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Universitas Lambung Mangkurat F-MIPA PS.
Farmasi
Nama : Muhammad Arief Rahman

NIM : 2111015210015
Kelompok : 1
LABORATORIUM
FARMAKOLOGI-TOKSIKOLOGI
BUKU KERJA PRAKTIKUM
FARMAKOKINETIKA
(JAE 502)
Disusun oleh
apt. Destria Indah Sari, M.Farm.

apt. Nurlely, M.Sc (Pharm).
apt. Okta Muthia Sari, M.Farm.
PROGRAM STUDI FARMASI

FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN
ALAM
UNIVERSITAS LAMBUNG MANGKURAT
2023
Buku Kerja Praktikum Farmakokinetika

Universitas Lambung Mangkurat F-MIPA PS.Farmasi
PERCOBAAN I
SIMULASI IN VITRO MODEL FARMAKOKINETIKA
Muhammad Arief Rahman

2111015210015
KELOMPOK 1
Mengetahui, Nilai Laporan Awal Nilai Laporan Akhir

Asisten
(Alya Nurwafa) Tanggal : Tanggal :

05 September 2022 14 September 2022

PERCOBAAN I
SIMULASI IN VITRO MODEL FARMAKOKINETIKA
Tujuan Umum : Memahami konsep farmakokinetika suatu obat

Alat dan Bahan :
Alat :
1. Beaker glass 1L/2L
2. Hotplate
3. Labu ukur 10 mL
4. Magnetic stirrer
5. Pipet tetes
6. Pipet ukur
7. Pipet volume 25 mL/30 mL
8. Propipet
9. Rak tabung reaksi
10. Spektrofotometer
11. Stopwatch
12. Tabung reaksi
Bahan :
1. Air suling
2. Rhodamin B
Ringkasan Cara Kerja/Tahapan Percobaan :

1. Pembuatan Larutan Baku Kerja Rhodamin B
Rhodamin B
• Ditimbang sebanyak 10 mg
• Dilarutkan dalam 100 mL aquadest sampai tanda
batas labu ukur 100 mL
• Diperoleh larutan baku induk 100 ppm
Larutan Baku
Induk 100 ppm
• Diambil 1 mL, dimasukkan ke labu ukur 10 mL

• Dilarutkan dengan aquadest sampai tanda batas

• Diperoleh larutan baku 10 ppm
Larutan Baku
Induk 10 ppm
• Diencerkan menjadi larutan 0,25; 0,5; 1; 2; 3; dan 5
μg/mL dengan labu ukur 10 mL
Hasil
2. Penentuan Panjang Gelombang Maksimum
Larutan Baku
Kerja 5 ppm
• Diamati nilai serapan pada panjang gelombang 530-

560 nm
• Dibuat kurva serapan pada kertas grafik berskala
sama
Hasil • Ditentukan λ maksimum
3. Pembuatan Kurva Baku
Larutan Baku
Kerja
• Diamati serapan pada panjang gelombang

maksimum yang telah didapat
• Dibuat kurva bakunya
Hasil
4. Simulasi Model Farmakokinetika In Vitro

Rute Intravaskular, kompartemen satu terbuka
Gelas Aqua
• Dikalibrasi gelas aqua 100 ml

• Diberi tanda
Air Suling
• Diisikan pada gelas beker secara kuantitatif, sesuai

nilai Vd 1,8 L
• Diletakkan di atas hotplate dan dijalankan stirrer
• Ditambahkan rhodamin B sesuai dosis yang sudah
ditentukan sebanyak 50 mL
Sampel Larutan
Rhodamin B
• Diambil setiap 3 menit CL 100 mL

• Dimasukkan CL yang diambil ke dalam gelas aqua
dan gantikan dengan aquadest 100 mL
• Dilakukan berulang sampai menit 30
• Dihentikan pada menit 30
• Diukur serapan pada panjang gelombang maksimum
• Dihitung parameter farmakokinetika
Hasil
Rute ekstravaskuler, kompartemen satu terbuka
Gelas Aqua
• Dikalibrasi gelas aqua 200 ml

• Diberi tanda
Air Suling
• Diisikan pada gelas beker secara kuantitatif, sesuai

nilai Vd 0,9 L
• Diletakkan diatas hotplate dan dijalankan stirrer
• Ditambahkan rhodamin B sesuai dosis yang sudah
ditentukan sebanyak 10 mL

Sampel Larutan
Rhodamin B
• Diambil setiap 3 menit CL 200 mL

• Dimasukkan CL yang diambil ke dalam gelas aqua
dan gantikan dengan aquadest 200 mL
• Ditambahkan rhodamin B 10 mL
• Diambil berulang sampai rhodamin B mencapai 50
mL atau sampai pada menit ke 15
• Diambil sesuai CL 200 mL pada menit ke 18 diganti
dengan aquadest 200 mL
• Dihentikan pada menit 18-30 untuk penambahan 10
mL
• Diukur serapan pada panjang gelombang maksimum
• Dihitung parameter farmakokinetika
Hasil
Pertanyaan :
1. Apa yang dimaksud dengan model farmakokinetika dan mengapa
diperlukan model farmakokinetika ? Sebutkan macamnya !
2. Apa yang dimaksud dengan volume distribusi dan klirens suatu obat ?
3. Parameter farmakokinetika yang mana yang dikaitkan dengan jumlah obat
dalam tubuh untuk pengukuran kadar obat dalam plasma ?
4. Jelaskan faktor dari timbulnya variabilitas kadar obat dalam plasma setelah
dosis yang sama diberikan kepada pasien yang berbeda !
Jawaban
1. Model farmakokinetika adalah model matematis yang disusun untuk
menggambarkan gerak obat dalam tubuh. Perlunya model farmakokinetika
adalah untuk mensimulasi kecepatan proses absorpsi, distribusi dan eliminasi
obat dalam tubuh serta menjelaskan konsentrasi obat dalam tubuh terhadap
waktu. Selain itu, model farmakokinetika digunakan juga untuk menemukan

parameter dengan cara menghubungkan konsentrasi dengan dosis yang

diberikan pada target (Mould & Upton, 2013). Kegunaan model
farmakokinetika :
a. Memprediksi kadar obat dalam plasma, jaringan, dan urin dengan rejimen
dosis apa pun.
b. Menghitung regimen dosis optimal untuk masing-masing kadar secara
individual
c. Memperkirakan kemungkinan akumulasi obat dan / atau metabolit
d. Mengevaluasi perbedaan tingkat atau luasnya ketersediaan antara
formulasi (bioekuivalensi).
e. Menjelaskan interaksi obat.
Macam-macam model farmakokinetika :
a. Model kompartemen
b. Model mammillary
c. Model catenary
d. Model farmakokinetik fisiologis
(Shargel & Yu, 2016).
2. Volume distribusi merupakan volume yang diperlukan untuk memuat semua
obat dalam tubuh secara homogen dengan konsentrasi yang sama dengan
dengan konsentrasi obat dalam darah, plasma atau cairan. Dengan kata lain
volume yang menunjukkan distribusi obat. Volume distribusi diperlukan
untuk menghitung bersihan obat (Rinidar et al., 2021). Sedangkan Klirens
obat adalah suatu ukuran eliminasi obat dari tubuh tanpa mempermasalahkan
mekanisme prosesnya. Klirens dapat didefinisikan sebagai volume bersihan
suatu obat dari tubuh per satuan waktu (mL/menit atau L/jam). Nilai klirens
dapat dipengaruhi oleh beberapa faktor fisiologi, seperti fungsi organ dalam
mengeliminasi obat dan kecepatan alir darah menuju organ eliminasi obat
(Pradana et al., 2013).
3. Volume distribusi merupakan suatu parameter yang berguna untuk menilai
jumlah relatif obat di luar kompartemen sentral atau jaringan. Jumlah total
obat dalam tubuh pada berbagai waktu pemberian dapat ditentukan dengan
mengukur konsentrasi obat dalam darah jika diketahuinya Vd suatu obat.

Distribusi bahan biologik biasanya terbatas di plasma dan cairan ekstraselular

karena umumnya bersifat polar dan bobot molekulnya besar. Protein dengan
bobot molekul di atas 30 kDa sangat lambat melewati kapiler pembuluh darah.
Distribusi bahan biologik dipengaruhi oleh ikatannya dengan protein plasma.
Konjugasi antibodi dengan protein plasma dapat menghambat metabolisme
antibodi dan meningkatkan efikasinya sebagai agen terapi (Suartini et al.,
2016).
4. Faktor dari timbulnya variabilitas kadar obat dalam plasma setelah dosis yang
sama diberikan kepada pasien yang berbeda yaitu fisiologi pasien, usia, berat
badan, jenis kelamin, dan status gizi akan mempengaruhi disposisi obat dan
harus dipertimbangkan. Selain itu, kondisi patofisiologis, seperti disfungsi
ginjal, penyakit hati, atau gagal jantung kongestif, dapat mengubah
farmakokinetik normal obat, dan dosis harus disesuaikan dengan hati-hati.
Pengaruh paparan jangka panjang terhadap obat pada pasien harus juga
diperhatikan termasuk kemungkinan penyalahgunaan obat oleh pasien. Selain
itu, faktor gaya hidup pribadi, seperti merokok, penyalahgunaan alkohol, dan
obesitas, merupakan masalah lain yang diketahui dapat mengubah
farmakokinetik obat serta kurangnya kepatuhan pasien (yaitu ketidakpatuhan
pasien) dalam meminum obat juga dapat menjadi masalah dalam mencapai
hasil terapi yang efektif (Shargel & Yu, 2016).
Pustaka
Mould, D. R & R. N. Upton. 2013. Basic Concepts in Population Modeing,
Simulation, and Model-Based Drug Development-Part 2: Introduction to
Pharmacokinetic Modeling Methods. Pharmacometrics & Systems
Pharmacology. 2: 1-14.
Pradana, D. A., F. Hayati & D. Sukma. 2013. Pengaruh Pra-Perlakuan Madu

Terhadap Farmakokinetika Eliminasi Rifampisin pada Tikus Wistar Jantan.
Jurnal Ilmiah Farmasi. 1: 18-28.
Rinidar., M. Isa & T. Armansyah. 2021. Pengantar Farmakologi: Analgesik-

Antipiretik-Anti Inflamasi. Syiah Kuala University Press, Aceh.
Shargel, L & A. B. C. Yu. 2016. Applied Biopharmaceutics and Pharmacokinetics

Seventh Edition. McGraw-Hill Education. New York City.

Suartini, I. G. A. A., I. Sendow, N. L. P. Agustini, A. Suprayogi, I. W. T. Wibawan

& I. G. N. K. Mahardika. 2016. Kinetika Immunoglobulin Kuning Telur
Antiparvovirus Anjing pada Anjing. Jurnal Veteiner. 17: 292-299.

Citation: CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e38; doi:10.1038/psp.2013.14
© 2013 ASCPT All rights reserved 2163-8306/12
www.nature.com/psp
Tutorial
Basic Concepts in Population Modeling, Simulation, and

Model-Based Drug Development—Part 2: Introduction to
Pharmacokinetic Modeling Methods
DR Mould1 and RN Upton1,2
Population pharmacokinetic models are used to describe the time course of drug exposure in patients and to investigate
sources of variability in patient exposure. They can be used to simulate alternative dose regimens, allowing for informed
assessment of dose regimens before study conduct. This paper is the second in a three-part series, providing an introduction
into methods for developing and evaluating population pharmacokinetic models. Example model files are available in the
Supplementary Data online.
CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e38; doi:10.1038/psp.2013.14; advance online publication 17 April 2013
BACKGROUND DATA CONSIDERATIONS
Population pharmacokinetics is the study of pharmacokinetics Generating databases for population analysis is one of the
at the population level, in which data from all individuals in a pop- most critical and time-consuming portions of the evaluation.2
ulation are evaluated simultaneously using a nonlinear mixed- Data should be scrutinized to ensure accuracy. Graphical
effects model. “Nonlinear” refers to the fact that the dependent assessment of data before modeling can identify potential
variable (e.g., concentration) is nonlinearly related to the model problems. During data cleaning and initial model evaluations,
parameters and independent variable(s). “Mixed-effects” refers data records may be identified as erroneous (e.g., a sudden,
to the parameterization: parameters that do not vary across transient decrease in concentration) and can be commented
individuals are referred to as “fixed effects,” parameters that out if they can be justified as an outlier or error that impairs
vary across individuals are called “random effects.” There are model development.
five major aspects to developing a population pharmacokinetic All assays have a lower concentration limit below which
model: (i) data, (ii) structural model, (iii) statistical model, (iv) concentrations cannot be reliably measured that should
covariate models, and (v) modeling software. Structural models be reported with the data. The lower limit of quantification
describe the typical concentration time course within the popu- (LLOQ) is defined as the lowest standard on the calibration
lation. Statistical models account for “unexplainable” (random) curve with a precision of 20% and accuracy of 80–120%.3
variability in concentration within the population (e.g., between- Data below LLOQ are designated below the limit of quantifi-
subject, between-occasion, residual, etc.). Covariate models cation. Observed data near LLOQ are generally censored if
explain variability predicted by subject characteristics (covari- any samples in the data set are below the limit of quantifica-
ates). Nonlinear mixed effects modeling software brings data tion. One way to understand the influence of censoring is to
and models together, implementing an estimation method for include LLOQ as a horizontal line on concentration vs. time
finding parameters for the structural, statistical, and covariate plots. Investigations4–7 into population-modeling strategies
models that describe the data.1 and methods to deal with data below the limit of quantification
A primary goal of most population pharmacokinetic model- (Supplementary Data online) show that the impact of cen-
ing evaluations is finding population pharmacokinetic param- soring varies depending on circumstance; however, methods
eters and sources of variability in a population. Other goals such as imputing below the limit of quantification concentra-
include relating observed concentrations to administered tions as 0 or LLOQ/2 have been shown to be inaccurate. As
doses through identification of predictive covariates in a tar- population-modeling methods are generally more robust to
get population. Population pharmacokinetics does not require the influence of censoring via LLOQ than noncompartmental
“rich” data (many observations/subject), as required for anal- analysis methods, censoring may account for differences in
ysis of single-subject data, nor is there a need for structured the results when applied to the same data set.
sampling time schedules. “Sparse” data (few observations/ It is worthwhile considering what the concentrations
subject), or a combination, can be used. reported in the database represent in vivo. Three major con-
We examine the fundamentals of five key aspects of pop- siderations of the data are of importance. First, the sampling
ulation pharmacokinetic modeling together with methods matrix may influence the pharmacokinetic model and its
for comparing and evaluating population pharmacokinetic interpretation. Plasma is the most common matrix, but the
models. extent of distribution of drug into RBC dictates (i) whether
Projections Research, Phoenixville, Pennsylvania, USA; 2Australian Centre for Pharmacometrics, University of South Australia, Adelaide, Australia.
1
Correspondence: DR Mould (drmould@pri-home.net)

Received 10 December 2012; accepted 18 February 2013; advance online publication 17 April 2013. doi:10.1038/psp.2013.14
21638306, 2013, 4, Downloaded from https://ascpt.onlinelibrary.wiley.com/doi/10.1038/psp.2013.14 by Nat Prov Indonesia, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Introductory Overview of Population Pharmacokinetic Modeling
Mould and Upton
2
whole blood or plasma concentrations are more informative; stages of model building (e.g., evaluating goodness of fit with
(ii) whether measured clearance (CL) should be referenced observed or simulated data) is reasonable. Changing default
to plasma or blood flow in an eliminating organ; or (iii) may values for optimization settings and convergence criteria
imply hematocrit or RBC binding contributes to differences in should not be considered unless the impact of these changes
observed kinetics between subjects.8 Second, whether the is understood.
data are free (unbound) or total concentrations. When free
concentrations are measured, these data can be modeled Comparing models
using conventional techniques (with the understanding that The minimum OFV determined via parameter estimation
parameters relate to free drug). When free and total concen- (OBJ) is important for comparing and ranking models. How-
trations are available, plasma binding can be incorporated ever, complex models with more parameters are generally
into the model.9 Third, determining whether the data must be better able to describe a given data set (there are more
parent drug or an active metabolite is important. If a drug has “degrees of freedom,” allowing the model to take differ-
an active metabolite, describing metabolite formation may be ent shapes). When comparing several plausible models, it
crucial in understanding clinical properties of a drug. is necessary to compensate for improvements of fit due to
increased model complexity. The Akaike information criterion
(AIC) and Bayesian information criterion (BIC or Schwarz cri-
SOFTWARE AND ESTIMATION METHODS
terion) are useful for comparing structural models:
Numerous population modeling software packages are avail- AIC = OBJ + 2 ⋅ np
able. Choosing a package requires careful consideration (1)
including number of users in your location familiar with the BIC = OBJ + np ⋅ Ln (N )
package, support for the package, and how well established
the package is with regulatory reviewers. For practical rea- where np is the total number of parameters in the model,
sons, most pharmacometricians are competent in only one and N is the number of data observations. Both can be used
or two packages. to rank models based on goodness of fit. BIC penalizes the
Most packages share the concept of parameter estimation OBJ for model complexity more than AIC, and may be pref-
based on minimizing an objective function value (OFV), often erable when data are limited. Comparisons of AIC or BIC
using maximum likelihood estimation.2 The OFV, expressed cannot be given a statistical interpretation. Kass and Raf-
tery13 categorized differences in BIC between models of >10
for convenience as minus twice the log of the likelihood, is
a single number that provides an overall summary of how as “very strong” evidence in favor of the model with the lower
closely the model predictions (given a set of parameter val- BIC; 6–10 as “strong” evidence; 2–6 as “positive” evidence;
ues) match the data (maximum likelihood = lowest OFV = and 0–2 as “weak” evidence. In practice, a drop in AIC or
best fit). In population modeling, calculation of the likelihood BIC of 2 is often a threshold for considering one model over
is more complicated than models with only fixed effects.2 another.
The likelihood ratio test (LRT) can be used to compare the
When fitting population data, predicted concentrations for
OBJ of two models (reference and test with more parame-
each subject depend on the difference between each sub-
ters), assigning a probability to the hypothesis that they pro-
ject’s parameters (Pi) and the population parameters (Ppop)
vide the same description of the data. Unlike AIC and BIC,
and the difference between each pair of observed (Cobs) and
models must be nested (one model is a subset of another)
predicted (ĉ ) concentrations. A marginal likelihood needs to
and have different numbers of parameters. This makes LRT
be calculated based on both the influence of the fixed effect
suitable to comparing covariate models to base models
(Ppop) and the random effect (η). The concept of a marginal
(Covariate models section).
likelihood is best illustrated for a model with a single popula-
As the OBJ depends on the data set and estimation
tion parameter (Supplementary Data online).
method used, the OBJ and its derivations cannot be com-
Analytical solutions for the marginal likelihood do not exist;
pared across data sets or estimation methods. The lowest
therefore, several methods were implemented approximat-
OBJ is not necessarily the best model. A higher order poly-
ing the marginal likelihood while searching for the maximum
nomial can be a near perfect description of a data set with the
likelihood. How this is done differentiates the many estima-
lowest OBJ, but may be “overfitted” (describing noise rather
tion methods available in population modeling software.
than the underlying relationship) and of little value. Polyno-
Older approaches (e.g., FOCE, LAPLACE) approximate
mial parameters cannot be related to biological processes
the true likelihood with another simplified function.10 Newer
(e.g., drug elimination), and the model will have no predictive
approaches (e.g., SAEM) include stochastic estimation, refin-
utility (either between data points or extrapolating outside the
ing estimates partially by iterative “trial and error.” All estima-
range of the data). Mechanistic plausibility and utility there-
tion methods have advantages and disadvantages (mostly
fore take primacy over OBJ value.
related to speed, robustness to initial parameter estimates,
and stability in overparameterized models and parameter
precision).11,12 The only estimation method of concern is the STRUCTURAL MODEL DEVELOPMENT
original First Order method in nonlinear mixed-effects model,
which can generate biased estimates of random effects. The The choice of the structural model has implications for
differences between estimation methods can sometimes be covariate selection.14 Therefore, care should be taken when
substantial. Trying more than one method during the initial evaluating structural models.
CPT: Pharmacometrics & Systems Pharmacology

Mould and Upton
3
Systemic models may be needed. Data from both single- and multidose stud-
The structural model (Supplementary Data online) is analo- ies may reveal saturable elimination if steady-state kinetics
gous to a systemic model (describing kinetics after i.v. dos- cannot be predicted from single-dose data. Graphical and/
ing) and an absorption model (describing the drug uptake into or noncompartmental analysis may show evidence of non-
the blood for extravascular dosing). For the former, though linearity: dose-normalized concentrations that are not super-
physiologically based pharmacokinetic models have a useful imposable; dose-normalized area under the curve (AUC) (by
and expanding role,15,16 mammillary compartment models are trapezoidal integration) that are not independent of dose;
predominant in the literature. When data are available from multidose AUCτ or Css that is higher than predicted by single-
only a single site in the body (e.g., venous plasma), concen- dose AUC and CL.
trations usually show 1, 2, or 3 exponential phases17 which Classically, saturable elimination is represented using the
can be represented using a systemic model with one, two, Michaelis–Menten equation.2 For a one-compartment model
or three compartments, respectively. Insight into the appro- with predefined dose rate:
priate compartment numbers can be gained by plotting log
concentration vs. time. Each distinct linear phase when log A
C=
concentrations are declining (or rising to steady state during V
a constant-rate infusion) will generally need its own compart- (2)
dA V ⋅C
= dose rate − max
ment. However, the log concentration time course can appear dt (k m + C )
curved when underlying half-lives are similar.
Mammillary compartment models can be parameterized where dA/dt is the rate of change of the amount of drug, Vmax
as derived rate constants (e.g., V1, k12, k21, k10) or preferen- is maximum elimination rate and km is concentration asso-
tially as volumes and CL (e.g., V1, CL, V2, Q12 where Q12 is ciated with half of Vmax. Note that when C << km, the rate
the intercompartmental CL between compartments one and becomes Vmax/km·C, where Vmax/km can be interpreted as the
two) and are interconvertible (Supplementary Data online). apparent first-order CL. When C >> km, rate becomes Vmax
Rate constants have units of 1/time; intercompartmental CLs (apparent zero-order CL). Vmax and km can be highly corre-
(e.g., Q12) have the units of flow (volume/time) and can be lated, making it difficult to estimate both as random effects
directly compared with elimination CL (e.g., CL, expressed parameters (Between-subject variability section). Broadly, km
as volume/time) and potentially blood flows. Volume and CL can be considered a function of the structure of the drug and
parameterization have the advantage of allowing the model the eliminating enzyme (or transporter) whereas Vmax can be
to be visualized as a hydraulic analog18 (Supplementary considered as a function of the available number of eliminat-
Data online). Parameters can be expressed in terms of half- ing enzymes (or transporters).
lives, but the relationship between the apparent half-lives and The contribution of saturable elimination to plasma con-
model parameters is complex for models with more than one centrations should be considered carefully in the context of
compartment (Supplementary Data online). For drugs with the drug, the sites of elimination for the drug, and the route
multicompartment kinetics, the time course of the exponen- of administration. For drugs with active renal-tubular secre-
tial phases in the postinfusion period depends on infusion tion, saturation of this process will decrease renal CL and
duration, a phenomenon giving rise to the concept of context increase concentrations above that expected from superpo-
sensitive half-time rather than half-life.19 sition. For drugs with active tubular re-absorption, saturation
When choosing the number of compartments for a model, of this process will increase renal CL and decrease concen-
note that models with too few compartments describe the trations below that expected from superposition.
data poorly (higher OBJ), showing bias in plots of residu-
als vs. time. Models with too many compartments show Absorption models
trivial improvement in OBJ for the addition of extra compart- Drugs administered via extravascular routes (e.g., p.o.,
ments; parameters for the additional peripheral compartment s.c.) need a structural model component representing drug
will converge on values that have minimal influence on the absorption (Supplementary Data online). The two key pro-
plasma concentrations (e.g., high volume and low intercom- cesses are overall bioavailability (F) and the time course
partmental CL, or the reverse); or parameters may be esti- of the absorption rate (the rate the drug enters the blood
mated with poor precision. stream).
An important consideration is whether assuming first-order F represents the fraction of the extravascular dose that
elimination is appropriate. In first-order systems, elimination enters the blood. Functionally, unabsorbed drug does not
rate is proportional to concentration, and CL is constant. contribute to the blood concentrations, and the measured
Doubling the dose doubles the concentrations (the principle concentrations are lower as if the dose was a fraction (F)
of superposition).2 Conversely, for zero-order systems, the of the actual dose. The fate of “unabsorbed” dose depends
elimination rate is independent of concentration. CL depends on administration route and the drug, but could include: drug
on dose; doubling the dose will increase the concentrations not physically entering the body (e.g., p.o. dose remaining in
more than two-fold. Note that elimination moves progres- the gastrointestinal tract), conversion to a metabolite during
sively from a first-order to a zero-order state as concentra- absorption (e.g., p.o. dose and hepatically cleared drug), pre-
tions increase, saturating elimination pathways. cipitation or aggregation at the site of injection (e.g., i.m., s.c.)
Pharmacokinetic data collected in subjects given a sin- or a component of absorption that is so slow that it cannot be
gle dose of drug are rarely sufficient to reveal and quanti- detected using the study design (e.g., lymphatic uptake of
tate saturable elimination, a relatively wide range of doses large compounds given s.c.).
www.nature.com/psp
Mould and Upton
4
Absolute bioavailability can only be estimated when con- may be dose dependent with higher doses associated with
current extravascular and i.v. data are available (assuming higher bioavailability. Conversely, if the active component is in
the i.v. dose is completely available, F = 100%). Both CL and the gut lumen to blood direction, higher doses may be associ-
F are estimated parameters (designated as θ), with F only ated with lower bioavailability. If the range of doses is insuf-
applying to extravascular data (Supplementary Data online). ficient to estimate parameters for a saturable uptake model
Although F can range between 0 (no dose absorbed) and 1 (e.g., Vmax, km) using DOSE or log(DOSE) as a covariate on F
(completely absorbed dose), models may be more stable if a may be an expedient alternative. In general, nonlinear uptake
logit transform is used to constrain F where LGTF can range affecting bioavailability can be differentiated from nonlinear
between ±infinity. elimination as the former shows the same half-life across a
range of doses, whereas the latter shows changes in half-life
LGTF = θ1 across a range of doses.
(3)
exp (LGTF) The representation of extravascular absorption is clas-
F =
(1+ exp (LGTF)) sically a first-order process described using an absorption
rate constant (ka). This represents absorption as a passive
In such models, the CL estimated for the i.v. route is the ref- process driven by the concentration gradient between the
erence CL; the apparent CL for the extravascular route (that absorption site and blood (Figure 1a). The concentration
would be estimated from the AUC) is calculated as CLi.v./F. gradient diminishes with time as drug in the absorption site
When only extravascular data are available, it is impossible is depleted in an exponential manner. An extension of this
to estimate true CLs and volumes because F is unknown. For model is to add an absorption lag if the appearance of drug
a two compartment model, e.g., estimated parameters are in the blood is delayed (Figure 1b). The delay can represent
a ratio of the unknown value of F: CL/F, V1/F, Q/F, and V2/F diverse phenomena from gastric emptying (p.o. route) to dif-
(absorption parameters are not adjusted with bioavailability). fusion delays (nasal doses). Because lags are discontinuous,
Although F cannot be estimated, population variability in F numerical instability can arise and transit compartment mod-
can contribute to variability in CLs and volumes making them els may be preferred.20
correlated (Supplementary Data online). “Flip-flop” kinetics occurs when absorption is slower than
If the process dictating oral bioavailability has an active elimination21 making terminal concentrations dependent on
component in the blood to gut lumen direction, bioavailability absorption, which becomes rate limiting. For example, for
a b
40 40
Absorption rate (mg/h)
30 Ka 30
LAG
0.1 0
0.15 0.25
20 0.2 20 0.5
0.25 1
0.3 2
0.35 3
10 10
0 0
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time after dose (h) Time after dose (h)
c d
40 40
30 Ktr 30
NCOMP
0.5 1
0.75 2
20 1 20 3
1.25 4
1.5 6
1.75 12
10 10
0 0
0 3 6 9 12 15 18 21 24 0 3 6 9 12 15 18 21 24
Time after dose (h) Time after dose (h)
Figure 1 Models of extravascular absorption. The time profile of absorption rate for selected absorption models. (a) A first-order absorption
model with different values of the absorption rate constant (ka). Absorption lag was 0.5 h in all cases. (b) A first-order absorption model with
different values of the absorption lag (LAG). Absorption rate constant was 0.5/h in all cases. (c) A three-compartment transit chain model with
different values of the transit chain rate constant (ktr). Note that decreasing the rate constant lowers the overall absorption rate and delays the
time of its maximum value. (d) Transit chain models with different numbers of transit chain compartments (NCOMP). The transit chain rate
constant was 1/h in all cases. Note that increasing the number of compartments introduces a delay before absorption, and functionally acts
as a lag. The dose was 100 mg in all cases (hence the area under the curve should be 100 mg for all models).

Mould and Upton
5
a one-compartment model, there are two parameter sets across all individuals evaluated, the individual η1 values are
that provide identical descriptions of the data, one with fast assumed to be normally distributed with a mean of 0 and
absorption and slow elimination, the other with slow absorp- variance ω2. This assumption is not always correct, and the
tion and fast elimination. Prior knowledge on the relative ramifications of skewed or kurtotic distributions for η1 will be
values of absorption and elimination rates is needed to distin- described later. The different variances and covariances of η
guish between these two possibilities. Drugs with rate-limiting parameters are collected into an “Ω matrix.”
absorption show terminal concentrations after an extravas- Pharmacokinetic data are often modeled assuming log-
cular dose with a longer half-life than i.v. data suggest. Flip- normal distributions because parameters must be positive
flop kinetics can be problematic when fitting data if individual and often right-skewed.22,23 Therefore, the CL of the ith sub-
values of ka and k10 (e.g., CL/V) “overlap” in the population. ject (CLi) would be written as:
It may be advantageous to constrain k10 to be faster than ka,
(e.g., k10 = ka +θ1) where θ1 is >0 if prior evidence suggests CLi = θ1 ⋅ exp (η1i )
(5)
flip-flop absorption.
where θ1 is the population CL and η1i is the deviation from the
Although first-order absorption has the advantage of being
population value for the ith subject. The log-normal function is
conceptually and mathematically simple, the time profile of
a transformation of the distribution of η values, such that the
absorption (Figure 1b) has “step” changes in rate that may be
distribution of CLi values may be log-normally distributed but
unrepresentative of in vivo processes, and this is a “change-
the distribution of η1i values is normal.
point” that presents difficulties for some estimation methods.
When parameters are treated as arising from a log-normal
For oral absorption in particular, transit compartment models
distribution, the variance estimate (ω2) is the variance in the
may be superior.20 The models represent the absorbed drug
log-domain, which does not have the same magnitude as the
as passing through a series of interconnected compartments
θ values. The following equation converts the variance to a
linked by a common rate constant (ktr), providing a continu-
coefficient of variation (CV) in the original scale. For small ω2
ous function that can depict absorption delays. The absorp-
(e.g., <30%) the CV% can be approximated as the square
tion time profile can be adjusted by altering the number of
root of ω2.
compartments and the rate constant (Figure 1c,d). As they
are coded by differential equations (Supplementary Data
online), transit compartment absorption models have lon- CV ( % ) = exp ω 2 − 1 ⋅ 100%
(6) ( )
ger run times than first-order models, which may outweigh
The use of OBJ (e.g., LRT) is not applicable for determin-
their advantages in some situations. The transit compartment
ing appropriateness of including variance components.24
model can be approximated using an analytical solution in
Variance terms cannot be negative, which places the null
some cases.20
hypothesis (that the value of the variance term is 0) on
the boundary of the parameter space. Under such circum-
STATISTICAL MODELS stances, the LRT does not follow a χ2 distribution, a neces-
sary assumption for using this test. Using LRT to include or
The statistical model describes variability around the struc- exclude random effects parameters is generally unreliable,
tural model. There are two primary sources of variability in as suggested by Wählby et al.25 For this reason, models with
any population pharmacokinetic model: between-subject different numbers of variance parameters should also not
variability (BSV), which is the variance of a parameter across be compared using LRT. Varying approaches to developing
individuals; and residual variability, which is unexplained vari- the Ω matrix have been recommended. Overall, it is best to
ability after controlling for other sources of variability. Some include a variance term when the estimated value is neither
databases support estimation of between-occasion variabil- very small nor very large suggesting sufficient information to
ity (BOV), where a drug is administered on two or more occa- appropriately estimate the term. Other diagnostics such as
sions in each subject that might be separated by a sufficient condition number (computed as the square root of the ratio
interval for the underlying kinetics to vary between occa- of the largest eigenvalue to the smallest eigenvalue of the
sions. Developing an appropriate statistical model is impor- correlation matrix) can be calculated to evaluate collinearity
tant for covariate evaluations and to determine the amount of which is the case where different variables (CL and V) tend
remaining variability in the data, as well as for simulation, an to rise or fall together. A condition number ≤20 suggests that
inherent use of models.2 the degree of collinearity between the parameter estimates
is acceptable. A condition number ≥100 indicates potential
BSV
instability due to high collinearity26 because of difficulties with
When describing BSV, parameterization is usually based on
independent estimation of highly collinear parameters.
the type of data being evaluated. For some parameters that
Skewness and kurtosis of the distributions of individual η
are transformed (e.g., LGTF), the distribution of η values may
values should be evaluated. Skewness measures lack of sym-
be normal, and BSV may be appropriately described using
metry in a distribution arising from one side of the distribution
an additive function:
having a longer tail than the other.27 Kurtosis measures whether
LGTFi = θ1 + η1i
(4) the distribution is sharply peaked (leptokurtosis, heavy-tailed)
or flat (platykurtosis, light-tailed) relative to a normal distribu-
where θ1 is the population bioavailability and η1i is the deviation. Leptokurtosis is fairly common in population modeling.
tion from the population value for the ith subject. Taken Representative skewed and kurtotic distributions are shown
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Mould and Upton
6
in Figure 2a,b, respectively. Metrics of skewness and kurtosis Initially, variance terms are incorporated such that cor-
can be calculated in any statistical package. Although refer- relations between parameters are ignored (referred to as a
ence values for these metrics are somewhat dependent on the “diagonal Ω matrix”):
package, they are mostly defined as 0 for a normal distribution.
Very skewed or kurtotic distributions can affect both type I and ω 2CL  CLi = θ1 ⋅ exp (η1i )
Ω =
(8) 2 
type II error rates27 making identification of covariates impracti-
 0 ω V Vi = θ 2 ⋅ exp (η2i )
cal, and can also impact the utility of the model for simulation.
Although the assumption of normality is not required during where ω2CL is the variance for CL and ω2v is the variance for dis-
the process of fitting data, it is important during simulation, at tributional volume. Models evaluating random effects param-
which random values of η are drawn from a normal distribu- eters on all parameters are frequently tested first, followed
tion. Therefore, if the distributions of η values are skewed or by serial reduction by removing poorly estimated parameters.
kurtotic, transformation is necessary to ensure that the η dis- Variance terms should be included on parameters for which
tribution is normal. Numerous transforms are available,28 with information on influential covariates is expected and will be
the Manly transform29 being particularly useful for distributions evaluated. When variance terms are not included (e.g., the
that are both skewed and kurtotic. An example implementation parameter is a fixed effects parameter), that parameter can
is shown below: be considered to have complete shrinkage because only the
median value is estimated (Between-subject variability sec-
LAM = θ1 tion). Thus, as with any covariate evaluation when shrinkage
(7)
ET 1 =
1 ( exp (η ⋅ LAM) − 1) is high, the probability of type I and type II errors is high and
covariate evaluations should be curtailed with few exceptions
LAM (e.g., incorporation of allometric or maturation models).
CL = θ 2 ⋅ exp (ET 1) Within an individual, pharmacokinetic parameters (e.g., CL
and volume) are not correlated.30 It is possible, for example,
where “LAM” is a shape parameter and “ET1” is the trans-
to alter an individual’s CL without affecting volume. How-
formed variance allowing η1 to be normally distributed.
ever, across a patient population, correlation(s) between
a b 0.8
1.6
Mode 0.7
1.4
Median 0.6
1.2
Mean 0.5
1.0
Density
Density
0.4
0.8
0.6 0.3
0.4 0.2
0.2 0.1
0.0 0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 −5 −4 −3 −2 −1 0 1 2 3 4 5
c d
0.4 4
2
0.3
Quantiles
Density
0
0.2
−2
0.1
−4
0.0
−4 −2 0 2 4 −4 −2 0 2 4
Quantiles of normal
Figure 2 Skewed and kurtotic distributions. (a) Shows distributions with varying degrees of skewness. For a normal distribution, the mode,
median, and mean should be (nearly) the same. As skewness becomes more pronounced, these summary metrics differ by increasing
degrees. (b) A range of kurtotic distributions. The red line is very leptokurtotic, the pink line is very platykurtotic (a uniform distribution is the
extreme of platykurtosis). (c) A frequency histogram of a leptokurtotic distribution. and (d) The quantile–quantile (q–q) plot of that distribution,
where the quantiles deviate from the line of unity indicates the heavy tails in the leptokurtotic distribution. For a normal distribution, the
quantiles should not deviate from the line of unity.

Mould and Upton
7
parameters may be observed when a common covariate combinations tend to show increased variability as a wider
affects more than one parameter. Body size has been shown range of simulated data (referred to as “inflating the variabil-
to affect both CL and volume of distribution.31 Overall, larger ity”). For covariance terms, unlike variance terms, the use of
subjects generally have higher CLs as well as larger volumes the LRT can be implemented in decision making because
than smaller subjects. If CL and volume are treated as cor- covariance terms do not have the same limitations (e.g.,
related random effects, then the Ω matrix can be written as covariance terms can be negative).
shown below:
BOV
ω2  Individual pharmacokinetic parameters can change between
Ω =  CL
(9)
2  study occasions (Supplementary Data online). The source
ωCL,V ω V  of the variability can sometimes be identified (e.g., chang-
where ωCL,V is the covariance between CL and volume of dis- ing patient status or compliance). Karlsson and Sheiner34
tribution (V). Thus the correlation (defined as r) between CL reported bias in both variance and structural parameters
and V, calculated as follows: when BOV was omitted with the extent of bias being depen-
dent on magnitude of BOV and BSV. Failing to account for
ω BOV can result in a high incidence of statistically significant
r = CL ,V
(10)
spurious period effects. Ignoring BOV can lead to a falsely
ωCL ⋅ ωV
optimistic impression of the potential value of therapeutic
Extensive correlation between variance terms (e.g., an r drug monitoring. When BOV is high, the benefits of dose
value ≥ ±0.8) is similar to a high-condition number, in that it adjustment based on previous observations may not trans-
indicates that both variance terms cannot be independently late to improved efficacy or safety.
estimated. Models with high-condition number or extensive BOV was first defined as a component of residual unex-
correlation become unstable under evaluations such as boot- plained variability (RUV)34 and subsequently cited as a com-
strap32 and require alternative parameterizations such as the ponent of BSV.35 BOV should be evaluated and included if
“shared η approach” shown below: appropriate. Parameterization of BOV can be accomplished
as follows:
CL = θ1 ⋅ exp (η1i )
(11)
V = θ1 ⋅ exp (θ 3 ⋅ η1i ) ( Occasion = 1) BOV = η1
If
If ( Occasion = 2 ) BOV = η2
where θ3 is the ratio of the SDs of the distributions for CL and (13)
volume (e.g., θ3 = ωV / ωCL ) and the variance of V (Var(V)) can BSV = η3
be computed as: CL = θ1 ⋅ exp (BSV + BOV)
(12)
Var(V ) = θ 2 ⋅ ω 2 Variability between study or treatment arms (such as
3 CL
crossover studies), or between studies (such as when an
The consequence of covariance structure misspecifica- individual participates in an acute treatment study and then
tion in mixed-effect population modeling is difficult to pre- continues into a maintenance study) can be handled using
dict given the complex way in which random effects enter the same approach.
a nonlinear mixed-effect model. Because population based
models are now more commonly used in simulation experi- Shrinkage
ments to explore study designs, attention to the importance The mixed-effect parameter estimation method (Software
of identifying such terms has increased. Under the extended and estimation methods) returns population parameters (and
least squares methods, the consequences of a misspecified population-predicted concentrations and residuals). Individ-
covariance structure have been reported to result in biased ual-parameter values, individual-predicted concentrations,
estimates of variance terms.33 In general, efficient estimation and residuals are often estimated using a second “Bayes”
of both fixed parameters and variance terms requires correct estimation step using a different objective function (also
specification of both the covariance structure as well as the called the post hoc, empirical Bayes or conditional estimation
residual variance structure. However, for any specific case, step). This step is more understandable for a weighted least
the degree of resulting bias is difficult to predict. Although squares Bayes objective function where for one individual
2 36
misspecification of the variance–covariance relationships in a population with j observations and a model with k param-
can inflate type I and type II error rates (such that important eters, OFV is represented as follows:
covariates are not identified, or unimportant covariates are
identified) specifying the correlations is generally less impor- OFV = Posterior + Prior
tant than correctly specifying the variance terms themselves. 2
 ^
 (14)
However, failure to include covariance terms can negatively  Cobs j − C j 
(θ − θk ,pop )
2
impact simulations because the correlation between param-
n   m
OFV = ∑ +∑
k
eters that is inherent in the data is not captured in the result- j =1 σ2 k =1 ωk2,pop
ing simulations. Thus, simulations of individuals with high CL
and low volumes are likely (a situation unlikely to exist in the A Bayes objective function can be used to estimate the
original data). Simulated data with inappropriate parameter best model parameters for each individual in the population
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Mould and Upton
8
by balancing the deviation of the individual’s model-predicted When shrinkage is high (e.g., above 20–30%),37 plotting
concentrations (Ĉ ) from observed concentrations (Cobs) and individual-predicted parameters or η values vs. a covariate
the deviation of the individual’s estimated parameter val- may obscure true relationships, show a distorted shape,
ues (θk) from the population parameter value (θk,pop). Eq. 14 or indicate relationships that do not exist.37 In exposure–
shows that for individuals with little data, the posterior term of response models, individual exposure (e.g., AUC from Dose/
the equation is smaller and the estimates of the individual’s CL) may be poorly estimated when shrinkage in CL is high,
parameters are weighted more by the population parameters thereby lowering power to detect an exposure–response
values than the influence of their data. Individual parameters relationship. Diagnostic plots based on individual-predicted
therefore “shrink” toward the population values (Figure 3). concentrations or residuals may also be misleading.37 Model
The extent of shrinkage has consequences for individual-pre- comparisons based on OBJ and population predictions are
dicted parameters (and individual-predicted concentrations). largely unaffected by shrinkage and should dominate model
evaluation when shrinkage is high.
Shrinkage should be evaluated in key models. The shrink-
a Shrinkage of individual-predicted clearance age can be summarized as follows:37
9 subjects with population CL = 2
SD (EBEη )
3.5
Shrinkage (η ) = 1 −
(15)
ω
3.0
where ω is the estimated variability for the population and SD
Post hoc individual clearance
2.5 is the SD of the individual values of the empirical Bayesian

Data size
Samples per subject = 1 estimates (EBE) of η.
2.0 Samples per subject = 3
Samples per subject = 8
Residual variability
1.5
RUV arises from multiple sources, including assay variability,
1.0
errors in sample time collection, and model misspecification.
Similar to BSV, selection of the RUV model is usually depen-
dent on the type of data being evaluated.
1 2 3 4
Common functions used to describe RUV are listed in
True individual clearance
Table 1. For dense pharmacokinetic data, the combined
b Shrinkage of individual-predicted concentrations additive and proportional error models are often utilized
9 subjects with population CL = 2
Samples per subject = 1 Samples per subject = 3 Samples per subject = 8
because it broadly reflects assay variability, whereas for
10.0
True CL = 0.5
1.0 Table 1 Common forms for residual error models
0.1
Residual error Formula
function
10.0
Concentration
Untransformed data
True CL = 2
1.0
Additive Y = f (θ ,Time ) + ε
0.1
10.0
Proportional Y = f (θ ,Time ) ⋅ (1 + ε )
True CL = 4
1.0 Exponential Y = f (θ , Time ) ⋅ exp ( ε )

0.1
Combined additive Y = f (θ ,Time ) ⋅ (1 + ε 1 ) + ε 2
3 6 9 12 15 18 3 6 9 12 15 18 3 6 9 12 15 18 and proportional
Time after dose Combined additive Y = f (θ , Time ) ⋅ exp ( ε 1 ) + ε 2
and exponential
Figure 3 The concept of shrinkage. A population of nine subjects Ln-transformed data
was created in which kinetics were one compartment with first-order
absorption and the population clearance was 2 (Ω was 14% and  θ y2 
σ was 0.31 concentration units). The subjects were divided into Additive Y = Log (f (θ , Time ) ) +   ⋅ε
 f (θ , Time )2  1
three groups with true clearances of 0.5, 2, or 4. Each subgroup  
where
was further divided into subjects with 1, 3, or 8 pharmacokinetic the variance of ε1 is fixed to 1 and θy is the addi-
samples per subject. The individual-clearances and individual- tive component of residual error
predicted concentrations for each subject were estimated using
Bayes estimation in NONMEM (post hoc step). (a) Post hoc Exponential Y = Log (f (θ , Time ) ) + (θ ) ⋅ ε
2
x 1
where the vari-
individual clearances vs. true clearance. Note that individual-
predicted clearances “shrink” towards population values as less ance of ε1 is fixed to 1 and θx is the proportional
data are available per subject and the true clearance is further from component of residual error
the population value. (b) Observed (symbols), population-predicted
(dashed line, based on population clearance), and individual-  θ y2 
Combined additive Y = Log (f (θ , Time ) ) +  θ x2 +  ⋅ ε1
predicted concentrations (solid line, based on individual-predicted 
 f (θ , Time )
2


and exponential
clearance). Note also that individual-predicted concentrations
“shrink” toward population-predicted concentrations values as less where the variance of ε1 is fixed to 1 and θx is the
data are available per subject and the true clearance is further from proportional component and θy is the additive
the population value. NONMEM, nonlinear mixed-effects modeling. component of residual error

Mould and Upton
9
pharmacodynamic data (which often have uniform variabil- Note that the data must be ln-transformed before evaluation
ity across the range of possible values), an additive residual and the resulting model-based predictions are also ln-trans-
error may be sufficient. Exponential and proportional mod- formed. The parameters, however, are not transformed. Using
els are generally avoided because of the tendency to “over- LTBS, the exponential residual error now enters the model
weight” low concentrations. This happens because RUV is using an additive type residual error function, removing the
proportional to the observation; low values have a corre- potential for η–ε interactions. The transform of the exponential
spondingly low error. For small σ2, the proportional model is error model removes the tendency for the exponential error
approximately equal to the exponential model. model to overweight the lowest concentrations as well. If addi-
Under all these models, the term describing RUV (ε) is tive or combined exponential and additive errors are needed
assumed to be normally distributed, independent, with a mean with LTBS, forms for these models are provided in Table 1.
of zero, and a variance σ2. Collectively, the RUV components The use of LTBS has some additional benefits: simulations
(σ2) are referred to as the residual variance or “Σ” matrix. As from models implementing this transform are always positive,
with BSV, RUV components can be correlated. Furthermore, which is useful for simulations of pharmacokinetic data over
although ε and η are assumed independent, this is not always extended periods of time. LTBS can improve numerical sta-
the case, leading to an η–ε interaction, (discussed later). bility particularly when the range of observed values is wide.
RUV can depend on covariates, such as when assays It should be noted that the OBJ from LTBS cannot be com-
change between studies, study conduct varies (e.g., out- pared with OBJ arising from the untransformed data. If the
patient vs. inpatient), or involve different patient populations LTBS approach does not adequately address skewness or
requiring different RUV models.38 This can be implemented kurtosis in the residual distribution, then dynamic transforms
in the residual error using an indicator or “FLAG” variable, should be considered.40
FLAG, defined such that every sample is either a 0 or 1,
depending on the assignment. In such cases, an exponential
COVARIATE MODELS
residual error can be described for each case as follows:
Y = f (θ ,t ) ⋅ (FLAG ⋅ exp (ε 1 ) + (1 − FLAG) ⋅ exp (ε 2 ) ) (16) Identification of covariates that are predictive of pharmaco-
kinetic variability is important in population pharmacokinetic
evaluations. A general approach is outlined below:
Because assay error is often a minor component of RUV,
other sources, with different properties should be considered. 1. Selection of potential covariates: This is usually based
Karlsson et al.39 proposed alternative RUV models describ- on known properties of the drug, drug class, or physiolo-
ing serially correlated errors (e.g., autocorrelation), which gy. For example, highly metabolized drugs will frequently
can arise from structural model misspecification, and time- include covariates such as weight, liver enzymes, and
dependent errors arising from inaccurate sample timing. genotype (if available and relevant).
As mentioned earlier, η and ε are generally assumed 2. Preliminary evaluation of covariates: Because run times
independent, which is often incorrect. For example, with can sometimes be extensive, it is often necessary to limit
proportional error model, it can be seen that ε is not inde- the number of covariates evaluated in the model. Covari-
pendent of η: ate screening using regression-based techniques, gen-
eralized additive models, or correlation analysis evaluat-
Y = f (θ ,η ,Time ) ⋅ (1 + ε )
(17) ing the importance of selected covariates can reduce the
number of evaluations. Graphical evaluations of data are
With most current software packages, this interaction can
often utilized under the assumption that if a relationship is
be accounted for in the estimation of the likelihood. It should
significant, it should be visibly evident. Example plots are
be noted that interaction is not an issue with an additive RUV
provided in Figure 4, however, once covariates are in-
model, and evaluation of interaction is not feasible with large
cluded in the model, visual trends should not be present.
RUV (e.g., model misspecification) or the amount of data per
3. Build the covariate model: Without covariate screen-
individual is small (e.g., BSV shrinkage or inability to distin-
ing, covariates are tested separately and all covariates
guish RUV from BSV).
meeting inclusion criteria are included (full model). With
As with BSV, it may be appropriate to implement a trans-
screening, only covariates identified during screening
form when the residual distribution is not normally distrib-
are evaluated separately and all relevant covariates are
uted, but is kurtotic or right-skewed. Failing to address these
included. Covariate selection is usually based on OBJ
issues can result in biased estimates of RUV and simulations
using the LRT for nested models. Thus, statistical sig-
that do not reflect observed data. Because RUV is dependent
nificance can be attributed to covariate effects and pre-
on the data being evaluated, both sides of the equation must
specified significance levels (usually P < 0.01 or more)
be transformed in the same manner. A common transform is
are set prior to model-based evaluations. Covariates
the “log transform both sides” (LTBS) approach.
are then dropped (backwards deletion) and changes to
Y = f (θ ,η , Time ) ⋅ exp (ε ) the model goodness of fit is tested using LRT at stricter
OBJ criteria (e.g., P < 0.001) than was used for inclu-
(
Ln (Y ) = Ln f (θ ,η , Time ) ⋅ exp (ε )
(18) ) sion (or another approach). This process continues until
all covariates have been tested and the reduced or final
Ln (Y ) = Ln (f (θ ,η , Time ) ) + ε
model cannot be further simplified.
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Mould and Upton
10
a b a Distribution density of residuals
0.5 0.5
0.0 0.4
IIVCL
0.0
IIVCL
−0.5
Distribution density
−0.5 0.3
Group
−1.0 i.v. 50 µg
−1.0 s.c. 50 µg
i.v. 200 µg
0.5 1.0 1.5 2.0 0.2 s.c. 200 µg
0 1
NCRCL
SEX
c d 0.1
0.5 0.5
0.0
0.0 0.0 −4 −2 0 2 4
IIVCL
IIVCL
Residual
−0.5 −0.5
b Q–Q normal plot of residuals
−1.0 −1.0 3
0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5

2
NWT NAGE
1
Figure 4 Graphical evaluations of covariates. Note that the Group
variance term is used to evaluate the effect of the covariate
Sample
i.v. 50 µg
and that normalized covariate values (designated with an 0 s.c. 50 µg
i.v. 200 µg
“N” preceding the covariate type) are used. For continuous s.c. 200 µg
covariates, a Loess smooth (red) line is used to help visualize −1
trends. For discrete covariates, box and whisker plots with the
medians designated as a black symbol are used. (a) A box and
−2
whisker plot evaluating sex; (b) evaluates the effect of normalized
creatinine clearance; (c) evaluates the effect of normalized
weight, and (d) evaluates the effect of normalized age. It should −3
be noted that there is a high degree of collinearity in these −3 −2 −1 0 1 2 3
covariates. CrCL, creatinine clearance; IIVCL, interindividual Theoretical
variability in CL; WT, weight.
Figure 5 Residual plots. Selected residual plots for an hypothetical
Models built using stepwise approaches can suffer from model and data set for a fentanyl given by two routes (i.v., s.c.)
selection bias if only statistically significant covariates are and two doses (50 and 200 μg) in 20 patients. (a) Distribution
density (which can be considered of a continuous histogram) of the
accepted into the model. Such models can also overesti- conditional weighted residuals (CWRES) conditioned with color on
mate the importance of retained covariates. Wählby et al.41 treatment group. The distribution should be approximately normally
evaluated a generalized additive models based approach vs. distributed (symmetrical, centered on zero with most values between
forward addition/backwards deletion. The authors reported −2 and +2). (b) A Q–Q plot of the CWRES conditioned with color on
treatment group. These lines should follow the line of identity if the
selection bias in the estimates which was small relative to the
CWRES is normally distributed. In both plots, deviations from the
overall variability in the estimates. expected behavior may suggest an inappropriate structural model
Evaluating multiple covariates that are moderately or highly or residual error model. Q–Q, quantile–quantile.
correlated (e.g., creatinine CL and weight) may also contrib-
ute to selection bias, resulting in a loss of power to find the a practical approach because only covariates important for
true covariates. Ribbing and Jonsson42 investigated this using predictive performance are included.
simulated data, and showed that selection bias was very
high for small databases (≤50 subjects) with weak covariate Covariate functions
effects. Under these circumstances, the covariate coefficient Covariates are continuous if values are uninterrupted in
was estimated to be more than twice its true value. For the sequence, substance, or extent. Conversely, covariates are
same reason, low-powered covariates may falsely appear to discrete if values constitute individually distinct classes or
be clinically relevant. They also reported that bias was negli- consist of distinct, unconnected values. Discrete covariates
gible if statistical significance was a requirement for covariate must be handled differently, but it is important for both types
selection. Thus stepwise selection or significance testing of of data to ensure that the parameterization of the covariate
covariates is not recommended with small databases. models returns physiologically reasonable results.
Tunblad et al.43 evaluated clinical relevance (e.g., a change Continuous covariate effects can be introduced into the
of at least 20% in the parameter value at the extremes of the population model using a variety of functions, including a
covariate range) to test for covariate inclusion. This approach linear function:
resulted in final models containing fewer covariates with a
minor loss in predictive power. The use of relevance may be (
CL = θ1 + ( Weight ) ⋅ θ 2 ⋅ exp (η1) (19) )
Mould and Upton
11
a
10.0 c
3.0 4
2
OBS conc
1.0 Group
i.v. 50 µg
CWRES
0 s.c. 50 µg
0.3
i.v. 200 µg
s.c. 200 µg
−2
0.1
−4
0.1 0.3 1.0 3.0 10.0 0 100 200 300 400 500 600
TAD (min)
PRED conc
b
10.0 d
4
3.0
2 Group
OBS conc
1.0
i.v. 50 µg
CWRES
0 s.c. 50 µg
0.3 i.v. 200 µg
s.c. 200 µg
−2
0.1
−4
0.1 0.3 1.0 3.0 10.0 0.0 0.5 1.0 1.5 2.0 2.5
IPRED conc PRED conc
Figure 6 Key diagnostic plots. Key diagnostic plots for an hypothetical data set for fentanyl given by two routes (i.v., s.c.) and two doses
(50 and 200 µg) in 20 patients. In each plot, symbols are data points, the solid black line is a line with slope 1 or 0 and the solid red line is a
Loess smoothed line. The LLOQ of the assay (0.05 ng/ml) is shown where appropriate by a dashed black line. (a) Observed concentration
(OBS) vs. population-predicted concentration (PRED). Data are evenly distributed about the line of identity, indicating no major bias in the
population component of the model. (b) OBS vs. individual-predicted concentration. Data are evenly distributed about the line of identity,
indicating an appropriate structural model could be found for most individuals. (c) Conditional weighted residuals (CWRES) vs. time after
dose (TAD). Data are evenly distributed about zero, indicating no major bias in the structural model. Conditioning the plots with color on group
helps detect systematic differences in model fit between the groups (that may require revision to the structural model or a covariate). (d)
CWRES vs. population-predicted concentration. Data are evenly distributed about zero, indicating no major bias in the residual error model.
CWERS, conditional weighted residuals; IPRED, individual-predicted concentration; LLOQ, lower limit of quantification; OBS, observed;
PRED, predicted; TAD, time after dose.
This function constitutes a nested model against a base Covariates can be normalized to the mean value in the data-
model for CL because θ2 can be estimated as 0, reducing the base, or more commonly to a reference value (such as 70 kg
covariate model to the base model. However, this parameter- for weight). This parameterization also has the advantage that
ization suffers from several shortcomings, the first is that the θ1 (CL) is the typical value for the reference patient.
function assumes a linear relationship between the parame-
CL = θ1 ⋅ ( Weight − 70 ) 2 ⋅ exp(η1 )
θ
ter (e.g., CL) and covariate (e.g., weight) such that when the Centered
covariate value is low, the associated parameter is correspond- (20) θ2
ingly low, which rarely exists. Such models have limited utility  Weight 
CL = θ1 ⋅   ⋅ exp(η1 ) Normalized
for extrapolation. The use of functions such as a power function  70 
(shown below) or exponential functions are common. Another
Holford31 provided rationale for using allometric functions with
liability of this parameterization is that the covariate value is
fixed covariate effects to account for changes in CL in pediat-
not normalized, which can create an “imbalance”: if the param-
rics as they mature based on weight (the “allometric function”).
eter being estimated is small (such as CL for drugs with long
half-lives) and weight is large, it becomes numerically difficult 0.75
 Weight 
to estimate covariate effects. Covariates are often centered or CL = θ1 ⋅   ⋅ exp (η1)
normalized as shown below. Centering should be used cau- (21)
 70 
tiously; if an individual covariate value is low, the parameter can  Weight 
become negative, compromising the usefulness of the model V = θ2 ⋅   ⋅ exp (η2 )
 70 
for extrapolation and can cause numerical difficulties during
estimation. Normalizing covariate values avoids these issues.
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Mould and Upton
12
1 2 3 4 5
3.0
1.0
0.3
0.1
6 7 8 9 10
3.0
1.0
0.3
Fentanyl (ng/ml)
0.1 Group
i.v. 50 µg
11 12 13 14 15 s.c. 50 µg
i.v. 200 µg
3.0
s.c. 200 µg
1.0
0.3
0.1
16 17 18 19 20
3.0
1.0
0.3
0.1
0 150 300 450 600 0 150 300 450 600 0 150 300 450 600 0 150 300 450 6000 150 300 450 600
Time after dose (min)
Figure 7 Goodness of fit plots. Goodness of fit plots for an hypothetical data set for fentanyl given by two routes (i.v., s.c.) and two doses
(50 and 200 µg) in 20 patients. Each panel is data for one patient. Symbols are observed drug concentrations, solid lines are the individual-
predicted drug concentrations and dashed lines are the population-predicted drug concentrations. The LLOQ of the assay (0.05 ng/ml) is
shown as a dashed black line. Each data set and model will require careful consideration of the suite of goodness of fit plots that are most
informative. When plots are not able to be conditioned on individual subjects, caution is needed in pooling subjects so that information is not
obscured and to ensure “like is compared with like”. LLOQ, lower limit of quantification.
Similarly, Tod et al.44 identified a maturation function (MF) grouped into two categories depending on results of visual
based on postconception age describing CL changes of acy- examination of the covariates. When polychotomous covari-
clovir in infants relative to adults. ates have an inherent order such as the East Coast Oncol-
ogy Group (ECOG) status where disease is normal at ECOG
Postconception age6.17 = 0 and most severe at ECOG = 4, then alternative functions
MF =
(22) 13.46.17 + Postconception age6.17 can be useful:
CLinfant = θ1 ⋅ MF ⋅ exp (η1)
CL = θ1 ⋅ exp (θ 2 ⋅ ( Covariate − 2 ) ) ⋅ exp (η1)
(24)
For both functions, covariate effects are commonly fixed to
published values. This can be valuable if the database being
evaluated does not include a large number of very young MODEL EVALUATIONS
patients. However, such models are not nested and cannot
be compared with other models using LRT; AIC or BIC are There are many aspects to the evaluation of a population
more appropriate. pharmacokinetic model. The OBJ is generally used to dis-
For discrete data, there are two broad classes: dichoto- criminate between models during early stages of model
mous (e.g., taking one of two possible values such as sex) development, allowing elimination of unsatisfactory models.
and polychotomous (e.g., taking one of several possible val- In later stages when a few candidate models are being con-
ues such as race or metabolizer status). For dichotomous sidered for the final model, simulation-based methods such
data, the values of the covariate are usually set to 0 for the as the visual predictive check (VPC)45 may be more useful.
reference classification and 1 for the other classification. For complex models, evaluations (e.g., bootstrap) may be
Common functions used to describe dichotomous covariate time consuming and are applied only to the final model, if at
effects are shown below: all. Karlsson and Savic46 have provided an excellent critique
of model diagnostics. Model evaluations should be selected
CL = θ ⋅ (θ )
Covariate
1 2 1 ⋅ exp (η ) to ensure the model is appropriate for intended use.
(23)
CL = θ1 ⋅ (1 + θ 2 ⋅ Covariate ) ⋅ exp (η1)
Graphical evaluations
Polychotomous covariates such as race (which have no The concept of a residual (RES, Cobs−Ĉ ) is straightforward
inherent ordering) can be evaluated using a different factor and fundamental to modeling. However, the magnitude of the
for each classification against a reference value or may be residual depends on the magnitude of the data. Weighted

Mould and Upton
13
residuals (WRES) normalize the residuals so that the SD is underestimate parameter uncertainty. Bootstrapping avoids
1 allowing informative residual plots. WRES is analogous to parametric assumptions made when computing CIs using
a Z-score for the deviation between the model prediction and other methods.
the data. The method for weighting is complex and depends Bootstrapping involves generating replicate data sets
on the estimation method,47 and hence a variety of WRES where individuals are randomly drawn from the original data-
have been proposed. WRES should be normally distributed, base and can be drawn multiple times or not drawn for each
centered around zero, and not biased by explanatory vari- replicate. In order to adequately reflect the parameter dis-
ables (Figure 5), which can be evaluated using histograms tributions, many replicates (e.g., ≥1,000) are generated and
and q–q plots of WRES conditioned on key explanatory evaluated using the final model, and replicate parameter
variables. Plots of WRES against time (Figure 6) should be estimates are tabulated. The percentile bootstrap CI are con-
evenly centered around zero, without systematic bias, and structed by taking the lower 2.5% and the upper 97.5% value
most values within −2 to +2 SDs (marking the ~5th and 95th of each parameter estimate from runs regardless of conver-
percentiles of a normal distribution). Systematic deviations gence status (with exceptions for abnormal terminations), as
may imply deficiencies in the structural model. Plots of WRES this interval should cover the true value of the parameter esti-
against population-predicted concentration (Figure 6) should mate ~ 95% of the time without imposing an assumption of
be evenly centered around zero, without systematic bias, with symmetry on the distribution.
most values within −2 to +2 SDs. Systematic deviations may Bootstrap replicates can be used to generate the CI for
imply deficiencies in the RUV model. Plots of observed vs. model predictions. For example, when modeling concentra-
population and individual-predicted concentration are also tions vs. time in adults and pediatrics, it may be necessary
shown in Figure 6. to show precision for average predictions within specific age
A fundamental plot is a plot of observed, population-pre- and weight ranges in pediatrics. In this case, the bootstrap
dicted and individual-predicted concentrations against time. parameters can be used to construct a family of curves rep-
These plots should be structured using combinations log- resenting the likely range of concentrations for patients with
scales, faceting, and/or conditioning on explanatory variables a given set of covariates.
to be as informative as possible. Plots of individual subjects
may be possible if the number of subjects is low (Figure 7), VPC
or subjects may be randomly selected. Individual-predicted VPCs generally involve simulation of data from the original or
concentrations should provide an acceptable representation new database50 and offer benefits over standard diagnostic
of the observed data, whereas the population-predicted con- plots.45 The final model is used to simulate new data sets
centrations should represent the “typical” patient (reflecting using the selected database design, and prediction intervals
the center of pooled observed data). (usually 95%) are constructed from simulated concentra-
tion time profiles and compared with observed data. VPC
Parameters: SE and confidence intervals can ensure that simulated data are consistent with observed
Most modeling packages report the precision of parameter data. VPC plots stratified for relevant covariates (such as
estimates, which is derived from the shape of the likelihood age or weight groups), doses, or routes of administration are
surface near the best parameter estimates.2 Precision can commonly constructed to demonstrate model performance
be expressed as SE or confidence intervals (CI) and are in these subsets. Numerous VPC approaches are available,
interconvertible: including the prediction-corrected VPC51 or a VPC utilizing
adaptive dosing during simulation to reflect clinical study
CI = Parameter_Value ± 1.96 ⋅ SE
(25) conduct.52
Precise parameter estimates are important (models with A related evaluation is the numerical predictive check
poor parameter precision are often overparameterized), but which compares summary metrics from the database (e.g.,
the level of precision that is acceptable depends on the size a peak or trough concentration) with the same metric from
of the database. For most pharmacokinetic databases, <30% simulated output.
SE for fixed effects and <50% SE for random effects are usu-
ally achievable (SE for random effects are generally higher CONCLUSION
than for fixed effects). It is important to quantify the precision
of parameters describing covariate effects, as these reflect There is no “correct” method for developing and evaluat-
the precision with which the covariate effect has been esti- ing population pharmacokinetic models. We have outlined
mated. CIs that include the null value for a covariate may a framework that may be useful to those new to this area.
imply the estimate of the covariate effect are unreliable. Population pharmacokinetic modeling can appear complex,
Bootstrap methods are resampling techniques that provide but the methodology is easily tested. When initially assessing
an alternative for estimating parameter precision.48 They are a method or equation, simplified test runs and plots using test
useful to verify the robustness of standard approximations data (whether simulated or subsets of real data) should be
for parameter uncertainty in parametric models.49 Although considered to ensure the method behaves as expected before
asymptotic normality is a property of large-sample analy- use. For formal analyses, a systematic approach to model
ses, in population pharmacometrics, the sample size is usu- building, evaluation, and documentation where each compo-
ally not large enough to justify this assumption. Therefore, nent is fully understood maximizes the chances of developing
CIs based on SE of the parameter estimates sometimes models that are “fit for purpose.”2 A good pharmacometrician
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Mould and Upton
14
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Jurnal Ilmiah Farmasi Vol. 10 No. 1 Tahun 2013
PENGARUH PRA-PERLAKUAN MADU TERHADAP FARMAKOKINETIKA

ELIMINASI RIFAMPISIN PADA TIKUS WISTAR JANTAN
1* 2 3
Dimas Adhi Pradana , Farida Hayati , Dian Sukma
Prodi Farmasi Fakultas Matematika dan Ilmu Pengetahuan Alam

Universitas Islam Indonesia
*email : adhi_pradana85@yahoo.com
ABSTRAK
the group II were given a single doses of
honey 7.65 mL/kg orally once daily for seven
Rifampisin merupakan salah satu th
days. On the day 8 the rats were given
obat yang dipergunakan sebagai terapi lini
concurrently with rifampicin dose of 50 mg/kg
pertama dalam pengobatan tuberkulosis.
orally. 0.2 mL of blood was taken through the
Tujuan dari penelitian ini untuk mengetahui
rats lateral tail vein at 0.25; 0.5, 1.0, 1.5, 2.0,
pengaruh pra-perlakuan pemberian madu
3.0; 4.0; 6.0; 8, 0; 10.0; 12.0, and 24.0 hours.
terhadap profil farmakokinetika fase eliminasi
Determination of rifampicin levels in plasma
rifampisin pada tikus Wistar Jantan. Dalam
were analyzed by HPLC at a wavelength of
penelitian ini hewan uji dibagi menjadi 2
244.6 nm. Parameters obtained from the two
kelompok, yaitu kelompok kontrol dan
groups were statistically analyzed through
perlakuan. Setiap kelompok terdiri dari 5
the normality test followed by unpaired t test
ekor tikus. Kelompok kontrol diberikan
with a level of 95%. The results showed that
rifampisin dosis tunggal 50 mg/kg tikus
the pretreatment of honey does not have a
sedangkan kelompok perlakuan diberikan
significant effect on the elimination phase of
madu 7,65 ml/kg secara oral sekali sehari
rifampicin based on t1/2
selama 7 hari dan pada hari ke-8 diberikan
rifampisin dosis 50 mg/kg BB tikus secara
Keywords: honey, HPLC, pharmacokinetics,
per oral. Sebanyak 0,2 ml sampel darah
rifampicin
diambil dari vena lateralis ekor pada 0.25;
0.5, 1.0, 1.5, 2.0, 3.0; 4.0; 6.0; 8, 0; 10.0;
12.0, dan 24.0 jam. Penetapan kadar PENDAHULUAN
rifampisin dalam plasma dilakukan dengan
metode HPLC pada panjang gelombang
244.6 nm. Parameter farmakokinetika fase Interaksi obat pada fase eliminasi
eliminasi yang ditetapkan adalah k, t ½, dan merupakan hal yang penting untuk diketahui
ClT. Hasil penelitian menunjukkan bahwa
pemberian pra perlakuan madu tidak karena terkait dengan efektivitas proses
mempengaruhi farmakokinetika fase metabolism dan atau ekskresi obat. Profil
eliminasi dari rifampisin.
farmakokinetika eliminasi suatu obat dapat
Keywords : HPLC, madu, farmakokinetika, berubah oleh adanya obat lain, obat herbal
ricampicin
bahkan makanan dan minuman. Hal
tersebut dapat disebabkan karena terjadinya
ABSTRACT
interaksi farmakokinetika yang dapat
Rifampicin is one of the first-line drug
merubah profil absorbsi, distribusi,
for the therapy of Tuberculosis (TB), which is
still commonly used. The aim of the study is metabolisme dan eksresi dari suatu obat
to determine the effect of pretreatment with
(Baxter, 2008). Rifampisin merupakan obat
honey on the pharmacokinetic profile of
orally administered rifampicin on male Wistar lini pertama yang berguna dalam
rats. In this study, animals were divided into
pengobatan TB (Debra et al, 1999).
two groups, each group consists of 5 rats.
The group I were given a single dose of Rifampisin merupakan obat yang bersifat
rifampicin 50 mg/kg orally as a control, while
18
19 | Dimas Adhi Pradana
autoinducer, hal ini dikarenakan rifampisin 10,92 ml/kg BB yang diberikan bersamaan
merupakan substrat CYP 3A4 sekaligus dengan parasetamol pada mencit betina
sebagai induktor kuat CYP 3A4 (Goodman & galur Swiss dapat menyebabkan kenaikan
Gilman’s, 2000). efek analgetika parasetamol (Trisnawati,
Madu merupakan minuman yang 2005). Pemberian madu juga dapat
memiliki nilai gizi tinggi dan berkhasiat untuk mempengaruhi profil farmakokinetika teofilin
mengobati berbagai penyakit. Setiap orang berupa penurunan nilai Ka dan Cp maks, serta
dapat mengonsumsi madu, baik anak-anak, peningkatan nilai Tmaks, Vd, dan ClT secara
dewasa, maupun orang tua (Suranto, 2004). bermakna (Paramitasari, 2012).
Madu memiliki pengaruh besar sebagai Beberapa uraian yang telah
nutraceutical. Madu juga memiliki aktivitas dikemukakan diatas melatarbelakangi
sebagai antimikroba, antivirus, dan penelitian tentang interaksi antara madu
antiparasit, antimutagenik, antitumor, dengan rifampisin yang dapat
berperan sebagai antioksidan, dan juga mempengaruhi profil farmakokinetika
memiliki efek antiinflamasi (Bogdanov et al, rifampisin khususnya fase eliminasi.
2008). Selain itu, madu juga diyakini dapat
meningkatan daya tahan tubuh pasien METODE PENELITIAN
tuberkulosis (Suranto, 2007). Kandungan
flavonoid pinocembrin, pinobanksin, chrysin, Alat – alat yang digunakan alat-alat
galangin, quercetin and luteolin pada madu gelas seperti kaca arloji, gelas beker, gelas
diketahui dapat menginduksi aktivitas enzim ukur, labu ukur, erlenmeyer, dan batang
sitokrom P450, terutama enzim CYP3A4, pengaduk, timbangan elektrik, stopwatch,
sehingga dimungkinkan dapat spatula, pipet volume, pipet ukur, mikropipet,
mempengaruhi metabolisme rifampisin spuit injeksi 1-5 mL, flacon, jarum oral,
(Tushar et al, 2006) ependorf, sentrifuge Hanil MF 80, vortex type
Penggunaan Rifampisin bersama 16700 mixer dan seperangkat alat
madu memungkinkan adanya interaksi spekrofotometer Shimadzu UV-Vis 1800,
sehingga dapat mempengaruhi profil HPLC Water e2695, detektor UV 2487 pada
farmakokinetika obat antituberkulosis 244,6 nm, kolom Sunfire C18 (5 µm) 4,6 x 150
terutama pada fase eliminasi. Penelitian mm, injektor SM7, perangkat lunak Empower
sebelumnya menunjukkan bahwa madu (versi 2.0, Waters Corporation).
dapat mempengaruhi profil farmakokinetika Bahan-bahan yang digunakan dalam
dan efektifitas beberapa obat. Sebuah penelitian ini meliputi rifampisin serbuk murni
penelitian mengenai pengaruh praperlakuan yang diperoleh dari PT Sanbe Farma, madu
madu terhadap profil farmakokinetika kelengkeng “Seribu Bunga”, kalium
sulfametazin pada tikus jantan menunjukkan dihidrogen fosfat (kualitas analisis, Merck),
bahwa madu dapat meningkatkan nilai Tmaks, asetonitril (kualitas ultra gradient solvent
Vd, t1/2, ClT, dan menurunkan harga ka, untuk HPLC, J.T. Baker), metanol (kualitas
Cmaks, AUC, dan k sulfametazin (Maulidah, solvent untuk HPLC, J.T.Baker), asam
2005). Penelitian lain mengenai pemberian askorbat (kualitas analisis, Merck), heparin
madu dosis 46 ml/kg BB; 8,19 ml/kg BB; dan

®
sodium (Inviclot ), asam ortofosfat (Merck) Rifampisin murni dilarutkan dalam metanol
dan akuabidestilata. hingga konsentrasi 5 μg/ml. Hasil larutan
Hewan uji dalam penelitian ini dimasukkan dalam vial injektor, diambil
adalah tikus putih (Rattus norvegicus) jantan secara autoinjeksi dalam jumlah 20 μL ke
galur Wistar yang diperoleh dari LPPT dalam HPLC dengan menggunakan kolom
(Laboratorium Penelitian dan Pengujian C18, fase gerak 0,05 M buffer fosfat :
Terpadu) UGM, berat badan tikus 180-250 g asetonitril (55:45 v/v) dengan laju alir 1,2
dan berumur 2-3 bulan. ml/menit dan panjang gelombang maksimum
Pada metode penetapan kadar yang sudah didapatkan sebelumnya,
rifampisin dalam darah dilakukan proses kemudian ditetapkan waktu retensi dan
sentrifuge dilakukan selama 5 menit dengan selektivitas rifampisin.
kecepatan 10.000 rpm. Analisis kadar 3. Penetapan persamaan kurva baku
rifampisin dalam darah dilakukan dengan rifampisin dalam darah
menggunakan HPLC metode fase terbalik Dalam pembuatan kurva baku dilakukan
(fase gerak bersifat polar dan fase diam pembuatan larutan stok rifampisin dengan
bersifat non polar), menggunakan kolom cara melarutkan 10 mg rifampisin dalam 10
ODS C18. Sebanyak 200 μl plasma dipipet ml metanol dan ditambahkan dengan 0,5
ke flacon kemudian tambahkan 400 μl mg/ml asam askorbat. Diambil sejumlah
asetonitril, vortex selama 1 menit dan rifampisin dari larutan stok kemudian
disentrifuse selama 30 menit dengan ditambahkan 0,2 ml plasma setelah
kecepatan 4.000 rpm. Darah diambil dikurangi volume larutan stock yang
beningannya dan dimasukkan kedalam vial ditambahkan untuk membuat konsentrasi
injektor lalu diinjeksikan ke HPLC sebanyak 0,5; 1; 2; 5, dan 10 μg/ml dalam 200 μl
20 μl secara auto injeksi. Fase gerak yang darah. Pengerjaan standar rifampisin
digunakan adalah 0,05 M buffer fosfat (pH dilakukan dalam range konsentrasi 0,5-10
2,6) : asetonitril (55:45 v/v) dengan laju alir (0,5; 1; 2; 5, dan 10) μg/ml dan tambahkan
1,2 ml/menit pada panjang gelombang 244,6 400 μL asetonitril. Vortex selama 1 menit
nm (Kumar et al, 2004). dan disentrifuse selama 30 menit dengan
kecepatan 4.000 rpm. Diambil beningannya
Uji pendahuluan dan diinjeksikan ke HPLC sebanyak 20 μl
a. Optimasi metode analisis secara auto injeksi. Regresi linear ditentukan
1. Penetapan panjang gelombang dengan analisis tinggi puncak terhadap
maksimum kurva konsentrasi. Linearitas ditentukan
Rifampisin dilarutkan dalam fase gerak 0,05 dengan koefisien korelasi (r) (Kumar et al,
M buffer fosfat : asetonitril (55:45 v/v) 2004).
dengan kadar 20 μg/ml kemudian di baca 4. Penetapan stabilitas rifampisin dalam
pada spektrofotometer UV-Vis dengan pelarut
panjang gelombang pada range 200-400 Diambil 200 µL darah dari hewan uji yang
nm. telah diberikan rifampisin secara oral dosis
2. Penetapan waktu retensi dan selektivitas 50 mg/Kg BB dan ditambahkan 400 µL
rifampisin asetonitril, kemudian divortex selama 1

menit dan disentrifuse selama 30 menit b. Penetapan dosis

dengan kecepatan 4.000 rpm, diambil Dosis rifampisin yang digunakan sesuai
beningannya. Larutan bening di simpan dengan dosis pada penelitian sebelumnya
0
pada suhu 2-8 C selama 24. Kadar yaitu 50 mg/kgBB tikus (setara dengan 560
rifampisin ditetapkan dengan HPLC pada mg/70 kg BB manusia) (Wahyono dkk,
jam ke-0 dan 24. Hasil yang diperoleh 2007), sedangkan dosis madu yang
dinyatakan sebagai persen degradasi dipergunakan sebesar 7,65 mL/kgBB tikus
rifampisin selama penyimpanan dalam (Trisnawati, 2005).
pelarut metanol dengan penambahan asam
askorbat (Anonim, 2009). c. Penetapan waktu sampling
5. Penentuan kriteria kecermatan Waktu sampling yang digunakan pada
(accurate) penelitian ini berdasarkan waktu sampling
Kadar rifampisin dalam darah dibuat penelitian sebelumnya yakni pada jam ke
dengan dengan cara melarutkan sejumlah 0,25; 0,5; 1; 1,5; 2; 3; 4; 6; 8; 10; 12; dan 24
tertentu rifampisin dalam 10 ml metanol dan (Wahyono & Hakim, 2005)
ditambahkan dengan 0,5 mg/ml asam
askorbat. Dibuat konsentrasi 5, 10 dan 15 HASIL DAN PEMBAHASAN
μl/ml dengan replikasi 3 kali. Divortex
selama 1 menit dan disentrifuse selama 30 Penelitian ini sudah memenuhi
menit dengan kecepatan 4.000 rpm lalu syarat secara etik dan mendapatkan surat
diambil beningannya dan diinjeksikan ke kelayakan etik (ethical clearance) nomor
HPLC sebanyak 20 μl secara auto injeksi 103/KEC-LPPT/V/2013 dari Komite Etik
(Anonim, 2009). Penelitian Hewan Coba Laboratorium
6. Penentuan kriteria ketepatan (precise) Penelitian dan Pengujian Terpadu (LPPT)
Nilai kesalahan acak mencerminkan presisi Universitas Gadjah Mada
yang diperoleh dalam suatu metode. 1. Penetapan panjang gelombang
Rentang penerimaan yang diperbolehkan maksimum rifampisin
untuk metode HPLC adalah kurang dari Penetapan panjang gelombang maksimum
15% (Anonim, 2009) (λmaks) rifampisin dibaca dengan
7. Penetapan batas deteksi dan menggunakan spektrofotometer UV-Vis.
kuantifikasi kadar rifampisin Hasil yang diperoleh menunjukkan bahwa
Penetapan batas deteksi dan kuantifikasi panjang gelombang maksimum rifampisin
dihitung melalui persamaan regresi linier terdapat pada panjang gelombang 244,6 nm
yang sudah didapat pada penetapan kurva

baku.

Gambar 1. Kromatogram spektrofotometri UV-Vis panjang gelombang Rifampisin
2. Penetapan waktu retensi
Gambar 2. Kromatogram rifampisin dalam darah (in vivo) sampel jam ke 8 pada tikus
kontrol 1 dengan fase gerak 0,05 M buffer fosfat : asetonitril (55:45 v/v)
Penetapan persamaan kurva baku

Berdasarkan hasil penelitian, retensi rifampisin dalam darah dilakukan
5,215 menit, sedangkan senyawa dengan membuat beberapa seri kadar
endogen darah terdekat memiliki waktu yang kemudian diukur luas area
retensi 3,451. dibawah puncak kromatogramnya
3. Penetapan persamaan kurva baku dengan menggunakan HPLC.
rifampisin dalam darah
Tabel 1. Pembacaan luas area dibawah puncak kromatogram

pada beberapa seri kadar rifampisin dalam darah
Seri Kadar
No Luas Area
(µg/mL)
1 0,5 9959
2 1 19153
3 2 47880
4 5 91928
5 10 197905

Persamaan kurva baku yang diperoleh 5. Penetapan kriteria akurasi dan presisi
adalah y = 19438,99676x+1440,71197; Tabel 1 menunjukkan nilai dari perolehan
x adalah kadar rifampisin dalam darah kembali, kesalahan sistematik, kesalahan
dan y adalah luas area dibawah puncak acak, dan HORRAT dari metode penetapan
kromatogram rifampisin hasil dari kadar rifampisin dalam darah pada penelitian
pengukuran dengan HPLC. Nilai r yang ini.
diperoleh adalah 0,9953. Nilai r pada Berdasarkan data yang tersaji pada
hasil regresi linier menunjukkan linieritas tabel 2, diperoleh informasi bahwa nilai rata-
yang baik karena mendekati satu rata perolehan kembali dan kesalahan acak
(Watson, 2000). dari masing-masing seri kadar masih
4. Penetapan kriteria sensitivitas memenuhi rentang yang diperbolehkan, yaitu
Kriteria yang digunakan untuk menilai untuk perolehan kembali sebesar 100 ±
sensitivitas metode pada penelitian ini 20%, sedangkan untuk kesalahan acak
adalah LOD, LOQ, dan LLOQ. sebesar kurang dari 15% untuk metode yang
Berdasarkan hasil perhitungan diperoleh menggunakan HPLC (Anonim, 2009). Nilai
nilai LOD sebesar 0,938 µg/mL, nilai HORRAT yang bernilai 0,01, 0,005, 0,89
LOQ sebesar 3,128 µg/mL, dan nilai menunjukkan bahwa metode yang
LLOQ sebesar 1,564 µg/mL. digunakan memiliki presisi yang baik, yaitu
kurangdari2.
Tabel 2. Nilai perolehan kembali, kesalahan sistematik, kesalahan acak, dan HORRAT pada
penetapan kadar rifampisin dalam darah
Kadar rifampisin
Kesalahan
Luas Recovery Kesalahan
Diketahui Terukur Sistematik HORRAT
Area (%) Acak (%)
(µg/mL) (µg/mL) (%)
79707 4,03 80,53 19,48

5 81130 4,10 81,99 18,01
80919 4,09 81,77 18,23 0,07 0,01
Rata-
4,07±0,04 81,43±0,79 18,57±0,79
rata±SD
201400 10,29 102,87 -2,87
10 200133 10,22 102,21 -2,21
200069 10,22 102,18 -2,18 0,03 0,003
Rata-
10,24±0,04 102,42±0,39 -2,42±0,39
rata±SD
259390 13,27 88,47 11,54
15 262664 13,44 89,59 10,41
242551 13,04 82,69 17,31 4,73 0,44
Rata-
13,04±0,56 86,91±3,70 13,09±3,70
rata±SD

Tabel 3. Persentase degradasi rifampisin dalam asetonitril (in vivo) pada pemberian rifampisin 50
mg/kg BB tikus setelah dilakukan penyimpanan dalam lemari pendingin
Jam ke- Luas area Kadar rifampisin dalam darah (µg/mL) Degradasi (%)
0 68872 3,468867 0
24 66997 3,372411 2,88
Berdasarkan nilai parameter kesalahan acak term. Setelah penyimpanan 24 jam, kadar
dan recovery, dapat disimpulkan bahwa rifampisin berkurang, namun persentase
metode analisis yang digunakan dalam degradasi yang terjadi tidak lebih dari 10%
penelitian ini memenuhi kriteria presisi dan sehingga kadar teofilin yang dianalisis masih
akurasi. stabil
6. Penetapan stabilitas rifampisin dalam 7. Hasil Uji Farmakokinetika
asetonitril Data penelitian farmakokinetika
Parameter untuk menilai stabilitas rifampisin yang diberikan peroral pada
rifampisin selama proses penyimpanan kelompok kontrol dan perlakuan masing-
adalah persentase degradasi. Uji stabilitas masing disajikan pada tabel 4.
rifampisin ini dilakukan selama 24 jam (short- Parameter farmakokinetika diperlukan
term temperature stability). untuk menginterpretasi perubahan-
Stabilitas obat dalam spesimen biologis perubahan disposisi obat di dalam tubuh
dapat dipengaruhi oleh beberapa faktor, seperti yang terwujud dalam perubahan nilai
diantaranya adalah sifat kimia obat, matriks, parameter. Parameter farmakokinetika terdiri
suhu, wadah dan kondisi penyimpanan. dari parameter primer, sekunder, dan
Tujuan dari penetapan stabilitas rifampisin turunan. Parameter primer terdiri dari ka, Vd,
dalam asetonitril adalah untuk mengetahui dan ClT, yang dipengaruhi oleh perubahan
kestabilan rifampisin yang terkandung salah satu atau lebih variabel fisiologis.
didalam spesimen biologis selama beberapa Parameter sekunder meliputi k, t1/2, dan tmaks
waktu jika disimpan dalam lemari pendingin dimana parameter-parameter tersebut
o
atau freezer dengan suhu 5-10 C dipengaruhi oleh perubahan parameter
dikarenakan penginjeksian sampel ke HPLC primer yang dikarenakan adanya perubahan
tidak memungkinkan untuk dibaca secara suatu variabel fisiologis, sedangkan
langsung dan selesai dalam satu hari. parameter turunan nilainya tidak hanya
Penetapan stabilitas rifampisin dalam bergantung pada parameter primer tapi juga
spesimen biologis dihitung sebagai dipengaruhi oleh dosis dan kecepatan
persentase degradasi terhadap kadar awal. pemberian obat, contohnya adalah AUC0-~,
Pemilihan waktu penetapan stabilitas 24 jam AUMC, Cpmaks, dan MRT.
dikarenakan penginjeksian sampel ke HPLC
setelah dipreparasi adalah maksimal 24 jam.
Jenis stabilitas yang digunakan adalah short

Tabel 4. Data kadar rifampisin dalam darah (µg/mL) pada kelompok kontrol dan perlakuan (n=5)
Kadar (µg/mL)
Waktu sampling
(jam ke-) Kelompok Kontrol Kelompok Perlakuan
(Rata-rata±SE) (Rata-rata±SE)
0,25 0,49±0,23 0,09±0,03

0,5 1,22±0,53 0,17±0,03
1 1,78±0,58 0,22±0,05
1,5 2,43±1,08 0,24±0,05
2 1,17±0,46 0,34±0,09
3 4,21±1,06 0,57±0,12
4 1,35±0,47 0,29±0,12
6 1,79±0,47 0,59±0,12
8 2,51±0,65 0,77±0,24
10 2,27±0,90 1,08±0,09
12 1,40±0,40 0,91±0,11
24 0,78±0,27 0,60±0,11
Keterangan :
- Kelompok kontrol adalah tikus yang diberikan rifampisin secara peroral dengan dosis
50 mg/kgBB
- Kelompok perlakuan adalah tikus yang diberikan madu peroral dengan dosis 7,65
mL/kgBB satu kali sehari selama tujuh hari berturut-turut dan pada hari kedelapan
diberikan bersamaan dengan rifampisin dosis 50 mg/kgBB
Tabel 5. Harga parameter farmakokinetika dan uji t tidak berpasangan untuk rifampisin pada
kelompok kontrol dan perlakuan
Nilai Parameter
Uji t tidak
Parameter
Kontrol Perlakuan % Beda berpasangan
Farmakokinetika
(Rata-rata±SE) (Rata-rata±SE) (nilai P)
k 0,08±0,02 0,05±0,01 -37,50 0,228

(/jam)
t1/2
11,67±2,80 18,19±2,97 +55,87 0,149
(jam)
ClT
0,93±0,17 1,76±0,37 +89,25 0,077
(L/jam)
*) perubahan nilai parameter yang signifikan (p<0,05)
eliminasi dipengaruhi oleh klirens dan
1. Tetapan laju eliminasi (K) volume distribusi (Hakim, 2011). Laju
Laju eliminasi suatu obat merupakan suatu eliminasi secara langsung mempengaruhi
ukuran yang berguna untuk menggambarkan besarnya waktu paruh eliminasi (t 1/2
eliminasi obat dari dalam tubuh. Laju eliminasi) (Shargel, 2005). Semakin besar

nilai K maka semakin singkat waktu paruh fisiologi, seperti fungsi organ dalam
eliminasi, semakin kecil nilai K maka mengeliminasi obat dan kecepatan alir darah
semakin lama waktu paruh eliminasi. menuju organ eliminasi obat (Hakim, 2011).
Semakin besar klirens, maka nilai K juga Hasil penelitian ini menunjukkan bahwa
makin besar, sehingga eliminasi obat dari praperlakuan madu dosis 7,65 mL/kgBB
dalam tubuh semakin cepat. Nilai K dapat ternyata mengakibatkan peningkatan klirens
ditentukan jika nilai klirens diketahui ataupun total rifampisin jika dibanding kelompok
dapat diketahui secara langsung dari nilai B kontrol, tetapi secara tidak signifikan
regresi linier log Cp (kadar obat dalam (p>0,05).
plasma) vs t (waktu) pada titik-titik eliminasi Hasil penelitian ini tidak
obat atau yang dianggap mewakili titik-titik menunjukkan peningkatan klirens total
eliminasi suatu obat. secara bermakna yang menunjukkan bahwa
Hasil penelitian ini menunjukkan bahwa metabolisme rifampisin tidak dipercepat
praperlakuan madu dosis 7,65 mL/kgBB akibat pemberian madu. Hal ini dapat terjadi
ternyata hanya sedikit menurunkan laju karena beberapa faktor, yang paling
eliminasi rifampisin secara tidak signifikan mungkin adalah karena prednison tidak
jika dibandingkan dengan kontrol(p>0,05). mampu mempercepat metabolisme teofilin.
2. Waktu paruh eliminasi (t1/2) Keterbatasan pada penelitian ini
Waktu paruh eliminasi diantaranya adalah nilai Cp yang diperoleh
menunjukkan lamanya waktu yang pada kelompok perlakuan sangat kecil,
diperlukan oleh sejumlah obat atau bahkan berada dibawah nilai LLOQ. Hal ini
konsentrasi obat untuk dapat tereliminasi disebabkan tidak dilakukannya pembuatan
menjadi setengahnya (berkurang menjadi kurva baku menggunakan kadar terendah
setengahnya) (Shargel, 2005). Nilai waktu sesuai nilai LLOQ yang diperoleh diawal
paruh eliminasi sangat tergantung kepada penelitian, selain itu tidak dilakukan
laju eliminasi obat, klirens total dan volume penyesuaian dosis setelah diketahui nilai Cp
distribusi (Hakim, 2011). Berdasarkan data yang dihasilkan berada dibawah LLOQ kurva
penelitian dapat diketahui bahwa pada baku, oleh karena itu perlu dilakukan
kelompok perlakuan mengalami peningkatan penyesuaian atau optimasi kembali terhadap
nilai t ½ walaupun tidak signifikan secara dosis rifampisin pada tikus. Hal lain yang
statistik. perlu diperhatikan adalah perlunya dilakukan
3. Klirens total (Clt) pembuatan kurva baku pada setiap
Klirens obat adalah suatu ukuran penetapan kadar rifampisin dalam darah.
eliminasi obat dari tubuh tanpa Berdasarkan hasil penelitian, terlihat bahwa
mempermasalahkan mekanisme prosesnya. madu dapat menunda absorpsi rifampisin
Eliminasi obat terdiri dari proses akibatnya nilai tmaks meningkat hingga berada
metabolisme dan ekskresi. Klirens dapat pada jam ke 8-10 waktu sampling, sehingga
didefinisikan sebagai volume bersihan suatu diperlukan cuplikan yang lebih sering antara
obat dari tubuh per satuan waktu (mL/menit jam ke-10 hingga jam ke-24 untuk
atau L/jam) (Shargel, 2005). Nilai klirens mengetahui apakah masih terdapat
dapat dipengaruhi oleh beberapa faktor peningkatan Cp diantara waktu tersebut.

Neal MJ.(2006), At a Glance Farmakologi

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Performance Liquid Chromatography
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Jurnal Veteriner Juni 2016 Vol. 17 No. 2 : 292-299
pISSN: 1411-8327; eISSN: 2477-5665 DOI: 10.19087/jveteriner.2016.17.2.292
Terakreditasi Nasional SK. No. 15/XI/Dirjen Dikti/2011 online pada http://ojs.unud.ac.id/php.index/jvet
Kinetika Immunoglobulin Kuning Telur

Antiparvovirus Anjing Pada Anjing
(KINETICS OF ANTICANINE PARVOVIRUS YOLK
IMMUNOGLOBULIN IN DOGS)
I Gusti Ayu Agung Suartini 1, Indrawati Sendow2,

Ni Luh Putu Agustini 3, Agik Suprayogi4,
I Wayan Teguh Wibawan5, I Gusti Ngurah Kade Mahardika6
1
Lab Biokimia, Fakultas Kedokteran Hewan, Universitas Udayana, 2Departemen Virologi, Balai
Besar Penelitian Veteriner, Badan Litbang Kementerian Pertanian- Bogor, 3Lab Bioteknologi
Balai Besar Veteriner Denpasar, 4Departemen Anatomi,Fisiologi dan Farmakologi, 5Departmen
Penyakit Infeksius dan Kesehatan Masyarakat. Institut Pertanian Bogor,
6
Lab Virologi, FKH Unud
Jl. Sudirman, Denpasar-Bali. Telp: 0361.223791,
Email: gaa.suartini@gmail.com
ABSTRAK
Studi kinetika immunoglobulin yolk (IgY)anti-canine parvovirus (CPV) telah dilakukan pada enam
ekor anjing umur 5-10 bulan. Immunoglobulin yolk diinjeksikan secara intravena dengan dosis 21 mg/10
kg bobot badan. Penentuan kadar IgY dalam darah dideteksi dengan metode enzym linied immunosorbent
assay (ELISA). Penelitian ini bertujuan untuk mengetahui kinetika IgY anti- CPV dalam darah anjing.
Hasil penelitian menunjukkan bahwa kinetika IgY mengikuti orde satu, sehingga perhitungan kinetika
selanjutnya dihitung berdasarkan hubungan antara ln kadar IgY terhadap waktu pengambilan serum.
Konstanta laju disosiasi IgY berkisar antara (0,007 sampai 0,015 jam). Konsentrasi IgY dalam darah
anjing (CPO)terdeteksi berkisar (0,746 sampai 0,992 mg/mL). Waktu paruh IgY berkisar antara (1,65
sampai 4,01 hari). Volume distribusi IgY berkisar antara (21,47 sampai 28,55 mL). Jumlah IgY yang ada
dalam tubuh anjing (area di bawah kurva) berkisar antara (42,60 sampai 142,00 mg/mL.jam). Lama
obat dalam tubuh anjing berkisar antara (3,08 sampai 8,51 hari). Clearance IgY berkisar antara (0,15
sampai 0,50 mL/jam) (0,29±0,11 mL/jam). Disimpulkan bahwa kinetika IgY anti-CPV dalam tubuh
anjing mengikuti kinetika orde satu dan satu kompartemen, terdistribusi hanya dalam darah dengan
waktu paruh 2,5 hari dan kemungkinan terakumulasi dalam tubuh lebih kecil jika dibandingkan dengan
IgG.
Kata-kata kunci : IgY, kinetika, canine parvovirus, anjing.
ABSTRACT
Kinetic study on Anti CPV IgY has been performed on six dogs aged 5-10 months. The IgY was injected
intravenously at dose of 21.4mg /10kg body weight. IgY levels in the blood were determined by ELISA. A
research was conducted to find out the kinetics of Anti CPV IgY in dogs blood. The kinetics of IgY was
calculated by using regression analysis to determine the association on the levels of IgY in serum against
time at injection. The results showed that kinetic parameters were calculated based on first order kinetics.
The constant elimination rate of IgY was at the range between 0.007 to 0.015 / h. IgY concentration in the
dogs blood was from 0.746 to 0.992 mg / mL. The half-life of IgY was from 1.65 to 4.01 / d. Volume
distribution of IgY was between 21.47 to 28,55 / mL. Total IgY in the dog bodies (AUC) was from 42,60 to
142,00 mg / mL.h. The duration of the IgY in the dog’s body was 3.08 to 8.51 days. Clearance time of IgY was
0.15 to 0.50 mL / h. In conclusion the kinetics of anti CPV IgY in dog’s body follow one compartment and
first order model, which are only distributed in the blood with the half-life at 2.5 days, and IgY has less
possibility to accumulate in the body compared to the IgG.
Keywords: IgY, kinetics, canine parvovirus, dogs.
292
IGA. Agung Suartini, et al Jurnal Veteriner
PENDAHULUAN tukan rute, interval dan dosis terapi, memper-

kirakan kadarnya dalam plasma, jaringan, urin
Terapi dan pencegahan penyakit menggu- dan kemungkinan akumulasi antibodi dalam
nakan antibodi dari kuning telur ayam atau tubuh (Shargel, 2004 ).
immunoglobulin yolk (IgY) terbukti efektif pada Canine parvovirus (CPV) adalah virus
manusia dan hewan (Carlender, 2002). infeksius penyebab diare berdarah dan
Informasi kinetika IgY sangat diperlukan untuk imunosupresif pada anjing (Hoskins, 1998).
memastikan dosis dan interval pemberian ketika Infeksi CPV sangat fatal pada anak anjing umur
digunakan untuk terapi. Nilai kinetika IgY enam minggu sampai enam bulan (Decaro et
hingga saat ini belum banyak dilaporkan. al. 2006). Mortalitas anak anjing dapat
Ketersediaan informasi tentang kinetika IgY mencapai 91 % pada infeksi CPV yang tidak
akan memudahkan aplikasinya secara in vivo. diterapi (Prittie et al. 2004). Penyebab utama
Ayam sangat potensial sebagai penghasil kematian anak anjing adalah septikemia dan
antibodi (Li et al., 2015). Ayam yang dehidrasi (Meunier et al. 1985). Tropisme virus
diimunisasi akan mentransfer antibodi dari parvo yaitu pada sel-sel yang aktif membelah
serum ke kuning telurnya sehingga IgY banyak seperti epitel saluran pencernaan, sumsum
terakumulasi dalam kuning telur (Janson et al., tulang dan jaringan limfoid (Martella et al.2004
1995). Syarat antibodi poliklonal untuk tujuan ). Terapi yang efek-tif untuk infeksi CPV belum
imunoterapi yaitu harus memiliki tingkat ditemukan hingga saat ini. Imunoterapi
kemurnian tinggi dan mudah diproduksi dalam menggunakan IgY anti-CPV nampaknya dapat
jumlah banyak (Schade et al., 2008 ). Syarat dicoba sebagai alternatif baru untuk menekan
ini ada pada IgY yang mudah dimurnikan angka kematian anjing akibat infeksi virus
menggunakan metode presipitasi sederhana parvo. Penelitian ini bertu-juan untuk
(Gassmann et al., 1990; Fernanda et al., 2014). mengetahui kinetika IgY anti-CPV dalam tubuh
Metode pemurnian antibodi yang mudah dan anjing. Data kinetika ini dapat digunakan
murah sangat diperlukan untuk produksi sebagai acuan menentukan dosis terapi dan
antibodi komersial (Kovac et al., 2004). interval pemberian terapi antibodi pada anjing
Imunoglobulin atau antibodi adalah protein yang terinfeksi CPV.
endogen yang dihasilkan oleh sel-sel B limfosit.
Struktur antibodi terdiri dari dua domain yaitu
domain variabel dan konstan. Domain variabel METODE PENELITIAN
berfungsi menetralisasi antigen sedangkan
domain konstan berikatan dengan berbagai Hewan Coba
reseptor Fc (Crystalization Fraction) (Simon dan Anak anjing lokal untuk hewan coba
Spath, 2003). Kedua domain tersebut berperan dipelihara di kandang hewan di Balai Besar
mengatur absorbsi, distribusi, eliminasi dan Veteriner Denpasar, Bali. Enam ekor anak
aktivitas antibodi (Desjarlais 2007; Nimmerjahn, anjing lokal dengan jenis kelamin jantan umur
2006). Berdasarkan struktur dan fungsinya antara 5-10 bulan dikandangkan terpisah
antibodi memiliki prospek untuk dikembangkan dengan ukuran 100 x 50 x 45 cm, diberi pakan
menjadi agen terapi yang potensial. anjing dog food dicampu dengan nasi, diberi
Antibodi memiliki karakteristik yang unik obat cacing (pyrantel) dan multivitamin. Anjing
yaitu bereaksi sangat spesifik terhadap antigen dimandikan dua kali dalam seminggu untuk
yang menginduksinya (Prittie, 2004). Hal ini menekan endoparasit dan ektoparasit. Anjing
menyebabkan ekspresi dan densitas antigen ditimbang bobot badannya untuk menentukan
dalam tubuh sangat berpengaruh terhadap dosis IgY anti CPV yang diinjeksikan secara
farmakokinetik, farmakodinamik, dan intravena. Anak anjing dipuasakan semalam
biodistribusi antibodi (Tabrizi et al.,2010). dan diberikan minum ad libitum. Serum anjing
Antibodi sebagai agen terapi memiliki diambil sebelum perla-kuan untuk memastikan
karakteristik stabil, selektif dan sangat spesifik. bahwa anjing tidak terinfeksi CPV.
Antibodi larut dalam darah, beredar lama dalam Pemeliharaan dan perlakuan hewan coba pada
tubuh, dan tidak dikonversi menjadi metabolit penelitian ini telah mengikuti aturan yang telah
toksik (Wang et al., 2008). Informasi kinetika ditetapkan pada Etical clearance yang
suatu antibodi dapat digunakan sebagai acuan dikeluarkan oleh Fakultas Kedokteran Hewan
terapi antibodi (Tabrizi et al., 2010). Data Universitas Udayana: No: 102A KE-PH/VIII/
kinetika antibodi bermanfaat untuk menen- 2011.
293
Produksi dan Pemurnian IgY anti-CPV Penentuan Kadar IgY dalam Serum Anjing
dari Kuning Telur Teknik ELISA digunakan untuk menge-
Senyawa IgY anti-CPV diproduksi dengan tahui adanya aktivitas IgY. Microtiter plate
cara vaksinasi ayam petelur galur Isabrown dilapisi dengan 100 uL antigen CPV. Plate
umur 20 minggu, dengan virus parvo dosis 213 kemudian diinkubasikan selama satu malam
HA unit yang sudah dilemahkan. Sebanyak pada suhu 37oC. Plate dicuci sebanyak tiga kali
0,5 mL virus diemulsikan dengan 0,5 mL dengan PBS-Tween. Plate diblok dengan 150
adjuvant Freund’s complete. Vaksinasi kedua, µL bovine serum albumin 1% selama 30 menit
ketiga dan keempat menggunakan 0,5 mL virus pada suhu 37oC. Plate dicuci sebanyak tiga kali
dan 0,5 mL adjuvant incomplete. Vaksinasi dengan PBS-T dan 100 µL sampel ditambahkan
pada ayam dilakukan sebanyak empat kali ke setiap lubang plate. Selanjutnya 100 µL
dengan interval dua minggu. Koleksi kuning antibodi yang telah dilabel dimasukan ke dalam
telur dilakukan ketika titer IgY sudah cukup lubang plate dan diinkubasikan selama satu jam
tinggi yaitu dua minggu setelah vaksinasi. pada suhu ruang, kemudian ditambahkan
Antibodi dalam kuning telur dimurnikan substrat peroksidase. Reaksi itu dihentikan
menggunakan Egg Stract Protein Purification setelah 10 menit, dengan menambahkan 50 µL
Kit (Promega). Karakterisasi dan konsentrasi 1,8 M H2SO4 dan plate dibaca dengan spectra
IgY dideteksi menggunakan Sodium Dodecyl Max pada panjang gelombang 450 nm
Sulfate Polyacrylamide Gel Electropho- (Carlander, 2002).
resis (SDS-PAGE) dan spektrofotometri
Nanodrop. Dosis IgY yang digunakan adalah Parameter yang Diamati
1000 Protektif Dose 50 (PD50) berdasarkan uji Parameter yang diamati pada penelitian ini
serum netralisasi IgY terhadap virus parvo adalah penurunan titer antibodi masing-masing
secara in vitro. serum sesuai interval waktu pengambilan
darah. Penurunan nilai optical density (OD)
Penentuan Dosis IgY anti-CPV dalam uji ELISA menunjukkan penurunan titer
Rataan bobot badan anak anjing yang antibodi sesuai selang waktu pengambilan
digunakan adalah 10 kg, diinjeksi dengan 1 mL serum. Nilai OD kemudian dicari kesetaraan
IgY anti-CPV 1000 PD50. Dosis protektif ini kadarnya. Satu OD setara dengan 1,14 mg/mL.
diperoleh berdasarkan hasil uji serum
netralisasi IgY terhadap virus parvo yang Analisis Data
dibiakan dalam sel feline kidney (Suartini et Kadar IgY pada masing-masing interval
al., 2014). Berdasarkan studi pendahuluan pengambilan serum versus waktu dianalisis
diketahui PD 50 setara dengan 1/256 kali menggunakan software Minitabs untuk
pengenceran konsentrasi IgY yang mengandung mengetahui orde eliminasi obat mengikuti orde
21,4 mg tiap mL. Anjing diinjeksi secara nol atau orde satu. Data tetapan disosiasi obat
intravena pada vena safena dengan 1 mL IgY. (K), kadar IgY tertinggi didalam darah (CPo),
Selanjutnya dilakukan pengambilan darah anak waktu paruh (t½), volume distribusi (Vd), area
anjing sebanyak 2 mL melalui vena safena di bawah kurva (AUC), Lama obat dalam tubuh,
dengan interval waktu sampling adalah 1, 3, 6, dan bersihan total/ atau clearance (Cl)
18, 27, 54, 72 dan 108 jam. Interval pengam- selanjutnya dihitung dengan rumus
bilan darah didasarkan pada waktu paruh IgY farmakokinetik sesuai dengan tipe eliminasi
dalam tubuh ayam yaitu 36 jam (Carlender, yang dimiliki oleh IgY (Gibaldi dan Perrier,
2002). Lama pengambilan darah untuk uji 1982).
kinetika suatu obat adalah 3-3,5 kali dari t1/2
obat tersebut atau minimal tiga titik untuk satu Penentuan Kinetika IgY
fase sampling (Lazuardi, 2010). Darah dibiarkan Berdasarkan analisis data kadar IgY versus
membeku dalam spuite dan disimpan di waktu diperoleh kinetika IgY model satu
refrigerator 4ºC semalam. Serum dipisahkan kompartemen dan orde satu. Persamaan
dengan cara disentrifuse pada kecepatan 1000 kinetika model satu kompartemen terbuka dan
rpm selama 10 menit. Masing-masing serum orde satu adalah : ln Cpt = ln Cpo- K.t, atau log
dipisahkan dan disimpan pada -70ºC hingga Cpt = log Cpo- K/2,303. t Tetapan disosiasi obat
digunakan untuk analisis kadar IgY dengan (K) menggambarkan hilangnya obat dari
uji Enzyme Linked Immunosorbent Assay sirkulasi sistemik. Konstanta disosiasi obat (K)
(ELISA). dihitung berdasarkan persamaan garis lurus
294
hubungan antara ln kadar terhadap waktu (t)

atau K = 0,693/t½. Waktu paruh (t½) adalah
waktu saat konsentrasi obat telah separuhnya
dieliminasi dari sirkulasi, dihitung berdasarkan
persamaan : t½= 0,693/K. Konsentrasi IgY
tertinggi dalam tubuh (Cp o ) dihitung
berdasarkan persamaan garis lurus ln Cpt = ln
Cp o – K.t. Volume distribusi (Vd) dihitung
berdasarkan persamaan (Vd) = dosis/Cpo. Area
di bawah kurva (AUC) adalah parameter yang
menggambarkan jumlah obat yang berhasil
diabsorpsi kedalam tubuh, mencerminkan
jumlah total obat aktif yang mencapai sirkulasi. Gambar 1. Kurva hubungan antara log kadar
to imunoglobulin-Y (IgY) anti conine
AUC = AUC tn + AUC ttn∞ = AUC to t ∞ . Lama parvovirus (CPV) terhadap waktu
obat dalam tubuh dihitung berdasarkan pengambilan darah.
persamaan ln Cpt = ln Cpo- K.t, t = ln Cpo/K. t
adalah waktu atau lamanya obat dalam tubuh. dalam tubuh anjing adalah (130,71 ± 43,84 jam).
Clearance total atau bersihan total (Cltot) adalah Konsentrasi IgY anti-CPV yang diekskresikan/
volume cairan tubuh yang dibersihkan dari obat bersihan (clearance) dari tubuh anjing adalah
per satuan waktu Cltot = K.Vd (0,29 ± 0,1 mL/jam).
Anjing yang diinjeksi IgY anti-CPV
intravena secara umum tidak menunjukkan
HASIL DAN PEMBAHASAN gejala toksik maupun anafilaksis. Hal ini telah
diantisipasi dengan melakukan ultrafiltrasi IgY
Hasil uji kinetika IgY anti-CPV disajikan dengan Minisart 0,20 µm untuk mencegah
pada (Tabel 1), menunjukkan bahwa rataan adanya bakteri penyebab pyrogen. Berku-
konstanta kecepatan disosiasi IgY anti-CPV rangnya konsentrasi IgY per satuan waktu
adalah (0,013 ± 0,003/jam). Konsentrasi IgY adalah tidak konstan (Gambar 1). Berdasarkan
anti-CPV tertinggi di dalam darah (Cpo) sesaat data tersebut teramati bahwa eliminasi IgY pada
setelah diinjeksikan secara intravena adalah anjing mengikuti kinetika orde satu (Shargel,
(0,95 ± 0,09 mg/mL). Waktu yang dibutuhkan 2004). Hal tersebut dapat dibuktikan dari
oleh IgY anti-CPV untuk dieliminasi separuhnya hewan uji no.1, bahwa berkurangnya
(t½) adalah (60,10 ± 18,96 jam). Volume distribusi konsentrasi IgY per jam pada jam ke 6-18 adalah
(Vd) IgY anti-CPV pada tubuh anjing adalah 0,033 mg/mL dan pada jam ke 18-27 adalah
(22,72 ± 2,61 mL). Area di bawah kurva (AUC) 0,006 mg/mL (Gambar 1). Nilai keduanya
atau konsentrasi IgY anti-CPV yang berhasil mempunyai selisih yang sangat jauh dan
diabsorbsi di dalam tubuh anjing adalah (84,79 menunjukkan berkurangnya konsentrasi per
± 31,23 µg/mL.jam). Lama IgY anti-CPV berada satuan waktu tidak konstan. Perhitungan
Tabel 1. Hasil perhitungan farmakokinetik imunoglobulin-Y (IgY) anti conine parvovirus (CPV)
Parameter Rataan Nilai Titer IgY anti CPV Standar Deviasi

dalam Tubuh Anjing
Konstanta Kec. Disosiasi/ Jam 0,007 – 0,015 0,013 ± 0,003

CP0 (ug/mL) 0,75 – 0,99 0,95 ± 0,09
Waktu Paruh (t1/2) (hari) 1,65 – 4,01 2,50 ± 0,79
Volume Distribusi (mL) 21,47 – 28,55 22,72 ± 2,61
Area Under Curve (ug/mL.jam) 42,60 – 142,00 84,79 ± 31,23
Lama Obat Dalam Tubuh (hari) 3,08 – 8,51 5,45 ± 1,83
Clearance (mL/jam) 0,15 – 0,50 0,29 ± 0,11
Keterangan : IgY = Immunoglobulin yolk CPV = canine parvovirus
295
kinetika selanjutnya didasarkan pada kinetika memperkuat dugaan bahwa IgY mengikuti
orde satu. Kinetika IgY yang mengikuti orde satu kinetika eliminasi orde satu (Gambar. 1). Nilai
juga diperkuat dengan nilai regresi hubungan kecepatan disosiasi memengaruhi waktu paruh
antara ln kadar versus waktu yang mendekati IgY dalam tubuh anjing. Tetapan laju disosiasi
-1 (-0,7 sampai -0,9) (Tabel 2). Perhitungan berbanding terbalik dengan waktu paruh.
parameter kinetika selanjutnya mengikuti Tetapan laju disosiasi yang rendah menunjuk-
persamaan ln Cpt = ln Cpo- K.t atau log Cpt = log kan waktu paruh obat semakin lama (Lazuardi,
Cpo- K/2,303. t. Cpt adalah kadar IgY pada waktu 2010).
tertentu, Cpo adalah kadar IgY mula-mula, K Waktu paruh IgY dalam tubuh anjing
adalah konstanta disosiasi obat, dan t adalah adalah (60,10±18,96 jam). Berdasarkan waktu
waktu. Waktu paruh (t½ ) IgY dihitung dengan paruh yang diperoleh, IgY dapat diberikan setiap
persamaan t ½ = 0,693/K dan bersihan/ clearance dua hari sekali atau lebih (60,1 jam : 24 jam =
(Cl) dihitung berdasarkan, Cl = K. Vd 2,5 hari). Interval penggunaan obat umumnya
Rataan konstanta kecepatan disosiasi IgY berjarak t½ dari obat tersebut (Shargel, 2004).
dalam tubuh anjing adalah (0,013 ± 0,003/jam). Dengan nilai waktu paruh dua hari lebih berarti
Hubungan antara log kadar antibodi terhadap IgY bisa diberikan dengan interval satu hari
waktu membentuk garis linear sehingga dengan dosis yang lebih kecil. Jika waktu paruh
Tabel 2. Nilai kinetika imunoglobulin-Y (IgY) anti conine parvovirus (CPV) berdasarkan interval
waktu pengambilan serum anjing
Hewan Waktu OD kadar In Kadar Konstanta CPO t½ Vd AUC Lama obat Clearance
coba (jam) Kec.disosiasi (mg/mL) (jam) (mL) (mg/mL.jam) dlm tubuh (mL/jam)
(/jam)
1 6 0,567 0,646 -0,437 -0,0175 0,746 39,6 28,55 42,6 73,88 0,5
18 0,509 0,580 -0,545
27 0,411 0,468 -0,759
36 0,339 0,386 -0,952
Rataan 0,457
stdev 0,101
2 1 0,464 0,528 -0,639 -0,0143 0,985 48,46 21,62 71 112,9 0,3
3 0,453 0,516 -0,662
6 0,430 0,490 -0,713
18 0,396 0,451 -0,796
27 0,308 0,351 -1,047
Rataan 0,410
Stdev 0,063
3 6 0,486 0,554 -0,590 -0,0118 0,988 58,73 21,56 85,2 129,4 0,25
18 0,411 0,468 -0,759
27 0,381 0,434 -0,835
Rataan 0,426
Stdev 0,054
4 3 0,552 0,620 -0,464 -0,0072 0,992 96,25 21,47 142 204,3 0,15
6 0,507 0,577 -0,549
18 0,485 0,552 -0,594
27 0,452 0,515 -0,664
Rataan 0,499
Stdev 0,042
5 1 0,488 0,556 -0,587 -0,0098 0,990 70,72 21,52 101,4 167,7 0,21
6 0,401 0,457 -0,783
18 0,387 0,441 -0,819
27 0,360 0,410 -0,891
Rata2 0,409
Stdev 0,055
6 3 0,579 0,660 -0,416 -0,0148 0,985 46,82 21,62 66,56 96,1 0,32
6 0,495 0,564 -0,573
18 0,447 0,509 -0,675
Rataan 0,507
Stdev 0,067
Keterangan: OD = optical density Vd = volume distribusi Ln = logaritma natural AUC = area under the curve
CPO = kadar IgY dalam darah anjing pada waktu ke-nol t½ = waktu paruh
296
IgY dibandingkan dengan waktu paruh IgG yaitu dengan mengukur konsentrasi obat dalam darah
27 hari (Carlender, 2002) berarti IgY memiliki jika diketahuinya Vd suatu obat. Distribusi
nilai kecepatan disosiasi yang lebih tinggi bahan biologik biasanya terbatas di plasma dan
dibandingkan IgG. Dengan demikian cairan ekstraselular karena umumnya bersifat
kemungkinan IgY untuk terakumulasi dalam polar dan bobot molekulnya besar. Protein
tubuh lebih kecil dibandingkan dengan IgG. dengan bobot molekul di atas 30 kDa sangat
Data waktu paruh IgY bermanfaat untuk lambat melewati kapiler pembuluh darah.
memperkirakan waktu lamanya IgY di dalam Distribusi bahan biologik dipengaruhi oleh
tubuh anjing, menentukan interval terapi dan ikatannya dengan protein plasma. Konjugasi
waktu tercapainya steady state ketika diberikan antibodi dengan protein plasma dapat
berulang. Waktu paruh antibodi tergantung menghambat metabolisme antibodi dan
dari dosis dan konsentrasi antigen (Lobo et al., meningkatkan efikasinya sebagai agen terapi
2004). Ketika konsentrasi antigen tinggi di (Baumann, 2006).
dalam tubuh maka waktu paruh antibodi Area di bawah kurva (AUC) dalam plasma
menjadi pendek, karena komplek antibodi- terhadap waktu adalah ukuran dari jumlah
antigen akan cepat dibersihkan dari darah. bioavailabilitas suatu obat. Nilai AUC dari IgY
Penurunan jumlah antigen dalam tubuh akan anti-CPV adalah (84,79 ± 31,23 mg/mL.jam).
menurunkan jumlah clearance sehingga waktu Lama obat dalam tubuh (t) IgY anti-CPV adalah
paruh antibodi diperpanjang (Baumann, 2006). : (130,71 ± 43,84 jam atau sekitar 2,1 kali waktu
Konsentrasi IgY anti-CPV sesaat setelah paruhnya.
injeksi (Cp0) adalah (0,95 ± 0,09 mg/mL). Data Antibodi memiliki nilai farmakokinetik yang
ini menunjukkan jumlah IgY tertinggi di darah berbeda-beda. Variasi farmakokinetik ini sangat
setelah diinjeksi. Bioavailabilitas antibodi yang berpengaruh terhadap nilai clearance atau
diaplikasikan secara intravena akan lebih besar bersihan suatu antibodi. Clearance atau ber-
dibandingkan jika diaplikasikan secara sihan jaringan hati dan ginjal sangat dipe-
intramuskuler dan subkutan. Adanya aktivitas ngaruhi oleh laju aliran darah dan kemampuan
proteolisis dan transit di saluran limfatik organ tersebut untuk metabolisme dan mengeli-
menyebabkan bioavailabilitas antibodi minasi obat. Kemampuan hati untuk memeta-
intramuskuler dan subkutan lebih rendah bolisme obat tergantung pada jumlah dan
dibandingkan secara intravena (Sarghel, 2004). kemampuan enzim hati untuk metabolisme
Rataan volume distribusi umumnya diukur (Baumann, 2006).
pada hewan yang memiliki persamaan umur, Rataan clearance IgY anti-CPV dari tubuh
bobot badan, dan jenis kelamin. Nilai volume anjing adalah 0,29 ± 0,11 mL/jam. Hal ini berarti
distribusi IgY anti-CPV pada penelitian ini rataan IgY anti CPV yang dibersihkan dari
adalah (22,72 ± 2,61 mL). Nilai Vd dapat darah anjing per jam berkisar antara 0,18-0,4
digunakan untuk memperkirakan dosis yang mL/jam. Nilai clearance suatu obat sangat
harus diberikan agar mencapai kadar yang berguna dalam menentukan dosis, interval
diinginkan, karena ada hubungan antara dosis pemberian dalam mencapai kadar atau steady
dan volume distribusi (dosis = Vd. Cp0). Cairan state yang diinginkan. Hubungan persamaan
tubuh anak anjing yang bobot tubuhnya sekitar tersebut, jika obat diberikan intravena adalah
10 kg adalah sekitar 500 mL. Nilai ini diperoleh Cl. Css = Dosis/T, dalam hal ini Css adalah
berdasarkan perhitungan, volume plasma konstanta steady state dan T adalah interval
adalah 5% dari bobot badan. Nilai Vd IgY lebih pemberian.
kecil daripada jumlah plasma dalam tubuh
anjing sehingga dipandang IgY hanya terdis-
tribusi dalam darah dan tidak masuk ke jari- SIMPULAN
ngan. Hal tersebut terjadi karena IgY adalah
suatu protein yang sulit menembus membran Kinetika IgY anti-CPV dalam tubuh anjing
sel. mengikuti model satu kompartemen dan orde
Volume distribusi merupakan suatu satu, interval terapi IgY anti-CPV adalah satu
parameter yang berguna untuk menilai jumlah kali sehari, dan berdasarkan konstanta
relatif obat di luar kompartemen sentral atau ketetapan disosiasi IgY anti-CPV tidak
jaringan. Jumlah total obat dalam tubuh pada berpotensi toksik.
berbagai waktu pemberian dapat ditentukan
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Universitas Lambung Mangkurat F-MIPA PS.

Nama : Muhammad Arief Rahman

BUKU KERJA PRAKTIKUM

apt. Destria Indah Sari, M.Farm.

PROGRAM STUDI FARMASI

Buku Kerja Praktikum Farmakokinetika

Muhammad Arief Rahman

Mengetahui, Nilai Laporan Awal Nilai Laporan Akhir

(Alya Nurwafa) Tanggal : Tanggal :

Buku Kerja Praktikum Farmakokinetika

Tujuan Umum : Memahami konsep farmakokinetika suatu obat

Ringkasan Cara Kerja/Tahapan Percobaan :

Buku Kerja Praktikum Farmakokinetika

• Dilarutkan dengan aquadest sampai tanda batas

2. Penentuan Panjang Gelombang Maksimum

• Diamati nilai serapan pada panjang gelombang 530-

3. Pembuatan Kurva Baku

• Diamati serapan pada panjang gelombang

4. Simulasi Model Farmakokinetika In Vitro

• Dikalibrasi gelas aqua 100 ml

Buku Kerja Praktikum Farmakokinetika

• Diisikan pada gelas beker secara kuantitatif, sesuai

• Diambil setiap 3 menit CL 100 mL

Rute ekstravaskuler, kompartemen satu terbuka

• Dikalibrasi gelas aqua 200 ml

• Diisikan pada gelas beker secara kuantitatif, sesuai

Buku Kerja Praktikum Farmakokinetika

• Diambil setiap 3 menit CL 200 mL

Buku Kerja Praktikum Farmakokinetika

parameter dengan cara menghubungkan konsentrasi dengan dosis yang

Buku Kerja Praktikum Farmakokinetika

Distribusi bahan biologik biasanya terbatas di plasma dan cairan ekstraselular

Pradana, D. A., F. Hayati & D. Sukma. 2013. Pengaruh Pra-Perlakuan Madu

Rinidar., M. Isa & T. Armansyah. 2021. Pengantar Farmakologi: Analgesik-

Shargel, L & A. B. C. Yu. 2016. Applied Biopharmaceutics and Pharmacokinetics

Buku Kerja Praktikum Farmakokinetika

Suartini, I. G. A. A., I. Sendow, N. L. P. Agustini, A. Suprayogi, I. W. T. Wibawan

Buku Kerja Praktikum Farmakokinetika

Basic Concepts in Population Modeling, Simulation, and

BACKGROUND DATA CONSIDERATIONS

Correspondence: DR Mould (drmould@pri-home.net)

CPT: Pharmacometrics & Systems Pharmacology

CPT: Pharmacometrics & Systems Pharmacology

CPT: Pharmacometrics & Systems Pharmacology

2.5 is the SD of the individual values of the empirical Bayesian

1.0 Table 1 Common forms for residual error models

1.0 Exponential Y = f (θ , Time ) ⋅ exp ( ε )

CPT: Pharmacometrics & Systems Pharmacology

a b a Distribution density of residuals

0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5

CPT: Pharmacometrics & Systems Pharmacology

CPT: Pharmacometrics & Systems Pharmacology

PENGARUH PRA-PERLAKUAN MADU TERHADAP FARMAKOKINETIKA

Prodi Farmasi Fakultas Matematika dan Ilmu Pengetahuan Alam

Jurnal Ilmiah Farmasi Vol. 10 No. 1 Tahun 2013

Jurnal Ilmiah Farmasi Vol. 10 No. 1 Tahun 2013

menit dan disentrifuse selama 30 menit b. Penetapan dosis

diambil beningannya dan diinjeksikan ke kelayakan etik (ethical clearance) nomor

HPLC sebanyak 20 μl secara auto injeksi 103/KEC-LPPT/V/2013 dari Komite Etik

(Anonim, 2009). Penelitian Hewan Coba Laboratorium

6. Penentuan kriteria ketepatan (precise) Penelitian dan Pengujian Terpadu (LPPT)

Nilai kesalahan acak mencerminkan presisi Universitas Gadjah Mada

yang diperoleh dalam suatu metode. 1. Penetapan panjang gelombang

Rentang penerimaan yang diperbolehkan maksimum rifampisin