CA1253300A - Indium-bleomycin complex ( suniii xxin-blmc) - Google Patents

Abstract

Abstract A new 111In-BLM complex designated 111In-BLMC
is described. The complex is characterized by a lack of capacity for binding to serum transferrin, a high selective affinity for viable tumor tissue, in vivo stability, improved activity ratios of tumor to tissues over known 111In-BLM complexes, tumor imaging flexi-bility and distinctness, and rapid clearance from the body. The new 111In-BLM Complex thus has clinical use as a radiopharmaceutical for combining radiotherapy and chemotherapy, and as a tumor-imaging agent for diagno-sis.

Description

~2S330~) Background of the Invention Field of the Invention Bleomycin is a well-known antitumor anti-biotic, commercially available as a mixture of bleo-mycin species differing only in a terminal amine.

While particular species such as bleomycin-A2 and bleo-mycin-B2 have been isolated and characterized, bleo-mycin is generally employed clinically as a mixture sold under a variety of trademarks such as BLENOXANE
(Bristol Laboratories). Bleomycin and its A2 and B2 species are characterized in U.S. Patent 4,339,426, issued July 13, 1982 to Meares, et al.
Bleomycin (BLM) has a selective affinity for a variety of tumors, and exerts its cytotoxic effects on susceptible tumor cells by inducing single-strand breaks in the cell DNA. In addition to its role as a chemotherapeutic, BLM has been chelated with a number of radionuclides for use as a radiopharmaceutical or tumor-imaging agent. These chelates have generally a wider application than BLM alone, as BLM does not exhibit cytoxicity against all tumor tissue to which it has a selective affinity; thus the chelates may func-Uo tion as a radiopharmaceutical, combining both radio-therapy and chemotherapy, or the BLM may merely func-tion as a vehicle for targeting the radionuclide on tumor tissue.

Description of the Prior Art Radiolabelled pharmaceuticals for radio-chemotherapy ideally have a high affinity for tumor tissue, deliver a high dosage of radioactivity to the tumor tissue and a minimal dosage to adjacent tissues, and function to sensitize the tissues to radiation.
Complexes of lllIndium (lllIn) with bleomycin mixtures and bleomycin-A2 or bleomycin-B2 which approach these criteria have been described. These known In-BLM
complexes have a high selective affinity for tumor tissue and are relatively safe. In emits both X-radiation and beta radiation, and bleomycin is believed to function as a radiosensitizer. These two properties are important in radiotherapy. lllIn also strongly emits gamma radiation (gamma energies of 173 and 247 kev), and has an effective half-life of 2.8 days; lllIn-BLM complexes are thus potentially useful as tumor-imaging agents. Unfortunately, however, known bleomycin chelates of lllIn bind to serum transferrin.
The body is thus broadly exposed to radiation, and the complex is consequently unsuitable for therapy or for use in tumor-imaging. The BLM chelate of 57Co has been _4_ i Z S 33 ~0 proposed as an alternative to In-BLM; however, the physical half-life of 57Co (270 days) ma~es its clini-cal use as a diagnostic agent impractical.

Summa~y of the Invention The invention comprises a new lllIn-BLM Com-plex designated 11 In-BLMC. The complex is character-ized by a lack of capacity for binding to serum trans-ferrin, a high selective affinity for viable tumor tissue, in vivo stability, improved activity ratios of tumor to tissues over known In-BLM complexes, tumor imaging flexibility and distinctness, and rapid clear-ance from the body. The new In-BLM Complex thus has clinical use as a radio-pharmaceutical for combining radiotherapy and chemotherapy, and for diagnosis as a tumor-imaging agent.

Brief Description of the Drawing Fig. 1 illustrates autoradiographs of TLC (a) and electrophoresis plates (b, c) for InCL3 and BLM
species labelled with 1 In (III). Lanes 2 are n _ BL Mc .
Fig. 2 is a radiochromatograph scan of an lllIn-BLMc TLC plate Fig. 3 illustrates autoradiographs of TLC (a) electrophoresis plates (b, c) of 1 In-BLMC solutions after storage.

12533~

Fig. 4 illustrates autoradiographs of TLC (a) and electrophoresis plates (b) of in vitro mixtures of urine with lllIn-BLMC and InC13.
Fig. 5 illus~rates autoradiographs of electro-phoresis plates of in ~itro and 1n vivo mixtures of plasma with lllIn-BLMC, 57Co-BLM, ~ n-BLM, and InC13.
Fig. 6 is a graphical illustration of tissue concentration of lllIn-BLMC and lllIn-BLM, 24 hrs after administration.
Fig. 7 graphically illustrates activity ratios of tumor to tissue in glioma-bearing mice for 1 In-BI,MC, lllIn-BLM-B2 and lllIn-BLM~
Fig. 8 graphically illustrates percent of retained activity over time (clearance) after admini-stration of lllInCL lllIn BLM B lllIn BLM
57cO-BLM~ and lllIn-BLMC-Fig. 9 is a series of gamma camera photo-graphs of tumor accumulation of radioactivity for lllIn-BLMC, 67Ga-citrate, and In-BLM, 4 hr. or 24 hr. after injection.
Fig. 10 illustrates autoradiographs of a TL5 plate (a) and electrophoresis plate (b) for 1In-labelled BLM species according to the prior art.
Fig. 11 is a graphic illus.ration of the effect of 0.9% NaCl, BLM and 11 In-BLMC on tumor size and host weight after intraperitoneal injection.

-6- lZS~300 Fig. 12 is a graphic illustration of the effect of 0.9% NaCl, BLM and ~ n-sL~Yc after into-tumor injection.
Fig. 13 is a series of microphotographs com-paring tumor size and characteristics after treatment ith lllIn-gLM (a) and lllIn_gLMc (b-e)-Fig. 14 is a graphic illustration describing lllIn_BLMc .

Detailed Description of the Invention The lllIn-BLMC product of the invention com-prises a purified species recovered from an unfraction-ated BLM mixture labelled with lllIn (III). The product is characterized (Fig. la, lane 2) by an Rf of 0.65 (thin layer chromatography on silica plates, employing 10~ aqueous ammonium acetate/methanol 1:1, v/v as eluant). Analysis of the thin layer chromato-grams by radiochromatograph scanning establishes a radiochemical purity of greater than 99% of the

2~ characterized product (Fig. 2). In-BLMC is distinguishable from 1 In-BLM, lll-BLM-A2 and In-BLM-B2 (-I, -II), and InC13 by gel electro-phoresis (5~ agarose, C02 - saturated 0.02M NaHC03), by migration of lllIn-BLMC toward the anode but a major part of In-BLM, lllIn-BLM-A2 and ll~In-BLM-B (-I, -II) toward the cathode, and lllInCL3 at the origin ~Fig. 1 b, c). lllIn-BLMC is further distinguishable lZ~3;~

from known BLM species including lllIn-BLM, In-BLM-A2, In-BLM-B2 (-I, -II), and InC13~ by a lack of binding capacity for serum transferrin.
Autoradiographs of TLC and electrophoresis plates for In-BLMC solutions stored at 4 C for three weeks showed no change in Rf for lllIn-BLMC (Fig 3 a,b:cf. Fig. 1).
~he In-BLMC complex of the present inven-tion is stable in vivo: TLC and electrophoresis patterns of urine receiving In-BLMC under in vivo conditions (1-4 or 24 hours after injection) are similar to those of in vitro mixtures of urine with 1 In-BLMC (Figs. 4(a) and (b), lanes 2 and 4). This is typical of both normal and tumor-bearing mice.
Electrophoretic patterns of in vitro mixtures of plasma with lllIn radiopharmaceuticals illustrate that lllIn of lllInC13 binds to serum transferrin in vi:ro, but -BLM, 57Co-BLM and In-BLMC do not (Fig. 5a -In-BLMC in lane 4; serum transferrin locates near the origin, slightly to anode side, not illustrated).
lllIn-BLMC and 7Co-BLM do not bind to serum trans-ferrin under in vivo conditions in plasma from mice not bearing a tumor, 1-3 hr. after injection (Fig.
5b- In-BLMC in lane 4, serum transferrin located again slightly to anode side of origin, not illus-trated).
The tissue distributions of In-BLMC, 12S~

In-BLM-B2 and In-BLM in glioma-bearing mice after injection of the pharmaceutical are summarized in Table I~ Concentrations in different tissues at 24 hr. are significantly lower for In-BLMC. Activity ratios of tumor to blood and to muscle for lllIn-BLMC are much higher than for In-~LM-B2 and In-BLM (Table II, Fig. 7). Rate of excretion (calculated from percent activity retained in normal mice) for In-BLMC is high (Fig. 8), reflecting the lllIn-BLMC lack of bind-ing capacity for serum transferrin.

Preparation and Characterization of In-BLMC
BLM mixture (BLENO~ANE) labelled with lllIn by conventional procedures fractionates into several components (Fig. 10). Autoradioaraphs of TLC plates are shown in E`ig. lOa. In-BLM separated into three major componerlts (Rf 0.75, 0.71 and 0.42), and six minor components (Rf: 0.78, 0.65, 0.63, 0.59, 0.54, and 0.48).
According to the process of the present inven-tion, a conventional bleomycin mixture is radiolabelled with lllIn(III) at a pH below about 3. The pH of the solution at t'he time the bleomycin and 1 In(III) are combined appears to be absolutely critical to the production of the lllIn-BLMC species described herein.
Under the correct process conditions, the several lllIn-labelled BLM components visible on an auto-i253~
g radiograph of a thin layer chromatogram of conventiona]
labelled BLM ~Fig. lOa) are replaced by a single, lllIn-labelled component (Rf 0.65) as illustrated in Fig. la. The mechanism of this shift is not known. A
possible partial explanation is that the component at Rf 0.65 in lane 4 of Fig. 10a is exclusively labelled with lllIn under the process conditions of the ~resent invention, and is thus the only component visible in the TLC autoradiograph in Fig. la. This is, however, merely a postulation at the present time (Example X
infra).
Lleomycin mixtures useful as starting mate-rials in the process of the present invention comprise mixtures of glycopeptide antibiotics isolated from the culture broth of Streptomyces verticillus by procedures of the type described by Umezawa (J. Antibiotics l9A:200, 1962~, exemplified by commercial bleomycin mixtures such as BLENOXANE. The bleomycin mixture is radiolabelled with lllIn (III) by combining bleomycin with a suitable solution of lllIn radionuclide, par-ticularly lllInC13, commercially obtainable in aqueous HCl at a pH of 1 to 3 (which maintains 1 1InC13 in solution). A diluent is employed, if necessary, so that the lllInC13 and BLM react at a pH of below about

3.5, preferably below about 3. At a reaction pH of from about 2 l:o about 3 (pHydrion paper), a substan-tially radiochemically pure In-BLMC product is ob-tained, as illustrated in Fig. l(a). At a pH lower than about 1.5 and higher than about 3.5, the product will be slightly contaminated with other lllIn-BLM
species; it will be progressively further contaminated as the pH decreases or increases in a typical reaction mixture. Since it is contemplated that the 1 InC13/BLM reaction product of the present invention will be directly usable in clinical applications, for these applications, at least, it is desirable to obtain a very pure lllIn-BLMC product, and a reaction pH of about 2 to 3 is recommended. In most clinical appli-cations according to the present invention, a slight amount of foreign lllIn-BLM species is not detrimental, and a reaction p~ of about 1.5 ~o 3.5, preferably about 1.5 to 3, is generally acceptable. BLM in either solid or solution form may be combined with lllInC13.
lllInC13 may be c:ombined with BLM in various concen-trations; dilute solutions having an lllInC13 concen-tration of about 2mCi/ml and concentrated solutions having an 111InC13 concentration of about 5mCi/Q.l ml are commonly commercially available, for example, from Medi-Physics, Emeryville, CA. The proportions of lllInC13 to BLM employed are determined by the desired specific activity of the product. It is generally desirable for cl nical applications that the BLM be substantially completely labelled, as BLM in large quantities is toxic; an excess of unlabelled BLM, or an - 1 1 - lZ533(~0 excess of unreacted lllIn(III), should be avoided by correct selection of reaction conditions or by subse-quent removal of excess reactant, if necessary. A
proportion of solid BLM, or its equivalent in solution form, of about 1.O mg. to about 25Jul to about 200,u1 of InC13 ~concentration 2mCi/ml, pH 1-3) will gener-ally be satisfactory, with preferred proportions of 1.0 mg. solid BLM to about 50Jul to lOOJul 11 InC13. Under the preferred proportions, combining solid BLM with 111InC13 at a concentration of 2mCi/ml, pH 1-3, w-ll generally produce a reaction solution having the requi-site pH. The pH of the reaction solution will vary, however, depending upon the pH of the reactants, and the exact amount of reactants employed. If the pH
requires adjustment, a diluent is employed which has sufficient alkalinity or acidity to bring the pH of the solutions, as combined, to a pH of below about 3.
Satisfactory results have not been obtained by adjust-ment of pH after the solutions are combined. Suitable diluents for pharmaceutical applications include physio-logical saline (e.g., 0.9% NaCl, about pH 5.7) to in-crease the pH, or HCl to lower the pH, or similar pharmaceutically acceptable diluents. The volume of acidifying or al~alinizing diluent required to produce the desired pH in the reaction solution will vary, depending upon the particular diluent employed, the relative alkalinity of the bleomycin mixture, the acid-lZ~3~

ity of the lllInC13 solution, and the amounts of both employed. In general, however, for the above-noted proportions of bleomycin to 1 InC13 (2mCi/ml, pH 1-3), a volume of 0.9% NaCl (about pH 5.7) at least about equal to the volume of 111InC13 solution will be re-quired. Generally, a ratio of 0.9% NaCl (about pH 5.7) ~o InC13 (2mCi/ml, pH 1-3) of about 1.5:1 to 4:1 v/v will be satisfactory. If concentrated InC13 is employed (e.g. pH 1-3, 5mCi/0.1 ml, Medi-Physics), a greater volume 0.9% NaCl to InC13 of about 4:1 to 8:1 v/v) of diluent will usually be required. In the absence of the pH-adjusting diluent, the pH of the reaction mixture is normally from about 4 to about 6, owing to the alkalinity of the bleomycin reactant.
Standard methods for preparing lllIn-labelled bleomycin such as by Thakur (Int. J. Radiat. Isotopes 24:
357-359, 1973) do not adjust the pH of the reaction solution; the bleomycin is thus typically radiolabelled at a pH of from about 4 to 6, with results as shown in Fig. lOa.

Tumor Imaging Gamma images of glioma-bearing mice show that

4 hours after i.v. injection (Fig. 9a,b) the tumor was more clearly visible with lllIn-BLMC than with lllIn-BLM, and accumulations of activity in liver and background lower for lllIn-BLMC than for 67Ga-citrate ~2S;3~

or In-BLM 24 hours after i.p. injection (Fig. 9c-e).
Ga-citrate is one of the few tumor-imaging agents presently employed clinically, and its applications are limited; the improved results obtained with lllIn-BLMC, which appears to have no serious disadvantages, will be of great clinical benefit.

Combined Radiotherapy and Chemotherapy Treatment of glioma-bearing mice with In-BLMC by standard proctocols produced a signi-ficant reduction in tumor size as compared to In-BLM-treated mice. Toxicity of In-BLMC (as reflected in measured host weight) was comparable to that of BLM alone, and only slightly higher than that of the NaCl control group (Figs. 11 and 12, Tables IV-V). Histopathology of the treated tumors showed that, in comparison to BLM-treated tumors, 1 In-BLMC-treated tumors were smaller and In-BLMC-treated tumors also evidenced newly formed granulation and connective tissue (Fig. 13 c-e).
The use of ~ n-BLMC as a radio-pharmaceutical (combined radiotherapeutic and chemo-therapeutic) is exemplified herein on glioma-bearing mice, employing standard mouse protocols. Based on these protocols, and standard clinical evaluations (Example IX, inrra)~ lllIn-BLMC is contemplated as an effective tumor-imaging agent and therapeutic drug in 12S3~r~0 the treatment and management of cancer in humans.
1 In-BLMC is particularly contemplated as useful in the treatment and management of gliomas in humans, as these tumors are not particularly sensitive to con-ventional radiotherapy. lllIn-BLMC of the present invention is also contemplated as applicable, however, to a broad range of tumors in addition to gliomas, such as squamous cell carcinomas and others described in the publication BLENOXANE (Sterile Bleomyin Sulfate).
Clinical Experience ~verview, ~ristol-Meyers Company, 1982. As previously noted, bleomycin has a known selective affinity for many tumors. For some, but not all of these tumors, bleomycin is a cytotoxic agent.
For others of these tumors, bleomycin may function as a radio-sensitizer, or have no apparent therapeutic value. lllIn-BLMC according to the present invention is potentially useful in the treatment and management of all tumors in humans for which bleomycin has a selec-tive affinity. For susceptible and for metastatic tumors, lllIn-BLMC may produce a combined radio-therapeutic and chemotherapeutic effect, with the bleo-mycin moiety acting as a cytotoxic agent and/or radio-sensitizer, and lllIn as a radio therapeutic. In other tumor~, the bleomycin moiety may function solely to deliver 11 In (III) to the target tissue, especially across the tumor cell membrane. In all of these appli-cations, the lllIn-BLMC properties of rapid clearance 1~533(~0 from the body, stability, low toxicity, and lack of binding to serum transferrin are especially relevant.

EXAMP~ES
MATERIALS AND METHODS
Example I.
Fractionation of BLM
BLM (BLENOXANE), Bristol Laboratories, Syracuse, N.Y.) was separated by thin-layer chroma-tography (TLC) on silica gel (Analtech). Fifteen units of BLM, dissolved in 15~ul of 0.9% NaCl, was applied in three spots or along one line to the plate, which was developed wi~h 10% ammonium acetate-methanol (1:1 v/v).
The positions of BLM fractions were located by ultra-violet light (254 nm). In order to identify the fractions from the relative sizes and positions after chromatography, it was important to apply the samples as spots, the relative amounts of A2 and B2 in the preparation being quoted by the manufacturer. The sections were extracted three times with 0.5% aqueous ammonium acetate-methanol (1:1) and the extracts were taken to dryness with nitrogen.

Example II
lllIn-labelling of Frac~ionated BLM and BLM (Prior Art) _ _ _ _ The fractions A2 and B2 and unfractionated BLM (Example I) were labelled with lllIn by employing a modification of the method of Thakur (Hou, et al. Eur.
J. Nucl. Med. in press; Int. J. Nucl. Med. & Biol., in press). lllInC1 was received in 0.45% to 0.9% NaCl solution that had been adjusted to a pH of 1-3 with HCl (Medi-Physics). It was taken to complete dryness with nitrogen gas at 55-60 C. BLM or BLM-A2 or -B2 in 0.9 NaCl was added with immediate mixing. The pH of the solutions of 1 In-BLM was between 4.0 and 4.5, and of lllIn-BLM-A2 and -B2 between 5.0 and 5.5 with pHydrion*
paper (Micro Essential Laboratory, 8rooklyn, N.Y.).
The higher pH for the fractions was caused by the ammon-ium acetate used for their extraction. The solutions of In-BLM-A2, -B2 and unfractioned lllIn-BLM were tested by TLC. Co-BLM was similarly labelled with the method used for 1 lIn-BLM.

Example III
Labelling Method to Obtain In-BLMC According to Present Invention 11 In-BLMC was prepared by mi~ing 25-200 ul 111InC13, received in 0.45 to 0.9% NaCl s~lution, ad-iusted to pH 1-3 with HCl ~Medi-Physics), with 1.0 mg of solid BLM or with BLM dissolved in 0.9~ NaCl. The final pH of the reaction solution was 2.0-3.0 with pHydrion paper (Micro Essential Laboratory).
In one specific preparation, 3 units solid BLM (BLENOXANE) was dissolved in 1.5-2.5 ml normal * a TrademarK

lZS33~}0 saline (0.9~ NaCl) solution, about pH 5.7); the BLM
solution was then combined with 1.0-1.5 ml 111InC13, received in 0.45 to 0.9~ NaCl solution, adjusted to pH
1-3 with HCl, at a concentration of 2mCi/ml. The resulting solution had a pH of between 2 and 3 and contained substantially radiochemically pure In-BLMC; i.e., insignificant amounts of other radio-labelled products are present. Solutions produced according to this method are directly usable for tumor imaging or radio- and chemotherapy as described in the following Examples; the proportions of lllInC13 em-ployed are adjusted to give a solution of a desired specific activity according to known principles of tumor imaging and radiotherapy.

Example IV
TLC and Electrophoresis Procedure Solutions of lllIn-BLM, lllIn-BLM-B , lllIn-BLM-A2, lllIn-BLMC and 57Co-BLM were analyzed by TLC using 10% ammonium acetate-methanol (1:1 v/v) as the eluant. After applying 0.2 ~1 of the samples, cold air was blown over the plate for 1-2 minutes. The plate was placed in a TLC cylinder containing freshly prepared eluant -and the cylinder immediately covered with a ~lass plate. All cylinders were enclosed in a sealed plastic bag together with an open beaker contain-ing eluant in order to maintain a saturated atmosphere.

One to three, 48-hour urine samples from normal or glioma-bearing mice given injections of 111 In-BLM or one of its fractions were also analyzed by this TLC
procedure. The same urine samples were also analyzed by gel electrophoresis on 5% agarose (Sigma or BRL low EEO) (Hoch, et al Anal Biochem. 78: 312-317, 1977).
The aqueous gel was equilibrated against Co2-saturated 0.02 M NaHCO3 and the electrode chambers contained CO2-saturated 0.1 M NaHCO3. The run was conducted for about 7 min. at 18 V/cm, so that bromphenol blue was displaced 15 mm. In vitro mixtures of S parts urine from untreated mice and 1 part of solution of 1 In-BLM
or its fraction were similarly analyzed immediately after mixing. The agarose gel plates for plasma sam-ples from normal mice collected 1-3 hr. after admini-stration of radiopharmaceutical were dried, auto-radiographed, and then stained with Coomassie Blue*for protein. The same gel electrophoresis procedure was used for In-BLM, In-BLMC, In-BLM-A2, In-BLM-B2 and 57Co-BLM; the dried plates were auto-radiographed or analyzed with a radiochromatogram scanner.

Example V
Tissue Distributions and Retention of Radioisotope Male 6- to 8-week old C57BL/6 mice received transplants of Glioma-26 in the left leg by a trocar a Trademark lZS33UO

and were given injections with radiopharmaceutical 6 days later. Another group of such mice received 1.5x105 tumor cells in 0.15 ml Hanks balanced salt solution in the left leg and were given injections with radiopharmaceutical 12 days later. lllIn-BLMC, lllIn-BLM-B2, or lllIn-BLM (35 uCi) from Examples II
and III were used. The tissue distributions were deter-mined 4 or 24 hr after the mice were given the radio-pharmaceutical. The tissues were weighed wet and counted in a scintillation counter.
Male 6-week-old C57BL/6 untreated, not tumor-bearing, mice were given intraperitoneal (ip) injections of 30-40 pCi of l11InC13, lllIn-BLM-B , lllIn-BLM, lllIn-BLMC or 57Co-BLM. At 1, 2, 4, 24, 48, and 72 hr. after injection of the radiopharmaceutical, the ani~al was made to urinate and then the whole body activity was measured in a dose calibrator (Fig. 8~.

Example VI
Urine and Plasma The activity distribution in 1-4-hr or 24-hr urine or 1-3-hr plasma from untreated or glioma-bearing mice after injection of radiopharmaceutical was determined by TLC and by gel electrophoresis (Example IV). Mixtures in vitro of urine or plasma from un-treated mice with In-BLMC, In-BLM-B2, or lllIn-BLM solution (5:1) (Examples II and III) were 12533(~) also analyzed (Example IV). After autoradiography, dried plasma electrophoresis gels were stained wi~.h Coomassie Blue for protein.

Example VII
Gamma Images - Tumor Imaging The 2-to 3-week-old glioma-bearing mice (tumor size: 1.0-1.5 cm) were given injections of lllIn-BLMc, 67Ga-citrate~ or In-BLM into the orbital sinus (or by ip injection). I~ages were taken with a gamma camera (Raytheon~ immediately, 1, 2, 4, 24, and 48 hr after injection by using a pinhole collimator (1/4 inch diameter), with the distance of 12 cm between mouse and collimator. lllIn-BLM, and lllIn-BLMC were obtained according to Examples II and III; 67Ga-citrate was commPrcially obtained from New England Nuclear (NEN), Boston, Massachusetts.

Example VIII
RadiotherapY and Chemotherapy Materials and Methods ...... _ .. _ 20 Prepa-ration of lllIn_BLMc Two to 3.2 ml of 111InC13 (concentration 2mCi/ml), receiveà in 0.45 to 0.9~ NaCl solution ad-justed to pH 1-3 with HCl (Medi-Physics), was mixed with 1.0 mg of solid BLM (Blenoxane, BRISTOL LAB) or with BLM dissolved in 0.9% NaCl. The final pH was * a Trademark 1.5-3.0 by pHydrion paper (Micro Essential Laboratory).
As a control, non-radioactive (cold) lllIn-BLMC was prepared similarly with 4-week-old 111InC13.

Radiotherapy and Chemotherapy Glioma 26 transplanted with a trocar in the left hind leg of C57 BL/6 male mice. Six days after transplantation (tumor age: 6 days) the mice were divided into 3 groups, and received intraperitoneal injections ( p groups) daily for 3 days: Group I (n=8) - 0.2 ml of 0.9% NaCl; Group II (n=5) - 0.004 mg of BLM/g body weight in 0.2 ml of 0.9% NaCl; Group III
(n=4) - 15uCi of In-BLMC carried by 0.004 mg BLM/g ~ody weight in G.2 ml of 0.9% NaCl).
Groups IV - VI received single intratumor injections (it groups) on the 13th day after trans-plantation: Group IV (n=3) - 0.7 ml of 0.9% NaCl/g tumor weight; Group V (n=3) - 0.5 mg of BLM in 0.7 ml of 0.9% NaCl/g tumor weight; Group VI (n=3) - 1.5mCi of In-BLMC carried by 0.5mg BLM in 0.7ml of 0.9% NaCl/g tumor weight.
Two control experiments were made with cold InC13 (InC13 that had been stored for 4 weeks and was practically nonradioactive, pH=1-3) and cold In-BLMC
(prepared with nonradioactive InC13 by the same method as that used for In-BLMC, final pH=1.5-3.0). At a tumor age of 6 days, p injections were given daily for 12533~0 3 days: Group VII (n=5) - 0.2ml of 0.9% NaCl; Group VIII (n=5) - In-BLMC carried by 0.0004mg BLMtg tumor weight in 0.2 ml of 0.9% NaCl. At a tumor age of 13 days, single doses were given it: Group IX (n=2) -0.7ml of 0.9% NaCl/g tumor weight; Group X (n=3) -InCl, 0.7ml/g tumor weight.
The tumor size (product of the three dimen-sions in mm3) and body weight were measured daily for 16 days following the first injection. Host weight was obtained by subtracting tumor weight from body weight, where tumor weight (g) =tumor size (dld2d3, mm3~ x 0.00053 Histopathology Several mice were killed on the 22nd day after tumor transplantation, and both tumors and tissues were sectioned for histology (H&E stain).

RESULTS
1 TLC and Electrophoresis .

TLC autoradiographs of InC13 and lllIn-BLMC solutions (Fig. la) show InC13 at the origin and the spot of In-BLMC at Rf 0.65. Electrophoresis autoradiographs of these solutions (Fig. lb,c) show 11 InC13 at the origin, lllIn-BLMC migrating toward the anode (lane 2), and 111In-BLM-B2 (-I, -II), -A2 and most of lllIn-BLM migrating toward the cathode (lanes 12533()0 3, 4, 5, 6).
Radiochromatogram scans of TLC plates showed the radiochemical purity of 1 In-BLMC to be 99~ (Fig.
2).
Autoradiographs of TLC and electrophoresis plates for lllIn-BLMC solutions that had been stored at 4C for 3 weeks or more showed no change (Fig. 3 a, b).
Autoradiographs of TLC plates of In-BLM
labelled according to the prior art and fractionated are shown in Fig. 10a. lllIn-BLM separated into three major components (Rf: 0.75 -lane 2, 0.71 -lane 3, desig-nated B -I, B2,-II) and 0.42 -lane 9, (A2)) and six minor components (Rf: 0.78, 0.65, 0.63, 0.59, 0.54 and 0.48). The latter appeared in autoradiography on longe. exposure. Autoradiographs of lllIn-BLM after electrophoresis (Fig. 10(b), a composite diagram from lllIn tagged fractions and many electrophoresis auto-radiographs) show on the cathodic of the origin two major components, lllIn-BLM-A2 (lane 9) and B2-II (lane 3), and near the origin 1 major component, B2-I (lane 2). On the anodic side four minor components were seen. The electrophoretic patterns of InC13, In-BLMC and of the separated fractions In-BLM-B~-I, -B2-II, A2 are shown in Fig. l(b,c).

2. Urine and Plasma TLC and electrophoresis patterns of urine receiving lllIn-BLMC under in vivo conditions (1-4 or 24 hr after injection~ were similar to those of in vitro mi~tures of urine with 11 In-BLMC, but differed from the patterns for lllInC13 (Figs. 4a,b). This was the case for both untreated and tumor-bearing mice (~ n-BLMC, lanes 2 and 4)-Electrophoretic patterns of in vitro mixtures of plasma with radiopharmaceutical solution show that In of lllInC13 binds to transferrin (lane 1), but In-BLM, 7Co-BLM and lllIn-BLMC do not (lanes 2-4) (Fig. 5a). Similarly, no binding to transferrin occurred under in vivo conditions in plasma from mice not bearing a tumor, 1-3 hr after injection of 1 In-BLMC (lane 4) or 7Co-BLM (lane 3) (Fig. 5B).
3. Tissue Distribution and Retention of Radioisotope The distributions of lllIn-BLMC, lllIn-BLM-B2 and In-BLM among tissues of glioma-bearing mice 4 hr and 24 hr after injection of the radiopharmaceutical are shown in Table I. At 24 hr, the concentrations (%
dose/g) in tumor, lung, liver, stomach, and kidney were significantly lower for lllIn-BLMC than for In-BLM, which, in turn, were lower than those for lllIn-BLM-B2.
Figure 6 shows that the concentrations (~ dose/g) in different tissues of lllIn-BLMC run almost parallel to those of lllIn_BLM
The activity ratios of tumor to tissue (Table II, Fig. 7~ show that the activity ratios of tumor to blood and to muscle for lllIn-BLMC of 13.1 and 12.4 lZS33(~(~

were significantly higher than those for lllIn-BLM-B2 (7.1 and 9.1) or lllIn-BLM (6.6 and 6.3) at 24 hr after injection of the radiopharmaceutical. Ratios of tumor to brain, heart, lung, stomach and femur were also significantly higher for lllIn-BLMC than for lllIn-BLM.
At 4 hr after ip injection (n=3), the activi-ty ratio of tumor to blood was 5.0 for 11 In-BLMC, which was significantly (P<0.005) higher than 1.5 for In-BLM (Table II). Table III shows that the activi-ty ratios of tumor to blood and brain for ip lllIn-BLMC
were significantly about twice as high at 24 hr than at 4 hr, while the ratios of tumor to muscle, lung and stomach were slightly, but not significantly, higher.
The percent activity retained in normal mice (n=2~ after injection of the radiopharmaceutical (Fig.
8) shows that the rate of excretion increased in the following order, reflecting decreasing binding capacity for transferrin~ InC13, 111In-BLM-B2, lllIn-BLM, 57Co-BLM, lllIn-BLMc-4. Gamma Images Gamma images of glioma-bearing mice given injections of lllIh-BLMC, 67Ga-citrate or 11 In-BLM
(Fig. 9) show that 4 hr. after injection (Fig. 9 a,b) the tumor was more clearly visible with In-BLMC-(Fig. 9b) than with In-BLM (Fig. 9a), and the accumulations of activity in liver and background were lower for lllIn-BLMC than those for lllIn-BLM. Fig. 9, c-e shows that 24 hr after i.p. injection the accumulation of radioactivity in the tumor was higher for In-BLMC (Fig. 9e) than that for 67Ga-citrate (Fig. 9d), or lllIn-BLM (Fig. 9c), and the activity of liver and background were lower for lllIn-BLMC than those for 57Ga-citrate or lllIn-BLM. The lllIn-BLMC
according to the invention is useful in known tumor imaging processes, such as those described in U.S.Patent 4,339,426, (supra); U.S. Patent 4,057,618 issued Nov. 8, 1977 to Salmon, et al; U.S. Patent 4,311,689, issued Jan. 19, 1982 to Ruddock; U.S. Patent 3,845,202 issued Oct. 29, 1974 to Tubis, et al; U.S.
Patent 4,360,509 issued Nov. 23, 1982 to Goedemans; and I5 U.S. Patent 4,017,596, issued April 12, 1977 to Loberg, et al.

5. Radiothera~y and Chemotherapy Tumor size and host weight in glioma-bearing mice after p therapy are shown in TABLE IV and Figure 11. During the observation interval, the tumor size in the 11 In-BLMC Group (III) was smaller by a factor of 1.8-3.8 when compared to that in the BLM Group (II) (p~0.~5 or p<0.001 until day 16) which, in turn, was smaller by a factor of 1.8-2.8 than that in the NaCl Group (I) (p<0.05 or p<0.001 until day 12 after the 12533~)0 first injection). The tumor size in the In-BLMC-treated group was smaller by a factor of 3.2-8.6 when compared to that in the NaCl Group. At day 16 after the first injection, the tumor size in the 1 In-BLMC group was 560 (range 240-1030)mm3, which was smaller than 198Q (range 1400-3290)mm in the BLM
Group, p<0.025; the tumor size in the BLM Group was smaller than that in the NaCl Group 4830 (range 2580-9180)mm (0.1 <p<0.2). During days 1 to 12 after the first injection, the host weiyht in the BLM Group was approximately 20% less than that in the NaC1 Group.
Toward the final days the difference in host weight was only about 10%. Host weights in the In-BLMC and BLM
groups were similar, and recovered to almost the original values.
TABBE V and Figure 12 show the therapeutic effect of lllIn-BLMC in the it group. The tumor size for the 1 lIn-BLMC Group (IV) decreased steadily for 3 days, then remained significantly smaller than that in the BLM Group (V) until the 9th day of therapy. On the 9th day of the experiment, the tumor size in the In-BLMC Group was 1060 (range 680-1810)mm , which was smaller than 2680 (range 2060-3210)mm3 seen in the BLM Group (p<0.05), and the latter was smaller than 6020 (range 4060-7070)mm3 in the NaCl Group (p<0.05).
The differences in the host weights for these groups were not significant.

12S;~300 Tumor size and host weigh for the two experiments with cold InC13 and In-BLMC are shown in TABLES VI and VII. In the p groups, during days 4 to 16 after the first injection, the tumor size was significantly smaller by a factor of 1.6-2.6 for cold In-BLMC (Group VIII) than for NaCl (Group VII, except for day 9 after the first injection, for which p=0.1).
During days 2-4 the host weight in the In-BLMC group was 10~ less than that in the NaCl group. In the it groups for cold InC13 (Group X) and NaCl (Group IX), the tumor sizes increased and were similar.

6. Histopathology Figure 13 a,b shows that the tumor size for the lllIn-BLMC group was smaller than that for the BLM
group, and that more necrotic tissue was present in tumors of the lllIn-BLMC ( p) group than in those of the BLM ( p) group. Figure 13c-e shows newly-formed granulation and connective tissue in the tumor of the p and it lllIn-BLMC groups, indicating replacement of tumor cells.
Liver and kidney morphology (not illustrated) were similar for the In-BLMC, BLM, and 0.9% NaCl groups (both p and it). Abnormalities were not found in these groups.
EFFICACY AND TOXICITY
The results indicate that 1 In-BLMC is more lZ53;:}C)0 effective than BLM alone in the treatment of glioma-bearing mice. lllIn-BLMC appears preferable to BLM alone because: 1) it reduced the tumor size in the p group to one-ninth of that for the NaCl group and in the it group to one-sixth of that for the NaCl group, while for BLM alone the figures were one-third and one-half; and 2) the necrotic area was increased to a greater extent by lllIn-BLMC than by BLM. To study the toxicity of lllIn-BLMC, the morphology of kidney and liver (which received the highest radiation dose) was investigated, and no differences were found among the In-BLMC, BLM and NaCl Groups. The host weight in the 11 In-BLMC and BLM groups did not differ. The host weights for BLM (Group II) was 20~ less than that for NaCl (Group I), but for In-BLMC (Group VIII) it was only 10% less than for NaCl (Group VII). The host weights had recovered in 15 days for BLM and recovery was complete in 3 days for In-BLMC. The quicker return of host weights suggest less toxicity for cold In-BLMC

than for BLM.

Human glioma, especially Grade III and IV
astrocytoma, is not particularly sensitive to radiotherapy. The mechanism of the effects of 11 In-BLMC on experimental gliomas is not known. In these experiments, the tumor sizes remained similar for cold InC13 (Group X) and for NaCl (Group IX, TABLE
VII), i.e., cold InC13 did not affect glioma. For cold 12S33~)0 In-BLMC (Group VIII), the tumor size became smaller than that for NaCl (Group VII) by a factor of 1.6-2.6 tTable VI), and for BLM (Group II) it became similarly smaller than that for NaCl (Group I) by a factor of 1.8-2.8 (Table IV). For radioactive 1 In-BLMC (Group III), the tumor size became smaller than that for NaCl (Group I) by a factor of 3.2-8.6 (Table IV). Thus, radioactive lllIn did play a major role in the effectiveness of In-BLMC against glioma. Moreover, in our experiments, the absor~ed dose of the tumor in the p group of lllIn-BLMC was about 60 rad, and this dose is not large enough for radiotherapy alone.
It has been reported that BLM has special affinity for ectodermal tissue and gliomas are of ectodermal origin. BLM also inhibits the growth of cultured human glioma cells. BLM is further known to be effective against squamous cell carcinomas, malignant lymphomas, testicular, cervical, and lung neoplasms, and head and neck cancers. It is believed that lllIn-BLMC will have a therapeutic effect on all known tumors susceptible to the pharmaceutical effects of BLM, or for which BLM has a selective affinity.
CLINICAL
It is of interest to compare the ratios of tumor dose to whole body dose received when treating with l11In-BLMC and the previously studied In-BLM.
Present data have shown that the concentrations -31- lZS~3~

(%dose/g) of lllIn-BLMC and lllIn-BLM in 12 different tissues vary almost in parallel (Fig. 6). The time integral of the whole body activity was 5.8 times as high for lllIn-BLM as that for lllIn-BLMC. The whole body dose then can be estimated to be 5.8 times as large for lllIn-BLM when compared with lllIn-BLMC.
In the described experiments, the mice were given 15 ~Ci of lllIn-BLMC carried by 4mg of BLM/kg body weight. The standard BLM dosage in the human is 0.25-0.5 mg/kg; ~his is smaller by a factor of 8-16 when compared to the doses used to treat the mice (4 mg BLM/kg). Therefore, it is proposed that one tenth of this dose may be effective for patients, i.e., 1.5mCi of In-BLMC carried by 0.4mg of BLM/kg body weight.
The radiation dose to kidney~ liver and red bone marrow was 1.64, 1.61 and 0.797 rad/mCi, respectively, for lllIn-BLM administered to patients. Therefore, it is estimated that the dose should be about 0.28, 0.28 and 0.14 rad/mCi for lllIn-BLMC. From the ratios of tumor to liver (1.9) and to blood (13.1) for lllIn-BLMC, the dose to tumor was estimated to be about 60 rad for a 60-k~ patient who would receive 1.5 mCi of lllIn-BLMC/kg body weight. It has been reported that doses of 3000 to 3500 rads for liver and 2000 rads for kidney, delivered over a 3- to 4-week period, and less than 200 rads to bone marrow are safe. Thus, if fractionated, 60-90 mCi (1.5 mCi/kg) of lllIn-BLMC per 12S3~00 -3~

dose would deliver a presumably safe dose of 15 times within a 3- to 4-week period; for a larger dose, given fewer times, the radiation would be even safer. A
standard clinical protocol for clinical evaluation of lllIn-sLMc is annexed hereto as Example IX.

i2S~

EXAMPLE IX
Clinical Evaluation of a New ~umor Imaging Agent n~ eOmycin complex (Protocol) 1. We have discovered a new lllIn-Bleomycin Complex (BLMC), which has high affinity to tumor, does not bind to transferrin and is stable in vivo. In tumor-bearing mice and rats, tumors were imaged more distinctly with the new lllIn-BLMC and 7Co BLM than with 67Ga-citrate.
The tissue distribution shows that lllIn-BLMC can substitute for 7Co-BLM and is much superior than 67~a-citrate; it was effective for combining radiotherapy and chemotherapy in glioma-bearing mice.
2. Animal Toxicity Studies:
Lewis rats, female, 160- 170 g, control group: i.p. 0.5 ml '~1 9% NaCl, n=4 test group: i.p 2mCi of In-BLMC carried by 5 mg BLM/kg in 0.5 ml 0.9% NaCl, n=3. This is equal to 10 times the planned human dose.
The rats were killed 2 weeks after lllIn-BLMC
and the organs were sectioned for histology. The morphology of liver, kidney, ovary, heart, lung, stomach, spleen, pancreas and femur were similar for the lllIn-BLMC and 0.9~ NaCl groups. Abnormalities were not found in these groups.
3. Studies on large ani~als for imaging:
One dog bearing veneral tumor, which was inoculated 1 year earlier, was given an i.v. injection :12S33()0 of In-BLMC 200 uCi carried by 0.1 unit BLM/Kg body weight, (the dose was 5 mCi lllIn-BLMC carried by 3 unit BLM). The dog was imaged 24 and 48 hours later.
In this observation period, all of the dog's three tumors showed activity uptake. Kidney and bladder had more intense activity than the tumors. At 48 hours, the background was less intense than at 24 hours.
4. Patient Selection Scintigraphic localization of lung cancer in a group of 5 patients. A comparison of 1 lIn-BLMC with 57Co-BLM.
5. Method of Labelling (for human use) 5-10 mCi lllC13 (receiv~d in 0.1 - 0.2 ml of 0.45%

to 0.9% NaCl solution, adjusted to pH 1-3 with HCl) was mixed with 3 unit of BLMl (Bristol Lab) dissolved in 0.8 ml of 0.9% NaCl. The final pH was 2.0 to 3.0 with pHydrion paper (Micro Essential Lab). This solution was prepared sterile.

6. Method of Imaging:

Gamma camera images are obtained at 1,2,4,24,48 hours after injection. Data will be stored on computer for later analysis.

7. Toxicity:

1. the minimally active dose is 2 unit of BLM, the regular dose of BLM for a patien~l~s 0.25-0.557nit/kg, and the dose BLM as carrier for In-BLM or Co-BLM
in clinical diagnosis is 5-15 units.

1253~0 BLM is contraindicated in patients who have demonstrated a hypersensitive or an idiosyncratic reaction to it.

8. Radiation Dosimetry Tissue distribution (~ dose~g) of lllIn-BLMC were calculated from the data for glioma-bearing mice (6 days after tumor transplantation), 1, 2, 4, 8 or 24 h after p injection. Initial concentrations were obtained by extrapolation to zero. The dose was calculated from the injected dose of 200JUCi lllIn/kg (12 mCi administered to a 60 kg patient, 4 ~Ci/20g for mice). Table A shows the parameters used for calculation.

lZS3300 Table A. Parameters for Dosimetry calculations Time Initial ~ Biological ~ 1/2 Dose Dose/ Dose x (~ dose/g) Curve 1 tO-4h) Curve 2 (4 h) RAD mCi lO(rad) _ Total * 100 0.5 24 0.03 0.003 0.3 Blood 10 0.3 14.4 0.02 0.002 0.2 Liver 1.7 0.6 20 0.02 0.002 0.2 Kidney 13 0.7 13.6 0.12 0.012 1.2 Femur 7.5 0-5 16.6 0.03 0.003 0.3 Testicle1.1 0.6 30 0.01 0.001 0.1 Urine 54 1.6 9.5 0.77 0.077 Urinary Bladder Wall <0.39** 0.04 <3.9 * Data frcm nonmal mice.
** From dose of urine divided by 2, because radiation is not isotropic *** Safety factor The absorbed dose for lmCi 57Co-bleomycin is 0.4 rad for liver, 2.0 rad for kidney and 1.4 rad for bladder . The total dcse of 12mCillk n-BLMC
and 1 mCi~7Co-BLM for crle patient w~uld be 0.42 rad for liver, 2.12 rad for kidney and 1.79 rad for bladder.

~533(~0 TABLE B. Dose Calculation for 1 In-BLMC
Radiation Iype Energy (keV) ~(g-rad/~Ci-h) .. ...... . .. _ In EC Decay (2.83 d 1) 1 (mun) = 0.10 Auger-L 2.72 0.0058 Auger-K 19.3 0.0065 Ce-K- 2 144.57 3 0.0259 Ce-L 2 167.26 3 0.0037 Ce-M NO- 2 170.51 3 O.oOo9 Ce-K- 3 218.679 20 0.0235 Ce-L- 3 241.372 20 0.0040 CE-NMO- 3 244.620 20 Q.0009 X-ray L 3.13 0.0005 X-ray K~ 22.98410 2 0.0116 X-ray K~12 23.17360 2 0.0220 X-ray K~ 26 0.0081 2 171.28 3 0.329 3 245.390 20 0.491 (g.rad/~ci.h) of lllIn: electron group 0.0712, low energy photon part: 0.0422 x 0.05 = 0.0021 higher energy photon part: 0.820 x 0.01 = 0.0082 total ~ (~G ) - 0.0815 g.radt~ci.h <Blood>
1) 0-4h:
D~se rate: 0.0815 (g.rad/~Ci.h) x 10% (~ dose/g) x 4 (~Ci)=G.033 rad/h Dose: 0.033 x ~ 4 e - 0.693 dt = 0.033 x 0.3 (e -0.~934- e -0.069 T 0.693 0.3 0.3 = 0.014 x (0.0000971 - 1) = 0.014 rad 2)~4h:
Dose rate: 0.0815 x 0.04% x 4 = 0.0001 Dose: 0.0001 x 14.4 = 0.002 In 2 <liver>
1) 0-4h:
Dose rate: 0.0815 x 1.7% x 4 = 0.006 rad/h Dose: 0.006 ~ 4 e - 0.693t T dt = - 0.006 x 0.6 (0.0098528-1) = 0.005 0.693 2) 4-~h Dose rate: 0.0815 x012~ x 4 = 0.0004 Dose: 0.0004 x 20 = 0.012 ~.693 1) + 2) : 0.02 rad ~' 12S330() <kldney>
1) 0-4h:
Do~e rate: 0.0815 x 13~ x 4 - 0~042 Do~e: 0.0~2 ~e-0.693t J. ~ tt - -0.042 x 0.7 (0.0190631 ~ 0.042 2) 4-~ h:
Do~e rate: O.OB15 x 1.3~ x 4 - 0.004 Do~e: 0.004 x 13.6_ - 0.079 0.693 1) ~ 2) : 0.12 r~d <fe~ur>
1) 0-4h Oo~e rate: 0.0815 x 7.5~ s 4 - 0.025 r~d/h Dose: 0.025 r e-0.693t ~ T dt---0.025 x 0.5 (0.0039109 - 1) - 0.018 r~
2) 4-9sh Do~e r~te. 0.0815 x 0.16% x 4 - O.OOOS rad/h Do~e: 0.0005 ~ 16.6 - 0.012 rsd 0.693 1) + 2) : 0.03 rad <testlcle>
1) 0-4h:
Do~e r~te: 0.0315 ~ s 4 - 0.004 rad/h Do~e: 0-004 ~c-O-6g3t ~, T dt- - 0.004 ~ 0.6 (0.0098528 - 1)-0.003 0.693 2) 4--oh:
Do~e rate: O.OôlS s 0.05~ s 4 - 0.0002 r~t/h Dosc: 0.0002 Y 30 - 0.009 r~d 0.693 1) + 2): 0.01 r-~
<url~e>
1) 0-4h Dose rate: 0.0815 x S4% x 4 - 0.18 r~d/h Do~e: 0.18 r~e-0~693t dt - - 0.18 x 1.6 (0.1768418~ 0.34 rad 0~693 2) 4-~Ch Dose rste: 0.0815 s 9.Sl s 4 - 0.031 r-~/h DOHe: 0.031 ~ 9.5 - 0.43 rad 0.693 1) ~ 2): 0.7~ r-d-12533()0 <Toeal Body>
1) 0-4h:
Dose rate: 0.0815 x 100~ x 4 - 0.016 r-dlh ~body ve~ght - ZOg Dose: 0.016 1~4e-o~693t J. T dt--0.326 x0.5 (0.0039109-1)-0.01 rsd 0.693 2) 4-~ h Dose r~te: 0.081S s 4Z ~c 4 ~ 0.001 rad/h Do~e: 0.001 x 24 - 0.02 0.693 1) + 2): 0.03 rsd ~2S~3UO

Example X
Description of In-BLMC
We have found that unfractioned IIIIn-BLM has

9 components (Fig 10). lllIn-BLMC has only one major component, the radio-chemical purity of which is 99%
(Fig. 1.2). Whether lllIn-BLMC is one of the components of lllIn-BLM mixture, or BLM that has undergone a structural change under the conditions (pH
= 2-3) when it chelates lllIn- is not known.
In the following experiments, 11 In-BLMC
solution, having a pH of 2.5, was adjusted to pH 4~5 with 0.05 NaOH. Then, lllInC13 was added to this solution. The autoradiographs of TLC and 5% agarose gel electrophoresis are shown in Fig. 14.
Fig. 14a shows that the Rf of 11 In-BLNC at pH 2.5 (lane 1) and of lllIn-BLMC at pH 4.5 (lane 2) are the same, 0.65. The patterns of lane 3, lllIn-BLMC
(in pH 4.5) to which was added 111InC13 (in Fig. 14 a) are similar to that of unfractionated lllIn-BLM in lane 4 except for the largest spot of Rf = 0.65. Fig. 14 b shows the electrophoretic patterns of these solutions.
A change in pH from 2.5 (lane 1) to 4.5 (lane 2) did not change the position of lllIn-BLMC, which migrated toward the anode. Addition of lllInC13 to the solution at pH 4.5 (lane 3) resulted in additional components that migrated toward the cathode, a known property of In-BLM-A2 and -B2, which are shown in ~ig. 14 c, 125~3()0 lane 4 and c.f. Fig. 1 c, lanes 3, 4, 5.
These results indicate that lllIn-BLMC may be the same material as that appearing as spot N.4 in the TLC of unfractionated lllIn-BLM (Fig. 14 c.f. Fig. 10).
This spot corresponds in position to a BLM component designated BLM-B3 (Umezawa et al.: J. Antibiotics, Ser. A 19: 2/82, 1966).

lZS~O

u ~ ~ _ A ~ D ~ D
~ ~ ~ ~ ~ `H~ l il . ~ .~ ~ D

3 D, D ~ i~l D --~ ;!S i~ ~ ~

8 ~ g ~ ~ t S.
. ~ ~ ~ ~æ ~

~ ~ ~ ~ ~S-~ ~, ~ ~ O ~ O ~ OO" V~
~ ~ ~ ~ ~ IE ~ ~ ~

~ 3 ~ ~

.~ ~ ~ 1~ ~ ~ ~, ~ ~ ~ ~ ~ ~ v~
~1 30~ ~ o~ o o o ~ .E
t ~ ~ b o o o ~! O O V~ ~ .
_ ~ ~ ~ ~ ~. ~_ ei P O _ D IC !~ O _ D 1~
~ .~ ~ V
æ~
3 v~v 4 3 lZS3300 ~æ ~ ~ ~
3 ~ H :2 ~ ~ H _J
~ . X ~;o~ ~

3 Y ~ .iz ~ r t :: ~ ~ ~ .
_ o ~ ~ ~ o o o a s ~ ~ ~' ~ ~ ~ .~
~ ~ ___ o _ ~ ~ e v _ .~ ~ ~ _ ~ o _ ~ ~ ~ _ oo ~ o-o ~
a E~ :~ ~ 8 ~ ~ ~ ~ ~ _ ~
o ~ _ o t ~ a V ~ "~

~ ~ ~ 7 ~ ~
n 1~ ;~ ~ ~ o ~.: o " ~ X ~
~ ~ ~ H ~ ~ H o_ _ ~
o 6 ~ ; . - ~r3c t-~

~ ~ V~ ~

lZS3300 ~ ~,, ~
U~ rl~r ,~ ~ ~ b oo ~
~ ~ ~ :~, ~ ' _~ ¦ ~ . N v~
(~ ~ t~ V

... N ~J

a~ ~ ~, .
~ ~ U:~ P.
.C ~ ~ , O

.~ m ~ a~ O ~

.
o ~ ~,~ V~

~r ~Z5~300 _ ~
_ 0--Ol~ rcooo~a~or~ ~
~ _ O----O O 00 _ _ _ _ _ r_ ~ ~ ~ ~ C
~ 11 +1 +1 +1 +1 +1 _1 +1 +1 +, +1 +1 +1 .1 +1 +1 +1 +1 _ _ m C ~-~u7~_~r _ _ ~ ~ ~ co co a> ai o~ (~ ,_ tl _ O~ O O O~ O a~
00000000000--O----O-- tD
~ 11 +1 +~ +1 +1 ~ +~ +1 +1 ~ +~ +1 +1 +1 +1 +1 +1 +1 _ ' o ~ ~ _ a) c Q~ m ~ i to _ 0 oi a~ c~ ~
3 _ 9 X _ ~ ) N ~ ~ O ~ ~'1 ~O a ~ a~--~ I~')-- O~
o B--~ _ _ _ _ _ _ _ _ o _ c~
I ~ +l +~ +1+1+1~ +~ +1+1~ +1~ +~ ~ +~ ~ +~ m Z c~--~t ~ 0 ~ C
0 ~ ~ N ~3 ~
v G O
' E _ a? ~ ;; ~~ .
o ~q ~D C +1 -H +,1 ~H +1 +1 ~ +1 ~1 +1 +1 ~1 +I t~ 1 ae ~ _ ~ c ~ ~ ~0 ~ 0 æ ~ o ~ ~ O
E _ ~ _ .--o S ~D tl - _ ~ æ ~
j~-- c~ m-- ~`~ ~ C9 ~ ~ } N ~ cn O ~ ~ _ ~ N tO O ~ ; g I ~ in B~ a~ ~ o ~ ~j O æ ~ j o~ v m ' E Z 1l ~--I +l +l +~ +1 +l +1 +1 +~ +~ +1 +1 o. _ 3 ~ ~ o ~ ~ ~ ~
E ~ .
.~ ~- ~c _ ~n ~n O C~ ~ I` ~ o> o - ~ ~ ~ ~ ~o o c~ ~ o m C O o ,, E

.o . _l _ E~

i~S'~ V

> a~ ~
V~" ~ C
~ ~ +l +l +l +l +l +l +l +l +~ ~ _ _ J c ~ _ _ ,_ ~ o~ ~ ~3 ~ ~ - "~

>~." (~i-_o_o--o-~1 +1 +1 +1 +1 +~ +1 +1 +1 +1 . ~ c ~r ~ ~ ~ ~
_ ~-- ~ 1 n ~ > o _ ~ o I ~ ~ ~ ~ 0 .
Z 11 +1 -H ~1 +1 -H +l +l +l +l +l a~
~e~ a ~ .
0 _ ~ E > ~ ~ ~ o ~ o ~ ~ ~ +~ +l ~ +l +l +l +~ ~ ~
E.- ~ ~ c ;~ } c~ o _ m ~ S ~" ~ ~ ~ $ ~ g c c ~i _ ~ ~ +1 +~ +1 +1 +1 +1 +~ ~ E
~ c 5 c ~ 8 I J ~ _ ~ C~ o ~ ' ~ ~r~ _ _ _ _ _ _ _. _ ~ e~ Z U +l +l ~1 +~ -tl ~ +l +~ ~ ~1 --c b ~ ~ v~ ~.

O - ~ V E, ._ .

12533V~

_ _ _ _ o _ o _ _ _ _ _ _ _ _ . o _ _ o O ll tl tl +I tl tl tl tl ~I tl I tl +I tl tl tl ~1 t~ m ~i ~ cn 0 o~ a~ 0 ~ ~ ~ ai u3 m C _ ~C - ,~>
.~ ~ tl +I ~ ~ ~ ~ +I t~ ~ +l +~ +l +l ~ ~ c ~o Z c ~ Co U~ ~ c 3 ~_ ~ æo~æo~ o c .
.q . 2 E _ . ~ - z E > --~ 3 æ 8 ~ ~ ~ ae ~ ~ ~ :Q~ ~ ~3 +~ + ~~ ~ æ '' ~ ~ ~' 0~
c~ c ~n _ ~_ E

E " C. v u~ ~ ~ _ æ ~ 0 ~ æ ~ ~
~C ae u~ z ~~ 5~ ~ ~ } v u~ E 0 ~.
E " ~_ _ o ~ Z; F ~

", _ _ o~ E o _I ~ C~

lZ533UO

~48--_ _ u~
~, X ~ tl +l t~ ~1 +1 1 +1 +I tl +
(n c- ~i~æ~ æ
C
C X
.~ = ~ ~ CO ~ O -- O ~ ~
3 ~ ~i ~tl ~ +i ~+1 +~ ~tl ~ tl +i :~ a~' ~ i 2, ~ OD 0 a~
., E
~ _ 'O` X~" +~+l~+~ + +
~ C_ _~ +~i m ' ) c x o ~) ~ __ ~ ~ U~
_ ~ ~D ~ ~itq ~D ~ ~ $ ~ o Z ~ ~ 11 +i ~ +1 +i +1 ~1 '0~ E z c E " ,_ ~ti c .~ ~ l l u H 0 C o _ N ~ ~ D 1~ 0 O>
~ _ E~

.

Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Substantially radiochemically pure 111Indium-bleomycin complex lacking binding capacity for serum transferrin.

2. In a diagnostic method for gamma imaging of tumors in mammals of the type wherein a gamma-emitting radionuclide is administered to the host as a tumor-imaging agent and the host is scanned with a gamma counter to visualize the tumor, the improvement comprising administering 111In-BLMC according to Claim 1 as the tumor-imaging agent.

3. The method of claim 2, wherein the tumor is a glioma.

4. The method of claim 2, wherein the mammal is a human.

5. A method for the preparation of an 111In-BLMC complex of bleomycin and 111Indium (III) lacking capacity for binding to serum transferrin, comprising reacting a bleomycin mixture derived from a culture broth of Streptomyces verticillus with 111InCl3 at a pH of from about 1.0 to about 3Ø

6. The method of claim 5, wherein 111InCl3 is in the form of an acid solution having a pH of from about 1 to about 3.

7. The method of claim 5, wherein a diluent is employed in admixture with one of the reactants used to adjust the pH
thereof.

8. The method of claim 5, wherein the pH of the reaction is from about 2 to about 3.

9. The method of claim 2, wherein the tumor-imaging agent comprises a solution consisting essentially of substantially radiochemically pure 111In-BLMC prepared by reacting an acidic solution of 111InCl3 and a bleomycin mixture at a reaction pH of from about 1.0 to about 3.

10. The method of claim 9, wherein the bleomycin is in solid form.

11. The method of claim 5, wherein bleomycin is in solid form.

12. The method of claim 9, wherein bleomycin and 111InCl3 are reacted in proportions of about 1.0mg. solid bleomycin to about 25ul to 200ul of 111InCl3 at a concentration of about 2mCi/ml.

13. The method of claim 9, wherein the proportion of 111InCl3 is from about 50ul to about 100ul.

14. The method of claim 9 wherein a pharmaceutically-acceptable diluent is premixed with one of the reactants used to adjust the pH of the reaction solution.

15. The method of claim 9, wherein 111InCl3 and bleomycin are reacted at a pH of from about 2 to about 3.

16. The method of claim 14, wherein the diluent is normal saline.

17. The method of claim 9, wherein unreacted bleomycin and/or unreacted 111In(III) is removed from the solution before it is administered.

18. The 111In-BLMC of claim 1 havlng an Rf of 0.65 (thin layer chromatography on silica plates, employing 10% aqueous ammonium acetate/methanol 1:1 v/v as eluant).