Infusion of N‐acetyl cysteine during hypoglycaemia in humans does not preserve the counterregulatory response to subsequent hypoglycaemia

Abstract Aim Administration of N‐acetyl cysteine (NAC) during hypoglycaemia will preserve the counterregulatory response to subsequent hypoglycaemia in healthy humans. Methods This was a randomized double‐blind cross over study where humans were given either a 60‐minute infusion of NAC (150 mg/kg) followed by a 4‐hour infusion of NAC (50 mg/kg) or saline starting 30 minutes before the initiation of a 2‐hour hypoglycaemic (HG) clamp at 8 am. After rest at euglycaemia for ~2 hours, subjects were exposed to a 2nd HG clamp at 2 pm and discharged home in euglycaemia. They returned the following day for a 3rd HG clamp at 8 am. Results Twenty‐two subjects were enrolled. Eighteen subjects completed the entire protocol. The epinephrine response during clamp 3 (171 ± 247 pg/mL) following clamp 1 NAC infusion was lower than the response during the clamp 1 NAC infusion (538 ± 392 pg/mL) (clamp 3 to clamp 1 NAC: P = .0013). The symptom response during clamp 3 (7 ± 5) following clamp 1 NAC infusion was lower than the response during the clamp 1 NAC infusion (16 ± 10) (clamp 3 to clamp 1 NAC: P = .0003). Nine subjects experienced rash, pruritus or nausea during NAC infusion. Conclusion We found no difference in the hormone and symptom response to experimental hypoglycaemia measured in subjects who were administered NAC as opposed to saline the day before. This observation suggests that further development of NAC as a therapy for impaired awareness of hypoglycaemia in patients with diabetes may be unwarranted.


| INTRODUC TI ON
Hypoglycaemia occurs frequently in the lives of patients with type 1 diabetes. Recurrent hypoglycaemia episodes closely spaced in time leads to impaired awareness of hypoglycaemia in which the counterregulatory response to hypoglycaemia is markedly reduced, and the first sign of a low blood sugar is neuroglycopenia. 1 Patients with impaired awareness of hypoglycaemia have a fourfold increased risk of experiencing severe hypoglycaemia, 2 which by definition is an event that requires the assistance of another person to treat. The fear of experiencing hypoglycaemia and of developing impaired awareness of hypoglycaemia is the primary barrier to achieving the level of glucose control necessary to prevent the microvascular complications of the disease and contributes to the mortality seen in patients with type 1 diabetes.
To address this critical issue in diabetes management, researchers have focused on developing ways to prevent and treat impaired awareness of hypoglycaemia. Initial efforts sought to prevent hypoglycaemia from ever occurring, with the thought that in the absence of recurrent hypoglycaemia the counterregulatory response would remain sufficiently robust to warn subjects of impending hypoglycaemia whenever a rare event occurred. Cranston et al 2

and Dagago Jack et al 3 both demonstrated that meticulous
attention to matching insulin doses to metabolic requirements over a 3 weeks period restored the counterregulatory response in well-controlled patients with type 1 diabetes, but such an approach has been very difficult to implement in clinical practice.
More recently, investigators have used information obtained in experimental studies designed to identify the pathways that link the detection of a single episode of hypoglycaemia to the blunting of the counterregulatory response during hypoglycaemia experienced in subsequent days. Such investigation has revealed the importance of opioid signalling during the first episode of hypoglycaemia and has demonstrated that blockade of opioid signalling using an intravenous administration of a drug during this first episode will preserve the counterregulatory response to hypoglycaemia the subsequent day. 4 However, acute oral administration of the opioid antagonist naltrexone did not have an impact on the counterregulatory responses in subjects with well-controlled type 1 diabetes 5 and 4 weeks of oral naltrexone therapy did not restore the counterregulatory response in patients with type 1 diabetes and impaired awareness of hypoglycaemia. 6 Future work will need to determine if the failure of naltrexone to restore the response was due to inadequate dosing or selection of patients who may be refractory to such restoration.
Reactive oxygen species generated in the brain of animals exposed to hypoglycaemia have also been linked to the blunting of the counterregulatory response to hypoglycaemia the following day. In a rodent model, administration of N-acetyl cysteine (NAC) during episodes of hypoglycaemia prevented the blunting of the counterregulatory response, possibly by preventing the hypoglycaemia induced rise in hypothalamic reactive oxygen species known to occur during acute episodes of hypoglycaemia. 7,8 Whether NAC will be beneficial in humans is unknown. NAC is a therapy that has long been FDA approved for the treatment of acetaminophen overdose, 9 is available as a nutritional supplement and has been used to treat a variety of human conditions including pulmonary, 10 renal 11 and psychiatric maladies. 12 The purpose of this study was to test the hypothesis that NAC will be effective in the prevention and treatment of impaired awareness of hypoglycaemia. In this investigation, we performed a proof of principle experiment to determine if the intravenous administration of NAC during experimental hypoglycaemia at the maximal dose approved for use in humans prevents the expected blunting in symptom scores and epinephrine secretion during subsequent hypoglycaemia in healthy volunteers using a standard 2-day protocol to induce impaired awareness of hypoglycaemia. 13 We also measured plasma and red blood cell levels of NAC during the infusion period to characterize NAC's pharmacokinetics. At the discretion of the investigator, additional doses of diphenhydramine were given to subjects who developed rash or pruritus.

| MATERIAL S AND ME THODS
In addition, ondansetron (4 mg) was administered intravenously to subjects who developed nausea.
At minute 30, a continuous infusion of insulin at a rate of 2.0 mU/ kg/min was started and blood glucose was allowed to fall to 50 mg/ dL. Insulin was given at this rate for 120 minutes during which blood glucose was clamped at 50 mg/dL using a variable infusion of glucose. Subjects were then brought to euglycaemia. At 4 hours, the continuous infusion of insulin was again started at a rate of 2.0 mU/ kg/min for 120 minutes. Blood glucose was again allowed to fall to were asked to rate their hypoglycaemic symptoms at baseline and every 15 minutes using a standardized questionnaire. 14 Part Two of the study was identical except the other treatment was given (saline was given if NAC was given during Part One; NAC was given if saline was given during Part One).
Plasma glucose concentration was measured in duplicate during the clamps using an Analox machine (Analox Instruments). The primary outcome was defined as the mean epinephrine recorded during minutes 60-75 of hypoglycaemia clamp 3; this is referred to below as the 'epinephrine response' to hypoglycaemia. A similar outcome was defined for total symptoms. If NAC prevents impaired awareness of hypoglycaemia, then the epinephrine response during the third hypoglycaemic clamp after NAC infusion will still be high, while the epinephrine response during the third hypoglycaemic camp after saline infusion will be blunted. Therefore, this within-person NAC minus saline difference in epinephrine response will be large and positive. Assuming a one-sided type I error of 5% and a power of 85%, this study was powered to detect a positive epinephrine response difference between NAC and saline of at least  The impact of NAC on free epinephrine levels in plasma was evaluated in plasma samples collected during euglycaemia and hypoglycaemia to determine if NAC has the potential to release protein-bound epinephrine and norepinephrine, causing the increase in unbound concentrations. Blood was collected from two subjects during their saline experiment's periods of euglycaemia and hypoglycaemia; plasma was separated via centrifugation. Plasma samples (n = 3-6 per condition) were spiked with 0, 10, 100 and 1000 μg/mL NAC, and epinephrine was measured as described above.

| RE SULTS
Twenty-two subjects were enrolled in the study. Two subjects de-

| D ISCUSS I ON
In this proof of principle study, we found no difference in the counterregulatory hormone and symptom response to experimental hypoglycaemia measured in subjects who were administered NAC as opposed to saline the day before. This observation suggests that Strengths of our study include a sufficiently large sample size to have confidence in our findings and evidence that the drug levels of NAC achieved were comparable with those associated with the treatment of acetaminophen overdose. 17 We also were able to induce blunting of the counterregulatory response to hypoglycaemia following exposure to recurrent hypoglycaemia using our protocol, an accomplishment that has been inconsistently noted in the literature. 18 One limitation was we performed our studies in normal controls rather than subjects with type 1 diabetes who experience the clinical syndrome of impaired awareness of hypoglycaemia. There is a growing appreciation that the exposure to recurrent hypo-and hyperglycaemia may be important in the pathogenesis of impaired awareness of hypoglycaemia in patients with diabetes. 19 Another limitation is that not all subjects remained at the target range of hypoglycaemia during the final 15 minutes of clamp 3, which necessitated data analysis using samples collected during minutes 60-75 of clamps one and three when all were maintained at target. Lucidi and colleagues have also observed such an increase in insulin resistance during an afternoon hyperinsulinaemic euglycaemic clamp that was performed after morning exposure to 3 hours of hyperinsulinaemia and hypoglycaemia. 20 In conclusion, NAC infusion during two episodes of experimental hypoglycaemia was without effect on the counterregulatory symptoms and hormone responses to a third episode of hypoglycaemia in healthy controls. This observation suggests this currently available antioxidant is unlikely to be an effective therapy to prevent or treat impaired awareness of hypoglycaemia in diabetes.

ACK N OWLED G EM ENTS
The authors acknowledge Rebecca Cote and Sarah Chapeau for regulatory and administrative support, Michelle Snyder for nursing support, and Usha Mishra for measurement of NAC concentrations.

CO N FLI C T O F I NTE R E S T
ERS has served as a consultant to Lilly, Sanofi, MannKind and Zucara.

AUTH O R CO NTR I B UTI O N S
AM, AK, LDC and ERS all contributed to the study design, acquisition of data, data analysis, data interpretation, drafting/revising the man-