A nearly fatal primary Epstein-Barr virus infection associated with low NK-cell counts in a patient receiving azathioprine: a case report and review of literature

Minna Honkila
Riitta Niinimäki
Mervi Taskinen
Outi Kuismin
Kaisa Kettunen
Janna Saarela
Sami Turunen
Marjo Renko
Terhi Tapiainen
A nearly fatal primary Epstein-Barr virus infection associated with low NK-cell counts in a patient receiving azathioprine: a case report and review of literature
10.1186/s12879-019-4022-3C A S E R E P O R T Open Access
Background: Symptomatic primary Epstein-Barr virus infection is a usually self-limiting illness in adolescents. We present a case of an adolescent who had been receiving azathioprine for inflammatory bowel disease for four years and developed a life-threatening primary Epstein-Barr virus infection successfully treated with rituximab.Case presentation: An 11-year-old girl presented with chronic, bloody diarrhea. Endoscopic biopsies confirmed a diagnosis of chronic ulcerative colitis with features of Crohn's disease. Azathioprine was initiated after one year due to active colitis. She responded well and remission was achieved. At the age of 16 years she developed a lifethreatening Epstein-Barr virus infection including severe multiple organ failure and was critically ill for 4 weeks in the intensive care unit. Natural killer cells were virtually absent in the lymphocyte subset analysis. Azathioprine was stopped on admission. She was initially treated with corticosteroids, acyclovir and intravenous immunoglobulin. Approximately 30 days after admission, she developed signs of severe hepatitis and pneumonitis and received weekly rituximab infusions for 8 weeks. Primary immunodeficiency was excluded by whole exome sequencing in two independent laboratories. Persistent viremia stopped when the natural killer cell count started to rise, approximately 90 days after the cessation of azathioprine. Conclusions: We found 17 comparable cases in the literature. None of the previous cases reported in the literature, who had been treated with azathioprine and developed either a severe or a fatal Epstein-Barr virus infection, underwent full genetic and prospective immunological workup to rule out known primary immunodeficiencies. Recently, azathioprine has been shown to cause rather specific immunosuppression, resulting in natural killer cell depletion. Our case demonstrates that slow recovery from azathioprine-induced natural killer cell depletion, 3 months after the stopping of azathioprine, coincided with the clearance of viremia and clinical recovery. Finally, our choice of treating the patient with rituximab, as previously used for patients with a severe immunosuppression and Epstein-Barr virus viremia, appeared to be successful in this case. We suggest testing for Epstein-Barr virus serology before starting azathioprine and measuring natural killer cell counts during the treatment to identify patients at risk of developing an unusually severe primary Epstein-Barr virus infection.

Background

Symptomatic primary Epstein-Barr virus (EBV) infection is a usually self-limiting illness presenting with fever, lymphadenitis, tonsillitis, hepatitis, and splenomegaly in adolescents and young adults. The duration of EBV viremia during primary EBV infection is rather short, while the median half-time for viral elimination is three days [1]. It is well known that after organ or allogenic hematopoietic stem cell transplantation (HSCT), or in patients with primary immunodeficiency, EBV primary infection or reactivation can cause uncontrolled and severe infection and lymphoproliferation. We present a case of an adolescent who had been receiving azathioprine for inflammatory bowel disease for four years who developed a life-threatening EBV infection. In addition, we performed a systematic review of the literature on azathioprine and EBV infection.

Case presentation

Methods

We first followed our patient up closely during severe EBV infection using repeated flow cytometry measurements to detect all lymphocyte subpopulations, including natural killer (NK) cell counts. The EBV viral load was measured using a quantitative nucleic acid amplification test (Fig. 1). Immunoglobulin (Ig) M antibodies against viral capsid antigen (VCA) and IgG antibodies to EBV nuclear antigen (EBNA) were measured. Whole exome sequencing (WES) was performed in two independent laboratories, the Institute for Molecular Medicine, Helsinki, Finland, as previously described [2], and Centogene, Germany (www.centogene.com), in order to detect any known primary immunodeficiency. Both the patient and her mother gave their written informed consent to the publication of this case history. We then performed a systematic literature search via PubMed in December 2016 using the terms "Epstein-Barr virus" and "azathioprine". A total 154 possible papers were identified for review, of which 13 were retrieved for detailed examination.

The patient

An 11-year-old girl presented with chronic, bloody diarrhea. Endoscopic biopsies confirmed a diagnosis of chronic ulcerative colitis with features of Crohn's disease. She was treated first with prednisolone and mesalazine, but azathioprine, 75 mg/day (2.5 mg/kg), was started after one year on account of continued active colitis. She responded well to this and remission was achieved. At the age of 16 years she was admitted to hospital on account of a high fever and poor general condition. Azathioprine was stopped on account of acute infection, as three-lineage cytopenia was detected on admission: her white blood cell count was 2.5 (4.5-11 × 10 9 /L), platelets 38 (200-450 × 10 9 /L) and hemoglobin 89 g/L (117-155 g/L). Splenomegaly, acute cardiac failure and acute hepatic failure, with 15% necrosis in a biopsy, were observed in the next few days. Symptoms of encephalitis were present, but a brain MRI was normal. Viral genome examinations by EBV polymerase chain reaction revealed > 350,000 copies/mL in plasma on admission and > 1 million copies/mL after 4 days of treatment (Fig. 1). A high viral load of > 8 million copies/mL was also detected in the liver biopsy specimen. Tests for IgM antibodies to VCA were positive on admission, whereas IgG antibodies to EBNA were negative, indicating acute EBV infection. She was treated once with rituximab and then with corticosteroids, acyclovir and intravenous immunoglobulin. She developed severe multiple organ failure, including cardiac, hepatic and respiratory failure, and was critically ill for 4 weeks in the intensive care unit. After this treatment in the intensive care unit, approximately 30 days after admission, the viral load started to increase again and she developed signs of severe hepatitis and pneumonitis, so that regular weekly rituximab infusions were given for a total of 8 weeks. Very low NK-cells were noted and the persistent viremia (detection level 300 copies/mL) stopped when the NK-cell count started to rise, approximately 90 days after the cessation of azathioprine (Fig. 1). During the course of illness, CD8+ T-cell counts were 1131 (220-1129 × 10 6 /L) on day 32, 2535 on day 70, 2487 on day 93 and 2537 on day 106. Due to the prolongation of viremia and hepatitis despite the discontinuation of azathioprine, WES was performed. This did not reveal any known primary immunodeficiency, but the patient was found to be carrying two rare heterozygous variants of the TLR3 and OAS1 genes, in which variants have previously shown to be associated with susceptibility to viral infections. TPMT genotype leading to low enzyme activity was excluded in this case after the patient's recovery. A high total IgG level (> 15 g/L) persisted for 6 months after clearance of the viremia, and the low CD19-cell count attributable to the rituximab treatment started to rise 6 months after cessation of the infusions. At the last follow-up visit, she was asymptomatic and healthy and had successfully returned to her studies 6 months after the onset of the EBV infection. She was still in remission with respect to her inflammatory bowel disease without any medication.

Systematic review of the literature

Examination of the 13 relevant publications yielded a total of 17 corresponding cases, mainly of adolescents or young adults with Crohn's disease or ulcerative colitis who had been treated with azathioprine and developed either severe or fatal EBV infections, or EBV-driven hemophagocytic lymphohistiocytosis or some other lymphoproliferative disorder, excluding lymphomas [3][4][5][6][7][8][9][10][11][12][13][14][15]. Four patients had died [4,7,10,14]. High EBV viremia (> 100,000 copies/mL) was documented in five patients [3,7,[10][11][12]. NK-cell counts had been made in two out of the 17 patients and either a low count or low NK-cell cytotoxicity had been noted during the acute phase of the illness in both patient cases [3,13]. CD8+ T-cell counts of the patients were not reported in the literature. Three patients had been successfully treated with repeated rituximab infusions [3,8,11]. Most patients had received corticosteroids and also acyclovir or ganciclovir. X-linked lymphoproliferative disease (XLP) was excluded in two male patients by genetic testing [7,10].

Discussion and conclusions

Azathioprine is widely used for the treatment of Crohn's disease and ulcerative colitis. Our case and systematic literature search confirm that a primary EBV infection can be extremely severe or even fatal for EBV-negative adolescents or young adults receiving azathioprine.

Azathioprine has recently been shown to cause rather specific immunosuppression, resulting in NK-cell [16,17], but not T-cell depletion [17]. Moreover, early-differentiated NK-cells seem to be critical in the immune control of primary EBV infection as this subset of NK-cells expands and restricts lytic EBV infection that is poorly controlled in infectious mononucleosis [18,19]. We repeatedly monitored our patient's lymphocyte subpopulation count using flow cytometry and noted an almost total loss of the NK-cell population, whereas the number of CD8+ T-cells were within the normal range. We were thus able to draw a clear time series showing that the clearance of EBV viremia coincided with the normalization of NK-cell counts three to four months after ceasing to administer azathioprine. This supports the idea that azathioprine caused a severe secondary immunodeficiency and a lack of NK-cells, resulting in an uncontrolled EBV infection and hyperinflammation, since NK-cells play a role in down-regulating inflammatory responses [20]. Thus the phenotype observed in our patient mimics the known pathomechanism of congenital hemophagocytic lymphohistiocytosis, which is a hyperinflammatory condition explained by low cytotoxic functioning of the NK-cells or T-cells on account of genetic defects [20]. The risk of hemophagocytic lymphohistiocytosis in children with Crohn's disease, often treated with thiopurines, has been estimated to be 100-fold in a population-based patient series of 5 cases with HLH and inflammatory bowel disease [4]. The risk of lymphoproliferative disorders, including lymphomas, has been reported in a large cohort of inflammatory bowel disease patients to be five-fold in those patients receiving thiopurines [21]. Our findings offer a possible explanation for this observed epidemiological risk.

Corticosteroids and acyclovir are recommended for the treatment of severe EBV infection. After an organ or HSCT EBV is known to cause a lymphoproliferative disorder which can be treated with rituximab, an anti-CD20 monoclonal antibody, in order to reduce B-cells and stop viremia, since B-cells are the main replication site of Epstein-Barr viruses during infection. In patients with XLP, a primary immunodeficiency leading to uncontrolled EBV infection, severe EBV infection has recently been treated with rituximab before HSCT. We chose to treat our patient with corticosteroids, acyclovir and one dose of rituximab at the onset of the disease. However, as the EBV viral load started to increase again after 1 month despite corticosteroid and acyclovir therapy and she simultaneously developed new signs of severe pneumonitis and hepatitis, we decided to administer rituximab weekly until viremia had been arrested, three months after admission. Donor-derived EBV-specific cytotoxic T-cells are a novel experimental treatment for uncontrolled EBV infections in patients with primary or secondary immunodeficiency [22], and we consequently considered treating our patient with EBV-specific cytotoxic T-cells before she started to respond well to rituximab treatment.

The previous cases reported in the literature had not undergone thorough WES to rule out undiagnosed known primary immunodeficiencies, and even though XLP was not a diagnostic alternative for a female patient, defects in the ITK, STK4, TNFRSF7 and CORO1A genes, for instance, can lead to a life-threatening EBV infection. If a primary immunodeficiency had been diagnosed, the patient could have been considered for treatment with EBV-specific cytotoxic T-cells as a bridge before HSCT. Rapid WES is thus a very useful diagnostic tool in cases of unusually severe EBV infection, in order to exclude primary immunodeficiencies, as these need to be treated with a prompt HSCT.

Our clinical case demonstrates that azathioprine-induced NK-cell depletion could be the reason for a severe, life-threatening primary EBV infection in this context. Achieving immune reconstitution may take several months after the cessation of azathioprine medication. All infectious disease specialists treating adolescents and young adult patients who have been receiving azathioprine should be aware of the risk. Testing for EBV serology before starting azathioprine could help to identify EBV-seronegative adolescents and young adults who might have a high risk of developing an unusually severe primary infection.

Fig. 1
1Epstein-Barr viremia and NK-cell counts after admission in a critically ill adolescent with a primary EBV infection and previously treated with azathioprine for inflammatory bowel disease. Azathioprine was stopped on day 1. NK-cell counts started to rise after 90 days and were normal after 120 days, coinciding with clearance of the viremia. During the course of illness, CD8+ T-cell counts were 1131 (220-1129 × 10 6 /L) on day 32, 2535 on day 70, 2487 on day 93 and 2537 on day 106

Abbreviations EBV: Epstein-Barr virus; EBNA: Epstein-Barr virus nuclear antigen; HSCT: Hematopoietic stem cell transplantation; Ig: Immunoglobulin; NK: Natural killer; VCA: Viral capsid antigen; WES: Whole exome sequencing
AcknowledgementsNot applicable.FundingThis case report received no funding.Availability of data and materialsAll data generated or analyzed during this study are included in this article. 23, FIN-90029 OYS, Oulu, Finland.2Authors' contributions MH, RN, ST, MR and TT participated in the clinical care of the patient. MT, OK, KK and JS advised and supervised the laboratory testing, interpretation and reporting. MH and RN collaborated in the writing of the case report, which was critically reviewed and edited by TT. All authors approved the final manuscript as submitted.Ethics approval and consent to participate Not applicable.Consent for publicationBoth the patient and her legal guardian gave their written consent to the publication of this case history.Competing interestsThe authors declare that they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
A prospective clinical study of Epstein-Barr virus and host interactions during acute infectious mononucleosis. H H BalfourJr, C J Holman, K M Hokanson, J Infect Dis. 192Balfour HH Jr, Holman CJ, Hokanson KM, et al. A prospective clinical study of Epstein-Barr virus and host interactions during acute infectious mononucleosis. J Infect Dis. 2005;192:1505-12.

Comparison of solution-based exome capture methods for next generation sequencing. A M Sulonen, P Ellonen, H Almusa, Genome Biol. 1294Sulonen AM, Ellonen P, Almusa H, et al. Comparison of solution-based exome capture methods for next generation sequencing. Genome Biol. 2011;12:R94.

A case of EBV driven haemophagocytic lymphohistiocytosis complicating a teenage Crohn's disease patient on azathioprine, successfully treated with rituximab. M P Fitzgerald, L Armstrong, R Hague, R K Russell, J Crohns Colitis. 7Fitzgerald MP, Armstrong L, Hague R, Russell RK. A case of EBV driven haemophagocytic lymphohistiocytosis complicating a teenage Crohn's disease patient on azathioprine, successfully treated with rituximab. J Crohns Colitis. 2013;7:314-7.

Association of Crohn's disease, thiopurines, and primary epstein-barr virus infection with hemophagocytic lymphohistiocytosis. V F Biank, M K Sheth, J Talano, J Pediatr. 159Biank VF, Sheth MK, Talano J, et al. Association of Crohn's disease, thiopurines, and primary epstein-barr virus infection with hemophagocytic lymphohistiocytosis. J Pediatr. 2011;159:808-12.

Primary EBV infection resulting in lymphoproliferative disease in a teenager with Crohn disease. D Gidrewicz, D Lehman, S Rabizadeh, F Majlessipour, M Dubinsky, J Pediatr Gastroenterol Nutr. 52Gidrewicz D, Lehman D, Rabizadeh S, Majlessipour F, Dubinsky M. Primary EBV infection resulting in lymphoproliferative disease in a teenager with Crohn disease. J Pediatr Gastroenterol Nutr. 2011;52:103-5.

EBV hepatitis in a young Crohn's disease patient on prolonged remission with azathioprine. E Angelucci, M Cesarini, E Caturelli, P Vernia, Inflamm Bowel Dis. 171Angelucci E, Cesarini M, Caturelli E, Vernia P. EBV hepatitis in a young Crohn's disease patient on prolonged remission with azathioprine. Inflamm Bowel Dis. 2011;17:E1.

Epstein-Barr virus-associated lymphoproliferative disorder in Crohn disease treated with azathioprine. S Ross, S K Rajwal, I Sugarman, J Pediatr Gastroenterol Nutr. 51Ross S, Rajwal SK, Sugarman I, et al. Epstein-Barr virus-associated lymphoproliferative disorder in Crohn disease treated with azathioprine. J Pediatr Gastroenterol Nutr. 2010;51:229-31.

Multifocal polyclonal Epstein-Barr virus-associated B-cell lymphoproliferative disorder secondary to azathioprine therapy successfully treated with rituximab. S Courby, B Fabre, D Salameire, Leuk Lymphoma. 51Courby S, Fabre B, Salameire D, et al. Multifocal polyclonal Epstein-Barr virus-associated B-cell lymphoproliferative disorder secondary to azathioprine therapy successfully treated with rituximab. Leuk Lymphoma. 2010;51:174-7.

Epstein-Barr virus and parvovirus B19 coinfection in a Crohn's disease patient under azathioprine. T Moreira, P Lago, M Salgado, R Pimentel, Inflamm Bowel Dis. 16Moreira T, Lago P, Salgado M, Pimentel R. Epstein-Barr virus and parvovirus B19 coinfection in a Crohn's disease patient under azathioprine. Inflamm Bowel Dis. 2010;16:905-6.

Fatal Epstein-Barr virus primo infection in a 25-year-old man treated with azathioprine for Crohn's disease. Y N'guyen, L Andreoletti, M Patey, J Clin Microbiol. 47N'guyen Y, Andreoletti L, Patey M, et al. Fatal Epstein-Barr virus primo infection in a 25-year-old man treated with azathioprine for Crohn's disease. J Clin Microbiol. 2009;47:1252-4.

Epstein-Barr virus-associated lymphoproliferation awareness in hemophagocytic syndrome complicating thiopurine treatment for Crohn's disease. C Serrate, M Silva-Moreno, P Dartigues, Inflamm Bowel Dis. 15Serrate C, Silva-Moreno M, Dartigues P, et al. Epstein-Barr virus-associated lymphoproliferation awareness in hemophagocytic syndrome complicating thiopurine treatment for Crohn's disease. Inflamm Bowel Dis. 2009;15:1449-51.

Cholestatic hepatitis, acute acalculous cholecystitis, and hemolytic anemia: primary Epstein-Barr virus infection under azathioprine. S Hagel, T Bruns, M Kantowski, P Fix, T Seidel, A Stallmach, Inflamm Bowel Dis. 15Hagel S, Bruns T, Kantowski M, Fix P, Seidel T, Stallmach A. Cholestatic hepatitis, acute acalculous cholecystitis, and hemolytic anemia: primary Epstein-Barr virus infection under azathioprine. Inflamm Bowel Dis. 2009;15:1613-6.

Hemophagocytic syndrome in an adolescent with Crohn disease receiving azathioprine and infliximab. K A Francolla, A Altman, F A Sylvester, J Pediatr Gastroenterol Nutr. 47Francolla KA, Altman A, Sylvester FA. Hemophagocytic syndrome in an adolescent with Crohn disease receiving azathioprine and infliximab. J Pediatr Gastroenterol Nutr. 2008;47:193-5.

Fatal infectious mononucleosis: a severe complication in the treatment of Crohn's disease with azathioprine. E F Posthuma, R G Westendorp, Van Der Sluys, A Veer, J C Kluin-Nelemans, P M Kluin, C B Lamers, Gut. 36Posthuma EF, Westendorp RG, Van der Sluys Veer A, Kluin-Nelemans JC, Kluin PM, Lamers CB. Fatal infectious mononucleosis: a severe complication in the treatment of Crohn's disease with azathioprine. Gut. 1995;36:311-3.

Reversible lymphoma in the setting of azathioprine therapy for Crohn's disease. L Larvol, J C Soule, T A Le, N Engl J Med. 331Larvol L, Soule JC, Le TA. Reversible lymphoma in the setting of azathioprine therapy for Crohn's disease. N Engl J Med. 1994;331:883-4.

Thiopurine treatment in patients with Crohn's disease leads to a selective reduction of an effector cytotoxic gene expression signature revealed by whole-genome expression profiling. G Bouma, J M Baggen, A A Van Bodegraven, Mol Immunol. 54Bouma G, Baggen JM, van Bodegraven AA, et al. Thiopurine treatment in patients with Crohn's disease leads to a selective reduction of an effector cytotoxic gene expression signature revealed by whole-genome expression profiling. Mol Immunol. 2013;54:472-81.

Thiopurine use associated with reduced B and natural killer cells in inflammatory bowel disease. J D Lord, D M Shows, World J Gastroenterol. 23Lord JD, Shows DM. Thiopurine use associated with reduced B and natural killer cells in inflammatory bowel disease. World J Gastroenterol. 2017;23:3240-51.

Role for early-differentiated natural killer cells in infectious mononucleosis. T Azzi, A Lünemann, A Murer, Blood. 124Azzi T, Lünemann A, Murer A, et al. Role for early-differentiated natural killer cells in infectious mononucleosis. Blood. 2014;124:2533-43.

Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection. O Chijioke, A Müller, R Feederle, Cell Rep. 5Chijioke O, Müller A, Feederle R, et al. Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection. Cell Rep. 2013;5:1489-98.

Hemophagocytic syndromes--an update. G E Janka, K Lehmberg, Blood Rev. 28Janka GE, Lehmberg K. Hemophagocytic syndromes--an update. Blood Rev. 2014;28:135-42.

Lymphoproliferative disorders in patients receiving thiopurines for inflammatory bowel disease: a prospective observational cohort study. L Beaugerie, N Brousse, A M Bouvier, Lancet. 374Beaugerie L, Brousse N, Bouvier AM, et al. Lymphoproliferative disorders in patients receiving thiopurines for inflammatory bowel disease: a prospective observational cohort study. Lancet. 2009;374:1617-25.

T cells for viral infections after allogeneic hematopoietic stem cell transplant. C M Bollard, H E Heslop, Blood. 127Bollard CM, Heslop HE. T cells for viral infections after allogeneic hematopoietic stem cell transplant. Blood. 2016;127:3331-40.


Revascularization Results in the Interventional Management of Stroke II Trial

T Tomsick
Interventional Management of Stroke II Investigators

J Broderick
Interventional Management of Stroke II Investigators

J Carrozella
Interventional Management of Stroke II Investigators

P Khatri
Interventional Management of Stroke II Investigators

M Hill
Interventional Management of Stroke II Investigators

Y Palesch
Interventional Management of Stroke II Investigators

J Khoury
Interventional Management of Stroke II Investigators

Revascularization Results in the Interventional Management of Stroke II Trial
10.3174/ajnr.A0843ORIGINAL RESEARCH
BACKGROUND AND PURPOSE: Our aim was to detail revascularization results, including impact on outcome and mortality, in the Interventional Management of Stroke (IMS) II trial. MATERIALS AND METHODS: IMS II was designed to obtain estimates of the efficacy and safety of reduced-dose intravenous recombinant tissue plasminogen activator (rtPA) followed by additional intra-arterial rtPA and low-energy sonography via the EKOS Primo Micro-Infusion Catheter at the occlusion in selected patients with ischemic stroke treated within 3 hours of onset. Revascularization outcomes were detailed and compared with modified Rankin Scale scores 0 -2, mortality outcomes, and results from IMS I.RESULTS: Complete recanalization at 60 minutes occurred in 12 of 29 (41.4%) sonography microcatheter-treated occlusions. Complete recanalization was achieved at 2 hours or procedure end in 20/29 (68.9%) in the ultrasound catheter-treated group, and final thrombolysis in cerebral infarction (TICI) 2/3 reperfusion was achieved in 18/29 (62.0%) ultrasound-treated subjects. Fifteen-minute angiograms demonstrated some recanalization in 69/145 (46.7%) sonography microcatheter treatment intervals, compared with 39/111 (35.1%) in IMS I treatments in 23 subjects with reliable 15-minute angiograms (P ϭ .046). Pooled IMS I-II data demonstrated that partial or complete recanalization occurred in 56/75 (74.6%) and good reperfusion (TICI 2/3) occurred in 46/75 (61.3%) of internal carotid artery T and M1 occlusions. Revascularization correlated with good outcome for TICI 2/3 reperfusion (P ϭ .0004), TICI 2B/3 reperfusion (P ϭ .0002), and arterial occlusive lesion 2/3 recanalization (P ϭ .03).CONCLUSION: IMS II provides evidence that the EKOS Primo sonography microcatheter exhibits a trend toward improved recanalization of the occlusion compared with a standard microcatheter and again confirms the correlation between recanalization and reperfusion with good clinical outcome and reduced mortality.

T he Interventional Management of Stroke (IMS) I trial suggested that the combined use of reduced-dose intravenous (IV) Activase (alteplase, recombinant tissue plasminogen activator [rtPA]; Genetech, South San Francisco, Calif), followed by microcatheter-delivered intra-arterial (IA) rtPA, was relatively safe and may be clinically useful in selected patients with acute ischemic stroke, as compared with similar subjects treated with a full dose of IV rtPA in the National Institute of Neurologic Disease and Stroke (NINDS) rtPA trial. 1 It had long been anticipated that multiple interventional methods might be applicable within an IV/IA rtPA paradigm and that these methods might further improve revascularization and clinical outcomes. 2 Encouraged by the safety and potential efficacy reports in a small feasibility trial of the MicroLysUS Sonography Catheter (EKOS Corp, Bothell, Wash) (hereafter referred to as the sonography microcatheter), the IMS Investigators initiated another phase II trial (IMS II) to further study its use following reduced-dose IV rtPA therapy. 3 This article reports in detail the revascularization, clinical, and safety outcomes in the IMS II trial, with emphasis on results obtained with the Primo sonography microcatheter (EKOS Corp), including general as well as detailed comparisons with revascularization outcomes from IMS I.

Methods

The IMS II study was a 13-center open-labeled single-arm pilot study, whose purpose was to determine the futility or lack thereof of the combined IV/IA therapy and to obtain preliminary estimates of the efficacy and safety of reduced-dose IV rtPA (0.6 mg/kg), 15% as a bolus with the remainder infused for 30 minutes, followed by additional rtPA (Յ22 mg) and low-energy sonography via the sonography microatheter at the site of the arterial occlusion in eligible patients who were younger than 81 years of age with acute large ischemic strokes (National Institutes of Health Stroke Scale [NIHSS] Ն 10) treated within 3 hours of symptoms onset. 4 The EKOS system consists of 2 components: 1) a single-use 3F sonography microcatheter with an end-hole infusion lumen incorporating a high-frequency (1.7 mHZ) cylindrical sonography transducer delivering sonography at low power (Ͻ0.45 W) into a 3.3F distal tip, and 2) a reusable control unit, which provides the sonography energy source and the user interface. Three models of the sonography microcatheter were used sequentially in the trial. The first model (SV2510), similar to that used in a feasibility trial, was intended for passage over a 0.010-inch microguidewire. When successful access occurred in only 2 of 6 subjects between January 26, 2003, and April 1, 2003, the IMS II Steering Committee temporarily stopped the trial to change to a 0.014-inch guidewire-based system (SV3014) and to upgrade the control unit. The trial was restarted May 5, 2003. However, unsuccessful access in 2 of 4 subjects again prompted study delay in anticipation of a new third model under development. This Primo model was used for the remainder of the trial between December 17, 2003, and April 12, 2005. This report will focus on the Primo sonography microcatheter efficacy. Details of study design, objectives, study protocol, sonography microcatheter and standard microcatheter administration of IA rtPA, and medical management and evaluation have recently been published and are not repeated here. 4 Revascularization was measured according to recanalization of the primary arterial occlusive lesion (AOL), and reperfusion was measured by a modified Thrombolysis in Cerebral Infarction (TICI) score. 5,6 AOL recanalization and the modified TICI perfusion were scored as grades 0 -3 ( Table 1). The protocol primary study revascularization end point was 100% complete recanalization of the primary occlusion at (AOL 3) at 60 minutes, compared with complete standard microcatheter thrombolysis at 60 minutes in the IMS I trial. Secondary revascularization measures included both AOL 3 recanalization and TICI 2/3 reperfusion at 120 minutes or at the end of the procedure, compared with 60 comparable occlusions (internal carotid terminus [ICA T], M1, M2, vertebrobasilar) in the IMS I trial.

In addition, any recanalization of the AOL by using a sonography microcatheter at each sequential 15-minute interval was recorded and compared with standard microcatheter recanalization at each sequential 15-minute interval from IMS I subjects with comparable occlusions as well as with standard microcatheter use in comparable IMS II occlusions.

The revascularization study end points were determined at the Clinical Coordinating Center at the University of Cincinnati by a central reader (T.T.). Operators were asked to ascribe Thrombolysis in Myocardial Infarction (TIMI) recanalization scores for their procedures, as had been recorded in IMS I. Operators' scores were compared with the scores of the central core lab.

The clinical efficacy measure of modified Rankin Scale (mRS) score 0 -2 outcome at 3 months and mortality were also recorded, and both these measures were further analyzed according to revascularization status. Revascularization end points were also compared with mRS score 0 -2 outcome and mortality from the IMS I study.

Finally, recanalization and reperfusion data from the sonography and standard microcatheter groups from IMS II and IMS I were pooled to estimate good outcome and mortality results that might be achieved with ICA T and M1 occlusions with the IV/IA rtPA paradigm.

As a primary safety measure, CT scans within the first 36 hours after completion of rtPA infusion were monitored for intracerebral hemorrhage (ICH) and contrast deposition, including contrast extravasation (CT attenuation Ͼ90 HU, hyperattenuation persisting at 24 hours) and contrast enhancement (high attenuation disappearing within 24 hours). 7 ICH was classified according to the European Cooperative Acute Stroke Study (ECASS) classification, subdivided into hemorrhagic infarction (HI) types 1 and 2 and parenchymal hematoma (PH) types 1 and 2. 8 Infarct, symptomatic ICH, and lesion (infarct ϩ ICH) volumes were retrospectively measured digitally via a manual region-of-interest analysis and reported (G. Ramadas, personal communication, August 2006). Vessel perforation, dissection, and subarachnoid hemorrhage were also recorded.

Angiograms were examined for reocclusion during the procedure as well as for new occlusions in previously uninvolved arterial distributions. Distal emboli in the middle cerebral artery (MCA) beyond an MCA occlusion could not be systematically identified because their pretreatment presence/absence was seldom confidently identified or excluded. Baseline and new emboli into the anterior cerebral artery (ACA) distribution with MCA and ICA T (obstructed ICA flow into the M1 and A1 segments) occlusion were recorded. Microcatheter contrast injections (MCI) were recorded and compared with contrast extravasation and ICH. 9 Catheter-tip temperature was recorded for all IMS II procedures by using the EKOS sonography microcatheter and reviewed postprocedure. Temperature graphs were compared with 15-minute control angiograms to identify whether observed temperature changes correlated with recanalization.

A National Institutes of Health-appointed Data and Safety Monitoring Board Committee reviewed all safety and outcome results of the IMS I and II Studies.

Results

Eighty-one subjects were entered into the IMS II trial (Fig 1). Twenty-six were treated with IV rtPA only, and 55 were treated with IV and IA rtPA. Among the 26 treated with IVonly, 2 improved clinically following IV rtPA and did not have arteriography, undergoing MR angiography (MRA) instead with no major AOL. Twelve others had no major treatable AOL at arteriography, 2 with no demonstrable AOL and 10 with distal MCA, ACA, or posterior cerebral artery occlusions not treated by the interventionist. Of these 14 with no major   AOL demonstrated on angiography or MRA, mRS 0 -2 was achieved in 9 (64.3%).

Of the remaining 12 IV-only subjects, 1 was discovered to have a high international normalized ratio and was excluded from IA treatment. One subject had coarctation of the aorta, and the ascending aorta could not be accessed in the usual retrograde transfemoral fashion for diagnostic arteriography and treatment. Three subjects were beyond the prescribed 5-hour initiation time; 2 had nonprotocol IA treatments other than rtPA, both with good outcomes. The other 7 subjects in the IV-only group had AOL eligible for IA treatment. Five had major intracranial occlusions distal to cervical ICA occlusions that could not be accessed due to inability to traverse the cervical ICA occlusions. One M2/3 occlusion was neither identified nor treated by the local treating interventionist. An early sonography microcatheter model would not traverse the distal ICA in another due to cervical and cavernous segment ICA tortuosity. MR spectroscopy 0 -2 outcomes were achieved in 3 of these latter 7 (42.9%) subjects not treated by IA therapy.

Fifty-five subjects were treated with both IV and IA rtPA (36 by sonography microcatheter and 19 by standard microcatheter). Thirty-five subjects had the final Primo sonography microcatheter model introduced. This catheter could not be advanced beyond tortuous segments of the ICA in 3 subjects (8.6%), who were then treated with a standard microcatheter. Three subjects were treated without sonography initiated: 2 regionally in the setting of ICA and vertebral artery dissection, respectively, and 1 when the catheter-control box interface cable connection failed. Twenty-nine subjects were treated with the Primo microcatheter with sonography activation. One of the 29 subjects had both an M2 AOL treated with the sonography microcatheter and a P2 occlusion treated with a standard microcatheter.

As noted, 19 subjects were treated by standard microcatheter, including 4 in whom the sonography microcatheter advancement had been unsuccessful. In addition, there were 1 ACA and 4 M3 occlusions in which the sonography microcatheter was not indicated. One other subject had an extremely tortuous cervical ICA and the appearance of intracranial atherosclerosis, in whom the sonography microcatheter was not indicated. Four other subjects had incomplete occlusions, and IA rtPA was administered via standard microcatheter. The occlusion of 1 subject was accessed after 5 hours, and a sonography microcatheter was not introduced. The 4 other subjects had sonography-microcatheter-eligible occlusions but were treated with a standard microcatheter, according to the discretion of the operator.

Revascularization Outcome

The primary study revascularization end point of complete recanalization of the primary AOL at 60 minutes was met in 12 of 29 (41.4%) patients with the Primo microcatheter with sonography activation. None of the 4 early sonography microcatheter models used achieved complete recanalization at 60 minutes. Complete recanalization of the primary AOL at 2 hours or by procedure end was achieved in 20/29 (68.9%) of the Primo microcatheter-treated group. Twenty-three control IMS I subjects had reliable 60-minute images for comparison of this primary end point, and 7 (30.4%) had complete recanalization (P ϭ .41). By the end of the procedure in IMS I, 32/60 (53.3%, P ϭ .08) patients with comparable AOLs (no M3, M4) exhibited complete recanalization (Fig 2). Recanalization in 14 patients with comparable IMS II AOL treated by standard microcatheter was compared with sonography microcatheter recanalization as well and demonstrated a cumulative 15-minute recanalization graph line that lies in close proximity to the 60-minute and 120-minute recanalization data points from the IMS I dataset (Fig 2).

Recanalization of the primary AOL leads to reperfusion into distal vessel segments. Final TICI 2/3 perfusion for the 29 Primo sonography microcatheter-treated subjects was 62.0%, compared with 56.8% for a comparable IMS I IA-treated group. No significant revascularization or outcome differences existed between IMS I and II.

Sequential 15-minute control IMS II angiograms demonstrated some recanalization, however small in degree, in 69/ 145 (46.7%) sonography microcatheter treatment intervals, compared with 28/65 (43.1%) comparable IMS II microcatheter treatments (P ϭ .09) and 39/111 (35.1%) comparable IMS I treatments in 23 subjects in whom reliable 15-minute angiograms were available (P ϭ .046).

Central core lab IMS II final TIMI revascularization scores demonstrated a 41% discrepancy between the core lab and the treating interventionist, with the treating interventionist usually scoring higher (better revascularization). Table 2 presents mRS 0 -2 outcomes for ICA T and M1 occlusion in IMS II and I subjects combined, according to multiple target revascularization end points. These pooled data demonstrate that partial or complete recanalization (AOL 2/3) occurred in 56/75 (74.6%), and good reperfusion (TICI 2/3) occurred in 46/75 (61.3%) of ICA T and M1 occlusions. Pooled data demonstrate that revascularization correlates with good outcome for TICI 2/3 reperfusion (P ϭ .0004), TICI 2B/3 reperfusion (P ϭ .0002), and AOL 2/3 recanalization (P ϭ .03) end points, compared with failure to achieve those end points, respectively. TICI 2A reperfusion correlated to better outcome than TICI 0/1 (P ϭ .03). In addition, a trend toward better mRS 0 -2 outcome was found for TICI 2B versus 2A reperfusion (P ϭ .08).

Revascularization Outcome versus mRS 0 -2 Clinical Outcome

Although only 10/23 (43.5%) M2 occlusions achieved grade 3 AOL recanalization, 16/23 (69.5%) achieved mRS 0 -2 outcome. Two subjects with M2 occlusions, both of whom recanalized and were discharged to home, were lost to follow-up and are included in the mRS Ͼ2 group. Comparison of the TICI 2/3 and AOL 2/3 end points as predictors of mRS 0 -2 outcome shows a sensitivity of 90.5% versus 95.2% and a specificity of 52% versus 34%, respectively.

Revascularization Outcome versus Mortality

Overall IMS II mortality was 16% of subjects, similar to the 16% mortality in IMS I. Of the 13 deaths, 4 were treated IV rtPA-only; 1 died secondary to symptomatic intracerebral hemorrhage (SICH) following failure to identify and treat an M2-3 occlusion. Three others died from sequelae of a large cerebral infarction: 1 of the 3 had an ICA occlusion that could not be traversed for IA therapy, a second was not treated after the 5-hour limit, and the third had coarctation of the aorta that could not be traversed for IA therapy. Of the 9 (16.3%) IV/IA deaths, 4 (44.4%) subjects did not recanalize to AOL grade 3, and 6 (66.6%) did not perfuse to TICI 2/3. Overall mortality and mortality in revascularized and nonrevascularized subjects with ICA T and M1 occlusions are further detailed in Table 2. Reduced mortality was associated with TICI 2/3 reperfusion (P ϭ .01), TICI 2B/3 reperfusion (P ϭ .05), and AOL 2/3 recanalization (P ϭ .03), compared with failure to meet those revascularization end points. Mortality in subjects failing to achieve either TIMI 2/3 reperfusion or AOL 2/3 recanalization was Ͻ40% for both end points.

Safety Outcomes

Symptomatic ICH occurred in 8/81 (9.9%) subjects with IMS II, including 1/26 (3.8%) treated with IV rtPA alone and 1 in a sonography microcatheter-treated subject without sonography applied. Six subjects in the sonography microcatheter group experienced symptomatic ICH, including the following: 1) One subject with M2 occlusion and baseline serum glucose concentrations of 400 mg/mL and creatinine concentrations of 2, who recanalized completely (AOL 3) to TICI 2B flow within 30 minutes.

2) One subject with a nonrecanalized M1 occlusion distal to the striate arteries who experienced severe cervical ICA spasm and additional thrombus development.

3) One subject with a nonrecanalized M1 occlusion distal to the striate arteries. 4) One subject with a T occlusion who recanalized within 30 minutes, with an unusual pattern of cortical/subarachnoid hemorrhage following on-the-table ICH. 5) Two subjects with recanalized M1 occlusion and angiographic evidence of pseudoaneurysm of the distal striate artery rupture following microcatheter contrast injections.

Of these 6 subjects, 5 had NIHSS Ն20. Four SICHs were PH1, and 2 were PH2, based on digital volume measurement. Mean hematoma volumes in these 6 SICHs measured 23.5 mL, representing 27.9% of lesion volume, compared with 25.2 mL, or 37.1% of lesion volume, for 6 nonultrasound IMS II and IMS I SICHs.

Three of the 7 SICHs had microcatheter injections. An early concern regarding a potential link between microcatheter injections and ICH with this paradigm was transmitted to investigators. 9 No direct vessel perforation, primary subarachnoid hemorrhage, or intracranial dissection was documented in IA-treated subjects. Four (7.3%) reocclusions (3 complete) occurred during the procedure in 55 IA-treated IMS II subjects. New emboli in the ACA distribution were identified in 1/22 (4.5%) M1/M2 occlusions and in 1/14 (7.1%) ICA T occlusions in IMS II.

Other Results

Retrospective temperature analysis provided 93 IMS II data points available with both arteriographic and temperature data for retrospective analysis in the sonography-treated subjects. Temperature decreases of as little as 0.2-0.3°C could be readily identified. Forty (41.9%) temperature decreases were identified. Forty-five (48.3%) recanalizations or microcatheter retractions were identified. Temperature analysis provided 81% sensitivity, 90% specificity, 85% positive predictive value, 87% negative predictive value, and 84.9% accuracy in identifying recanalization or catheter retraction on the following arteriographic sequence.

Available 24-hour CT lesion volumes demonstrated no significant differences in IMS II infarct volumes compared with IMS I subjects overall or to those treated by combined IV/IA methods.

Discussion

The IMS II trial was designed to study the safety and potential efficacy of a sonography microcatheter in the recanalization of acute intracranial artery occlusions in acute ischemic stroke, following reduced-dose IV rtPA therapy. Both clinical and technical factors contributed to the actual use of a sonography microcath- eter in only 33 subjects. Clinical exclusions for AOL location and etiology (atherosclerotic occlusion, dissection) occurred. Technical problems (difficulty and failure in catheter passage, connector cable problem, etc) further limited sonography administration and evaluation in several subjects. Other potentially eligible subjects were excluded on the basis of clinical, angiographic, or other operator-defined exclusion factors. When used per protocol, the Primo sonography microcatheter demonstrated a trend toward achieving faster and more complete AOL recanalization compared with standard microcatheter use in IMS II, with an incomplete 60-minute dataset from IMS I and with the complete 120-minute dataset from IMS I (Fig 2), though these differences were not statistically significant. It might be unduly optimistic to expect that new revascularization methods would achieve a statistical benefit compared with a widely accepted active therapy in a small trial.

Standard microcatheter use in IMS I and II allowed mechanical manipulation of thrombus, including microcatheter and microguidewire passage and drug delivery distal to the occlusion initially, as well as 15-minute guidewire/microcatheter advancement. On the other hand, sonography microcatheter use was limited to proximal bolus and drip infusion, with no guidewire manipulation beyond that required for initial and subsequent microcatheter advancement. Figure 2 suggests an early recanalization equivalency or advantage for the standard microcatheter; this potential advantage may actually be a mechanical one due to microcatheter/guidewire manipulation of favorably predisposed occlusions in the setting of IV rtPA activity. Allowing microguidewire/sonography microcatheter manipulation in a similar fashion may further enhance recanalization potential and may lead to greater in revascularization effect.

Several other important observations are suggested from Fig 2. In addition to the apparent initial 15-to 30-minute equivalence of standard microcatheter use, Fig 2 also demonstrates that the initial 15-minute period actually achieves the greatest recanalization effect for both the sonography microcatheter (21%) and the standard microcatheter (24%) in IMS II overall. This also may be due to mechanical manipulation of favorably predisposed occlusions. It also demonstrates a relatively linear increase in recanalization effect with time, suggesting that failure to achieve recanalization in any time period does not predict failure in subsequent periods. In addition, the 60-minute recanalization data points of 23 IMS I subjects with reliable images and the 120-minute data points of 53 IMS I subjects with T, M1, M2, or vertebrobasilar occlusions lie in close proximity to the 15-minute line of 14 IMS II standard microcatheter cases. This suggests reproducible results in achieving complete recanalization with the standard microcatheter.

Identification of 14/81 (17.2%) subjects without AOL qualifying for IA sonography microcatheter thrombolysis confirmed pre-IMS and IMS I study observations: IV rtPA may recanalize some vessels early, leading to normal findings on arteriograms (n ϭ 2) or distal occlusions (n ϭ 10) (Fig  1). 1,10 Any comparison of recanalization rates by using IAonly revascularization methods with the combined approach in IMS I and II with studies of must take into account this early recanalization achieved after IV therapy.

It is not possible to accurately compare recanalization and reperfusion results achieved with the combined IV/IA rtPA ther-apy paradigm with other reported IA revascularization methods in which treatment was initiated later. The 78% AOL 3 recanalization and 83.3% TICI 2/3 reperfusion of isolated M1 occlusion in IMS II, comparable with 78% for both measures in IMS I, may represent a true recanalization and reperfusion benefit compared with results obtained with r-pro-urokinase treatment in the Prolyse in Acute Cerebral Thromboembolism (PROACT II) study (67% TIMI 2/3 for M1 and M2 occlusions) and with the Multimechanical Embolus Removal in Cerebral Ischemia (MERCI) retriever in the MERCI and Multi-MERCI trials (45% and 54% TIMI 2/3, respectively). 11,12 However, it still remains uncertain that revascularization end point definitions and application in the IMS studies are comparable with other revascularization studies.

A relationship between both AOL and TICI revascularization end points and mRS 0 -2 outcome was demonstrated for ICA T and M1 occlusion ( Table 2). TICI 2/3 reperfusion better predicted good outcome than did primary AOL 2/3 recanalization. To further analyze the TICI 2 reperfusion end point in hope of identifying an acceptable or optimal stopping point for revascularization, we also categorized subjects as TICI 2A, with Ͻ50% MCA distribution reperfusion, or TICI 2B, with recanalization with flow Ͼ50% (Tables 1 and 2). TICI 2B/3 perfusion best predicted good outcome statistically (P ϭ .0002), more robustly than TICI 2/3 (P ϭ .0004). There was a trend for TICI 2B superiority to TICI 2A in predicting good outcome (P ϭ .08). AOL 2/3 recanalization (partial or complete recanalization with some flow) also predicted good outcome (P ϭ .027), suggesting that recanalization with some flow predicts good outcome nearly as well as more complete reperfusion. On the basis of these observations, safe revascularization appears to be an appropriate surrogate end point for the IV/IA treatment paradigm. It is not clear that a link between no revascularization and poor outcome is due totally to the no revascularization effect itself. Procedural factors (eg, contrast deposition, saline deposition, micro-and distal emboli, parenchymal hematoma, and even hemorrhagic transformation of injured brain) during prolonged failed revascularization procedures may contribute to the lack of good outcomes observed with this treatment paradigm.

Good outcomes with M2 occlusion were not dependent on revascularization, with 13/17 IMS (76%) I and II isolated M2 subjects achieving good outcome despite incomplete recanalization and reperfusion. This likely relates in part to the much smaller volume of ischemic brain with branch occlusions as compared with occlusions of major proximal arteries.

Of interest, operators scored their revascularization efforts more optimistically than the core lab. Differences between a TICI score of 2 and 3 are of questionable practical significance because these 2 scores are commonly viewed together as significant revascularization and correlate variably with increase in good outcome (Table 2). Nevertheless, it raises a legitimate question of accepting unadjudicated prospective data regarding important end points as a determinant of efficacy. In addition, this observation raises questions regarding revascularization end point terminology, definition, convention, and application. "Good" recanalization (AOL 2/3) in 46/75 (61.3%) and "good" reperfusion (TICI 2/3) in 56/75 (73.6%) of ICA T and M1 occlusions suggest that both measures are legitimate determinants of revascularization, but 12% difference confirms that they are not the same.

Sonography microcatheter activity measurably increases catheter-tip temperature. Monitoring this effect as a safety measure provided a unique opportunity to examine the effect of recanalization or catheter retraction on catheter-tip temperature. Retrospective correlation of catheter-tip temperature decrease with recanalization suggests that observing tip temperature during a procedure might prospectively allow identification of recanalization between strictly prescribed angiogram intervals and, therefore, might allow the revascularization procedure to be shortened. Retrospective analysis of temperature changes was not complete for all subjects. However, where available, the correlation between a decrease in temperature and recanalization or catheter retraction from the occlusion was quite strong. Whereas some recanalization was identified in 47.6% of subjects and temperature decreases were identified in 41% of subjects, treatment time might be reduced accordingly if real-time changes in temperature decrease might be prospectively identified and acted upon (Fig 3).

Higher ICH and contrast deposition rates with microcatheter injections suggest that technical factors may be important in clinical outcomes. 9 A correlation between MCI and ICH, as well as PH, has also been found in a thrombolysis registry analysis. 13 Higher ICH rates with the sonography microcatheter use were confounded by this relationship. Demonstration of rupture points of distal lenticulostriate arteries in several instances negated focal sonography or focal tissue temperature changes as responsible for these ICH cases. No single etiologic mechanism seems responsible for the observed ICH. A multifactorial contribution of rtPA, heparin, contrast deposition, and microcatheter injection pressure/volume is hypothesized.

It is not unreasonable to set a target for good outcomes at 50% or greater in patients with major stroke treated with the IV/IA paradigm applied as rapidly as possible, with limited imaging selection. mRS 0 -2 outcomes were achieved in 45% of IMS II overall, compared with 42% of IMS I subjects and with 39% in the treated group of the NINDS study, despite significantly quicker time to IV thrombolytic therapy in the latter. Six AOLs eligible for an IA therapy did not receive it for a variety of reasons. Subjects with M2 occlusions, with baseline NIHSS 11 and 21, were lost to follow-up but were included in the mRS Ͼ2 group. It is reasonable to expect that monitoring catheter-tip temperature may accelerate sonography-assisted thrombolysis procedures. Other further advances in revascularization with improved thrombus-removal devices, balloon angioplasty, and/or removable or implantable stents may play a part in further improving interventional outcomes. The IMS III trial, in which 900 subjects will be randomized to either full-dose IV rtPA or to a reduced-dose IV rtPA, plus IA therapy with either the Cordis Neurovascular (Cordis, Miami Lakes, Fla), Neurowave Catheter (EKOS, Bothell, Wash), Merci Retriever Device (Concentric Medical, Mountain View, Calif), or other devices that may be introduced as their approval status becomes defined, will have the opportunity to test this hypothesis.

Conclusions

IMS II provides further evidence that the EKOS Primo sonography microcatheter demonstrates a promising trend toward improved recanalization of the AOL when compared with a standard microcatheter. A correlation between recanalization and reperfusion with outcome is again confirmed. The role of IV/IA revascularization therapy will be further tested against full-dose IV therapy alone in the IMS-III trial.

Perfusion past the initial obstruction but limited distal branch filling with little or slow distal perfusion 2A ϭ Perfusion of less than half of the vascular distribution of the occluded artery (eg, filling and perfusion through 1 M2 division) 2B ϭ Perfusion of half or greater of the vascular distribution of the occluded artery (eg, filling and perfusion through 2 or more M2 divisions) 3 ϭ Full perfusion with filling of all distal branches II. Posttreatment AOL recanalization grade 0 ϭ No recanalization of the occlusion 1 ϭ Incomplete or partial recanalization of the occlusion, with no distal flow 2 ϭ Incomplete or partial recanalization of the occlusion, with any distal flow 3 ϭ Complete recanalization of the occlusion with any distal flow * The modified TICI reperfusion score was essentially equivalent to TIMI score applied in IMS I, with grade 2 further divided into A and B for post hoc analysis.

Fig 1 .
1Patient flow and treatment group designation for 81 subjects entered into IMS II.

Fig 2 .
2Graph depicts total cumulative complete grade 3 AOL recanalization at each 15-minute interval for the EKOS Primo sonography microcatheter (n ϭ 29) and the standard microcatheter in IMS II (n ϭ 14). Standard microcatheter recanalization data points at 60 and 120 minutes are also depicted. IMS I data are incomplete at 60 minutes, with 23 confirmed control arteriograms. Sixty-and 120-minute IMS I data points closely approximate the line of IMS II microcatheter recanalization. The open circle emphasizes an early equivalence of standard microcatheter to the sonography (US) microcatheter. The greatest recanalization effect (Ͼ20%) is within the first 15 minutes of microcatheter placement.

Fig 3 .
3Temperature (T) chart correlation to recanalization in M2 occlusion. Upper left: control arteriogram demonstrates EKOS sonography microcatheter placed in the M2 occlusion (white arrow) just before treatment time 0. Upper right: 15-minute control arteriogram demonstrates microcatheter tip retraction into the M1 segment (white arrow). Note the temperature decrease of approximately 0.2°C during the 15-minute time interval, indicating cooling of the catheter by flowing blood. Lower right: oblique 30-minute arteriogram with catheter-tip position (white arrow) proximal to recanalization of the M2 segment, with distal emboli (AOL 3 recanalization, TICI 2 perfusion). Note the temperature drop of approximately 0.4°C during the 15-minute time interval. The downward spikes of T indicate cooling by contrast injection. The spike at 25 minutes was an additional injection to confirm maintenance of catheter position, where no recanalization was yet identified. deg C indicates degrees celsius.

Table

Table 2 :
2mRS 0 -2 outcomes and mortality with 75 ICA T (n ؍ 29) M1 (n ؍ 46) occlusions according to TICI reperfusion and AOL
recanalization in IMS I and II*

mRS 0-2
Mortality

IMS I
IMS II
Total I & II
IMS I
IMS II
Total I & II

TICI 2/3
10/25 (40%)
11/21 (52.4%)
21/46 (45.6%)
P ϭ .0004
4/25 (20%)
1/21 (4.7 %)
5/46 (10.9%)
P ϭ .01
No TICI 2/3
0/16 (0%)
2/13 (15.4%)
2/29 (6.9 %)
5/16 (31.3%)
5/13 (38.4%)
10/29 (34.5%)
TICI 2B, 3
7/15 (46.7%)
8/11 (72.7%)
15/26 (57.7%)
P ϭ .0002
2/15 (13.3%)
0/11 (0%)
2/26 (7.7%)
P ϭ .05
No TICI 2B, 3
3/26 (11.5%)
5/23 (21.7 %)
8/49 (16.3 %)
7/26 (25.9%)
6/23 (26.1%)
13/49 (26.5%)
AOL 2/3
10/30 (34.5%)
11/26 (42.3%)
21/56 (37.5%)
P ϭ .027
5/30 (16.6%)
3/26 (11.5%)
8/56 (14.3%)
P ϭ .03
No AOL 2/3
0/11 (0%)
2/8 (25%)
2/19 (10.5%)
4/11 (36.3%)
3/8 (37.5%)
7/19 (36.8%)
Overall
10/41 (24.4%)
13/34 (38.2%)
23/75 (30.6%)
9/41 (21.9%)
6/34 (17.6%)
15/75 (20%)

* These data includes 25 (33%) subjects with ICA bifurcation occlusion or stenosis Ն 70%. Statistical comparisons of 3 dichotomized revascularization end points are included.

Combined intravenous and intra-arterial rtPA for acute ischemic stroke: the interventional management of stroke study. Stroke. 35The IMS study investigatorsThe IMS study investigators. Combined intravenous and intra-arterial rtPA for acute ischemic stroke: the interventional management of stroke study. Stroke 2004;35:904 -12

Sensitivity and prognostic value of early CT in occlusion of the middle cerebral artery. T A Tomsick, AJNR Am J Neuroradiol. 15Tomsick TA. Sensitivity and prognostic value of early CT in occlusion of the middle cerebral artery. AJNR Am J Neuroradiol 1994;15:16 -18

North American clinical experience with the EKOS MicroLysUS infusion catheter for the treatment of embolic stroke. B R Mahon, G M Nesbit, S L Barnwell, AJNR Am J Neuroradiol. 24Mahon BR, Nesbit GM, Barnwell SL, et al. North American clinical experience with the EKOS MicroLysUS infusion catheter for the treatment of embolic stroke. AJNR Am J Neuroradiol 2003;24:534 -38

The interventional management of stroke (IMS) II study. Ims Ii Trial, Investigators, Stroke. 38EpubIMS II Trial Investigators. The interventional management of stroke (IMS) II study. Stroke 2007;38:2127-35. Epub 2007 May 24

Revascularization end points in stroke interventional trials: recanalization versus reperfusion in IMS-I. P Khatri, J Neff, J P Broderick, Stroke. 36Khatri P, Neff J, Broderick JP, et al. Revascularization end points in stroke interventional trials: recanalization versus reperfusion in IMS-I. Stroke 2005;36:2400 -03

Trial design and reporting standards for intra-arterial cerebral thrombolysis for acute ischemic stroke. R T Higashida, A J Furlan, Stroke. 34Higashida RT, Furlan AJ. Trial design and reporting standards for intra-arte- rial cerebral thrombolysis for acute ischemic stroke. Stroke 2003;34:1923-24

Contrast enhancement and contrast extravasation on computerized tomography after intraarterial thrombolysis in patients with acute ischemic stroke. W Yoon, J J Seo, J K Kim, Stroke. 35Yoon W, Seo JJ, Kim JK, et al. Contrast enhancement and contrast extravasa- tion on computerized tomography after intraarterial thrombolysis in pa- tients with acute ischemic stroke. Stroke 2004;35:876 -81

Hemorrhagic transformation of ischemic brain tissue: asymptomatic or symptomatic?. C Berger, M Fiorelli, T Steiner, Stroke. 32Berger C, Fiorelli M, Steiner T, et al. Hemorrhagic transformation of ischemic brain tissue: asymptomatic or symptomatic? Stroke 2001;32:1330 -34

Microcatheter contrast injections during intraarterial thrombolysis increase intracranial hemorrhage risk. R Khatri, J Broderick, J Khoury, Stroke. 37622Khatri R, Broderick J, Khoury J, et al. Microcatheter contrast injections during intraarterial thrombolysis increase intracranial hemorrhage risk. Stroke 2007;37:622

Combined IV and intraartrial thrombolysis for acute ischemic stroke. M L Flaherty, D Woo, B Kissells, Neurology. 64Flaherty ML, Woo D, Kissells B, et al. Combined IV and intraartrial thrombol- ysis for acute ischemic stroke. Neurology 2005;64:386 -88

Safety of mechanical thrombectomy and intravenous tissue plasminogen activator in acute ischemic stroke: results of the Multi Mechanical Embolus Removal in Cerebral Ischemia (MERCI) trial, part I. W S Smith, AJNR Am J Neuroradiol. 27Smith WS. Safety of mechanical thrombectomy and intravenous tissue plas- minogen activator in acute ischemic stroke: results of the Multi Mechanical Embolus Removal in Cerebral Ischemia (MERCI) trial, part I. AJNR Am J Neuroradiol 2006;27:1177-82

Safety and efficacy of mechanical embolectomy in acute ischemic stroke: results of the MERCI trial. W Smith, Stroke. 36EpubSmith W. Safety and efficacy of mechanical embolectomy in acute ischemic stroke: results of the MERCI trial. Stroke 2005;36:1432-38. Epub 2005 Jun 16

Microcatheter contrast injections during intraarterial thrombolysis increase parenchymal hematoma risk: registry experience. R Khatri, P Khatri, J Khoury, Stroke. 38454Khatri R, Khatri P, Khoury J, et al. Microcatheter contrast injections during intraarterial thrombolysis increase parenchymal hematoma risk: registry ex- perience. Stroke 2007;38:454


A Formulation Study of 5-Aminolevulinic Encapsulated in DPPC Liposomes in Melanoma Treatment
2016. 2016

Ming-Wei Lin
Center for Stem Cell Research
Kaohsiung Medical University
80756KaohsiungTaiwan

Yaw-Bin Huang
Center for Stem Cell Research
Kaohsiung Medical University
80756KaohsiungTaiwan

School of Pharmacy
Kaohsiung Medical University
80756KaohsiungTaiwan

Chun-Lin Chen
Center for Stem Cell Research
Kaohsiung Medical University
80756KaohsiungTaiwan

Department of Biological Science
National Sun Yat-sen University
80424KaohsiungTaiwan

Pao-Chu Wu
School of Pharmacy
Kaohsiung Medical University
80756KaohsiungTaiwan

Chien-Ying Chou
School of Pharmacy
Kaohsiung Medical University
80756KaohsiungTaiwan

Ping-Ching Wu
Department of Biomedical Engineering
National Cheng Kung University
701TainanTaiwan

Institute of Oral Medicine and Department of Stomatology
College of Medicine
National Cheng Kung University Hospital
National Cheng Kung University Tainan 701
Taiwan

Medical Device Innovation Center
Innovation Center of Medical Devices and Technology
National Cheng Kung University Hospital
Taiwan

National Cheng Kung University
701TainanTaiwan

Corresponding authors: Shih-Ya Hung
Graduate Institute of Integrated Medicine
College of Chinese Medicine
Department of Biomedical Engineering
China Medical University
40402TaichungTaiwan

National Cheng Kung University
701TainanTaiwan


Shih-Ya Hung
Graduate Institute of Integrated Medicine
College of Chinese Medicine
China Medical University
40402TaichungTaiwan

Division of Colorectal Surgery
Medical University Hospital
40447TaichungChina, Taiwan

Corresponding authors: Shih-Ya Hung
Graduate Institute of Integrated Medicine
College of Chinese Medicine
Department of Biomedical Engineering
China Medical University
40402TaichungTaiwan

National Cheng Kung University
701TainanTaiwan

A Formulation Study of 5-Aminolevulinic Encapsulated in DPPC Liposomes in Melanoma Treatment

Int. J. Med. Sci
1372016. 201610.7150/ijms.15411Received: 2016.03.01; Accepted: 2016.05.08;I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f M Me ed di ic ca al l S Sc ci ie en nc ce es s Research Paper http://ivyspring.com/terms for terms and conditions.5-aminolevulinic acidDPPC liposomesmelanomaphotodynamic therapyskin cancer
Photodynamic therapy (PDT) is a widely used technique for epithelial skin cancer treatment. 5-aminolevulinic acid (5-ALA) is a drug currently used for PDT and is a hydrophilic molecule at its physiological pH, and this limits its capacity to cross the stratum corneum of skin. Since skin penetration is a key factor in the efficacy of topical 5-ALA-mediated PDT, numerous strategies have been proposed to improve skin penetration. Yet this problem is still ongoing. The results of a previous study showed a low rate of 5-ALA encapsulated in liposomes (5.7%) that were 400 nm in size. In the present study, we used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes as vehicles and tested their delivery efficacy of 5-ALA-medicated PDT both in vitro and in vivo. Our data shows that 5-ALA encapsulated in 0.1 or 0.5% DPPC liposomes (5-ALA/DPPC) had a better encapsulated rate (15~16%) and were smaller in size (84~89 nm). We found the 5-ALA/DPPC formulation reduced cell viability, mitochondria membrane potential, and enhanced intracellular ROS accumulation as compared to 5-ALA alone in melanoma cells. Furthermore, the 5-ALA/DPPC formulation also had better skin penetration ability as compared to the 5-ALA in our ex vivo data by assaying 5-ALA converted into protoporphyrin IX (PpIX) in the skin of the mice that were experimented on. In melanoma xenograft models, 5-ALA/DPPC enhanced PpIX accumulation only in tumor tissue but not normal skin. In conclusion, we found DPPC liposomes to be good carriers for 5-ALA delivery and believe that they may prove useful in 5-ALA-mediated PDT in the future.

Introduction

Skin cancer is the most common human cancer with over a million cases detected every year [1]. Risk factors for skin cancer include ultraviolet (UV) radiation from sun exposure, receiving an organ transplant, HIV, genetic factors, etc. [2]. Melanoma is the deadliest form of skin cancer. It is an aggressive, therapy-resistant malignancy of melanocytes and inclined to metastasize quickly into the lymphatic system and other organs such as the lungs, the liver, the brain, or the bones [3,4]. Incidences of melanoma have been steadily increasing worldwide, resulting in an increasing public health problem; and current Ivyspring International Publisher chemotherapy; and chemo-immunotherapy regimes have shown little clinical benefit with no improvement in overall survival [4].

Photodynamic therapy (PDT) is a popular and non-invasive treatment based on selectively accumulating a non-toxic photosensitizing drug (e.g., 5-aminolevulinic acid and methyl aminolevulinate), a drug which will preferentially accumulate in tumorous tissue rather than normal tissue [5]. Subsequent activation by 635 nm light irradiation converts 5-ALA into protoporphyrin IX (PpIX) and initiates a photodynamic reaction via generating highly reactive singlet oxygen ( 1 O 2 ), and this process results in tumor cell death [6]. As a novel therapy, PDT has the advantages of being able to select for tumor cells and not destroy normal cells, and of not being limited by the location or size of skin lesions [7]. PDT has proven to have several benefits including low morbidity, minimal functional disturbance, better cosmetic outcomes, and the ability to repeatedly be used many times in one instance [8,9]. PDT is a successful treatment for non-melanoma skin cancers in clinical practice. [7] More and more doctors are using PDT to cure patients with skin cancer, especially elderly patients [7].

There are now increasing numbers of photosensitizing prodrugs being used clinically; and 5-aminolevulinic acid (5-ALA)-mediated PDT is one of the fastest developing areas in PDT [6]. 5-ALA is a naturally occurring delta amino acid that is ultimately converted in to PpIX, the immediate precursor of heme [5]. Topical 5-ALA PDT has shown significant efficacy in superficial skin cancer, actinic keratosis, psoriasis, cutaneous T-cells lymphoma, basal cell carcinoma, and squamous cell carcinoma [10][11][12][13]. However, 5-ALA is a 167.6 g/mol hydrophilic molecule with a low penetration rate through intact skin. [14] Thus, recent investigations have focused on the development of carriers of 5-ALA that are able to penetrate intact skin. Enhancement of 5-ALA skin penetration via the improvement of the transdermal drug delivery system is a key factor in the success of topical applied PDT [15]. Enhancing techniques to increase transdermal drug delivery include chemical modification of 5-ALA, pretreatment with physical or chemical penetration enhancers (e.g., lasers, ultrasound, DMSO, EDTA), and penetration using different vehicles (e.g., lotion, liposomes) [15]. Of the aforementioned, liposomes are one of the best delivery systems for low-molecular-weight drugs, DNA, siRNA, etc. [16]. Liposomes are membrane-like phospholipid bilayers with a water phase inside that can carry both lipophilic and hydrophilic agents and are generally accepted in various delivery strategies for the systemic or topical administration of drugs [17]. However, low 5-ALA encapsulation in liposomes has been reported, with Pierre et al demonstrating that 5-ALA-entrapped in stratum corneum lipid liposomes (SCLLs) formulation was only 5.7% [18].

Since PDT is now a well-established treatment modality for cutaneous carcinomas and is based on the administration of a light-activated drug followed by the illumination of the pathological area, the treatment of metastatic melanoma remains a therapeutic challenge [19]. The aim of this study is to evaluate the in vitro and in vivo efficacy of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes formulation in the treatment of melanoma and gain more information about DPPC liposomes and their interaction with 5-ALA. The objective of this investigation was to create DPPC liposomes formulations as delivery systems that would be able to overcome the poor penetration of 5-ALA. WST-1 cell proliferation assay, ROS production and cellular mitochondrial membrane potential measurements were used to determine the photo-cytotoxicity of 5-ALA encapsulated in DPPC liposomes on melanoma cancer cells when exposed to PDT. The amount of 5-ALA skin penetration, as well as skin retention from DPPC liposomes, was investigated. Melanoma xenograft mouse models were employed to confirm liposomal-based PDT efficiency in vivo.

Materials and Methods

Materials

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was obtained from Avanti (USA). Cholesterol was purchased from Sigma-Aldrich (USA). Other chemicals used in the study were reagent grade. Chemical structures of 5-ALA and DPPC are present in Figure 1A.

Preparation of 5-ALA encapsulated in DPPC liposomes (5-ALA/DPPC)

5-ALA encapsulated in DPPC liposomes

(5-ALA/DPPC) was prepared according to the modified thin film hydration method [20]. DPPC and cholesterol were dissolved in chloroform and methanol (3:1, v/v) in 250 mL round bottom flasks. The mixture was evaporated in a rotary evaporator above transition temperature, and solvent traces were removed under a vacuum overnight. The film was hydrated with 0.5% 5-ALA aqueous solution above lipid transition temperature for 30 minutes. The vesicle suspension was dispersed by a probe sonicator (UP50H, Germany) at 30% amplitude for 15 minutes. For the diffusion experiments, DPPC liposomes loaded with 0.5% 5-ALA were prepared under the same conditions.

Characterization of 5-ALA encapsulated in DPPC liposomes

The particle size and zeta potential of 5-ALA encapsulated in DPPC liposomes were measured by laser light-scattering with a helium-neon laser at 630 nm (Zetasizer 3000HS A , Malvern, UK) at 25 °C. The polydispersity index (PI) was used to measure size distribution. The entrapment efficiency of 5-ALA in DPPC liposomes was employed with an ultracentrifuge method (CS120GXL, Hitachi) at 60,000 g and 4 °C for 30 minutes. 5-ALA was analyzed by HPLC following centrifugation.

Morphological determination of 5-ALA DPPC liposomes by transmission electron microscopy

The 5-ALA/DPPC (0.5% DPPC) was stained with a phosphotungstic acid (1%) solution and titrated with a copper wire. After it had dried, its morphological structure was determined by transmission electron microscopy (TEM; Joel model JEO 2000 EX II, Japan)

Cell culture

Murine melanoma cells (B16F10) were obtained from the America Type Culture Collection (ATCC). B16F10 cells were allowed to grow to confluence using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin).

PDT and WST-1 cell cytotoxicity assay

Cell cytotoxicity of PDT was assessed using a WST-1 cell proliferation kit (Roche). Briefly, cells were seeded with 5 x10 4 cells/well in a culture medium containing 5-ALA or 5-ALA/DPPC in 96-well microplates 4 hours before assay. 5-ALA in this culture medium was removed and replaced by PBS. An instrument with a light-emitting diode (LED) a red light source at 630 nm was used for PDT. A light dose of 50 J/cm 2 was delivered over 20 min. Cell viability was measured 24 h later by WST-1 colorimetric assay with a microplate ELISA reader.

Reactive oxygen species (ROS) measurement

PDT-treated cells were then incubated with 1 µM superoxide-sensitive dye dihydroethidium. The ROS production during PDT was determined by FACScan flow cytometry (Becton Dickinson).

Detection of mitochondrial transmembrane potential

PDT-treated cells were incubated with 0.1 µM Mitotraker (Invitrogen) for 30 min at 37 o C in the dark.

The cells were washed with warm PBS, and Mitotraker fluorescence intensity was determined by FACScan flow cytometry (Becton Dickinson).

Confocal laser scanning microscopy

For confocal microscopy assay, the mice were treated with either the control (PBS), 5-ALA, 5-ALA/DPPC (0.5% DPPC). These were the three groups for our experiment. After 4 hours, the skin where the drug was administrated was harvested and fixed with 4% paraformaldehyde. Then tissue samples were sliced into 5 µm sections by frozen section and mounted with Vectashield media (Vector Laboratories) for laser confocal microscopy observation (model FV1000, Olympus).

Melanoma xenograft mouse models

Six-week-old male BALB/c nu/nu nude mice were housed in a specific pathogen-free environment. The B16F10 cells were harvested, washed, and suspended in PBS. Then, 5 x 10 5 cells were injected subcutaneously into the right flank regions of the mice. The mice were then checked every day for the appearance of tumor, and the sizes of the tumors were determined by measuring 2 diameters with a caliper after PDT. Tumor volume (TV) was estimated using the equation TV = (length x width) 2 /2 and expressed as a % of control. Three mice were included in each of the three groups.

Results

Characteristics of 5-ALA encapsulated in DPPC liposomes (5-ALA/DPPC)

After using the thin film method by addition of cholesterol as a stabilizer, the data of mean diameter (size), polydispersity index (representing the particle size), zeta potential, and entrapment efficiency of 5-ALA/DPPC were measured. As shown in Table 1, the size of 5-ALA/0.1% DPPC and 5-ALA/0.5% DPPC were smaller than 5-ALA alone (0.5% final), with both being under 100 nm. Moreover, polydispersity indexes were within 0.31 to 0.36 in the 5-ALA, 5-ALA/0.1% DPPC and 5-ALA/0.5% DPPC formulations. The 5-ALA/DPPC formulations achieved a cationic surface charge of 5-ALA that meant a tendency towards increasing contact with the cell membrane. The zeta potentials of the 5-ALA, 5-ALA/0.1% DPPC, and 5-ALA/0.5% DPPC liposomal formulations were about 3.40 ± 0.80, 6.60 ± 0.50 and 15.10 ± 0.70 mV, respectively. The encapsulation of 5-ALA/0.1% DPPC and 5-ALA/0.5% DPPC were similar (Table 1). This data indicates that DPPC enhances 5-ALA entrapment efficiency to 15-16%, which is a substantial increase as compared to stratum corneum lipid liposomes (SCLLs, 5.7%) [18]. In vitro analysis 5-ALA/DPPC produced photo-toxicity 5-ALA administration with PDT produces PpIX accumulation in B16F10 melanoma cells in a dose-dependent manner [21]. Here, we studied whether DPPC as the 5-ALA carrier changes the PpIX accumulation in B16F10 cells. The photo-toxicity of 5-ALA-induced PpIX accumulation in B16F10 melanoma cells was determined by WST-1 to detect cell viability after PDT. As figure 2A shows, the 5-ALA-treated cells' viability was reduced as compared to the control. 5-ALA/DPPC had lower cell viability than 5-ALA alone. The cell viability in 5-ALA and 5-ALA-DPPC were 52 ± 12.8 % and 33 ± 9.4 %, respectively. It has been shown that ROS production after PDT leads to DNA fragmentation, mitochondria damage, and cell death [22]. Figures 2B & 2C show 5-ALA/DPPC-treated cells as having higher ROS production and lower mitochondrial membrane potential than 5-ALA-treated B16F10 cells. These data suggest that 5-ALA/DPPC has a higher photo-toxicity than 5-ALA in vitro.

Morphology of 5-ALA/DPPC

Since the physicochemical characteristics of 5-ALA/0.1% DPPC and 5-ALA/0.5% DPPC were similar, we further used TEM to study the morphology of 5-ALA/0.5% DPPC. Figure 1B shows that 5-ALA/0.5% DPPC had a relatively uniform size distribution which was consistent with the particle size data (89.80 ± 2.40 nm) that we obtained using laser sizing.

5-ALA/DPPC-induced PpIX accumulation in ex vivo mice skin model

We further performed confocal microscopy to assay the 5-ALA-induced PpIX accumulation after PDT in the mice skin. The accumulation of PpIX emits a characteristic red fluorescence when exposed to light of the appropriate wavelength. Figure 3 shows confocal images obtained from mechanical skin sections of control-(PBS-), 5-ALA-and 5-ALA/DPPC (0.5% DPPC)-treated mice. After incubation for 4 hours, the red fluorescence intensity from PpIX of 5-ALA/DPPC-treated skin was obviously stronger than that of 5-ALA. This result indicates that the skin penetration ability of 5-ALA/DPPC was enhanced as compared to the 5-ALA treatment.

5-ALA/DPPC in the mouse xenograft tumor model

The in vivo mouse xenograft tumor model was established by subcutaneous implantation of B16F10 cells in nude mice. The results showed that the 5-ALA and 5-ALA/DPPC-treated group both had smaller tumor volume than the control group but those differences were not statistically significant within 3 days ( Figure 4A). We further analyzed PpIX accumulation in both tumor and healthy skin tissues. Figure 4B shows that PpIX contents in tumor tissues were higher in both 5-ALA and 5-ALA/DPPC (0.5% DPPC) groups but not in healthy skin tissues. Furthermore, the 5-ALA/DPPC group had a higher PpIX content in tumor tissue as compared to the 5-ALA group ( Figure 4B). This indicates that 5-ALA/DPPC has better skin penetration ability than 5-ALA alone, and this allows for PpIX accumulation in tumor tissue in vivo but we need more mice to assay the influence of 5-ALA/DPPC with respect to tumor volume.

Discussion

5-ALA is a drug currently used for PDT and has been approved by the U.S. Food and Drug Administration. 5-ALA is a hydrophilic molecule at its physiological pH, and this limits its capacity to cross the stratum corneum of skin. This is concerning because skin penetration is a key factor in the efficacy of topical 5-ALA-medicated PDT [5,15]. Numerous strategies have been proposed to improve penetration, and strategies such as l-menthol and its derivative are effective to enhance 5-ALA skin permeation [23]. Yet this problem is still ongoing. Therefore, improving the delivery efficacy for 5-ALA penetration is an urgent matter in 5-ALA-mediated PDT.

As mentioned before, the efficiency of ALA-liposome distribution to tumors is limited by several factors including the physicochemical property of the drugs and the relatively poor perfusion of skin. The lipid components of DPPC liposomes are predominantly DPPC and cholesterol. Based on their biphasic property, DPPC liposomes can act as carriers for hydrophilic drugs such as 5-ALA. Due to the hydrophilic properties of 5-ALA, it can be entrapped in the aqueous phase of liposomes and this brings about the limitation of the entrapment efficiency [18]. Therefore, DPPC was substituted as components of liposomes to increase the entrapment efficiency. In this study, we used DPPC liposomes as vehicles and tested their delivery efficacy of 5-ALA-medicated PDT both in vitro and in vivo. We found that the 5-ALA/DPPC formulation reduced cell viability, mitochondria membrane potential, and enhanced intracellular ROS accumulation as compared to 5-ALA alone in vitro. In addition, the 5-ALA/DPPC formulation also showed better skin penetration ability as compared with 5-ALA in our ex vivo data. In the mouse xenograft tumor models, 5-ALA/DPPC enhanced PpIX accumulation in tumor tissues only and not in healthy skin. Although it reduced tumor volume, it did not do so to a statistical significant extent as compared to 5-ALA. This may be due to the xenograft tumor cell number, tumor depth, or the day of measurement; the exact reason for this still requires further investigations.

The skin distribution of topically applied 5-ALA depends on many parameters, such as drug permeability through the stratum corneum, as well as diffusion through the epidermis and dermis (which are related through compound structures), formulation, release from the vehicle, local drug clearance, and the conversion rate of 5-ALA into PpIX [24]. Methods for improvement of 5-ALA delivery in PDT are based on three concepts: (a) formulation changes to enhance penetration or chelate iron; (b) pretreatment with physical penetration enhancers via ultrasound, laser or iontophoresis; and (c) chemical modification of 5-ALA to increase uptake or selective cleavage [5]. Liposomes are one of the best drug delivery systems for low-molecular-weight drugs, imaging agents, peptides, proteins, and nucleic acids [16]. Liposomes are microscopic vesicles consisting of one or more membrane-like phosphoid bilayers surrounding an aqueous medium with a particle size from 20 to 500 nm. [17] Pierre et al [18] worked on a delivery system for 5-ALA based on liposomes with a lipid composition of cremide (50%), cholesterol (28%), palmitic acid (17%), and colesteryl sulfate (5%). These SCLL encapsulation rate of 5-ALA was 5.7% with a 400 nm particle size. Another report showed that the PDT efficacy and the accumulation of 5-ALA-induced PpIX in murine thymic lymphoma cells increased with a decrease in the average diameter of DPPC liposomes. [25] In the present report, we have shown that the entrapment efficiency of 5-ALA/DPPC was about 15~16% with a particle size of around 84 to 89 nm, suggesting that 5-ALA encapsulated in DPPC liposomes has a better encapsulation rate and are smaller in size than those encapsulated in SCLLs.

In conclusion, 5-ALA-based PDT in the treatment for cancer has attracted much attention, and improving the delivery efficacy for 5-ALA penetration through the skin is urgent in 5-ALA-mediated PDT as its penetration is quite poor without any aid. In the present study, we found DPPC liposomes are good carriers for 5-ALA delivery and may prove useful in 5-ALA-mediated PDT in the future.

Figure 1 .
1The morphology of 5-ALA/DPPC by transmission electron microscopy (TEM). Molecular structures of 5-aminolevulinic acid (5-ALA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). (B) The TEM image shows the morphology of 5-ALA encapsulated in DPPC (0.5%, 5-ALA/DPPC) which was stained with 1% phosphotungstic acid before being analyzed by TEM. Scale bar = 500 nm.

Figure 2 .
2In vitro analysis 5-ALA/DPPC produced photo-toxicity. The production of reactive oxygen species after photodynamic therapy leads to DNA fragmentation, mitochondria damage, and cell death. Here, we used DPPC (0.5%) as the 5-ALA carrier (5-ALA/DPPC) to study PpIX accumulation in B16F10 cells. (A) 5-ALA-treated B16F10 cells decreased in cell viability as compared to the control. And 5-ALA/DPPC-treated cells had lower cell viability as compared to 5-ALA-treated cells. The cell viability of 5-ALA and 5-ALA/DPPC groups were 52 ± 12.8 % and 33 ± 9.4 %, respectively. (B) & (C) show 5-ALA/DPPC-treated cells had a higher ROS production and lower mitochondrial membrane potential than 5-ALA-treated B16F10 cells. *, p<0.05.

Figure 3 . 5 -
35ALA/DPPC-induced PpIX accumulation in ex vivo mice skin model. Since the accumulation of PpIX emits a red fluorescence, we performed a confocal microscopy to assay the 5-ALA-induced PpIX accumulation after PDT in the skin of the mouse. After 4 hours of incubation, the PpIX intensity (red fluorescence) from 5-ALA/DPPC (0.5% DPPC)-treated skin was stronger than that in 5-ALA. Scale bar = 100 µm. Here we used DAPI (blue) to stain the nuclei in the skin.

Figure 4 . 5 -
45ALA/DPPC in the mouse xenograft tumor model. The in vivo mouse xenograft tumor model was established by subcutaneous implantation of B16F10 cells in nude mice. (A) The tumor volume of the 5-ALA and the 5-ALA/DPPC (0.5% DPPC)-treated groups both had smaller tumor volume than the control group yet didn't show statistically significance from Day 1 to Day 3. (B) PpIX accumulation in tumor and healthy skin tissues shows that the PpIX contents in tumor tissues were higher in both the 5-ALA and 5-ALA/DPPC groups but not in healthy skin tissues. Furthermore, the 5-ALA/DPPC group had a higher PpIX content in tumor tissue as compared to the 5-ALA only group.

Table 1 .
1Physicochemical characteristics of 5-ALA-liposomeFormulation
Size (nm)
PI
Zeta (mV)
EE (%)
Blank
114.20 ± 1.70 0.36 ± 0.06
3.40 ± 0.80
-
0.1% 5-ALA/DPPC 84.50 ± 8.08 0.31 ± 0.10
6.60 ± 0.50
15.23 ± 1.01
0.5% 5-ALA/DPPC 89.80 ± 2.40 0.36 ± 0.01
15.10 ± 0.70 16.43 ± 2.53

PI: polydispersity index; EE: encapsulation efficiency

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AcknowledgementsCompeting InterestsThe authors have declared that no competing interest exists.
Epidemiology of melanoma and nonmelanoma skin cancer. A C Geller, G D Annas, Semin Oncol Nurs. 19Geller AC, Annas GD. Epidemiology of melanoma and nonmelanoma skin cancer. Semin Oncol Nurs. 2003; 19: 2-11.

Skin cancer and new treatment perspectives: a review. M C Simoes, J J Sousa, A A Pais, Cancer Lett. 357Simoes MC, Sousa JJ, Pais AA. Skin cancer and new treatment perspectives: a review. Cancer Lett. 2015; 357: 8-42.

Malignant melanoma in the 21st century, part 1: epidemiology, risk factors, screening, prevention, and diagnosis. S N Markovic, L A Erickson, R D Rao, R H Weenig, B A Pockaj, A Bardia, Mayo Clin Proc. 82Markovic SN, Erickson LA, Rao RD, Weenig RH, Pockaj BA, Bardia A, et al. Malignant melanoma in the 21st century, part 1: epidemiology, risk factors, screening, prevention, and diagnosis. Mayo Clin Proc. 2007; 82: 364-80.

New therapeutic options for advanced non-resectable malignant melanoma. S Stadler, K Weina, C Gebhardt, J Utikal, Adv Med Sci. 60Stadler S, Weina K, Gebhardt C, Utikal J. New therapeutic options for advanced non-resectable malignant melanoma. Adv Med Sci. 2015; 60: 83-8.

Photodynamic therapy of skin cancer: controlled drug delivery of 5-ALA and its esters. R F Lopez, N Lange, R Guy, M V Bentley, Adv Drug Deliv Rev. 56Lopez RF, Lange N, Guy R, Bentley MV. Photodynamic therapy of skin cancer: controlled drug delivery of 5-ALA and its esters. Adv Drug Deliv Rev. 2004; 56: 77-94.

5-aminolevulinic acid induce apoptosis via NF-kappaB/JNK pathway in human oral cancer Ca9-22 cells. H M Chen, C M Liu, H Yang, H Y Chou, C P Chiang, M Y Kuo, J Oral Pathol Med. 40Chen HM, Liu CM, Yang H, Chou HY, Chiang CP, Kuo MY. 5-aminolevulinic acid induce apoptosis via NF-kappaB/JNK pathway in human oral cancer Ca9-22 cells. J Oral Pathol Med. 2011; 40: 483-9.

Efficacy and safety of topical ALA-PDT in the treatment of EMPD. Y Gao, X C Zhang, W S Wang, Y Yang, H L Wang, Y G Lu, Photodiagnosis Photodyn Ther. 12Gao Y, Zhang XC, Wang WS, Yang Y, Wang HL, Lu YG, et al. Efficacy and safety of topical ALA-PDT in the treatment of EMPD. Photodiagnosis Photodyn Ther. 2015; 12: 92-7.

Usefulness of Photodynamic Therapy as a Possible Therapeutic Alternative in the Treatment of Basal Cell Carcinoma. P Savoia, T Deboli, A Previgliano, P Broganelli, International journal of molecular sciences. 16Savoia P, Deboli T, Previgliano A, Broganelli P. Usefulness of Photodynamic Therapy as a Possible Therapeutic Alternative in the Treatment of Basal Cell Carcinoma. International journal of molecular sciences. 2015; 16: 23300-17.

Photodynamic therapy (PDT) of cancer: from local to systemic treatment. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the. J M Dabrowski, L G Arnaut, European Society for Photobiology. 14Dabrowski JM, Arnaut LG. Photodynamic therapy (PDT) of cancer: from local to systemic treatment. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology. 2015; 14: 1765-80.

Photodynamic therapy with violet light and topical 6-aminolaevulinic acid in the treatment of actinic keratosis, Bowen's disease and basal cell carcinoma. A T Dijkstra, I M Majoie, J W Van Dongen, H Van Weelden, W A Van Vloten, J Eur Acad Dermatol Venereol. 15Dijkstra AT, Majoie IM, van Dongen JW, van Weelden H, van Vloten WA. Photodynamic therapy with violet light and topical 6-aminolaevulinic acid in the treatment of actinic keratosis, Bowen's disease and basal cell carcinoma. J Eur Acad Dermatol Venereol. 2001; 15: 550-4.

Photodynamic therapy for nonmelanoma skin cancers. Current review and update. N C Zeitouni, A R Oseroff, S Shieh, Mol Immunol. 39Zeitouni NC, Oseroff AR, Shieh S. Photodynamic therapy for nonmelanoma skin cancers. Current review and update. Mol Immunol. 2003; 39: 1133-6.

Topical 5-ALA photodynamic therapy for the treatment of cutaneous T-cell lymphoma. J A Leman, D C Dick, C A Morton, Clin Exp Dermatol. 27Leman JA, Dick DC, Morton CA. Topical 5-ALA photodynamic therapy for the treatment of cutaneous T-cell lymphoma. Clin Exp Dermatol. 2002; 27: 516-8.

Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of delta-aminolaevulinic acid. C Fritsch, P Lehmann, W Stahl, K W Schulte, E Blohm, K Lang, Br J Cancer. 79Fritsch C, Lehmann P, Stahl W, Schulte KW, Blohm E, Lang K, et al. Optimum porphyrin accumulation in epithelial skin tumours and psoriatic lesions after topical application of delta-aminolaevulinic acid. Br J Cancer. 1999; 79: 1603-8.

Pain associated with photodynamic therapy using 5-aminolevulinic acid or 5-aminolevulinic acid methylester on tape-stripped normal skin. S R Wiegell, I M Stender, R Na, H C Wulf, Arch Dermatol. 139Wiegell SR, Stender IM, Na R, Wulf HC. Pain associated with photodynamic therapy using 5-aminolevulinic acid or 5-aminolevulinic acid methylester on tape-stripped normal skin. Arch Dermatol. 2003; 139: 1173-7.

Enhancement techniques for improving 5-aminolevulinic acid delivery through the skin. L W Zhang, Y P Fang, J Y Fang, Dermatologica Sinica. 29Zhang LW, Fang YP, Fang JY. Enhancement techniques for improving 5-aminolevulinic acid delivery through the skin. Dermatologica Sinica. 2011; 29: 1-7.

Liposome drugs' loading efficiency: a working model based on loading conditions and drug's physicochemical properties. D Zucker, D Marcus, Y Barenholz, A Goldblum, J Control Release. 139Zucker D, Marcus D, Barenholz Y, Goldblum A. Liposome drugs' loading efficiency: a working model based on loading conditions and drug's physicochemical properties. J Control Release. 2009; 139: 73-80.

Nano-or submicron-sized liposomes as carriers for drug delivery. J Y Fang, Chang Gung Med J. 29Fang JY. Nano-or submicron-sized liposomes as carriers for drug delivery. Chang Gung Med J. 2006; 29: 358-62.

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study. M B Pierre, A C Tedesco, J M Marchetti, M V Bentley, BMC Dermatol. 15Pierre MB, Tedesco AC, Marchetti JM, Bentley MV. Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study. BMC Dermatol. 2001; 1: 5.

Photodynamic treatment induces cell death by apoptosis or autophagy depending on the melanin content in two B16 melanoma cell lines. A Sparsa, S Bellaton, T Naves, M O Jauberteau, J M Bonnetblanc, V Sol, Oncol Rep. 29Sparsa A, Bellaton S, Naves T, Jauberteau MO, Bonnetblanc JM, Sol V, et al. Photodynamic treatment induces cell death by apoptosis or autophagy depending on the melanin content in two B16 melanoma cell lines. Oncol Rep. 2013; 29: 1196-200.

Influence of lipid composition on the phase transition temperature of liposomes composed of both DPPC and HSPC. Drug development and industrial pharmacy. J Chen, D Cheng, J Li, Y Wang, J X Guo, Z P Chen, 39Chen J, Cheng D, Li J, Wang Y, Guo JX, Chen ZP, et al. Influence of lipid composition on the phase transition temperature of liposomes composed of both DPPC and HSPC. Drug development and industrial pharmacy. 2013; 39: 197-204.

Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma. Ickowicz Schwartz, D Gozlan, Y Greenbaum, L Babushkina, T Katcoff, D J Malik, Z , Br J Cancer. 90Ickowicz Schwartz D, Gozlan Y, Greenbaum L, Babushkina T, Katcoff DJ, Malik Z. Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma. Br J Cancer. 2004; 90: 1833-41.

Cell death pathways in photodynamic therapy of cancer. P Mroz, A Yaroslavsky, G B Kharkwal, M R Hamblin, Cancers (Basel). 3Mroz P, Yaroslavsky A, Kharkwal GB, Hamblin MR. Cell death pathways in photodynamic therapy of cancer. Cancers (Basel). 2011; 3: 2516-39.

Enhancement in skin permeation of 5-aminolevulinic acid using l-menthol and its derivatives. Drug development and industrial pharmacy. Y Tokuoka, M Suzuki, Y Ohsawa, A Ochiai, M Ishizuka, N Kawashima, 34Tokuoka Y, Suzuki M, Ohsawa Y, Ochiai A, Ishizuka M, Kawashima N. Enhancement in skin permeation of 5-aminolevulinic acid using l-menthol and its derivatives. Drug development and industrial pharmacy. 2008; 34: 595-601.

Dosimetry model for photodynamic therapy with topically administered photosensitizers. L O Svaasand, P Wyss, M T Wyss, Y Tadir, B J Tromberg, M W Berns, Lasers Surg Med. 18Svaasand LO, Wyss P, Wyss MT, Tadir Y, Tromberg BJ, Berns MW. Dosimetry model for photodynamic therapy with topically administered photosensitizers. Lasers Surg Med. 1996; 18: 139-49.

Size and surface charge effect of 5-aminolevulinic acid-containing liposomes on photodynamic therapy for cultivated cancer cells. Drug development and industrial pharmacy. T Kosobe, E Moriyama, Y Tokuoka, N Kawashima, 31Kosobe T, Moriyama E, Tokuoka Y, Kawashima N. Size and surface charge effect of 5-aminolevulinic acid-containing liposomes on photodynamic therapy for cultivated cancer cells. Drug development and industrial pharmacy. 2005; 31: 623-9.