In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters.

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Heterogeneity of delta-aminolevulinic acid-induced protoporphyrin IX fluorescence in human glioma cells and leukemic lymphocytes.

Delta-aminolevulinic acid (ALA)-PDT efficacy is particularly dependent on the quality of protoporphyrin IX (PpIX)-induced synthesis. The purpose of this study was to determine the ability of cells from two human cancer types to synthesise PpIX after ALA administration. Biopsies of glioma cells have been obtained from patients with glioblastomas that have or have not been given ALA IV (ex vivo incubation). Peripheral blood lymphocytes, obtained from leukemic patients, have also been ALA-incubated in vitro. In glioma cells, fluorescence heterogeneity was extensive either in ALA infused patients or in ex vivo ALA incubated cells. Mean intensities after 3 h were 110 cts (range 0-340) and 1000 cts (range 0-3600). Similar results were found in leukemic lymphocytes where cell fluorescence varied from 0 to 480 cts with a percentage of fluorescent cells varying with time and from one patient to another. Furthermore, PpIX was not detectable in two patients with CLL. These observations suggest that a marked heterogeneity of ALA uptake and/or PpIX synthesis exists in a given human cancer cell population particularly after systemic administration. Improvements for ALA transformation into PpIX are strongly recommended to ensure the efficacy of ALA/PpIX-PDT.

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography.

Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15mgkg(-1). Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000cps for 5mgkg(-1), 6h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7min) detectable only after injection of at least 15mgkg(-1). Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination.

Synthesis, and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for photodynamic therapy.

This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.

Effects of photodynamic therapy on adhesion molecules and metastasis.

Photodynamic therapy (PDT) induces among numerous cell targets membrane damage and alteration in cancer cell adhesiveness, an important parameter in cancer metastasis. We have previously shown that hematoporphyrin derivative (HPD)-PDT decreases cancer cell adhesiveness to endothelial cells in vitro and that it reduces the metastatic potential of cells injected into rats. The present study analyzes the influence of PDT in vivo on the metastatic potential of cancers cells and in vitro on the expression of molecules involved in adhesion and in the metastatic process. Photofrin and benzoporphyrin derivative monoacid ring A (BPD) have been evaluated on two colon cancer cell lines obtained from the same cancer [progressive (PROb) and regressive (REGb)] with different metastatic properties. Studies of BPD and Photofrin toxicity and phototoxicity are performed by colorimetric MTT assay on PROb and REGb cells to determine the PDT doses inducing around 25% cell death. Flow cytometry is then used to determine adhesion-molecule expression at the cell surface. ICAM-I, MHC-I, CD44V6 and its lectins (àHt1.3, PNA, SNA and UEA) are studied using cells treated either with BPD (50 ng/ml, 457 nm light, 10 J/cm2) or Photofrin (0.5 microgram/ml, 514 nm light, 25 J/cm2). Changes of metastatic patterns of PROb cells have been assessed by the subcutaneous injection of non-lethally treated BPD or Photofrin cells and counting lung metastases. First, we confirm the metastatic potential reduction induced by PDT with respectively a 71 or 96% decrease of the mean number of metastases (as compared with controls) for PROb cells treated with 50 ng/ml BPD and 10 or 20 J/cm2 irradiation. Concerning Photofrin-PDT-treated cells, we find respectively a 90 or 97% decrease (as compared with controls) of the mean number of metastases for PROb cells treated with 0.5 microgram/ml Photofrin and 25 or 50 J/cm2 irradiation. Then, we observe that CD44V6, its lectins (àHt1.3, PNA, SNA) and MHC-I are significantly decreased (compared with the other molecules tested) in PROb and REGb cells after both BPD and Photofrin PDT treatment. These modifications in adhesion-molecule expression, particularly of CD44V6, can thus account only for part of the decrease in the metastatic potential of PDT-treated cancer cells. Changes in adhesion-molecule expression induced by PDT are only transient, implying that the rate of metastatic reduction is probably not linked simply to these changes.

Delta-aminolevulinic acid-induced fluorescence in normal human lymphocytes.

Endogenously generated protoporphyrin IX (PpIX) from exogenous delta-aminolevulinic acid (ALA) has the photodynamic capacity to inactive cancer cells of different origins. The aim of this study was to characterize the ability of normal lymphocytes to transform ALA into PpIX in order to appreciate through further studies changes in pathologic lymphocytes. We investigated in this study PpIX synthesis by normal human lymphocytes using a confocal laser microspectrofluorometer. Live lymphocytes were identified by monoclonal antibody fluorescent labeling. B and T lymphocytes synthesized PpIX (80-100 counts), with a maximum being reached after 4 h ALA incubation. When T subpopulations of lymphocytes were labeled, T4 and T8 changes in fluorescence kinetics were similar, reaching a maximum after 5 h ALA incubation. The influence of monoclonal antibody labeling on this delayed increase for maximum fluorescence is considered. Phytohemagglutinin (PHA, incubation for 72 h) lymphocyte stimulation induced a 100% increase in PpIX fluorescence for T lymphocytes, whereas pokeweed mitogen activation produced an increase of about 50% in the B- or T-lymphocyte signal. Finally, the scanning fluorescence image clearly indicated the inhomogeneity of cytoplasmic ALA-induced PpIX fluorescence, which was probably due to the distribution of mitochondria. The influence of this heterogeneity on PpIX photosensitivity effects is discussed.

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT.

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.

Potential efficacy of a delta 5-aminolevulinic acid bioadhesive gel formulation for the photodynamic treatment of lesions of the gastrointestinal tract in mice.

A delta 5-aminolevulinic acid (ALA) bioadhesive gel has been developed and evaluated in an in-vivo mouse model for photodynamic treatment of gastric cancer or Barrett's oesophagus. Four gels were tested: noveon AA-1, keltrol T, lutrol and blanose. An initial in-vitro study of gel adhesion showed that noveon and keltrol had longer polyethylene transit times than lutrol and blanose. In-vivo assays indicated that protoporphyrin IX was synthesized by gastric mucosa when ALA-noveon and ALA-lutrol were used (preferable results for noveon). Keltrol was eliminated from the study after these investigations. Only ALA-noveon gel was retained for studies of the relationship between ALA dose and fluorescence. Fluorescence measurements in-vivo showed that ALA concentration and application time had an influence on protoporphyrin IX synthesis. Maximum intensity (2091 counts s-1) was found with 2 mg mL-1 ALA, and fluorescence intensities differed with application time, reaching 1805 counts s-1 after 240 min. ALA-noveon, showing good adhesion and enabling efficient diffusion of ALA at a pH < 6, was considered the best formulation for maintaining ALA stability.

Photodynamic treatment of normal endothelial cells or glioma cells in vitro.

Photodynamic therapy is based on the interaction of a sensitizer (hematoporphyrin derivative) selectively retained by tumor cells, which becomes toxic after light exposure. We studied the influence of exogenous prostaglandins and indomethacin on photodynamic therapy of normal human endothelial cells and glioma cells. Although differing in origin and kinetic properties, endothelial cells exhibited photodynamic therapy sensitivity quite comparable to that of C6 cells. However, in contrast to studies performed using radiotherapy, exogenous prostaglandins decreased rather than protected the surviving fraction of both cell types treated by photodynamic therapy. Indomethacin, a potent inhibitor of endogenous prostaglandin synthesis, increased the surviving fraction of C6 glioma cells but not that of endothelial cells. Exogenous or endogenous prostaglandins seem to influence in vitro photodynamic therapy in a different way than does radiotherapy.

Integrated imaging system for stereotactic neurosurgery.

Talairach's method remains the most universal of all stereotactic methods. It makes it possible to go back to an examination left interrupted, but above all, it provides multiple lateral and coronal approaches in matters of epilepsy, radio-isotopes or photobiology. The advances achieved in modern imaging methods, notably CT and MRI, and the performance of modern computers have enabled us to devise an integrated imaging system meant to accelerate and make feasible calculations of penetrating trajectories, according to the position of the target as previously defined by CT and MRI sections, and taking into account the position of vessels given by angiography. The principal options we selected were: transfer onto video tapes of teleangiographic films and introduction of CT and MRI images obtained either from films or directly in digits on cartridges or networks. The system includes an advanced PC-type microcomputer with 8 memories of 1,024/1,024 images: 4 mega octets random access memories and a hard disc of 150 mega octets; 2 high-resolution screens to present the images, dialogue tools (alphanumeric screen, keyboard, trackball) and the usual peripherals. Our system performs three main functions: it acquires images to create or complete the patient's records; it exploits the images by calculating the parameters required for operations; it handles the records through the various peripherals of the system. Our system can retrace the proportional 3D squaring according to the CA-CP distance calculated from ventriculography or MRI, and calculates the position of the SEEG electrodes. The stereoscopic effect is obtained by means of glasses with liquid crystal obturator from two angiographic series: one orthogonal, the other 6 degrees out of phase. The daily use of this system on more than 50 patients has shown that it is accurate and reliable, irrespective of the makes of CT and MRI machines.

[Sequential scintiscans. An easy method for testing C.S.F. shunts]. / La scintigraphie séquentielle Méthode simple de contrôle des dérivations de L.C.R.

Different procedures have been since 1965 by the authors to test with isotopes the patency of shunts for hydrocephalus. The authors now measure the time 99m Technetium takes to appear in the thyroid gland after injection in the flushing device. This method shows easily and very quickly, with no computing, the dynamics of C.S.F. shunts. No hospitalisation is necessary and it is often possible to determine the cause of the disturbance in the shunt.

[Bone integration within calcium phosphate ceramic by creation of posterior inter-articular lumbar spine fusions in dogs]. / Etude de l'intégration osseuse d'une céramique de phosphate de calcium par réalisation d'arthrodèses lombaires inter-articulaires postérieures chez le chien.

Following earlier experiments in which several calcium phosphate ceramics were tested, the aim of this study was to evaluate bone integration within a macroporous biphasic calcium ceramic (M.B.C.P.) in comparison to autologous bone grafts, by producing posterior lumbar spine fusions in dogs. Five dogs were used. 40 posterior lumbar joints were exposed; 38 articular surfaces were removed, 27 M.B.C.P. and 5 autologous bone grafts were implanted; 6 joints were kept free of implant for control purposes. Fixation with a metal rod was performed using Luque's method. Tetracycline was used for a double marking of bone growth. Joints were removed at 4 weeks in 1 dog, 8 weeks in two and 13 weeks in the remaining 2 dogs. Demineralized and non demineralized sections were examined. After 1 month, sections with M.B.C.P. showed early signs of mineralization of the scar tissue between the biomaterial and one of the facets of the joint. Fluoroscopy revealed an absence of bone growth in the pores at the center of the ceramic implant. After 3 months, a number of M.B.C.P. blocks had become integrated into both sides of the joint; however some microfractures in the biomaterial with discontinuity between the mineralized areas was seen. Macroporous calcium phosphate ceramic leads to revascularization allowing bone reconstruction. Similarities in the kinetics and mechanisms of bone integration between the ceramic and autologous bone grafts are demonstrated.

[Use of stereotaxic neurosurgery in the treatment of pain in cervicofacial cancers]. / Apport de la neuro-chirurgie stéréotaxique dans le traitement des algies des cancers cervico-faciaux.

Mesencephalic tractotomy employing the stereotaxic method was conducted 12 times in 11 patients with pain due to cervicofacial cancer only partially relieved by high doses of opiates. Immediate results were spectacular in 10 cases and partial on the other 2 occasions. However, relief was not permanent even in patients in whom the greatest improvement was obtained, and the usual analgesics had to be prescribed after 3 months. The operation, which requires only a short period of anesthesia, was always well supported, complications being anesthesia of one side of the body in 2 patients and convergence paralysis in two cases, one of which was transient. Mesencephalic tractotomy constitutes a new weapon for the treatment of diffuse cervicofacial cancer pain not responding to major medical treatment. Elective thermocoagulation of the Vth and IXth nerves was also successfully employed for systematized pains.

[Intraneural synovial cyst in the peroneal nerve. Case report (author's transl)]. / Kyste synovial intraneural du sciatique poplite externe. A propos d'un cas.

The authors report one case of paralysis of the peroneal nerve due to an intraneural synovial cyst, in connection with the superior tibio-fibular joint. After an anatomic work and a complete revue of the literature, etiopathogenic and diagnostic problems seem to be resolved, and this justifies their terminologic choice.

[Comparative study of complications observed in ventriculo-atrial and ventriculo-peritoneal derivations. Apropos of 106 cases]. / Etude comparative des complications observées dans les dérivations ventriculo-atriales et ventriculo-péritonéales. (A propos de 106 cas)

In this study that concerns 106 cases, authors insist on the frequency of the infectious and mechanical complications. They make a review of the etiologies and of the various materials used by themselves and study successively the mechanical, infectious and mixed complications, sector by sector. They drive, at each level, conclusions concerning the results with their subsequent lessons. Having recalled, in short, the principles of supervision of any child bearer of a "valve", they conclude by developing their present idea of the "ideal derivation" by on the base of the material submitted to experimentation. They insist on the value of the ventricular-peritoneal derivations in the new-born.

[Value of angiography in the diagnosis of peripheral nerve tumors]. / Intérêt de l'angiographie dans le diagnostic des tumeurs des nerfs périphériques

In current practice, arteriograpy is already acknowledged, at the present time, as a method of primary importance which cannot be dispensed withfor diagnosis, prognosis and treatment. By opposition, considering pathology of the peripheric nerves, arteriography is often overlooked before chirurgical exploration though it affords the same advantages as in the remaining fields of pathology. Technically our preference goes to selective angiography by direct puncture of humeral or femoral artery. Arteriography will permit:-Visualisation of the location of the tumor close to the major vessels;-Evidence of the exact site of the efferent and afferent pedicles;-Discovery of other localisation. Mostly arteriography helps preoperatively to histological diagnosis. It must be recalled that benign neurinomas often assume a misleading aspect on the tumorography with stagnation of the contrast medium which might suggest malignancy. These benign tumors have three peculiar features: 1. Arteries are observed to stretch out around the lesion but never to narrow. 2. There is no explosive vascularisation ; hypervascularisation appears progressively, is located on the periphery of the lesion, and occupies but a part of the whole area. 3. Veins are not invaded but pushed apart and duly appear in normal timing. This peculiar aspect is quite the opposite of what can be observed in malignant tumors, which are, by far, less frequent.

[Contribution of arteriography to the investigation of peripheral nerve tumours (author's transl)]. / Apport de l'artériographie dans les tumeurs nerveuses tronculaires des membres.

Based on their personal experience of the use of arteriography in 11 patients, the authors describe the angiographic appearances of both benign and malignant peripheral nerve tumours. They emphasise the reasons for errors, and the criteria enabling the precise nature of the lesion to be determined pre-operatively in the majority of cases. Other contributions supplied by angiography are discussed.

[Stereotaxic treatment of expanding cysts in craniopharyngiomas by endocavitary beta-irradiation (Re-186; Au-198; Y-90)]. / Traitement stéréotaxique des kystes expansifs de crânio-pharyngiomes par irradiation endocavitaire bêta (Re 186; Au 198; Y 90).

The authors report the results of 17 intracystic injection of colloidal 186 rhenium, 2 colloidal 198 gold, and 1 colloidal 90 yttrium for endocavitary treatment of 18 cystic craniopharyngiomas (16 pts during a period ranging from January 1975 till July 1982. Follow-up studies ranging from 12 to 72 months (m: 36 m) revealed that all craniopharyngiomas cysts were effectively treated with cessation of fluid formation, progressive shrinkage of the formerly expansive cysts, and finally cyst obliteration in 75% of the cases. No early or late side-effects were observed during the entire observation period. Late reexpansion of one craniopharyngioma cyst, observed at 11 months, was successfully treated by a second injection. Leakage of colloid isotope into the CSF spaces during the "test" or "therapeutic" injections occurred in 18% of the global number of injections, however no clinical complications were observed. On the basis of a clinical dosimetric study a 30 000 rads wall-dose, not exceeding 40 000 rads is actually considered as the safer dose for endocavitary treatment of craniopharyngioma.

[Tactical approach to tumours of the sacro-coccygeal region. Report of five cases (author's transl)]. / Tactique d'approche des tumeurs de la région sacro-coccygienne. A propos de cinq cas.

The name of sacro-coccygeal tumors is used generally for various tumors embryologically different in their origin. Rare, but not exceptional in number, these sacro-coccygeal tumors may have various locations and different sizes which make diagnosis more or less difficult. Benign in the first months of life, risks of malignancy will increase with age. Systematical intrarectal exploration will help not to overlook pelvic forms and will recognize pelvic extension in other types. Once found out, the extension of these sacro-coccygeal tumors should be estimated by complementary examination choosen according to the presumed type of tumor and it is to be regretted that myelography should be so often neglected. Treatment should always be surgical with one piece aphotesis if possible; good preparation and exact reanimation of the child are necessary because sometimes surgical treatment may be a very severe aggression for small babies. Constant surgical and biological overall should be maintained for many years in order to discover as early as possible any local relapse or metastasis.

[Perineal pain and lesions of the internal pudendal nerves]. / Douleur périnéale et souffrance des nerfs honteux internes.

A number of chronic pain syndromes in the perineal area can be related to pudendal nerves suffering. The constancy of symptoms among various patients, and in duration for a particular one, alterations revealed by electrophysiologic studies, pain relief by diagnostic blocks, data from anatomic studies, preliminary results of medical and surgical applied therapies, give consistent arguments for possible organic lesions of pudendal nerves.