Biodistribution of Benzoporphyrin Derivative in Tumor BearingRats by Laser Induced Fluorescence Spectroscopy.

Sandor G. Van', Mango Stavridi1, Thanassis Papaioannou1,Theodore G. Papazoglou3, Vani R. Pergadia1, Michael C. Fishbein2,

David Wolfson2 and Warren S. Grundfest1

'Laser Research and Technology Development Department,2Department of Pathology and Laboratory Medicine,

Cedars-Sinai Medical Center, Los Angeles, California 90048Telephone: (310) 855-5854, FAX: (310) 655—1778

3Foundation for Research and Technology-Heilas (FO .R . T . H.)Institute of Electronic Structure & Laser Laser and

Applications Division, Heraklion, Crete, GreeceTelephone: (30—81) 210—035 FAX: (30—81) 239—735

ABSTRACT

The goal of this study was to detect the presence ofbenzoporphyrin derivative—monoacid (BPD-MA) in tissues of a tumorbearing animal model. Eighty one Lobund-'Wistar rats, inoculatedwith Pollard rat adenocarcinoma cells, were used. This animalmodel exhibits unique predictable, unilateral, metastatic spread.The animals were injected intravenously with 0.75mg/kg of BPD-MA.A Helium-Cadmium (HeCd) laser (442nm, 17mW) was used as anexcitation source and coupled to a 400pm core diameter fiber.Following laparotomy, exploration of the abdominal and inguinalarea was performed with laser induced fluorescence. Fluorescencespectra cf the primary tumor, bilateral lymph nodes, and variousorgans were recorded. Fluorescence measurements were conductedfour hours post injection. The spectra obtained werecharacterized by a broadband autofluorescence (54Onm) and acharacteristic peak of BPD-MA (690nm) . The ratio offluorescence intensities (690nm to 540nm) was used as an index ofthe presence of the drug. The data reveal that BPD-MAconcentration was higher in lymph nodes with neoplasm than ineither primary tumors or inflammatory, or normal lymph nodes.Overall the BPD-MA concentration was higher in lymph nodes thanin the skin, kidney, large bowel, muscle or spleen. Skinexhibited the lowest fluorescence intensity ratio, indicative ofa lower drug concentration in this tissue. On the other hand,liver, small bowel and stomach showed much higher fluorescenceintensity ratios than all other tissues. These results areconsistent with results obtained using radiolabelledphotosensitizers for biodistribution analysis. In summary, ourresults suggest that laser induced fluorescence spectroscopy mayprovide a alternative way of assessing the biodistribution ofBPD-MA or other photosensitizers.

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I • INTRODUCTION

Small foci of nietastases from printary neoplasras are often hard todetect. Current diagnostic niethods for nialignancy detectionoften fail to detect inetastases because they are microscopic insize and hidden within tissues or organs.

Porphyrins have been known to selectively accumulate inneoplastic tissue [1). These chromophotes also emit acharacteristic dual-peaked red fluorescence after being exposedto light of the appropriate wavelength. Lipson in 1961, used twoproperties of porphyrins to develop a priTnary tunior detectionsysten (2 ] . Dougherty then took advantage of HematoporphyrinDerivative's (HPD) photosensitizing properties to eradicatetumors. This opened the door for further investigation toimprove HPD's function as a potential method for cancer treatment[3].

Termed, "Hentatoporphyrin Derivative Photodynaniic Therapy," HPD'smechanism in cancer therapy is based on its affinity formalignant tumors relative to other tissues. When injectedintravenously, HPD localizes at higher levels in malignant tumortissues than in normal tissues [4 ,5 ] . HPD is then activated bylight to catalyze the production of singlet oxygen oxidizedunsaturated carbon-carbon bonds in amino acids and fatty acids.The ensuing loss of structural integrity of cellularmacromolecules results in a cytocidal effect and eventual tumornecrosis [3).

It is obvious that a photosensitizer, in order to be suitable forPhotodynamic Therapy (PDT) , must be non—toxic in therapeuticdoses. Use of HPD or its derivatives in cancer treatment is avery exciting and rapidly evolving development. Several laserbased devices have demonstrated the ability to distinguishmalignant tissue from normal tissue [6 , 7 ) . However spectroscopicanalysis of fluorescence signals provides a more accurate andsensitive method to distinguish tissue types.

In Laser Induced Fluorescence Spectroscopy (LIFS) , laserirradiation generates a fluorescence signal that can be collectedand returned to a spectroscopic analyzer. Tissue detection isnow possible by analysis of the fluorescence spectrum in respectto its intensity at certain wavelengths [8,9]. These studieswould be facilitated provided that there is a method capable ofanalyzing the biodistribution of the drug. Radiolabelledphotosensitizers are widely used for this purpose [10,11,12].This study investigates the feasibility of laser inducedfluorescence spectroscopy to detect the relative biodistributionof BPD-'MA in a tumor bearing rat model with unilateral muetastaticspread.

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II. MATERIALS AND METHODS

A HeCd (442nxn) low power laser (Omnichronie, Inc., Chino, CA) wasused for tissue excitation during abdominal exploration. Thelaser output was reflected at a right angle by a dichroic mirror(HR/442nm -HT>5OOnm, CVI, New Mexico) and coupled into a 400nncore diameter fiberoptic probe. The distal end of the fiber wasused for tissue scanning. The excitation output of the laser atthe distal end of the fiber was 17mW. Fluorescence was collectedby the same fiber and was transmitted through the dichroicbeamsplitter and guided via a fiber bundle to a 0.5 spectrograph(bOg/mm diffraction gratingwith a slit width of 5Ojnn). Anintensified 1024 element linear diode array detector (EG & G1422G, Princeton, NJ) was used. A longpass glass filter (Schott

1 1 PA) was used to block the excitation light.Figure 1 shows the setup for acquisition and analysis of theflourescence signal with an optical multichannel analyzer (OMAIII, EG & G, Princeton, NJ).

Surface spectra of primary tumor (PT) , skin (5K) , muscle (MS),liver (LV) , kidney (KY) , spleen (SP) , stomach (ST) , small bowel(SB) and large bowel (LB) were obtained. Lymph nodes (LN) werealso irradiated as follows: right side - inguinal (RIN), iliac(RILN) , para-aortic (RPAR) , sub-'renalis (RSBR) , supra-renalis(RSPR) , left side iliac (LILN) , and the mesenterium (MC I &11) . Each lymph node was evaluated histologically, and the lymphnodes were classified as either normal (NLN) , inflammatory (ILN)or metastatic with neoplasm in the lymph node (NELN) as shown inFigure 2. Fluorescence intensities of BPD-MA at 690nm and theintensity of autofluorescence at 540nm were obtained from 3 sitesof each organ. The corresponding intensity ratio and I 693nm/ I540nm were used as an index to measure the fluorescence intensityof photosensitizer.

Benzoporphyrin derivative monoacid ring A (Quadra LogicTechnologies Inc., Vancouver, Canada) was administeredintravenously (0.75mg/kg) to 81 male Lobund-Wistar rats, 4 hoursprior to surgical exploration.

All tumors were subcutaneously implanted by inoculating 10 5viable cells of Pollard rat prostatic adenocarcinoma (PA-Ill)into the right flank of each animal (Lobund Laboratory, NotreDame University , ID) [ 13 ] . This model was selected because thetumor is known to metastasize uniformly and spontaneously fromextravascular sites only through ipsilateral lymphatic channels.Because of this neoplasm's unique, predictable, spread, thecontralateral side of the animal could be used as a control. Inaddition, cancer detection is facilitated in this model becauserats with PA-Ill cells survive beyond forty days followinginoculation without evidence of physical impairment.

The animals were anesthetized intraperitoneally with Ketamine40mg/kg and Xylazine 5.0mg/kg. LIFS exploration of the abdominal

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and inguinal area were performed following midline laparotoniy.The fiber was in contact with the target tissue.

The lyxaph nodes were evaluated through: a) visual inspection;b) in situ volume determination with a digital caliper (Ultra-Call Mark III, Fred K. Fowler Co., Inc., Newton, MA) and,c) evaluation of the fluorescence/intensity ratio. Theintraoperative lymph node evaluation is shown in Table I.

Sensitivity and specificity of the LIFS biodistributionmeasurements were computed using a Logistic Regression Analysis,BMDP Statistical Software [14). Briefly, sensitivity was definedas the percentage of animals with disease that tested positive,or A/ (A+C). Specificity was defined as the percentage of animalswithout disease that tested negative, or D/ (B+D). Inflammatoryand normal tissues (non-metastatic) were scored as negative.Metastatic tissues were scored as positive. (Figure 2)

DISEASE

+ -

TEST + A B

TEST - C D

B is a false positive. C is a false negative.

III. RESULTS

Figure 3 shows the spectra for BPD-MA from photosensitizertreated animals and the presence of photosensitizer in terms ofintensity ratios (I 693nm/ I 540nm) of each organ.

Table II summarizes the intensity ratio for lymph nodescategorized by histology. As shown, the metastatic lymph nodesdisplayed a greater intensity ratio than normal or inflamedlymph nodes. Table III summarizes the intensity ratio for lymphnodes categorized by location. On the right site along with theaorta the lymph nodes exhibited a greater intensity ratio. Theresults of the intensity ratio for organs, primary tumor andlymph nodes are summarized in Table IV. The skin exhibited thelowest intensity ratio. Tumor, iriflamatory and normal lymphnodes exhibited a higher ratio than skin, kidney, muscle, largebowel and spleen. The ratios for liver, small bowel and stomach

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were higher than the ratios for other tissues. The intensityratio of lymph nodes with neoplasni was higher than that of allother tissues except the liver. Table V shows the sensitivity /specificity for rnetastatic lymph nodes versus non-metastatic(normal + inflamatory) lymph nodes as evaluated by the methodsstated in Table I. At 80% of the rats with disease that aretest positive, the error of the detection method (visualisation& volume & ratio) is only 1%.

Iv. DISCUSSION AND CONCLUSION

The relative biodistribution of Benzoporphyrin derivativemonoacid ring A was determined in terms of laser inducedfluorescence of the drug. Skin tissues emitted the lowestfluorescence/intensity ratios, indicative of the small drugamount retained by these tissues. The greater fluorescenceintensity ratio exhibited by the lymph nodes on the right sidemay be because of the high incidence of neoplasni there due tothe unique unilateral spread of Pollard rat adenocarcinoma. Theprimary tumor retains higher drug amounts for some tissues,however, not higher for all tissues tested. The data revealthat BPD-:MA concentration was higher in lymph nodes withneoplasm than primary tumors, and inflammatory and normal lymphnodes. Overall, the corresponding ratios of the lymph nodes werehigher than those obtained in skin, kidney, large bowel, muscleand splee:n. The liver, small bowel and stomach intensity ratioswere higher than those for all other tissues. This may be dueto the BPD-MA elimination pathway through the liver, bile andsmall bowel.

These results are consistent with results obtained by Richter etal. [12] using radiolabelled photosensitizers forbiodistribution analysis. The present approach eliminates thehazards associated with radiolabelling. Additionally, it isless timeconsuming and allows for immediate results. Hence,the optical biopsy of neoplasra using laser induced fluorescencedetection and exogenic flourophores (BPD-MA) may be analternative method to radiographic modality. It is a sensitiveand nove:L technique that is able to localize small foci ofmalignant tissue.

V • ACKNOWLEDGEMENTS

This work was funded by the Hellmnan Foundation and the MedallionGroup of Cedars-Sinai Medical Center. The authors would like tothank Dr. M. Pollard from Loburid Laboratory for the uniqueanimal model and QLT Phototherapeutics Inc. for Photofrin®.

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VI. REFERENCES

1. Policarcl A., Etudes sur les aspects offerts par de tunteursexperintentales exaniinees a las luntiere de Woods. C.R.SeancSoc.Biol. , Vol. 91: 142324 (1924).

2 . Lipson R. L . et al . , The use of derivative of hematoporphyrinin tumor . J . Nat . Cancer Inst . , Vol . 26 : 1-11(1961).

3 . Dougherty T . J. , Photodynainic therapy . Status and potential.Oncology, Vol. 3, 7: 67 (1989).

4 S Dougherty T . J. , Photosensitizers: therapy and detection ofmalignant tumors. Photochemistry and Photobiology, Vol.45,6: 879 (1987).

5 . Montan S . , Multicolor imaging and contrast enhancement incancertumor localization using laser induced fluorescencein heinatoporphyrin derivative bearing tissue. OpticsLetters, Vol.10, 2: 56—58 (1985).

6. Profio E., Fluorescence bronchoscopy for localization ofcarcinonia in situ. Med.Phys. , Vol. 10, 1: 35-39 (1983).

7 . Kato H . , et al . , Early detection of lung cancer by nteans ofheniatoporphyrin derivative fluorescence and laserphotoradiation. Clinics in Chest Medicine, Vol.6, 2: 337353 (1985).

8 . Svanberg K . , Fluorescence studies of hentatoporphyrinderivative in nornial and malignant rat tissue. CancerResearch, Vol. 46: 3806—3808 (1986).

9. Van S.G., et al., Intraoperative Metastases Detection byLaser Induced Fluorescence Spectroscopy. Optical Methods ofTumor Treatment and Early Diagnosis Mechanisnts andTechniques. SPIE Proc Vol. 1426: 111—120 (1991).

10 . Bugelski P . J. , Porter C . W . , Dougherty T .J.,Autoradiographic Distribution of DehematoporphyrinDerivative in normal and tumor tissue of the mouse. CancerResearch, Vol. 41: 4606 (1981).

11 Gomer C. J., Dougherty T. J., Determination of [3H) and [14C]Hematoporphyrin Derivative distribution in malignant andnormal tissue. Cancer Research, Vol. 39: 146 (1979).

12. Richter A. M., Ceruti-Sola S., Sternberg E. D., Dolphin D.,Levy J.G., Biodistribution of tritiated Benzoporphyrinderivative (3H-BPD-MA), a new potential photosensitizer, innormal and tumor bearing mice. J. Photochem. and Photobiol.,B. Biology, Vol. 5: 231 (1990).

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13. Pollard M. R., Luckert P. H., Transplantable metastasizingprostate adenocarcinomas in rats. J. Natl. Cancer Inst.,Vol. 54: 643-649 (1975).

14. BMDP Statistical Software Manual, University of CaliforniaPress Berkely (1993).

O.5m Spectrograph

Figure 1. The setup for acquisition and analysis of theflourescence signal with an optical inultichannelanalyzer (OMA III, EG & G, Princeton, NJ).

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Lens #2

OMA

Lens #1

Fiber #1

OMA Console

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Figure 2. A: Normal lymph node. B: Sinus histiocytosis(inflammatory). C: Prominent germinal center(inflammatory). D: Function of lumph node metastasis inthe top center. Note large pleomorphic nuclei withprominent nucleoli. (Hematoxylin eosin, originalmagnification 40X).

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C..,

0()

Wavelength (nm)

Figure 3. After excitation by blue light (442nm), BPD-MA emitsa characteristic red fluorescence peak at approximately690 nm. The fluorescence intensities at 540 nm (auto-flourescence) and 690 nm (BPD-MA) were simultaniouslymonitored in rats.

(Excitation : 442 nm)80

70

60

50

40

30

20

10

0

350 400 450 500 550 600 650 700 750 800

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TABLE I

VISUAL VISUAL & VOLUME VISUAL & RATIO VISUAL & VOLUME & RATIO

TABLE II

INTENSITY RATIO ANALYSIS OF BPD-MA IN LYMPH NODES

RATIO SUMMARY FOR LYMPH NODES BY HISTOLOGY

HISTOLOGY NUMBER OF RATIO MEAN RATIO SDOBSERVATION

INFLAMMATORY LN 614 3.67 4.02METASTATIC UI 206 6.26 4.91NORMAL LN 200 3.06 2.30

TABLE III

INTENSITY, RATIO ANALYSIS OF BPD-MA IN LYMPH NODES

RATIO SUMMARY FOR LYMPH NODES BY LOCATION

LOCATIONS NUMBER OF RATIO MEAN RATIO SDOBSERVATION

Left Iliac Lymph Node (LILN) 230 5.22 4.76)4esenteric I LN (MC I) 229 2.45 1.80Mesenteric II LN (MC II) 220 2.37 1.79

Right Iliac Ui (RILN) 233 5.86 5.33

Right Inguinal LN (RIN) 18 4.72 2.41'Right Para-Aortic Ui (RPAR) 24 7.40 6.00

Right Sub-Renalis UI (RSBR) 45 4.50 2.94

Right SupraRenalis LN (RSPR) 11 4.26 3.19

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TABLE IV

THE FLUORESCENCE INTENSITY RATIO FOR ORGANS

RATIO SUMMARY FOR , PRIMARY TUMOR AND LYMPH NODES

SITE NUMBER OF RATIO MEAN RATIO SDOBSERVATION*

Skin (5K) 214 0.09 0.03Kidney (KY) 226 1.16 0.43Muscle (MS) 228 1.84 1.05Large Bowel (LB) 231 1.87 0.75Spleen (SP) 229 2.25 1.27Normal Lymph Nodes (NLN) 200 2.98 1.96

Inflammatory Lymph Nodes (ILN) 614 3.34 2.78Lymph Nodes (N & I & NE) (LN) 1020 3.79 3.05Primary Tumor (PT) 215 4.14 2.19Stomach (ST) 227 4.28 1.89Small Bowel (SB) 224 4.44 3.78Neoplasm in Lymph Nodes (NELN) 206 5 • 88 3 • 69Liver (LV) 227 13.60 6.17

Number of observations included replicates for each

TABLE V

SENSITIVITY/ SPECIFICITY FOR METASTATIC VS •

rat.

NON-METASTATIC(NORMAL + INFLAMMATORY) LYMPH NODES FOR VARIOUS MODELS

USING LOGISTIC REGRESSION

INTRAOPERATIVE EVALUATION OF LYMPH NODES

SENSITIVITY SENSITIVITY/ SPECIFICITY

VISUAL VISUAL + VOLUME VISUAL + RATIO VISUAL + VOLUME+ RATIO

70% 68.5/99.9 72.5/97.2 70.0/99.977% 96.6 77.5/99.5 77.0/96.6 77.0/99.880% 80.5/90.7 80.5/94.1 80.0/99.090% 89.0/85.5 91.0/75.5 91.0/88.095% 97.0/41.7 95.0/51.6 94.5/77.0

Table V illustrates that the error of detection was only 1% for the 80% ofdiseased animals that tested positive.

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*

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