A locus for bipolar affective disorder on chromosome 4p.

The main clinical feature of bipolar affective disorder is a change of mood to depression or elation. Unipolar disorder, also termed major depressive disorder, describes the occurrence of depression alone without episodes of elevated mood. Little is understood about the underlying causes of these common and severe illnesses which have estimated lifetime prevalences in the region of 0.8% for bipolar and 6% for unipolar disorder. Strong support for a genetic aetiology is found in the familial nature of the condition, the increased concordance of monozygotic over dizygotic twins and adoption studies showing increased rates of illness in children of affected parents. However, linkage studies have met with mixed success. An initial report of linkage on the short arm of chromosome 11 (ref. 4) was revised and remains unreplicated. Reports proposing cosegregation of genes found on the X chromosome with bipolar illness have not been supported by others. More recently bipolar disorder has been reported to be linked with markers on chromosomes 18, 21, 16 and a region on the X chromosome different from those previously suggested. We have carried out a linkage study in twelve bipolar families. In a single family a genome search employing 193 markers indicated linkage on chromosome 4p where the marker D4S394 generated a two-point lod score of 4.1 under a dominant model of inheritance. Three point analyses with neighbouring markers gave a maximum lod score of 4.8. Eleven other bipolar families were typed using D4S394 and in all families combined there was evidence of linkage with heterogeneity with a maximum two-point lod score of 4.1 (theta = 0, alpha = 0.35).

Erythropoiesis equivalence, pharmacokinetics and immune response following repeat hematide administration in cynomolgus monkeys.

Hematide is a synthetic PEGylated peptidic erythropoiesis stimulating agent (ESA) that is presently being developed for the correction of anemia in patients with chronic renal failure. Unlike currently marketed ESAs, Hematide does not possess any sequence homology to erythropoietin (EPO) and has not elicited moribund immune responses in animal safety studies thereby allowing the generation of a robust safety package. Animals administered marketed ESAs develop anti-EPO antibodies that null the effect of the administered ESA and neutralize endogenous EPO, resulting in severe anemia that precludes the interpretation of chronic safety studies. The primary objective of this study is to determine whether Hematide-specific antibodies are generated when male monkeys are exposed to high Hematide doses (10 mg/kg, intravenous [IV] and subcutaneous [SC]) administered at frequent dosing intervals (every two weeks) for a total of 9 doses; secondary objectives are to evaluate whether developed antibodies impact pharmacokinetics (PK) and pharmacology. In this study, no Hematide-specific antibodies were detected. Hematide exhibits a prolonged plasma half-life and slow clearance by either IV or SC administration. Hematide induced significant erythropoiesis with reticulocytosis and subsequent increases in red blood cells, hematocrit and hemoglobin (Hgb) levels. No erythropoietic differences were noted between the IV and the SC dosed groups with mean +/- SD Hgb levels of 20.9 +/- 2.5 and 20.3 +/- 2.1 g/dL, respectively, occurring on Day 48, corresponding to Hgb increases of 6.5 and 6.7 g/dL, respectively, over pre-dose levels. In conclusion, Hematide is a potent erythropoiesis stimulating agent that exhibits plasma persistence in monkeys. Similar erythropoietic responses were produced following IV and SC administration. The absence of antibody development suggests that Hematide, at the doses and regimen described, has a low immunogenic potential in cynomolgus monkeys.

Photoangioplasty: An emerging clinical cardiovascular role for photodynamic therapy.

Photodynamic therapy (PDT) has been studied and applied to various disease processes. The potential of PDT for selective destruction of target tissues is especially appealing in cardiovascular disease, in which other existing interventional tools are somewhat nonselective and carry substantial risk of damage to the normal arterial wall. Enthusiasm for photoangioplasty (PDT of vascular de novo atherosclerotic and, potentially, restenotic lesions) is fueled by more effective second-generation photosensitizers and technological advances in endovascular light delivery. This excitement revolves around at least 4 significant attributes of light-activated therapy: the putative selectivity and safety of photoangioplasty, the potential for atraumatic and effective debulking of atheromatous plaque through a biological mechanism, the postulated capability to reduce or inhibit restenosis, and the potential to treat long segments of abnormal vessel by simply using fibers with longer light-emitting regions. The available nonclinical data, coupled with the observations of a new phase I trial in human peripheral atherosclerosis, suggest a promising future for photoangioplasty in the treatment of primary atherosclerosis and prevention of restenosis.

Reduction of vein graft disease using photodynamic therapy with motexafin lutetium in a rodent isograft model.

BACKGROUND: Motexafin lutetium (Lu-Tex) is a photosensitizer that targets atheromatous plaque. Subsequent photoactivation (photodynamic therapy [PDT]) induces local cytotoxic effects. The aim of the present study was to investigate whether Lu-Tex targets vein graft intimal hyperplasia and whether subsequent photoactivation attenuates the disease process. METHODS AND RESULTS: The subcellular localization of Lu-Tex and postillumination viability were studied in cultured human vein graft smooth muscle cells. Inferior vena cava-grafted rats were injected with Lu-Tex (10 mg/kg) 4 or 12 weeks after grafting. Biodistribution was assessed in a subgroup 24 hours after administration. Light therapy (742 nm) was performed 24 hours after Lu-Tex injection by illuminating intraperitoneally placed isografts using a laparotomy. Animals were divided into the following 4 groups: PDT (n=15), Lu-Tex injection and laparotomy (n=13), light treatment (n=14), and laparotomy only (n=13). Grafts were harvested 14 days after treatment for histochemical analysis. Lu-Tex localized within subcellular organelles of smooth muscle cells, and subsequent photoactivation induced cell death via apoptosis. The Lu-Tex concentrations present in the vein grafts were 9.3 times higher than those in the normal inferior vena cava. Postoperative PDT at 4 weeks after surgery significantly reduced the intima/media ratio, whereas treatment at 12 weeks did not reduce the intima/media ratio. Activated macrophages were observed 4 weeks after grafting; however, a significant reduction occurred in these cells by 12 weeks. The mechanism by which PDT works may be related to the presence of activated macrophages. CONCLUSIONS: PDT significantly reduces the intima/media ratio in the early phase of vein graft disease. Lu-Tex-mediated PDT may be a viable method for the attenuation of atherosclerotic disease in vein grafts.

Photodynamic therapy with motexafin lutetium induces redox-sensitive apoptosis of vascular cells.

Motexafin lutetium is a photosensitizer that accumulates in atherosclerotic plaque and, after activation by far-red light, produces cytotoxic singlet oxygen. The combination of photosensitizer and illumination, known as photodynamic therapy (PDT), has been shown to reduce atheroma formation in animal models and is under clinical investigation. However, the effects of PDT with motexafin lutetium on isolated vascular cells are unknown. This study was designed to characterize the effects of PDT on vascular cell viability and to define the cell-death pathway for this agent. Fluorescence microscopy of RAW macrophages and human vascular smooth muscle cells revealed time-dependent uptake of motexafin lutetium. Illumination of motexafin lutetium-loaded cells with 732-nm light (2 J/cm(2)) impaired cellular viability and growth (IC(50) 5 to 20 micromol/L). Depletion of intracellular glutathione potentiated (P=0.035) and the addition of antioxidant N-acetylcysteine attenuated (P=0.002) cell death, suggesting that the intracellular redox state influences motexafin lutetium action. PDT was associated with the loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, and caspase activation. PDT promoted phosphatidylserine externalization and induced apoptotic DNA fragmentation, with the number of apoptotic cells increasing from 7+/-2% to 34+/-3% of total cells. Reducing plaque cellularity by the induction of apoptosis may be one mechanism by which PDT reduces plaque burden, possibly modulates plaque vulnerability, and inhibits restenosis in vivo.

Motexafin gadolinium: a redox active drug that enhances the efficacy of bleomycin and doxorubicin.

The effect of motexafin gadolinium (MGd), a redox mediator, on tumor response to doxorubicin (Dox) and bleomycin (Bleo) was investigated in vitro and in vivo. MES-SA human uterine sarcoma cells were studied in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. Rif-1, a murine fibrosarcoma cell line, was studied using a clonogenic survival assay. Tumor growth delay assays were performed using the EMT-6 murine mammary sarcoma cell line in BALB/c mice. MGd (25-100 microM) produced dose-dependent enhancement of Bleo cytotoxicity to MES-SA cells. The IC(50) for Bleo was reduced by approximately 10-fold using 100 microM MGd. In clonogenic assays using Rif-1 cells, MGd enhanced the activity of Bleo approximately 1000-fold. This effect was shown to be mediated, in part, by MGd inhibition of potentially lethal damage repair. MGd enhanced the tumor response to bleomycin and Dox in vivo. MGd had no significant effect on the systemic exposure to Dox (expressed in terms of the plasma area under the curve, 0-24 h) and did not increase Dox myelosuppression. MGd enhanced the effectiveness of the redox active drugs, Bleo and Dox.

Photodynamic therapy of B16F10 murine melanoma with lutetium texaphyrin.

Photodynamic therapy (PDT) of pigmented melanoma has generally been unsuccessful because of insufficient light penetration in such tissues. In this study, the responsiveness of the heavily pigmented B16F10 murine melanoma to lutetium texaphyrin (PCI-0123), a water-soluble sensitizer with strong absorbance in the near infrared (700-760 nm), was examined. These studies were carried out in both normal and ApoE deficient C57BL/6 mice. The latter strain exhibits a lipoprotein profile more like humans (low density lipoprotein > high density lipoprotein) than rodents (high density lipoprotein >> low density lipoprotein). Under optimal conditions of drug dose, light dose, and interval between drug administration and irradiation--the median survival time of C57BL/6 tumor bearing mice was approximately doubled (29 d) compared with tumor bearing control animals (13 d). The life-span of the ApoE knockout mice was greater (33 d) than the C57BL/6 animals (23 d) when irradiation occurred 3 h after administration of a 10 micromol per kg drug dose. The greater efficacy of PDT in the ApoE deficient mice was associated with more rapid clearance of drug from the blood, greater accumulation of sensitizer in tumor tissue, and substantially greater drug binding to the very low density lipoprotein/low density lipoprotein plasma fraction. Confocal laser scanning microscopy showed that the predominant subcellular site of photosensitizer binding was to melanosomes; costaining was performed with Mel-5. Melanosomes are susceptible to oxidative stress. Photo-oxidation, mediated by PCI-0123 PDT, could potentially overload an already highly oxidized stressed state leading to cell death. The good tissue penetration depth achieved by PCI-0213 mediated PDT and the activation of melanosomes makes PDT of pigmented melanoma, for the first time, clinically relevant.

Effect of density-gradients on the binding of photosensitizing agents to plasma proteins.

The binding of photosensitizing agents to low-density lipoprotein is considered an important factor in tumor localization. We examined the affinity of a group of photosensitizers, varying in charge and hydrophobicity, for LDL, under conditions designed to determine whether the high salt concentration involved in conventional KBr gradients affected the results. Density-gradients containing KBr vs D2O were evaluated; the latter can delineate VLDL and LDL from other plasma components, while the KBr gradient readily resolved VLDL, LDL, HDL and albumin. Distribution of the photosensitizers to plasma fractions was assessed, along with the effect of Cremophor EL, an emulsifier used for formulation of water-insoluble drugs. Both the D2O and KBr gradients provided similar results with regard to the affinity of anionic, neutral or cationic photosensitizers for LDL. The use of Cremophor EL for drug formulation was associated with an altered electrophoretic lipoprotein profile. In some cases, affinity of CRM-solubilized sensitizers for plasma components varied with the density-gradient employed. The high salt concentration used in density-gradient fractionation had little effect on the affinity of photosensitizing agents to low-density lipoprotein but may introduce artifacts when emulsifiers are used in drug formulation.

Interactions of Solutol HS 15 and Cremophor EL with plasma lipoproteins.

Two emulsifying agents, Solutol HS15 and Cremophor EL, were compared with regard to their effects on human plasma lipoproteins in vitro and on mouse plasma lipoproteins in vitro and in vivo. Both agents promoted binding of a hydrophobic photosensitizing agent (C8KC) to a circulating plasma species of low bouyant density. Persistence of this material was greater with Cremophor than with Solutol. Experiments carried out with labeled Solutol indicated that the vehicle itself is a component of this new species. High concentrations of either vehicle ( > or = 0.06%) led to decreased electrophoretic mobility of human LDL and HDL in vitro. In the mouse, a different effect was observed, resulting in complex changes in electrophoretic mobility of plasma lipoproteins. The plasma half-life of C8KC in the circulation of the mouse was correlated with the persistence of an altered electrophoretic lipoprotein pattern. Since Solutol and C8KC showed similar half-lives, this result suggests that the plasma half-life of the sensitizer is correlated with the persistence of the vehicle. While Solutol and Cremophor were designed to be vehicles for drug formulation, they also influence persistence of some drugs in the circulation.

In vivo animal studies with gadolinium (III) texaphyrin as a radiation enhancer.

PURPOSE: Gadolinium texaphyrin (Gd-Tex, PCI-0120) is an expanded porphyrin that has demonstrated radiation enhancement. In this study, we evaluated the radiation enhancement and biolocalization of Gd-Tex in three animal tumor models. METHODS AND MATERIALS: EMT6, SMT-F, and MCa tumors were established intramuscularly or subcutaneously. Gd-Tex and other metallotexaphyrins were administered prior to single or multiple fractions of radiation. 14C-labeled Gd-Tex was used for biolocalization studies. RESULTS: Gd-Tex, in combination with radiation, produced significant tumor growth delay compared to irradiated control groups in both single and multifraction radiation studies. Gd-Tex radiation enhancement was observed only when the drug was given before, but not after irradiation. Several metallotexaphyrins, identical except for the metal ion, were studied in the EMT6 tumor model including gadolinium (Gd), lutetium (Lu), europium (Eu), yttrium (Y), and cadmium (Cd) texaphyrin complexes. Only Gd-Tex produced radiation enhancement. Biodistribution studies using 14C-labeled Gd-Tex demonstrated drug selectivity and retention in tumors growing intramuscularly compared to uninvolved muscle and plasma. CONCLUSIONS: Gd-Tex produces reproducible radiation enhancement in a variety of in vivo tumor models. This drug's unique radiochemistry, tumor selectivity, and in vivo activity suggests possible mechanisms of action not addressed by in vitro assay methods.

Photodynamic therapy with motexafin lutetium (Lu-Tex) reduces experimental graft coronary artery disease.

BACKGROUND: Motexafin lutetium (Lu-Tex) is a photodynamic therapy (PDT) agent that localizes in atheromatous plaque in which it can be activated by far-red light. Lu-Tex biolocalization was examined in graft coronary artery disease (GCAD) with a rodent allograft model. After photoactivation, the effect on intimal proliferation was assessed. METHODS: A PVG to ACI rat heterotopic heart transplantation model was used. Lu-Tex (10 mg/kg) was intravenously administered 90 days after transplantation. Photoactivation was performed 24 hr after Lu-Tex administration. A light-emitting diode, central wavelength of 742 nm, was used to illuminate the intraperitoneally placed allografts via a laparotomy (light fluence of 75 J/cm2 at a power density of 75 mW/cm2). Animals were divided into four groups according to postoperative treatments: PDT with Lu-Tex injection and light illumination (n=21), Lu-Tex injection and laparotomy (n=14), laparotomy with light only (n=14), and laparotomy only (n=16). GCAD was quantitatively assessed 14 days after treatments. RESULTS: Lu-Tex localized in atherosclerotic plaque in vessels with GCAD. PDT significantly reduced both the percent of affected vessels and intimal proliferation compared to all other control study groups. alpha-Smooth muscle cell actin and anti-rat macrophage antibody-positive areas were significantly reduced within the neointima in allografts treated with PDT compared to all other study groups. CONCLUSIONS: PDT significantly reduced atherosclerotic lesions of GCAD. Lu-Tex-mediated PDT may, therefore, be a potential method for treating accelerated atherosclerosis associated with transplantation.

Biodistribution of haematoporphyrin analogues in a lung carcinoma model.

In an attempt to identify novel compounds useful for the optimization of Photodynamic Therapy (PDT), the tissue localization of new synthetic porphyrins was compared with Photofrin II in nude mice xenografted with a human small cell lung cancer (POVD). Three haematoporphyrin analogues were selected for this study based on prior in vitro photosensitivity screening of a series of 15 such derivatives, as well as on the basis of improved localization in C6 gliomas in mice. Two of the porphyrins yielded better tumour:normal lung ratios than Photofrin II and, of these two, one (P13) is known to exhibit good photosensitization properties both in vitro and in vivo, and is therefore a good candidate as a lead compound for the development of porphyrins suitable for the photodynamic treatment of lung tumours.

Limb ischaemia after intra-arterial injection of Temazepam gel: histology of nine cases.

AIMS: To record the histopathological findings associated with intra-arterial injection of Temazepam gel by nine drug misusers. METHODS: Standard histological examination and immunocytochemistry for endothelial markers (factor VIII related antigen, Ulex europaeus lectin) were carried out. RESULTS: Intra-arterial injection of Temazepam gel may cause severe vascular injury and lead to amputation of fingers or limbs. Histological changes include myocyte necrosis, interstitial oedema, extensive arterial, venous, and capillary thrombosis, and sometimes vasculitis, endothelial swelling, and denudation. CONCLUSIONS: Inadvertent injection of Temazepam gel into arteries may cause catastrophic ischaemic damage, possibly as a result of toxic effects on endothelial cells.

Lutetium texaphyrin (Lu-Tex): a potential new agent for ocular fundus angiography and photodynamic therapy.

PURPOSE: To investigate the suitability of lutetium texaphyrin (lu-tex) as a fluorescence imaging agent in the delineation of retinal vascular and choroidal vascular diseases. The utilization of an efficient fluorescent molecule that is also a photosensitizer represents a unique opportunity to couple diagnosis and therapy. METHODS: Fundus fluorescence angiography comparing lu-tex (motexafin lutetium, Optrin, Pharmacyclics Inc, Sunnyvale, California) with the conventional angiographic dyes, sodium fluorescein, and indocynanine green (ICG), was performed on the eyes of normal and laser-injured New Zealand white rabbits. Plasma pharmacokinetic data and plasma protein binding were assessed in addition to light microscopy of the retina in both imaged and laser-injured eyes. RESULTS: Normal retinal and choroidal vasculature was well delineated by lu-tex angiography. Experimentally induced choroidal and retinal vascular lesions were enhanced by lu-tex and demonstrated different staining patterns than fluorescein or ICG, particularly at the margins of the lesions. Lu-tex cleared rapidly from the plasma, with 39.7% bound to the high-density lipoprotein (HDL) fraction while 15.8% was bound to the low-density lipoprotein (LDL) fraction. No evidence of retinal toxicity after dye administration was observed by either ophthalmoscopy and fundus photography or by light microscopy. CONCLUSION: Lu-tex angiography is a potentially valuable method for retinal vascular and choroidal vascular evaluation, and it has advantages over fluorescein and ICG angiography. The same agent could conceivably be used for both the identification of abnormal vasculature and subsequent photodynamic treatment.

Fractionation of Cremophor EL delineates components responsible for plasma lipoprotein alterations and multidrug resistance reversal.

Treatment of cancer patients with 3-h infusions of taxol formulated with Cremophor EL resulted in a marked decrease in the electrophoretic mobility of all plasma lipoproteins. Cremophor was fractionated by reverse-phase chromatography to determine which components were responsible for this behavior. Effects of different Cremophor fractions on reversal of multidrug resistance, amino acid transport, and cytotoxicity also were evaluated using murine leukemia cells in culture. Lipoprotein alterations were caused by Cremophor components of intermediate hydrophobicity, which also antagonized amino acid transport and decreased viability of murine leukemia cells. Cremophor components responsible for reversal of multidrug resistance were of greater hydrophobicity. The lipoprotein-altering components of Cremophor could be selectively removed without affecting either taxol solubilization or multidrug-resistant reversal.

Impaired accumulation of a cationic photosensitizing agent by a cell line exhibiting multidrug resistance.

Transport and accumulation of copper benzochlorin iminium salt (CDS1), a cationic photosensitizing agent, were examined using the P388/ADR murine leukemia, which exhibits the MDR (multidrug resistance) phenotype, and the wild-type parent cell line, P388. The recent availability of radioactive CDS1 permitted kinetic studies at drug levels in the submicromolar range. Exclusion of CDS1 by P388/ADR cells could be demonstrated, indicating that this agent is a substrate for the outward transport system associated with MDR. These results have implications with regard to the efficacy of cationic photosensitizers against this common neoplastic phenotype. The CDS1 was readily accumulated by P388 cells and by P388/ADR cells when the outward transport system was inhibited. Under these conditions, CDS1 was tightly bound and could not be washed out even when the outward transport system was reactivated.

Biodistribution and PDT efficacy of a ketochlorin photosensitizer as a function of the delivery vehicle.

C8KC is a new ketochlorin photosensitizer that must be formulated with an emulsifier because of its poor water solubility. In this report, we compare properties of Cremophor EL (CRM) and Tween 80 as delivery vehicles for C8KC. Unlike Tween 80, CRM altered the physical properties of both human and mouse plasma lipoproteins, resulting in decreased electrophoretic mobility of the individual lipoproteins along with the formation of a lipoprotein degradation product: a phospholipid fraction of low buoyant density. In human plasma, where there was sufficient low-density lipoprotein (LDL) for a distinction to be made, CRM caused a shift in binding of a ketochlorin from albumin to LDL and the degraded lipoprotein fraction. In mice bearing the RIF tumor, the use of CRM for drug formulation was associated with longer plasma and tissue persistence of C8KC, and enhanced photodynamic therapy (PDT) efficacy. These results indicate the importance of both sensitizer and vehicle as determinants of PDT efficacy.

Sites of photodamage in vivo and in vitro by a cationic porphyrin.

Localization and photodynamic efficacy of a monocationic porphyrin (MCP) were assessed using murine leukemia cells in culture. This sensitizer localized at surface membrane loci and catalyzed selective photodamage to membrane structures. Although both cationic and hydrophobic, this porphyrin was not recognized by the multidrug transporter, which excludes many cationic agents from cells that express multidrug resistance. Photodynamic studies with the murine radiation-induced fibrosarcoma tumor model indicated moderate photosensitization of neoplastic lesions in vivo at 3 h, but not at 24 h after sensitizer administration. Pharmacokinetic studies indicate that plasma levels, not tissue levels were the major determinant of photodynamic therapy (PDT) response. Consistent with this observation, vascular damage and disturbances of tissue perfusion followed PDT. These effects were more pronounced in tumor-bearing skin than in normal skin. The therapeutic response to MCP appeared to be related mainly to secondary, probably vascular, effects.

Subcellular localization of porphyrins using confocal laser scanning microscopy.

The in vitro subcellular distribution patterns of 10 porphyrins, varying in hydrophobicity and charge, were studied using confocal laser scanning microscopy on two cell lines (V79 and C6 glioma cells) for incubation times up to 24 h. All of the porphyrins were taken up rapidly by both cell lines and distinct classes of subcellular distribution patterns were observed: general cytoplasmic staining; localization in lysosomes (usually associated with general cytoplasmic staining); localization in mitochondria (and general cytoplasmic staining); localization in mitochondria with subsequent uptake into lysosomes. Structure-localization relationships which have emerged are that porphyrins with dominantly cationic side chains localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes.

Localization and efficacy analysis of the phototherapeutic lutetium texaphyrin (PCI-0123) in the murine EMT6 sarcoma model.

Lutetium texaphyrin (PCI-0123) is a pure, water-soluble photodynamic therapy (PDT) agent that is activated by tissue-penetrating far red light. The sensitizer is highly fluorescent and exhibits a strong, broad emission signal at 750 nm. In vitro cellular uptake studies revealed an increase in sensitizer retention with incubation time. Confocal laser scanning microscopy demonstrated that the intracellular localization site of PCI-0123 is the lysosomes. Ensuing illumination of the EMT6 cells led to lysosomal breakup, extensive cytoplasmic blebbing and subsequent cell death. Noninvasive spectral imaging analysis of PCI-0123 fluorescence depicted selective drug uptake, compared to surrounding normal tissue, in EMT6 mammary sarcomas syngeneic to BALB/c mice. The PCI-0123 PDT was shown to effectively treat the EMT6 murine sarcoma. Irradiation (732 nm light) 3 h postintravenous injection of 10 mumol PCI-0123 per kg gave 100% cures (no evidence of cancer), whereas light exposure at 5 h resulted in 75% cures. Hematoxylin and eosin histologic examination of photoirradiated tumors indicated apoptosis of the EMT6 neoplasms at early times post-PDT progressing, with time, to extensive necrotic areas. Gel electrophoresis of extracted photoirradiated tumors showed the typical apoptotic DNA ladder pattern that increased in intensity following PDT treatment.