CA2075491A1

CA2075491A1 - Low molecular weight carbohydrates as additives to stabilize metal oxide compositions

Info

Publication number: CA2075491A1
Application number: CA002075491A
Authority: CA
Inventors: Ernest V. Groman; Lee Josephson
Original assignee: Individual
Current assignee: Advanced Magnetics Inc
Priority date: 1990-02-06
Filing date: 1991-02-05
Publication date: 1991-08-07
Also published as: ATE143814T1; EP0517740A1; EP0517740B1; WO1991012526A1; DE69122608D1; DE69122608T2; US5102652A; EP0517740A4; JPH05504149A; JP3172175B2

Abstract

ABSTRACT OF THE DISCLOSURE This invention relates to compositions comprising a colloidal or particulate metal oxide which are stabilized by low molecular weight carbohydrates. The carbohydrates are characterized by the fact that (a) they are not retained on the surface of the metal oxide based on the equilibrium room temperature dialysis of about 2 ml of the metal oxide composition at 0.2 M metal concentration against deionized water; and (b) they impart sufficient stability to the metal oxide compositions such that the compositions can withstand heat stress without perceptible aggregation as determined by a prescribed test procedure.

Description

WO 91/12526 PCl/U~i9~ )770 -1- 2~7~

LON MOLECULAR ~EIGHT CARBO~YDRATES AS
ADDITIVE8 TO 8'rA~3ILIZE META~ o:l~ID15 COMPO5ITIONS

l. BACKGROUND OF THE INVENTION

l.l TECHNICAL FIELD

This invention relates to compositions comprising a colloidal or particulate metal oxide dissolved or suspended in a liquid carrier to which a soluble low molecular weight carbohydrate has been added.
It has been discovered that the low molecular weight carbohydrate imparts significant stability to the 25 compositions over a wide range of conditions without modi~ying the surface of the metal oxide. As such, the low molecular weight additives are useful in the formulation of diverse metal oxide products, including magnetic resonance contrast agents, anemia-treating 30 pharmaceuticals and ferrofluids.

l.2 BACKGROUND ART
`:
The advent of magnetic resonance imaging in medicine 3~ has led to the investigation of a wide range of materials .

WO91/~2526 P~T/VS91/~770 2- 2 ~

as magnetic resonance (MR) contrast agents. Some of the materials investigated are colloidal or particulate in nature. When colloidal or particulate materials are used as parenteral MR contrast agents, the presence of large 5 particles or aggregates can be life-threatDning to the subject recipient. In addition, considerations of consumer convenience, and the economic desirability of manufacturing a small number of large lots, require both a long shelf life and the storage of the 10 colloid/particulate materials at ambient temperatures.
The develop~ent of commercial parenteral MR contrast aqents based on colloidal and particulate active ingredients requires that the desirable physical properties of the colloid/particulate material be 15 maintained over a wide range of conditions.
So-called lyophobic colloids and particulate solutions (colloids/particulates with water repelling surfaces) exhibit a general tendency to form high molecular weight aggregates or frank particles upon 20 storage. An example of this phenomenon includes the observation of aggregate formation when superparamagnetic iron oxide is subjected to autoclaving conditions (see Figure 5 of U.S. Pat. No. 4,827,945 incorporated above by reference). Addition of a polycarboxylate, such as 25 citrate, prevents this undesirable aggregation. However, it is difficult to make ~he citrate-stabilized fluids isotonic. An advantage of the low molecular weight carbohydrate stabilizers of the current invention is that they can be used to adjust the osmotic pressure of the 30 administered fluid over a wide range. In particular, they can be added to produce an isotonic fluid.
A common approach to the problem of instability in lyophobic colloids and particulate solutio~s involves the binding of certain agents to the surface o~ the colloid 35 or particulatè, so as to provide increased compatibility W~91/12526 PCT/U~91/~770 - ~7~

between the very large surface area of the colloid/
part.iculate (i.~., large surface area per gram of colloid/particulate~ and the solvent. This compatibility between surface and .solvent leads to increased stability 5 of the colloid/particulate upon autoclaving and/or storage. Polymeric, high molecular weight agents such as dextran (Hasegawa et al., U.S. Pat. No. 4,101,435;
Molday, U.S. Pat. No. 4,452,773 hoth incorporated herein by reference~, bovine serum albumin (Owen, U.S. Pat. No.
10 4,795,698 incorporated hsrein by reference) and organosilane (Whitehead, U.S. Pat. NoO 4,695,392 incorporated herein by re~erence) have been used to coat (or otherwise associate with) and presumably to stabilize colloid/particulate solutions. Currently known polymeric 15 stabilizing agents typically have molecular weights above about 5,000 daltons. However, one significant problem encountered in the association of polymers with the surface of the colloid or particulate is that the polymers fr~-quently dissociate from the surface upon 20 prolonged storage or under high temperatures. Such ~` dissociation directly and significantly decreases the stability of the colloid/ particulate solutions.
~ Dextran/magnetite is an example of a particulate ; solution specifically noted to be stabilized by the 25 polymeric dextran (see ~asegawa et al., U.S. Pat. No.
4,101,435, column 4, lines 9-43). Several workers have used dextrans of various molecular weights as ingredients ~` in the synthesis of magnetic colloids or particles (see Hasegawa et al., U.S. Pat. No. 4,101,435; Molday, U.S.
30 Pat. No. 4,454,773; Schroder U.S. Pat. No. 4,501,726 incorporated herein by reference). The resulting - complexes of dextran and iron oxide have varying sizes and structures, but all have molecular weights of at least absut 500,000 daltons. The incorporation of high 35 molecular weight dextran into magne~ic particles or W~9l/12~26 PCT/US~1/~770 -~- 2~7~

colloids may, however, cause some patients ~o experience adverse reactions to the dextran, when such complexes are administered as parenteral MR contrast agents. These adverse reactions may also in part be due to the previously discussed problem of the high molecular weight polymers such as dextran which dissociate from the metal oxide colloid or particle upon prolonged storage or under high temperatures, leaving the metal oxide free to aggregate.
Similarly, a stable colloidal complex of ferric hydroxide and partially depolymerized dextran has been used in the treatment of iron-deficiency aneamia (Herb, U.S. Pat. No. 2,885,393; London, et al.~ U.S. Pat. No.
2,820,740 and Re 24,642 all incorporated herein by 15 reference). The most suitable range in molecular weight of the partially depolymerized dextran for injection was found to be 30,000 to 80,000 daltons or lower. (Herb, U.S. Pat. No. 2,885,393 col. 2 line 1-7).
Ferrofluids involve another example of the 20 stabilization of magnetic colloids/particulates throu~h surface modification. Typically, low molecular weight (less than 5,000 daltons) detergents are bound to the surface of a particulate solution of magnetic iron oxide (Rosensweig, R., Scientific American, October 1982, pp.
25 136-145; Khalafalla, U.S. Pat No. 4,208,294; Kovac U.S.
Pat. No. 3,990,981, all incorporated herein by reference).
A final approach to the stabilization of colloids involves the addition of polymeric agents to the solvent.
30 Such agents can adsorb to the surface of the colloid in a weak, revexsible fashion, changing the surface characteristics sufficiently to enhance stability. There are several problems with adding free polymer as a stabilizing agent for colloids, and in particular for the 35 stabilization of colloids or particles for parenteral WV91/12526 PCT/US91/~770 ~75~

ad~inistration (e.g., injection). First, upon storage the free polymer may aggregate, producing a ]iquid with unacceptable physical properties. This aggregation can occur when a polymeric stabilizer is employed that is 5 capable of gelation or aggregation over the storage period. Polymers that have been used for stabili~ing colloids that exhibit the property of gelation are gelatin and high molecular weighL dextran. Second, after injection, adverse reactions to free polymer are 10 possible. For example, injection of dextran as a plasma expander is associated with adverse reactions (Mishler, J.H., Clinics in Haemotology 13:75-92 (19~4) incorporated herein by reference).

2. SU~RY OF T~E INVENTION

Accordingly, an object of this invention is to provide a method of stabilizing colloidal or particulate metal oxide compositions without significant surface 20 modification of the metal oxide.
A further object of this invention is to provide colloidal or particulate metal oxide compositions useful as parenteral MR contrast agents i~ animal and human s~bjects which are highly stable to prolonged storage and 25 autoclaving.
A still further object of this invention is to provide parenterally administrable colloidal sr particulate iron oxide compositions useful in the treatment of iron anemia in animal and human subjects and 30 which are hi~hly stable to prolonged storage and autoclaving.
A still further object of this invention is to provide improved, stable water-based ferrofluid compositions for use in non medical applications~
These and other objec~s are achieved by the addition ., 2 ~ 7 ~
W0 91J125~6 - 6 - PCT/U~91/00770 of an effective amount of certain sol1Jble low molecular weight carbohydrates to the liquid carrier phase of metal oxide compositions.

3. DESCRIPTION OF THE INVENTION

We have discovered that the ~tability of known colloidal or par-ticulate metal oxide compositions can be significantly increased by adding a stabilizer comprising one or more soluble low molecular weight 10 carbohydrates to the liquid carrier phase of such compositions. The liquid carrier phase may comprise a buffer and a preservative. These carbohydrates are characterized by the fact that a) they are not retained on the surface o~ the metal oxide based on the 15 equilibrium room temperature dialysis of about - 2 milliliters of the metal oxide composition at 0.2 M
metal concentration against deionized water; and b) they impart sufficient stability to the metal oxide compositions such that the compositions can withstand 20 heat stress without perceptible aggregation. ~he ability of a colloid to withstand the deleterious e~fects of storage can be observed over a variety of times and temperatures. A common practice within the pharmaceutical industry is to analyze the stability of a material for short periods of time, and at temperatures above ambient temperature. In this way formulations of greater or lesser stability can be screened and more stable formulation~ selected. The selection of low molecular weight carbohydrates as stabilizers of metal oxide colloids at elevated temperatures is demonstrated in Tables I and II.
After selection of the most stable compositions ~rom such screening studies, the rate of deterioration of a pharmaceutical can be determined at several differ-ent, elevated temperatures. Data concerning the rate of deterioration at various elevated temperatures i5 obtained and used to calculate the Arrhenius activation Wo 91t~25~6 PCr/US91/0~7~0 --7 ~
2 ~

energy, which in turn is used to estimat~ the stability of the pharmaceutical undex conditions of storage by a customer, usually 0-30~C. (See pages 1a-31 of "Chemical Stability of Pharmaceuticals: A Handbook for PharmacistsN K.A. Connors, G.L. Amidon and V.J. Stella, Wiley & Sons, New York, 1986 which is incorporated by reference). Thus, the low molecular weight carbohydrate stabilizers of the invention can be assumed to exhibit some degree of stabilizing action for metal oxide colloids when these colloids are stored at any temperature.
The low molecular weight carbohydrates of the invention can be used to stabilize the colloidal materials used as parenterally administered MR contrast agents in U.s. Pat. No. 4,770,183 and U.s. Pat. No.

4,827,945 incorporated above by reference. These colloidal materials are used to obtain an in vivo MR
image of an organ or tissue of an animal or human subject. Preferred colloidal materials used as a parenterally administered M~ contrast agent which can be stabilized by the low molecular weight carbohydrates according to this invention are superparamagnetic materials which comprise biodegradable superparamagnetic iron oxides. The biodegradable superparamagnetic iron ~5 oxide is characteri~ed by biodegradation in an animal or human subject within about 2 weeks or less after administration, as evidenced by a return of the proton relaxation rates of the organ or tissue to preadministration levels. The biodegradable superparamagnetic iron oxide can be coated by or associated with a high molecular weight polymeric substance such as those discussed below.
The low molecular weight carbohydrates can also be used to stabilize solutions/suspensions of other colloidal or particulate materials that have been used as WO9l/l~26 PCr/US91/00770 207~

MR contrast agents, and which have been parenterally administered. These include dextran-magnetite (R.L.
Magin et al., Society for Magnetic Resonance in Medicine (1987~ P. 538 incorpor~ted herein by reference), magnetic 5 carbohydrate matrix type particles (A. Hemmingsson et al., Acta Radiologica 2~:703 705 (19~7) incorporated herein by reference), and albumin microspheres (D.J.
Widder et al., Amer. J. Roent. 148:399-404 (1987) incorporated herein by re~erence~. Other colloidal or 10 particulate metal oxides in solution/suspension, such as those disclosed in U.S. Patents Nos. 4,101,435;
4,452,773; 4,795,698; 4,695,392; and 4,501,726, incorporated above by reference, can be stabilized by these low molecular weight carbohydrates as well.
The low~molecular weight carbohydrates of the invention can effectively stabilize metal oxide compositions where the metal oxide surface is uncoated or coated by (or unassociated or associated with) a high molecular weight polymer such as dextran having a 20 molecular weight of about 5,000 to about 500,000 daltons, starch having a molecular weight of about 5,000 to about 500,000 daltons, polysaccaride having a molecular weight of about 5,030 to about 500,000 daltons, bovine serum albumin or organosilane.
Representative examples of the metal oxide include, but are not limited to, iron oxide, chromium oxide, cobalt oxide, manganese oxide, iron oxyhydroxide, chromium oxyhydroxide, cobalt oxyhydroxide, manganese oxyhydroxide, chromium dioxide, other transition metal 30 oxides as well as mixed metal oxides. Additionally, the particle size of the metal oxides must necessarily be below 0.8 micron to pass the below-described stability test.
The low molecular weight carbohydrates of the 35 invention preferably have a molecular weight of less than W09l/12~26 PCT/US9~/~770 _g_ 2~7~9~
5,000 daltons, most pre~erably l,ooo daltons or less.
The preferred concentrations of the carbohydrates of the invention which effectively impart stabilization to the c~rrier phase of the metal oxide composition is in the 5 range of about O.OOl M to about 2 M, most preferably about 0.05 M to about 0.5 M.
Some preferred low molecular weight stabilizing agents include, but are not limited to, mannitol, sorbitol, glycerol, inositol, dextran l (Pharmacia Inc., 10 Piscataway, N.J.) and ascorbate. In the case of dextran l, which has a molecular weight of about l,000 daltons, the same compound can both stabilize the colloid or particulate suspension against unwanted physical changes and block possible adverse reactions. The simultaneous 15 injection of~dextran l and a complex of dextran and the magnetic iron oxide decreases adverse reactions to high molecular weight dextran alone.

4. EXAMPLES
Experimental examples supporting the use of low molecular weight carhohydrates as stabilizing agents for metal oxide compositions are presented below. Example l sets forth one type of stress test for screening useful 25 low molecular weight carbohydrate stability agents.
Example 2 examines the ability of various low molecular weight carbohydrates to stabilize colloidal superparamagnetic iron oxide. Example 3 demonstrates that mannitol, taken as representative of the low 30 molecular weight stabilizing agents of the invention, is not retained in association with the metal oxideO As a result, the stabilizing agents of the invention are ; believed to exert their effects in a manner different from other stabilizing agents which are retained on the 35 metal oxide surface. Example 4 describes the use of low '~

WO9l/1252~ PCT/U~1/~77~

~ ~ 7 ~
molecular weight carbohydrates to ~urther stabilize colloidal compositions containing a dextran iron complex which can be used for the treatment of iron-deficiency anemia. Example 5 describes the use of low molecular 5 weight carbohydrates to further stabilize aqueous-based ferrofluids.

4.1 EX~MPLE_l Stress Test to Screen for Low Molecular Weiqht Carbohydrate Stabilizin~ Agents ~ convenient stress test ~or selecting carbohydrates for their ability to stabilize metal oxide colloids or 15 particulate suspensions against undesirable changes in physical state is afforded by autoclaving (i.e. holding at about 121 degrees centigrade for about 30 minutes) the metal oxide in a liquid carrier phase to which the carbohydrate has been added, followed by filtration 20 t~rough a 0.8 micron filter. ~ fully stabilized metal oxide composition passes through the filter, while compositions undergoing undesirable changes in physical properties are retained o~ the filter. ~ designation of "fail" is given to those compositions in which the metal 25 oxides aggregated, producing colored (fully dark brown to black) filters. ~ designation of "pass" is given to compositions that maintained their physical state and upon filtration yielded colorless or wllite filters.
designation of "intermediate" is given to those 30 compositions yielding filters that retain significant metal oxide but which exhibit incomplete coverage of the filter.

;,`~

SlJElSTlTUTE SHEET

WO~I/125Z6 ~CT/~S9~/~770 2 ~
4.2 EXAMPLE 2 The ~bility of Selected Low Mo~ecular Weight Carbohydrates to Stabilize Superparamaq~eti~ Collo1ds Table I shows the effect of a variety of low mGlecular weight agents on the filterability of an ~utoclaved superparamagnetic colloid. ~he colloid is a superparamagnetic fluid of iron oxid~ associated with l0 dextran (MW = 10,000-15,000 daltons) having 11 milligrams iron per milliliter (nmg Fe/ml") at pll ~.~ prepared according to example 7.10 of U.S. Pat. No. 4,~27,945, except that the heating step was omitted in step 7.1~.2.
Specifically, five liters of a solution containing 15 755 grams ("g") FeCl3.6El2O and 320 g FeC12.4~l2O was added slowly to 5 liters of 16% Nil4011 containing 2500 g dextran (MW =10,000-15,000 daltons). The iron salt solution was ~dded over 5 minutes during which time the base was vigorously stirred during addition. ~ blac]c magnetic 20 slurry was formed. ~fter centrifugation, the supernatant was diluted to a total volume o~ 20 liters with deionized sterile water and the resultant solution was dialyzed against ammonium citrate buffer by use of a ~ollow fiber dialyzer/concentrator, model DC 1~ ~MICON Corp., 25 Danvers, Mass.) ~he ammonium citrate buffer is 10 mM
citrate, adjusted to pll ~.2 Witll Nl1401~. ~he dialyzer cartridge had a 100,000 dalton molecular weigllt cutoff, permitting removal of dextran. Ultrafiltration was accomplished in a noncontinuous fashion, reducing the ~0 volume from 20 to 5 liters ~nd adding 16 liter volumes Or solution. Five volumes of 16 liter~ of deionized, water were added. After this ultrafiltration step, the colloid (39.3 mg Fe/ml) was ~iltered through a 3 micron filter and then diluted with distilled water to 35 yield a concentration of 11.2 mg Fe/ml.

elUlE~TlTUTE ~SHEET
:

WO91J12525 PCT/US91/~770 ~12- 2~7~491 The low molecular weight agent is khen added to the colloid. In most cases the concentration of low molecular weight carbohydrate was 325 mM or about isotonic with blood. The concentration of the low 5 molecular weight carbohydrate in the final stabilized colloid can be from about 0.001 M to about 2 M.
To perform the test, 10 ml of colloid i5 autoclaved at 121 degrees centigrade for 30 minutes and then filtered over a 0.8 micron filter (Gelman Sciences Inc., 10 Ann Arbor, MI), followed by visual examination of the filter. Filters were rated as described in Example l and results are shown in Table I.

1~
i `:
~ 35 W091/12526 PCT/U~91/~770 2~7~9~

TABLE I

Effect of Low Molecular Agents on Stability of Superparamaqnetic Colloid Autoclaved at 121C
S ~
Agent Concentration Colloid Quality water only fail galactose 325 mM fail mannose 325 mM fail fructose 325 mM fai1 maltose 325 mM intermediatP
sucrose 325 mM fail lactose ~25 mM fail ` ribose 325 m~ fail glucosamine 325 mM fail dextran 1* lO0 mg/ml pass ~ acetate 325 mM fail : 20 PEG-300 lO0 mg/ml fail threitol 325 mM intermediate ~` gluconate ~25 mM pass/intermediate citrate 25 mM pass tartrate 325 mM pass mannitol 325 mM pass :: sorbitol 325 mM pass ascorbate 325 mM pass .
T-10 dextran 100 mg/ml fail NaCl 250 mM fail * Dextran 1 is dextxan with a molecular weight of about l,000 daltons. The solution, supplied for injection by the manuPacturer, was diluted from 150 mg/ml, and the ~inal solution contained about 0. 06 M NaCl.
: 35 .~ .

WOgl/12526 P~T/U~91/~770 ~7~

Several concJusions car. be made from Table I.
As expected, based on earlier observations for dextran-ass~ciated lron oxide colloids (see Figure 5 of u.s Pat.
No. 4,827,945), the failure to add a stabilizing agent to 5 the pxesent dextran associated iron oxide colloid (i.e., water only), resulted in massive, adverse changes in physical state (i.e., failure of the filtration stress test). As demonstrated previously, citrate can stabilize the colloid to autoclavi.ng by being retained on the 10 surface of the iron oxide (i.e. ferric oxyhydroxide, see Col. 28 of U.S. Pat. No. 4,827,945). Addition of polymeric dextran (MW=10,000) was ineffective in stabilizing the colloid, but the addition of dextran 1 was highly effective. No attempt was made to distinguish 15 between the aggregation of ~he superparamagnetic iron oxide and/or ths~ aggregation (or gelation) of the added stabilizing agent as the cause of poor filtration ~ characteristics.
- Linear polyalcohol type compounds stabilized 20 the superparamagnetic colloids, even in cases where the corresponding cyclical hemiacetal monosaccharide was ineffective. This observation was highly unexpected.
For example, mannitol passed while mannose failed the stress test. Similarly, the linear polyol acid gluconate ~5 gave satisfactory results while closely related monosaccharides like glucose and galactose gave poor results. Two compounds that were not linear polyalcohol type agents gave satisfactory results; they were ascorbate and dextran 1.
The stabilizing e~fects exerted by the low molecular weight agents of the invention can be observed under a variety of storage conditions, i.e. different times and temperatures. Table II shows an experiment demonstrating that the stabilizing effects of low 35 molecular weight carbohydrates noted at 121~C (Table I) s WO91/1~526 PC~/US91/~770 -15- 2~

can also be observed after storage for 3 days at 55'C.
In Table II, the colloid of Table I was used and handled in the same manner as in ExamplP 2, except for the heating conditions. Quality was assessed by filtration 5 as described in Example l.

lU

3~

W~91/125~6 PCT/US91/~770 ~75~9~
TABL~ II

Effect of Low Molecular Weight Agents on Stability of Superparamaqnetic Colloid At 55 C.

- Agent Concentration Colloid Quality ._ _ _ . . .. .. ~ .. . __ .. .. ..

10 water fail - acetate 325 mM fail mannose 325 mM fail mannitol 3~5 mM pass .... ~

4.3 EXAMPLE 3 :```
The Failure of Mannitol to be Retained On The Surface of Su~_paramaqnetic I_on Oxide - To investigate whether the stabilization of ~ superparamagnetic iron oxide involves the retention of ~- the stabilizing agents on the surface of the iron oxide used in Table I, mannitol was selected for study. The retention of mannitol by the superparamagneti~ iron oxide was studied by the equilibrium dialysis technique (D.
Freifelder l'Physical Biochemistry: Applications to Biochemistry and Molecular Biology," W.H. Freeman, San Francisco, 1976 p. 518 incorporated herein by reference).
30 If mannitol is stabilizing the colloid by adsorbing onto the surface of the colloid, 14C-labelled mannitol should be retained by the superparamagnetic iron oxide colloid when examined by the equilibrium dialysis technique. A
membrane with a 12-14 kilodalton cutoff was used which 35 permits mannitol to escape from the bag but retains WO91/1~5Z6 PC~/U~91/~770 -17- 2~7~9~

superparamagnetic iron oxide within the bag.
Three samples were prepared:

A. Superparamagnetic dextran-associated iron oxide colloid (as above), at 11 mg Fe/ml, plus 0.325 M
mannitol containing 5 microcuries 14C mannitol (CFA-238, Amersham Corp., Arlington Heights, IL).

. Mannitol but no superparamagnetic iron 10 OXide .

C. Superparamagnetic dextran-associated iron oxide with mannitol as in sample A but autoclaved at 121 degrees centigrade for 30 minutes.
Samples of approximately 2 mls were placed in an appropriate length of dialysis tubing (Spectra/Por 2, 12,000-14,000 daltons, molecular weight cutoff, Spectrum Medical Industries, Los Angeles, CA) and sealed with 20 clips- The samples were then placed into 725 ml of deionized ("DI") water containing a magnetic stirring bar. The samples were dialyzed with gentle stirring using the magnetic stirring bar. After at least 24 hours, a 2 ml sample of dialysate was taken for analysis and the dialysate was replaced with a fresh 725 ml of DI
water. In all, three volumes of dialysate were collected. After dialysis was completed, the retentate containing the iron oxide colloid was dissolved in concentrated HCl and brought to 25 mls with DI water.
30 The 2 ml samples of each 725 ml dialysate and a 2 ml sample of retentate were each added to 15 ml of scintillation cocktail (NEF-952, E.I. DuPont de Nemours &
Company, Boston, MA) and radioactivity determined in a Packard TriCarb Scintillation counter. The results are 35 shown in Table III.

6 PCT/U~gl/~770 -18- ~7~9~

TABLE III

The Lack of Retention of Mannitol Upon Dialysis With SUperPara~aqnetiC Iron Oxide Sample A B C
Autoclaved no no yes ~` Volume ~ml ) 2 2 1.8 Membrane 12-14 K12-14 K1~-14 K
(a) Dialysate #1 204087519614881659163 CPM #2 6525 15950 7125 ~3 383 0 4500 Total ~ 15 Dialysa~e 204776319774381670788 -~ Retentate 355 6338 18375 Total . Recovery 204811819837751689163 Theory(b) 195880019055001647720 Total(C) Recovery 105 104 103 ~`~ % Retained(d) 0, 02 0.32 1.09 . ~ Sample C was autoclaved at 121-C for 30 minutes before dialysis.
(a) N#l~ represents the first 725 ml volume of DI
water; #2 and #3 represent the second and third 725 ml volume of DI water.
(b) nTheory" is the total counts of the sample before dialysis (c) % Total Recovery = total recovery theory (d) % Retained = retentate total recovery :` :

.

WO91/12~26 PCT/US91/~770 2 ~

Table III shows the results for the distribution of mannitol between ~ialysate and retentate. The CPM's are corrected for background. The theoretical value for total counts is based on measurement of a sample be~ore 6 dialysis as described above. The percentage activity remaining in the retentate is based on the total recovered activity.
About 99% or more of the mannitol was present in the dialysate, r~gardless of whether the colloid was 10 autoclaved (unautoclaved column A, autoclaved column C).
When the combination o~ superparamagnetic colloid and mannitol is subjected to the extreme condition of autoclaving (column C), a small amount of degradation of mannitol results. This is believed to account for the 15 small amount of l4C retained in the dialysis bag after autoclaving (l.09%).
Both before and after the temperature and time stress to which the colloid is subjected, there is no formation of a complex between superparamagnetic iron oxide and mannitol. Thus, the low molecular weight carbohydrate stabilizers of the invention do not bind to (or become a coating for~ the high molecular weight metal oxide colloid~
The results in Table III indicate that a 25 mannitol-superparamagnetic iron oxide complex does not exist as a definable entity. Rather, the presence of mannitol in the carrier phase changes the properties of the fluid in such a way that the stability o~ the colloid is enhanced. It should be realized that the binding of 30 mannitol to the superparamagnetic iron oxide colloid can only be asc~rtained in relation to some experimental techni~ue, which measures binding in~eractions of equal to or greater than some specific strength, i.e., weaker interactions than can be measured are always possible.
35 The equilibrium dialysis method we have used, (room WO91/12526 PCT/US91/~770 ~20- 2~75~

temperature dialysls of 2 ml colloid at 0.2 ~ iron against a large volume water) is a standard, easy to perform test of association between a colloid (or particle) on the one hand and a low molecular weight stabilizing agent on the other.
- ~uffers such as ~ris and/or preservatives such - as phenol can be added in conjunction with the low molecular weight stabilizers that are the subject of this invention.
The inabllity of mannitol to be retained by the superparamagnetic iron oxide upon dialysis contrasts with the retention of citrate exhibited by the same colloid.
We have previously noted the ability of superparamagnetic iron oxide colloids to retain citrate (sea Table IV of U.S. Pat. ~o: 4,827,945).

4.4 EXAMPLE 4 The Ability of Selected Low Molecular Weight Carbohydrates to Stabilize Compositions For The Treat~ent of Iron-Deficiency Anemia ~he colloidal therapeutic compositions for th~ treatment of iron-deficiency aneamia containing, by way of illustration, a dextran-iron complex as disclosed in 1~erb U.S. Pat. No. 2,~85,393 and London, et al. U.S.
Pat. No. 2,820,740 and Re. 24,642, incorporated herein by reference, can be further stabilized by the presence of the low mGlecular weight carbohydrates of the present invention at concentrations from about O.OOl M to about 2 M as demonstrated by subjecting such low molecular weight carbohydrate-stabilized composition to either stress test set forth in Example l and observing a "passing" result.

.

WO9l/l2~26 PCT/VS~/~770 -21- 2~7~

: 4.5 EXAMPLE 5 The Ability of Selected Low Molecular : Weight Carbohydrates To Stabilize S Aq~ous-Based Ferrofluids The aqueous-based ferrofluids, as described, by way of illustration, in Khalafalla, et al.
U.S. Pat. No. 4,208,294 and Kelley, U.S. Pat. No.
4,019,994, hoth incorporated herein by reference, can be further stabilized by the presence of the low molecular : weight carbohydrates of the present invention at concentrations from about 0.001 M ~o about 2 M as demonstrated by subjecting such low molecula~ weight carbohydrate-stabilized oomposition to either stress test set forth in Example 1 and okserving a "passing" result.
, ~ .

: The compositions disclosed can be varied : 20 in a number of ways. The description is intended to illustrate the principles of using the low molecular weight carbohydrate stabilizers for metal oxide colloid and particulate compositions. It is understood that changes and variations can be made therein without departing from the scope of the invention as defined in the following claims.

.

~ 5 :`
: `

:: , , .

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An improved parenterally administrable composition, comprising a colloidal or particulate biodegradable superparamagnetic metal oxide in a physiologically acceptable carrier, which metal oxide is biodegraded by a subject within about two weeks or less after administration, as evidenced by a return of proton relaxation rates of an affected organ or tissue of said subject to preadministration levels and which is filterable through a 0.8 micron filter, wherein the improvement comprises the addition to said carrier of:
an effective amount of a stabilizer which comprises a physiologically acceptable, soluble, low molecular weight carbohydrate, which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts improved physical stability to said composition as determined by heating said composition at about 55°C for about 3 days and then filtering said composition through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

2. An improved parenterally administrable composition, comprising a colloidal or particulate biodegradable superparamagnetic metal oxide in a physiologically acceptable carrier, which metal oxide is biodegraded by a subject within about two weeks or less after administration, as evidenced by a return of proton relaxation rates of an affected organ or tissue of said subject to preadministration levels and which is filterable through a 0.8 micron filter, wherein the improvement comprises the addition to said carrier of:
an effective amount of a stabilizer which comprises a physiologically acceptable, soluble, low molecular weight carbohydrate, which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts improved physical stability to said composition as determined by autoclaving said composition at about 121°C for about 30 minutes and then filtering said composition through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

3. An improved parenterally administrable composition, comprising a colloidal or particulate ferromagnetic or paramagnetic metal oxide in a physiologically acceptable carrier, which metal oxide is filterable through a 0.8 micron filter, wherein the improvement comprises the addition to said carrier of:
an effective amount of a stabilizer which comprises a physiologically acceptable, soluble, low molecular weight carbohydrate, which carbohydrate is selected from the group consisting of glycerol, inositol, a dextran having a molecular weight of about 1,000 daltons and an ascorbate, and which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts improved physical stability to said composition as determined by heating said composition at about 55°C for about 3 days and then filtering said composition through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

4. An improved parenterally administrable composition, comprising a colloidal or particulate ferromagnetic or paramagnetic metal oxide in a physiologically acceptable carrier, which metal oxide is filterable through a 0.8 micron filter, wherein the improvement comprises the addition to said carrier of 4 an effective amount of a stabilizer which comprises a physiologically acceptable, soluble, low molecular weight carbohydrate, which carbohydrate selected from the group consisting of glycerol, inositol, a dextran having a molecular weight of about 1,000 daltons and an ascorbate, and which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts improved physical stability to said composition as determined by autoclaving said composition at about 121°C for about 30 minutes and then filtering said composition through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

5. The composition of claim 1, 2, 3 or 4 in which said metal oxide has been coated by or is associated with a high molecular weight polymer.

6. The composition of claim 5 in which said high molecular weight polymer is selected from the group consisting of dextran having a molecular weight of about 5,000 to about 500,000 daltons, starch having a molecular weight of about 5,000 to about 500,000 daltons, polysaccharide having a molecular weight of about 5,000 to about 500,000 daltons, bovine serum albumin and organosilane.

7. The composition of claim 1, 2, 3 or 4 in which wherein said metal oxide is a transition metal oxide.

8. The composition of claim 1, 2, 3 or 4 in which said metal is selected from the group consisting iron, chromium, cobalt, manganese and mixed metals thereof.

9. The composition of claim 5 in which said colloidal or particulate metal oxide is selected from the group consisting of a dextran-magnetite, a magnetic carbohydrate matrix type particle and an albumin microsphere.

10. An improved MR contrast agent composition parenterally administrable to an animal or human subject comprising (a) a superparamagnetic metal oxide in a physiologically acceptable carrier, which metal oxide is selected from the group consisting of biodegradable superparamagnetic iron oxide associated with a polymeric substance, said biodegradable superparamagnetic iron oxide being characterized by biodegradation in said subject within about 2 weeks or less after administration, as evidenced by a return 60207.1 of the proton relaxation rates of an organ or tissue of said subject to preadministration levels; and (b) mannitol at a concentration between about .001 M and 2 M.

11. The composition of claim 1, 2, 3 or 4 in which the low molecular weight carbohydrate is present at a concentration of about 0.001 M to about 2 M.

12. The composition of claim 1, 2, 3 or 4 wherein said low molecular weigh carbohydrate has a molecular weight below 5,000 daltons.

13. The composition of claim 12 wherein said low molecular weight carbohydrate is a linear polyalcohol carbohydrate.

14. The composition of claim 13 wherein said linear polyalcohol carbohydrate is selected from the group consisting of mannitol, sorbitol, glycerol and inositol.

15. The composition of claim 1, 2, 3 or 4 wherein said low molecular weight carbohydrate is dextran having a molecular weight of about 1,000 daltons.

16. The composition of claim 1, 2, 3 or 4 wherein said low molecular weight carbohydrate is mannitol.

17. The composition of claim 1, 2, 3 or 4 wherein said low molecular weight carbohydrate is ascorbate.

18. The composition of claim 1, Z, 3 or 4 in which said carrier further comprises a buffer.

19. The composition of claim 1, 2, 3 or 4 in which said carrier further comprises a preservative.

20. An improved water-based ferrofluid composition, comprising a colloidal or particulate biodegradable superparamagnetic metal oxide in a suitable carrier, which metal oxide is biodegraded by a subject within about two weeks or less after administration, as evidenced by a return of proton relaxation rates of an affected organ or tissue of said subject to preadministration levels, wherein the improvement comprises addition to the carrier of:
an effective amount of a stabilizer which comprises a physiologically acceptable, soluble, low molecular weight carbohydrate, which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by heating said composition at about 55°C for about 3 days and then filtering said composition through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

21. An improved water-based ferrofluid composition, comprising a colloidal or particulate biodegradable superparamagnetic metal oxide in a suitable carrier which metal oxide is biodegraded by a subject within about two weeks or less after administration, as evidenced by a return of proton relaxation rates of an affected organ or tissue of said subject to preadministration levels, wherein the improvement comprises addition to the carrier of:

an effective amount of a stabilizer which comprises an acceptable, low molecular weight carbohydrate, which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about, 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by autoclaving said composition at about 121°C for about 30 minutes and then filtering said composition through a 0.8 micron filter, whereafter no precipitate is visible on said filter.

22. An improved water based ferrofluid composition, comprising a colloidal or particulate ferromagnetic or paramagnetic metal oxide in a suitable carrier, wherein the improvement comprises addition to the carrier of:
an effective amount of a stabilizer which comprises a physiologically acceptable, soluble, low molecular weight carbohydrate, which carbohydrate is selected from the group consisting of glycerol, inositol, a dextran having a molecular weight of about 1,000 daltons and an ascorbate, and which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by heating said composition at about 55°C for about 3 days and then filtering said composition through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

23. An improved water-based ferrofluid composition, comprising a colloidal or particulate ferromagnetic, or paramagnetic metal oxide in a suitable carrier wherein the improvement comprises addition to the carrier of:
an effective amount of a stabilizer which comprises an acceptable, low molecular weight carbohydrate, which carbohydrate is selected from the group consisting of glycerol, inositol, a dextran having a molecular weight of about 1,000 daltons and an ascorbate, and which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by autoclaving said composition at about 121°C for about 30 minutes and then filtering said composition through a 0.8 micron filter, whereafter no precipitate is visible on said filter.

24. A method for satbilizing a biodegradable superparamagnetic metal oxide composition comprising a colloidal or particulate metal oxide in a liquid carrier, which metal oxide is biodegraded by a subject within about two weeks or less after administration, an evidenced by a return of proton relaxation rates of as effected organ or tissue of said subject to preadministration levels, which method comprises adding an effective amount of stabilizer to said carrier which stabilizer comprises a soluble, low molecular weight carbohydrate, which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by heating said composition at about 55°C for about 3 days and then filtering said composition for reducing anemia through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

25. A method for stabilizing a metal oxide composition comprising a colloidal or particulate biodegradable superparamagnetic metal oxide in a liquid carrier, which metal oxide is biodegraded by a subject within about two weeks or less after administration, as evidenced by a return of proton relaxation rates of an affected organ or tissue of said subject to preadministration levels, which method comprises adding an effective amount of stabilizer to said carrier which stabilizer comprises a soluble, low molecular weight carbohydrate, which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by autoclaving said composition at about 121°C for about 30 minutes and then filtering said composition through a 0.8 micron filter, whereafter no precipitate is visible on said filter.

26. A method for stabilizing a metal oxide composition comprising a colloidal or particulate ferromagnetic or paramagnetic metal oxide in a liquid carrier, which method comprises adding an effective amount of stabilizer to said carrier which stabilizer comprises a soluble, low molecular weight carbohydrate, which carbohydrate is selected from the group consisting of glycerol, inositol, a dextran having a molecular weight of about 1,000 daltons and an ascorbate, and which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by heating said composition at about 55°C for about 3 days and then filtering said composition for reducing anemia through a 0.8 micron filter, whereafter substantially no precipitate is visible on said filter.

27. A method for stabilizing a metal oxide composition comprising a colloidal or particulate ferromagnetic or paramagnetic metal oxide in a liquid carrier, which method comprises adding an effective amount of stabilizer to said carrier which stabilizer comprises a soluble, low molecular weight carbohydrate, which carbohydrate is selected from the group consisting of glycerol, inositol, a dextran having a molecular weight of about 1,000 daltons and an ascorbate, and which carbohydrate (a) is not retained on the surface of said metal oxide based on the equilibrium room temperature dialysis of about 2 ml of said composition at 0.2 M metal concentration against deionized water;
and (b) imparts stability to said composition as determined by autoclaving said composition at about 121°C for about 30 minutes and then filtering said composition through a 0.8 micron filter, whereafter no precipitate is visible on said filter.

28. A method for obtaining an in vivo MR
image of an organ or tissue of an animal or human subject which comprises parenterally administering to such subject the composition of claim 1, 2, 3 or 4.

29. A method for reducing anemia in an animal or human subject the composition of claim 1, 2, 3 or 4.