Global Medicine and Therapeutics

Research Article

ISSN: 2516-7065

The role of antioxidant proteins in improvement of autism
core symptoms in two patients with tuberous sclerosis
treated with mTOR inhibitor everolimus
Kunio Yui1*, Hitomi Sasaki1, George Imataka2 and Toru Okanishi3

Department of Urology, Fujita Health University School of Medicine, Aichi 470-1192, Japan
Department of Pediatrics, Dokkyo Medical University, Mibu 321-0293, Tochigi, Japan3
Department of Neuropediatrics, Seirei Hamamatsu General Hospital, Hamamatsu, 430-8558, Sizuoka, Japan
1
2

Abstract
Background: The neuropsychiatric clinical manifestations of tuberous sclerosis complex (TSC) include social and behavioral impairment, which are similar to thecore symptoms of autism spectrum disorder (ASD). The present study examined the clinical effects of the mTOR inhibitor everolimus for 24 weeks on social andbehavioral symptoms, and its relation of serum levels of the copper- and iron-binding antioxidants, such as ceruloplasmin (Cp) and transferrin (Tf), and superoxide
dismutase (SOD) in a pre-pubercent boy and a girl child with TSC.
Case presentation: MRI revealed a SEGA located at the foramen of Monro in the two patients and a renal angiomyolipoma (AML) on these patient’s kidney in the
one patient. Everolimus apparently improved the patients’ social as well as behavioral impairments over the course of a 24-week treatment. Slight reductions in sizeof SEGA and AML in one patient, and slightly reduced SEGA in another patient. Serum Cp and Tf levels were gradually increased in response to the improvementof autistic symptoms.
Conclusion: Increased antioxidant activity may contribute to the improvement in the ASD social as well as behavioral symptoms with a 24-week treatment of t
everolimus in two cases with TSC.

Introduction
Tuberous sclerosis (TSC) is a rare multi-system monogenic
hamartomatous disorder that affects almost every organ in the body[1]. Patients with TSC have neurological deficits including seizures andbehavioral and psychiatric deficits including autism spectrum disorder(ASD) with TSC-specific lesions such as subependymal giant cell
astrocytomas (SEGA) and renal angiomyolipomas (AML) [2,3].
Clinical characteristics of ASD in young patients with TSC prented
impaired social interaction including impairments of gestures, eyecontact, responsive social affect, and shared enjoyment [4], andbehavioral symptoms such as repetitive and stereotyped behavior [5,6].
These social and behavioral symptoms are completely similar to DSM5 criteria for the core social deficits and restricted, repetitive patterns of
behavior [7]. Approximately 60% of patients with TSC are diagnosed
with ASD [8].
Tuberous sclerosis complex 1 and tuberous sclerosis complex 2
are the tumor-suppressor genes and inactivating mutation in one ofthese genes may be resulted in hyperactivity of the manmalian targetof rapamycin (mTOR), inducing the tumorous symptoms in TSC [811]. Results from both human and animal studies have suggested thatmTOR inhibitors improve deficits in social interaction and abnormal
autistic behavior in human [5,6] and in TSC mouse models [12].
However, there are a few studies of the clinical effect of the mTOR
inhibitor everolimus on ASD symptoms. A previous review articles
suggested that mutation of TSC 1 and TSC2 were characterized byhyperactivity of mTOR, but the contribution of this mechanism to

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doi: 10.15761/GMT.1000101

development of ASD still unclear [13,14]. Moreover, according to aprevious review article, animal models with heterozygous mutations in
Tsc1 or Tsc2 exhibited ASD-like social deficit in the absence of corticallesion [14]. Thus, other neurobiological mechanisms may contributeto the development of ASD symptoms in TSC. Thus, Importantly, themTOR pathway is closely related oxidative stress [15] or antioxidant
capacity [16]. Oxidative stress-induced reactive oxygen species [17]enhanced mTOR [15]. Particularly, copper-related antioxidants, suchas ceruloplasmin (Cp) [18] and iron-related transferrin (Tf) [19] areclosely related to mTOR signaling. For example, Cp expression may berelated to activation of mTOR pathway [18]. Tf uptake modulates themTOR signaling pathway [20]. Thus, the ASD symptoms may dependon other neurobiological mechanisms.
This study attempted to examine the possibility that everolimus
treatment may improve the ASD symptoms in relation to increased
serum levels of Cp and Tf levels in two cases with TSC

Correspondence to: Kunio Yui MD PhD, Visiting Professor, Department ofUrology, Fujita Health University, Toyoake, Aichi, Japan, Tel: 81 78 791 8981;
Fax: 81 79 237 8755; E-mail: yui16@bell.ocn.ne.jp
Key words: Tuberous sclerosis, mTOR inhibitor, everolimus, autism spectrum
disorder, ceruloplasmin, transferrin
Received: February 02, 2018; Accepted: February 12, 2018; Published: February
15, 2018

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Yui K (2018) The role of antioxidant proteins in improvement of autism core symptoms in two patients with tuberous sclerosis treated with mTOR inhibitor
everolimus

Case presentation
Case 1 (patient A)
Subject characteristics: The patient A was 11 years old male patientwith TSC accompanied with the core ASD symptoms. He was born at
37 weeks of gestation by normal delivery with 2,856 g. At the age of 13
months, the patient experienced first episode of typical infantile spasms,
which is an age-related epileptic encephalopathy, and catastrophic
status [21], consisting of a sudden extension of the axis and proximal
limb musculature with a duration of 30 minutes, and the patient was
treated with predonine and benzodiazepine for four months. From
the age of 17 months, the patient was treated with various types ofantiepileptics, including sodium valproate and carbamazepine until 12
years of age, and his epileptic seizure never recurred.

MRI findings
At the age of 8 years, brain magnetic resonance imaging (MRI)
showed a subependymal giant cell astrocytoma (SEGA) (2.1cm x
4.0cm) located at the right foramen of Monro within a coronal T2weighted image (Figure 1a, white arrow). At the age of 9 years old, hereceived keyhole craniotomy for this SEGA. Eight months after his
keyhole operation (at the age of 10 years), MRI revealed small SEGA
(0.5 X 0.4 cm) on the left foramen of Monro (Figure 1b, white arrow),and two small renal angiomyolipomas (AML) (both were 0.8 cm X 0.8cm) (Figure 1c) on the patient’s left kidney and multiple renal cystes on
the right kidney (Figure 1c, small white arrows). These MRI findings
including two major diagnostic criteria such as a SEGA and a AML met
the diagnostic criteria for TSC [22] without genomic analysis.

ASD symptoms
The patient never exhibited any deficits in social interaction andrepetitive behaviors until 5 years of age. From 6 to 10 years of age, thepatient’s impaired social interaction gradually developed. However,the patient did not receive any psychosocial or psychotherapeutic
treatment because the attending pediatrician’s doctors recognized his
social and communication deficits as organic TSC-related secondary
symptoms.
The patient never responds to many forms of non-verbal
communication, including facial expressions and engage in eye
contact with his family members and others because he was unable
to understand their needs. He often showed delayed speech andsometimes created neologisms and idiosyncratic language. His
speech was confined to narrow topics of expertise. Thus, he gradually

withdrew into his repetitive play and behavior. He exhibited delayed
acquisition of motor skills, and had difficulty with motor coordination,
postural control, and imitating the movements of other people. He had
a consistent habit of washing his face in the morning and maintained
this habit. When his routine was changed, he usually entered a state ofpanic. Because of his severe impairment in reciprocal social interaction,
he was moved from a normal school to a special needs school at the ageof 10 years.

Diagnosis of ASD
At the age of 11 years, the patient A participated in our clinical trial
at the Department of Pediatrics at Dokkyo Medical University, Japan.
Diagnostic evaluation was conducted using the Autism Diagnostic
Interview-revised (ADI-R) and Autism Diagnostic Interview Schedule(ADOS). This assessment was performed by two psychiatrists who
specialize in ASD. The ADI-R is a standardized, semi-structured,investigator-based interview for parents or caregivers of individuals
with ASD [23]. The ADI-R was used to diagnose ASD in many
individuals aged 4 and 18 years of age [24]. The ADOS was a semistructured, standardized assessment designed for use in the diagnostic
evaluation of individuals with suspected autism spectrum disorder(ASD) [25]. His ADI-R scores were above the diagnostic cutoff scoresfor reciprocal interaction (his score was 18, cutoff = 10), the verbal (hisscore was 14; cutoff = 8) and non-verbal communication (his score
was 10; cutoff = 7) and stereotyped behavior (his score was 4; cutoff= 13). The patient’s total score on ADOS module 2 algorithm showed
17.0, which was greater than the total scores of the ADOS (8.94 ± 9.71)
recently reported in 33 adolescents with high-functioning autism [26].
These findings indicated a definite ASD diagnosis. These findings
indicating a ASD diagnosis.

Intelligence test
At the age of 10 years, he received intelligent test using the Wechsler
Intelligence Scale for Children [27]. His total Intelligence Quotient(IQ) was 67, which was defined as borderline IQ [28].
Assessment of ASD symptoms
The patient’s ASD severity was assessed using the ADOS.
Social impairment was assessed using the Social Communication
Questionnaire (SCQ) and Social Responsiveness Scale (SRS) [29]. The
SCQ is a measure for parents/caregivers to assess their child’s reciprocalsocial interaction, communication, and repetitive, stereotyped domains[30] based on the ADI-R. The SRS is highly feasible for the quantitative
ascertainment of autistic social impairment [31]. Behavioral symptoms

Figure 1. Brain and kidney MRI findings of the patient A. a MRI revealed a subpendeymal giant cell astrocytoma (SEGA) located at the right foramen of Monro (white arrow); b Brain
MRI after keyhole operation, most of the SAGE was removed. At the 10 years old, small SEGA (0.5 x 0.4cm) was detected at the left foramen of Monro after two years and eight months(white arrow); c MRI showed left two low-density areas with negative Hounsfield unit values inside the kidney, suggesting a renal angiomyolipoma (AML) (white arrows) and four multiple
renal cystes (small arrows).

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Yui K (2018) The role of antioxidant proteins in improvement of autism core symptoms in two patients with tuberous sclerosis treated with mTOR inhibitor
everolimus

were assessed using the Aberrant Behavior Checklist (ABC). The ABC
is intended to evaluate treatment responses in psychopharmacologicaland behavioral intervention trials for children and adolescents withmental retardation [32] and normal IQ levels [33]. As shown in figure
2, the patient’s total score on the ADOS was greater than the total scorein 33 adolescents with high-functioning autism [25]. The patient A’s
SRS and ABC total scores were 125 and 113, respectively. These SRS
and ABC scores were greater than those of ASD individuals with an age
range of 13-27 years (SRS, 120.12; ABC, 60.14) [34]. The SCQ cutoff
score was compared with established cutoffs for ASD screening (scores≥15) [35] mal range (cut-off score ≥15).
Collectively, his social and behavioral symptoms were within
at least the moderate-severe range. Other evaluations, including an
ophthalmologic and dental examinations at the pretreatment and posttreatment screenings revealed no abnormalities.

Biochemical measurement
mTOR inhibition and vascular endothelial growth factor-D(VEGF) expression were closely associated with synergistic antitumor
activity [36]. Serum VEGF-D was used as a useful biomarker formonitoring treatment responses to the mTOR inhibitor sirolimus [37].
Moreover, the mTOR is closely associated with antioxidant
capacity [16,19,20,38,39], serum levels of antioxidant proteins such as
ceruloplasmin (Cp), transferrin (Tf) and superoxide dismutase (SOD)
were measured by the SRL. Ltd. (Tokyo, Japan).

Serum assay for serum VEGF-D
Blood samples were centrifuged at 1400 × g for 10 min within 30 minof collection. The samples were stored at −80°C until assay by ELISA(the Quantikine Human VEGF kit). The limits of sensitivity of the
ELISA are 31.2-2,000 pg/ml and sensitivity were 5 pg/ml (Funakoshi,Co., Ltd) [40]. All of the samples were measured in duplicate.

Serum assay for everolimus
Serum everolimus levels were determined after liquid extraction bya liquid chromatography-mass spectroscopy method with lower limitsof quantification for everolimus of 0.3 ng/mL [41].

Serum assay for ceruloplasmin, transferrin and superoxide
dismutase
Whole-blood samples were collected in EDTA tubes byvenipuncture and immediately placed on ice in a refrigerator. The
specialists at SRL, Inc (Tokyo, Japan) measured the plasma levels of
PUFAs, Cp, Tf and SOD. Seum Cp levels were measured using a Bering
BN ⅡNephelometer (Siemens Healthcare Diagnostics K.K., USA) [42].

Serum levels of SOD were assayed using an SOD Assay Kit (Takara
Bio, Tokyo) according to the cytochrome c method [43]. Serum Tflevels were estimated using a standard turbidimetric assay and an
automated biochemical analyzer (JCA-BM8000 series, JEOL Ltd.,Tokyo, Japan) [44].

Results of these assays
Serum levels of VEGF were 252.6 pg/ml at baseline and gradually
increased to peak values (315.1 pg/ml) at 12 weeks and then gradually
decreased from 16 (289.5 pg/ml) to 24 weeks (247.4 pg/ml) after
treatment. Serum everolimus levels were 9.58 ng/ml at 12 weeks,
increased to 18.1 ng/ml at 16 weeks, and then decreased to 7.32 ng/ml
at 24 weeks after treatment.
Importantly, serum Cp levels gradually increased from baseline(19 mg/dl) to 16 weeks (32 mg/dl) and then remained higher than thebaseline levels (Figure 3, upper panel). Serum Tf levels were gradually
increased from baseline (282 mg/dl) to peak values at 16 weeks (386mg/dl), and then gradually decreased at 24 weeks (288 mg/dl), in
accordance with symptom improvement (Figure 3, upper panel). While,serum SOD levels were slightly increased at 8 weeks of treatment, andthen slightly decreased at 16 weeks, suggesting less contribution to theefficacy of everolimus (Figure 3, upper panel).

Treatment
Keyhole Surgery in SEGAAt the age of 10 years old, he received keyhole craniotomy for
SEGA. “keyhole” method is conducted in the case of sufficient for
exposing large areas deep in tissue, enabling adequate removal anddecompression [45]. After this operation, although ASD symptoms
were not improved, progressive augmentation of the ASD symptoms
was not detected.

Treatment with everolimus and serum everolimus levels
The patient A was treated with 4.4 mg /day of everolimus for 24
weeks. The patient A’s body surface area was 1.47 m2, and thus themedian maintenance dosage of everolimus was 4.4 mg/m2/day basedon 3 mg/m2 in the Novartis indication. Serum levels during themaintenance dose were 9.58 ng/ml at 12 weeks, 18.1 ng/ml at 16 weeks,
15.1 ng/ml at 20 weeks, and 7.3 ng/ml at 24 weeks after the treatment.
Ointment was applied because of a slight degree of inflammation of
his oral mucosa as a common side effect of everolimus, however, nosevere or life-threatening side effects related to everolimus treatment
were observed. Everolimus induced remarkable improvement in thesocial and behavioral symptoms without dose adjustment during thetreatment.

Figure 2. Brain and kidney MRI findings of the patient B. a MRI revealed a subpendeymal giant cell astrocytoma (SEGA) located at the right foramen of Monro (white arrow); b Brain MRI
revealed four subpendeymal nodules (white arrow).

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Yui K (2018) The role of antioxidant proteins in improvement of autism core symptoms in two patients with tuberous sclerosis treated with mTOR inhibitor
everolimus

Figure 3. Total scores of Social Responsiveness Scale (SRS), Aberrant Behavior Checklist (ABC), Social Responsiveness Scale (SRQ) and Autism Diagnostic Observation Schedule(ADOS) in patient A.

Clinical symptoms after everolimus treatment
Everolimus treatment induced aooarent improvement in impaired
social cognition, social interaction, and mutual communication. Threemonths after the treatment, the patient A was able to understand andfollow some verbal commands issued by his mother and classmates,
and frequently communicated with his family members and classmates.
After four months, repetitive motions, such as swaying, or twirling
disappeared. He also had less trouble understanding other people’s feelings.
The patient A’s SRS, ABC, SCQ and ADOS total scores were
gradually decreased, indicating a decrease in the social and behavioral
consequences of ASD symptoms. Interestingly, the SRS and ABC total
scores decreased from 126 before treatment to 76 (40 % decrease), andfrom 113 before treatment to 54 following treatment (54 % decrease),
respectively compared with baseline. Thus, everolimus treatment
remarkably improved the patient’s social as well as behavioral
symptoms.
There was slight size reduction (0.3 cm) of the patient’s SEGA on
the right foramen of Monro after the 24-week treatment (Figure 2b).
The sizes of two AML were slightly reduced (0.4 cm) after 24 weeks oftreatment.

Case 2 (patient B)
Patient Characteristics
The second patient (Patient B) was a 6-year-old girl with TSC
accompanied by the core ASD symptoms and seizures. She was bornby spontaneous vaginal delivery and had a birth weight of 2060 g. She

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received phototherapy because of neonatal jaundice at the time of birth.
At the age of 6 months, she exhibited infantile spasms associated withage-related epileptic encephalopathy and developed a catastrophic
status [21] consisting of sudden extension of the axis and proximal
limb musculature for a duration of 2 to 3 minutes. At the age of 13
months, she experienced her first episode of complex partial seizures,
which are a presenting sign in many young children with TSC [1]. Her
seizures consisted of sudden loss of consciousness for 2 minutes andoccurred several times a week. The patient was treated with different
types of anticonvulsants, such as sodium valproate and carbamazepine.
Her epilepsy gradually increased in frequency and severity, with apoor response to the antiepileptic drugs. Her electroencephalogram
frequently showed spikes and spike-and-wave complexes at the central
areas (Cz and C3), which are sometimes recognized in patients withcomplex partial seizures [46].

MRI findings
At the age of 6 months, MRI revealed SEGA (0.5 X 0.5 cm) locatedat the right foramen of Monro within a coronal T2-weighted image(Figure 4, white arrow), and subpendymal nodules located at besideinterventricular form. There was no AML in her kidney. These MRI
findings including two major diagnostic criteria such as a SEGA andsubpendymal nodules met the diagnostic criteria for TSC [22]. Thus,
the diagnosis was confirmed without genomic analysis.

Autism spectrum disorder symptoms
From the age of 7 months, the patient B gradually developed
impairment of social interaction. She did not respond to many forms

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Yui K (2018) The role of antioxidant proteins in improvement of autism core symptoms in two patients with tuberous sclerosis treated with mTOR inhibitor
everolimus

Figure 4. Total scores of Social Responsiveness Scale (SRS), Aberrant Behavior Checklist (ABC), Social Responsiveness Scale (SRQ) and Autism Diagnostic Observatiob Schedulen(ADOS) in patient B.

of nonverbal communication by facial expressions, physical gestures,or eye contact. She was unable to interpret and understand the interests
and needs of other children. She often engaged in “parallel play” at theedge of a group rather than joining in cooperative play and did not
engage in pretend play. She exhibited delayed speech and struggled
to make sense of nonverbal forms of communication. She found it
difficult to form and sustain friendships with other children becauseof her difficulties with social and emotional responsiveness. She wasvery sensitive to the sound of fireworks and starting pistols. Although
she desired to form friendships with other children, she exhibited
inappropriate friendliness and lack of awareness of others’ interests
and needs because she was unable to understand their needs. Thus,
she worked upon other children regardless of their dislike. She also
sometimes created fictional tales by talking to herself in a loud voicein her home and in kindergarten during lectures. The patient wanted
to spend time playing by herself at the edge of a group. She was often
unable to understand what her teachers and classmates said, and herteachers sometimes provided guidance to help her understand.
With respect to behavioral symptoms, the patient B usually was
not able to engage in group games, imitate others, collaborate, or use
their toys in creative ways. Her play also tended to have a persistentsensorimotor or ritualistic quality. Her behaviors were characterized
by repetitive and stereotyped patterns of activities such as repetitive
drawing of faces of children for approximately 30 minutes. She
exhibited delayed acquisition of motor skills and had difficulty withmotor coordination, postural control, and imitating the movements ofother people. She was unable to run along lines and was very sensitive
to starting pistols in sport meetings.

Psychometric evaluation
At age 5 years, the patient B participated in our clinical trial at
the Department of Pediatrics at Seirei Hammatsu General Hospitalin Japan and she took psychometric tests. Her ADI-R scores wereabove the autism diagnostic cutoff scores for qualitative abnormalities
in reciprocal interaction (her score was 13, cutoff = 10), qualitative
abnormalities in communication for both the verbal (her score was 14;
cutoff = 8) and non-verbal total scores (her score was 9; cutoff = 7), andrepetitive/stereotyped patterns of behavior was low the cut-off score(her score was 4; cutoff = 3). These ADI-R scores indicated confirm adiagnosis of ASD.

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As shown in figure 5, the patient B’s total score on the ADOS
module 2 algorithm showed a communication score of 10.0, which was
greater than the total scores of the ADOS (8.94 ± 9.71) reported in 33
adolescents with high-functioning autism [25]. Her SRS and ABC total
scores were 80 and 36, respectively. The total SRS and ABC scores werelower than those of ASD individuals with an age range of 13-27 years(SRS, 120.12; ABC, 60.14) [34]. The patient B’s social and behavioral
ASD symptoms appeared to be less severe. As the SCQ cutoff score wascompared with established cutoffs for ASD screening (scores of ≥15)[35], the patient B’s SCQ score of 10 was within the normal range. At
the age 6 years she received intelligent test using the WISC for Children[27]. Her total Intelligence Quotient was 61, which was defined asborderline IQ [28].
Collectively, her social and behavioral symptoms were within themoderate range of ASD symptoms. Other evaluations revealed no
additional abnormalities.

Biochemical measurement
The patient B’s serum level of VEGF-D was 336.9 pg/ml at baseline
and 548.7 ng/ml at week 8, and gradually increased to peak values ofvalue of 571.4 pg/ml at 16 weeks, then decreased to 501.5 pg/ml at 24
weeks after treatment. Thus, the peak serum VEGF level was higher inpatient B than in patient A.
As shown in figure 4, serum Cp levels gradually increased frombaseline (20 mg/dl) to 16 weeks (29 mg/dl) and then remained higher
than the baseline levels at 24 weeks (29 mg/dl) (Figure 5 lower panel).
Serum Tf levels were gradually increased from baseline (297 mg/dl)
to peak value at 24 weeks (390 mg/dl) in accordance with symptom
improvement (Figure 3, lower panel). Thus, it is important to notethat serum Cp and Tf levels were gradually increased 16-24 weeks oftreatment, in accordance with symptom improvement in the patient B.
While, serum SOD levels tended to decrease from the baseline (4.7 pg/ml) to 24 weeks of treatment, suggesting no contribution to the efficacy
of everolimus (Figure 3, lower panel).

Treatment with everolimus and serum everolimus levels
After the physical screening, patient B received 1.9 mg of
everolimus every day for 24 weeks. The patient’s body surface area
was 0.63 m2, based on 3 mg/m2 in the Novartis indication. The serum
everolimus levels during maintenance dosing were 7.0, 7.5, and 7.9 ng/

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Figure 5. Serum levels of ceruloplasmin (Cp), transferrin (Tf) during the treatment with everolimus for 24 weeks in patient A and patient B.

ml at 12, 16, and 24 weeks after treatment, respectively. One week after
the everolimus treatment, she suffered from mild lung inflammation,
but she immediately recovered by steroid treatment. She was also
treated with topical ointment because of slight inflammation of her oral
mucosa, which is a common side effect of everolimus. However, nosevere or life-threatening side effects related to everolimus treatment
were observed.

Clinical symptoms after everolimus treatment
The everolimus treatment induced apparent improvements in hersocial interactions and verbal and nonverbal communication skills.
She showed gradual language development without a dose adjustment
during the 24 weeks of treatment. She was able to appropriately
communicate with classmates and adults and showed pretend playskills. Thus, she was gradually able to enjoy school life.
The patient B’s SRS and SQC scores gradually decreased,
indicating improvement of the social impairment. In particular, theSRS and SCQ total scores decreased from 80 before treatment to 44
following treatment (decrease of 45%), and from 19 before treatment
to 10 following treatment (47% decrease), respectively comparedwith baseline. Thus, the everolimus treatment induced remarkable
improvements in the patient B’s social impairment. While, the ABC
total score was not changed.
The size of the SEGA was also slightly reduced (-0.2 cm), and that ofsubpendymal nodules were not changed after the everolimus treatment.

Discussion
This study presented for the first time that everolimus treatment
for 24 weeks apparently improved the core social as well as behavioral
symptoms of ASD in two patients. The core ASD symptoms appearedwith a SEGA and AML in Patient A, and with SEGA and subpendymal
nodules in Patient B. These findings indicate that the core ASD
symptoms may be related to the presence of a SEGA. Importantly, thepresent findings indicate that the increased serum Cp and Tf levels may
have contributed to the improvements in the core ASD symptoms.
In patient A, the serum everolimus levels were 18.10 and 7.32 ng/
ml at 16 and 24 weeks after treatment, respectively. In patient B, theserum everolimus levels were 7.0 and 7.9 ng/ml at 16 and 24 weeks
after treatment, respectively. A previous study of the pharmacokinetics
of everolimus revealed a mean everolimus blood level of 10.1± 0.7 ng/
ml at 20 weeks after treatment in 25 healthy adolescents [41]. Thislevel is similar to those in-patient A, indicating adequate levels of
everolimus. With respect to the patient B, considering that maintaining
the everolimus trough in the range of 3 to 8 ng/ml was associated withgood efficacy and safety profiles [47], the serum everolimus levels (7.07.9 mg/ml) may be appropriate for improvement of social impairment.

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The SRS total scores decreased by 40 to 45% in these two patients.
As a 50% reduction in the initial states was considered as achieving aclinically significant improvement [48], everolimus treatment showeda trend toward significantly improved the patient’s ASDl symptoms.
Thus, proper treatment with everolimus may exert a life-enhancing
clinical effect on the core social as well as behavioral symptoms.
Similarly, a previous study showed that everolimus treatment improvedsocial interaction; cognition; attention; behavioral deficits such as
swaying, twirling, and preoccupation with flowing water; and seizures[49]. This finding may support our present case study.
In this study, the serum VEGF-D levels gradually increased frombaseline (252.6 pg/ml) to a peak value of 315.1 pg/ml at 12 weeks in thepatient A, and serum VEGF levels gradually increased from baseline(426.7 pg/ml) to a peak value (571.4 pg/ml) at 16 weeks in the patient
B (Figure 4). A previous study reported that the average serum levelof VEGF in 19 normal healthy children was 306.1 ± 39. 4 pg/ml [50].
Although the patient B’s serum VEGF-D levels at baseline were higher
than the range reported in normal children, the serum VEGF-D levels
gradually increased between 8 and 16 weeks. Considering that the serum
VEGF-D levels are considered a biomarker of the treatment response[51], gradually increases in the serum VEGF-D levels observed in thepatient B and the patient A indicate the response to everolimus.
The reduction in SEGA size was associated with a decrease in seizure
frequency with everolimus treatment in in 28 children aged 3 years orolder [2] and in 3-year-old children [52]. The effects of a reduction in
the AML size on clinical symptoms was not reported [53]. The patient
A’s slight reduction in AML (-14.3%) may be no apparent relationshipwith the symptom improvement. Although many studies found a
50% reduction in the SEGA, several studies reported slight decreasein SEGA size after everolimus treatment [14,54]. As the variable
effects of everolimus on reduction of SEGA size reported in theseprevious studies, small size reduction observed in the two cases may
be reasonable. Considering that the therapeutic benefit of everolimus
may prevent the development of SEGA and AML [53,55], the ASD may
depend on other neurobiological mechanisms of everolimus.
Importantly, many previous studies indicated an increase inantiocVEGF as exogene oxidant capacity. For example, VEGF-D
deficiency may induce oxidative stress [56]. Moreover, exogenous
stimulation of incVEGF-D cells induces an antioxidant response in
human endothelial cells [57]. Everolimus attenuates oxidative stressby altering antioxidant levels [16] (Das) or reversing the accumulationof oxidative stress-related reactive oxygen species [58] (Dermit).
Thus, elevated VEGF-D levels may be related to enhanced antioxidant
capacity. It is important to note that everolimus attenuates oxidative
stress by altering antioxidant levels [16] or reversing the accumulation

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Yui K (2018) The role of antioxidant proteins in improvement of autism core symptoms in two patients with tuberous sclerosis treated with mTOR inhibitor
everolimusof oxidative stress-related reactive oxygen species [58]. Everolimus
may therefore increase antioxidant capacity. Everolimus treatment
elevated serum VEGF-D levels in this study, increasing antioxidant
capacity. Collectively, the everolimus treatment increase may berelated to an increase serum antioxidant levels such as increased
serum Cp and Tf levels. Of reference, Cp is the main copper-binding
protein in blood plasma [59] and an important serum antioxidant[60]. Increased intracellular copper levels suppressed mTOR signaling[61] and copper treatment downregulated mTOR signaling [62]. With
respect to increased serum Tf levels, the mTOR signaling pathway
modulates Tf uptake [20], and mTOR regulates iron homeostasis
through the Tf receptor [19]. Additionally, the Tf receptor may be
useful for measuring intracellular changes in mTOR activity [63].
Taken together, everolimus may increase the antioxidant capacity as
shown by the increased serum Cp and Tf levels. With respect to theassociation between Cp and Tf, the transfer of iron was regulated byboth CP and Tf and that the activity of Cp was modulated by Tf [64].
Cp plays an important role in iron metabolism [65]. Those previous
data support the present finding that everolimus might act on mTOR
signaling by increasing the serum Cp and Tf levels. The main role of
SOD is to prevent the formation of aggressive reactive oxygen species[60], which were induced by excess oxidative stress [66], suggesting lesscontribution of the serum SOD levels to the efficacy of everolimus.

Conclusion
The present study first revealed that a 24-week everolimus
treatment increased activity of antioxidant such as Cp and Tf, and thus
may contribute to the apparent improvement in the social as well as
behavioral ASD symptoms in the prepubercent boy and a girl childwith TSC

Contribution of authors
All authors met the criteria for this journal’s authorship credit in
the author guide
Each author’s name next to the appropriate category.
Study conception and design: Kunio Yui
Acquisition of data: George Imataka, Touru Okanishi and Kunio Yui
Analysis and interpretation of data: George mataka, Touru
Okanishi and Kunio Yui
Drafting of manuscript: Kunio Yui, Hitomi Sasaki
Critical revision: Hitomi Sasaki, George Imataka

Funding sourceThis study was supported by a small research grant such as Grantin-Aid for Scientific Research (C) (2014-2016) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.

Conflict of interest
The authors are not currently applying for any patents relating to
the content of the manuscript, and did not receive reimbursements,
fees, funding, or salary from an organization that holds or has applied
for patents relating to the content of the manuscript. The authors
declare that they have no conflict of financial and non-financial interest.

Acknowledgements
The authors thank patients’ mothers for providing each patient
medical information

Glob Med Therap, 2018

doi: 10.15761/GMT.1000101

Ethical statements
All the procedures in this study were in accordance with the ethicalstandards of Dokkyo Medical University, Tochigi, Japan (NO. 27014,
Japanese Medical Association, Clinical Trial Registration (JMASIIA00258), and with the 1964 Declaration of Helsinki. All procedures
were performed at the Department of Psychiatry, Dokkyo Medical
University. Written informed consent was obtained from the patient’s
mother because the patient was unable to understand informed
consent. Because the patient was minor child, the patient’s motherprovided her written consent for the publication of this case report on
the behalf of the patient.
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Copyright: ©2018 Yui K. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

Glob Med Therap, 2018

doi: 10.15761/GMT.1000101

Volume 1(1): 9-9