Preclinical evaluation of 5-aminolevulinic acid-based photodynamic therapy for canine transitional cell carcinoma.

As a prelude to photodynamic therapy, 5-aminolevulinic acid (ALA) was given orally to healthy dogs. ALA-induced protoporphyrin IX (PpIX) fluorescence significantly increased in the mucosa of the urinary bladder in an ALA dose-dependent fashion. Vomiting occurred after ALA administration in 70% of the dogs but did not affect PpIX fluorescence. ALA-based photodynamic therapy (PDT) of the urinary bladder in healthy dogs caused only submucosal oedema within the bladder wall. No haematologic or serum biochemistry abnormalities were observed after ALA administration. Microscopic haematuria was observed in all the dogs after PDT but was mild and self limiting. ALA-based PDT was administered to six dogs with transitional cell carcinoma (TCC) of the lower urinary tract. ALA-based PDT resulted in tumour progression-free intervals from 4 to 34 weeks in five dogs; one dog with pre-existing hydronephrosis died shortly after PDT. Dogs with TCC represent an outbred, spontaneous, tumour model for developing PDT protocols for humans with bladder cancer.

Er:YAG laser modification of root canal dentine: influence of pulse duration, repetitive irradiation and water spray.

The aim of this study was to determine the effects of varying parameters of Er:YAG laser irradiation with and without water spray cooling on root canal dentine in vitro. After horizontally removing tooth crowns from extracted human teeth, roots were axially sectioned into thin slices, exposing the root canal surface. An Er:YAG laser delivered 10-30 J/cm(2) into a 0.4-mm diameter laser spot on the root canal surface. Single pulses of different lengths (80-280 micro s) were applied with and without water spray cooling/irrigation, and sequences of three pulses at a repetition rate of 30 Hz were applied at selected pulse parameters. The irradiated samples were investigated using both confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). At most irradiation conditions, the root canal dentine surface was ablated. Three-dimensional images from CLSM revealed that the cavity walls were not smooth. Depths of the cavities revealed significant differences between the cavities. No debris was observed at the surface of cavities at any irradiation condition. Strong melting and recrystallisation, or unusually flat surfaces with open dentinal tubules were obtained with sequences of three pulses without water cooling. CLSM is an effective tool for investigation of laser effects on root canal dentine. By varying the irradiation conditions, the Er:YAG laser can induce different modifications of root canal surface, which may be very interesting for root canal preparation.

Fluorescence detection of cervical intraepithelial neoplasia for photodynamic therapy with the topical agents 5-aminolevulinic acid and benzoporphyrin-derivative monoacid ring.

OBJECTIVE: The aim of this study was to determine whether 2 photosensitizers, benzoporphyrin-derivative monoacid ring and 5-aminolevulinic acid, are selectively absorbed by dysplastic cervical cells after topical administration. STUDY DESIGN: This phase I clinical trial involved 18 women with biopsy-proven cervical intraepithelial neoplasia at the Beckman Laser Institute, Irvine, Calif. Colposcopically directed cervical biopsy specimens obtained after 1.5, 3, or 6 hours of exposure to a randomly assigned photosensitizer were evaluated for selective drug absorption with hematoxylin and eosin staining and fluorescence microscopy. RESULTS: After exposure to 5-aminolevulinic acid, cervical tissue showed maximal fluorescence in dysplastic cells relative to normal cells, with negligible stromal fluorescence. According to our detection methods benzoporphyrin-derivative monoacid ring demonstrated nonselective, diffusion-driven uptake, with fluorescence appearing in the superficial cells, followed by nonselective drug absorption in the remaining cells and stroma of the epithelium. CONCLUSION: Our data demonstrated selective absorption of 5-aminolevulinic acid by dysplastic cervical cells. This agent therefore represents a promising photosensitizing prodrug for the treatment of cervical intraepithelial neoplasia with photodynamic therapy.

Morphological study of the effects of CO2 laser emitted at 9.3 microm on human dentin.

OBJECTIVE: The purpose of this study was to investigate the effects of dentin ablation using a carbon dioxide (CO2) laser emitted at 9.3 microm by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). BACKGROUND DATA: There have been no reports on effects of CO2 laser irradiation emitted at 9.3 microm on dentin by SEM and CLSM. METHODS: Thirty extracted human teeth showing no clinical signs of caries were used. All teeth were horizontally sectioned to approximately 200 microm thickness and sections were irradiated using a 9.3 microm CO2 laser at different parameters as follows: 26 mJ [energy density (ED) 53.0 J/cm2] and 30 mJ (ED 61.1 J/cm2). After laser irradiation, samples were treated with sodium hypochlorite, stained using rhodamine-123, and observed with CLSM followed by SEM procedures. RESULTS: No craters or cracks were observed, but many small molten and rehardened particles were documented on the sample surface using SEM. Some small cracks were seen in the subsurface layer, and some patent dentinal tubules were detected using CLSM. CONCLUSION: These results suggest that laser irradiation at these parameters affected the sample surface only (less than 20 microm) and would be less harmful to thermal damage of dental pulp for dentin ablation.

Highly selective targeting of ovarian cancer with the photosensitizer PEG-m-THPC in a rat model.

Photodynamic therapy (PDT) uses light to activate a photosensitizer that has been absorbed or retained preferentially by cancer cells after systemic administration. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21,23-[H]-porphyrin (PEG-m-THPC), was evaluated to target selectively unresectable pelvic ovarian cancer bulks. Our goals were two-fold: (1) to establish an ovarian cancer model suitable for the development of debulking techniques and (2) to characterize the pharmacokinetics and tumor selectivity of PEG-m-THPC by fluorescence microscopy. NuTu-19 ovarian cancer cells were injected into the caudal part of the right psoas muscle of Fisher rats. Five weeks later, 30 mg/kg body weight of PEG-m-THPC was injected intravenously. Necropsy was performed between 4 and 10 days following drug application, and fluorescence of the tumor and various abdominal organs was measured. All rats developed bulky pelvic tumors with an average diameter of 2.6 cm (+/- 0.6 SD). Tumor masses were encompassing and infiltrating pelvic organs in a similar manner to ovarian cancers in humans. Fluorescence of cancer tissue was maximal 8-10 days following drug application. At 8 days, the tumor-to-tissue ratio was 40:1 (+/- 12 SE) for most abdominal organs. We conclude that this tumor model may be used for the study of new pelvic debulking techniques, and that the tumor selectivity of PEG-m-THPC is exceptionally high 8 days after drug application. Based on these data, we are currently developing a PDT-based minimally invasive debulking technique for advanced ovarian cancer.

Preclinical study in cats of the pro-photosensitizer 5-aminolevulinic acid.

OBJECTIVE: To determine whether feline cells were able to convert 5-aminolevulinic acid (ALA) to protoporphyrin IX (PpIX) in vivo and in vitro, whether i.v. administration of ALA to healthy cats resulted in adverse effects, and whether PpIX accumulated in a squamous cell carcinoma (SCC) of a cat. ANIMALS: 4 healthy adult cats and 1 adult cat with a cutaneous SCC. PROCEDURE: In vitro production of PpIX was determined by incubating Crandell feline kidney cells with ALA. Effects of ALA administration and in vivo production of PpIX were determined by administering ALA (100, 200, or 400 mg/kg of body weight) to healthy cats and collecting skin biopsy specimens for up to 24 hours after drug administration. Blood samples were collected for CBC and serum biochemical analyses, and necropsies were performed. Accumulation of PpIX in a SCC was determined by treating a cat with a facial SCC with ALA and collecting specimens of the tumor and adjacent grossly normal skin. RESULTS: Incubation of ALA with feline cells resulted in time- and dose-dependent cytoplasmic accumulation of PpIX in vitro. After i.v. ALA administration, PpIX was detected in all tissues examined, with the highest fluorescence intensity in epithelia and in squamous cell carcinoma. The tumor-to-skin fluorescence intensity ratio was 5. All cats developed hepatotoxicoses. CONCLUSIONS AND CLINICAL RELEVANCE: Results from this limited number of cats suggest that ALA may be a useful photosensitizer in cats, but that doses > 100 mg/kg, i.v., may not be safe.

Laser-micropipet combination for single-cell analysis.

Due to its potential for exquisite mass detection limits and resolving power, capillary electrophoresis is used for biochemical measurements on single cells; however, accurate measurements of many physiological parameters require sampling strategies that are considerably faster than those presently available. We have developed a laser-based technique to lyse single, adherent, mammalian cells on millisecond time scales. The cellular contents are then introduced into a capillary where electrophoretic separation and detection are performed. Improved temporal resolution of biological measurements results from the extremely rapid lysis made possible by this method. Additionally, the cell is not perturbed by mechanical or electrical stresses prior to sampling. Such disturbances can alter cellular physiology, resulting in inaccurate measurements. The fast cell lysis, the absence of cellular stresses prior to lysis, and the application to adherent mammalian cells are significant refinements to CE-based measurements on single cells. With this laser-micropipet combination, it will be possible to measure the intracellular concentration of molecules that change on subsecond to second time scales, for example, substrates of many cellular enzymes.

Uptake of the photosensitizer benzoporphyrin derivative in human endometrium after topical application in vivo.

STUDY OBJECTIVE: To determine both the time leading to maximum endometrial drug uptake and distribution of the photosensitizer benzoporphyrin derivative-monoacid ring A (BPD-MA) after intrauterine instillation (Canadian Task Force classification ). DESIGN: Assessment of histology specimens (Canadian Task Force classification I). SETTING: University-based facility. PATIENTS: Twenty-two women scheduled for hysterectomy. INTERVENTIONS: We instilled 1.5 ml of a 2 mg/ml of BPD-MA-Hyskon solution into the uterine cavity of 22 women before hysterectomy. The fluorescence induced was measured by fluorescence microscopy on frozen sections of uterine samples from 20 of 22 patients. Systemic uptake of BPD-MA was determined in plasma of six patients by spectrofluorometry. MEASUREMENTS AND MAIN RESULTS: The BPD-MA-induced fluorescence was maximum 1 hour after instillation, with significantly higher uptake in endometrial glands than in underlying stroma. Hormonal endometrial stimulation correlated with fluorescence intensity: atrophy < secretory phase < proliferative phase. Strongest fluorescence was seen in endometrial cancer. Drug uptake by endometrial glands was found at a depth of 2 mm from the surface. Systemic uptake of BPD-MA was under the detection level of 2 ng/ml after application. CONCLUSION: Fluorescence in human endometrial glands suggests that selective destruction of human endometrium with photodynamic therapy may be possible 1 hour after topical application of BPD-MA for benign and malignant lesions. No systemic drug uptake, side effects, or major technical difficulties were detected. Limited penetration of the drug and selective uptake by endometrial glands provided a high degree of safety for endometrial ablation.

In vivo fluorescence detection of ovarian cancer in the NuTu-19 epithelial ovarian cancer animal model using 5-aminolevulinic acid (ALA).

The purpose of this study was to determine whether in vivo fluorescence detection of protoporphyrin IX (PpIX) could be used to identify intraperitoneal micrometastases of epithelial ovarian carcinoma after application of 5-aminolevulinic acid (ALA). ALA was applied intraperitoneal at different concentrations (25, 50, and 100 mg/kg) and iv (100 mg/kg) to immunocompetent Fischer 344 rats bearing a syngeneic epithelial ovarian carcinoma. At different time intervals after ALA administration (1.5, 3, and 6 hr) the peritoneal cavity was illuminated with ultraviolet (uv) light. In vivo fluorescence of PpIX initially was determined by direct visualization. Subsequently ex vivo measurements were made with a slow-scan, thermoelectrically cooled CCD camera. Red in vivo fluorescence was observed in ovarian micrometastases smaller than 0.5 mm in 100% of the ALA-administered animals independent of time interval, drug concentration, or route of administration. The intensity of the fluorescence was concentration dependent as strong fluorescence was consistently found only above 25 mg/kg ALA. Ex vivo tumor to peritoneum fluorescence yield peaked 3 hr after administration of a 100 mg/kg intraperitoneal dose. Direct visualization of in vivo fluorescence after ALA application may improve the detection of intraperitoneal ovarian cancer micrometastases.

Selective photosensitizer localization in the human endometrium after intrauterine application of 5-aminolevulinic acid.

OBJECTIVE: Our purpose was twofold: to determine the distribution of the endogenous photosensitizer protoporphyrin IX in the uterus and to ascertain the time interval leading to maximal endometrial fluorescence after intrauterine instillation of 5-aminolevulinic acid. STUDY DESIGN: One milliliter of a 400 mg/ml 5-aminolevulinic acid-Hyskon solution was instilled into the uterine cavity of 27 women before hysterectomy. On frozen sections of uterine samples 5-aminolevulinic acid-induced fluorescence was measured with fluorescence microscopy. RESULTS: 5-Aminolevulinic acid-induced fluorescence could first be detected in the superficial endometrial glands 75 minutes after drug injection. In the endometrial gland stumps fluorescence intensity peaked 4 to 8 hours after 5-aminolevulinic acid instillation and was > 48 times higher than in the underlying myometrium. CONCLUSIONS: Fluorescence in the endometrial glands suggests that selective photodynamic destruction of the endometrium may be possible 4 to 8 hours after intrauterine 5-aminolevulinic acid instillation.

Fluorescence imaging studies for the disposition of daunorubicin liposomes (DaunoXome) within tumor tissue.

Unilamellar liposomes that retain their contents in the systemic circulation can alter the pharmacokinetics of anticancer agents in favorable ways. It has long been recognized that certain liposome compositions may increase the local drug concentration substantially above that achievable with a free drug. We report here that liposomes can alter the in vivo disposition of an entrapped drug not only on a macroscopic but also on a microscopic scale. We show through in vitro studies that intact liposomes composed of distearoylphosphatidylcholine and cholesterol and containing daunorubicin (DaunoXome) are taken up into P1798 tumor cells. These liposomes produce an enhanced cytotoxicity relative to the free drug for incubation times longer than about 8 h. For in vivo studies, we developed and used a noninvasive fluorescence imaging technique to follow the accumulation of liposomal daunorubicin within murine tumors. With this method, we show that the maximum concentration of the available liposomal drug in tumors exceeds that of the free drug, and additionally, liposomal daunorubicin persists at high levels for several days. Total liposome-delivered drug fluorescence from whole tumor extracts peaks at about 8 h. In comparison, the fluorescence intensity of daunorubicin demonstrate persistent high levels of daunorubicin fluorescence within cells and throughout the tumor masses. Free daunorubicin, in contrast, transiently achieves modest levels of fluorescence and rapidly drops to background within a few h. These results indicate distinct mechanisms for the localization of free and liposomal daunorubicin, suggesting that liposmal daunorubicin can provide sustained intracellular levels of the drug within the tumor.

Selective photosensitizer distribution in vulvar condyloma acuminatum after topical application of 5-aminolevulinic acid.

OBJECTIVE: Our purpose was to determine the feasibility of selective photosensitization of vulvar condylomas by use of tropical application of 5-aminolevulinic acid. STUDY DESIGN: In vivo fluorescence was assessed and biopsy specimens of condylomas were taken for fluorescence microscopy in 24 patients at different times after application of 2.5% 5-aminolevulinic acid ointment or 20% 5-aminolevulinic acid cream. RESULTS: Both in vivo fluorescence imaging and fluorescence microscopy showed selective fluorescence of condylomas of the labia minora and vestibule only within short time intervals, because fluorescence of poorly keratinized normal epithelium was induced by both 5-aminolevulinic acid formulations. In non-hair-bearing skin, lesional fluorescence remained highly selective. Fluorescence microscopy showed that 90 minutes after drug application peak selectivity in epithelial lesional fluorescence was significantly higher with 2.5% 5-aminolevulinic acid ointment (4.5 +/- 0.9) than it was with 20% cream (2.1 +/- 0.2). CONCLUSION: Selective fluorescence of vulvar condyloma acuminatum can be induced by nonselective topical 5-aminolevulinic acid application. Studies evaluating selective photodynamic destruction of condylomas are justified.

Photosensitization of the rat endometrium following 5-aminolevulinic acid induced photodynamic therapy.

BACKGROUND AND OBJECTIVE: The impact of photodynamic therapy (PDT) on the endometrium following topical application of 5-aminolevulinic acid (ALA) was studied in a rat model. Study Design/ MATERIALS AND METHODS: Fluorescence microscopy revealed peak ALA to protoporphyrin IX (Pp IX) conversion 3-6 hours after application. Significantly higher Pp IX levels were observed in the endometrial glands compared with endometrial stroma and myometrium. RESULTS: Histological studies showed PDT-induced endometrial destruction with atrophy 7-10 weeks after treatment. Reproductive performance studies demonstrated significant implantation failure in the treated uterine horns compared with controls. The number of implantation sacs in the treated and untreated horns was 0.4 +/- 0.3 and 8.9 +/- 1.0, respectively (P < 0.01). CONCLUSION: We conclude that the high rate of Pp IX conversion in the endometrial glands can be exploited to treat dysfunctional uterine bleeding with PDT. In addition, this concept may also be applied to study endometrial regeneration and embryo implantation mechanisms.

Photodynamic therapy of ocular melanoma with bis silicon 2,3-naphthalocyanine in a rabbit model.

PURPOSE: To investigate bis (tri-n-hexylsiloxy) silicon 2,3-naphthalocyanine (SINc; 0.5 mg/kg) for photodynamic therapy of an experimental ocular melanoma in pigmented rabbits. METHODS: SINc was dissolved in canola oil by heating, emulsified with Tween 80, and administered by ear vein. Pharmacokinetics were studied in frozen tumor sections by fluorescence microscopy using a charge coupled device, camera-based, low-light detection system with digital image processing at 1 and 24 hours. A Ti:sapphire laser and a microlens were used to deliver the light (770 nm; 40 mW/cm2; 20 J/cm2). A control rabbit received light without SINc. RESULTS: Localization studies of SINc showed intravascular distribution shifting to a tumor stromal and perivascular distribution 24 hours after treatment. Tissue thermal damage after irradiation was minimal in the control. Exudative retinal detachments were not observed. Tumor destruction was observed, with sharp demarcation to a depth of 3.5 mm. CONCLUSIONS: Tumor light penetration was good at 770 nm, and thermal effects from the exciting light alone were minimal. Photodynamic therapy with SINc resulted in localized tumor destruction reflecting the light beam path without damage to adjacent tissue or intraocular complications.

Photodynamic laser cyclodestruction with chloroaluminum sulfonated phthalocyanine (CASPc) or Photofrin (PII) vs. Nd:YAG laser cyclodestruction in a pigmented rabbit model.

BACKGROUND AND OBJECTIVE: To investigate Photofrin (PII) and CASPc for photodynamic therapy (PDT) of the ciliary body in rabbits. STUDY DESIGN/MATERIALS AND METHODS: PII (10 mg/kg) or CASPc (1 mg/kg) was given by ear vein. Pharmacokinetics were studied in frozen sections by fluorescence microscopy (CCD camera based low light detection system with digital image processing) at 1 and 24 h (8 rabbits; 16 eyes). Laser light was delivered (argon pumped dye laser; 630 and 675 nm; 8 rabbits; 16 eyes) by contact fiberoptic. To compensate for iris attenuation, irradiance was 125 mW/cm2 (20, 40, 80, or 160 J/cm2). Controls (4 rabbits; 8 eyes) received laser light without photochemicals (OD) and for comparison, continuous wave Nd:YAG laser by fiberoptic (0.8-1.2J; OS). RESULTS: Localization studies showed intravascular distribution with some selective ciliary body distribution at 24 h (PII > CASPc). Rabbits treated with PII or CASPc exhibited variable amounts of gross ciliary body edema, infarction, and necrosis by 24-48 h. This response was not seen in PDT control tissues; damage was seen in the iris and ciliary body, with partial vacuolization of the pigment epithelium. CONCLUSION: PDT may offer a more selective approach to ciliary body destruction. A small but significant thermal effect was seen during PDT from melanin photon uptake with damage to iris and ciliary body. Thermal damage and potential interaction with ocular visual pigments may limit use of these photochemicals and wavelengths for PDT of the ciliary body.

Photodynamic destruction of endometrial tissue with topical 5-aminolevulinic acid in rats and rabbits.

OBJECTIVE: The goal of this study was to determine the optimal parameters for photodynamic endometrial destruction with topically applied 5-aminolevulinic acid, a precursor for the endogenous synthesis of the fluorescent photosensitizer protoporphyrin IX. STUDY DESIGN: 5-Aminolevulinic acid pharmacokinetics were measured in rat and rabbit models by analyzing tissue frozen sections 3 to 12 hours after topical administration. Dose-response studies were conducted for 100 to 400 mg/ml 5-aminolevulinic acid. Photodynamic therapy was performed intraluminally, and tissue morphologic features were evaluated 3 and 7 days after treatment. RESULTS: Peak fluorescence was observed 3 hours after topical administration. Glandular fluorescence significantly exceeded stromal and myometrial in all studies, particularly for 200 mg/ml 5-aminolevulinic acid. Histologic studies revealed persistent epithelial destruction with minimal regeneration. CONCLUSION: Topical 5-aminolevulinic acid photodynamic therapy can be used for highly effective, long-lasting destruction of endometrial epithelium. However, optical dosimetry can vary, particularly in the rabbit model, and this appears to have an impact on long-term reepithelialization.

Benzoporphyrin derivative: a potent photosensitizer for photodynamic destruction of rabbit endometrium.

OBJECTIVES: To determine the optimal pharmacokinetic characteristics for photodynamic endometrial destruction using topically applied benzoporphyrin derivative and to evaluate long-term morphologic effects in a rabbit model. METHODS: Benzoporphyrin derivative pharmacokinetics were measured by analyzing frozen tissue sections 1.5-12 hours following topical administration. Photodynamic therapy was induced intraluminally 1.5 hours after drug application, and tissue morphology was evaluated by light and scanning electron microscopy 1 and 4 weeks after treatment. RESULTS: The highest glandular and stromal fluorescence was observed 1.5 hours after topical administration. Relative fluorescence of the endometrial glands significantly exceeded that of stroma and myometrium mainly at 1.5 and 3 hours (P < .0001). Histology revealed persistent epithelial destruction with minimal regeneration. Stroma resembled scar tissue in the majority of sections. The bordering myometrium was loosened and invaded by connective tissue. CONCLUSION: Topically applied benzoporphyrin derivative can be used for highly effective, long-lasting photodynamic destruction of rabbit endometrium. However, optical dosimetry can vary, particularly in the rabbit model, and this appears to affect long-term reepithelialization.