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Haematoporphyrin derivatives: Distribution in a living organism
J. Photochem.
Photobiol.
341
B: Biol., 16 (1992) 341-346
Haematoporphyrin organism
derivatives:
A. F. Mirono?, A. S. Seylanovb, and A. Ju Nockel”
distribution
J. A. Seylanovb,
in a living
V. M. Pizhik’,
“M. V. Lomonosov Institute of Fine Chemical Technology, Moscow (Russian %stitute of Medical Radiology, Erevan (Armenia) ‘Instihcte of Medical Radiobiology, filuga (Russian Federation)
(Received
April 13, 1992; accepted
I. V. Deruzhenko” Federation)
June 4, 1992)
Abstract Pharmacokinetics of accumulation in organs and tissues was studied for two haematoporphyrin-based photosensitizers. These sensitizers, haematoporphyrin derivative (HpD) and an oligomeric haematoporphyrin (OHp), contained different amounts of monomeric fraction (25% and 5% respectively) and in OHp the macrocycles were bonded together with ether bonds. OHp was shown to accumulate in tumours in higher amounts than HpD. The maximal tumour to tissue concentration ratio for OHp was 6.7 observed 54 h after injection; the same ratio for HpD was 2.8 after 48 h.
Photodynamic therapy, haematoporphyrin derivatives, haematoporphyrin, accumulation in tumours, gel chromatography, liquid chromatography, mass spectrometry.
Keywords:
oligomeric high performance
1. Introduction The photodynamic therapy (PDT) and laser fluorescence diagnostics of cancer are based on the selective accumulation of photosensitizing dyes in tumour cells and tissues. Medical practitioners mainly use the photosensitizers haematoporphyrin derivative (HpD), Photofrin II and Photosan, which are mixtures of porphyrin monomers and oligomers. The porphyrin macrocycles in the oligomers are linked together by ether, ester or C-C bonds [l]. To increase the selective accumulation of the photosensitizer in the tumour the HpD needs to be purified from the monomeric components [2]. Alternatively, the oligomers could be chemically synthesized by a regiospecific procedure [3]. The preferential localization of oligomers in the tumour is apparently caused by their lipophilicity and capacity to bind to low-density lipoproteins (LDLs) in the blood stream [4]. Cancer cells have heightened LDL receptor activity in comparison with most normal cells [S]. Cancer cells can actively bind LDLs enriched with photosensitizer. We have studied the distribution in tumour and normal tissues of two preparations based on haematoporphyrin. One preparation was a conventional HpD sample and the other contained a much smaller amount of the monomeric fraction and a higher percentage of oligomers, in which the porphyrin macrocycles were bound with ether bonds.
342
2. Materials
and methods
HpD was prepared by a previously described procedure via haematoporphyrin diacetate. The content of monomers was 23% * 2% [6]. The other preparation, an oligomeric haematoporphyrin (OHp), was synthesized by the coupling of haematoporphyrin trifluoroacetate with an unsubstituted haematoporphyrin according to a procedure described in ref. 3. Both components were used as dimethyl esters and the product obtained was saponified with an alkali to give a water-soluble OHp. Using gel chromatography according to ref. 7 we have shown that the monomeric fraction in the starting OHp as well as in the saponified product did not exceed 5% (Fig. 1). The mass spectrum of OHp (Fig. 2) was recorded on a time-of-flight mass spectrometer MSBX (SELMI, Ukraine, Sumy) with a 252Cf ion source at a positive polarity at an accelerating voltage of 20 kV. The mass spectrum shows that OHp, in addition to dimers (m/z, 1145.8, 1163.4) and trimers (m/z, 1727.7, 1745.5) (the presence of dimers and trimers could also be deduced from the gel chromatography experiment), contains higher oligomers (m/z, 2310.9, 2890.5, 3471.9, 3489.9, 4061.8, 4642.7, 5245.6, 5834.6) up to ten-membered compounds. High performance liquid chromatography (HPLC) was performed using an instrument with gradient system (“Gilson”). Fluorescence and absorbance were registered simultaneously on a detector (Gilson 121) and UV/Vis Holochrome. The column was a Supercosil reverse-phase LC18 “Zorbax ODS” (4.6 mmX250 mm; particle size 5 pm; DuPont). The mobile phases were: (A) 8.3 mM phosphate buffer plus 3 mM tetrabutylammonium phosphate (TBAP), pH 3.5; (B) methanol plus 3 mM TBAP. The gradient time from eluent A to B was 10 min; the column was then eluted for another 15 min with solvent B. The flow rate was 1.3 ml min-‘. The accumulation kinetics were studied using female rats with a sarcoma 45 tumour transplanted on the left flank of the animals. HpD and OHp were injected intravenously at a concentration of 2.5 mg kg-’ body weight. HpD was dissolved in distilled water; OHp was used in a 5% water solution of NaHCO,, pH 8-8.5. The dyes were extracted from tumours, muscle tissues, liver, kidney and blood at predetermined intervals by the procedure described in ref. 8.
I
’
in
20
ELUTION
30
40 VOLUME
50 ml
’
to ELUTI
20 ON
30
40
Fig. 1. Gel chromatography of the oligomeric haematoporphyrin introduced
as methyl ester;
(B) as free acid.
50
a’
VOLUME
(OHp): (A) starting OHp
343
22500
15000
r
kbkz.7
5266 r- 5UM.b
7500
0 M/z Ii
CYU”tN
1
.
3B
1500
1250
kobl.8 b
1000
750
P
500
250
0 lcao
15m
n/z Fig. 2. Mass spectrum
of the oligomeric
Statistical analysis Software, USA).
was performed
haematoporphyrin.
with the computer
program
GraphPAD
(JCI
3. Result and discussion Table 1 shows the content of the photosensitizers in the tumour, normal tissues and organs. The highest concentration of photosensitizers was observed for liver and kidneys at 36 h after injection. The dye content then began to decrease and at 54 h after injection it was 50% and 70% of the maximal value respectively. However, the level of HpD and OHp in the liver and kidneys was still high, and higher than that
344 TABLE
1
HpD and OHp distribution in organs and tissues deviation was less that 15% of the mean values) Tissue
Time after injection
(porphyrin
(pg) and tissue (g); standard
(h)
24
36
48
54
60
72
OHP Liver Kidneys Tumour Muscle Blood
76.00 36.00 18.00 4.80 25.60
97.80 35.80 19.30 6.30 16.80
50.80 24.30 17.10 3.50 2.50
49.30 26.20 16.10 2.40 1.20
43.80 21.00 9.60 3.00 0.90
39.80 17.10 9.50 2.90 0.80
HPD Liver Kidneys Tumour Muscle Blood
32.40 15.60 14.80 6.80 12.70
36.40 17.30 13.10 5.00 10.40
21.90 12.10 6.85 2.40 2.60
18.50 12.80 6.88 3.30 2.00
17.50 11.40 3.80 2.30 1.80
9.50 5.30 3.10 2.10 0.90
in the tumour tissue even at 72 h after injection. The dye concentration in the blood decreased more rapidly and was almost undetectable at injection. The best experimental picture was obtained at about 36-54 h. It should be noted that the absolute concentration of OHp in the tumour was significantly higher than that of HpD. A comparison of the dye elimination kinetics from the liver, kidneys, blood, tumour and muscle tissues shows that elimination is slower for OHp. This delay is greater for the tumour tissue. For HpD the maximal tumour to tissue concentration ratio was 2.8 as observed 48 h after injection; the same ratio for OHp was 6.7 after 54 h. The low concentration of OHp in blood compared with HpD favours the luminescent contrasting of the tumour and PDT. The advantages of OHp result from the low content of the monomeric fraction, the high level of polymerization, and probably from the nature of bonding between the macrocycles. The HPLC elution profile of HpD is shown in Fig. 3. The absolute intensity of the fluorescence and absorption of the separated HpD fractions significantly decreases as a function of the elution time. However, the ratio of the spectral characteristics (absorption/fluorescence) for each peak is not constant and increases for fractions with longer elution time. These changes in spectral characteristics reflect the fact that the increase in the degree of polymerization of the dye and the increase in its lipophilicity result in a decrease in the fluorescence intensity [9]. The elution profile of HpD depends on the size of the oligomers; this is further confirmed by the OHp elution profile (Fig. 4) which shows that it contains a smaller amount of monomeric fraction. The oligomeric fraction in OHp is much more abundant than in HpD (Fig. 3). On the basis of these results it can be concluded that the accumulation of HpD and OHp in tissues depends on the degree of polymerization of the preparation, its lipophilicity and composition. The differences in the absolute concentrations of HpD
345
0’.00
s’.oo
,;o! 00
lsl.oo
20100
25!00
min
AB#-tUOR :1 -LO, Z-3.2, 3-2.7, 4- 6.1 Fig. 3. HPLC elution profile of HpD. Detection at 365 nm. Fluorescence detection at emission excitation, 305-395 nm; emission, 420-650 nm. HpD fractions are indicated by numbers (1, 2, 3 - non identified ones, 4 - oligomers). -, Fluorescence; ---, absorption.
FLGUKESCENCE
Fig. 4. HPLC elution profile of alkali-treated 305-395 nm; emission, 420-650 nm.
OHp. Fluorescence
detection
at emission excitation,
and OHp in the tumour (6.9 and 16.1 pg kg-’ tissue respectively) indicate the effectiveness of OHp for PDT. The slow elimination of OHp from the tumour tissue enhances the accumulation selectivity of this dye in the tumour tissue. Another positive feature of OHp is its fast elimination from normal tissue in comparison with HpD. Therefore the dose of the preparation can be lower (OS-l.0 mg kg-’ body weight) and skin photosensitization will be less important.
346
References 1 R. Bonnett Adv.
and M. C. Berenbaum,
Exp. Med.
Biol.,
160 (1983)
HpD-a
study of its components
and their properties,
241-250.
2 K. R. Weishaupt,
T. J. Dougherty and W. R. Potter, Purified hematoporphyrin derivative for diagnosis and treatment of tumor, Patent WO 84/01382 - C1.C 07 D, 1984. 3 A. F. Mironov, A. N. Nizhnik, I. V. Deruzhenko and R. Bonnet& Regiospecific synthesis of ether-bonded oligomers of haematoporphyrin IX and its relatives, Tetrahedron Z-&t., 31 (1990) 6409-6412. 4 A. Barel, G. Jori, A. Perin,
5 6 7 8
9
P. Romandini, A. Pagnan and S. Biffanti, Role of high-, lowand very low-density lipoproteins in the transport and tumor-delivery of hematoporphyrin in vivo, Cancer Lett., 32 (1986) 145-150. G. Jori, Photodynamic therapy of solid tumors, Radiat. Phys. Chem., 30 (1987) 375-380. A. F. Mironov, A. N. Nizhnik and A. Y. Nockel, On the nature of chemical bonds in haematoporphyrin derivative, .J. Photochem. Photobiol. B: Biol., 6 (1990) 337-341. A. F. Mironov, A. N. Nizhnik and A. Y. Nockel, Hematoporphyrin derivatives: an oligomeric composition study, J. Photochem. Photobiol. B: Biol., 4 (1990) 297-306. M. L. Pantelides, J. V. Moore and N. J. Blacklock, A comparison of serum kinetics and tissue distribution of Photofrin II-following intravenous and intraperitoneal injection in the mouse, Photochem. Photobiol., 49 (1989) 67-70. R. Rotomskis, E. J. Van De Meent, T. J. Aartsma and A. J. Hoff, Fluorescence spectra of haematoporphyrin and haematoporphyrin-diacetate aggregates in buffer solution,J. Photochem. Photobiol.
B: Biol., 3 (1989)
369-377.
Photobiol.
341
B: Biol., 16 (1992) 341-346
Haematoporphyrin organism
derivatives:
A. F. Mirono?, A. S. Seylanovb, and A. Ju Nockel”
distribution
J. A. Seylanovb,
in a living
V. M. Pizhik’,
“M. V. Lomonosov Institute of Fine Chemical Technology, Moscow (Russian %stitute of Medical Radiology, Erevan (Armenia) ‘Instihcte of Medical Radiobiology, filuga (Russian Federation)
(Received
April 13, 1992; accepted
I. V. Deruzhenko” Federation)
June 4, 1992)
Abstract Pharmacokinetics of accumulation in organs and tissues was studied for two haematoporphyrin-based photosensitizers. These sensitizers, haematoporphyrin derivative (HpD) and an oligomeric haematoporphyrin (OHp), contained different amounts of monomeric fraction (25% and 5% respectively) and in OHp the macrocycles were bonded together with ether bonds. OHp was shown to accumulate in tumours in higher amounts than HpD. The maximal tumour to tissue concentration ratio for OHp was 6.7 observed 54 h after injection; the same ratio for HpD was 2.8 after 48 h.
Photodynamic therapy, haematoporphyrin derivatives, haematoporphyrin, accumulation in tumours, gel chromatography, liquid chromatography, mass spectrometry.
Keywords:
oligomeric high performance
1. Introduction The photodynamic therapy (PDT) and laser fluorescence diagnostics of cancer are based on the selective accumulation of photosensitizing dyes in tumour cells and tissues. Medical practitioners mainly use the photosensitizers haematoporphyrin derivative (HpD), Photofrin II and Photosan, which are mixtures of porphyrin monomers and oligomers. The porphyrin macrocycles in the oligomers are linked together by ether, ester or C-C bonds [l]. To increase the selective accumulation of the photosensitizer in the tumour the HpD needs to be purified from the monomeric components [2]. Alternatively, the oligomers could be chemically synthesized by a regiospecific procedure [3]. The preferential localization of oligomers in the tumour is apparently caused by their lipophilicity and capacity to bind to low-density lipoproteins (LDLs) in the blood stream [4]. Cancer cells have heightened LDL receptor activity in comparison with most normal cells [S]. Cancer cells can actively bind LDLs enriched with photosensitizer. We have studied the distribution in tumour and normal tissues of two preparations based on haematoporphyrin. One preparation was a conventional HpD sample and the other contained a much smaller amount of the monomeric fraction and a higher percentage of oligomers, in which the porphyrin macrocycles were bound with ether bonds.
342
2. Materials
and methods
HpD was prepared by a previously described procedure via haematoporphyrin diacetate. The content of monomers was 23% * 2% [6]. The other preparation, an oligomeric haematoporphyrin (OHp), was synthesized by the coupling of haematoporphyrin trifluoroacetate with an unsubstituted haematoporphyrin according to a procedure described in ref. 3. Both components were used as dimethyl esters and the product obtained was saponified with an alkali to give a water-soluble OHp. Using gel chromatography according to ref. 7 we have shown that the monomeric fraction in the starting OHp as well as in the saponified product did not exceed 5% (Fig. 1). The mass spectrum of OHp (Fig. 2) was recorded on a time-of-flight mass spectrometer MSBX (SELMI, Ukraine, Sumy) with a 252Cf ion source at a positive polarity at an accelerating voltage of 20 kV. The mass spectrum shows that OHp, in addition to dimers (m/z, 1145.8, 1163.4) and trimers (m/z, 1727.7, 1745.5) (the presence of dimers and trimers could also be deduced from the gel chromatography experiment), contains higher oligomers (m/z, 2310.9, 2890.5, 3471.9, 3489.9, 4061.8, 4642.7, 5245.6, 5834.6) up to ten-membered compounds. High performance liquid chromatography (HPLC) was performed using an instrument with gradient system (“Gilson”). Fluorescence and absorbance were registered simultaneously on a detector (Gilson 121) and UV/Vis Holochrome. The column was a Supercosil reverse-phase LC18 “Zorbax ODS” (4.6 mmX250 mm; particle size 5 pm; DuPont). The mobile phases were: (A) 8.3 mM phosphate buffer plus 3 mM tetrabutylammonium phosphate (TBAP), pH 3.5; (B) methanol plus 3 mM TBAP. The gradient time from eluent A to B was 10 min; the column was then eluted for another 15 min with solvent B. The flow rate was 1.3 ml min-‘. The accumulation kinetics were studied using female rats with a sarcoma 45 tumour transplanted on the left flank of the animals. HpD and OHp were injected intravenously at a concentration of 2.5 mg kg-’ body weight. HpD was dissolved in distilled water; OHp was used in a 5% water solution of NaHCO,, pH 8-8.5. The dyes were extracted from tumours, muscle tissues, liver, kidney and blood at predetermined intervals by the procedure described in ref. 8.
I
’
in
20
ELUTION
30
40 VOLUME
50 ml
’
to ELUTI
20 ON
30
40
Fig. 1. Gel chromatography of the oligomeric haematoporphyrin introduced
as methyl ester;
(B) as free acid.
50
a’
VOLUME
(OHp): (A) starting OHp
343
22500
15000
r
kbkz.7
5266 r- 5UM.b
7500
0 M/z Ii
CYU”tN
1
.
3B
1500
1250
kobl.8 b
1000
750
P
500
250
0 lcao
15m
n/z Fig. 2. Mass spectrum
of the oligomeric
Statistical analysis Software, USA).
was performed
haematoporphyrin.
with the computer
program
GraphPAD
(JCI
3. Result and discussion Table 1 shows the content of the photosensitizers in the tumour, normal tissues and organs. The highest concentration of photosensitizers was observed for liver and kidneys at 36 h after injection. The dye content then began to decrease and at 54 h after injection it was 50% and 70% of the maximal value respectively. However, the level of HpD and OHp in the liver and kidneys was still high, and higher than that
344 TABLE
1
HpD and OHp distribution in organs and tissues deviation was less that 15% of the mean values) Tissue
Time after injection
(porphyrin
(pg) and tissue (g); standard
(h)
24
36
48
54
60
72
OHP Liver Kidneys Tumour Muscle Blood
76.00 36.00 18.00 4.80 25.60
97.80 35.80 19.30 6.30 16.80
50.80 24.30 17.10 3.50 2.50
49.30 26.20 16.10 2.40 1.20
43.80 21.00 9.60 3.00 0.90
39.80 17.10 9.50 2.90 0.80
HPD Liver Kidneys Tumour Muscle Blood
32.40 15.60 14.80 6.80 12.70
36.40 17.30 13.10 5.00 10.40
21.90 12.10 6.85 2.40 2.60
18.50 12.80 6.88 3.30 2.00
17.50 11.40 3.80 2.30 1.80
9.50 5.30 3.10 2.10 0.90
in the tumour tissue even at 72 h after injection. The dye concentration in the blood decreased more rapidly and was almost undetectable at injection. The best experimental picture was obtained at about 36-54 h. It should be noted that the absolute concentration of OHp in the tumour was significantly higher than that of HpD. A comparison of the dye elimination kinetics from the liver, kidneys, blood, tumour and muscle tissues shows that elimination is slower for OHp. This delay is greater for the tumour tissue. For HpD the maximal tumour to tissue concentration ratio was 2.8 as observed 48 h after injection; the same ratio for OHp was 6.7 after 54 h. The low concentration of OHp in blood compared with HpD favours the luminescent contrasting of the tumour and PDT. The advantages of OHp result from the low content of the monomeric fraction, the high level of polymerization, and probably from the nature of bonding between the macrocycles. The HPLC elution profile of HpD is shown in Fig. 3. The absolute intensity of the fluorescence and absorption of the separated HpD fractions significantly decreases as a function of the elution time. However, the ratio of the spectral characteristics (absorption/fluorescence) for each peak is not constant and increases for fractions with longer elution time. These changes in spectral characteristics reflect the fact that the increase in the degree of polymerization of the dye and the increase in its lipophilicity result in a decrease in the fluorescence intensity [9]. The elution profile of HpD depends on the size of the oligomers; this is further confirmed by the OHp elution profile (Fig. 4) which shows that it contains a smaller amount of monomeric fraction. The oligomeric fraction in OHp is much more abundant than in HpD (Fig. 3). On the basis of these results it can be concluded that the accumulation of HpD and OHp in tissues depends on the degree of polymerization of the preparation, its lipophilicity and composition. The differences in the absolute concentrations of HpD
345
0’.00
s’.oo
,;o! 00
lsl.oo
20100
25!00
min
AB#-tUOR :1 -LO, Z-3.2, 3-2.7, 4- 6.1 Fig. 3. HPLC elution profile of HpD. Detection at 365 nm. Fluorescence detection at emission excitation, 305-395 nm; emission, 420-650 nm. HpD fractions are indicated by numbers (1, 2, 3 - non identified ones, 4 - oligomers). -, Fluorescence; ---, absorption.
FLGUKESCENCE
Fig. 4. HPLC elution profile of alkali-treated 305-395 nm; emission, 420-650 nm.
OHp. Fluorescence
detection
at emission excitation,
and OHp in the tumour (6.9 and 16.1 pg kg-’ tissue respectively) indicate the effectiveness of OHp for PDT. The slow elimination of OHp from the tumour tissue enhances the accumulation selectivity of this dye in the tumour tissue. Another positive feature of OHp is its fast elimination from normal tissue in comparison with HpD. Therefore the dose of the preparation can be lower (OS-l.0 mg kg-’ body weight) and skin photosensitization will be less important.
346
References 1 R. Bonnett Adv.
and M. C. Berenbaum,
Exp. Med.
Biol.,
160 (1983)
HpD-a
study of its components
and their properties,
241-250.
2 K. R. Weishaupt,
T. J. Dougherty and W. R. Potter, Purified hematoporphyrin derivative for diagnosis and treatment of tumor, Patent WO 84/01382 - C1.C 07 D, 1984. 3 A. F. Mironov, A. N. Nizhnik, I. V. Deruzhenko and R. Bonnet& Regiospecific synthesis of ether-bonded oligomers of haematoporphyrin IX and its relatives, Tetrahedron Z-&t., 31 (1990) 6409-6412. 4 A. Barel, G. Jori, A. Perin,
5 6 7 8
9
P. Romandini, A. Pagnan and S. Biffanti, Role of high-, lowand very low-density lipoproteins in the transport and tumor-delivery of hematoporphyrin in vivo, Cancer Lett., 32 (1986) 145-150. G. Jori, Photodynamic therapy of solid tumors, Radiat. Phys. Chem., 30 (1987) 375-380. A. F. Mironov, A. N. Nizhnik and A. Y. Nockel, On the nature of chemical bonds in haematoporphyrin derivative, .J. Photochem. Photobiol. B: Biol., 6 (1990) 337-341. A. F. Mironov, A. N. Nizhnik and A. Y. Nockel, Hematoporphyrin derivatives: an oligomeric composition study, J. Photochem. Photobiol. B: Biol., 4 (1990) 297-306. M. L. Pantelides, J. V. Moore and N. J. Blacklock, A comparison of serum kinetics and tissue distribution of Photofrin II-following intravenous and intraperitoneal injection in the mouse, Photochem. Photobiol., 49 (1989) 67-70. R. Rotomskis, E. J. Van De Meent, T. J. Aartsma and A. J. Hoff, Fluorescence spectra of haematoporphyrin and haematoporphyrin-diacetate aggregates in buffer solution,J. Photochem. Photobiol.
B: Biol., 3 (1989)
369-377.