C肽在1型糖尿病的替代治疗研究
DOI: 10.2337/dc06-1274 © 2007by the American Diabetes Association
C-Peptide Replacement Therapy and Sensory Nerve Function in Type 1 Diabetic Neuropathy1Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden Address correspondence and reprint requests to Karin Ekberg, PhD, Creative Peptides, Fogdevreten 2, SE-171 77 Stockholm, Sweden. E-mail:karin.ekberg@creativepeptides.se RESEARCH DESIGN AND METHODS—This was an exploratory, double-blinded,randomized, and placebo-controlled study with three study groupsthat was carried out at five centers in Sweden. C-peptide wasgiven as a replacement dose (1.5 mg/day, divided into four subcutaneousdoses) or a dose three times higher (4.5 mg/day) during 6 months.Neurological examination and neurophysiological measurementswere performed before and after 6 months of treatment with C-peptideor placebo. RESULTS—The age of the 139 patients who completed theprotocol was 44.2 ± 0.6 (mean ± SE) years andtheir duration of diabetes was 30.6 ± 0.8 years. Clinicalneurological impairment (NIA) (score >7 points) of the lowerextremities was present in 86% of the patients at baseline.Sensory nerve conduction velocity (SCV) was 2.6 ± 0.08SD below body height–corrected normal values at baselineand improved similarly within the two C-peptide groups (P<0.007). The number of patients responding with a SCV peak potentialimprovement >1.0 m/s was greater in C-peptide–treatedpatients than in those receiving placebo (P< 0.03). In theleast severely affected patients (SCV < 2.5 SD below normalat baseline,n= 70) SCV improved by 1.0 m/s (P< 0.014 vs.placebo). NIA score and vibration perception both improved withinthe C-peptide–treated groups (P< 0.011 andP<0.002). A1C levels (7.6 ± 0.1% at baseline) decreasedslightly but similarly in C-peptide–and placebo-treatedpatients during the study. CONCLUSIONS—C-peptide treatment for 6 months improvessensory nerve function in early-stage type 1 diabetic neuropathy. Abbreviations:CMAP, compound muscle action potential amplitude • MCV, motor nerve conduction velocity • NIA, neuropathy impairment assessment • QST, quantitative sensory testing • SCV, sensory nerve conduction velocity • SCVi, sensory nerve conduction velocity measured at initial potential deflection • SCVp, sensory nerve conduction velocity measured at peak potential • VPT, vibration perception threshold C-peptide replacement in animal models of type 1 diabetes isaccompanied by improved nerve function and amelioration of diabetes-inducedmorphological changes (12,13). Specifically, C-peptide treatmentresults in decreased paranodal swelling and demyelination, decreasedaxonal degeneration, reduced frequency of axoglial dysjunction,and augmented regenerative activity (13). Accompanying thesebeneficial effects of C-peptide is a partial restoration ofthe diabetes-induced reduction in Na+,K+-ATPase activity ofthe nerves (13,14). Moreover, C-peptide is known to have a stimulatoryeffect on endothelial nitric oxide synthase (15,16), therebyaugmenting endoneurial blood flow (7,17). C-peptide also exertsneurotrophic effects and has an inhibitory effect on cellularapoptosis (for review, see ref.18). Little information is availableregarding the effect of C-peptide on nerve function in patientswith type 1 diabetes, but after 3 months of C-peptide replacementin patients with subclinical neuropathy, sensory nerve conductionvelocity was substantially improved by 2.7 m/s (19). C-peptideadministration is also reported to result in improvement ofautonomic nerve function in type 1 diabetes (20). The aim of the present study was to examine whether C-peptideexerts a beneficial effect on peripheral nerve functional abnormalitiesin patients with type 1 diabetes and established peripheralneuropathy. Specifically, the effect of 6 months of C-peptidetreatment on sensory nerve conduction velocity and other earlysigns of diabetic neuropathy in the lower extremities was investigated. All patients were informed of the nature, purpose, and possiblerisks of the study before consenting to participate. The protocolwas approved by the institutional human ethics committee ofthe Karolinska Institute and of the investigational sites andby the Swedish Medical Product Agency. The study was conductedin accordance with good clinical practice guidelines and theprinciples of the Declaration of Helsinki. The study was carried out in a double-blinded, placebo-controlled,randomized fashion with three study arms: treatment with C-peptidelow dose (1.5 mg/day corresponding to physiological replacement),C-peptide high dose (4.5 mg/day), or placebo (diluent) for 6months. After giving their informed consent, the patients underwentphysical examination, including an electrocardiogram, measurementof blood pressure, and clinical chemistry laboratory testing.In addition, samples for measurement of C-peptide plasma concentrationwere collected. Furthermore, neurological and neurophysiologicalexaminations were carried out, as described below. In patientswho fulfilled the inclusion criteria, neurophysiological evaluationand quantitative sensory testing (QST) were repeated on anotherday (2–14 days after the first assessment), and the meanof the two assessments was used as baseline value. Thereafter,the patients were randomly assigned to one of the three studygroups and instructed to take the trial medication four timesdaily as subcutaneous injections of 20% of the daily study medicationdose in conjunction with their regular insulin administrationin the morning, at lunch, and at dinner and 40% of the dailydose at bedtime for a total of 6 months. Human C-peptide wasproduced recombinantly by Creative Peptides (Stockholm, Sweden).Every 6 weeks, the patients met with the study nurse for reviewof drug compliance and for safety assessments. After 6 monthsof treatment, assessments of neurophysiological variables andQST were repeated and performed in duplicate (on separate days2–14 days apart), and a neurological evaluation was performed.Thereafter, the study was ended, and the trial medication wasdiscontinued. Patients’ compliance was checked by reviewof the patients’ diaries and visual control of the returnedmedication vials. As an additional check, C-peptide concentrationsin plasma samples taken after 3 months of treatment and at theend of the study were reviewed (evaluation performed after databaseclosure). Randomization and blinding of the trial medication were performedby the Karolinska Hospital Pharmacy, and source data verificationwas monitored by an external monitor (PharmAid, Stockholm, Sweden).The study was carried out at five centers in Sweden: KarolinskaUniversity Hospital at Huddinge and Solna, Lundby Hospital (neurophysiologicalassessments at the Sahlgrenska Hospital) in Gothenburg, LinköpingUniversity Hospital in Linköping, and Uppsala UniversityHospital in Uppsala. Sensory function and neurophysiological assessments Neurological examination and symptom assessment Analyses Statistical methods The predetermined primary analysis was to evaluate the changein SCV from baseline to 6 months in the per protocol patientpopulation and to compare the effect of placebo with that ofC-peptide, i.e., the low- and the high-dose groups combined.Power analysis was performed on the basis of previous publishedresults (19), reporting a significant improvement (+2.7 m/s)in sensory nerve conduction velocity after 3 months of C-peptidereplacement in type 1 diabetes patients with subclinical neuropathyand a common SD for the two groups (active and placebo) of 4.07m/s. It was estimated that The characteristics of the 139 patients (61 women and 78 men)in the different study groups who completed the study showedno statistically significant differences for any of the baselinevariables. Patients were, on average, 44.2 ± 0.6 yearsof age and had a diabetes duration of 30.6 ± 0.8 years.Their body height was 174.1 ± 0.9 cm, and BMI was 25.0± 0.2 kg/m2. Regarding known microvascular complications,54% of the patients reported having signs or symptoms of peripheralneuropathy, 45% had simplex retinopathy, and 41% had proliferativeretinopathy, whereas only 13% reported microalbuminuria and2% reported proteinuria. The patients’ average insulindose was 0.64 ± 0.01 IU · kg–1·24 h–1and the level of glycemic control, as reflectedby the A1C, was similar in the three groups, with an averageA1C of 7.6 ± 0.1%. During and after the 6 months of treatment,there were no significant differences in A1C between the groups.However, A1C decreased 0.21 ± 0.09 (P< 0.01) and0.03 ± 0.12% (NS) within the C-peptide low- and high-dosegroups, respectively, and 0.42 ± 0.10% (P< 0.001)in the placebo group. The C-peptide plasma concentration atbaseline was 0.02 ± 0.00 nmol/l, and the levels weremeasured again on two occasions, after 3 and 6 months of treatment.These results confirmed exposure to C-peptide in the patientsin the C-peptide low- and high-dose groups. During the studythere were no adverse drug reactions or adverse events thatcould be related to the trial medication nor were there anysignificant changes in safety variables (blood chemistry andvital signs). The baseline SCV in the sural nerve in the diabetic patientswas significantly reduced compared with normal (Table 1); SCVpwas, on average, 2.6 ± 0.08 SD below normal, SCVi was3.2 ± 0.08 SD below normal, and action potential was4.6 ± 0.29 µV. Similarly, the MCV in the peronealnerve was also significantly reduced in the patients (–2.9± 0.10 SD) (Table 1). There was no significant differencebetween the three treatment groups at baseline. QST revealedmore markedly elevated thresholds in the feet than in the lowerlegs, especially to vibration (Table 1) and cold stimulation(cold temperature threshold in the feet 3.2 ± 0.19 SDabove normal and in the lower legs 2.2 ± 0.15 SD). Thecorresponding values for heat stimulation were 1.2 ±0.08 and 0.8 ± 0.09 SD. Pathological neurological findingsassessed in the neurological examination (NIA >7 points)were present in 86% of the patients at baseline; the averageNIA score was 18.0 ± 0.86 points. Of the randomly assignedpatients, 35% reported subjective symptoms from the lower limbsat baseline, and six of these patients reported having symptomsincluding sensation of pain.
There was no statistically significant difference between theresponses in the C-peptide low- and high-dose groups after 6months (Table 1). Accordingly, the low- and high-dose C-peptideresults were combined in the continued analyses. There was significantimprovement from baseline for SCVp (P< 0.007) and SCVi (P< 0.001) in the C-peptide–treated patients, but thesechanges were not significantly different from those of the placebogroup. The number of responders, defined as patients with animprovement in SCVp of >1 m/s (23), was significantly greaterin the group receiving active treatment compared with thosereceiving placebo (37 and 19%;P< 0.032; Pearson  2test).With a study duration of no more than 6 months it was anticipatedthat the patients who were least affected at baseline may havea greater potential for improvement. Thus, a subgroup analysiswas performed in the half of the patients who showed the leastaffected nerve conduction velocity at baseline (cutoff equalto the median SCVp, i.e., >–2.5 SD,n= 70; this analysisincluded 21, 31, and 18 patients in the placebo, low-dose, andhigh-dose groups, respectively, and with demographics similarto those of the entire group). In this group C-peptide administrationfor 6 months induced an improvement in SCVp of 1.03 m/s greaterthan that of the placebo group (C-peptide 0.61 ± 0.25m/s and placebo –0.42 ± 0.29 m/s,P< 0.014)(Fig. 1). The corresponding improvement in SCVi in the C-peptide–treatedpatients was even greater (1.27 ± 0.36,P< 0.001)than that in SCVp. Analysis of the number of responders pertreatment group in this patient population, i.e., patients withan improvement in SCVp >1 m/s, showed that there were 39%responders in the active group and 5% in the placebo group (P< 0.004).
The MCV decreased during the study period (Table 1). After 6months of C-peptide administration, there were statisticallysignificant improvements for VPT (P< 0.01) and NIA score(P> 0.01) within the C-peptide–treated group. Theplacebo group showed no significant changes in VPT or NIA score.No significant changes were observed for temperature perceptionor symptoms in any of the groups during the study (data notpresented).
The present study shows for the first time in type 1 diabeticpatients with established clinical neuropathy that C-peptideadministration results in improvement of early neurologicalabnormalities that accompany type 1 diabetes. After 6 monthsof administration of C-peptide, there was a significant improvementin SCV, especially in patients whose nerve function was lessaffected at baseline (1.03 m/s improvement for the C-peptidepatients vs. placebo,P< 0.014). A1C decreased slightlyduring the study, but this was most marked in the placebo group.Consequently, the improvements in nerve function after C-peptidewere not related to changes in glycemic control. We have previouslydemonstrated a positive effect of C-peptide on SCV in type 1diabetic patients with subclinical neuropathy (19). In the presentstudy, the patients’ mean duration of diabetes was threetimes longer, and the patients had more marked nerve dysfunction,probably including not only circulatory and metabolic changesbut also varying degrees of structural changes. The differencein magnitude of SCV improvement emphasizes the importance ofearly intervention (23–25). The beneficial effect of C-peptide on SCV was accompanied bysignificant improvements in VPT and in the NIA score withinthe C-peptide groups, although the latter changes were not statisticallysignificantly different from those in the placebo group. Theeffect on VPT is well in agreement with the finding in SCV,because vibration perception is mediated primarily by largemyelinated fibers such as the sural nerve. The NIA score representsthe sum of several nerve qualities, in which large fiber functionsrepresent the greater proportion of the score. Cold and heatperceptions, on the other hand, are mediated by the small A- In the present study C-peptide was administered four times dailyin two different total doses. The low dose (1.5 mg/day) wascalculated to represent a physiological replacement of C-peptide.Measurements of plasma concentrations confirmed expected exposuresto C-peptide, although in a slightly lower range than anticipated.This may be related to less than full compliance with the treatmentregimen, even though the predetermined criteria for compliancewere fulfilled by the patients. The basis for the multiple C-peptidedose regimen used in the present study was to achieve a minimaleffective plasma concentration for as many hours of the dayas possible. This goal may have been achieved to a greater extentin the high-dose group, which may help to explain the tendencytoward slightly, but not significantly, greater improvementin the higher dose group. In fact, the higher dose was not expectedto result in a greater effect; the basis for this theory restsin studies of C-peptide binding to cell membranes (26) showinghalf saturation already at 0.3 nmol/l and full saturation at0.9 nmol/l in several cellular systems. Thus, raising the concentrationabove this level would not be expected to elicit any furtherphysiological effects. In fact, dose dependency for C-peptidein the concentration range of 0–1.0 nmol/l has been demonstratedin vitro as well as in vivo in rats and humans (27–29),but with concentrations above the physiological level the responseshave not been greater. The findings for the higher dose groupthus provide support for the view that C-peptide should be givenas a physiological replacement. As in our previous study (19), there was no improvement in MCVafter the 6 months of C-peptide treatment. This lack of improvementmay be related to the short study duration in relation to theinitial conduction velocity deficit. In fact, lack of improvementin MCV was reported also for another intervention attempt involving3 months’ treatment duration (30), but with more prolongedtreatment a tendency for improved MCV was noted (31). The deteriorationrate of nerve functional measures is not linear and differsamong types of nerves (32). It is conceivable that the differentresponses in motor and sensory nerves may be related to varioussensitivity to factors such as hypoxia, reduced Na+,K+-ATPaseactivity, sorbitol accumulation, and response to growth factors(33). In summary, C-peptide treatment in replacement doses for 6 monthsimproves sensory nerve function in patients with diabetic neuropathyand mild to moderate nerve conduction abnormalities. The effectwas most marked in the patients who had the least amount ofdisease at the onset of the study, as can be expected for astudy that is of short duration at least in the context of diabeticneuropathy. Finally, the study results emphasize the need forearly intervention in this disorder.
In addition to the authors, the C-peptide study group includesthe following participating investigators: Sten Andersson, MD,Karolinska University Hospital, Solna, Stockholm, Sweden; MikaelElam, MD, PhD, Department of Neurophysiology, Sahlgrenska Hospital,Gothenburg, Sweden; Eva Svanborg, MD, PhD, and Nicola Reiser,MD, Department of Neurophysiology, Linköping UniversityHospital, Linköping, Sweden; Anna Sjölin, MD, PhD,Anna Stenborg, MD, and Roland Flink, MD, PhD, Departments ofMedicine, Neurology, and Neurophysiology, respectively, UppsalaUniversity Hospital, Uppsala, Sweden; Erik Moberg, MD, PhD,Per Oskarsson, MD, PhD, Tomas Andersson, MD, PhD, Benjamin RibaltaStanford, MD, Martin Engvall, MD, and Cecilia Bungerfeldt, MD,Departments of Medicine, Neurophysiology, and Neurology, KarolinskaUniversity Hospital, Huddinge, Stockholm. In addition, two biostatisticiansare included in the group: Björn Jonsson, PhD, and AndersLindeberg, MSc, Stockholm, Sweden.
A table elsewhere in this issue shows conventional and SystèmeInternational (SI) units and conversion factors for many substances. DOI: 10.2337/dc06-1274. Clinical trial reg. no. NCT00278980,clinicaltrials.gov. The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked "advertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact. Received for publication June 20, 2006. Accepted for publication August 30, 2006.
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INTRODUCTION
RESEARCH DESIGN AND METHODS--
10 cm below theknee) for evaluations of the vibration perception thresholds(VPTs). Heat and cold temperature thresholds were determinedusing the Marstock technique with a temperature-regulated probe(Thermotest; Somedic or Medoc Advanced Medical Systems) (
) and placebo administration (n= 21,
) in the least affected half of the patients, i.e., those with SCVp >–2.5 SD at baseline. SCVi represents the fastest population of the axons in the nerve, whereas SCVp provides an estimate of the average conduction velocity.Pvalues below the columns refers to within-group changes.
fibers and C-fibers, respectively. In the present study, therewere no detectable effects by C-peptide on small nerve fiberfunction, and it is conceivable that these fibers respond differentlyto intervention. This is in contrast to the finding of a beneficialpreventive effect on nociceptive function after C-peptide administrationin animals (
