- Email: [email protected]
Clinical Porcine Islet Xenotransplantation Under Comprehensive Regulation
Clinical Porcine Islet Xenotransplantation Under Comprehensive Regulation S. Matsumotoa,*, P. Tanb, J. Bakerc, K. Durbinb, M. Tomiyaa, K. Azumaa, M. Doia, and R.B. Elliottb a Otsuka Pharmaceutical Factory, Naruto, Japan; bLiving Cell Technologies, Auckland, New Zealand; cCentre for Clinical Research and Effective Practice, Middlemore Hospital, Auckland, New Zealand
ABSTRACT Background. Xenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors. This is the first report of phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand. Methods. Newborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed. Encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs. The patients received encapsulated islets of 5,000 (n ¼ 4; group 1), 10,000 (n ¼ 4; group 2), 15,000 (n ¼ 4; group 3), or 20,000 (n ¼ 2; group 4) islet equivalents per kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events. To assess graft function, transplant estimated function (TEF) scores were calculated. Sufficient or marginal numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice as an in vivo functional assay. Results. There were 4 serious adverse events, of which 3 were considered to be possibly related to the procedure. Tests for porcine endogenous retrovirus DNA and RNA were all negative. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. The average TEF scores were 0.17, 0.02, 0.01, and 0.08 in groups 1, 2, 3, and 4 respectively. The in vivo study demonstrated that a sufficient number of the transplanted group reversed diabetes with positive porcine C-peptide. Conclusions. Transplantation of encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients. The mouse in vivo assessment data demonstrated certain graft function.
W
ITH the current improvement of clinical allogeneic islet transplantation, the shortage of human donors for allogeneic islet transplantation will be serious issue [1]. Xenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors [1]. According to the international xenotransplantation association consensus statement, clinical islet xenotransplantation needs to be conducted under an appropriate regulatory framework [2]. This is the first report of a phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand [3].
MATERIALS AND METHODS Clinical Trial This study was approved by the Minister of Health under the regulatory framework for xenotransplantation and by the Northern Regional Ethics Committee (NTX/07/06/064). The study was registered at ClinicalTrials.gov (NCT00940173). Porcine islets were obtained from newborn piglets (Auckland Islet Pigs; Diatranz Otsuka). The donor pig herd was maintained in *Address correspondence to Shinichi Matsumoto, 115 Kuguhara, Tateiwa, Muya-cho, Naruto, Tokushima 772-8601, Japan. E-mail: [email protected]
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2014.06.008
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1992
Transplantation Proceedings, 46, 1992e1995 (2014)
CLINICAL PORCINE ISLET XENOTRANSPLANTATION a designated pathogen-free facility and screened for an extensive panel of infectious agents, including porcine endogenous retrovirus (PERV) [4]. Newborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed under good manufacturing practice as previously described [5]. Neonatal porcine islets were counted for determining islet equivalents (IEQ) and encapsulated with the use of alginatepoly-L-ornithine-alginate (APA) as previously described [5]. APA-encapsulated neonatal porcine islets were cultured for an additional 3 weeks before transplantation. From October 2009 to March 2011, encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs at the Counties Manukau Surgical Centre (Auckland, New Zealand). All patients had intensive insulin therapy for at least 8 weeks before transplantation. The patients were assigned to 4 dose groups and received encapsulated islets of 5,000 (n ¼ 4; group 1), 10,000 (n ¼ 4; group 2), 15,000 (n ¼ 4; group 3), or 20,000 (n ¼ 2; group 4) IEQ/kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events assessed on a regular basis. To assess the function of transplanted encapsulated porcine islets, transplant estimated function (TEF) scores were also calculated [6].
In Vivo Function Test To assess the function of APA-encapsulated neonatal porcine islets, in vivo function tests were conducted in diabetic mice. Newborn piglets were provided from Kadoi Ltd (Ibaraki, Japan). Four piglets were shipped to TransGenic Inc (Kobe, Japan) for surgical procedures and then anesthetized and bled. Pancreata were procured with the use of sterile technique, and procured pancreata were preserved with cold ET-Kyoto solution and shipped to Otsuka Pharmaceutical Factory Inc (Naruto, Japan). Cold preservation time was w3 hours. Neonatal islet isolation, counting islets for IEQ, and encapsulation were conducted at Otsuka Pharmaceutical Factory with the use of the same protocol as the living cell technologies protocol [5]. APA-encapsulated neonatal porcine islets were cultured for an additional 3 weeks before transplantation. Sufficient (10,000 IEQ; n ¼ 5) or marginal (4,000 IEQ; n ¼ 5) numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice. Saline solution as the transplant vehicle was transplanted into diabetic B6 mice as a negative control (n ¼ 3). In addition, a no-treatment group was included as healthy control (n ¼ 3). Two hundred mg/kg body weight streptozotocin was injected into the abdominal cavities of B6 mice. Nonfasting blood glucose >300 mg/dL was considered to be diabetic. After transplantation, blood glucose levels <200 mg/dL on 2 consecutive occasions were considered to be a reversal of the diabetes. Oral glucose tolerance tests were also conducted on day 24 after transplantation with surviving mice. The blood was sampled from the tail vein before and 5, 15, 30, 60, 90, and 120 minutes after
1993 an oral injection of 1 g/kg body weight dextrose. Porcine C-peptides were measured from transplanted B6 mice at day 32 after transplantation as well as from normal healthy mice.
Statistic Analysis Differences among the 4 groups were assessed with the use of analysis of variance. Differences between baseline and 1 year were assessed with the use of Student t test. A P value of <.05 was considered to represent a significant difference.
RESULTS Clinical Outcomes
Patients’ characteristics are presented in Table 1. Each group consists of 4 patients except for group 4. Overall average recipients age was 52.7 2.3 years old (mean SE), duration of diabetes 21.2 2.1 years, body weight 74.2 3.4 kg, and body mass index 25.2 1.0 kg/m2. There were no significant difference among the groups, but the average body weight in group 3 was >75 kg and in group 4 was >8 5 kg. There were 4 serious adverse events, including hypersensitivity, post-procedural discomfort, anxiety, and depressed mood, of which 3 were considered to be possibly related to the procedure. However, all of these events resolved without residual effects, although treatment for the depression was continued. Tests for PERV DNA and RNA were negative in recipients’ blood samples taken before transplantation and at weeks 1, 4, 8, 12, 24, and 52 after transplantation in all patients. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups, especially in group 1 and 2, where the numbers of unaware hypoglycemia were less than one-half those of the other groups after 1 year (Table 2). Average HbA1c and daily total insulin dose at baseline and at 1 year after transplantation as well as TEF score are presented in Table 3. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. In group 4, 1 patient did not measure HbA1c at 1 year, so that datum was missing. In Vivo Study Outcome
All 3 diabetic mice without transplantation died within 2 weeks with high blood glucose (Fig 1). Four out of 5 mice reversed diabetes when transplanted with sufficient numbers but none when transplanted with marginal numbers of islets. Even sufficient transplantation could not reduce blood glucose levels to normal healthy levels.
Table 1. Patient Characteristics in Each Group (Mean ± SE) Group
1 2 3 4
Age (y)
51.0 50.8 53.3 59.0
6.0 4.1 3.7 4.1
Sex (F/M)
3/1 1/3 2/2 0/2
Body Weight (kg)
69.6 71.7 76.4 84.1
6.7 3.6 9.2 5.4
BMI (kg/m2)
24.4 23.2 26.2 28.7
2.7 1.4 1.3 1.0
Abbreviations: BMI, body mass index; T1DM, type 1 diabetes mellitus; IEQ, islet equivalents.
Duration of T1DM (y)
18.8 24.0 17.5 28.5
3.1 4.8 4.1 2.0
Transplanted Islet Yield (IEQ/kg)
5,057 10,416 14,456 19,822
84 613 334 716
1994
MATSUMOTO, TAN, BAKER ET AL
Table 2. Number With Unaware Hypoglycemia at Baseline (L4 to L1 wk) and at 1 Year (49e52 wk), Mean ± SE Baseline
1 2 3 4
11.5 13.0 9.8 24.5
1 y After Tx
4.2 4.4 7.5 8.5
4.8 2.8 9.0 14.0
1.5 2.1 4.0 8.0
Abbreviation: Tx, transplantation.
Blood glucose level (mg/dL）
Group
600 500
400 300
200 100
0
Oral glucose tolerance tests were conducted with surviving mice: all 5 sufficient-transplanted mice, 4 marginaltransplanted mice, and all 3 normal healthy control mice. Sufficient-transplanted mice showed excellent glucose metabolic profile (Fig 2), whereas marginal-transplanted mice showed a diabetic pattern (Fig 2). Three out of 5 in the sufficient-transplant group showed positive C-peptide, with a mean positive C-peptide of 0.15 0.02 ng/mL. None of marginal-transplant group or normal healthy mice had positive porcine C-peptide. DISCUSSION
This is the 1st report for clinical islet xenotransplantation under the comprehensive regulatory framework in New Zealand. There were limited serious adverse events without serious residual effects. Therefore, intraperitoneal transplantation of APA-encapsulated neonatal porcine islets seems to be a safe procedure. Importantly, PERV examination showed complete negativity for PERV infection in this study. A previous PERV survey study including >150 patients receiving porcine spleen perfusion, skin transplantation, islet transplantation, artificial liver perfusion, and renal perfusion also demonstrated complete negativity for PERV infection [7]. Thus the present study was congruent with earlier findings. Regarding efficacy, groups 1 and 2 showed the effect of reducing unaware hypoglycemia. The international xenotransplantation association consensus statement mentioned that hypoglycemia unawareness could be a justified target for islet xenotransplantation. This is because hypoglycemia unawareness is the major risk factor for severe hypoglycemia which is potential life threatening [8]. The statement also mentioned that it is necessary to balance the risks of immunosuppression and problems with life-long monitoring with the benefit of reduction of unaware hypoglycemia. In this study, we did not use any immunosuppressive drugs, which is an advantage of this study.
0
5
10
15 20 Days after transplantation
25
30
Fig 1. Nonfasting blood glucose of diabetic B6 mice with sufficient number (open squares; n ¼ 5) and marginal number (solid diamonds; n ¼ 4e5) of APA-encapsulated islet transplantation, without transplantation (solid circles; n ¼ 1e3), and healthy mice (open circles; n ¼ 3). In calculating the data, >600 mg/dL was recorded as 600 mg/dL. Results are shown as mean SD.
On the other hand, the effect of reducing HbA1c or total insulin dose was not apparent. Previously it was demonstrated that secretory unit of islet transplant objects (SUITO) index was an excellent assessment tool for clinical allogeneic islet transplantation [9,10]. The SUITO index is calculated with C-peptide and glucose levels. After allogeneic islet transplantation, a low SUITO index group effectively prevents severe hypoglycemia, even when other clear clinical benefit was not clear [10]. In the present study, we had not established a reliable method to measure porcine C-peptide in human serum; therefore, we used the TEF score, which can be calculated with the use of HbA1c and daily insulin dose [6]. A TEF score of 0.3 is equivalent to SUITO index 15 and TEF score 0.5 is equivalent to SUITO index 30 [6]. Overall, the average TEF scores in this study were low, which indicated that the majority of the patients were in a low SUITO index group. The clinical features in this study were congruent with the clinical features after allogeneic islet transplantation with a low SUITO index. However, the highest TEF score of 1 patient in group 1 was 0.58, which was equivalent to SUITO index 40 [6]. A SUITO index of >26 was previously associated with freedom from insulin therapy after allogeneic islet transplantation [9,10]. This patient’s insulin dose was less than one-half the basal dose at 1 year after transplantation, with HbA1c <7%. To assess the potency of APA-encapsulated neonatal porcine islets, we transplanted them into diabetic mice. The sufficient-number (10,000 IEQ) transplant group was able to reverse diabetes, but not the marginal-number (4,000 IEQ) transplant group. Transplantation of 2,500 IEQ
Table 3. Average HbA1c and Daily Total Insulin Dose at Baseline and at 1 Year After Transplantation (Mean ± SE) HbA1c (%) Group
1 2 3 4
Daily Insulin Dose (Units)
Baseline
1 y After Tx
7.35 0.51 7.63 0.22 7.80 0.37 6.6
7.38 7.65 7.53 7.30
0.43 0.21 0.28 0.16
Abbreviation: TEF, transplant estimated function.
Base
49.5 31.1 41.8 56.6
37.4 5.2 7.0 1.92
1 y After Tx
TEF Score (mean SE)
0.17 0.15 0.02 0.03 0.01 0.05 0.08
37.4 29.5 37.9 59.9
6.2 3.4 4.1 4.47
CLINICAL PORCINE ISLET XENOTRANSPLANTATION
1995
of islet isolation and encapsulation process might improve the clinical outcomes.
Blood glucose level (mg/dL)
450 400 350 300
REFERENCES
250 200 150 100 50 0
0
20
40
60
80
100
120
Time (minute)
Fig 2. Blood glucose profile of 1-g oral glucose tolerance test in normal healthy mice (open circles; n ¼ 3) and diabetic B6 mice with sufficient number (open squares; n ¼ 5) and marginal number (solid diamonds; n ¼ 4) of APA-encapsulated islet transplantation. Results are shown as mean SD.
human islets was sufficient to reverse diabetes in diabetic nude mice [11], and transplantation of 10,000 IEQ/kg human islets was sufficient to achieve insulin independence in a clinical setting [12]. When we extrapolate these findings to APA-encapsulated neonatal islets, 40,000 IEQ/kg is necessary to achieve insulin independence. Therefore, relatively low efficacy in this study might be due simply to insufficient beta-cell mass. On the other hand, we did not find apparent dose effects in this clinical trial. Further optimization of the islet isolation process and/or encapsulation process are necessary to improve efficiency. In a preliminary study, optimization of islet isolation and encapsulation improved in the in vivo assay. We plan to implement these optimizations into a clinical trial. CONCLUSION
Transplantation of APA-encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients under intensive insulin therapy. In vivo assessment data demonstrated certain graft function. Further optimization
[1] Matsumoto S. Islet cell transplantation for type 1 diabetes. J Diabetes 2010;2:16e22. [2] Cozzi E, Tallacchini M, Flanagan EB, et al. The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetesdchapter 1: key ethical requirements and progress toward the definition of an international regulatory framework. Xenotransplantation 2009;16:203e14. [3] World Health Organization. Second WHO global consultation on regulatory requirements for xenotransplantation clinical trials. Geneva: Switzerland; October 17-19, 2011. [4] Garkavenko O, Croxosn MC, Irgang M, et al. Monitoring for presence of potentially xenotic viruses in recipients of pig islet xenotransplantation. J Clin Microbiol 2004;42:5353e6. [5] Hillberg AL, Kathirgamanathan K, Lam JBB, et al. Improving alginate-poly-L-ornithine-alginate capsule biocompatibility through genipin crosslinking. J Biomed Mater Res Part B 2013;101B:258e68. [6] Caumo A, Maffi P, Nano R, et al. Comparative evaluation of simple indices of graft function after islet transplantation. Transplantation 2011;92:815e21. [7] Paradis K, Langford G, Long Z, et al., XEN 111 Study Group. Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 1999;285:1236e41. [8] O’Connell PJ. The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetesdchapter 6: patient selection for pilot clinical trials of islet xenotransplantation. Xenotransplantation 2009;16:249e54. [9] Matsumoto S, Yamada Y, Okitsu T, et al. Simple evaluation of engraftment by secretory unit of islet transplant objects (SUITO) for living donor and cadaveric donor fresh or cultured islet transplantation. Transplant Proc 2005;37:3435e7. [10] Takita M, Matsumoto S. SUITO index for evaluation of clinical islet transplantation. Cell Transplant 2012;21:1341e7. [11] Shimoda M, Itoh T, Iwahashi S, et al. An effective purification method using large bottles for human pancreatic islet isolation. Islets 2012;4:398e404. [12] Matsumoto S, Takita M, Chaussabel D, et al. Improving efficacy of clinical islet transplantation with iodixanol-based islet purification, thymoglobulin induction, and blockage of IL1-beta and TNF-alpha. Cell Transplant 2011;20:1641e7.
ABSTRACT Background. Xenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors. This is the first report of phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand. Methods. Newborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed. Encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs. The patients received encapsulated islets of 5,000 (n ¼ 4; group 1), 10,000 (n ¼ 4; group 2), 15,000 (n ¼ 4; group 3), or 20,000 (n ¼ 2; group 4) islet equivalents per kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events. To assess graft function, transplant estimated function (TEF) scores were calculated. Sufficient or marginal numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice as an in vivo functional assay. Results. There were 4 serious adverse events, of which 3 were considered to be possibly related to the procedure. Tests for porcine endogenous retrovirus DNA and RNA were all negative. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. The average TEF scores were 0.17, 0.02, 0.01, and 0.08 in groups 1, 2, 3, and 4 respectively. The in vivo study demonstrated that a sufficient number of the transplanted group reversed diabetes with positive porcine C-peptide. Conclusions. Transplantation of encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients. The mouse in vivo assessment data demonstrated certain graft function.
W
ITH the current improvement of clinical allogeneic islet transplantation, the shortage of human donors for allogeneic islet transplantation will be serious issue [1]. Xenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors [1]. According to the international xenotransplantation association consensus statement, clinical islet xenotransplantation needs to be conducted under an appropriate regulatory framework [2]. This is the first report of a phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand [3].
MATERIALS AND METHODS Clinical Trial This study was approved by the Minister of Health under the regulatory framework for xenotransplantation and by the Northern Regional Ethics Committee (NTX/07/06/064). The study was registered at ClinicalTrials.gov (NCT00940173). Porcine islets were obtained from newborn piglets (Auckland Islet Pigs; Diatranz Otsuka). The donor pig herd was maintained in *Address correspondence to Shinichi Matsumoto, 115 Kuguhara, Tateiwa, Muya-cho, Naruto, Tokushima 772-8601, Japan. E-mail: [email protected]
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2014.06.008
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1992
Transplantation Proceedings, 46, 1992e1995 (2014)
CLINICAL PORCINE ISLET XENOTRANSPLANTATION a designated pathogen-free facility and screened for an extensive panel of infectious agents, including porcine endogenous retrovirus (PERV) [4]. Newborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed under good manufacturing practice as previously described [5]. Neonatal porcine islets were counted for determining islet equivalents (IEQ) and encapsulated with the use of alginatepoly-L-ornithine-alginate (APA) as previously described [5]. APA-encapsulated neonatal porcine islets were cultured for an additional 3 weeks before transplantation. From October 2009 to March 2011, encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs at the Counties Manukau Surgical Centre (Auckland, New Zealand). All patients had intensive insulin therapy for at least 8 weeks before transplantation. The patients were assigned to 4 dose groups and received encapsulated islets of 5,000 (n ¼ 4; group 1), 10,000 (n ¼ 4; group 2), 15,000 (n ¼ 4; group 3), or 20,000 (n ¼ 2; group 4) IEQ/kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events assessed on a regular basis. To assess the function of transplanted encapsulated porcine islets, transplant estimated function (TEF) scores were also calculated [6].
In Vivo Function Test To assess the function of APA-encapsulated neonatal porcine islets, in vivo function tests were conducted in diabetic mice. Newborn piglets were provided from Kadoi Ltd (Ibaraki, Japan). Four piglets were shipped to TransGenic Inc (Kobe, Japan) for surgical procedures and then anesthetized and bled. Pancreata were procured with the use of sterile technique, and procured pancreata were preserved with cold ET-Kyoto solution and shipped to Otsuka Pharmaceutical Factory Inc (Naruto, Japan). Cold preservation time was w3 hours. Neonatal islet isolation, counting islets for IEQ, and encapsulation were conducted at Otsuka Pharmaceutical Factory with the use of the same protocol as the living cell technologies protocol [5]. APA-encapsulated neonatal porcine islets were cultured for an additional 3 weeks before transplantation. Sufficient (10,000 IEQ; n ¼ 5) or marginal (4,000 IEQ; n ¼ 5) numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice. Saline solution as the transplant vehicle was transplanted into diabetic B6 mice as a negative control (n ¼ 3). In addition, a no-treatment group was included as healthy control (n ¼ 3). Two hundred mg/kg body weight streptozotocin was injected into the abdominal cavities of B6 mice. Nonfasting blood glucose >300 mg/dL was considered to be diabetic. After transplantation, blood glucose levels <200 mg/dL on 2 consecutive occasions were considered to be a reversal of the diabetes. Oral glucose tolerance tests were also conducted on day 24 after transplantation with surviving mice. The blood was sampled from the tail vein before and 5, 15, 30, 60, 90, and 120 minutes after
1993 an oral injection of 1 g/kg body weight dextrose. Porcine C-peptides were measured from transplanted B6 mice at day 32 after transplantation as well as from normal healthy mice.
Statistic Analysis Differences among the 4 groups were assessed with the use of analysis of variance. Differences between baseline and 1 year were assessed with the use of Student t test. A P value of <.05 was considered to represent a significant difference.
RESULTS Clinical Outcomes
Patients’ characteristics are presented in Table 1. Each group consists of 4 patients except for group 4. Overall average recipients age was 52.7 2.3 years old (mean SE), duration of diabetes 21.2 2.1 years, body weight 74.2 3.4 kg, and body mass index 25.2 1.0 kg/m2. There were no significant difference among the groups, but the average body weight in group 3 was >75 kg and in group 4 was >8 5 kg. There were 4 serious adverse events, including hypersensitivity, post-procedural discomfort, anxiety, and depressed mood, of which 3 were considered to be possibly related to the procedure. However, all of these events resolved without residual effects, although treatment for the depression was continued. Tests for PERV DNA and RNA were negative in recipients’ blood samples taken before transplantation and at weeks 1, 4, 8, 12, 24, and 52 after transplantation in all patients. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups, especially in group 1 and 2, where the numbers of unaware hypoglycemia were less than one-half those of the other groups after 1 year (Table 2). Average HbA1c and daily total insulin dose at baseline and at 1 year after transplantation as well as TEF score are presented in Table 3. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. In group 4, 1 patient did not measure HbA1c at 1 year, so that datum was missing. In Vivo Study Outcome
All 3 diabetic mice without transplantation died within 2 weeks with high blood glucose (Fig 1). Four out of 5 mice reversed diabetes when transplanted with sufficient numbers but none when transplanted with marginal numbers of islets. Even sufficient transplantation could not reduce blood glucose levels to normal healthy levels.
Table 1. Patient Characteristics in Each Group (Mean ± SE) Group
1 2 3 4
Age (y)
51.0 50.8 53.3 59.0
6.0 4.1 3.7 4.1
Sex (F/M)
3/1 1/3 2/2 0/2
Body Weight (kg)
69.6 71.7 76.4 84.1
6.7 3.6 9.2 5.4
BMI (kg/m2)
24.4 23.2 26.2 28.7
2.7 1.4 1.3 1.0
Abbreviations: BMI, body mass index; T1DM, type 1 diabetes mellitus; IEQ, islet equivalents.
Duration of T1DM (y)
18.8 24.0 17.5 28.5
3.1 4.8 4.1 2.0
Transplanted Islet Yield (IEQ/kg)
5,057 10,416 14,456 19,822
84 613 334 716
1994
MATSUMOTO, TAN, BAKER ET AL
Table 2. Number With Unaware Hypoglycemia at Baseline (L4 to L1 wk) and at 1 Year (49e52 wk), Mean ± SE Baseline
1 2 3 4
11.5 13.0 9.8 24.5
1 y After Tx
4.2 4.4 7.5 8.5
4.8 2.8 9.0 14.0
1.5 2.1 4.0 8.0
Abbreviation: Tx, transplantation.
Blood glucose level (mg/dL）
Group
600 500
400 300
200 100
0
Oral glucose tolerance tests were conducted with surviving mice: all 5 sufficient-transplanted mice, 4 marginaltransplanted mice, and all 3 normal healthy control mice. Sufficient-transplanted mice showed excellent glucose metabolic profile (Fig 2), whereas marginal-transplanted mice showed a diabetic pattern (Fig 2). Three out of 5 in the sufficient-transplant group showed positive C-peptide, with a mean positive C-peptide of 0.15 0.02 ng/mL. None of marginal-transplant group or normal healthy mice had positive porcine C-peptide. DISCUSSION
This is the 1st report for clinical islet xenotransplantation under the comprehensive regulatory framework in New Zealand. There were limited serious adverse events without serious residual effects. Therefore, intraperitoneal transplantation of APA-encapsulated neonatal porcine islets seems to be a safe procedure. Importantly, PERV examination showed complete negativity for PERV infection in this study. A previous PERV survey study including >150 patients receiving porcine spleen perfusion, skin transplantation, islet transplantation, artificial liver perfusion, and renal perfusion also demonstrated complete negativity for PERV infection [7]. Thus the present study was congruent with earlier findings. Regarding efficacy, groups 1 and 2 showed the effect of reducing unaware hypoglycemia. The international xenotransplantation association consensus statement mentioned that hypoglycemia unawareness could be a justified target for islet xenotransplantation. This is because hypoglycemia unawareness is the major risk factor for severe hypoglycemia which is potential life threatening [8]. The statement also mentioned that it is necessary to balance the risks of immunosuppression and problems with life-long monitoring with the benefit of reduction of unaware hypoglycemia. In this study, we did not use any immunosuppressive drugs, which is an advantage of this study.
0
5
10
15 20 Days after transplantation
25
30
Fig 1. Nonfasting blood glucose of diabetic B6 mice with sufficient number (open squares; n ¼ 5) and marginal number (solid diamonds; n ¼ 4e5) of APA-encapsulated islet transplantation, without transplantation (solid circles; n ¼ 1e3), and healthy mice (open circles; n ¼ 3). In calculating the data, >600 mg/dL was recorded as 600 mg/dL. Results are shown as mean SD.
On the other hand, the effect of reducing HbA1c or total insulin dose was not apparent. Previously it was demonstrated that secretory unit of islet transplant objects (SUITO) index was an excellent assessment tool for clinical allogeneic islet transplantation [9,10]. The SUITO index is calculated with C-peptide and glucose levels. After allogeneic islet transplantation, a low SUITO index group effectively prevents severe hypoglycemia, even when other clear clinical benefit was not clear [10]. In the present study, we had not established a reliable method to measure porcine C-peptide in human serum; therefore, we used the TEF score, which can be calculated with the use of HbA1c and daily insulin dose [6]. A TEF score of 0.3 is equivalent to SUITO index 15 and TEF score 0.5 is equivalent to SUITO index 30 [6]. Overall, the average TEF scores in this study were low, which indicated that the majority of the patients were in a low SUITO index group. The clinical features in this study were congruent with the clinical features after allogeneic islet transplantation with a low SUITO index. However, the highest TEF score of 1 patient in group 1 was 0.58, which was equivalent to SUITO index 40 [6]. A SUITO index of >26 was previously associated with freedom from insulin therapy after allogeneic islet transplantation [9,10]. This patient’s insulin dose was less than one-half the basal dose at 1 year after transplantation, with HbA1c <7%. To assess the potency of APA-encapsulated neonatal porcine islets, we transplanted them into diabetic mice. The sufficient-number (10,000 IEQ) transplant group was able to reverse diabetes, but not the marginal-number (4,000 IEQ) transplant group. Transplantation of 2,500 IEQ
Table 3. Average HbA1c and Daily Total Insulin Dose at Baseline and at 1 Year After Transplantation (Mean ± SE) HbA1c (%) Group
1 2 3 4
Daily Insulin Dose (Units)
Baseline
1 y After Tx
7.35 0.51 7.63 0.22 7.80 0.37 6.6
7.38 7.65 7.53 7.30
0.43 0.21 0.28 0.16
Abbreviation: TEF, transplant estimated function.
Base
49.5 31.1 41.8 56.6
37.4 5.2 7.0 1.92
1 y After Tx
TEF Score (mean SE)
0.17 0.15 0.02 0.03 0.01 0.05 0.08
37.4 29.5 37.9 59.9
6.2 3.4 4.1 4.47
CLINICAL PORCINE ISLET XENOTRANSPLANTATION
1995
of islet isolation and encapsulation process might improve the clinical outcomes.
Blood glucose level (mg/dL)
450 400 350 300
REFERENCES
250 200 150 100 50 0
0
20
40
60
80
100
120
Time (minute)
Fig 2. Blood glucose profile of 1-g oral glucose tolerance test in normal healthy mice (open circles; n ¼ 3) and diabetic B6 mice with sufficient number (open squares; n ¼ 5) and marginal number (solid diamonds; n ¼ 4) of APA-encapsulated islet transplantation. Results are shown as mean SD.
human islets was sufficient to reverse diabetes in diabetic nude mice [11], and transplantation of 10,000 IEQ/kg human islets was sufficient to achieve insulin independence in a clinical setting [12]. When we extrapolate these findings to APA-encapsulated neonatal islets, 40,000 IEQ/kg is necessary to achieve insulin independence. Therefore, relatively low efficacy in this study might be due simply to insufficient beta-cell mass. On the other hand, we did not find apparent dose effects in this clinical trial. Further optimization of the islet isolation process and/or encapsulation process are necessary to improve efficiency. In a preliminary study, optimization of islet isolation and encapsulation improved in the in vivo assay. We plan to implement these optimizations into a clinical trial. CONCLUSION
Transplantation of APA-encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients under intensive insulin therapy. In vivo assessment data demonstrated certain graft function. Further optimization
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