DliMEDE-DTPA-PARA AGNETIC CONTRAST AGENTS FOR MR IMAGING, APPARATUS AND METHODS 01 DESCRIPTION

TECHNICAL FIELD

This invention relates to MR contrast agents, and more particularly to homologs of Amide DTPA-PM contrast

05 agents.

BACKGROUND

Schering (3,129,906 Germany) by Gries, Rosenberg, and einstien teaches the incorporation of paramagnetic metals into diethylene triamine pentaacetic acid (DTPA) forming

10 chelates useful as contrast agents in magnetic resonance imaging. The contrast agent DTPA-(GdΙII) as taught by

Schering is insoluble in water and requires the addition of cations **C+" (amines such as glucamine, N- methylglucamine, etc.) as shown below: The charge

15 balance of the Schering DTPA-Gd(III) ion is:

Schering DTPA-Gd(III) Charge Balance

C+ C+ DTPA . Gd

+1 +1 -5 +3 = 0

The resulting contrast agent has three ion particles in

20 solution for each paramagnetic atom (a particle to PM ratio of 3:1). A paramagnetic metal with a valence of two, such as Mn, would require an additional glucamine ion:

Schering DTPA-Mn(II) Charge Balance

25 C+ C+ C+- DTPA Mn

+1 + +1 +1 -5 +3 = 0 raising the PM to particle ratio to 4:1.

These contrast agents raise the in vivo ion concentration and disturb the local osmolarity balance.

30 The osmolarity is normally regulated at about 300 milliosmols per liter. Increasing the osmolarity with injected ions, causes water to collect within the unbalance region which dilutes the ion concentration.

35

SUMMARY

It is therefore an object of this invention to provide improved amide contrast agents for MR imaging.

It is another object of this invention to provide MR amide contrast agents which have a high stability, a low toxicity and are physiologically tolerable.

It is a further object of this invention to provide amide contrast agents in pharmacological form with a low osmolarity. It is a further object of this invention to provide amide contrast agents which are in vivo responsive.

It is a further object of this invention to provide amide contrast agents which are organ selective.

It is a further object of this invention to provide amide contrast agents which cause surface highlighting of the small and large intestine.

It is a further object of this invention to provide a method of manufacturing such amide contrast agents.

It is a further, object of this invention to provide a method of using such amide contrast agents.

It is a further object of this invention to provide an MR system employing such amide contrast agents.

Briefly, these and other objects of the present invention are accomplished by providing a chemically stable physiologically tolerable contrast agent in a pharmacological state, for in vivo use during diagnostic magnetic resonance (MR) imaging. The contrast agent enhances the MR image of a subject within the MR scanning magnetic field. A paramagnetic metal ion PM(+Z) having an atomic charge of Z locally affects the MR scanning magnetic field to reduce the Tl relaxation time of local protons within the subject. The contrast agent contains a

triamine chelator DTPA ¹ securely polar bonded around the PM(+Z) ion at a plurality of coordination points, and has the form:

I + I CH2 H CH2

I I I

DTPA' = N ^» CH2-CH2—N—CH2-CH2—N PM(Z) I I I

CH2 CH2 CH2 I I I c=o C=0 C=0 0 0 0 for chemically isolating the PM(+Z) ion from the in vivo environment. The contrast agent also contains a functional amide group of the form:

0 -C-N-(CH2) (n-l)-CH3,

H wherein "n" is an integer from 0 to 16 indicating the number of Carbon atoms in the

Carbon-Hydrogen portion of each amide group. The functional amide may be a homo-diamide or a hetero- diamide. The Amide-DTPA'-Pf-I contrast agent is dispensed in a a pharmaceutically acceptable vehicle means such as water. The Carbon-Hydrogen portion to the amide compound becomes associated with water of hydration which increases the paramagnetic strength of the contrast agent. The PM ion may have a valence of +3 and produce a contrast agent molecule of zero net charge. The PM ion may have a valence of +2 and require an inert cation IN having an atomic charge to produce a molecule with a zero net charge. The paramagnetic metal ion PM(+Z) is at least one ejen-ent selected frcr. the Transition Elements 24-29 or the Lanthanide Elements 57-71.

BRIEF DESCRIPTION OF THE DRAWING

Further objects and advantages of the present paramagnetic contrast agents, and the method of manufacture and use thereof, will become apparent from the following detailed description and drawing in which.

Figure 1A is a diagram showing the chelate structure and water of hydration of a Diamide-DTPA-PM(Z) contrast agent in which Z=+3;

Figure IB is a diagram showing the chemical structure of the Diamide-DTPA-PM contrast agent of Figure 1A;

Figure 1C is a diagram showing the chemical structure of a general Diamide-DTPA-P (Z) contrast agent in which Z=+2;

Figure 2 is a diagram showing the anhydride ammonium hydroxide production of Dimethyl-DTPA-PM(Z) in which Z=+3; Figure 3 is a diagram showing the anhydride butyl amine production of Dibutyl-DTPA-PM(Z) in which Z=+2;

Figure 4A is a colon shown in cross section (from the "Atlas of Descriptive Histology" Reith-Ross p 121) ? Figure 4B is a planar schematic drawing of an MR image of a colon showing surface highlighting by Diamide- DTPA-PM;

Figure 4C is a perspective schematic drawing of an MR image of a colon showing surface highlighting by Diamide- DTPA-PM of occulted and non-occulted surfaces _?

Figure 5 is a cut-away perspective view of an MR system showing the motion platform and subject using Diamide-DTPA-PM paramagnetic contrast agents; and

Figure 6 is a flow chart showing a method of using the Diamide-DTPA-PM paramagnetic contrast agents.

01 DIAMINE-DTPA-PM CONTRAST AGENTS (Figure 1A IB 1C) The present paramagnetic contrast agents are amide homologs of the DTPA-PM chelate, having the general chemical name diamido acetyl - diethylene triamine 05 triacetic acid (or Diamide-DTPA) . The probable physical chelation structure of Diamide-DTPA-PM is a classic octahedron (8 faces, 6 apexes) as shown in Figure 1A. The Diamide-DTPA homologs are strong chelators having six polar bond coordination points 104 (three nitrogen points 10 104:N and three oxygen points 104:0) which enclose the paramagnetic ion PM(Z) on all sides.

Diamide-DTPA-PM has the general chemical structure shown in Figure IB. The homologs thereof have similar structures with a specific number "n" of carbons in the 3.5 Carbon-Hydrogen portion of the amide group. The number of Carbons in the methylene CH2 chain between the -CONH- active group and the terminal methylene -CH3, is "n-l".

Two of the original five DTPA acetic acid groups have become amide groups "A". In general: 20 Diamide-DTPA-PM = 2A-DTPA'-PM where A is a general amide group of the form: 0 A = -C-N-(CH2) (n-l)-CH3, H 25 and DTPA' is a modification of Schering DTPA of the form:

I I

CH2 H CH2

I I I

DTPA' = N—CH2-CH2— —CH2-CH2—

30

CH2 CH2 CH2

C=0 C=0 C=0 O O O 35 and PM is a paramagnetic metal ion. The elimination of the two acetic acid groups reduces the ion charge of the DTPA chelator from five to three.

Paramagnetic ions having a valence of Z=+3 as shown in Figure 1A and IB, produce a diamide contrast agent of the general form:

Diamide-DTPA-PM(+3) = 2A-DTPA'-PM(+3) . This Type III contrast agent has a zero net charge as tabulated belows

Diamide-DTPA-PM(+3) Charge Balance 2A DTPA' PM (+0) + (-3) + (+3) = 0. The particle (osmolarity) to paramagnetic (molar celaxivity) ratio for Diamide-DTPA-PM(+3) type contrast agents (Z=+3) is 1:1. The Diamide-DTPA-PM(Z) contrast agents forme around plus III paramagnetic metals can be prepared in highly concentrated solutions while retaining isotonicit-y with body fluids. The Schering DTPA-PK{+3) has a particle to paramagnetic ratio of 3:1, and can only be made in isotonic solutions at substantially lower concentrations. Therefore, greater volumes of the Schering DTPA-PM(+3) need be injected into animals or humans to obtain the same paramagnetic effect.

Paramagnetic ions having a valence of Z=2, produce amide contrast agents of the general form:

Diamide-IN-DTPA-PM(+2) = 2A-IN-DTPA'-PM(+2) where IN is a suitable inert ion, such as a simple mineral salt cation (Na+, Li+, etc.) or an organic ion such as Methyl glucamine or N-methyl glucamine, having a charge of plus one (see Figure 1C) . This Type II contrast agent also has a zero net charge as tabulated below: Diamiάe-IN-DTPA-PK(+2) Charge Balance 2A IN DTPA ¹ PM

(+0) + (+1) + (-3) + (+3) = 0. The particle to paramagnetic ratio for the IN-Diamide- DTPA-PM(+2) contrast agents is 2:1, producing a low osmolarity impact.

The above Diamide-DTPA-PM Type III and Type II contrast agents have a paramagnetic effect similar to the Schering DTPA-PM. For example. Methyl Amide DTPA-Gd(III) requires a concentration of about 3.31 mM to produce a Tl relaxation time of 67 msec (10 MHz field strength, using an RADX) . The concentration of Schering DTPA-Gd(III) required to produce a similar result is about 3.16. t'ethyl Amide DTPA-Gd(III) has about the same paramagnetism of Schering DTPA-Gd(III) . Possibly the water of hydration 108 (see Figure 1A) which collects around the amide CH2 chains offers a reliable souxce of protons (H+) 110 for resonanting with the applied MR fields. Protons 110 have a high probability of being present within the local magnetic field of the PM ions. These protons form a class of protons for MR imaging which is distinct from random in vivo protons. The prolonged association time of bound water 108, and the close proximity of protons 110 to the FM ion, establishes a definite and distinct Tl relaxation time vbich iε lor-rer than the Tl for random protons. As a result, protons 110 provided by the water of hydration appear at a higher intensity in the MR image.

METHOD OF MANUFACTURE (Figures 2 and 3)

A general anhydride-diamide method is suitable for . making each homolog of the amide family of DTPA'-PM contrast agents. In the example below the paramagnetic ion is provided by Fe(III)-(Cl)3, for chelation into dimethyl amide (n=l) . However, other paramagnetic ions in other forms may be employed for chelation into other amide homologs.

Step 1) FORMATION of Amide-DTPA (see Figure 2) Mix 1-5 grams dianhydride DTPA (obtained from Sigma Chemical Co, St Louis MO) into 50-150 mL of 5 percent (v/v) NH4-OH . (ammonium hydroxide) in water. Fixed ratios of NaOH/DTPA are not required, Precise so long as excess NH40H is provided. Step 2) HEAT the solution for several hours (overnight) at reflux temperature, to produce the amide derivative

Dimethyl-DTPA (n=l) plus water. Higher homologs of Diamide-DTPA may be formed using the corresponding higher homolog of alkyl amines for the reactant. Chloroform may be used as the solvent for higher homologs. Formation of the Dibuty -DTPA (n= ) cia ide hor-a.olog is shown in Figure 3.

Step 3) REMOVE the excess solvent, by vacuum rotary evaporation leaving an Diamide-DTPA crystal residue. Step 4) MIX the Diamide-DTPA residue in an FeC13 water solution of stoichiometric proportions, to form Diamide-DTPA-(Fe+3) plus 3HC1. Type II metals will require an inert cation (IN) which may be added to the solution' at this point.

Step 5) REMOVE the HCl

A) by evaporation using a rotary evaporator.

B) by neutralization using NaOH or NH40H.

C) by chromatograpy using a silica gel column. Step 6) REMOVE the water by vacuum-freezing to form a highly stable.form of Diamide-DTPA-PM.

Step 7) DISPERSE the Amide-DTPA-PM in suitable vehicle to provide a pharmacological form. Water is a suitable vehicle for dissolving the lower homologs of Diamide-DTPA-PM (n less than 10) . Higher homologs are hydrophobic and form an emulsion with water. These higher homologs have the same density as water and therefore do not settle out. The isodense character of the homologs of Diamide-DTPA-PM permits a wide range of water:homolog ratios.

AMIDE FAMILY (n=0 to n=16)

The amide family of DTPA'-PM contrast agents include the homo-diamides (n=n') structure and the hetero-diamides (n not equal to n') structure.

Name of Amide n,n' Properties of Interest

Diamide-DTPA-PM 0 ,0 Excellent

Methyl-DTPA-PM 1,1 renal and

Ethyl-DTPA-PM 2,2 blood-brain

Propyl-DTPA-PM 3,3 barrier contrast

Butyl-DTPA-PM 4,4 agent.

Pentyl-DTPA-PM 5,5 Demonstrates renal

Hexyl-DTPA-PM 6 _f 6 and hepatobiliary

Heptyl-DTPA-PM 7,7 imaging.

Octy1-DTPA-PM 8,8 Also shows cardiac

Nonyl-DTPA-PM 9,9 imaging of infarctions

Decyl-DTPA-PM 10,10 and ischemic lesions.

to 16,16

Diamide-Stearyl- Passes into the DTPA-PM 0,16 Cardiac system imaging,

The hetero-diamides have one short CH2 chain (n=l or more), and one long CH2 chain (n=16 or less). A single long hydrophobic chain, together with the charged DTPA' moiety, renders the chelate an isosteric substitute for fatty acids; and produces substantial tissue levels of the chelate in those organs which have efficient fatty acid uptake systems such as the myocardium.

ORGAN SELECTIVE (Figure 4A 4B 4C)

Venously introduced contrast agents are immediately distributed throughout the circulatory system for imaging. Organs such as the kidney, brain, liver, and heart receive substantial blood flow; and provide selective images which are agent enhanced.

A ide-DTPA-PM has a prolonged circulation time due to its high stability. The Amide contrast agent is less affected by ensymes degradation than simple ion-DTPA chelates (Schering) . In addition, the higher homologs of Amide-DTPA-PM tend to be less polar, and to bind more to serum proteins, further increasing their circulation time. They tend to be extracted from circulation by the liver and excreted in the hepatobiliary system. The amide contrast agent passes through the bile duct (controlled by the ampulla of Vater) and is absorbed into the colon. The Amide contrast agents are suitable for imaging the hepatobiliary (gall bladder) system.

Figure 4A is a cross sectional view of the colon 440. The diamide appearing along the convoluted inner surface of the colon wall 442 is slowly brushed away by the lu inal content .444. The high viscosity of the contrast agent prevents it from immediately mixing with the luminal content 444. Because the washout rate is slower than the excretion rate, the agent accumulates in a film or layer 446 along the inner surface of colon 440.

The paramagnetic properties of amide enriched layer 446 establishes a shorter Tl relaxation time for the local protons within the layer. In the resulting MR image, amide layer 4 _.6 is displayed at a higher intensity, highlighting the inner surface of the colon 440. Surface highlighted images are particularly useful in studying

01 those disease processes involving changes in mucosal transit such as malabsorption, non-tropical sprue, ulceratine colitis, regional enteritis etc. The luminal content is not amide enriched and appears grey or dark

05 (unenhanced) along with the background tissue.

Figure 4B shows a schematic MR image of the colon in cross-section, and Figure 4C shows a schematic MR image of the colon in perspective. Both simple planar views and the complex perspective views can be computer generated

10 from the MR data. The surface amide accumulation 446 appears bright and outlines of the inner surface colon 440 unimpedded by the luminal content. This surface effect is especially noticeable in perspective view 4C which reveals the front surface 448-F, and both the unoccultec back

15 surface 448-B and occulte back surface 448-0. The thin amide layer Aβ en the front surface has a transparent characteristic which permits the occulted back surface to be viev/eά. The display intensity of the region of overlap between the front surface 448-F and occulted back surface

20 448-0 is the summation of the separate intensities.

The lower homologs tend to be more polar and remain in solution longer. These homologs are kidney selective and suitable for imaging the kidney, ureter, and bladder. The higher homologs are fatty acid analogs and are

25 thus extracted by the heart along with the regular fatty acids. These homologs (n=7 and greater) are cardiac .selective and suitable for imaging the cardiac system and cardiac related functions.

Oral introduction of the Diamide-DTPA-PM contrast

30 agent requires a higher volume. The agent fills the luminal channel of the digestive system for providing a volume or bulk MR image.

3

STABLE-POWDER STATE

The stable powder state of the Diamide-DTPA-PM contrast agents have an indefinite shelf life, and is the preferred state for shipping and storage. The contrast agent in water solution (or other solvent) is packaged in small storage vials, and frozen under a vacuum. The low pressure sublimates the solvent, leaving crystals of the contrast agent. The vial is sealed to prevent entry of external contaminants, and to to preserve the internal vacuum. The resulting freeze-dried, vacuum sealed powder, is highly stable and free from environmental degradation effects.

FEAPMACOLOGICAL-SOLUTION STATE Prior to injection, the stable-powdered contrast agent may be raise to the pharmacological state by the addition of a suitable solvent such as water, serum, albumin solutions, or saline. A typical injectable composition contains about lOmg human serum albumin (1 percent USP Parke-Davis) and from about 10 to 500 micrograms of Diamide-DTPA-PM material per milliliter of 0.01 M phosphate buffer (pE 7.5) containing 0.9 percent NaCl. The pE of the aqueous solutions may range between 5-9, preferably between 6-8. The storage vial may have twin compartments containing the desired amounts of powdered Diamide-DTPA-PM and solvent for a single application. When the seal between the compartments is broken, the Diamide-DTPA-PM goes into solution at the desired concentration for immediate use. The Diamide- DTPA-PM solution mixes readily with the in vivo fluids.

PARAMAGNETIC EXAMPLES

Paramagnetic material PM may be any paramagnetic element, molecule, ion or compound having a combined valance of "Z". Paramagnetic material PM includes at least one of the following elements: Ions of Transition Elements

Cr(III) 24 (Chromium) Co(II) 27 (Cobalt)

Mn(II) 25 (Manganese) Ni(II) 28 (Nickel)

Fe(III) 26 (Iron) Cu(III) 29 (Copper) Fe(II) 26 (Iron) Cu(II) 29 (Copper)

Ions of Lanthanide Elements • .

La(III) 57 (Lanthanum) Gd(III) 64 (Gadolinium) Ce(III) 58 (Cerium) Tb(III) 65 (Terbium) Pr(III) 59 (Praseodymium) Dy(III) 66 (Dysprosium) Nd(III) 60 (Neoάymium) Ho(III) 67 (Holmiu ) Pm(III) 61 (Promethiu ) Er(III) 68 (Erbium) Sm(III) 62 (Samarium) Tm(III) 69 (Thulium) Eu(III) 63 (Europium) Yb(III) 70 (Ytterbium)

Lu(III) 71 (Lutetiu ) Gd has the highest paramagnetic property; but is a costly and highly toxic in the free state. Placing the Gd within the chelator produces a physiologically tolerable form of Gάj but also reduces paramagnetic effect of the Gd. The chelate structure tends to shield the paramagnetic ions and prevents close proximity to local H+ protons. Fe and Mn have a high paramagnetic property and excellent physiological tolerance. Both of these paramagnetic ions are normally present in the physiological environment.

GENERAL MR SYSTEM (Figure 5)

Magnetic resonance (MR) imaging system 500 has two magnetic components which scan subject 504 for obtaining MR data enhanced by the presence of contrast agent 508. Bulk magnetic field Mz from Z field source 510 causes paramagnetic particles such as local hydrogen protons within the subject to aline with the Z axis. Periodic or rotating field J.xy fro ZY field generator 514 extends between XY electrodes 516. The subject to be scanned is positioned on support platform 520 and moved through the magnetic fields by drive 522. Rotating field Mxy is tuned to cause resonant precession of the local protons within the tissue of interest. Each local proton precesses about the Z axis in response to a particular frequency of rotating field Mxy. When rotating field Mxy is removed, the precessi g protons decay back into alinement with Mz. The decay period of the local protons (spin lattice relaxation time Tl) varies between organs and between conditions within the same organ. Tumor tissue tends to have a longer Tl than healthy tissue. The presence of the paramagnetic metal.ions FM causes a shortening of the proton Tl, without substantially affecting T2 (spin-spin relaxation time) . The energy of precession is released forming a free induction signal. Grid detector 526 senses the decay signals which are stored and processed by data processer system 530. to form an image 532 on monitor 536. The metal ion in the contrast agent are not directly imaged by the MR system.

The imaging system is further disclosed in Scientific American, May 1982, pages 78-88, and "NMR A Primer for Medical Imaging" by Wolf and Foρp Slack Eook Division (ISBN 0-943432-19-7), which disclosures are hereby incorporated by reference.

METHOD OF USE (Figure 6) Figure 6 shows a method of imaging subject 504 with MR system 500 employing an paramagnetic contrast agent 508. Step 1) PROVIDING a physiologically tolerable contrast agent 508 in the form: 2A-DTPA-PM(+Z) . If initially in powder form, the 2A-DTPA-PM contrast agent must be dispensed into a suitable carrier vehicle.

Step 2) INTRODUCING the 2A-DTPA-PM contrast agent into subject 508 (preferably by intravenous injection) . Step 3) WAITING for the amide functional groups to cooperate with the in vivo environment. Step 4) IMAGING the subject Vith HE ε}εteir 5C0 to obtain an enhance MR image. Comparison or subtraction imaging, requires an initial step of providing data from a prior MR imaging, and the final step of subtraction comparing the prior MR image with the current MR image. A historical base line image from the subjects file ιr.ay be employe as the prior image. Alternatively, a current MR image made without the use of a contrast agent may be employed.

INDUSTRIAL APPLICABILITY

It vj l be apparent to those skilled in the art that the ckjectε of this invention have been achieved as described hereinbefore by providing an improved physiologically tolerable contrast agents with a high stability, and a low toxicity. The contrast agent has a high paramagnetic effect due to the amide water of hydration, and a low osmolarity due to the amide bonding. The variability of the amide structure permits a range of vivo response and organ selection, including surface selectivity of the colon.

CONCLUSION Clearly various changes may be made in the structure and embodiments shown herein without departing from the concept of the invention. Further, the features of the embodiments shown in the various Figures may be employed with the embodiments of the other Figures.

Therefore, the scope of the invention is to be determined by the terminology of the following claims and the legal equivalents thereof.