UJI PRAKLINIK

OBAT HERBAL

Mae Sri Hartati wahyuningsih

Departemen farmakologi dan Terapi

Fakultas Kedokteran

UGM

NAPRALERT (1985) Loub, W. D. J. Chem. Inf. Comput. Sci. 25:99-103

• approximately 3,500 new chemical structures from natural sources were reported.

• 2,618 were obtained from higher plants

• 512 from lower plants (lichens, filamentous fungi, and bacteria)

• 372 from other sources (marine organisms, protozoa, arthropods, and chordates)

Approaches to Drug Discovery from Plants

• look for new chemical constituents and hope to find a biologist who is willing to test each substance with whatever pharmacological test is available

• collect every readily available plant, prepare extracts, and test each extract for one or more types of pharmacological activity

vinca alkaloids, vincristine sulfate (childhood leukemia) and vinblastine sulfate Vincristine (Hodgkin's disease)

Vincristine • Vincristine was discovered by Gordon H. Svoboda at the Lilly

Research Laboratories. • In January 1958, Svoboda submitted an extract of the

Madagascan periwinkle plant [Catharanthus roseus (L.) G. Don] to a pharmacological screening program at Lilly (Farnsworth, 1982).

• This was the fortieth plant that he selected for inclusion in the program.

• Vincristine was marketed in the United States in 1963, less than 5 years after a crude extract of C. roseus was observed to have antitumor activity.

• In 1985, total domestic and international sales of vincristine (as Oncovin$) and vinblastine (as Velban$) were approximately $100 million, 88% of which was profit for the company (G. H. Svoboda)

medicinal folkloric information

• what correlation, if any, exists between the current medical use of the 119 drugs and the alleged medical uses of the plants from which they were derived?

• 74% of the 119 chemical compounds used as drugs have the same or related use as the plants from which they were derived.

• This does not mean that 74% of all medical claims for plants are valid, but it surely points out that there is a significance to medicinal folklore that was not previously documented

• ABC Pharmaceutical Corporation hire one or two physicians to travel to Africa, Borneo, New Caledonia, or other exotic areas to live with the people for a year or so.

• During this period, Drs. U. Canduit and 1. M. Reliant would observe the witch doctors treating patients and then would make their own diagnoses of each patient and conduct follow-up observations on outcome.

• When improvement is noted, they would record which plants had been used to treat the patients.

• These plants would then be collected and sent to the Research Laboratory of the ABC Pharmaceutical Corporation located in Heartbreak, Colorado, for scientific studies

Pharmaceutical Corporation

Drug Development Stages

• Discovery

• Product Selection

• Preclinical

• Clinical

– Phase I

– Phase II

– Phase III

Cost Per Stage

• Discovery - $.5M

• Product Selection - $.5M

• Preclinical - $1- 1.5M

• Clinical

– Phase I - $5-10M

– Phase II - $10-20M

– Phase III - $30-50M

• Product Launch - $5-10M

Time and Financial Constraints

The drug development process is time-consuming and expensive:

• R&D is time consuming—It takes 10 to 15 years to bring a new drug to market.

• From 5,000 to 10,000 compounds must be screened to yield 1 potentially successful drug.

• 10% or $15 to 30 billion is spent for preclinical studies today.

• 15% will be spent on preclinical studies in the next three to five years.

Reasons For Drug Failure

39%

30%

11%

5%

5%10%

Pharmacokinetics Lack of efficacy

Animal toxicity Commercial reasons

Adverse effects in man Miscellaneous

Source: 198 NCEs in clinical development by large UK companies, 1964–1985.

80%

80% can be detected in preclinical phase

Pengembangan Obat

• Uji Preklinik

– Uji Farmasetika

– UjiFarmakologi

– UjiToksikologi

• Uji Klinik

fase 1, 2 and 3

• Postmarketing surveilance

– (fase 4 uji klinik)

Uji Preklinik

• Uji Farmasetika

* Kekeringan, * Kestabilan, dll.

• Uji Farmakologi

* Awal , * Lanjut

• Uji Toksikologi

* Akut, * Subakut, * Kronik

* Teratogenik, Hepatotoksik, dll.

JAMU

OBAT HERBAL TERSTANDARD

Peran Uji Preklinik dalam Pengembangan Obat Herbal

FITOFARMAKA

Uji Preklinik

UJI KLINIK

Uji Farmasetika

• Stabilitas zat aktif ekstrak sebelum dan sesudah pengeringan.

• Stabilitas fisik sediaan kapsul dan tablet dan stabilitas kimiawi zat aktif.

• Persyaratan Farmasetika untuk sediaan kapsul dan tablet.

• Sifat zat tambahan inert, baik fisika-kimia maupun fisiologis

Uji Farmakologi

Uji Farmakologi Awal

• Apakah ada efek yang

diinginkan • Apakah ada efek yang tak

diinginkan - efek samping

- efek toksik

Uji Farmakologi Lanjut

• Dimana tempat kerjanya

• Bagaimana mekanisme kerjanya

Pharmacological Screening

• Molecular Level – Receptor binding, enzyme activity,

Cytochrome P-450.

• Cellular/Tissue Level – Cell function (tissue culture), isolated tissues.

• Organ/System Level – Identify primary and secondary targets.

Early Pharmacological study / screening

Invivo model - analgesic

- antipyretic

- antiinflamation

- sedatif / hypnotic

- antihypertensiv

- diuretic

- hypoglicemia

- antiulcer

Hot plate procedure

- Heat stimulus

- does not affect the mechanoreceptors

- easy to be controled

- easy to be applied to the moving subject (animal)

* mobile subject hot plate procedure

• immobile subjects immersion radiant heat

procedure

Uji Analgesic

Hot plate procedure

- Observations

- 30 minutes after injection (of testing substance)

- place the animals on the hot place

(constant temperature, 50o – 60o C) to

- observe licking latency or jumping latency tR

(disregard kicking and dancing)

- remove animal from hot plate

(as soon as they exhibit licking and jumping)

Hot plate procedure

- Calculate

- mean and SD (of the jump latency for control group

and treatment groups)

- % of animal showing an analgesic response

(for each group)

- plot the dose – response curve ED-50

(% animal showing the analgesic activity)

- calculate the ED-50

Uji Antiinflamasi

Uji efek antiinflamasi

• Tujuan : efek antinflamasi • Hewan coba : rat atau mencit. • Parameter : volume odem, index arthritis, ekspresi Cox-2, TNF alfa , interleukin 6. • Induksi : akut : 0,1 ml karagenin 1% kronis: 0,1 ml CFA (Complete Freund’s Adjuvant)

Pengukuran volume odem

• Plethysmograph air raksa

• Plethysmograph elektrik

Langkah pengukuran • Persiapan : NaCl fisiologis • Kalibrasi : standard volume • Pengukuran : volume odem

Analisa : membandingkan nilai AUC volume odem

INDEKS ARTHRITIS ((Courtenay et al.,1980).

Gejala Skor

Bengkak dan kemerahan pada satu jari 0.25

Bengkak dan kemerahan pada dua jari

atau lebih

0.50

Bengkak pada foot pad (telapak kaki) 0.75

Bengkak dan kemerahan pada jari-jari.

dan bengkak pada telapak kaki

1.00

• Pembuatan preparat Cox-2 dan Cox-1

• Pengecatan preparat Cox-2 dan Cox-1

Ekspresi ( + ) : plasma warna coklat

Ekspresi ( - ) : plasma warna biru

UJI EFEK APHRODISIAK

( SEXUAL BEHAVIOUR )

• Dilakukan pada fase gelap (12.30 – 17.00).

• Obat diberikan 1 jam sebelum uji

• Ditempatkan pada kotak uji, terbuat dari kaca, ukuran 50 X 60 cm atau 25 X 40 cm.

• Tikus betina dalam keadaan siap kawin dimasukkan kedalam kotak uji.

• Diamati perilaku seksualnya

Istilah perilaku seksual

• Mounting rat : tikus dalam posisi kawin tapi tidak melakukan intromisi

• Intromission rat : tikus melakukan intromisi tapi tidak mengeluarkan sperma

• Ejaculat : beberap kali intromisi diikuti pengeluaran semen

Parameter perilaku seksual

• Mounting latency = ML

• Mounting Frequency = MF

• Intromission Latency = IL

• Intromission Frequency = IF

• Ejaculatory Latency = EL

• Post Ejaculatory Interval = PEI

Antisecretory test

• Aim of the experiment To know the effect of a testing compound in rat gastric acid secretion. • The stomach was removed and suspended in an organ bath. • The gastric lumen was perfused with unbuffered mucosal solution • allowed to stabilize for 1 h and perfusate was spilled out. • The perfusate was allowed to flow continuously, and collected for 10

minutes duration. • H+ concentration was measured by mean of titration with 0.002 N NaOH

and phenolphtalein as indicator, as basal H+ concentration. • Add the testing compound to the unbuffered mucosal solution for 30

minutes. • After that, 736.4 µg/kgBW histamine or 0.3072 µg/kgBW pentagastrin or

726.8 µg/kgBW acetylcholine is added to the the unbuffered mucosal solution for 80 minutes to induce gastric secretion.

• Perfusate from gastric lumen were collected every 10 minutes and H+ concentration were measured by mean of titration.

• The elevation of H+ concentration was calculated in percentage, and was expressed as means + SEM.

Termostat dan sirkulator

organbath

buffered serosal

unbuffered mucosal

air 37oC

oksigen

carbogen

Gambar 4 : sekresi ion H lambung rat terisolasi

0

1

2

3

4

5

6

7

8

9

0.000001 0.00001 0.0001

kadar histamin (uM)

ka

da

r i

on

H p

ad

a p

erfu

sat

(mE

q) kontrol

simetidin 0,1 uM

simetidin 1 uM

simtidin 10 uM

omeprazol 0,01 uM

omeprazol 0,1 uM

omeprazol 1 uM

EA|PK 15 mg

EAPK 30 mg

EAPK 60 mg

Musa balbisiana Colla

Diuretic activity Lipschitz et al, 1943 • male albino rats weighing between 120-150 g, deprived of

food and water for 18 hours prior to the experiment, were divided in eight groups of six rats in each.

• The first group of animals, serving ascontrol, received normal saline (25 ml/kg, p.o.); the second group received furosemide (10 mg/kg, p.o.) in saline (14); other groups received doses of extract (200and 400mg/kg) or extract fractions (200mg/kg each), in normal saline.

• Immediately after admistration, the animals were placed in metabolic cages (2 per cage), specially designed to separate urine and faeces, keptat 20°C±0.5°C.

• The volume of urine collected wasmeasured at the end of 5 h.

• During this period, no food and water was made available to animals.

• The parameters taken were total urine volume,concentration of Na+, K+ and Cl. in the urine.Na+ K+

Laxative activity Capasso et. al.

• (18) on rats of either sex, fasted for 12 h before the experiment, but with water provided ad libitum.

• The animals were divided into eight groups of six in each. • The animal groups were administered orally either with

vehicle (1% Tween-80 solution in normal saline, 25 ml/Kg), reference standard drug, agar-agar (300 mg/kg, p.o.) in saline (11) or doses of extract (200 and 400mg/kg) or extract fractions (200mg/kg each).

• Immediately after dosing, the animals were separately placed in cages suitable for collection of faeces.

• After 8h of drug administration, the faeces were collected • and weighed. • Thereafter, food and water were given to all rats and

faecal outputs were again weighed after a period of 16 h.

Uji Antihipertensi

• Hewan coba: hipertensi spontan

hipertensi buatan

# silver clip di arteria renalis

# alkohol

• Metode pengukuran tekanan darah:

tail cuff method

• Langkah2 pengukuran

Aklimatisasi hewan coba

Kalibrasi

Pengukuran tekanan darah

Pengukuran tekanan darah: tail cuff method

• Prinsip sama dengan sphigmomanometer manusia

• Tekanan naik s/d 300 mmHg

• Diturunkan perlahan

• Nadi pertama sebagai tanda tekanan sistolik

• dapat digunakan pada tikus, mencit, anjing, primata kecil.

Pengukuran Tekanan Darah Mencit

• Holder

• Blood pressure analyzer

• Paper recorder /

monitor

Uji Farmakodinamik tahap awal

Model invitro - antispasmodik

- bronkodilator

- antiaritmia

- spasmolitik

- antiskresi asam lambung

- dsbg.

Efek ekstrak pace pada jantung kelinci terpisah

UJI TOKSIKOLOGI

UJI TOKSISITAS AKUT

Tujuan

• Menetapkan potensi toksisitas akut (LD50).

• Menilai berbagai gejala klinis, spektrum efek toksik, dan mekanisme kematian

Hewan coba

• Paling tidak 1 spesies

• Biasanya spesies pengerat (mencit atau tikus) – Mencakup kedua jenis kelamin

– 4-6 kelompok

– Tiap kelompok minimal 4 ekor jantan & 4 ekor betina

• Spesies bukan pengerat, tiap kelompok minimal 2 ekor (anjing, babi)

Perlakuan terhadap hewan coba

• Dosis tunggal • Besar dosis bertingkat, ditingkatkan berdasarkan faktor

logaritmik, dengan ratio tertentu misalnya 1,2; 1,5; atau 2 sampai batasyang masih memungkinkan untuk diberikan

• Cara pemberian sesuai dengan cara penggunaan • Ditentukan LD50, yaitu dosis yang menyebabkan kematian

(dosis lethal) pada 50% hewan coba • Perlu dosis yang menyebabkan kematian >50% hewan coba • Bila tidak dapat ditentukan LD50, maka diberikan dosis lebih

tinggi, dan sampai dosis maksimal yang masih mungkin diberikan pada hewan coba

Pengamatan

• Dimulai sejak persiapan • Jangka waktu pengamatan 7-14 hari (dapat lebih lama pada pemulihan gejala

toksik) • 24 jam pertama diamati terus menerus (bantuan video) • Yang diamati:

– Kematian – Gejala-gejala klinis terutama yang berkaitan dengan organ vital (ginjal, hati,

hemopoetik) – Hewan yang mati diotopsi – Hewan yang hidup sampai batas akhir pengamatan diotopsi – Hewan yang menunjukkan efek toksik tetap dikorbankan bermanfaat untuk

diamati terjadi tidaknya pemulihan – Berat badan – Persentase kematian – Patologi organ (makroskopis, mikroskopis)

• Analisis statistik dengan metode yang sesuai

MACAM

PERLAKUAN :

5 macam dosis

Persentase kematian ----- LD50

Keadaan fisik, tanda-tanda

toksisitas

ADG (Average Daily Gain)

Desain Penelitian

Variabel bebas

Gambaran histopatologis

Variabel terikat

Uji Toksisitas akut

Mencit atau tikus putih

dewasa (12-14 minggu)

Kelompok 1

Dosis I

Kelompok 3

Dosis III

Kelompok 4

Dosis IV

Kelompok 5

Dosis V

Kelompok 6

kontrol

Kelompok 2

Dosis II

CARA PENELITIAN

Uji Toksisitas Akut

Diamati 24 jam,

tentukan LD50

Puasa 12-14 jam

Pengamatan

dilanjutkan

sampai 14 hari

UJI TOKSISITAS JANGKA PANJANG

TUJUAN

• Mengetahui spektrum efek toksik

• Hubungan dosis dan toksisitas pada pemberian berulang dengan jangka waktu lama

• Obat diberikan berulang, setiap hari sepanjang waktu tertentu

– Uji subakut/subkronik (1-3 bulan)

– Kronik (3-6 bulan)

• Akumulasi, toleransi, metabolisme dan kelainan khusus di organ atau sistem organ tertentu dapat dipelajari.

Lama uji toksisitas jangka panjang didasarkan pada lama penggunaan obat yang akan diuji:

Masa

penggunaan

klinis

Masa pemberian

obat uji

Dosis tunggal

atau <2 minggu

2 minggu – 1

bulan

Dosis berulang

(1-4 minggu)

4 minggu – 3

bulan

Dosis berulang

(1-6 minggu)

3- 9 bulan

Dosis berulang

(>6 bulan)

9-12 bulan

• Hewan coba: tikus atau mencit

• Perlakuan: persiapan seperti pada uji toksisitas akut

• Cara pemberian, frekuensi, interval, lama pemakaian perlu dipertimbangkan hal-hal: – Penggunaan empirik di masyarakat (untuk Obat

Tradisional)

– Rencana maksud pemanfaatannya kelak

– Hasil pengamatan uji toksisitas akut

Dosis

• Lazim digunakan 3 peringkat dosis (ditentukan dengan memepertimbangkan aktivitas farmakologik dan hasil uji toksisitas akut)

• Dosis tertinggi diupayakan yang dapat menimbulkan efek toksik (perubahan hematologik, biokimia, anatomi, histolgi) namun mayoritas harus dapat bertahan hidup.

• Dosis paling rendah harus mendekatidosis efektif sesuai dengan spesies yang digunakan dalam pengujian

• Tanda-tanda umum diamati setiap hari

• Berat badan, makan, minum diamati secara periodik (misalnya tiap 1 minggu)

• Berat badan: sebelum pemberian oabt

Gejala-gejala klinis diamati pada hari ke-0 (sebelum obat diberikan) dan selanjutnya diamati setiap hari. Berat badan diukur pada saat sebelum obat diberikan dan selanjutnya diukur tiap 1 minggu, sehingga dapat dihitung pertambahan rerata berat badan per hari (ADG= Average Daily Gain). Jumlah makanan, jumlah minum diukur sebelum obat diberikan dan selanjutnya diukur tiap 1 minggu, sehingga dapat diukur rerata jumlah masukan makanan dan minuman per hari. Volume urin dan berat tinja diukur sebelum obat diberikan dan selanjutnya diukur tiap 1 minggu (dilakukan juga pengamatan terhadap kualitas urin dan tinja) Pemeriksaan darah rutin, kadar kalium, GOT, GPT, ureum, kreatinin, albumin, globulin, protein total dilakukan pada hari ke-0, ke-45, dan hari ke-91. Pada hari ke-91 semua hewan coba dikorbankan, diperiksa organ dalamnya secara makroskopis dan dibuat preparat untuk selanjutnya dilakukan pemeriksaan histopatologi.

UJI TOKSISITAS KHUSUS

• Mutagenik

• Teratogenik

• Karsinogenik

Pelaksanaan dilakukan secara selektif berdasarkan hal-hal:

• Formula obat beralasan untuk diantisipasi karena dicurigai berisi kandungan zat kimia yang potensial untukmenimbulkan salah satu efek khusus

• Potensial digunakan oleh perempuan dalam batas usia subur,

perlu dipertimbangkan kemungkinan berefek teratogenik. Kemungkinan terjadinya efek teratogenik, sebagai akibat aktifitas suatuzat kimia dapat juga terjadi pada sistem reproduksi laki-laki dalam masa usia subur

• Dalam masyarakat didapatkan prevalensi gejala atau penyakit tertentu yang secara epidemiologik menunjukkan terkat dengan penggunaan suatu formula oabat tertentu

UJI TERATOGENIK

• Tujuan:

Mengetahui efek teratogenik dengan mengetahui ada tidaknya janin cacat, resorpsi, atau janin yang mati

Hari ke-0 kehamilan adalah pada waktu ditemukan sperma dalam vulva Apabila pada hewan coba terlihat tanda-tanda akan keguguran atau melahirkan prematur harus dikorbankan dan diotopsi. Janin diambil melalui pembedahan sehari sebelum perkiraan hari kelahiran. Dilakukan pengamatan terhadap jumlah resorption site, resorpsi, maserasi, janin yang mati, janin yang hidup, berat janin yang hidup, panjang, adanya kelainan, cairan amnion, plasenta.

Uji Teratogenik

THANK YOU