NZ620329B2 - Method for dissolution testing of solid compositions containing digestive enzymes - Google Patents
Method for dissolution testing of solid compositions containing digestive enzymes Download PDFInfo
- Publication number
- NZ620329B2 NZ620329B2 NZ620329A NZ62032912A NZ620329B2 NZ 620329 B2 NZ620329 B2 NZ 620329B2 NZ 620329 A NZ620329 A NZ 620329A NZ 62032912 A NZ62032912 A NZ 62032912A NZ 620329 B2 NZ620329 B2 NZ 620329B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- dissolution
- lipase
- medium
- pancrelipase
- dissolution medium
- Prior art date
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- PYGXAGIECVVIOZ-UHFFFAOYSA-N dibutyl decanedioate Chemical compound CCCCOC(=O)CCCCCCCCC(=O)OCCCC PYGXAGIECVVIOZ-UHFFFAOYSA-N 0.000 description 1
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- 230000029087 digestion Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
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- 238000001952 enzyme assay Methods 0.000 description 1
- 230000003628 erosive Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000019836 ficin Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000001087 glyceryl triacetate Substances 0.000 description 1
- 235000013773 glyceryl triacetate Nutrition 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229920000639 hydroxypropylmethylcellulose acetate succinate Polymers 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001771 impaired Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000005414 inactive ingredient Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000001264 neutralization Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 239000008029 phthalate plasticizer Substances 0.000 description 1
- 108010020708 plasmepsin Proteins 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000003918 potentiometric titration Methods 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 239000004208 shellac Substances 0.000 description 1
- 229940113147 shellac Drugs 0.000 description 1
- 235000013874 shellac Nutrition 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 230000001225 therapeutic Effects 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- QZCLKYGREBVARF-UHFFFAOYSA-N tributyl 2-acetyloxypropane-1,2,3-tricarboxylate Chemical compound CCCCOC(=O)CC(C(=O)OCCCC)(OC(C)=O)CC(=O)OCCCC QZCLKYGREBVARF-UHFFFAOYSA-N 0.000 description 1
- ZFOZVQLOBQUTQQ-UHFFFAOYSA-N tributyl 2-hydroxypropane-1,2,3-tricarboxylate Chemical compound CCCCOC(=O)CC(O)(C(=O)OCCCC)CC(=O)OCCCC ZFOZVQLOBQUTQQ-UHFFFAOYSA-N 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2545/00—Reactions characterised by their quantitative nature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
Abstract
Disclosed is a process for measuring an amount of digestive enzymes released from a composition in a dissolution medium comprising using fluorescence spectroscopy for measuring the amount of digestive enzymes released from the solid composition in the dissolution medium.
Description
METHOD FOR DISSOLUTION TESTING OF SOLID COMPOSITIONS CONTAINING DIGESTIVE
ENZYMES
Field of the Invention
The invention is directed to a process for measuring the amount of digestive enzymes
released from a solid composition in a dissolution medium by fluorescence spectroscopy. The
invention is also directed to a combined method for measuring both the dissolution and the
gastroresistance of a solid composition comprising pancrelipase.
Background of the Invention
A solid pharmaceutical composition or dosage form, such as a tablet or capsule, is generally
composed of a mixture of active ingredient(s) and excipient(s). The reproducibility of the
adsorption of an active ingredient (drug) from a solid composition form after oral administration
depends on several factors such as the release of the drug from the composition and the dissolution
or solubilization of the drug under physiological conditions. Because of the critical nature of the
release of the drug from the composition and the dissolution or solubilization of the drug, a
dissolution test is highly relevant to the prediction of the in-vivo performance of a drug. Drug
approving authorities such as the FDA and EMA often require pharmaceutical companies to
determine the drug release characteristics of any new pharmaceutical composition in order to obtain
approval. These tests can also be required as an USP quality parameter, to assess batch-to-batch
quality of a pharmaceutical composition, for accepting products, waiving bioequivalence
requirements or supporting requests for other bioequivalence requirements than the recommended.
Various protocols have been developed for conducting the in-vitro dissolution tests and are
routinely applied for both product development and quality control. Drug dissolution testing is
mostly conducted using recommended compendia methods and apparatus, such as the U.S.
Pharmacopoeia and the European Pharmacopoeia e.g. USP 34 <71 1> and EP 7.2, 2.9.3 .
Dissolution media typically used in such tests are for example water and buffers such as phosphate
buffers or citrate buffers. Different types of dissolution apparatus, based on different stirring
methods are available commercially and are recognized by the compendia methods. These
apparatus include: paddle, basket, flow-through, and reciprocating cylinder. While exact procedures
(protocols) and apparatus vary, all drug dissolution test methods involve placing the pharmaceutical
composition or dosage form into a dissolution medium and applying some stirring to the dissolution
medium in order to promote disintegration and dissolution of the drug under test.
The dissolution medium and the detection method for determining the amount of the
released drug in the dissolution medium depends upon (is chosen according) the chemical nature of
the drug, and physical and stability considerations are also of great importance in making the
appropriate choices.
The test contained in the pancrelipase Delayed Release Capsule, USP Monograph for the
determination of digestive enzymes release from pharmaceutical oral dosage forms, such as
pancrelipase delayed released capsules is based on the specific measurement of lipase activity. Such
method requires a long analysis time and is affected by several drawbacks. The main drawback is
the instability of the marker-lipase in the dissolution medium, more precisely in the enteric stage
buffer (pH 6.0) dissolution medium; the extent of lipase degradation needs to be established and a
correction factor is then introduced into the dissolution calculation to account for lipase activity loss
during the test. Furthermore, the complexity of the method (both in the reagent / substrates
preparation and the analytical determination) increases significantly the variability of the results and
worsens the intra/ inter laboratories results' reproducibility. Moreover, lipase assay has a narrow
linearity range (8-16 USP units /mL): this represents a significant limitation since the assay covers
only capsule strengths ranging between 6,400 and 12,800 USP UI /capsule and therefore the single
unit testing approach cannot be performed. The long analysis time in the current method limits the
possibility of using it for determining a multi-point dissolution profile.
There are no method /procedure describing how to overcome these drawbacks for
determining the release of digestive enzymes from a solid composition.
The digestive enzymes, such as pancrelipase and other pancreatic enzymes products (PEPs)
can be administered to patients suffering from exocrine pancreatic insufficiency (EPI); the
administration of digestive enzyme supplements allows patients to more effectively digest their
food.
Exocrine pancreatic insufficiency (EPI), of which the FDA estimates that more than 200,000
Americans suffer, involves a physiological disorder wherein individuals are incapabile of properly
digesting food due to a lack of digestive enzymes made by their pancreas. That loss of digestive
enzymes leads to disorders such as the maldigestion and malabsorption of nutrients, which lead to
malnutrition and other consequent undesirable physiological conditions associated therewith. These
disorders are common for those suffering from cystic fibrosis (CF) and other conditions
compromising the exocrine function of the pancreas, such as pancreatic cancer, pancreatectomy,
and pancreatitis. The malnutrition can be life threatening if left untreated, particularly in the case of
infants and CF patients, and the disorder can lead to impaired growth, a compromised immune
response, and shortened life expectancy.
Digestive enzymes, such as pancrelipase and other pancreatic enzymes products (PEPs), can
be administered to at least partially remedy EPI. The administered digestive enzymes provide for
patients to be able to more effectively digest their food.
Pancreatic enzymes, which have been used in the treatment of EPI to compensate for lost
digestive function, have been in use for more than 60 years. Their use until recently was not subject
modern regulatory guidelines governing drug approvals based on safety, and efficacy, and
manufacturing controls. Recently, pancreatic enzyme replacement therapies have become the
subject of US and European regulatory authority initiatives that require that marketed pancreatic
enzyme products to go through the current drug approval process in order to remain in commerce.
Zenpep®, Creon® and Pancreaze® are three products that successfully went through the process set
by the FDA and are approved for marketing in the United States. In other territories/countries
where similar initiatives are still proceeding or have not been implemented as yet, a variety of
pancreatic enzyme products are still available.
Capsules containing digestive enzymes such as pancrelipase have been developed for oral
administration. However, if a patient is unable to swallow the capsules, each capsule can be opened
and the contents sprinkled on a small amount of food, usually a soft, acidic food (such as
commercially available applesauce) and administered orally to the patient with a spoon.
Alternatively, such medications may be administered orally for infants and children, using a syringe
device containing the contents suspended in a medium amenable to administration thereby.
The pancrelipase products are generally labeled as containing three enzyme classes, lipase,
amylase, and protease, and the levels or potency of which are listed. These enzymes catalyze the
hydrolysis of fats into glycerol and fatty acids, starch into dextrin and sugars, and proteins into
amino acids and derived substances. Digestion is, however, a complex process involving many
other enzymes and substrates that contribute to correct digestive functioning and producing the full
range of digestive products. Other enzymes contained in pancrelipase include trypsin,
carboxypeptidases, elastases, phospholipases, and cholesterases amongst others and various co-
factors and coenzymes. These substances are produced naturally in the pancreas and also contribute
to correct digestive functioning.
Pancrelipase is typically prepared from porcine pancreatic glands, although other sources
can also be used, for example those described in U.S. 6,051,220, U.S. 2004/0057944, U.S.
2001/0046493, and WO2006044529, each of which is herein incorporated by reference in its
entirety for all purposes.
Pancreatic enzymes show optimal activity under near neutral and slightly alkaline
conditions. Under gastric conditions, pancreatic enzymes may be inactivated with a resulting loss in
biological activity. Therefore, exogenously administered enzymes are generally protected against
gastric inactivation and remain intact during their transit through the stomach and into the
duodenum. Therefore, it is desirable to coat pancreatic enzymes. Pancreatic lipases are the most
sensitive to gastric inactivation and are key enzymes in the treatment of malabsorption. Lipase
activity is typically monitored to determine the stability of an enzyme composition containing
lipase. The entire contents of U.S. 7,658,918 issued to Ortenzi et al. is expressly incorporated by
reference in its entirety herein for all purposes, and describes stable digestive enzyme compositions
and explains that certain particulate medications, administered orally, are designed to pass through
the stomach of the patient and thereafter to release within the intestines. The administration of a
proper dosage of such particulate medications to patients, particularly infants and children, should
be as accurate as possible.
Unfortunately, no process for measuring the amount of digestive enzymes released from a
solid pharmaceutical composition or dosage form with good precision and good sensitivity, and that
is ready to be implemented in different laboratories has been described.
Brief Summary of the Invention
The invention is directed to a process for measuring the amount of digestive enzymes
released from a solid composition in a dissolution medium by fluorescence spectroscopy. The
invention is also directed to a combined method for measuring both the dissolution and
gastroresistance of a solid compositions comprising pancrelipase.
Brief Description of the Figures
Figure 1. Dissolution profile of pancrelipase beads (Zenpep minitablets) - protease assay
(mean curve).
Figure 2. Dissolution profile of pancrelipase beads (Zenpep minitablets) - lipase assay
(mean curve).
Figure 3. Dissolution profile of pancrelipase beads (Zenpep minitablets) - total proteins
assay by fluorescence spectroscopy (mean curve).
Figure 4a. Dissolution profiles of pancrelipase beads (Zenpep minitablets): enzyme-
specific measurements vs fluorometric determination of total proteins content.
Figure 4b. Dissolution profiles of pancrelipase beads (Zenpep minitablets): lipase assay vs
fluorometric determination of total proteins content.
Figure 4c. Dissolution profiles of pancrelipase beads (Zenpep minitablets): protease assay
vs fluorometric determination of total proteins content.
Figure 5. Dissolution profile of pancrelipase compositions (Zenpep and Creon ).
Detailed Description of the Invention
The present invention is directed to a process for measuring the amount of digestive
enzymes released from a solid composition in a dissolution medium by fluorescence spectroscopy.
The amount is measured as % of digestive enzymes released from the solid composition or dosage
form or single unit form.
In another embodiment of the process of the invention, the solid composition is a
formulation comprising pancrelipase, more particularly it is an enteric coated pancrelipase
composition comprising pharmaceutically inactive excipients.
In another embodiment the process comprises the steps of: (a) allowing the solid
pancrelipase composition to release the digestive enzymes in a dissolution medium, (b) reading the
fluorescence to measure the amount of digestive enzymes in the medium.
In another embodiment of the invention the dissolution medium is water, HC1 solution,
simulated gastric fluid, buffer solution, simulated intestinal fluid, or aqueous or buffer solution
containing at least one surfactant.
In another embodiment the dissolution medium consists of at least two media that are
applied sequentially. A two staged dissolution test may also be carried out with the present process.
The first stage is an acid stage and the dissolution medium is an aqueous medium having acid pH,
such as pH ranging from about 1 to about 4.5, or from about 1 to about 2, or of about 1.2. The
second stage is performed in a second dissolution medium which is an aqueous buffer solution
having pH above 5, or between about 5.5 and about 6.8, or about 6.
In the method according to the invention the technique used for detecting the digestive
enzymes released from a composition in a dissolution medium is fluorescence spectroscopy.
Molecules have various states referred to as energy levels. Fluorescence spectroscopy is
primarily linked to electronic and vibrational states. Generally, the species being examined has a
ground electronic state, and an excited electronic state of higher energy. Within each of these
electronic states there are various vibrational states. In fluorescence spectroscopy the species is first
excited, by absorbing a photon, from its ground electronic state to one of the various vibrational
states in the excited electronic state. The molecule then drops down to one of the various vibrational
levels of the ground electronic state again, emitting a photon in the process. As molecules may drop
down into any of several vibrational levels in the ground state, the emitted photons will have
different energies, and thus frequencies. The fluorescence response of a protein is due to the
presence of the amino acids containing aromatic moiety (tryptophan, tyrosine, or phenylalanine).
Fluorescence response of a protein is generally obtained with an excitation wavelength of 280 nm.
Most of the fluorescence emissions in the proteins are due to excitation of tryptophan residues, with
a minor contribution of tyrosine and phenylalanine.
The fluorometric process herein disclosed is based on the measurement of total proteins
content of digestive enzymes (pancrelipase, API) released in a dissolution medium from a
composition or dosage form comprising said enzymes. The phrase "total proteins" used herein
identifies all the proteins that are released by the drug product, that is all the proteins present in the
starting solid composition such as lipases, proteases and amylases. In order to get a direct value of
the released API, the standard preferably used in this method is prepared with the same lot of the
tested drug product, but grinding and pouring it into the same dissolution medium to obtain the
100% API dissolved. Dissolution of the tested batch is measured as fraction percent towards the
standard preparation.
The process of the present invention can be applied to pancrelipase solid compositions that
may comprise pharmaceutically inactive excipients, such as any suitable oral dosage form that
contains digestive enzymes. Non-limiting examples of suitable dosage forms include tablets,
capsules, sachets or single units. In a particular embodiment, the dosage form is a capsule. Each
dosage form contains digestive enzyme beads (also called units) of API (drug). For the present
invention the digestive enzyme beads are any kind of particulates. The term "bead" includes
granule, particle, tablet, sphere, minitablet, microtablet, microparticle, microsphere,
minimicrosphere, microcapsule, and micropellet,. The bead may be any suitable particle size or
shape; particularly having a size range of about 50 to about 5,000 mih , more particularly they can
have a nominal (e.g., mean) particle diameter in the range of about 2 to about 5 mm, or of less than
about 2 mm, for example about 1-2 mm. "Minimicrosphere" have the smallest median size of 1.15
mm or "microtablet" have highest median size at 2.63 mm are also suitable for the present process.
The beads can have an average size of less than about 800 mih , preferably less than 500 mih ,
preferably of about 400 mih to about 600 mih or of about 250 mih to about 500 mih . These beads may
have a volume diameter (d(v,0.1) (defined as the diameter where 10% of the volume distribution is
below this value and 90% is above this value) of not less than 400 mih and a volume diameter
d(v,0.9), (defined as the diameter where 90%> of the volume distribution is below this value and
% is above this value) of not more than 900 mih .
All the digestive enzymes beads, more particularly pancrelipase enzyme beads, suitable for
the preparation of pharmaceutical products may be coated by an enteric layer. In embodiments
where pancrelipase cores are surrounded by an enteric coating the coating acts as a barrier,
protecting the drug from the acidic environment of the stomach and substantially prevents the
release of the medication before it reaches the small intestine. Suitable combinations of enteric
coating compositions with other coating compositions can be used to provide the desired type of
control over drug release or therapeutic effects. The enteric coating includes at least one enteric
polymer and further excipients. The phrase "enteric polymer" means a polymer that protects the
digestive enzymes from gastric contents, for example a polymer that is stable at acidic pH, but can
break down rapidly at higher pH or a polymer whose rate of hydration or erosion is slow enough to
ensure that contact of gastric contents with the digestive enzymes is relatively minor while it is in
the stomach, as opposed to the remainder of the gastro-intestinal tract. Non-limiting examples of
gastro-resistant polymers are cellulose acetate phthalate, hydroxypropylmethyl cellulosephthalate,
hydroxypropyl methylcellulose acetate succinate, polyvinylacetate phthalate, copolymers of
methacrylic acid, esters of methylmethacrylate, and shellac. These polymers are commercially
available with different brand names, such as: Cellacefate® (cellulose acetate phthalate), Eudragit®
L100, S100, L30D, FS30D, L100-55 (copolymers of methacrylic acid), Aquateric® (cellulose
acetate phthalate), Aqoat® (hydroxypropyl methylcelluloacetate succinate), HP55® (hydroxypropyl
methylcellulose phthalate). Preferably the enteric coating comprises: 10-20 wt. % of at least one
enteric polymer; wherein each said wt. % is based on the total weight of the coated particles. The
coating may further comprises a lipophilic agent, such as a C6-C30 lipophilic low molecular weight
molecule selected from the aliphatic carboxylic acids and alcohols, preferably a C14-C18
carboxylic acid or alcohol, such as stearic acid, myristic acid, myristic alcohol, or stearyl alcohol.
Other optional ingredients of the coating are plasticizers, anti-tacking agents (such as talc,
magnesium stearate, colloidal silicon dioxide and combinations thereof; further optionally a low
viscosity ethylcellulose). Non-limiting examples of suitable plasticizers include triacetin, tributyl
citrate, triethyl citrate, acetyl tri-n-butyl citrate, diethyl phthalate, dibutyl sebacate, polyethylene
glycol, polypropylene glycol, castor oil, acetylated mono- and di-glycerides, cetyl alcohol, myristil
alcohol, and mixtures thereof. The preferred plasticizer is a non-phthalate plasticizer or mixtures
thereof.
The coated stabilized digestive enzyme particles can then be formulated into capsules. A
particular dosage form of stabilized digestive enzyme particles is a capsule filled with enteric coated
pancrelipase enzymes beads. Capsules containing the enterically coated pancrelipase enzymes
comprised of hydroxypropylmethylcellulose having a water content of about 6 wt% or less are a
particular embodiment for a dosage for; more particularly having a water content of about 4 wt% or
less; further particularly having a water content of about 2 wt% or less .
The term "digestive enzyme" used herein denotes an enzyme in the alimentary tract which
breaks down the components of food so that they can be taken or absorbed by the organism. Non-
limiting examples of digestive enzymes include pancrelipase (also referred to as pancreatin), lipase,
co-lipase, trypsin, chymotrypsin, chymotrypsin B, pancreatopeptidase, carboxypeptidase A,
carboxypeptidase B, glycerol ester hydrolase, phospholipase, sterol ester hydrolase, elastase,
kininogenase, ribonuclease, deoxyribonuclease, a-amylase, papain, chymopapain, glutenase,
bromelain, ficin, b -amylase, cellulase, b -galactosidase, isomaltase, and mixtures thereof. They are
obtained through extraction from pancreas or pancreatic juices or produced artificially or obtained
from sources other than pancreas such as from microorganisms, bacteria, mold, fungi, plants or
other animal tissues, genetically modified microorganisms, fungi or plants.
The terms "pancrelipase" or "pancrelipase enzymes" or "pancreatin" denotes a mixture of
several types of enzymes, including amylase, lipase, and protease enzymes, or mixture thereof
having pancreatic origin. Pancrelipase is commercially available, for example from Nordmark
Arzneimittel GmbH, Scientific Protein Laboratories LLC or Sigma Aldrich; and similar extracts
from porcine, bovine or other mammalian sources may be used. Examples of commercial
pancrelipase formulations include Zenpep, Viokace, Ultrase, Creon, Pancreaze, and Panzytrat; more
particularly, Zenpep capsule for oral administration contains enteric coated beads (1.8-1.9 mm for
750, 3,000, 5,000 USP lipase units, 2.2-2.5 mm for 10,000, 15,000, 20,000, 25,000 and 40,000 USP
lipase units).
The term "lipase" denotes an enzyme that catalyzes the hydrolysis of lipids to glycerol and
simple fatty acids. Examples of lipases suitable for the present invention include, but are not limited
to animal lipase (e.g., porcine lipase), bacterial lipase (e.g., Pseudomonas lipase and/or
Burkholderia lipase), fungal lipase, plant lipase, recombinant lipase (e.g., produced via recombinant
DNA technology by a suitable host cell, selected from any one of microorganisms, bacteria, yeast,
fungi, plants, insects or mammalian host cells in culture, or recombinant lipases which include an
amino acid sequence that is homologous or substantially identical to a naturally occurring sequence,
lipases encoded by a nucleic acid that is homologous or substantially identical to a naturally
occurring lipase-encoding nucleic acid, etc.), synthetic lipase, chemically-modified lipase, and
mixtures thereof. The term "lipids" broadly includes naturally occurring molecules including fats,
waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides,
triglyceridses, phospholipids, etc.
The term "amylase" refers to glycoside hydrolase enzymes that break down starch, for
example a -amylases, b -amylases, g -amylases, acid a-glucosidases, salivary amylases such as
ptyalin, etc. Amylases suitable for use in the present invention include, but are not limited to animal
amylases, bacterial amylases, fungal amylases (e.g., Aspergillus amylase, for example, Aspergillus
oryzae amylase), plant amylases, recombinant amylases (e.g., produced via recombinant DNA
technology by a suitable host cell, selected from any one of microorganisms bacteria, yeast, fungi,
plants, insects or mammalian host cells in culture, or recombinant amylases which include an amino
acid sequence that is homologous or substantially identical to a naturally occurring sequence,
amylases encoded by a nucleic acid that is homologous or substantially identical to a naturally
occurring amylase-encoding nucleic acid, etc.), chemically modified amylases, and mixtures
thereof.
The term "protease" refers generally to enzymes (e.g., proteinases, peptidases, or proteolytic
enzymes) that break peptide bonds between amino acids of proteins. Proteases are generally
identified by their catalytic type, e.g., aspartic acid peptidases, cysteine (thiol) peptidases,
metallopeptidases, serine peptidases, threonine peptidases, alkaline or semi-alkaline proteases,
neutral and peptidases of unknown catalytic mechanism. Non-limiting examples of proteases
suitable for use in the present invention include serine proteases, threonine proteases, cysteine
proteases, aspartic acid proteases (e.g., plasmepsin) metalloproteases and glutamic acid proteases.
In addition, proteases suitable for use in the present invention include, but are not limited to animal
proteases, bacterial proteases, fungal proteases (e.g., an Aspergillus melleus protease), plant
proteases, recombinant proteases (e.g., produced via recombinant DNA technology by a suitable
host cell, selected from any one of bacteria, yeast, fungi, plant, insect or mammalian host cells in
culture, or recombinant proteases, which include an amino acid sequence that is homologous or
substantially identical to a naturally occurring sequence, proteases encoded by a nucleic acid that is
homologous or substantially identical to a naturally occurring protease-encoding nucleic acid, etc.),
chemically modified proteases, and mixtures thereof.
The pancrelipase enzymes of the compositions or oral dosage forms analyzed in the present
invention can include one or more lipases (i.e., one lipase, or two or more lipases), or one or more
amylases (i.e., one amylase, or two or more amylases), or one or more proteases (i.e., one protease,
or two or more proteases), as well as mixtures of these enzymes in different combinations and
ratios.
Lipase activities in the compositions or dosage forms to be analyzed by the process of the
present invention can be from about 650 to about 45,000 IU (USP method), from about 675 to about
825 IU, from about 2,700 to about 3,300 IU, from about 4,500 to about 5,500 IU, from about 9,000
to about 11,000 IU, from about 13,500 to about 16,500 IU, from about 18,000 to about 22,000 IU,
from about 22,500 to about 27,500 IU, from about 36,000 to about 44,000 IU and all ranges and
subranges there between. Lipase activities can be of about 750, about 3,000, about 4,200, about
,000, about 6,000, about 10,000, about 10,500, about 15,000, about 16,800, about 20,000, about
21,000, about 24,000, or about 25,000, or about 40,000 IU (USP method) or multiple thereof.
Amylase activities in the compositions or dosage forms can be from about 1,600 to about 6,575 IU
(USP method), from about 6,000 to about 225,000 IU, for example from about 6,400 to about
26,300 IU, from about 10,700 to about 43,800 IU, from about 21,500 to about 87,500 IU, from
about 32,100 to about 131,300 IU, from about 42,900 to about 175,000 IU, from about 53,600 to
about 218,700 IU and all ranges and subranges there between. Protease activities in the
compositions or dosage forms can be from about 1,250 to about 3,850 IU (USP method), from
about 5,000 to about 130,000 IU, for example from about 5,000 to about 15,400 IU, from about
8,400 to about 25,700 IU, from about 16,800 to about 51,300 IU, from about 25,000 to about
77,000 IU, from about 33,500 to about 102,600 IU, from about 41,800 IU to about 128,300 IU and
all ranges and subranges there between. Combined enzyme compositions include the following: (A)
the lipase activity can range from about 675 to about 825 IU, the amylase activity from about 1,600
to about 6,575 IU, and the protease activity from about 1,250 to about 3,850 IU (USP method); (B)
the lipase activity can range from about 2,700 to about 3,300 IU, the amylase activity from about
6,400 to about 26,300 IU, and the protease activity from about 5,000 to about 15,400 IU (USP
method); (C) the lipase activity can range from about 4,500 to about 5,500 IU, the amylase activity
from about 10,700 to about 43,800 IU, and the protease activity from about 8,400 to about 25,700
IU (USP method); (D) the lipase activity can range from about 9,000 to about 11,000 IU, the
amylase activity from about 21,500 to about 87,500 IU, and the protease activity from about 16,800
to about 51,300 IU (USP method); (E) the lipase activity from about 13,500 to about 16,500 IU, the
amylase activity from about 32,100 to about 131,300 IU, and the protease activity from about
,000 to about 77,000 IU (USP); (F) the lipase activity can range from about 18,000 to about
22,000 IU, the amylase activity from about 42,900 to about 175,000 IU, and the protease activity
from about 33,500 to about 102,600 IU (USP); and (G) the lipase activity can range from about
22,500 to about 27,500 IU, the amylase activity from about 53,600 to about 218,700 IU, and the
protease activity from about 41,800 IU to about 128,300 IU (USP). Also the lipase activity in the
compositions or doages forms to be analyzed by the process of the invention can range from about
,000 PhEur lipase units to about 40,000 PhEur lipase units, it may be about 5,000, or about 10,000,
or about 15,000 or about 20,000 or about 30,000 or about 40,000 PhEur lipase units.
In another embodiment of the present invention also single units containing a fraction of the
above listed amylase activities can also be analyzed with the present procedure.
In another embodiment of the present invention also single units containing a fraction of the
above listed amylase activities can also be analysed with the present process.
The ratio of amylase/lipase activities in the compositions or dosages forms can range from
about 1 to about 10, such as from about 2.38 to about 8.75 (enzymatic assay is performed according
to USP). This ratio can range from about 1 to about 8, such as from about 1.86 to about 5.13
(enzymatic assay is performed according to USP), or the ratio can be about 1, about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, or about 10.
The inactive ingredients of the product include croscarmellose sodium, hydrogenated castor
oil, colloidal silicon dioxide, microcrystalline cellulose, magnesium stearate, hypromellose
phthalate, talc, and triethyl citrate. Every dose of Aptalis Pharma's preparations provides patients
and physicians with a consistent amount of the main pancreatic enzymes lipase, protease, and
amylase due to their highly stable formulation. Capsules can be opened and the content split to
individually titrate the dose.
Another embodiment of the invention, the process comprises the steps of: (a) allowing the
solid pancrelipase composition to release the digestive enzymes in the dissolution medium, (b)
reading the fluorescence to detect the enzymes and measure the amount of digestive enzymes in the
medium. The dissolution test is carried out using the dissolution equipments described in the
compendia USP or EMA methods or using all these equipments and protocols that are known and
applied by the experts of the field. The dissolution medium is chosen among different solutions
suitable for the testing of total proteins dissolution such as water, HC1 solutions, simulated gastric
fluids, buffer solutions, simulated intestinal fluids, aqueous or buffer solutions containing
surfactants. Buffer solutions may be for example phosphate buffers or citrate buffers.
In a particular embodiment, the dissolution medium consists of at least two dissolution
media that are used sequentially (two stages). The first dissolution medium is an aqueous medium
has an acid pH between about 1 and 4.5, particularly between about 1 and about 2, more particularly
at a pH of aboutl .2 (acid stage) and the second dissolution medium is aqueous solution having pH
of above about 5.0, particularly between about 5.5 and about 6.8, more particularly at a pH of about
6 (buffer enteric stage).
In another embodiment of the invention the first dissolution medium is an aqueous medium
having pH between about 1 and about 4.5 and the second dissolution medium is aqueous buffer
solution having pH above about 5.
In another embodiment the first dissolution medium is an aqueous medium having pH
between about 1 and about 4.5 and the second dissolution medium is aqueous buffer solution
having pH between about 5.5 and about 6.8.
In another embodiment of the invention the first dissolution medium is an aqueous medium
having pH between about 1 and about 2 and the second dissolution medium is aqueous buffer
solution having pH above about 5.
In another embodiment of the invention the first dissolution medium is an aqueous medium
having pH between about 1 and about 2 and the second dissolution medium is aqueous buffer
solution having pH between about 5.5 and about 6.8.
In another embodiment of the invention the first dissolution medium is an aqueous medium
having pH of about 1.2 and the second dissolution medium is aqueous buffer solution having pH of
about 6.
In a further embodiment the process comprises the steps of a) adding the solid pancrelipase
composition in the first dissolution medium (acid stage), b) transferring the suspension to the
second dissolution medium (enteric stage), c) allowing the release of the digestive enzymes, d)
sampling aliquots of dissolution medium, e) reading fluorescence at 346 nm, d) calculating the
amount of digestive enzymes released. Calculation is performed as reported in the Examples.
When applying dissolution tests to a drug product, USP requires the calculation of "Q"
values. These Q values are correlated to the labeled potencies and the current acceptance criteria are
fixed as 75% of the lipase labeled activity.
The fluorometric process of the present invention, even though not specific for enzymatic
activity measurement, shows full correlation to enzymatic dissolution profiles, thus demonstrating
that the total proteins release is strictly correlated to the enzymes release. Therefore "Q" value can
be calculated for the present method in the following way: Q" value in the dissolution method with
fluorometric measurement:
Q = % dissolution x batch lipase assay (USP-U/cps)
labeled lipase Activity (USP-U/cps)
where: % dissolution is % of API released, calculated as indicated in the analytical procedure; batch
lipase assay is the batch lipase activity; labeled lipase activity: lipase activity indicated in the drug
product label.
While the fluorometric method disclosed herein showed full correlation with enzymatic
activity measurement while conducting the buffer enteric stage dissolution test of pancrelipase
compositions and is therefore proposed as method substituting the lipase activity enzymatic test,
this method cannot detect any gastroresistance problem occurring during the dissolution at the
acidic stage, since total proteins fluorometric measurement is not affected by acid permeation
through the membrane as it is lipase assay. The lipase activity is strongly decreased in case acidic
juice permeates through the protective membrane. The current USP test (lipase activity
measurement) cumulates at the end of the dissolution at the buffer enteric stage the effects of
potential weak gastro-resistance at the acidic stage with lipase dissolution and degradation
phenomena, occurring at the buffer enteric stage.
Therefore, another embodiment of the present invention is a process for measuring the
amount (%) of digestive enzymes released from a solid pancrelipase composition in dissolution
medium by fluorescence spectroscopy combined with a gastroresistance test, wherein the
gastroresistance is measured by determining the residual lipase activity of the product by the
specific lipase assay method, after acidic medium exposure (acid stage). In this embodiment the two
tests (dissolution FL test and gastoresistant GR test) can be carried out in each order.
FL test: the dissolution test performed in two stages (acid and enteric). In the first acid stage
dissolution medium is an aqueous medium having acid pH between about 1 and about 4.5,
preferably pH between about 1 and about 2, preferably a pH of about 1.2 (acid stage); in the second
enteric stage the dissolution medium is aqueous buffer solution having pH above about 5,
preferably pH between about 5.5 and about 6.8, preferably pH of about 6 (buffer enteric stage). The
gastro-resistance test is performed in aqueous medium having acid pH between about 1 and about
4.5, preferably pH between about 1 and about 2, preferably pH of about 1.2. The fluorometric test
measures the digestive enzymes (API or drug) released at the end of the enteric stage; for the
acceptance criteria, the Q value can be calculated by the ratio batch released lipase activity / labeled
lipase activity.
GR test: the gastroresistance test is performed under the conditions of the acid stage of the
dissolution test (the dissolution medium is an aqueous solution having acidic pH between about 1
and about 4.5, particularly at pH between about 1 and about 2, more particularly at a pH of about
1.2, where the % gastroresistance is measured by the determination of residual lipase activity of the
product, after exposure to the acidic medium, with the lipase assay method the acceptance criteria
will be those indicated for the acid stage of delayed-release dosage forms in USP.
From the foregoing description and the experimental part, it can be seen that the present
fluorimetric process of analysis provides several important advantages.
The invention provides a simple and fast procedure because the time required for reagents
preparation is significantly reduced and no specific analytical expertise in enzyme assay is required.
Hence, the analytical transfer is very easy. The proposed method is easier and faster to be
performed than the lipase assay and the time required for reagents preparation is significantly
reduced.
The marker (total proteins) is stable in the dissolution medium and therefore no correction
factor for compensation for degradation (as required in the lipase assay method) is needed in the
calculation. In fact, the marker assayed in the fluorometric measurement (total proteins) shows less
than 3% degradation after 30 min in enteric stage medium buffer pH 6, at 37°C; whereas the lipase
enzymatic activity measured with the current method shows about 11% degradation in the enteric
stage medium buffer pH 6, at 37°C.
The new method is also precise and has good sensitivity such that the quantitation limit
/linearity range is suitable also for single unit testing. The fluorometric method exhibits better
performance characteristics than the lipase enzymatic assay in terms of working concentration,
which is 0.3 lipase USP units 3 m g pancrelipase/mL; which is about 1/50 of the working
concentration of lipase assay; the linearity range is 10-200% of working concentration; the
precision is not more than 2,0% either for repeatability and intermediate precision (as measured in
the dissolution results of six different lots of pancrelipase formulations, Zenpep®).
Moreover, with such process a testing multi-point (>3) dissolution profile can be obtained.
It is also shown in the experimental part that the invented fluorometric non-specific
procedure of detection of total proteins content is equivalent, in terms of performance, to the two
enzyme-specific assays based on protease activity and on lipase activity (current compendia
method), on the basis of a the comparison of dissolution profiles obtained using the three
measurement methods.
It is to be understood that both the foregoing general description and the following detailed
description are exemplary, but are not restrictive, of the invention.
Examples
Equipments, materials and methods
Equipment: LS 50B Fluorescence Spectrometer (Perkin Elmer), LS 55 Fluorescence
Spectrometer (Perkin Elmer), Lambda 20 UV-VIS Spectrometer (Perkin Elmer), 786 Titrando
Potentiometric Titration System (Metrohm), VK-7025 dissolution bath (Vankel), Premier 5100
dissolution bath (Distek), USP Apparatus 1-basket (for acid stage); USP Apparatus 2- paddle (for
enteric stage).
Reagents for dissolution test. Acid stage medium (pH 1.2): Place 2.00 g of sodium chloride
in 800 mL purified water and stir until complete solubilization. Add 7 mL 37% HC1 and mix.
Adjust the pH of the solution to 1.20 ± 0.05 with 1 N HC1 or 1 N NaOH. Dilute to 1000 mL with
purified water; check the pH and adjust to 1.20 ± 0.05 with 1N HC1 or 1N NaOH, if needed.
Reagents for dissolution test. Enteric stage medium (pH 6.0): Place 9.20 g monobasic
potassium phosphate and 2.00 g sodium chloride in 800 mL purified water and stir until complete
solubilization. Adjust the pH of the solution to 6.00 ± 0.05 with 1 N NaOH. Dilute to 1000 mL with
purified water; check the pH and adjust to 6.00 ± 0.05 with 1N HC1 or 1N NaOH, if needed.
All examples are carried out using enterically coated pancrelipase beads, either pancrelipase
minitablets (MTs) or microtablets (MCTs), which are a blend of pancrelipase raw material and
excipients (e.g., croscarmellose sodium, hydrogenated castor oil, colloidal silicon dioxide,
microcrystalline cellulose, and magnesium stearate) coated with the enteric polymer hypromellose
phthalate (HP55); these MTs and MCTs are contained in HPMC capsules and are marketed under
the name Zenpep®. The skilled artisan will recognize that alternative enteric polymers and
excipients may be used in the enterically coated pancrelipase beads.
The measurement of lipolytic activity is carried out with a method based on the compendia
procedure of lipase assay described in the pancrelipase USP monograph, which is based on the
titration, by means of pH-stat method, of the free fatty acids formed from the hydrolysis of
esterified fatty acids in the substrate used (olive oil). It is based on the following principle: lipase
catalyses the hydrolysis of the triglycerides which leads to the formation of free fatty acids (FFA).
The titration of the formed FFA according to time provides for the determination of the enzymatic
activity of lipase, which can be expressed in units: 1 U = 1 miho ΐ of formed FFA per minute. The
reaction occurs by maintaining a steady pH value through an experimental system that provides for
the addition of NaOH (titrant) when the pH value changes compared to a fixed value (pHstat
method). The quantity of added titrant according to time corresponds to the quantity of FFA formed
by the lipase action on the triglycerides. Provided to work with a suitable quantity of substrate and
under experimental conditions where the enzyme is stable, a linear kinetics for the FFA formation
according to time can be obtained. The curve slope {added titrant = f (volume (mL)/ time
(minutes))} gives the lipase enzymatic activity.
The measurement of proteolytic activity is carried out according to the compendia procedure
described in the pancrelipase USP monograph.
Example 1 Preparation of standard solution of drug product
The standard solution is prepared with the same lot of drug product present in the dosage
form under analysis. An amount of pancrelipase beads (Zenpep® minitabs or microtabs) equivalent
to 7,000 USP lipase units, is accurately weighed, in a mortar. Add 5-6 mL of enteric stage medium
and grind until a complete dispersion of the product is obtained. The suspension is transferred into a
500 mL volumetric flask. The mortar is rinsed 2-3 times with few mL of enteric stage medium and
the liquid is transferred into the 500 mL volumetric flask. Enteric stage medium is added into the
volumetric flask up to final total 500 mL and the mixture is stirred for 10 minutes. The aliquots that
are sampled from the dissolution medium are further diluted 1:50 with enteric stage medium. With
this dilution the final concentration of API is about 0.3 USP lipase units (about 3 m g
pancrelipase/mL). This last dilution is carried out in the dissolution test - endpoint only and in the
dissolution test - multipoint (dissolution profile).
Example 2. Dissolution test
800 mL of the acid stage medium is added in each vessel of the dissolution bath equipped
with basket apparatus; the dissolution medium is equilibrated at 37°C. An amount of pancrelipase
beads (Zenpep minitabs or microtabs) equivalent to 11,200 USP lipase units (14 USP lipase
units/mL) is weighted and six independent samples are prepared in this way and placed in the
baskets. The apparatus is operated at 100 rpm. After 1 hour the baskets are removed from the
medium, rinsed with a few milliliters of water and the content of each basket is transferred in a
corresponding vessel containing 800 mL of the enteric stage medium at 37°C of the dissolution bath
equipped with paddle apparatus. The apparatus is operated at 100 rpm. After 30 min an aliquot of
the dissolution medium from each vessel is sampled for measuring the API released. In the
determination of the dissolution profile, 2.5 mL aliquots of the dissolution medium in the enteric
stage are sampled at 10, 12, 15, 18, 30 min; no medium replacement is done during the test, the loss
of volume being taken into account in the calculation by the correction factors below.
Table 1 Correction factors (FC) for the calculation of dissolution profile
Example 3. Determination of the digestive enzymes (API, active ingredient) released in the
dissolution test by fluorescence spectroscopy (total proteins assay)
Each aliquot of dissolution medium sampled from each basket as described in Example 2 is
diluted 1:50 with the enteric stage medium. The diluted solutions are read in the fluorescence
spectrometer with the following operative parameters: 1 cm pathlength quartz cuvette; excitation
wavelength: 280 nm; emission wavelength (measurement): 346 nm, excitation slit: 6.0. The target
concentration of the marker (API = pancrelipase; 100% released) at the endpoint: 0.3 USP lipase
units/mL or about 3 m g pancrelipase/mL; is obtained with the following calculation:
USP units in the vessel (% APIin DP formulation) , .
Lipase 0.72
^g/mg)
- — - x 1000
Potencyof DrugProduct(LipaseUSP U/mg) Dilution(ml)
The amount of released API is determined against a standard solution prepared with the
same lot of the drug product as described in Example 1.
For dissolution test- the endpoint the calculation is done with the following formula.
% API released =
For dissolution test - the multipoint (dissolution profile) the calculation is done with the
following formula:
EsMp X W s < VsMP c c
% A PIreleased= 100 FC
Wherein: E S is fluorescence reading (emission at 346 nm) of the sample, subtracted of the blank;
E S is fluorescence reading (emission at 346 nm) of the standard, subtracted of the blank; W S is
TD MP
the sample weight (mg); W S is the standard weight (mg); V S is the dilution volume of the
TD MP
sample (mL); V S is the dilution volume of the standard (mL); FC is the correction factor (see
Table 1).
USP units in the vessel (% APIin DP formulation) , .
Lipase 0.72
^g/mg)
- — - x 1000
Potencyof DrugProduct(LipaseUSP U/mg) Dilution(ml)
Example 4. Validation study of the total proteins assay by fluorescence spectroscopy
The performance characteristics of the fluorometric determination of total proteins content
in the API released from pancrelipase composition (Zenpep® formulation) in the dissolution test is
evaluated by the following parameters: specificity, linearity, accuracy, precision, quantitation limit,
stability of sample and standard solution, demonstration of the completeness of the extraction in the
preparation of standard solution and the results are summarized in Table 2.
Table 2. Validation data of fluorimetric method
With the validation data obtained, it is here shown that the proposed fluorometric method for
the determination of total proteins content in the dissolution test of pancrelipase dosage forms
(Zenpep® formulation) is suitable for the intended use.
Example 5. Dissolution profile of pancrelipase beads (Zenpep minitabs with three measurement
methods: total proteins content by fluorescence spectroscopy (non-specific assay), the proteolytic
activity by protease assay (enzyme specific assay), and the lipolytic activity by lipase assay
(enzyme specific assay)
The dissolution test is performed according to method described above (see Example 2), by
sampling 2.5 mL aliquots at: 10, 12, 15, 18 and 30 min. No medium replacement is done during the
test. The comparison of the three processes is performed according to the SUPAC approach
(Guidance for Industry "SUPAC MR: Modified release solid oral dosage forms. Scale-up and post-
approval changes: chemistry, manufacturing, and controls, in vitro dissolution testing, and in vivo
bioequivalence documentation" Center for Drug Evaluation and Research (CDER), September
1997) for the demonstration of similarity of dissolution profiles by means of f2 test; to generate the
required number of data for each of the three measurement methods twelve independent samples
(sample = amount of pancrelipase beads, Zenpep® minitabs equivalent to 11,200 UI) are analyzed,
in groups of three samples per run, four runs in total.
Example 5.1 Dissolution profile of pancrelipase beads (Zenpep minitablets) by protease assay
The individual dissolution values and overall average at each tested timepoint are
summarized in Table 3; the mean curve is shown in Figure 1.
Table 3. Dissolution profile data (protease assay)
Example 5.2. Dissolution profile of pancrelipase beads (Zenpep minitablets) by lipase assay
The individual dissolution values and overall average at each tested timepoint are
summarized in Table 4; the mean curve is shown in Figure 2. The correction factor of 1.125 is used
in the calculation to compensate the lipase degradation during the dissolution test.
Table 4. Dissolution profile data (lipase assay)
Example 5.3 Dissolution profile of pancrelipase beads (Zenpep minitablets) by fluorimetric
determination of total proteins content
The individual dissolution values and overall average at each tested timepoint are
summarized in Table 5; the mean curve is shown in Figure 3.
Table 5. Dissolution profile data (total proteins assay by fluorescence spectroscopy)
Example 5.4 Comparison of the dissolution profiles obtained with the three measurement methods
The average dissolution data of pancrelipase beads (Zenpep® minitablets), measured at each
timepoint with the three assay methods, are summarized in Table 6.
Table 6. Average dissolution data of pancrelipase beads (Zenpep minitablets) obtained with lipase
assay, protease assay, and fluorimetric determination of total proteins
% dissolution (average ± SD of n = 12 independent samples)
Test 10 min 12 min 15 min 18 min 30 min
11 ±4 CV =36% 44±8 CV =18% 103±4 CV =4%
3±2 CV =67% 75±7 CV =9%
protease assay
±2 CV =40% 13±4 CV = 31% 42±6 CV =14% 73±7 CV =10% 97±4 CV =4%
lipase assay
total proteins
assay by
±2 CV =40% 13 ±3 CV = 23% 45±6 CV =13% 74±5 CV =7% 96±2 CV =2%
fluorescence
spectroscopy
The three dissolution profiles show a nearly complete overlapping, as illustrated in the
graphical comparison of Figures 4a-c; in particular, lipase assay and fluorometric assay mean
curves are completely superimposable, while protease mean curve only differs at the endpoint
showing a value > 100%. Also the CVs of each series of data is quite similar among the three
measurement methods, with the lower values exhibited by the fluorometric assay.
The SUPAC approach used to evaluate the equivalence of the performance of a drug product
after and before changes (similarity test £2 on the dissolution profiles of the products to compare) is
used to show the equivalence of the new proposed method for the fluorometric determination of
total proteins in the dissolution test of Zenpep® formulation with the one currently used (lipase
assay) and with the other enzyme-specific measurement (protease assay).
To apply the similarity test £2 on the dissolution profiles, the FDA guidelines (Guidance for
Industry "SUPAC MR: Modified release solid oral dosage forms. Scale-up and postapproval
changes: chemistry, manufacturing, and controls, in vitro dissolution testing, and in vivo
bioequivalence documentation" Center for Drug Evaluation and Research (CDER), September
1997; Guidance for Industry - dissolution testing of Immediate Release Solid Oral Dosage Forms,
Center for Drug Evaluation and Research (CDER), August 1997) indicate some conditions that
should be fulfilled:
a. use the mean dissolution values (n = 12) from both curves at each time interval,
b. only one measurement should be considered beyond 85% dissolution point,
c. the average difference at any sampling time point should not be greater than 15%
between the dissolution profiles,
d. to allow use of mean data, the percent coefficient of variation at the earlier time
point should not be more than 20%, and at other time points should not be more than
%.
The requirements a, b and c are fulfilled in the present dissolution data; however, CV values
greater than those allowed (d) were observed at the first three timepoints (10, 12, 15 min) for all the
measurement methods evaluated. The observed high variability of these timepoints can be
explained with the very low concentration of the analyte at the first sampling time (10 min) and
with the intrinsic variability of the formulation in the narrow range of elapsed time for the further
timepoints at 12 and 15 minutes, taking into account that the 100% release of the formulation is
obtained in a very short time (30 minutes) in the dissolution test conditions.
Based on the above consideration, the f2 test is applied anyway, assuming that the observed
similar variability in the three measurement methods did not alter significantly the average curves
obtained, showing full overlap. A further evaluation is then made by taking into account the last
three timepoints (1530 min), in order to verify if the f2 test would pass even with the data
which fulfill completely the SUPAC requirements.
Example 5.4.1. Comparison: fluorimetric determination content vs lipase assay
The 2 test for the dissolution profiles of fluorimetric determination vs lipase assay
(reference), showed a similarity of 87.4% between the two curves when all the timepoints are
considered, while the similarity was 83.3% when the calculation is made on the last three
timepoints.
Example 5.4.2. Comparison: fluorimetric determination of total proteins content vs protease assay
The f2 test for the dissolution profiles of fluorimetric determination vs
protease assay (reference), showed a similarity of 72.3% between the two curves when all the
timepoints were considered, while the similarity was 68.6% when the calculation was made on the
last three timepoints. Generally, f2 values greater than 50 (50-100) ensure sameness or equivalence
of the two curves and, thus, of the performance of the test. According to the results obtained in the
comparison of dissolution profiles by means of f2 test is therefore possible to state that the
fluorometric determination of total proteins content is, in all respects, equivalent to the enzyme-
specific methods in the measurement of API release in dissolution test of pancrelipase formulations
(Zenpep® formulations).
Example 6. Comparison of validation data
To complete the comparison between the new fluorimetric test and the known accepted
lipase activity enzymatic assay (USP method) , Table 7 is included.
Table 7 Comparison of Validation Data
(a): target concentration for lipase assay = 12 lipase units/mL; (b): target concentration for FL
assay = 0.3 lipase units/mL (3 m g DS/mL); (c): 1 lot of DP, six independent samples/run; (d):
6 lots of DP, six independent samples/run for each lot
Example 7. Dissolution test by fluorimetric spectroscopy on pancrelipase beads.
The fluorimetric method as described in the above examples is carried out also with other
pancrelipase formulations present on the market with the name Creon® having different dosage
strengths. The results are compared with those obtained for pancrelipase beads marketed as
Zenpep® and are reported in Figure 5. The differences in the behavior are in agreement with the
different strengths of the formulations and the different surface areas of the particles of the two
formulations (Zenpep®, Creon®). Two beads sizes are used for different Zenpep® strengths, the
smaller ones (about 1.8 mm average diameter), showing faster dissolution profiles, are used to fill
,000 UI capsules, while the bigger ones (about 2.4 average diameter) are used to fill 20,000 UI
capsules. Creon® beads are about 1 mm average diameter, but significantly more irregular than
Zenpep®s beads, thus providing faster dissolution profiles than Zenpep®, but less reproducible
from batch to batch (all Creon® strengths use the same granules).
Example 8. Dissolution test combined with gastro-resistance test: FL-test plus GR- test
FL-test : dissolution test with fluorimetric measurement
800 mL of the acid stage medium are placed in each vessel of the dissolution bath equipped
with basket apparatus (USP Apparatus 1- basket); the dissolution medium is (acid stage)
equilibrated at 37°C. An amount of pancrelipase beads corresponding to 11,200 USP lipase units
(10 capsules of Zenpep® minitabs or microtabs) is weighed and transferred into each basket of
Apparatus 1. The apparatus is operated at 100 rpm. After 1 hour the baskets are removed from the
medium, rinsed with a few milliliters of water and the content of each basket is transferred into the
dissolution vessels containing 800 mL of the enteric stage medium at 37°C of the dissolution bath
equipped with paddle apparatus (USP Apparatus 2). The apparatus is operated at 100 rpm. After 30
min a 10-mL portion of the solution under test is removed, transferred to a test tube and equilibrated
at r.t. A further dilution with enteric stage medium is made to obtain the proper concentration (about
0.3 USP units/mL). The solutions are stored in ice/water bath and manually shaken before reading.
The diluted solutions are read in the fluorescence spectrometer with the following operative
parameters: 1-cm pathlength quartz cuvette; excitation wavelength:= 280 nm; emission wavelength
(measurement) = 346 nm; Excitation slit: = 6.0.
The target concentration of the marker (API = pancrelipase; 100% released) in the diluted
test solution is 0.3 USP lipase units/mL.
The standard solution is prepared as described in Example land is stored in ice/water bath
under stirring until reading.
The amount of release of the drug from the pancrelipase solid composition is calculated as
follows.
Calculations are carried out for bulk minitabs/microtabs.
.. . .. LCxPS x VC .. .
% dissolutio n = 100
LS x PC x VS
Calculations are carried out for capsules.
,. , . LCx PS x VC USx PM
% dissolution = x x
LS x PC x VS UL
Wherein: LC is fluorescence reading (emission at 346 nm) of the sample; LS fluorescence reading
(emission at 346 nm) of the standard; PS is standard weight (mg); PC is sample weight (mg); US is
potency of the standard (lipase USP units/mg, batch lipase assay); PM is mean weight of the
capsule content (mg/capsule); UL is labeled content of lipase in the individual dosage unit (USP
units/capsule); VC is dilution volume of the sample (mL); VS is dilution volume of the standard
(mL).
GR test : Gastroresistance test (with lipase assay)
800 mL of the acid medium are placed in each vessel (USP Apparatus 1 -Basket) of
the dissolution bath equipped with basket apparatus and then the dissolution medium is equilibrated
at 37°C. An amount of pancrelipase beads corresponding to 11,200 USP lipase units (10 capsules of
Zenpep® minitabs or microtabs) is weighed and transferred into each basket of Apparatus 1. The
apparatus is operated at 100 rpm. After 1 hour the baskets are removed from the medium and the
samples are rinsed by dipping briefly (for about 5 seconds) the baskets in a 1,000 mL beaker
containing 900 mL of cold purified water at 4°C, and this process is repeated three times, without
changing the rinsing medium. The content of each basket is quantitatively transferred to a ceramic
mortar, add 5-6 mL of cold purified water. The sample is ground until a complete dispersion of it is
obtained, quantitatively collected into a 200 mL volumetric flask and the mortar well is rinsed. This
operation is repeated 2-3 times. Purified water is added to final total volume. A further dilution with
cold purified water to obtain the proper concentration (about 14 USP units/mL, which is theoretical
max concentration available assuming 100% gastroresistance of the product) is done. The sample
solution is prepared immediately before the titration and is kept under stirring in an ice/water bath.
The standard solution is prepared as follows: an amount of USP Pancreatin lipase
RS, or pancrelipase working standard, corresponding to 6,000 USP Units is accurately weighted and
added into a ceramic mortar, about 5-6 mL of purified water are added and followed by grinding.
The liquid from the mortar is poured into a 500-mL volumetric flask and the mortar is also rinsed.
This operation is repeated 2-3 times. Purified water is added to total final volume. The standard
solution is prepared immediately before the titration and is kept under stirring in an ice/water bath.
The final concentration of the standard solution is about 12.0 USP Units lipase/mL.
For the lipase assay: 30 mL of the substrate emulsion (at 37° ± 0.1 °C) are poured in
the jacketed vessel of the titrator, and stirred by a magnetic stirrer, maintaining the temperature at
37° ± O. C. 0.1N NaOH is added to adjust the pH to 9.20-9.23 potentiometrically. 1.0 mL of the
sample or standard solution are added and followed by addition of 0.1N NaOH for 5 minutes to
maintain the pH at 9.0.
The calculations are done with the following formula:
AVMSxDS PC UC
where: VMC is volume of 0.1N NaOH consumed per min by the sample (mL/min); AVMS is
average volume of 0.1 N NaOH consumed per min by the standard (mL/min); PC is sample weight
(mg), PS is weight of the standard (mg); DC is dilution of the sample (mL); DS is dilution of
standard (mL); US is potency of the standard (lipase USP Units/mg); UC is batch lipase assay (USP
lipase units/mg).
The acceptance criteria according to USP, <71 1> dissolution - Delayed Release
forms (acid stage), Acceptance Table 3 are:
Level 1 acceptance criteria: GR% shall be greater or equal to 90% for each
individual unit;
Level 2 acceptance criteria: the average value of the GR of the 12 units (6 units in
level 1, 6 units in level 2) is not less than 90% and no individual unit is lower than
75%;
Level 3 acceptance criteria: the average value of the GR of the 24 units (6 units in
level 1, 6 units in level 2, 12 units in level 3) is not less than 90%>, and no individual
units is lower than 75%.
Based on all the above reported data and considerations, the fluorometric measurement of
total proteins content as marker of the digestive enzymes released in dissolution testing of
pancrelipase beads is a reliable and advantageous alternative to the current accepted compendia
measurement method based on the assay of lipase activity.
We
Claims (16)
1. A process for measuring an amount of digestive enzymes released from a composition in a dissolution medium comprising using fluorescence spectroscopy for measuring the amount of digestive enzymes released from the solid composition in the dissolution medium.
2. The process of claim 1, wherein the composition is a pancrelipase solid composition.
3. The process of claim 2, wherein the pancrelipase solid composition is an enteric coated pancrelipase composition.
4. The process of claim 1 or 2, wherein the dissolution medium is water, HCl solution, simulated gastric fluid, buffer solution, simulated intestinal fluid, aqueous or buffer solution containing at least one surfactant.
5. The process of claim 2, 3 or 4, comprising the steps of: a) allowing the solid pancrelipase composition to release the digestive enzymes in the dissolution medium, b) reading the fluorescence to measure the amount of digestive enzymes in the medium.
6. The process of claim 2, 3, 4, or 5, wherein the dissolution medium consists of at least two media that are applied sequentially.
7. The process of claim 6, comprising the steps of: a) adding the solid pancrelipase composition in a first dissolution medium, b) transferring the suspension in a second dissolution medium, c) allowing the release of the digestive enzymes, d) sampling aliquots of dissolution medium, e) reading fluorescence at 346 nm, f) calculating the amount of digestive enzymes released.
8. The process of claim 7, wherein the first dissolution medium is an aqueous medium having pH between about 1 and about 4.5 and the second dissolution medium is aqueous buffer solution having pH above about 5.
9. The process of claim 7 or 8, wherein the first dissolution medium is an aqueous medium having pH between about 1 and about 2. 206469NZ_claims_20151019_PLH
10. The process of claim 7, 8 or 9, wherein the second dissolution medium is aqueous buffer solution having pH between about 5.5 and about 6.8.
11. The process of claim 7, 8, 9 or 10, wherein the first dissolution medium is an aqueous medium having pH of about 1.2 and the second dissolution medium is aqueous buffer solution having pH of about 6.
12. The process of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein the pancrelipase compositions have a total of 750, 3,000, about 4,200, about 5,000, about 6,000, about 10,000, about 10,500, about 15,000, about 16,800, about 20,000, about 21,000, about 24,000, about 25,000, about 40,000 USP lipase units or multiple thereof, or about 5,000, about 10,000, about 15,000 about 20,000 about 30,000, about 40,000 PhEur lipase units or multiple thereof.
13. The process of claim 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 combined with a gastroresistance test, wherein the residual lipase activity of the composition in aqueous medium having pH between about 1 and about 4.5 is measured by the specific lipase assay method.
14. The process of claim 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 combined with a gastroresistance test, wherein the residual lipase activity of the composition in aqueous medium having pH between about 1 and about 2 is measured by the specific lipase assay method.
15. The process of claim 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 combined with a gastroresistance test, wherein the residual lipase activity of the composition in aqueous medium having pH of about 1.2 is measured by the specific lipase assay method.
16. The process of any one of claims 13 to 15, wherein the measuring of the amount of digestive enzymes released from the solid composition in dissolution medium by fluorescence spectroscopy is carried out either before or after the gastroresistance test. 206469NZ_claims_20151019_PLH
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161521227P | 2011-08-08 | 2011-08-08 | |
US61/521,227 | 2011-08-08 | ||
PCT/IB2012/054050 WO2013021359A1 (en) | 2011-08-08 | 2012-08-08 | Method for dissolution testing of solid compositions containing digestive enzymes |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ620329A NZ620329A (en) | 2015-11-27 |
NZ620329B2 true NZ620329B2 (en) | 2016-03-01 |
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