C-Peptide Levels and Insulin Independence Following Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus FREE

Accumulated clinical experience indicates that there is an inverse association between beta-cell function and chronic complications of type 1 diabetes mellitus (DM)—the higher the C-peptide levels (an indirect measure of viable beta-cell function), the lower the incidence of microvascular complications of type 1 DM.¹ Since the establishment of the autoimmune etiology of type 1 DM in the late 1970s, many clinical trials analyzing the effects of different types of immune interventions demonstrated that beta-cell preservation is an achievable target in different degrees.^2,3

Based on the theory of possible reconstitution of immune tolerance after “immunologic reset” with autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT),⁴ in 2007, we reported a phase 1/2 trial evaluating the safety and metabolic effects of autologous nonmyeloablative HSCT in 15 patients with newly diagnosed type 1 DM.⁵ The majority of patients became insulin free with no further immune suppressive medications. However, it was suggested that subsequent insulin independence was a prolonged honeymoon period due to dietary and exercise changes associated with close posttransplant medical observation.^6,7

Herein, we report updated results of the extended group of 23 patients transplanted from January 2004 through April 2008, followed up from 7 to 58 months (mean, 29.8 months and median, 30 months) after treatment, and monitored for C-peptide levels to determine if posttransplant insulin independence was due to improved endogenous beta-cell function independent of dietary or lifestyle alterations.

Between November 2003 and April 2008, 160 patients were screened for enrollment. Of those patients, 71 fulfilled the inclusion criteria and were personally interviewed, 23 patients opted to participate, and all 23 were subsequently enrolled and observed until December 2008 at the Bone Marrow Transplantation Unit of the School of Medicine of Ribeirão Preto, Ribeirão Preto, Brazil. The majority of screened patients were male and most patients interested in the study were excluded for not fulfilling protocol criteria or for not accepting the potential adverse effects of the study protocol. Inclusion criteria were patients of both sexes, aged 12 to 35 years, with a clinical and laboratory diagnosis of type 1 DM during the previous 6 weeks confirmed by measurement of serum levels of anti–glutamic acid decarboxylase antibodies.

Exclusion criteria included positive serology for human immunodeficiency virus; hepatitis B or C; underlying hematologic, nephrologic, cardiac, psychiatric, or hepatic disease; or pregnancy. After the first patient was enrolled, selection criteria were adjusted to transiently exclude diabetic ketoacidosis onset and to avoid glucocorticoid use in the immunosuppression regimen.

Race/ethnicity was self-reported and was assessed because of the diversity of the Brazilian population along with the prevalence of black and white biracialism. The study protocol was approved by both Committees of Ethics in Research of the University Hospital of the School of Medicine of Ribeirão Preto and of the Brazilian Ministry of Health. An informed consent according to the Declaration of Helsinki was signed by all patients and by their parents when the patients were younger than 18 years.

The main outcome measure was to determine C-peptide levels during the mixed-meal tolerance test, before, and at different times following transplantation. Other end points included morbidity and mortality from transplantation, temporal changes in exogenous insulin requirements (daily dose and duration of usage), and serum levels of glycated hemoglobin A_1c (HbA_1c).

Blood samples for C-peptide were collected in the fasting state and every 30 minutes during a 2-hour mixed-meal tolerance test. The morning and evening doses of insulin were withheld the day before the test, which was repeated at pretreatment, 6 months, 12 months, and then yearly following autologous nonmyeloablative HSCT. Serum C-peptide levels were measured by radioimmunoassay using commercial kits (Diagnostic Systems Laboratories Inc, Webster, Texas). Blood samples for HbA_1c were collected after 8-hour fasting at pretreatment and every 3 months, and measured by low-pressure liquid chromatography.

All patients were encouraged to self-monitor blood glucose at least twice daily (before and 2 hours after different meals and/or at 3 AM) indefinitely after discharge from the hospital. Insulin titration was based on fasting glucose levels before meals and 2 hours after meals with target blood glucose levels of less than 120 mg/dL and less than 140 mg/dL, respectively (to convert to millimoles per liter, multiply by 0.0555). The dose of insulin was reduced by 1 to 2 IU/mL (to convert to picomole per liter, multiply by 6.945) if patients presented clinical findings of hypoglycemia and/or blood glucose levels of less than 90 mg/dL.

Standard recommendations for lifestyle modification (performing physical activities and a low-sugar diet) after autologous nonmyeloablative HSCT were made to all patients irrespective of exogenous insulin use. Intensive insulin therapy was the treatment of choice for all patients who needed exogenous insulin. All changes in insulin doses were ordered by one of the endocrinologists of the team (C.E.B.C.).

Peripheral hematopoietic stem cells were mobilized with cyclophosphamide and granulocyte colony-stimulating factor (Leucin, Laboratory Bergamo, São Paulo, Brazil). Cyclophosphamide (2 g/m²) was infused in 2 doses 12 hours apart in 250 mL of saline solution over 1 hour. Uroprotection was achieved with intravenous saline infusion at 250 mL/h, initiated 4 hours before cyclophosphamide infusion and continued for 16 hours. Mesna (sodium 2-mercaptoethanesulfonate, 4 g/m²) was infused over 24 hours to bind toxic cyclophosphamide metabolites in the bladder. Granulocyte colony-stimulating factor (10 ug/kg/d) was injected subcutaneously starting 1 day after cyclophosphamide infusion and continuing until leukapheresis was completed.

Leukapheresis using a continuous-flow blood cell separator was initiated when the rebounding CD34 cells reached 10 cells/μL of whole blood. Apheresis was continued daily until the number of harvested progenitor cells reached a minimum of 3.0 × 10⁶ CD34 cells per kilogram body weight. Unmanipulated peripheral blood stem cells were frozen in 10% dimethyl sulfoxide in a rate-controlled freezer and stored in the vapor phase of liquid nitrogen.

Conditioning was achieved with cyclophosphamide and antithymocyte globulin. Cyclophosphamide was administered intravenously in divided doses of 50 mg/kg/d over 1 hour on days 5, 4, 3, and 2 before stem cell infusion. Rabbit antithymocyte globulin (thymoglobulin, Genzyme Polyclonals SAF, Marcyl’Etoile, France) was administered at a dose of 0.5 mg/kg/d on day 5 before, and at a dose of 1 mg/kg/d on days 4, 3, 2, and 1 before stem cell infusion. Except for the first patient, prophylaxis of antithymocyte globulin reactions was performed with dexchlorpheniramine (6 mg orally) avoiding the use of glucocorticoids. Stem cell infusion was performed on day 0 and granulocyte colony-stimulating factor (5 μg/kg/d) was administered subcutaneously from day 5 after stem cell infusion until neutrophil count was more than 1000/μL.

Multiple comparisons of total area under the curve (AUC) of serum C-peptide levels measured during the mixed-meal tolerance test (during fasting and at 30, 60, 90, and 120 minutes) were made by using a model of multiple regression of mixed effects for periods 0, 6, 12, and 24 months posttransplantation. To present the mean variation of HbA_1c levels with time, a model of linear regression of random effects was constructed by using the following equation: y = β₀ + β₁ × log (time) + β₂ × [log (time)]², in which each parameter represents a random effect in each patient. These models are characterized to present residuals that are normally distributed. Data analysis was completed by using PROC MIXED, SAS statistical software, version 8 (SAS Institute Inc, Cary, North Carolina). Statistical significance was set a priori at P < .05.

Twenty-three patients aged 13 to 31 years (mean, 18.4 years) were enrolled in the study between November 2003 and April 2008. Individual demographic characteristics and follow-up variables are shown in Table 1. This is a very selected group of patients, predominantly male, with a short duration of disease and mostly without previous diabetic ketoacidosis. All patients presented clinical findings of hyperglycemia associated with a mean blood glucose level of 398.6 mg/dL (range, 130-793 mg/dL) at diagnosis and mean anti–glutamic acid decarboxylase antibody level of 24.9 U/mL (range, 1.1-102.0 U/mL). Mean body mass index (calculated as weight in kilograms divided by height in meters squared) at diagnosis was 19.7 (range, 16.6-23.4). Nine patients presented both HLA haplotypes characteristic of increased risk for type 1 DM⁸ and 14 patients presented 1 of those haplotypes.

Mean time from diagnosis to mobilization was 37.7 days (range, 24-56 days) and mean duration of hospital stay for transplantation (from conditioning to discharge) was 18.6 days (range, 15-24 days). Mean number of infused CD34 cells was 10.52 × 10⁶/kg (range, 4.98-23.19 × 10⁶/kg). Neutrophil engraftment (>500/μL) occurred between days 8 and 11 after transplantation (mean, 9.3 days) and platelet engraftment (>20 000/μL) was detected between day 0 and day 18 after transplantation (mean, 10.4 days).

The majority of the adverse effects related to treatment were mild and included nausea, vomiting, fever, and alopecia (Table 2). With regard to severe adverse effects, 2 patients presented bilateral nosocomial pneumonia that completely responded to intravenous broad-spectrum antibiotics. There was no mortality. During long-term follow-up, there was 1 case each of Graves disease, transient hypergonadotropic hypogonadism, and autoimmune hypothyroidism. Nine patients had posttransplant oligospermia. Two patients fathered children 2 years after transplantation.

Of the 23 included patients, 20 experienced time free from insulin (12 continuously and 8 transiently). In the majority of patients, insulin suspension occurred soon before or after stem cell infusion, ranging from 6 days before to 34 days after the infusion (mean, +2 days). Patients remained continuously insulin free for a mean time of 31 months (range, 14-52 months) (Table 1). One patient went more than 4 years without exogenous insulin use, 4 patients for at least 3 years, 3 patients for at least 2 years, and 4 patients for at least 1 year. In the patients in the continuously insulin-free group, mean value of HbA_1c was 8.0% at pretreatment and 5.4%, 5.7%, 5.7%, 5.5%, and 6.0%, respectively, at 3, 12, 24, 36, and 48 months after transplantation (P < .001 between pretreatment and all the follow-up values). Mean (SE) AUC of C-peptide levels before transplantation (225.0 [75.2] ng/mL per 2 hours) showed a significant increase at 24 months after transplantation (785.4 [90.3] ng/mL per 2 hours, P < .001) and at 36 months after transplantation (728.1 [144.4] ng/mL per 2 hours, P = .001) (Figure).

Eight patients became transiently free from exogenous insulin for a mean 17.7 months (range, 6-47 months). Of the 8 patients that restarted insulin, 4 resumed insulin after an upper respiratory tract infection. Daily insulin doses after insulin resumed were small, ranging from 0.1 to 0.3 IU/kg, to maintain good glucose control. In spite of the insulin use, mean (SE) AUC of C-peptide levels before transplantation (148.9 [75.2] ng/mL per 2 hours) also showed a significant increase at 36 months after transplantation (546.8 [96.9] ng/mL per 2 hours, P = .001) that was sustained at 48 months following transplantation (413.9 [124.7] ng/mL per 2 hours, P = .22, between 36 and 48 months after transplantation) (Figure).

Two patients (patient 2 and patient 4) became transiently insulin free for 47 and 43 months, respectively. After 4 and 2 months of resuming insulin and using daily insulin doses of 0.25 and 0.20 IU/kg, respectively, we opted to add sitagliptin (a dipeptidyl peptidase 4 inhibitor) 100 mg/d orally. Both became completely insulin free again after 2 months for patient 2 and 1 month for patient 4, and both remained free from exogenous insulin for 5 and 6 months, respectively. In patient 2, AUC of C-peptide levels presented increasing values from initial diagnosis (53.6 ng/mL per 2 hours) until 36 months following transplantation (470.4 ng/mL per 2 hours). Soon before insulin resumption, the AUC decreased to 135.9 ng/mL per 2 hours. Seven months after sitagliptin use, AUC of C-peptide levels reached levels of 530.2 ng/mL per 2 hours. In patient 4, the same occurred with AUC of C-peptide levels (ie, increasing from initial diagnosis [134.6 ng/mL per 2 hours] until 36 months following transplantation [812.5 ng/mL per 2 hours]). At 43 months after transplantation, before insulin resumption, the AUC of C-peptide levels was 153.2 ng/mL per 2 hours and 6 months after sitagliptin use, the AUC of C-peptide levels was 1069.6 ng/mL per 2 hours.

Only 3 patients did not experience any time free from insulin. One patient presented with diabetic ketoacidosis at diagnosis and received glucocorticoids to prevent rabbit antithymocyte globulin reactions, 1 developed diabetic ketoacidosis before enrollment, and 1 had inadvertently received corticosteroids (300-mg hydrocortisone) along with stem cell infusion. None of the patients achieved HbA_1c levels of less than 7%, in spite of progressive increase in daily insulin doses (>0.8 IU/kg/d).

Many study protocols have been developed to try to interdict the autoimmune process involved in type 1 DM disease process. Although some promising studies have suggested improvement in beta-cell function, no intervention has resulted in a definitive increase of beta-cell function.^3,9- 18 Moreover, although some studies showed sustained beta-cell function, cases of exogenous insulin suspension were rare.

In 2007,⁵ we reported intriguing findings of a group of 15 patients with newly diagnosed type 1 DM, with a mean follow-up of 18.8 months. Thirteen of 15 patients experienced continuous time free from insulin and 1 patient became transiently insulin free. However, one of the limitations of our previous study was the short period that the patients were followed up as well as lack of convincing C-peptide data to confirm a treatment effect rather than a prolonged honeymoon effect from close medical follow-up and physician-directed changes in lifestyle. In our current study, we showed that 20 of 23 patients became insulin free (12 continuously and 8 transiently) for periods as long as 4 years associated with good glucose control. To confirm that this is a treatment effect that improves beta-cell mass rather than a prolonged honeymoon phase, we now report the long-term outcome of C-peptide levels.

In the group of continuous insulin-free individuals, the increment of C-peptide levels occurred up to 24 months after transplantation and were maintained until at least 36 months. In the group of transiently insulin-free individuals, the increments still reached significance at 36 months with sustained increase until at least 48 months. The C-peptide data confirm increased endogenous insulin production as the predominate mechanism of euglycemic control in both patients who remained insulin free as well as those who needed to restart insulin at reduced doses. Because late complications of diabetes on the microvascular compartment are inversely related to C-peptide levels,¹ it is probable that even those patients who resumed insulin are at lower risk for long-term diabetes complications.

Recent studies using antibodies against CD3,^14,15 heat shock protein,¹⁶ rabbit anti-lymphocyte globulin,¹⁷ and alum-formulated glutamic acid decarboxylase¹⁸ showed only transient increase followed by slower declines of C-peptide levels along the follow-up. Moreover, almost all patients required exogenous insulin use.

Two patients who resumed insulin administration had an increase in C-peptide levels after the use of oral dypeptidil peptidase 4 (sitagliptin), a novel class of oral antihyperglycemic agents.¹⁹ Dypeptidil peptidase 4 inhibitors enhance meal-stimulated active glucagonlike peptide 1 and glucose-dependent insulinotropic polypeptide levels, improve measures of beta-cell function, suppress glucagon levels produced by alpha cells, and increase beta-cell mass in rodent models.^20- 22 The increase of C-peptide levels in those patients may not completely explain the metabolic improvement observed since the period of sitagliptin prescription to insulin suspension was too short. One alternative explanation is that sitagliptin promoted a rapid suppression of glucagon levels in parallel with further increase in insulin production. In addition, sitagliptin may also have immunoregulatory function in autoimmune insulitis.²³ All these results must be confirmed in a larger group of patients with a longer follow-up.

With regard to late adverse effects, 3 patients developed late endocrine dysfunctions that could be caused by autoimmune dysregulation associated with the transplant procedure²⁴ or by autoimmune polyendocrine syndrome frequently associated with type 1 DM.²⁵ Nine patients developed cyclophosphamide-related oligospermia after transplantation. Regarding the early adverse effects, 2 patients had bilateral pneumonia and most patients presented fever, rash, or urticaria. Those adverse effects are not negligible but may outweigh long-term risks of type 1 DM.

In conclusion, autologous nonmyeloablative HSCT was able to induce prolonged and significant increases of C-peptide levels associated with absence of or reduction of daily insulin doses in a small group of patients with type 1 DM. We showed that 20 of 23 patients became insulin free (12 continuously and 8 transiently) for periods as long as 4 years associated with good glucose control and acceptable incidence of adverse effects. At the present time, autologous nonmyeloablative HSCT remains the only treatment capable of reversing type 1 DM in humans. Randomized controlled trials and further biological studies are necessary to confirm the role of this treatment in changing the natural history of type 1 DM.