Background In cancer surgery, lymphadenectomies are often performed with either or both the aim of eliminating cancer disease which has spread to lymph nodes and staging the disease.

The intervention may be made by open surgery or by so-called laparoscopy. The surgeon generally identifies the fat tissue strings which from experience are known to include the lymph nodes and which are located close to the large blood vessels, and above all the veins.

The outcome of this type of surgery may vary depending on if the surgeon can identify the single lymph nodes in the fat masses, since these have a color and consistency which are very like those of the fat.

The lymphadectomic preparations are sent to the pathology department, usually for freeze sectioning, i. e. rapid diagnostics, for the determination of if, or not, there is cancer in the lymph nodes by a combination of examination by visual macroscopy and palpation. This is then followed by a so-called cryostatic sectioning, staining and microscopic inspection for the purpose of identifying cancer metastases in the lymph nodes.

Based on the result of this procedure, the subsequent treatment of the patient is decided.

The pathological inspection for localization of the lymphnodes in the fat tissue preparations obtained by resection is often very difficult due to the slight difference in the consistency and color.

There are some techniques disclosed in the prior art which aid the identification of lymph nodes and the staging of cancer. Many of these are based on radiation responsive instruments and radiolabeled locators. (See e. g. Offodile R., et al. Minimally Invasive Breast Carcinoma Staging using Lymphatic Mapping with radiolabeled dextran. Cancer, 1998 May, 82: 9,1704-8; Albertini J. J., et al. Intraoperative Radiolymphoscintigraphy Improves Sentinel Lymph Node Identification for Patients with Melanoma. Annals of Surgery, 1996, Vol. 223, No. 2,217-225.) Another approach to visualize lymph nodes was injection of a vital dye at the primary breast cancer site, and the axillary incision was standardized to approximately 5 minutes after the injection. (Guiliano A. E., et al. Lymphatic Mapping and Sentinel Lymphadenectomy for Breast Cancer. Annals of Surgery, 1994, Vol. 220, No. 3,391-401.)

Description of the invention The present invention provides a non-radioactive contrast agent, and a method, for facilitating ocular identification and inspection of lymph nodes. The contrast agent comprises a conjugate of a macromolecule and a reactive dye. The conjugate is arrested by phagocytic cells in the lymph nodes and is kept there by virtue of the macromolecule while the dye provides the coloring. The macromolecule prolongs the duration of the conjugate in the lymph nodes in relation to unconjugated dye molecules, which pass pretty quickly through the lymph nodes after a short accumulation therein.

The conjugate of the invention may be injected interstitially to an area close to lymph nodes of interest in an individual, i. e. primarily to an area of a primary cancer. The conjugate is then transported by the lymphatic tract to lymph nodes and is arrested and accumulated therein. The time interval between the injection and the incision may vary depending on the type and size of the conjugate, but will preferably be in the range of 30 min to 2 days.

The conjugate of the invention is preferably water-soluble or colloidal, and it is injected in the form of a solution or suspension in a physiologically acceptable vehicle, such as saline.

Thus, one aspect of the invention is directed to a contrast agent for facilitating ocular identification and inspection of lymph nodes, which comprises a non-carcinogenic non-toxic biodegradable conjugate of a macromolecule and a reactive dye.

In an embodiment of this aspect of the invention the macromolecule is selected from macromolecules known to undergo receptor mediated phagocytes.

In another embodiment the conjugate has a large enough size to be arrested by the sinusoidal histiocytes of lymph nodes.

In a preferred embodiment the macromolecule is selected from the group consisting of hyaluronan, dextrans, glycogens, denaturated albumin molecules, and so-called sulfur colloids, and the reactive dye is selected from the group consisting of triazine based reactive dyes, such as Cibachron dyes or Procion dyes, and pyrimidinyl based reactive dyes, such as Rectone dyes, and reactive alkene dyes, such as Ramazol dyes, patent violet, trypan blue, and isosulfan blue.

In a most preferred embodiment the macromolecule is a dextran having a molecular weight (weight average molecular weight) of from 1 000 to 500 000, preferably from 10 000 to 100 000, and the reactive dye is Cibachron Blue F3G-A, Ciba Brilliant R 2Gp or Reactonmarin blau GRL.

In a further preferred embodiment the degree of substitution expressed as mmol of dye per gram of conjugate is between 0.01-1.0, preferably between 0.05 and 0.5.

Another aspect of the invention is directed to a method of facilitating ocular identification and inspection of lymph nodes comprising interstitial injection, to an area close to lymph nodes of interest in an individual, of a solution or suspension of a contrast agent, which contrast agent comprises a non-carcinogenic non-toxic biodegradable conjugate of a macromolecule and a reactive dye, in an amount providing a color to the lymph nodes which are visible to the naked eye after permitting the conjugate to localize in the lymph nodes and after incision.

The individual referred to may be an animal or human patient.

Also in this aspect of the invention, preferably the macromolecule is selected from macromolecules known to undergo receptor mediated phagocytes or the conjugate has a large enough size to be arrested by the sinusoidal histiocytes of lymph nodes.

Further, preferably the macromolecule is selected from the group consisting of hyaluronan, dextrans, glycogens, denaturated albumin molecules, and so-called sulfur colloids, and the reactive dye is selected from the group consisting of triazine based reactive dyes, such as Cibachron dyes or Procion dyes, and pyrimidinyl based reactive dyes, such as Rectone dyes, and reactive alkene dyes, such as Ramazol dyes, patent violet, trypan blue, and isosulfan blue. Preferably the macromolecule is a dextran having a molecular weight of from 1 000 to 500 000, and the reactive dye is Cibachron Blue F3G-A, Ciba Brilliant R 2Gp or Reactonmarin blau GRL, and preferably the degree of substitution expressed as mmol of dye per gram of conjugate is between 0.01-1.0.

The invention will now be illustrated by the following examples and experiments, but these should not be considered limiting to the scope of the invention defined in the claims.

Preparation of conjugates Derivatives of dextran with dyes J. F. Kennedy (Advan. Carbohydr. Chem.) teaches that commercial triazinebased reactive dyes react with polysaccharides to form stable coloured derivatives. Blue dextran 2000 (Amersham Pharmacia Biotech, Uppsala, Sweden) is a water-soluble derivative of dextran prepared from Cibachron Blue.

Example 1. Blue dextran 70 Dextran T70 (6 g; Amersham Pharmacia Biotech, Uppsala, Sweden) is dissolved in water (25 ml). Cibachron blue F3 G-A, (0. 6 g; Ciba-Geigy, V. Frölunda, Sweden) is added with stirring followed by 0.15 ml 50% (w/v) sodium hydroxide. The mixture is stirred at 40°C overnight and precipitated by adding 99% ethanol (75 ml). The supernatant is decanted and the residue is redissolved in water (50 ml), neutralised with dilute hydrochloric acid and

precipitated by adding ethanol (150 ml). The reprecipitation is repeated until a clear supernatant is obtained and the product is free from non-bound dye. The product is obtained as a powder by redissolving the residue in water (50 ml) and precipitating in ethanol (500 ml).

The blue precipitate is filtered and dried in vacuo at 50°C.

Yield, 5.1 g Substitution; 0.08 mmol Cibablue/g blue dextran Example 2. Blue dextran 70 (high substitution) Dextran T70 (10 g; Amersham Pharmacia Biotech) is dissolved in water (150 ml).

Cibachron blue F3 G-A (2.6 g) is added with stirring followed by 4 ml 6N sodium hydroxide.

The mixture is stirred at 40°C overnight and precipitated by adding 99% ethanol (250 ml).

The supernatant is decanted and the residue is redissolved in water (150 ml), neutralized with dilute hydrochloric acid and precipitated by adding ethanol (250 ml). The reprecipitation is repeated until a clear supernatant is obtained and the product is free from non-bound dye. The product is obtained as a powder by redissolving the residue in water (100 ml) and precipitating in ethanol (1.5 L). The blue precipitate is filtered and dried in vacuo at 50°C.

Yield, 9.6 g Substitution; 0.19 mmol Cibablue/g blue dextran Example 3. Blue dextran 10 Dextran T10 (10 g; Amersham Pharmacia Biotech) is dissolved in water (150 ml).

Cibachron blue F3 G-A (1.0 g) is added with stirring followed by 4 ml 6N sodium hydroxide.

The mixture is stirred at 40°C overnight and precipitated by adding 99% ethanol (500 ml).

The supernatant is decanted and the residue is redissolved in water (100 ml), neutralised with dilute hydrochloric acid and precipitated by adding ethanol (300 ml). The reprecipitation is repeated until a clear supernatant is obtained and the product is free from non-bound dye. The product is obtained as a powder by redissolving the residue in water (100 ml) and precipitating in ethanol (1 L). The blue precipitate is filtered and dried in vacuo at 50°C.

Yield, 9.2 g Substitution; 0.1 mmol Cibablue/g blue dextran Example 4. Blue dextran 40 Dextran T40 (10 g; Amersham Pharmacia Biotech) is dissolved in water (150 ml).

Cibachron blue F3 G-A (1.0 g) is added with stirring followed by 4 ml 6N sodium hydroxide.

The mixture is stirred at 40°C overnight and precipitated by adding 99% ethanol (500 ml).

The supernatant is decanted and the residue is redissolved in water (100 ml), neutralised with dilute hydrochloric acid and precipitated by adding ethanol (300 ml). The reprecipitation is

repeated until a clear supernatant is obtained and the product is free from non-bound dye. The product is obtained as a powder by redissolving the residue in water (100 ml) and precipitating in ethanol (1 L). The blue precipitate is filtered and dried in vacuo at 50°C.

Yield, 9.1 g Substitution; 0.11 mmol Cibablue/g blue dextran Example 5. Blue dextran 70 Dextran T70 (6 g; Amersham Pharmacia Biotech) is dissolved in water (30 ml).

Reactonmarin blau GRL (0.6 g; Ciba-Geigy, V. Frölunda, Sweden) is added with stirring followed by 0.15 ml 50% (w/v) sodium hydroxide. The mixture is stirred at 40°C overnight and precipitated by adding 99% ethanol (75 ml). The supernatant is decanted and the residue is redissolved in water (50 ml), neutralised with dilute hydrochloric acid and precipitated by adding ethanol (150 ml). The reprecipitation is repeated until a clear supernatant is obtained and the product is free from non-bound dye. The product is obtained as a powder by redissolving the residue in water (50 ml) and precipitating in ethanol (500 ml). The blue precipitate is filtered and dried in vacuo at 50°C.

Yield, 3.9 g Substitution; 0.06 mmol Reactonmarin blau/g blue dextran Example 6. Red dextran 70 Dextran T70 (10 g ; Amersham Pharmacia Biotech) is dissolved in water (150 ml).

Ciba Brilliant R 2GP (1.0 g ; Ciba-Geigy, V. Frolunda, Sweden) is added with stirring followed by 4 ml 6N sodium hydroxide. The mixture is stirred at 40°C overnight and precipitated by adding 99% ethanol (500 ml). The supernatant is decanted and the residue is redissolved in water (100 ml), neutralised with dilute hydrochloric acid and precipitated by adding ethanol (300 ml). The reprecipitation is repeated until a clear supernatant is obtained and the product is free from non-bound dye. The product is obtained as a powder by redissolving the residue in water (100 ml) and precipitating in ethanol (1 L). The red precipitate is filtered and dried in vacuo at 50°C. Yield, 9.9 g Experiments The following experiments illustrates the usefulness of the invention.

Experiment 1 Animal used in the experiment: Pig, 20 kg A nearly saturated aqueous solution of Blue dextran 70 (Example 2), 0.25 ml per kg body weight was injected into the groin. Three hours later, the animal was sacrificed, and regional lymph nodes in the lower part of pelvis were identified and found to be blue.

Experiment 2 Animal used in the experiment: Mouse 50 g A nearly saturated aqueous solution of Blue dextran 70 (Example 2), 0.25 ml per kg body weight was injected into the root of the tail. The next day, the mouse was sacrificed, and regional lymph nodes were identified and found to be blue.