Photodiagnosis and Photodynamic Therapy (2006) 3, 147—155

Photodynamic therapy of diseased bone Stuart K. Bisland PhD, Bsc (Hons) ∗, Shane Burch Ontario Cancer Institute, University Health Network, University of Toronto, 610 University Avenue, Toronto, Ont., Canada M5G 2M9 Available online 2 May 2006 KEYWORDS Bone; Cancer; Bacterial infection; Osteomyelitis; Photodynamic therapy; Bioluminescence; Computed tomography

Summary Objective: Photodynamic therapy (PDT) defines the light activation of a photosensitizing compound, leading to the generation of cytotoxic, reactive oxygen species. We are investigating the unique application of PDT for diseased bone. Methods: Bioluminescent, human breast cancer cells (MT-1) were injected intracardially into athymic rats. At 2—3 weeks following injection rats developed diffuse bone metastases that were visible using an in vivo bioluminescence imaging system. Metastatic lesions within vertebrae and long bones were treated with differing regimens of 690 nm laser light (25—150 J) following injection of benzoporphyrinderivative monoacid (BPD-MA) and the tumour response assayed histologically upon sacrifice. Subsequent large animal studies involved light propagation studies in porcine vertebrae and BPD-MA-PDT treatment of large primary osteosarcomas in canine. Canine were subject to high dose PDT (500 J/cm). A second rat model involved bacterial infection in bone. Bioluminescent Staphylococcus aureus (S. aureus) were grown as biofilms onto wires, implanted into rat tibia and exposed to 5-aminolevulinic acid (ALA)-PDT delivered percutaneously with 635 nm laser light (75 J/cm2 ). Response to therapy was monitored as changes in bioluminescence signal and colony forming assay upon sacrifice. Results: PDT (150 J) proved effective in ablating metastatic lesions within rat femur and lumbar vertebrae. Porcine studies confirmed the average depth of light penetration into trabecular bone as 0.16 ± 0.04 cm with an average incident fluence rate of 4.3 mW/cm2 , however, the necrotic/non-necrotic interface extended 0.6 cm out from the treatment fiber. In canine a single, high dose treatment created wellcircumscribed margins of effect encompassing the entire 3—4 cm osteosracoma. Finally, ALA-PDT proved effective against S. aureus biofilms in bone, although recurrent infection did occur in some. Conclusions: Results support the application of PDT to the treatment of primary or metastatic lesions within bone. Secondly, ALA-PDT may be useful as a treatment for osteomyelitis with repeat or chronic treatment regimens. © 2006 Elsevier B.V. All rights reserved.

Introduction ∗

Corresponding author. Tel.: +1 416 946 4501 5916; fax: +1 416 946 6529. E-mail address: [email protected] (S.K. Bisland).

Photodynamic therapy (PDT) defines the oxygendependent photochemical reaction that occurs

1572-1000/$ — see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(06)00036-6

148 upon light-mediated activation of a photosensitizing compound that leads to the generation of cytotoxic, reactive oxygen species, predominantly, singlet oxygen [1]. Our interest in delivering PDT into bone has focused on both cancer and microbial infections. We first established the concept and feasibility of using benzoporphyrin-derivative monoacid (BPD-MA)-PDT for treating metastatic lesions within vertebrae or long bones using a small animal model [2]. Over 100,000 bone metastases are identified in North America each year and of those an estimated 30—40,000 cases of metastatic breast cancer lesions occur in the spine [3,4]. Yet, despite this alarming statistic, the frontline approach for treating such cancers remains unsatisfactory and the related diagnosis is often met with poor prognosis. Presently, radiation therapy (RT) is considered the mainstay of treatment for ambulatory patients, while surgery is reserved for those experiencing collapse or neurological compromise. However, RT provides only limited relief from pain, composite to cord compression, offers no stability to the spine and can adversely affect the capacity for soft tissue to repair following treatment. This in turn translates into a 3-fold increase in morbidity and mortality following surgical intervention [5—7]. It is therefore imperative to establish that PDT, if it is to be considered as an alternative to radiation, can offer targeted tumour ablation without collateral damage to nearby spinal cord and little obstruction to subsequent wound repair processes within soft tissue. We subsequently assessed light dosimetry and PDT effects in large animal spine [8] and confirmed the effects of PDT in non-tumour bearing bone together with vertebroplasty. Bone is also susceptible to bacterial infection following trauma or surgery and we are interested in using 5-aminolevulinic acid (ALA)-PDT to treat Staphylococcus aureus (S. aureus)-derived infections in bone. Bacterial infection in bone, as well as other pathogenic microorganisms including fungi and mycobacteria, can lead to osteomyelitis, an acute or chronic inflammation of bone and bone marrow [9,10]. S. aureus is implicated in 60—90% of childhood osteomyelitis and greater than 55% in adults [11,12]. Whether by etiology or clinical presentation, the predominant factor predicting the progression of the disease and therapeutic outcome, regardless of anatomical location, is vascularity. Blood-borne bacteremia may result from trivial traumas involving skin lacerations or bruising, while avascular foci resulting from peripheral vascular disease, burns or soft tissue disease are usually prone to contiguous infection of neighbouring tissues. This is particularly true for diabetics or patients with sickle cell anaemia, for whom the

S.K. Bisland, S. Burch incidence of skeletal infection is increased approximately 20—1000 times, respectively [13,14]. The rationale for ALA seems justified; bacteria are highly metabolic, rapidly dividing cells that produce lots of porphyrin-derived photosensitizing metabolites, much like tumour cells. Indeed, the results of our studies with S. aureus-biofilm laden wires implanted into rat tibiae were conclusive: that single treatments of ALA-PDT could provide transient abatement of infection in bone [9]. The question of recurrent infection still remains to be addressed and for bacterial infections in particular, it will also be necessary to confirm that PDT can be delivered as an effective regimen without increasing the likelihood of contiguous spread of the infection following damage to surrounding soft tissues. One approach that we are developing termed metronomic PDT (mPDT) involves a protracted, low drug and light dose regimen which may circumvent the risk of recurring infection and ALA is uniquely apt for this with high oral bioavailability, low toxicity and good selectivity. The risk of collateral damage to surrounding normal tissues is also reduced following ALA-mPDT and in rat brain the number of glioma cells that die via apoptosis following mPDT is significantly increased as compared with the single or fractionated PDT regimens [15]. It will be of interest to establish whether ALA-mPDT protocols can be devised to combat persistent bacterial infections. There are very few reports describing the use of PDT to treat structural lesions within bone [16,17]. Similarly, PDT treatment of biofilm-producing S. aureus in vivo although described by other groups [18], has not been conducted in bone to model osteomyelitis. An integral part of these studies has been the continued refinement of practical approaches relating to light delivery and imaging modalities that are conducive to the eventual use of PDT for treating bone-related diseases in the clinic. This paper describes the experimental methodologies and results thus far, together with a discussion of these results and some of the practical considerations for integrating this approach into the clinic.

Experimental models of PDT in bone Bone metastases Materials and methods We used a similar rat model to that first described by Engebraaten and Fodstad [19] for initiating human breast cancer-derived (MT-1) metastatic lesions within bone except for our studies, we transformed the MT-1 cells to express the luciferase

Photodynamic therapy of diseased bone gene and display resistance to neomycin antibiotic [20]. Luciferase-mediated bioluminescence was initiated following intraperitoneal injection of Luciferin substrate at 30 mg/kg body weight. Thus, transformed MT-1 cells (MT-1Luc ) could be selectively cultured in vitro and visualized in vivo using bioluminescence imaging (IVIS, Xenogen Corp., Alameda, CA, USA) and quantified as total flux (photons/steradian/cm2 ) using Living ImageTM software (Xenogen). Typically, within 16—21 days following intracardiac injection of MT-1Luc cells (2 × 106 cells in 200 ␮L), rats (rnu/rnu, 150 g) had developed multi-focal metastatic bone disease in spine and femur that were subsequently treated with intravenous BPD-MA (2 mg/kg), followed 3 h later by low (25 J) or high (150 J) dose laser light (690 ± 5 nm). A total of 38 rats were treated including controls (no drug, 150 J) at the 3-h time-point, each group consisting of at least n = 5 animals. Light was administered via parapedicular placement of an optical fibre (200 ␮m diameter) adjacent to the diseased vertebra. Accurate placement was proffered by low intensity X-ray fluoroscopic imaging. The lytic degradation of bone was assessed using either fluoroscopy or FaxitronTM (Faxitron Xray Corp., Wheeling, IL, USA) in live animals or microcomputer tomography (␮CT) of euthanized animals. Results It is interesting to observe that, as is often seen in humans, these rats display metastatic disease throughout the spine and that is responsible for the rapid onset of morbidity in these rats a few weeks following injection. Fig. 1(I) A and C show bioluminescent lesions (arrows) prior to PDT within the spine and femur, respectively. Bioluminescence is almost entirely lost 48 h following PDT [Fig. 1(I) B and D]. The IVIS is shown in Fig. 1(II). All lesions targeted for light treatment with 25 or 150 J at 3 h post drug administration showed a response. The light only and drug only controls did not have an effect in any of the lesions. One hundred and fifty joules resulted in a 99.8% decrease in tumour growth at 48 h post-treatment while 25 J reduced tumour growth by 66% as compared to control lesions. The extent of tumour-induced osteolysis was poorly discerned using either Faxitron or fluoroscopy, particularly in spine, however ␮CT provided very clear 2 or 3-dimensional reconstructed images of the osteolysis in tumour-bearing vertebrae [Fig. 1(III; see arrows)]. Fluoroscopy was very useful for assisting with fibre(s) placement onto target, bioluminescent vertebrae and was further aided by surface markers and a stereotactic grid for guidance [Fig. 1(IV)] although, it is also

149 noteworthy to consider Kodak’s (Eastman Kodak Company, Rochester, NY, USA) new combined Xray/bioluminescence imaging station (In vivo FXTM ) that allows precise co-registration of these images. Subsequent histological analysis of treated lumbar vertebrae confirmed the eradication of tumour as well as bone marrow 48 h following BPD-MAPDT. Of clinical importance, was the propensity in 13/33 (39% of animals) for unilateral or bilateral paralysis following similar PDT protocols in thoracic vertebrae, highlighting the need to protect the spinal cord from PDT-induced damage at relatively short drug/light intervals, when the BPD-MA is still present within the vasculature. Paralysis was not seen when irradiating at later time points (24 h post BPD-MA injection) even with comparatively high light dose, 150 J/cm2 .

Light dosimetry in porcine vertebrae Materials and methods Mature Landrace swine (45 kg, n = 5) were used to address the technical feasibility of fibre placement and photodynamic response using intravenous BPD-MA (6 mg/m2 ) in normal cancellous bone. Transpedicular placement of optical fibers inside the vertebra(e) was conducted using a cone beam CT unit with C-arm (Siemens PowerMobil) and flat panel detector to allow large (256 × 256 × 192), high resolution (2048 × 1536 pixels at 194 ␮m pixel pitch), 3-dimensional (D) reconstructions from 2D projections [21] with numerous fields of view (see Fig. 2a—f). Light propagation through the vertebral body was assessed using a 2 cm cylindrically diffusing fibre inserted through the left pedicle into the vertebral body. Light of as 690 nm was delivered at 150 mW/cm and detected by a second isotropic detector fibre implanted into the same vertebral body via the right pedicle [8]. Fibres were orientated such that their tips were angled approximately 90◦ to one another with ≤0.2 cm separation. The histology for contra-lateral vertebrae that did not receive light was also examined. Light measurements were recorded upon incremental retraction of the detector probe. Results Subsequent assimilation of these measurements with the histology of PDT-treated vertebrae (48 h post-treatment) revealed a necrotic radius of 0.59 ± 0.02 cm out from the delivery fiber and a local fluence of 9.3 J/cm2 at the necrotic/nonnecrotic interface [8]. Contra-lateral drug only control vertebrae did not display any necrosis (not shown). These results are very encouraging and confirm the feasibility of delivering light into tra-


S.K. Bisland, S. Burch

Figure 1 (I) Photographs of bioluminescent MT-1Luc lesions within spine and femur of rnu/rnu rats (see arrows) before (A and C) and after (B and D) BPD-MA-PDT. (II) The IVIS bioluminescence detector. (III) Micro-CT images provide vivid assessment of osteolytic lesions secondary to tumour infiltration were evident along the rat spine. (IV) Target vertebrae were irradiated upon precise paraventricular placement of an optical fiber using fluoroscopy.

becular bone, not surprising given its highly scattering, cavernous network.

PDT treatment of primary bone cancers in canine Materials and methods We have recently conducted studies that demonstrate the use of BPD-MA-mediated PDT to successfully treat primary osteosarcomas in the radii of dogs. Patient dogs (n = 7) were treated at the University of California Veterinary College in Davis, USA with a single acute PDT regiment. BPD-MA (0.4 mg/kg) was administered as an intravenous infusion over 10 min followed 5 min later

by laser light (690 ± 5 nm). A single control animal that received light only treatment has also been included to confirm the PDT effect. Light was delivered into the tumour located within the medullary cavity of the distal radius using an optical fibre (0.94 mm diameter) with cylindrically diffusing terminals 5 cm in length. The total delivered fluence was 500 J at a rate of 200 mW/cm. The response of tumours to PDT was assessed at 48 h post-treatment using MRI. Patient dogs were also scheduled for limb amputation allowing histological analysis of the treatment site. Results Following only a single treatment at the prescribed PDT dose all tumours responded to therapy. An

Photodynamic therapy of diseased bone


Figure 2 (a) Cone beam CT unit with C-arm and flat panel detector. (b) Axial reconstruction showing transpedicular placement of the delivery fiber held in place using a custom-designed bone screw device (arrow). (c) 3D reconstruction showing a cannula in vertebral body of L1. (d—f) alternate, 2D views of inserted cannula.

example of MR images taken before and after PDT treatment is shown in Fig. 3a and b. The osteosarcoma fills the entire circumference of the medullary cavity extending from the lower-radius to the wrist. Following treatment (Fig. 3b) the margins of treatment conform very closely with the pre-operative MR of the tumour. Histology from the control animal confirmed no effect with light alone (Fig. 3c). Treatment resulted in haemorrhagic necrosis with few remaining tumour cells (Fig. 3d). It is very encouraging to witness the extent of PDTinduced tumour cell death following a single, high dose treatment with no evidence of collateral toxicity or morbidity in the treated canine. It is clear that PDT can provide remarkable tumour control even for these large, bulk tumours and if used in combination with surgical resection and/or repeat PDT regiments, could provide complete tumour eradication using what is essentially a minimally invasive approach. Ultimately, it is hoped that PDT will be able to offer limb salvage for similar large primary lesions in bone.

Rat model of bacterial infection in bone It is perhaps even more tempting to consider the application of PDT not only for cancers within bone but also as a therapy for bacterial infections within

bone. There have been a number of reports describing the application of PDT as an anti-microbial therapy in vitro and in vivo [22]. We are however the first group to describe the treatment of SA biofilms within bone directly, although similar implants have been described previously [23]. We again adopted fluoroscopy and bioluminescence as our primary imaging tools in a rat model of osteomyelitis (see Fig. 4a—d, below). A bioluminescent strain of the Gram-positive S. aureus was grown as biofilms onto lengths (0.5 cm) of wire and implanted into the medullary cavity of rat tibia (n = 5). Ten days following implant PDT was administered involving intraperitoneal injection of ALA (300 mg/kg) followed 4 h later by exposure to light (635 ± 10 nm; 75 J/cm2 ). An acute regiment of PDT resulted in a temporary but significant (paired Student’s ttest) decrease in the percentage (of pre-treatment value) bioluminescence at 24 h (p = 0.0071), which was lost at 48 h (p = 0.112) post-treatment as compared to contra-lateral tibia that received no light (Fig. 4d). In all cases an increased bioluminescence was evident within days of treatment indicating recurrence of the S. aureus infection and this often occurred in regions well beyond the initial infection site. Despite this however, the actual number of viable S. aureus colony forming units (CFU/mL) surviving within the isolated tibia was typically


S.K. Bisland, S. Burch

Figure 3 (a) Axial T1 weighted gadolinium enhanced MR scan of a canine radius before PDT treatment. The osteosarcoma complete fills the medullary cavity extending from the lower radius to the wrist. (b) An MR scan 48 h following a single PDT treatment using BPD-MA reveals a vast area of PDT-induced cell death spanning the entire breadth of the tumour. (c) Histological examination of haematoxylin and eosin stained control slices reveal the chaotic, highgrade osteosarcoma with no effect by light treatment alone. (d) The PDT effect, resulted in pronounced haemorrhagic necrosis throughout the treatment site with few remaining tumour cells.

reduced indicating that the recurrent infection was largely confined to the surrounding muscle and tissues and not the bone. In order to determine the CFU/mL, tibia were excised and cleared of soft tissues before being mashed into slurry containing bacterial growth medium using a mortar and pestle. It is likely, therefore, that the preponderance for re-infection is to some extent determined by the PDT-induced damage to neighbouring soft tissues. We hypothesize that we can perhaps minimize the extent of collateral damage to neighbouring tissues by delivering a mPDT regimen directly onto the infection [15]. The rational for ALA-mediated PDT extends from the elevated levels of porphyrin that can result, namely coproporphyrinogen (III) in

Gram-positive strains and protoporphyrin IX (PpIX) in Gram-negative strains [24]. Indeed, many bacteria virtue of their rapid metabolism, display heightened endogenous porphyrins that could potentially facilitate specificity using PDT without the addition of exogenous substrate although, this remains to be seen. ALA is an appropriate compound for mPDT as it can be administered chronically via the drinking water at therapeutic doses without side effects and its mechanism of action is governed by the metabolic state of the cells. It remains to be clarified as to whether our ability to treat S. aureus biofilms within bone offers any selectivity toward the bacterial cells versus the resident bone marrow cells since established biofilms are known to

Photodynamic therapy of diseased bone


Figure 4 (a) IVIS image of bioluminescent Xen-29 biofilms 24 h post implantation into the right and left tibia of a rat. (b) Fluoroscopy confirmed the precise location of the same biofilm-coated wires within the tibia (see arrows). (c) An excised tibia clearly demonstrates the presence of viable bacterial infection confined to the wire. (d) Relative light units (RLU; percentage of bioluminescence before treatment) revealed a statistically significant difference between untreated () and treated () tibia at 24 h but not at 48 h post-treatment (n = 4).

be largely senescent when compared to planktonic cultures.

Discussion and conclusions Our studies demonstrate that the application of PDT as treatment for diseased bones including metastatic lesions and microbial infection is possible and therapeutically efficacious. Moreover, PDT is not hampered by the limitations associated with current regiments of treatment including collateral damage following radiation and the increased resistance of microorganisms to antibiotics. The use of PDT to treat primary and secondary cancerous lesions within bone via minimally invasive procedures offers great potential and the rational for developing PDT, certainly as a palliative therapy

for patients recalcitrant to current regimens, seems very promising indeed. For bone metastases, it will be important, however, to establish safe and reproducible outcomes regarding fibre placement into the spine and response to treatment. Both of these will require choreographed pre-treatment planning in which optimized fibre(s) placement is calculated according to the light dosimetry of the vertebrae and tumour. Currently, we are initiating our photodynamic action while the photosensitizer BPD-MA is still within the vasculature. It is proposed that given the high vascularity of bone and tumour alike, the PDT-induced lesion will be maximal and since preservation of the surrounding bone marrow is not a prerequisite, and its eradication together with the tumour may actually prove to be more efficacious [8], it is unlikely that a cellular targeted therapy could be as effective. We are, however, conducting

154 extensive studies of the pharmacokinetics related to BPD-MA in tumour-bearing vertebrae in order to optimize the interval of time between drug and light delivery. Ultimately, patients with metastatic disease seldom present with localized lesions, it is more common that the disease is both diffuse and multi-focal. It is imperative therefore to develop strategies that allow for prolonged or repeat treatments. One strategy that we are investigating is mPDT. It could be proposed that implantable light sources that deliver low dose drug and light into the tumour may be utilized in such a way as to allow the patient greater management of his or her disease without the draw backs of debilitating side effects. Despite the fact that our initial focus has been breast cancer-derived metastatic disease in spine, the epidemiological and etiological will undoubtedly encompass a large number of potential cancerrelated indications including, multiple myeloma, chondrosarcoma, hemingioma, and Ewing’s sarcoma. Anti-microbial PDT has been conducted since the early 1970s, however, only recently has there been renewed interest in the concept of using endogenous porphyrins to treat bacteria [22,25]. A number of studies have now confirmed the anti-microbial action of ALA-PDT and it is becoming increasing evident that distinct differences do exist between Gram-positive and Gram-negative strains as far as how the ALA is metabolized [24]. Indeed, the fact that coproporphyrinogen III is largely responsible for the photo-inactivation of Gram-positives like S. aureus and is not accumulated within eukaryotic cells, could be a means of increasing therapeutic selectivity. Furthermore, the excitation spectra for PpIX and coproporphyrinogen III at the near infrared region may be sufficiently distinct to allow selective excitation of one metabolite and not the other [24]. The incidence of osteomyelitis in humans is increasing both as a primary complication following trauma and/or surgery and as a secondary complication associated with other underlying disease, such as diabetes. Furthermore, prosthetic implants, of which there is increasing use, are highly susceptible to bacterial contamination. Our in vivo studies address the use of ALA-PDT to treat S. aureus-derived biofilms grown onto a small length of metallic wire and implanted into rat tibia in an attempt to mirror the scenario of prosthetic implant in patients. A number of issues relating to these studies remain to be verified, including the levels of coproporphyrinogen III that result following ALA administration in the S. aureus biofilm, the penetration of ALA into cells within the biofilm layers as compared to cells grown as

S.K. Bisland, S. Burch planktonic cultures. It will be important to quantify the propagation of light (at 615—635 nm) into long bones as well as through the biofilms in order that we can optimize the delivery of light and extend the current paradigm of treatment protocols to include the use of mPDT in an attempt to reduce the risk of recurrence and minimize damage to surrounding tissues. We must confirm that mPDT is effective in treating bacterial infections within bone without promoting the spread of infection into soft tissues, as appears to be the case following the acute PDT regimens. Finally, although the endpoints of our current studies do not assess the progression of osteomyelitis per se, but rather the persistence of bacterial infection, it will be important for future studies to confirm directly that ALA-PDT can prevent or significantly reduce the likelihood of osteomyelitis progression in this model. In this regard, we have recently acquired a digital FaxitronTM unit (MX-20 digital; Faxitron X-ray Corp.) that will allow us to follow the progression of osteolytic degradation within the bone. The development of PDT strategies for treating bone diseased bone must be conducive to the clinic and suitably compliant to address the varied clinical scenarios that will arise. Imaging tools that provide intra-operative navigation for placement of fibers, validation of response and registration for re-location upon follow-up will be pivotal for PDT acceptance into clinic. Cone beam CT appears to be a very strong candidate for generating 3dimenional images of the target tissue(s). It will be important to integrate modalities for measuring response, perhaps magnetic resonance imaging, at least for soft tissues (tumours) and bone marrow. We are currently focused on providing a multimodal platform for treatment planning based on physiological parameters governing photodynamic effect such as photosensitizer concentration, tissue oxygenation and light together with co-registration of pre- and intra-operative imaging to allow accurate fibre placement. Ultimately, one would hope that on-line treatment could be provided with real-time feedback of response. Despite the many software packages that exist for finite element interpolation and intra-operative navigation, few reports have yet to demonstrate unequivocally the application of intraoperative feedback parameters for optimizing therapy. We must therefore further explore the integration of modern imaging and navigational technologies that allow for co-registration of pre- and intraoperative treatment planning strategies with realtime feedback of therapeutic response to provide a safe and effective PDT treatment.

Photodynamic therapy of diseased bone

Acknowledgements The authors wish to acknowledge the tremendous technical assistance of Dr. Annie Linn (Princess Margaret Hospital) throughout these studies. We also wish to thank Dr. Brian Wilson, also at Princess Margaret Hospital, for his continued mentoring with the design and planning of the work. Finally, we wish to acknowledge Dr. Julia Levy and the team at Quadra Logics Technologies (QLT) Inc., Vancouver, BC, Canada for their continued moral and, importantly, financial support throughout these studies.

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Photodynamic therapy of diseased bone.

Photodynamic therapy (PDT) defines the light activation of a photosensitizing compound, leading to the generation of cytotoxic, reactive oxygen specie...
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