetic susceptibility by, for example, providing a large number of ionic groups that enable binαmg of compounds by ionic interactions, improving biocompatibility ana biodegradability, intensifying diagnostic and therapeutic effect, simplifying the technology of manufacturing the magnetically controlled composition, and ensunng its guaranteed long-term storage capabilities without changing the desired charactenstics

This is achieved by using suitable composite, iron-ceramic particles, as a magnetically susceptible matenal for a magnetically controlled composition The particles are disk and sphencally shaped, approximately 0 1 to 10 0 μm diameter, and contain 1 0% to 95 0% ceramic (or a denvatized ceramic) and 5 0% to 99 0% iron, by iron They are obtained by jointly deforming (i e , milling) a mixture of iron and ceramic powders

Adsorption occurs on the surface, or modified surface, of the particle so the drug is readily available and capable of incorporation at the treatment site

The powders are combined in a planetary ball, or attntion mill with a solvent (e g ethanol) The resulting composite powder is then sieved or magnetically separated to obtain the desired fraction of product, and correspondingly, the desired magnetic susceptibility The biologically active agent or diagnostic aid is adsorbed to or deposited on to the composite and administered to the patient m a suspension of the composite in a stenle diluent

The methods of use include methods for localized in vivo diagnosis or treatment of disease providing a magnetically responsive iron-ceramic earner having adsorbed thereon a biologically active substance selected for its efficacy m diagnosing or treating the disease, and injecting the earner into the body of a patient For example, the earner is injected b> inserting delivery means into an artery to within a short distance from a body site to be treated and at a branch or branches (preferably the most immediate) to a network of artenes carrying blood to the site The earner is injected through the delivery means into the blood vessel. Just pnor to injection, a magnetic field is established extenor to the body and adjacent to the site with sufficient field strength to guide a substantial quantity ofthe injected earner to, and retain the substantial quantity of the earner at, the site Preferably, the magnetic field is of sufficient strength to draw the earner into the soft tissue at the site adjacent to the network of vessels, thus avoiding substantial embohzation of any of the larger vessels by the carrier particles. See, for example, U.S. Provisional Application Ser. No. 60/160,293, which is incorporated herein by reference.

It is therefore an object of this invention to provide a highly magnetically responsive composition for optionally carrying biologically active substances and methods of production and use thereof.

It is another object of this invention to provide a magnetically responsive carrier for biologically active substances that has high magnetic responsiveness, yet is durable during storage and use.

It is another object of this invention to provide a magnetically responsive composition comprising particles approximately 0.1 to 10.0 μm in diameter, each iron-ceramic particle containing 1.0% to 95.0% ceramic (or a ceramic derivative) and 5.0% to 99.0% iron, by mass.

It is still another object of this invention to provide a composition utilized for localized in vivo diagnosis or treatment of disease including a carrier with composite iron- ceramic particles approximately 0.1 to 10.0 μm in diameter, each iron-ceramic particle containing 1.0% to 95.0% ceramic (or a ceramic derivative) and 5.0% to 99.0% iron, by mass, and having adsorbed thereon one or more optional biologically active substances selected for efficacy in diagnosing and/or treating a particular disease.

With these and other objects in view, which will become apparent to one skilled in the art from the following description, this invention resides in the novel construction, combination, arrangement of parts and methods substantially as hereinafter described, and more particularly defined by the appended claims, it being understood that changes in the precise embodiment of the herein disclosed invention are meant to be included as they come within the scope of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1 is a magnified photograph (X1000) of composite iron-silica particles FIG. 2 is a magnified photograph (X3000) of composite iron-silica particles. FIG. 3 is a flow diagram ofthe production process of this invention. FIG. 4 is a Doxorubicm binding curve for an iron-silica gel composite

FIG. 5 is Doxorubicm binding curve for an iron-C18 composite FIG. 6 is a Scanning Electron Microscopy photograph showing the morphology of iron-hydroxyapatite particles.

FIG. 7 is the same frame as m FIG. 6, with momtonng of backscatter to show iron in white and hydroxyapatite in black

FIG 8 is the spectra of the particle shown in FIG 6 confirming that the white spots are composed of iron.

FIG. 9 is the spectra ofthe particle shown in FIG. 6 confirming that the black spots are composed of hydroxyapatite. FIG. 10 is particle size analysis of hydroxyapatite particles using light scattenng technique.

FIG.l 1 is a magnetic susceptibility curve of an iron-hydroxyapatite microparticle using magnetometer technique.

FIG. 12 is a Langmuir isotherm plot for iron-hydroxyapatite. FIG. 13 is a Langmuir isotherm plot for hydroxyapatite (no iron)

FIG. 14 is a doxorubicm desorption profile for iron-hydroxyapatite. FIG. 15 shows labeling of iron-hydroxy apatite particles with Indium 1 1 1 by direct incubation and stability in different media.

FIG. 16 shows labeling of iron-hydroxyapatite particles with Indium 111/oxyqumoline and stability in different media.

DETAILS OF THE INVENTION

The invention is a composite particle compnsed of 1 0% to 95 0% a ceramic ₍or ceramic denvative) and 5 0% to 99 0% iron, by mass With compositions having less than 1 0% ceramic, the binding capacity of a particle is decreased to the point of being largely ineffective for carrying biologically active substances With compositions of greater than

95 0% ceramic, the magnetic susceptibility is generally reduced beyond an effective level for targeting biologically active substances in vivo The particles are disk and sphencally shaped, approximately 0 1 to 10 0 μm in diameter

The term "ceramic" means a natural or synthetic porous, adsorptive matenal It is usually, but not necessanly an oxide or mixed oxide, wherein the oxide is metallic or non- metallic It is usually, but not necessanly inorganic It is usually, but not necessanly without a crystalline structure Examples of synthetic ceramic matenals include, but are not limited to tπcalcium phosphate, hydroxyapatite, aluminum hydroxide, aluminum oxide, aluminum calcium phosphate, dicalcium phosphate dihydrate, tetracalcium phosphate, macroporous tnphasic calcium phosphate, calcium carbonates, hematite, bone meal, apatite wollastonite glass ceramics and other ceramic or glass matnces Also included are polymers that have a degree of crystallmity that will support pores and adsorption Examples of such polymers mclude, but are not limited to polyethylenes, polypropylenes, and polystyrenes Appropnate matenals based upon these parameters will be apparanent to any person having ordinary skill m the art A table of examples follows

Oxide Non -metallic Amorphous

Silica Y Y Y

Hydroxyapatite Y N Y

Zeolites Y N N

Aluminas Y N Y

Diamond N Y N

Also included in the definition of "ceramic" are silica and silica denvatives (including, but not limited to octadecycl silane [Cι₈], octyl silane [C₈], hexyl silane [Co], phenyl silane [C₆], butyl silane [C₄], aminopropylsilane [NH₃C₃], cyano nitnle silane [CN], tnmethylsilane [Ci], sulfoxyl propyl silane [SO₄C J, dimethylsilane [Ci], acidic cation- exchange coating [SCX . basic quaternary ammonium anion exchange coating [SAX], dihydroxypropyl silane [diol]), into a composite particle 0.1 - 10.0 um in diameter. By wav of example, the following silicas are useful for forming the composites of the invention.

Eka Nobel Kromasil®

Packing Particle Pore Pore Surface Carbon Phase Bonded End Material Shape Size Volume Area Load Type Phase Cap & Sιze (A) (ml/g) (m²/g) (%) Coverage (μm) (μmol/m²)

Kromasil S.5,7,10, 100 0.9 340 (elemental Silica 13.16 analysis)

Kromasil S.5,7,10, 100 0.9 340 4.7 Monomen-c 4.3 CI 13, 16

Kromasil S.5,7,10, 100 0.9 340 Monomeπc Yes C4 13,16

Kromasil S.5,7,10, 100 0.9 340 12 Monomenc 3.6 Yes C8 13,16

Kromasil S,5,7,10, 100 0.9 340 19 Monomeπc 3.2 Yes C18 13,16

EM Science ticle Fore Fore Surface Ca bonGεc jnε Par rDor Phase trie

Shape Size Volume Area Load Type Phase

Mate τrιiaall Cap

&Sιze (A) (mlg) (m^:/gj (%) Coverage

(μm) (μmol/m")

Lichrosorb Si

60 1,5,10 60 550 No

Lichrosorb Si

100 1,5,10 100 420 No

Lichrosorb RP-18 1,5,10 60 150 16.0 Monomeπc 1.55 No Lichrosorb 1,5,10 60 9.0 M<Λomenc 0.78 No RP-8

Lichrosorb 1,5,10 60 0.7 550 12 . 2.5 Yes RP-select B

LichrospherSi S.3,5, 60 0.95 650 No

60 10

LichrospherSi S.5,10 100 125 420 No 100

Lichrospher S.3,5, 60/100 5 350 12.5 4.1 No

RP-8 10

Lichrospher S, 60/100 1.25 350 13 -1 ι Yes

RP-8E/C 3,5,10

Lichrospher S,3,5, 100 1.25 350 214 3.9 No

RP-18 10

Lichrospher S.3,5, 100 1.25 350 21.5 Yes

RP-18 EC 10

Lichrospher S.3,5, 100 1.25 350

CN 10

Lichrospher S.3,5, 100 1.25 350 4.5 3.8

NH2 10 3,5, 100 1.25 350 8.3 4.0

Lichrospher S, 3,5, 60 0.9 360 12.0 Yes

RP-select B 10 End

Packing Particle Pore Porε Surface Carbon Phase Bonded se Cap Matenal Shape Size Volume Area Load Type Pha & Size (A) (ml/g) (πr/g) (%) Coverage (μm) (umol/πr)

Inertsil S, 5 150 . 320 0 - - No

Silica

Inertsil S, 5 150 - 320 18.5 Monomeπc 3.23 Yes

ODS-2

Inertsil S, 3, 5 100 - 450 15 Monomeπc - -

ODS-3

Inertsil C8 S, 5 150 - 320 10.5 Monomeπc 3.27 Yes

Inertsil S.5 100 - 450 10 Monomeπc - Yes

C8-3

Inertsil Ph S. 5 150 - 320 10 Monomeπc 2.77 Yes

(Phenyl)

Inertsil S, 5 100 - 450 10 Monomeπc - Yes

Ph-3

(Phenyl)

Inertsil C4 S, 5 150 - 320 7.5 Monomeπc 3.77 Yes

Monomeπc * Yes

Inertsil S, 5 80 - 450 16 8θA

Inertsil S, 10 100 - 350 14 - - -

Vvdac/The Separations Group

Packing Particle Pore Pore Surface Carbon Phase Bonded End Matenal Shape Size Volume Area Load Type Phase Cap & Sιze (A) (ml/g) (m²/g) (%) Coverage (μm) (μmol/m^:)

Vydac SD. 5, 300 0.6 90 8 Polymeric 4.16 Yes 201TP 10 C18

Vydac SD, 5, 300 0.6 90 8 Polyermic 4.16 Yes 218TP 10 C18

Vydac SD, 5, 300 0.6 90 3 Polymeric 4.89 Yes 214TP 10 C4

Vydac S, 5, 10 80 0.8 450 13.5 - 1.53 - 201HS CI S

Waters

Packing Particle Pore Pore Surface Carbon Phase Bonded End Material Shape Size Volume Area Load Type Phase Cap & Size (A) (ml/g) (πr/g) (%) Coverage (μm) (μmol/m^")

μBondapak 1, 10 125 1.0 330 10 Monomeric 1.46 Y^'es C18 μBondapak I, 10 125 1.0 330 2.08 Yes Phenyl μBondapak I. 10 125 1.0 330 .:> 1.91 No NH2 μBondapak I. 10 125 1.0 330 2.86 Yes CN μPorasil I. 10 125 1.0 330 No Silica

Novapak S, 4 60 120 3.41 Yes C18

Novapak S, 4 60 0.3 120 2.34 Yes Phenyl

Novapak S, 4 60 0.3 120 1.65 Yes

CN

Novapak S, 4 60 0.3 120 No Silica

Resolve CI 8 S, 5, 10 90 0.5 175 10 2.76 No

Resolve C8 S. 5, 10 90 0.5 175 2.58 No

Resolve CN S, 5, 10 90 0.5 175 2.53 No

Resolve S, 5, 90 0.5 175 0 No Silica 10

Spherisorb S, 3, 5, 80 0.5 220 No Silica 10

Spherisorb S, 3, 5, 80 0.5 220 Monomeric 1.47 Partial

Packms Panicle Pore Porε Surface Carbon Phase Bonded Enc

Material Shape Size Volume Area Load Type Phase Cap

& Sιze (A) (ml/g) (m^:/g) (%) Coverase

(μm) (μmol/m^:)

Spherisorb S, 3, 5, 80 0.5 220 12 Monomenc 2.72 Yes ODS-2 10

Spherisorb S, 3, 5, 80 0.5 220 Monomeric 2.51 Yes C8 10

Spherisorb S, 3, 5, 80 0.5 220 Monomeric 2.27 Yes C6 10

Spherisorb S, 3, 5, SO 0.5 220 Monomeric 1.08 Partial

Phenyl 10

Spherisorb S, 3, 5, 80 0.5 220 3.5 Monomeric 2.37 No

CN 10

Spherisorb S, 3, 5, 80 0.5 220 Monomeric 1.58 No NH2 10

Spherisorb S, 5, 80 0.5 220 No SAX 10

Spherisorb S, 5, 80 0.5 220 SCX 10

Symmetry S 100 340 19 5.09 Yes

YMC

Packing Particle Pore Pore Surface Carbon Phase Bonded End

Matena Shape Size Volume Area Load Type Phase Cap

1 & Size (A) (ml/g) (πr/g) (%) Coverase

(μm) (μmol/m^")

C18-A S, 120 1.0 -300 17 Monomeπc - γ_es

3,5,7,10,15

CI S- S, 120 1.0 -300 17 Monomeric - γ_es

AM 3,5,7, 10,15

+

ODS- S, 120 1.0 -300 16 Monomeπc - Yes

AQ 3,5,7,10,15

C8 S, 120 1.0 -300 10 Monomeric - γ_es

3,5,7, 10,15

+

Phenyl S, 120 1.0 -300 9 Monomeπc - γ_es

3,5,7,10,15

+

C4 S, 120 1.0 -300 7 Monomeric - γ_es

3,5,7,10,15

+

Basic S, - - - - Monomeric - Yes

3,5,7,10, 15

+

Note: Bonded phase coverage calculated as per Sander, L.C., and Wise, S.A., Anal. Chem., 56: 504- 510, 1984. Material characterisncs obtained from literature published by the material manufacturer or an authorized representative thereof.

The powders are mixed in a planetary ball, or attntion mill in the presence of a πquiα for example, ethanol, to inhibit oxidation of the iron The liquid may also serve as a lubncant dunng the milling of the iron and ceramic powder, to produce the appropnate particle size distribution It also may reduce compacting of the ceramic dunng processing As a result, the porosity of the ceramic deposits in the composition is maintained so as to maximize adsorption capacity ofthe particles

The mixture is put into a standard laboratory planetary ball, or attntion mill of the type used in powder metallurgy The mill holds canisters containing the iron and ceramic powders, ethanol, and metal or metal alloy balls of vanous diameters For example, the mill can have 6 mm diameter balls composed of case hardened metal carbide An appropnate amount of a liquid (e g , ethanol), is added for lubncation Depending on the type used, the mill is run between 2 and 14 hours at speeds of 100 φm to 1000 φm It is believed that mill speeds over 1000 φm could create an undesirable quantity of overly small particles Appropπate liquids and milling conditions are easily determined by any person having ordinary skill m the art.

After joint deformation of the iron-ceramic mixture, the particles are removed from the mill and separated from the grinding balls, for example, by a strainer The particles may be re-suspended in ethanol and homogenized to separate the particles from one another The ethanol is removed, for example, by rotary evaporation, followed by acuum drying Any suitable drying technique may be employed, for example, in a vacuum oven (purging )

Particles should be handled so as to protect against oxidation of the iron, for example, a nitrogen environment.

The resulting dned powder may then be sieved or magnetically separated to obtain the desired fraction of product providing the desired magnetic susceptibility and therapeutic or diagnostic binding capacity The product is then packaged into dosage units in a nitrogen- purged glove box and terminally steπhzed Any suitable stenhzation technique may be employed For example, the iron-ceramic particles may be sten zed using gamma irradiation and the aqueous solution of excipients may be stenhzed by autoclave

When ready for use, the biologically active agent or agents are adsorbed to or precipitated onto the composite The composite, with the active agent adsorbed, is administered to the patient m a suspension of the composite in a steπle diluent The iron-ceramic Darticies are useful as a earner for dehvenng one or more aαsorbeα biologically active substances to specific body sites under control of an external magnetic field As used herein, the term "biologically active substance" includes substances useful for in vivo medical diagnosis and/or treatment Biologically active substances include, but are not limited to, antineoplastics, blood products, biological response modifiers, anti-fungals, antibiotics, hormones, vitamins, proteins, peptides, enzymes, dyes, anti-allergies, anti-coagulants, circulatory agents, metabolic potentiators, antituberculars, antivirals, antianginals, anti-inflammatones, antiprotozoans, antirheumatics, narcotics, opiates, diagnostic imaging agents, cardiac glycosides, neuromuscular blockers, sedatives, anesthetics, as well as paramagnetic and radioactive particles Other biologically active substances may include, but are not limited to, monoclonal or other antibodies, natural or synthetic genetic matenal ana prodrugs As used herein, the term "genetic matenal" refers generally to nucleotides and polynucleotides, including nucleic acids, RNA and DNA of either natural or synthetic ongin. including recombinant, sense and antisense RNA and DNA Types of genetic matenal may include, for example, genes earned on expression vectors, such as plasmids. phagemids, cosmids, yeast artificial chromosomes, and defective (helper) viruses, antisense nucleic acids, both single and double stranded RNA and DNA and analogs thereof Also included are proteins, peptides and other molecules formed by the expression of genetic matenal For in vivo diagnostic imaging, the type of detection instrument available is a ma_jor factor in selecting a given radioisotope The radioisotope chosen must have a type of decav that is detectable for a given type of instrument Generally, gamma radiation is required Still another important factor in selecting a radioisotope is that the half-life be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deletenous radiation with respect to the host is minimized. Selection of an appropnate radioisotope would be readily apparent to one having average skill in the art Radioisotopes which may be employed include, but are not limited to ^99mTc, ^{l 42}Pr, ^{l 61}Tb, '^S6Re, and ^{l 88}Re Additionally, typical examples of other diagnostically useful compounds are metallic ions including, but not limited to ' "in. ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹TI Furtheimore, paramagnetic elements that are particularly useful in magnetic resonance imaging and electron spin resonance techniques include, but are not limited to ^ Gd, "Mn, ¹⁶²Dy, ⁱ²Cr, and ⁵⁶Fe It is also noteα tnat raαioisotopes are also useful m radiation therapy tecnmques Generally, alpha and beta radiation is considereα useful for therapy Examples of therapeutic compounds mclude, but are not limited to ²P, ¹⁸⁶Re, ¹⁸⁸Re, ^{l 23}I ¹²ⁱI, ⁹⁰Y, ^{l 66}Ho, "³Sm, ^{, 42}Pr, ¹⁴³Pr, ¹⁴⁹Tb, ^16,Tb, ^{ι π}In, ⁷⁷Br, ²¹²Bι, ²¹³Bι, ²²³Ra, ²¹⁰Po, ^{, 9;}Pt, ^19imPt, ²^Fm, ^{, 6i}Dy, ¹⁰⁹Pd, ¹²¹Sn, ^{12 /}Te, and ²¹ Αt The radioisotope generally exists as a radical within a salt, however some tumors and the thyroid may take up iodine directly The useful diagnostic and therapeutic radioisotopes may be used alone or m combination

The iron-ceramic composite particle suφasses previous inventions by utilizing metallic iron that has a higher magnetic susceptibility than iron oxides that facilitates and expedites mobility to the treatment site Advantages over current iron-carbon composite products include surface binding versatility, as well as biocompatibility and biodegradation properties of ceramics that are relatively well known

As a general pnnciple, the amount of any aqueous soluble biologically active substance adsorbed can be increased by increasing the proportion of ceramic in the particles up to a maximum of about 50% by mass of the composite particles without loss of utility of the particles in the therapeutic treatment regimens descπbed in this application In many cases it has been observed that an increase the amount of adsorbed biologically active substance is approximately linear with the increase in ceramic content However, as ceramic content increases, the susceptibility, or responsiveness, of composite particles to a magnetic field decreases, and thus conditions for their control in the body worsen (although adsoφtion capacity increases) Therefore, it is necessary to achieve a balance in the iron ceramic ratio to obtain improved therapeutic or diagnostic results To increase the amount of agent given dunng a treatment regimen, a larger dose of particles can be administered to the patient, but the particles cannot be made more magnetic by increasing the dose Appropnate ratios may be determined by any person having average skill m the art.

It has been determined that the useful range of iron ceramic ratio for particles intended for use in in vivo therapeutic treatments as descnbed in the application is, as a general rule, from about 99 1 to about 5 95 for example about 80 20 to about 60 40 The maximum amount of the biologically active substance that can be adsorbed in the composite lromceramic earner particles of any given concentration of ceramic will also differ depending upon the chemical nature of the biologically active substance, and, in some cases, the type of ceramic used in the composition Any person naving ordinary skill in the art will be able to determine the proper ratio for the desired application

Because it is convenient to prepare and market the earner particles in a dry form, the excipients may be prepared m dry form,and one or more dry excipients are packaged together with a unit dose of the earner particles A wide vanety of excipients may be used, for example, to enhance adsoφtion or desoφtion, or to increase solubility The type and amount of appropnate dry excipients will be determined by one of skill in the art depending upon the chemical properties ofthe biologically active substance Most preferably, the package or kit containing both the dry excipients and dry earner particles is formulated to be mixed with the contents of a vial containing a unit dose of the drug and sufficient amount of a biologically compatible aqueous solution, such as saline, as recommended by the drug manufacturer, to bπng the drug to a pharmaceutically desirable concentration Upon mixture of the solution containing the dilute drug with the contents of the kit including the dry components (ι e the dry earner particles and dry excipients), the drug is allowed to adsorb to the earner particles. forming a magnetically controllable composition containing a therapeutic amount of the biologically active substance adsorbed to the earner particles that is suitable for m vivo therapeutic or diagnostic use

Alternatively, a liquid kit may be employed. Here, the earner particles are contained as one unit, for example, in a vial, while the aforementioned excipients are contained m another unit m the form of an aqueous solution At the time of administration, the ferroceramic particles are mixed with the contents of a vial containing a unit dose of the drug and sufficient amount of a biologically compatible aqueous solution, such as saline, as recommended by the drug manufacturer, to bnng the drug to a pharmaceutically desirable concentration. Subsequently, the resulting particles having the biologically active substance adsorbed thereon, are mixed with yet another unit containing the excipients in aqueous solution Any suitable stenhzation technique may be employed For example, the ferroceramic particles may be stenhzed using gamma irradiation and the aqueous solution of excipients may be stenhzed by autoclave Use of autoclave undesirably oxidizes the ferroceramic particles Also, when the biologically active agent to be adsorbed to or deposited onto the microparticles is soluble in an aqueous medium, the buffer used can have an impact on the overall binding Any person having ordinary skill m the art would oe able to determine the most appropnate buffer

A diagnostic or therapeutic amount of biologically active substance adsorbed to the earner particles will be determined by one skilled m the art as that amount necessary to effect diagnosis or treatment of a particular disease or condition, taking into account a vanety of factors such as the patient^'s weight, age, and general health, the diagnostic or therapeutic properties of the drug, and the nature and seventy of the disease

A number of considerations are involved m determining the size of earner particles to be used for any specific therapeutic situation The choice of particle size is determined in part by technological constraints inherent in producing the particles under 0 2 μm in size In addition, for particles less than about 1 0 μm m size, the magnetic control in blood flow and the carrying capacity is reduced Relatively large particle sizes can tend to cause desirable or undesirable embohzation of blood vessels dunng injection either mechanically or by facilitating clot formation by physiological mechanisms The dispersion may coagulate, which makes injections more difficult, and the rate at which biologically active substances desorb from the particles in the targeted pathological zones may decrease The method (such as is descnbed below) of milling together a mixture of iron and ceramic powders produces an irregularly shaped form with a granular surface for the particles, and results in a particle population having an average major dimension of about 0 1 μm to about 5 0 μm Because the iron in the particles descnbed in this invention is not in the form of an iron oxide, as is the case in certain previously disclosed magnetically controlled dispersions, the magnetic susceptibility, or responsiveness, of ferroceramic particles is maintained at a high level

The lronxeramic particles are charactenzed by particles of iron and particles of ceramics bound together The two components are maintained as individual entities The charactenstic substructure of the particles formed dunng the process of joint deformation of the mechanical mixture of iron and ceramic powders, also increases the magnetic susceptibility of iron inclusions m ferroceramic particles as compared with iron particles having other types of substructure Because of the large surface of ceramic deposits in the particles, the adsorbed biologically active substance can compnse about 100% r 50% by weight, relative to the ceramic fraction of the particle, that being vaπable from about 5% to 95% of the initial particle mass, ana most preferably from 15% to 60% In different terms, this can oe up to about 200 mg of adsorbed biologically active substance per gram of particles Therefore, in use, much less of the earner is injected to achieve a given dose of the biologically active substance or, alternatively, a higher dosage of the biologically active substance per injection is obtained than is the case with some previously known earners

The following descnbes a method for producing small quantities of the ferroceramic composition of this invention, it being understood that other means and mechanisms besides milling could be conceived of for jointly deforming iron and ceramic powders, which compnse the essential starting elements for production of the earner. The procedure utilized exerts mechanical pressure on a mixture of ceramic and iron particles to deform the iron particles and develop a substantial substructure, which captures the ceramic The formation of the ferroceramic particles is accomplished without the addition of heat in the process (although the mixture heats up dunng the mechanical deformation step), and is conducted m the presence of a liquid, for example ethanol, to inhibit oxidation of the iron and to assure that the particles produced are clean (stenle). The liquid may also serve as a lubncant duπng the milling ofthe iron and ceramic powder, and may reduce compacting of ceramic dunng processing. As a result, the density of the ceramic deposits m the composition is maintained so as to maximize adsoφtion capacity ofthe particles

As the joint deformation of the particles and ceramics continues, there develops at the interface ofthe two solids a third phase compnsed of a molecular mixture of iron and ceramic. This interface stabilizes the particle such that it is durable to stenhzation and in vivo use. This interface is expected to form with other types of ferro particles, such as ferrocarbon, as molecular mixtures of iron and carbon exist in nature or can be formed by smelting, for example, cementite and steel Ferroceramic mixtures are not commonly known or manufactured such that a molecular mixture may be found at the interface ofthe two substances

For example, to produce particles having an average of about 75 25 iron ceramic ratio by mass, one part of substantially pure iron particles having average diameters from 0 1 um to 5 μm in size are mixed with about 0 1 to 1 0 parts by weight of substantially pure ceramic granules (typically about 0 1 μ to 5 0 μm in diameter) The iron particles and ceramic granules are mixed vigorously to achieve good distnbution throughout the volume Each biologically active substance should be evaluated individually with the various types of ceramics in order to determine the optimum reversible ceramic binding. Factors such as pH, temperature, particulate size, salts, solution viscosity and other potentially competing chemicals in solution can influence adsoφtion capacity, rate, and desoφtion parameters. The mixture is put into a standard laboratory planetary ball, or attrition mill of the type used in powder metallurgy. For example, the mill can have 6 mm diameter balls. An appropriate amount of a liquid, for example ethanol, is added for lubrication. The mixture is milled for between 1 and 12 hours, or for the time necessary to produce the particles heretofore described. Depending on the mill used, the speed of the mill may be anywhere in the range from about 100 φm to about 1000 φm (typically about 300 φm. After joint deformation of the iromceramic mixture, the particles are removed from the mill and separated from the grinding balls, for example, by a strainer. The particles maybe resuspended in ethanol and homogenized to separate the particles from each other. The ethanol is removed, for example, by rotary evaporation, followed by vacuum drying. Any suitable drying technique may be employed. Particles should be handled so as to protect against oxidation of the iron, for example, in a nitrogen environment.

After drying, the particles should be collected according to appropriate size. For example, the particles may be passed through a 20 μm sieve and collected in an air cyclone to remove particles larger than 20 μm. The cyclone only collects particles of a certain size and density, providing a method for removing fines and loose ceramic. The sieved particles may be packaged under nitrogen and stored at room temperature.

Particles may be subaliquoted into dosage units, for example, between 50 and 500 mg per dose, and may be further overlayed with nitrogen, for example. Dosage units may be sealed, for example, with butyl rubber stoppers and aluminum crimps. Dosage units may then be sterilized by appropriate sterilization techniques, for example, gamma irradiation between 2.5 and 4.0 Mrads. Other sterilization techniques may also be used, for example, dry heat and electrobeam sterilization.

When ready for use, or before packaging if the carrier is to be prepared with a preselected biologically active substance already adsorbed thereon, about 50 mg to 150 mg (about 75 mg to about 100 mg is preferred to be absolutely assured of maximum adsoφtion) of the biologically active substance in solution is added to 1 gram of the carrier. When ready for application to a patient, the combination is placed into suspension (for example, in 5 to 10 ml) of a biologically compatible liquid such as water or saline utilizing normal procedures. Example 1

A composite particle composed of silica gel and iron was manufactured and preliminary characterization was performed. Characterization included particle sizing analysis (light scattering technique), surface area, pore size analysis, scanning electron microscopy and doxorubicin binding. Tests show that 95% of the final product has particles that are less than 1.11 m and have a mean (volume) diameter of 0.92 m. Results from surface area analysis show the iron-silica gel composite to have a total surface area of 48 m²/g and a total pore volume of 0.19 cc/g. SEM pictures reveal discrete particles made of both iron and silica gel components (Figures 1 and 2). Preliminary doxorubicin binding assays (Figure 4) show correlation between the concentration of bound (Q) and unbound (C) doxorubicin.

Doxorubicin Binding for Iron-Silica Gel Composite

120

D) & 00 80

^■a c 60 o ε 40 o 20

Q 0

200 400 600 800 1000

Cunbound (μ9 DOX/ml solution)

Example 2

A composite particle composed of silica-C18 and iron was manufactured and preliminary characterization was performed. Characterization included particle sizing analysis (light scattering technique) and doxorubicin binding. Tests show that 95% of the final product has particles that are less than 1.60 m and have a mean (volume) diameter of 1.58 m. Preliminary doxorubicin binding assays (Figure 5) show a linear correlation between the concentration of bound (Q) and unbound (C) doxorubicin.

Doxorubicin Binding on Iron-Silica C18 Composite

60 50 CD

^■α E 40 O o 30 o

CO Q α. 20 σ i^> 10

200 400 600 800 1000

C (Unbound) [ug DOX/ml solution)

Example 3

In order to bind a biologically active substance for targeted delivery, initially, the structure of the agent would be evaluated. Pachtaxel, for example, contains three -OH groups and three benzene nngs. Using the information contained in Table 1 , binding would be attempted using those denvatives for benzene nngs and -OH groups. First-line silica derivatives would include bare silica, C8 and C18. Second-line derivatives would include phenyl, CI, C2, C4 and C6. Additional silica denvatives would be tested based on the results from experiments. The derivatives should be easily determinable by any person having ordinary skill in the art. Neoplastic agents may be especially useful with the particles of the invention. Examples of other useful neoplastic agents are exemplified in Table 2.

Table 1 : Examples of Functional Characteristics of Agents and Silica Denvatives

Table 2: Agents Useful in Neoplastic Disease

Agent Agent Name Trade Name Abbr.

HORMONE AND ESTKOtlENS Diethylstilbestrol DES HORMONE INHIBITORS Conjugated Estrogens Premaπn Ethinyl Estradiol Estmyl

ANDROC.ENS Testosterone propionate TES Fluoxymesterone Halotestm, Ora-

Testryl, Utandran

PROGESTINS 17-Hydroxyprogesterone Delalutm caproate Medroxyprogesterone acetate Provera Meeestrol acetate Meeace

LEUPR LIDE Lupron

Goserelin acetate Zoladex

ADRENO ORTICOSTEROIDS Predπisone

^■uVπESTROiltNS Tamoxifen Nolvadex I

HORMONE SYNTHESIS INHIBITORS Aminoglutethimide Elipten, Cytadren 1 j

ANTIANDROCENS Flutamide Eulexiπ 1

Example 4

The adsorption capacities of hydroxyapatite panicles and the iron-hydroxyapatite composite particles were determined by a doxorubicin binding assay. The Langmuir adsorption isotherms were determined from doxorubicin binding data at several concentrations and the total drug loading capacities were calculated from the inverse of the slope of the isotherms. Figure 12 shows the isotherm for the iron-hydroxyapatite composite particles, which had a total capacity of 33 micrograms doxorubicm per milligram particles. Figure 13 shows the isotherm for the hydroxyapatite alone, which has a binding capacity of 53 micrograms doxorubicin per milligram particles. The difference in the drug binding capacity between the hydroxyapatite and the iron-hydroxyapatite composite material is due to the difference in compositions of these samples: the composite material of this example has ~ 25% per weight of hydroxyapatite.

2 - 0

1000 c

0 ϋ 00 0 •ICC 0 ecc- 3

Example 5

Iron-hydroxyapatite composite particles were loaded with doxorubicin by soaking the particles in a concentrated aqueous solution of the drug. The desorption profile was determined in a semi-dynamic assay by measuring the amount of doxorubicin released from the particles incubated in aliquots of human plasma at 37°C. Figure 14 shows that the drug is effectively released from the microparticles as a function of time.

Amount Onαrbβd va. Time July 27, 1999

'a

-* 15O 0 • £

I ^:

- I □

3

■

0.0 i

Example 6

Iron-hydroxyapatite micro particles were incubated with Indium-I l l m PBS for 30min at 37° C and 1400rpm. The labeling efficiency was determined by comparing the amount of radioactivity in the incubation with the bound radioactivity after two washes with PBS. The inset in Figure 15 shows the resulting labeling efficiencies, which were approximately 60% after the second wash. The stability of the labeled particles was tested in both PBS and human plasma at 37°C. For each time point, the total activity of the sample was compared with the activity in the supernatant, After 12 days, the iron-hydroxyapatite micro particles in PBS retained more than 95% of the Indium-1 11 and the stability in of the particles in plasma was about 90%. These results demonstrated that the microparticles are easily labeled with Indium cation and that the labeling is very stable in human plasma.

Tιme [h]

Example 7

The previous experiment was repeated using an Indium complex instead of the Indium salt. Indium- 1 1 1-oxyquinoline complex was used in the incubation step after being prepared by well know methods. The efficiency and stability were determined as described previously and the results are shown in Figure 16. The labeling efficiency increased to over 90% after the second wash. The stability of the Indium-oxyquinoline labeled micro particles is very similar to the direct labeling, with more than 95% of the radioactivity remaining bound after 12 days in PBS and about 90% of the radioactivity still bound after 12 days in plasma. Thus, Indium complex can also be directly labeled in a very stable manner onto the particles.

Time [hi

Claims

What Is Claimed:

1. A magnetically responsive composition comprising particles including iron and ceramic or a derivative thereof, wherein the ratio of ceramic:iron is in the range from about 1% to 95%o ceramic to 5% to 99% iron, and wherein the diameter of each particle is approximately 0.1 to 10.0 μm.

2. The composition of claim 1 wherein the ceramic comprises silica.

3. The composition of claim 2, wherein the silica is a macroporous silica gel, having pores in the range from about 2 A to about 500 A.

4. The composition of claim 2, wherein the silica is derivatized with octadecylsilane, having pores in the range from about 2 A to about 500 A.

5. The composition of claim 1 wherein the ceramic is hydroxyapatite.

6. The composition of claim 5 wherein the hydroxyapatite has pores in the range from about 250 Λ to about 1200 A.

7. The composition of claim 1 wherein the biologically active agent is a selected from the group consisting of chemotherapeutic agents, radioisotopes, genetic materials, contrast agents, dyes, and derivatives or combinations thereof.

8. A kit for administering a biologically active substance to an in vivo site in a patient comprising a unit dose of ferroceramic, each particle including a ratio of iron to ceramic in the range from about 99: 1 to 5:95.

9. A kit for administering a biologically active substance to an in vivo site in a patient comprising a receptacle containing: a) unit dose of dry ferroceramic particles, each particle including a ratio of iron to ceramic in the range of about 99:1 to 5:95; and b) one or more dry excipients.

10. A kit for administering a biologically active substance to an in vivo site in a patient comprising: a) a first receptacle comprising a unit dose of ferroceramic particles, each particle including a ratio of iron to ceramic in the range from about 99:1 to 5:95; and b) a second receptacle comprising an aqueous solution comprising one or more excipients.

11. The kit of claim 8, 9, or 10, wherein the excipients include a biologically compatible polymer for stabilization after the particles are combined with the aqueous solution.

12. The kit of claim 8, 9, or 10, wherein the excipients include mannitol, sorbitol, sodium carboxy methyl cellulose, polyvinyl pyrrolidone or combinations thereof.

13. The kit of claim 8, 9, or 10, wherein the contents ofthe kit are combined with a commercially prepared formulation of a biologically active substance.

14. The kit of claim 10 wherein the aqueous solution comprises at least one buffer.

15. The kit of claim 8, 9, or 10, wherein the unit dose of ferroceramic particles has been sterilized by means of gamma irradiation, dry heat or electron beam.

16. The kit of claim 10, wherein the aqueous solution comprising the excipients has been sterilized by means of autoclave.

17. A method of sterilizing a composition comprising iron-silica particles comprising the use of gamma inadiation.

18. A method for localized in vivo delivery of a biologically active agent comprising: a) adsorbing a biologically active agent onto a magnetically responsive carrier composition comprising iron and ceramic; b) injecting the carrier having the adsorbed biologically active agent into a patient; and c) establishing a magnetic field exterior to the patient and adjacent to a desired site, wherein the magnetic field is of sufficient strength to guide and retain at the site a portion ofthe carrier.

19. The method of claim 18 wherein the injecting step is via intra- arterial.

20. The method of claim 18 wherein the desired site is a tumor.

21. The method of claim 18 wherein the biologically active agent is selected from the group consisting of a diagnostic, a therapeutic, a bifunctional and combinations thereof.