METHOD FOR THE DIAGNOSIS AND TREATMENT OF INFLAMMATION

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

This invention is directed to methods of diagnosing inflammation sites in an individual.

DESCRIPTION OF THE BACKGROUND ART

Inflammation occurs as a consequence of tissue damage. This tissue damage can result from micro- bial invasion, auto-immune processes, tissue infec¬ tion, allograft rejection, or such hurtful or destructive external influences as heat, cold, radiant energy, electrical or chemical stimuli, or mechanical trauma. Whatever the cause or bodily site, the inflammatory response is quite similar, consisting of a complicated set of functional and cellular adjustments, involving the microcirσula- tion, fluid shifts, and inflammatory cells (leuko¬ cytes) . When tissue damage occurs, soluble chemi¬ cal substances are elaborated which initiate the inflammatory response. The inflammatory response

consists of a complex series of events which may be summarized as:

1. A local increase in blood- flow, with capillary dilatation and increased permeability to the fluid components of the blood;

2. A localized exudation of fluid at the site of injury, including the proteins of the plasma that normally leave the capil¬ laries at a relatively low rate;

3. The exudation of leukocytes from the capillaries into the inflammation site. This exudate initially consists primarily of polymorphonuclear leukocytes, followed by monocytes, lymphocytes, and plasma cells. These leukocytes produce a vari¬ ety of mediators that control the extent and duration of the inflammatory re¬ sponse, and have a series of receptors on their surfaces available to react to the host of chemical mediators and proteins that are part of the inflammatory fluid. Such leukocyte receptor-mediator or protein interactions are important in controlling leukocyte function within the inflammatory site.

The identification and characterization of the sites of inflammation are an important part of medical and veterinary practice. In the case of infectious causes of inflammation, it is frequently necessary to search for "hidden sites of inflamma¬ tion" in individuals who present with clinical syn¬ dromes no more specific than fever and weight loss.

Similarly, in patients with auto-immune disease such as rheumatoid arthritis or allograft rejection as causes of inflammation, identification of the site(s) and extent of inflammation and its changes with therapy are an important part of medical and veterinary practice. Not surprisingly, then, much effort has been expended and many techniques deve¬ loped in an attempt to assess the site(s) and extent of the inflammatory process. These tech¬ niques include conventional x-ray techniques, computerized axial tomographic scanning (CAT scan¬ ning) , and a variety of radionuclide scans. (Sutt- on, A Textbook of Radiology and Imaging. 3rd Ed. , Churchill Livingston, 1980; Clinical Nuclear Medi¬ cine. Maysey et al.. ed. , W.B. Sanders, 1983.) Examples of radionuclide scans which have been utilized are:

1. 67Gallium, which when injected into an animal or a human binds to the plasma protein transferrin and tends to localize at sites of chronic inflammation.

2. Indium labeled endogenous granulo- cytes, which when re-injected into the host will tend to accumulate at the site of inflammation; and

3. Radiolabeled chelates which pass into the extracellular fluid and can possibly then accumulate at such sites of fluid accumu¬ lation as those associated with inflam¬ mation;

4. Thallium scan or so-called first pass radionuclide angiogram to assess areas of increased blood flow.

All of these techniques, on occasion, may provide useful information, but are not adequate because of both false positive and false negative results. A more sensitive and specific means of delineating the anatomical localization of sites of inflammation, particularly one that could be per¬ formed serially to assess the response to therapy, is greatly to be desired.

SUMMARY OF THE INVENTION

The present invention relates to a substan¬ tially non-invasive method of diagnosing sites of inflammation.

The present inventors have discovered that when immunoglobulins are allowed to contact both inflamed and non-inflamed sites in an individual, they tend to accumulate at the inflamed sites. Further, it was surprisingly discovered that the accumulation at the site of inflammation is not dependent upon the epitopic specificity of the immunoglobulins used. This effect, the concentra¬ tion of immunoglobulin at the site of inflammation and its use in diagnostic imaging, has not been previously recognized. In other words, non-speci¬ fic immunoglobulins or mixtures thereof can be used.

The present invention thus relates to an in vivo method of detecting an inflammation site in an individual. This method comprises administering to the individual a detectably labeled immunoglobulin or fragment thereof, wherein the immunoglobulin substantially accumulates at the site when the site

is inflamed. Preferably, the immunoglobulin does not substantially accumulate at the site when the site is not inflamed.

DESCRIPTION OF THE DRAWINGS

Figure 1: Imaging of inflammation with radiola¬ beled preparations.

Comparative imaging results of three animals with inflammation due to injection of viable bacte¬ ria in the left thigh. Each animal was injected with a mixture of three radiolabeled preparations and imaged at various times.

Figure 2: Imaging of inflammation with radiola¬ beled preparations.

Comparative imaging results of three animals with inflammation due to injection of viable bac¬ teria in the left thigh. Each animal was injected with a mixture of three radiolabeled preparations and imaged at various times.

Figure 3: Imaging of inflammation with non-speci¬ fic murine immunoglobulin.

This figure shows the kinetics of radiolabeled immunoglobulin in two animals with inflammatory sites in the left thigh due to injection of viable bacteria. Although the inflammation site is clea¬ rly detectable at 24 and 48 hours post-injection, the site is no longer detected by 160 hours post- injection.

Figure 4: Imaging of inflammation in human patie- nt.

I munoglobulin scan of human patient showing localization of ycotic aneurysm in femoral graft.

Figure 5: Imaging of inflammation in human pati¬ ent.

Immunoglobulin scan of human patient indica¬ ting a diverticular abscess.

Figure 6: Imaging of inflammation in human pa¬ tient.

Immunoglobulin scans pre- and post-treatment of human patient with enteritis demonstrating the absence of inflammation after treatment.

Figure 7: Imaging of inflammation in human pa¬ tient.

Diagnosis of site of inflammation in groin of human patient by radiolabeled immunoglobulin imag¬ ing technique.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The terms "inflammation" or "inflamed site" are used to denote conditions occurring in an individual due to tissue damage, regardless of the underlying cause or etiology. The concept is more fully described above in the Background Art.

The term "individual" is meant to include both animals and humans.

The immunoglobulins of the invention can be either polyclonally or monoclonally derived, the important factor being that the epitopic specifi¬ city of the immunoglobulin used is irrelevant to enable it to localize at the inflammation site.

Polyclonal immunoglobulin preparations can be derived directly from the blood of the desired animal species. Thus, in the case of humans, polyclonal immunoglobulin preparations can be prepared from outdated units of blood utilizing protocols known or readily asσertainable to those of ordinary skill of the art. Such products are commercially available (Sandoz Limited; Cutter Laboratories) *nd are conventionally used in the treatment of immunodeficiency states, but not in diagnosis. In addition, if desired, polyclonal immunoglobulin preparations may be prepared from the blood of immunized individuals of the desired species following immunization with any of a va¬ riety of antigens followed by harvesting of the blood and processing it according to defined tech¬ niques. A distinctive advantage of non-specific, polyclonal immunoglobulin preparations is that by preparing immunoglobulin from the same species into which it will be injected, immune reactions across species barriers are prevented and repeated injec¬ tions of the same product are less likely to cause side-effects. It should be emphasized that cross- species injections can be done. However, their use might increase the incidence of untoward reactions such as anaphylactic reactions, febrile reactions, and/or the generation of an immune response to the foreign immunoglobulin protein that will block its effective use, as well as endanger the health of the patient. The avoidance of such reactions adds greatly to the appeal of using an immunoglobulin preparation which is from the same species as that being diagnosed.

The nature of the immunoglobulins used is such that they need not have epitopic specificity for the inflamed site, but they nevertheless accumulate substantially at the inflamed site. Thus, immuno¬ globulin, having essentially any epitopic specific¬ ity, will work in the invention.

Monoclonal immunoglobulins which can be used according to the method of the invention can be prepared using hybridoma fusion techniques (Kohler et_al. , European Journal of Immunology .6:292, 1976) or can be derived from known secreting myeloma cell lines such as those available from depositories such as the American Type Culture Collection. As with the polyclonal immunoglobulin preparation, no antigenic or epitopic specificity is needed for the monoclonal immunoglobulin preparation to function effectively in this method. As a consequence, monoclonals of any specificity can be used.

In detecting an in vivo inflammation site in an individual, the detectably labeled immunoglo¬ bulin is advantageously given in a dose which is • diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled immunoglobulin administered is sufficient to enable detection of the site of inflammation when compared to the background signal.

Generally, the dosage of detectably labeled immunoglobulin for diagnosis will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, counterindications, if any, and other variables, to be adjusted by the individual physician. Dosage

can vary from 0.01 mg/kg to 2,000 mg/kg, preferably 0.1 mg/kg to 1,000 mg/kg.

The term "immunoglobulin or a fragment there¬ of" as used in this invention is meant to include intact molecules as well as fragments thereof, such as, for example, the Fc fragment, which is capable of accumulating at the site of inflammation.

Similarly, the term "Fc portion or part there¬ of" as used in this invention is meant to include intact Fc fragments as well as portions of the Fc fragment capable of accumulating at the site of inflammation.

The term "diagnostically labeled" means that the immunoglobulin has attached to it a diagnosti¬ cally detectable label.

There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include radioactive isotopes and paramagnetic isotopes. Those of ordinary skill in the art will know of other suit¬ able labels for binding to the immunoglobulins used in the invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the immunoglobulin can be done using standard techniques common to those of ordinary skill in the art.

For diagnostic in vivo imaging, the type of detection instrument available is a major factor in selecting a given radionuclide. The radionuclide chosen must have a type of decay which is detec¬ table for a given type of instrument. In general, any conventional method for visualizing diagnostic

imaging can be utilized in accordance with this invention.

Another important factor in selecting a radio¬ nuclide for in vivo diagnosis is that the half-life of a radionuclide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host is minimized. Ideally, a radionuclide used for in vivo imaging will lack a particulate emission, but produce a large number of photons in a 140-200 keV range, which may be read¬ ily detected by conventional gamma cameras.

For in vivo diagnosis, radionuclides may be bound to immunoglobulin either directly or indi¬ rectly by using an intermediary functional group. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to immunoglobulins are diethylenetriaminepentaa- cetiσ acid (DTPA) and ethylenediaminetetracetic acid (EDTA) . Typical examples of metallic ions which can be bound to immunoglobulins are ^ c, ¹²³ I, mm ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga, ¹²⁵ I, ⁶⁸ Ga, ⁷² As, ⁸⁹ zr, and ²⁰¹ T1.

The immunoglobulins used in the method of the invention can also be labeled with paramagnetic isotypes for purposes of in vivo diagnosis. Ele¬ ments which are particularly useful (as in Magnetic Resonance Imaging (MRI) techniques) in this manner include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe.

Alternatively, the method of the invention can be used to monitor the course of inflammation in an individual. Thus, by measuring the increase or decrease in the size or number of inflammatory

sites it would be possible to determine whether a particular therapeutic regimen aimed at ameli¬ orating the cause of the inflammatory process, or the inflammatory process itself, is effective.

Another embodiment of the invention includes a method for diagnosing the specific underlying cause of the inflammation at the site. In this method an individual suspected of having an inflammatory site is first administered a diagnostically effective amount of immunoglobulin, as previously described. This detectably labeled immunoglobulin may be of the same or different species as the individual to which it is being administered. The individual suspected of having an inflammatory site is then imaged to determine the presence of a site of inflammation. If the individual is found to have an inflammatory site, the individual is then given an antibody preparation(s) specific for the under¬ lying cause of the inflammation which is suspected. This specific antibody can be from an individual of the same, or a different, species to that of the individual having the inflammatory site. After determining the specific cause of the inflammatory site it is then possible to administer a thera¬ peutic agent, such as therapeutically labeled antibody specific for the underlying cause of the inflammatory process at the inflammation site.

There are distinct advantages to this embo¬ diment of the invention, utilizing the sequential use of non-specific immunoglobulin from an individ¬ ual of the same species as that suspected of having the inflammatory site, followed by the administra¬ tion of specific antibody to define and identify

the nature of the underlying cause of the inflam¬ matory response. The potential advantages of this sequential strategy include the following:

1. The immunoglobulin preparation used to determine whether any inflammation at all is present is non-sensitizing to the recipient individual since it is from an individual of the same species. Thus, there is little in the way of an adverse allergic or immunologiσ reaction by the recipient to the immunoglobulin prepa¬ ration.

2. If the inflammation is not found with the immunoglobulin preparation of 1. above, it is not necessary to administer the detectably labeled specific antibody. Thus, in the situation where detectably labeled specific antibody is unavailable from an individual of the same species as that of the recipient, the recipient will not be exposed to potentially sensitizing amounts of foreign antibody protein. At present, specific antibodies, particu¬ larly monoclonal antibodies, are chiefly of murine origin and, thus, may possibly excite an adverse immunologic response in a non-murine recipient. Such adverse immunologic responses limit the number of times a given recipient individual can be exposed to a specific antibody prepara¬ tion from a different species. There¬ fore, exposure to such specific antibody of a different species should be res-

tricted to situations of maximum clinical benefit.

The term "therapeutically conjugated" means that a specific antibody used in the just-described preferred method of the invention is conjugated to a therapeutic agent. The therapeutic agents used in this matter act directly upon the underlying cause of the inflammation. Examples of therapeutic agents which can be coupled to the specific anti¬ bodies used according to the method of the inven¬ tion are drugs, radioisotopes, lectins, toxins, and antimicrobials.

Lectins are proteins, usually isolated from plant material, which bind to specific sugar moie¬ ties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. Ricin is a toxic lectin which has been used immunotherapeutically. This is accomplished by binding the alpha-peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site-specific delivery of the toxic effect.

Toxins are poisonous substances produced by plants, animals, or microorganisms that, in suffi¬ cient dose, are often lethal. Diphtheria toxin is a protein produced by Corynebacteriu diphtheriae. This toxin consists of an alpha and beta subunit which, under proper conditions, can be separated. The toxic component can be bound to antibody and used for site-specific delivery to the primary underlying cause of the inflammatory response.

Examples of radioisotopes which can be bound to specific antibody for therapeutic purposes, used

______ according to the method of the invention, are I,

¹³¹ I, ⁹ °Y, ⁶⁷ CU, ²¹⁷ Bi, ²¹¹ At, ²¹² Pb, ⁷ SC, and ¹⁰⁹ Pd.

Anti- icrobials are substances which inhibit such infectious microorganisms as bacteria, virus¬ es, fungi, and parasites. These anti-microbials can be any of those known to those of ordinary skill in the art.

Other therapeutic agents which can be coupled to specific antibodies used according to the method of the invention are known, or can be easily ascer¬ tained, by those of ordinary skill in the art.

Preparations of the imaging immunoglobulins for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propyleneglycol, polyethyleneglycol, vegetable oil such as olive oil, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, parenteral vehicles including sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intra¬ venous vehicles include fluid and nutrient repleni- shers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preserv¬ atives and other additives may also be present, such as, for example, antimicrobials, anti-oxi- dants, σhelating agents, and inert gases and the like. See, generally. Remington's Pharmaceutical Science. 16th ed. , Mac Eds, 1980.

This invention can be utilized to delineate inflammation at a wide variety of body sites and to

diagnose inflammation resulting from a variety of causes such as, but not limited to, infections with parasites, microbes, viruses or fungi; trauma; autoimmune processes; or tumors. The invention is also useful as a means to evaluate the efficacy of, and responses to, therapeutic treatment of inflam¬ mation. The diversity of body sites at which inflammation may be identified are exemplified by, but not limited to, muscle, vascular walls, abdo¬ men, groin, and other organ sites.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLE 1

IMAGING OF INFLAMMATION WITH RADIOLABELED PREPARATIONS

Deep thigh inflammation was created in 250 gm Sprague-Dawley rats by the injection of 10 ⁸ viable E. coli, Staphylococcus aureus, or Pseudomonas aeruginosa suspended in 0.1 ml of phosphate-buf¬ fered saline. Twenty-four hours later, at a time when each animal had clearly evident inflammation of the thigh, each animal was injected via the tail vein with 65 ug of a commercially available, intra¬ venous, polyclonal human immunoglobulin preparation (Sandoglobulin ) labeled with 100 uCi of ¹²⁵ I

(Iodogen ^R , Pierce Chemical Co., Rockford, II.) ;

100 uCi of 67Ga; and 65 ug of human albumen labeled

with 100 uCi of ^e. Thus, each animal was injected with three different isotopes, enabling three different approaches to inflammation imaging to the same animal via a gamma counter. Following injections of these various agents, images were obtained at 1, 3, 19, and 24 hours. Figures 1 and 2 illustrate two representative experiments involv¬ ing three animals in each experiment. Note that the same three animals were imaged at each time point. These images show that there was essen¬ tially no imaging of the inflammatory lesion with - 67Ga (considered the standard inflammatory scanning technique) ; minimal transient imaging with the ^Tc labeled albumen; but obvious, increasing visualization of the site of inflammation with the I-labeled polyclonal human immunoglobulin pre¬ paration.

In addition to imaging, radioactive counts and count densities were determined over the inflamed site and the non-inflamed contralateral thigh for each isotope at each of the time points of imaging. These values are expressed in Tables 1 and 2 (which correspond to Figures 1 and 2, respectively) . The same results were observed quantitatively: a transient accumulation at the site of inflammation with albumen; minimal if any accumulation with gallium; and a more sustained, increasing accu¬ mulation of labeled immunoglobulin.

Table 2*

Animal/Tine Gallium-67 I-Immunoglobulin ^99a Tc-Albuoin

#4/1 hr inflamed leg 937/12 2085/27 2051/27 uninflamed leg 290/6 633/12 599/13

#4/3 hr inflamed leg 868/11 3032/41 1853/25 uninflamed leg 369/11 696/10 434/6

#4/19 hr inflamed leg 567/4 4576/39 1208/13 uninflamed leg 286/4 603/9 406/6

#4/24 hr inflamed leg 638/5 4437/36 980/7 uninflamed leg 249/3 1262/13 385/4

05/1 hr inflamed leg 800/12 1580/24 1556/24 uninflamed leg 621/7 935/10 891/10

#5/3 hr inflamed leg 1025/12 2257/28 1388/17 uninflamed leg 282/6 575/13 357/8

#5/19 hr inflamed leg 730/6 4268/30 1300/11 uninflamed leg 308/3 656/7 389/4

#5/24 hr inflamed leg 723/6 3226/29 755/6 uninflamed leg 345/4 668/8 280/3

Table 1 (contd.)

#3/1 hr inflamed leg 683/7 1791/19 2054/23 uninflamed leg 370/5 438/6 758/10

#3/3 hr inflamed leg 831/11 1792/24 1195/16 uninflamed leg 238/4 319/5 273/5

#3/19 hr inflamed leg 677/5 4491/35 1014/12 uninflamed leg 307/3 638/7 341/3

#3/24 hr inflamed leg 754/5 4148/28 897/8 uninflamed leg 232/3 503/6 240/3

*Data expressed as counts in area of interest (numerator)/count density in counts/pixel (denoninator).

Table 2*

125

Animal/Time Gallium-67 I-Insjunoglobulin "°Tc-Albumin

#4/1 hr inflamed leg 937/12 2085/27 2051/27 uninflamed leg 290/6 633/12 599/13

#4/3 hr inflamed leg 868/11 3032/41 1853/25 uninflamed leg 369/11 696/10 434/6

#4/19 hr inflamed leg 567/4 4576/39 1208/13 uninflamed leg 286/4 603/9 406/6

#4/24 hr inflamed leg 638/5 4437/36 980/7 uninflamed leg 249/3 1262/13 385/4

#5/1 hr inflamed leg 800/12 1580/24 1556/24 uninflamed leg 621/7 935/10 891/10

#5/3 hr inflamed leg 1025/12 2257/28 1388/17 uninflamed leg 282/6 575/13 357/8

#5/19 hr inflamed leg 730/6 4268/30 1300/11 uninflamed leg 308/3 656/7 389/4

#5/24 hr inflamed leg 723/6 3226/29 755/6 uninflamed leg 345/4 668/8 280/3

Table 2 (contd. )

#6/1 hr inflamed leg 1315/14 2658/29 2519/28 uninflamed leg 226/3 369/6 385/6

#6/3 hr inflamed leg 1245/12 3640/36 2353/23 uninflamed leg 301/5 507/8 371/6

#6/19 hr inflamed leg 609/6 8161/52 1431/17 uninflamed leg 205/2 282/3 294/4

#6/24 hr inflamed leg 702/5 7354/50 749/9 uninflamed leg 287/4 455/6 215/3

*Data expressed as counts in area of interest (numerator)/count density in counts/pixel.-(denominator).

EXAMPLE 2

In other experiments, carried out as in Exa¬ mple 1, In coupled to the human polyclonal immunoglobulin instead of 125I, yielded excellent images that were brighter and appeared more promp-

125 tly than did the I-labeled material. This shows that the imaging method is not restricted to a single label.

EXAMPLE 3

A "dose-response" experiment was carried out uti .li.zi.ng 125I-labeled polyclonal human immunoglo¬ bulin, in which doses of 65 ug, 650 ug, 5.8 mg, and 122 mg were utilized per animal. The procedures of Example 1 were used. In these experiments, the amount of radioactivity was kept constant (100 uCi) , and increasing amounts of "cold" immuno¬ globulin were added to increase the amount of protein injected. Thus, quantities approximately equivalent to a standard dose, lOx standard, 10Ox standard, and lOOOx standard, were injected. All of these concentrations yielded similar images and similar counts over time, suggesting that the receptors for the immunoglobulin were not saturated even at a lOOOx dose schedule. Because of the absence of epitopic specificity of the immunoglobu¬ lin preparation used, the accumulation of labeled immunoglobulin at the site of inflammation is not due to conventional recognition of epitopic deter¬ minants.

EXAMPLE 4

INFLAMMATION SITE IMAGING USING NON-SPECIFIC MURINE MONOCLONAL ANTIBODY

Deep thigh inflammation was created in Sprag- ue-Dawley rats as previously described above in Example 1. Twenty-four hours later, at a time when each animal clearly evidenced inflammation in the injected thigh, 65 ug of murine monoclonal antibody labeled with 100 uCi of either ¹²⁵ I or ^1:L1 In were injected intravenously via the tail vein. The murine monoclonal antibodies employed in ^* these experiments were of the IgGτ_ or IgG ₂ subclass and were of three epitopic specificities. The two epitopic specificities which were used were:

1. IgGi or IgG ₂ antibodies directed against an arsenate hapten not found in mammalian species or bacteria.

2. IgGτ_ antibodies specific for lipid A of E. coli.

In these experiments, the anti-arsenate mono¬ clonal antibodies were injected into animals having either IL. coli. S. aureus, or P_j_ aeruginosa infec¬ tion. The anti-E. coli monoclonal antibody was injected into animals bearing staphylococcal or Pseudomonas infection.

All of these labeled immunoglobulin prepara¬ tions gave similar results. In all animals there appeared an image 1-4 hours post-injection, which peaked in intensity 24-48 hours post-injection, and disappeared completely by 96-120 hours. A repre¬ sentative experiment utilizing the IgGτ_ anti-arsen-

ate antibody in an animal with a Pseudomonas infec¬ tion is shown in Figure 3.

These studies demonstrate that a wide variety of non-specific monoclonal immunoglobulins can be used in this method.

EXAMPLE 5

COMPARISON OF NON-SPECIFIC IMMUNOGLOBULIN IMAGING TO SPECIFIC ANTIBODY IMAGING

Pseudomonas aeruginosa Type I deep thigh infection in Sprague-Dawley rats was created as described above. Twenty-four hours later, 0.65 ug of 125I-labeled monoclonal antibodies of one of two possible antigenic specificities were injected intravenously via the tail vein. Half of the ani¬ mals were injected with a murine monoclonal IgGτ_ antibody specific for Type I P^_ aeruginosa. The remaining animals were injected with murine mono¬ clonal IgGτ_ antibody specific for arsenate hapten which is an antigen not found in mammalian tissues or bacteria. The anti-arsenate antibody is thus a control preparation with no epitopic specificity for this animal model. Serial images were taken with the gamma camera at 1, 4, 24, 48, 72, 96, 120, 144, and 168 hours after injection of the radio- labeled monoclonal antibodies. In the first twen¬ ty-four hours the images obtained with the specific and non-specific antibodies were identical with images appearing as early as 1-4 hours post-injec¬ tion. However, beginning at 48 hours, the non¬ specific (anti-arsenate) image began to fade, and had totally disappeared by 72-96 hours. In con¬ trast, the specific (anti-P. aeruginosa) image con-

tinued to increase in intensity, peaking at 96-120 hours, and not disappearing until 166 hours had elapsed. Thus, it is possible to differentiate between the non-specific accumulation of immunoglo¬ bulin at an inflammation site from the reaction of specific antibody with the underlying cause of the inflammatory response, by following the persistence of the image with time. Specific antibodies remain at the inflammation site longer.

EXAMPLE 6

INFLAMMATION SITE IMAGING USING IMMUNOGLOBULIN

FRAGMENT

Deep thigh inflammation was created in Sprag- ue-Dawley rats as described in Example 1 above. After 24 hours when each animal had clear evidence of inflammation, they were injected with 65 ug of either ¹²⁵ I-labeled Fc fragment (100 uCi) , ¹²⁵ ι- labeled Fab fragment (100 uCi) , or 125I-labeled polyclonal human immunoglobulin (100 uCi, Sando- globulin ^R ) . The immunoglobulin fragments were derived from a polyclonal human immunoglobulin preparation (Sandoglobulin ^R ) using standard tech¬ niques. The animals were then scanned using a gamma camera as described in Example 1. It was found that while the labeled Fc fragments and labeled immunoglobulins gave similar results, both of these preparations gave considerably stronger and more sustained images than the images produced by the labeled Fab fragments. This finding further supports the fact that accumulation of immunoglobu¬ lin at the site of inflammation is unrelated to the

epitopic specificity of the immunoglobulin since the Fc fragment has no epitopic binding capability.

EXAMPLE 7

Commercially available polyclonal intravenous human immunoglobulin preparations marketed by Sandoz, Cutter, and Hyland (all licensed as thera¬ peutic preparations) can be utilized interchange¬ ably as imaging reagents. Localized inflammation was produced in 200 gram Sprague-Dawley rats by the injection of 10 ⁸ EL. coli into the left back thigh. Twenty-four hours later, 0.25 mg/kg of the test immunoglobulin preparation, labeled with iodine-125 by the Iodogen ^R bead technique (100-150 uCi) was injected intravenously. Serial images were then taken 4, 24, and 48 hours post-injection of the radiolabeled immunoglobulin with a gamma camera. In all cases with each of the immunoglobulin pre¬ parations, an image of the site of inflammation was seen 4 hours post-injection, with an image of increasing intensity observed 24 hours post-injec¬ tion. There was no difference in the images achie¬ ved with the different immunoglobulin preparations with this method of applying a radiolabel. When indium-Ill is utilized as the radiolabel after coupling of diethylenetriaminepenta-acetic acid to the immunoglobulin, the Hyland and Sandoz immuno¬ globulin preparations yield distinctly better images than does the Cutter preparation. These results indicate that the imaging method is effec¬ tive with immunoglobulins from various sources and utilizing different radionuclides.

EXAMPLE 8

The non-specific immunoglobulin scan can be utilized in a variety of inflammatory states such as those due to a variety of infectious agents or the non-infectious inflammatory agent turpentine. In each of these studies, approximately 10 ⁸ organ¬ isms, or 0.5 cc of turpentine, was injected into the thigh of 100 gram Sprague-Dawley rats. Twenty- four hours later, 0.25 mg/kg of a polyclonal intra¬ venous human immunoglobulin preparation (in all these studies the Sandoz preparation Sandoglobulin ^R was used) labeled with either I or ^ι:L1 In, as previously described, was injected intravenously. Images were then taken with a gamma camera 4, 24, and 48 hours later. Inflammation was induced in these studies by E. coli. Klebsiella pneumoniae. Staphylococcus aureus. Candida albicans. Pseudomo¬ nas aeruginosa. Bacteriodes fragilis. or turpen¬ tine. In all cases, imaging was observed, commen¬ surate with the degree of inflammation present, as early as 4 hours post-injection and with intensi¬ fying images observed 24-48 hours post-injection. These results, utilizing a non-microbial form of inflammation, different classes of bacteria, and a fungus, demonstrate that this technique is truly non-specific and capable of diagnosing inflammation resulting from diverse causes.

EXAMPLE 9

The non-specific immunoglobulin scan can detect inflammation at different anatomical sites. a. Unilateral nephrectomies were carried out in two groups of rats. In one group, the operation was done sterilely. In the other group, the nep- hrectomy sites were inoculated with 10 ⁸ E_j_ coli. Both groups of animals were injected with In- labeled Sandoglobulin ^R 48 hours later, with serial scans then carried out with the gamma camera. In every instance, scans taken 24 hours post-injection could clearly distinguish sterile post-operative surgically induced inflammation from deep surgical wound infection. b. Intra-abdominal abscesses were created with Bacteriodes fragilis or mixed bowel bacteria according to standard methods. Approximately 7-10 days later. In-labeled Sandoglόbulin ^R was admi¬ nistered intravenously and serial images obtained with the gamma camera. In each instance, the scans demonstrated the site of the intra-abdominal abs¬ cess. c. Pneumocyctis carinii pneumonia is an opportunistic pneumonia observed in immunocomprom- ised patients, particularly those with AIDS. Early diagnosis, and assessment of response to therapy, particularly in those patients with equivocal chest x-ray, is an important clinical need. Model Pne¬ umocyctis pneumonia was created in rats by placing them on a protein-deficient diet and treating them with daily cortisone and tetracycline for two months. This resulted in the development of Pneum-

ocyctis pneumonia. At a time when these animals' chest x-rays and gallium scans (the current tech¬ nique employed clinically) were negative, but pathology revealed clear-cut Pneu ocystis pneumon¬ ia, the In-Sandoglobulin ^R scans were positive 24-48 hours post-injection. When these animals were treated with effective anti-Pneumocystis therapy (trimethoprim-sulfamethόxazole) repeat scans improved, commensurate with the pathologic evidence of improvement in the pneumonia. These results indicate the usefulness of the method of the present invention to monitor the response of the individual to treatment.

EXAMPLE 10

Anti-inflammatory therapies do not influence the utility of this scanning technique. Groups of animals were pre-treated for 5-7 days with one of the following anti-inflammatory regimens: methyl- prednisilone, 5 mg IP daily; indomethacin 10 mg/kg- /day IV; and acetylsalicylic acid administered in the drinking water to achieve a blood level of 15- 25 mg/liter (therapeutic levels used in treatment of humans with rheumatoid arthritis) . Model thigh infection was then created with E_ _s . coli as des¬ cribed in Example 1, and 24 hours later (while the anti-inflammatory therapy was continued) ¹¹¹ In- labeled Sandoglobulin ^R was injected intravenously, with serial scans then carried out. These studies showed no significant effect of the anti-inflamma¬ tory treatment on the images obtained.

EXAMPLE 11

The Fab and Fc studies described in Example 6 above were repeated utilizing model infection created with either E. coli or Klebsiella pneumo- niae. In every instance, the Fab fragment gave no images, whereas the Fc fragment gave images similar to those obtained with immunoglobulins. These re¬ sults were identical to those seen in Example 6.

EXAMPLE 12

Radiolabeled non-specific human immunoglobulin was used to image localized inflammation in 15 human patients. There were no false positives or negatives. The technique has proven to be useful in" diagnosing intramuscular infection, skeletal infection, and intra-abdominal and pelvic infec¬ tion. In all these studies, a single polyclonal human immunoglobulin preparation has been employed, Sandoglobulin ^R , labeled with In after coupling with diethylenetriaminepentaacetic acid by the carboxy-carbonic anhydride method (Krejcarek and Tucker, Biochem. and Biophys. Res. Coromun. 77:581- 585 (1977)). A dose of 1.9 mCi/patient was admini¬ stered intravenously with several images carried out over the 24 hours post-injection. It should be noted in all these studies that In non-specifi- cally accumulates in the liver and kidney. This is a function of the indium label, not the immuno¬ globulin, as shown by animal studies in which iodine-labeled immunoglobulin does not accumulate

in the liver whereas indium-labeled immunoglobulin does.

EXAMPLE 13

A 62-year-old woman with extensive vascular disease underwent a left femoral artery-inferior tibial artery bypass graft on 10/31/86; on 11/18/86 she underwent a right femoral-popliteal arterial bypass graft; and on 12/2/86 a thrombectomy of the right femoral artery. Over the next two weeks, she had a low-grade fever, with minimal findings at her operative site. On 12/16/86, she underwent an immunoglobulin scan which revealed a clear-cut accumulation in the lower portion of her right femoral graft (Figure 4) of radiolabeled immuno¬ globulin 6 hours post-injection of the reagent. Emergency exploration of this site that evening revealed a mycotic aneurysm of her graft. This was removed, and grew Pseudomonas aeruginosa. In this instance, the scan provided life-saving information non-invasively with no side effects. The results demonstrate the effectiveness of immunoglobulin imaging to diagnose and localize inflammation in the vascular wall.

EXAMPLE 14

A middle-aged woman presented with fever, lower abdominal pain, and a palpable mass on rectal examination. A computerized tomographiσ (CT) study of her abdomen revealed no abnormality. An immuno¬ globulin scan revealed a clear-cut accumulation of

radiolabeled immunoglobulin in the lower abdomen as early as 3 hours post-injection (Figure 5) . Surgi¬ cal exploration revealed a large diverticular abscess at the anatomical site outlined by the immunoglobulin scan.

EXAMPLE 15

A middle-aged man with longstanding regional enteritis presented with fever and tenderness in his right upper quadrant just below his liver. CT study was non-diagnostic. Initial immunoglobulin scan demonstrated an inflammatory mass just below his liver (Figure 6a) corresponding to the site of pain. He was treated with steroids and broad- spectrum antibiotics with resolution of his symp¬ toms. At the completion of therapy, a repeat immunoglobulin scan revealed no remaining area of inflammation (Figure 6b) . Therapy was stopped, and the patient remained well. This result demon¬ strates that immunoglobulin imaging is effective not only as a diagnostic tool but also to determine the efficacy of treatment.

EXAMPLE 16

A middle-aged man presented in mid-July 1986 with a left groin mass, fever, and chills of 3-4 days' duration. On 7/12/86, he underwent percuta¬ neous drainage of the groin mass, which yielded purulent material that grew small amounts of mixed bowel flora. He was treated with 7 days of intra¬ venous cefazolin therapy. The initial immunoglo-

bulin scan on 7/16/86 revealed a residual inflam¬ matory mass (Figure 7a) . No further therapy was carried out. The patient continued to drain puru¬ lent material from his left groin and to experience discomfort for the next four months. A repeat immunoglobulin scan on 11/5/86 revealed a more extensive inflammatory mass in the same area (Fig¬ ure 7b) . Subsequent surgical exploration revealed an extensive inflammatory mass extending from the sig oid colon into the groin and up into the retro- peritoneum. Thus, the immunoglobulin imaging technique is also effective in following the course of inflammation over a prolonged period.