Reduced albumin-cobalt binding with transient myocardial ischemia after elective percutaneous transluminal coronary angioplasty: a preliminary comparison to creatine kinase-MB, myoglobin, and troponin I.

BACKGROUND: Previous reports suggest that ischemic conditions rapidly reduce the capacity of human albumin to bind exogenous cobalt. A new assay based on human albumin-cobalt binding (ACB) may help detect early myocardial ischemia. We investigated altered ACB during the first 24 hours after transient ischemia induced during elective percutaneous transluminal coronary angioplasty (PTCA). We then compared ACB assay results with creatine kinase isoenzyme (CK-MB), myoglobin, and cardiac troponin I (cTn-I) values after PTCA. METHODS AND RESULTS: In 41 patients undergoing elective PTCA, plasma samples were tested for the ACB assay, CK-MB, myoglobin, and cTn-I before, immediately after, and 6 and 24 hours after PTCA. Thirteen additional patients served as a control group with albumin-cobalt assays performed before and after diagnostic coronary catheterization without angioplasty. ACB assay results demonstrated a significant mean percent difference (10.1%) immediately after PTCA compared with baseline (P < .000001) and returned to baseline by 6 hours after PTCA. ACB assay differences immediately after PTCA were significantly greater than in the control group (10.1% vs -0.9%, P < .001). Mean CK-MB, myoglobin, and cTn-I values were not elevated above baseline immediately after PTCA but were significantly elevated above baseline 6 and 24 hours after PTCA. CONCLUSIONS: These preliminary results suggest that human albumin undergoes a significant reduction in its capacity to bind exogenous cobalt soon after transient coronary occlusion during human PTCA and before significant elevations of CK-MB, myoglobin, or cTn-I. Further confirmatory investigations are warranted to determine if the ACB assay is a useful diagnostic test for early myocardial ischemia.

Laser dosimetry studies in the prostate.

OBJECTIVE: To review the currently available data of photodynamic therapy (PDT) optical dosimetry for possible prostatic applications. SUMMARY BACKGROUND DATA: PDT is a new cancer treatment modality often used as an alternative tumor treatment method. Recently, PDT has been suggested as an alternative therapy for prostatic carcinoma and BPH. METHODS: PDT: utilizes light and a preadministered photosensitizer drug to achieve localized tumor control. This article reviews currently available data on optical dosimetry of PDT in both human and canine prostates. RESULTS: At 630 nm, a common wavelength used for Photofrin PDT, results indicate that light penetration is similar in cancerous and normal prostatic tissue. Because of limited light penetration, multiple fiber irradiation is necessary if eradicating the entire prostate glad is the ultimate goal. The available data also show that dynamic changes occur in light fluence rate distribution during PDT irradiation. CONCLUSIONS: PDT can be used to destroy prostatic tissue. Real-time optical dosimetry is necessary if accurate lesion volume control is desired.

Sentinel lymph node metastasis in experimental melanoma: relationships among primary tumor size, lymphatic vessel diameter and 99mTc-labeled human serum albumin clearance.

BACKGROUND: This study was designed to investigate the relationships among primary tumor size, lymphatic vessel diameters, the incidence of sentinel lymph node (SLN) metastasis and lymphatic clearance from murine footpad melanomas. METHODS: Lymphatic clearance (LC) of [99mTc]HSA from the middle of the footpad of syngeneic C57BL/6 mice, with or without primary melanomas (sizes varying from 1 to 5 mm in anteroposterior diameter), was quantitated using a gamma scintillation detection system. Lymphatic vessel diameters (LD) were measured after injection of aniline blue dye into footpad tumors. The incidence of SLN, femoral lymph node (FLN), and lung metastases was recorded. RESULTS: Metastasis to SLNs increased as tumor growth progressed (r = 0.976, p = 0.001), and there was a correlation between tumor size and both FLN (p = 0.041) and lung (p = 0.055) metastases. There was also a correlation between lymph node metastasis and LC (r = 0.83, p = 0.04) and LD (r = 0.84, p = 0.04). CONCLUSIONS: These studies support the hypothesis that lymph flow and LD is increased in experimental murine melanomas and this relates to both primary tumor size and to lymphatic and hematogenous metastasis.

Lymphatic diameters and radionuclide clearance in a murine melanoma model.

OBJECTIVES: To determine the clearance of a radionuclide from various sizes of footpad melanomas via lymphatics and to measure the diameters of these vessels. DESIGN: Nonrandomized animal study. SETTING: A hospital research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Female mice were injected in the right rear footpad with B16 F10 cells that were allowed to grow to either 1, 2, 3, 4, or 5 mm in anteroposterior diameter. Clearance from feet with or without tumors of injected technetium Tc 99m human serum albumin (99mTcHSA) was assessed for 200 minutes. Calf lymphatic diameters were measured using aniline blue dye. RESULTS: The clearance of the injected 99mTcHSA from mouse footpads without tumors was 1.26 +/- 0.18 x 10(-4) mL/min x cm3 of tissue. Clearance increased 2.24-fold to 2.82 +/- 0.12 x 10(-4) mL/min x cm3 of tissue from 1-mm tumors and to 6.20 +/- 0.08, 6.11 +/- 0.13, 6.91 +/- 0.58, and 7.23 +/- 0.48 x 10(-4) mL/min x cm3 of tissue from 2-, 3-, 4-, and 5-mm tumors, respectively (P < .05). Calf lymphatic diameters increased from 75.41 +/- 9.72 microns in naive nontumor-bearing mice to 93.51 +/- 7.12, 111.61 +/- 27.07, 126.69 +/- 25.20, 124.43 +/- 24.75, and 127.44 +/- 25.35 microns in mice bearing 1-, 2-, 3-, 4-, and 5-mm tumors, respectively (P < .01). CONCLUSIONS: There was a size-dependent, direct correlation between increasing tumor size in the footpad and increasing diameter of lymphatics draining the footpad. Clearance of injected 99mTcHSA from these tumors also exhibited a similar positive correlation with tumor size.

Changes in in vivo optical properties and light distributions in normal canine prostate during photodynamic therapy.

The optical absorption and transport scattering coefficients of normal prostate tissue have been measured in vivo in dogs. The measurements were made at 630 nm before and during treatment by Photofin photodynamic therapy using interstitial optical fiber fluence-rate detectors. Corresponding measurements were made ex vivo, at 1 week after treatment, in the contralateral lobe. The optical properties were derived by applying a diffusion theory model to the fluence rates measured at two different source-detector fiber distances. While the in vivo pretreatment and in vivo contralateral post-treatment absorption and scattering values are self-consistent and in agreement with published data, significant changes were observed in the light fluence rates, and hence in the derived optical properties, during light irradiation. The possible causes of such changes are considered, and the implications for light dosimetry in photodynamic therapy are discussed.

Interstitial photodynamic therapy in the canine prostate.

OBJECTIVE: To determine the depth of tissue destruction and the minimum light dose required for necrosis in interstitial photodynamic therapy (PDT), as a prerequisite for the investigational therapy of patients. MATERIALS AND METHODS: Seven adult beagle dogs were given 2 mg/kg of the photosensitizer Photofrin intravenously and two controls received none. After 24 h, 450 J/cm of 630 nm wavelength laser light was delivered interstitially to the prostate via a 2 cm long diffuser fibre. Seven single-fibre treatments were performed in five sensitized dogs and two single-fibre treatments in the controls. The two remaining sensitized dogs had two fibres placed 10 mm apart within the prostate to determine the coalescence of PDT lesions. The penetration depth of light was measured in all prostates, and after PDT the extent of necrosis was assessed histologically. RESULTS: The mean (standard deviation, SD) radius of PDT destruction around each diffuser was 5.3 (1.4) mm and PDT lesions overlapped in prostates treated with two fibres placed 10 mm apart. There was no observable tissue damage in the controls. The mean (SD) minimum light dose required for PDT necrosis was 84 (64) J/cm2. Differences among animals in the light penetration depth were small, with a mean of 2.14 (0.2) mm, and did not correlate with the depth of necrosis (P = 0.07). Bleeding around the optical diffuser fibre impeded light penetration. CONCLUSION: Interstitial PDT in the canine prostate using Photofrin produced modest volumes of tissue necrosis. The minimum light dose required to induce necrosis was variable because bleeding was unpredictable in relation to the optical fibre.

Sequencing of combined hyperthermia and photodynamic therapy.

Photodynamic therapy (PDT) and hyperthermia are two alternative tumor treatment modalities currently being investigated in clinical trials. It has been suggested that, due to the differences in cell-killing mechanisms, synergetic tumor responses may be achieved if the two modalities are combined in appropriate sequences. This hypothesis is tested in the current study by delivering graded PDT doses during a transient tumor reoxygenation period after a hyperthermia treatment, or delivering graded hyperthermia doses when the tumor becomes acidic and hypoxic after a PDT treatment. The results indicate that the latter combination sequence has a profound effect on tumor response. While treating the tumors with PDT followed by hyperthermia evokes a synergetic tumor response, reversing the sequence results only in an additive effect. Possible mechanisms associated with tissue oxygenation are discussed.

Damage threshold of normal rat brain in photodynamic therapy.

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT-induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence-rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy-induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 10(16) photons/cm3. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.

Tumor oxygenation changes post-photodynamic therapy.

Tumor oxygenation after a photodynamic therapy (PDT) treatment is a critical factor for understanding the posttreatment metabolic pathway of the tumor. It also provides important information for designing combination therapy of PDT and other oxygen-dependent anticancer modalities. In this study, mammary carcinoma in flank and hind leg of C3H mice were subjected to PDT at either subcurative or curative level (12.5 mg/kg Photofrin; 200 or 600 J/cm2, respectively). The before and post-PDT tumor oxygenation was measured with an oxygen-sensitive microelectrode. The data revealed that tumor oxygenation at the time of PDT has a profound effect on posttreatment tumor oxygenation, which may largely be due to an interplay between direct PDT cytotoxicity and PDT damage to the tumor microvasculature. Transient reoxygenation occurred after PDT, which may provide a window for improved combination therapy for other oxygen-dependent modalities.

PO2 in irradiated versus nonirradiated tumors of mice breathing oxygen at normal and elevated pressure.

PURPOSE: To determine if prior tumor irradiation influences tumor pO2 changes in mice breathing oxygen (100%) at normal and elevated pressure. METHODS AND MATERIALS: Single-point pO2 measurements were performed in nonirradiated and previously irradiated (72 h) isotransplanted MCaIV tumors in C3H/Sed mice. Continuous recordings were performed at the same tumor locus under air breathing, followed by 100% oxygen and oxygen at three atmospheres pressure. Following decompression and induction of pentobarbital anesthesia, the procedure was repeated at the same locus. Six nonirradiated and five irradiated tumors were evaluated under the three gas breathing conditions +/- anesthesia. RESULTS: The mean, median, and range of pO2 values did not differ under air-breathing conditions in the nonirradiated vs. previously irradiated tumors. However, prior irradiation substantially enhanced the tumor pO2 increase when the inspired gas phase was switched from air to 100% oxygen at 1 or 3 atmospheres pressure. In four of six nonirradiated tumors, 100% oxygen breathing resulted in a pO2 increase of < 4 mmHg; in the irradiated tumors, the minimum increase was 16 mmHg. Pentobarbital anesthesia did not significantly influence the results obtained. CONCLUSION: These data indicate that the efficacy of oxygen breathing increases during tumor treatment, and suggests that oxygen breathing is a simple nontoxic method for reducing or eliminating radiobiologic hypoxia during therapy.

Neuronal injury after photoactivation of photofrin II.

Photodynamic therapy has been used in the management of patients with malignant brain tumors even though the effects of this form of treatment on the adjacent normal brain are incompletely characterized. The authors examined, in sequential experiments, morphologic alterations affecting the cerebral cortex in rats injected with Photophrin II and exposed to light. Initially, minimal cell alterations, including cisternal swelling of both endoplasmic reticulum and Golgi apparatus, involved only neurons located in the superficial layers of the cerebral cortex exposed to light. These changes spread, over a period of several hours, from the surface to the bottom of the cortex and eventually involved the entire cortical segment exposed to light. The earliest structural signs of lethal injury to neurons developed over a period of 18 hours after porphyrins had been photoactivated and astrocytes had been severely damaged. Signs of lethal injury to neurons included an increase in the number of mitochondrial cristae and appearance of amorphous electron-dense deposits within swollen mitochondria. The appearance of these alterations was followed by segregation of intracytoplasmic organelles and fragmentation of nuclear and cytoplasmic membranes. The tissue changes, including those involving neurons, eventually progressed to coagulation necrosis at 48 hours. These observations suggest that prophyrins injected to rats (48 hours before photoactivation) cause swelling and necrosis of astrocytes. This is followed by neuronal necrosis, which appears at two time intervals; the initial neuronal necrosis occurs after the astrocytic disintegration. A second type of neuronal alteration appears after microvessels become thrombosed and ischemia is likely to develop.

The effect of light fluence rate in photodynamic therapy of normal rat brain.

This paper reports the effect of incident light fluence rate on the depth to which necrotic lesions are produced by photodynamic therapy (PDT) in the brains of normal Fisher rats. The rats were injected intraperitoneally with Photofrin (12.5 mg kg-1) 48 h prior to PDT with a fixed incident fluence of 35 J cm-2. The treatment was performed at 10, 50, 100, and 200 mW cm-2 and also in a periodic manner (30 s "on" at 100 mW cm-2, 30 s "off"). The depth to which necrosis occurred was determined 24 h after treatment by microscopic examination of tissue sections. No differences were found in the depth to which necrosis was produced by any of the five irradiation schedules. This finding is discussed in the context of other published dose-rate experiments.

Lymph flow from murine footpad tumors before and after sublethal hyperthermia.

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starling's Law.

Effects of light beam size on fluence distribution and depth of necrosis in superficially applied photodynamic therapy of normal rat brain.

The light fluence distributions of 632.8 nm light incident on the exposed surface of normal rat brain in vivo have been measured using an interstitial, stereotactically-mounted optical fiber detector with isotropic response. The dependence of the relative fluence rate on depth and the spatial distribution of fluence were compared for incident beam diameters of 3 and 5 mm. The fluence rate at depth of 1-6 mm along the optical axis within the brain tissue was approximately 70% greater for a 5 mm diameter beam than for a 3 mm beam, at the same incident fluence rate, although the plots of the relative fluence rate vs depth were parallel over the depth range 1-6 mm. The depths of necrosis resulting from photodynamic treatment of brain tissue using the photosensitizer Photofrin and irradiation by 632 nm light with 3 and 5 mm incident beams were also measured. The observed difference in necrosis depths was consistent with the measured difference in fluence. The importance of beam size in photodynamic treatment with small diameter incident light fields is discussed.

Variations in pO2 and pH response to hyperthermia: dependence on transplant site and duration of treatment.

It has been clearly established that changes in intratumor pO2 and pH occur following hyperthermia, and it has been hypothesized that these changes may, in some way, be related to the ultimate response (i.e., cure) of the lesion. The purpose of this study was twofold: first, to examine the changes in intratumor pH during the course of a hyperthermia treatment at biologically related end point "doses"; second, to examine the response of pO2 after treatment in a different lesion transplant site. During hyperthermia treatment of the tumor transplanted in the leg, intratumor pH was found to drop from a control value of 6.74 +/- 0.17 to 6.47 +/- 0.13 within 15 min following the start of treatment. The values then remained relatively constant throughout the remainder of the treatment (either 1 or 2 h at 43.5 degrees C). Following the subcurative (10% tumor cures at 30 days; 60 min at 43.5 degrees C) treatment the pH began to rise immediately, while after the higher dose (60% tumor cures at 30 days; 120 min at 43.5 degrees C) a slight rise in pH was followed by a continuous drop in pH for up to 4 h, as we have reported previously. Oxygen response in the two transplant sites (leg and flank) was found to be remarkably different even though the tumor cure rate was identical for a given hyperthermia "dose" in terms of time and temperature. In the leg, only very low levels of oxygen can be measured in the tumor 24 h after treatment with either "dose" studied (all measured pO2 values less than or equal to 5 mm Hg). In the flank, the tumor response is dependent on hyperthermia "dose." Only 28% of measured oxygen values are less than or equal to 5 mm Hg 24 h following a subcurative "dose," while 4 h following the higher "dose" there is a nonsignificant trend toward hypoxia (approximately 65% of values less than or equal to 5 mm Hg) with a subsequent shift toward reoxygenation. These latter observations are contrary to results reported previously and tend to contradict some current theories regarding the physiological mechanisms associated with hyperthermia treatment.

Photoactivated Photofrin II: astrocytic swelling precedes endothelial injury in rat brain.

Light activation of circulating hematoporphyrin derivatives has been used in the treatment of selected brain tumors. The effects of this photodynamic therapy on the non-neoplastic, adjacent brain tissue are incompletely characterized. We studied in adult Fisher rats the time-dependent (1 hour to 7 days) effects of photoactivated Photofrin II. Our protocol was comparable to that used in the treatment of human brain tumors. Structural and functional changes spread from the treatment surface and from the center to the periphery to involve the entire cerebral cortex exposed under a 5 mm craniectomy. The sequential changes spreading from the surface to the deepest cortical layer involve first astrocytes (1 hour), then endothelial cells and, ultimately, neurons. Thrombi were first noted in the microvasculature after 18 hours and coagulation necrosis of the entire area at risk occurred only after 48 hours. The results suggest that the photosensitizing agent crosses the intact blood-brain barrier and enters the astrocytic compartment where it becomes cytotoxic upon light activation. A comparison between the focal brain lesions of photodynamic therapy and those induced by middle cerebral artery occlusion suggests that cell damage evolves along different paths in these two forms of brain injury.

In vivo 31P NMR study of combined hyperthermia and photodynamic therapies of mammary carcinoma in the mouse.

Although the sequence and time interval effects of combined photodynamic therapy (PDT) and hyperthermia tumor treatments have been studied using survival curves, tumor regrowth, and cloning assays, the metabolic response to combined treatment measured by nuclear magnetic resonance (NMR) spectroscopy has not yet been clarified. In this study, mammary carcinoma in the flank of C3H mice was subjected to PDT (12.5 mg/kg Photofrin II, 632 +/- 1 nm at 200 J/cm2) and water bath hyperthermia (43.5 degrees C, 30 min) with no delay or 4 h delay between treatments. In vivo 31P-NMR spectroscopy was employed to measure energy metabolism and pH of the tumors before and serially after treatment for up to 1 week. The data revealed significant differences in the time course of high energy phosphate levels between treatment combinations, which may reflect the biological effectiveness of the combined treatments. Our observations indicate that 31P-NMR spectroscopy can be used to evaluate the metabolic response of tumors to treatment with combined PDT and hyperthermia.

1H magnetic resonance imaging of normal brain tissue response to photodynamic therapy.

1H Magnetic resonance imaging (MRI) was used to study the effects of photodynamic therapy (PDT) on normal rat brain (n = 5) using T1-, T2-, diffusion-, and proton density (rho)-weighted images. Rats received intraperitoneal injections of 12.5 mg/kg of Photofrin II, and 48 hours later the dural area over the frontal cortex was treated with 35 J/cm2 of light (632 +/- 1 nm). The T1-, T2-, and diffusion-weighted images revealed an evolving high contrast region of brain that corresponded to the PDT-treated area. Lesioned brain exhibited significant increases in T1 and T2 relaxation times at 1 day (P less than 0.01) and 3 days (T1, P = 0.018; T2, P less than 0.01) after treatment, compared with the contralateral equivalent volume of nonlesioned brain. Water proton diffusion coefficient (DW) in the lesioned area decreased at 1 day (P = 0.026) and increased at 3 days (P = 0.012) compared with nonlesioned brain. An increase in the proton density ratio (rho D/rho O) from PDT (rho D) versus nonlesioned side (rho O) was found 3 days after PDT treatment (P = 0.03). The data indicate that the biophysical parameters obtained from magnetic resonance imaging scans, T1, T2, DW, and proton density, can be used to monitor changes in an evolving photochemically induced lesion.

Dose-dependent thermal response of tumor pH and energy metabolism evaluated by in vivo 31P NMR spectroscopy and microelectrodes.

In vivo 31P NMR spectroscopy and pH microelectrodes were employed to measure the energy metabolism and pH of a mammary carcinoma in the flank of the C3H mouse before and serially up to a week after various hyperthermia treatments. Water bath hyperthermia was used to treat the tumor at 43.5 degrees C for 30 min (TCD0/30, NMR measurement only), 1 h (TCD10/30), and 2 h (TCD60/30), respectively. The data indicate that, except at 4 h after TCD60/30 treatment, all pH values measured by NMR (pHn) were significantly higher (P less than or equal to 0.001) compared to pH values measured by microelectrodes (pHe) at all treatment levels and times. The magnitude of the difference between pHn and pHe (delta pH) was significantly decreased from the pretreatment level only at 4 h after hyperthermia treatment (0.51 pH units for TCD60/30 and 0.21 pH units for TCD10/30). The ratio of beta-nucleoside triphosphate to inorganic phosphate (beta-NTP/Pi) and pHn were more sensitive to hyperthermia treatment than pHe. The beta-NTP/Pi ratio failed to recover to the pretreatment ratio after 1 or 2 h hyperthermia treatment, while a total recovery was observed within 72 h for 30 min hyperthermia treatment. Our data suggest that the temporal profile of beta-NTP/Pi, pHn, and delta pH may be indicative of the biological outcome of hyperthermia treatment.

Development of lymph node and pulmonary metastases after local irradiation and hyperthermia of footpad melanomas.

C57BL/6 mice with syngeneic B16-F10 melanomas were treated 7 days after tumor inoculation into the footpad with local hyperthermia (HT) of 43.5 degrees C for 90 min. A combination of local 30 Gy X-irradiation (XRT) given 2, 4 or 12 h after HT cured the primary tumor in 34/35 mice, with irreversible damage to normal foot tissues in most of the animals. When 7.5, 10 or 15 Gy XRT were delivered 4, 18 or 24 h after HT, there were only a small number of cures and also a much smaller incidence of irreversible normal tissue damage. HT alone resulted in a significant (P less than 0.001) increase in metastases to regional lymph nodes (RLN) and the lungs. The 'curative' doses of combined XRT and HT resulted in a significant (P less than 0.001) decrease in metastasis to RLN and to the lungs. Conversely, subcurative doses of combined therapy resulted in an increase in RLN and lung metastasis (P less than 0.001). Abdominal lymph node metastasis, not usually seen in control mice, is markedly increased after HT alone or in combination with subcurative XRT (P less than 0.001). The overall survival of mice treated with HT alone is decreased (P less than 0.0028). The survival of mice treated with HT followed 4, 18 or 24 h later with 10 Gy XRT is further decreased (P less than 0.0025). These data show that subcurative HT, or XRT plus HT, increases the incidence of spontaneous metastasis in this syngeneic mouse melanoma model. Curative doses prevent this effect on metastasis, but there is an unacceptable incidence of irreversible damage to the tumor-bearing foot. The cause(s) of this phenomenon are not known.