Radiotoxicologie, Radiochimie, Radiopharmacie FARM...

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Radiotoxicologie, Radiochimie, RadiopharmacieFARM 3200

Cours n° 7

RADIOPHARMACIE (1) Introduction et critères de qualité

Prof. Bernard Gallez

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RadiopharmaceutiquesGénéralités

Radiopharmaceutique

= préparation pharmaceutique, contenant un ou plusieurs radionucléides, utilisée en médecine humaine ou vétérinaire, pour le diagnostic in vivo ou in vitro, ou pour la thérapie

Cas le plus courant:

nucléide radioactif sous forme non scellée, destiné à l’administration à un patient pour des raisons de diagnostic ou de thérapeutique

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RadiopharmaceutiquesTypes de préparations

Produits enregistrés prêts à l’emploi

Nucléides contenu dans une formulation « stable »

Produits enregistrés « semi-préparés »

Générateurs

Kits

Produits « maison » (non enregistrés)

Radiopharmaceutiques PET non enregistrés

Anticorps, peptides radiomarqués

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RadiopharmaceutiquesRéponse pharmacologique

Les composés doivent être radiomarqués avec une activité spécifique suffisamment hautepour minimiser les possibilités d’effets pharmacologiques

En principe, pas d’effet pharmacologique, sauf émanant d’allergie potentielle à un composant particulier ou modulation pharmacologique de la distribution radiopharmaceutique

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RadiopharmaceutiquesInteractions médicamenteuses

Interactions désirées

Utilisation d’un médicament en vue d’induire une réponse pharmacologique qui influence la performance fonctionnelle d’un organe ou d’un tissu, cette performance étant mesurée à l’aide de la biodistribution du radiopharmaceutique

Interactions non-désirées

Modification non voulues des performances d’un radiopharmaceutique par la prise de médicaments

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RadiopharmaceutiquesInteractions médicamenteuses

Exemples d’interactions désirées

Perfusion myocardique

Dipyridamole, adénosine, dobutamine

Perfusion cérébrale

Acétazolamide (Diamox)

Etudes rénales

Furosémide, captopril

Etudes hépato-biliaires

Cholecystokinine

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RadiopharmaceutiquesInteractions médicamenteuses non désirées

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RadiopharmaceutiquesFormes des préparations

Solutions vraies

Dispersions colloïdales

Suspension particulaires

Gaz

Aérosols (liquides dispersés en phase gazeuse)

Gélules

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RadiopharmaceutiquesFacteurs affectant la Biodistribution

Perfusion

Débit sanguin régional

Perméabilité capillaire

Diffusion à travers membranes biologiques

Taille particulaire

Interactions cellulaires

Transport actif

Transport facilité

Phagocytose

Interactions/biotransformations intracellulaires

Elimination

Rénale

Bile

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Quality control parameters

Radioactivity tests- radioativity

- identification of the nuclide

- radionuclidic purity

- radiochemical purity

- specific radioactivity

Specific tests for some radiopharmaceuticals- Physiological distribution

Usual tests for injectable solutions

- chemical purity

- sterility

- apyrogenicity

- pH

- isotonicity

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Quality criteriaRadioactivity

Definition : radioactivity of a preparation = number of nucleardesintegrations or transformations per unit time.

Units (International System) : Becquerel (Bq) = 1 nucleartransformation per second

1 Ci = 3.7 1010 Bq = 37 GBq

1 mCi = 3.7 107 Bq = 37 MBq

1µCi = 3.7 104 Bq = 37 kBq

1 GBq = 27.027 mCi

1 MBq = 27.027 µCi

1 kBq = 27.027 nCi

Absolute radioactivity measurements require a specialized laboratorybut identification and measurement of radiation can be carried out comparatively and relatively by the use of radioactive standards.

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The absolute measurement of radioactivity of a given samplemay be carried out if the decay scheme of the radionuclide isknown, but in practice many corrections are required to obtainaccurate results.

It is common to carry out the measurement with the aid ofprimary standard source.

Primary standards may not be available for short-livedradiopharmaceuticals.

For these radionuclides, the instruments may be calibrated usinga secondary standard (e.g. 57Co (T=270 days) can be used as secondary standard for 99mTc).

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Counters

- Ionisation chambers and Geiger-Müller counters

> beta/gamma emitters

- Solid scintillators or semiconductor counters

> gamma emitters

- Liquid-scintillation counters

> low-energy beta emitters

Whatever apparatus is used, for an accuratecomparison of radioactive sources, it is essential for samples to be measured under identical conditions.


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Quality criteriaRadioactivity / Specific problems

Quenching in liquid scintillationMeasurements should be corrected for light-quenching effects

Internal standardization

External standardization

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Quality criteriaRadioactivity / Specific problems

Background

All measurements of radioactivity should be corrected by substracting the background activity due to radioactivity in theenvironment and to spurious signals generated in the equipmentitself

Dead time of counters

When measurements are made at high levels of radioactivity, itmay be necesary to correct for the finite resolving time of thedetector

Calibrations

The following factors should be taken into account:

Calibration factor

Sample geometry factor

Dynamic range accuracy

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Statistics in counting

The results of determinations of radioactivity shows variations which mainly derive from the random nature ofnuclear transformations. A sufficient number of countsshould be registered in order to compensate for variations in the number of transformations per unit of time. Thestandard deviation is the square root of the counts, so atleast 10000 counts are necessary to obtain a relative standard deviation of not more than 1 per cent.

Reference to a time

All statements of radioactive content should be accompaniedby a statement of the date and the time at which themeasurment was made (and reference to a time zone if exported)

The radioactivity of a solution is expressed per unit volume to give the radioactive concentration

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Quality criteriaIdentification of the radionuclide

The radionuclide is identified by:

Measuring the half-life of the radionuclide

and/or

Determining the nature and energy ofthe radiation

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Quality criteriaIdentification of the radionuclideHalf-lifeRadioactive decay

Radioactivity decays at an exponential rate which ischaracteristic for each radionuclide

The curve of exponential decay is described by the equation:

At = Ao.e-λt

At = the radioactivity at time t

Ao = the radioactivity at time = 0

λ = the decay constant characteristic of each radionuclide

E = the base of Napierian logarithms

o Half-life

The time in which a given quantity of radionuclide decays to half its initial value.

The half-life (T1/2) is related to the decay constant (λ) by theequation:

T1/2 = ln 2 / λ (ln 2 = 0.693)

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The half-life is measured with a suitable detectionapparatus. The radioactivity chosen, having regard to experimental conditions, must be sufficiently high to allowdetection during several presumed half-lives, but should belimited to minimize count rate defects and effects such as dead time losses.

The same source is measured in the same geometricalconditions at intervals usually corresponding to half of thehalf-life throughout a time equal to about three half-lives.

A graph is drawn with time as the abcissa and thelogarithm of the instrument response as the ordinate.

The calculated half-life should not differ by more than5 per cent from the half-life stated in thepharmacopeia.

Quality criteriaIdentification of the radionuclideHalf-life

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The nature and energy of the radiation emitted may bedetermined by several procedures including theconstruction of an attenuation curve and the use ofspectrometry

The attenuation curve is often used for analysis of β-

radiation

Liquid scintillation counting and spectrometry canbe used for α and β- emitters

γ spectrometry is used to identify radionuclides withgamma rays and detectable X-Rays.

Quality criteriaIdentification of the radionuclideNature and energy of radiations

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Attenuation curve

The attenuation curve is drawn for β- emitters when nospectrometer for beta rays is available. This methodof estimating the maximum energy of beta radiations gives only an approximate value.

The source is placed in front of the window of GM counter or a proportionalcounter. The count rate is measured. Between the source and the counterare placed, in succession, at least six aluminium screens of increasing massper unit area within such limits that, with a pure beta emitter, this count rate is not affected by the addition of further screens. A graph is drawn in thesame manner for a standardized preparation. The mass attenuationcoefficients can be calculated from the median parts of the curves which are practically curvilinear.


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Attenuation curve

The mass attenuation coefficient mx, expressed as square centimeters per milligram, depends on the energy of the betairradiation and on the nature and the physical properties of thescreen. It therefore allows beta emitters to be identified. It iscalculated using the equation:

µm = ln A1 – ln A2

m2 – m1

m1 = mass per unit area of the lightest screen

m2 = mass per unit area of the heaviest screen

A1 = count rate for mass per unit area m1

A2 = count rate for mass per unit area m2


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Gamma spectrometry

The preferred detector for gamma and X-rays spectrometry isa germanium semi-conductor detector. A thallium-activated sodium iodide scintillation detector can also be usedbut this has a much lower energy resolution

The detector needs to be calibrated because the detectionefficiency is a function of the energy of the gamma and the X-ray as well as the form of the source and the source-to-detector distance. The detection efficiency may be measuredusing a calibrated source of the radionuclide to be measuredor a graph efficiency against gamma and X-rays energy maybe constructed from a series of calibrated sources of variousradionuclides.


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Gamma spectrometry

The gamma and X-Ray spectrum of a radionuclide whichemits gamma and X-rays is unique to that nuclide and ischaracterized by the energies and the number of photons ofparticular energies emitted per transformation.

This property can be used to identify which radionuclides are present in a source and the amounts of each. It facilitates theestimation of the degree of radionuclidic impurity by detectingpeaks other than expected


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Gamma spectrometry


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Quality criteriaRadionuclidic purity

Definition :

the ratio, expressed as a percentage, of the radioactivityof the radionuclide concerned to the total radioactivity ofthe radiopharmaceutical preparation. The relevant radionuclidic impurities are listed with their limit in theindividual monographs.

In most of the cases, to state the radionuclidic purity of a radiopharmaceutical preparation, the radioactivities andthe identities of every radionuclide present must beknown.

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Most generally used method :

- gamma spectrometry (semiconductors)

but : not suitable for alpha/beta emitters impurities

Other methods :

- half-lives determination

- « tricks » : use of screen (see 99Mo in 99mTc)

The gamma-ray spectrum should not be significantlydifferent from that a standardized preparation.


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Specific set of limits may exist for specific impurities, depending on:

Radiotoxicity of the radionuclide

Production procedure99Mo produced by fission different from 99Mo produced by neutron activation

These requirements are not in themselves sufficient to ensurethat the radionuclidic purity of a preparation is sufficient for human use.

Depending on the procedures used, the contaminants can be foreseen.

The preparations should be examined for impurities of long half-lifedecay.

In cases of two or more positron emitting radionuclides need to be identifiedand differentiated, half-life determinations need to be carried out in addition to gamma spectrometry.

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Due to differences in the half-lives of the differentradionuclides present in a radiopharmaceuticalpreparation, the radionuclidic purity changes withtimes.

The requirement of the radionuclidic puritymust be fulfilled throughout the period ofvalidity.

It is sometimes difficult to carry out these tests before authorising the release for use of the batch when the half-live is short. The test then consitutesa control of the quality of production.


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The determination of half-life is a marker ofradionuclidic purity of a preparation

Example15O: T=2 min13N: T=10 min#1 100% 15O#2 99% 15O + 1% 13N#3 98% 15O + 2% 13N

Time (min)

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Quality criteriaRadionuclidic purityImportance of the production procedure

99Mo for 99mTc generators

- 98Mo(n,γ)99Mo

- nuclear fission of uranium and extraction : fission products

Development of new radionuclides in PET centers

e.g 86Y (β+,T=14.6h) produced by reaction (p,n) on a target of 86Sr using protons with energy of 16 MeV. If protons of 30 MeV, the reaction (p,2n) becomesimportant and leads to 85Y (β+/CE, 5h) whichdesintegrates to 85Sr(CE, 65d).

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Importance of other contaminants coming from impurities in thetarget :

Target in Havar®. Havar contains 52Cr, 59Ni, 96Mo

Usual productions:

Nuclide produced Target Reaction

18F H218O 18O(p,n)18F

13N H216O 16O(p,α)13N

15O 14N214N(d,n)15O

11C 14N214N(p, α)11C

Identification at KUL (Leuven) and at UCL (Louvain-la-Neuve) of several contaminants in 18F and 13N, but not in 15O neither in 11C:

52Mn coming probably from 52Cr(p,n)52Mn56Co 59Ni(p, α)56Co96Tc 96Mo(p,n)96Tc

No impurity if the target is a gas. Impurities present when transfer

possible to a liquid target

Quality criteriaRadionuclidic purityImportance of the production procedure

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Quality criteriaRadiochemical purity

Definition:

the ratio, expressed as a percentage, of the radioactivity ofthe radionuclide concerned which is present in theradiopharmaceutical preparation in the stated chemical form, to the total of radioactivity of that radionuclide present in theradiopharmaceutical preparation. The relevant radiochemicalimpurities are generally listed with their limit in the individualmonographs.

The determination of radiochemical purity requiresseparation of the different chemical substances containingthe radionuclide and estimating the radioactivity associatedwith the declared chemical substance

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Origin of radiochemical impurities:

Radionuclide production

Subsequent chemical procedures

Incomplete preparative separation

Chemical changes during storage

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In principle, any method of analytical separation may beused in the determination of radiochemical purity. Themonographs for radiopharmaceutical products may includepaper chromatography, thin-layer chromatography, electrophoresis, size-exclusion chromatography, gaschromatography, liquid chromatography.

In hospital environment, paper chromatography andTLC are mostly used.

In paper and TLC chromatography, it s preferable not to dilute the preparation to be examined except

If the radioactivity amount is too high and can lead to loss due to the dead time of the counter

A carrier is needed to avoid unspecific adsorption on thesupport


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After the development of the chromatography, thesupport is dried.

The positions of the radioactivity along the support isdetermined using different possible ways:

Autoradiography

Phosphorimager / Instant imager

Multi-channel position sensitive counters

Moving counters along the chromatogram

Suitable collimated counters (γ camera)

By cutting the strips and couting each portion


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The position of the spots or areas permit the chemicalidentification by comparison with solutions of the samechemical substances (non radioactive) using a suitabledetection method

Radioactivity is measured by integration using plottinginstrument. When the strips are cut into portions, the ratios of the quantities of radioactivity measured give the ratio ofconcentrations of the radioactive chemical species


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Exampleo-[123/131I]-iodohippurate

Tubular secretion markerExample of preparation: exchange of aromatic iodine catalyzed by cupperRadiochemical purityTLC on silicagel.Eluent: toluene/n-butanol-Acetic acid/water 80/20/4/1Carrier: o-iodohippuric acid, o-iodobenzoic acid, potassium iodine

Origin Front

I- HIP OIB

Synthesis Desired DegradationImpurity Product Product

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For several radiolabelled compounds, there is a needto use more than one chromatography system to knowthe RCP

Example: HMPAO-99mTc

System 1: ITLC-Methylethylketone

Rf 0.8-1: TcO4- + HMPAO-Tc

Rf 0 : Hydrolyzed Tc + secondary complexes

System 2: ITLC – NaCl 0.9%

Rf 0.8-1: TcO4- + HMPAO-Tc + secondary complexes

Rf 0: Hydrolyzed Tc


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Quality criteriaSpecific radioactivity

Definition: the radioactivity of a nuclide per unit mass ofthe element or of the chemical form concerned

Specific radioactivity is usually calculated taking intoaccount the radioactive concentration (radioactivity per unit volume) and the concentration of the chemical substance being studied, and verification that the radioactivity is onlyattributable to radionuclide (radionuclidic purity) and thechemical species (radiochemical purity) concerned.

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Quality criteriaPhysiological distribution

A physiological distribution test is prescribed, if necessary, for certain radiopharmaceutical preparations.

The distribution pattern of radioactivity observed in specified organs, tissues or other body compartments ofan appropriate animal species (usually rats or mice) can bea reliable indication of the expected distribution in humansand thus the suitability for the intended purpose.

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The individual monographs prescribe the detailsconcerning the performance of the test and thedistribution requirements which must be met for theradiopharmaceutical preparation.

A biodistribution conforming the requirements will assure appropriate distribution of the radiopharmaceuticalpreparation to the intended biological target in humans andlimits its distribution to non-target areas.

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This test is applied when the physico-chemicalcharacterization of the radiolabelled compound is notfully described, and thus the relationship betweenchemical structure and biological behavior ispoorly understood (e.g. colloids labelled withtechnetium, diphosphonate complexes,…)

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In general, the distribution is performed as follows:

Each of three animals is injected intravenously with the preparation to betested. If relevant, the species, sex, strain, and weight or age of theanimals is specified. The test injection should be theradiopharmaceutical as it is intended for human use. Productscomprising reagents for reconstitution should be reconstituted accordingto the manufacturer’s instructions. In some cases, dilution immediatelybefore the injection may be necessary.

The injection will normally be made via the intravenous route for whichpurpose the caudal veins are used. Other veins such as the saphenous, femoral, jugular or pudendal veins may be used in special cases. Animalsshowing extravasation of the injection should be rejected from the test.


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In general, the distribution is performed as follows:

At the specified time after injection, the animals are killed by an appropriatemethod and dissected. Selected organs and tissues are assayed for theirradioactivity. The physiological distribution is calculated and expressed in terms of the percentage of radioactivity which is found in each of the selectedorgans or tissues. For this purpose, the radioactivity in an organ may berelated to the injected radioactivity calaculated from the radioactive content ofthe syringe measured before and after injection (minus the radioactivity foundat the point of injection). For some radiopharmaceutical preparations, it maybe appropriate to determine the ratio of the radioactivity in weighted samplesof selected tissues (radioactivity per gram).

To meet the requirements of the test, the distribution of radioactivity in atleast 2 of the 3 animals must comply with all the specified criteria (Importance of the « re-test » procedure)


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Quality criteriaChemical purity

The determination of chemical purity requires identification and quantification of individual chemical constituents or impurities present in the radiopharmaceutical preparation

These tests include the quantification of

Normal consituents of a labelling kit (quantification of ligand, ofstannous tin)

Impurities (aluminium present in technetium generator eluates)

Synthesis precursors or catalysts (kryptofix in synthesis of [18F]-FDG

Residual solvents (synthesis of PET tracers)

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Quality criteriaChemical purity

These tests should be conducted in

Industries manufacturing radiopharmaceuticals

PET centers

Several tests are important to perform in nuclearmedicine centers such as the detection of aluminium in generator eluates

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Quality criteriaSterility

Radiopharmaceutical preparations for parenteraladministration must be prepared using precautionsdesigned to exclude microbial contamination and to ensure sterility

Special difficulties:

Small size of batches: sometimes limited to one or a fewsamples

Radiation hazards

Direct inoculation method: result known after several days

Not possible to await the results before release

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Generally, possibility for waiting the decrease of the

radioactivity and performing the sterility assay later

Test = control of quality of the production

Need for a performance test on the procedure used

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Direct transfer to test media or membrane filtration under aseptic conditions:

Fluid thioglycollate medium (bacteria) 30 to 35°C

Tryptic soy broth (fungi) 20 to 25°C

Use of positive and negative controls !

Reading after a few days (turbidity)

Alternative method: Membrane filtration technique

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Shortcomings of the standard methods: time factor!

Recent developments in rapid microbiology methods:

Qualitative tests: Yes/No answer to the question ofmicrobial contamination

Quantitative tests: numerical value for the microbialcontents

Identification tests: identity of microorganisms

Detection of cells by labeling with fluorophores, cellauto-fluorescence, cell by-products, capillaryelectrophoretic method,…

Hope for a response about sterility before the batch release

Under evaluation by the FDA


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Quality criteriaBacterial endotoxins - Pyrogens

Endotoxins = polysaccharides from bacterial membranes (Gram -)

Water soluble

Heat stable

Filtrable

Importance for working in aseptic conditions along theproduction process

All glassware and equipment (when possible) should be heated at200°C for at least 2 hours before production

Problems:

Fever

Leukopenia, pain in joints, headache, specially in immunodepressedpatients

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Quality criteriaBacterial endotoxins - PyrogensAssay:

LAL test (Limulus Amoebocyte Lysate)

Gel formation in the presence of endotoxins

! Operating procedures (meticulous handling necessary: many

false positive results)

! Interference by inhibition or activation

If LAL impossible, increase of temperature observed after

IV injection to 3 rabbits (Eur. Pharm.:Pyrogens)

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Novel pyrogen tests based on the human fever reaction

Proposed alternative to LAL and tests on rabbits

Why?

Limitations of the present tests:

LAL: limited to Gram – (missing Gram + and fungal pyrogens)

Rabbits: use of animals

New tests based on:

Release of fever mediators such as IL-1

In vitro test with human cells

Not yet approved in many countries

In discussion at the European Pharmacopeia

For more information, see ATLA 30, S2, 49-51, 2002

Quality criteriaBacterial endotoxins - Pyrogens

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Quality criteriaOther tests

pH

Should be in the physiologically acceptable range (5-8) andoptimized for the stability of the preparation

! Specially important to check in PET centers when operatingprocedures include extreme pH conditions at one particular step(hydrolysis step)

Physical appearance / Particulate contamination

All products for administration by injection (except colloids andaggregates) should be free from particulates

Visual examination

Strict control of the glassware

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Particle size

Lung perfusion studies: MAA and microspheres (20-80 microns)

RES: coloids (less than 1 micron)

Light scattering / Photon correlation techniques

In routine: filtration through polycarbonate membranes withcontrolled etched pores

Isotonicity

Osmometers (cryoscopic decrease or change in vapour tensions)

Quality criteriaOther tests

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