Infectious Diseases

ISSN: 2374-4235 (Print) 2374-4243 (Online) Journal homepage: http://www.tandfonline.com/loi/infd20

The mechanisms of delayed onset type adverse
reactions to oseltamivir
Rokuro Hama
To cite this article: Rokuro Hama (2016): The mechanisms of delayed onset type adverse
reactions to oseltamivir, Infectious Diseases
To link to this article: http://dx.doi.org/10.1080/23744235.2016.1189592

Published online: 02 Jun 2016.

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Date: 02 June 2016, At: 05:40

INFECTIOUS DISEASES, 2016
http://dx.doi.org/10.1080/23744235.2016.1189592

REVIEW ARTICLE

The mechanisms of delayed onset type adverse reactions to oseltamivir
Rokuro Hama

Downloaded by [Dr Rokuro Hama] at 05:40 02 June 2016

Non-Profit Organization, Japan Institute of Pharmacovigilance, Osaka, Japan

ABSTRACTARTICLE HISTORY

Oseltamivir is recommended for the treatment and prophylaxis of influenza in persons at higher risk for
influenza complications such as individuals with diabetes, neuropsychiatric illnesses, and respiratory, cardiac, renal, hepatic or haematological diseases. However, a recent Cochrane review reported that reduction of antibody production, renal disorders, hyperglycaemia, psychiatric disorders, and QT prolongation
may be related to oseltamivir use. The underlying mechanisms are reviewed. There is decisive evidence
that administration of a clinically compatible dose of oseltamivir in mice challenged by a respiratorysyncytial virus (RSV) that lacks a neuraminidase gene showed symptom-relieving effects and inhibitionof viral clearance. These effects were accompanied by decreased level of T cell surface sialoglycosphingolipid (ganglioside) GM1 that is regulated by the endogenous neuraminidase in response to viral challenge. Clinical and non-clinical evidence supports the view that the usual dose of oseltamivir
suppresses pro-inflammatory cytokines such as interferon-gamma, interleukin-6, and tumour necrosis
factor-alpha almost completely with partial suppression of viral shedding in human influenza virus infection experiment. Animal toxicity tests support the clinical evidence with regard to renal and cardiac disorders (bradycardia and QT prolongation) and do not disprove the metabolic effect. Reduction of
antibody production and cytokine induction and renal, metabolic, cardiac, and prolonged psychiatricdisorders after oseltamivir use may be related to inhibition of the host’s endogenous neuraminidase.
While the usual clinical dose of zanamivir may not have this effect, a higher dose or prolonged administration of zanamivir and other neuraminidase inhibitors may induce similar delayed reactions, including
reduction of the antibody and/or cytokine production.

Received 23 March 2016
Revised 1 April 2016
Accepted 5 April 2016
Published online 1 June 2016

Introduction
Neuraminidase inhibitors (NIs) are expected to reduce complications of influenza, especially in persons at higher risk for
influenza complications,[1,2] and oseltamivir is included in the
Model List of Essential Medicines.[3] Persons at higher risk
include those with diabetes, neuropsychiatric illnesses, andrespiratory, cardiac, renal, hepatic, or haematological diseases.[1,2] However, serious neuropsychiatric adverse reactions to oseltamivir, including sudden deaths and abnormal
behaviours leading to accidental death, have been reported
since the drug was introduced into medicine.[4–8] In Japan,
oseltamivir has been contraindicated in principle for children
and adolescents aged 10 to 19 years since 2007 due to concern about the risk of abnormal behaviours.[4–7] Adverse
reactions to oseltamivir include sudden and delayed onset
types and others.[6,7] Sudden death is another concern about
oseltamivir use.[5–8]
The Cochrane’s neuraminidase inhibitors team showed in
its latest review [9] that neuraminidase inhibitors (NIs), including oseltamivir and zanamivir, have symptom-relieving effects
in adults, but it is unclear whether the same effects occur inchildren. Oseltamivir reduced a four-fold increase in antibody
titre.[9,10] Evidence of reduction in hospitalization was notshown for either NI. Oseltamivir did not reduce complications,
including pneumonia classified as serious or which led to
study withdrawal. Both NIs reduced ‘‘laboratory confirmed
CONTACT Rokuro Hama

gec00724@nifty.com

Antibody; endogenous
neuraminidase; oseltamivir;
pro-inflammatory cytokines;
QT prolongation; renal
impairment

symptomatic influenza infection,’’ but not influenza-like illness. Oseltamivir increased risk of nausea, vomiting, headache, psychiatric, renal, and diabetic/hyperglycaemic events,
and pain in limbs, but zanamivir did not. Oseltamivir reduced
diarrhoea and apparent cardiac events, but induced prolonged QTc time (Bazett correction).[9]This review discusses the biological base of adverse reactions, in particular, those of the delayed onset type.

Brief description of neuraminidase inhibitors
Neuraminidase inhibitors and medical use
Neuraminidase inhibitors (NIs) comprise inhaled zanamivir(Relenza, GlaxoSmithKline), oral oseltamivir (Tamiflu, Gilead
Sciences, F. Hoffman-La Roche and Chugai in Japan), parenteral peramivir [11] (BioCryst Ltd and Shionogi), inhaled laninamivir (Daiichi Sankyo Co. Ltd),[12] and others still underdevelopment.[13] Zanamivir and oseltamivir have been marketed in the US since 1999, and subsequently in other countries including Japan. Peramivir and laninamivir were put on
the market in Japan in January and September 2010,
respectively.
F. Hoffman-La Roche (Roche) donated oseltamivir to theWorld Health Organization (WHO), and the WHO decided
to recommend stockpiling neuraminidase inhibitors#702, Ueshio 5-1-20, Tennoji-ku, Osaka 543-0002, Japanß 2016 Society for Scandinavian Journal of Infectious Diseases

KEYWORDS

Downloaded by [Dr Rokuro Hama] at 05:40 02 June 2016

2

R. HAMA(especially oseltamivir) for use in the event of a pandemic.[14]Prior to the emergence of influenza A/H1N1 in 2009, governments worldwide stockpiled 220 million treatments of oseltamivir, swelling cumulative sales since the start of 2003 to CHF
7.6 billion.[15] The use of NIs has increased dramatically since
the outbreak of A/H1N1 in April 2009.
Between the first approval in the US and the 2006/2007
season, approximately 48 million patients received a prescription for oseltamivir worldwide,[16] while 4 million patients
received a prescription for zanamivir.[17] Japan, the US, andthe other countries consumed 76%, 21%, and 3% of theworld total of oseltamivir, respectively.[16] For children (suspension), 85% of the world total was consumed in Japan.[16]Zanamivir was rarely prescribed (less than 1% of total antiviral prescriptions for influenza) in Japan before the Japanese
Ministry of Health, Labour and Welfare (MHLW) contraindicated oseltamivir use in patients aged 10 to 19 years in
March 2007.[4,6]. After this restriction, prescriptions of oseltamivir decreased substantially. After laninamivir was put on
the market in Japan in September 2010, its share increased
rapidly. In the 2013/14 season, the prescription shares of oseltamivir, zanamivir, laninamivir, and peramivir were 36%, 19%,
42%, and 3% in Japan, respectively.[18]Mode of action and pharmacokinetics in non-infectious
state
Although NIs may reduce the ability of the virus to penetrate
the mucus in the very early stage of infection,[19–24] theirmain mechanism of action is thought to lie in their ability tointerfere with the release and spread of progeny influenza
virus from infected host cells by inhibiting neuraminidase of
influenza viruses.[21,23,24] It is generally believed that oseltamivir most likely reduces symptom duration by reducing viralload, and via the spread and release of cytokines.[25]However, the full prescription information of oseltamivir(revised in April 2010) states ‘‘The concentrations of oseltamivir
carboxylate required for inhibition of influenza virus in cell culture were highly variable depending on the assay method used
and the virus tested. (. . .) The relationship between the antiviral
activity in cell culture, inhibitory activity in the neuraminidase
assay, and the inhibition of influenza virus replication inhumans has not been established’’.[24] It is not clearly statedthat the mechanisms of symptom relief are derived from thereduction of viral load.
Oseltamivir phosphate (OP) is an ethyl ester prodrug
that requires ester hydrolysis for its conversion to theactive form of the neuraminidase inhibitor, oseltamivir carboxylate (OC). The brand name drug of OP capsule (Tamiflu
capsule) contains 75 mg of oseltamivir expressed as freebase (OT), which is compatible with 98.6 mg of OP. OP dissociates in the gastrointestinal tract to form OT, which isabsorbed and metabolized into OC by hepatic carboxylesterase (hCE).
In healthy volunteers, the area under the curve (AUC) of
OP is 3 to 5% that of OC. The penetration of OP across the
blood–brain barrier (BBB) is restricted (less than 10%) by

P-glycoprotein (P-gp) in mature and non-infectedanimals.[26–28]
When healthy adult volunteers were administered with75 mg of OT (equivalent to 98.6 mg of OP), approximate average Cmax(ng/mL), Tmax (h), AUC (ng.h/mL), and elimination
half-life (t1/2:h) of OP were as follows, respectively: 60 ng/mL,
0.7–2 h, 150-200 ng.h/mL, and 1.2–1.9 h. For OC, they were200–300 ng/mL, 4–5 h, 3000–4000 ng.h/mL, and 5–10 h.[29]The pharmacokinetic (PK) parameters in healthy children
aged 3 years or older are not very different from those in
adults. In the 5 patients with decreased creatinine clearance(<30 mL/min), after 6 days administration of 100 mg of OT (asfree form), PK parameters (±SD) of OC were as follows: Cmax:
4052 (±1519) ng/mL, Tmax: 5.20 (±1.11), AUC0-12:43086 ng.h/mL (±18068), and t1/2:16.1 (±2.69).[29]In the 12 patients who were haemo-dialyzed, PK parameters (±SD) after 75 mg of OT were as follows: Cmax: 2131(±533) ng/mL, Tmax: 27.3 (±6.9), AUC0-last: 106314 (±26,029)
ng.h/mL, and t1/2: 159 (these data were calculated before thenext dialysis, 48 h after the previous dialysis; SD was not
given).[29] Thus, almost all OC is secreted in urine, and dose
adjustment is necessary if the patients’ creatinine clearance isless than 30 ml/min.[24,29,30]

Evidence on adverse reactions to oseltamivir inhumans and in animals
To date, it is postulated that adverse effects of oseltamivir
include sudden onset type, delayed onset type, andothers.[6,7] This chapter mainly summarizes the delayed onset
type adverse reactions to oseltamivir reported in the clinical
trials and epidemiological studies, which may help to explain
possible adverse effects of other neuraminidase inhibitors. A
very brief review of the sudden onset type neuropsychiatricadverse effects of oseltamivir is also provided.

Sudden onset type neuropsychiatric reactions
The sudden onset type reactions include nausea, vomiting,and hypothermia, as well as neuropsychiatric reactions suchas abnormal behaviours, hallucination, and sudden respiratoryarrest followed by cardiac arrest and death.[6,7,31] These
appear very shortly (from less than 1 h to 24 h at most) after
the first dose of oseltamivir, and disappear rapidly unlessthey induce respiratory arrest and sudden death, or sequelae.[6,7,31] They may disappear even if oseltamivir is continuously taken, although symptoms may reappear if the drug istaken several times. The underlying mechanisms of suddenonset type reactions are discussed elsewhere.[6,31]

Delayed onset type reactions with prolonged time forrecovery
Delayed onset type reactions include disorders of variousorgans and systems such as renal, metabolic, cardiac, hepatic, haematological, immune, nervous, psychiatric, and general systems (fatigue or malaise). Most of the reactions of
this type appear at least a few days after commencement

INFECTIOUS DISEASES

Downloaded by [Dr Rokuro Hama] at 05:40 02 June 2016of oseltamivir intake, although QTc prolongation is closely
related to the plasma concentration of oseltamivir carboxylate, even at first dose (details described below). Durationof symptoms from these reactions, especially neuropsychiatric ones, tends to be prolonged: for months or evenyears.[6]Inhibition of antibody production and reinfection
Oseltamivir significantly reduced the odds of patients having
a 4-fold antibody rise, by almost 20% (risk ratio by 8%),according to a meta-analysis of 8 reports (10 studies).[9,10]
Heterogeneity was not significant (I2 ¼ 4%).
Attenuation of secretory IgA (sIgA) was moremarked.[32–35] Sawabuchi et al. reported that lower induction of sIgA against the influenza A virus was observed inchildren treated with oseltamivir in comparison with children
treated without oseltamivir. The odds of a child’s sIgA level
increasing more than 10-fold were non-significantly lower inchildren treated with oseltamivir (2/12) than in children without oseltamivir (3/3): odds ratio is 0.17 (95%CI: 0.01, 2.39,
p ¼ 0.13) (calculated from the data shown in the Figure 1 ofRef. [32]).
Anti-influenza A virus sIgA attenuation was observed inchildren treated both with oseltamivir or with zanamivir.[33]These findings are consistent with evidence from animal
tests using sub-clinical doses of oseltamivir in influenza A/
H1N1 infected mice.[34,35] Non-significant slight reductionof haemagglutinin (HA) specific IgG antibody in the serumand spleen was reported, while HA specific secretory IgA
antibody (sIgA Ab) in nasal wash and bronchoalveolar fluids(BALF) was significantly reduced: by approximately 80% on
day 12.[34]In human clinical trials, zanamivir at the usual dosage did
not reduce antibody (anti-HA Ab) production,[9] but attenuated sIgA antibody significantly.[33] In a double blind, placebo controlled trial with healthy volunteers designed toinvestigate the effect of zanamivir treatment (20 mg/day for
14 days) on the humoral immune response to influenza, thezanamivir group responded with significantly lower antibody
titres to the H1N1.[36] Levels of pro-inflammatory cytokines
including IL-6, TNF-a, IFN-c, and other chemokines were
almost completely suppressed in the viral challenge randomized controlled trial (RCT) using a very high dose (600 mg) of
intravenous zanamivir before inoculation of influenza virus inhuman adults.[37]Shinahara et al. [33] reported: ‘‘Even under the spread of a
new virus subtype in 2009/2010, only 8.6% of the children of
the no-treatment group were re-infected. However, the proportions of children treated the previous year with oseltamivir andzanamivir who developed re-infection in 2009–2010 were significantly higher at 37.3% and 45.0%, respectively (p < 0.01), thanthose of the no-treatment group.’’Several cases of re-infection with the same influenza virus
within one season were reported.[38,39] Kopel et al. [38]reported a 13-year-old boy with cerebral palsy who threetimes had episodes of fevering with positive 2009A/H1N1
influenza detected by the RT-PCR method. He was treatedwith the standard dose (75mg b.i.d for 5 days) of oseltamivir

3

at the time of the first episode. A second course of oseltamivir was administered for 10 days with the dosage adjusted
for age and doubled from that of the previous regimen. His
HI titres were high, but the level of secretory IgA was notdetermined.

Renal impairment
Dose-related histological change in the renal tubules,
increased urine volume, and increased relative weights of theliver and kidneys were observed at various doses in various
animals treated with oseltamivir for various periods.[29]For example, in the 6-month rat toxicity test, renal weights(both relative and absolute) increased, and histological examination revealed degenerating and regenerating changes in
the renal tubular epithelia, basement membranes, andBowman capsules; vacuolization in the renal tubular epithelia;
and mineralization in the highest dose group (761 mg/kg).
These histopathological findings were not reversed at 8
weeks after cessation of oseltamivir intake. In the mediumdose group (152 mg/kg), renal relative and absolute weights
increased, and vacuolization in the renal tubular epithelia wasobserved.[29] According to the ‘‘Guidance for Industry’’ [40]for a person weighing 60 kg, conversion factors for Human
Equivalent Dose (HED) for mouse, rat, ferret, marmoset monkey, rabbit, and dog are 12.3, 6.2, 5.3, 6.2, 3.1, and 1.8,
respectively. Hence 152 mg/kg in rat is 24.5 mg/kg expressedby HED, NOAEL for renal impairment was 38 mg/kg. HED for
38 mg/kg is 6.1 mg/kg converted by body surface area and5.5 mg/kg converted by AUC level of OC. These are 2.4 and2.2 times higher than the usual dose for a person weighing
60 kg. Renal impairment in the highest-dose oseltamivir group
was accompanied by increased water intake, increased leukocyte count, and increased bilirubin, BUN, creatinine, urine volume, and NAG/creatinine ratio.
Metabolic disorders: hyperglycaemia and diabetes
Increased glucose level in the highest dose group (1522 mg/kg) was observed in a rat oral 2-week test. The data were notdescribed, but this increased glucose level was accompanied
by increased leukocyte count, dose-related significant increaseof BUN (p < 0.01), increased weights of the kidney and theliver, mineralization of tubules of renal medulla (male: 8/10),
and mild-to-moderate accumulation of lung alveolar
macrophages.[29]Cardiac disorders: bradycardia and QT prolongation
Oseltamivir decreased heart rate in the 9-month repeated toxicity test using marmoset monkeys. The average heart rateduring treatment period with oseltamivir was 328 beats/min,
which was an 11% and 16% decrease compared with thecontrol group (368 beat/min) and the average of baseline
and recovery phase (392 beat/min), respectively.[29]In an experiment using beagle dogs to test the effects on
cardiac functions such as QT time,[41] mean baseline QTcintervals (msec ± SE) were 417 ± 16 in the control (vehicle)
group (n ¼ 4) and 374 ± 2 in the oseltamivir carboxylate (OC)4

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Downloaded by [Dr Rokuro Hama] at 05:40 02 June 2016group (n ¼ 4). This difference was significant (p ¼ 0.0372)
according to the summary data t-test. Other evidence suggested that variation was higher in the control group(p ¼ 0.005; Bartlett’s test).[42] The average ± standard error of
QTc interval of anesthetized dogs intravenously infused with
100 mg/kg OC over 30 min significantly increased (390 ± 4)
compared with that before infusion (376 ± 2). The average
QTc recovered (374 ± 6) at 1.5 hours after discontinuation, but
the standard error became larger. QTc prolongation is closely
related to the serum concentration of OC.[29,41,42] The reasons for such large and systemic imbalances are not known,
but they are unlikely to have occurred by chance.
Bradycardia and QT prolongation that occurred in the animal tests were also observed in the RCTs in humans, and theoccurrence was closely related to the timing of the increasein the concentration of oseltamivir carboxylate.[9]Delayed onset and prolonged type psychiatric symptoms
The present author reported the following case of psychiatric
reaction.[6] A 15-year-old junior high school boy with a bodytemperature of 39.2 C due to influenza B diagnosed by rapid
testing took Tamiflu 75 mg b.i.d. for 5 days. His body temperature was normalized on day 5, but he felt lethargic. Afterhe took the last (10th) dose of Tamiflu in the morning on day
6, he went to school, sat on his desk, and began to sing
loudly during a lesson. He could not communicate with hisclassmates. He seemed to be delirious. Four days after this
episode, his parents took him to a general hospital. Before
admission, he commented, ‘‘There are insects on my mask,’’ asign of visual hallucinations. After admission, he tried to pullout his venous lines and attempted to go home, shouting‘‘This is not a hospital but a nursing home for elderly people.’’He could not wait until his turn for examination and rushed
out of the hospital into the street, where he narrowly avoided
being run over by a car. On day 16 he was discharged, andon days 19 and 20 he came to know that he had behaved
abnormally from records on his mobile phone. It took 13
days after the beginning of abnormal behaviour for his psychiatric symptoms to be completely resolved.[6]Among the four study reports of prophylaxis RCT analyzed
in the systematic review,[9] the case of longest recovery time
was Subject 23639/3122 in trial WV15825.[43] The narrativedescription on module 1 of the clinical study report stated asfollows:
This 69-year-old female was hospitalized on study day 8 becauseof paranoid schizophrenia. Her medical history included paranoid
schizoaffective disorder, hypertension, and coronary artery disease.
Her medications included ketoconazole, amlodipine, haloperidol
and lorazepam. On study day 8 she ran away from her place ofresidence, but she was found and transferred to hospital for
medical treatment. Study medication was discontinued on study
day 8. Paranoid schizophrenia of severe intensity was diagnosed.
She subsequently absconded from the hospital and was found on
study day 15 with moderate concussion. She was again
hospitalized, for 10 days. The paranoid schizophrenia resolved
within 68 days and was considered unrelated to study medication.

In the prophylaxis RCTs, 11 and 2 cases of psychiatricevents with late onset and prolonged recovery (14 days andlonger) were reported in the oseltamivir and placebo groups,

respectively. The pooled odds ratio was 3.37 (95%CI:
1.11–10.23, p ¼ 0.032, I2 ¼ 0%).
No animal study was performed to confirm the associationof psychotic reactions of delayed onset with prolonged
recovery.

Other adverse effects (pneumonia, wheezing, gastric
bleeding, and others)
Three of the six rats treated with intravenous OC (at 12 times
higher level than clinical area under the curve) for 2 weeks
developed acute alveolitis.[29] Of the 3, 1 exhibited wheezing
on day 14 and was sacrificed the next day. Diffuse haemorrhagic alveolitis (pneumonia) and pulmonary microvascularthromboembolism were observed in this animal. The safe
level of intravenous OC dose is lower than twice the AUC of
the usual clinical human dose.
In the marmoset monkey 7-day oral toxicity studies,[29] allfour animals treated with a 127-times-higher-than-HED doseof OT were sacrificed within 4 days (1 on day 2 and 3 on day4) because they were near death after severe vomiting, sleep,
hypoactivity and collapse. Macroscopic reddening of thestomach mucosa and histologically mucosal bleeding witherosions, ulcers, and atrophy were observed in the stomachs
of all the animals.[29]The safety index (animal AUC0–24 with no toxicity byhuman average AUC0–24 when taking 75 mg capsule b.i.d.) is
3 for the 4-week toxicity studies in rats, 3 for the 6-monthoral toxicity studies in rats, 8 for the 2-week oral toxicity
study in rats, and 10 for the marmoset monkey 7-day oral
toxicity study.

Potential adverse reactions to oseltamivir and to otherneuraminidase inhibitors
Potential adverse reactions to oseltamivir and to the otherneuraminidase inhibitors are summarized in Table 1.

Mechanisms for symptom relief and the host’s
endogenous neuraminidase
Mechanisms for symptom relief are mainly discussed in thissection, and those for delayed onset type reactions are mainly
discussed in the next section. Both mechanisms seem to berelated to inhibition of the host’s neuraminidase.

Inhibition of the host’s neuraminidase and symptom
reliefSymptom relief in RSV-infected mice by oseltamivir
Decisive evidence is shown by Moore et al.,[44] who
reported that administration of a clinically compatible doseof oseltamivir in mice challenged by a respiratory syncytial
virus (RSV) that lacks a neuraminidase gene showed symptom-relieving effects (decreased weight loss) andinhibition of viral clearance. These effects were accompanied by decreased level of CD8þ T cell surface sialoglycosphingolipid (ganglioside) GM1, which is regulated by theINFECTIOUS DISEASES

5

Table 1. Spectrum of adverse reactions to oseltamivir and other neuraminidase inhibitors.
SystemSymptom group

Sudden onset type reactions (only to oseltamivir)
Digestive
Gastrointestinal
Central nervous system
Mild to moderate symptoms
Psychiatric symptoms

Respiratory suppression
Delayed onset and/or prolonged type reactions (all neuraminidase inhibitors)
Immune/inflammatory/infectious
Antibody

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Cytokine
Cell
Renal

Reinfection etc.
Histology/function

Metabolic
Cardiac
NeurologicalPsychiatricDiabetic
Contraction
Nociceptor
Delayed onset/prolonged type

Digestive
OthersGI tract

endogenous sialidase/neuraminidase in response to viralchallenge, along with suppression of cytokines expression.
To date, no such study has been conducted for zanamivir,
laninamivir or peramivir.

Decreased GM1 ganglioside and suppression of
pro-inflammatory cytokines
In the human phase II randomized controlled trial with experimental infection,[25] pro-inflammatory cytokines including IL6, TNF-a, and IFN-c were completely suppressed by oseltamivir administered 28 h after the experimental inoculation of
the influenza virus, while reduction of viral titre in nasal lavages was partial.
Attenuating induction of pro-inflammatory cytokines
including IL-6, TNF-a, and IFN-c is related to decreased
secretion by immune cells including dendritic cells,[45] bypolymorphonuclear
leukocytes,[46]andbyCD8þ Tcells.[44,47] Reduced cytokine induction is derived fromdecreased expression of GM1 ganglioside in these cells
related to inhibition of the host’s endogenous neuraminidase (or sialidase),[44–47] especially Neu 3 (the third subtype of neuraminidase mainly expressed in plasma
membrane).[45]Decreased GM1 ganglioside and pain
Crain et al. [48] reported that oseltamivir at 100 to 1000 times
lower HED than the clinical dose may affect the host’s neuraminidase and reduce endogenous GM1 ganglioside, leading
to some reactions in the host. They suggest that ‘‘Clinicaladministration of oseltamivir at doses that result in inhibition of
influenza may also have an additional effect by decreasing GM1
levels in nociceptive neurons’’.[48]Symptoms and reactions
Nausea, vomiting,
Hypothermia, sleep, headache, vomiting, bulging of fontanelle
Sensory disturbances, impairment of cognition, abnormal behaviour (suppressed or excitatory), unconsciousness, paranoia, delusion, hallucination,
psychosis, depression, aggression, agitation, delirium, suicidal (ideation,
attempt, complete)
Cyanosis, difficulty of breathing, hyperpnea, hypopnea, irregular breath,
respiratory failure, respiratory arrest, cardiorespiratory arrest and death
Reduction of antibody production, especially secretory IgA at respiratory
tract
Attenuated induction of cytokine and chemokines (IL-6, IFN-c, TNF-a, etc.)
Reduction of inflammatory cells in nasal wash and lung, reduction of CD8þ
T cell in lung
Reinfection of influenza, pneumonia and exacerbation of other infections
Degenerating and regenerating changes in the renal tubular epithelia andBowman capsules, increased urine volume and kidney weight, proteinuria, etc.
Hyperglycaemia, exacerbation of diabetes mellitus, new onset diabetes
Decreased heart rate, bradycardia, QT prolongation
Pain in limbs or other parts of the body
Delayed onset psychiatric symptoms (abnormal behaviours, psychosis
hallucination, delusion, agitation, schizophrenic reactions,depression, etc.)
Bleeding
Bleeding (hepatic and/or haematological impairment)

Animal infection model: symptoms, inflammatory/
cytokine response, and viral load
Ferret model: reduction of febrile, inflammatory responsewith little viral load change
The ferret model is one of the best animal models for human
influenza infection. Roche used this model and reported asfollows in the protocol (Module II) of most clinical study
reports for treatment randomized controlled trials [43*a]:
Adult ferrets (four per group) were inoculated with a virulent
influenza strain. Ro-0796 was administered orally at a dose ofeither 5mg/kg or 25-mg/kg b.i.d. for 3 days starting 2 h post
exposure. A control group of four ferrets received vehicle alone. In
this experiment, Ro 64-0796 was shown to reduce the febrile
response and reduce the number of inflammatory cells in nasal
washing in a dose dependent manner. However, neither dose wasdemonstrated to reduce the viral titres obtained from the lungs ornasal washings of infected animals.
Ro-0796 refers to oseltamivir phosphate.

According to the published paper of oseltamivir,[49] thearea under the curve (AUC) of the viral load of both 5 mg/kg/
day and of 25-mg/kg/day oral doses and peak viral load in
the 5 mg/kg/day group were not reduced significantly,
although average peak viral titres of the 25 mg/kg/day group
was reduced significantly.
In contrast, AUC of the febrile response was reduced significantly in both oseltamivir dose groups, dose-dependently.
In addition, the total number of inflammatory cells in nasal
wash obtained from infected animals in both oseltamivir dosegroups was continuously reduced for more than 96 h.
The conversion factor for ferret dose to human equivalentdose (HED) for a person weighing 60 kg in the ‘‘Guidance forIndustry’’ [40] is 5.3. Therefore, 5 mg/kg and 25-mg/kg for
adult ferrets correspond to approximately 1 mg/kg and 5 mg/

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kg for humans. These are almost equivalent doses and 4 to 5
times higher than the human single dose of oseltamivir(75 mg/60kg).
Hence, the clinical dose of oseltamivir may reduce febrile
response and the number of inflammatory cells in a nasal
washing without significant reduction of viral titres obtained
from the lungs or nasal washings of infected animals.
*a: Module II of the most CSRs for adult treatment RCTs
including 2 pivotal RCTs (WV15670, WV15671) and others(WV15673/15697, WV15707, WV15708, WV15730, WV15758,
WV15759/15871, WV15799, WV15812/872, WV15819/15876/
15978, WV15825), except 3 CSRs (M76001, WV16277 andML16369). These full CSRs are available in ref [43] http://datadryad.org/resource/doi:10.5061/dryad.77471.

Mouse model: mild influenza and lack of evidence of
reduction of viral load
Oral administration of 10 mg/kg of OP per day caused a 100fold reduction in lung homogenate viral titres in mice
infected with a 90% lethal dose of some strains of influenza
A or B viruses, and enhanced survival.[29,49] Similar experiments were reported for peramivir.[50–52]
However, in a study by Wong et al. [53] using mice
infected with mild influenza (inoculated with a non-lethal
dose of influenza virus), which is a better model for testing
the effects of oseltamivir in human seasonal influenza, aclinicallycompatible
doseofoseltamivir(10 mg/kg ¼ approximately 0.8 mg/kg as HED) administered (in 3 different experiments) at 4 hours before inoculation, 24 h after
inoculation, or 48 h after inoculation showed no significant
effect on viral titres at day 5 post-inoculation.
Wong et al. [53] observed that oseltamivir markedly andsignificantly reduced lung inflammatory cell response andinduction of pro-inflammatory cytokines and chemokines
such as TNF-a, IL-1b, IL-6, granulocyte–macrophage colonystimulating factor (GM-CSF), keratinocyte-derived chemokine(KC), macrophage inflammatory protein-1a (MIP-1a), andmonocyte chemotactic protein-1 (MCP-1) whether administered prophylactically or therapeutically. However, these wereaccompanied by small non-significant effects on viral titres.
Based on these findings, the researchers discussed the possibility of intrinsic anti-inflammatory effects of oseltamivir.[53]No animal study has been conducted of the infection
model with mild and non-lethal doses of the influenza virus
for zanamivir, laninamivir, or peramivir. Only animal studies of
the infection model using lethal doses of the influenza virus
are available.
In vitro findings showing inhibitory effects on
immune cells
Peripheral T-lymphocytes from healthy adult whole blood
were incubated with antigen presenting cells (APCs) pre-sensitized with influenza viruses and were tested for their proliferation ability with and without oseltamivir carboxylate.
Proliferation of the T-lymphocytes was suppressed by 15%
and 20% when incubated with 1 lM and 10 lM of oseltamivir
carboxylate, respectively, compared with the control.[29]Concentration of oseltamivir carboxylate (OC) of 1 lM is compatible with the human clinical concentration of OC.[29,30]
The Pharmaceuticals and Medical Devices Agency (PMDA)
and the Summary Basis of Approval (SBA) did not refer toany published paper for these findings.[29,30] No publishedpapers with these data could be found.

Mechanism for delayed onset type reactions
Delayed onset and prolonged type of psychiatric andother neurological symptoms
Psychiatric and nervous symptoms that occur in the very earlyphase of the treatment such as acute behavioural change andrespiratory depression leading to death may be due to theeffects of unmetabolized oseltamivir phosphate (OP) on thecentral nervous system (CNS). If OP has affinity to NMDA
receptors [31,54,55] and is used for an extended period oftime, it may induce schizophrenic reactions in humans, as
shown in the prophylaxis RCTs of oseltamivir,[9,43] by a mechanism similar to that of the sudden onset type reactions.
The symptoms that occur in the late phase of treatment
with prolonged duration, such as psychosis, confusion, andaggression, and are frequently observed in the prophylaxis trials (shown in the section ‘‘Other adverse effects (pneumonia,
wheezing, gastric bleeding, and others)’’) may also be due tothe effects of oseltamivir carboxylate (OC) on CNS. Pain in thelimbs [9] may also be induced by both the mechanisms.
Izumi et al. reported that systemic injection of oseltamivir(50 mg/kg i.p.) significantly altered the duration of loss oflightning reflex following ethanol injection in rats. Ethanol
injection in the presence of oseltamivir also resulted inenhanced hypothermia.[56] Izumi et al. also reported thatcombination of oseltamivir with other neurostimulants alter
synaptic plasticity and this may contribute to behaviouralchanges associated with the drug.[57]As described in section ‘‘Cardiac disorders: bradycardia andQT prolongation’’, QT prolongation is closely related to theplasma concentration of oseltamivir carboxylate.
Taking these into account, it may be possible that oseltamivir carboxylate directly alters the cell excitability of both neurons and heart muscles, although it is not known whether thealteration is derived from inhibition of the host’s endogenous
neuraminidase or from other mechanisms, including effects onother receptors or enzymes. Among receptors or enzymes thatwere tested by Lindeman et al.,[58] those that showed apparent dose-related increase are listed in Table 2.
Muraki et al. [55] demonstrated that oseltamivir, but notoseltamivir carboxylate, directly blocks human neuronal nicotinic acetylcholine receptors. Hiasa et al. [59] found that oseltamivir, but not oseltamivir carboxylate, competitively andselectively inhibited human MAO-A. They estimated the Ki
value to be 25 to 28 lM, and IC50 was shown to be between
50 to 100 lM in their paper, while Lindeman et al. reported
that both oseltamivir and oseltamivir carboxylate lacked clinically relevant pharmacological activities on a panel of 155
other molecular targets, including MAO-A. Differing results
between the study by Lindeman et al. and those by Muraki

INFECTIOUS DISEASES

7

Table 2. Activity of OP and OC against molecular targets of high relevance for mood, cognition and behaviour in binding or functional
assay (data are extracted from Ref. [58]).
Inhibition (% control)

concentration of OT

concentration of OC

Target of molecule (receptor etc.)/method of assay

3lM

30lM

3lM

30lM

Adenosine A receptor

8

13

15

27

L-type Ca2þ channel (diltiazem site)

14

41

10

9

Naþ channel (site 2)

11

38

n.i.d.

9

NMDA-type glutamate receptor (PCP)

14

23

21

12
n.i.d.

AMPA-type glutamate receptor

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Inhibition (% control)

417

n.i.d.

Glycine-site on NMDAR (strychnine-insens.)

n.i.d.

n.i.d.

1

24

Kainate-type glutamate receptor

n.i.d.

1410

10

Muscarinic receptor M4

n.i.d.

n.i.d.

1

10

Neuropeptide Y receptor Y1

8

6

n.i.d.

11

Nociceptin receptor

9

8

8

16

Norepinephrine transporter

n.i.d.

n.i.d.

5

11

Nicotinic acetylcholine receptor (a4b3, BGTX insens.)

5

n.i.d.

n.i.d.

11

hERG potassium channel (Kv11.1)

n.i.d.

17

8

16

Sigma receptor (non-selective)

934

n.i.d.

3

GABA transporter

15

15

n.i.d.

17

Serotonin 5-HT2A receptor
Serotonin 5-HT4e receptor

4n.i.d.

13
13

15
n.i.d.

n.i.d.
n.i.d.

Chemokine receptor 1 (CD191)

3

2

2

8

Chemokine receptor 3 (CD193)

10

18

15

12

CXC chemokine receptor 2 (IL-8B)

1

2

11

16

Data are extracted from Ref. [57]. n.i.d.: No inhibition detected, BGTX: alpha-bungarotoxin.
Note that maximum concentration of OP and OC tested was 30 lM.
Dose-dependent increase is not denied for shaded cells.

et al. or Hiasa et al. may be derived from the different assay
methods used.
Accordingly, it is possible that there exist target receptors
or enzymes that oseltamivir carboxylate specifically acts on.

Attenuated antibody production, immune suppression
and re-infection
Attenuated antibody production [32–34] may be derived fromthe same mechanisms as those for reduction of cytokine
induction related to inhibition of the host’s neuraminidase
due to decreased expression of GM1 ganglioside in various
immune cells.[44–47] These findings are supported by thefact that both oseltamivir and zanamivir reduce antibody production at certain doses both in humans and other animals.
Marois et al. [47] demonstrated that influenza specificCD8þ effector T cell recruitment was reduced up to 81% in
the lungs of mice treated with oseltamivir (5 or 50 mg/kg
twice daily; EC50 49 nM in vitro) compared to saline controls.
They also showed that oseltamivir administration significantly
decreased the pools of tissue-resident and circulating effector
memory (93.7%) and central memory CD8þ T cells (45%) compared to saline controls. During heterologous secondary infection, a decreased memory CD8þ T cell pool combined with
reduced generation of secondary influenza-specific effectors
in the lymph nodes resulted in 10-fold decreased CD8þ Tcell
recall responses, which increased mouse morbidity anddelayed viral clearance. Furthermore, they reported that antiviral administration led to a significant 5.7-fold decreased

production of functional anti-influenza antibodies. They summarized that oseltamivir treatment affects the kinetics, magnitude, and nature of innate, adaptive, and memory immune
responses during the mouse-adapted influenza (PR8;H1N1)
infection in the mouse model. They suggested that administration of oseltamivir in infected individuals might reduce thegeneration of protective immunity against reinfection and,thus, lead to the development of disease.[47]The evidence of re-infection in the subsequent season[33] or within the same season [38,39] supports theadverse effect of both neuraminidase inhibitors on the
immune system.
These findings are also consistent with the evidence on
the mechanism of action of oseltamivir from animal models,[49,53] a randomized controlled experimental human influenza study,[25] and in vitro findings showing inhibitory
effects on immune cells.[29]Other adverse reactions: renal, metabolic, cardiac,
prolonged psychiatric and bleeding disorders,pneumonia, etc
In mammalian cells, four types of sialidase (neuraminidase)
have been identified. They are classified according to theirmajor intracellular localization as intralysosomal sialidase(NEU1), cytosolic sialidase (NEU2), plasma membrane-associated sialidases (NEU3), and mitochondrial sialidase(NEU4).[60,61]
Hepatic NEU3 may be associated with sensitivity to insulin
and glucose tolerance through modification of ganglioside

8

R. HAMA

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composition and peroxisome proliferator-activated receptor
gamma signaling.[62]Clinical administration of oseltamivir at doses that result ininhibition of influenza may also have an additional effect by
decreasing GM1 levels in various cells, including immune
cells,[44–47] nociceptive neurons,[48] insulin or peroxisome
proliferator-activated receptor gamma signaling,[62] and possibly other important cells in the kidney, liver, heart, or central
nervous system.
The evidence from these reports suggests that reductionof human endogenous sialidase (neuraminidase) activity byoseltamivir carboxylate may cause delayed onset type adverse
reactions to neuraminidase inhibitors. These include not only
inhibition of antibody and pro-inflammatory cytokine induction, but also prolonged neuropsychiatric reactions, hyperglycaemia, renal and hepatic impairment, pneumonia, andexacerbation of infection, such as re-infection of influenza,
gastrointestinal tract haemorrhage, and others.

Difference between oseltamivir and otherneuraminidase inhibitors in delayed onset typereactionsSufficient plasma concentration of oseltamivir carboxylate, ametabolite of orally administered oseltamivir phosphate, actson the host’s endogenous neuraminidase to reduce (or suppress) the immune response even at the dose of 20 mg b.i.d.
for 5 days.[25] However, bioavailability of inhaled zanamivir is11%, estimated using the data of area under the curve (AUC)
from inhalation and intravenous administration over 30 min
while peak concentration (Cmax) was 3.1% of that of intravenously administered zanamivir.[63] According to the datafrom the summary basis of approval of zanamivir inJapan,[64] bioavailability of inhaled zanamivir is calculated tobe approximately 9 to 72% (geometric mean ¼ 25%). The low
bioavailability of zanamivir may be the major reason why
reduction of antibody rise was not observed in the systematic
review of zanamivir.[9]However, if zanamivir is administered at a high dose or foran extended period, or if the patient is very susceptible,
inhaled zanamivir might reach a concentration high enoughto reduce the immune response. In fact, 20 mg/day of inhaledzanamivir for 14 days showed significant reduction of antibody titres to the H1N1 compared with a placebo.[36] Levelsof various pro-inflammatory cytokines and chemokines were
almost completely suppressed in the viral challenge RCT
using a very high dose (600 mg) of intravenous zanamivir
before inoculation of influenza virus in human adults.[37]Although no report which examined the suppression ofcytokine induction by laninamivir was found in either publishedpapers or the summary basis of approval,[65] several reports
indicated that pro-inflammatory cytokine induction was suppressed by peramivir administration in the animal models of
lethal influenza virus infection.[49–51] Prolonged median survival was reported in these animal infection models with almost
100% mortality. However, prolonged survival was not stated in
the ferret infection model of laninamivir.[65] Moreover, viraltitres in the nasal wash were higher at 72 h after inoculation of

influenza virus in ferrets administered with inhaled laninamivir
4 h after inoculation compared with vehicle control, while they
were lower at 24 and 48 h after inoculation.[65]The AUC at the maximum animal dose of zanamivir(191 lg h/mL after intravenous 90 mg/kg) [64] in rats was only
2.2 times higher than that of healthy human male adults [63](86.6 lg h/mL after intravenous administration of 600 mg over
30 min). The maximum dose of inhaled laninamivir in the toxicity tests was 7.4 times higher than the usual clinical dose
based on the AUC.[65] Hence, it may be hard to detect renal
toxicity of zanamivir and laninamivir using existing animal
toxicity studies.
According to the Japanese summary basis of approval,[66]the kidney is a toxic target organ of peramivir. NOAEL of peramivir for renal toxicity in rabbits was 100 mg/kg, which is
32 mg/kg in HED (conversion factor 3.1) or 2.7 times higher
than the maximum clinical dose of peramivir (600 mg/50 kg).
The safety margin for renal toxicity was estimated as 3.0 and
2.1 based on the AUC in single-dose and multiple-dose (7days) oral rabbit toxicity studies.[66] It is very important touse sensitive animals to detect early signs of toxicity.[67] For
example, canaries were used by miners to detect theincreased level of carbon monoxide.[68]Inhibitory effects on antibody production have beenreported for oseltamivir and zanamivir. Inhibitory effects ofcytokines or chemokines have been reported for oseltamivir,
zanamivir, and peramivir. Toxicities in kidneys have beenreported for oseltamivir and peramivir. On the other hand,
toxicities affecting other cells and cell functions, including
effects on ganglioside GM1, have not been investigated,
except for oseltamivir.
Based on these findings, it can be said that neuraminidase
inhibitors in general may act as inhibitors of the host’s
endogenous neuraminidase and thereby induce various
delayed onset type reactions as shown in the case of
oseltamivir.

Summary and conclusion
Neuraminidase inhibitors (NIs) are generally believed torelieve influenza symptoms by inhibiting the neuraminidaseof the influenza virus. However, a clinically compatible doseof oseltamivir relieves symptoms in mice infected with RSVthat lacks neuraminidase. The underlying mechanisms ofthese phenomena are explained by the inhibition of thehost’s endogenous neuraminidase, leading to reduction ofGM1 ganglioside in immune cells. This may cause subsequent
reduction of pro-inflammatory cytokines such as IL-6, interferon, and TNF-a. These mechanisms may be closely related
to symptom relief without significant reduction of viral load.
They also cause attenuated antibody production of secretory
IgA antibodies and plasma HI antibodies of influenza, andmay be the cause of re-infection of influenza within the sameor the subsequent seasons.
Mechanisms for delayed onset type reactions with prolonged course, such as psychosis and other psychiatric reactions, and renal, metabolic (hyperglycaemia or diabetes), andINFECTIOUS DISEASES

cardiac reactions (QT prolongation), may be related to inhibition of the host’s endogenous neuraminidase.

[15]
[16]

Disclosure statement
Rokuro Hama was a co-recipient of a UK National Institute for HealthResearch grant (HTA 10/80/01, Update and amalgamation of two
Cochrane reviews: neuraminidase inhibitors for preventing and treating
influenza in healthy adults and children (www.nets.nihr.ac.uk/projects/hta/
108001). RH wrote two books published in 2008 about the harm of oseltamivir and antipyretics. He provided scientific opinions and expert testimony on 14 adverse reaction cases related to oseltamivir for theapplications by their families for adverse reaction relief by PMDA
(Pharmaceuticals and Medical Devices Agency) and in the lawsuits forrevocation of the PMDA’s decision concerning with these reactions. Mostof the cases were reported in reference.[6]

[17]

[18]

[19]

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