AU615184B2 - Method of targeting tumors in humans - Google Patents

Description

g S F Ref: 55915 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATI 5 8 4

(ORIGINAL)

FOR OFFICE USE: Class Int Class

I

Complete Specification Lodged: Accepted: Published: 0 4 t Priority: Related Art: Name and Address of Applicant: 1 1^ Vestar, Inc.

939 East Walnut Street Pasadena California 91106 UNITED STATES OF AMERICA Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia CC CC Address for Service: Complete Specification for the invention entitled: Method of Targeting Tumors in Humans The following statement is a best method of performing it full description known to me/us of this Invention, including the 5845/3 170/173 METHOD OF TARGETING TUMORS IN HUMANS BACKGROUND OF THE INVENTION Field of the Invention

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This invention relates to a method of targeting tumors in humans,-by the use of micellular particles such as phospholipid vesicles. More particularly, the invention relates to'a method of introducing neutral or charged phospholipid micellular particles containing a radiolabeled marker into a patient to a diagnose such tumors.

I I Description of Prior Art Before various abnormalities in a patient's body can be diagnosed and treated, it is often necessary to locate the abnormalities. This is particularly true of abnormalities such as malignant tumors since the treatment is often on a localized basis. Thus, the location of the malignant tumor must be identified so that therapy can be directed to such cancer cells for treatment.

1-7ious attempts have been made over an extended number of years to identify specific locations, such as tumors, by simple techniques. For example, it would be desirable to identify the location of cancer cells by a simple method 170/173 involving the localization of a particular chemical at the.

specific site. It would also be desirable to treat the cancer by introducing modified chemicals into the patient's body and having such chemicals move to specific locations to combat the cancer cells at such locations. In spite of such attempts, however, simple delivery systems for targeting tumors in humans do not exist as yet.

Placing a chemotherapeutic drug in the body orally, subcutaneously or intravenously can result in harm to the normal :::.cells in the body which take up the drug and a worsening in the .patient's condition, without achieving the desired reduction in tumor cell activity. In the past, this toxicity to normal cells in the patient's body has been a major disadvantage in the treatment of tumors with chemotherapeutic agents. The lack of efficacy of such chemotherapy is also attributable to the failure of the freely circulating drug, to localize within tumor cells before it is excreted or taken up by other cells in the body.

Prior attempts to improve treatment of tumors by j .chemotherapeutic agents have included encapsulation of such agents wifthin biodegradable phospholipid micellular particles in the form of vesicles or liposomes. Encapsulation is thought to reduce potential toxicity from the circulating drugs.

Researchers have also sought to utilize such encapsulation to selectively target tumors within a body for delivery of chemotherapeutics. However? until the invention disclosed in application Serial No. 663,503, filed October 22, 1984, "Method of Targeting a Specific Location in a Body," and its parent application Serial No. 363,593, filed March 30, 1982, efforts to locate or treat tumor cells with drug-encapsulating targeting 170/173 The inability to provide a satisfactory particletargeting method is believed to be due to the nature of the solid tumors and their metastases which are located in extravascular tissues. Thus; to accomplish targeting of intravenously injected radiolabelled or chemotherapeutic particles to the tumor cells, the particles must leave the normal circulation by crossing the blood vessel membranes to enter the extravascular tissues. This movement is known as "extravasation". In addition the encapsulated agent must cross the tumor cell membrane. Normally, small substances such as small molecular weight proteins and Smembrane-soluble molecules can cross cell membranes by a process known.as passive diffusion. However, passive diffusion will not -te i allow sufficient accumulation of larger particles carrying dru'gs within cells to reach therapeutic levels. Additionally, cells t can actively transport materials across the membrane by a process such as pinocytosis wherein extracellular particles are engulfed Sby the membrane and released inside the cell. Entry of encapsulating particles into individual cells may occur by pinocytosis.

S iS Progress in targeting tumors with chemotherapeutic drugs has been hampered by the inability to accomplish and detect movement of drug carriers across blood vessel membranes. In the usual case', large structures such as drug encapsulating vesicles cannot escape from blood vessels such as capillaries, and thus remain in circulation.

An understanding of extravasation, however, requires an examination of the structure of the vascular morphology of a tumor. Various blood vessels are associated with tumors, in particular capillaries. It is now known that tumor capillaries 170/173 fenestrations, as a result of tumor cell growth patterns. H.I.

Peterson, Vascular and Extravascular Spaces in Tumors: Tumor Vascular Permeability, Chapter III, Tumor Blood Circulation, H.I.

Peterson, Ed. (1979). Studies of tumor capillary permeability reveal morphologic variations in the capillaries which allow some substances to cross the capillary membrane. Such variations include defects in vascular endothelium from poor cell differentiation, or breaks in vascular walls as a result of invading tumor cells. H.I. Peterson, supra. Notwithstanding such knowledge of tumor vascular morphology, researchers such as Peterson have concluded that transport of large molecules or materials across the tumor capillary wall occurs as a result of passive diffusion and that "concentrations of active drugs sufficient for therapeutic effect are difficult to reach". H.I.

Peterson, supra, at 83.

Prior to such morphologic studies, early reports S suggested that vesicles might undergo transcapillary passage across the capillary membranes into humor cells. G. Gregoriadis, Liposomes in Biolbgical Systems, Gregoriadis, Ed., Ch 2, (1980).

However, available data indicated that the vesicles were unstable in vivo and that the radiolabel may have leaked, thus apparently prompting the two alternative theories of longer circulation of vesicles in the blood with release of drugs at a slower rate or interaction of the liposomes with the capillary walls without crossing the wall surface, which would result in the appearance of drugs at the tumor sites, but without drugs within tumor cells. Id. Other researchers simply concluded that the vesicles do not penetrate vascular walls after intravenous administration. B. Ryman et al., Biol. Cell, Vol. 47, pp. 71-80 Li- 0 170/173 Thus, although the prior art has recognized that.

vesicles carrying radiolabel markers or therapeutic drugs must cross vascular barriers to reach tumor cells, the experience of the art has taught that intravenous administration is noE effective to deliver the vesicles to extravascular tumor cells.

In the aforesaid application, Serial No. 663,503, and parent application, Serial No. 363,593, "Method of Targeting a Specific Location in a Body", the disclosures of whi4ch are incorporated herein by reference, a method is provided for targeting tumors in vitro and in animals, specifically, mice. In the present invention, a method is provided for enhancing extravasation of radiolabelled particles to tumor cells within humans, for the identification of such tumor sites.

0000 e a, o 0 000 0 0 0 SUMMARY OF THE INVENTION In the method of this invention, phospholipid micellular particles such as:vesicles that are pure (more than approximately 98% pure) neutral phospholipid molecules are incorporated into small (less than 2000A 0 micelles as a component of the external surface. The phospholipid molecules are radiolabelled to enhance the identity and the diagnosis of the tumor at the specific site.

In-lll labelled vesicles were injected intravenously into 13 patients with diagnoses of terminal cancer. Following intravenous injections of up to 275 mg of lipid and 500 microcuries of In-lll, vesicles were rapidly taken up by the liver and spleen and a median of 12.5% of the injected vesicles remained within circulation at 24 hours. In no patient did symptoms -6develop related to vesicles following administration. Twelve of the thirteen patients had tumors imaged by this scan.

According to a first embodiment of this invention there is provided a method of targeting tumors in humans comprising providing vesicles of less than about 2000A comprising chemically pure phospholipid molecules, incorporating a radioactive agent into said vesicles, and introducing an effective amount of said vesicles Into the bloodstream of a human to obtain movement of the vesicles to the tumor.

According to a second embodiment of this invention there is provided a method of targeting tumors in humans comprising providing chemically pure phospholipid vesicles of less than approximately 2000Ao, incorporating a radioactive agent into such vesicles, and introducing such vesicles into 0,0, the bloodstream of a human to provide a lipid dose level of about milligrams to about 1 gram and to obtain movement of the vesicles to the tumor.

0" According to a third embodiment of this invention there is provided a S method of targeting tumors in humans, comprising providing chemically pure 0oo0 phospholipid vesicles of less than about 2000AO, incorporating Indium-lll vinto such vesicles, and introducing such vesicles into the bloodstream of a human to provide a lipid dose level of about 40 milligrams to about 1 gram and a dose level of radioactive agent within the range of about 0.5 to about 2 millicuries and to obtain movement of the vesicles to active tumor cells.

0 According to a fourth embodiment of this invention there is provided oO2 5 a process of preparing a pharmaceutical composition for the diagnosis or treatment of tumors in humans, especially breast cancer, oat cell carcinoma, prostatic carcinoma, renal cell-carcinoma, colon carcinoma or oo mto malignant lymphoma, comprising mixing an effective amount of vesicles of less than 200 nm comprising chemically pure phospholipid molecules, said vesicles having incorporated a radioactive agent, with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

According to a fifth embodiment of this invention there is provided a process of preparing a pharmaceutical composition for imaging or treatment of tumors in bone, lymph node and soft tissue including mediastinum, lung, liver and spinal cord, comprising mixing an effective amount of vesicles of less than 200 nm comprising chemically pure phospholipid molecules, said vesicles having a radioactive agent, with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

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0UU _II 0 6A According to a sixth embodiment of this Invention there is provided a process of preparing a pharmaceutical composition for the diagnosis of treatment of metastatic cancers arising from primary breast cancer, oat cell carcinoma, prostatic carcinoma, renal cell carcinoma or colon carcinoma in humans, comprising mixing an effective amount of vesicles of less than 200 nm comprising chemically pure phospholipid molecules, said particles having incorporated a radioactive agent, with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

DETAILED DESCRIPTION OF THE INVENTION As used herein, "micellular particle" and "micelles" refer to particles which result from aggregation of amphihilic molecules. In this invention preferred amphiphiles are biological lipids. Micelles are S water-soluble aggregates of molecules with hydrophobic and hydrophilic 0o S portions (so-called amphiphilic molecules) which associate spontaneously.

,:15 Such micelles can be in the form of small spheres, ellipsoids or long So:* cylinders, and can also consist of bilayers with two parallel layers of amphiphilic molecules. Such bilayered micelles usually take the shape of S spherical vesicles with an internal aqueous compartment. Useful compositions of these micelles include phospholipid molecules in the structure.

"Vesicle" refers to a micelle which is in a generally spherical form, often obtained from a lipid which forms a bilayered membrane and is referred to as a "liposome". Methods for forming these vesicles are, by S now, well known in the art. Typically, they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine, and may include other materials such as neutral lipids, for example, cholesterol, and also surface modifiers such as positively or negatively charged compounds.

09 The phospholipid molecules may constitute distearoyl phosphatidylcholine. The stability of the distearoyl phosphatidylcholine J 30 micelles may be enhanced by the incorporation of TMS/341x 170/173 cholesterol. Positively charged molecules such as stearylamine or aminomannose or aminomannitol derivatives of cholesterol or negatively charged molecules such as dicetyl phosphate may also be incorporated into the vesicles.

When phospholipid micelles are introduced into the blood stream, the micelles move to the specific locations of cancerous growth in the patient's body. To enhance movement of the phospholipid vesicles to the specific locations, positively charged phospholipid vesicles may first be introduced into the Spatient's blood-stream to block the macrophages or other phagocytic cells in the patient's body. The positively charged molecules bound to such phospholipid vesicles may be an aminomannose or-aminomannitol derivative of cholesterol. Concurrently or after a suitable period of time-such as approximately one (1) hour, other phospholipid vesicles may be introduced into the patient's blood stream to move to the specific locations in the S body. Such phospholipid vesicles may include cholesterol and may It, be neutral or may be positively charged as by the inclusion of a stearylamine or aminomannose or aminomannitol derivative of cholesterol or -may be negatively charged as by the inclusion of a dicetyl phosphate.

When the phospholipid vesicles are introduced into the body to target tumors, indium-lll may be used as the labelling.

agent. The indium-lll may be chelated to a suitable material such as nitrilotriacetic acid (NTA). NTA is advantageous because it forms a relatively weak bond with the indium-Ill. As a result, when the phospholipid vesicles reach the tumor and are lysed, the nitrilotriacetic acid is displaced by proteins at the tumor. Since the proteins form a stronger bond with indium-lll, 1, r F: I 1l 0 S70/173 (in excess of 24 hours), which provides for easy identification of the tumor over the extended period of time.

Materials and Methods Liposome Preparation. Small unilamellar vesicles (SUV) with the ionophore A23187 were prepared from distearoyl phosphatidycholine (DSPC), cholesterol dicetyl phospuiate (DP), -stearylamine (SA) and the 6-aminomannose and 6-aminomannitol (AML) derivatives of cholesterol, according to previous o O aeo methods. Briefly, chloroform solutions of 10 mg lipid with the following molar ratios: DSPC:Ch, 2:1; DSPC:Ch:X, 4:1:1 where X=SA,.DC or AML; and DSPC:Ch:AM, 8:3:1, were evaporated to dryness under nitrogen (N 2 and further dried under vacuum overnight. Each tube was filled with 0.6 ml 5mM phosphate 0* *0 buffered 0.9% saline, pH. 7.4(PBS), containing .mM nitrilotriacetic acid (NTA). and sonicated under N 2 for 5 to minutes with a sonicator equipped with a titanium microtip.

Liposomes were annealed at 60 0 C for 10 minutes and S centrifuged at 300 x g for five to ten minutes. Liposomes were yJ separated from unencapsulated NTA with a 30 x 1.5 cm Sephadex Gcolumn. Liposome size was determined by laser light scattering. All vesicle types were shown by laser light scattering microscopy to have a mean diameter less than 0.1 microns (1000AO). For example, DSPC:Ch vesicles had a mean diameter of 528A°. However, vesicles as large as approximately 2000 Angstroms are believed to be satisfactory in obtaining the desired results of this invention, although the preferred range is approximately 500 to about 700A°.

The vesicles obtained as described above are chemi- L I iiu k m ntiva v i-hnt;I_ pl-q mpan p m.-Prri,1 1 -i 170/173 which constitute phospholipid vesicles are more than 98% pure.

For example, when the phospholipid chemical added is distearoyl phosphatidylcholine, this material is used at more than 98% purity. The same constraint holds for other components, such as cholesterol, which compose the vesicle. The phospholipid vesicles obtained as described above are stable when injected into experimental animals.

The aminomannose and aminomannitol portions of these derivatives of cholesterol extend externally from the phospho- 00.0 lipid particles. Thus, when such derivatives are incorporated or associated into the surfaces of vesicles or other micelles, an o 0 o amine moiety is provided that extends approximately 5-15 Angstroms, preferably about 10 Angstroms, beyond the surface of 0 0 the micelles. In the case of vesicles, it appears that the appropriate molecular design comprises a hydrophobic portion o" which serves to anchor the molecule within the vesicular bilayer, and a linking portion which is at least mildly hydrophilic which spans the requisite distance between the hydrophobic region and the amino functional group. The hydrophilicity is apparently required-to prevent the link from internalizing within the 0 e bilayer also and thus serves to "extend" the amine from the surface. An example of a successful extended amine within the context of this invention is a 6-aminomannose cholesterol derivative such as, for example, 6-(5-cholesten-3-/--yloxy)hexyl- 6-amino-6-deoxyl-l-thio-,CD-mannopyranoside. In this example, the cholesterol portion provides the hydrophobic moiety, while the aminomannose is relatively hydrophilic. Other embodiments are also possible: other amino sugars attached to other cholesterol derivatives, for example, are equally suitable as x> ie ninf t-bphvdronhilic and hvdror)hobic 170/173 portions. Polyamines and polyamino acids which can be bound covalently or associated by other means to the vesicle or other micelle surface may also be used.

The amino derivatives and cholesterol tend to impart stability to the phospholipid vesicles. Cholesterol may be included in the range of approximately 0% to 50% of cholesterol by weight and the remainder constituting the phospholipids. .The charged molecules such as the stearylamine, the dicetyl phosphate and the aminomannose and aminomannitol derivatives of cholesterol 1000 °000 may be includedin the range of 0% to 20% by weight of the o00 0000 o o0 charged molecules and the remainder constituting the o co. phospholipids.

0400 o0.o The chemically pure liposome compositions discussed above are quite stable to leakage in vitro and in vivo. However, So, phospholipid mixtures such as egg lecithin form more fluid 0 0 S°o, membranes than pure phospholipids. As a result, liposomes from .ao natural lecithin mixtures are less stable to leakage of their 0 000 contents than pure phopholipids.

0o In-lll Loading Procedure. Loading of In-Ill into oo~ o oo reformed liposomes was facilitated by the presence of A23187 in the lipid bilayer. In-1ll was loaded into liposomes at 60-80 0

C

in accordance with the procedure described by. Mauh and Gamble, Anal. Biochem. 94, 302-307 (1979). Incubations were terminated by the addition of 10mM ethylenediaminetetraacetic acid (EDTA) in mM phosphate buffered 0.9% sodium chloride, pH 7.4 (PBS), and free In-Ill was separated from the loaded liposomes by chromatography on Sephadex G-50. Up to 90% of the added In-1ll could be incorporated into preformed liposomes by this technique, and specific activities of up to 300 uCi/mg lipid have been obtained.

170/173 All patients diagnosed by the process of this invention had biopsy-proven malignant disease diagnosed as incurable and a life-expectancy of less than two years. Patients received a dose of 500 gCi of In-Ill in varying amounts of vesicles (45-275 mg) such that the relationship between kinetics of distribution and clearance to lipid dose could be determined. The vesicles were of the following formulation: S Per 100 mg lipid mq L-<K-distearoyl/phosphatidylcholine (DSPC) 80.70 Cholesterol 19.30 i Nitrilotriacetic Acid (trisodium salt) 0.03 In-111C1 3 (9Ci) (250 1000 See Table 1) Ionophore A23187 0.10 Patients were tested..at the dosage levels indicated n Table 1. Three patients were admitted to each dose level until dose level three was attained. If toxicity was observed in any of the dose levels prior to level three, eight additional .96 0 patients-were to be entered at that dose level to determine adverse reaction frequency.

Twenty-four and forty-eight hours following the intravenous administration of vesicles over a three minute period, whole body and regional imaging was performed utilizing gamma camera-dedicated computer systems. Window settings were adjusted to include both 172 KeV and 247 KeV energy peaks for In-ill emissions.

170/173 TABLE 1 DOSAGE LEVEL LEVEL LIPID DOSE 11l Tn DOSE 00)0 0009

I

1 0 C

C

000.

0 o 0 C 00 50 mg 100 mg 200 mg 200 mg 200 mg 500 jCi 500 gaCi 500 71ii 750 g.Ci* 1000 p1Ci* *dosage level not used to date Tests were perfformned. according to the schedule in Table 2.

170/173 TABLE 2 PARAMETERS FOLLOWED Prein- 1 4 8 24 48 72 jection Hours Hours Hours Hours Hoturs Hours 4 r Examination X X X X r> Scan X X X X (optional) (optional) CBC,Differential X X Platelet Count X X Chemistry-Profile X X and electrolytes (SMA 18) Chest X-Ray X X Serum Complement X X Urinalysis X X SBlood Sample for: X X X X X X oo° -Radioactivity Urine Sample for X X X X Radioactivity Stool Sample for X Radioactivity

RESULTS

A. DESCRIPTION OF PATIENTS/DIAGNOSES Thirteen patients (nine men and four women) were treated and the results were analyzed at the time of this report. Their ages ranged between 39-80. Patients were diagnosed as having orimarv cancers of the followinq sites: 170/173 Site No. of Patients Lung 3 Breast 3 Prostate 3 Colon 1 Pancreas 1 Kidney 1 Lymphoma 1 a All patients except two (prostate cancer and oat cell

*CAB

Slung cancer) had prior treatment for their cancer: ablative S*o0 surgery, radiation therapy, chemotherapy and hormonal therapy were variously used to treat specific patients. Certain patients .presented signs and symptoms that were known or suspected to be secondary to their -cancers bone pain and anemia).

B. DOSAGE DATA S" -Patients received intravenous injections of vesicles containing 45-275 mg of lipid and in all cases; 500 microcuries of indium-1ll. For kinetic studies, blood, urine and stool samples were collected over the first three days. Whole body and regional gamma camera images are obtained at 1-48 hours.

C. SAFETY DATA None of the 13 patients developed symptoms within 72 hours after administration of the vesicles which were judged to be attributable to the test article. Patient #2 complained of

I

170/173 weakness at 48 hours, having been recently placed in a new analgesic, Vistaril. Patient #3 complained of dizziness lasting minutes which developed 8 hours post-injection. A neurological exam performed at 24 hours was within normal limits. The same patient's eosinophil count rose from 1% at baseline to 7% at 48 hours. Patient #6 developed a two degree increase in temperature and an increase in pulse (T 97.4, P 64 at baseline; T 99.9, P 90 at 8 hours). The temperature rise began at four hours and continued through 72 hours. The surgical insertion of hIckman catherter was judged to be the likely Setiology. At 48 hours the same patient creatinine showed a ,slight increase from 0.7 mg/dl. to 0.9 mg/dl. Patient #9 showed an increase in glucose from baseline of 135 mg/dl to 194 mg/dl at 24 hours. Patient #13 showed an incr.ease in eosinophils from 0% at baseline to 5% at 48 hours.

Otherwise, vital signs and physical examination, as well as blood and urine tests showed no change over the 72 hours following administration of the vesicles. Chest X-rays revealed o no evidence of altered aeraticn or vascularity within the lungs.

D. RADIOPHARMMACOKINETIC RESULTS Radiokinetic determinations indicate that the liposomes were rapidly taken up by the liver and spleen. Within the first one to four hours, there was a large amount of radioactivity remaining within the blood stream as well, but by 24 hours after injection, a significant amount of vesicles had left the blood 'stream. A median of 12.5% of the injected vesicles remained within the circulation at 24 hours. Urinary excretion was quite small (median 0.95%) and fecal excretion was insignificantly

L

170/173 The radiation exposure to the whole body was 0.29 rads median. The patient with the highest amount of radiation exposure had only 0.30 rads whole body radiation. The dose limiting radiation exposures were liver and spleen. Usually liver exposure was slightly higher (7 patients) although in patients, spleen exposure was slightly higher. The median exposure to the liver was 2.3 rads with the greatest exposure being 4.7 rads and the median exposure to the spleen was 1.6 rads ,:with the highest exposure being 4.8 rads.

At 1-4. hours after administration, images demonstrated a vascular pool. At 24 and 48 hours, the liver and spleen had 9999 accumulated significant amounts of radioactivity, and the vasculate pool was minimal (exception patient #4 where the vascular pool remained high even at 48 hours.) 9 e4 E. RESULTS AND IRAGING EFFICACY *to.

Twelve of the thirteen patients had tumors imaged by this scan, including three T tients with breast cancer, three *:patienyts with oat tell carcinoma of the lung, three patients with prostatic -carcinoma, one patient with carcinoma of the rectum, one patient with carcinoma of the kidney, and one patient with malignant Lymphoma. Only in the patient with carcinoma of the pancreas was there no tumor image seen, and in that patient, the site of the tumors on the peritoneal surface was less than 5 mm.

Accumulating all of the organs which had tumor demonstated by standard techniques, 22 such organ sites were involved the tumor. Of those, 20 had tumors imaged by scans (false positive rate=9%). The patient with carcir'oma of the pancreas did not have peritoneal soft tissue tumors identified, and a 170/173 brain which hadpreviously been heavily irradiated had no evidence of brain metastasis on liposome scan.

In the 82 instances of organ systems that were not clinically involved with tumor by standard techniques, there was 1 instance of imaging (of bone) by liposome scan (false positive rate=1.2%). The accuracy rate for vescan was 101/104 or 97%.

Organs which were imaged successfully by the vesicle scan included bone, lymph node, soft tissue including mediastinum, lung, liver, and spinal cord. A single patient with a brain tumor previously treated with radiation therapy was not 4 able to be imaged.- Four cases of unsuspected tumors were observed. In patients with oat cell carcinoma, one patient with an unsuspected o o meningeal metastasis was identified,-and another patient with an o. unsuspected liver metastasis was identified. One patient with carcinoma of the breast showed involvement diffusely over the right chest, and subsequently developed a malignant pleural effusion which was not present It the time of the vesicle scan.

SOne patient .with darcinoma of the prostate had a heterogeneous uptake ofliposomes in the liver, and subsequently developed N malignant hepatomegaly.

The results described above are summarized in Table 3 which follows: -4

A

0O 0 0 o 0 0 o 0 0 0 0 o 0 0 0 000 0 0 0 0 o 0 0 S C o 0 000 0 0 00 0 0 0 0 0 a .000 Ct 0 0 a 00. 0 000 0 o 0 0 0 0 0 0 0 0 0 TABLE '3- Blood in pt:D 01 02 04 06 07 08 09 11 12 13 Disease Prostate Pancreas Breast Breast Lung, Oat Cell Breast Colon Prostate Kidney Lung, Oat Cell Prostate Lung, Oat Cell Lymphoma prior* Treatment

R,C,H

R, C C, H

C

R, C Dose Lipid 62.6 44.5 56.8 82.1 80 98.9 135 102 273 200 R, H R, C

H

C,H

R, C In-1il 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.53 0.5 0.5 4h 65.7 15 .0 40.4 86 67.6 42 11.8 50.6 51

ND

5.9

ND

2 4h 28.5 1.3 12.5 64 31.7 12. 2 6.4 27.1 22.4 5.5 9.6 2.0 14. 4 Exc.

Urine 1.4 0.7 1.2 0.9 3.0 1.0 0.6 0.6 1.0 Radiation Exposures Whole Liver Spleen Body 2.0 0.6 0.27- 4.7 3.1 0.30 2.5 2.2 0.z2 8 0.8 1.2 0.28 1.5 1.6 0.30 2.8 3.4 1.4 2.2 2.1 3.1 4.8 1.4 1.5 1.5 0.29 0 0 0.29 0.20 0.29 0.29 173 276 200 0.2 2.0 0.7 2.3 1. 1 3.8 1.8 2.4 4.4 0.30 2 0e. 000 a a no S 0 a C a na a S a at* a SOS a a C a.

ota C a. 0 Detection Bone M4arrow Bone 0.31 TP 0.22 TN 0.28 TN 0.34 TP 0.38 TN TABLE 3 (Contd.)_ of tumor in organ** Soft Pleura Node Tissue Lung Liv--zi Braij TN TN TN TP TN TN FN TN TN TN TN TP TP TN TN TN TP TN TN TN TP TN TN TP FN Spinal Cord n Comments Autopsy Conf.

0 CT scan false in Pancreas Pleural effusion 1 month Toxicat.

0 0 0 0 0 later 0.28 0.16 0.32 0.36 0.19 0.29 0.25 0.28 i 170/173 As indicated above, the patients were given intravenous injections of up to approximately 275 milligrams of lipid and 500 microcuries of Indium-1. Other dose levels, however, are within the scope of the invention. Thus, the lipid dose may vary from approximately 40 milligrams to about 1 gram, with a range of about 100 to about 700 milligrams being preferred, and from about 200 to approximately 500 milligrams being especially preferred.

The particular dose level of lipid will be determined on a case by case basis, with the amount being sufficient to present enough vesi'cles for tumor targeting and at the-same time kept to as "6 small an amount as reasonably possible since the vesicles itt constitute a foreign object in the human body.

0* il In the examples herein reported, the dose of radiolabelled substance was 500 microcuries of Indium-111. It is to S be understood, however, that other dose levels and other radiolabelled substances may. be utilized. Thus, for example, radiolabelled materials such as gallium 67 (Ga-67), technetium 99m (Tc-99m), and iodine 131 (1-131), may be utilized for *.imaging. It should also be understood that the particular dose ""level of-radiolabelled substance will vary depending upon the specific substance utilized, as well as upon the preliminary diagnoses of the condition of the patient. Accordingly, with Indium-1l1, the dose level will typically range between approximately 0.5 to about 2.0 millicuries, whereas with gallium 67 and iodine 131, the dose will ordinarily be between about 2 and approximately 5 millicuries. With technetium 99m, however, which is known to be excreted much more readily and extensively than other radioactive elements, the dose will vary from approximately 5 to about 20 millicuries.

ii/ i 170/173 It will also be clear that the method of this invention, in addition to targeting tumors as described, is applicable in determining whether particles designed for therapy would move to a specific tumor site for a given patient. Moreover, it has been found that the method of this invention is particularly advantageous in that it does not image arthritis or inflamations as do other imaging techniques, and that only active tumor cells are imaged rather than cells that have been treated as by Og -radiation or chemotherapy.

o eo Although this invention has been described *s00 with reference to particular applications, the principles ooooo0 000000 involved are susceptible to other applications which will be apparent to those skilled in the art. The invention 0 00 0 0 is accordingly to be limited only by the scope of the o o 0 00 appended claims.

00oo o00 0 0 0 0 00 0000

Claims (30)

1. A method of targeting tumors in humans comprising providing vesicles of less than about 2000A° comprising chemically pure phospholipid molecules, incorporating a radioactive agent into said vesicles, and introducing an effective amount of said vesicles into the bloodstream of a human to obtain movement of the vesicles to the tumor.

2. A method according to claim 1 wherein the agent emits gamma radiation.

3. A method according to claim 2 wherein the agent comprises Indium-Ill.

4. A method according to claim 1 wherein the radioactive agent is Gallium 67, Technetium 99m or Iodine 131. 0 5. A method according to any one of claims 1 to 4 wherein said phospholipid molecules constitute distearoyl phosphatidylcholine.

6. The method set forth in claim 5 wherein cholesterol is incorporated with said phospholipid molecules to enhance the stability of said vesicles. 0 7. The method of any one of claims 1 to 6 wherein charged molecules are attached externally to said vesicles.

8. The method of claim 7 wherein said small, chemically pure phospholipid molecules are neutral and wherein the charged molecules are positively or negatively charged.

9. The method of any one of claims 1 to 8 in which said vesicles S are introduced into the bloodstream to provide a lipid dose level of 00°o, between approximately 40 milligrams to about 1 gram. The method of claim 9 in which said lipid dose is between about 100 and about 700 milligrams.

11. The method of claim 10 in which said lipid dose is between approximately 200 and about 500 milligrams.

12. The method of claim 3 in which said vesicles are introduced into the bloodstream to provide a dose level of between about 0.5 and about 2 millicuries of radioactive agent.

13. The method of claim 4 in which the vesicles are introduced into the bloodstream to provide a dose level of radioactive agent within the range of about 2 to about 5 millicuries.

14. The method of claim 4 in which said vesicles are introduced into the bloodstream to provide a dose level of radioactive agent of about 5 to about 20 millicuries. 22 A method of targeting tumors in humans comprising providing chemically pure phospholipid vesicles of less than approximately 2000A", incorporating a radioactive agent into such vesicles, and introducing such vesicles into the bloodstream of a human to provide a lipid dose level of about 40 milligrams to about 1 gram and to obtain movement of the vesicles to the tumor.

16. The method of claim 15 in which said radioactive agent comprises Indium-lll and said vesicles are introduced into the bloodstream to provide a dose level of radioactive agent within the range of about 0.5 and about 2 millicuries.

17. The method of claim 15 in which said radioactive agent is Gallium 67 or Iodine 131 and said vesicles are introduced into the S bloodstream to provide a dose level of radioactive agent within the range of about 2 to about 5 millicuries. 18, The method of claim 15 in which said radioactive agent is a ao o° Technetium 99m and said vesicles are introduced into the bloodstream to provide a dose level of radioactive agent within the range of about 5 to about 20 millicuries.

19. The method of any one of claims 15 to 18 in which lipid dose is between about 100 and about 700 milligrams. The method of claim 19 in which said lipid dose Is between 00 approximately 200 and about 500 milligrams.

21. The method of any one of claims 15 to 20 wherein said phospholipid vesicles constitute distearoyl phosphatidylcholine.

22. The method of claim 21 wherein cholesterol is incorporated with 0 0 0 said phospholipid vesicles to enhance the stability of said vesicles.

23. The method of any one of claims 1 to 22 in which said tumor is a 0::Oo malignant breast cancer, oat cell carcinoma, prostatic carcinoma, renal cell carcinoma, colon carcinoma or malignant lymphoma.

24. The method of any one of claims 1 to 23 in which said vesicles Sre less than approximately 1000A 0 The method of claim 24 in which said vesicles are from about 500 to about 700A 0

26. A method of targeting tumors in humans, comprising providing chemically pure phospholipid vesicles of less than about 2000A°, incorporating Indium-1ll into such vesicles, and introducing such vesicles into the bloodstream of a human to provide a lipid dose level of about milligrams to about 1 gram and a dose level of radioactive agent within the 23 range of about 0.5 to about 2 milli curies and to obtain movement of the vesicles to active tumor cells.

27. A process of preparing a pharmaceutical composition for the diagnosis or treatment of tumors in humans, especially breast cancer, oat cell carcinoma, prostatic carcinoma, renal cell-carcinoma, colon carcinoma or malignant lymphoma, comprising mixing an effective amount of vesicles of less than 200 nm comprising chemically pure phospholipid molecules, said vesicles having incorporated a radioactive agent, with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

28. A process of preparing a pharmaceutical composition for imaging or treatment of tumors in bone, lymph node and soft tissue including mediastinum, lung, liver and spinal cord, comprising mixing an effective amount of vesicles of less than 200 nm comprising chemically pure o o phospholipid molecules, said vesicles having a radioactive agent, with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

29. A process of preparing a pharmaceutical composition for the diagnosis of treatment of metastatic cancers arising from primary breast cancer, oat cell carcinoma, prostatic carcinoma, renal cell carcinoma or colon carcinoma in humans, comprising mixing an effective amount of vesicles of less than 200 nm comprising chemically pure phospholipid molecules, said particles having incorporated a radioactive agent, with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. The process according to any one of claims 27 to 29 wherein the S radioactive agent emits gamma radiation. S o 31. The process according to claim 29 wherein the radioactive agent comprises Indium-1ll.

32. The process according to any one of claims 27 to 29 wherein the oo~ radioactive element is Gallium 67, Technetium 99m or Iodine 131.

33. The process according to any one of claims 27 to 29 wherein said phospholipid molecules constitute distearoyl phosphatidyl choline.

34. The process according to claim 33 wherein cholesterol is included in said phospholipid molecules to enhance the stability of said vesicles. The process according to any one of claims 27 to 29 wherein charged molecules are attached to said vesicles.

36. The process according to claim 33 wherein said small chemically pure phospholipid molecules are neutral and the vesicles further contain positive or negatively charged molecules. 24

37. The process according to any one of claims 27 to 29 wherein the composition is composed of a first group of vesicles comprising chemically pure phospholipid molecules, having positively or negatively charges molecules extending externally from the vesicles, and a second group of neutral vesicles of less than 200 nm, comprising chemically pure phospholipid molecules, said vesicles having incorporated a radioactive agent.

38. The process according to any one of claims 27 to 29 wherein said vesicles provide a lipid dose level of hetween 40

39. The process according to any one of claims 27 to 38 vesicles are less than 100 nm. The process according to any one of claims 27 to 38 vesicles are from 50 to 70 nm.

41. A method of targeting tumors in humans which method substantially as herein described with reference to any one of and 37 mg to 275 mg. wherein said wherein said is the examples. :ce t l 99 C I If DATED this NINTH day of AUGUST 1990 Vestar, Inc. Patent Attorneys for the Applicant SPRUSON FERGUSON TMS!