Effect of delivery system on the pharmacokinetics and tissue distribution of bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC), a photosensitizer for tumor therapy.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a representative of a group of naphthalocyanine derivatives with spectral and photophysical properties that make them attractive candidates for photodynamic therapy (PDT). Tissue distributions were studied in tumor-bearing rats as a function of delivery system and time following administration. The tumor model was an N-(4-[5-nitro-2-furyl]-2-thiazolyl) formamide (FANFT)-induced urothelial cell carcinoma transplanted into one hind leg of male Fischer 344 rats; isoBOSINC was delivered to the rats by intravenous injection of 0.50 mg/kg of body weight as a suspension either in 10% Tween 80 in saline (Tween) or 10% (Cremophor EL + propylene glycol) in saline (Cremophor). The isoBOSINC was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin. Quantitation was by a high-performance liquid chromatographic technique with detection that utilizes the native fluorescence of the naphthalocyanine derivative. Independent of the delivery system, the dye was retained in tumors at higher concentrations than in normal tissues, except for spleen and liver. The isoBOSINC retention in tumors was high and was vehicle dependent. For Tween, the maximal ratio of dye in tumor versus peritumoral muscle occurred 12 h after injection; for Cremophor, the maximal ratio occurred later, 336 h postinjection. When the drug was delivered in Tween, isoBOSINC in serum showed two compartment kinetics: half-lives of about 2 and 11 h were found for the distribution and the elimination phases, respectively. When Cremophor was the vehicle, the elimination half-life was about 20 h, and one compartment kinetics was observed. The latter findings may explain the generally higher levels of the dye attained by the tissues at later times with Cremophor as the vehicle. An interesting exception was that after 7 and 14 days postinjection in Tween, the levels of dye found in testes were six- to seven-fold higher than those found after Cremophor delivery. Levels of dye were very low or not detectable in the brain. Optimal parameters for PDT of tumors with this novel photosensitizer are clearly time- and vehicle-dependent, and future PDT studies will need to incorporate these modulators.

Photodynamic therapy of tumors: effects of hematoporphyrin derivative on normal rat intestine.

Hematoporphyrin derivative (HpD) is a complex mixture of dicarboxylic porphyrins in addition to dimers, oligomers and aggregates of variable sizes. The ability of this mixture (and enriched preparations thereof) to be retained by tumors and to sensitize them to destruction by light has led to worldwide studies of the treatment modality called photodynamic therapy (PDT). Understanding how PDT affects normal tissues surrounding the tumor is of crucial importance. Prior studies have documented that normal intestinal blood flow can be disrupted by HpD PDT. In addition, mucosal and submucosal damage to normal rat jejunum was observed following HpD PDT. The present study was designed to correlate the above observed changes with local levels of porphyrins. Three groups of Fischer 344 rats were injected with 0 (controls), 10 or 20 mg HpD/kg body weight. Twenty-four hours after the drug was injected, jejunal segments were excised and blood samples taken. Jejunal contents were obtained by saline perfusion and analyzed for porphyrins alongside the jejunal homogenates and the bloods. Jejunal segments of animals injected with 10 or 20 mg HpD/kg b.w. had porphyrin levels some 4- and 9-fold above controls (controls m +/- S.D. = 0.25 +/- 0.12 micrograms/g wet weight). For each injected dose the jejunal perfusates contained 2- and 3-fold higher levels of porphyrins (in transit) as the controls (controls, m +/- S.D. = 1.48 +/- 0.66 micrograms/g w.w.). In the general circulation, only 0.6% or 0.4% of the respective injected doses remained. After HpD i.v. injections, high levels of porphyrins were found in the small intestine and in transit.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetic and tissue distribution studies of the photosensitizer bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) in normal and tumor-bearing rats.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a representative of a group of naphthalocyanine derivatives with spectral and photophysical properties that make them attractive candidates for photodynamic therapy (PDT). Tissue distributions were studied in normal and in tumor-bearing rats as a function of time following intravenous injection of isoBOSINC as a suspension in 10% Tween 80 in saline. The dose studied was 0.25 mg/kg of body weight. The compound isoBOSINC was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin, and quantitated by a high-performance liquid chromatographic technique. The tumor model, an N-(4-[5-nitro-2-furyl]-2-thiazolyl)formamide (FANFT)-induced urothelial cell carcinoma, was transplanted into the hind legs of Fischer 344 rats. The dye was retained in tumors at higher concentrations than in all tissues and organs examined, except for spleen and liver. The highest concentration ratio of dye in tumor versus peritumoral muscle (24.5) occurred 9 h after injection. Serum clearance of isoBOSINC showed similar kinetic behavior for both groups of rats, with a t 1/2 of elimination of approximately 10 h. At 7 and 14 days postinjection, the levels of dye found in testes were generally higher than in most other tissues, except spleen and liver. Concentrations of isoBOSINC were either very low or not detectable in rat brain. Trace amounts of the dye were excreted in the urine, and by day 14 approximately 17% of the dose was accounted for in the feces. The significant levels of the drug in tumors, as well as the excellent ratios of tumor-to-muscle concentration observed, have promising implications for PDT of tumors.

Photosensitizers for tumor therapy: determination of bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) in rat tissue and serum by high-performance liquid chromatography.

Bis(di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine (isoBOSINC) is a synthetic potential photosensitizer for tumor therapy. A new method, which combines solvent extraction and several purification steps, has been developed to determine its presence in tissues. Separation and quantitation of isoBOSINC is done by high-performance liquid chromatography on a silica column with toluene as a mobile phase and using fluorescence detection (lambda ex = 365 nm, lambda em = 750 nm). For recovery studies, isoBOSINC was added to muscles at levels of 0.067 and 0.67 micrograms/g; the mean recoveries were 100%, with coefficients of variation of 6.1 and 6.4%, respectively. For liver samples, the amounts added were 0.67 and 6.7 micrograms/g and for serum 0.67 and 6.70 micrograms/ml. The mean recoveries for liver were 86 and 93%, with coefficients of variation of 7.7 and 4.4%, respectively. For serum, the mean recoveries were 99 and 96%, with coefficients of variation of 2.6 and 6.9%, respectively. Due to its low detection limit and selectivity, the method is appropriate for pharmacokinetic as well as tumor uptake studies following in vivo exposure to isoBOSINC. Preliminary data on tissue distribution of the photosensitizer in normal rats are also presented.

A dose response analysis of purpurin derivatives used as photosensitizers for the photodynamic treatment of transplantable FANFT induced urothelial tumors.

Rats engrafted with transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) induced urothelial tumors were treated with purpurin derivatives and red light (greater than 590 nm., 360 joules/cm.2). Treated and control tumors were harvested 12 days after treatment and dry weights compared. At doses of 5.0 micrograms./gm. and 2.5 micrograms./gm. body weight, the purpurins designated NT1 and NT2, when combined with light, caused statistically significant (p less than 0.02) tumor regression when compared to light shielded controls. At 1.0 micrograms./gm. body weight, NT1 and light also induced significant tumor regression (p less than 0.02). Purpurins, which have strong absorption bands above 650 nm. and can be synthesized with a high degree of purity, appear to have potential as photosensitizers for photodynamic therapy.

Morphological study of the combined effect of purpurin derivatives and light on transplantable rat bladder tumors.

Purpurin derivatives, a group of synthetic photosensitizers, were tested for their photodynamic activity against transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced urothelial tumors growing in male Fischer 344 rats. Histological examination of tumors in animals treated with the purpurin derivatives and red light (greater than 590 nm, 360 joules/cm2) revealed tumor cell necrosis 24 h after completion of therapy. Control tumors showed no histological change. Purpurins have a major absorption peak in the red region (greater than 650 nm) of the visible spectrum, a region with good tissue penetration, and purpurins can be synthesized with a high degree of purity. This study demonstrates the potential of purpurin derivatives as photosensitizers for photodynamic cancer therapy.

Photodynamic treatment of transplantable bladder tumors in rodents after pretreatment with chloroaluminum tetrasulfophthalocyanine.

Chloroaluminum tetrasulfophthalocyanine (AlPCS) was used as a photosensitizer for the photodynamic treatment of transplantable N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) induced urothelial tumors. Two groups of six rats each were injected with AlPCS (three micrograms./gm. body weight) and 24 hours after injection underwent photodynamic treatment with red light (greater than 590 nm., 360 joules/cm.2). Tumors examined four hours (Group I) and 24 hours (Group II) after the completion of phototreatment showed extensive hemorrhagic necrosis. Tumors treated with AlPCS alone showed no changes. In two other groups of six rats each, blood flow to tumors treated with AlPCS alone (Group III) and AlPCS plus light (Group IV) was measured using the radioactive microsphere technique. AlPCS plus light resulted in a significant decrease (p less than .05) in tumor blood flow within 10 minutes of completion of phototreatment while AlPCS alone had no effect on tumor blood flow. These findings are similar to those observed when higher doses (10 micrograms./gm. to 20 micrograms./gm. body weight) of hematophorphyrin derivative (HpD) and light were used for phototreatment of FANFT induced tumors. AlPCS is a stable sulfonated derivative of tetraazotetrabenzoporphyrin which absorbs maximally in the red portion of the visible spectrum, a region with good tissue penetration properties. These studies suggest the AlPCS may be a useful new agent for photodynamic therapy of cancer.

Histological study of the effect of hematoporphyrin derivative photodynamic therapy on the rat jejunum.

Hematoporphyrin derivative photodynamic therapy is evolving as a local treatment for neoplastic disease. The emphasis of previous research has been on the determination of mechanisms of tumoricidal activity and defining the tumoricidal porphyrin component in hematoporphyrin derivative. The effect of hematoporphyrin derivative photodynamic therapy on normal tissue has received little attention. In the following study we examined the morphological changes of normal rat intestine exposed to hematoporphyrin derivative and light. In this model a segment of rat jejunum was exposed to red light (greater than 590 nm; 360 J/cm2) 24 or 72 h after the i.v. administration of hematoporphyrin derivative (5 or 10 micrograms/g body weight). Control groups received either no treatment, hematoporphyrin derivative only, or light only. Four h after treatment, intestinal segments were removed and examined by light microscopy. Segments treated with hematoporphyrin derivative and light showed extensive sloughing of the mucosa and submucosa with sparing of the muscular and serosal layers. It appears that hematoporphyrin derivative photodynamic therapy is capable of causing mucosal and submucosal damage to normal rat jejunum at these doses of light and hematoporphyrin derivative.

Jejunal blood flow after exposure to light in rats injected with hematoporphyrin derivative.

Experiments were performed to determine the effect of hematoporphyrin derivative (HPD) photodynamic therapy on blood flow to normal rat intestine. A segment of rat jejunum was exposed to red (greater than 590 nm) light (200 mW/cm2) 24 h after the i.v. administration of HPD. Blood flow to the light exposed segment was determined using the radioactive microsphere technique while blood flow to an adjacent light shielded segment of intestine served as an internal control. Animals were divided into six groups of six each: Group I, no HPD, no light; Group II, light, no HPD; Group III, HPD (20 micrograms/g body weight), no light; and Group IV, HPD (20 micrograms/g body weight), light. Blood flow in these four groups was determined 10 min after completion of a 30-min exposure to light. Only in Group IV was there a statistically significant decrease (P less than 0.005) in blood flow to the segment treated with HPD and light. In Groups V [HPD (20 micrograms/g body weight), light] and VI [HPD (10 micrograms/g body weight), light] blood flows were determined 24 h after exposure to light. In both of these groups there was also a significant (P less than 0.05) decrease in blood flow in the segment treated with HPD and light. This study demonstrates that normal intestinal blood flow can be disrupted by HPD photodynamic therapy.

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells.

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.

Localization of hematoporphyrin derivative in injured bladder mucosa. An experimental study.

Hematoporphyrin derivative, a fluorescent mixture of porphyrins, has the putative property of being retained in neoplastic tissue after systemic administration. This preferential retention is the basis for the use of this agent as a tumor localizer and tumor photosensitizer. The retention of hematoporphyrin derivative in non-neoplastic but regenerating urothelium has not been reported. In this study, thermal urothelial injury was induced in Fischer 344 rats. Animals were then injected intravenously with hematoporphyrin derivative at 3 days, 1, 2, 3 and 6 weeks after injury. Photography under ultraviolet illumination was used to detect porphyrin fluorescence in the bladder mucosa. Up to 3 weeks after injury porphyrin fluorescence was detectable in areas of inflammation and hyperplasia around the area of injury. This study suggests that in this experimental model HpD fluorescence is not specific for neoplasia.

Hematoporphyrin derivative photochemotherapy of experimental bladder tumors.

Recent studies have shown that disruption of tumor blood flow is a major consequence of hematoporphyrin derivative photochemotherapy. A series of experiments was undertaken on the transplantable N-(4-(5-nitro-2-furyl)-2-thiazolyl)-formamide induced urothelial tumor in Fischer 344 rats to determine a dose response for both hematoporphyrin derivative and light. Tumor blood flow was used as the biologic criteria of response. Hematoporphyrin derivative doses of 10 micrograms./gm. body weight or above were necessary to cause a significant decrease in tumor blood flow when the tumors were illuminated with 360 joules/cm.2 of noncoherent red light (greater than 590 nm.). With a constant hematoporphyrin derivative dose of 20 micrograms./gm. body weight, significantly lower tumor blood flows were observed with fluences of 240 joules/cm.2 and above. In order to correlate dose response to tumor regression, experiments were done in which tumor dry weights were determined 3 weeks after completion of photochemotherapy (360 joules/cm.2). Hematoporphyrin derivative doses of 10 micrograms./gm. body weight or above were necessary to induce tumor regression. These studies support the hypothesis that disruption of tumor blood flow is a tumoricidal mechanism of hematoporphyrin derivative photochemotherapy.

Blood flow in transplantable bladder tumors treated with hematoporphyrin derivative and light.

Following hematoporphyrin derivative (HPD) photochemotherapy, blood flow to transplantable N-[4-(5-nitro-2-furyl)-2-thia-zolyl] formamide-induced urothelial tumors was determined by a radioactive microsphere technique using either 103Ru or 141Ce. Two tumors were implanted s.c. on the abdominal wall of Fischer 344 weanling rats. HPD (10 mg/kg body weight) was administered 24 hr prior to phototherapy (red light, greater than 590 nm; 360 J/sq cm). One of the two tumors was shielded from light exposure and served as an internal control. Blood flows were determined in control animals that received no treatment (Group 1), HPD only (Group 2), or light only (Group 3). In Groups 4 and 5, animals received the combination of HPD and light but differed in the time interval between treatment and blood flow determinations (10 min and 24 hr, respectively). Only blood flow to tumors treated with HPD and light showed a significant decrease (p less than 0.05) when compared with their internal controls both at 10 min (Group 4) and 24 hr (Group 5) after completion of phototherapy. These studies suggest that disruption of tumor blood flow may be an important mechanism of action of this method of cancer therapy.

The diagnosis of acute intermittent porphyria. Usefulness and limitations of the erythrocyte uroporphyrinogen I synthase assay.

In acute intermittent porphyria (AIP) the inherited metabolic defect residues in a partial enzyme deficiency at the uroporphyrinogen I synthase (URO-S) step of heme biosynthesis. Assay of this enzyme in erythrocytes is increasingly used for diagnosis of the genetic defect. Erythrocyte URO-S activity was measured by three laboratories in members of 14 AIP kindreds and found helpful for diagnosing the asymptomatic carrier state, since activity of the enzyme was usually distributed bimodally. However, a definite diagnosis of the carrier state was not always possible with this assay: approximately 7% of a total of 340 determinations were inconclusive. Repeat enzyme assays, enzyme assays by more than one laboratory, quantitative urine porphobilinogen determinations, and studies of other family members all aided in arriving at a firm diagnosis for most of those in whom a single erythrocyte URO-S measurement was inconclusive. Because the enzyme is unstable, blood specimens should be stored frozen if immediate assay is not possible.