Time-related decline of human islet allograft (TX) function in generally immunosuppressed type 1 diabetic patients (1–5) has led us, after years of preclinical study (6), to initiate a phase 1 pilot clinical trial of microencapsulated TX into 10 nonimmunosuppressed patients with type 1 diabetes, under permission and surveillance by the Italian Ministry of Health (file no. 19382, PRE 805, 5 September 2003).

Human islet procurement

Human islets were isolated from single-donor pancreases according to the Edmonton protocol (1). Only preparations complying with standard quality control criteria (1) were considered for TX. The islets were cultured for 24 h in HAM F12 (Celbio, Milano, Italy), supplemented with antibiotics and 1.25% human albumin (Kedrion Spa, Milano, Italy) at 37°C in 95% air/CO2.

Microencapsulation

The islets were washed and thoroughly mixed with 1.6% endotoxin- and pyrogen-free sodium alginate (Stern Italia, Milan, Italy) that had been highly purified according to U.S. Pharmacopeia. Upon extrusion through a microdroplet generator (droplets generated by combination of air shears and mechanical pressure by a peristaltic pump), microdroplets containing the islets, upon collection in a CaCl2 bath, turned into gel microbeads. The beads, containing 1–2 islets and measuring an average 500 μm in diameter, were sequentially double-coated with 0.12% and 0.06% poly-l-ornithine (Sigma) and finally with 0.04% sodium alginate (Stern) (7).

Patient recruitment

Ten patients with long-standing type 1 diabetes on intensive insulin therapy regimens (lispro prebreakfast, lunch, and supper and glargine presupper) and undetectable serum C-peptide responses (sCPRs) were tested for complete blood chemistry, parameters of diabetes control (GHb, daily blood glucose profiles, and insulin requirement), anti–GAD 65 antibodies, and islet cell antibodies. Chest X-ray and abdominal echography were also performed.

Pre- and post-TX patient assessment

The patients were maintained on euglycemia overnight before TX by supplementing exogenous insulin as required. After TX, the patients were monitored hourly for blood glucose (range: 80–150 mg/dl) and insulin requirement. sCPR samples, assayed by radioimmunoassay (sCPR sensitivity: 0.06 ng/ml [intra-assay CV 3.7–4.5%, interassay CV 4.4–5%]) (TechnoGenetics, Milan, Italy) in our laboratory, were drawn twice daily for the first 7 days and weekly thereafter, both in basal and 90 min after meals. A 75-g oral glucose tolerance test (OGTT) was scheduled for patient 1 at 60 days post-TX. At 7 days post-TX, the patients were discharged and instructed to continue blood glucose home self-monitoring, with adjustments of insulin requirement. At 30 days post-TX, control abdominal echography was scheduled.

Encapsulated human islets for TX

A total of 400,000 and 600,000 microencapsulated islets (islet equivalents normalized to 150 μ) were grafted in patient 1 and patient 2, respectively. The final TX volume included 50 ml of microcapsules diluted in 100 ml of saline. Empty microcapsules did not exceed 5% of total.

Site of TX and delivery procedure.

At the Department of Radiology and Imaging, with the patients under local anesthesia (2% xylocaine) but no systemic sedation, the central abdominal region, under echography guidance, was punctured by a 14-gauge needle and therein injected with a few milliliters of saline to create a virtual space within the peritoneal leaflets for microcapsule delivery by a 60- ml plastic syringe over 15 min.

Both patients showed a rise in sCPR levels several weeks post-TX, amelioration of their mean daily blood glucose levels, and a progressive decline in exogenous insulin consumption. GHb also decreased throughout months of post-TX follow-up. At 60 days post-TX, an OGTT in patient 1 showed a biphasic C-peptide response, compatible with the presence of differentiated islet β-cells. At 1 year (patient 1) and 6 months (patient 2) of post-TX follow-up, which is in progress, sCPR was still detected in these recipients (Table 1).

Our long-standing experimental background in the field has enabled us to initiate a pilot clinical trial of microencapsulated human islet TX into nonimmunosuppressed patients with type 1 diabetes. Current data seems to show that the TX procedure is simple, noninvasive, painless, and devoid of side effects. Although the patients were unable, thus far, to withdraw exogenous insulin, we believe that decline in GHb, disappearance (patient 1) of frequent weekly hypoglycemic episodes, and responsiveness to OGTT (patient 1) overall indicated TX metabolic function. Adjustments in grafted viable islet cell mass dosing might further improve clinical results in the remaining eight case subjects.

Table 1—

Patients 1 and 2 pre- and post-TX

Patient 1Patient 2
Pre-TX severe hypoglycemia (events/week) 4 1 
Post-TX severe hypoglycemia (events/week) 0 0 
Duration of type 1 diabetes (years) 25 20 
Pre-TX mean blood glucose (mg/dl)* 275 ± 98 235 ± 78 
Post-TX mean blood glucose (mg/dl)   
    6 months 115 ± 56 155 ± 44 
    1 year 167 — 
Pre-TX sCPR (ng/ml) Undetectable Undetectable 
Post-TX sCPR (ng/ml) (4–12 weeks)   
    4 weeks Pre-meal: 0.1; Post-meal: 0.8 Pre-meal: 0.2; Post-meal: 0.8 
    8 weeks Pre-meal: 0.2; Post-meal: 0.9 Pre-meal: 0.25 
    12 weeks — Post-meal: 1.1 
Post-TX serum C-peptide (ng/ml)   
    6 months Pre-meal: 0.1; Post-meal: 0.4 Pre-meal: 0.35; Post-meal: 0.8 
    1 year Post-meal: 0.25 — 
Daily exogenous insulin (IU) Pre-TX 32 37 
Daily exogenous insulin (IU) Post-TX (4–48 weeks)   
    4 weeks 13 17 
    24 weeks — 27 
    48 weeks 20  
Pre-TX GHb (%) 10 9.5 
Post-TX GHb (%) (4–48 weeks)   
    16 weeks 6.2 7.2 
    24 weeks — 7.3 
    48 weeks 7.7 — 
Anti-GAD 65 antibodies Negative Negative 
Islet cell antibodies Negative Negative 
sCPR (ng/ml)†   
    0 min 0.075 — 
    30 min 0.1 — 
    60 min 0.2 — 
    90 min 0.12 — 
    120 min 0.23 — 
    180 min 0.18 — 
    240 min 0.07 — 
Patient 1Patient 2
Pre-TX severe hypoglycemia (events/week) 4 1 
Post-TX severe hypoglycemia (events/week) 0 0 
Duration of type 1 diabetes (years) 25 20 
Pre-TX mean blood glucose (mg/dl)* 275 ± 98 235 ± 78 
Post-TX mean blood glucose (mg/dl)   
    6 months 115 ± 56 155 ± 44 
    1 year 167 — 
Pre-TX sCPR (ng/ml) Undetectable Undetectable 
Post-TX sCPR (ng/ml) (4–12 weeks)   
    4 weeks Pre-meal: 0.1; Post-meal: 0.8 Pre-meal: 0.2; Post-meal: 0.8 
    8 weeks Pre-meal: 0.2; Post-meal: 0.9 Pre-meal: 0.25 
    12 weeks — Post-meal: 1.1 
Post-TX serum C-peptide (ng/ml)   
    6 months Pre-meal: 0.1; Post-meal: 0.4 Pre-meal: 0.35; Post-meal: 0.8 
    1 year Post-meal: 0.25 — 
Daily exogenous insulin (IU) Pre-TX 32 37 
Daily exogenous insulin (IU) Post-TX (4–48 weeks)   
    4 weeks 13 17 
    24 weeks — 27 
    48 weeks 20  
Pre-TX GHb (%) 10 9.5 
Post-TX GHb (%) (4–48 weeks)   
    16 weeks 6.2 7.2 
    24 weeks — 7.3 
    48 weeks 7.7 — 
Anti-GAD 65 antibodies Negative Negative 
Islet cell antibodies Negative Negative 
sCPR (ng/ml)†   
    0 min 0.075 — 
    30 min 0.1 — 
    60 min 0.2 — 
    90 min 0.12 — 
    120 min 0.23 — 
    180 min 0.18 — 
    240 min 0.07 — 

Data are means ± SD unless otherwise indicated.

*

Mean blood glucose: mean of the blood glucose levels recorded over 24 h ± SE.

†

By 75-g OGTT.

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A table elsewhere in this issue shows conventional and Système International (SI) units and conversion factors for many substances.

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DIABETES CARE
2006