Magne Kongshaug

5-Aminolevulinic acid-based photodynamic therapy : Clinical research and future challenges

Photodynamic therapy (PDT) for cancer patients has developed into an important new clinical treatment modality in the past 25 years. PDT involves administration of a tumor‐localizing photosensitizer or photosensitizer prodrug (5‐aminolevulinic acid [ALA], a precursor in the heme biosynthetic pathway) and the subsequent activation of the photosensitizer by light. Although several photosensitizers other than ALA‐derived protoporphyrin IX (PpIX) have been used in clinical PDT, ALA‐based PDT has been the most active area of clinical PDT research during the past 5 years. Studies have shown that a higher accumulation of ALA‐derived PpIX in rapidly proliferating cells may provide a biologic rationale for clinical use of ALA‐based PDT and diagnosis. However, no review updating the clinical data has appeared so far.

5-Aminolevulinic Acid-Based Photodynamic Therapy: Principles and Experimental Research

The iron(I1) complex of protoporphyrin IX (PpIX)? (heme) is bound to different proteins to form key biomolecules (hemoproteins) such as hemoglobin, myoglobin, cytochromes, catalase, peroxidase and tryptophan pyrrolase. The lives of the cells and of the body as a whole is therefore crucially dependent upon the biosynthesis and metabolism of porphyrins. Almost all types of cells of the human body, with the exception of mature red blood cells, are equipped with a machinery to synthesize heme. In the first step of the heme biosynthetic pathway 5-aminolevulinic acid (ALA) is formed from glycine and succinyl CoA. The synthesis of ALA is regulated by the amount of heme in the cell. The last step in the formation of heme is the incorporation of iron into PpIX and takes place in the mitochondria under the action of the enzyme, ferrochelatase. By adding exogenous ALA, PpIX may accumulate because of the limited capacity of ferrochelatase. Porphobilinogen deaminase (PBGD) is another enzyme that is active in the heme synthesis pathway (catalyzing the formation of uroporphyrinogen from porphobilinogen [PBG]). The activity of this enzyme is higher in some tumors (1-3), while that of ferrochelatase is lower (2-7), so that PpIX accumulates with some degree of selectivity in tumors. Because PpIX is an efficient photosensitizer, ALA has been introduced as a drug for clinical photodynamic therapy (PDT) of cancer (8,9). Photodynamic therapy involves systemic administration of a tumor-localizing photosensitizer and its subsequent activation by light of an appropriate wavelength to create a pho-

Increased binding of chlorin e6 to lipoproteins at low pH values

It is well known that the extracellular pH in tumors is lower than that of normal tissue. This has been proposed to be one of the reasons for the tumor selective uptake of several photosensitizers. Photosensitizers like chlorin e(6) are bound to blood components and delivered to different sites in the organism. Thus, the effect of pH on their interaction with human plasma needs to be studied in order to understand a possible role of the acidic microenvironment in tumors for the drug distribution. Increasing amounts of human plasma in the sample resulted in a gradual red shift of the fluorescence emission maxima of chlorin e(6), indicating binding of the drug to some of the plasma components. Titration showed that the drug-plasma interaction was pH-dependent. The titration curve had an inflection point at 7.4+/-0.1. The relative distribution of the drug among plasma components, as found after ultracentrifugation of chlorin e(6)-doped plasma in a salt gradient, showed more binding of the drug to nonlipoproteins than to lipoprotein classes at both pH values studied (6.5 and 7.4). A decrease in the pH was connected with a significant increase in drug-lipoprotein binding. The pH of the environment affects chlorin e(6)-plasma interaction and the distribution of the drug among different plasma components. The results of this study indicate a possible role of the acidic microenvironment in tumors for the preferential uptake and retention of several photosensitiziers.

Binding of drugs to human plasma proteins, exemplified by Sn(IV)-etiopurpurin dichloride delivered in cremophor and DMSO.

1. The mode-delivery-effect upon the binding of Sn(IV)-etiopurpurin dichloride (SnET2) in human plasma has been studied by ultracentrifugation, combined with absorption and fluorescence spectroscopy. SnET2 was delivered to plasma either in Cremophore EL (CRM) or in dimethyl sulfoxide (DMSO). To facilitate interpretation, optical, conductivity and aggregation properties of SnET2 were obtained for various solutions. 2. The second order rate constant for the aggregation of SnET2 monomers seemed to be remarkably small, of the order of 10(3) M-1 min-1. 3. SnET2 was bound as monomeric entities. Such entities had environmental-sensitive fluorescent properties dependent on the type of protein or solvent (DMSO, CRM, H2O) with which they interacted. 4. SnET2 showed saturable binding with high density subfraction(s) of high density lipoproteins and with one or more high density proteins. Complete or substantial saturation was achieved at the SnET2 level of 3.5 micrograms/ml. Such binding might be mediated by apolipoprotein D and alpha 1-acid glycoprotein. 5. There was little effect of SnET2 concentrations (3.5-35 micrograms SnET2/ml) upon the plasma binding of SnET2, irrespective of the mode of delivery. 6. The percentages of SnET2 bound to low density lipoproteins (LDL), high density lipoproteins (HDL), and high density proteins (HDP) were 10, 70 and 20%, respectively, for delivery in DMSO. The value for LDL also includes binding with very low density lipoproteins (VLDL). For delivery in CRM the corresponding values were 20, 50 and 30%. Apparently, CRM interacted with HDL entities and reduced their affinity for SnET2. 7. The distribution pattern of SnET2 among lipoproteins reflects interactions with apoproteins and/or with surface phospholipids rather than with core lipid constituents of lipoproteins. 8. Conductivity measurements showed that SnET2 was partly an ionic entity in water. 9. The plasma binding of SnET2 is compared with the corresponding binding of other drugs, both tetrapyrroles and nontetrapyrroles.

pH-Dependent Modification of Lipophilicity of Porphyrin-type Photosensitizers

Abstract Structural modifications of photosensitizers (changes in protonation, ionic state and aggregation state) under different environmental conditions should be precisely determined to understand the interaction of the photosensitizers with biological systems. In the present study partition coefficients of hematoporphyrin IX (HpIX), disulfonated meso-tetra-phenylporphine, meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin in the 1-octanol–phosphate buffer system were determined in the pH region 4.0–8.0. Only the partition coefficients of HpIX and mTHPP were found to be pH dependent. Computer processing of fluorimetric titration data was applied to estimate pKa values of the imino nitrogens of mTHPP. Monoprotonated species of mTHPP seem to be unstable or nonexistent. The possibility that both imino nitrogens of this dye are protonated according to a common pKa is proposed. The pKa value of the imino nitrogens of mTHPP was found to be 2.99 ± 0.04 after the application of a model taking aggregation of the drug into account. The contributions of various aqueous ionic species of mTHPP as functions of pH were calculated and compared with partition coefficients.

Hematoporphyrin diethers--V. Plasma protein binding and photosensitizing efficiency.

1. Binding of added hematoporphyrin (HP) ethers to human plasma proteins and lipoproteins has been investigated by ultracentrifugation. 2. The binding to low density lipoproteins (LDL) has been discussed in terms of photosensitized tumor growth delay of tumors and HPLC-retention time, i.e. degree of polarity. 3. The LDL-binding data show a uniform relationship to sensitizing efficiency and degree of polarity, the only exception being HP-diamyl ether. No such uniform relationship exists for less related dyes, such as HP, tetraphenylporphyrin tetrasulfonate and HP-dimethyl ether.

Separation of lipoproteins, albumin and γ-globulin by single-step ultracentrifugation of human serum. Application I: Binding of hematoporphyrin to human serum and to albumin

Previous studies of the serum binding of the photosensitizer hematoporphyrin (Hp) have given widely different results. The serum binding of Hp is therefore further illuminated by experiment and discussion. Ultracentrifugal separation of serum is improved and applied to study the binding of Hp to human serum and HSA. The observed distribution of Hp among the serum proteins is compared with the distribution expected from available association constants for Hp binding with individual proteins. The lipoprotein classes and the two major high density proteins (HDP), albumin and gamma-globulin, were separated in a NaCl-KBr gradient by single spin ultracentrifugation (SW 40; 30,000 rpm). HSA- and HDP-bound Hp were similarly distributed in the centrifuged gradient. Centrifugation of Hp-doped HSA separated the unbound Hp (75%) and the HSA-bound Hp (25%). The present association constant for the Hp-HSA complex (10(3)/M) was much lower than earlier published ones (10(6)/M) found by other techniques. The association of Hp with HDP in serum was much stronger than the association of Hp with the isolated HSA (electrophoretic grade). The estimated ratio of HSA-bound to LDL-bound HP in serum was at least 25 times larger than the experimental value. The percentage of LDL-bound Hp decreased with increasing Hp concentration. The serum binding of Hp is the same as that found previously using another rotor and another salt gradient (70.1 Ti, 70,000 rpm, NaCl-CsCl). LDL has high-affinity-low-capacity binding sites for Hp. HSA is the major HDP protein that binds Hp in human serum. The strength of the HSA-Hp complex may depend on the batch of HSA used and upon the absence/presence of other proteins. Proteins may interact in serum in manners that affect the binding of certain drugs. Neither the type of gradient salt nor the field of gravity affected the serum binding of Hp.

A simplified preparation of hematoporphyrin-diethers—IV

Abstract 1. 1. A relatively simple technique is described for the synthesis of hematoporphyrin ethers, from dimethyl to dioctyl, based upon reaction under prescribed conditions of temperature and time of the porphyrin with the relevant alcohol containing 20% (w/v) H2SO4 and subsequent hydrolysis of ester functions. 2. 2. Yields are good and means of purification, where necessary, are described. 3. 3. The procedures should be suitable for large scale production and are relatively inexpensive.

Localizing and photosensitizing mechanism by tetra(3-hydroxyphenyl)porphin in vivo on human malignant melanoma xenografts in athymic nude mice

Observations on time-dependent localization of tetra(3-hydroxyphenyl)porphin (3-THPP) in human malignant melanoma transplanted to athymic nude mice from 1 to 120 h after intraperitoneal (i.p.) 10 mg kg−1 b.w. administration were made by means of fluorescence microscopy. Fluorescence was found on the membrane of the melanoma cells and in the cytoplasm with a peak fluorescence intensity at 24 h post-injection of 3-THPP. The growth of the tumour cells was obviously inhibited at an early stage after PCT. Morphological changes of the tumour at various intervals after treatment by PCT with 3-THPP were also observed. Diffuse degeneration of the tumour cells with swelling of mitochondria and endoplasmic reticulum, heterochromatin aggregation and margination, etc., and subsequently diffuse necrosis with little or no the background of tumorous vascular response were found at an early stage after PCT. On the other hand, it was also observed that the necrosis of the melanoma areas was caused as a consequence of tumorous vascular injury at a later stage after PCT. Thus, two tumoricidal processes caused by PCT with 3-THPP were seen: a direct phototoxic action on tumour cells at an early stage after PCT and an indirect effect secondary to tumorous vascular injury at a later period after PCT.

Biodistribution, pharmacokinetic, and in-vivo fluorescence spectroscopic studies of photosensitizers

Some key data concerning the pharmacokinetics of PCT photosensitizers are reviewed. The following topics are discussed: The binding of photosensitizers to serum proteins, and the significance of LDL binding for tumor localization, the distribution of sensitizers among different tissue compartments and the significance of extracellular proteins and other stromal elements, such as macrophages, low tumor pH, leaky vasculature and poor lymphatic drainage for tumor selectivity of drugs, the retention and excretion of sensitizers, and intracellular pharmacokinetics. Furthermore, the usefulness of fluorescence measurements in the study of sensitizer pharmacokinetics is briefly discussed. A key observation is that 1O2 has a short radius of action. Since practically all PCT sensitizers act via the 1O2 pathway, only targets with significant sensitizer concentrations can be damaged. A given number of 1O2 entities generated in different organelles (mitochondria, lysosomes, plasma membrane, etc.) may lead to widely different effects with respect to cell inactivation. Similarly, sensitizers localizing in different compartments of tissues may have different photosensitizing efficiencies even under conditions of a similar 1O2 yield.