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Photodynamic therapy of spontaneous animal tumors using the active component of hematoporphyrin derivative (DHE) as photosensitizing drug: Clincal results
101
Cancer Letters, 38 (1987) lOl- 105 EIsevier Scientiic Publishers Ireland Ltd.
PHOTODYNAMIC THERAPY OF SPONTANEOUS ANIMAL TUMORS USING THE ACTIVE COMPONENT OF HEMATOPORPHYRIN DERIVATIVE (DHE) AS PHOTOSENSITIZING DRUG: CLINCAL RESULTS
RENATO CHELI’, FLAMINIO and RINALDO CUBEDDUb
ADDIS*,
CARLO
M. MORTELLARO*,
DIEGO
FONDA*
‘Clinica Chirurgica Veterinaria, Universid degli Studi, Via PO&O 7 and *Centro Ekttronica Qwntistica e Strumentazione Ekttnzmicq C.N.R, Instituto di Fisico, Politecnico, P.za L-eonwdo da Vinci 32,ZQl33 Milan0 (Italy) (Received 18 February 1987) (Revised version received 26 May 1987) (Accepted 1 June 1987)
SUMMARY
Photodynamic therapy was performed on 25 primary spontaneous tumors in dogs and cats. The animals were injected with 2.5 m&g body wt. of tumor localizing fraction of hematoporphyrin derivative and treated 48 h later with laser light at 631 nm. In 5 cases the treatment was performed on the tumor bed after surgical excision of the tumor mass. An evaluation of clinical results is presented and discussed. Complete remission was obtained in 19 cases and partial remission in 6 cases.
INTRODUCI’ION
Photodynamic ,therapy using hematoporphyrin derivative (HpD) is a new experimental method of treating tumors. This therapy has been applied both in humans [1,2] and in veterinary clinics [3,4] with promising results. HpD is known to be a mixture of several porphyrins and its chemical composition is not completely determined. Recently it has been shown that it is possible to separate a tumor localizing fraction (commonly referred as DHE di-hematoporphyrin ether or ester) which is currently thought to be ester- and/or ether-linked porphyrins with two or more hematoporphyrin rings so linked [5]. On the basis of our previous experiences with HpD phototherapy in veterinary oncology [4], we have applied this treatment protocol to 25 primary spontaneous tumors in dogs and cats using DHE at half dose to evaluate its therapeutic efficacy as compared to that of HpD. 01987 Elaevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
03043835187608.50
102 MATERIALS
AND METHODS
The animals to be treated were clinically examined and selected on the basis of tumor localization and size. Biopsy was performed on all neoplasias before and after the treatment for histological evaluation. Radiographs were taken both of thoracic area in order to look for metastatic lesions, and topically when the tumors were near to bony structures. DHE was kindly provided by Photofrin Medical Inc. (Raritan, NJ, U.S.A.) under the trade name of Photofrin II. The animals were injected intravenously with 2.5 mg/kg body wt. and exposed to the laser light after 46 h. When required a second irradiation was performed after 10 days from the injection. The light source was a Rhodamine B dye laser continuously pumped by an Argon laser and tuned at 631 nm wavelength with an output power up to 2 W. The irradiaton was carried out coupling the light to an optical fiber (400 ).un diameter), ususlly applied externally to the tumor. In one case the fiber was inserted in a flexible endoscope to perform intracavity irradiation. In a second case the laser beam was coupled to two optical fibers which were subsequently inserted through two l&gauge needles into the tumor mass located inside the tibial bone. Laser energy fluence was determined from the light intensity measured at the end of the fiber, the irradiation time and the area of the tumor surface. According to the size and shape of each tumor, the light could be applied to two or more different locations, In the case of fiber insertion, the energy fluence was evaluated on the basis of the estimated tumor volume. During the irradiation the animals were usually under general inhalation anesthesia. All subjects were treated with the consent of the owners and were returned to them after each irradiation In a few cases the temperature of the tumor mass was evaluated during the irradiation at the tumor surface by a thermocouple (Exacon mod. C-N3), in order ,to evidentiate possible hyperthermic effects. Although this type of measurement does not provide information about the temperature distribution inside the tumor, it gives indications of the maximum temperature values and avoids the required perturbations and bleeding of the tumor mass caused by the insertion of a thermocouple. Therapeutic effects were determined on the basis of clinical evaluation, supported by histological and radiological features and based on either the reduction or the disappearance of both tumor masses and possible recurrences. Each case was provided with a complete photographic documentation. RESULTS
Photodynamic therapy was performed on 20 primary tumors of 14 dogs and 6 cats and on 5 tumor beds of as many dogs after surgical excision of the tumor mass, as outlined respectively in Tables 1 and 2. One DHE
103 TABLE 1 PDT AFTER i.v. INJECTION
OF 2.5 mg/kg DHE [2]
CR = Complete response; SR = Significant sponse (60 - 40% reduction). Histologicai type
response
Location/size
(over 60% reduction);
Energy fluence
(Zxnun) Nose
(13) Squamous cell carcinoma (dog) (14) Sebaceous glands adenoma (dog) (15) Malignant giant cell tumor (dog) (16) Malignant melanoma (dog)
(1.2 cm? Ear (5 cm’) Tibia1 bone (10 cm? Hard palate (3 cm?
(17) Fibroepithelioma (dog) (18) Squamous cell carcinoma (dog) (191Eosinophiic granuloma (cat) (20) Adenocarcinoma (dog)
Oral cavity (1 cm? Gastric mucosa (6 cm? Lips (0.6 cm? Oral cavity (3 cm?
PR = Partial Re-
Response (foiIow-up) (&)
580 x 10’
348
CR (12 months)
160 x 30’
288
CR (9 months)
130 x 25’
195
PR
800 500 400 750
11’ 20’ 40’ 15’
528 600 960 675
SR
CR (8 months)
400 x 10’
240
PR
x x x x
1000 x
8’
480
CR (6 months)
330 x
5’
100
CR (6 months)
TABLE 2 PDT AFTER EXERESIS
i.v. INJECTION
CR = Complete response; Histological type
(1) Adenocarcinoma (dog) (2) Malignant Schwannoma (dog) (31Fibrosarcoma (dog) (4) Sweat glands carcinoma (cat) (5) Sebaceous glands adenocarcinoma (dog)
OF 2.5 mg/kg DHE TUMOR TREATED
PR = Partial response Location/size
Lip (1.5 cm21 tongue Oral cavity (2 cm? Ear (0.8 cm2) Gum (12 cm2)
(60-4096
AFTER
SURGICAL
reduction).
Energy fluence
Response
g
(fo’owup)
x min) (-YY)
100 x 11’
66
CR (9 months)
400 x
4’
96
CR
400 x
5’
120
PR
600 x
3’
108
CR (14 months)
85 x 20’
102
CR (6 months)
104
administration was sufficient in all treatments except for case nos. 1 and 6 (3 administrations on day 1, 40 and 60) and case nos. 3 and 16 (2 administrations on day 1,60). No sideeffects were observed in the general conditions of the subjects after DHE injection. In respect to location, size and histological type, the response of the neoplasias after DHE administration and laser irradiation resulted similar to that induced by photochemotherapy with HpD. In the case where the temperature of the tumors was measured, an appreciable increase was observed up to 43OC. This result seems to indicate that a hyperthermic effect is usually present together with the photodynamic one at the dose rates used in our protocol. Hyperthermia seems to have a synergic but not predominant effect. In fact, no therapeutic response was ever achieved by light treatment alone. This is also confirmed by the cases reported in Ref. 6 where a very limited tumor regression was obtained independently on the light dose when the HpD was administered at the dose of 2.5 m&kg body wt. (i.e. half of the optimized dose). It is worth noting however, that according to our previous experiences, a complete eradication of the tumor was observed only when the light was administered at high intensity in a short time. The therapeutic responses described in Table 1 indicate that a complete eradication of the tumor was obtained in 15 cases, while in 5 only significant or partial reduction of the neoplasia was achieved. In case nos. 1, 2 and 3 however we used a different Photofrin II lot respect to all the others. The large number of treatments needed in case no. 1 and the limited therapeutic response of the other two cases could be possibly due to a damaging of the drug during the shipment, since it was received unfrozen. Also case no. 5 required several treatments but in this subject the particular location of the neoplasia, i.e. on the glands and inside the urethra, suggested this therapeutic procedure to prevent a possible stenotic complication. The partial response of case no. 15 was in agreement with the limited therapeutic effectiveness of PDT on bone tumors, as reported in the literature [3,4] and verified also by us using HpD. The evaluation of the size of the squamous cell carcinoma in the gastric mucosa (case no. 18) was done mainly by endoscopic inspection and it was not possible to determine the real tumor dimensions. The post-mortem examination performed 1 month after the treatment, showed that the tumor was largely infiltrated into the gastric wall in an area much larger than the irradiated one, so that the partial response to PDT obtained was justified by an insufficient irradiation. A limited light penetration and consequently an insufficient light dose can explain the SR of case no. 16. In fact, although very high energy fluence was shed on the tumor surface, the black pigmentation of this neoplasia limited the therapeutic effectiveness of the treatment. It is worth noting that the partial response obtained in the last 3 cases discussed seems to be related more to a lack of the requirements for successful PDT than to a failure of DHE itself.
105
Table 2 shows the therapeutic results obtained in 5 cases of surgically excised tumors, where the treatment was performed on the supposed residual neoplastic area. According to the protocol used with HpD [4], the energy fluence is lower than that used in the other reported cases due to the reduced depth of the lesions. In 4 cases no recurrence was observed while in case no. 3 the therapy was unable to prevent the tumor regrowth and the recurrence appeared 1 month after the irradiation. It is worth noting however that all the treated tumor types present early recurrence after surgery alone. The clinical results obtained with PDT using DHE at the dose of 2.5 mgl kg body wt. seem to indicate that this drug is almost as efficient as HpD at the dose of 5 mglkg body wt. This confirms that DHE is the component most active in the HpD mixture. Photofrin II presents therefore the advantage of a reduced posology, but seems to be, in our experience, more delicate to manipulate being more sensitive to the temperature variations. For all the other clinical aspects as the general conditions of the subjects, the side-effects and the evolution of the tumor mass after irradiation, it behaves like HpD [4]. However, the occurrence of few cases with limited clinical response suggests a slight increase of the drug dose up to 3 or 3.5 mgkg. REFERENCES Dougherty, T.J., Lawrence, G., Kaufman, J.E., Boyle, D.G., Weishaupt, K.R. and Goldfarb, A. (1979) Photoradiation in the treatment of recurrent breast carcinoma. J. Natl. Cancer Inst., 62, 231-237 Dahhnan, A., Wile, A.G., Burns, R.G., Mason, G.R., Johnson, F.M. and Bems, M.W. (1983) Laser photoradiation therapy of cancer. Cancer Res., 43, 436-434 Dougherty, T.J., Grindey, G.B., Fiel, R., Weishaupt, K.R. and Bovle, D.G. (1975) Photoradiation therapy: cure of animals tumors with hematoporphyrin and light. J. Natl. Cancer Inst., 65, 116-122. CheIi, R., Addis, F., MorteIlaro, C.M., Fonda, D., Andreoni, A. and Cubeddu, R. (1984) Hematoporphyrin derivative photochemotherapy of spontaneous animal tumors: clinical results with optimised drug dose. Cancer Letters, 23,61-66. Kessel, D. and Cheng, M. (1985) On the preparation and properties of dihematoporphyrin ether, the tumor locahsing component of HpD. Photochem. Photobiol., 41, 277-282. Cheli, R., Addis, F., Moxtdlaro, CM, Fonda, D., Andreoni, A. and Cubeddu, R. (1984) HpD phototherapy on spontaneous tumors in dog and cat. In: Phorphyrins in Tumor Phototherapy, pp. 261-258. Editors: A. Andreoni and R. Cubeddu, Plenum Press, New York, London.
Cancer Letters, 38 (1987) lOl- 105 EIsevier Scientiic Publishers Ireland Ltd.
PHOTODYNAMIC THERAPY OF SPONTANEOUS ANIMAL TUMORS USING THE ACTIVE COMPONENT OF HEMATOPORPHYRIN DERIVATIVE (DHE) AS PHOTOSENSITIZING DRUG: CLINCAL RESULTS
RENATO CHELI’, FLAMINIO and RINALDO CUBEDDUb
ADDIS*,
CARLO
M. MORTELLARO*,
DIEGO
FONDA*
‘Clinica Chirurgica Veterinaria, Universid degli Studi, Via PO&O 7 and *Centro Ekttronica Qwntistica e Strumentazione Ekttnzmicq C.N.R, Instituto di Fisico, Politecnico, P.za L-eonwdo da Vinci 32,ZQl33 Milan0 (Italy) (Received 18 February 1987) (Revised version received 26 May 1987) (Accepted 1 June 1987)
SUMMARY
Photodynamic therapy was performed on 25 primary spontaneous tumors in dogs and cats. The animals were injected with 2.5 m&g body wt. of tumor localizing fraction of hematoporphyrin derivative and treated 48 h later with laser light at 631 nm. In 5 cases the treatment was performed on the tumor bed after surgical excision of the tumor mass. An evaluation of clinical results is presented and discussed. Complete remission was obtained in 19 cases and partial remission in 6 cases.
INTRODUCI’ION
Photodynamic ,therapy using hematoporphyrin derivative (HpD) is a new experimental method of treating tumors. This therapy has been applied both in humans [1,2] and in veterinary clinics [3,4] with promising results. HpD is known to be a mixture of several porphyrins and its chemical composition is not completely determined. Recently it has been shown that it is possible to separate a tumor localizing fraction (commonly referred as DHE di-hematoporphyrin ether or ester) which is currently thought to be ester- and/or ether-linked porphyrins with two or more hematoporphyrin rings so linked [5]. On the basis of our previous experiences with HpD phototherapy in veterinary oncology [4], we have applied this treatment protocol to 25 primary spontaneous tumors in dogs and cats using DHE at half dose to evaluate its therapeutic efficacy as compared to that of HpD. 01987 Elaevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
03043835187608.50
102 MATERIALS
AND METHODS
The animals to be treated were clinically examined and selected on the basis of tumor localization and size. Biopsy was performed on all neoplasias before and after the treatment for histological evaluation. Radiographs were taken both of thoracic area in order to look for metastatic lesions, and topically when the tumors were near to bony structures. DHE was kindly provided by Photofrin Medical Inc. (Raritan, NJ, U.S.A.) under the trade name of Photofrin II. The animals were injected intravenously with 2.5 mg/kg body wt. and exposed to the laser light after 46 h. When required a second irradiation was performed after 10 days from the injection. The light source was a Rhodamine B dye laser continuously pumped by an Argon laser and tuned at 631 nm wavelength with an output power up to 2 W. The irradiaton was carried out coupling the light to an optical fiber (400 ).un diameter), ususlly applied externally to the tumor. In one case the fiber was inserted in a flexible endoscope to perform intracavity irradiation. In a second case the laser beam was coupled to two optical fibers which were subsequently inserted through two l&gauge needles into the tumor mass located inside the tibial bone. Laser energy fluence was determined from the light intensity measured at the end of the fiber, the irradiation time and the area of the tumor surface. According to the size and shape of each tumor, the light could be applied to two or more different locations, In the case of fiber insertion, the energy fluence was evaluated on the basis of the estimated tumor volume. During the irradiation the animals were usually under general inhalation anesthesia. All subjects were treated with the consent of the owners and were returned to them after each irradiation In a few cases the temperature of the tumor mass was evaluated during the irradiation at the tumor surface by a thermocouple (Exacon mod. C-N3), in order ,to evidentiate possible hyperthermic effects. Although this type of measurement does not provide information about the temperature distribution inside the tumor, it gives indications of the maximum temperature values and avoids the required perturbations and bleeding of the tumor mass caused by the insertion of a thermocouple. Therapeutic effects were determined on the basis of clinical evaluation, supported by histological and radiological features and based on either the reduction or the disappearance of both tumor masses and possible recurrences. Each case was provided with a complete photographic documentation. RESULTS
Photodynamic therapy was performed on 20 primary tumors of 14 dogs and 6 cats and on 5 tumor beds of as many dogs after surgical excision of the tumor mass, as outlined respectively in Tables 1 and 2. One DHE
103 TABLE 1 PDT AFTER i.v. INJECTION
OF 2.5 mg/kg DHE [2]
CR = Complete response; SR = Significant sponse (60 - 40% reduction). Histologicai type
response
Location/size
(over 60% reduction);
Energy fluence
(Zxnun) Nose
(13) Squamous cell carcinoma (dog) (14) Sebaceous glands adenoma (dog) (15) Malignant giant cell tumor (dog) (16) Malignant melanoma (dog)
(1.2 cm? Ear (5 cm’) Tibia1 bone (10 cm? Hard palate (3 cm?
(17) Fibroepithelioma (dog) (18) Squamous cell carcinoma (dog) (191Eosinophiic granuloma (cat) (20) Adenocarcinoma (dog)
Oral cavity (1 cm? Gastric mucosa (6 cm? Lips (0.6 cm? Oral cavity (3 cm?
PR = Partial Re-
Response (foiIow-up) (&)
580 x 10’
348
CR (12 months)
160 x 30’
288
CR (9 months)
130 x 25’
195
PR
800 500 400 750
11’ 20’ 40’ 15’
528 600 960 675
SR
CR (8 months)
400 x 10’
240
PR
x x x x
1000 x
8’
480
CR (6 months)
330 x
5’
100
CR (6 months)
TABLE 2 PDT AFTER EXERESIS
i.v. INJECTION
CR = Complete response; Histological type
(1) Adenocarcinoma (dog) (2) Malignant Schwannoma (dog) (31Fibrosarcoma (dog) (4) Sweat glands carcinoma (cat) (5) Sebaceous glands adenocarcinoma (dog)
OF 2.5 mg/kg DHE TUMOR TREATED
PR = Partial response Location/size
Lip (1.5 cm21 tongue Oral cavity (2 cm? Ear (0.8 cm2) Gum (12 cm2)
(60-4096
AFTER
SURGICAL
reduction).
Energy fluence
Response
g
(fo’owup)
x min) (-YY)
100 x 11’
66
CR (9 months)
400 x
4’
96
CR
400 x
5’
120
PR
600 x
3’
108
CR (14 months)
85 x 20’
102
CR (6 months)
104
administration was sufficient in all treatments except for case nos. 1 and 6 (3 administrations on day 1, 40 and 60) and case nos. 3 and 16 (2 administrations on day 1,60). No sideeffects were observed in the general conditions of the subjects after DHE injection. In respect to location, size and histological type, the response of the neoplasias after DHE administration and laser irradiation resulted similar to that induced by photochemotherapy with HpD. In the case where the temperature of the tumors was measured, an appreciable increase was observed up to 43OC. This result seems to indicate that a hyperthermic effect is usually present together with the photodynamic one at the dose rates used in our protocol. Hyperthermia seems to have a synergic but not predominant effect. In fact, no therapeutic response was ever achieved by light treatment alone. This is also confirmed by the cases reported in Ref. 6 where a very limited tumor regression was obtained independently on the light dose when the HpD was administered at the dose of 2.5 m&kg body wt. (i.e. half of the optimized dose). It is worth noting however, that according to our previous experiences, a complete eradication of the tumor was observed only when the light was administered at high intensity in a short time. The therapeutic responses described in Table 1 indicate that a complete eradication of the tumor was obtained in 15 cases, while in 5 only significant or partial reduction of the neoplasia was achieved. In case nos. 1, 2 and 3 however we used a different Photofrin II lot respect to all the others. The large number of treatments needed in case no. 1 and the limited therapeutic response of the other two cases could be possibly due to a damaging of the drug during the shipment, since it was received unfrozen. Also case no. 5 required several treatments but in this subject the particular location of the neoplasia, i.e. on the glands and inside the urethra, suggested this therapeutic procedure to prevent a possible stenotic complication. The partial response of case no. 15 was in agreement with the limited therapeutic effectiveness of PDT on bone tumors, as reported in the literature [3,4] and verified also by us using HpD. The evaluation of the size of the squamous cell carcinoma in the gastric mucosa (case no. 18) was done mainly by endoscopic inspection and it was not possible to determine the real tumor dimensions. The post-mortem examination performed 1 month after the treatment, showed that the tumor was largely infiltrated into the gastric wall in an area much larger than the irradiated one, so that the partial response to PDT obtained was justified by an insufficient irradiation. A limited light penetration and consequently an insufficient light dose can explain the SR of case no. 16. In fact, although very high energy fluence was shed on the tumor surface, the black pigmentation of this neoplasia limited the therapeutic effectiveness of the treatment. It is worth noting that the partial response obtained in the last 3 cases discussed seems to be related more to a lack of the requirements for successful PDT than to a failure of DHE itself.
105
Table 2 shows the therapeutic results obtained in 5 cases of surgically excised tumors, where the treatment was performed on the supposed residual neoplastic area. According to the protocol used with HpD [4], the energy fluence is lower than that used in the other reported cases due to the reduced depth of the lesions. In 4 cases no recurrence was observed while in case no. 3 the therapy was unable to prevent the tumor regrowth and the recurrence appeared 1 month after the irradiation. It is worth noting however that all the treated tumor types present early recurrence after surgery alone. The clinical results obtained with PDT using DHE at the dose of 2.5 mgl kg body wt. seem to indicate that this drug is almost as efficient as HpD at the dose of 5 mglkg body wt. This confirms that DHE is the component most active in the HpD mixture. Photofrin II presents therefore the advantage of a reduced posology, but seems to be, in our experience, more delicate to manipulate being more sensitive to the temperature variations. For all the other clinical aspects as the general conditions of the subjects, the side-effects and the evolution of the tumor mass after irradiation, it behaves like HpD [4]. However, the occurrence of few cases with limited clinical response suggests a slight increase of the drug dose up to 3 or 3.5 mgkg. REFERENCES Dougherty, T.J., Lawrence, G., Kaufman, J.E., Boyle, D.G., Weishaupt, K.R. and Goldfarb, A. (1979) Photoradiation in the treatment of recurrent breast carcinoma. J. Natl. Cancer Inst., 62, 231-237 Dahhnan, A., Wile, A.G., Burns, R.G., Mason, G.R., Johnson, F.M. and Bems, M.W. (1983) Laser photoradiation therapy of cancer. Cancer Res., 43, 436-434 Dougherty, T.J., Grindey, G.B., Fiel, R., Weishaupt, K.R. and Bovle, D.G. (1975) Photoradiation therapy: cure of animals tumors with hematoporphyrin and light. J. Natl. Cancer Inst., 65, 116-122. CheIi, R., Addis, F., MorteIlaro, C.M., Fonda, D., Andreoni, A. and Cubeddu, R. (1984) Hematoporphyrin derivative photochemotherapy of spontaneous animal tumors: clinical results with optimised drug dose. Cancer Letters, 23,61-66. Kessel, D. and Cheng, M. (1985) On the preparation and properties of dihematoporphyrin ether, the tumor locahsing component of HpD. Photochem. Photobiol., 41, 277-282. Cheli, R., Addis, F., Moxtdlaro, CM, Fonda, D., Andreoni, A. and Cubeddu, R. (1984) HpD phototherapy on spontaneous tumors in dog and cat. In: Phorphyrins in Tumor Phototherapy, pp. 261-258. Editors: A. Andreoni and R. Cubeddu, Plenum Press, New York, London.