Photodiagnosis and Photodynamic Therapy (2007) 4, 13—25

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Research and development of photodynamic therapy photosensitizers in China De-Yu Xu PhD ∗ Shanghai Institute of Red-Green Photosensitizers, Xiang Yin Road #20, 4th floor, Shanghai 200433, The People’s Republic of China Available online 13 November 2006

KEYWORDS Photosensitizer; Photodynamic therapy; China

Summary Photodynamic therapy (PDT) started in the People’s Republic of China in the early 1980s after hematoporphyrin derivative (HpD) was used in China as PDT photosensitizer in clinical PDT for the treatment of various cancers. Since then, numerous domestic photosensitizers have been synthesized and evaluated. In general, the research and development of PDT photosensitizers in China can be divided into two stages. Firstly, attention was focused on the development of mixed porphyrin preparations similar to HpD and Photofrin II, and the second stage was searching for new photosensitizers with definite structures. In the past 2 decades three mixed porphyrin preparations and a series of new photosensitizers with different structures were developed and entered into formal clinical trials in China. This manuscript will introduce past research and development activities in China and present published preclinical and clinical data of some promising photosensitizers. © 2007 Published by Elsevier B.V.

Introduction It is well known that modern photodynamic therapy (PDT) is established along with the discovery and development of new photosensitizers and light sources. In China, PDT basic studies and clinical trials started in the early 1980s after learning of the successful work of Dougherty [1]. At first, Photofrin II and domestically produced hematoporphyrin derivatives (HpD) were used in China as PDT photosensitizer in the treatment of various cancers. Hereafter the Chinese began their own studies on searching for new photosensitizers. In general, there might be two stages in the research and development of new photosensitizers in mainland China. Firstly, attention was focused on the development for mixed porphyrin preparations similar to HpD and Photofrin II, and the second stage was searching for new photosensitizers with definite structures. Moreover, during the past 2 decades



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three mixed porphyrin preparations and a series of new photosensitizers with different structures have been developed and used in mainland China. Here, a brief introduction of the research and development of domestic PDT photosensitizers and published preclinical and clinical data of some promising domestic photosensitizers will be presented.

Mixed porphyrin preparations There were three mixed porphyrin preparations developed for antitumor PDT during the early 1980s in China. One called Beijing Hematoporphyrin Derivative (BHpD, Aibulin or HPS) which was prepared by Beijing Institute of Pharmaceutical Industry [2]. The other was called Yangzhou Hematoporphyrin Derivative (YHpD or HA308) and prepared by Yangzhou Biochemical Medicinals Co. Ltd., and the third named Photocarcinorin (PsD-007) and prepared by Xu et al. BHpD has been used in clinical PDT for the treatment of various cancers with permission from National Ministry of Health. Its commercial brand name is Hematoporphyrin

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D.-Y. Xu

Injection. Like HpD, it is a mixed porphyrin preparation, but the chemical composition and structures of its main components have not yet been published. YHpD can be prepared using protoporphyrin as the starting material and purified by means of P-10 Biogel chromatographic separation. It is an imitative product of Photofrin II. YHpD consists of different porphyrins, and five of them have been isolated and their structures are identified as methyl esters. YHpD appears as a single dark purple band on Biogel P-10 column. HPLC analysis shows that its main components are hematoporphyrin, 3(or 8-)hydroxyethyl-8-(or 3-)vinyldeuteroporphyrin (HVD), di-hematoporphyrin ethers (DHE, accounted for about 26% of the total amount), 3-(or 8-)-(1-hydroxyethyl)8-(or 3-)(1ethoxyethyldeuteroporphyrin) and protoporphyrin [3—6]. Comparative studies show that the photodynamic effect of YHpD is more potent than that of Photofrin II [7]. PsD-007 is relatively new. It received the regulatory approval from the Army Health Administration of China in 1983 after the completion of several preclinical and clinical studies and has been used widely in China for the treatment of cancer [8—18]. Its preparation, chemical composition, structure of main active components, photophysicochemical properties, antitumor photobiological activities, principal pharmacotoxicological parameters and clinical efficacies will be presented in detail in the following sections.

Preparation, chemical composition and structures of the main components of PsD-007 Authors note that commercial hematoporphyrin (Hp) contains almost all the active components of HpD but in much lower concentration, suggesting the active components may be formed during the preparation and separation of Hp. PsD-

Table 1

007 is prepared directly from hemin by removal of the iron ion and addition of hydrogen bromide to the vinyl groups of protoporphyrin and subsequently producing the key intermediate 3,8-di(1-bromoethyl)-deuteroporphyrin, which is hydrolyzed to a mixture of porphyrins consisting of hematoporphyrin and its mono- or dioxyacetyl derivatives, 3(or 8-)-(1-hydroxyethyl)-8(or 3)-vinyldeuteroporphyrin and protoporphyrin. The later two are formed through hydration and dehydration equilibrium of hematoporphyrin. It is a purple red solid. Akyloxy fission reactions take place when this solid is refluxed with methanol and the oxyacetyl derivatives of Hp are subsequently transformed to their corresponding methoxy derivatives: 3-(or 8-)-(1-hydroxyethyl)-8(or 3-)-(1-methoxyethyl)-deuteroporphyrin (MHD), 3-(or 8-)-(1methoxyethyl)-8-(or 3-)-vinyldeuteroporphyrin (MVD) and di(1-methoxyethyl)deuteroporphyrin (DMD) (Table 1). This is the crude product of PsD-007. It can be obtained in a yield of 30% after removal of most of the hematoporphyrin presented in the crude product through chromatography. Fig. 1 shows the reactions in the preparation of PsD-007 [14]. UV—Vis absorption and fluorescence emission spectra of aq. solution of sodium PsD-007 and other two mixed porphyrin preparations Photofrin II and YHpD have been determined. The Soret peak of PsD-007 differs significantly from those of the other two at the concentration of 10 ␮g/ml (Table 2). The ability of PsD-007 and its main active components for photogenerating singlet oxygen has been evaluated according to the increasing intensities of ESR signal of nitroxyl free radical products. The relative quantum yields of singlet oxygen are: PsD-007: 1 ± 0.07, MVD: 1.10 ± 0.10 — maximum possible yield of singlet oxygen is 1.0, MHD: 0.75 ± 0.06 and DMD: 0.70 ± 0.06, respectively [15]. Data of photooxidation of NADPH in D2 O by PsD-007 and its main active components are listed in Table 3 [14].

Chemical composition of PsD-007 and structures of its main components

Name

Structures

RT in HPLC (min)

R1 3-(or 8)-(1-Methoxyethyl)-8-(or 3)-hydroxyethyldeuteroporphyrin (MHD) 3-(or 8)-(1-Methoxyethyl)-8-(or 3)-vinyldeuteroporphyrin (MVD) 3-(or 8)-(1-Hydroxyethyl)-8-(or 3-)vinyldeuteroporphyrin (HVD) 3,8-di-(1-Methoxyethyl)deuteroporphyrin (DMD) Hematoporphyrin (Hp) Protoporphyrin (Pp)

R2

CH(OCH3 )CH3

or CH(OH)CH3

CH(OH)CH3

CH(OCH3 )CH3

2.879

CH(OCH3 )CH3

or CH CH2

CH CH2

CH(OCH3 )CH3

3.901

CH(OH)CH3

or CH CH2

CH CH2

or CH(OH)CH3

2.991

CH(OCH3 )CH3

CH(OCH3 )CH3

CH(OCH3 )CH3

CH(OCH3 )CH3

3.390

CH(OH)CH3 CH CH2

CH(OH)CH3 CH CH2

CH(OH)CH3 CH CH2

CH(OH)CH3 CH CH2

2.610 4.571

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Figure 1 Reaction of the synthesis of PsD-007. 1: Hemin; 2: 3,8-di(1-bromoethyl)-deuteroporphyrin: 3: hematoporphyrin; 4: 3(or 8)-(1-oxyacetylethyl)-8(or 3)(1-hydroxyethyl)deuteroporphyrin; 5: 3,8-di(1-oxyacetylethyl)deuteroporphyrin; 6: 3(or 8)-(1hydroxyethyl)-8(or 3)-vinyldeuteroporphyrin; 7: protoporphyrin; 8: 3(or 8)-(1-oxyacetylethyl)-8(or 3)vinyldeuteroporphyrin; 9: 3(or 8)-(1-hydroxyethyl)-8(or 3)vinyldeuteroporphyrin; 10: 3(or 8)-(1-hydroxyethyl)-8(or 3)-(1-methoxyethyl)deuteroporphyrin; 11: 3.5di(1-methoxyethyl)deuteroporphyrin.

Antitumor photodynamic activity Table 2 The Soret peak and absorbance of PsD-007, Photofrin II and YHpD

Soret peak (nm) Absorbance

PsD-007

Photofrin II

YHpD

375 1.357

363 1.120

360 1.638

In vitro studies showed that 50% photoinactivations (IC50 ) of liver cancer cells (SMMC-7721) and gastric cancer cells (NGCC-8310) could be achieved at dose levels of 10 ␮g/ml and 6 J. At the same dose the photoinactivation of both cancer cell lines by Photofrin II was only 20% [16,17]. Data in Table 4 are the IC50 of human uterocervical cancer cells

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D.-Y. Xu

Table 3

Photooxidation of NADPH in D2 O %NADPH degradation at different concentrations of tested compound (×10−6 mol/l)

DMD MHD PsD-007 YHpD

MVD

44.54

29.69

19.79

13.20

8.80

63.25 ± 2.62 60.13 ± 1.03 66.23 ± 3.69 76.26 ± 0.66

78.81 ± 0.73 63.34 ± 0.22 76.95 ± 1.28 83.95 ± 0.69

85.05 ± 0.55 74.43 ± 0.66 86.73 ± 1.10 89.30 ± 0.43

86.81 ± 0.23 78.81 ± 0.51 88.40 ± 1.06 91.40 ± 0.40

99.39 ± 0.52 87.74 ± 0.79 93.14 ± 1.00 95.52 ± 0.47

24.59

16.38

10.92

7.28

4.87

69.93 ± 0.92

79.36 ± 1.77

85.18 ± 1.81

89.66 ± 0.27

97.32 ± 0.61

Table 4 Photoinactivation of Hela cells by the main active components of PsD-007a Component

IC50 (␮g/ml)

95% Confidence limit

Regression equation

MVD DMD MHD YHpD

8.92 15.51 6.30 11.70

8.06—9.87 4.15—16.97 5.56—7.11 10.16—12.73

Y = 3.63X − 1.56 Y = 4.71X − 0.85 Y = 3.84X + 1.69 Y = 3.91X + 0.56

a

Light source: argon ion laser (488, 514.5 nm); light dose: 4.28 ± 0.8 J/cm2 .

(Hela cells) by main active components of PsD-007 in vitro using YHpD as a reference [14]. Antitumor effect of PsD007 mediated PDT has also been demonstrated in various animal models. Experimental results from transplanted Sarcoma S180 , uterocervical cancer U14 in KM mice and Lewis lung cancer in C57 mice indicated a strong dose escalating effect [18]. To further identify the active ingredients of PsD007, the antitumor activity of several purified components were tested against Sarcoma S180 tumor. Results confirmed that MVD, DMD and MHD had a strong antitumor effect.

Pharmacotoxicological studies Acute toxicity [19—21]: Median lethal dose of PsD-007 in mice was determined using HpD as a reference. The i.v. LD50 and 95% CL of PsD-007 and HpD are shown in Table 5. Minimal lethal dose (MLD) of 171 mg/kg and approximate lethal dose (ALD) of 114 mg/kg have been determined in dogs. Observations of general conditions, toxic effects (daily), body weight and EKG (weekly) were recorded in a course of 14

Table 5

LD50 of PsD-007 and HpD in mice

Drug

Batch number

LD50 (mg/kg)

Confidence limit (95%, mg/kg)

HpD PsD-007 PsD-007 PsD-007

— 830421 830519 840413

140 202* 218* 210*

111—168 176—229 201—235 186—236

*

days post i.v. injection of 30, 60 and 120 mg/kg b.w. All the intoxication manifestations were similar to those seen in porphyria and they were dose-related and reversible. Laboratory tests showed that RBC, Hb and hematocrit were reduced; MCV, MCH and MCHC were somewhat abnormal, but there were no obvious changes in reticulocyte, leukocyte, differential leukocyte and platelet counts; nor in serum alkaline phosphatase, thymol turbidity test, serum billirubin, LDH, creatinin and glucose except that the activities of sGPT and PHI were temporarily elevated; urine pH remained in normal range and urinary protein increased in high dosage groups. EKG showed temporary S-T segment descending and T-wave inverting in high dosage groups. Histological examinations showed that damage to the liver and kidney were more obvious in high dosage groups. Subacute toxicity [20,21]: PsD-00 was given to rats and dogs at dose levels of 60, 30 or 15 mg kg−1 d−1 , respectively, for 14 consecutive days. The intoxication manifestations and changes in hematology, liver and renal function, EKG and organ weight were found to be similar to those observed in acute toxicity tests. They were dose-related, reversible and predicable. The principal pharmakokinetic parameters have been determined in rabbits post i.v. administration. They were: ˛ = 0.2634 h−1 , ˇ =0.0302 h−1 , A = 38.00 ␮g/ml, B = 9.17 ␮g/ml, K2,1 = 0.0755 h−1 , k1,2 = 0.1128 h−1 , t1/2 (˛) = 2.99 h and t1/2 (ˇ) = 22.95 h [9]. The pharmacokinetic equation therefore could be described as: C = A e−˛t + B e−ˇt or C = 38.00 e−0.2634t + 9.17 e−0.3021 . Phototoxic reactions of PsD-007 have been evaluated in KM mice at dose levels of 2.5, 5.0 and 10 mg/kg. Manifestations of phototoxicity observed in the normal tissues in mice included erythema, edema and necrosis of the ears. Death could occur in high dose groups. However, in comparison to HpDs and chloropromazine, phototoxic reactions of PsD-007 were relatively milder and disappeared more rapidly [22].

p < 0.01, compared with HpD.

Clinical trial data A phase I clinical trial was performed in Shanghai Changhai Hospital—–the affiliated hospital of Second Military Medical University in 23 patients (32—80 years old) suffering from carcinomas of skin, lungs, bladder, esophagus or nasopharynx. All the volunteers were subjected to a skin allergy test which had to be proved negative before drug administration. Patients were protected from direct exposure

Photosensitizer R & D in China

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to sunlight or any other strong light post drug administration. No side effects or obvious changes in clinical examinations and laboratory tests were reported [23]. Pharmacokinetic study was conducted in 11 patients using a fluorospectrometer. Data indicated a three-compartment model as determined by the drug—time curve through Akaike’s Information Criterion (AIC). Principal pharmacokinetic parameters of PsD-007 were: t1/2 = 0.26 ± 0.5 h; t1/2˛ = 4.52 ± 1.04 h; t1/2ˇ = 32.9 ± 5.31 h. It suggested that a single dose of 5 mg/kg was safe and sufficient, therefore being used in phase II trial. A phase II trial was performed in seven hospitals and on 145 patients suffering from cancers of bladder, lungs, esophagus, cervixuteri, stomach, colon, rectum, tongue, bucca, palate, lip, nasopharynx, jaw, tonsil, heel, brain and superficial cancers (basal cell, squamous, tarsal gland carcinomas and melanocarcinoma). PDT was carried out by argon laser irradiation after i.v. injection of PsD-007 (5 mg/kg). The preliminary results suggested that the efficiency of PDT for these cases was 95% except for the brain tumors. The rate of complete response (CR) was about 60% in a short-term

follow-up. The PDT effects observed on many of the superficial carcinoma were impressive, especially in basal cell carcinoma of glabella or palpebral margin due to the fact that not only did CR appear with tumor disappearance but also no scar was left on the treated site. In the treatment of bladder cancer, CR and partial response (PR) was 83% (15/18) and 16% (3/18), respectively. During follow-up, recurrences occurred within 3—9 months in three patients and all disappeared after repeat treatments. Besides, CR was observed in four cases of nasopharyngeal carcinoma who had failed radiotherapy and no recurrence was found in two cases during a follow-up of 18 months [24].

New photosensitizers with confirmed structures Hematoporphyrin ethers A series of hematoporphyrin ethers have been synthesized and tested by Xu et al. [11,25,26]. Their photodynamic

Table 6 Photoinactivation of human intestinum crassum cells (SW-1116) and leukeamia cells (K-562) by different hematoporphyrin ethers (␮g/ml) in vitro

No. of compound

1 2 3 4 5 6 7 8 9 10 11 12

50% Photoinactivation (IC50 ± 95% CL)

Structure R1

R2

SW-1116 cells

K-562 cells

C 2 H5 CH2 CH CH2

R1 R1 R1 R1 R1 C2 H5 ,(H) n-C3 H7 ,(H) i-C3 H7 ,(H) CH2 CH CH2 ,(H) CH2 CH2 OCH3 ,(H) CH2 CH2 OC2 H5 ,(H) CH2 CH2 OnC4 H9 ,(H)

1.057 ± 0.144 1.471 ± 0.070 1.012 ± 0.056 1.019 ± 0.070 1.399 ± 0.084 1.663 ± 0.111 2.487 ± 0.318 3.602 ± 0.476 3.716 ± 0.336 6.673 ± 0.681 7.628 ± 0.390 1.180 ± 0.050

0.464 ± 0.07 0.775 ± 0.176 0.932 ± 0.056 0.711 ± 0.050 0.366 ± 0.046 1.760 ± 0.098 3.573 ± 0.427 2.039 ± 0.136 3.743 ± 0.144 6.014 ± 0.678 4.133 ± 0.423 0.633 ± 0.043

6.215 ± 0.628

5.753 ± 0.841

1.053 ± 0.066 1.588 ± 0.100 2.051 ± 0.159 2.007 ± 0.266 4.117 ± 0.130 0.750 ± 0.065 0.997 ± 0.047 0.987 ± 0.055 1.100 ± 0.510 4.805 ± 0.510

0.970 ± 0.062 1.004 ± 0.050 0.722 ± 0.065 1.989 ± 0.166 1.584 ± 0.310 0.435 ± 0.037 0.534 ± 0.105 0.687 ± 0.065 0.615 ± 0.058 4.670 ± 0.066

CH2 CH2 OC2 H5

Cyclohexyl n-Butyl H,(C2 H5 ) H,(n-C3 H7 ) H,(i-C3 H7 ) H,(CH2 CH CH2 ) H,(CH2 CH2 OCH3 ) H,(CH2 CH2 OC2 H5 ) H,(CH2 CH2 OnC4 H9 )

13 14 15 16 17 18 19 20 21 22 HpD

H,(CH2 -Phenyl) H,(Cyclopentyl) H,(Cyclopentyl) H(Phenyl) CH3 ,(n-C4 H9 ) CH3 ,(Cyclopentyl) CH3 ,(CH2 ,CH CH2 ) CH3 ,(CH3 -Phenyl)

CH2 -Phenyl,(H) Cyclopentyl,(H) Cyclohexyl,(H) Phenyl,(H) n-C4 H9 ,(CH3 ) Cyclopentyl,(CH3 ) CH2 CH CH2 ,(CH3 )

CH2 -Phenyl,(CH3 )

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D.-Y. Xu

Figure 2

Structures of synthesized BpD.

effects were evaluated on leukeamia cells (K-562) and intestinum crassuim cancer cells (SW-1116). Some of them showed strong photodynamic effect (Table 6). Among them hematoporphyrin ethers 3- or 8-monomethyl ether (HMME) was studied in great depths since the early 1990s [27,28]. It has been approved by the State FDA for clinical trials for the treatment of port-wine stains (PWS).

Synthetic chlorins A series of new types of chlorins, such as dihydromesoporphyrin and dihydrodeuteroporphyrin, have been synthesized by reduction of mesoporphyrin and deuteroporphyrin, respectively. Animal study showed that the photodynamic effects of these synthetic chlorins were much higher than those of the corresponding porphyrins [31].

Benzoporphyrin derivatives Meso-tetra-substituted porphines Benzoporphyrin derivatives monoacid ring A (BpD-MA) and seven other new benzoporphyrin derivatives (Fig. 2) have been synthesized and tested in transplanted tumors by Yu et al. [29,30]. At least five of them, i.e. BpD-MA, 8-Me-BpD, 3-Me-BpD, 8-He-BpD and BpD-MB, showed strong antitumor effect in mice model. Photodynamic effects of BpDs with reduced ring A (e.g. BpD-MA, BpD-DA, 8-Me-BpD) and 8-HeBpD were higher than those of the corresponding reduced ring B (e.g. BpD-MB, Me-BpD) and 3-Me-BpD and 3-He-BpD. Enhanced photodynamic effect could be achieved if the vinyl group in position 3 or 8 was replaced by an alkyloxy or hydroxy group.

Figure 3

A series of new meso-tetra-substituted porphines (Fig. 3) has been synthesized by Wang and Xu [32]. The photosensitizing ability and antitumor photodynamic activity of the synthetic porphines were reported by Chen et al. [33] and Liu et al. [34]. Results of the determination of the singlet oxygen yield, sensitizing effect on photooxidation of NADPH in deuterium oxide, and photodynamic effect on transplanted animal tumors for some of these porphines are listed in Tables 7—9. The ability of singlet oxygen generation of most of the porphines tested was somewhat higher than those of mixed porphyrin preparations

Synthesis of meso-tetra-(substitutedaryl)porphines.

Photosensitizer R & D in China

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Table 7 Singlet oxygen yields of different porphines determined by means of ESR spectrometry

Table 8 in mice

Photodynamic effect of porphines on Sarcoma S180

Porphines

Dose (mg/kg)

Light dose (J/cm2 )

Cured/ treated

I-1a I-2a I-3a I-4a PsD-007

5 5 5 5 5

180 180 180 180 180

3/7 1/8 0/8 3/7 8/10

a

See structure in Table 7.

Degradation products of chlorophyll a

Porphines

Structure

I-1 I-2 I-3 I-4 I-5 I-6 II-1 II-2 II-3 YHpD

In view of the fact that chlorophyll and its degradation products exhibit the basic structure skeleton of chlorin, and their absorption in the red band of light is almost one order higher than that of porphyrins. Therefore, a lot of attention has been focused on the research and development of new photosensitizers from degradation products of chlorophyll a. The first chlorophyll preparation used for PDT in China was a mixture of degradation products of chlorophylls from silkworm excrement and was called CPD4 [35—38]. Although CPD4 has been used in some clinical trials, it might not be ideal for clinical PDT due to its complexity and undefined chemical compositions. It contains almost all the possible degradation products of chlorophyll a and chlorophyll b as well as other byproducts. These, of course, make critical limitation for its use in PDT. The main products of degradation of chlorophyll a are pheophorbide a, pyropheophorbide a, chlorin e6 and purpurin 18. Fig. 4 shows an outline of the degradation reactions of chlorophyll a. A series of derivatives of chlorophyll a degradation products such as chlorin e6 , purpurin 18 and chlorin p6 has been synthesized and tested by the authors [39—49]. Preliminary tests show that all these derivatives are active but some possess strong photodynamic effect. Chlorin e4 — a decarboxylation of chlorin e6 — exhibits extremely strong in vitro and in vivo photodynamic effects. A mixture of pyropheophorbide a and pheophorbide a could be prepared from acid degradation of silkworm excrement crude chlorophylls extract. However, it is hard to

1

Relative O2 yield

R1

R2

OCH2 COOH OCH2 COOH OCH3 CH3 SO3 Na N+ (CH3 )3  Cl SO3 Na SO3 Na

H OCH3 SO3 Na SO3 Na H H SO3 Na Cl Br

1.38 ± 0.10 1.19 ± 0.07 1.08 ± 0.08 1.18 ± 0.08 7.40 ± 1.04 1.25 ± 0.08 0.22 ± 0.02 1.23 ± 0.08 0.95 ± 0.05 1.00 ± 0.07

as HpD and PsD-007; among them TPPS showed the highest yield of singlet oxygen generation. Results obtained from determination of the sensitizing effect of porphines on the photooxidation of NADPH in deuterium oxide were parallel to the data of their abilities of singlet oxygen generation. On the contrary, the photodynamic effects of those porphines on transplanted animal tumor were significantly lower than that of HpD and PsD-007. In addition, a delayed phototoxic reaction on the normal skin of the tested animals could still be noticeable 1-month post porphine administration.

Table 9

Photooxidation of NADPH by different meso-tetra-(substitutedphenyl)porphines in D2 O

Compound

1 2 3 4 5 6 7 8 Reference drug

Structure

%NADPH degradation at different concentrations of tested compound (×10−6 mol/l)

R1

R2

44.54

4-SO3 Na 4-OCH2 COONa 3-OCH3 3-SO3 Na 3-SO3 Na 3-CH2 N-morpholinyl 3-CH2 N-piperidinyl 3-CH2 N(C2 H5 )2 YHpD PsD-007

H H 4-OCH2 COONa 4-OCH3 4-CH3 4-OCH3 4-OCH3 4-OCH3

0.85 33.47 67.47 21.53 55.25 86.29 19.24 26.78 76.27 66.23

29.69 ± ± ± ± ± ± ± ± ± ±

0.67 1.10 0.66 5.40 0.37 1.27 0.52 0.47 0.66 3.69

19.79

1.23 ± 0.45 71.36 27.95 63.84 92.33 35.93 42.49 83.95 76.95

± ± ± ± ± ± ± ±

1.10 5.29 0.44 1.91 0.41 0.69 0.69 1.28

4.70 35.49 74.54 32.81 72.02 94.31 50.96 54.11 89.30 86.73

13.20 ± ± ± ± ± ± ± ± ± ±

0.40 1.62 0.82 4.74 0.54 1.46 0.67 0.54 0.43 1.10

14.38 41.23 78.84 41.01 70.52 96.32 64.57 62.67 91.04 88.40

8.80 ± ± ± ± ± ± ± ± ± ±

5.97 3.00 1.70 4.63 0.33 0.17 1.68 0.90 0.40 1.06

34.73 43.19 84.52 55.12 80.86 98.08 79.57 76.36 95.52 93.14

± ± ± ± ± ± ± ± ± ±

5.27 1.54 0.17 2.11 0.70 0.78 0.22 0.44 0.47 1.00

20

D.-Y. Xu

Figure 4

An outline of the degradation of chlorophyll a from silkworm excrement.

Photosensitizer R & D in China

Figure 5

21

Process of the ring V cleavage of pheophorbide a in the synthesis of chlorin e6 -amides.

separate them one from another due to similarity in structure and polarity [40]. By the reaction of the mixture directly with amines without separation through the cleavage of pentanone rine (V) of pheophorbide a, 12 chlorin e6 -6-amides were obtained by Zhang and Xu (Fig. 5 and Table 10) [45]. However, pyropheophorbide a failed in such a reaction, thus could be easily separated from the reaction mixture and this simplifies the reaction procedure. It provides a new method for the synthesis of chlorin e6 amides. Structures of main compounds have been confirmed by FAB-MS, H1 NMR, IR and UV—vis spectra. The sensitizing effects of 12 chlorin e6 amides were determined by photooxidation of NADPH in deuterium oxide. PDT efficacy of four chlorin e6 amides was evaluated on transplanted S180 sarcoma in mice [46]. The photosensitizing abilities of these chlorin e6 amides tested were much higher than that of HpD at the wavelength of 630 nm. The following chlorin e6 amides might become promising PDT photosensitizers: chlorin e6 -6-N-ethylamide-10b-methyl ester, chlorin e6 -6-N-propylamide-10b-methyl ester, chlorin e6 6-N-(2-hydroxyethyl)amide-10b-methyl ester and chlorin e6 -6-N-(2-hydroxypropyl)amide. A series of chlorin e6 ethers has been synthesized by Fang and Xu (Fig. 6) [43]. An intermediate of chlorin e6 ethers, i.e. chlorin e6 -C15 -monomethyl ester (CMME), might be a promising candidate for new PDT photosensitizers [44].

Metal phthalocyanines Some of the water soluble nickel, cobalt, copper, zinc and aluminium phthalocyanines have been synthesized and

Table 10

Chlorin e6 amides

No. of compounds

Structure R1

1 2 3 4 5 6 7 8 9 10 11 12

H H H H H H H H CH2 CH3

H

R2 CH2 CH3 CH2 CH2 CH3 CH(CH3 )2 CH2 CH2 CH2 CH3 CH2 -C6 H5 Cyclohexyl CH2 CH2 OH CH2 CH(OH)CH3 CH2 CH3 —(CH2 )5 — —(CH2 )4 — NH-C6 H5

22

D.-Y. Xu

Figure 6

Figure 7

Reactions in the synthesis of chlorin e6 ethers.

Structure of hypocrellin A (HA), hypocrellin B (HB) and ceroosponin.

tested by Chen and Xu in the late 1980s. Although it was demonstrated that five synthetic phthalocyanines could be uptaken into the human tongue squamous cancer cells (TCA113), the rate of uptake was relatively low. They also found that the phthalocyanines mainly distributed in the cytoplasm and not in the nucleus and their affinities with tumor cells were relatively strong [50]. A new zinc phthalocyanine substituted with sulfonic acid and phthalimide group called Photocyanine (2,9-dipotassium-sulfonate-16,23-dimethylenephthalimidophthalocyanine or ZnPcS2 P2 K2 ) has been synthesized and tested by Huang et al. [51—55]. In vivo data showed that photodynamic effects of ZnPcS2 P2 K2 on both Sarcoma S180 and uterine cervix U14 were significantly higher than that of HpD. The central ions, ring substituents and axial ligand of phthalocyanines could affect photodynamic effects. It was reported that the central ions significantly influence the ability of singlet oxygen production and the order of which was as follows: ZnPc > AlClPc > CrPc > MnPc > CoPc > NiPc > FeClPc > FePc. The amphipathy might be a promising factor in phthalocyanine’s PDT application [56].

ical properties of HA and HB are listed in Table 11 [57]. There are three visible light bands which belong to the absorption bands of ␲, ␲* transition of pyrelenequinone ring and intermolecular charge transform of pyrelenequinone pigments (Figs. 8 and 9). There are two fluorescence peaks in its dilution; the principle peak is a neutral fluorescent molecular emission band and the shoulder peak is a zwitterions emission band produced from intramolecular proton transfer of neutral molecules. Another shoulder peak shown in the concentrated solution is a fluorescence peak of exicemers (Fig. 10). It has been reported that hypocrellin is a novel type of singlet oxygen sensitizer and might be a useful photosensitizer for tumor PDT [58—61]. Experimental results indicated that HA and HB were more potent than that of HpD for pho-

Perylenequinones (PQ) Hypocrellins are 3,10-dihydroxy-4,9-perylenequinone derivatives from the fungi Hypocrella Bambusae (B. et Br) parasitized in Sinarundiaria sp. The content of hypocrellins in Hypocrella Bambusae (B. et Br) is up to 4% by total weight. The photoactive components of hypocrellins are hypocrellin A (HA) and hypocrellin B (HB) (Fig. 7). The Chinese have developed several unique approaches for the preparation of HA and HB. Photophysical and photochem-

Figure 8 The absorption spectra of HA (—–), HB (—··—) and ceroosponin (- - -) in benzene.

Photosensitizer R & D in China

23

Figure 9

Absorption spectra of HA and HB in DMF.

Table 11 Photophysical and photochemical parameters of HA and HB HA

HB

KF (s−1 )

1.4 × 108

9.1 × 107 6 × 107

˚F , Fluorescence quantum yield ˚isc , Intersystem crossing quantum yield  s , Singlet life time (ns) Es , Singlet energy (kcal/mol) ˚st , Singlet quantum yield Kic , Intraconversion constant (s−1 )

0.14

0.06



0.18

0.98

0.66

48.5

47.7

0.86

0.76

Summary

8.6 × 108

1.1 × 1010

7.7 × 108 ˚TS , Triplet quantum yield

0.03



0.80 0.78

0.72 0.77

KTS (s−1 )

7.8 × 103



 T , Triplet life time (␮s)

4.5 (with O2 ) >0.13 (without O2 ) 4.2 4—6

ET , Triplet energy (kcal/mol)

todynamic killing of MDAMB 543 human mammary carcinoma cells [62].

2.7 × 109 1.7 × 108

Kist , Intersystem crossing constant (s−1 )

Figure 10 Fluorescence spectra of HA (—–), HB (—– —–) and ceroosponin (- - -) in benzene.

41.0

40.2

42.5

180.75 kJ/aml

Research and development of photosensitizers or photosensitizing drugs used for PDT are one of the new developing fields in medicinal chemistry. Along with the development of clinic PDT protocols, it has become an urgent topic for the medicinal chemist to study and produce photosensitizers that have definite structure, high photosensitizing ability, selectively localized in the targeted tissue, low-toxic and relative high absorbance in the longer wavelength. In the past 2 decades three mixed porphyrin preparations have been developed and used in cancer PDT in China [63,64]. Several new photosensitizers with definite structures including porphyrin derivatives, meso-tetra-substituted phenyl porphins, metal phthalocyanines, derivatives of chlorophyll a degradation products and hypocrellin A derivatives have been prepared and studied in detail. Among these, Hemporfin (3or 8-monomethyl ether of hematoporphyrin or HMME) is now entering clinical trials with the permission of Chinese State FDA. Another new photosensitizer, 2,9-dipotassiumsulfonate-16,23-dimethylenephthalimido-phthalocyanine or Photocyanine, has now completed preclinical studies and has been submitted to the State FDA for PDT clinical trials.

24 In accordance with the current status and our experiences, some suggestions may be useful for the development of new photosensitizers to be used in PDT: (1) it seems not appropriate to develop mixed porphyrin preparations with indefinite composition as new PDT photosensitizers because they not only cannot effectively control the quality, but also carry difficulties for the investigation of their mode of action. These, of course, also make great limitations for developing them as new remedies. Therefore, attention must be focused on searching for new photosensitizers with confirmed structure which may be used safely and effectively in PDT and easily achieve quality control. (2) Development of new antitumor-PDT photosensitizers should be independent to the photodiagnostic agents for tumorlocalization, because the principles of these two functions are completely different. (3) In principle, the development of a new PDT photosensitizer must go through some necessary steps as currently used for new drugs which include design, synthesis, separation, purification and identification of the title compound, experimental screening for efficacy, and studies of preclinical pharmacotoxicology and mode of actions. Effort may be made for changing the lack of data of structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) of photosensitizers under investigation. (4) Last but not least, chlorophyll a degradation products chlorins and porphyrins derived from hemin may be the preferential objects in developing new drugs for antitumor-PDT in the near future in China. Because they exhibit the same molecular skeleton as natural chlorophyll or hemin, the absorbance of derivatives of chlorophyll a degradation products in 640—670 nm is approximately one order higher than those of HpD and Photofrin II. Furthermore, natural chlorophyll and hemin are common ingredients of human food. They can be degraded and metabolized in the human body. So we have reasons to consider that some of the degradation products of both chlorophylls and hemin are familiar to the human body and, therefore, toxicity of them may be relatively low at some extent. In addition, there are abundant resources of both chlorophyll (Silkworm excrement) and hemin in China.

Acknowledgements The author would like to thank Dr. Zheng Huang for his helpful discussion and Ms. Sue Huang for her editorial assistance on this manuscript.

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Research and development of photodynamic therapy photosensitizers in China.

Photodynamic therapy (PDT) started in the People's Republic of China in the early 1980s after hematoporphyrin derivative (HpD) was used in China as PD...
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