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Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model: a novel hematoporphyrin derivative
Biochimie 70 (1988) 251-258 ,~ Soci6t6 de Chimie biologique/Elsevier, Paris
251
Research article
Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model" a novel hematoporphyrin derivative * Mohamed EL-FAR I, M. ABOU-EL-ZAHAB ~, M. GHONEIM 2 and E. IBRAHIM 2
1Faculty of Science, Chemistry Dept., and :Urology and Nephrology Centre, Mansoura University, Egypt (Received 25-6-198L accepted after revision 4-11-1987)
S u m m a r y - Application of laser irradiation with porphyrin(s) or their derivatives for the destruction of tumors in humans requires preliminary studies of their localization in normal and malignant tissues. A novel derivative of hematoporphyrin (HP) was prepared. The newly developed hematoporphyrin (DHP) was administered to Fisher rats with bladder tumors and showed greater accumulation in the tumoral tissues. Comparative data oh (HP) and (DHP) are presented and discussed in light of the enhanced tumor porphyrin uptake caused by these agents. The homogeneous intense fluorescence noted with DHP-treated animals suggests that total tumor kill curative therapy will be more feasible. The study paves the way to refining increased porphyrin augment phototherapy and laser application in the field of oncology. bladder cancer / hematoporphyrin derivative / phototherapy / porphyrins
Introduction The interaction of porphyrins with normal and malignant cells is the subject of intensive investigations. Porphyrins have been known to display a selective affinity for neoplastic tissues. This property prompted the use of porphyrins as tumor localizers and has been exploited both for diagnosis and treatment. Diagnosis is dependent upon the property of porphyrins to fluoresce when excited by blui~ light. Thus, endoscopic excitation of premalignant lesions containing hematoporphyrin derivative (HPD) has led to their detection in the bladder. On the other hand, the ability of porphyrins to cause irreversible photodynamic
damage to cells and tissues, upon radiation with visible light, was utilized to induce the regression of tumors, i.e., photoradiation therapy (PDT). Initial reports on PDT of human bladder tumors go back to 1975, when Kelly et ai. [1, 2] reported on the successful photodynamic destruction of bladder tumor tissue by means of endoscopically controlled exposure to a mercury vapor lamp following the intravenous administration of HDP. Several thousand patients have now been treated with generally promising results [3]. Porphyrins are a group of heteroaromatic compounds, whose nucleus is relatively large and hydrophobic, and hence porphyrins tend to be only slightly soluble in water, unless func-
*An abstract of this work was accepted for presentation at the Clayton Foundation Conference on Photodynamic Therapy, held in Los Angeles, CA, U.S.A., February 1987. Abbreviation: FANFr: N-[4o(5-nitro-2-furyl)-2-thiazolyl] formamide.
252
M. El-Far et ai.
tional groups conferring water solubility have purposely been introduced. To overcome this, Lipson and Baldes [4] described the preparation of hematoporphyrin derivative [HPD]. They treated hematoporphyrin (HP) with sulfuric acid in acetic acid to give a solid material named HPD. Before use, it should be treated with alkali which was found to cause chemical changes in the solid material [5]. Alkali-treated hematoporphyrin derivative was first employed in this way by two groups: Dougherty et ai. [6] and Kelly et ai. [1]. Since that time, there has been a great deal of interest in photodynamic therapy, i.e., the use ofporphyrin as photosensitizers plus laser light in the treatment of tumors in patients. Application of HPD in clinical oncology was initiated by Dougherty and coworkers, and the activity in this field has rapidly expanded to its present high level. El-Far and Pimstone [7-11] were the first to demonstrate that uroporphyrin I (UP.I) and uroporphyrin Ill (UP.Ill) were efficiently and selectively retained in a transplantable mouse mammary carcinoma. They also showed that UP.I alone can effect photodynamic necrosis of tumor at high intensities using an argon dye laser. Their findings were recently confirmed by other groups [12-14]. El-Far and Pimstone [9, 11] using 28 different porphyrins having different chemical structures and side chains, found that only HPD, tetraphenylporphinesulfonate (TPPS4), tetracarboxyphenylporphine (TCPP), and UP.I and UP.III could be selectively retained in transplantable mouse mammary carcinoma. These 4 porphyrins differ significantly and considerably in their properties. HPD, like its crude parent compound HP, is a complex mixture of dicarboxylic porphyrins. Moan et al. [15] using high performance liquid chromatography to separate HP demonstrated the presence of 3 major porphyrin fractions and at least 17 additional minor components. On the other hand, UP has 8 aliphatic carboxylic side chains, while TPPSa has 4 sulfonatophenyl groups and TCPP has 4 carboxylic groups. Clearly much more information on the relationship between the structures of porphyrins and their selective uptake and localization in tumor cells and normal cells is needed. Such information should allow us to design a porphyrin with very high specificity towards the malignant cells, i.e., maximal retention in tumors with minimal retention in other normal tissues, especially skin.
The present work was undertaken to determine the tumor localizing potential of a newly developed porphyrin derivative from HP, using a bladder tumor model. Additional carboxylic groups were purposely added in an effort to explore the effects of certain specific functional groups on the tumor localizing efficiency and uptake of porphyrin by malignant cells. Previous studies by El-Far and Pimstone [7] showed that tumor uptake of porphyrin may in part be governed by a poor binding affinity to circulating plasma proteins. Also, selective porphyrin retention may be due to a relatively greater affinity for tumor protein constituents [10].
Materials and methods Chemical compounds
HP was obtained from porphyrin products (Logan, UT, U.S.A.). A stock solution was prepared in a final concentration of 5 mg/ml. Porphyrin material was first dissolved in 0.1 N NaOH. After 1 h of stirring at room temperature the solution was neutralized to pH 7.2 using 0.1 N HC! and then adjusted to its final concentration with 0.9% NaCI solution [16]. Developed hematoporphyrin preparations
HP was condensed with ethyl diazoacetate in ether solution and in the presence of HCI to give the developed hematoporphrin. Ethyl diazoacetate (EDA). was prepared according to the method of Sllberrad [17]. Briefly, a few crystals of sodium acetate were dissolved in 20 ml of water. Ethyl glycine hydrochlor i d a ( l f l o~ a n d 7 ~ g n f ~ n c l i n m n i t r i t a w a r a t h a n a d d . . . .
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ed. The reaction mixture was placed in an ice container until the temperature fell to about 0°C. Two drops ofsulfuric acid (10%) and then 20 ml ofether were added. As soon as the reaction had taken place, the ethereal solution of EDA was removed (run off). Then the EDA solution in ether was adjusted to 20 ml using fresh diethyl ether. 50 mg of l i P were dissolved in 1 ml of conc. HCI and cooled to 0°C. The cold solution of EDA was added to the acidic IIP solution. DHPI was prepared by adding 1 ml of ethereal solution containing EDA to 50 mg of l i P in liCl. DliP2 was prepared by adding 2 ml of ethereal solution containing EDA to another 50 mg of l i P in HCI. DHP3 was prepared by adding 3 ml of ethereal solution containing EDA to another 50 mg of l i P in acidic solution. In all cases, the condensation reaction between l i p acidic solution and ethereal solution containing EDA should be carried out under strict cold conditions" the temperature should be kept around 0°C. In one experiment with DHPb condensation was carried out at room temperature" upon adding ethereal EDA'to the HP solution, the
253
A novel H P derivative
temperature rose to over 50°C. The resulting preparation was named (DHPI)*. After condensation took place, the reactant materials were left to stand at room temperature for 2 days in the dark, and then evaporated to dryness using an electric fan. The resulting dry product was then subjected to NaOH and prepared as described before. The porphyrin solutions were kept in a dark glass bottle at 4°C for 5 weeks before use. In some experiments, we varied the concentration of NaOH due to the presence of a more acidic character in our developed HP preparations. Indeed, our condensation reaction between EDA and HP would be in agreement with the results of Johnson et al. [18] who reported the condensation of EDA with cobalt octaethyl porphyrin in acidic medium. The scheme of our one reaction flask could be presented as follows" 1) HCI + NH2CH2COOC2H5 + NaNO2 -HC____~I CIN=N-CH2COOC2H5 2) ~NHCIN=N=CH2-COOC2H5-HCI_~ N2 ~N-CH2-COOC2H2 ~HC! N-CH2-COOH Tumor model The tumor model was the transplantable bladder tumor in Fisher rats. Primary bladder tumors induced in Fisher 344 inbred rats by F A N F T were transplanted into syngeneic rats by orthotopic injection of bladder tumor cells into the bladder submucosa. A majority of tumors were well-differentiated transitional-cell carcinomas. The technique of this orthotopic implantation as well as usefulness of this tumor model for bladder cancer studies were previously described by Ibrahiem et al. [19]. Approximately 2 months after tumor transplantation, the rats were given an intra-cardiac injection of either the original HP or the developed derivatives dis-
solved as described before and given at the desired dosage in a volume of about 0.1 ml. At that time, none of the tumors exhibited necrosis as determined by routine histological studies. At specified times after the injections, the rats were killed and the t u m o r and other tissues were rapidly excised. A m i n i m u m of 4 tumor-bearing animals were used for testing each porphyrin in most cases. Porphyrin localization was assessed at specified times after injection. The rats were killed, tumor and other tissues were rapidly excised. The tissues were then weighed and analyzed immediately. Tumors were excised in toto, divided in two with a scalpel so that the surface and cross section, red porphyrin fluorescence could be assessed by the naked eye following photoexcitation with a blackray long wavelength ultraviolet lamp (Model B-100A, UV Products Inc., CA, U.S.A.). Porphyrin fluorescence was similarly assessed in the skin and other organs. Porphyrins were extracted and quantitated according to the technique of El-Far and Pimstone [7]. Briefly, each tissue was blended and homogenized with 1 N aqueous perchloric acid and methanol (1:1, v/v) in the dark, using an electric homogenizer with a teflon pestle. The homogenate was then centrifuged and the tissue precipitate was re-extracted until no fluorescence was observed under UV light. The combined supernatant was filtered through Whatman no. 1 filter paper and its volume recorded. The supernatant content was quantitated fluorometrically and determined, using the porphyrin under study as the standard under optimal conditions for photoexcitation and fluorescence emission.
Results and Discussion T h e r e s u l t s are s u m m a r i z e d a n d p r e s e n t e d in T a b l e s I a n d II. P o r p h y r i n c o n t e n t in tissues is e x p r e s s e d as ~ g i g wet weight. O b v i o u s t u m o r f l u o r e s c e n c e was n o t e d in all a n i m a l s injected with d i f f e r e n t p o r p h y r i n p r e p a r a t i o n s . In t h e g r o u p o f a n i m a l s i n j e c t e d with D H P preparat i o n s 1,2 a n d 3, we n o t e d t h a t D H P was p r e s e n t
Table I. Tissue porphyrin contents in/~g/g wet weight 24 h post-injection with a dose of 20 mg/kg of body weight. Material (5 mg/ml)
Bladder tumor
Skin
HP DHPI* DHPI DHP2
3.7 3.1 6.8 12.4
1.7 1.8 3.1 3.2
_ _ ± ±
2.1 a (7) b 1.2 (5) 3.7 (5) 1.7 (4)
-4- 0.7 ± 0.6 ± 0.6 ± 0.5
Liver (7) (5) (5) (5)
8.2 9.0 12.9 21.6
± ± ± ±
Kidney 3.7 2.1 2.2 1.7
(6) (5) (6) (4)
a Mean + S.D. b Numbers between parentheses = number of samples analyzed (1 sample/rat).
5.1 5.6 11.1 22.5
± 1.7 ± 2.2 _+ 2.2 +__ 1.7
Spleen (6) (5) (6) (4)
3.2 2.4 3.0 8.4
± 2.2 ± 0.6 _ 1.4 __. 1.7
(7) (5) (6) (4)
M. El-Far et al.
254
Table il. Tissue porphyrin contents in ag/g wet weight at 15 h post-injection with a dose of 15 mg/kg of body
weight. Material (5 mg/ml)
Bladder tumor
Skin
HP DHPI DHP2 DHP3
5.4 __. 2.2a (4) b 9.2 + 0.9 (3) 8.3 _ 4.2 (4) 18.2 __. 1.6 (2)
2.3 __ 1.1 3.9 _ 0.6 3.9 +_ 1.1 4.2 __+0.6
(3) (4) (3) (2)
Liver
Kidney
Spleen
"8.7 _____4.6 (4) 11.1 _ 2.3 (4) 12 +_.4.8 (4) 14.3 ___ 1.2 (2)
4.8 _____3.8 (4) 9.4 _+ 1.3 (4) 14.4 _+ 1.6 (4) 17.2 +_ 2 (2)
4.3 _____2.2 (4) 5.9 _ 1.9 (4) 6.3 ___ 1.6 (4) 5 ___ 1.5 (2)
a Mean _+S.D. b Numbers between parentheses = number of samples analyzed (1 sample/rat).
in tumor tissues in amounts far in excess to that observed with a commerical HP preparation. Table I shows that the porphyrin contents in tumors of animals treated with DHP3 were about 3-fold greater that those in tumors from HP-treated animals. Also, porphyrin contents and tumor uptake were found to be increased with increasing reaction molar ratios, i.e., DHP3 was more highly represented in the tumor than DHP2 and DHPn. We have also noted that DHPI obtained under cold conditions of preparation showed a distribution different from that obtained and prepared under higher temperature. The latter preparation showed exactly the same distribution as that observed for the HP solution. This would simn!v ..... .- , demonstrate that the chemical condensation did occur under cold conditions, as recommended, and enhanced uptake in the tumor would be mainly attributed to the chemical change which may be due to the presence of more carboxylic groups added to the porphyrin skeleton. It was interesting to note that the naked eye distribution of tumor fluorescence was altered when DHP preparations 1, 2 and 3 were used. Tumor fluorescence in animals treated with DHP2 and DHP3 is homogeneous through out the tumor and more intense, whereas with commercially prepared HP, heterogeneous fluorescence and intensity were observed. This is not a novel observation, we have previously noted that tumor fluorescence with HPD in 30% of the cases was not homogeneous (patchy) but was more intense in the periphery than in the center of the tumor [7, 9, 11]. Similar observations have been reported in rodent tumors, following HPD injection [20] as well as in human patients with bronchial carcinoma receiving HPD [21]. We believe that this patchy
fluorescence might be correlated to the remarks made by Dougherty [22], as it is not clear how the biological phenomena of localization and photosensitization are related to the porphyrin compt, nents in the mixt.ures, or why apparently similarly prepared injectable solutions show different biological activities. The observations of Kreimer-Birnbaum et al. [23] concerning HPD are consistent with the previous findings. DHP preparations showed a greater porphyrin concentration in the tumor and no patchy fluorescence was observed. The clinical benefit of using DHP is the enhancement of tumor porphyrin uptake. Since it is known that HPD does not cause uniform tumor fluorescence, islands of viable tumor tlnl~ilL
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therapy. The homogeneous intense fluorescence noted in our experimental animals given DHP suggests that total tumor kill and curative therapy will be more feasible than with current techniques. Some investigations have focused on the time dependency of the ratio between the tissue porphyrin concentration in the tumor and correlate it to the skin, where the primary interaction between the incident radiation and the phototreated patients takes place in skin tumors. Analysis of our results demonstrate that at 15 h post-injection of 15 mg/kg, a substantial difference existed between the a~:)ur~t~ ofporphyrin in the tumor and the skin (Table I). The tumor to skin ratio with commercial HP is 2.37, while, in the case, of DHP3 it is 4.30, i.e., an increase of 44%. A substantial difference was also observed between the amounts of porpl:yrin in the tumor and skin, 24 h post-injection of 20 mg/kg (Table II). In this particular case, the tumor to skin ratio with commercial HP was 2.16, while it was 3.85 with DHP2. It is worth mentioning that the ratio was
255
A novel H P derivative
herty [16] injected 3H- and 14C-labeled HPD into tumor-bearing mice and found that the concentration of HPD was higher in liver and kidney than in the tumor. On the other hand, the porphyrin load in the skin, in cases of DHP preparations, can be effectively reduced and partially eliminated after the diagnostic and therapeutic maneuvers are completed by administration of oral charcoal or by means of a blood exchange technique. We have recently demonstrated that unwanted porphyrins can effectively and significantly be eliminated from the circulatory system and, as a consequence, reduce skin porphyrins [26]. Previous reports suggest that porphyrin localization in tumors may depend upon the relative concentrations of lipophilic and hydrophobic molecules administered• It has been shown that the affinity of porphyrins for cultured cells and their subcellular distribution is dependent upon their hydro- or lipo-solubility [27] On the other hand, Jori et al., [14] have extended the study in vivo and showed that i.p. injection of liposome-bound porphyrins into
also increased by nearly the same value as found in the lower dose (15 mg/kg, 15 h). Further studies are under way to identify the optim u m conditions. It is well known that the dose of the injected porphyrin could be a key factor in its distribution among different organs [24]. Similar conclusions h a v e been previously reported by Musser et al. [25] who investigated TCPP and TPPS4 in mice bearing solid L1210 tumors and sarcoma 180 tumors• They concluded that lowering the dose of TPPS4 did not alter the overall distribution pattern but did improve the t u m o r : t i s s u e ratios, while TCPP had the opposite effect. This may suggest that an investigation at earlier time periods following injection might have been better when low doses of DHP were used. In general, the tumor retained considerably more of the DHP preparations (Tables I and II) than the commercially prepared HP over the 15 and 24 h periods (Fig. 1). Our results also demonstrate that liver and kidney tissues retain greater quantities than the tumor of all preparations of DHP as well as commercial HP. Previously, Gomer and Doug-
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DHPI
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~-
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Fig. 1 Bladder tumor porphyrin cuntents over 15 and 24 h periods.
256
M. El-Far et al.
mice bearing a MS-2 fibrosarcoma resulted in a remarkably more efficient tumor targeting than that obtained with the administration of the same porphyrin dissolved in a homogeneous aqueous solution. They indicated also that the subcellular distribution of liposome-delivered porphyrin is also dependent upon its solubility properties, thus confirming Kessel's findings. El-Far and Pimstone [7, 9, 11] reported the superiority of uroporphyrin, a water soluble porphyrin, over HPD in tumor localization in this regard, using a transplantable mammary carcinoma in the BALB/c mouse as the tumor model. Our present results in this regard demonstrate that tumor porphyrin uptake is greater with the newly developed hematoporphyrin which is richer in hydrophilic characteristics, than commercial HP. A major disadvantage of HPD is that it is not fully chemically characterized. A number of theories have been proposed as to the metabolic basis for tumor uptake of HPD and its major active component. Many investigators have sought to separate the components of HPD, characterize them and identify which ones localize in and photosensitize neoplastic tissue. Perhaps the most provocative and perplexing studies were those reported'by Berenbaum et al. [28] and Dougherty [29]. The early studies by Berenbaum et al. [28] suggested the presence of ether linked dimers or oligomers in the h,,-~n,il,llllUl
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[30] showed that at least 60% of the product consists of porphyrins which are not localizers • hematoporphyrin (HP), hydroxyethylvinyldeuteroporphyrin (HVD), and protoporphyrin (PP). In 1984, he suggested that the major tumor localizing component of HPD is a dihematoporphyrin ether. In 1986, two groups confirmed the presence of the ether-linked HP dimer [31, 32]. The chemical and biological studies by Musselman et al. [30] also suggested the presence of the ether bond and not an ester. While this is an attractive possibility it would not explain why the 8 carboxylic porphyrin (UP.I) shows a superiority over other porphyrins, as reported by El-Far and Pimstone and supported by others. Thus, we propose that DHP could be classified as a 4-carboxylic-HP, but it is richer in a fraction of porphyrin localizer. In agreement with the dimer proposed by others, it is tempting to speculate that a DHP dimer fraction could consist of two DHP molecules joined via an ether linkage (Fig. 2). This porphyrin dimer would contain 8 carboxylic
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groups like UP, but their distribution is different. There have been many reports on the reverse-phase HPLC analysis of HPD [5, 15, 28] and significant difficulties were reported by some of them in identifying the active component in its mixture due to the retention of a major part of HPD by the column during separation. That is why we did not try to fully investigate the chemical changes. This study was designed only to look at the effect of EDA condensation of HP on porphyrin localization and retention in the tumor. Our thin-layer chromatography studies showed that the major fractions of DHP have lower Rs than that observed with a commercial HP preparation (not shown). It is worth mentioning that we did not chemically characterize (DHP) preparations, but believe that this complex mixture of different carboxylic porphyrins may explain why uroporphyrin (8-COOH) show better localization than HPD and'also why heptacarbox-
A novel H P derivative
ylic porphyrin isomer I (7-COOH) has been shown to be a good t u m o r localizer as recently reported by El-Far et al. [33]. Our findings reported herein have opened the door to future research to synthesize more selective porphyrins and to improve the efficacy of phototherapy using lasers. The amplification of tumor porphyrin levels by D H P has a major potential clinical application in the treatment of tumors for which tumor localization is of lesser importance, for example, ocular tumors. Some techniques allow light to be aimed with such accuracy through the lens of the eye, that the targeting of light energy allows no stray light to fall upon adjacent n o n - t u m o r tissue. The same advantages apply to photoradiation therapy, where light is administered interstitially, i.e., through a fiberoptic embedded in the tumor. Since the extent of necrosis is a function both of the. log of the total energy administered in joules and the dose ofporphyrin given [34], a spectacular therapeutic advance could be obtained due to the enhancement of tumor porphyrin uptake in using DHP preparations. This study will pave the way to refining porphyrin augmented phototherapy using lasers and the usefulness of new porphyrins or their derivatives as diagnostic markers of early cancer. Clearly, more information is needed on DHP d o s e - r e s p o n s e and toxicity to different tissues with and without light. At-~-nrttirag t,~ th~ ninn~,~rino et,,ttv taFRpnenn and his recent report [35], porphyrins, such as HPD have been investigated for their possible use in the diagnosis and treatment of h u m a n bladder cancer and shown to be effective in treating some types of it. Ultimately, more information on the relationship between the structures of porphyrins, especially uptake and retention in tumoral and normal tissues is needed. Such information would permit the deliberate design of more porphyrins with superior retention in tumors and minimal affinity for other tissues or organs. • i~el~egVI
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Acknowledgments Dr. M. El-Far wishes to express his thanks to Professor Kevin Smith, Department of Chemistry, University of California, Davis, U.S.A., for reviewing the chemistry part in this paper and for his keen interest at all times. The Fulbright grant awarded to Dr. ElFar towards presenting this work is also greatly appreciated.
257
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Zorat P. & Calzavera F. (1979) Tumori 65,. 425 25 Musser D.A., Wagner J.M. & Datta-Gupta N. (1978) J. NatL Cancer Inst. 61, 1397 26 El-Far M., lbrahim H. & Ghoneim M. (1986) Proc. 1st Chem. Conf., Fac. Sci. Mansoura University. Egypt, pp. 93=107 27 Kessel D. (1981) Cancer Res. 41,'1318 28 Berenbaum M.C., Bonnet R. & Scouridcs P.A. (1982) Br. J. Cancer 45, 571 29 Dougherty T.J. (1984) in : Porphyrin in Tumour Phototherapy (Andreoni A. & Cubeddu R. eds.), Plenum Press, New York, p. 25 30 Dougherty T.J., Boyle D.G., Weishaupt K.R., Henderson B.A., Potter W.R., Bellnier D.A. & Wityk K.E. (1983) in: Porphyrin Photosensitiza-
31 32
33 34 35
tion (Kessel D. & Dougherty T.J., eds.), Plenum Press, New York, p. 3 Musselman B.D. & Chang C.K. (1986) in: Po "phyrin Photosensitization Workshop, L.A., 26-27 June 1986 Abstr. 34 Scourides P.A., Bohmer R.M., Kaye A., Ngu M., Zhu S., Gogerly R. & Morstyn G. (1986) in:Porphyrin Photosensitization Workshop, L.A., 26-27 June 1986 Abstr. 46 El-Far M.A. (198.7) 2nd Congress of the European Society ,for Photobiology, Italy, September 1987, Abstr. 175 Pimstone N.R., Homer I.J., Shaylor-Billings J. & Gandhi S.N. (1982) Lasers Med. Surg. 337, 60 Benson R.C. (1986) Eur. UroL 12, Suppl. 1, 47
251
Research article
Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model" a novel hematoporphyrin derivative * Mohamed EL-FAR I, M. ABOU-EL-ZAHAB ~, M. GHONEIM 2 and E. IBRAHIM 2
1Faculty of Science, Chemistry Dept., and :Urology and Nephrology Centre, Mansoura University, Egypt (Received 25-6-198L accepted after revision 4-11-1987)
S u m m a r y - Application of laser irradiation with porphyrin(s) or their derivatives for the destruction of tumors in humans requires preliminary studies of their localization in normal and malignant tissues. A novel derivative of hematoporphyrin (HP) was prepared. The newly developed hematoporphyrin (DHP) was administered to Fisher rats with bladder tumors and showed greater accumulation in the tumoral tissues. Comparative data oh (HP) and (DHP) are presented and discussed in light of the enhanced tumor porphyrin uptake caused by these agents. The homogeneous intense fluorescence noted with DHP-treated animals suggests that total tumor kill curative therapy will be more feasible. The study paves the way to refining increased porphyrin augment phototherapy and laser application in the field of oncology. bladder cancer / hematoporphyrin derivative / phototherapy / porphyrins
Introduction The interaction of porphyrins with normal and malignant cells is the subject of intensive investigations. Porphyrins have been known to display a selective affinity for neoplastic tissues. This property prompted the use of porphyrins as tumor localizers and has been exploited both for diagnosis and treatment. Diagnosis is dependent upon the property of porphyrins to fluoresce when excited by blui~ light. Thus, endoscopic excitation of premalignant lesions containing hematoporphyrin derivative (HPD) has led to their detection in the bladder. On the other hand, the ability of porphyrins to cause irreversible photodynamic
damage to cells and tissues, upon radiation with visible light, was utilized to induce the regression of tumors, i.e., photoradiation therapy (PDT). Initial reports on PDT of human bladder tumors go back to 1975, when Kelly et ai. [1, 2] reported on the successful photodynamic destruction of bladder tumor tissue by means of endoscopically controlled exposure to a mercury vapor lamp following the intravenous administration of HDP. Several thousand patients have now been treated with generally promising results [3]. Porphyrins are a group of heteroaromatic compounds, whose nucleus is relatively large and hydrophobic, and hence porphyrins tend to be only slightly soluble in water, unless func-
*An abstract of this work was accepted for presentation at the Clayton Foundation Conference on Photodynamic Therapy, held in Los Angeles, CA, U.S.A., February 1987. Abbreviation: FANFr: N-[4o(5-nitro-2-furyl)-2-thiazolyl] formamide.
252
M. El-Far et ai.
tional groups conferring water solubility have purposely been introduced. To overcome this, Lipson and Baldes [4] described the preparation of hematoporphyrin derivative [HPD]. They treated hematoporphyrin (HP) with sulfuric acid in acetic acid to give a solid material named HPD. Before use, it should be treated with alkali which was found to cause chemical changes in the solid material [5]. Alkali-treated hematoporphyrin derivative was first employed in this way by two groups: Dougherty et ai. [6] and Kelly et ai. [1]. Since that time, there has been a great deal of interest in photodynamic therapy, i.e., the use ofporphyrin as photosensitizers plus laser light in the treatment of tumors in patients. Application of HPD in clinical oncology was initiated by Dougherty and coworkers, and the activity in this field has rapidly expanded to its present high level. El-Far and Pimstone [7-11] were the first to demonstrate that uroporphyrin I (UP.I) and uroporphyrin Ill (UP.Ill) were efficiently and selectively retained in a transplantable mouse mammary carcinoma. They also showed that UP.I alone can effect photodynamic necrosis of tumor at high intensities using an argon dye laser. Their findings were recently confirmed by other groups [12-14]. El-Far and Pimstone [9, 11] using 28 different porphyrins having different chemical structures and side chains, found that only HPD, tetraphenylporphinesulfonate (TPPS4), tetracarboxyphenylporphine (TCPP), and UP.I and UP.III could be selectively retained in transplantable mouse mammary carcinoma. These 4 porphyrins differ significantly and considerably in their properties. HPD, like its crude parent compound HP, is a complex mixture of dicarboxylic porphyrins. Moan et al. [15] using high performance liquid chromatography to separate HP demonstrated the presence of 3 major porphyrin fractions and at least 17 additional minor components. On the other hand, UP has 8 aliphatic carboxylic side chains, while TPPSa has 4 sulfonatophenyl groups and TCPP has 4 carboxylic groups. Clearly much more information on the relationship between the structures of porphyrins and their selective uptake and localization in tumor cells and normal cells is needed. Such information should allow us to design a porphyrin with very high specificity towards the malignant cells, i.e., maximal retention in tumors with minimal retention in other normal tissues, especially skin.
The present work was undertaken to determine the tumor localizing potential of a newly developed porphyrin derivative from HP, using a bladder tumor model. Additional carboxylic groups were purposely added in an effort to explore the effects of certain specific functional groups on the tumor localizing efficiency and uptake of porphyrin by malignant cells. Previous studies by El-Far and Pimstone [7] showed that tumor uptake of porphyrin may in part be governed by a poor binding affinity to circulating plasma proteins. Also, selective porphyrin retention may be due to a relatively greater affinity for tumor protein constituents [10].
Materials and methods Chemical compounds
HP was obtained from porphyrin products (Logan, UT, U.S.A.). A stock solution was prepared in a final concentration of 5 mg/ml. Porphyrin material was first dissolved in 0.1 N NaOH. After 1 h of stirring at room temperature the solution was neutralized to pH 7.2 using 0.1 N HC! and then adjusted to its final concentration with 0.9% NaCI solution [16]. Developed hematoporphyrin preparations
HP was condensed with ethyl diazoacetate in ether solution and in the presence of HCI to give the developed hematoporphrin. Ethyl diazoacetate (EDA). was prepared according to the method of Sllberrad [17]. Briefly, a few crystals of sodium acetate were dissolved in 20 ml of water. Ethyl glycine hydrochlor i d a ( l f l o~ a n d 7 ~ g n f ~ n c l i n m n i t r i t a w a r a t h a n a d d . . . .
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ed. The reaction mixture was placed in an ice container until the temperature fell to about 0°C. Two drops ofsulfuric acid (10%) and then 20 ml ofether were added. As soon as the reaction had taken place, the ethereal solution of EDA was removed (run off). Then the EDA solution in ether was adjusted to 20 ml using fresh diethyl ether. 50 mg of l i P were dissolved in 1 ml of conc. HCI and cooled to 0°C. The cold solution of EDA was added to the acidic IIP solution. DHPI was prepared by adding 1 ml of ethereal solution containing EDA to 50 mg of l i P in liCl. DliP2 was prepared by adding 2 ml of ethereal solution containing EDA to another 50 mg of l i P in HCI. DHP3 was prepared by adding 3 ml of ethereal solution containing EDA to another 50 mg of l i P in acidic solution. In all cases, the condensation reaction between l i p acidic solution and ethereal solution containing EDA should be carried out under strict cold conditions" the temperature should be kept around 0°C. In one experiment with DHPb condensation was carried out at room temperature" upon adding ethereal EDA'to the HP solution, the
253
A novel H P derivative
temperature rose to over 50°C. The resulting preparation was named (DHPI)*. After condensation took place, the reactant materials were left to stand at room temperature for 2 days in the dark, and then evaporated to dryness using an electric fan. The resulting dry product was then subjected to NaOH and prepared as described before. The porphyrin solutions were kept in a dark glass bottle at 4°C for 5 weeks before use. In some experiments, we varied the concentration of NaOH due to the presence of a more acidic character in our developed HP preparations. Indeed, our condensation reaction between EDA and HP would be in agreement with the results of Johnson et al. [18] who reported the condensation of EDA with cobalt octaethyl porphyrin in acidic medium. The scheme of our one reaction flask could be presented as follows" 1) HCI + NH2CH2COOC2H5 + NaNO2 -HC____~I CIN=N-CH2COOC2H5 2) ~NHCIN=N=CH2-COOC2H5-HCI_~ N2 ~N-CH2-COOC2H2 ~HC! N-CH2-COOH Tumor model The tumor model was the transplantable bladder tumor in Fisher rats. Primary bladder tumors induced in Fisher 344 inbred rats by F A N F T were transplanted into syngeneic rats by orthotopic injection of bladder tumor cells into the bladder submucosa. A majority of tumors were well-differentiated transitional-cell carcinomas. The technique of this orthotopic implantation as well as usefulness of this tumor model for bladder cancer studies were previously described by Ibrahiem et al. [19]. Approximately 2 months after tumor transplantation, the rats were given an intra-cardiac injection of either the original HP or the developed derivatives dis-
solved as described before and given at the desired dosage in a volume of about 0.1 ml. At that time, none of the tumors exhibited necrosis as determined by routine histological studies. At specified times after the injections, the rats were killed and the t u m o r and other tissues were rapidly excised. A m i n i m u m of 4 tumor-bearing animals were used for testing each porphyrin in most cases. Porphyrin localization was assessed at specified times after injection. The rats were killed, tumor and other tissues were rapidly excised. The tissues were then weighed and analyzed immediately. Tumors were excised in toto, divided in two with a scalpel so that the surface and cross section, red porphyrin fluorescence could be assessed by the naked eye following photoexcitation with a blackray long wavelength ultraviolet lamp (Model B-100A, UV Products Inc., CA, U.S.A.). Porphyrin fluorescence was similarly assessed in the skin and other organs. Porphyrins were extracted and quantitated according to the technique of El-Far and Pimstone [7]. Briefly, each tissue was blended and homogenized with 1 N aqueous perchloric acid and methanol (1:1, v/v) in the dark, using an electric homogenizer with a teflon pestle. The homogenate was then centrifuged and the tissue precipitate was re-extracted until no fluorescence was observed under UV light. The combined supernatant was filtered through Whatman no. 1 filter paper and its volume recorded. The supernatant content was quantitated fluorometrically and determined, using the porphyrin under study as the standard under optimal conditions for photoexcitation and fluorescence emission.
Results and Discussion T h e r e s u l t s are s u m m a r i z e d a n d p r e s e n t e d in T a b l e s I a n d II. P o r p h y r i n c o n t e n t in tissues is e x p r e s s e d as ~ g i g wet weight. O b v i o u s t u m o r f l u o r e s c e n c e was n o t e d in all a n i m a l s injected with d i f f e r e n t p o r p h y r i n p r e p a r a t i o n s . In t h e g r o u p o f a n i m a l s i n j e c t e d with D H P preparat i o n s 1,2 a n d 3, we n o t e d t h a t D H P was p r e s e n t
Table I. Tissue porphyrin contents in/~g/g wet weight 24 h post-injection with a dose of 20 mg/kg of body weight. Material (5 mg/ml)
Bladder tumor
Skin
HP DHPI* DHPI DHP2
3.7 3.1 6.8 12.4
1.7 1.8 3.1 3.2
_ _ ± ±
2.1 a (7) b 1.2 (5) 3.7 (5) 1.7 (4)
-4- 0.7 ± 0.6 ± 0.6 ± 0.5
Liver (7) (5) (5) (5)
8.2 9.0 12.9 21.6
± ± ± ±
Kidney 3.7 2.1 2.2 1.7
(6) (5) (6) (4)
a Mean + S.D. b Numbers between parentheses = number of samples analyzed (1 sample/rat).
5.1 5.6 11.1 22.5
± 1.7 ± 2.2 _+ 2.2 +__ 1.7
Spleen (6) (5) (6) (4)
3.2 2.4 3.0 8.4
± 2.2 ± 0.6 _ 1.4 __. 1.7
(7) (5) (6) (4)
M. El-Far et al.
254
Table il. Tissue porphyrin contents in ag/g wet weight at 15 h post-injection with a dose of 15 mg/kg of body
weight. Material (5 mg/ml)
Bladder tumor
Skin
HP DHPI DHP2 DHP3
5.4 __. 2.2a (4) b 9.2 + 0.9 (3) 8.3 _ 4.2 (4) 18.2 __. 1.6 (2)
2.3 __ 1.1 3.9 _ 0.6 3.9 +_ 1.1 4.2 __+0.6
(3) (4) (3) (2)
Liver
Kidney
Spleen
"8.7 _____4.6 (4) 11.1 _ 2.3 (4) 12 +_.4.8 (4) 14.3 ___ 1.2 (2)
4.8 _____3.8 (4) 9.4 _+ 1.3 (4) 14.4 _+ 1.6 (4) 17.2 +_ 2 (2)
4.3 _____2.2 (4) 5.9 _ 1.9 (4) 6.3 ___ 1.6 (4) 5 ___ 1.5 (2)
a Mean _+S.D. b Numbers between parentheses = number of samples analyzed (1 sample/rat).
in tumor tissues in amounts far in excess to that observed with a commerical HP preparation. Table I shows that the porphyrin contents in tumors of animals treated with DHP3 were about 3-fold greater that those in tumors from HP-treated animals. Also, porphyrin contents and tumor uptake were found to be increased with increasing reaction molar ratios, i.e., DHP3 was more highly represented in the tumor than DHP2 and DHPn. We have also noted that DHPI obtained under cold conditions of preparation showed a distribution different from that obtained and prepared under higher temperature. The latter preparation showed exactly the same distribution as that observed for the HP solution. This would simn!v ..... .- , demonstrate that the chemical condensation did occur under cold conditions, as recommended, and enhanced uptake in the tumor would be mainly attributed to the chemical change which may be due to the presence of more carboxylic groups added to the porphyrin skeleton. It was interesting to note that the naked eye distribution of tumor fluorescence was altered when DHP preparations 1, 2 and 3 were used. Tumor fluorescence in animals treated with DHP2 and DHP3 is homogeneous through out the tumor and more intense, whereas with commercially prepared HP, heterogeneous fluorescence and intensity were observed. This is not a novel observation, we have previously noted that tumor fluorescence with HPD in 30% of the cases was not homogeneous (patchy) but was more intense in the periphery than in the center of the tumor [7, 9, 11]. Similar observations have been reported in rodent tumors, following HPD injection [20] as well as in human patients with bronchial carcinoma receiving HPD [21]. We believe that this patchy
fluorescence might be correlated to the remarks made by Dougherty [22], as it is not clear how the biological phenomena of localization and photosensitization are related to the porphyrin compt, nents in the mixt.ures, or why apparently similarly prepared injectable solutions show different biological activities. The observations of Kreimer-Birnbaum et al. [23] concerning HPD are consistent with the previous findings. DHP preparations showed a greater porphyrin concentration in the tumor and no patchy fluorescence was observed. The clinical benefit of using DHP is the enhancement of tumor porphyrin uptake. Since it is known that HPD does not cause uniform tumor fluorescence, islands of viable tumor tlnl~ilL
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therapy. The homogeneous intense fluorescence noted in our experimental animals given DHP suggests that total tumor kill and curative therapy will be more feasible than with current techniques. Some investigations have focused on the time dependency of the ratio between the tissue porphyrin concentration in the tumor and correlate it to the skin, where the primary interaction between the incident radiation and the phototreated patients takes place in skin tumors. Analysis of our results demonstrate that at 15 h post-injection of 15 mg/kg, a substantial difference existed between the a~:)ur~t~ ofporphyrin in the tumor and the skin (Table I). The tumor to skin ratio with commercial HP is 2.37, while, in the case, of DHP3 it is 4.30, i.e., an increase of 44%. A substantial difference was also observed between the amounts of porpl:yrin in the tumor and skin, 24 h post-injection of 20 mg/kg (Table II). In this particular case, the tumor to skin ratio with commercial HP was 2.16, while it was 3.85 with DHP2. It is worth mentioning that the ratio was
255
A novel H P derivative
herty [16] injected 3H- and 14C-labeled HPD into tumor-bearing mice and found that the concentration of HPD was higher in liver and kidney than in the tumor. On the other hand, the porphyrin load in the skin, in cases of DHP preparations, can be effectively reduced and partially eliminated after the diagnostic and therapeutic maneuvers are completed by administration of oral charcoal or by means of a blood exchange technique. We have recently demonstrated that unwanted porphyrins can effectively and significantly be eliminated from the circulatory system and, as a consequence, reduce skin porphyrins [26]. Previous reports suggest that porphyrin localization in tumors may depend upon the relative concentrations of lipophilic and hydrophobic molecules administered• It has been shown that the affinity of porphyrins for cultured cells and their subcellular distribution is dependent upon their hydro- or lipo-solubility [27] On the other hand, Jori et al., [14] have extended the study in vivo and showed that i.p. injection of liposome-bound porphyrins into
also increased by nearly the same value as found in the lower dose (15 mg/kg, 15 h). Further studies are under way to identify the optim u m conditions. It is well known that the dose of the injected porphyrin could be a key factor in its distribution among different organs [24]. Similar conclusions h a v e been previously reported by Musser et al. [25] who investigated TCPP and TPPS4 in mice bearing solid L1210 tumors and sarcoma 180 tumors• They concluded that lowering the dose of TPPS4 did not alter the overall distribution pattern but did improve the t u m o r : t i s s u e ratios, while TCPP had the opposite effect. This may suggest that an investigation at earlier time periods following injection might have been better when low doses of DHP were used. In general, the tumor retained considerably more of the DHP preparations (Tables I and II) than the commercially prepared HP over the 15 and 24 h periods (Fig. 1). Our results also demonstrate that liver and kidney tissues retain greater quantities than the tumor of all preparations of DHP as well as commercial HP. Previously, Gomer and Doug-
20 Z
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HP
DHPI
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~-
16
z
14
z
12
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==
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.= ,,,= .=
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Fig. 1 Bladder tumor porphyrin cuntents over 15 and 24 h periods.
256
M. El-Far et al.
mice bearing a MS-2 fibrosarcoma resulted in a remarkably more efficient tumor targeting than that obtained with the administration of the same porphyrin dissolved in a homogeneous aqueous solution. They indicated also that the subcellular distribution of liposome-delivered porphyrin is also dependent upon its solubility properties, thus confirming Kessel's findings. El-Far and Pimstone [7, 9, 11] reported the superiority of uroporphyrin, a water soluble porphyrin, over HPD in tumor localization in this regard, using a transplantable mammary carcinoma in the BALB/c mouse as the tumor model. Our present results in this regard demonstrate that tumor porphyrin uptake is greater with the newly developed hematoporphyrin which is richer in hydrophilic characteristics, than commercial HP. A major disadvantage of HPD is that it is not fully chemically characterized. A number of theories have been proposed as to the metabolic basis for tumor uptake of HPD and its major active component. Many investigators have sought to separate the components of HPD, characterize them and identify which ones localize in and photosensitize neoplastic tissue. Perhaps the most provocative and perplexing studies were those reported'by Berenbaum et al. [28] and Dougherty [29]. The early studies by Berenbaum et al. [28] suggested the presence of ether linked dimers or oligomers in the h,,-~n,il,llllUl
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[30] showed that at least 60% of the product consists of porphyrins which are not localizers • hematoporphyrin (HP), hydroxyethylvinyldeuteroporphyrin (HVD), and protoporphyrin (PP). In 1984, he suggested that the major tumor localizing component of HPD is a dihematoporphyrin ether. In 1986, two groups confirmed the presence of the ether-linked HP dimer [31, 32]. The chemical and biological studies by Musselman et al. [30] also suggested the presence of the ether bond and not an ester. While this is an attractive possibility it would not explain why the 8 carboxylic porphyrin (UP.I) shows a superiority over other porphyrins, as reported by El-Far and Pimstone and supported by others. Thus, we propose that DHP could be classified as a 4-carboxylic-HP, but it is richer in a fraction of porphyrin localizer. In agreement with the dimer proposed by others, it is tempting to speculate that a DHP dimer fraction could consist of two DHP molecules joined via an ether linkage (Fig. 2). This porphyrin dimer would contain 8 carboxylic
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II
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groups like UP, but their distribution is different. There have been many reports on the reverse-phase HPLC analysis of HPD [5, 15, 28] and significant difficulties were reported by some of them in identifying the active component in its mixture due to the retention of a major part of HPD by the column during separation. That is why we did not try to fully investigate the chemical changes. This study was designed only to look at the effect of EDA condensation of HP on porphyrin localization and retention in the tumor. Our thin-layer chromatography studies showed that the major fractions of DHP have lower Rs than that observed with a commercial HP preparation (not shown). It is worth mentioning that we did not chemically characterize (DHP) preparations, but believe that this complex mixture of different carboxylic porphyrins may explain why uroporphyrin (8-COOH) show better localization than HPD and'also why heptacarbox-
A novel H P derivative
ylic porphyrin isomer I (7-COOH) has been shown to be a good t u m o r localizer as recently reported by El-Far et al. [33]. Our findings reported herein have opened the door to future research to synthesize more selective porphyrins and to improve the efficacy of phototherapy using lasers. The amplification of tumor porphyrin levels by D H P has a major potential clinical application in the treatment of tumors for which tumor localization is of lesser importance, for example, ocular tumors. Some techniques allow light to be aimed with such accuracy through the lens of the eye, that the targeting of light energy allows no stray light to fall upon adjacent n o n - t u m o r tissue. The same advantages apply to photoradiation therapy, where light is administered interstitially, i.e., through a fiberoptic embedded in the tumor. Since the extent of necrosis is a function both of the. log of the total energy administered in joules and the dose ofporphyrin given [34], a spectacular therapeutic advance could be obtained due to the enhancement of tumor porphyrin uptake in using DHP preparations. This study will pave the way to refining porphyrin augmented phototherapy using lasers and the usefulness of new porphyrins or their derivatives as diagnostic markers of early cancer. Clearly, more information is needed on DHP d o s e - r e s p o n s e and toxicity to different tissues with and without light. At-~-nrttirag t,~ th~ ninn~,~rino et,,ttv taFRpnenn and his recent report [35], porphyrins, such as HPD have been investigated for their possible use in the diagnosis and treatment of h u m a n bladder cancer and shown to be effective in treating some types of it. Ultimately, more information on the relationship between the structures of porphyrins, especially uptake and retention in tumoral and normal tissues is needed. Such information would permit the deliberate design of more porphyrins with superior retention in tumors and minimal affinity for other tissues or organs. • i~el~egVI
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Acknowledgments Dr. M. El-Far wishes to express his thanks to Professor Kevin Smith, Department of Chemistry, University of California, Davis, U.S.A., for reviewing the chemistry part in this paper and for his keen interest at all times. The Fulbright grant awarded to Dr. ElFar towards presenting this work is also greatly appreciated.
257
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31 32
33 34 35
tion (Kessel D. & Dougherty T.J., eds.), Plenum Press, New York, p. 3 Musselman B.D. & Chang C.K. (1986) in: Po "phyrin Photosensitization Workshop, L.A., 26-27 June 1986 Abstr. 34 Scourides P.A., Bohmer R.M., Kaye A., Ngu M., Zhu S., Gogerly R. & Morstyn G. (1986) in:Porphyrin Photosensitization Workshop, L.A., 26-27 June 1986 Abstr. 46 El-Far M.A. (198.7) 2nd Congress of the European Society ,for Photobiology, Italy, September 1987, Abstr. 175 Pimstone N.R., Homer I.J., Shaylor-Billings J. & Gandhi S.N. (1982) Lasers Med. Surg. 337, 60 Benson R.C. (1986) Eur. UroL 12, Suppl. 1, 47