WO2011088513A1

WO2011088513A1 - A method for measuring interstitial volume in organs and tissues

Info

Publication number: WO2011088513A1
Application number: PCT/AU2011/000062
Authority: WO
Inventors: James Charles Moon
Original assignee: Equilibrium Imaging Limited; Resonance Health Analysis Services Pty Ltd
Priority date: 2010-01-20
Filing date: 2011-01-20
Publication date: 2011-07-28

Abstract

The present disclosure provides a method of measuring interstitial volume reflecting diffuse fibrosis, oedema or amyloidosis and its reciprocal, cell volume, in body organs. The method comprises obtaining first image signal intensity values (by computerised tomography or magnetic resonance imaging) for blood and at least a portion of one or more organs in a subject at an area of interest. Thereafter an extracellular contrast agent (gadolinium or iodine based) is administered to the subject in such a way that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium condition. Thereafter second image signal intensity values for blood and one or more organs in the subject at the area of interest are obtained. Further, the method comprises the step of determining the interstitial volume by determining a volume of distribution and its reciprocal, cell volume of the contrast agent for at least the portion of the one or more organs by comparing to the blood volume of distribution using the first and second image signal intensity values.

Description

A METHOD FOR MEASURING INTERSTITIAL VOLUME

IN ORGANS AND TISSUES

Field of the Invention

The present invention relates to a method for measuring an interstitial volume, such as that associated with fibrosis in organs or tissues.

Background of the Invention

Fibrosis is a final common endpoint in virtually all

pathological processes in human organs and tissues. For example, in the heart, focal fibrosis resulting from myocardial infarction is the leading cause of death and heart failure in the world. Additionally, in a multitude of illnesses and in senescence, the interstitium is expanded by fibrosis and cellularity is reduced, resulting in organ dysfunction and disease. For example, diffuse interstitial fibrosis plays a key role in cardiac diseases such as hypertension,

cardiomyopathies and valve disease, leading to heart failure and death. Similarly, diffuse fibrotic liver disease is the clinical result of chronic hepatitis and alcohol abuse.

Fibrosis may be assessed in vivo by magnetic resonance imaging (MRI) using the late gadolinium enhancement (LGE) contrast technique. The fibrosis leads to an increased volume of distribution . for gadolinium and there is late enhancement of the fibrosis by MRI. However, this technique is only

applicable to the assessment of focal fibrosis. There have been attempts to measure ^' diffuse fibrosis using contrast MRI after a contrast bolus, but these have been confounded by kinetic changes as the contrast washes through the tissue which require sophisticated modelling and multiple assumptions. Presently, the primary method for assessing diffuse fibrosis is tissue sampling via biopsy, which is invasive, attracts both morbidity and mortality, suffers from sampling errors and is not possible in several disease sites. Although some noninvasive techniques do exist, for example ultrasound and biochemical markers in liver fibrosis, these techniques are often indirect and crucially are organ specific. Accordingly, the ability to assess the interstitial volume of an organ, in particular diffuse fibrosis, using a reproducible, non- invasive technique in any organ is highly desirable.

Summary of the Invention

Embodiments of the present invention relate to a non- invasive method of measuring interstitial volume and may be used to measure a level of fibrosis in an organ, including diffuse fibrosis, amyloidosis and cell volume.

Accordingly, in a first aspect, the present invention provides a method of measuring interstitial volume, the method

comprising the steps of:

obtaining first image signal intensity value^'s for blood and at least a portion of one or more organs in a subject at an area of interest; thereafter

administering to the subject an extracellular contrast agent in such a way that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium condition; thereafter

obtaining second image signal intensity values for blood and one or more organs in the subject at the area of interest; and

determining the interstitial volume by determining a volume of distribution of the contrast agent for at least the portion of the one on more organs compared to the blood using the first and second image signal intensity values.

For example, the interstitial volume may be indicative of a level of amyloidosis or fibrosis, such as diffuse or focal fibrosis or oedema.

In one embodiment, the step of administering to the subject an extracellular contrast agent comprises administering a bolus of a contrast agent followed by a continuous infusion of a contrast agent. In this case the continuous infusion of the contrast agent typically is conducted such that the equilibrium condition is substantially maintained during the continuous infusion of the contrast agent. The continuous infusion may be an infusion that is separate to the bolus infusion.

Alternatively, the continuous infusion may be an extension of the bolus infusion. In this case the second image intensity values typically are obtained during the continuous infusion. The equilibrium will be reached and typically maintained during the continuous infusion of. the contrast agent. A period of time within which the equilibrium is achieved depends on a size of the bolus, its rate of delivery, the dose rate of the

continuous infusion and the time delay between bolus and continuous infusion of the contrast agent.

Further, the step of administering to the subject an.

extracellular contrast agent may comprise administering a bolus of contrast agent (without further infusion) and imaging at the equilibrium point - the point where concentrations of contrast in tissue are at an inflection point and cease rising and start to fall. This point, the transient equilibrium point may be extended for a period of time that is sufficiently long for the obtaining second image signal intensity values, by altering the bolus injection rate and bolus profile. This alteration in bolus delivery is also designed to smooth out recirculation blood effects. Consequently, the second image signal intensity values are in this case typically obtained at transient

equilibrium conditions after bolus injection and typically without any further infusion of contrast agent. The second image signal intensity. values are typically obtained at a first maximum of a signal associated with organ tissue after

administering the bolus (the transient equilibrium point) .

The step of administering to the subject an extracellular contrast typically comprises administering the contrast agent in vivo.

The step of determining the interstitial volume may comprise obtaining a blood sample from the subject and determining the haematocrit level.

In another embodiment, the step of using the first and second image signal intensity values to determine the volume comprises calculating the volume using the following equation (or a derivative thereof) :

OV_d =(1 - haematocrit) x (OSIV2-OSIV1) / (BSIV2-BSIV1)

where OV_d is the volume of distribution of contrast agent for an organ; OSTV2 is an organ image signal intensity value

associated with the second image signal intensity values OSIV1 is an organ image signal intensity value associated with the first image signal intensity values, BSIVl is a blood image signal intensity value associated with the first image signal intensity values and BSIV2 is a blood image signal intensity value associated with the second image signal intensity values. The first and second image signal intensity values for blood and one or more organs typically are derived from an image, such as from an image obtained by magnetic resonance imaging (MRI) . Each image intensity value may be related to an imaging signal strength either at a region or pixel of an image or signals integrated over a region of interest or portion thereof .

In another embodiment, the volume of distribution is converted to and expressed as the amount of fibrosis, amyloid or oedema (expressed as a percentage of the organ or tissue) , based on prior biopsy data in the disease in question. Alternatively, the cell volume may be derived - the reciprocal of these percentages (1 minus percentage of fibrosis, for example)

The step of obtaining first and second image signal intensity values may comprise MRI. In this embodiment, the contrast agent may comprise a gadolinium chelated by an agent such as Diethylenetriaminepentaacetate (DTPA) or variants of DTPA. Further, in this embodiment the image signal intensity value may be associated with an MRI rate constant for longitudinal spin relaxation after excitation referred to as "Tl" . The method typically also comprises correcting image signal intensity values or an image for fluctuations associated with the heart rate of the subject.

In an alternative embodiment, the step of obtaining first and second image signal intensity values for blood and one or more organs in a subject comprises computerised tomography. In this case the step of obtaining first and second image signal intensity values for blood and one or more organs in a subject typically comprises administering an iodine based contrast agent .

In a second aspect, the present invention provides a method of diagnosing a disease, the method comprising the steps of:

obtaining first image signal intensity values for blood and at least a portion of one or more organs at an area of interest in a subject suspected of having the disease; thereafter

administering to the subject an extracellular contrast agent such that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium

condition; thereafter

obtaining second image signal intensity values for blood and one or more organs in the subject associated with the area of interest; and

determining an interstitial volume by determining a volume of distribution of the contrast agent for at least the portion of the one or more organs using the first and second image signal intensity values; and

comparing the determined interstitial volume with an interstitial volume in a corresponding organ in a healthy subject to determine the presence of the disease.

In a third aspect, the present invention provides a method of monitoring progression of a disease, the method comprising the steps of:

obtaining first image signal intensity values for blood and at least a portion of one. or more organs at an area of interest in a subject diagnosed with the disease; thereafter

administering to the subject an extracellular contrast agent such that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is., at the area of interest, substantially at an equilibrium

condition; thereafter

obtaining second image signal intensity values for blood and one or more organs in the subject associated with the area of interest;

comparing the determined interstitial volume with an interstitial volume in a corresponding organ in a healthy subject to determine progression of the disease.

In one embodiment, the disease is a disease associated with fibrosis or amyloidosis.

The method in accordance with any one of the preceding aspects may comprise analysing the volume of distribution of the contrast agent to provide information concerning a percentage of fibrosis or amyloidosis - in a specific organ and wherein providing that information comprises calibration with

information obtained from human biopsies. The calibration with information obtained from human biopsies may comprise a curve fitting procedure.

In a fourth aspect the present invention provides a method of determining cell volume, the method comprising the steps of: obtaining first image signal intensity values for blood and at least a portion of one or more organs in a subject at an area of interest; thereafter

administering to the subject an extracellular contrast agent in such a way that diffusion of the contrast agent between the blood and at least the portion of the one or moire organs is, at the area of interest, substantially at an equilibrium condition; thereafter

subtracting the determined interstitial volume from a volume of the area of interest to determine the volume that is substantially free of contrast agent and that is indicative of the cell volume in the region of interest.

The method in accordance with the fourth aspect of the present invention may comprise steps in accordance with embodiments of first, second and third aspects of the invention.

The invention will be more fully understood from the following description of specific embodiments of the invention. The description is provided with reference to the accompanying - drawings .

Brief Description of the Drawings

Figure 1 (a) shows a graph of measured Tl values for RR intervals illustrating effects of mathematical heart rate correction;

Figure 1(b) shows a graph of volume of distribution Vd(m) as detected using cardiovascular magnetic resonance (CMR) with and without heart rate correction set out against collagen volume fraction (CVF) ; Figure 2 (a) shows a schematic of a cardiovascular magnetic resonance (CMR) protocol;

Figure 2 (b) shows a graph illustrating a measurement of a volume of distribution Vd(m) using cardiovascular magnetic resonance (CMR) . This graph shows blood and myocardium signal intensities plotted against inversion time before and after contrast agent administration. The ratio of the change in myocardial: blood signal multiplied by Vd(b) (1-haematocrit) gives the Vd(m) ;

Figure 3 (a) shows a graph illustrating blood and myocardial magnetic resonance (TI) measurements after a contrast agent bolus infusion only;

Figure 3 (b) shows a graph illustrating blood and myocardial magnetic resonance (TI) measurements after a contrast agent bolus infusion and subsequently steady state achieved by continuous contrast agent infusion;

Figure 4 shows histology images for three biopsies from aortic stenosis patients illustrating the range of fibrosis present;

Figure 5 shows a plot illustrating examples of achieved equilibrium contrasts for equilibrium MRI scanning;

Figure 6 (a) to (d) shows plots illustrating transient

equilibrium conditions that were achieved by tailoring a 'bolus infusion profile without continuous infusion reducing

recirculation (Figure 6 (c) illustrates optimised conditions and Figure 6 (d) illustrates a case in which no transient

equilibrium is obtained when the bolus input rate is too low); Figure 7 illustrates MRI results for measured myocardial volume of distribution against histological Collagen Volume Fraction (CVF) for the volume of distribution (Vd(m) ) correlating with CVF in aortic stenosis (left, n=18) , hypertrophic

cardiomyopathy (middle, n=8) and the combined population^' (right, n=26) ;

Figure 8 represents blood equilibrium contrast obtained using a bolus plus infusion of CT iodine contrast agent, in this case iodohexol ;

Figure 9 represents an examples of equilibrium contrast

Computer Tomography (CT) images that were obtained after infusion of iodohexol at equilibrium condition as illustrated in Figure 8 ; and

Figure 10 - 13 show graph illustrating examples of monitoring and predictin diseases (fibrosis, cell volumes and focal fibrosis quantification) using methods in accordance with embodiments of the present invention

Detailed Description of Specific Embodiments

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of histology and medical imaging, which are within the skill of the art. Such techniques are described in the literature. See, for example, Ross et al., "Histology: Text and Atlas", 3rd Ed., Williams & Wilkins, 1995; Markus Rudin, "Molecular imaging: basic

principles and applications in biomedical research" , Imperial College Press, 2005; and "Molecular Imaging and Contrast Agent Database (MICAD) " [database online] . Bethesda (MD) : National Library of Medicine (US), NCBI; 2004- {current year}. Available from: http://micad.nih.gov. Embodiments of the present invention are directed towards a method of measuring interstitial volume.

The "interstitium" or "interstitial space" refers to the space between cells in a tissue or organ. Accordingly, "interstitial volume" refers to the amount of interstitial space within a tissue or organ.

Interstitial expansion occurs as the results of a number of pathological conditions, for example, amyloidosis and fibrosis (diffuse and focal) . The term . "amyloidosis" refers to an excess of amyloid proteins deposited in tissue and organs.

"Amyloid proteins" are insoluble fibrilar proteins

characterized by a cross-beta sheet quaternary structure.

There are several different types of amyloidosis including Primary Amyloidosis (AL) , Secondary Amyloidosis (AA) , Familial Amyloidosis, Cutaneous Amyloidosis, Cerebral/Central Nervous System Amyloid Diseases, Heavy Light Chain Amyloidosis (AH) , Beta- 2 -Microglobulin Amyloidosis (B2M - Dialysis Related) , and Localized Amyloidosis.

The term "fibrosis" refers to an excess or thickening of connective tissue within a tissue or organ. When cells within an organ are injured or die due to viral infection, toxins, trauma, or other factors, such as senescence, the immune system is activated and the repair process is initiated. The injury or death of cells stimulates inflammatory immune cells to release cytokines, growth factors, and other chemicals. These chemical messengers direct support cells in the organ to activate and produce collagen, glycoproteins (such as

fibronectin) , proteoglycans, and other substances. These substances are deposited in the organ and form connective tissue. At the same time, the process of breaking down or degrading collagen may be impaired. In a healthy organ, the synthesis (fibrogenesis) and breakdown . (fibrolysis) of

connective tissue are in balance. Accordingly, -fibrosis occurs when excessive connective tissue is synthesised faster than it can be broken down and removed from the organ.

Fibrosis may be defined as either "focal fibrosis" or "diffuse fibrosis". The term "focal fibrosis" refers to fibrosis that results in connective tissue build-up in a discrete area and is commonly referred to as "scarring" . Causes of focal fibrosis include, for example, myocardial infarction in the heart and irritants such as silica and coal dust in the lungs. In contrast, "diffuse fibrosis" refers to fibrosis that is disseminated throughout an organ or tissue. Diffuse fibrosis occurs as a result of the normal aging process and may also be caused, for example, by chronic hepatitis in the liver and diabetes mellitus in the kidneys. While the methods of the some embodiments of the present invention may be used to measure both focal and diffuse fibrosis ("total fibrosis") in an organ, the present methods are especially useful in

measuring diffuse fibrosis. For focal fibrosis, a utility of a test is in the detection of residual cell volume,

distinguishing total replacement fibrosis (scar) , from focal fibrosis with residual cells within it.

The process of interstitial expansion, as described above, occurs in almost all organs of the body and as such the method of embodiments of the present invention is not tissue or organ specific, but may be applied to any organ within an animal. The term "organ" refers to a group of tissues that perform a specific function or group of functions . Examples of animal organs may include, but are not limited to the heart, lungs, brain, eve, stomach, spleen, bones, muscles, pancreas, kidneys, liver, intestines, skin, lymph nodes, urinary bladder and sex organs (for example ovary and prostate).

Embodiments of the present invention use the volume of

distribution of an extracellular contrast agent in an organ to measure the level of interstitial volume in the organ by noninvasive medical imaging methods. An "extracellular contras agent", as used herein, refers to an extracellular substance used to enhance the contrast of structures or fluids within the body in medical imaging. The term "extracellular" refers to substances that leave the vascular compartment, do not bind tissues and are not actively transported into cells. There are many different types of contrast agents, all of which are known to those skilled in the art.

Gadolinium-based contrast agents are suitable for use iri the present invention with magnetic resonance imaging and include Gadolinium-Diethylenetriaminepentaacetate (GD-DTPA) ,

Gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) , Gadolinium 1,4, 7-triscarboxymethyl-l, 4 , 7, 10- tetraazacyclododecane (Gd-HP-D03A) , 4RS) - [4 -carboxy-5 , 8, 11- tris (carboxymethyl) -l-phenyl-2-oxa-5, 8, ll-triazatridecan-13- oato(5-)] gadolinate (2-) dihydrogen compound with 1-deoxy-l- (methylamino) -D-glucitol (1:2) (Gd-BOPTA) , Gadolinium

diethylenetriaminepentaacetate-bis (methylamide) (Gd-DTPA-BMA) and Gadolinium [8 , 11-bis (carboxymethyl) -14- [2- [ (2- methoxyethyl) amno] -2-oxoethyl] -6-oxo-2-oxa-5, 8 , 11, 14- tetraazahexadecan-16-oato(3-) ] (Gd-DTPA-BMEA) . In some embodiments, the contrast agent used is GD-DTPA.

Iodinated contrast agents are also suitable for use in the present invention with X-ray imaging or computed tomography (CT) and include Diatrizoate (Hypaque 50), Metrizoate (Isopaque Coronar 370) , Ioxaglate (Hexabrix) , Iopamidol (Isovue 370) , Ioxilan (Oxilan) , Iohexol (Omnipaque 350) and Iopromide

Iodixanol (Visipaque 320) . In some embodiments, the contrast agent used is Iohexol, (see example 11 discussed below) .

When. the extracellular contrast agent is injected intravenously into a subject as a bolus the blood/plasma concentration rises and the contrast agent leaks into the interstitium. As discussed above, extracellular contrast agents do leave the blood but cannot enter the functional cells of organs or become bound to these cells. At a time point shortly after the bolus is administered (approximately 1 to 2 minutes) , the rising interstitial concentration and the falling plasma concentration are transiently equal i.e. there is no net exchange between the two compartments (".the equilibrium point") . After this point the plasma concentration falls due to renal excretion and the gradient reverses, with the contrast agent passing out of the interstitium back into the plasma and then out of the body.

In one example, before and after a contrast agent is

administered to a subject, first and second "image signal intensity values" from regions of interest in the body of the subject are obtained using a medical imaging device. The image signal intensity value may be related to signal strength either at a region of an image or integrated over an image . The image signal intensity values will vary with the contrast agent and medical imaging device used. For example, if magnetic

resonance imaging (MRI) is be used, a gadolinium-based contrast agent is administered. The image signal intensity values for

MRI are associated with "Tl", which is the MRI rate constant for longitudinal spin relaxation after excitation.

Accordingly, in some, embodiments, the image signal intensity values obtained are associated with Tl. Conversely, if computer tomography (CT) will be used, the contrast agent used is iodine-based. The image signal intensity values for CT are associated with a "Hounsfield Unit" (HU) , which is the

standardised measurement of how much the X-Ray beam is

absorbed.

DUe to the extracellular nature of the contrast agent, the image signal intensity value or values for a particular organ usually is directly dependent on the concentration of contrast agent in the interstitium within the organ, which in turn is directly related to the level of fibrosis in the organ. At equilibrium, discussed supra, the difference in the image signal intensity value or values from the blood/plasma and from an organ of interest is a direct reflection of differences in the volume of distribution of the contrast agent.

Accordingly, embodiments of the present invention obtain image signal intensity values when the diffusion of the contrast agent between the blood and organs is substantially at an equilibrium condition.

While diffusion of the contrast agent between the blood and organs to substantially an equilibrium condition may be established through any means, in some embodiments diffusion of the contrast agent between the blood and organs to

substantially an equilibrium condition may be established either by administering a bolus of contrast media followed by a continuous infusion of contrast media (at a dose lower than the bolus) or by imaging at or around the transient 'equilibrium point' (see above) without a continuous infusion.

Administration of the continuous infusion may be started at any time after the bolus is administered and continued until the second image signal intensity value is obtained. In a

particular embodiment, the continuous infusion is started about 15 minutes after the bolus, sometimes termed a 'primed

infusion' and the second image signal intensity value is obtained between about 45 and about 80 minutes after the bolus was administered.

In order to reach equilibrium rapidly, the continuous infusion may be started immediately after or contemporaneously with a smaller and slower bolus delivery for equilibrium in as little as 5 to 10 minutes using either iodine contrast and CT or gadolinium contrast and MRI (this will be illustrated below with reference to Figure 5, 8 and 9) .

Imaging may also occur without continuous infusion after the bolus infusion. In this case a bolus injection may be shaped to create a smooth peak for measurement at this transient

equilibrium point (illustrated below with reference to Figure 6) .

Numerous test have been conducted in order to confirm that the above described contrast agent protocols (bolus infusion followed by lower dose continuous infusion or bolus . infusion shaped to achieve transient equilibrium) results in equilibrium in the blood stream ("blood - blood equilibrium") and .between blood stream and tissue ("blood - tissue equilibrium" ) . These have been conducted in normal subjects and patients with a variety of conditions and in different organs.

Embodiments of the present invention comprise obtaining a image value (or values) for the blood and an image_. signal intensity value (or values) for one or more organs of a subject before a contrast agent is administered to the subject and after diffusion of the contrast agent between the blood and organs at substantially an equilibrium condition has been established. As discussed supra, these image signal intensity values may be used to determine the contrast agent volume of distribution for an organ, which correlates with the amount of fibrosis in the organ. In some embodiments, the contrast agent volume of distribution for an organ ("organ V_d" ) is calculated using the following equation:

OVd =(1 - haematocrit) x (0SIV2-0SIV1) / (BSIV2 - BSIV1)

where OV_d is the volume of distribution of contrast agent for an organ; OSIV2 refers to the image signal intensity value

obtained for an organ of interest while diffusion of the contrast agent between the blood and organs approaches or is at an equilibrium condition. OSIV1 refers to the image signal intensity value obtained prior to the administration of any contrast agent. Similarly, the term "BSIV2" refers to the image signal intensity value obtained for blood while diffusion of the contrast agent between the blood and organs approaches or is at an equilibrium condition, while the term "BSIV1" refers to the image signal intensity value obtained for blood prior to the administration of any contrast agent. This equation may be additionally refined with terms to reflect more complex aspects of signal change, for example, from the

variance of (1-ihaematocrit) from Vd(b) , blood protein content, factors relating to the relative hydration of interstitial proteins, termed fibrosis.

In order to determine the blood contrast volume of distribution a sample of blood is taken from the subject and the haematocrit level (1 -haematocrit) determined. Tests for determining the haematocrit ^' level in a blood sample are well known to those skilled in the art, for example, a Complete Blood Count test may be used.

In some embodiments, after a bolus of gadolinium-based contrast agent^■ is^' administered, a late gadolinium enhancement (LGE) image is obtained.

As detailed above, expansion of the interstitium through processes such as fibrosis has a direct relationship with key disease parameters and/or the progression of disease.

Accordingly, embodiments of the present invention also provide methods of diagnosing and monitoring the progression of diseases associated with interstitial expansion. Examples are shown in Figure 10 and 11. The term "disease associated with interstitial expansion" as used herein, refers to any

impairment of normal physiological function of an organ as a result of an expansion of the interstitium, for example, accumulation of excess of connective tissue in the organ, ie. fibrosis. Using the methods described above, diagnosis of a disease may be achieved by determining the interstitial volume in an organ of a healthy individual and comparing that to the interstitial volume in the same- organ of an individual

suspected of having a disease associated with interstitial expansion. A level of interstitial volume higher than that in the healthy individual would indicate the presence of disease. Figure 12 for example (which will be illustrated in more detail below) shows very high VDm levels in cardiac amyloidosis compared to aortic stenosis, for example, and massive

interstitial expansion in myocardial infarction.

Similarly, disease progression may be monitored by periodically assessing the interstitial volume of an organ of an individual diagnosed with a disease associated with interstitial

expansion. Increasing interstitial volume would indicate that the disease is progressing, whereas decreasing interstitial volume would indicate that the disease is regressing. An example of disease monitoring over time is illustrated in Figure 13 described below) .

Interstitial expansion may be associated with diffuse fibrosis, amyloidosis, focal scar or oedema. Examples of diffuse fibrosis, amylodosis and focal scar imaging are shown in Figure 12. Diseases associated with an expansion of interstitial volume include, for example, hypertension, cardiomyopathies, valve disease,, acute and chronic infarction, myocarditis, cirrhosis, hepatitis, atherosclerosis, asthma, scleroderma, pulmonary. fibrosis, diabetes mellitus, Alzheimer syndrome, Down syndrome, hereditary cerebral hemorrhage with amyloidosis, Medullary thyroid carcinoma, isolated atrial amyloidosis, pituitary amyloid and cutaneous amyloidosis.

The method may also comprise obtaining a measure for an age of an organ. For example, fibroses, such as diffuse fibrosis, may be characterised in the above-described manner and the

resulting data may be compared with those of a library of age specific corresponding data previously obtained so that an effective age of the organ may be determined. Further, the fibrosis may be characterised in a plurality of organs of a subject and data analysis can provide information concerning effective relative organ ages. This is also illustrated in Example 13 for the heart.

In one embodiment of the present invention there is provided a method of determining a cell volume. In this embodiment the method comprises determining the distribution volume of contrast agent in the above-described manner and subtracting that volume from the total volume associated with an area of interest to obtain a measure for the volume that is substantially free of contrast agent and corresponds to the cell volume. Changes in the ratio of cell/interstitial volume may therefore be tracked over time, a example being shown in Figure 11 (described below in the context of example 12) , where organ changes are shown to be due to cell volume reduction rather than fibrosis reduction.

The invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are

illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above. In particular, while the invention is described in detail in relation to cardiovascular magnetic resonance and the

measurement of diffuse myocardial fibrosis, it will be clearly understood that the findings herein are not limited to the heart per se, but also encompass the measurement of fibrosis in any organ and by any method described, supra.

Example 1: Tl measurement technique

Gd-DTPA is an MRI extracellular contrast agent that potently shortens Tl. To develop the measurement technique of Tl, phantom samples were manufactured using agar jelly (10 g/1) and pipetted drops of Gd-DTPA and MnC12 to produce phantoms with varying Tl and T2 properties. These were imaged using the standard IR FLASH (Slice thickness 8.0 mm, TR 950 ms, TE 5.17 ms) with varying heart rates, segmentation and repeat times with the gold standard being Tl relaxometry (single line readout, TR>5x Tl) .

Multiple different readout combinations were tried iteratively. It was found that a segmented readout reduced measured Tl (time to inversion) compared to relaxometry, but provided the segmentation and flip angle were relatively low (21 segments, a=21°), the difference from measured to true Tl was kept to 5%.

The change in Tl was measured before and after a steady state infusion of Gd-DTPA. This is given by:

(1/Tl) ost ^■= (1/TlJpre + (1/Tl) Gd-DTPA

where

(l/Tl)Gd-DTPA = Rl [Gd-DTPA]

Post = measured Tl after administering the contrast agent, pre = Tl prior to administering the contrast agent, Rl = Tl relaxivity of Gd-DTPA (known)

At- contrast equilibrium, extracellular [Gd-DTPA] is the same in blood and myocardium. Vd(b) is known (1-haematocrit) so Vd(m) is :

Vd(m) = (l-haematocrit) x (1/Tl) myo. post - (1/Tl) myo. pre

(l/Tl)blood.post - (1/Tl) blood. re

Example 2: Heart rate correction

Figure 1 shows a graph illustrating the effects of mathematical heart rate correction. The heart rate at the time of the Tl measurement sequence heavily influences the measured. Tl. A spoiled gradient echo inversion recovery technique was used and the derived Tl for incomplete longitudinal recovery was corrected using the following method:

After a 180° inversion pulse, M₂ (longitudinal magnetisation) recovers exponentially from -1 to +1:

Mz = 1 - 2e-^t/T1 For breath-hold cardiac imaging the choice of TR (repetition time) is restrained to a multiple of the RR (the time interval between cardiac contractions) . During a segmented inversion recovery sequence, after a dummy pulse, M_z reaches a 'steady state' somewhat less than 1, assuming the TR is regular. This is "/" , the restoration factor. At steady state, magnetisation recovers from - f to f during time TR: / = 1 - (1+f) e^{"ra T1}

Furthermore, when the image shows no signal (M_z=0) , at time Tl' , magnetisation has recovered from -/ to zero

(0 = 1 - (l+ )e -^TI'/T1) .

Tl can be isolated from the above equation:

Tl = Tl' / ln(l+f)

f can be derived in the following manner:

/ =. 1 - (i+/)¹-^TR/TI'

This gives equations for both / and Tl, which can be solved for

This method was proven in phantom experiments (see Figure 1) . At the limit, when TR and Tl are similar, imaging ghosting, is present and the correction breaks down. In post-hoc analysis, when Tl (corrected and uncorrected) were compared to CVF from histology removing the correction caused the r² to fall from 0.80 to 0.54.

Example 3: Cardiovascular magnetic resonance (CMR) Protocol

The CMR protocol is outlined in Figures 2 (a) and (b) . CMR was performed using a 1.5-T magnet (Avanto, Siemens Medical

Solutions) . Within a standard clinical scan, a Tl measurement sequence was performed prior to administering the contrast agent (8 breath-holds) . After a bolus of Gadoteric acid (0.1 mmol/kg, Dotarem, Guerbet S.A.) and standard LGE imaging, the scan was finished and the patient removed from the scanner. At 15 minutes after the bolus, the infusion was started. At any time between 45 and 80 minutes post bolus, the patient was returned to the scanner and the Tl measurement repeated (with re-piloting, a total of 9 breath-holds) . Some patients (n=8 and 8 normal volunteers) remained in the scanner to test contrast equilibrium and some (n=22) had the Tl measurements performed twice to assess repeatability.

The Tl measurement used a standard late gadolinium enhancement (LGE) sequence. This is a multi-breath-hold, spoiled gradient echo inversion recovery (IR) sequence with the following parameters: slice thickness 8 mm, TE=4.6ms, a=21°, field of view 340 x 220 mm (transverse plane) , sampled matrix size 256 x 105, 21 k-space lines acquired every other RR interval (21 segments), spatial resolution 1.3 x 2.1 x 8 mm, no parallel imaging .

Tl measurement was performed pre- and post-equilibrium contrast using inversion times (Tl) of 140 milliseconds, then 200 to 800 milliseconds in 100 millisecond increments (see Figure 3 (a) and (b) ) . Regions of interest were drawn in the blood and septum at the biopsy site. Mean signal intensities were plotted and a curve fitting technique used to find the null point Tl' . A heart rate correction algorithm corrected for incomplete longitudinal recovery, and phantom work demonstrated that the combination of low flip angle, short read-out, and no parallel imaging minimally altered the derived Tl compared to Tl relaxometry. Key factors used in the methodology are: a) the use of null points which are coil location and scanner image scaling independent; b) pre and post scanning with a single change - equilibrium contrast (post contrast scan used with repeat heart rate recording, (mean RR interval 875ms±220ms vs. 901ms±198ms) , meticulous iso-centering and re-importing of the pre-scan sequence) and, c) the use of the blood: myocardial ratio such that any systematic changes affecting both tissue signals may affect their signal ratio less.

Example 4 : Achieving contrast equilibrium conditions for MRI measurements

Figure 3 illustrates blood and myocardial magnetic resonance measurements (a) after bolus infusion only and (b) after a subsequent steady has been achieved by continuous contrast agent infusion. Contrast equilibrium was achieved by primed infusion (loading bolus followed by slow continuous infusion) . The induction of an early steady state was optimized by

iteration of infusion rate and timing, with confirmation in 8 study patients and 8 normal volunteers, who remained in the scanner for the duration of the infusion. For rapid within- subject Tl measurement a TI (time to inversion) scout sequence (TR =45, TE= 1.27, slice thickness 8 mm, Flip angle 30°, TI range (105 - the RR interval) phases >14) in the transverse plane was used to measure Tl' (Tl prime, the Tl without heart rate or readout correction) repeated every 5 minutes . Steady state was defined as occurring when the variation in Tl' on serial measurements within an individual was within 5% of the final 10 minutes of Tl' measurements.

It was attempted to achieve contrast equilibrium within 1 hour. This was achieved using a 0.1 mmol/kg Gd-DTPA bolus (Dotarem, Guerbet, SA) , followed by a 15. minute pause then an infusion at a rate of 0.0011 mmol/kg/hour (equivalent to 0.1 mmol/kg over 90 minutes) . In the subset of patients (n=8, and 8 normals) who remained in the scanner during equilibration, contrast equilibrium (see Figures 3 (b) ) was achieved within 30 and 45 minutes of the contrast bolus in 90% and 100% of subjects respectively. The mean time to contrast equilibrium for myocardium, blood and their ratio were 19±8, 26±10 and 20+8 minutes respectively. A margin for error was incorporated. The mean length of infusion was 40±7.9 minutes, the time for the second scan acquisition (9 breath-holds) was 5 minutes ± 42 seconds and the total Gd-DTPA contrast dose was

0.14+0.009mmol/kg.

Although achievement of equilibrium is robust in the heart and liver, the above described infusion protocol involves a bolus of gadolinium followed by a delay of 15 minutes before the infusion is commenced. Imaging then occurs at 40 minutes. Any reduction in protocol time would improve, patient throughput and experience. In order to accelerate achievement of

equilibrium, bolus size and infusion timing was changed such that equilibrium conditions were achieved within approximately 10 minutes. An adequate state of equilibrium was defined when consecutive Tl measurements from regions of interest placed in the right lobe of the liver and terminal ileum differ by less than 10*. In 5 volunteer subjects adequate hepatic equilibrium was possible within 10 minutes with a smaller bolus (6ml) and an immediate infusion, which is illustrated in Figure 5. The effect of this change was tested by measuring the Tl of the liver and bowel using RI (inversion recovery experiment) in normal volunteers .

Alternatively, imaging may also be conducted without requiring a further infusion after the bolus infusion. To avoid

recirculation peaks, the bolus injection is in this case

"shaped" to create a smooth^" peak for measurement at a transient equilibrium point. Examples of the results of measurements obtained that way are shown in Figure 6 (a to d) , which

illustrates bolus concentrations. In this embodiment, a transient equilibrium is present when organ concentration levels cease to rise and start to fall. At that point, blood and organ concentrations are basically the same. However, this occurs at a time of blood contrast recirculation, seen best in 6(a), top left. To minimise this, the input function is altered, here being slowed until 6 (c) (bottom left) where recirculation effects are substantially reduced (the transient equilibrium is achieved after approximately 75 seconds where the myocardium' signal is peaked) . If this is over done (6d, bottom right) , no early organ inflection point is obtained.

Example 5: Clinical Validation

The in vivo measurements were validated against human operative myocardial biopsy in patients with aortic stenosis.

Consecutive pre-operative patients were invited to participate, unless they had uncontrolled tachyarrhythmia, a

contraindication to CMR or severely impaired renal function

(GFR <30 mL/min) . A second disease model studied, hypertrophic cardiomyopathy with outflow tract obstruction requiring myectomy. Patients with LGE in the myectomy or biopsy site

(n=5) were excluded from analysis. Blood samples were taken at the time of CMR to measure Vd(b) (1 - haematocrit) .

Example 6 : Histology

For histological analysis, an intra-operative deep myocardial biopsy (Trucut needle) was taken in aortic stenosis and the myectomy specimen was used in hypertrophic cardiomyopathy and images of examples are shown in Figure 4. Samples were fixed immediately in 10% buffered formalin, embedded in paraffin and stained with picro-sirius red. High power magnification (200x) digital images excluding subendocardial„pr perivascular areas underwent automated image analysis (macro written in imageJ version 1.42) . A combination of standard deviation from mean signal and isodata automatic thresholding derived the collagen area expressed as a percentage of total myocardial area, excluding fixation artefact. On average 12 high power fields were assessed per biopsy and the mean % fibrosis and fibrosis heterogeneity (coefficient of variation between fields) were obtained.

All biopsies were uneventful. The mean histological CVF was 20.5% in aortic stenosis (inter-subject range 6% to 39.8%, SD=11.0, n=18) , and 17.1% in hypertrophic cardiomyopathy

(inter- subject range 10.4% to 30.6%, SD = 7.37, n=8) (results are illustrated in Figure 4) .

Example 7; Statistics

Inter-study repeatability for the CMR measurement and fibrosis measurement of Tl was assessed by calculating the coefficient of variation (equal to the SD of the difference between two measurements over the mean of the two measurements, expressed as a percentage) . Correlation was assessed using Pearson's test (SPSS) .

Example 8 : Equilibrium-CMR Measurement of Fibrosis

Embodiments of the "equilibrium contrast cardiovascular magnetic resonance" (EQ-CMR) technique are. based on three elements: a bolus of the extra-cellular contrast agent

Gadolinium (Gd-DTPA) followed by continuous infusion to achieve blood: myocardial contrast equilibrium; a blood test to measure blood contrast volume of distribution (one minus the

hematocrit) and CMR before and after contrast equilibrium to measure changes in tissue signal (Tl measurement with heart rate correction. This allows for the calculation of the myocardial contrast volume of distribution, Vd(m), which reflects the amount of fibrosis. This technique provides the ability to measure diffuse fibrosis in the heart, and other organs .

In this validation study, fibrosis 'was quantified in the septum at the point of cardiac biopsy. Of 31 patients, 19 (11 aortic stenosis and 8 HCM) patients had focal fibrosis detected as LGE . In five, this was in the potential area of biopsy and, as pre-specified, these patients were excluded from further analysis. In the 26 patients without LGE in the biopsy area, Vd(m) measured by equilibrium CMR correlated strongly with CVF: aortic stenosis: r2=0.86, p<0.001; hypertrophic cardiomyopathy: r2=0.62, p<0.02 _/ combined r2=0.80, p <0.001 (5). The linear correlation equation for this relation was CVF% = 197 x Vd(m) - 36. This is illustrated in Figure 7.

Example 9 : Measurement Repeatability

In a subset of patients (n=22) , CMR measurement and analysis were repeated. Intra-study CMR reproducibility was 1%, SD <1%. For CVF assessment, automated image analysis reproducibility was 5%. However, within the same biopsy, CVF extent was not uniform between high power fields with a mean normalized (to CVF %) standard deviation of 37% in aortic stenosis and 41% in hypertrophic cardiomyopathy across an average of 12 high power fields -per biopsy. Example 10: Achieving of Contrast Equilibrium for Computer Tomography (CT) and acceleration thereof

Figure 8 illustrates a blood equilibrium contrast obtained using a bolus plus infusion of iodohexol (CT contrast agent) . The data represented in Figure 8 were calculated from blood samples measured using High performance liquid chromatography (HPLC) . As can be seen from Figure 8, contrast equilibrium was achieved after a relatively short period of time. Figure 9 shows examples of Equilibrium Contrast images obtained using CT. Blood, liver and heart equilibrium contrast CT scanning analyses were performed using the above -described rapid iodohexol infusion (see also Figure 8) . Regions of interest have been drawn in the heart, liver and blood (with exclusion of vessels in the liver) . However, nearly all organs are at equilibrium condition (bowel wall, fat, skeletal muscle, spleen) and regions could be drawn in any of these organs as they are all present in the image. Here, the haematocrit was 0.354, and the volume of distribution was calculated as 0.236 and 0.357 in liver and heart respectively.

Example 11: Equilibrium contrast CT scanning in heart and liver

After defining the basic lohexol infusion protocol, a modified infusion protocol was tested for 10 patients (of differing body mass index, already undergoing single-phase contrast enhanced CT for clinical indications) using CT. A 4cm pre-contrast spiral through the top of the liver to include the cardiac apex (120kV, 200mAs) was performed. After the clinical scan and bolus infusion, the subsequent iodine infusion was given and the 4cm spiral repeated through the cardiac apex and liver at our derived time for steady state and then at two further 10 minutes intervals (radiation dose is approximately 18 times less than a standard liver CT (5.3mSv) . Regions of interest were drawn in the liver, myocardium and aortic blood,

attenuation measured in Hounsfield units (HU) and plotted against time, and volumes of distribution calculated, which is illustrated in Figure 9.

Example 12: Disease monitoring and predicting

Figure 10 illustrates the clinical importance of equilibrium C R results in 2 diseases: Aortic stenosis (a), showing volume of distribution predicts exercise capacity (n=69) and (b) anthracycline cardiotoxicity (n=6) where volume of distribution tracks left atrial area.

Figure 11 represents an example of disease monitoring and the detection of cell volume changes after intervention. In this example 60 Aortic stenosis patients have been re- scanned after 6 months and a percentage fibrosis and cells volume quantified. The graphs represented in Figure 11 show, for the first time non-invasively, that LVH regression at 6 months post aortic valve replacement is cellular regression rather than fibrosis regression.

Figure 12 represents volume of distribution measurement in different forms of extracellular expansion: Diffuse fibrosis (conditions: aortic stenosis or hypertrophic cardiomyopathy) ; diffuse infiltration with amyloid (condition: TTR and AL systemic amyloidosis) ; and focal fibrosis (scar)

quantification: (condition: chronic infarction) . For

comparison, measurements for healthy subjects (wide age range) are included. Figure 13 shows data illustrating a relationship between the results of equilibrium CMR measurements and age. Even though the age range of the subjects was limited, a trend may be recognizable .

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the

protocols and reagents which are reported in the publications and which might be used in connection with the invention.

Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Claims

The Claims:

1. A method of measuring interstitial volume, the method

comprising the steps of:

obtaining first image signal intensity values for blood and at least a portion of one or more organs in a subject at an area of interest; thereafter

administering to the subject an extracellular contrast agent in such ^"a way that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium condition; thereafter

obtaining second image signal intensity values for blood and one or more organs in the subject at the area of interest; and determining the interstitial volume by determining a volume of distribution of the contrast agent for at least the portion of the one or more organs compared to the blood using the first and second image signal intensity values.

2. The method according to claim 1, wherein the step of

administering to the subject an extracellular contrast agent comprises administering a bolus of contrast agent followed by a continuous infusion of contrast agent.

3. The method of claim 2 wherein the continuous infusion of the contrast agent is conducted -such that the equilibrium condition is substantially maintained during the continuous infusion of the contrast agent.

4. The method of claim 2 or 3 wherein the continuous infusion is an infusion that is separate to the bolus.

5. The method of claim 2 or 3 wherein the continuous infusion is an extension of the bolus.

6. The method of any one of claims 2 to 5 wherein the second image intensity values are obtained during the continuous infusion.

7. The method of claim 1 wherein the step of administering to the subject an extracellular contrast agent comprises

administering a bolus of contrast agent and a quantity of bolus infused per time is selected such that an equilibrium condition is achieved for a period of time that is sufficiently long for the obtaining second image signal intensity values.

8. The method of claim 7 wherein the second image signal intensity values are obtained at transient equilibrium conditions after bolus injection.

9. The method of claim 8 wherein the second image signal intensity values are obtained at a first maximum of a signal associated with myocardium after administering the bolus.

10. The method of any one of the preceding claims wherein the step of administering to the subject an extracellular contrast comprises administering the contrast agent in vivo.

11. The method according to any one of the preceding claims, wherein the interstitial volume is indicative of the level of fibrosis.

12. The method according to claim 11, wherein the fibrosis is diffuse fibrosis.

13. The method according to claim any one of claims 1 to 10, wherein the interstitial volume is indicative of the level of amyloidosis.

14. The method according to any one of the preceding claims, wherein the step of determining an interstitial volume comprises obtaining a blood sample from the subject and determining the haematocrit level .

15. The method according to any one of the preceding claims, wherein the step determining an interstitial volume comprises calculating the volume using the following equation or a

derivative thereof:

OV_d =(1 - haematocrit) x(OSIV2-OSIVl) / (BSIV2 - BSIV1)

where OV_d is the volume of distribution of contrast agent for an organ; 0SIV2 is an organ image signal intensity value associated with the second image signal intensity values-; OSIV1 is an organ image signal intensity value associated with the first image signal intensity values, BSIVl is a blood image signal intensity value associated with the first image signal intensity values and

BSIV2 is a blood image signal intensity value associated with the second image signal intensity values.

16. The method according to any one of the preceding claims, wherein the first and second image signal intensity values for blood and one or more organs are derived from an image .

17. The method according to any one of the preceding claims, wherein the step of obtaining first and second image signal intensity values for blood and one or more organs in a subject^, comprise magnetic resonance imaging (MRI) .

18. The method according to claim 16, wherein each image signal intensity value is associated with an MRI rate constant for longitudinal spin relaxation after excitation referred to as "Rl" .

19. The method according to any one of claims 1 to 16, wherein the step of obtaining first and second image signal intensity values for blood and one or more organs in a subject comprise computer tomography (CT) .

20. The method according to claim 19, wherein the contrast agent is iodine based.

21. The method according to any one of claims claim 17 to 20 comprising correcting image signal intensity values or an image for fluctuations associated with the heart rate of the subject.

22. The method according to any one of claims 16 to 18, wherein the contrast agent is gadolinium DTPA based.

23. The method according to claim 2, wherein the second image signal intensity value is obtained between about 10 and about 80 minutes after the bolus was _ administered.

24. The method according to claim 2, further comprising the step of obtaining an image of one or more organs using late gadolinium enhancement (LGE) after the bolus is administered and before the continuous infusion is started.

25. The method according to any one of the preceding claims, wherein the organ is selected from the group consisting of heart, lung, brain, eye, stomach, spleen, bone, muscle, pancreas, kidney, liver, intestine, skin, lymph node, urinary bladder, ovary and prostate.

26. A method of diagnosing a disease, the method comprising the steps of:

administering to the subject an extracellular contrast agent such that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium condition; thereafter obtaining second image signal intensity values for blood and one or more organs in the subject associated with the area of interest; and

determining an interstitial volume by determining a volume of distribution of the contrast agent for at least the portion of the one or more organs using the first and second image signal intensity values,- and; and

comparing the determined interstitial volume with an

interstitial volume in a corresponding organ in a healthy subject to determine the presence of the disease.

27. A method of monitoring progression of a disease, the method comprising the steps of:

obtaining first image signal intensity values for blood and at least a portion of one or more organs at an area of interest in a subject diagnosed with the disease; thereafter

administering to the subject an extracellular contrast agent such that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium condition; thereafter obtaining second image signal intensity values for blood and one or more organs in the subject associated with the area of interest;

comparing the determined interstitial volume with an

interstitial volume in a corresponding organ in a healthy subject to determine progression of the disease.

28. The method according to claims 26 or 27, wherein the disease is a disease associated with fibrosis.

29. The method according to claims 26 or 27, wherein the disease is a disease associated with amyloidosis.

30. The method according to any one of the preceding claims comprising analysing the volume of distribution of the contrast agent to provide information 'concerning a percentage of fibrosis or amyloidosis in a specific organ and wherein providing that information comprises calibration with information obtained from human biopsies .

31. The method of claim 30 wherein the calibration with

information obtained from human biopsies comprises a curve fitting procedure.

32. A method of determining cell volume, the method comprising the steps of :

obtaining first image signal intensity values for blood and at least a portion of one or more organs in a subject at an area of interest; thereafter administering to the subject an extracellular contrast agent in such a way that diffusion of the contrast agent between the blood and at least the portion of the one or more organs is, at the area of interest, substantially at an equilibrium condition; thereafter^"

obtaining second image signal intensity values for blood and one or more organs in the subject at the area of interest;

determining an interstitial volume from using the first and second image signal intensity values; and

subtracting the determined interstitial volume from a volume of the area of interest to determine the volume that is

substantially free of contrast agent and that is indicative of the cell volume in the region of interest.