Use of AMI-227 as an oral MR contrast agent

Magnetic Resonance Imaging, Vol. 12, No. 4, pp. 631-639, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

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l Original Contribution

USE OF AMI- AS AN ORAL MR CONTRAST AGENT

JAMES ROGERS, JEROME LEWIS, AND LEE JOSEPHSON

Advanced Magnetics Inc., 61 Mooney Street, Cambridge, MA 02138, USA

We report the use of an ultrasmall superparamagnetic iron oxide colloid AMI- as an oral contrast agent. Due to the small size, AMI- has a larger effect on Tl than larger superparamagnetic iron oxides colloids like ferumoxsil. At 2 T, AMI- had an RJR, of 11.4 compared with an RJR1 for the superparamagnetic iron oxide ferumoxsil of 179. The Rls of the two agents were 7.1 and 1.6 mM-r s-l, for AMI- and ferumoxsil, respectively. Due to its smaller RJR,, orally administered AMI- can produce brightening or darkening of the lumen of the the GI tract, depending on instrument parameters. At 1 mM Fe, image brightening (2 T, TR = 300, TE = 25) or image darkening of the GI tract (2 T, TR = 1500, TE = 80) was obtained. The ability of AMI- to produce either image brightening or darkening suggests it may be useful as an MR contrast agent for the GI tract.

Keywords: Contrast media; Ultrasmall superparamagnetic iron oxide: Oral; Brightening; Darkening.

INTRODUCTION

The need for gastrointestinal (GI) magnetic resonance (MR) contrast agents for abdominal MR imaging to be- come widespread has been discussed.‘*2 A wide variety of materials have been examined as GI MRI contrast agents which have been classified according to their miscibility with water, and their ability to act as negative or positive contrast agents (see Table 1 of Ref. 2). Miscible brightening agents include soluble paramagnetic chelates such as Gd-DTPA. Miscible darkening agents include superparamagnetic iron oxide colloids like ferumoxsil, and oral magnetic particles, sometimes called OMP (e.g., Nycomed). (Perumoxsil is the generic USAN name for AMI-121, silicone coated superparamagnetic iron oxide.) Miscible brightening agents function to shorten T, and increase the signal intensity of the lumen of the GI tract with T, -weighted sequences. Miscible darkening agents, like ferumoxsil, shorten T,, and decrease signal intensity with T2-weighted imaging sequences.

In the present study we report the use of the ultrasmall superparamagnetic iron oxide AMI-227. AMI- 227 is also known as BMS 180549 and its parenteral applications are under clinical development by Bristol- Myers Squibb. We have compared AMI- with the larger superparamagnetic iron oxide colloid ferumox-

sil, now in clinical development as an oral contrast agent,3 and with Gd-DTPA. Because of its ability to increase or decrease image signal intensity at a single concentration, AMI- can brighten or darken images of the GI tract as a function of pulse sequence.

METHODS

Ah41-227 was made by a minor modification of methods previously described.4 By size exclusion chromatogra- phy on Sephadex 4B column (Pharmacia, Piscataway, NJ), the colloid was found to be a single peak with an apparent volume of 720 kDa, corresponding to a diameter of approximately 19 nm. Its magnetic properties are derived from superparamagnetic iron oxide crystals with a mean crystal diameter of 4.3-6.2 nm by X-ray diffraction and transmission electron micros- copy. The superparamagnetic iron oxide core is cov- ered with T-IO dextran.

The ferumoxsil was the superparamagnetic iron oxide colloid used in clinical studies.3 Gd-DTPA was made as described elsewhere.’ The three agents above were each diluted in 0.2 M Tris, 0.5% carboxymethyl cellulose (CMC) with a pH of 8.5 for relaxivity measurements, signal intensity studies, and imaging studies. The CMC prevented the ferumoxsil from settling, while the Tris was present to act as a buffer against

RECENED g/14/93; ACCEPTED 12/14/93.

631

Address correspondence to James Rogers.

632 Magnetic Resonance Imaging 0 Volume 12, Number 4, 1994

stomach acid. For imaging studies, the concentrations used were 1.0 mM Fe, 3.14 mM Fe, and 1 .O mM Gd for AMI-227, ferumoxsil, and Gd-DTPA, respectively.

Spin-lattice and spin-spin relaxation times, T, and T2 respectively, were measured with dilutions of the three agents in the media above. Relaxation times were obtained on a General Electric CSI-II, 2.0 T, 45 cm imaging spectrometer (GE NMR Instruments, Inc. Fre- mont, CA) operating at 85.47 MHz. using a 12 mm, 7 turn, homemade solenoid coil. T, was measured with a fast inversion recovery Fourier transform (FIRFT) technique.6 The 90”-pulse width was optimized for each sample. T, values were derived from a plot of the MR signal intensity vs. the inversion time, TI, which was fitted to a three-parameter exponential function. T2 measurements were obtained using the Hahn spin- echo technique. T2 was derived from plot of the MR signal intensity vs. the echo time (TE), which was fit to a two parameter exponential function.

The increase in relaxation rates (11 T,) with increas- ing millimolar concentration of metal was analyzed by a linear least squares regression fit using Eq. (1). The spin lattice relaxivity (R,) or spin-spin relaxivity (R2) was determined as the slope of Eq. (1).

l/T, = l/TceO + R (millmolar concentration) (1)

For measurements of MR signal intensity, data was acquired as a function of contrast agent concentration at three spin-echo pulse sequences, TRITE 300125, 500/30, and 1500/80. Image signal intensity was measured with the instrument software and with a region of interest that encompassed 90 pixels. The observed signal intensity @I,,,) was normalized by subtracting background noise from the signal intensity and divid- ing by the signal intensity of the carrier media (SI,) less the background noise (Eq. (2)).

SI SI - noise

obs = SI, - noise

(2)

To obtain theoretical values of signal intensity, re- ferred to as SItheor, values of l/T, and l/T2 were determined from the relaxivities (Table l), the contrast agent concentration, and Eq. (1). SItheor was then calculated from the standard spin-echo Eq. (Eq. (3)) using

Table 1. Relaxivity of contrast agents at 2 T (mM s)-’

Agent

AMl-227 Ferumoxsil Gd-DTPA

RI R2 R~/RI

7.1 81.1 11.4 1.6 286 179 4.8 5.1 1.06

the TE and TR chosen and the calculated relaxation times.

SI = H+[( 1 _ 2e-‘T’R-TE/2’/T~ + e-TW7i je-TE/T2]

(3)

Male CD rats, (Charles River Laboratories, Wil- mington, MA), housed in accordance with DHHS pub- lication No. 85-23, revised 1985 “Guide for the care and use of laboratory animals,” were placed in metabolic cages with water provided ad lib and fasted for 2 days prior to imaging. The rats weighed 250-350 g. A total of 15 rats, including rats imaged without contrast agent, were used in these studies. Each rat was given two 5-ml doses of the specific contrast agent by gavage, 90 and 30 min prior to imaging. Fifteen minutes before imaging, the rats were anesthetized with 100 mg/kg of Inactin (Promanta, Hamburg, Germany) administered intraperitoneally (IP) and then placed supine on a acrylic platform along with a vegetable oil phantom. The coil was tuned and the magnet shimmed before data acquisition. Interleaved 8 slice multislice spin- echo (SE) images were acquired with a field of view of 150 mm, slice thickness of 3 mm, and a slice separa- tion of 6 mm (center to center).

RESULTS

The relaxivities for the contrast agents examined are given in Table 1. R2/R, for AMI- was 11.4, between values of 1.06 for Gd-DTPA and 179 for SPIO. Correlation coefficients for the fit of relaxation rates to Eq. (1) were >0.99 in all cases.

The observed and calculated signal intensities (SIob, and SItheor) are plotted vs. concentrations of AMI-227, Gd-DTPA, and ferumoxsil for three pulse sequences in Figs. l-3, respectively. SItheor is shown as the lines, while SI&, are presented as data points.

At concentrations between about 0.1 and 0.7 mM iron as AMI-227, the signa intensity increased (i.e., SI > I), with the T,-weighted 300/25 or moderately T, -weighted 500/30 pulse sequences (Fig. 1). At concentrations greater than about 1 mM, a decrease in signal intensity was observed at all pulse sequences. With AMI-227, the heavily T2-weighted spin-echo pulse sequence decreased signal intensity over a wide concentration range. Also shown on Fig. 1 is the SItheor obtained with a 300/12 pulse sequence, which could not be obtained with our imager. With AMI-227, the mag- nitude of the brightening and concentration range of brightening was highly dependent on pulse sequence. The maximal signal intensity increase was about two- fold with the 500/30 pulse sequence, about 2.5-fold with the 300/25 pulse sequence, and about four-fold

AMI- as an oral MR contrast agent 0 J. ROGERS ET AL. 633

4.50 -

. /. 300125 Ohs.

4.00 -- / \ I’ ‘\ 0 500/30 Obs.

l 1500/80 Obs.

-_- 300/I 2 Theory

~ 300125 Theory

--- 500130 Theory

\ ------- ‘\ 1500/80Theory

‘\

0.50 -- ‘\\ \

* \

‘*.

0.00 ‘._._A _ I

I I I - I 0.00 0.50 1 .oo 1.50 2.00 2.50 3.00 3.50

Concentration (milliMolar Fe)

Fig. 1. Effect of MI-227 on observed and theoretical signal intensity with spin-echo sequences of 3OW25, X0/30, and 1500/80. Also shown is the theoretical signal intensity for the 300/12 pulse sequence.

0 500/30 Obs.

. 1500/80 Obs.

___ 300125 Theory

0.00 0.50 1 .oo 1.50 2.00 2.50 3.00 3.50

Concentration (milliMolar Fe)

Fig. 2. Effect of ferumoxsil on observed and theoretical signal intensity with spin-echo sequences of 300/25,500/30, and 1500/80.


6.00

5.00

4.00

5

g

j 3.00

0 i iii

2.00

1.00

0.00

.

-- - -?_ ----- .

---__ ---------

--------*

I I I / I I i

n 300125 Obs.

0 500130 Obs.

l 1500180 Obs.

~ 300125 Theory

--- 500130 Theory

-------1500180Theoly

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

Concentration (miMiMolar Gd)

Fig. 3. Effect of Gd-DTPA on observed and theoretical signal intensity with spin-echo sequences of 300/25,500/30, and 1500/80.

with the 300/12 pulse sequence. In addition, the concentration range over which brightening occurred be- came broader as TE was reduced.

Ferumoxsil produced a decrease in signal intensity (Fig. 2) over a wide range of concentrations and pulse sequences. The signal intensity resulting from various concentrations of Gd-DTPA (see Fig. 3) behaved as expected.’ At short TEs and TRs (Ti weighting) signal intensities increased, that is, brightening occurred, and over a wide concentration range. At higher concentrations (>2 mM) and with the 1500/80 sequence, the T2 term of Eq. (3) dominated, and signal intensity decreased.

Anatomical detail in the vicinity of the GI tract is difficult to discern without contrast agents, as illus- trated by the 300/25 pulse sequence image of Fig. 4 and the 1500/80 image of Fig. 5. After administration of AMI-227, and with the T1-weighted 300/25 pulse sequence, the ability of AMI- to increase signal intensity was evident as a brightening of the lumen of the GI tract (Fig. 6). On the other hand with a 1500/80 pulse sequence, the ability of AMI- to decrease signal intensity was evident as a darkening of the lumen of the GI tract (Fig. 7). Thus, a specific concentration of AMI- acted as a brightening or darkening con-

trast agent for the lumen of the GI tract, a feature further discussed below.

The AMI- enhanced images of the GI tract were compared with the images obtained when Gd-DTPA or ferumoxsil was administered to rats in a similar fash- ion. As previously reported,3 the lumen of the GI tract was darkened by the administration of ferumoxsil (Fig. 8). As previously reported,’ the lumen of the GI tract was brightened due to the administration of Gd- DTPA (Fig. 9).

DISCUSSION

We examined the effects of AMI-227, Gd-DTPA, and ferumoxsil on relaxation rates (Table I), on the signal intensity of phantoms (Figs. l-3), and on MR images after oral administration (Figs. 6-9). The relaxivities obtained were consistent with previously reported values under slightly different conditions, with Gd-DTPA having the lowest RJR, of 1.06, ferumoxsil the highest of 179 for SPIO, and AMI- intermediate with a value of 11.4. AMI- produced darkening or brightening of the lumen of the GI tract which depended on pulse sequence. With a Ti-weighted spin-echo brightening was observed, while with a Tz-weighted spin-echo sequence darkening was observed.

Fig. 4. Rat abdomen and GI tract with a spin-echo 300125 sequence

Fig. 5. Rat abdomen and GI tract with a spin-echo 1500180 sequence.

Fig. 6, Brightening effect of AMI- on GI tract with a spin-echo 300/25 sequence

Fig. 7. Darkening effect of AM1-227 on Gl tract with a spin-echo 1500/80 sequence.

Fig. 8. Darkening effect of ferumoxsil on GI tract with a spin-echo 1500/80 sequence.

Fig. 9. Brightening effect of Gd-DTPA on GI tract with a spin-echo 300/25 sequence


AMI- increased or decreased image signal intensity as a function of pulse sequence and concentration, as shown in Fig. 1. With concentrations between approximately 0.1 mM and 0.7 mM, and with the Tl -weighted 300125 sequence or moderate T, -weighted 500/30 sequence, AMI- substantially increased signal intensity, that is, values greater than 1.5 were obtained. However, with the Tz-weighted 1500/80, AMI- decreased signal intensity, with a 50% reduction occurring at about 0.25 mM iron. Also shown on Fig. 1 is the theoretical effect of AMI- with the 300/12 pulse sequence. TEs of I 12 ms are readily attainable on many clinical imaging systems, including the so-called Turbo- flash imaging. Thus it is expected that if AMI- were used with current short TE type clinical imaging systems, both the intensity of the brightening and concentration range over which brightening occurred would exceed that attained in our imaging studies.

Over a wide concentration range, ferumoxsil decreased signal intensity at all pulse sequences employed (Fig. 2). A Iower concentration of ferumoxsil than AMI- was required for signal reduction; for exam- ple, with the 500/30 pulse sequence, a 50% reduction in signal occurred with 1 .O mM AMI- vs. 0.1 mM for ferumoxsil.

Gd-DTPA produced a greater increase in signal intensity than AMI- with 300/25 or 500/30 pulse sequences; peak signal intensity increased three- to six- fold for Gd-DTPA, compared to a more modest 1.5- to 2.5-fold for AMI-227, (compareFigs. 1 and 2). Gd- DTPA also produced an increase in signal intensity over a wide concentration range, from approximately 0.25 mM to at least 3 mM (Fig. 2).

With MR phantoms, the comparison of SI,i,, and

%heor indicated a close correspondence between the two for most pulse sequences and contrast agents. Da- vis suggested several possibilities for discrepancies between observed and theoretically generated MR signal intensities.’ We believe the differences observed reflect instrument limitations, that is, nonideal pulses and gra- dients, imperfect magnet shimming, and environmen- tal effects.

The selective T, enhancing effects of Gd-DTPA (Table 1, Fig. 3) and selective T2 enhancing effects of ferumoxsil (Table 1, Fig. 2) seem to preclude their use as brighten/darkening type agents like AMI-227. Gd- DTPA and ferumoxsil either brighten or darken MR image over wide concentration ranges, as expected from their values of RI and R2 and the theoretical signal intensity predicted by Eq. (3). AMI- has effects on both Rl and R2 as evident from its RI/R2 of 11.4, which lies between the 179 of ferumoxsil and 1.04 of Gd-DTPA. As a result of its intermediate RI/R2, AMI- exhibits pulse sequence-dependent brighten-

ing/darkening with phantoms and in imaging studies. However, the relaxation enhancing properties of Gd- DTPA and ferumoxsil make them well suited as brightening or darkening agents, respectively (see Ref. 2 and references contained therein).

A potential advantage of AMI- as an oral contrast agent is that it can be formulated without thickening agents, which may serve to enhance palatability. With a volume median diameter of 200 nm,3 the phar- maceutical drug product using ferumoxsil as an active ingredient employs carboxymethyl cellulose to retard settling. A second miscible darkening agent under clinical development, oral magnetic particles (OMP), uti- lizes a magnetic particle 3-4 pm in diameter, and also requires thickening agents.’ To facilitate comparison of different agents, carboxymethylcellulose was used in all studies here. However, with a solution diameter of 19 nm, AMI- does not settle upon standing and will not require thickening agents.

A significant issue with AMI- is whether it will provide uniform and useful brighten and darkening, given the variable dilution in the human GI tract. In the current study with a 300/25 pulse sequence, the brightening effect of AMI- was obtained after oral administration at a concentration of 1 mM Fe of AMI- 227 (Fig. 6), indicating some dilution occurred, (see Fig. 1). However, as TE is reduced, brightening becomes greater and occurs over a wider concentration range (Fig. 1). Our results suggest that useful brightening with AMI- will be particularly pronounced with com- mon, clinical imagers utilizing TEs of 12 ms or shorter. We further hypothesize that reliable darkening with AMI- will occur with heavily Tz-weighted, spin- echo, or gradient-echo pulse sequences. Obviously these hypotheses must be tested in the clinical situation.

The ability of the attapulgite to darken and brighten the lumen of the GI tract has been noted.” A T,- weighted gradient-echo sequence using a very short TE (lOU2.3) was used. Attapulgite darkened the lumen of the GI tract with Ti-weighted (400/14) and T2- weighted (2500/100) spin-echo sequence, obtained at 1.5 T, while AMI- brightened with the 300125 images at 2 T. Thus, AMI- may brighten at somewhat longer TEs than attapulgite. In addition, attapulgite is a clay and, unlike AMI-227, settles readily on standing. Hence the physical forms of the ultrasmall, superparamagnetic colloid AMI- and particulate, diamag- netic, agents like attapulgite, kaolin, and bentonite are quite different.

AMI- is capable of brightening or darkening MR images when administered orally, as shown here, or IV. When present in the vascular compartment, the ability of a AMI- to brighten images with T, -weighted pulse sequences has been recently noted.“*‘* Subse-

AMl-227 as an oral MR contrast agent 0 J. ROGERS ET AL. 639

quent uptake of AMI- by phagocytes deceased signal intensity with &-weighted pulse sequences.”

Darkening type GI contrast agents are limited in their inability to differentiate the hypointense intestinal wall from gas or the opacified intestinal lumen.’ But utilizing brightening effects of AMI- with a TI - weighted image, regions of gas could be delineated since they would remain dark. Brightening-type GI agents such as Gd-DTPA increase lumen signal intensity and permit discrimination between the lumen and the intestinal wall or fatty tissue, but can also increase ghost- ing artifacts.’ The ability of AMI- to exhibit image brightening or darkening could offer the clinician the flexibility to chose the sequence best suited to potential pathology of interest, a flexibility that could be ex- ercised during the imaging protocol itself.

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Use of AMI-227 as an oral MR contrast agent

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