final report_andré riscado

University of Lisbon

Faculty of Pharmacy

Department of Galenic Pharmacy and Pharmaceutical Technology

University of Milan

Faculty of Pharmacy

Biopharmaceutics and Pharmaceutical Technology Laboratory

Professor Gazzaniga Research Group

Organic Acids in Hot Melt Extrusion: Improving the Rates of Dissolution of a pH

Dependent Weak Base in the Small Intestine

André Miguel de Sousa Riscado

Master in Pharmaceutical Sciences

ERASMUS Research Programme

2014

University of Milan

Faculty of Pharmacy

Department of Pharmaceutical Sciences

Biopharmaceutics and Pharmaceutical Technology Laboratory

Professor Gazzaniga Research Group

Organic Acids in Hot Melt Extrusion: Improving the Rates of Dissolution of a pH

Dependent Weak Base in the Small Intestine

André Miguel de Sousa Riscado

Supervisors: Dr. Matteo Cerea, PhD and Dr. Anastasia Foppoli, PhD

Co-supervisor: Professor João F. Pinto

Master in Pharmaceutical Sciences

ERASMUS Research Programme

2014

Organic Acids in Hot Melt Extrusion: Improving the Rates of Dissolution of a pH Dependent Weak Base in the Small Intestine

André Miguel de Sousa Riscado – ERASMUS Research Program ii

INDEX:

1. Acknowledgements 1

2. List of Abbreviations 2

3. List of Figures 4

4. Abstract 6

5. Introduction 9

5.1. Solubility and Bioavailability 9

5.2. Enhancing Solubility 9

5.3. Solid Dispersions 11

5.3.1. Techniques 11

5.3.2. Types of Solid Dispersions 11

5.3.3. Commercialised Medicines 12

5.4. Hot Melt Extrusion 12

5.4.1. Applications of Hot Melt Extrusion 14

5.4.2. Materials used in the Extrusion Process 14

5.4.3. Equipment and Process 16

5.5. Microenvironmental pH 18

5.5.1. pH Modifiers 19

5.6. Active Substance 21

6. Materials and Methods 22

6.1. Materials 22


André Miguel de Sousa Riscado – ERASMUS Research Program iii

6.2. Methods 22

6.2.1. Preliminary Tests 22

6.2.2. Dissolution Tests 23

6.2.3. Extrusion Process 24

6.2.4. pH Modification 25

6.2.5. DSC Analysis 26

6.2.6. Thermogravimetric Analysis 27

6.2.7. Evaluation of Possible Degradation 28

6.2.8. Assessment of Formulation B Dissolution Rates 29

7. Results and Discussion 30

7.1. Optimum wavelength 30

7.2. Particle Size 30

7.3. Hot Melt Extrusion of Binary Mixture 30

7.4. Dissolution Test of Binary Extrudates in SGF 31

7.5. Dissolution Test of Binary Extrudates and Physical Mixture in PBS 33

7.6. pH Modification 34

7.7. Hot Melt Extrusion with Citric Acid 35

7.8. Dissolution Test of the Formulations Containing Citric Acid 36

7.9. DSC Analysis of a Citric Acid Sample and Formulations A and B 40

7.10. Thermogravimetric Analysis 41

7.11. Hot Melt Extrusion with Fumaric Acid 44

7.12. Dissolution Test of the Formulations Containing Fumaric Acid 45

7.13. DSC Analysis of Fumaric Acid Sample and Formulation D 46


André Miguel de Sousa Riscado – ERASMUS Research Program iv

7.14. Evaluation of possible Degradation 48

7.15. Assessment of Formulation B Dissolution Rates 51

8. Conclusions 53

9. Future Work 54

10. Bibliography 55

11. Annex 58


André Miguel de Sousa Riscado – ERASMUS Research Program 1

1. AKNOWLEDGEMENTS

Throughout my personal and professional path and growth, I developed the

conceptualization of myself as a human being, my flaws and my virtues, my

weaknesses and my strengths, my wishes and dreams, my goals and my purposes. At

this moment, as I am about to graduate, I would like to leave a special thank you to all

the people that influenced me as a person, that touched me in a certain way, that

drove me through the journey that led me here. This said, I would like to emphasize

the wonderful persons that constitute my family, all the love, the support, the

motivation, the persistence, the patience, the help, I owe them everything.

Regarding my ERASMUS program, first of all a special appreciation to my

professor and co-supervisor João Pinto for arranging this opportunity and making it

work. Also a thank you note to Professor Andrea Gazzaniga that allowed me to work in

his laboratory and cooperate with his team.

To my tutors PhD Anastacia Foppoli and PhD Matteo Cerea, thank you for

always having the doors to your office opened, thank you for the kindness and

accommodating way that you received me and my colleague Marisa, thank you for the

help, the support, the advices, the expertise and for being excellent at what you do.

A sincere gratitude to my friend Andrea Murdocco, that was always available to

answer my questions and to share his knowledge, thank you for the excellent

recommendations.

To my good friend Marisa, whom I shared from the beginning this wonderful

adventure, thank you for always being there for me, those 3 months were perfect and

you were one of the main reasons.

I was very fortunate to meet all the persons I’ve met during my staying in Italy,

true friendships were built. To all of them a deep thank you and a “see you soon”.



2. LIST OF ABBREVIATIONS

AA

Adipic Acid

API Active Pharmaceutical Ingredient

CA Citric Acid

CA-MH Citric Acid Mono-Hydrated

CC Calibration Curve

DSC Differential Scanning Calorimetry

EC Ethyl Cellulose

FA Fumaric Acid

GI Gastro Intestinal

GMP Good Manufacturing Practices

GRAS Generally Recognized as Safe

HME Hot-Melt Extrusion

HPC Hydroxypropyl Cellulose



HPMC Hydroxypropylmethyl Cellulose

KVa64 Kollidon Va64

NCE New Chemical Entity

PBS Phosphate Buffer Solution

PEG Polyethylene glycol

PM Physical Mixture

RPM Rotations per Minute

SA Succinic Acid

SGF Simulated Gastric Fluid

TA Tartaric Acid

TGA Thermogravimetric Analysis

Tg Glass Transition Temperature

UV Ultraviolet

∆P Pressure Variation



3. LIST OF FIGURES

Figure 1 - Drug Release profile of Lex-B powder in Simulated Gastric Fluid medium.

Three vessels contain powder particles with a size-range of 180-250 µm and three

vessels contain unknown particle size powder.

Figure 2 – Comparison between Kollidon Va64 and Soluplus, as polymers for Hot-Melt

Extrusion, and its influence in the rates of dissolution of Lex-B.

Figure 3 – Comparison between Hot-Melt Extrudates and Physical Mixture of Lex-

B/Kva64. Purple: HME; Green: PM.

Figure 4 - Drug Release profile of HME Lex-B/KVa64 (Vessels 1-3) and PM Lex-B/KVa64

(Vessels 4-6) in Phosphate Buffer medium.

Figure 5 – Drug Release profile of Formulation A1 and B1 extrudates in PBS medium,

with 2 drops of Tween 20.

Figure 6 - Drug Release profile of Formulation C extrudates (vessels 1, 2 and 3) and of

Physical Mixture of Lex-B and Citric Acid (vessels 4, 5 and 6).

Figure 7 – Differential Scanning Calorimetry Analysis; Purple: Citric Acid Mono-

Hydrated (CA-MH); Red: Physical Mixture of Formulation A1; Black: Physical Mixture of

Formulation B1; Green: Extrudates of Formulation A1; Blue: Extrudates of Formulation

B1.

Figure 8 – Thermogravimetric Analysis of a Citric Acid Mono-Hydrated sample.

Temperature range from 25°C to 250°C, with a ramp of 10°C/minute.

Figure 9 – TGA of a Citric Acid sample. Temperature range from 25°C to 155°C, with a

ramp of 10°C/minute. Isothermal analysis at 155°C for 20 minutes.

Figure 10 – TGA of a sample of Fumaric Acid. Temperature range from 25°C to 250°C

with a ramp of 10°C/minute.



Figure 11 - Drug Release profile of extrudates of Formulation D (vessels 1, 2 and 3) and

of physical mixture of Formulation D (vessels 4 and 5) in PBS medium with 2 drops of

Tween 20, at 37°C.

Figure 12 - DSC Analysis to a FA sample (Black); HME Formulation D (Blue); PM

Formulation D (Red).

Figure 13 – DSC Analysis of Lex-B (Black); KVa64 (Red); PM of Lex-B/KVa64 (Green) and

HME of Lex-B/KVa64 (Blue).

Figure 14 – Scan performed in Methanol with a solution containing Lex-B.

Figure 15 – Calibration Curve of Lex-B in Methanol: y= 9.9399x – 0.0055; to obtain the

real title of Formulation B, C and D.

Figure 16 – Calibration Curve of a Solution containing Lex-B, Citric Acid and Kva64 in

Methanol: y= 9.5774 + 0.0098; to obtain the real title of Formulation B.

Figure 17 – Calibration Curve of Formulation B in Phosphate Buffer: y = 46.92x –

0.0088.

Figure 18 – Drug Release profile of Formulation B, using the data from the first

dissolution test but with a different Calibration Curve.



4. ABSTRACT

This report consists on the initial steps and studies of a more global work that

intends, as a final aim, to come up with a formulation that must allow an initial release

burst in the stomach and then a continuous release in the intestine, of a weak base

with a pH dependent solubility. This compound is highly insoluble (1.2 mg/L) in the

small intestine (pH circa 6.8). For confidential reasons, the active substance will be

denominated as Lex and some results can’t be published. The present work will only

focus on the pre-formulation studies made in order to enhance the drug solubility at

high pH and make its release pH independent.

For this purpose, the Hot-Melt Extrusion (HME) process, in which a solid

dispersion is formed, seemed more suitable and was successfully carried out. Two

polymers were tested as carriers and the best one (HPC polymer KVa64) was chosen.

The extrusion was performed in a counter rotating twin extruder at a

Temperature of 155°C and 30 rpm. The extrudates were characterised based on their

physical characteristics, drug release, crystallinity, and drug degradation.

Nonetheless, the dissolution rates were not sufficiently increased, especially in

high pH environments. Hence, conjugated with the HME process, pH modifiers,

particularly Citric Acid and Fumaric Acid, were added to the formulation in order to

increase the plasticization of the polymer and to create an in-situ micro-environment

with a favourable pH that would enhance the drug’s dissolution rates.

By the end of the process, it was possible to obtain homogeneous extrudates

that led to a considerable 50% drug release, in spite of showing stability problems.

In conclusion, the use of Organic Acids in HME proved to be successful in

respect to the increase of Lex-B dissolution rates. It has a synergist effect within a solid

dispersion formed through the HME process. However future studies need to discover

the optimal proportion between Organic Acid and Polymer and they also have to be

pointed towards solving the degradation issue that results in a drug loss of

approximately 5% after one week in storage.

Keywords: Solubility; pH-dependent; Hot-Melt Extrusion; Polymer; Acid



RESUMO

Este relatório consiste nas etapas iniciais de um estudo mais global que

pretende, como propósito final, formular um comprimido que permita uma libertação

imediata de uma porção da substância activa no estômago, seguida de uma libertação

prolongada desta mesma ao longo do intestino. A substância activa consiste numa

base fraca com uma solubilidade dependente do pH e que é altamente insolúvel (1,2

mg / L) no intestino delgado (pH cerca de 6,8). Por razões confidenciais, a substância

activa será denominada de Lex e certos resultados não poderão ser apresentados.

Neste relatório apenas serão abordadas as tentativas de aumentar as taxas de

dissolução do fármaco num pH elevado e de tornar a sua libertação pH dependente.

Para atingir tais resultados, procedeu-se a uma extrusão (Hot-Melt Extrusion)

de uma mistura binária entre polímero e substância activa. Dois polímeros foram

testados como transportadores, sendo escolhido aquele que conduziu a melhores

taxas de dissolução de Lex, tendo sido o polímero HPC (KVa64).

O processo de extrusão foi realizado num “counter rotating twin extruder” a

uma temperatura de 150°C e a 30 rpm. Os extrudidos foram caracterizados com base

nas suas características físicas, taxas de libertação do fármaco, estado cristalino dos

compostos e estabilidade do fármaco.

No entanto, os resultados não foram satisfatórios, nomeadamente a pH

elevado, e surgiu a necessidade de aumentar as taxas de dissolução de uma outra

maneira. Assim, conjugando com o processo de HME, procedeu-se à adição de

modificadores do pH à mistura binária. Ácidos orgânicos, tais como o ácido cítrico e o

ácido fumárico, para além aumentarem a plastificação do polímero, foram relatados

com possuidores dessa função de modulação, originando um microambiente com um

pH favorável que envolve a formulação e que permite uma melhor dissolução do

fármaco.

Os resultados mostraram que, através de um processo de extrusão de uma

mistura de Lex-B/Ácido Cítrico/KVa64 (10:15:75), foi possível obter extrudidos

homogéneos que permitiram taxas de dissolução de 50%, o que é uma quantidade

considerável. No entanto foi possível denotar problemas de estabilidade.

Concluindo, a utilização de ácidos orgânicos no processo de HME, provou ser

bem sucedida quanto ao aumento das taxas de dissolução de Lex. Existe um efeito

sinérgico ao incluirmos estes modeladores do pH no núcleo de uma dispersão sólida



formada pelo processo HME. No entanto, são necessários mais estudos por forma a

apurar-se a proporção ideal entre o ácido orgânico e o polímero. As atenções também

precisam de ser apontadas no sentido de resolver o problema de degradação, que

resulta numa perda de fármaco de aproximadamente 5%, após uma semana de

armazenamento.

Palavras-chave: solubilidade pH dependente; Hot-Melt Extrusion; Polímero; Ácido



5. INTRODUCTION

5.1. Solubility and Bioavailability

Solubility, the phenomenon of dissolution of a certain solute in a solvent, in

order to give a homogenous system, is one of the most important parameters in order

to achieve the desired concentration of a certain drug in systemic circulation. When

the oral route is chosen, the drug must be completely dissolved in the Gastro Intestinal

fluid in order to facilitate the drug’s absorption, allow better bioavailability and ensure

the effectiveness of the drug treatment. [1]

Low aqueous solubility and dissolution rates remain one of the most

challenging aspects and are a major problem encountered during pre-formulation

studies for the development of new chemical entities (NCEs) as well as for generic

development, since more than 40% of the NCEs developed by the pharmaceutical

industry are practically insoluble in water. [2]

This poses a problem because more and more often drugs are conceived and

formulated to be orally delivered due to the ease of administration, the high patient

compliance, reduced costs, easy production, good stability and less sterility

constraints. So, this poor bioavailability, due to poor aqueous solubility and low

dissolution rates must be overcome.

5.2. Enhancing Solubility

Proper selection of solubility enhancement method is the key to ensure the

goals of a good formulation like good oral bioavailability, reduced frequency of dosing,

better patient compliance and also low cost of production. Selection of the method for

solubility enhancement depends upon drug characteristics like solubility, chemical

nature, melting point, absorption site, physical nature, pharmacokinetic behaviour,

dosage form requirement like tablet or capsule formulation, strength, immediate, or

modified release and regulatory requirements like maximum daily dose of any

excipients and/or drug, excipients approval and analytical accuracy. [2]



Several techniques are being used to enhance the solubility and rates of

dissolution of poorly soluble drugs. They comprise physical and chemical modifications

and the selection of the best method depends on the drug’s properties, site of

absorption and required dosage form characteristics.

Regarding the physical modifications, particle size reduction (micronization p.e

usually accomplished by milling) is the most common procedure; as a particle becomes

smaller, the surface area to volume ratio increases and allows greater interaction with

the solvent, which causes an increase in the rates of dissolution. It is an efficient,

reproducible, and economic mean of solubility enhancement but it may induce drug

degradation.

Nanosuspensions, solid dispersions and crystal engineering are also always

taken in account. Indeed, considering the last one, metastable polymorphic

modifications and the amorphous state all have a higher free energy than the crystal

state and therefore, have a higher apparent solubility, so these forms should be used

in formulations of poorly water soluble compounds. The downside of such high energy

states, however, is their thermodynamic instability that may lead to their reconversion

into more stable physical states, affecting the solubility and dissolution rates. [3]

Concerning chemical modifications: change of pH, complexation (with

cyclodextrin p.e), salt formation and pro-drugs are common methods. [1]

The use of surfactant has also proven effective and it is considered a

miscellaneous method. Surfactants reduce surface tension and improve the dissolution

of lipophilic drugs in aqueous medium. Surfactant also improves wetting of solids and

increases the rate of disintegration of solid into finer particles. [2]

This report focuses on particle size reduction, use of surfactant, solid

dispersion, crystal engineering and change of pH.



5.3. Solid Dispersions

As for the solid dispersion technique, strategy includes complete removal of

drug’s crystallinity, and molecular dispersion of the poorly soluble compound in a

hydrophilic polymeric carrier [3]. It refers to a group of solid products consisting of at

least two different components, generally a hydrophilic matrix with a hydrophobic

drug completely and homogeneously dispersed. The increase in dissolution rate for

solid dispersions can be attributed to a number of factors, which include reduction in

particle size, absence of aggregation or agglomeration of fine crystallites of the drug,

the possible solubilisation effect in the polymer, improved wettability and

dispersability of the drug and transition of the drug’s crystalline state into its

amorphous form. [4]

The carrier’s molecular weight and composition, the drug crystallinity and the

particle porosity and wettability have to be taken in account during the formulation of

the solid dispersion, as they impact in the active pharmaceutical ingredient’s (API’s)

bioavailability increment, in the reduction of the side effects and in the extension of

the drug’s duration of action in the body. [5]

5.3.1. Techniques

To produce a solid dispersion it’s important to overcome the crystal lattice of

the drug, bringing it to a higher energy state – the amorphous state. Moreover, the

drug and the polymer need to be blended and co-dispersed. [6] Various techniques can

be used to obtain a solid dispersion such as: Hot-Melt Method; Solvent Evaporation

Method and Hot-Melt Extrusion [2], this last one being the chosen process in this

report.

5.3.2. Types of Solid Dispersions

Based on the API’s molecular state distributed in the carrier phase and based

on its thermal properties, such as melting point and glass transition temperature (Tg),

solid dispersions can be categorized as: crystalline solid suspensions, amorphous solid

dispersions and amorphous solid solutions. Crystalline solid dispersions are systems



wherein the crystalline drug substance is dispersed into an amorphous carrier matrix;

this type of dispersion is generally designed to achieve controlled drug release profiles

for highly soluble drugs. Amorphous solid dispersions occur when the melt extruded

drug/polymer is cooled at a rate that doesn’t allow the drug to recrystallize; it not only

offers the inherent free energy benefits of an amorphous system, but also provides

maximum specific surface area and higher saturation solubility, which ultimately

increases drug solubility. In amorphous solid solutions the drug is molecularly

dissolved in the polymeric carrier matrix and exhibits a single glass transition

temperature. [7]

5.3.3. Commercialized Medicines

Recently, some new solid dispersion formulations have entered the market

such as Kaletra (Abbott), Intelence (Tibotec), Certican (Novartis), Isoptin SR-E (Abbott),

Nivadil, Prograf (Fujisawa Pharmaceutical Co., Ltd) and Rezulin (Sankyo). All of these

new formulations utilize amorphous polymers as a carrier. [3]

5.4. Hot-Melt Extrusion (HME)

The extrusion process started to be used in the plastic industry in the mid-

nineteenth century. Since 1930, the industrial application of HME received

considerable attention from the pharmaceutical field with the aim of producing

different dosage forms such as tablets, capsules, films, and implants for oral,

transdermal and transmucosal routes. [8]

HME consists in the physical mixture of a hydrophobic drug, a hydrophilic

carrier and other excipients, [2] further heated until the molten state (needing no

solvents) while being intensively mixed (causing de-aggregation of the drug and

distributing it uniformly in the polymer matrix) inside a machine called extruder and

exiting under pressure, through a small die, into granules, cylinders or films. [9] [10]

This process occurs under controlled conditions that can be modulated and so

are called operational parameters, such as temperature, mixing (RPM or screw speed),



feed-rate, time of extrusion and pressure (Torque) being this last one directly

proportional to the melt viscosity (η) of the molten feedstock. Both screw speed and

feeding rate are related to shear stress and mean residence time which will affect the

dissolution rate and stability of the final products. [9] [11] The application of elevated

screw speeds is advantageous due to shorter residence times of the drug inside the

barrel hence a reduced exposure to high temperatures, and also in terms of process

output rates and efficiency.

This process generates enough energy by friction and heating to overcome the

crystal lattice and soften the polymer. [6] It is a very advantageous process since it is

very simple and effective and it doesn’t require the use of solvents, therefore reducing

possible stability problems and reducing the number of procedure steps, which is also

an economical value point. [9] [12] Compared to the traditional fusion method, this

technique offers the possibility of continuous production, which makes it suitable for

large-scale production. Furthermore, the product is easier to handle because at the

outlet of the extruder the shape can be adapted to the next processing step without

grinding. [2]

Just like in the traditional fusion process, miscibility of the drug and the matrix

can be a problem. High-shear forces, resulting in high local temperature and also the

programmed heating itself (as part of the procedure) in the extruder, are a problem for

heat sensitive materials [2] and can cause drug degradation, even with a very short

exposition inside of the extruder. It has other disadvantages, mainly due to the short

number of ideal polymers available, besides the difficult cleaning of the machine and

some good manufacturing practices (GMP) issues.

By the end of the extrusion, depending on the reologic characteristics and

desired dosage form, the extrudates can be milled and taken to a spheronizator, if the

purpose is to create pellets that are, after, conserved in plastic bags full of nitrogen. If

the goal is to make pills, we should freeze the extrudates and then use a hammer mill

to form a powder that is further compressed into pills.



This process generates a homogeneous solid solution or dispersion, with the

dissolution of the active substance in the inert matrix of the polymer (carrier) and also

presupposes the drug’s transition from its crystalline state to an amorphous one. All of

this leading to an improvement of the dissolution rates and bioavailability of the drug,

allowing a possible controlled release of the drug and avoiding the bad taste, masking

it and contributing to a higher patient compliance. However, it undertakes the risk of

formation of supersaturated solid solutions. [3]

5.4.1 Applications of Hot-Melt Extrusion

The number of patents and publications relevant to pharmaceutical

applications are increasing and to date, FDA has approved several hot-melt extruded

products including Rezulin®, KALETRA®, NORVIR® and ONMEL® and some medical

devices and implants like NuvaRing®, IMPLANON®, and OZURDEX® [13] [14]

5.4.2. Materials used in the extrusion process

Polymers or Carriers: Melting substances that can be processed at relatively not

very high temperatures due to the thermal sensitivity of many drugs (not very high

melting point); its characteristics such as glass transition temperature (Tg) and

hygroscopicity influence the processing conditions as well as control the active

substance release from the drug dosage form. Both active substance and polymer

need to be compatible and the polymer must not be toxic at all. [6] The most common

polymers used in HME include cellulosic polymers like hydroxypropyl cellulose (HPC),

ethyl cellulose (EC - Ethocel) and hydroxypropylmethyl cellulose (HPMC - Methocel). [9]

[13]

For example: HPC (Klucel) releases the active substance by diffusion and

erosion; it is a non-ionic hydrophilic polymer with dual solubility for aqueous and

organic solvents; the solubilisation of the compound on these polymers is dependent

on the size of its chains (the polymer’s); the release rate of the active substance is

controlled choosing the proper size of the chain of the polymer, the bigger chains



leading to a slower release of the active substance; the polymers with lower molecular

weight have lower viscosity and thus less Torque associated.

The polymers that, melted, have relatively low viscosity and a high thermic

conductivity, exhibit a more efficient solid dispersion. The melting point, the glass

transition temperature, the molecular weight and the viscosity of the polymer when

melted, must be studied and known.

In order to obtain high stability in the supersaturated solid dispersion the

carrier should have certain properties such as thermal stability and thermoplasticity,

water solubility, being inert and generally recognized as safe (GRAS). The presence of

functional groups that are either donors or acceptors for hydrogen bonds is an

additional benefit, since specific interactions increase the solid solubility of the drug

into its carrier and also seem to play an important role in inhibiting phase separation

and crystallization of the drug from a glass solution. [3]

Functional Excipients: They modulate the API drug release by changing the

porosity or tortuosity of the drug release form. Examples are the viscosity agents

incorporated in the polymeric matrixes (in order to reduce the initial burst for

example); citric acid, lactose, sodium bicarbonate, microcrystalline cellulose, starch

and manitol (this last one increases the dissolution rate of the API because it leads to a

bigger porosity due to the formation of micro pores).

Plasticizers: Low molecular weight substances that generate more flexible

polymers due to inter-molecular strengths between the polymer and the plasticizer

that reduce the glass transition temperature and lower the viscosity, increasing the

free volume between the polymeric chains. The use of plasticizers allows us to work

with more ideal operational parameters such as lower temperature and lesser Torque

as well as a higher number of rotations per minute (by lowering the shear forces

needed to extrude the polymer) [12] [13]. As expected, they have to be compatible and

stable with the polymer. Examples are: Triacetim, Citrate esters, PEG, Surfactants,

Mineral Oil, Citric Acid and Vitamin E.



5.4.3 Equipment and Process

Focusing now on the extrusion machine and the technology associated, there

are two sorts of methods:

- RAM (Piston) Extrusion, based on the principle of high positive pressures and

used in high value materials due to its high precision, although it has the inconvenient

of limited fusion capacity.

- The Screw Extrusion is based on shear stress and intense mixture. If instead of

one, there are present two screws, the machine is denominated as Twin Extruder. In

this last case, the screws can additionally be Co-Rotating (same direction) or Counter-

Rotating (opposite direction). [9] We have been focusing on the screw extrusion and will

continue to since it is the technology used in this study.

The equipment used in HME comprises an extruder, auxiliary equipment,

downstream processing equipment and other monitoring tools such as temperature

gauges, screw-speed controller, an extrusion torque monitor and pressure gauges used

for evaluation of performance and product quality. [13] The extruder is usually

composed of a feeding hopper (gravimetric or volumetric feeding), temperature

controlled-barrels, single or twin screws and the die. [6] [8] [9] Additional systems include

mass flow feeders to accurately introduce materials into the feed hopper, process

analytical technology to measure extrudates properties (spectroscopic systems), liquid

and solid side stuffers, vacuum pumps to degassing extrudates, pelletizers and

calendaring equipment. [6] [9] All the material used in the extruder equipment is made

of stainless steel and should be resistant to abrasion, corrosion and adhesion. [13] See

annex 1.

The extruder includes barrels enclosing single or twin screws which transports

and force the melt through a die, giving it a particular shape. [6] The one or two rotating

screws (co-rotating or counter rotating) are inside a stationary cylindrical barrel that is

manufactured in sections to reduce the blend residence time. These sectioned parts

are then bolted or clamped together and the temperatures are controlled by electrical

heating bands and monitored by thermocouples. [7]



The screws have three discrete zones: feed zone, melting or compression and a

metering zone. The pressure within the feed zone is low in order to allow the feeding

from the hopper and to gentle mix the API with the polymer and other excipients. [8] [9]

When the mixture reaches the compression zone, the pressure starts gradually

increasing along the length, removing the entrapped air and allowing us to get a

homogeneous extrudate [8] [9]. In the last zone there is a stabilization of the

effervescent flow of the matrix which ensures the extruded product has a uniform

thickness, shape, and size. [8]

According to the type and complexity of the process, the screws should rotate

at a selected predetermined speed to compensate the torque and shear generated

from both the material being extruded and the screws being used. [8] Different

parameters should be considered to characterize the screws. The common one is the

length/diameter (L/D) ratio, which typically ranges from 20 to 40:1. Typical pilot plant

extruders have diameters ranging 18–30 mm, whereas production machines are much

larger with diameters typically exceeding 50 mm. [7] Other parameters are residence

time, self-wiping screw profile, minimum inventory and versatility. [7]

The Twin Screw Extrusion process, used in this study, has plenty of advantages

over the Single Screw Extrusion such as easier material filling of the machine, better

dispersion of the API in the carrier and faster process with less overheating and

residence time, being for these reasons, the first choice when preparing drug

dispersions for solubility enhancement. [15] Nevertheless, “it is mechanically complex

and very expensive.” (Repka et al.2002)

Comparing Co and Counter-Rotating Screws, the last one (which is the one used

during these experiments) allows higher shear strengths and better dispersion of the

particles, as they subject materials to very high shear forces while the material is

squeezed through the gap between the two screws as they come together. [9]

However, air retention (visible on the extrudates) occurs more often. It also has a more

reduced output, a more limited rotating speed, a difficult cleaning and suffers bigger



wearing (screws and barrel), comparing to Co-Rotating Screws. Summing up, Co-

rotating twin screws are industrially the most important extruder types. [9]

We can further classify the process as: non-intermeshing or fully intermeshing

extrusion, being the first one used when there are high quantities of volatile

compounds to be processed (allows large volume de-volatization via a vent opening).

Non-intermeshing co-rotating twin extruders are used when highly viscous materials

need to be quickly removed from the machine. The second one is “the most popular”

(Thiele, 2003), it prevents localized material overheating within the extruder (since it

minimizes the nonmotion), and is also self-wiping. The material doesn’t rotate along

the screw (first in/first out principle). The most common type of extruder usually used

is the co-rotating intermeshing twin extruder. [9]

5.5. Microenvironmental pH

In clinical practice, conditions in the GI tract of patients have an impact on the

oral absorption of drugs, and the intraluminal pH varies widely from highly acidic in the

stomach to around pH6 in the small intestine. In addition, the secretion of gastric acid

and the gastric emptying time have been shown to be influenced by various factors,

such as age, food, disease and even medicines. [1] Weakly ionizable acidic and basic

compounds exhibit pH dependent solubility changing their solubility as a function of

the surrounding pH; [16] usually they have low oral absorption with high variability

depending on the patient’s condition. [1]

Examples of such drugs include verapamil hydrochloride, papaverine

hydrochloride, dipyridamole and trimethoprim. [16]

Microenvironmental pH can be described as the pH of the saturated solution in

the immediate vicinity of the drug particles. [16] The pH-modification approach involves

altering the microenvironmental pH in the diffusion area by dissolving acidic or basic

pH-modifier excipients in the formulation to create a favorable microenvironmental

state, [1] thus bringing the pH inside the formulation to a value where drug solubility is

higher, increasing the drug’s dissolution. [16] PH-Modulation at the diffusion layer can



remarkably increase the saturation solubility of the drug at the solid interface, leading

to increased drug dissolution. [1] See annex 2.

This strategy is intended to be applied to conventional tablet and capsule

dosage forms, which are commercially still the most widely used dosage form. [16]

Modulating the level as well as the duration of a suitable pH value in the

microenvironmental area constitutes a key factor for the improvement of drug

dissolution and it depends on the physicochemical properties of the pH-modifier,

specifically strength and solubility. So, the relative effectiveness of pH modifiers can be

evaluated by the measurement of microenvironmental pH; the measurement of the

residence time of the pH-modifier in the formulation and also its release; and finally

the measurement of the improvement in drug dissolution. [16]

Several methods are used for measuring the microenvironmental pH, such as

indicator dyes, that change color with a change in pH. These dyes can be incorporated

into the formulation. For example, thymol blue, which is red at pH < 2.8 and yellow at

pH > 2.8, was used by Varma et al. for measuring the acidity of oxybutynin matrix

tablets containing FA. A surface pH electrode is also an available and used technique.

[16]

5.5.1. pH Modifiers

This pH modulation can be achieved using pH modifiers, which can be organic

acids (widely used to modulate the release of weak basic drugs), alkalizing agents

(used in case of weak acid drugs) and also acidic polymers (compared to organic acids,

enteric polymers have the added advantage of slower release from the matrix due to

their lower solubility and higher molecular weight). [17]

Several organic acids have often been used as acidifiers, to modulate the

release of weak basic drugs, which is this report’s case. Citric Acid (CA), Fumaric Acid

(FA), Succinic Acid (SA) and Tartaric Acid (TA) are some examples. [1] In acidic media,

the organic acid serves as inert filler and the drug diffuses out owing to the acid’s

inherent high solubility. However, in alkaline pH, organic acids dissolve to decrease the



pH of the microenvironment and create an acidic drift in the direct vicinity of the drug.

This increases the solubility of the drug, thus leading to a higher concentration

gradient and a higher driving force for diffusion. [16]

The pH-modifier should be selected and its amount optimized on the basis of its

characteristics and compatibility with the drug substance and designed formulation [1]

(influence on the polymer viscosity and effect on the overall osmotic pressure [16]). The

concentration of pH-modifiers in formulation impacts not only on drug dissolution but

also on its manufacturability and stability. A large amount of pH-modifier could lead to

high drug dissolution, but could also induce poor manufacturability or poor stability in

some cases. [1]

One of the characteristics of the pH-modifiers is that they shouldn’t dissolve so

quickly from the matrix tablet and should remain in sufficient quantity until the drug

has been completely released and solubilized. [1] In fact, one of the limiting factors in

enhancing the drug solubility by using pH-modifiers is the aqueous solubility of the

acids themselves. [18]

Ideally, organic acids should have increased acid strength (low pKa) and

relatively low solubility in the lower pH range so that they have greater residence time

in the matrix and can provide low pH in it for longer periods. [16] However, to much low

solubility could be also prejudicial, not allowing the achievement of the intended pH,

so in conclusion, there is an optimal solubility that allows reaching the desired pH but

without diffusing too quickly. [17]

Some molecular interactions among the functional groups of drug and pH-

modifier that could be Van der Waal’s type or hydrogen bonding could greatly

contribute for the supersolubilization [19] and prevention of the drug precipitation after

drug dissolution as well as enhancement of its dissolution. [1]

The limitations of the current methodology and the inherent difficulties

associated with the heterogeneity of a solid system make modulation of the

microenvironmental pH an empirical endeavor. [17]



5.6. Active Substance

Lex exists in the form of a hydrochloride salt or as a base. This last one will be

the one used in the studies that will follow, thus starting to be named as Lex-B. Its

melting temperature is around 118-120°C and, opposing Lex-Salt, it is soluble in DMSO,

Chloroform, Methanol (237.5 mg/ml) and in Simulated Gastric Fluid (SGF) (circa 148

mg/L). It is very poorly soluble in Water (0.1 mg/ml) and in Phosphate Buffer (PBS)

(circa 1 mg/L). It is also referred to be very unstable with light.



6. MATERIALS AND METHODS

6.1. Materials

For confidential reasons, it will not be provided any info on Lex-B.

Soluplus was purchased to BASF and the batch used is 08358475L0.

HPC Kollidon Va64 was purchased to BASF and the batch number is

70299536W0.

Citric Acid was acquired to ACEF and the batch that the laboratory possessed

was the number H0219009.

Fumaric Acid was attained at Develo Pharma with the batch number

5C314155G.

Tween 20 was obtained from CRODA and the batch that was used during

studies was 2503PP3990.

6.2. Methods

6.2.1 Preliminary Tests

In order to discover the optimum wavelength in which Lex-B absorbs in water,

Simulated Gastric Fluid (SGF) and Phosphate Buffer (PBS), scans were performed,

dissolving a small portion of Lex-B in the different mediums and obtaining readings of

absorbance with different wavelengths (of a manually set range of values) using UV

analysis (Perkin-Elmer, Lambda 25, UV/VIS Spectrometer) with quartz cells of 1cm.

Weighings were performed in a balance (Mettler PC440, Delta Range®).

To do the Calibration Curve in PBS, 25.02 mg of Lex-B were weighted in a

balance (Mettler PC440, Delta Range®) into 50 ml of Methanol (0.5004 mg/ml) being

this the mother solution, from which dilutions of 1:100 (0.005004 mg/ml); 1:200

(0.002502 mg/ml) and 1:2000 (0.0002502 mg/ml) were made in PBS. This generated 3

points of the Curve, which graphic and equation are visible in annex 3. As for the



Calibration Curve in SGF, the mother solution consisted in 25.5 mg of Lex-B to which 50

ml of Methanol were added. From this solution, dilutions in SGF were made in order to

obtain 6 points with concentrations ranging from 0.00255 mg/ml to 0.0306 mg/ml. The

graphic and equation are also presented in annex 4. Readings were obtained through

UV analysis (Perkin-Elmer, Lambda 25, UV/VIS Spectrometer) at 242nm, using quartz

cells of 1cm.

6.2.2. Dissolution Tests

To evaluate the particle size effect, a dissolution test in 1000 ml of SGF

medium, with 2 drops of Tween 20 (approximately 27 mg) to allow the particles to be

completely wet, was carried out for 6 hours. Six vessels, with paddles rotating at 100

rpm (Distek, Dissolution System 2100B, North Brunswick, NJ) as indicated by the

European Pharmacopeia, 7th Edition, paddle method (Eur.Ph.2.9.3) in sink conditions

at 37°C were used and samples were taken at specific programmed times using

(through an online method) a pump (IPC, ISMATEC, Switzerland) that fills the

spectrophotometer cell’s volume for reading and replaces the same volume in the

medium almost instantaneously. Each vessel contained circa 14 mg of Lex-B. Vessels 1,

2 and 3 contained Lex-B in the size-range of 180-250 µm and the other 3 had unknown

particle size powder of Lex-B.

As for the binary extrudates, the dissolution was conducted for 3 hours exactly

in the same sink conditions as the previous one, with 1000 ml SGF as medium. Three

vessels were used for Lex-B/KVa64 binary extrudates and the other three for Lex-

B/Soluplus binary extrudates and each vessel contained 140 mg of sample in the form

of a powder with 180-250 µm, which corresponds to 14 mg of Lex-B.

To compare the Lex-B/KVa64 binary extrudates with its physical mixture, it was

executed another dissolution test with the same processing conditions as the previous

one. It was added circa 140 mg of extrudates to 3 vessels and circa 140 mg of physical

mixture powder to the other 3 vessels containing 1000 ml SGF. The test went on for 3

hours. Because the powder would most certainly either float, either aggregate, it was



decided that 2 drops of Tween 20 should be added. This exact same test was also

carried out in PBS with 2 drops of Tween 20 and went on for 5 hours.

Concerning the dissolution tests on the extrudates of the formulations

containing Citric Acid, the conditions were again the same as the previous tests. In one

of the tests, formulations A and B were evaluated in 1000 ml PBS with 2 drops of

Tween 20, for 3 hours. Six vessels were used, each with circa 140 mg of extrudates (3

for each formulation) in the form of powder with 180-250 µm.

In the other test, three vessels with circa 140 mg each of Formulation C

extrudates and three vessels with 56 mg each of physical mixture of Citric Acid and

Lex-B (3:1) were used.

The dissolution tests on the formulations containing Fumaric Acid were

performed in PBS with 2 drops of Tween 20, with samples being collected for three

and a half hours and in the same conditions as the previous dissolution tests. Three

vessels had approximately 140 mg of extrudates in the form of a powder with 180-

250µm and the other 2 vessels contained 140 mg of the physical mixture.

6.2.3. Extrusion Process

All the extrusions were conducted in a fully intermeshing counter-rotating twin-

screw extruder (HAAKE Minilab II, Thermo Scientific), visible in annex 5. The HAAKE

Minilab extruder was developed for the compounding of small volume samples. It’s

useful for research, test different materials and to develop new formulations in a small

scale. The processing conditions were the following: Temperature of 155°C and 30

rpm. After ensuring that the machine was clean, by making a first extrusion using only

the polymer that will act as a carrier, followed the addition of the physical mixture

previously prepared from the materials that were conserved in an oven (Reciterm,

ISCO; Italy) for 24 hours at 40°C. The feeding started when all the working parameters

were at the set values and was performed in a continuous way, to prevent air entrance

and it was stopped when the material started to flush throughout the die. At this

point, the cycle function was activated and, 5 minutes after significant different



pressures are detected in the pressure sensors, the flush function was activated again

and the material was collected.

Regarding the extrusion of the binary mixture, either with KVa64 or with

Soluplus, the proportion of API and Polymer was 1:9, respectively (justified regarding

an experience with Lex-Salt and PEG 6000 in which the proportion 1:10 showed best

results [4]) and for each polymer, a total amount of 20 grams of physical mixture was

prepared.

The formulations containing Citric Acid experienced the exact same extrusion

conditions. As for the use of Fumaric Acid, a mixture of Lex-B/Fumaric Acid/KVa64

(1:2:7), was formulated and named Formulation D, that was further extruded in the

same previous stated processing conditions.

6.2.4. pH Modification

Because the rates of dissolution of Lex-B/KVa64 in PBS were very poor, the

studies proceeded with the addition of CA. In order to know how to incorporate Citric

Acid Mono-Hydrate in our formulation, the literature regarding the “Use of Citric Acid

in oral dosage forms to improve the release rates of Diltiazem Hydrochloride from a

Eudragit RS matrix by HME” was accessed. According to this source, the best ratio

between drug and polymer that led to best dissolution rates was 1:4, respectively. The

amount of Citric Acid used should be of circa 10-30%, in order to achieve best

processing conditions of HME, more specifically reduction of the T°C of extrusion and

possibility of increasing the rotation speed of the screws without raising the

temperature.

Regarding the dissolution rates, and maintaining the polymer at 60%, using

API:CA at 3:1, 2:2 and 1:3, resulted in drug releasing of 76.5%, 83,09% and 83,38%,

respectively and comparing with 54,54% using just API and Polymer, after 12 hours. To

sum up, the optimum formulation was 2:2:6 Active Substance, Citric Acid and Polymer,

respectively. 30% of Citric Acid doesn´t lead to significant improvements comparing to



20%, possibly because of its limited solubility in the polymer, thus the effect of

plasticization and drug dispersion is not improved. [20]

Although different active substance and different polymer, and without any

more data, these data were the starting point to perform the following formulations:

Formulation A: Lex-B: Citric Acid: Polymer (10:30:60)

Formulation B: Lex-B: Citric Acid: Polymer (10:15:75)

Formulation C: Lex-B: Citric Acid: Polymer (10:7.5:82.5)

6.2.5. DSC Analysis

Characterization of the extrudates included Thermogravimetric Analysis and

Differential Scanning Calorimetry.

Considering the DSC Analysis, it is commonly used to detect phase

transformations including melting, miscibility, glass transitions and re-crystallization of

melt extrudates [21]. In this case it evaluates the effect of the solid dispersion on Lex-B.

It’s a thermal analysis technique that measures enthalpy changes in samples due

to changes in their chemical or physical properties as a function of temperature and

time [18]. It consists in two cells, one reference cell and another sample cell. Associated

to these cells are two electrodes transmitting between them a constant electric

potential.

The machine, which is DSC-1 Stare System (Mettler Toledo, Switzerland)

connected to the Star software in the computer, induces the cell heating (both

reference, supposedly empty, and the sample one) in a gradual way. These cells,

where our sample is collocated, are 40μl perforated and covered aluminum AL-

Crucibles (ME-27331) pans. Whenever some event that presupposes structural

rearrangement related to stability/instability phenomena’s and that consumes or

generates energy (heat) leading to change of temperature, it results in an alteration of

the electric potential between both cells since the temperature becomes different in

each cell. Two situations are possible: in the presence of an exothermic event, the



sample releases energy, raising the cell temperature; in the presence of an

endothermic event (as degradation), the sample takes energy, reducing the cell

temperature. For each situation, the machine answers in a way that the reference and

sample cell maintain the constant and gradual temperature raising (programmed

slope). To do so, the machine possesses two circuits with a resistance that transforms

electric energy in heat and vice-versa. For endothermic processes, the machine

calculates, hypothetically, the energy that would have to provide in order to reach the

same temperature of the reference cell. In exothermic processes, the machine

removes heat from both cells until the sample cell reaches the temperature that would

supposedly have with the programed slope and then calculates, hypothetically, the

energy necessary to provide to the reference cell so it would reach the temperature

correspondent to the slope programmed.

This hypothetical energy that is provided corresponds to the peaks in the

graphs. Meanwhile, the chamber where the samples are inserted is purged with

Nitrogen in order to drag gases that may be produced by sample heating and also to

distribute the temperature in a homogeneous way through the entire cell, to avoid

areas where the temperature is superior to others.

In all the analysis the process was always the same, where samples were

accurately weighed in a balance (Mettler PC440, Delta Range®) and heated in sealed

aluminium pans at a rate of 10°C/ min between 25°C and 250°C, under nitrogen

atmosphere. Empty aluminium pan was used as a reference.

6.2.6. Thermogravimetric Analysis

It is used to evaluate the possible degradation of the active substance and

excipients, which are subjected to a high temperature during HME. It consists in a

thermal balance that is subjected to increase heating. Weight loss vs. Temperature is

the evaluated parameter. The machine used was the model TGA 2050, from TA

instrument.



Referring to the TGA of Citric Acid, a powder sample of approximately 10mg CA

was accurately weighed in a balance (Mettler PC440, Delta Range®), placed into an

aluminum pan and inserted inside the furnace of the above described machine. The

percentage weight loss of the samples was monitored from 25 to 250°C employing a

heating rate of 10C°/min.

To better evaluate the extent of Citric Acid degradation at the extrusion

temperature, a powder sample of approximately 10mg CA was placed inside the

furnace and the percentage weight loss of the samples was monitored from 25 to

155°C employing a heating rate of 10°C/min. Then, an isothermal analysis at 155°C for

20 minutes was conducted.

6.2.7. Evaluation of possible degradation

A title was performed in order to access the amount of Lex-B present in the

extrudates from all formulations. It was used the same quantity of extrudates as in the

dissolution test and, as medium, methanol was chosen, to insure that all the drug

present was dissolved.

Similarly to what was accomplished in the preliminary tests, being the medium

methanol, there was the need to perform a screening with Lex-B on this medium.

Noticing that Citric Acid interferes in the readings, two Calibration Curves were

prepared, one to access the title of formulations A and C and the other to gain better

perception on Lex-B concentration in formulation B.

With both CC, the titles were executed. The samples prepared contained a

theoretical quantity of 28 mg/L of Lex-B.

Formulation B did not need a blank solution; Formulation A had a blank

solution consisting in 8.4 mg of Citric Acid in 100 ml of Methanol; and the blank

solution corresponding to Formulation C consisted in 2.1 mg of Citric Acid in 100 ml of

Methanol.



After analysing the results of the first title, to better understand if the possible

degradation occurs during the HME process or during the storage, it was made another

extrusion of formulation B and D. The title of its extrudates, as well as their physical

mixture, was performed in the same day, in MeOH and using the same spectrum. The

theoretical concentration is of 28 mg/L. For formulation B, the blank consisted in 4.2

mg of Citric Acid in 100 ml MeOH and for formulation D the blank consisted in 5.6 mg

of Fumaric Acid, also in 100 ml of MeOH. The polymer KVa64 had already proven that

it doesn’t interfere in the absorbance.

To evaluate the stability of the drug another title was performed after one

week. Because they had bigger concentrations of Lex-B, the first part of the extrudates

of Formulation B and D as well as the physical mixtures were chosen for this study and

were prepared in solutions with a final theoretical Lex-B concentration of 28 mg/L,

using MeOH as medium and reading with the same spectrophotometer.

For formulation B, the blank consisted in 4.2 mg of Citric Acid in 100 ml MeOH

and for formulation D the blank consisted in 5.6 mg of Fumaric Acid, also in 100 ml of

MeOH.

6.2.8. Assessment of Formulation B Dissolution Rates

A Calibration Curve with the API content of formulation B was made in

Phosphate Buffer in the same day as the extrusion was performed, in order to confirm

the veracity of the results of the dissolution rates previously shown. Four points were

chosen: From the Mother Solution, which consisted in 140 mg of extrudates (in the

form of a powder of 180-250 µm) of Formulation B in 500 ml of Phosphate Buffer, 4

dilutions were made, being these 1:2 (0.014mg/ml of Lex-B); 1:4 (0.007mg/ml); 1:5

(0.0056mg/ml) and 1:10 (0.0028mg/ml).



7. RESULTS AND DISCUSSION

7.1. Optimum wavelength

Previous work had shown that the solubility of Lex-B is influenced and

considerably variable, depending on the pH of the medium. In order to know the Lex-B

content of future samples, there was the need to perform Calibration Curves in

different mediums, being the ones of interest the Simulated Gastric Fluid and

Phosphate Buffer. But first it was necessary to discover the wavelength at which the

active substance had better absorption, therefore several scans were made in

Phosphate Buffer, SGF and Water and it was concluded that there are two optimum

wavelengths where Lex-B absorbs. These two peaks are at 242 nm and at 359 nm, the

first being better since the absorbance is higher and so, the mistake associated with

these readings is lower. Hence, 242 nm will be the wavelength chosen to make the

calibration curves, as well as all readings in the UV Spectrum.

7.2. Particle Size

After the CC’s were established, the first aspect that needed to be accessed,

regarding the dissolution of Lex-B, was if different particle sized powder showed

different dissolution rates. Accordingly, a dissolution test was accomplished and no

floating was observed, though some aggregates were formed in 2 vessels.

As it can be observed in the graphic further down, the use of Lex-B powder

particles in the size-range of 180-250 µm brings no significant improvements in the

dissolution rates. Even so, from this moment forward, studies would be carried out

using particles in this specific size-range.

7.3. Hot Melt Extrusion of Binary Mixture

The next step on improving the rates of dissolution of Lex-B consisted on

performing a Hot Melt Extrusion. For this matter, two polymers were chosen: Soluplus

and Kollidon Va64. Their capacity of forming a solid dispersion with Lex-B and increase

its dissolution rates was tested. Regarding the extrusion, Torque and ∆P were quite

similar for both polymers and the extrudates showed equivalent characteristics such as



Figure 1 - % Drug Release profile of Lex-B powder in SGF medium. 3 Vessels containing powder

particles with a size-range of 180-250µm and 3 Vessels containing unknown particle size powder.

being easily breakable, this eases the milling process necessary to obtain 180-250µm

powder for the dissolution tests that were next performed.

7.4. Dissolution Test of Binary Extrudates in SGF

As shown in the graphic, KVa64 showed better results on improving the rates of

dissolution of Lex-B. It reached the 85% (average) drug release in SGF after 3h and 84%

just in the first minutes. This was a crucial aspect in the choice of this polymer as the

carrier to be used in the HME process, in the next studies.

Because an extrusion is costing and time consuming, the necessity of this

process must be evaluated. To do so, a dissolution test with the physical mixture of

KVa64 and Lex-B in the same ratio and amount as used for extrusion, was

accomplished in the same processing conditions. As the graphic below indicates, the

physical mixture shows results that are not even better than the ones obtained with a

simple dissolution of Lex B powder and are certainly not as good as the ones obtained

with the extrudates, proving the benefits of extrusion.



Figure 3 - Comparison between Hot-Melt Extrudates and Physical Mixture of Lex-B/Kva64. Purple: HME; Green: PM

Figure 2 – Comparision between Kollidon Va64 and Soluplus, as polymers for HME , and its influence in the rates of dissolution of Lex-B



Figure 4 - % Drug Release profile of HME Lex-B/KVa64 (Vessels 1-3) and PM Lex-B/KVa64 (Vessels 4-

6) in PBS medium

7.5. Dissolution Test of Binary Extrudates and Physical Mixture in PBS

Now, this same experiences should be performed in PBS, the difficult and

challenging medium, where the extrusion process can be compared and proven to be

beneficial to the dissolution rates of Lex-B or not.

From this experiment, it can be concluded that, both extrudates and PM,

showed equally very poor results. The two drops of surfactant were not enough,

because aggregation continued to occur. The released amount using the extrudates

was circa 5-6% of the total amount.

Therefore, it was necessary to find another way to complement the HME

process and improve the rates of dissolution, some excipient that could be combined

with the polymer and active substance.



7.6. pH Modification

The pH-modification of a formulation could be a promising approach to

overcome the poor oral absorption of drugs with pH-dependent solubility [1]. In this

way, the next step consisted in the addition of an organic acid that could create an

acidic microenvironment, advantageous for the dissolution of the drug.

Comparing the acids, Siepe et al. studied the release of dipyridamole from

matrices containing organic acids (Fumaric Acid, Citric acid, Succinic Acid and Adipic

acid at 20% w/w). After 4 h of dissolution, CA and SA were almost completely released

(CA - 95.6%, SA - 93.9%), whereas a significant part of the initial amount of FA (28.4%)

was still present. The drug release values correlated with the order of organic acid

released being highest for FA.

FA, followed by CA, is most effective in modulating the micro-environmental pH

as it has higher acid strength (low pKa) and lower solubility, as a result of which it

remains in the matrix for a longer period of time.

Even so, Citric Acid was chosen. Mitra and co-workers explained that the

reason for selecting CA as a suitable pH-modifier was that it can reduce

microenvironmental pH efficiently; it has an acceptable safety profile after oral

administration and it has also high aqueous solubility (1,330 mg/ml in water). [1] It is

relatively stable at high temperatures and has a melting point of 153°C and boiling

point of 175°C. “Citric Acid Mono-Hydrated has been widely used as an acidifying

agent in solid oral dosage forms” (Siepe et al, 2006; Tatavarti and Hoag, 2006) and,

from all the acids in equation, it is the only one reported to be used successfully as a

pH modifying agent in HME [1].

It was also already shown that it could create micro pores that would increase

the surface of contact between the medium of dissolution and our API and, even more,

this organic acid also promotes thermal processability, matrix integrity by plasticization

of the polymer as well as better dispersion of the drug in it for the same reasons. [20]



Having chosen and theoretically justified the use of Citric Acid, complementary

thermal analysis to future extrudates should be done, in order to assure their stability

and certify this organic acid as the right choice. Regardless the results, an alternative

should be tested. From several organic acids that can be included in an oral dosage

form for humans, with similar pKa of Citric Acid (3.1) like Succinic Acid (Melting point –

184°C; pKa1 – 4.16; pKa2 – 5.61); Adipic Acid; Acetic Acid (pKa - 4.7); Ascorbic Acid and

Fumaric Acid, the choice fell upon the last since it has the most similar pKa and

because of all the above mentioned [22] [23]. See annex 6.

Studies proceeded in order to be able to see and compare the results of this

addition.

7.7. Hot Melt extrusion with Citric Acid

Having in mind that the extrusion conditions are the same used for the previous

extrusions, as previously said, concerning Formulation A, the ∆P/P1 values were 7/18

after 5 minutes of cycle and the extrudates were yellow, smooth, crystalline, glassy

aspect, easily breakable and bubbly in an homogeneous way, as seen in annex 7, figure

1.

Formulation B had the ∆P/P1 values of 9/21 after 5 minutes of cycle and the

extrudates were similar to formulation A, being yellow, glassy, smooth and easily

breakable but less bubbly as seen in annex 7, figure 2,3 and 4.

Formulation C had the ∆P/P1 values of 9/23 after 5 minutes of cycle and its

extrudates were characterized as yellow, crystalline, smooth, fragile, little sticky and

with few bubbles.

The bubbles are an important characteristic and they appear either because of

air incorporation in the mixture, water release due to the use of Citric Acid Mono-

Hydrated or degradation of Citric Acid and effervescent reaction. The last two

hypotheses are supported by the fact that the amount of bubbles seems proportional

to the quantity of Citric Acid present in the Formulation. However it must be confirmed

by Thermogravimetric Analysis and Differential scanning Calorimetry.



Figure 5 – % Drug Release profile of Formulation A1 and B1 extrudates in Phosphate Buffer medium, with 2 drops of Tween 20

So, with this processing conditions previously stated (same used to attain the

first extrudates) it was possible to obtain good extrudates that will undergo a

dissolution test to see the effect of the Citric Acid in the formulation. Two different

dissolution tests were made:

7.8. Dissolution Test of the formulations containing Citric Acid

In the first one, formulations A and B were tested. By the first minute, in some

vessels, there was a white powder that through the second minute was already

dissolved. The pH of the medium was measured before and after the dissolution to

access if the Citric Acid would alter the bulk pH, which it didn’t.

As it can be observed in the graphs, inclusion of Citric Acid allows an increase in

the percentage of drug release from circa 6% to circa 70% and 90% for formulation A

and B respectively, which means a 15 fold increase. In the first five minutes

formulation A had reached the 60% drug release and formulation B about 70%.



But how to explain the difference between both formulations? Well first of all

before this dissolution test it was proved that Citric Acid doesn’t interfere with the

reading, as well as the polymer, because they don’t absorb significantly at 242 nm in

this medium, which means that these values are only due to Lex-B dissolution

(Formulation A has 30% Citric Acid and 60% KVa64 whereas Formulation B has 15%

and 75% respectively).

At the beginning, it would be expectable the formulation with most Citric Acid

percentage to have better dissolution rates, but it didn’t happen that way, maybe

because there is an optimum percentage of Citric Acid above which the dissolution

rates are worse due to a lower percentage of polymer in the formulation. Remember

that the polymer is crucial for the improvement of dissolution since it allows the API to

dissolve on it, forming a solid solution. So the more polymer is present, the more API

can be dissolved. Another explanation is the possibility of degradation of Citric Acid,

leading to an unpredictable concentration of it by the end of the extrusion, and so

formulation B could actually have more Citric Acid content than formulation A. Further

studies should provide the answer.

In order to better understand the optimal percentage of Citric Acid to be used,

the second dissolution test was performed, in the same conditions as the previous one,

with extrudates of formulation C (3 Vessels with circa 140 mg each) and physical

mixture (3 Vessels with 56 mg each) of Citric Acid and Lex-B (3:1), to access if the use

of only Citric Acid mixed with the API improves dissolution.

In vessels 1 to 3, containing the extrudates, the materials dissolved quickly and

it could be seen in vessel 2 that there was a jelly substance sticking to the end of it, but

that after 5 minutes was dissolved. In vessels 4-6, the powder of the physical mixture

didn’t dissolve easily and some aggregates were formed, besides some powder sticking

to the bottom of the vessels and also some floating.



Figure 6 - % Drug Release profile of Formulation C extrudates (vessels 1-3) and Physical Mixture of Lex-B and Citric Acid (vessels 4-6)

The graphics show that the physical mixture of Citric Acid and Lex-B proved to

be more effective than the extrudates containing polymer and Lex-B only, which

possibly leads to the conclusion that the acidic in-situ microenvironment created by

the Citric Acid has impact on the dissolution of Lex-B but, because it is only a simple

mixture, the contact between the organic acid and the API is not at an extent that

would be considered significant, as it is on the extrusion, that is a solid solution.

Regarding the extrudates, formulation C led to 80% drug release after 2 hours.

This formulation is the second best in terms of improvement of the dissolution rates

comparing to the other two, and is the one with less Citric Acid.

What future studies need to accomplish is the determination of the optimal

Organic Acid/Polymer ratio, and for that purpose, maybe create a formulation with

10% acid.

Concerning the mechanism by which dissolution rates are improved, future

studies should be pointed towards the understanding of it. Now we can only formulate

certain hypotheses such as the creation of an in-situ acidic microenvironment with a



proper pH for the dissolution of our substance (the acidity of citric acid is sufficient to

maintain a low pH in the microenvironment of the API. Hence, a higher ratio of the API

will be present in the more soluble ionized form, resulting in a faster drug release);

Citric Acid and Lex-B can form a salt that improves the dissolution of Lex-B, although

the poor acidity of CA would pose an obstacle, corroborating this theory [20] [24] ;

increasing amounts of Citric Acid can also lead to a decrease (or even total loss) in

crystallinity of the soluble components, intensifying the amorphous character and

improving rates of dissolution; improved drug dispersion in the plasticized polymer

because of the plasticizer effect of Citric Acid in the polymer can also enhance its

solubility; increased polymer permeability and pore formation (the high aqueous

solubility of this acid will result on its rapid dissolution from the matrix, contributing to

the increase in the polymer’s porosity and drug diffusion through the water-filled

porous network) (Espinoza et al. and Peng et al., 2001) [20]

The first scenario seems more expectable and studies should go towards its

confirmation. In summary, the addition of CA MH as a release modifier and processing

aid to an insoluble drug–polymer system enabled the extrusion of an amorphous

matrix system exhibiting enhanced dissolution properties.

After the dissolution tests, emerged the need to confirm that there wasn’t any

Citric Acid or API degradation and that Lex-B concentration remained the theoretical

one of 14 mg/L. As a result, it was performed a DSC and a TGA as well as a title.



7.9. DSC Analysis of a Citric Acid Sample and Formulations A and B

Regarding the DSC analysis, which graphic is presented below, the first aspect

to emphasize is the fact that there are no peaks suitable to integrate.

Concerning the Sample of Citric Acid, there is clearly an endothermic “belly”

(large base peak) at around 50°C that most certainly indicates loss of water adsorbed

in the surface of the powder. There is another endothermic “peak” at 120°C which can

indicate loss of water (CA MH contains water molecules in its structure) and also

fusion. At circa 170°C we see another endothermic “peak” that surely indicates

degradation.

Comparing now the DSC of physical mixture and extrudates A1 and B1, and

having in consideration the conservation of the extrudates samples in a desiccator with

an atmosphere of P2O5, we can say that the hot melt extrusion process eliminates the

water content, that is still present in the physical mixture, in a proportional way,

depending on the content of Citric Acid. The fusion peak is present in all samples. The

physical mixture of formulation A, with bigger content of Citric Acid, shows a more

expressive degradation peak, as it was expectable. The endothermic “belly” at 50°C

disappears in HME and in the Physical Mixture. Being the theoretical content of Citric

Acid, the same both in physical mixture and in the extrudates, and comparing the

extent of the degradation peak present in both, especially in formulation A, it is

plausible to affirm that some degradation occurs or that, in some way and at some

level, a salt is formed between Citric Acid and Lex-B and so the temperature of

degradation changes, reducing the intensity of this peak.



!$AG_CA STD 16-4-14

AG_CA STD 16-4-14, 14,7800 mg

!$AG_HME LEXB/KVA64/CA 1/6/3

AG_HME LEXB/KVA64/CA 1/6/3, 13,0200 mg

!$AG_HME LEXB/KVA64/CA 1/7.5/1.5

AG_HME LEXB/KVA64/CA 1/7.5/1.5, 13,2200 mg

!$AG_PM LEXB/KVA64/CA 1/6/3 A1

AG_PM LEXB/KVA64/CA 1/6/3 A1, 12,4600 mg

!$AG_PM LEXB/KVA64/CA 1/7.5/1.5 B1

AG_PM LEXB/KVA64/CA 1/7.5/1.5 B1, 10,4300 mg

Wg -1

2

°C30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

^ exo

ST ARe SW 10. 00Lab: MET T LER

Figure 7 – DSC Analysis; Purple: Citric Acid Mono-Hydrated; Red: Physical Mixture of formulation A1; Black: Physical Mixture of formulation B1; Green: Extrudates of formulation A1; Blue: Extrudates of formulation B1

7.10. Thermogravimetric Analysis

Facing this possibility of Citric Acid degradation, to make sure it doesn’t occur

during the HME process and to see if the temperature of 155° could be maintained for

a while without losing Citric Acid, a TGA on a Citric Acid Mono-Hydrated sample was

performed and the results observed. As shown in the graphic, and in conformity with

the DSC analysis, there is a loss of weight of about 9,2% at 115°C, that doesn’t reach a

“plateau” because, at around 175°C, a bigger slope can be seen and the weight loss is

increased until about 90%. Citric Acid Mono-Hydrated has circa 9,2% of his weight

constituted by water molecules, so possibly, the weight loss that started at 115°C can

be described as water loss, which would explain the bubbles on the extrudates. To be

sure, it was next performed a more specific and precise analysis at the range of 115°C

for a longer time and with a lower increase in temperature per minute, allowing the

slope of weight loss to reach a “plateau”. In addition, a Karl Fisher test should be done

in order to access the water content of a CA sample.



Moreover, the TGA, also in accordance to the previous DSC, shows possible CA

degradation (80% weight loss) at 175°, that although higher than our working

temperature, it is quite close to it so it was better to do a TGA with an isothermal

analysis at 155° for 20 minutes and see the results.

The TGA was made and, as showed in the graphic, no significant degradation

occurs. The Citric Acid used was conserved in the oven for several weeks, reducing the

chances of being hydrated, opposing the one used in the first TGA, that was stored

under no specific conditions and so, was most certainly hydrated. Following this

thought, there isn’t any loss of weight at 115°C, which will complement our hypothesis

of that being the evaporation of the water content present in the sample used in the

first TGA. If this proved the presence of water content on the Citric Acid Mono-

Hydrated used on the first HME, the bubbles found in the extrudates would surely be

caused by water release. Another aspect is that, at 155°C, for 5 minutes, which is the

cycle time, there is only 2% weight loss. Nevertheless, we have to consider that since

the material enters the extruder until it gets out, more or less 10 minutes pass, and in

that case, 3.5% weight loss occurs. At the end of the 20 minutes, there is 5% of weight

loss. In accordance to literature, where it is described the melting point of 153°C, it

was observed that the rest of our sample had melt and was in the form of a

transparent liquid, instead of a brown one that would indicate burning.

The degradation or weight loss that is seen, by itself doesn’t represent a big

problem, but alerts us for the problems of using CA in processes that last longer than

20 minutes. This to conclude that, although 155°C doesn’t correspond to the

degradation temperature of Citric Acid, some loss of material happens and so, there

must be a rigorous control of the time that the material stays in the extruder. Future

studies should focus on lowering the temperature of extrusion when using

formulations with Citric Acid.



Figure 9 – TGA of a Citric Acid Sample. Range of temperature from 25°C to 155°C, with a ramp of 10°C/minute. Isothermal analysis at 155°C for 20 minutes

.

Figure 8 – TGA of a Citric Acid Mono-Hydrated Sample. Temperature range from 25°C to 250°C with a ramp of 10°C/minute



Figure 10 – TGA of a sample of Fumaric Acid. Temperature range from 25°C to 250°C with a ramp of 10°C/minute

Taking now in consideration the use of Fumaric Acid and since there is no

literature regarding the use of it in HME process, a TGA was essential to see if the

processing conditions at the extrusion could be maintained. As seen in the figure

above, besides having no water associated, the temperature of degradation of circa

178°C is similar to Citric Acid. According to literature, its melting point is 287°C.

7.11. Hot Melt Extrusion with Fumaric Acid

The HME process at 155°C and 30 rpm resulted in ∆P/P1 values of 3/6 and

generated extrudates coloured yellow, fragile, glassy, also with bubbles (which is very

odd possibly meaning air retention) and not very homogeneous, as seen in annex 7,

Figure 5 and 6.



0

10

20

30

40

50

60

70

80

90

100

-5 10 25 40 55 70 85 100 115 130 145 160 175 190 205 220 235 250

% Drug Release Correct

Chan. 1 HME

Chan. 2 HME

Chan. 3 HME

Figure 11 - % Drug Release profile of extrudates of Formulation D in PB with 2 drops of Tween 20, at 37°C

7.12. Dissolution test of the formulations containing Fumaric Acid

Then followed a dissolution test, where it could be observed that inside the

vessels with the extrudates, there was no floating and, similarly to Citric Acid, also all

the powder was dissolved in the first minutes. A slight “web”, maybe due to the

polymer, was formed. As expectable, in vessels 4 and 5, containing the physical

mixture, there was floating. To check if the pH bulk had changed with the addition of

Fumaric Acid, thus enhancing solubility of our API, or if an acidic micro-environment

was created, a pH measurement was performed in the medium of vessels 2 and 4 and

compared with the medium pH measured before adding the samples and starting the

dissolution. There was no change observed.

The drug release percentage at plateau is around 50% and, so, this formulation

showed worse results when comparing to Citric Acid in the attempt of improving the

dissolution rates of Lex-B, most probably due to a lower strength ability to change the

pH micro-environment.



7.13. DSC Analysis of Fumaric Acid Sample and Formulation D

To complement the analysis on the effect of Fumaric Acid, it was executed a

DSC to a Fumaric Acid sample and to Extrudates and Physical Mixture of Formulation

D.

Analysing the graphic above, several hypotheses can be formulated: Starting

with the sample of Fumaric Acid, although it has a quite high fusion point (287°C),

there is a very intense peak around 200ªC that can indicate degradation, which,

happening before the melting temperature is very rare, only if maybe it reacts with

some substance due to bad storage. Also, a deficient storage can cause isomeric

interconversion into Maleic Acid, which has a melting point of 135°C and this way, the

peak that is seen could be the fusion peak. Even if significantly lower than described in

literatures, this could also actually be the fusion peak of Fumaric Acid.

!&AG_HME LEXB/FA/KVA64 1/2/7

AG_HME LEXB/FA/KVA64 1/2/7, 8,9800 mg

!&AG_PM LEXB/FA/KVA64 1/2/7

AG_PM LEXB/FA/KVA64 1/2/7, 9,0100 mg

!&AG_FA STD 9-5-14

AG_FA STD 9-5-14, 14,1200 mg

Wg -1

0,5

°C30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

^ exo


Figure 12 - DSC Analysis to a FA sample (Black); HME Formulation D (Blue); PM Formulation D (Red)



PM LEX B/KVA64

HME LEX B/KVA64

KVA64 STD

LEX B STD

Wg -1

0,5

°C30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

^ exo


Figure 13 – DSC Analysis of Lex-B (Black); KVa64 (Red); PM Lex-B:KVa64 (Green); HME Lex-

B:KVa64 (Blue)

Analysing the physical mixture of Formulation D, the same fusion peak can be

seen, as well as a “belly” at the range of 60-110°C, indicating loss of water, since the

samples were not kept on a desiccator under P2O5 atmosphere.

Moving on with the analysis and now comparing the HME of Formulation D, it

can be seen that the fusion peak (in a lesser extent, maybe indicating some

degradation) and the water “belly” are still present. However, there are two new

peaks, at 45°C and 58°C. The one at 45°C represents the glass transition temperature,

it is called in fact “relaxation enthalpy” and usually happens with aged amorphous

compounds, which, in order to increase flexibility and space between chains,

consumes heat and generates an endothermic peak. This peak that is also seen in the

graphic below appears also in the DSC of polymer alone and also in the DSC of Physical

Mixture and Extrudates of Polymer and API alone. Nonetheless, its absence in the DSC

of Physical Mixture of Formulation D cares of explanation.

The peak at 58°C corresponds to a very low fusion point that occurs in result of

an eutectic mixture formed between Lex-B and Polymer.



7.14. Evaluation of possible degradation

As a final procedure, a title was performed in order to access the amount of

Lex-B present in the extrudates from all formulations and see if API degradation

occurred in the HME. The title would also allow the determination of the real

dissolution rates of our formulations (since 100% is considered the theoretical quantity

of 14 mg).

As previously said, this process started with a screening to know the optimal

wavelength on which Lex-B absorbs in methanol. As the figure below shows, there

were two peaks, at 237 nm and 353 nm, being the last one more reliable. Then, and

having in account the interference of CA in this medium, two CC were made and

presented below.

-0,5

0

0,5

1

1,5

2

2,5

3

0 200 400 600 800

Scan Methanol



~

Figure 14 – Scan performed in Methanol with a solution containing Lex-B

Figure 15 – Calibration Curve of Lex-B in Methanol: y= 9.9399x – 0.0055; to obtain the real title of Formulation A, C and D

y = 9,5774x + 0,0098 R² = 0,9999

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0 0,01 0,02 0,03 0,04 0,05

Series1

Linear (Series1)

Figure 16 – Calibration Curve of a Solution containing Lex-B, Citric Acid and Kva64 in Methanol: y= 9.5774 + 0.0098; to obtain the real title of Formulation B



With both Calibration Curves, it was possible to correlate the content of Lex-B

present in the extrudates that were in storage for more or less 1 month. The solutions

prepared contained a theoretical quantity of 28 mg/L of Lex-B, and hoping that there

was no degradation, the results correlated should be similar to this amount

And so, the title showed that Formulation A had 15.81 mg/L of Lex-B;

Formulation B had 21.02 mg/L and Formulation C had 18.83 mg/L.

This clearly indicates that there isn’t only some loss of API due to transfers

between recipients, there is clearly degradation. To understand if this degradation

occurs during the HME process or during the storage, another extrusion was made,

this time only of formulation B (because it presented best results) and of formulation D

(because it contains a different organic acid). The title of its extrudates, as well as their

physical mixture, was performed in the same day, so if there is degradation, clearly it

occurs during the HME process. Besides clarifying this matter of possible degradation,

this essay also had the intent to see how the API content is distributed along the

extrudates. The theoretical concentration is of 28 mg/L.

Taking this in account, and regarding formulation B, the physical mixtures

presented 27.48 mg/L; the first part of the extrudates had 25.34 mg/L; the middle part

23.81 mg/L and the last part to come out of the extruder showed 22.58 mg/L. Hence,

the physical mixture showed a concentration similar to the theoretical one (a bit lower

possibly due to loss of material during recipient transfers or during the weight process)

and this is what we should considerer the maximum content when comparing the

extrudates that, although presented lower content, it is just circa 2-5 mg lower which

simply could indicate loss of material in the walls of the extruder. It can also be said

that the API is more concentrated in the first part of the extrudates coming out of the

extruder.

As for Formulation D, and taking in consideration the use of Fumaric Acid in the

Blank solution, its physical mixture has a result even a bit higher than the theoretical

content (28.5 mg/L). However, when comparing the extrudates, the possibility of API

degradation seems certain; in the extrudates collected in the beginning of the process,



there was a 7 mg loss (21.31 mg/L) and by the end of the process, the extrudates

presented only 61.86% (17.63 mg/L) of the physical mixture content.

One week later, with the intent of understanding the stability of Lex-B during

storage, in the dark, for the same formulation’s extrudates and PM, a new title was

made, using the same spectrum, so the results could be comparable.

So, the Lex-B content on the physical mixture and extrudates after one week in

storage were the following: Physical mixture of formulation B presented 30.16 mg/L;

Formulation B had 26.23 mg/L; Physical mixture of formulation D showed 38.68 mg/L

and finally formulation D exhibited 19.85 mg/L.

As it can be seen, due to an analytical error or a spectrum malfunction, the

readings were significantly higher than acceptable (the maximum concentration should

be of 28 mg/L). Because all values were certainly inflated, there was the need of

executing normalization and transforming the readings of the physical mixtures (from

the previous title and this one) in 100%. So, the same day the extrusion was made, the

extrudates of formulation B had 92.2% of the concentration of the physical mixture

and had 86.97% after one week (approx. 5% degradation). The extrudates of

formulation D had 74.8% of Lex-B comparing to the physical mixture content in the

same day and after one week they had 51.32 % (approx. 23% degradation).

These results raise some concern, as they show high instability of Lex-B during

storage, especially for formulation D. Future studies concerning the stability of the

drug in storage should be taken in consideration, as well as ways to reduce its

instability.

7.15. Assessment of Formulation B Dissolution Rates

A Calibration Curve with the API content of formulation B was made in

Phosphate Buffer in the same day as the extrusion was performed, in order to confirm

the veracity of the results of the dissolution rates previously shown. So, using the data

from the previous dissolution test associating it with the new calibration curve resulted

in the graphic below.



Figure 18 – % Drug Release profile of Formulation B, using the data from the first dissolution test but with a different Calibration Curve

Figure 17 – Calibration Curve of Formulation B in Phosphate Buffer: y = 46.92x – 0.0088

It is quite clear that the drug percentage release is significantly lower than the

first results, but we can’t rely entirely in these data due to matters already discussed.

To access the maximum amount of Lex-B present in the extrudates and to obtain the

correct percentages of drug release, and having in consideration its instability in

storage, it should, in order to have best and precise results, be performed the title as

soon as the extrusion is finished and, right after, a dissolution test.



8. CONCLUSIONS

To sum up, the use of Organic Acids blended with the API and Polymer in a HME

process proved to be successful in increasing the drug’s rates of dissolution in PBS.

Considering parameters such as rates of dissolution, mechanism of action and

degradation and stability, the best organic acid between Citric and Fumaric Acid is

definitely the first one.

Regarding Citric Acid, the best proportion verified in this work happened with

formulation B: (10:15:75) API/CA/Polymer, however the ideal proportion may be yet

to be found and studies should focus on finding it, nonetheless one thing is sure, a

higher amount of Citric Acid doesn’t necessarily mean a better performance on

increasing the rates of dissolution.

This formulation led to a 15 fold increase in the rates of dissolution comparing

to the HME of a binary mixture (Lex-B/KVa64) in PBS. However it showed stability

problems with 8% degradation during the extrusion and 5% degradation while in

storage, for one week.

Although the mechanism of action isn’t perfectly clear, it most certainly relates

to the creation of an in situ microenvironment, or micro-pore formation, the acid could

also act as a plasticizer and even intensify the amorphous character of the mixture,

thus improving the rates of dissolution.

Since the correct way to determine the title of a solution as well as the real

dissolution profile is to perform these two operations as soon as the extrudates are

produced, we can’t entirely rely on the first results of the dissolution rates. The last

results indicate a 50% drug release, which is still a highly considerable amount that still

allows the following end statement: Organic Acids greatly enhances the solubility of

weak base dependent pH drugs while applied in a HME process.



9. FUTURE WORK

The art of formulation is merging into the science of physical laws to give a

result that is useful, cost-effective, does not require complex processing steps and,

overall, aims to provide therapeutic benefit in the form of pH-independent release

(MPH).

There is still much work to do regarding the topics discussed in this report,

much more digging and research to elaborate. For a start, concerning the extrusion

process, testing different working parameters (as different T°C or rpm) is required and

possibly beneficial since it may lead to more optimal extrusion conditions generating a

better solid dispersion.

There are many mechanisms by which organic acids enhance the rates of

dissolution. Studies should be pointed towards their understanding and should go

forward on this matter in order to discover and perfect the mechanism, thus

optimizing the rates of dissolution of our drug, in a pH independent manner.

Also more assessments should be done in order to find the optimal ratio

between Lex-B, polymer and acid that can lead to a maximum % drug release and

dissolution from the formulation.

Overcoming the problem of degradation that occurs during storage is also a

major issue that deserves attention and efforts. Preventing this degradation is crucial.

This report focused only on the solubility enhancement but, as was already

mentioned, the global purpose is to design and construct a formulation that would

allow an initial burst of Lex-B in the stomach and then a gradual release through the

intestine. This means that most certainly an enteric coating would have to be used and

so, more excipients should be added and compatibility studies as well as drug release



assays have to be performed. This pre-formulation development is far from finished

and should definitely be continued.

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11. ANNEX

Annex 1 - Schematic diagram of a single screw extruder

Annex 2 – Mechanism of action of pH-Modifiers



Annex 4 – Calibration Curve of a Lex-B Solution in SGF

Annex 3 – Calibration Curve of a Lex-B Solution in Phosphate Buffer



Annex 5 – Extruder HAAKE Minilab II, Thermo Scientific

Annex 6 - Main characteristics of Organic Acids that could possibly be used as a pH modifier excipient



Annex 7:

Figure 1 – Extrudates of Formulation A

Figure 2 - Extrudates of Formulation B; First part coming out of the extruder

Figure 3 - Extrudates of Formulation B; Second significant part coming out of the extruder

Figure 4 - Extrudates of Formulation B; Last significant part coming out of the extruder



Figure 5 - Extrudates of Formulation D; First part coming out of the extruder

Figure 6 - Extrudates of Formulation D; Last part coming out of the extruder

final report_andré riscado

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