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Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma
Cancer Letters 141 (1999) 29±38
Topical and intratumoral photodynamic therapy with 5aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma Adriana Casas a, HaydeÂe Fukuda a, Roberto Meiss b, Alcira M. del C. Batlle a,* a
Centro de Investigaciones sobre Por®rinas y Por®rias (CIPYP) FCEyN (University of Buenos Aires) and CONICET, Ciudad Universitaria, PabelloÂn II, 2do piso, (1428)Capital Federal, Argentina b Departamento de PatologõÂa, I.E.O., Academia Nacional de Medicina, Las Heras 3092, (1425)Capital Federal, Argentina Received 4 July 1998; received in revised form 1 March 1999; accepted 1 March 1999
Abstract One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a signi®cant synthesis of porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradiation was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2±4 h after intratumoral administration of 30 mg ALA/cm 3. The pattern of tumour response evaluated as the delay of tumour growth was similar following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour growth delay. These ®ndings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: d-Aminolevulinic acid; Photodynamic therapy (PDT); Subcutaneous transplantable tumour; Topical application; Intratumour injection
1. Introduction Photodynamic therapy (PDT) is a cancer treatment based on the accumulation of a porphyrin-related photosensitizer in tumour cells, and their subsequent destruction on exposure to visible light. Singlet * Corresponding author at: Viamonte 1881 10A, 1056 Buenos Aires, Argentina. Fax: 1 54-1-8117447. E-mail address: [email protected] (A.M. del C. Batlle)
oxygen species are produced, causing damage to membranes and organelles, leading to cell death and tumour ablation [10]. One of the most promising substances for PDT is 5-aminolevulinic acid (ALA), a haem precursor which induces endogenous accumulation of porphyrins, mainly protoporphyrin IX (PpIX), in malignant tissues. Biological membranes are considered as critical targets for cell killing by PDT; damage to the vascular endothelium resulting in tissue/tumour ischaemia is
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00079-8
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an additional mechanism leading to tumour necrosis [24]. PDT has also been shown to induce apoptosis in vivo [28] and in vitro [9]; however, the apoptotic response to PDT seems to depend on both the photosensitizer and the cell line [18]. Due to its water solubility ALA can be given either by intravenous injection [12], oral administration [8], topically [13], and there are a few reports on its intratumoral (i.t.) application [3,6,7]. In topical PDT limited penetration of ALA and inadequate distribution of induced porphyrins might be a possible drawback for this modality. Recently, Peng et al. [20] demonstrated that the systemic administration of ALA led to more selective and homogeneous accumulation of ALA-induced porphyrins in the tumour. Clinical trials have already been performed using topically [2,26] or systemically [15] applied ALA, with good results. Topical ALA-PDT has also been shown to be effective in the treatment of premalignant conditions such as keratoses and in psoriasis [1,25]. Experimental models have shown that i.t. injection was more effective than or at least equally effective to the other two routes of ALA administration [3,6,7]. We have shown that the i.p. and i.t. injection of ALA could produce a signi®cant synthesis of porphyrins in the mammary transplantable adenocarcinoma M2 [6]. In the present study we investigated the effects of laser irradiation on the growth of a subcutaneous implanted tumour after topical or i.t. application of ALA. The amount of ALA leading to the highest accumulation of porphyrins in the tumour and the time point of the highest tumour/skin ratio of porphyrin levels, to attain optimum ef®ciency of PDT were determined. Tumour response was evaluated following tumour growth pro®les, delay growth indexes and light microscopy examination of PDTtreated areas.
2. Materials and methods 2.1. Chemicals ALA was purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals were of analytical grade.
2.2. Animals Male BALB/c mice, 12 weeks old, weighing 20±25 g were used. They were provided with food (Purina 3, Molinos RõÂo de la Plata) and water ad libitum. A mammary adenocarcinoma [23] (M2, Hospital Roffo, Buenos Aires) was propagated by serial transplantation into male BALB/c mice. Non-necrotic tumour material for inoculation was obtained by sterile dissection of large ¯ank tumours. A 1-mm 3 sample of macroscopically viable tumour, which is equal to approximately 2 £ 105 cells, was injected s.c. under the dorsal ¯ank of each mouse. The take rate of the tumours following transplantation was nearly 100%. Under these controlled conditions the implant size did not vary by more than 10%. Animals were treated in accordance with guidelines established by the Animal Care and Use Committee of the Argentine Association of Specialists in Laboratory Animals (AADEALC), in full accord with the UK Guidelines for the Welfare of animals in Experimental Neoplasia [27]. 2.3. Preparation and administration of ALA The hydrochloric salt of ALA was dissolved in sterile water (pH 6.5) at a concentration of 100 mg/ ml and used immediately for i.t. administration. For topical application ALA was prepared daily as a 10, 20, 30 or 40% formulation in a cream (Genargen Argentinaw). 2.4. Pharmacokinetics of ALA-induced porphyrins in tissues Tumours with a volume of approximately 400±450 mm 3 were used for pharmacokinetic studies. For timeresponse kinetics mice were given either 20 mg of ALA per cm 3 of tumour tissue intratumorally, or 50 mg of a 20% ALA cream by topical application. At different times between 1 and 24 h after ALA administration (®ve mice for each point) animals were sacri®ced. For dose-response kinetics, 50 mg of a cream containing 10, 20, 30 or 40% ALA were applied topically on tumours of the same uniform size rubbing over the whole surface of the tumour during a period of 60 s. For the i.t route, 10, 20, 30, 40 and 80 mg ALA/cm 3 tumour were injected (®ve mice for each
A. Casas et al. / Cancer Letters 141 (1999) 29±38
point). Animals were killed 3 h after topical administration and 4 h after i.t. injection. The skin overlying the tumour (SOT) was carefully removed from the tumour itself. Samples of normal skin (NS) previously shaved were excised from the contralateral ¯ank of the tumour-bearing mice. 2.5. Tissue extraction of porphyrins The tissue samples were homogenized in a 4:1 solution of ethyl acetate/glacial acetic acid mixture. The homogenates were centrifuged for 30 min at 3000 £ g, and the supernatants shaken with an equal volume of 5% HCl [5]. Extraction with HCl was repeated until negative ¯uorescence in the organic layer. Porphyrins were spectrophotometrically determined in the aqueous fraction measuring absorbances at 380, 430 nm and the Soret band [22]. 2.6. Laser and irradiations A rhodamine dye laser (Model DL30, Oxford Lasers) pumped by a copper vapour laser (CU15A, Oxford Lasers) tuned to 630 nm was used. The light was focused into a 400-mm diameter optical ®bre and the cut end of the ®bre was positioned to provide a 3.5-cm diameter light spot, producing a treatment area of uniform intensity. The output power from the ®bre was measured with a power meter (Model LM100XL, Coherent, Auburn, CA) before each application. Total doses of 97 J/cm 2 were delivered using a ¯uence rate of 80 mW/cm 2 over 20 min. Light doses up to 100 J/cm 2 do not cause additional hyperthermic effects, which may in¯uence the ef®cacy of PDT; therefore, higher light doses were not used (Casas et al., unpublished results). 2.7. Treatment protocol for ALA-based PDT Eight days after implantation, when tumours reached the appropriate size of 70±100 mm 3, the SOT was shaved and a 20% ALA cream was topically applied. Ten days after implantation, when tumours reached the size of 180±200 mm 3, they were intratumorally injected with 30 mg ALA/cm 3 tumour. Animals were anaesthetized by i.p. injection of 70
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mg/kg ketamine hydrochloride (Parke Davis, Argentina, S.A.) and 6 mg/kg xylazine (RompuÂnw, Bayer Argentina S.A.) and lesions were super®cially irradiated. During treatment normal tissue surrounding the tumour was shielded with a piece of black plastic, leaving exposed a peritumoral margin of 3 mm. Body temperature was monitored with a rectal probe (Tinitalk II, Gemini Dataloger). Normal shaved skin was exposed to both ALA and light for histological examination. 2.8. Assessment of tumour response The longer (l) and shorter (w) perpendicular axes and height (h) of each tumour were determined with callipers prior to or after PDT daily up to day 25 after implantation to evaluate the delay of tumour growth. Tumour volume was calculated using the formula l £ w £ h £ 0:5, where 0.5 is a correction factor empirically determined [21]. To assess the response to treatment two indexes were de®ned: D24 (tumour volume 24 h after PDT/tumour volume before PDT) and D48 (tumour volume 48 h after PDT/tumour volume before PDT). 2.9. Histological studies Prior to and 4, 9, 24, 48 and 72 h, up to 6 days after PDT, samples of tumour with its surrounding skin and samples of the NS were excised. They were extended, sliced, ®xed in 10% buffered formalin, embedded in paraf®n, sectioned, stained with haematoxylin and eosin and examined by light microscopy. The presence of tumour tissue, necrosis and cells with morphological features of apoptosis was evaluated. Epidermal and dermal damage, vascularity changes and presence of lymphocytic in®ltration were also investigated. 2.10. Statistical analysis The unpaired t-test was used to establish the significance of differences between groups. Differences were considered statistically signi®cant when P , 0:05.
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Fig. 1. Concentration of ALA-induced porphyrins in tumour, normal skin and skin overlying the tumour as a function of time after topical application of ALA cream. At different times after tumour topical application of 50 mg of 20% ALA cream, tissues were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
3. Results 3.1. Kinetics of porphyrin biosynthesis in tumour, normal skin and skin overlying the tumour after topical or intratumoral administration of ALA Fig. 1 shows the kinetics of porphyrin accumulation after topical application of ALA. The highest amount of porphyrins (0.6 mg/g tissue) was found in the tumour tissue 2±3 h after administration of ALA, then concentration decreased rapidly. The ALA-
Fig. 2. Concentration of ALA-induced porphyrins in tumour, normal skin and skin overlying the tumour as a function of time after intratumoral administration of ALA. At different times after intratumoral injection of 20 mg/cm 3 ALA, tissues were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
induced porphyrin accumulation in NS (0.4 mg/g tissue) was highest 90 min after ALA application. A delay in peak levels of porphyrins was observed in the SOT, reaching a maximum concentration of 1.1 mg/g tissue, 8 h after ALA application. When ALA was given intratumorally (Fig. 2), the highest amount of tumoral porphyrins (2 mg/g tissue) was found between 2 and 4 h after drug administration. Normal and tumour-overlying skin showed a
A. Casas et al. / Cancer Letters 141 (1999) 29±38
Fig. 3. Porphyrin accumulation in tissues after topical application of various concentrations of ALA. Three hours after application of 50 mg of a cream containing 10, 20, 30 or 40% ALA, tumour (X), normal skin (P) and skin overlying the tumour (O) were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
similar pattern of porphyrin synthesis. Maxima were observed at 2 and 4 h after ALA injection (1.2 and 1 mg/g tissue, respectively). After reaching the peak, the porphyrin concentration declined rapidly near to basal levels in tumour and NS by 24 h, in either route of ALA administration, but not in SOT tissue. Tumour to NS porphyrin concentration ratios were approximately 3 between 2 and 3 h after topical application of ALA and nearly 2 between 4 and 6 h after i.t. administration. Conversely, tumour/SOT porphyrin values were higher for the i.t. route, reaching a maximum of nearly 3 between 4 and 6 h after, compared to 0.86 for the topical application at the same interval.
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Fig. 4. Porphyrin accumulation in tissues after intratumoral administration of various concentrations of ALA. Four hours after injections of 10, 20, 30, 40 and 80 mg ALA/cm 3 tumour tissue, tumour (X), normal skin (P) and skin overlying the tumour (O) were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
Normal skin porphyrins peaked at 30% ALA, and total accumulation was always lower than tumoral porphyrins. Tumour to NS and SOT porphyrin concentration ratios reached their maximum levels after 20% ALA (2.64 and 0.89, respectively). When ALA was i.t. injected (Fig. 4), the maximum accumulation of porphyrins was found with a 30-mg ALA/cm 3 injection. Both NS and SOT exhibited a saturation pattern above 40 mg ALA/cm 3. The tumoral levels of ALA-induced porphyrins were always higher than those of NS and SOT. At 30 mg ALA/cm 3, tumour to NS and SOT ratios were also maximal (2.7 and 2.4, respectively).
3.2. ALA-induced accumulation of porphyrins
3.3. Effectiveness of ALA-based PDT in delaying tumour growth
The amount of porphyrins generated in tumour after topical application reached a plateau with a 20% ALA cream (Fig. 3). SOT showed almost an identical pattern, although the amount of porphyrins was higher.
The effectiveness of ALA-induced porphyrins as photosensitizers for PDT was determined by assessing the extent of tumour growth after one, two and three PDT applications. Two response indexes were de®ned, measuring the
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A. Casas et al. / Cancer Letters 141 (1999) 29±38
Table 1 Tumour response indexes after a single application of ALA-PDT a
Control (n 7) Topical ALA (n 12) i.t. ALA (n 5)
Å ) D24 (X
Å ) D48(X
1.71 ^ 0.84 0.96 ^ 0.39 b 0.75 ^ 0.21 d
2.56 ^ 1.10 0.87 ^ 0.65 c 0.94 ^ 0.44 b
a As a measurement of tumour response to topical and i.t. ALAPDT, two indexes were de®ned: D24 and D48 (see Section 2). D24 and D48 control indexes were calculated as volume ratios between day 10 and days 9 and 8, respectively, after implantation of tumours in non-treated mice. Results are presented as means ^ standard deviations. b **P , 0:01 compared to the corresponding control. c *P , 0:007 compared to the corresponding control. d ***P , 0:03 compared to the corresponding control.
ratios between tumour volumes before and after treatment, since the rapid growth of M2 tumour does not allow the evaluation of survival or of tumour doubling times.
Fig. 5. Time-dependence of tumour growth after topical ALA-PDT. Tumours with an initial volume of 70±100 mm 3 were topically treated with 20% ALA cream and 3 h later they were exposed to 630 nm light at a ¯uence rate of 97 J/cm 2. Animals were treated with one (P), two (O) or three (B) doses of ALA-PDT. Arrows indicate the day of treatment. X, mean of six control curves (neither ALA nor light). Growth curves of tumours with the same response indexes are represented (D24 0:70).
Fig. 6. Time-dependence of tumour growth after intratumoral ALA-PDT. Tumours with an initial volume of 180±200 mm 3 were intratumorally injected with 30 mg ALA/cm 3 tumour and 4 h later they were exposed to 630 nm light at a ¯uence rate of 97 J/ cm 2. Animals were treated with one (P), two (O) or three (B) doses of ALA-PDT. Arrows indicate the day of treatment. X, mean of six control curves (neither ALA nor light). Growth curves of tumours with the same response indexes are represented (D24 0:80).
D24 and D48 indexes for both topical and intratumoral ALA-PDT are shown in Table 1. After a single application both indexes were lower than those of the untreated tumours, indicating that if not a complete reduction, a delay of tumour growth occurs. After two and three PDT applications similar indexes were obtained and no signi®cant differences between either route of ALA administration were evidenced (data not shown). Irradiation on ALA injected intratumorally appeared to induce a greater reduction in tumour volume earlier. An index of 0.75 was observed at 24 h and 0.94 at 48 h, but differences between times were not statistically signi®cant. In order to compare the response to repetitive PDT, tumour growth curves with the same D24 index were used (Figs. 5 and 6). Animals received either one, two or three successive treatments of topical and intratumoral ALA-PDT, respectively. A single application induced a clear tumour growth delay for both topical and intratumoral ALA administration. A second PDT treatment applied 48 h later and a third one, applied 6 days later induced a further delay in tumour growth. It is noteworthy that tumour
A. Casas et al. / Cancer Letters 141 (1999) 29±38
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Fig. 7. Morphological images of apoptotic cells after ALA-based PDT treatment. Histological section of tumour treated 24 h after topical ALAPDT, showing condensation of an eosinophilic cytoplasm and a basophilic nuclei. Cap-like condensed chromatin at one nuclear pole appear in some cells. Similar images were observed for the i.t. route 48 and 72 h after treatment. None of these features were obtained in control tissues.
height was the axis most dramatically reduced after PDT. The D24 and D48 indexes were always similar for the same tumour after the ®rst, second or third PDT dose. The application of ALA or light alone did not cause any measurable effects on tumour growth. In all cases core body temperature throughout laser treatment decreased from 33 to 298C during irradiation, due to anaesthesia. 3.4. Macroscopical analysis and histological studies Normal skin exhibited neither macroscopic nor microscopic changes with PDT treatment. The same results were observed for tumour or skin exposed to either ALA or light alone at all times analyzed. After 24 h of ALA-based PDT, administered by either route, macroscopically necrotic zones, gross tumour volume reduction, ulceration and eschar formation were induced. At 0, 4 and 9 h after treatment no microscopic changes in either tumour tissue or its overlying skin were observed. At 24 h preserved tumour tissue with
scanty foci of necrosis and cells with apoptotic images up to a depth of 4 mm from the epidermis were seen in both i.t. and topically ALA-PDT treated samples (Fig. 7). Necrotic cells with vacuolization of the cytoplasm, pycnotic appearance of the nucleus and loss of cellularity were seen. Epidermal with slight keratosis, dermal oedema and vascular dilation in the SOT were also observed. At 48±72 h there was an expansion of tumour in®ltrating the dermis and epidermis with keratinization and sloughing of the SOT. Haemorrhagic foci and large areas of necrosis and of apoptotic cells in tumour tissue were also seen. From day 5 after PDT, connective ®brovascular tissue surrounding the tumour, rich in neo-formed vessels with wall enlargement, vascular stasis and congestion was developed. No lymphocytic in®ltration was ever found in any microscopic studies performed. Non-shielded peritumoral skin of mice, having been treated three times with topical ALA-PDT, remained free of macroscopically visible tumour for 10 days, and exhibited hair basophilic, amorphous destruction inside the follicle which correlated with visible alopecia.
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A. Casas et al. / Cancer Letters 141 (1999) 29±38
4. Discussion It has already been demonstrated that porphyrins can be synthesized and accumulated in different tissues such as tumour and skin of mice given i.t. injection of ALA [6]. Here, we have shown that topical or intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma induced a signi®cant synthesis of porphyrins and a consequent sensitization to laser light. Although complete remissions were not achieved with the present light dose regimen, periods up to 10 days free of macroscopically visible tumour were sometimes observed both with the i.t. and topical modes of ALA application (data not shown). For both routes of ALA administration a sort of saturation pattern was observed in the porphyrinsynthesizing capacity of the tissues, indicative of the existence of particular regulatory mechanisms in the metabolism of ALA-induced porphyrins in tumourbearing animals [17]. It is noteworthy that this ALA dose-response saturation in tumour is evidenced when the drug is administered by either route. It was achieved with 50 mg of 20% ALA cream, which corresponds to a total amount of 25 mg ALA/cm 3 tumour, and with 30 mg ALA/cm 3 in the case of i.t. application. The total amount of porphyrins in tumour tissue was ®ve times higher when ALA was i.t. injected, and tumour/SOT porphyrin concentration ratios were higher. However, no signi®cant differences in the actual ef®cacy of photosensitization were observed, in agreement with the results of Cairnduff et al. [3] for an intradermally located murine adenocarcinoma. These ®ndings may be explained by either the existence of a saturating amount of ALA-formed porphyrins necessary to produce the photodynamic damage [14] or alternatively by an uneven distribution of porphyrins in different tumour layers which eventually result in a similar or equivalent amount of porphyrins directly exposed to light in either route of ALA administration. According to tumour/skin porphyrin concentration ratios and total amounts of porphyrins accumulated in tumour tissue, irradiation would be optimal between 2 and 3 h after topical application of 20% ALA cream and 2±4 h after intratumoral administration of 30 mg ALA/cm 3.
Maximal accumulation of porphyrins was observed 3 h after topical ALA application, in agreement with Henderson et al. [11] and Peng et al. [19], who found that ¯uorescence of ALA-induced porphyrins in murine adenocarcinomas peaked between 3 and 5 h post-topical application. Induced anoxia by vascular damage does not seem to lead to an impairment of repetitive-PDT ef®cacy, since the same tumour response is maintained along successive treatments. It appears that continuous applications of PDT would photosensitize surviving cells, which could recover their oxic stage by means of the neo-formed vessels provided by the surrounding ®brovascular reparation tissue. Another interesting hypothesis is that proposed by Moan and Sommer [16], who stated that the anoxic tumour cells are inactive in secondary reactions, probably due to nutrient deprivation caused by breakdown of the circulatory system. Unlike most currently used cancer therapies, the response to PDT treatment was constant for the same tumour, indicating that no resistance was acquired. As a minor drawback, hair loss in mice surviving 10 days after the third PDT treatment was observed. This was also addressed by Divaris et al. [4] after exposure of ALA i.p. injected animals to white light as a consequence of a high porphyrin concentration in the hair follicle. The ®nding of alopecia in only these longsurviving animals indicated that almost a week is necessary to develop follicle damage in addition to the application of three doses of PDT. The absence of a cellular immune response after ALA-PDT is also noticeable, as is the presence of morphological features of apoptotic cells. These apoptotic cells may derive either from direct tumour cell killing or from vascular damage. This paper reports on the topical ALA-PDT application in a subcutaneous carcinoma leading to subsequent tumour growth delay. These ®ndings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas. Indeed, lack of resistance of this mammary adenocarcinoma to repetitive ALA-PDT applications encourages the use of ALAbased photodynamic therapy as a promising alternative in the routine treatment of tumours resistant to cytostatic drugs.
A. Casas et al. / Cancer Letters 141 (1999) 29±38
Acknowledgements This research was supported by grants from the Argentine National Research Council (CONICET), the Association for International Cancer Research (AICR)-UK and the Alberto J. Roemmers Foundation, Argentina. The authors are very grateful to Victoria Castillo for her skilful technical assistance. A.M. del C.B. and H.F. hold the posts of Superior and Associate Researchers at the CONICET. A.C. is a CONICET Research Fellow.
References [1] W. Boehncke, R. Sterry, R. Kaufmann, Treatment of psoriasis by topical photodynamic therapy with polychromatic light, Lancet 343 (1994) 801 (Letter). [2] F. Cairnduff, M. Stringer, E. Hudson, D. Ash, S. Brown, Super®cial photodynamic therapy with topical 5-aminolevulinic acid for super®cial primary and secondary skin cancer, Br. J. Cancer 69 (1994) 605±608. [3] F. Cairnduff, D.J. Roberts, B. Dixon, S.B. Brown, Response of a rodent ®brosarcoma to photodynamic therapy using 5aminolaevulinic acid or polyhematoporphyrin, Int. J. Radiat. Biol. 67 (1995) 93±99. [4] D. Divaris, J. Kennedy, R. Pottier, Phototoxic damage to sebaceous glands and hair follicles of mice after systemic administration of 5-aminolaevulinic acid correlates with localized protoporphyrin IX ¯uorescence, Am. J. Pathol. 136 (1990) 891±897. [5] J. Falk, Porphyrins and metalloporphyrins, Biochim. Biophys. Acta Library, 2, Elsevier, Amsterdam, 1964. [6] H. Fukuda, S. Paredes, A. Batlle, Tumour-localizing properties of porphyrins. In vivo studies using free and liposome encapsulated aminolevulinic acid, Comp. Biochem. Physiol. 102B (1992) 433±436. [7] H. Fukuda, S. Paredes, A. Casas, F. Chueke, A. Batlle, Potential of liposome-entrapped aminolevulinic acid in cancer therapy. Effect of prior injection of empty liposomes and different routes of administration, Cancer J. 5 (1992) 295±299. [8] E.W. Grant, A.J. Hopper, A.J. MacRobert, P.M. Speight, S.G. Bown, Photodynamic therapy of oral cancer: photosensitization with systemic aminolevulinic acid, Lancet 342 (1993) 147±148. [9] X.-Y. He, R.A. Sikes, S. Thomsen, L.W. Chung, S.L. Jacques, Photodynamic therapy with Photofrin II induces programmed cell death in carcinoma cell lines, Photochem. Photobiol. 59 (1994) 468±473. [10] B.W. Henderson, L. Vaughan, D. Bellnier, H. Van Leengoed, P.G. Johnson, A.R. Oseroff, Photosensitization of murine tumour, vasculature and skin by 5-aminolevulinic acidinduced porphyrin, Photochem. Photobiol. 62 (1995) 780± 789.
37
[11] B.W. Henderson, T.J. Dougherty, How does photodynamic therapy work?, Photochem. Photobiol. 55 (1992) 145±157. [12] H. Heyerdahl, I. Wang, D.L. Lin, R. Berg, S. AnderssonEngels, Q. Peng, J. Moan, S. Savnberg, K. Svanberg, Pharmacokinetic pattern studies on 5-aminolevulinic acid-induced Protoporphyrin IX accumulation in tumours and normal tissues, Cancer Lett. 112 (1997) 225±231. [13] J. Kennedy, R. Pottier, Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy, J. Photochem. Photobiol. B. 14 (1992) 275±292. È berriegler, In-vitro investigation of ALA[14] B. Krammer, K. U induced protoporphyrin IX, J. Photochem. Photobiol. B. 36 (1996) 121±126. [15] P. Mlkvy, H. Messmann, J. Regula, M. Cories, M. Paner, C.E. Millson, A.J. MacRobert, S.G. Bown, Sensitization and photodynamic therapy (PDT) of gastrointestinal tumours with 5aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX). A pilot study, Neoplasma 42 (1995) 109±113. [16] J. Moan, S. Sommer, Oxygen dependence of the photosensitizing effect of hematoporphyrin derivative in NHIK 3025 cells, Cancer Res. 45 (1985) 1608±1610. [17] N. Navone, C. Polo, R. Dinger, A. Batlle, Heme regulation in mouse mammary adenocarcinoma and liver of tumourbearing mice. I. Effect of allyl-isopropylacetamide and veronal on 5-aminolevulinate synthetase, cytochrome P450 and cytochrome oxidase, Int. J. Biochem. 22 (1990) 1005± 1012. [18] B.B. Noodt, K. Berg, T. Stokke, Q. Peng, J.M. Nesland, Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX, Br. J. Cancer 74 (1996) 22± 29. [19] Q. Peng, J. Moan, T. Warloe, J.M. Nesland, C. Rimington, Distribution and photosensitizing ef®ciency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma, Int. J. Cancer 52 (1992) 433±443. [20] Q. Peng, T. Warloe, J. Moan, H. Heyerdahl, H.B. Steen, J.M. Nesland, K.E. Giercksky, Distribution of 5-aminolevulinic acid-induced porphyrins in nodulo-ulcerative basal cell carcinoma, Photochem. Photobiol. 62 (1995) 906±913. [21] G. Peters, J. Nadal, H. Pinedo, Diurnal variation in the therapeutic ef®cacy of 5-¯uorouracil against murine colon cancer, In Vivo 1 (1987) 113±118. [22] C. Rimington, Spectral absorption coef®cients of some porphyrins in the Soret band region, Biochem. J. 75 (1960) 620±623. [23] A. Scolnik, M. Rubio, L. Colombo, R. Comolli, R. Caro, Further studies on the histamine metabolism in the M2 adenocarcinoma, Biomed. Pharmacother. 38 (1984) 465±467. [24] W.M. Star, H.P. Marijnissen, A.E. Van Der Berg-Block, J.A. Versteeg, K.A. Franken, H.S. Reinhold, Destruction of rat mammary tumour and normal tissue microcirculation by HPD photoradiation observed in vivo in sandwich observation chambers, Cancer Res. 46 (1986) 2532±2540. [25] M. Stringer, P. Collins, D. Robinson, G. Stables, R. SheehanDare, The accumulation of Protoporphyrin IX in plaque psoriasis after topical application of 5-aminolevulinic acid indi-
38
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cates a potential for super®cial photodynamic therapy, J. Invest. Dermatol. 107 (1996) 76±81. [26] R.M. Szeimies, S. Karrer, A. Sauerwald, M. Landthaler, Photodynamic therapy with topical application of 5-aminolevulinic acid in the treatment of actinic keratoses: an initial clinical study, Dermatology 192 (1996) 246±251. [27] UK-Coordinating Committee on Cancer Research (1988),
UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia. UKCCCR, London. [28] J. Webber, Y. Luo, R. Crilly, D. Fromm, D. Kessel, An apoptotic response to photodynamic therapy with endogenous protoporphyrin in vivo, J. Photochem. Photobiol. B 35 (1996) 209±211.
Topical and intratumoral photodynamic therapy with 5aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma Adriana Casas a, HaydeÂe Fukuda a, Roberto Meiss b, Alcira M. del C. Batlle a,* a
Centro de Investigaciones sobre Por®rinas y Por®rias (CIPYP) FCEyN (University of Buenos Aires) and CONICET, Ciudad Universitaria, PabelloÂn II, 2do piso, (1428)Capital Federal, Argentina b Departamento de PatologõÂa, I.E.O., Academia Nacional de Medicina, Las Heras 3092, (1425)Capital Federal, Argentina Received 4 July 1998; received in revised form 1 March 1999; accepted 1 March 1999
Abstract One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a signi®cant synthesis of porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradiation was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2±4 h after intratumoral administration of 30 mg ALA/cm 3. The pattern of tumour response evaluated as the delay of tumour growth was similar following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour growth delay. These ®ndings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: d-Aminolevulinic acid; Photodynamic therapy (PDT); Subcutaneous transplantable tumour; Topical application; Intratumour injection
1. Introduction Photodynamic therapy (PDT) is a cancer treatment based on the accumulation of a porphyrin-related photosensitizer in tumour cells, and their subsequent destruction on exposure to visible light. Singlet * Corresponding author at: Viamonte 1881 10A, 1056 Buenos Aires, Argentina. Fax: 1 54-1-8117447. E-mail address: [email protected] (A.M. del C. Batlle)
oxygen species are produced, causing damage to membranes and organelles, leading to cell death and tumour ablation [10]. One of the most promising substances for PDT is 5-aminolevulinic acid (ALA), a haem precursor which induces endogenous accumulation of porphyrins, mainly protoporphyrin IX (PpIX), in malignant tissues. Biological membranes are considered as critical targets for cell killing by PDT; damage to the vascular endothelium resulting in tissue/tumour ischaemia is
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00079-8
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an additional mechanism leading to tumour necrosis [24]. PDT has also been shown to induce apoptosis in vivo [28] and in vitro [9]; however, the apoptotic response to PDT seems to depend on both the photosensitizer and the cell line [18]. Due to its water solubility ALA can be given either by intravenous injection [12], oral administration [8], topically [13], and there are a few reports on its intratumoral (i.t.) application [3,6,7]. In topical PDT limited penetration of ALA and inadequate distribution of induced porphyrins might be a possible drawback for this modality. Recently, Peng et al. [20] demonstrated that the systemic administration of ALA led to more selective and homogeneous accumulation of ALA-induced porphyrins in the tumour. Clinical trials have already been performed using topically [2,26] or systemically [15] applied ALA, with good results. Topical ALA-PDT has also been shown to be effective in the treatment of premalignant conditions such as keratoses and in psoriasis [1,25]. Experimental models have shown that i.t. injection was more effective than or at least equally effective to the other two routes of ALA administration [3,6,7]. We have shown that the i.p. and i.t. injection of ALA could produce a signi®cant synthesis of porphyrins in the mammary transplantable adenocarcinoma M2 [6]. In the present study we investigated the effects of laser irradiation on the growth of a subcutaneous implanted tumour after topical or i.t. application of ALA. The amount of ALA leading to the highest accumulation of porphyrins in the tumour and the time point of the highest tumour/skin ratio of porphyrin levels, to attain optimum ef®ciency of PDT were determined. Tumour response was evaluated following tumour growth pro®les, delay growth indexes and light microscopy examination of PDTtreated areas.
2. Materials and methods 2.1. Chemicals ALA was purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals were of analytical grade.
2.2. Animals Male BALB/c mice, 12 weeks old, weighing 20±25 g were used. They were provided with food (Purina 3, Molinos RõÂo de la Plata) and water ad libitum. A mammary adenocarcinoma [23] (M2, Hospital Roffo, Buenos Aires) was propagated by serial transplantation into male BALB/c mice. Non-necrotic tumour material for inoculation was obtained by sterile dissection of large ¯ank tumours. A 1-mm 3 sample of macroscopically viable tumour, which is equal to approximately 2 £ 105 cells, was injected s.c. under the dorsal ¯ank of each mouse. The take rate of the tumours following transplantation was nearly 100%. Under these controlled conditions the implant size did not vary by more than 10%. Animals were treated in accordance with guidelines established by the Animal Care and Use Committee of the Argentine Association of Specialists in Laboratory Animals (AADEALC), in full accord with the UK Guidelines for the Welfare of animals in Experimental Neoplasia [27]. 2.3. Preparation and administration of ALA The hydrochloric salt of ALA was dissolved in sterile water (pH 6.5) at a concentration of 100 mg/ ml and used immediately for i.t. administration. For topical application ALA was prepared daily as a 10, 20, 30 or 40% formulation in a cream (Genargen Argentinaw). 2.4. Pharmacokinetics of ALA-induced porphyrins in tissues Tumours with a volume of approximately 400±450 mm 3 were used for pharmacokinetic studies. For timeresponse kinetics mice were given either 20 mg of ALA per cm 3 of tumour tissue intratumorally, or 50 mg of a 20% ALA cream by topical application. At different times between 1 and 24 h after ALA administration (®ve mice for each point) animals were sacri®ced. For dose-response kinetics, 50 mg of a cream containing 10, 20, 30 or 40% ALA were applied topically on tumours of the same uniform size rubbing over the whole surface of the tumour during a period of 60 s. For the i.t route, 10, 20, 30, 40 and 80 mg ALA/cm 3 tumour were injected (®ve mice for each
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point). Animals were killed 3 h after topical administration and 4 h after i.t. injection. The skin overlying the tumour (SOT) was carefully removed from the tumour itself. Samples of normal skin (NS) previously shaved were excised from the contralateral ¯ank of the tumour-bearing mice. 2.5. Tissue extraction of porphyrins The tissue samples were homogenized in a 4:1 solution of ethyl acetate/glacial acetic acid mixture. The homogenates were centrifuged for 30 min at 3000 £ g, and the supernatants shaken with an equal volume of 5% HCl [5]. Extraction with HCl was repeated until negative ¯uorescence in the organic layer. Porphyrins were spectrophotometrically determined in the aqueous fraction measuring absorbances at 380, 430 nm and the Soret band [22]. 2.6. Laser and irradiations A rhodamine dye laser (Model DL30, Oxford Lasers) pumped by a copper vapour laser (CU15A, Oxford Lasers) tuned to 630 nm was used. The light was focused into a 400-mm diameter optical ®bre and the cut end of the ®bre was positioned to provide a 3.5-cm diameter light spot, producing a treatment area of uniform intensity. The output power from the ®bre was measured with a power meter (Model LM100XL, Coherent, Auburn, CA) before each application. Total doses of 97 J/cm 2 were delivered using a ¯uence rate of 80 mW/cm 2 over 20 min. Light doses up to 100 J/cm 2 do not cause additional hyperthermic effects, which may in¯uence the ef®cacy of PDT; therefore, higher light doses were not used (Casas et al., unpublished results). 2.7. Treatment protocol for ALA-based PDT Eight days after implantation, when tumours reached the appropriate size of 70±100 mm 3, the SOT was shaved and a 20% ALA cream was topically applied. Ten days after implantation, when tumours reached the size of 180±200 mm 3, they were intratumorally injected with 30 mg ALA/cm 3 tumour. Animals were anaesthetized by i.p. injection of 70
31
mg/kg ketamine hydrochloride (Parke Davis, Argentina, S.A.) and 6 mg/kg xylazine (RompuÂnw, Bayer Argentina S.A.) and lesions were super®cially irradiated. During treatment normal tissue surrounding the tumour was shielded with a piece of black plastic, leaving exposed a peritumoral margin of 3 mm. Body temperature was monitored with a rectal probe (Tinitalk II, Gemini Dataloger). Normal shaved skin was exposed to both ALA and light for histological examination. 2.8. Assessment of tumour response The longer (l) and shorter (w) perpendicular axes and height (h) of each tumour were determined with callipers prior to or after PDT daily up to day 25 after implantation to evaluate the delay of tumour growth. Tumour volume was calculated using the formula l £ w £ h £ 0:5, where 0.5 is a correction factor empirically determined [21]. To assess the response to treatment two indexes were de®ned: D24 (tumour volume 24 h after PDT/tumour volume before PDT) and D48 (tumour volume 48 h after PDT/tumour volume before PDT). 2.9. Histological studies Prior to and 4, 9, 24, 48 and 72 h, up to 6 days after PDT, samples of tumour with its surrounding skin and samples of the NS were excised. They were extended, sliced, ®xed in 10% buffered formalin, embedded in paraf®n, sectioned, stained with haematoxylin and eosin and examined by light microscopy. The presence of tumour tissue, necrosis and cells with morphological features of apoptosis was evaluated. Epidermal and dermal damage, vascularity changes and presence of lymphocytic in®ltration were also investigated. 2.10. Statistical analysis The unpaired t-test was used to establish the significance of differences between groups. Differences were considered statistically signi®cant when P , 0:05.
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Fig. 1. Concentration of ALA-induced porphyrins in tumour, normal skin and skin overlying the tumour as a function of time after topical application of ALA cream. At different times after tumour topical application of 50 mg of 20% ALA cream, tissues were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
3. Results 3.1. Kinetics of porphyrin biosynthesis in tumour, normal skin and skin overlying the tumour after topical or intratumoral administration of ALA Fig. 1 shows the kinetics of porphyrin accumulation after topical application of ALA. The highest amount of porphyrins (0.6 mg/g tissue) was found in the tumour tissue 2±3 h after administration of ALA, then concentration decreased rapidly. The ALA-
Fig. 2. Concentration of ALA-induced porphyrins in tumour, normal skin and skin overlying the tumour as a function of time after intratumoral administration of ALA. At different times after intratumoral injection of 20 mg/cm 3 ALA, tissues were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
induced porphyrin accumulation in NS (0.4 mg/g tissue) was highest 90 min after ALA application. A delay in peak levels of porphyrins was observed in the SOT, reaching a maximum concentration of 1.1 mg/g tissue, 8 h after ALA application. When ALA was given intratumorally (Fig. 2), the highest amount of tumoral porphyrins (2 mg/g tissue) was found between 2 and 4 h after drug administration. Normal and tumour-overlying skin showed a
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Fig. 3. Porphyrin accumulation in tissues after topical application of various concentrations of ALA. Three hours after application of 50 mg of a cream containing 10, 20, 30 or 40% ALA, tumour (X), normal skin (P) and skin overlying the tumour (O) were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
similar pattern of porphyrin synthesis. Maxima were observed at 2 and 4 h after ALA injection (1.2 and 1 mg/g tissue, respectively). After reaching the peak, the porphyrin concentration declined rapidly near to basal levels in tumour and NS by 24 h, in either route of ALA administration, but not in SOT tissue. Tumour to NS porphyrin concentration ratios were approximately 3 between 2 and 3 h after topical application of ALA and nearly 2 between 4 and 6 h after i.t. administration. Conversely, tumour/SOT porphyrin values were higher for the i.t. route, reaching a maximum of nearly 3 between 4 and 6 h after, compared to 0.86 for the topical application at the same interval.
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Fig. 4. Porphyrin accumulation in tissues after intratumoral administration of various concentrations of ALA. Four hours after injections of 10, 20, 30, 40 and 80 mg ALA/cm 3 tumour tissue, tumour (X), normal skin (P) and skin overlying the tumour (O) were excised and porphyrins extracted as detailed in Section 2. Each data point represents the average of ®ve determinations. Error bars show standard deviations.
Normal skin porphyrins peaked at 30% ALA, and total accumulation was always lower than tumoral porphyrins. Tumour to NS and SOT porphyrin concentration ratios reached their maximum levels after 20% ALA (2.64 and 0.89, respectively). When ALA was i.t. injected (Fig. 4), the maximum accumulation of porphyrins was found with a 30-mg ALA/cm 3 injection. Both NS and SOT exhibited a saturation pattern above 40 mg ALA/cm 3. The tumoral levels of ALA-induced porphyrins were always higher than those of NS and SOT. At 30 mg ALA/cm 3, tumour to NS and SOT ratios were also maximal (2.7 and 2.4, respectively).
3.2. ALA-induced accumulation of porphyrins
3.3. Effectiveness of ALA-based PDT in delaying tumour growth
The amount of porphyrins generated in tumour after topical application reached a plateau with a 20% ALA cream (Fig. 3). SOT showed almost an identical pattern, although the amount of porphyrins was higher.
The effectiveness of ALA-induced porphyrins as photosensitizers for PDT was determined by assessing the extent of tumour growth after one, two and three PDT applications. Two response indexes were de®ned, measuring the
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Table 1 Tumour response indexes after a single application of ALA-PDT a
Control (n 7) Topical ALA (n 12) i.t. ALA (n 5)
Å ) D24 (X
Å ) D48(X
1.71 ^ 0.84 0.96 ^ 0.39 b 0.75 ^ 0.21 d
2.56 ^ 1.10 0.87 ^ 0.65 c 0.94 ^ 0.44 b
a As a measurement of tumour response to topical and i.t. ALAPDT, two indexes were de®ned: D24 and D48 (see Section 2). D24 and D48 control indexes were calculated as volume ratios between day 10 and days 9 and 8, respectively, after implantation of tumours in non-treated mice. Results are presented as means ^ standard deviations. b **P , 0:01 compared to the corresponding control. c *P , 0:007 compared to the corresponding control. d ***P , 0:03 compared to the corresponding control.
ratios between tumour volumes before and after treatment, since the rapid growth of M2 tumour does not allow the evaluation of survival or of tumour doubling times.
Fig. 5. Time-dependence of tumour growth after topical ALA-PDT. Tumours with an initial volume of 70±100 mm 3 were topically treated with 20% ALA cream and 3 h later they were exposed to 630 nm light at a ¯uence rate of 97 J/cm 2. Animals were treated with one (P), two (O) or three (B) doses of ALA-PDT. Arrows indicate the day of treatment. X, mean of six control curves (neither ALA nor light). Growth curves of tumours with the same response indexes are represented (D24 0:70).
Fig. 6. Time-dependence of tumour growth after intratumoral ALA-PDT. Tumours with an initial volume of 180±200 mm 3 were intratumorally injected with 30 mg ALA/cm 3 tumour and 4 h later they were exposed to 630 nm light at a ¯uence rate of 97 J/ cm 2. Animals were treated with one (P), two (O) or three (B) doses of ALA-PDT. Arrows indicate the day of treatment. X, mean of six control curves (neither ALA nor light). Growth curves of tumours with the same response indexes are represented (D24 0:80).
D24 and D48 indexes for both topical and intratumoral ALA-PDT are shown in Table 1. After a single application both indexes were lower than those of the untreated tumours, indicating that if not a complete reduction, a delay of tumour growth occurs. After two and three PDT applications similar indexes were obtained and no signi®cant differences between either route of ALA administration were evidenced (data not shown). Irradiation on ALA injected intratumorally appeared to induce a greater reduction in tumour volume earlier. An index of 0.75 was observed at 24 h and 0.94 at 48 h, but differences between times were not statistically signi®cant. In order to compare the response to repetitive PDT, tumour growth curves with the same D24 index were used (Figs. 5 and 6). Animals received either one, two or three successive treatments of topical and intratumoral ALA-PDT, respectively. A single application induced a clear tumour growth delay for both topical and intratumoral ALA administration. A second PDT treatment applied 48 h later and a third one, applied 6 days later induced a further delay in tumour growth. It is noteworthy that tumour
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Fig. 7. Morphological images of apoptotic cells after ALA-based PDT treatment. Histological section of tumour treated 24 h after topical ALAPDT, showing condensation of an eosinophilic cytoplasm and a basophilic nuclei. Cap-like condensed chromatin at one nuclear pole appear in some cells. Similar images were observed for the i.t. route 48 and 72 h after treatment. None of these features were obtained in control tissues.
height was the axis most dramatically reduced after PDT. The D24 and D48 indexes were always similar for the same tumour after the ®rst, second or third PDT dose. The application of ALA or light alone did not cause any measurable effects on tumour growth. In all cases core body temperature throughout laser treatment decreased from 33 to 298C during irradiation, due to anaesthesia. 3.4. Macroscopical analysis and histological studies Normal skin exhibited neither macroscopic nor microscopic changes with PDT treatment. The same results were observed for tumour or skin exposed to either ALA or light alone at all times analyzed. After 24 h of ALA-based PDT, administered by either route, macroscopically necrotic zones, gross tumour volume reduction, ulceration and eschar formation were induced. At 0, 4 and 9 h after treatment no microscopic changes in either tumour tissue or its overlying skin were observed. At 24 h preserved tumour tissue with
scanty foci of necrosis and cells with apoptotic images up to a depth of 4 mm from the epidermis were seen in both i.t. and topically ALA-PDT treated samples (Fig. 7). Necrotic cells with vacuolization of the cytoplasm, pycnotic appearance of the nucleus and loss of cellularity were seen. Epidermal with slight keratosis, dermal oedema and vascular dilation in the SOT were also observed. At 48±72 h there was an expansion of tumour in®ltrating the dermis and epidermis with keratinization and sloughing of the SOT. Haemorrhagic foci and large areas of necrosis and of apoptotic cells in tumour tissue were also seen. From day 5 after PDT, connective ®brovascular tissue surrounding the tumour, rich in neo-formed vessels with wall enlargement, vascular stasis and congestion was developed. No lymphocytic in®ltration was ever found in any microscopic studies performed. Non-shielded peritumoral skin of mice, having been treated three times with topical ALA-PDT, remained free of macroscopically visible tumour for 10 days, and exhibited hair basophilic, amorphous destruction inside the follicle which correlated with visible alopecia.
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4. Discussion It has already been demonstrated that porphyrins can be synthesized and accumulated in different tissues such as tumour and skin of mice given i.t. injection of ALA [6]. Here, we have shown that topical or intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma induced a signi®cant synthesis of porphyrins and a consequent sensitization to laser light. Although complete remissions were not achieved with the present light dose regimen, periods up to 10 days free of macroscopically visible tumour were sometimes observed both with the i.t. and topical modes of ALA application (data not shown). For both routes of ALA administration a sort of saturation pattern was observed in the porphyrinsynthesizing capacity of the tissues, indicative of the existence of particular regulatory mechanisms in the metabolism of ALA-induced porphyrins in tumourbearing animals [17]. It is noteworthy that this ALA dose-response saturation in tumour is evidenced when the drug is administered by either route. It was achieved with 50 mg of 20% ALA cream, which corresponds to a total amount of 25 mg ALA/cm 3 tumour, and with 30 mg ALA/cm 3 in the case of i.t. application. The total amount of porphyrins in tumour tissue was ®ve times higher when ALA was i.t. injected, and tumour/SOT porphyrin concentration ratios were higher. However, no signi®cant differences in the actual ef®cacy of photosensitization were observed, in agreement with the results of Cairnduff et al. [3] for an intradermally located murine adenocarcinoma. These ®ndings may be explained by either the existence of a saturating amount of ALA-formed porphyrins necessary to produce the photodynamic damage [14] or alternatively by an uneven distribution of porphyrins in different tumour layers which eventually result in a similar or equivalent amount of porphyrins directly exposed to light in either route of ALA administration. According to tumour/skin porphyrin concentration ratios and total amounts of porphyrins accumulated in tumour tissue, irradiation would be optimal between 2 and 3 h after topical application of 20% ALA cream and 2±4 h after intratumoral administration of 30 mg ALA/cm 3.
Maximal accumulation of porphyrins was observed 3 h after topical ALA application, in agreement with Henderson et al. [11] and Peng et al. [19], who found that ¯uorescence of ALA-induced porphyrins in murine adenocarcinomas peaked between 3 and 5 h post-topical application. Induced anoxia by vascular damage does not seem to lead to an impairment of repetitive-PDT ef®cacy, since the same tumour response is maintained along successive treatments. It appears that continuous applications of PDT would photosensitize surviving cells, which could recover their oxic stage by means of the neo-formed vessels provided by the surrounding ®brovascular reparation tissue. Another interesting hypothesis is that proposed by Moan and Sommer [16], who stated that the anoxic tumour cells are inactive in secondary reactions, probably due to nutrient deprivation caused by breakdown of the circulatory system. Unlike most currently used cancer therapies, the response to PDT treatment was constant for the same tumour, indicating that no resistance was acquired. As a minor drawback, hair loss in mice surviving 10 days after the third PDT treatment was observed. This was also addressed by Divaris et al. [4] after exposure of ALA i.p. injected animals to white light as a consequence of a high porphyrin concentration in the hair follicle. The ®nding of alopecia in only these longsurviving animals indicated that almost a week is necessary to develop follicle damage in addition to the application of three doses of PDT. The absence of a cellular immune response after ALA-PDT is also noticeable, as is the presence of morphological features of apoptotic cells. These apoptotic cells may derive either from direct tumour cell killing or from vascular damage. This paper reports on the topical ALA-PDT application in a subcutaneous carcinoma leading to subsequent tumour growth delay. These ®ndings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas. Indeed, lack of resistance of this mammary adenocarcinoma to repetitive ALA-PDT applications encourages the use of ALAbased photodynamic therapy as a promising alternative in the routine treatment of tumours resistant to cytostatic drugs.
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Acknowledgements This research was supported by grants from the Argentine National Research Council (CONICET), the Association for International Cancer Research (AICR)-UK and the Alberto J. Roemmers Foundation, Argentina. The authors are very grateful to Victoria Castillo for her skilful technical assistance. A.M. del C.B. and H.F. hold the posts of Superior and Associate Researchers at the CONICET. A.C. is a CONICET Research Fellow.
References [1] W. Boehncke, R. Sterry, R. Kaufmann, Treatment of psoriasis by topical photodynamic therapy with polychromatic light, Lancet 343 (1994) 801 (Letter). [2] F. Cairnduff, M. Stringer, E. Hudson, D. Ash, S. Brown, Super®cial photodynamic therapy with topical 5-aminolevulinic acid for super®cial primary and secondary skin cancer, Br. J. Cancer 69 (1994) 605±608. [3] F. Cairnduff, D.J. Roberts, B. Dixon, S.B. Brown, Response of a rodent ®brosarcoma to photodynamic therapy using 5aminolaevulinic acid or polyhematoporphyrin, Int. J. Radiat. Biol. 67 (1995) 93±99. [4] D. Divaris, J. Kennedy, R. Pottier, Phototoxic damage to sebaceous glands and hair follicles of mice after systemic administration of 5-aminolaevulinic acid correlates with localized protoporphyrin IX ¯uorescence, Am. J. Pathol. 136 (1990) 891±897. [5] J. Falk, Porphyrins and metalloporphyrins, Biochim. Biophys. Acta Library, 2, Elsevier, Amsterdam, 1964. [6] H. Fukuda, S. Paredes, A. Batlle, Tumour-localizing properties of porphyrins. In vivo studies using free and liposome encapsulated aminolevulinic acid, Comp. Biochem. Physiol. 102B (1992) 433±436. [7] H. Fukuda, S. Paredes, A. Casas, F. Chueke, A. Batlle, Potential of liposome-entrapped aminolevulinic acid in cancer therapy. Effect of prior injection of empty liposomes and different routes of administration, Cancer J. 5 (1992) 295±299. [8] E.W. Grant, A.J. Hopper, A.J. MacRobert, P.M. Speight, S.G. Bown, Photodynamic therapy of oral cancer: photosensitization with systemic aminolevulinic acid, Lancet 342 (1993) 147±148. [9] X.-Y. He, R.A. Sikes, S. Thomsen, L.W. Chung, S.L. Jacques, Photodynamic therapy with Photofrin II induces programmed cell death in carcinoma cell lines, Photochem. Photobiol. 59 (1994) 468±473. [10] B.W. Henderson, L. Vaughan, D. Bellnier, H. Van Leengoed, P.G. Johnson, A.R. Oseroff, Photosensitization of murine tumour, vasculature and skin by 5-aminolevulinic acidinduced porphyrin, Photochem. Photobiol. 62 (1995) 780± 789.
37
[11] B.W. Henderson, T.J. Dougherty, How does photodynamic therapy work?, Photochem. Photobiol. 55 (1992) 145±157. [12] H. Heyerdahl, I. Wang, D.L. Lin, R. Berg, S. AnderssonEngels, Q. Peng, J. Moan, S. Savnberg, K. Svanberg, Pharmacokinetic pattern studies on 5-aminolevulinic acid-induced Protoporphyrin IX accumulation in tumours and normal tissues, Cancer Lett. 112 (1997) 225±231. [13] J. Kennedy, R. Pottier, Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy, J. Photochem. Photobiol. B. 14 (1992) 275±292. È berriegler, In-vitro investigation of ALA[14] B. Krammer, K. U induced protoporphyrin IX, J. Photochem. Photobiol. B. 36 (1996) 121±126. [15] P. Mlkvy, H. Messmann, J. Regula, M. Cories, M. Paner, C.E. Millson, A.J. MacRobert, S.G. Bown, Sensitization and photodynamic therapy (PDT) of gastrointestinal tumours with 5aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX). A pilot study, Neoplasma 42 (1995) 109±113. [16] J. Moan, S. Sommer, Oxygen dependence of the photosensitizing effect of hematoporphyrin derivative in NHIK 3025 cells, Cancer Res. 45 (1985) 1608±1610. [17] N. Navone, C. Polo, R. Dinger, A. Batlle, Heme regulation in mouse mammary adenocarcinoma and liver of tumourbearing mice. I. Effect of allyl-isopropylacetamide and veronal on 5-aminolevulinate synthetase, cytochrome P450 and cytochrome oxidase, Int. J. Biochem. 22 (1990) 1005± 1012. [18] B.B. Noodt, K. Berg, T. Stokke, Q. Peng, J.M. Nesland, Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX, Br. J. Cancer 74 (1996) 22± 29. [19] Q. Peng, J. Moan, T. Warloe, J.M. Nesland, C. Rimington, Distribution and photosensitizing ef®ciency of porphyrins induced by application of exogenous 5-aminolevulinic acid in mice bearing mammary carcinoma, Int. J. Cancer 52 (1992) 433±443. [20] Q. Peng, T. Warloe, J. Moan, H. Heyerdahl, H.B. Steen, J.M. Nesland, K.E. Giercksky, Distribution of 5-aminolevulinic acid-induced porphyrins in nodulo-ulcerative basal cell carcinoma, Photochem. Photobiol. 62 (1995) 906±913. [21] G. Peters, J. Nadal, H. Pinedo, Diurnal variation in the therapeutic ef®cacy of 5-¯uorouracil against murine colon cancer, In Vivo 1 (1987) 113±118. [22] C. Rimington, Spectral absorption coef®cients of some porphyrins in the Soret band region, Biochem. J. 75 (1960) 620±623. [23] A. Scolnik, M. Rubio, L. Colombo, R. Comolli, R. Caro, Further studies on the histamine metabolism in the M2 adenocarcinoma, Biomed. Pharmacother. 38 (1984) 465±467. [24] W.M. Star, H.P. Marijnissen, A.E. Van Der Berg-Block, J.A. Versteeg, K.A. Franken, H.S. Reinhold, Destruction of rat mammary tumour and normal tissue microcirculation by HPD photoradiation observed in vivo in sandwich observation chambers, Cancer Res. 46 (1986) 2532±2540. [25] M. Stringer, P. Collins, D. Robinson, G. Stables, R. SheehanDare, The accumulation of Protoporphyrin IX in plaque psoriasis after topical application of 5-aminolevulinic acid indi-
38
A. Casas et al. / Cancer Letters 141 (1999) 29±38
cates a potential for super®cial photodynamic therapy, J. Invest. Dermatol. 107 (1996) 76±81. [26] R.M. Szeimies, S. Karrer, A. Sauerwald, M. Landthaler, Photodynamic therapy with topical application of 5-aminolevulinic acid in the treatment of actinic keratoses: an initial clinical study, Dermatology 192 (1996) 246±251. [27] UK-Coordinating Committee on Cancer Research (1988),
UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia. UKCCCR, London. [28] J. Webber, Y. Luo, R. Crilly, D. Fromm, D. Kessel, An apoptotic response to photodynamic therapy with endogenous protoporphyrin in vivo, J. Photochem. Photobiol. B 35 (1996) 209±211.