IN VIVO FLUORESCENCE OF TUMORS AFTER TREATMENT WITH DERIVATIVES OF HEMATOPORPHYRIN

Abstract— Administration of a mixture of porphyrins termed HPD (hematoporphyrin derivative) to mice bearing the Lewis lung tumor leads to preferential accumulation of fluorescence at tumor loci in vivo after 48 h. HPLC analysis shows that the fluorescent species consist of hematoporphyrin and its dehydration products. But injection of these porphyrins does not lead to fluorescence localization. The intracellular fluorescence which is observed apparently arises from intracellular degradation of the tumor-localizing component of HPD. These fluorescent species represent only a small fraction of the total accumulated porphyrin pool; a larger weakly-fluorescent porphyrin pool is also present, and may be the major factor in tumor photosensitization.     

PROBING THE STRUCTURE OF HPD BY FLUORESCENCE SPECTROSCOPY

Fluorescence emission spectra indicate that oligomers containing both hematoporphyrin and its dehydration products (vinyl porphyrins) comprise the tumor-localizing fraction of HPD. In the relatively polar solvent methanol, the vinyl porphyrins exhibit reduced fluorescence yields while the hematoporphyrin residues are relatively resistant to fluorescence quenching by Fe+3. In the less polar solvent tetrahydrofuran, fluorescence from oligomeric vinyl porphyrins was enhanced, and Fe+3-induced quenching of oligomeric hematoporphyrin promoted. These, together with other studies in biological systems, suggest a substantial degree of interaction among the porphyrin units contained in these oligomers, as a function of the polarity of the environment.     

CHEMISTRY OF HEMATOPORPHYRIN-DERIVED PHOTOSENSITIZERS

Abstract The hematoporphyrin-derived tumor-localizing preparation HPD consists of porphyrin monomers, which are'inactive'(not tumor-localizing), and a dimer/oligomer fraction which is responsible for the localizing phenomenon. In an organic solvent system, gel-exclusion chromatography can separate HPD into fractions containing porphyrin monomers, dimers or oligomers. The relative amount of the dimer/oligomer fraction of HPD was a function of the pH of the mixture used to transform HP mono/di acetate to HPD. HPD prepared by the'Upson'procedure contained dimer/oligomer linkages which are labile to 1 M NaOH (in 50% tetrahydrofuran), and are reduced by LiAlH₄ to alcohols. These properties are characteristic of esters. But a commercial product, Photofrin II, contained approx. 50% of material refractory to both reagents described above. This behavior is characteristic of an ether linkage. These observations show that the nature of the linkage joining the porphyrin units is sensitive to conditions employed in HPD preparation. Tumor localization derives, in part, from affinity of these oligomers for plasma lipoprotein, and is associated with conformational alterations characteristic of these porphyrin-porphyrin linkages.     

On the preparation and properties of dihematoporphyrin ether, the tumor-localizing component of HPD

Abstract— A porphyrin mixture derived from hematoporphyrin and termed HPD (hematoporphyrin derivative) is used for localization and therapy of tumors. The major localizing component of HPD has been provisionally identified as a dihematoporphyrin ether (DHE). In this study, we describe HPLC procedures for analytical and preparative separation of HPD components. Optimal conditions for DHE formation were determined. A comparison of absorbance and fluorescence spectra in different solvents indicate that DHE aggregates are substantially more stable than are the analogous hematoporphyrin structures. Studies on the interaction between DHE and tumor cells indicate an initial binding of the drug at lipophilic membrane sites, followed by gradual relocation to intracellular loci.     

PROPOSED STRUCTURE OF THE TUMOR-LOCALIZING FRACTION OF HPD (HEMATOPORPHYRIN DERIVATIVE)

Abstract— Synthesis of the tumor-localizing preparation HPD (hematoporphyrin derivative) results in the formation of dimers and oligomers of hematoporphyrin joined by labile linkages. Studies with HPD and an HPD analog containing the chlorin analog of mesoporphyrin suggest the presence of two different linkages, either of which yields a tumor-localizing product. One such linkage is apparently derived from a hematoporphyrin-based oligomer present in commercial preparations of hematoporphyrin as an impurity. The other linkage is formed during the chemical steps leading to the conversion of hematoporphyrin to "HPD".     

PORPHYRIN ACCUMULATION BY ATHEROMATOUS PLAQUES OF THE AORTA

Abstract A complex mixture of porphyrins termed hematoporphyrin derivative (HPD) has been clinically useful for tumor localization. When sections of human aorta containing atheromatous plaques were incubated with HPD, accumulation of fluorescent porphyrin was observed within the plaques. Analytic studies showed that the plaques had accumulated hematoporphyrin (HP), which is substantially more hydrophilic than that HPD fraction generally associated with tumor localization. Fluorescence spectra suggest that the plaque binding sites of HP resemble the relatively aqueous micelles formed by the detergent sodium dodecylsulfate. This result has implications for tumor-localization procedures, since accumulation of hydrophilic porphyrins by tumors has been reported.     

THE STRUCTURE OF THE ACTIVE MATERIAL IN HEMATOPORPHYRIN DERIVATIVE

Abstract The structure of the active material in hematoporphyrin derivative is shown to be a condensation polymer of hematoporphyrin linked by ether functional groups. When a mixture of the monoacetates and the diacetate of hematoporphyrin is treated with dilute sodium hydroxide solution a polymeric fraction is formed which constitutes the active material of hematoporphyrin derivative. This fraction is stable to basic hydrolysis using conditions which are shown to hydrolyse porphyrin esters, but is hydrolysed by acidic conditions which cleave porphyrin ethers as well as esters. When hematoporphyrin diacetate is similarly treated with base a polymeric fraction is formed which is hydrolysed by both acidic and basic conditions showing it to be ester linked. This ester linked polymer is unstable in aqueous solution at pH 7 and converts to the polyether within 2 days at room temperature.     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

Abstract
The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5–6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero-and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N -methylated porphyrins show clearance times in the vicinity of 15–22 h. Monoaspartyl chlorin e₆ shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5-6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero- and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N-methylated porphyrins show clearance times in the vicinity of 15-22 h. Monoaspartyl chlorin e6 shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

Synthesis of porphyrin dimers fused with a benzene unit

Bicyclo[2.2.2]octadiene-connected pyrrolo-porphyrins have been prepared by an inverse-type [3+1] porphyrin synthesis of a bicyclo[2.2.2]octadiene-fused dipyrrole with a tripyrrane dicarbaldehyde. Another [3+1] porphyrin synthesis of pyrrole-connected porphyrins with the same or other tripyrrane dicarbaldehydes gave bicyclo[2.2.2]octadiene-bridged diporphyrins, the central metals and/or peripheral substituents of which were different. Thermal decomposition of the bicyclo[2.2.2]octadiene skeleton to a benzene moiety gave pi-system-fused porphyrin dimers in a highly pure form.     

Carotenohematoporphyrins as Tumor-Imaging Dyes. Synthesis and In Vitro Photophysical Characterization*

Multichromophoric dyes for use in tumor imaging have been synthesized and photophysically characterized. Structurally, these dyes are dyads and triads that consist of one or two carotenoid polyenes covalently attached to hematoporphyrin (HP) or hematoporphyrin dimethyl ester (HPDME) moieties via ester linkages. The ground-state absorption of each compound shows that the electronic interaction between the chromophores is small. The fluorescence quantum yield for the dyad monocar-oteno- HPDME is 0.033 and the dicaroteno-HPDME triads have yields between 0.016 and 0.007, all of which are reduced with respect to the parent compound HPDME (0.09). Global analysis of the transient fluorescence decays of the dyads and triads requires two exponential components (?5–6ns and ?1–2ns) to fit the data, while a single exponential component with a lifetime of 9.3 ns describes the decay data of the parent HPDME. Possible mechanisms for the observed porphyrin fluorescence quenching by the nearby carotenoid are discussed. Nanosecond transient absorption reveals a carotene triplet with maximum absorption at 560 nm and a 5.0 μs lifetime. No transient was detected at 450 nm, indicating rapid (10 ns) triplet energy transfer from the hematoporphyrin to the carotenoid moieties in fluid as well as in rigid media. The yield of triplet energy transfer from the porphyrin to the carotenoid moiety is unity. Singlet oxygen, O₂(¹δ_g), studies support the transient absorption data, as none of these compounds is capable of sensitizing O₂(¹δ_g). Liposome vesicles were used to study the photophysical characteristics of the dyes in phospholipid membranes. Singlet oxygen was not sensitized by the dyads and triads in liposomes. Transient absorption measurements suggest that the triads are substantially aggregated within the phospholipid bilayer, whereas aggregation in the dyads is less severe.     

Synthesis of three-dimensionally arranged porphyrin arrays via intramolecular meso-meso coupling

The synthesis and photophysical properties of three-dimensionally arranged porphyrin arrays with through-space electronic communication are reported. 1,3,5-Trioxamethylphenylene bridged Zn(II) porphyrin trimer 3 was coupled by Ag(I)-promoted oxidative coupling reaction to give porphyrin cage 5 comprising three meso-meso linked diporphyrins, which was then transformed by oxidation with DDQ and Sc(OTf)₃ into porphyrin cage 7 comprising three fused diporphyrins. Intramolecular meso-meso coupling reaction was applied to porphyrin pentamer 11 to provide porphyrin array 12 consisting of a porphyrin core flanked by two meso-meso linked diporphyrins. Further oxidation of 12 with DDQ and Sc(OTf)₃ afforded triply stacked porphyrin array 13 that is comprised of a porphyrin core flanked by two porphyrin tapes. UV-vis-NIR absorption and fluorescence spectra of 5, 7, 12, and 13 showed their distorted conformations and electronic interaction within the stacked porphyrin arrays.     

PHOTODYNAMIC INACTIVATION OF R3230AC MAMMARY CARCINOMA IN VITRO WITH HEMATOPORPHYRIN DERIVATIVE: EFFECTS OF DOSE, TIME, and SERUM ON UPTAKE and PHOTOTOXICITY

Abstract Primary cultures of the R3230AC mammary adenocarcinoma were used for pharmacokinetic studies of hematoporphyrin derivative (HPD), a preparation containing several porphyrin species and useful as a photoactivatable anti-tumor agent. Uptake of HPD in vitro was shown to be time-, dose- and temperature-dependent with an apparent plateau reached at 2 - 4 h. An increase in the amount of serum in the medium progressively reduced the amount of HPD taken up by the cells; at a level of 10% serum, uptake of HPD was reduced by >95%. The time-course of efflux of HPD from these cells demonstrated a complex pattern, with an initial rapid component followed by a more gradual rate of efflux up to 4 h. Assessment of photoradiation-induced cytotoxicity was performed by a method developed to quantitatively measure trypan blue exclusion. Relative cytotoxicity was determined by use of heat-killed cells as a standard. At two different concentrations of HPD, cytotoxicity was dependent on light exposure time. The presence of serum, which reduced uptake of HPD was correctable to reduced cytotoxicity. Based on the amount of light exposure to produce 50% cell kill, an order of potency was obtained for HPD > hematoporphyrin > hydroxyethylvinyldeuteroporphyrin in vitro. This order of potency correlated with the relative proportion of hydrophobic components as estimated by HPLC analysis. The results indicate that HPD is an effective cytotoxic agent in vitro in a well-differentiated mammary adenocarcinoma model.     

Synthesis of Zn-, Mn-, and Fe-Containing Mono- and Heterometallated Ethanediyl-Bridged Porphyrin Dimers

New ethanediyl-bridged unsymmetrical mono- and heterometallated dimers of 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphyrin (H₂oep) containing transition-metal ions (Mn and Fe) were synthesized by a facile stepwise metallation/demetallation process under mild conditions. The novel metallation strategy initially involved the predominant insertion of Zn into one of the two porphyrin rings of the free-base dimer, followed by the incorporation of Mn or Fe into the other porphyrin ring under exceptionally mild conditions, giving corresponding heterometallic dimers; the subsequent removal of Zn yielded mono-transition-metal dimers. The emission spectrum of the monozinc dimer predominantly exhibited fluorescence bands of the free-base porphyrin component, indicating a very efficient energy-transfer process. Conversely, emission of the free-base or Zn porphyrin component of transition metal containing dimers was strongly quenched due to photoinduced electron transfer.     

PHOTOSENSITIZATION BY SYNTHETIC DIPORPHYRINS AND DICHLORINS in vivo AND in vitro

A group of polycarboxylic diporphyrins, two dichlorins and a porphyrin-chlorine dimer, with rings linked by methylene groups, were examined to help identify structures which can mediate photodynamic tumor eradication in vivo. Among the features sought were short persistence of normal tissue photosensitization and substantial absorbance at wavelengths longer than 630 nm. Both objectives were achieved, with pertinent structure-activity relationships partly characterized. The relative hydrophobicity of the different sensitizers was an important determinant of their accumulation in cell culture, but not of in vivo effectiveness. These compounds showed affinity for protein and high-density lipoprotein components of serum. Their distribution may be mediated by a different mechanism than that which occurs with more hydrophobic sensitizers like hematoporphyrin derivative which have greater affinity for low-density lipoproteins and less for protein components of serum, as compared with the products examined in this study.     

DETERMINANTS OF PHOTOSENSITIZATION BY MONO-L-ASPARTYL CHLORIN e6

The mono-N-aspartyl derivative of chlorin e6 (MACE) is a new photosensitizer being examined for use in anti-neoplastic photodynamic therapy. Studies were carried out to identify unique aspects of MACE localization by murine leukemia L1210 cells in vitro. Octanol/water partitioning studies were used to quantitate the hydrophobicity of MACE and two analogs, chlorin e6 and mesochlorin. Sites of cellular localization of these dyes were probed by fluorescence studies, and by examining loci of photodamage. These studies indicate that MACE, a hydrophilic dye, partitions to cytoplasmic loci. Data obtained with chlorin e6, a more hydrophobic dye, are consistent with binding at both membrane and cytoplasmic sites. A substantially more hydrophobic product, meso-chlorin, binds primarily to the cell membrane. While the tumor-localizing porphyrin product HPD binds to plasma LDL less than HDL, MACE and CE are predominantly bound to plasma protein and HDL. Patterns of distribution and localization of MACE differ substantially from those observed with HPD and other hydrophobic sensitizers. Phototoxic effects of MACE could not be specifically attributed to membrane or mitochondrial damage.     

Pharmacokinetics of Photogem using fluorescence monitoring in Wistar rats

In this study we investigated the pharmacokinetics of a hematoporphyrin derivative (Photogem) in Wistar rats using the fluorescence spectroscopy to evaluate the drug distribution in liver, kidney and skin tissues. The detection system is composed of a 532 nm exciting laser, a Y-type catheter for light delivery and collection, a monochromator and a computer for data acquisition. The analysis of the fluorescence spectra was based on the intensity of porphyrin emission bands from specific tissues of the investigated organ. A simple transport model is proposed to determine the accumulation and elimination times for each type of investigated tissue. The obtained results show the viability of the fluorescence spectroscopic technique for the drug concentration monitoring in different target tissues and related pharmacokinetics. These effects should be considered before any in vivo study of Photodynamic Therapy using Photogem.     

Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin, pheophorbide a and chlorin e6 in ethanol and aqueous solution

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.     

Time-gated fluorescence spectroscopy of porphyrin derivatives and aluminium phthalocyanine incorporated in vivo in a murine ascitic tumour model.

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4-6 h.     

Time-gated fluorescence spectroscopy of porphyrin derivatives incorporated into cells

Time-gated fluorescence spectroscopy was performed on the tumour-localizing fraction (TLF) of haematoporphyrin derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 micrograms ml-1 of TLF for various time periods; they were then washed and resuspended in buffer. Fluorescence decay measurements and time-integrated and time-gated spectra were then obtained from the cell suspensions. Similar experiments were repeated using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a consequence of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to monomers and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is more pronounced for HPD. The results obtained seem to indicate that both the composition of the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the incorporated sensitizer.