FLUORESCENCE DIAGNOSTICS OF CANCER USING delta -AMINO LEVULINIC ACID

Abstract: A procedure for the detection of malignant tumours using the agent delta -amino levulinic acid (hydrochloride) as a tumour marker is described. The tissue is exposed to the agent either by intravenous injection or by application of an unguent over superficial tumours. After a suitable time delay fluorescence is induced in the tissue using an excitation source, e.g. a laser. Cancer then emerges with a strong and specific fluorescence peak at 635 nm. By dividing this red peak intensity by the intensity of the blue-green autofluorescence from the tissue an improved demarcation is obtained and also immunity to variations in many experimental parameters.

Description: Fluorescence diagnostics of cancer using δ-amino levulinic acid Field of the invention Cancer diseases are very widespread and have a major social and socio-economic impact. The incidence of cancer has shown a steady increase during the last tens of years. It is generally accepted that an early cancer diagnosis and a radical treatment is of great importance for the prognosis for the patient. Conventional diagnostic methods for cancer comprise different types of X-ray investigations, endoscopic investigations with biopsy sampling in the hollow organs of the body, cytological puncture of resistances, as well as smear cytology. The present invention describes the use of δ-amino levulinic acid (abbreviated ALA below) as a specifically tumour seeking agent, to be visualized through its specific fluorescence on irradiation with a laser or other suitable radiation source. A point monitoring or an imaging fluorescence system can be used for the detection. ALA, intravenously administered, or applied as an unguent over superficial tumours, is very selectively taken up by tumour cells, in which the substance is transformed into protoporphyrin IX, which exhibits a very strong and characteristic fluorescence in the red wavelength region. This procedure can be used, e.g. for the detection of early bronchial or bladder cancer and for the demarcation of the extension of a tumour for assuring a radical tumour treatment. Background for the invention Since long it is known that certain agent that are intravenously injected can be accumulated in tumours. If photo sensitizing agents are utilized such a tumour marking can be used in two ways, for diagnostics thorugh the fluorescence of the agent and for tumour treatment by photodynamic techniques, where the photo- excited agent transfers its energy to oxygen molecules, that make a transition from their normal triplet state to an aggressive singlet state. The release of singlet oxygen selectively in the tumour causes tissue necrosis. During the last few years the commercially available agent Photofrin (Quadra Logic Technologies, Vancouver, Canada) has been injected and used clinically for the double purpose just described. Experimentally it has been found, that the selectivity for a tumour over surrounding tissue seldom exceeds a factor of 2-3, which limits the power of the method both for diagnostics and for therapy. It is possible to increase the discrimination for diagnostics by using also the autofluorescence of the tissue in the blue-green spectral region. This is utilized in procedures and equipment described in the Swedish patent # 455646 (Fluorescence device) and the European patent application # 0411104 (Improvements in diagnosis by means of fluorescent light emission from tissue). Also other agents, such as phtalocyanines, chlorines, purpurines and bensopo hyrin exhibit an accumulation in tumour tissue, but also for these the selectivity is limited, which reduces their value. Normal sensitizers have the difficulty that also the skin generally is sensitized, calling for the patient to be protected from strong day light for a period of about 4 weeks. Description of the invention The present invention is based on our observation, in animal experiments as well as clinically, that the agent δ-amino levulinic acid is an extremely efficient fluorescence marker for tumours. This is true for intravenous injection as well as for superficial treatment with ALA unguent direct on the tumour. The small non-fluorescing ALA molecules penetrate the cell membrane of malignant tumour cells and undergo in the mitochondria parts of the haem cycle, leading to the formation of protoporphyrin IX. Fluorescence can be induced by illumination by radiation from, e.g. a pulsed laser. This can be a dye laser pumped by a nitrogen laser. An especially suitable excitation wavelength is 405 nm, where porphyrins have their absorption maximum. The radiation can be conducted to the tissue through a quartz fibre, which can also be used to collect fluorescence light and to conduct it to a spectral analyzer. This can be a spectrometer equipped with a diode-array detector or a more simple instrument based on optical filters. Tumours are marked by the occurrence of a fluorescence peak at 635 nm and a weaker peak at 710 nm. A demarcation ratio (the ratio between the specific fluorescence signal in a tumour and the signal in close-lying normal tissue) of about 10 or better can be obtained using this naturally occurring molecule in the body. It has no known side effects. A strong tumour demarcation is obtained after intra-venous injection if the investigation is performed about 1/2 hour after injection. ALA can also be prepared in an unguent base (about 20 % ALA) and be spread over superficial tumours and be kept in position using an occlusion bandage. The fluorescence investigation is performed 2-8 hours after the application. Apart from exhibiting an increase in the specific red fluorescence, a decrease in the blue-green fluorescence from endogenous chromophores is observed in tumours. An especially effective form of diagnostics for ultraviolet or violet excitation is to evaluate the background-free fluorescence at 635 nm and divide it by the fluorescence intensity at about 470 nm. Such a ratio is dimensionless and therefore the signal will be independent of the measurement distance, the angle of incidence for the light, and fluctuations in the excitation and detection equipment. The procedure given in this invention can also be used in combination with an imaging cancer detection system, which has been described in the Swedish patent # 455646. A simultaneous recording of images in three different fluorescence colours is then made (635 nm, 600 nm and 470 nm). An image intensifier, which is activated in synchronism with the exciting light source, is used for the recording. A new, generalized and tumour enhancing image is obtained by subtracting the 600 nm image (background) from the 635 nm image, followed by a division of the result by the blue image, pixel by pixel. Cancer detection with fluorescence techiques using ALA is illustrated in Figure 1, where 1 indicates the excitation source, 2 indicates optical components to bring the exciting light to the tissue, 3 indicates the tissue that has been exposed to ALA, 4 indicates optical components for conducting the fluorescence light to a wavelength-dividing system 5 placed in front of the detection system 6. Finally, 7 indicates a signal processing system for tumour recognition based on the fluorescence properties. The system proposed enables strongly improved possibilities for early detection of malignant tumours, also small tumours in the hollow organs of the body, and for clear demarcation of diseased tissue from surrounding normal tissue. No side effects occur. The efficiency for this form of cancer detection is illustrated by the following three non-restricting examples. Example 1. A patient with basal cell carcinoma of the forehead was investigated with the method described in this invention. 6 hours before the fluorescence investigation the tumour areas were exposed to an unguent containing ALA in hydrochloric form (Porphyrin Products, Inc., Logan, Utah; catalogue # A 167). The unguent also covered a 10 mm wide zone of normal tissue. The fluorescence detection equipment used, as an excitation source, a Laser Science model VSL 337 pulsed nitrogen laser, pumping a Laser Science dye laser operating at a wavelength of 405 nm. The radiation from the dye laser was focussed by a lens into a quartz fibre. The other end of this fibre was put in contact with the tissue. Fluorescence light was collected through the same fibre, and after passing a dichroic beam splitter, used for injecting the laser light into the fibre, the fluorescence light was focussed on the entrance slit of a Jobin-Yvon 0.25 m grating monochromator equipped with an EG&G Optical Multichannel Analyser (OMA) HI system. Huorescence spectra from a basal cell cancer tumour and from close-lying, ALA-exposed normal skin are shown in Figure 2. Using the intensity of the characteristic red fluorescence a demarcation ratio of 50 between the tumour and the normal tissue is obtained. If instead the ratio between the red and the blue- green fluorescence is used a demarcation ratio of 150 is obtained. Example 2. A patient with widespread local metastases of breast cancer was investigated with the same equipment and the same procedure as was described in Example 1. In Figure 3 fluorescence recordings 6 hours after application of the ALA unguent are shown for a skin area with lymphangitic metastatic breast cancer growth and for a close-lying normal skin area, also exposed to the ALA unguent. In this case the demarcation ratio is 13 and 25, respectively. Example 3. An experimental animal tumour model (human adenocarcinoma induced in the muscle of Wistar/Furth rats) was investigated with the method described in the present invention. The rat was intravenously injected with ALA hydrochloride in saline at a concentration of 30 mg/kg bodyweight. The rat was killed 30 minutes after the injection and the exposed tumour and the surrounding muscle were investigated using the same fluorescence set-up as was described in Example 1. Fluorescence signals from tumour and surrounding muscle are shown in Figure 4. A demarcation ratio of 9 is obtained using the characteristic red fluorescence. In this particular case, a division by the blue fluorescence intensity does not yield any further demarcation, for the excitation wavelength chosen, but still the advantages of recording a dimensionless quantity, as discussed above, are retained. Figure captions Figure 1. Schematic arrangment for fluorescence detection with ALA. Figure 2. Fluorescence recordings for basal cell carcinoma and normal tissue for a patient that was exposed to an ALA unguent in the tumour area. Figure 3. Fluorescence recordings for metastasis of breast cancer and normal tissue for a patient that was exposed to an ALA unguent in the tumour area. Figure 4- Fluorescence recordings for an experimental animal tumour and surrounding normal tissue after intravenous injection of ALA.

Claims: Claims 1. A method for the detection of malignant tumours by fluorescence characterized by a substance, δ- aminolevulinic acid, which is supplied to the tissue and that the ratio between the specific peak of fluorescence intensity in red and in the blue-green is used to enhance the detection of tumours in investigations where the excitation is made in the ultra violet, violet or blue spectral range. 2. The method as recited in claim 1, characterized in that the applied substance is δ-aminolevulinic acid hydro-chloride. 3. The method as recited in claim 1, characterized in that the substance is a salve which is applied on to the suspicious malignant tissue. 4. The method as recited in claim 3, characterized in that the fluorescense examination is performed between 2 and 8 hours after the salve is applied. 5. The method as recited in claim 1, characterized in that the substance is intravenously administered. 6. The method as recited in claim 5, characterized in that the substance is injected with a concentration of 0.5 to 50 mg per kilo body weight. 7. The method as recited in claim 5, characterized in that the fluorescence examination is performed between 15 minutes to 2 hours after the injection. 8. The method as recited in claim 1, characterized in that the fluorescence examination is performed with laser excitation. 9. The method as recited in claim 1, characterized in that the fluorescence examination is performed with a conventional light source. 10. The method as recited in claim 1, characterized in that the fluorescence examination is performed with a fibre optic point monitoring system with excitation and detection through the same fibre. 11. The method as recited in claim 1, characterized in that the fluorescence examination is performed with a multi-color imaging system with subsequent data handling system.

Inventors: BERG ROGER, JOHANSSON JONAS, SVANBERG KATARINA, SVANBERG SUNE, BERG, ROGER, JOHANSSON, JONAS, SVANBERG, KATARINA, SVANBERG, SUNE
Applicants: BERG ROGER, JOHANSSON JONAS, SVANBERG KATARINA, SVANBERG SUNE, BERG, ROGER, JOHANSSON, JONAS, SVANBERG, KATARINA, SVANBERG, SUNE
Application year: 1992
Main classification: A61K49
IPC codes: A61B5, A61K31, A61K49