Martin Ochsner

Photophysical and photobiological processes in the photodynamic therapy of tumours

Photodynamic therapy (PDT) is an innovative and attractive modality for the treatment of small and superficial tumours. PDT, as a multimodality treatment procedure, requires both a selective photosensitizer and a powerful light source which matches the absorption spectrum of the photosensitizer. Quadra Logics Photofrin, a purified haematoporphyrin derivative, is so far the only sensitizer approved for phase III and IV clinical trials. The major drawbacks of this product are the lack of chemical homogeneity and stability, skin phototoxicity, unfavourable physicochemical properties and low selectivity with regard to uptake and retention by tumour vs. normal cells. Second-generation photosensitizers, including the phthalocyanines, show an increased photodynamic efficiency in the treatment of animal tumours and reduced phototoxic side effects. At the time of writing of this article, there were more than half a dozen new sensitizers in or about to start clinical trials. Most available data suggest a common mechanism of action. Following excitation of photosensitizers to long-lived excited singlet and/ or triplet states, the tumour is destroyed either by reactive singlet oxygen species (type II mechanism) and/or radical products (type I mechanism) generated in an energy transfer reaction. The major biological targets of the radicals produced and of singlet oxygen are well known today. Nucleic acids, enzymes and cellular membranes are rapidly attacked and cause the release of a wide variety of pathophysiologically highly reactive products, such as prostaglandins, thromboxanes and leukotrienes. Activation of the complement system and infiltration of immunologically active blood cells into the tumorous region enhance the damaging effect of these aggressive intermediates and ultimately initiate tumour necrosis. The purpose of this review article is to summarize the up-to-date knowledge on the mechanisms responsible for the induction of tumour necrotic reactions.

Light scattering of human skin: A comparison between zinc(II)— phthalocyanine and photofrin II®

Zinc(II)-phthalocyanine is the active component of the liposomal formulation CGP 55847 which showed a highly activity in photodynamic therapy studies on a variety of animal tumours (K. Schieweck et al., SPIE Conf. Proc., 2078 (1994) 107-118). The photophysical properties of zinc(II)-phthalocyanine have been studied in detail and compared with those of Photofrin II(R), the only sensitizing agent approved so far for Phase III and IV clinical trials (M. Ochsner-Bruderer, Inaugural Dissertation, University of Basle, 1994). As will be shown in a series of papers, the main photophysical properties of zinc(II)-phthalocyanine are significantly better than those of Photofrin II(R) (M. Ochsner-Bruderer, Inaugural Dissertation, University of Basle, 1994). In this paper we especially consider the effect of the absorption wavelength on the penetration of light into the human skin. The results clearly show that the longer absorption wavelength of zinc(II)-phthalocyanine causes a deeper penetration of light into the human skin as compared with Photofrin II(R). In addition to this, the higher extinction coefficient (epsilon S) lowers the zinc(II)-phthalocyanine dose required to induce a tumour necrosis.

Protein kinase C inhibitors potentiate angiotensin II-induced phosphoinositide hydrolysis and intracellular Ca2+ mobilization in renal mesangial cells.

Stimulation of mesangial cells with angiotensin II leads to rapid phosphoinositide hydrolysis and subsequent mobilization of intracellular Ca2+. Previous studies indicated that activation of protein kinase C (PKC) triggers a negative-feedback signal, which limits phosphoinositide turnover. By comparing the relative susceptibility of PKC isoenzymes to phorbol ester-induced down-regulation with the down-regulation of the functional cell response, i.e. feedback inhibition of inositol trisphosphate production, we inferred that PKC-alpha and PKC-delta are candidates for regulating phosphoinositide hydrolysis in mesangial cells. To test this hypothesis further, we examined the effects of inhibitors of PKC, that are reportedly not active on PKC-delta, on angiotensin II-stimulated phosphoinositide degradation and Ca2+ mobilization. Pretreatment of mesangial cells with the PKC inhibitors staurosporine and K252a potently augmented inositol trisphosphate and 1,2-diacylglycerol formation as well as Ca2+ mobilization in response to angiotensin II. These results suggest that PKC-alpha, but not PKC-delta, is the most likely candidate mediating feedback inhibition of angiotensin II-stimulated phosphoinositide turnover in mesangial cells.

Potentiation of angiotensin II-stimulated phosphoinositide hydrolysis, calcium mobilization and contraction of renal mesangial cells upon down-regulation of protein kinase C.

Long‐term pretreatment of rat mesangial cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) down‐regulated protein kinase C activity and potentiated the angiotensin II‐induced inositol trisphosphate (InsP3) formation. This increased response to angiotensin II occurred without a significant change in the receptor number or K d value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4α‐phorbol 12,13‐di‐decanoate was without effect on angiotensin II‐stimulated InsP3 generation. Long‐term pretreatment with TPA also increased the angiotensin II‐induced mobilization of Ca2+ and the subsequent contraction of mesangial cells.

Nafenopin, a hypolipidemic and non-genotoxic hepatocarcinogen increases intracellular calcium and transiently decreases intracellular pH in hepatocytes without generation of inositol phosphates

Addition of nafenopin (30-300 microM to 45Ca2+ preloaded cultured hepatocytes caused a rapid and concentration-dependent increase in 45Ca2+ efflux in a manner similar to vasopressin, as evidenced by the loss of radioactivity from the cells. In contrast to vasopressin, addition of nafenopin to [3H]inositol prelabelled hepatocytes in culture did not increase [3H]inositol phosphate production. When added simultaneously with vasopressin, nafenopin inhibited the vasopressin-stimulated [3H]inositol phosphate production. In hepatocyte suspensions isolated from rats treated for 1 week with a carcinogenic dose of nafenopin (1000 ppm in their daily food) the incorporation of [3H]inositol into the phosphoinositide fraction, particularly phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, was much less than that in hepatocytes isolated from untreated rats. The vasopressin-stimulated [3H]inositol phosphate production was also decreased. Experiments with hepatocyte suspensions preloaded with Ca2+ or pH sensitive fluorescent indicators demonstrated that addition of nafenopin caused an increase in intracellular free Ca2+ and transient acidification of the cells. The increase in [Ca2+]i was decreased by only about 25% when extracellular calcium was removed indicating that nafenopin mainly mobilizes Ca2+ from intracellular stores. The recovery to basal pH was amiloride-sensitive indicating the importance of Na+/H+ exchange in pH recovery after intracellular acidification. Amiloride also inhibited DNA synthesis induced by nafenopin and by epidermal growth factor in cultured hepatocytes; but this effect occurred concomitantly with inhibition of basal DNA synthesis. We suggest that hepatic Ca2+ mobilization induced by nafenopin may play an important role in the mechanism by which nafenopin exerts its physiological as well as its tumour promotive activity upon chronic treatment with carcinogenic doses.

CGP 55 847, liposome-delivered zinc(II)-phthalocyanine as a phototherapeutic agent for tumors

Zinc(II)-phthalocyanine (Zn-Pc) was chosen for development as a second-generation photosensitizer for photodynamic therapy (PDT) of tumors and for benign conditions because of its advantageous chemical and photophysical properties. Zn-Pc displayed good selectivity for malignant tissue in pharmacokinetic studies with Meth-A-sarcoma-bearing BALB/c mice when injected in a dose of 0.125 mg/kg, delivered by CGP 55 847. Intravenous doses of Zn- Pc ranging from 0.032 to 0.375 mg/kg caused tumor necrosis and, subsequently, cure of Meth-A-sarcoma-bearing mice when phototreatment was performed 48 hours after injection of CGP 55 847. Intravenous injection of Zn-Pc into hairless mice in doses ranging from 0.1 to 1.0 mg/kg caused dose- and time-dependent phototoxicity. We conclude that the promising pharmacological properties of liposomally delivered Zn-Pc, along with its advantageous chemical and photophysical properties, warrant the development of CGP 55 847 as a candidate drug for photodynamic therapy of tumors in humans.

Synthesis of axially disubstituted octakis-alkoxy-germanium(IV) phthalocyanines

A series of new octakis-substituted Germanium-phthalocyanines for use in photodynamic therapy of tumors has been synthesized. Starting from 1,4-dialkoxy-2,3-dicyano-benzene the metal-free octakis-alkoxy-phthalocyanine 13 was prepared. The metallation to the corresponding octakis-alkoxy-GeCl2-phthalocyanine 15 was done with a GeCl4-DMF complex. Alkaline hydrolysis gave quantitatively the octakis-alkoxy-Ge(OH)2- phthalocyanine 16. By substitution of the hydroxy groups with alcohols, thiols and silanols the axially disubstituted phthalocyanines were obtained. The most important photophysical properties for PDT (Q-bands, singlet oxygen quantum yields and (Phi) f) are reported.

Simultaneous measurement of Ca2+ transients and changes in the cell volume and microviscosity of the plasma membrane in smooth muscle cells. Evaluation of the effect of formoterol.

The effects of the beta 2-adrenoceptor agonist formoterol (50 nM) on the angiotensin II (20 nM)-induced Ca2+ response and changes in the cell volume and microviscosity of the plasma membrane of vascular smooth muscle cells were studied. Applied as a model substance for the stimulation of the phosphoinositide-phospholipase C pathway, angiotensin II has been used to simulate the bronchospasm of smooth muscle in asthma. Our results demonstrated that angiotensin II-induced smooth muscle contraction primarily involves an InsP3-mediated release of Ca2+ from intracellular stores and, to a minor extent, an enhanced influx of Ca2+ through the plasma membrane. Both the Ca2+ response and the contractile reaction were strongly antagonized by pretreatment of the cells with 50 nM formoterol. The protective effect of formoterol on smooth muscle contractions is proposed to be mainly related to a direct stimulation of beta 2-adrenoceptor-coupled cAMP generation. Moreover, it is predicted that the interaction between the beta 2-adrenoceptor glycoprotein and adenylate cyclase will be enhanced following a formoterol-associated decrease in the microviscosity of the plasma membrane.

Simultaneous registration of Ca2+ transients and volume changes in rat mesangial cells: Evaluation of the effects of protein kinase C down-regulation

The intracellular free Ca2+ concentration ([Ca2+]i) could be correlated with the contractile response in rat mesangial cells using an apparatus which measured both biochemical processes simultaneously. Long-term pretreatment of mesangial cells with 12-O-tetradecanoly-phorbol 13-acetate (24 h, 500 nM) increased the (20 nM) angiotensin II-induced mobilization of Ca2+ and led to an enhanced and sustained contraction of the cells. The contractile response was delayed by approximately 3.5 s with respect to the intracellular increase in Ca2+ concentration. The simultaneous registration of Ca2+ transients and cell contractions confirms that [Ca2+]i is the major determinant of the angiotensin II-mediated mesangial cell contraction.

Simultaneous measurement of Ca2+ transients and of membrane depolarizations in synaptosomes.

Calcium and membrane potential sensitive dyes have been widely used to study the biochemical effects of the intracellular calcium concentration and of the membrane potential on diverse biochemical processes. However, due to the discontinuous measurement techniques applied, it was until now impossible to get an insight into the sequence and dynamics of the induced biological reactions. In order to study the relationship between the intracellular calcium concentration and the membrane potential, an apparatus was developed capable of measuring both biological processes simultaneously. Potassium chloride induced changes of the synaptosomal membrane potential and of the intracellular calcium concentration are presented.