Triheptanoin for Glucose Transporter Type I Deficiency (G1D) Modulation of Human Ictogenesis, Cerebral Metabolic Rate, and Cognitive Indices by a Food Supplement

Research

Original Investigation Triheptanoin for Glucose Transporter Type I Deficiency (G1D) Modulation of Human Ictogenesis, Cerebral Metabolic Rate, and Cognitive Indices by a Food Supplement

Juan M. Pascual, MD, PhD; Peiying Liu, PhD; Deng Mao, BS; Dorothy I. Kelly, MA; Ana Hernandez, MS; Min Sheng, PhD; Levi B. Good, PhD; Qian Ma, MD, PhD; Isaac Marin-Valencia, MD, MS; Xuchen Zhang, MD; Jason Y. Park, MD, PhD; Linda S. Hynan, PhD; Peter Stavinoha, PhD; Charles R. Roe, MD; Hanzhang Lu, PhD

Supplemental content at IMPORTANCE Disorders of brain metabolism are multiform in their mechanisms and jamaneurology.com manifestations, many of which remain insufficiently understood and are thus similarly treated. Glucose transporter type I deficiency (G1D) is commonly associated with seizures and with electrographic spike-waves. The G1D syndrome has long been attributed to energy (ie, adenosine triphosphate synthetic) failure such as that consequent to tricarboxylic acid (TCA) cycle intermediate depletion. Indeed, glucose and other substrates generate TCAs via anaplerosis. However, TCAs are preserved in murine G1D, rendering energy-failure inferences premature and suggesting a different hypothesis, also grounded on our work, that consumption of alternate TCA precursors is stimulated and may be detrimental. Second, common ketogenic diets lead to a therapeutically counterintuitive reduction in blood glucose available to the G1D brain and prove ineffective in one-third of patients.

OBJECTIVE To identify the most helpful outcomes for treatment evaluation and to uphold (rather than diminish) blood glucose concentration and stimulate the TCA cycle, including anaplerosis, in G1D using the medium-chain, food-grade triglyceride triheptanoin.

DESIGN, SETTING, AND PARTICIPANTS Unsponsored, open-label cases series conducted in an academic setting. Fourteen children and adults with G1D who were not receiving a ketogenic diet were selected on a first-come, first-enrolled basis.

INTERVENTION Supplementation of the regular diet with food-grade triheptanoin.

MAIN OUTCOMES AND MEASURES First, we show that, regardless of electroencephalographic spike-waves, most seizures are rarely visible, such that perceptions by patients or others are inadequate for treatment evaluation. Thus, we used quantitative electroencephalographic, neuropsychological, blood analytical, and magnetic resonance imaging cerebral metabolic rate measurements.

RESULTS One participant (7%) did not manifest spike-waves; however, spike-waves promptly decreased by 70% (P = .001) in the other participants after consumption of triheptanoin. In addition, the neuropsychological performance and cerebral metabolic rate increased in most patients. Eleven patients (78%) had no adverse effects after prolonged use of triheptanoin. Three patients (21%) experienced gastrointestinal symptoms, and 1 (7%) discontinued the use of triheptanoin.

CONCLUSIONS AND RELEVANCE Triheptanoin can favorably influence cardinal aspects of neural function in G1D. In addition, our outcome measures constitute an important framework for the evaluation of therapies for encephalopathies associated with impaired intermediary metabolism. Author Affiliations: Author affiliations are listed at the end of this article. Corresponding Author: Juan M. Pascual, MD, PhD, Rare Brain Disorders Program, The University of Texas Southwestern Medical Center, JAMA Neurol. 2014;71(10):1255-1265. doi:10.1001/jamaneurol.2014.1584 5323 Harry Hines Blvd, Mail Code Published online August 11, 2014. 8813, Dallas, TX 75390.

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lucose is the principal cerebral metabolic substrate un- whereas other interventions, such as the modified Atkins diet, der normal circumstances. Glucose provides energy have not been subject to rigorous investigation in G1D. Thus, G (adenosine triphosphate) to neural cells and carbon for most therapeutic efforts have been limited to early diagnosis neurotransmitter production, one of the numerous biosyn- and initiation of a ketogenic diet during childhood.16,28 thetic reactions that the brain carries out, via glycolysis and In contrast with this form of therapy,studies2,29 have shown the tricarboxylic acid (TCA) cycle.1 However, glucose metabo- that nutrients with additional potential to refill TCA cycle in- lism also fulfills other, less-acknowledged cerebral biosyn- termediates via anaplerosis, such as triheptanoin (an edible tri- thetic requirements, which are no less important from a clini- glyceride of the 7-carbon fatty acid heptanoate that can yield cal standpoint when they remain unmet,2 such as anaplerosis 3 molecules of heptanoate and other anaplerotic metabolites (ie, the replenishment of intermediates that are normally lost in the liver30), offer therapeutic benefit in diseases in which from the TCA pool). Thus, a myriad of cerebral energetic and metabolic precursor depletion or overuse is suspected or iden- carbon fluxes, many of which are poorly understood tified. Dietary triheptanoin gives rise (after hepatic metabo- quantitatively,3 stem from brain glucose availability. This is, lism) to plasma heptanoate and to 5-carbon ketone bodies (β- in turn, contingent on the activity of the facilitative mem- ketopentanoate and β-hydroxypentanoate),30 all of which can brane glucose transporter type I (GLUT1), which permits glu- penetrate31 and be readily metabolized by the brain (eFigure cose to cross the blood-brain barrier and subsequently transit in the Supplement), as has been shown32,33 in rodents includ- into, and probably through, astrocytes. ing G1D mice. Neoglucogenesis can also be observed after hep- Human GLUT1 deficiency (G1D) due to mutation of the gene tanoate infusion, and this process may act synergistically with SLC2A1 (OMIM 606777) is associated with partial loss of func- the other heptanoate metabolites in brain glucose–deficient tion (ie, GLUT1 activity is never abolished) and results in de- states, such as G1D.33 Heptanoate metabolism exerts anticon- creased brain glucose accumulation.4 Glucose transporter type vulsant effects in an unrelated animal model of experimental I deficiency manifests as an encephalopathy frequently (but not pilocarpine-induced epilepsy and refills depleted brain TCA exclusively) characterized by medication-refractory infantile- cycle intermediates.34 Therefore, the rationale for the present onset seizures,5 diminished encephalic mass, intellectual dis- work combines potential biochemical benefits,2,29 experience ability, and complex (ie, multiform) motor disturbances (spas- with other neurometabolic disorders that we have treated with ticity, ataxia, chorea, dystonia, and combinations thereof).6-9 triheptanoin,30,35,36 and our ex vivo G1D rodent model evi- This encephalopathy is often accompanied by a reduced cere- dence of the metabolic effects of triheptanoin on the brain,37 brospinal fluid glucose concentration, even though cerebro- all in the context of limited therapeutic alternatives. spinal fluid glucose abundance primarily reflects secretion by the choroid plexus and exceeds the concentration typical of the 10 brain’s interstitial space. Notably, a series of important neu- Methods ral function defects in G1D is independent of blood-brain barrier GLUT1 dysfunction and can be ameliorated by increasing This study was conducted with institutional review board the brain tissue glucose concentration, as has been shown11-13 approval of The University of Texas Southwestern Medical in the G1D mouse. We noted that these findings might bear rel- Center and was referenced under the US Food and Drug evance to human G1D,14 thus inviting treatment development Administration Investigational New Drug 59303 (sponsor- based on the enhancement of brain glucose influx and/or the investigator C.R.R.). Written informed consent was obtained supply of an effective substitute substrate. from each participant older than 18 years, and informed con- In this regard, the ketogenic diet, which is used for the treat- sent was obtained from one parent or guardian of all younger ment of G1D solely on the basis of biochemical assumptions and participants. Assent was obtained from children between 10 empirical observations and has not been the subject of a con- and 18 years. Travel and lodging costs up to a total of $500 trolled clinical trial in G1D,15 ameliorates some movement dis- per family were reimbursed for each visit. orders and epilepsy in two-thirds of the patients who receive it (J.M.P., unpublished data, 2012).16,17 Unfortunately, neuro- Participants logic deficits, particularly those centered on other aspects of A summary of patient age, SLC2A1 G1D causative mutation, and movement coordination (eg, ataxia and dysarthria) and cogni- other factors is given in eTable 1 in the Supplement. Study par- tion, tend to persist while the diet is used, with a significant ticipants were recruited (1) from the Rare Brain Disorders Pro- portion of patients experiencing recurrent or incompletely gram at The University of Texas Southwestern Medical Cen- treated abnormalities.18 The remaining one-third of the pa- ter and Children’s Medical Center Dallas, including existing tients who are poorly responsive to the diet face an even more patients and those responding to our official website announce- problematic clinical course. The diet, given with the therapeu- ment, and (2) via public announcement by the Glut1 Defi- tic intent of stimulating brain metabolism in G1D, may act as ciency Syndrome Foundation. Patients enrolled included males an anticonvulsant through several potential mechanisms,19,20 and females, ages 2 years to 28 years, and English or Spanish including the production of acetyl-coenzyme A that can stimu- speakers. Participants were consecutively enrolled from all eli- late the neural TCA cycle,21,22 but the diet’s mode of action has gible patients who contacted us. There was no consideration not been investigated in G1D. In addition to insufficiently al- given to geographic location (including the United States and leviating all incapacitating features of G1D,18 ketogenic diets are Canada), disease severity, or selection factors other than those not universally tolerable,23-27 and few adults receive them, listed here. Exclusion criteria included the current use of a ke-

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togenic diet. Participants with metal implants incompatible Figure 1. Flow Diagram of Study Visits and Procedures with exposure to a research magnetic field or unable to toler- ate magnetic resonance imaging (MRI) because of anxiety were Screening excluded from the MRI procedures. Participants who were not Informed consent and genotyping Physical and neurologic examination previously genetically verified for G1D received genotyping via the National Institutes of Health Office of Rare Diseases Re- search Collaboration, Education, and Test Translation (CETT) Visit 1: Baseline (fasting) Fasting glucose and other safety blood testing program for G1D at the University of Texas Southwestern Medi- Neuropsychological testing cal Center and Children’s Medical Center Dallas. Because ap- EEG MRI (optional) proximately 15% of patients with G1D do not harbor identifi- Administration of triheptanoin able pathogenic mutations in SLC2A1,38 one patient, who Wait 60-90 min exhibited clinical symptoms, a decreased cerebrospinal fluid Random glucose measurement EEG glucose concentration, and a characteristic brain fludeoxyglu- Neuropsychological testing 4,39 MRI (optional) cose F 18 positron emission tomography scan indicative of Start daily triheptanoin therapy G1D, was enrolled despite normal clinically available DNA se- quencing of SLC2A1 and standard chromosomal microarray Visit 2: 3-mo follow-up analysis. No medications were changed immediately before or Physical and neurologic examination during the study for any participant. Fasting glucose and other safety blood testing Neuropsychological testing EEG (optional) MRI (optional) Dietary Supplement Discontinue triheptanoin therapy Triheptanoin is a naturally occurring fat40 that is readily syn- thesized from castor bean oil for use in the human food industry as an additive to dairy products or as an emollient in Visit 3: 6-mo follow-up/study exit Physical and neurologic examination 41 cosmetics. In the United States, food-grade triheptanoin first Fasting glucose and other safety blood testing Neuropsychological testing received orphan designation for the treatment of fatty acid oxi- Exit study dation disorders after work performed under our Investiga-

tional New Drug 59303 status (sponsor-investigator, C.R.R.), Each patient participated in 3 visits. A limited number of patients underwent and, more recently (2012), it achieved an equivalent legal sta- electroencephalogram (EEG) and magnetic resonance imaging (MRI) at the tus in the European Union for the treatment of very-long- 3-month follow-up visit. chain 3-hydroxyacyl-coenzyme A-dehydrogenase deficiency (designation EU/3/12/1081). tional electrode was placed directly on the skin of the chest. Participants received open-label adjunctive supplemen- All preparation equipment coming into contact with the skin tation with food-grade triheptanoin, a medium-chain triglyc- was discarded after a single use, and electrodes and caps were eride of heptanoic acid that is colorless and odorless. Trihep- disinfected after use following standardized University of Texas tanoin was manufactured in oil form (Sasol Germany GmbH) Southwestern Medical Center guidelines. and permitted, per the manufacturer (product information Baseline (resting) EEG recordings after overnight (≥8 hours) documentation, version 4.02; revision January 16, 2008), for fasting (except for water and medications) were obtained for 30 applications in the food industry as a food additive (butter to 45 minutes. Baseline neuropsychological testing, lasting ap- marker-fat) and commercialized as Spezialöl 107 (European In- proximately 30 minutes, occurred before baseline EEG record- ventory of Existing Commercial Substances Chemical Ab- ings were performed. Parents and patients (when sufficiently stracts Service numbers: 620-67-7/210-647-2; minimum 95% mature) were asked to note all visible seizures, and their obser- triheptanoate and 99% 7-carbon fatty acid as determined by vations were contrasted with EEG recordings. After a suffi- gas chromatography by the manufacturer). The triheptanoin ciently informative baseline EEG (ie, defined as a tracing con- oil used in this study was purchased from the Sasol North taining a steady rate of spike-wave discharges) (Figure 2)had American distributor. been established and recorded (for a minimum of 15 minutes), participants consumed a single dose of triheptanoin oil over 1 Procedures to 3 minutes in amounts ranging from 15 to 60 mL according to The experimental procedures are illustrated in Figure 1. Each body weight (0.75-1.0 g/kg). Electroencephalographic monitor- patient underwent a pretreatment medical history, complete ing was continued for at least an additional 90 minutes after par- physical and neurologic examinations, general analytical labo- ticipants consumed the oil. Neuropsychological testing was re- ratory evaluation (following overnight fasting), electroencepha- peated approximately 90 to 120 minutes after oil ingestion. Blood logram (EEG), and neuropsychological evaluation. Partici- samples were collected from each participant before and at the pants who consented to imaging also underwent a 30-minute conclusion of the EEG. A seizure rate, defined as the total du- MRI examination of the total rate of oxygen consumption by ration of EEG-recorded spike-wave seizures divided by the total the brain (ie, the cerebral metabolic rate [CMRO2]). duration of the EEG recording, was calculated for the baseline All participants underwent EEG recording. The EEG was period (before triheptanoin oil consumption) and posttrihep- acquired by placing an MRI-compatible electrode cap contain- tanoin period (from 5 minutes after triheptanoin oil consump- ing 32 active electrodes except where noted below. An addition to the conclusion of the EEG recording).

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Figure 2. Electroencephalogram (EEG)-Captured Spike-Wave Seizures in Patients With Glucose Transporter Type 1 Deficiency

A B

Seizure 1 s Fp1 onset Duration Fp2 F3 F4 C3 Cz C4 P3 P4 O1 O2 500 µV F7 F8 Seizure state T7 T8 P7 Nonseizure state P8 Fz Cz C Pz Oz Transient FC1 period FC2 Before oil After oil CP1 Seizure state CP2 FC5 FC6 Nonseizure state CP5 CP6 TP9 TP10 0 500 1000 1500 2000 2500 3000 3500 4000 4500 POz Time, s

A, An example segment of an EEG recording containing 3 spike-wave seizure participant. Spike-wave periods are represented as bar plots against an EEG periods. Electrode positioning followed standard abbreviated nomenclature. time course in the setting of a nonseizure baseline state. The pink bar indicates Vertical bars indicate 1 second. B, Illustration of the identification of seizure triheptanoin (oil) administration. duration. C, The seizure-nonseizure binarized time course of a representative

Participants who consented to imaging underwent MRI be- short administration time, and availability of parallel forms, fore and after triheptanoin administration. The second MRI allowing for quick readministration without the concern of took place 60 to 90 minutes following triheptanoin consump- practice effects.50 Administration of the tests took approxi- tion. This time interval was selected to capture the peak me- mately 30 to 45 minutes per testing session. tabolism of triheptanoin in blood as separately determined by us in 11 participants (C.R.R., unpublished data, 2006). Mag- 3-Month Triheptanoin Consumption netic resonance imaging was performed before and after tri- To investigate its long-term effect on outcome measures, triheptanoin consumption and immediately after each session heptanoin oil was administered for 3 months at approxi- of neuropsychological testing, followed by a 5-minute rest pe- mately 1 g/kg of body weight per day, divided into 4 doses per riod. The EEG was recorded continuously during the MRI scan. day, given 30 to 60 minutes before a routine meal. This dose Magnetic resonance imaging included a routine axial T1- represents approximately 30% of the total dietary fat. Partici- weighted sequence to establish that brain configuration was pants followed additional dietary modifications with the goal normal in all participants and, more importantly, allowed for of maintaining a constant total daily fat intake and body weight. CMRO2 quantification,42-47 with a test-retest CMRO2 reliabil- A reduction in simple carbohydrate consumption was insti- ity of less than 5% error. In brief, using phase-contrast MRI and tuted with the intent of lowering the glycemic index so as to T2-relaxation underspin tagging MRI measurements, the minimize insulin release, interference with ketogenesis, or ex- whole-brain arteriovenous oxygenation gradient was deter- cessive weight gain. Daily caloric and protein intake re- mined and used to calculate brain oxygen extraction with the mained unmodified. aid of full-brain volumetric measurements excluding cerebrospinal fluid. Scan acquisition time was 5 minutes per MRI. No Long-term Follow-up sedation or stimulation was administered. Participants were evaluated at 3 months and 6 months for fol- Neuropsychological testing consisted of the Peabody Pic- low-up testing and posttreatment advice as necessary. Trihep- ture Vocabulary Test, Fourth Edition,48 (PPVT-4) and the Ex- tanoin was discontinued at the 3-month visit. Follow-up vis- pressive Vocabulary Test, Second Edition (EVT-2).49 These 2 its included complete physical and neurologic examinations, tests were chosen for their ease of administration, relatively general analytical laboratory analyses, and neuropsychologi-

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cal testing. Additional CMRO2 determination or an EEG was perienced significant (10%) weight gain at 2 months that did performed as described below for select participants. not lead to discontinuation. This patient did not follow the rec- ommended dietary and nutritional advice by decreasing ex- Longer-term (Indefinite) Consumption of Triheptanoin tra sources of fat and simple sugars. Two patients (14%) expe- All participants who reported clinical benefit and requested rienced diarrhea and/or digestive discomfort within days of to continue triheptanoin beyond the 3-month visit were re- treatment initiation, but these symptoms resolved by reduc- moved from the initial research protocol. To continue trihep- ing the dose of triheptanoin by one-half and gradually increas- tanoin for an indefinite period, patients voluntarily submit- ing the amount to the target levels over several days. ted a signed letter requesting continuation and then were enrolled in a second institutional review board–approved pro- Correlation Between EEG and Absences tocol, agreeing to provide semiannual blood tests and gen- All 14 patients were observed by their primary caretaker while eral physical examination reports. Additional contact was undergoing EEG monitoring. Patients considered to be ma- maintained with each of these participants via telephone or ture and their caretakers were asked to report all absence events secure, Health Insurance Portability and Accountability Act– that they noticed. Patients and caretakers had no access to the compliant e-mail communications as needed. EEG tracing and were visually supervised in a separate room through a window by the EEG recording operator. The EEG op- Statistical Analysis erator (P.L. and D.M.) and an expert neurologist (J.M.P.) noted The primary outcome of our study was a reduction in electro- the correlation between reported absence or any other poten- graphic seizures as measured by seizure rate (total time of the tial seizure events and the EEG tracing. All of the participants seizures divided by the total EEG-recorded time) and in- who manifested spike-waves, including the caretakers, under- creased neurocognitive performance as measured by the PPVT-4 reported EEG spike-wave seizures. For 7 patients, more than and EVT-2. Secondary outcomes included CMRO2. Descriptive 50% of spike-wave seizures lasting longer than 3 seconds (ie, statistics are provided for demographic and clinical measures. EEG absence seizures that are typically documented in clini- Wilcoxon matched-pairs signed rank tests were performed on cal neurophysiological reports) were not noticeable by the ob- EEG and CMRO2 data before and after triheptanoin consump- server as absences or other abnormal behavior. For 4 partici- tion at baseline and at baseline and 3-month measures of EEG, pants, no absence or other abnormal manifestation was neuropsychological measures, and blood analyses (total cho- reported despite the occurrence of abundant spike-wave sei- lesterol, high-density lipoprotein cholesterol, low-density li- zures lasting longer than 3 seconds. poprotein cholesterol, triglycerides, glucose, and β-hydroxybutyrate levels). SPSS, version 20 (SPSS Inc), was used to analyze Immediate Response to Therapy these data using a 2-sided P value (unless otherwise noted). EEG Recordings The EEG recordings were analyzed as described in previous work51-53 with the exception that only the clinically relevant Results aspects of the EEG (ie, those that are part of a standard EEG report) were analyzed. After the standard consent procedure, Patient Characteristics triheptanoin was urgently initiated in the inpatient setting in Fourteen patients with a G1D diagnosis were enrolled (eTable 1 2 of the 14 patients (14%) because of unmanageable epilepsy in the Supplement). The median age at enrollment was 12.5 years (absence seizures more frequent than hourly). Thus, baseline (range, 2-27 years), and 6 of 14 patients (43%) were female. Most research EEG recordings were not performed in these pa- patients identified themselves as white (12 [86%]) and non- tients as described above and were not included in the quan- Hispanic (13 [93%]); 1 patient (7%) was African American and 1 titative EEG analysis. However, these patients were receiving patient (7%) was white and Asian. The median age at the onset EEG monitoring just as triheptanoin was initiated, and its ad- of symptoms based on 13 (93%) patients was 8.5 months (range, ministration was followed by the termination of absences and 1 month to 30 months), and the median age at diagnosis based a decrease in EEG spike-waves within 2 hours after trihepta- on 11 (79%) patients was 7.3 years (range, 2-27 years). Patients noin ingestion. Of the 12 remaining patients, 1 patient (8%) ex- were enrolled from the United States and Canada. hibited no EEG-documented or observable absence seizures Of the 14 patients enrolled in the trial, 5 participated in at baseline for the duration of the EEG recording and was ex- imaging. These patients were older than 15 years. The 9 pa- cluded from the EEG data analysis. The overall self-reported tients who elected not to participate in imaging did so for rea- or parent-reported absence seizure rate was below 20% of all sons including the inability to remain immobile owing to move- electrographic spike-wave seizures regardless of EEG seizure ment disorder or anxiety (5 [36%]), immaturity (2 [14%]), metal frequency or duration. implants preventing exposure to research MRI (1 [7%]), and per- Electrographic spike-wave seizures were precisely cap- sonal choice (1[7%]). tured by EEG recording (Figure 3). There were no other types of EEG-documented seizures in any of the patients. The sei- Adverse Events zure rate was calculated both before and after triheptanoin ad- No patient experienced serious or unexpected adverse events. ministration for each participant by dividing the total dura- One patient (7%) discontinued triheptanoin therapy after 3 tion of recorded spike-wave seizures by the total duration of weeks owing to gastric discomfort. One other patient (7%) ex- the EEG recording (eTable 2 in the Supplement and Figure 2).

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Figure 3. Seizure Rate Reduction After Acute Triheptanoin Oil Consumption

A B Seizure rate Seizure rate (except GD002) GD001 GD002 0.9 0.20 GD003 GD005 0.18 0.8 GD006 0.16 GD008 0.7 GD009 0.14 GD011 0.6 GD012 0.12 GD013 0.5 A, Seizure rate of all participants GD014 0.10 before and after triheptanoin oil 0.4 consumption. B, Data representing 0.08 the seizure rate of all participants 0.3 0.06 except GD002 (who exhibited the largest seizure rate) before and after 0.2 0.04 triheptanoin oil consumption. The 0.1 0.02 vertical axis (note the different ranges) represents the fractional seizure rate 0 0 in both panels. Participant GD007 Before After Before After manifested no seizure or other EEG Triheptanoin Triheptanoin Triheptanoin Triheptanoin abnormalities, and triheptanoin had no effect on his EEG.

Figure 4. Neuropsychological Indices in Patients With Glucose Transporter Type I Deficiency (G1D) After Triheptanoin Food Supplementation

A B GD001 GD003 100 110 GD005 GD006 90 GD007 90 GD009 80 GD011 GD012 70 70

60 50 50

40 30

EVT-2 EVT-2 EVT-2 PPVT-4 PPVT-4 PPVT-4 1A 1B 2 1A 1B 2

Vocabulary performance improved acutely and over long-term follow-up with (PPVT-4 2). PPVT-4 and EVT-2 scores were below normal for the age ranges and triheptanoin supplementation. The neuropsychological scores of all 8 participants increased at subsequent time points in rigorously statistically significant fashion. before and after triheptanoin use are represented. Standardized Peabody Picture The interquartile ranges (IQRs) for the PPVT-4 scaled scores at each of the 3 time Vocabulary Test, Fourth Edition (PPVT-4), and Expressive Vocabulary Test, points were 59-75, 62-86, and 61-84, respectively. PPVT-4 scores improved Second Edition (EVT-2), ratings were obtained in the fasting state (baseline) at significantly over time (Friedman test; P = .03). The IQRs for the EVT-2 scaled time 0 minutes (PPVT-4 1A) and 60 minutes (PPVT-4 1B) following triheptanoin scores at each of the 3 time points were 60-80, 57-84, and 65-89, respectively. ingestion and then after 3 months of daily triheptanoin supplementation EVT-2 scores improved significantly over time (Friedman test, P = .02).

As shown in Figure 2B, the duration of each spike-wave seg- At the baseline visit, patients underwent testing while fast- ment was delimited by the onset of the first spike and the re- ing and then again approximately 1 hour after ingesting tri- turn to the baseline rhythm immediately after the last wave. heptanoin. During this visit on the PPVT-4 evaluation, the re- The rates of seizures before and after triheptanoin oil admin- ceptive vocabulary improved in 7 of 8 participants (88%) and istration at baseline decreased in all patients (P = .001) (Figure 3). worsened in 1 participant (12%) (P = .04). On the EVT-2 evaluation, 5 of 8 participants (62%) improved and 2 participants Neuropsychological Indices (25%) worsened (P = .12). Overall, 8 of 8 participants showed Eight patients (57%) completed baseline neuropsychological improved scores on some aspect of neuropsychological test- testing. The first 3 participants did not undergo neuropsycho- ing between the fasting baseline evaluation and testing an hour logical testing owing to logistic limitations, and 2 partici- after administration of triheptanoin, but these changes were pants (14%) were urgently enrolled as inpatients as described not significant (Figure 4). above. Therefore, the PPVT-4 and EVT-2 tests could not be administered to them. In addition, 1 patient completed baseline Blood Glucose neuropsychological testing but did not return for testing at 3 Blood glucose levels were measured 90 to 120 minutes after months and was therefore excluded from the analyses. In all, triheptanoin consumption at the baseline visit and compared the PPVT-4 and EVT-2 could not be administered in 6 patients. with fasting levels collected earlier that morning. A Wilcoxon

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matched-pairs signed rank analysis yielded a significant de- Figure 5. Magnetic Resonance Imaging–Measured Cerebral Metabolic crease (fasting glucose median, 87 mg/dL; posttriheptanoin glu- Rate (CMRO2) in Patients Before and After Acute Triheptanoin Oil (C7) cose median, 81 mg/dL; P = .02) (to convert glucose to milli- Consumption moles per liter, multiply by 0.0555). 180 Before C7 175 Cerebral Metabolic Rate After C7 170 Five patients completed imaging at baseline. The median per- 5-6 mo after C7 centage change in CMRO2 from baseline following the first ad- 165 ministration of triheptanoin was 5.9%, but this change was not 160

significant (P = .31). Three participants (60%) experienced an 155

increase in the cerebral metabolic rate, and 2 (40%) experi- 150

enced small changes (Figure 4). 145 CMRO2, µmol/100 g/min 140 Response at 3-Month Follow-up 135 Neuropsychological Testing 130 When baseline fasting PPVT-4 and EVT-2 scores were com- M, 14 y M, 16 y F, 17 y M, 21 y M, 28 y pared with scores at the 3-month follow-up, immediate gains GD008 GD009 GD013 GD011 GD007 (ie, those detected after the first triheptanoin dose) were main- In participants GD009 and GD007, CMRO2 was measured again 5 to 6 months tained on the PPVT-4 (ie, all 7 patients [88%] who exhibited after triheptanoin oil consumption. The reference values for CMRO2 have been immediate improvement continued to manifest similar im- measured by us in a broad adult age range and are sex dependent (ie, females provement at the 3-month follow-up). Immediate gains were exhibit higher CMRO2 than males). At ages 21 and 28 years, the CMRO2 also maintained on the EVT-2, with 2 additional patients (25%) reference values for men are 149 and 154 μmol/100 g/min, respectively. The SD for none of our normal CMRO2 measurements exceeded 12.5 μmol/100 g/min demonstrating improvement, for a total of 7 of 8 patients (88%) (unpublished findings). showing improvement at the 3-month follow-up. Significant improvement in the PPVT-4 and EVT-2 scores were observed from baseline to the 3-month follow-up (P = .04 and P =.02, heptanoin therapy at that time owing to weight gain. Ten par- respectively) (Figure 4). ticipants (91%) signed informed consent to continue treatment with triheptanoin as described above. Of those 10 Laboratory Tests participants, 1 patient (7%) discontinued triheptanoin after 5 Blood glucose, total cholesterol, high-density lipoprotein cho- months of use because of lack of efficacy, although parental lesterol, low-density lipoprotein cholesterol, triglycerides, and reporting indicated nonadherence. All other participants con- β-hydroxybutyrate levels were compared between baseline and tinued to receive triheptanoin without adverse effects at the the 3-month follow-up using Wilcoxon signed rank tests. All time of the last assessment for a total of 185 patient-months. values were within the laboratory reference ranges. There were Longer-term blood glucose determinations as described no significant differences (P > .05; not shown). in the Methods section were performed at least semiannually for all patients who continued to receive triheptanoin. No ab- Cerebral Metabolic Rate normality and no significant change in glycemia were noted Two patients (14%) participated in imaging at the 5- to 6-month on these tests, suggesting that triheptanoin, administered with follow-up evaluation under a separate institutional review board– a concurrent reduction in simple carbohydrates, does not ex- approved protocol. The first of these participants (designated ert its effects via a change in blood glucose levels unless any GD007), who had manifested no EEG-documented seizures and extra production of glucose is balanced with an equally in- no change in the CMRO2 after triheptanoin ingestion, demon- creased rate of consumption. strated a 17% increase in the CMRO2 from fasting baseline levels. The second patient (designated GD009) manifested no significant change in the CMRO2 relative to a previous significantly Discussion increased CMRO2 after triheptanoin initiation (Figure 5). This study was motivated by 2 reasons. First, the pharmaco- Continuation of the Medical Food Supplementation Regimen logic treatment of G1D-associated seizures is generally inef- Of the 14 participants, 12 (86%) completed 3 months of trihep- fective. A potential role for several specific antiepileptic drugs tanoin consumption and 11 (78%) returned for the 3-month fol- was suggested by the spike-wave EEG pattern often identi- low-up. One participant (7%) discontinued triheptanoin ear- fied in G1D seizures, which is typical of some absence lier than stipulated owing to intolerable gastric discomfort, and epilepsies.54,55 Yet this approach has proven to be ineffective.16 1 (7%) discontinued triheptanoin because of parent-reported In practice, antiepileptic drug use and discontinuation in G1D lack of efficacy. Of the 12 participants who completed 3 months remains subordinate to trial-and-error interventions.16,38,56-58 of therapy, 1 individual (7%) declined to return for follow-up Second, we noted that, in normal conditions, an impor- owing to financial hardship. tant brain glucose–derived anaplerotic process is catalyzed by Of the 11 participants (79%) who returned for the 3-month pyruvate carboxylase inside the astrocyte,59,60 which de- follow-up, only 1 individual (9%) elected to discontinue tri- pends on glycolysis (ie, on pyruvate generation) to refill TCA

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cycle precursors and maintain byproduct output including glu- task. Perhaps the most striking finding was in the very young tamate, glutamine, and γ-aminobutyric acid.61 Neurons may participants (aged 2 and 4 years) who were significantly de- carry out only a modest degree of anaplerosis via the malic layed in all aspects of intellectual and motor function; they enzyme.62 In contrast, common (ie, natural) dietary fats are achieved rapid improvements in developmental milestones, oxidized into only even–carbon number ketone bodies (β- meeting them at age-appropriate intervals. hydroxybutyrate and acetoacetate),22 which leads to acetyl- coenzyme A generation, but cannot sustain anaplerosis be- Cerebral Metabolic Rate cause the even 4-carbon-number ketone bodies are fully The CMRO2 is diminished in G1D (Figure 5). In contrast with the converted into water and carbon dioxide in the TCA cycle.21,29 cerebral metabolic rate characteristic of healthy individuals who Thus, any ketogenic diet in use today, especially when it re- were the same age as participants in the present study,63 pa- sults in relative hypoglycemia, represents a rudimentary— if tients with G1D demonstrate decreased but uniquely different not a restrictive—treatment for G1D when considered from this CMRO2 values. This phenomenon may reflect the broad phe- perspective. Even–carbon number ketones, generated from notypic diversity of our case series, of which the CMRO2 is but common dietary fat or a ketogenic diet, ameliorate seizures but one aspect. Because G1D is a lifelong genetic disorder that af- are not anaplerotic, as underscored by the fact that the 2 key fects the brain in early infancy,the maximum achievable CMRO2 metabolic roles of glucose are (1) energy production by oxida- level is unknown should patients experience full restoration of tion of acetyl-coenzyme A and (2) anaplerosis by providing py- glucose influx after the disorder becomes symptomatic for an ruvate for carboxylation. Ketogenic diets can fulfill the first role, extended developmental period of their lives or even if the age- but the diet’s fat cannot meet the second role. In contrast, stud- normal CMRO2 level can be exceeded as the consequence of a ies have shown that mouse brain nuclear magnetic resonance favorable therapeutic effect. It is not plausible, nor was it at- and mass spectrometry32 indicate that heptanoate is anaple- tempted, to safely advance general correlations between CMRO2 rotic because it can satisfy both roles.33 Further observations and EEG spike-wave frequency or neuropsychological indices; made in transgenic antisense G1D mice,13 which appeared nor- this endeavor was outside the scope and inferential power of mal at birth but later developed frequent spontaneous sei- the present study. Therefore, only individual observations de- zures, decreased brain weight, spasticity, and ataxia (thus, serve remarks. Of 5 participants who underwent CMRO2 deter- closely mimicking the most commonly recognized human G1D mination, 3 (patients GD008, GD009, and GD011) demon- syndrome), also converge on the potential use or overuse of strated rapid (ie, consistent with the cerebral metabolism of alternative metabolic sources by the brain in G1D. In the state triheptanoin metabolites), significant increases (Figure 5). The of depleted brain glucose accumulation and spontaneous sys- CMRO2 of patients GD013 and GD007 did not increase after acute temic ketosis typical of G1D mice, whole-brain TCA cycle in- triheptanoin ingestion. Patient GD013 exhibited spike-wave sei- termediates and neurotransmitter contents are preserved, un- zures that were abolished by triheptanoin (Figure 3), and pa- dermining the hypothesis of global brain energy failure while tient GD007 did not exhibit spike-wave seizures before or after suggesting that TCA refilling proceeds via enhancement of al- triheptanoin use. The robustness of the CMRO2 measure- ternative metabolic fluxes, such as fatty acids.13 This was il- ments prompts an explanatory framework for these single- lustrated for heptanoate as noted above.33 patient observations. The simplest interpretation of all of the results reported above, should they prove to be a general fea- Clinical Outcomes ture of G1D, is that triheptanoin metabolism may lead to in- As indicated by the blood analytical values, there was no sig- creased oxygen consumption only while the brain undergoes nificant change in glucose levels or lipid levels, which indi- a reduction of ictogenesis. In parallel to this contention, we hy- cates that the addition of triheptanoin to the diet is safe and pothesize that, when ictogenesis is abolished by triheptanoin metabolically neutral as assessed by these analyses. or absent at baseline, triheptanoin exerts little or no effect on Because we identified a prohibitively small detection rate CMRO2. Because the relationship between ictogenesis and of absences by mere observation, EEG recordings served as the whole-brain oxygen consumption is unknown (as determined most appropriate quantifiable outcome. The reduction in EEG by the global encephalic CMRO2 reported in the present study) spike-wave seizures and neuropsychological results of recep- and because G1D-associated spike-wave seizures may be caused tive and expressive vocabulary were favorably affected by tri- by aberrant thalamocortical synchronization rather than global heptanoin. Other nonclinical data and nonsystematic evi- or multifocal epileptogenesis,13 further work currently in prog- dence also support the clinical improvement noticed by many ress will aim to elucidate CMRO2 changes in regional ictogenic patients. The voluntary continuation rate (11 of 14 patients at G1D tissue. In addition, the significant increase in CMRO2 for 3 months and 10 of 14 patients at 6 months) is indicative of im- patient GD007 after 6 months of treatment is notable. Despite provement. Several parents spontaneously reported that other the absence of epilepsy as detected by observation and by EEG, caregivers (school teachers, speech therapists, and physical this finding is compatible with triheptanoin-induced long- therapists) who were unaware of the patients’ study partici- term changes in the nonepileptic G1D brain. pation noted marked improvement in both motor and cognitive skills. This is consistent with improvement on the vocabu- Potential Therapeutic Mechanisms of Triheptanoin lary tests, which measure not only language ability but also in Human G1D general intellectual function, because patients are required to Glucose supports brain metabolism, and neurometabolic dis- maintain attention and concentration for the duration of the eases involving dysfunctional glucose metabolism are often

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associated with intractable absence seizures.5 Additional mol- have been in use for decades (eg, EEG, quality of life, and in- ecules, such as fatty acids from ketogenic diets and their de- tellectual assessments). The best treatments include those that rivative ketone bodies, can partially substitute for glucose ex- target causes, as we believe triheptanoin does. However, ob- cept that (1) normally or postprandially increased glycemia servation need not be synonymous with causation: absence sei- interferes with ketosis, necessitating that canonical keto- zures, such as those noted in G1D, are temporally well- genic diets are paradoxically, for brain states associated with circumscribed impairments of consciousness associated with decreased glucose flux,64 carbohydrate restricted; (2) mito- visually observable manifestations.72 During a typical epi- chondrial fatty acid oxidation can compete with glucose sode of absence, EEG recording reveals repetitive spike- metabolism65; and (3) all food-derived fats and ketone bodies waves that often last more than 3 seconds.73 However, observ- contain even-chain carbons that are fully consumed in the TCA able absences, EEG recordings, and sustained attention can be cycle and excreted as water and carbon dioxide, thus yielding differentially affected by drug treatment,74,75 supporting the no net carbon to compensate for the natural loss of meta- limited causality clause described above. We anticipate that fur- bolites.2,29,66 This compensation is normally provided by ther ways of assessing epilepsy will emerge and have thus fol- anaplerosis, which in the brain stems principally from glu- lowed a focused approach here. cose via carbon-donor reactions67 that are unavailable in the ketogenic diet. In contrast with ketogenic diets, the metabo- Implications for Further Trials: Is Triheptanoin a Drug, lism of triheptanoin generates both even-carbon and 5-car- Prodrug, or a Medical Food? bon ketones (β-hydroxypentanoate and β-ketopentanoate) in The International Union of Pure and Applied Chemistry defi- the liver, and the latter are anaplerotic,29,68,69 potentially af- nition of a prodrug is a compound that undergoes biotransfor- fording superior benefit. Studies13 have shown that G1D is as- mation before exerting pharmacologic effects. Prodrugs are sociated with cerebral carbon depletion in mice, and the re- thus canonical drug precursors containing specialized non- sults of the present trial support prior findings37 of reduced toxic protective groups used in a transient manner to alter or cerebral oxygen consumption (CMRO2) in man. eliminate undesirable properties in the parent molecule. In contrast, the term medical food, as defined in section 5(b) of the Ictogenesis, Spike-Waves, and Absence Epilepsy in G1D Orphan Drug Act (21 U.S.C. 360ee [b] [3]), identifies any food In the present study, we attempted to follow a mechanistic ap- that is formulated to be consumed or administered enterally proach by targeting what we believe to be factors that repre- under the supervision of a physician and is intended for the sent primary causal events. This framework is largely based on specific dietary management of a disease or condition for which our work illustrating that decreased glucose flux leads to ce- distinctive nutritional requirements, based on recognized sci- rebral metabolic deficit that, in turn, differentially affects ex- entific principles, are established by medical evaluation. In this citatory and inhibitory synaptic balance, as well as on the fact sense, triheptanoin fulfills the requirements of a medical food. that these aberrant phenomena are poised to be relevant to disease pathogenesis based on the plausible direct relationship be- Limitations and New Questions tween metabolism, synaptic function,70 and epileptogenesis.71 One patient discontinued triheptanoin because of gastroin- Furthermore, these phenomena are favorably modifiable in pa- testinal intolerance; this unintended effect has been ob- tients with G1D and in mouse brain slices by increasing blood served with oil consumption and can be ameliorated by glucose or ketone body availability. We have observed that varying the administration schedule or by administering tri- (1) G1D is associated with diminished cerebral glucose flux (re- heptanoin in solid form or in an emollient, such as sugar-free sulting in decreased brain acetyl-coenzyme A); (2) there is no and fat-free yogurt or pudding. intrinsic neuronal excitability dysfunction or gross structural The age range of the patients enrolled was 2 through 27 abnormalities as suggested by the rapid reversibility of abnor- years, and this heterogeneity in ages implies heterogeneity in malities shown on the human EEG and of key synaptic cur- brain metabolism rates. The dose administered (1 g/kg), al- rent abnormalities with glucose or ketone bodies; and (3) brain though appropriate for older adolescents and adults, did not glucose is used for anaplerosis, and stimulating this process may take into account the younger ages, and therefore higher me- be of additional benefit when glucose availability is de- tabolism, of some of the participants. Future trials using bio- creased. Given these observations, we reasoned that provid- logically relevant age stratification can help to assess the ef- ing both a source of acetyl-coenzyme A and a source of anaple- fect of age on outcomes over changing levels of metabolism rotic carbon (via triheptanoin metabolism) results in a treatment through a person’s development. strategy based on disease mechanisms and one that antiepileptic drugs or the ketogenic diet do not target. This argumen- tation represents a reductionist approach such that comple- Conclusions mentary interventions targeting secondary excitability or other abnormalities may be needed, as is the case with most neuro- The effect of dietary food-grade triheptanoin on central dis- logic diseases. In line with the necessarily reductionist ap- ease-relevant phenomena is manifest in a broad age range of proach outlined above, we selected the EEG as an important patients with G1D-associated epilepsy exhibiting varying de- outcome. However, there are many nonobservable biological grees of disease severity and not receiving a ketogenic diet. To aspects in epilepsy; consequently, the field continues to be our knowledge, this is the first systematic study of a thera- driven by the most reliable and accessible indicators, which peutic agent in G1D. Based on the glucose dependence of the

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abnormalities cited above, we reasoned that, if triheptanoin formance, suggesting that triheptanoin is effective in amelio- operates on mechanisms relevant to disease pathogenesis, the rating the brain glucose depletion state associated with G1D therapeutic outcome should be promptly noticeable (ie, con- encephalopathy. The results establish that triheptanoin is en- sistent with the intestinal absorption, hepatic metabolism, and dowed with sufficient potential to favorably and signifi- brain penetration of triglyceride metabolites) and sustained cantly affect outcome measures directly relevant to G1D and while the therapy is maintained. All patients with G1D who encourage further trials. The new trials should include young were receiving triheptanoin experienced decreased spike- infants and patients with nonconvulsive forms of G1D and ge- wave seizures, and several patients exhibited a rapid increase netically unrelated encephalopathies associated with de- in the cerebral metabolic rate (ie, those with the highest fre- creased glucose metabolism or abundance, as noted in sev- quency of spike-waves) and improved neuropsychological per- eral neurodegenerative diseases.

ARTICLE INFORMATION supported by NIH grant NS065640. Dr Lu is 330.338/H327]. Paper presented at: Society for Accepted for Publication: May8,2014. supported by NIH grants NS067015 and Neuroscience Annual Meeting; Chicago, IL; October MH084021. 19, 2009. Published Online: August 11, 2014. doi:10.1001/jamaneurol.2014.1584. Role of the Funder: The funding organizations had 13. Marin-Valencia I, Good LB, Ma Q, et al. Glut1 no role in the design and conduct of the study; deficiency (G1D): epilepsy and metabolic Author Affiliations: Rare Brain Disorders Program, collection, management, analysis, and dysfunction in a mouse model of the most common Department of Neurology and Neurotherapeutics, interpretation of the data; preparation, review, or human phenotype. Neurobiol Dis. 2012;48(1):92-101. The University of Texas Southwestern Medical approval of the manuscript; and decision to submit 14. Akman CI, Engelstad K, Hinton VJ, et al. Acute Center, Dallas (Pascual, Kelly, Good, Ma, Marin- the manuscript for publication. Valencia, Zhang, Roe); Department of Physiology, hyperglycemia produces transient improvement in glucose transporter type 1 deficiency. Ann Neurol. The University of Texas Southwestern Medical REFERENCES Center, Dallas (Pascual); Department of Pediatrics, 2010;67(1):31-40. The University of Texas Southwestern Medical 1. Pascual JM, Wang D, Lecumberri B, et al. GLUT1 15. Wang D, Pascual JM, Yang H, et al. Glut-1 Center, Dallas (Pascual, Marin-Valencia); Eugene deficiency and other glucose transporter diseases. deficiency syndrome: clinical, genetic, and McDermott Center for Human Growth and Eur J Endocrinol. 2004;150(5):627-633. therapeutic aspects. Ann Neurol. 2005;57(1):111-118. Development/Center for Human Genetics, The 2. Marin-Valencia I, Roe CR, Pascual JM. Pyruvate 16. 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