Drug Discoveries & Therapeutics. 2024; 18(2):260-264.

Brief Report260

DOI: 10.5582/ddt.2024.01049

Determination of anamorelin concentration in human plasmausing a simple high-performance liquid chromatographyultraviolet detection method
Takeo Yasu1,5,*, Nanami Iwatuki1, Yoshito Gando1, Yasuhiko Matumoto2,5, Masahiro Masuo3,
Mikio Shirota4, Masayoshi Kobayashi3,51Department of Medicinal Therapy Research, Education and Research Unit for Comprehensive Clinical Pharmacy, Meiji Pharmaceutical University,Tokyo, Japan;
2
Department of Microbiology, Meiji Pharmaceutical University, Tokyo, Japan;
3
Department of Respiratory Medicine, Tokyo Metropolitan Bokutoh Hospital, Tokyo, Japan;
4
Department of Pharmacy, Tokyo Metropolitan Toshima Hospital, Tokyo, Japan;
5
Bokutoh Hospital-Meiji Pharmaceutical University Joint Research Center, Tokyo, Japan.

SUMMARY

Anamorelin, a non-peptide ghrelin analog and growth hormone secretagogue, is a novel oral drug
used to treat cancer cachexia. Patients with cancer cachexia frequently use several drugs andanamorelin is a substrate of cytochrome P450 (CYP) 3A4; therefore, drug-drug interactions withCYP3A4 inhibitors and inducers pose a clinical problem. In this study, we aimed to determine
the concentration of anamorelin in human plasma using a simple high-performance liquid
chromatography-ultraviolet (HPLC-UV)-based method. The analysis involved extracting a 200-μL
plasma sample and protein precipitation using solid-phase extraction. Anamorelin was isocraticallyseparated using a mobile phase consisting of 0.5% potassium dihydrogen phosphate (pH 4.5) and
acetonitrile (61:39, v/v) on a Capcell Pack C18 MG II column (250 mm × 4.6 mm) at a flow rate of 1.0
mL/min and monitored at a detection wavelength of 220 nm. The calibration curve was linear withina plasma concentration range of 12.5-1,500 ng/mL, with a coefficient of determination of 0.9999.
The intra- and inter-day coefficients of variation were 0.37-6.71% and 2.05-4.77%, respectively. The
accuracy of the assay and recovery were 85.25-112.94% and > 86.58%, respectively. This proposed
HPLC-UV method is simple and can be applied in clinical settings.

Keywords

cancer cachexia, drug-drug interactions, clinical settings

1. Introduction
Anamorelin, a non-peptide ghrelin analog and growth
hormone secretagogue, is a novel oral drug used to treat
cancer cachexia (1). Approximately 80% of patientswith advanced cancer develop cancer cachexia, which
is characterized by involuntary weight loss frequently
accompanied by anorexia and altered metabolism (2).
Anamorelin has been reported to increase appetite,overall body weight, lean body mass, and muscle
strength in clinical studies involving patients with cancer
experiencing cancer cachexia (3,4). However, thistherapy for cancer cachexia is approved and marketed
only in Japan. Anamorelin is a substrate of cytochrome
P450 (CYP) 3A4; therefore, drug-drug interactions
with CYP3A4 inhibitors and inducers pose a clinicalproblem. A three-fold increase in the plasma values forthe area under the plasma concentration-time curve overa 24-h dosing interval was observed with ketoconazole,a CYP3A4 inhibitor (5). Patients with cancer cachexia
use several medications and herbs more frequently
than the general population to improve cancerassociated symptoms and quality of life, decrease
cancer progression, and reduce the side effects ofchemotherapy (6). However, such concomitant use andits effects on the pharmacokinetics of anamorelin havenot been fully studied. Hyperglycemia, hepatotoxicity,and depression of the stimulant conduction system havebeen reported as common adverse events of anamorelin(7). Furthermore, the exposure-response and exposuresafety relationships of anamorelin levels in plasma
have not been reported. We hypothesize that monitoring
anamorelin levels in the blood will aid in determining
the relationship between its blood levels and efficacy

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Drug Discoveries & Therapeutics. 2024; 18(2):260-264.

or safety. Following this, anamorelin treatment can beeffectively optimized in patients with cancer cachexia
in clinical settings. To date, liquid chromatography
coupled with tandem mass spectrometry and highperformance liquid chromatography-ultraviolet (HPLCUV) have not been used to determine anamorelin
concentration in human plasma. HPLC-UV offers many
advantages over other detection systems, such as low
operational cost, versatility, and simple operation. In
this study, we aimed to measure anamorelin levels inhuman plasma using a HPLC-UV-based method thatcan be applied in clinical practice.
2. Materials and Methods
2.1. Standards and reagents
Anamorelin and ketoconazole were purchased from
Sigma-Aldrich (St. Louis, MO, USA) and Tokyo
Chemical Industry Co. (Tokyo, Japan), respectively.
HPLC-grade acetonitrile, methanol, and water wereobtained from Kanto Chemical Co., Inc. (Tokyo,Japan) and potassium dihydrogen phosphate (KH2PO4)
was purchased from FUJIFILM Wako Pure ChemicalCorporation (Osaka, Japan). An Oasis hydrophiliclipophilic balance (HLB) extraction cartridge waspurchased from Waters Corp. (Milford, MA, USA).
Human plasma and EDTA-2Na were purchased fromCosmo Bio Co., Ltd. (Tokyo, Japan).
2.2. HPLC apparatus and analytical conditions
The HPLC apparatus consisted of a pump (PU4180; Jasco, Tokyo, Japan), UV detector (UV-4075;Jasco, Tokyo, Japan), and autosampler (AS-4550,Jasco, Tokyo, Japan). Separation was performed on aoctadecylsilyl analytical column (Capcell Pack C18
MG II, 250 mm × 4.6 mm i.d., 5 µm, Osaka Soda,Tokyo, Japan). The detection wavelength was 220 nm
and the mobile phase consisted of 0.5% KH2PO4 (pH4.5) and acetonitrile (61:39, v/v) with a flow rate of 1.0mL/min.
2.3. Preparation of stock and working solutions
Anamorelin and ketoconazole stock or working
solutions were diluted in methanol and then stored at−60℃ in the dark until use.
2.4. Sample preparation
A 10-µL anamorelin working solution was vortexedwith 200 µL plasma for 1 min. Anamorelin-spiked
plasma (200 µL) was mixed with 10 µL of the internal
standard (IS; 10 µg/mL ketoconazole) and 800 µL
water, vortexed for 30 s, and applied to the preconditioned Oasis HLB cartridges. The cartridges

were washed with 1 mL of water and 60% methanol
in water (v/v). The analytes were eluted with 1 mL of
100% methanol and vacuum-evaporated to dryness at
80°C using a rotary evaporator. The dried residues werereconstituted with 50 µL methanol, and a 20-µL aliquot
was injected into the HPLC system.
2.5. Calibration and Method validation
The calibration concentrations of anamorelin were12.5, 25, 100, 500, 1,000, and 1,500 ng/mL. Methodvalidation was based on the Guidelines for theValidation of Methods for the Quantitative Analysisof Biological Samples by the US Food and Drug
Administration (FDA) (8). The recovery and accuracyof the assay were determined at the calibration
concentrations of anamorelin (12.5–1,500 ng/mL). The
assay precision was evaluated by analyzing five sets ofcontrol samples on the same day (intra-day) and fivedifferent days (inter-day) at concentrations of 12.5, 25,
100, 500, 1,000, and 1,500 ng/mL.
2.6. Sample stability
Anamorelin stability in plasma samples was evaluatedat three different concentrations (12.5, 100, and 1,500
ng/mL). Bench-top stability was evaluated usingsamples stored at 20°C for 6 h (n = 5). The stability ofthe processed samples was evaluated after storage at
4°C for 24 h (n = 5). Long-term stability was evaluatedusing samples stored at −60°C for 4 weeks (n = 5). Thefreeze and thaw stability were evaluated using samples
thawed after storage at −60°C after three freeze-thaw
cycles (n = 5).
2.7. Clinical application
Blood samples were collected after obtaining written
informed consent from a patient with non-small-celllung cancer (NSCLC) receiving anamorelin. Plasma
samples were obtained by centrifuging the blood
samples at 3,000× g for 5.5 min. Plasma and serum
were stored at −80°C until analysis. This study wasapproved by the Institutional Review Board of Tokyo
Metropolitan Bokutoh Hospital (approval number: #05–101) and conducted in accordance with the Declarationof Helsinki. Concomitant medications used by thepatient were acetaminophen, loxoprofen, rabeprazole,naldemedine, magnesium oxide, oxycodone,prochlorperazine, trichlormethiazide, amlodipine, andinsulin.
3. Results and Discussion
In the present study, we developed a sensitive
HPLC-UV-based method to determine anamorelin
concentrations in human plasma in a clinical setting.

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Drug Discoveries & Therapeutics. 2024; 18(2):260-264.
The chromatograms of blank plasma and anamorelinin plasma (100 ng/mL) are provided in Figure 1A and1B, respectively. The retention times for anamorelin
and the IS were 10.2 and 17.4 min, respectively, andthere were no interfering peaks (Figure 1B). The sixpoint standard calibration curve for anamorelin waslinear over the 12.5–1500 ng/mL concentration range.
The equation for the calibration curve was y = 0.0041x+ 0.0026 (r2 = 0.9999), where y and x are the peakheight ratio and anamorelin concentration in plasma(ng/mL), respectively. The lower limit of quantification
for anamorelin and the recovery were 6.25 ng/mL and> 86.58%, respectively. At these concentrations, theranges of intra- and inter-day coefficients of variation
were 0.37–6.71% and 2.05–4.77%, respectively (Table1). The stability of anamorelin in plasma was assessed

under various conditions (Table 2). Anamorelin was
not significantly degraded, and the final concentration
was within 85.25–112.94% of the theoretical values.
The matrix effects were assessed by comparing thepeaks of the quality control sample in the mobile
phase with those of the supernatant from the extracted
blank plasma. No significant peak suppression orenhancement was observed. Therefore, the intra- andinter-assay variations, accuracy, recovery, and stability
under various conditions complied with the FDA
guideline recommendations (8). In Japanese patientswith NSCLC, the median maximum concentration(C max) and trough concentration (C trough) of 100 mg
anamorelin on days 7, 21, and 42 at multiple doses
were 1,120 ± 922 and 26.1 ± 25.2 ng/mL, respectively(9). In the present study, a concentration range of 12.5–

Figure 1. Chromatograms of the (A) blank plasma sample, (B) plasma sample containing 100 ng/mL anamorelin, and (C) plasma sample
from a patient treated with 100 mg anamorelin (294.9 ng/mL).

Table 1. Intra- and inter-day accuracy and precision results for anamorelin samples
Intra-day (n = 5)

Anamorelin added(ng/mL)

12.525100500
1000
1500

Inter-day (n = 5)
Recovery (%)

Mean ± SD(ng/mL)

CV (%)

Accuracy (%)

Mean ± SD(ng/mL)CV (%)

Accuracy (%)

12.51 ± 0.37
26.75 ± 1.61
96.51 ± 5.31
474.05 ± 22.65
975.71 ± 65.45
1395.42 ± 58.66

0.37
6.02
5.50
4.78
6.71
4.20

100.11107.00
96.51
94.81
97.57
93.03

13.34 ± 0.64
27.13 ± 0.87
100.68 ± 3.54
493.29 ± 10.10
991.27 ± 38.61
1468.47 ± 35.57

4.77
3.20
3.512.05
3.90
2.42

106.73108.51100.68
98.66
99.13
97.90

89.49
86.58
98.14
91.46
94.31
98.62

CV, coefficient of variation; SD, standard deviation.

Table 2. Stability analyses under various conditions (n = 5)
Anamorelin added(ng/mL)

12.5
100
1500

Stability condition (%)
Benchtop
mean ± SD

Processed sample
mean ± SD

4 weeks
mean ± SD

Freeze-and-thaw
mean ± SD

97.69 ± 7.95
101.69 ± 3.44
108.28 ± 6.36

112.94 ± 4.90
95.40 ± 2.58
104.19 ± 6.03

99.26 ± 5.88
87.32 ± 3.07
85.25 ± 1.06

108.55 ± 5.09
98.93 ± 1.90
103.92 ± 1.29

SD, standard deviation.

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Drug Discoveries & Therapeutics. 2024; 18(2):260-264.

1,500 ng/mL was covered using our method, indicating
its suitability for the evaluation of Cmax and Ctrough ofanamorelin in a clinical setting.
A 76-year-old man with NSCLC who received
carboplatin + nab-paclitaxel + atezolizumab wasstarted on anamorelin (100 mg/day) for anorexia. The
loss of appetite improved after 5 days of anamorelin
treatment, and body weight increased from 60.8 kg to62.1 kg after 7 days of anamorelin treatment, indicating
the effectiveness of anamorelin. At the initiation ofanamorelin administration, the patient's aspartate
aminotransferase (AST) and alanine aminotransferase(ALT) levels were 28 and 34 IU/L, respectively.
However, blood tests showed AST and ALT levels of
98 IU/L (grade 2 based on the Common Terminology
Criteria for Adverse Events [ver.5.0]) and 187 IU/L
(grade 3), respectively, on day 28 (Figure 2). The patient
was admitted to the hospital for close examination ofliver damage. Anamorelin-induced hepatotoxicity wassuspected; therefore, anamorelin was discontinued afteradmission. Seven days after anamorelin discontinuation,
the patient's AST and ALT levels improved to normalvalues, and the patient was diagnosed with anamorelininduced hepatotoxicity. A plasma sample of anamorelin
obtained on day 28 showed a value of 294.9 ng/mL
4 h after administration. The time for anamorelin toreach the maximum serum concentration ranges from
0.5 to 2.0 h (10), indicating its presence (half-life of
1–2 h) in the blood. In the ONO-7643-04 study by Ono
Pharmaceutical, the mean level of anamorelin in theblood 4–6 h after multiple 100 mg doses was 385 ± 324
ng/mL in Japanese patients with NSCLC (9). Comparedwith the patient in the present study, those in the ONO7643-04 study had higher blood anamorelin levels 4–6
hours after administration. There are no reports on therelationship between anamorelin levels in the bloodand hepatotoxicity; however, there have been reportsof moderately elevated but normalized AST and ALTlevels in patients after anamorelin discontinuation (11),as in the patient in this study. Notably, only one casewas studied; therefore, there may be no relationshipbetween anamorelin blood levels and hepatotoxicity.
In two phase III trials, the most frequent drug-related
adverse events were hyperglycemia (4.2–5.3%). Onlya few drug-related Grade ≥ 3 AEs were observed inboth studies (0.9% and 2.7%, respectively) (3,4). In aphase 1 study, nine healthy male volunteers received
10, 25, and 50 mg oral doses of anamorelin, and serum
growth hormone levels increased significantly in adose-dependent manner (12). Therefore, hyperglycemia
may be associated with anamorelin levels in theblood. In the future, we aim to evaluate the exposureresponse and exposure-safety relationships in casesof hyperglycemia, a characteristic adverse event of
anamorelin, in a large sample size using our assay.
Nevertheless, the therapeutic range of anamorelin
remains unclear. The number of older patients withcancer cachexia has increased with population aging,
resulting in increased anamorelin use. Our proposed
method can be applied to patients receiving anamorelinand can be used to explore the therapeutic target range.
Our reported method has a limitation, as it wasapplied to samples from only one patient. During fullvalidation in blank human plasma, amamorelin andbiogenic components were completely separated.
Furthermore, no interferential peak near anamorelin
was observed in six different lots of blank humanplasma. However, in this one patient's plasma, withseparation between anamorelin and the biogenic
component being 1.0 (Figure 1C). Therefore, it may benecessary to reconsider the measurement conditions.
Nevertheless, an interference peak near anamorelin inthis case did not affect the quantification of anamorelin.
It is therefore necessary to measure samples from alarge number of patients in the future and assess foreign
substances, such as concomitant medications andindividual patient biogenic components.
In conclusion, we developed a simple HPLC-UVbased method to determine anamorelin concentrationin human plasma. This method is simple and can beused to evaluate interactions between anamorelinand concomitant drugs in clinical settings. FurtherFigure 2. Clinical course of anamorelin for cancer cachexia in a patient with non-small cell lung cancer. The straight line indicates aspartatetransaminase (AST), whereas the dashed line indicates alanine transaminase (ALT).

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Drug Discoveries & Therapeutics. 2024; 18(2):260-264.
studies using this assay are required to elucidate therelationship between anamorelin blood levels and its
clinical efficacy and safety.
Acknowledgements
The authors thank the staff of the Department ofClinical Laboratory, Tokyo Metropolitan Bokutoh
Hospital, for sample management.
Funding: None.
Conflict of Interest: The authors have no conflicts ofinterest to disclose.
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Received June 29, 2024; Revised July 30, 2024; Accepted
August 19, 2024.
*Address correspondence to:
Takeo Yasu, Department of Medicinal Therapy Research,Education and Research Unit for Comprehensive ClinicalPharmacy, Meiji Pharmaceutical University; 2-522-1, Noshio,Kiyose, Tokyo 204-8588, Japan.
E-mail: yasutakeo-tky@umin.ac.jp
Released online in J-STAGE as advance publication August25, 2024.

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